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https://openalex.org/W2108961704
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https://europepmc.org/articles/pmc3723919?pdf=render
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Dosimetric accuracy and clinical quality of Acuros XB and AAA dose calculation algorithm for stereotactic and conventional lung volumetric modulated arc therapy plans
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Radiation oncology
| 2,013
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cc-by
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RESEARCH Open Access Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 * Correspondence: kroon.p@bvi.nl
1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB
Tilburg, the Netherlands
Full list of author information is available at the end of the article © 2013 Kroon et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Petra S Kroon1*, Sandra Hol2 and Marion Essers1 Abstract Introduction: The main aim of the current study was to assess the dosimetric accuracy and clinical quality of
volumetric modulated arc therapy (VMAT) plans for stereotactic (stage I) and conventional (stage III) lung cancer
treatments planned with Eclipse version 10.0 Anisotropic Analytical Algorithm (AAA) and Acuros XB (AXB) algorithm. Methods: The dosimetric impact of using AAA instead of AXB, and grid size 2.5 mm instead of 1.0 mm for VMAT
treatment plans was evaluated. The clinical plan quality of AXB VMAT was assessed using 45 stage I and 73 stage III
patients, and was compared with published results, planned with VMAT and hybrid-VMAT techniques. Results: The dosimetric impact on near-minimum PTV dose (D98%) using AAA instead of AXB was large (underdose
up to 12.3%) for stage I and very small (underdose up to 0.8%) for stage III lung treatments. There were no
significant differences for dose volume histogram (DVH) values between grid sizes. The calculation time was
significantly higher for AXB grid size 1.0 than 2.5 mm (p < 0.01). The clinical quality of the VMAT plans was at least
comparable with clinical qualities given in literature of lung treatment plans with VMAT and hybrid-VMAT
techniques. The average mean lung dose (MLD), lung V20Gy and V5Gy in this study were respectively 3.6 Gy, 4.1%
and 15.7% for 45 stage I patients and 12.4 Gy, 19.3% and 46.6% for 73 stage III lung patients. The average
contra-lateral lung dose V5Gy-cont was 35.6% for stage III patients. Conclusions: For stereotactic and conventional lung treatments, VMAT calculated with AXB grid size 2.5 mm
resulted in accurate dose calculations. No hybrid technique was needed to obtain the dose constraints. AXB is
recommended instead of AAA for avoiding serious overestimation of the minimum target doses compared to the
actual delivered dose. Keywords: AAA, Acuros XB, Plan quality, Stage I lung cancer, Stage III lung cancer, VMAT Dose calculations All calculations were performed using the treatment
planning system (TPS) Eclipse version 10 with beam
algorithms AAA and AXB (Varian Medical Systems,
Palo Alto, CA), which was installed on a standard
clinical workstation (Dell T5500) with dual 2.40 GHz
quad-core Intel processors (E5620), 24 GB RAM, and a
64 bit Windows 7 operating system, in a distributed
calculation framework (DCF) network of 3 worksta-
tions. The dose reporting mode dose-to-medium Dm
was selected for AXB. Immediately after clinical introduction of the Varian
Eclipse software, we also clinically introduced VMAT for
lung SBRT stage I NSCLC as well as for lung stage III
treatments. For this clinical introduction, we investigated
the dosimetric accuracy and quality of stereotactic and
conventional VMAT planning in Eclipse using AXB and
AAA. Routinely, for all our patients, we perform pre-
treatment verification measurements using an ionization
chamber in the isocentre, combined with film measure-
ments in the isocentre plane. Background could achieve at least comparable clinical plan qualities
and skin dose levels than intensity modulated radio-
therapy (IMRT) [10-12] and can successfully be used for
stereotactic body radiotherapy (SBRT) for patients with
stage I Non-Small-Cell Lung Cancer (NSCLC) [2,3,5]. Volumetric modulated arc therapy (VMAT) has been
shown to be a powerful technique for irradiation of many
treatment sites with obtaining higher dose conformity to
the tumor while decreasing intra-fraction movements
because of shorter delivery times [1-8]. Reliable and accu-
rate dose delivery can be obtained using VMAT as shown
by pre-treatment dosimetric plan validations [9]. VMAT In case of stage III large tumor lung cancers, it is diffi-
cult to limit doses to organs at risks (OARs) such as heart
and lung. High doses are preferred since local control in-
creased significantly (p = 0.02) when patients are treated
with higher doses than 64 Gy [13]. Rengan et al. [13]
stated that the median survival time for patients treated to
64 Gy or higher was 20 months versus 15 months for * Correspondence: kroon.p@bvi.nl
1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB
Tilburg, the Netherlands
Full list of author information is available at the end of the article Page 2 of 8 Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 those treated to less than 64 Gy. Advanced planning tech-
niques, IMRT and VMAT, have been shown to be able to
increase the therapeutic dose with equal toxicity profiles
compared to three-dimensional conformal radiotherapy
(3DCFRT) [14]. Unfortunately, it is not always possible to
deliver doses higher than 60 Gy to the planning target
volume (PTV) using 3DCFRT, IMRT and VMAT because
of dose limiting organs [14]. De Bree-Balk et al. [14] stated
that possibly further improvements could be made by
using hybrid techniques which combine two static fields
with IMRT or VMAT, as also investigated by Verbakel
et al. [15], who made a comparison between conventional
static field plans, IMRT, hybrid-IMRT, VMAT and hybrid-
VMAT; and by Chan et al. [16], who compared between
3DCFRT, VMAT and hybrid-VMAT. In both studies the
VMAT plans consisted of at least 2 arcs and the hybrid
plans of a combination of two static fields and IMRT or
VMAT. They have concluded that hybrid techniques are
superior in dosimetric outcomes for treating stage III lung
tumours compared to the other techniques. Background The treat-
ment planning for these studies has been performed using
Varian Eclipse treatment planning version 8 or 10 with
Anisotropic Analytical Algorithm (AAA). stage III lung tumors (average PTV 690 cm3), Fogliata et al. [26] have illustrated that AAA leads to a monitor unit
underestimation of approximately 1-2% relative to AXB
grid size 2.5 mm using a treatment planning comparison
between 3DCFRT, IMRT and VMAT. Consequently, this suggests that AAA could overesti-
mate the minimum target dose, which leads to lower
target coverage than the prescribed dose, in case AXB
represents the real dose distribution. Kan et al. [27] have
illustrated that AXB was more accurate in predicting
secondary build-up near and beyond air/tissue interfaces
than AAA, using a comparison with measurements. Bush et al. [22] have shown that AXB was capable of
modelling radiotherapy dose deposition in the low dens-
ity regions. Dose distributions calculated by AXB were
in good agreement with BEAMnrc/DOSXYZnrc Monte
Carlo dose calculations. The purpose of this study was threefold. First, we
wanted to justify the assumption that AXB represents bet-
ter the real dose distribution than AAA by cross-checking
the finding of Bush et al. [22] with measurements, for
calculation grid sizes of 1.0 mm and 2.5 mm. Second, we
investigated the dosimetric impact of using AAA instead
of AXB, and grid size 2.5 mm instead of 1.0 mm for
VMAT treatment plans for stage I and stage III lung
tumors. Third, we investigated whether using AXB calcu-
lations in Eclipse version 10.0, VMAT results in improved
treatment plans compared to VMAT and hybrid-VMAT
plans published in literature, again for stage I (lung stereo-
tactic) and stage III (conventional lung) treatments. We have recently introduced the Varian Eclipse software
version 10, with the AAA as well as the Acuros XB (AXB)
algorithm for photon dose calculations in our institute. AXB solves the linear Boltzmann transport equation e.g.,
[17]. The dosimetric accuracy of AXB has already been in-
vestigated in several studies [18-22]. Fogliata et al. [19,21]
have concluded that AXB gives acceptable characteristics
in homogeneous media for small and large fields (range
0.8×0.8 to 40.0×40.0 cm2) using comparisons of AXB with
AAA and measurements. In heterogeneous situations, the
AXB algorithm has been shown to provide a valid and ac-
curate alternative to Monte Carlo calculations for field
sizes ranging from 2.5×2.5 to 30.0×30.0 cm2 [18,20,22]. Treatment planning For the algorithm comparison, optimal VMAT plans
were prepared using AAA algorithm grid size 2.5 mm
consisting of two 180 degrees arcs using the VMAT
optimization tool. Dose prescription was 54 Gy in 5 frac-
tions to the 80% isodose, which covered at least 99% of
the PTV, for stage I, and for stage III 66 Gy in 33 frac-
tions, where at least 99% of the PTV received 90% of the
prescribed dose. For stage III, an additional constraint of
D≤1% = 72.6 Gy (110% of prescribed dose) was used and
the dose to OARs, such as lung and heart, was opti-
mized. The plans were recalculated using the same beam
settings and monitor units as the AAA grid size 2.5 mm
treatment plans for AAA grid size 1.0 mm, AXB grid
size 2.5 mm and AXB grid size 1.0 mm. Therefore, it is now shown by simulations and measure-
ments that calculations using AXB represent better the
actual delivered dose distribution in case of narrow beams
and heterogeneous situations than AAA. Of course, in
most patients real lung density will be larger than the low
density taken in this study (ρ = 0.03 g cm-3), and differences
between AXB and AAA will be smaller as also stated by
Bush et al. [22]. In the VMAT optimization process, a Clinical Protocol
was used (and if necessary, optimized interactively) with
Optimization Objectives for the PTV, heart, lungs and
spinal cord (with a margin of 5 mm). Most dose volume
histogram (DVH) objectives had a priority of 50, whereas
the minimum and maximum PTV dose, defined as D100%
and D0%, had a priority of 250 and the maximum spinal
cord dose (10 Gy for stage I and 44 Gy for stage III) had a
priority of 150 or 200. For stage III VMAT plans, a MLD
objective of 12 Gy with a priority of 200 was applied; and
a contra-lateral lung dose V5Gy-cont objective of 30% or
25% was used with a priority of respectively 450 or 500 for
the 73 stage III patients. This study used the volume of
both lungs minus internal target volume (ITV) for the cal-
culation of MLD, and volume of both lungs minus PTV Justification of the assumption that AXB represents the
real dose distribution The measurements and
Eclipse dose calculations were performed for a field size of
1.0×1.0 cm2 and 4.0×4.0 cm2 using a 6 MV photon beam
(Clinac 2100iX equipped with a Millenium 120-MLC,
Varian Medical Systems, Palo Alto, CA) of 200 monitor
units and a source to skin distance (SSD) of 100 cm. Justification of the assumption that AXB represents the
real dose distribution The analyses in this study to cross-check the findings by
Bush et al. [22] confirmed that AXB is much more ac-
curate in heterogeneous situations than AAA (Figure 1). For single fields, there were no large differences between
grid size 2.5 mm and grid size 1.0 mm. The relative
doses were much higher for AAA than AXB in the mid-
dle low density layer (ρ = 0.03 g cm-3) of the interface
phantom. The difference in relative doses were even
larger than 20% for a field size of 1.0×1.0 cm2 and larger
than 10% for a field size of 4.0×4.0 cm2. Bush et al. [22]
have also published large relative dose differences, e.g. larger than 30% for a very low density media (ρ =
0.001 g cm-3) and a field size of 4.0×4.0 cm2. Justification of the assumption that AXB represents the
real dose distribution It was already shown by Gete et al. [23] that AAA cal-
culations can slightly overestimate the minimum PTV
dose relative to Monte Carlo calculations with BEAMnrc/
DOSXYZnrc for stage I lung tumors (PTV range between
19 to 62 cm3) with forward planning with multiple static
non-coplanar conformal fields. It has also been shown by
VMAT comparison studies that AXB leads to a slightly
more accurate dose distribution than AAA [24,25]. For Before performing the main parts of our study, we vali-
dated the findings of Bush et al. [22] that AXB repre-
sents the actual dose delivery by performing percentage
depth dose (PDD) measurements using EBT2 film (ISP,
Wayne, NJ) in a simple heterogeneous interface phan-
tom, which consisted of three layers: upper and lower of
polystyrene with a density of ρ = 1.05 g cm-3 and thick-
ness of 5 and 7 cm, respectively, and a middle layer of Page 3 of 8 Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 for V20Gy, and V5Gy. Most of the time, we applied a
“Normal Tissue Objective” with a fall-off of 4 for stage I
stereotactic treatments and of 1 for stage III conven-
tional lung treatments. A help volume with a margin of
5 mm around the PTV was introduced with also re-
latively high priorities when OARs were not spared suf-
ficiently. It was almost never necessary to include
constraints for the other OARs. foam, with a low-density ρ = 0.03 g cm-3 and a thickness
of 8 cm. This very low density was chosen to investigate
the accuracy of both algorithms in a very extreme situ-
ation analogue to Bush et al. [22]. The measurements and
Eclipse dose calculations were performed for a field size of
1.0×1.0 cm2 and 4.0×4.0 cm2 using a 6 MV photon beam
(Clinac 2100iX equipped with a Millenium 120-MLC,
Varian Medical Systems, Palo Alto, CA) of 200 monitor
units and a source to skin distance (SSD) of 100 cm. foam, with a low-density ρ = 0.03 g cm-3 and a thickness
of 8 cm. This very low density was chosen to investigate
the accuracy of both algorithms in a very extreme situ-
ation analogue to Bush et al. [22]. Patient selection Dose volume histograms were produced for all plans in
order to analyze the doses to the PTV and OARs. The
statistical differences were tested for the VMAT treat-
ment plans between AAA grid size 2.5 mm, AAA grid
size 1.0 mm, AXB grid size 2.5 mm and AXB grid size
1.0 mm. The statistical significance of the differences
was tested with a paired two-tailed student t-test with
significant level p < 0.05. Eight stage I and seven stage III lung patients were se-
lected to investigate the dosimetric impact of using
AAA instead of AXB, and grid size 2.5 mm instead of
1.0 mm, for dose calculations of VMAT treatment plans. The average PTV volume was 24.0 cm3 (range 5.1 to
56.9 cm3) for stage I and 418.9 cm 3 (range 140.3 to
762.6 cm3) for stage III tumors. OARs were delineated
including heart and contra-lateral lung. The clinical
quality of the VMAT plans was assessed using 45 stage I
lung patients (average PTV 35.4 cm3; range 5.5 to
175.4 cm3 ) and 73 stage III lung patients (average PTV
344.4 cm3; range 25.1 to 1069.0 cm3). Dosimetric impact of algorithms and grid sizes on clinical
lung VMAT plans Dose volume histograms were produced for all plans so
that the relative differences between algorithms (AAA and
AXB) and grid sizes (2.5 and 1.0 mm) could be analyzed. The dose calculation time by AAA grid size 1.0 mm in
comparison to AAA grid size 2.5 mm, AXB grid size
2.5 mm and AXB grid size 1.0 was much larger. For ex-
ample for stage I, the calculation times were 7, 10 and 3
times larger with AAA grid size 1.0 mm in comparison to Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 Page 4 of 8 Figure 1 Measured and calculated percentage depth dose curves in a slab-phantom. Predicted percentage depth dose curves by AAA and
Acuros XB grid sizes 2.5 and 1.0 mm compared to measured data using a slab-phantom with (top) 1.0x1.0 cm2 and (bottom) 4.0x4.0 cm2 6 MV
AP photon beams. Figure 1 Measured and calculated percentage depth dose curves in a slab-phantom. Predicted percentage depth dose curves by AAA and
Acuros XB grid sizes 2.5 and 1.0 mm compared to measured data using a slab-phantom with (top) 1.0x1.0 cm2 and (bottom) 4.0x4.0 cm2 6 MV
AP photon beams. AAA grid size 2.5 mm, AXB grid size 2.5 mm and AXB
grid size 1.0 mm. We have decided to focus on the dose
distribution differences between AAA grid size 2.5 mm
and AXB grid size 2.5 mm; and on the dose distribution
differences between AXB grid size 2.5 mm and AXB grid
size 1.0 mm since AAA grid size 1.0 mm will probably
never be used clinically and the differences with AAA grid
size 2.5 mm were very small. AAA grid size 2.5 mm, AXB grid size 2.5 mm and AXB
grid size 1.0 mm. We have decided to focus on the dose
distribution differences between AAA grid size 2.5 mm
and AXB grid size 2.5 mm; and on the dose distribution
differences between AXB grid size 2.5 mm and AXB grid
size 1.0 mm since AAA grid size 1.0 mm will probably
never be used clinically and the differences with AAA grid
size 2.5 mm were very small. a result, AAA grid size 2.5 mm treatment plans might
be clinically approved with the real V20Gy being slightly
too high. Dosimetric impact of algorithms and grid sizes on clinical
lung VMAT plans The differences in lung doses calculated by
AAA and AXB were patient dependent since they were
dependent on field size, location of the target inside the
lung and the density of the lung. Depending on the ac-
tual combination of field size, target location and lung
density, AAA can over- or underestimate the lung dose,
as also stated by Bush et al. [22]. The relative dose differ-
ences between both models for the other OARs were
small. However, there was a large difference in calculation
time between both algorithms. The dose calculation times
required by AAA grid size 2.5 mm were about 2 times the
calculation times required by AXB grid size 2.5 mm. A summary of the relative dose differences between
algorithms and grid sizes is given in Table 1. For individual
stage I lung patients differences occurred in dose distribu-
tions between AXB and AAA. The maximum difference
of near-minimum PTV dose (D98%) between AXB grid
size 2.5 mm and AAA grid size 2.5 mm was −7.1 Gy
corresponding to a relative difference of −12.3%, which
indicated a serious underdosage of the delivered dose
when this patient would have been planned with the AAA
algorithm (Table 1, Figure 2). This finding is similar to re-
sults published by Gete et al. [23]. They observed a 12.8%
lower minimum PTV dose using Monte Carlo simulations
than using AAA version 8.6 calculations for one patient
plan. These relative differences can be explained by the
significantly improved accuracy of AXB under the con-
ditions of electronic disequilibrium compared to AAA. AAA only predicts little secondary build-up at regions
beyond low-density media like lung [22,26,27]. In case of stage III patients, the difference in PTV
doses were smaller than in case of stage I patients
(Table 1, Figure 1) due to, e.g. larger fields and tumor
sizes, resulting in smaller errors in the AAA algorithm. The differences in OAR doses were also small. There
was again a significant (p < 0.01) difference in calculation
time between AAA and AXB. The average final dose
calculation time was respectively, 6 and 4 minutes, for
AAA grid size 2.5 mm and AXB grid size 2.5 mm. Dosimetric impact of algorithms and grid sizes on clinical
lung VMAT plans Table 1 Relative differences between algorithms and grid sizes for lung cancer patients Table 1 Relative differences between algorithms and grid sizes for lung cancer patients
Relative difference
p-value
Relative Table 1 Relative differences between algorithms and grid sizes for lung cancer patients
Relative difference
p-value
Relative d
(AXB2.5 -AAA2.5)/ AAA2.5×100%
(AXB1.0 - AXB2.5 *Significant relative difference tested by two-tailed student t-test. that the plan qualities were at least comparable. For
example, McGrath et al. [3] have published an average
MLD for stage I tumors of 4.6 Gy (with dose prescrip-
tion: 99% of the PTVITV has to receive more than
43.2 Gy) with VMAT. In our study the average MLD
was 3.6 Gy (with a higher dose description: 99% of the
PTV has to receive more than 54.0 Gy). stage I patients with grid size 2.5 mm and 1.0 mm, re-
spectively, and 4 and 27 minutes for stage III patients
with grid size 2.5 mm and 1.0 mm. Therefore, we recommend using AXB grid size 2.5 mm
for VMAT planning of lung tumors, since this results in
accurate dose values with acceptable calculation times. For stage III patients, the clinical quality of treatment
plans is a trade-off between high doses to gross tumour
volume and limiting treatment related pneumonitis
(TRP). Doses higher than 64 Gy are preferred [13] which
could be obtained for all 73 clinical patients in this
study. Simultaneously, lung doses should be minimized
since these influenced the post-radiation acute TRP. Sev-
eral studies describe analyses to determine indicators for
predicting TRP [29-33]. Different predictors were indi-
cated in these studies, e.g. V5Gy-cont by Song et al. [29],
V5Gy by Wang et al. [30], MLD and V30Gy by Kim et al. [31], and V10Gy by Shi et al. [32] and Spych et al. [33]. Song et al. [29] have shown with an extensive multivari-
ate analysis including V5Gy, V10Gy, V13Gy, V15Gy, V20Gy
and MLD for total lung, ipsilateral lung and contra- Dosimetric impact of algorithms and grid sizes on clinical
lung VMAT plans Only small significant differences were found between
AXB grid size 2.5 mm and AXB grid size 1.0 mm for
near-maximum PTV dose (D2%) (Table 1, Figure 2),
while the calculation time will increase drastically when
grid size 1.0 mm was used instead of grid size 2.5 mm. The average calculation time was 2 and 8 minutes for AXB showed a slightly but significant (p < 0.05) higher
V20Gy (volume of both lungs minus PTV with a dose of
20 Gy or more) value than AAA for stage I patients. As Page 5 of 8 Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 Table 1 Relative differences between algorithms and grid sizes for lung cancer patients
Relative difference
p-value
Relative difference
p-value
(AXB2.5 -AAA2.5)/ AAA2.5×100%
(AXB1.0 - AXB2.5)/ AXB2.5×100%
Average ± SDV (min-max)
Average ± SDV (min-max)
Stage I (N = 8)
PTV
D98%
−3.2% ± 4.0% (−12.3% – 0.5%)
0.06
0.7% ± 1.3% (−0.8% – 3.1%)
0.19
D2%
0.2% ± 1.2% (−2.1% – 1.3%)
0.69
0.9% ± 0.4% (0.3% – 1.4%)
<0.01*
Dmean
−0.6% ± 2.2% (−4.9% – 1.6%)
0.46
0.2% ± 0.5% (−0.6% – 1.2%)
0.45
Total lung
V5Gy
1.3% ± 1.9% (−1.9% –3.2%)
0.12
0.1% ± 0.6% (−0.8% – 1.1%)
0.73
V20Gy
2.0% ± 2.3% (0.0% – 5.7%)
0.05*
−0.7% ± 1.2% (−2.7% – 0.0%)
0.17
MLD
0.0% ± 0.5% (−0.7% – 0.7%)
0.60
0.1% ± 0.2% (0.0% – 0.4%)
0.08
Time
Calculation
−39.2% ± 13.5% (−50.3% – –8.2%)
<0.01*
304.7% ± 48.7% (226.9% – 345.8%)
<0.01*
Stage III (N = 7)
PTV
D98%
−0.3% ± 0.7% (−0.8% – 1.3%)
0.33
0.2% ± 0.8% (−1.4% – 0.9%)
0.57
D2%
−0.6% ± 2.0% (−2.1% – 3.0%)
0.47
0.6% ± 0.4% (−0.1% – 1.0%)
<0.01*
Dmean
−0.8% ± 0.7% (−1.6% – 0.3%)
0.02*
0.1% ± 0.3% (−0.5% – 0.4%)
0.40
Total lung
V5Gy
−1.8% ± 1.7% (−4.8% – 0.3%)
0.03*
−0.4% ± 0.7% (−1.5% – 0.4%)
0.16
V20Gy
0.2% ± 0.7% (−0.5% – 1.5%)
0.69
0.0% ± 0.0% (0.0% –0.0%)
1.00
MLD
−0.5% ± 0.6% (−1.3% – 0.0%)
0.09
0.2% ± 0.3% (0.0% – 0.6% )
0.17
Time
Calculation
−31.4% ± 9.9% (−44.4% – –17.9%)
<0.01*
556.9% ± 91.1% (412.3% – 646.0%)
<0.01*
*Significant relative difference tested by two-tailed student t-test. Clinical quality of VMAT The dose volume quantities of 45 stage I and 73 stage III
lung patients planned with VMAT AXB grid size 2.5 mm
were analyzed. The average MLD, total lung V20Gy and
total lung V5Gy were 3.6 Gy, 4.1% and 15.7% for stage I
and 12.4 Gy, 19.3% and 46.6% for stage III. The pre-
scribed dose of 66 Gy could be delivered to all patients. The MLD was between 16.0 and 18.5 Gy for 15% of the
stage III patients, for all other patients, the MLD was
lower than 16 Gy. It was difficult to compare the obtained plan qualities
with literature since different patients were used. How-
ever, when we compared the dose volume quantities
with published studies about VMAT techniques for stage
I and stage III NSCLC patients [2,3,16,28], we concluded Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 Page 6 of 8 Figure 2 Dose volume histogram comparisons of PTV doses between algorithms and grid sizes. Comparison of dose volume histograms
of the planning target volume (PTV) between AAA grid size 2.5 mm, Acuros XB grid size 2.5 mm and Acuros XB grid size 1.0 mm of (top) a stage
I and (bottom) a stage III lung patient. The black vertical lines indicate the prescription: The dose that has to cover 99% of the PTV, respectively,
54.0 Gy for stage I and 59.4 Gy for stage III. Figure 2 Dose volume histogram comparisons of PTV doses between algorithms and grid sizes. Comparison of dose volume histograms
of the planning target volume (PTV) between AAA grid size 2.5 mm, Acuros XB grid size 2.5 mm and Acuros XB grid size 1.0 mm of (top) a stage
I and (bottom) a stage III lung patient. The black vertical lines indicate the prescription: The dose that has to cover 99% of the PTV, respectively,
54.0 Gy for stage I and 59.4 Gy for stage III. lateral lung that V5Gy-cont was the only remaining signifi-
cant factor associated with TRP. They concluded that
V5Gy-cont should be kept as low as possible and they sug-
gested a cut-off value of 60% since incidences of grade ≥3
pneumonitis were 35% and 0%, respectively, for V5Gy-cont ≤
60% and V5Gy-cont > 60% (p = 0.01). for only one patient. Kim et al. g
0Gy
95%
0 %
2 [15] Use the volume of both lungs minus PTV for V20Gy; PTV V95% > 97% and PTV V107% < 5%.
3 g
20Gy
5Gy
98%
y
2%
y
4 This study uses the volume of both lungs minus ITV for MLD, and volume of both lungs minus PTV for V20Gy and V5Gy.
PTV V90% > 99% and PTV V110% ≤1%. y
3 [16] Use the volume of both lungs minus PTV for MLD, V20Gy and V5Gy; D98% > 57.0 Gy and D2% < 64.2 Gy. erage values and standard deviations are given.
4] Use the volume of both lungs minus CTV for MLD and V20Gy; PTV V95% > 99% and PTV V107% < 1%. Average values and standard deviations are given.
1 [
]
h
l
f b
h l
f
d
d Average values and standard deviations are given.
1 [14] Use the volume of both lungs minus CTV for MLD and V20Gy; PTV V95% > 99% and PTV V107% < 1%. 5 Median instead of average value is given. g
20Gy;
95%
107%
e of both lungs minus PTV for V20Gy; PTV V95% > 97% and PTV V107% < 5%. Average values and standard deviations are given Conclusions We investigated the quality of VMAT treatment plans
using Eclipse treatment planning system version 10.0 for
stage I and III lung patients. All plans consisted of 2
partial arcs of 180 degrees. We showed that the AXB
calculation algorithm was preferable to AAA since pos-
sible PTV underdosage, as a result of inaccurate AAA
calculations, can be avoided. In addition, the calculation
time was much shorter for AXB. For clinical VMAT
lung plans, the quality and accuracy of AXB grid size
2.5 mm was comparable with AXB grid size 1.0 mm. However, the calculation time increases drastically when
grid size 1.0 mm was used. 7. Mancosu P, Castiglioni S, Reggiori G, Catalano M, Alongi F, Pellegrini C,
Arcangeli S, Tozzi A, Lobefalo F, Fogliata A, Navarria P, Cozzi L, Scorsetti
M: Stereotactic body radiation therapy for liver tumours using
flattening filter free beam: dosimetric and technical considerations. Radiat Oncol 2012, 7:16. 7. Mancosu P, Castiglioni S, Reggiori G, Catalano M, Alongi F, Pellegrini C,
Arcangeli S, Tozzi A, Lobefalo F, Fogliata A, Navarria P, Cozzi L, Scorsetti
M: Stereotactic body radiation therapy for liver tumours using
flattening filter free beam: dosimetric and technical considerations. Radiat Oncol 2012, 7:16. 8. Scorsetti M, Bignardi M, Alongi F, Fogliata A, Mancosu P, Navarria P, Castiglioni S,
Pentimalli S, Tozzi A, Cozzi L: Stereotactic body radiation therapy for abdominal
targets using volumetric intensity modulated arc therapy with RapidArc:
Feasibility and clinical preliminary results. Acta Oncol 2011, 50:528–538. Feasibility and clinical preliminary results. Acta Oncol 2011, 50:528–538. 9. Fogliata A, Clivio A, Fenoglietto P, Hrbacek J, Kloeck S, Lattuada P, Mancosu P,
Nicolini G, Parietti E, Urso G, Vanetti E, Cozzi L: Quality assurance of RapidArc
in clinical practice using portal dosimetry. Br J Radiol 2011, 84:534–545. 10. Seppala J, Suilamo S, Kulmala J, Mali P, Minn H: A dosimetric phantom
study of dose accuracy and build-up effects using IMRT and RapidArc in
stereotactic irradiation of lung tumours. Radiat Oncol 2012, 7:79. 11. Bertelsen A, Hansen O, Brink C: Does VMAT for treatment of NSCLC
patients increase the risk of pneumonitis compared to IMRT? – A
planning study. Acta Oncol 2012, 51:752–758. The VMAT plans were compared to published treat-
ment planning studies. The clinical VMAT AXB grid size
2.5 mm plans obtained in this study were at least compar-
able to the published planning studies, e.g. Authors’ contributions PK designed the set-up of the study, performed the data collection and
analyses, carried out the comparisons and drafted the manuscript. SH
generated most of the treatment plans. ME supervised the project, helped to
draft the manuscript, participated in the design of the study, data collection,
treatment planning and analyses. All authors read and approved the final
manuscript. 15. Verbakel WFAR, Van Reij E, Ladenius-Lischer I, Cuijpers JP, Slotman BJ, Senan
S: Clinical application of a novel hybrid intensity-modulated
radiotherapy technique for stage III lung cancer and dosimetric
comparison with four other techniques. Int J Radiation Oncol Biol Phys
2012, 83(Suppl 2):297–303. Received: 8 February 2013 Accepted: 3 June 2013
Published: 24 June 2013 Received: 8 February 2013 Accepted: 3 June 2013
Published: 24 June 2013 Received: 8 February 2013 Accepted: 3 June 2013
Published: 24 June 2013 Received: 8 February 2013 Accepted: 3 June 2013
Published: 24 June 2013 study were at least comparable to the plans obtained
with VMAT and hybrid-VMAT discussed in literature
[14-16]. Historically,
all
our
stage
III
plans
were
normalised as: 99% of the PTV is covered by 90% of the
prescribed dose (66 Gy). Our normalisation is in close
agreement with the recommended normalisation method
by ICRU 83 [34], to the mean PTV dose. For our stage III
patients, the mean PTV dose was 101.2% ± 1.4%. This im-
plies that our normal tissue DVH values will even be
slightly better when we use ICRU 83 for normalisation in
the future. References
V Zhang GG, Ku L, Dilling TJ, Stevens CW, Zhang RR, Li W, Feygelman V:
Volumetric modulated arc planning for lung stereotactic body
radiotherapy using conventional and unflattened photon beams: a
dosimetric comparison with 3D technique. Radiat Oncol 2011, 6:152. 6. Wolff D, Stieler F, Welzel G, Lorenz F, Abo-Madyan Y, Mai S, Herskind C,
Polednik M, Steil V, Wenz F, Lohr F: Volumetric modulated arc therapy
(VMAT) vs. serial tomotherapy, step-and-shoot IMRT and 3D-conformal RT
for treatment of prostate cancer. Radiother Oncol 2009, 93(Suppl 2):226–233. 7. Mancosu P, Castiglioni S, Reggiori G, Catalano M, Alongi F, Pellegrini C,
Arcangeli S, Tozzi A, Lobefalo F, Fogliata A, Navarria P, Cozzi L, Scorsetti
M: Stereotactic body radiation therapy for liver tumours using
flattening filter free beam: dosimetric and technical considerations. Radiat Oncol 2012, 7:16. 5. Zhang GG, Ku L, Dilling TJ, Stevens CW, Zhang RR, Li W, Feygelman V:
Volumetric modulated arc planning for lung stereotactic body
radiotherapy using conventional and unflattened photon beams: a
dosimetric comparison with 3D technique. Radiat Oncol 2011, 6:152. 5. Zhang GG, Ku L, Dilling TJ, Stevens CW, Zhang RR, Li W, Feygelman V:
Volumetric modulated arc planning for lung stereotactic body
radiotherapy using conventional and unflattened photon beams: a
dosimetric comparison with 3D technique. Radiat Oncol 2011, 6:152. 6. Wolff D, Stieler F, Welzel G, Lorenz F, Abo-Madyan Y, Mai S, Herskind C,
Polednik M, Steil V, Wenz F, Lohr F: Volumetric modulated arc therapy
(VMAT) vs. serial tomotherapy, step-and-shoot IMRT and 3D-conformal RT
for treatment of prostate cancer. Radiother Oncol 2009, 93(Suppl 2):226–233. 6. Wolff D, Stieler F, Welzel G, Lorenz F, Abo-Madyan Y, Mai S, Herskind C,
Polednik M, Steil V, Wenz F, Lohr F: Volumetric modulated arc therapy
(VMAT) vs. serial tomotherapy, step-and-shoot IMRT and 3D-conformal RT
for treatment of prostate cancer. Radiother Oncol 2009, 93(Suppl 2):226–233. Acknowledgements 16. Chan OSH, Lee MCH, Hung AWM, Chang ATY, Yeung RMW, Lee AWM: The
superiority of hybrid-volumetric arc therapy (VMAT) technique over
double arcs VMAT and 3D-conformal technique in the treatment of
locally advanced non-small cell lung cancer – a planning study. Radiother
Oncol 2011, 101:298–302. Acknowledgements
The authors thank E. Gerrits, P. Korthuis, A. Rog, L. Vugts, J. van Wershoven
and M. van Wieren for their assistance during the measurements. The authors thank E. Gerrits, P. Korthuis, A. Rog, L. Vugts, J. van Wershoven
and M. van Wieren for their assistance during the measurements. Competing interest Competing interest Competing interest
The authors declare no conflict of interest. p
g
The authors declare no conflict of interest. 14. De Bree-Balk I, Van Hinsberg MGE, Van Veelen LR: High-dose radiotherapy
in inoperable nonsmall cell lung cancer: comparison of volumetric arc
therapy, dynamic IMRT and 3D conformal radiotherapy. Med Dosim 2012,
37(Suppl 4):353–357. Conclusions planned with
hybrid-VMAT, therefore being a quick and easy alterna-
tive for this technique. 12. Holt A, Van Vliet-Vroegindeweij C, Mans A, Belderbos JS, Damen EMF:
Volumetric-modulated arc therapy for stereotactic body radiotherapy of
lung tumors: a comparison with intensity-modulated radiotherapy
techniques. Int J Radiation Oncol Biol. Phys. 2011, 81(Suppl 5):1560–1567. 13. Rengan R, Rosenzweig KE, Venkatraman E, Koutcher LA, Fox JL, Nayak R,
Amols H, Yorke E, Jackson A, Ling C, Leibel S: Improved local control with
higher doses of radiation in large-volume stage III non-small-cell lung
cancer. Int J Radiation Oncol Biol Phys 2004, 60(Suppl 3):741–747. References
V 1. Vanetti E, Clivio A, Nicolini G, Fogliata A, Ghosh-Laskar S, Agarwal JP, Upreti
RR, Budrukkar A, Murthy V, Deshpande DD, Shrivastava SK, Dinshaw KA,
Cozzi L: Volumetric modulated arc radiotherapy for carcinomas of the
oro-pharynx, hypo-pharynx and larynx: a treatment planning
comparison with fixed field IMRT. Radiother Oncol 2009, 92:111–117. 2. Verbakel WFAR, Senan S, Cuijpers JP, Slotman BJ, Lagerwaard FJ: Rapid
delivery of stereotactic radiotherapy for peripheral lung tumors using
volumetric intensity-modulated arcs. Radiother Oncol 2009, 93:122–124. 3. McGrath SD, Matuszak MM, Yan D, Kestin LL, Martinez AA, Grills IS:
Volumetric modulated arc therapy for delivery of hypofractionated
stereotactic lung radiotherapy: a dosimetric and treatment efficiency
analysis. Radiother Oncol 2010, 95:153–157. 1. Vanetti E, Clivio A, Nicolini G, Fogliata A, Ghosh-Laskar S, Agarwal JP, Upreti
RR, Budrukkar A, Murthy V, Deshpande DD, Shrivastava SK, Dinshaw KA,
Cozzi L: Volumetric modulated arc radiotherapy for carcinomas of the
oro-pharynx, hypo-pharynx and larynx: a treatment planning
comparison with fixed field IMRT. Radiother Oncol 2009, 92:111–117. p
2. Verbakel WFAR, Senan S, Cuijpers JP, Slotman BJ, Lagerwaard FJ: Rapid
delivery of stereotactic radiotherapy for peripheral lung tumors using
volumetric intensity-modulated arcs. Radiother Oncol 2009, 93:122–124. 3. McGrath SD, Matuszak MM, Yan D, Kestin LL, Martinez AA, Grills IS:
Volumetric modulated arc therapy for delivery of hypofractionated
stereotactic lung radiotherapy: a dosimetric and treatment efficiency
analysis. Radiother Oncol 2010, 95:153–157. This study illustrates that it is possible with two 180
degree arcs to obtain clinical plan qualities compared to
VMAT and hybrid-VMAT plans described in literature. Generating acceptable plans using VMAT with two arcs of
180 degrees required only 1 hour for plan optimization and
dose calculations. Consequently, our clinical VMAT plans
show comparable clinical plan quality as hybrid VMAT
techniques, therefore being a quick and easy alternative. y
4. Ong C, Verbakel WFAR, Cuijpers JP, Slotman BJ, Lagerwaard FJ, Senan S:
Stereotactic radiotherapy for peripheral lung tumors: A comparison of
volumetric modulated arc therapy with 3 other delivery techniques. Radiother Oncol 2010, 97:437–442. 4. Ong C, Verbakel WFAR, Cuijpers JP, Slotman BJ, Lagerwaard FJ, Senan S:
Stereotactic radiotherapy for peripheral lung tumors: A comparison of
volumetric modulated arc therapy with 3 other delivery techniques. Radiother Oncol 2010, 97:437–442. 5. Clinical quality of VMAT [31] have used a MLD cut-
off value of 16 Gy and indicated that the actual incidence
of lung toxicity of grade ≥2 was 8% for MLD ≤16 Gy and
54% for MLD > 16 Gy (p < 0.01). Wang et al. [30] have de-
termined an incidence of grade ≥3 TRP at 1 year of 13%
for MLD ≤16.5 Gy and of 36% for MLD >16.5 Gy (p =
0.02). We have obtained a MLD ≤16.5 Gy for 90.4% of the
patients and the maximum MLD was 18.3 Gy. In this study, a V5Gy-cont planning objective of 30% or
25% was used with priority of 450 or 500 for the 73 stage
III patients planned with VMAT. Adding this objective
decreased significantly the MLD, V5Gy and V5Gy-cont (p <
0.01) from 12.7 Gy, 53.5% and 47.7% to 12.4 Gy, 46.6%
and 35.6%, respectively. The V5Gy-cont was larger than 60% The lung dose values, MLD, V20Gy, V5Gy and V5Gy-cont
were compared to lung dose values published in the
literature using VMAT and hybrid-VMAT techniques. It
is shown in Table 2 that the clinical VMAT plans of this Table 2 Comparison of stage III VMAT and hybrid-VMAT plans
Study
Method
N
Dprescibed [Gy]
VPTV [cm3]
MLD [Gy]
V20Gy [%]
V5Gy [%]
V5Gy-cont [%]
De Bree-Balk et al. [14]1
VMAT
20
66
8385
20.0
36.6
NA
69.6
Verbakel et al. [15]2
VMAT
14
66
779
NA
30.3 ± 5.7
NA
44.6 ± 9.0
H-VMAT
14
66
779
NA
30.1 ± 5.8
NA
36.2 ± 15.0
Chan et al. [16]3
VMAT
24
60
508
14.4 ± 2.9
25.4 ± 6.0
64.0 ± 15.4
NA
H-VMAT
24
60
508
14.0 ± 2.9
23.3 ± 5.3
59.5 ± 16.7
NA
This study4
VMAT
73
66
344
12.4 ± 3.5
19.3 ± 6.8
46.6 ± 10.6
35.6 ± 7.1
This study4 VPTV > 500 cm3
VMAT
13
66
678
14.5 ± 2.1
22.1 ± 6.4
52.1 ± 10.3
38.7 ± 10.0 Table 2 Comparison of stage III VMAT and hybrid-VMAT plans
Study
Method
N
D
[Gy]
V
[ Table 2 Comparison of stage III VMAT and hybrid-VMAT plans Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 Page 7 of 8 Page 7 of 8 Author details
1D
f 1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB
Tilburg, the Netherlands. 2Department of Radiotherapy, Institute Verbeeten,
Brugstraat 10, 5042 SB, Tilburg, the Netherlands. 17. Vassiliev ON, Wareing TA, McGhee J, Failla G, Salehpour MR, Mourtada F: Validation
of a new grid-based Boltzmann equation solver for dose calculation in
radiotherapy with photon beams. Phys Med Biol 2010, 55:581–598. 17. Vassiliev ON, Wareing TA, McGhee J, Failla G, Salehpour MR, Mourtada F: Validation
of a new grid-based Boltzmann equation solver for dose calculation in
radiotherapy with photon beams. Phys Med Biol 2010, 55:581–598. 1Department of Medical Physics, Institute Verbeeten, Brugstraat 10, 5042 SB
Tilburg, the Netherlands. 2Department of Radiotherapy, Institute Verbeeten,
Brugstraat 10, 5042 SB, Tilburg, the Netherlands. Page 8 of 8 Page 8 of 8 Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 Kroon et al. Radiation Oncology 2013, 8:149
http://www.ro-journal.com/content/8/1/149 18. Han T, Mikell JK, Salehpour M, Mourtada F: Dosimetric comparison of
Acuros XB deterministic radiation transport method with Monte Carlo
and model-based convolution methods in heterogeneous media. Med Phys 2011, 38(Suppl 5):2651–2664. 19. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Accuracy of Acuros XB
and AAA dose calculation for small fields with reference to RapidArc
stereotactic treatments. Med Phys 2011, 38(Suppl 11):6228–6237. 20. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Dosimetric eva 20. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Dosimetric evaluation of
Acuros XB advanced dose calculation algorithm in heterogeneous
media. Radiat Oncol 2011, 6:82. 21. Fogliata A, Nicolini G, Clivio A, Vanetti E, Mancosu P, Cozzi L: Dosimetric
validation of the Acuros XB advanced dose calculation algorithm:
fundamental characterization in water. Phys Med Biol 2011, 56:1879–1904. 22. Bush K, Gagne IM, Zavgorodni S, Ansbacher W, Beckham W: Dosimetric
validation of Acuros XB with Monte Carlo methods for photon dose
calculations. Med Phys 2011, 38(Suppl 4):2208–2221. 23. Gete E, Teke T, Kwa W: Evaluation of the AAA treatment planning algorithm
for SBRT lung treatment: comparison with Monte Carlo and homogeneous
pencil beam dose calculations. J Med Imaging Radiation Sci 2012, 43:26–33. 24. Fogliata A, Scorsetti M, Navarria P, Catalano M, Clivio A, Cozzi L, Lobefalo F,
Nicolini G, Palumbo V, Pellegrini C, Reggiori G, Roggio A, Vanetti E, Alongi F,
Pentimalli S, Mancosu P: Dosimetric comparison between VMAT with
different dose calculation algorithms and protons for soft-tissue sarcoma
radiotherapy. Acta Oncol 2013, 52:545–552. 25. Author details
1D
f Han T, Followill D, Mikell J, Repchak R, Molineu A, Howell R, Salehpour M,
Mourtada F: Dosimetric impact of Acuros XB deterministic radiation
transport algorithm for heterogeneuous dose calculation in lung cancer. Med Phys 2013, 40(Suppl 5):2651–2664. 26. Fogliata A, Nicolini G, Clivio A, Vanetti E, Cozzi L: Critical appraisal of
Acuros XB and Anisotropic Analytic Algorithm dose calculation in
advanced non-small-cell lung cancer treatments. Int J Radiation Oncol Biol
Phys 2012, 83(Suppl 5):1587–1595. 27. Kan MWK, Leung LHT, Yu PKN: Verification and dosimetric impact of Acuros XB
algorithm on intensity modulated stereotactic radiotherapy for locally
persistent nasopharyngeal carcinoma. Med Phys 2012, 39(Suppl 8):4705–4714. 28. Scorsetti M, Navarria P, Mancosu P, Alongi F, Castiglioni S, Cavina R, Cozzi L,
Fogliata A, Pentimalli S, Tozzi A, Santoro A: Large volume unresectable
locally advanced non-small cell lung cancer: acute toxicity and initial
outcome results with rapid arc. Radiat Oncol 2010, 5:94. 29. Song CH, Pyo H, Moon SH, Kim TH, Kim DW, Cho KH: Treatment-related
pneumonitis and acute esophagitis in non-small-cell lung cancer
patients treated with chemotherapy and helical tomotherapy. Int J Radiation Oncol Biol Phys 2010, 78(Suppl 3):651–658. 30. Wang S, Liao Z, Wei X, Liu HH, Tucker SL, Hu C, Mohan R, Cox JD, Komaki R:
Analysis of clinical and dosimetric factors associated with threatment-
related pneumonitis (TRP) in patients with non-small-cell lung cancer
(NSCLC) treated with concurrent chemotherapy and three-dimensional
conformal radiotherapy (3D-CRT). Int J Radiation Oncol Biol Phys 2006,
66(Suppl 5):1399–1407. 31. Kim M, Lee J, Ha B, Lee R, Lee K, Suk Shu H: Factors predicting radiation
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6(Suppl 5):756–763. Author details
1D
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and take full advantage of: 34. ICRU Report 83: Prescribing recording and reporting photon beam intensity
modulated radiation therapy (IMRT). Oxford: Oxford University Press; 2010. 34. ICRU Report 83: Prescribing recording and reporting photon beam intensity
modulated radiation therapy (IMRT). Oxford: Oxford University Press; 2010. 34. ICRU Report 83: Prescribing recording and reporting photon beam intensity
modulated radiation therapy (IMRT). Oxford: Oxford University Press; 2010. • Convenient online submission doi:10.1186/1748-717X-8-149
Cite this article as: Kroon et al.: Dosimetric accuracy and clinical quality
of Acuros XB and AAA dose calculation algorithm for stereotactic and
conventional lung volumetric modulated arc therapy plans. Radiation
Oncology 2013 8:149. doi:10.1186/1748-717X-8-149
Cite this article as: Kroon et al.: Dosimetric accuracy and clinical quality
of Acuros XB and AAA dose calculation algorithm for stereotactic and
conventional lung volumetric modulated arc therapy plans. Radiation
Oncology 2013 8:149.
|
https://openalex.org/W4236569036
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https://www.biodiversitylibrary.org/itempdf/50506
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English
| null |
American game-bird shooting, by George Bird Grinnell ... with colored plates of ruffed grouse and bobwhite, forty-eight full-page portraits of game birds and shooting scenes, and many cuts in text.
| null | 1,910
|
public-domain
| 158,504
|
CopyriGHT, 1910, BY
FOREST AND STREAM PUBLISHING COMPANY \
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~ AMERICAN
GAME-BIRD
SHOOTING
By George Bird Grinnell
Author of PAWNEE HERO STORIES
BLACKFOOT
LODGE TALES
#¥ THE STORY OF
THE INDIAN # THE JACK SERIES #
AMERICAN DUCK SHOOTING, etc., etc.
With Colored Plates of Ruffed Grouse
and Bobwhite,
Forty-eight Full-page
Portraits of Game Birds and Shooting
Scenes, and
many
Cuts
in
Text
NEW YORK
Forest and Stream Publishing
Company
269465 AMERICAN
GAME-BIRD
SHOOTING
By George Bird Grinnell
Author of PAWNEE HERO STORIES
BLACKFOOT
LODGE TALES
#¥ THE STORY OF
THE INDIAN # THE JACK SERIES #
AMERICAN DUCK SHOOTING, etc., etc. With Colored Plates of Ruffed Grouse
and Bobwhite,
Forty-eight Full-page
Portraits of Game Birds and Shooting
Scenes, and
many
Cuts
in
Text
NEW YORK
Forest and Stream Publishing
Company
269465 269465 PREFACE OWPULE
AND MGQ@w
PL ETE
work on American upland shooting
has as yet been written. Volumes
have been published, each of which
covers some section of what 1s really
a large subject, but from the broader sportsman’s
viewpoint no one of them is complete. Of these works,
the most useful was written by the veteran naturalist
and sportsman, D. G. Elliott, whose “Game Birds of
North America’ deals chiefly with the natural history
side of the grouse and quails, and touches only lightly
on the methods by which they are pursued for sport. It seems time now that a book should be written,
covering the whole subject of upland American game-
bird shooting; that is, the shooting of those birds in
which the pointing dog is the assistant to the gun—
the turkeys, grouse and quail, and the American wood-
cock and Wilson’s snipe, called also “English” and
“jack” snipe, a bird of continental distribution. Some years ago I brought together a large amount O Some years ago I brought together a large amount vii vii PREFACE of material on duck shooting in North America, and
the gratifying reception met by that volume encour-
ages me to believe that there 1s place for a companion
work, which shall appeal especially to men who tramp
the uplands with gun and dog. They may perhaps
welcome a book which shall deal with their favorite
sport, and shall touch on it as practiced in different
parts of the country. No two men use the terms shooting and hunting in
the same way, and if the question could be submitted
to a general conference of sportsmen, wide differences
of opinion as to what constitutes a game bird would
of course be found to exist. In certain sections of the
country, and among certain classes of people, bluejays
and woodpeckers are regarded as legitimate objects of
the gunner’s pursuit; if they are less highly esteemed
than quail and prairie chicken, it is only because they
weigh less. For the purposes of this book I have considered as
game birds only the species that are commonly hunted
with dogs. This naturally throws out of the list many
birds which offer good sport and are excellent for the
table. Such are many of the plovers and beach birds,
the Bartramian sandpiper, and all the rails. PREFACE In the preparation of the volume I have not hesi-
tated to draw for information on all available sources, Vili Vili PREFACE and I have striven to bring together much material of
interest
to the sportsman which is now scattered
through many volumes and periodicals, and is thus not
easily accessible. The plan of the book does not differ essentially from
that of “American Duck Shooting.”
Its first part is
devoted to descriptions of the birds and their hab-
its, the second part to the various methods employed
in taking the birds and the aids to shooting—dogs, guns
and ammunition; while in the third is discussed the
shooting of the future. I have received at several hands valuable assistance,
which I desire here to acknowledge. Dr. A. K. Fisher,
of the Biological Survey of Washington, a keen sports-
man and experienced ornithologist, kindly read over
the manuscript. Mr. B. Waters, distinguished as a
crack shot, and especially learned in the hunting dog,
is chiefly responsible for the chapters especially de-
voted to dog and gun. To the kindness of Mrs. Ver-
non Bailey and of the Houghton, Mifflin Co. I owe the
excellent plates of the scaled quail and Mearns’ quail,
taken from her “Handbook of Birds of the Western
United States.”
Messrs. Little, Brown & Company
give me permission to reproduce several cuts from
Baird,
Brewer
and Ridgway’s
“North
American
Birds.”
The Forest and Stream Publishing Company 1X PREFACE has allowed me to enrich the book with a wealth of
illustrations not always to be had in such a volume. The colored plates of the ruffed grouse and Virginia
quail, so admirably depicted by Mr. Louis Agassiz
Fuertes, I use by the kind permission of the Biological
Survey of the Department of Agriculture, who also
let me have the excellent figures of the sage grouse and
of Gambel’s quail. Dr. C. F. Hodge, of Clark Um-
versity, Worcester, Mass, has furnished me with a
number of beautiful photographs of his ruffed grouse
and bobwhites. Sportsmen and ornithologists will find here much
that is familiar, but, I hope, also something that ts new. Gro. BirD GRINNELL. Gro. BirD GRINNELL. New York, October, r9Io. New York, October, r9Io. CONTENTS PAGE
|S LR
CIEE) B72 Gi bs WISNER
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LutFO) 33 YB I jf Coe BU
has I AR
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Pa Xvii AMERICAN GAME BIRDS PAGE
rE CG HEN 2) 1 2 eR Sd 0 2d
DS
a
ee
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er
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NVO OSC OC Aon
isk oatca any iMrn Me dN se er RAM cute Len hte
6
ACMUERICANIN SNEP EG rele uate insen Usa eis eui sat Be aaa Maa lly 30
ICIS
RNIN GC EO) WS PB URIS ese Wee iced ac am eile tei
Wala acletenesg
40
AE
AINE REC AING GUA Doe ot inltaee dagtekince
wk ecrennls
47
OVATE VE OBWHIEN SPAR TRIDGE Ren cmceeenioatineie
49
INEASIKE DS OB IWATE Reema
eietn ceeacstatoleval al
spevaeelay aro
ceieie
61
VIO WA TING © UATE erctetaattrerverercinoheoterere
aleve ote alert tateve eis 76
SCALED MO WATE prsercperctenraieia
svertalee custavercncieresicleotcten tel ore 81
CALIFORNIA OUATEY VALLEY (OUATIe
Joc 'sa clon ote sere 85
GAMBERIS)
©OUADET
pis celc alee ical cleseminblorale
sl
airetece
QI
INVASSENAG ORIN EBARINS 1) OAT EN weieealocialvelelitoielee
sree 100
SEE ACN i RUC
AIN TI GiIRI@ IS Bek ior salsieieclstelarte
cee sishe 114
DUSKY GROUSE Se iieise a cottctee Se ole ds ale elele sel atcteslete II5
CANADA GROUSE, SPRUCE PARTRIDGE........-0e2005
125
I RANIKDENGS «GROUSE c 24 sssis poireieecersieleleler'e
toe ele are! oles 135
RUPPED GROUSE; PARTRIDGEvs
secs os scicisiieiccic
clewlolois 139
X1 CONTENTS PAGE PAGE
THE AMERICAN GROUSE—(Continued). WALLOW! PTARMIGAN! oo. 0c)c cissica ree ine eee
ae
168
ROCK /PTARMIGANG ..0./5iiscrelcclontos
iets Sarcoma eens er 187
WHITE-TAILED -PTARMIGAN.<cc
cite neenneesehenaieee
193
PINNATED: ‘GROUSE. isis cos
te
ee ee ee
ne 206
SHARP-TAILED -GROUSERHonasceeaeee
rete ntene 251
SAGE ~ GROUSE su. dont a Soctig clement
ea 272 UPLAND SHOOTING PAGE
UPLAND SHOOTING 2.6 2. 2
ee Cee
ee eee 303
WOODCOCK SHOOTING b.c.sccke aoe ceeaiee hee eee
305
SNIPE) ‘SHOOTING
serwseisle cies Seat oa lee helene eee ee
oe 319
QUAIL SHOOTING 4h.:cclenne:
ciclis le eee eee 332
BOBWHITE sacs Soa on eases Secu
ols Se
ee
Ee 332
VALLEY (QUATED 2a
Se cetee sion tennis enein Goer: 350
GAMBEL’S ‘OUAIL wos
e
on deen
sake cere
le 352
MOUNTAIN (OUATL 2 Goce tebe
ene Ge
ee ae er 353
SCALED (OUATE. nine Se teks c Manin Oe
Lee 354
MEARNS’ “OUAIU. (cic taro lncnten teed eee
eee 357 PAGE
UPLAND SHOOTING 2.6 2. 2
ee Cee
ee eee 303
WOODCOCK SHOOTING b.c.sccke aoe ceeaiee hee eee
305
SNIPE) ‘SHOOTING
serwseisle cies Seat oa lee helene eee ee
oe 319
QUAIL SHOOTING 4h.:cclenne:
ciclis le eee eee 332
BOBWHITE sacs Soa on eases Secu
ols Se
ee
Ee 332
VALLEY (QUATED 2a
Se cetee sion tennis enein Goer: 350
GAMBEL’S ‘OUAIL wos
e
on deen
sake cere
le 352
MOUNTAIN (OUATL 2 Goce tebe
ene Ge
ee ae er 353
SCALED (OUATE. nine Se teks c Manin Oe
Lee 354
MEARNS’ “OUAIU. (cic taro lncnten teed eee
eee 357
SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350
RUPFED
}\GROUSE® aici feidic ceneere stein aes SR oe
eee 359
DUSKY! GROUSE. se iic cine seven ines a hee He
eee 384
ELARIWIGAN: SHOOTIN Giacccucessingnnone
senor eee 302
WIELO
Wi PPAR MIGAN(. cro o:5 <teicitea’s S alata seine oo ocelot 392
SHOOTING THE PRATRIE GROUSE.¢.ns teen 308
PINNATED (GROUSE. : vistscde cle oo. o6 eo
aoeieea ec eee
eee 309
SHARP-TATLED
GROUSE.0
«ss 04 cans ea eloe ee ee
eee 412
SAGE GROUSE, os.05.0.4 voen'se bc cei» ce tbiceec eel Eeeenes 416 SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350
RUPFED
}\GROUSE® aici feidic ceneere stein aes SR oe
eee 359
DUSKY! GROUSE.
se iic cine seven ines a hee He
eee 384 SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350
RUPFED
}\GROUSE® aici feidic ceneere stein aes SR oe
eee 359
DUSKY! GROUSE. se iic cine seven ines a hee He
eee 384 SHOOTING THE WOODS GROUSE... eeeeeeeeeee 350
RUPFED
}\GROUSE® aici feidic ceneere stein aes SR oe
eee 359
DUSKY! GROUSE. se iic cine seven ines a hee He
eee 384
ELARIWIGAN: SHOOTIN Giacccucessingnnone
senor eee 302
WIELO
Wi PPAR MIGAN(. cro o:5 <teicitea’s S alata seine oo ocelot 392
SHOOTING THE PRATRIE GROUSE.¢.ns teen 308
PINNATED (GROUSE. : vistscde cle oo. o6 eo
aoeieea ec eee
eee 309
SHARP-TATLED
GROUSE.0
«ss 04 cans ea eloe ee ee
eee 412
SAGE GROUSE, os.05.0.4 voen'se bc cei» ce tbiceec eel Eeeenes 416 CONTENTS PAGE
AON ATEST MOY ON TUM CUR RN
A
Sei
ere en a
422 ED SLOP SOO PUNG sh twee
een le 437
SPOMEENG aera age
ey sect enrc
te
eee tiene MALIN), oe. 457
GONSPANDIBORDS. Bitches sak
cect. sera
ua) Roky
462
DISVe S
ac ti
SSRIS Sirsa
ans
Sun anna
ies aha) pea 470 THE SHOOTING OF THE FUTURE
PAGE PAGE
RLOOKSDAGKWARD
2. Mh
bike kit! Avtar MANES Ua
SII
TRANSPLANTING AMERICAN GAME BIRDS................... 527
DESTRUCTION AND PROTECTION.<..2/0)-2-0.00005,..00000
6. 536
ONCLUSION ne es tees
Gein
te cle
ih
eee
De
555 Xili Xili PART TO FACE PAGE
Rurrep Grouse,
(Colored: Plate):..<..... 6-5. Frontispiece
WIG ODCOCKHON WNIEST Ys ier:dioidssieici els ls 010) oleleusisliars evekelers slelevarclsrateterare
Nest OF WOODCOCK......... 4 Nevahs\4 sajtettacelo oualaovtane arene retaliate eliaiske
Nest oF WILSON’S SNIPE..... Bi, Mista ave Whotala ctalateleaisietetete
thetote
MouNTATN OMA asceescen sidtcsouetordcaveteral
e
toredetelele le ener ener hore ae
SCALED) WATE eerie AOU GNOC TaC silavekaelecgialeynens
CAVIRORNEAW@UAME Spine aeiel andisib.a seleuel sis) otTAtetensiohs avelonsvelaretele
tetera
NEST OF CALIFORNIA QUAIL....... svavsliermveysholelaievers clerclelettus orevere
GAMBEL S@OUADR ieee acca
slate Vel sterereness
eet tosobeteke sie
MassENA OR MEARNS’ QUAIL....... SHAS
HS CULO MEMES
STERRAVOUSKY,
GROUSE AND) MOUNGesiaciiee cilia cleiclterecieiectel:
DD WSK is GROUSEN. Gies ciors eaere ers sveonreeiae
=e eresetoreve
craieta mete e
CANADA Grouse. (After Radubonts s Plate). Bet Ser beeders faveilors terre
STRUTTING RUEFED) GROUSES {aspen
en cae Wialelevmrere Sieeronsete Sodtee
THE DRUMMING GROUSE..........-. Yale ih lotactsterebane Eis eiores
NESTOR IRUREED. GROUSE a domeuleoceercten
chic neas Sieseverteen
DAY=DREAMING oie scnicelereres ARS acess Syole evaretetesats ab. etelleis wets
RURRED) GROUSETON INIEST Oe aarmeiecietemteisieiinel
sete stoveteealnereuays
WTLLOWJE
TAR MiIGAN «DN IWAUGAS KeNuatnetslstelelctelere
e
eferele ssehionmeraens
WHITE-TAILED PTARMIGAN.. Veal
Sravdce aliens aves sieve re isteieternee
PINNATED GROUSE AND Ween
Ree aia vis) arate Sa tg calle leuctaverere
SHARP-TAILED GROUSE. (ined einen si Blate)
Nevetrs
WVGTAD oR OKIE Yi ARUN IG eraleteteteteretotele toisiare aie’ ototeloreterete lavereternetete
15
27 Xiv LIST OF ILLUSTRATIONS PAGE PART 1 TO FACE PAGE
BOBWHITE IN Potato Fietp. (Colored Plate)............. 305
ViooncocK) CROUGHING:.:. 5.5. 01s. Thais Ua ein eens Serereralehelsteleey
[307
SETTER POINTING SNIPE....... WE
SieYanata chaleteters PAIOSIOC
OR ane 325
ONPEROIND las cisjelee alos aratacelavegieashas
eicic ale alerts aeerericisioe ey Gat
SHO OMMN
GUNES SEA TNIE Sh cpl sale terete valsis seve term: oraterels eis raleteicreveln oars 345
SETTER POINTING, WITH BIRD IN MOUTH........... Naletelewter GOL
RUFFED GROUSE Cock, REARED IN DOMESTICATION........ 377
DUSiveE GROUSE RON! DHE IVLOUNDATNS suericisiecinisiecis
civic celelsicierl,
S07,
WVitireTe OVW
WAR MOKA Sa tet ctceclel teevetel
ene ia
fie Mislenatarstes sewies she gOS
PRAIRIE CHICKEN SHOOTING IN KANSAS........ Rede ae etrelisy OS
POINTERS HAST ONY GHICKENGEayelcicisin
ss
icicee ticle caletere ot
oretere ae ALT
WILD TURKEYS IN THE PINES..... clare oleracea se setae
AS
SELTER STANDING A RUPFED (GROUSE...)
-2
ues
ee oe Ayes Bate pAzaL
DELIVERING YEE) tESURD). fel cte lo pias’ s cle loicieislotioe
6. etejole FR
taal ela 487
SHOOTING IN Mississippi. (Painting by Edmund Osthaus). 503 PART, IIL TO FACE PAGE
BonwaHrre Carck,/TBREE WEEKS ‘ORDs)i«
wos syigce scioeresiee « S11
EUROPEAN OWADI soit cise tieieleas
is 4 ele ays
ae oreitvtetatrake
Wiel sts AAA ace
Salis
asRi Dusky (GROUSE-PHEASANT:.. <4 iecesc.elsinee's
ss cies eens
519
EUROPEAN PARTRIDGE... ace
stole
ele Siatuvavincatal
Clabes cetera petals 521
DoOURE ET EVANDEDY OF) OUATE We sjesmele
acre Hae
vel be evotetel
ey bas 537
WINTER QUARTERS OF Dr. HopGE’s QUAIL........ susthalstete 539
RUFFED GROUSE CHIcKs Just HATCHED....... Sr
eae sea calcite 541
BEEDING BOBWHITE) HICKS... ...05+0.+-<- Bieta etepeteheaheienve
teks 545
BOBWHITE REARED BY Dr. C. F. Hopce..... syotevertiektehentetens
547
RUFFED GROUSE FEEDING FROM HAND.).. csicciccccssescness SSE XV XV LINE (‘CUTS IN TEx PAGE
RANGE OF THE WOODCOCK: | (Map): .cuscciee aniline cee
ig
BILe, Foor, TAIL. AND WING OF BOBWHITEs. + -2o2ceeccoee en
ho
BIEL AND) FOOT. OF: MOUNTAIN: ©UATI pce
ced cree sericiice
hike
enya
BIELPAND) HOOT OF SCALED OUATE Ann eae Wave oe este
MO? Bic AND FooT OF CALIFORNIA QUAIL... .105-> cemeuniinsc
GOO
Bier, Foor AND’ WING OF MEARNS’ QUAIL. secs cemeene cee
07,
BiLit, Foot, Tart AnD W1NG oF Dusky GROUSE.......... 116
WAIL OF, CANADA, (GROUSE. jee ceeelececetiat
ees eee eee cO
‘(AIC OF FRANKLIN'S, GROUSE! cheese ccc dee ee eee
ee
Briuxi, Foot, Tain anD WING OF RUFFED GROUSE.......--. 140
BILL, Foot, Tart AND WING OF WHITE-TAILED PTARMIGAN. 104
Bit, Foot, TaAiL AND WING OF PINNATED GROUSE..... 568
Alay)
BiLt, Foot, TArL AND WING OF SHARP-TAILED GROUSE.... 252
Briiy, Foot, Par AND WING OF SAGE GROUSE..-2e6s)eeee) 273
Biti, Foot, TAIL AND WING OF WILD TURKEY............ 283 XVi INTRODUCTION The astonishingly rapid increase of population in
the United States has resulted in an equally startling
decrease in America’s larger fauna, especially in those
mammals, birds and fishes which are useful for food. The story of the extermination of large animals over
vast areas is familiar to all, and men not yet beyond
middle life have themselves seen the extermination of
food birds over much of the country east of the Mis-
sissipp1 and north of the Ohio River. Even young
men can remember when the prairie chickens abounded
in Iowa, Minnesota and Nebraska, in regions where
now there are but few. As the game became more scarce the importance of
preserving it began gradually to be appreciated. Yet
in a thickly settled region it is very difficult to effi-
ciently protect the game. Obviously, the best way to
accomplish this is to interest the general public in it,
to point out the economic value of the game birds,
and to secure for the authorities, whose work it is to
enforce the laws, the backing of public opinion in be-
half of those laws. XVii INTRODUCTION The number of people who are trying to accom-
plish something in this good work in America
is
greater now than ever before, and the promise of good
results is brighter than it has been. On the other
hand, in many sections the stock of native birds is
pitifully reduced,
so that while once the only per-
sons who used the shotgun were the field shooters,
to-day the number of men who shoot afield is small
by comparison with those who use the shotgun at clay
targets thrown into the air from a trap. The good sportsman—good in the sense of being
devoted to the ethics of sport, and in the sense of being
successful in his pursuit of game—wishes to know all
he can about the life ways of the beautiful birds that
he seeks for, and in getting together the material for
this book I have endeavored to keep this fact con-
stantly in mind. XVili Limicole. The great order Limicole stands between the galli-
naceous birds on the one hand, and the herons, cranes,
rails, and their allies on the other. This order, known
as shore birds, or the plover-snipe group, is a large and
cosmopolitan order. The species are usually of small
size and the legs are long, as is usually—but not always
—the bill. In certain of its characters the skeleton
resembles those of the gulls and auks. The birds live
in open places on the ground and usually near water
or in moist situations—though to this rule there are
marked exceptions. The young leave the nest and run
about as soon as hatched; in other words, as the natur-
alists say, they are pracocial. The eggs are few in
number, averaging about four. The food is chiefly
insects, worms, small shellfish or other soft animals
which are found in sand or mud. The wings as a rule
are long, flat and pointed, but are sometimes rounded. The tail is usually short. The head is almost always
I I I AMERICAN GAME BIRD SHOOTING 2 covered with feathers and in some cases there is a dis-
tinct crest. The bill varies greatly, from short in some species to
long in others. In one species it turns up, in another
is bent to one side, often it is curved downward, and
sometimes it is oddly expanded at the end, or it may
be compressed to a knife shape. Usually it is covered
with a soft skin, is often much swollen and in some
cases is a very sensitive organ of touch. The nostrils
open above ina groove. They are never feathered. The sexes are usually alike, though they differ in the
phalaropes, the jacana and the European ruff. The
breeding dress is often quite different from that of the
autumn, and some species are extremely showy in their
spring plumage. Within this order are found two great families that
are peculiarly familiar to American gunners. These are
the Charadriude, or plover family, with a dozen or
fifteen species, and the Scolopacide, or snipe family,
which contains more than forty species, and in which
are found the only two here to be considered. Besides
these two great families, there are other smaller ones,
which include the oyster catchers and turnstones, the
avocet and stilt, and the phalaropes. Limicole. Not very distantly related to this group is the bustard Not very distantly related to this group is the bustard
family of the Old World, some of which reach a size
near that of the American wild turkey. This bustard
family, according to Coues, perfectly connects the shore
birds with the Alectorides, or cranes, rails and their
allies. SNIPE-LIKE BIRDS 3 The only birds that we have to consider in this con-
nection belong to the snipe family, which may be sep-
arated into several groups. All snipe-like birds have
long bills usually covered with a sensitive skin, which
is soft throughout and furnishes to the bird a useful
organ of touch. While the bill is long, it is never wide
—though in one or two cases expanded at the tip; the
nostrils are short, narrow slits. The toes are usually
four, though in two or three cases there are but three. Usually they are separated, but in a few species they are
palmated or semi-palmated. The neck and legs are usu-
ally long and the legs seldom feathered down to the
tarsal joint. Most of the birds belonging to this family
are of small size, but occasionally, as in the curlews, the
birds are as large as a small domestic fowl. Snipe and sandpipers are exceedingly gregarious, Snipe and sandpipers are exceedingly gregarious,
traveling
in flocks whose numbers
can
hardly be
counted. This makes them especially subject to danger
from gunners, and the birds being gentle and unsus-
picious may often return to the decoys over and over
again after being shot at. Over-shooting and lack of
enforced protection has almost put an end to the shore-
bird shooting on a great part of the Atlantic coast. build in or near marshy Snipe-like birds usually build in or near marshy
places or by water, and as a rule lay four eggs. The
voice is a shrill, but often sweet, whistle, readily imi-
tated and used to lure the birds to decoys. Ornitholo-
gists state that there are about ninety well-marked
species of these birds, which are divided
into five
groups, the first containing the woodcock and snipe, the AMERICAN GAME BIRD SHOOTING 4 second the godwits, the third the sandpipers, the fourth
the tattlers, and the fifth the curlews. Limicole. In the woodcock and true snipe the eye is placed high In the woodcock and true snipe the eye is placed high
up on the head, so that the ear is below it, and, as Dr. Coues says, if the brain be examined it will be found
curiously tilted over so that its anatomical base looks
forward. The bill is straight and is much longer than
the head. It is deeply grooved almost to its very end,
where it is conspicuously swollen and soft. The soft,
sensitive covering of the bony bill is abundantly supplied
with nerves, and the bill is an instrument of touch by
which the bird feels in the soft earth where it feeds for
the food which it desires. Not only is the bill used
as a direct probe, but it is capable of being somewhat
bent in one way or another. Any one who has ever
compared the bill of the dried skin of a woodcock or a
snipe with that of a fresh specimen has probably noticed
that the dried bill is much shrunken and is also pitted,
showing where the more or less thin skin which covers
the bill has shrunken into the pits in its bony substance. Woodcock and the snipe differ from many of their al- Woodcock and the snipe differ from many of their al-
lies in not being gregarious. They do not gather in
great flocks and so cannot be destroyed in immense
numbers. On the other hand, they are very simple,
gentle birds, and the woodcock living in cover is easily
shot, though the snipe is better able to care for himself. The godwits are large birds with long, grooved bills The godwits are large birds with long, grooved bills
slightly turned up instead of down. They frequent wet
meadows and marshes as well as bays and estuaries, and SNIPE-LIKE BIRDS iE are as truly bay and shore birds as are the sandpipers. There are not many species. The sandpipers are a large group, and among them
are some species with peculiar bills. The bill is soft
and sensitive, though less so than in the two previous
groups. All the sandpipers are extremely gregarious,
and while they vary somewhat in size, as a rule they
are small birds. The tattlers, of. Limicole. which the winter and the summer yel-
lowleg and willet are familiar examples, are larger
than the sandpipers and longer legged. In these birds
the bill is less sensitive than in the sandpipers. They
are not less gregarious than the previous group, and are
noted for their restlessness and for the noise they
make, whence their common name. It is a large
group. T The curlews are distinguished by a down-turned sen-
sitive bill of great length, and in most cases by their
large size. These also are noisy birds and remind one
somewhat of the godwits, which nearly equal some of
the curlews in size. Both godwits and curlews are
found all over the world. The flesh of all the The flesh of all these birds is usually very delicious,
though of course varying in excellence with the foods
of the species. Some of these which feed along the
shore and largely on shellfish are not particularly well
flavored, while the woodcock and snipe are among the
most delicate of our game birds. Philohela minor Philohela minor The American woodcock is a small bird, weighing
only from five to nine ounces. He is eleven or twelve
inches in length, and of this the bill occupies from
two and one-half to three inches. The plumage below
is rich russet-brown, paling, on the upper breast, sides
of the neck and forehead, to ashen-gray. The crown
is black, with two or three crosslines of tawny, and the
back is curiously mottled with tawny, ash-gray and
black, the latter predominating. The tail feathers are
black, barred with tawny, their tips smoky-gray on
the upper side and snow-white beneath. The legs and
feet are pale flesh-color, the bill dark horn-color at
the tip, becoming paler at the base, and the large, soft,
humid eyes are brown. The range of the woodcock is from Canada south
to the limits of the United States and west to beyond
the Mississippi River, but the high, dry plains of the
trans-Missouri region limit the extension of his range
westward, for he is a bird that loves moisture and cool,
dark thickets. Huis range is shown on the accompany-
ing little map taken from Dr. Fisher’s article, in the
Year Book of the Department of Agriculture for Igo1. 6 6 WOODCOCK 7 The English woodcock,
so called—by which
is
meant the woodcock of Europe—is twice the bulk of
the American bird, and though in a general way sim-
ilar in color, the pattern is so different that the two
could never be confused, even though they were of the
same size. This European bird has occasionally been RANGE OF THE WOODCOCK RANGE OF THE WOODCOCK taken in America. Any sportsman who may chance to
kill a woodcock twice the size of the ordinary bird,
paler in color and distinctly cross-barred, should by
all means have it at once examined by a naturalist; and
if unable to reach a naturalist, he should have the skin
preserved, and should report the occurrence. Many of the woodcock pursue their winter journe Many of the woodcock pursue their winter journeys
as far south as the Gulf of Mexico, while others winter
much farther to the north. Philohela minor In fact, it is not altogether AMERICAN GAME BIRD SHOOTING 8 unexampled for them to winter as far north as Con-
necticut, provided they can have a certain amount of
shelter and the weather is mild enough, so that their
feeding grounds are not sealed up by the frost. On
the other hand, the birds which do not go farthest to
the southward may occasionally suffer by unexpected
and severe freezes, such as took place in February,
1899, to be spoken of further on. Wherever it goes,
the woodcock starts early for its summer home and
is often found breeding in New Jersey in the month
of March. The woodcock is thus one of the early spring mi-
grants, and, soon after his arrival, which is usually
in March or early April, according to the weather,
he makes his presence known, to those who understand
where and when to listen for him, by the curious
night song with which he wooes his mate. This song
has been interestingly described by Walter H. Rich
in the Journal of the Maine Ornithological Society
for June, 1910. He says: “As we walked along in the dusk,
. . . my com-
panion called my attention to a bird note which was
new to him. I listened
a moment, and then said, ‘The
first night hawk, I think.’
An instant later I doubted,
and at once my doubt was confirmed when there
sounded again the nasal p-a-a-nck! p-a-a-nck! of a
woodcock. Soon we heard him whistle somewhere on
wing, and presently he appeared flying about, high up
under a particularly bright star, where he sang and
trilled and twittered. I do not remember having ever WOODCOCK 9 before heard a woodcock make so much, so varied and
so good music. It was a veritable song, and many a
so-called songbird does not possess such a repertoire
as did he. “The night was clear and cloudless, with no moon,
but bright with starlight, so that there was no difficulty
in following every development. 1 think he went
through his performance at least a dozen times, with
intervals of rest between of not more than a minute. On each occasion he came to earth near by us, always
within fifty yards, and nearly always within thirty feet,
seeming to have some curiosity as to our presence and
intentions, but showing very little fear. Philohela minor In one in-
stance he had almost settled upon the ground, within
three feet of me, before noticing my motionless figure;
either that, or, as may be possible, he was examining
his disturbers at close range, for, flying first close to
my companion, and making a half circle about his
head, he approached me, poised an instant within hand
reach, and then came to earth not over ten feet away. “When I approached him as he rested upon the
ground he made a peculiar guttural note, which I can-
not describe better than to say that it was like the faint,
muffled b-o-o-mp of a bullfrog. I stood quietly wait-
ing until he should resume, which he presently did,
jerking out, with much bobbing of head and twitching
of tail, a series of nasal, rasping quancks! After a
little he whistled away again, circled, twittered, sang,
and dropped to earth. He must have made eight or AMERICAN GAME BIRD SHOOTING Io ten flights before I left him, and from all indications
was ready to make as many more. “A week later a party of four of us made an excur- “A week later a party of four of us made an excur-
sion into the cover to listen to my twilight musician,
and, though the wind was high and conditions seemed
somewhat unfavorable, we were treated to a very fine
exhibition of his powers. “This evening there were two birds present, and at “This evening there were two birds present, and at
times we had both in the air together. Once there
came a sudden whistling of wings, and we saw one bird
chase the other out of this territory. Pursuer and pur-
sued were very plainly visible as they darted and
twisted over the tops of the pines on the western bor-
der of the cover. Presently one returned and resumed
the serenade. I wondered whether this performance
was a part of the courtship, or whether my little friend
was driving an interloper away from his lady’s case-
ment. If the latter was the case, no serious harm
seems to have resulted from the combat, if such it
was, since we heard what was probably the intruder
performing a little distance away a short time after. “One of my companions carefully timed several of “One of my companions carefully timed several of
the flights. ~%*Some years ago I contributed to the Century Magazine arti-
cles on the woodcock and the snipe, from which, by the cour-
tesy of the Century Company, I am permitted to make extracts
for the present chapters on those birds, Philohela minor They ranged from forty-five to fifty-five
seconds in duration, divided approximately as follows:
first, a period of whistling, circling flight while the
bird mounted to his proper height—anywhere between
sixty and one hundred yards, as nearly as I could esti-
mate it in the uncertain light—lasting about thirty-five
seconds; second, another period of short but somewhat WOODCOCK DE varying extent, when he piped a few preparatory notes
at little intervals before starting into his full tide of
song; third, the song itself, filling perhaps fifteen sec-
onds; and finally, the dart to earth, with wings half
closed, made in silence. The intervals between flights
ranged between thirty and sixty seconds. “The song notes were as clear and liquid as a bobo- “The song notes were as clear and liquid as a bobo-
link’s, and fully as musical. During the production
of these the whistle of the wings could be plainly heard
as a sort of accompaniment to the vocalism.” I think no such good description of the woodcock's
song has before been given. Many observers mention
only a single note, somewhat like the ordinary call
of the night hawk, which he continues for some time,
and then suddenly pitches downward from his height,
and drops into cover. Here the female is waiting for
him, and about her he struts, with head thrown back,
wings trailing and tail spread, a parody on the turkey
cock of the farmyard.*
The nest is a rude structure
of dead leaves and grass, and is usually placed under
a fence, or by a log in some thick swamp, or perhaps
on a tussock or bit of high ground in an alder run. The eggs are generally four in number, and are of a
dull cream color, marked with large spots of bright
or dull brown. In No. 2 of Vol IX, of the Bulletin of the Buffalo
Society of Natural Sciences, are found “Notes on AMERICAN GAME BIRD SHOOTING 12 the American Woodcock,” by Ottomar Reinecke, of
Buffalo, IN. Y: Mr. Reinecke—as have many other people familiar
with the bird—points out that it is one of the earliest
of the birds to nest. Sometimes this early nesting is
followed by unhappy consequences, as in cases which
took place in the spring of 1904. The author says:
“Tn our locality the 13th of April is usually the time
to look for full sets of woodcock’s eggs. Philohela minor That year
was no exception to the rule, although at the time re-
ferred to we had been visited by a heavy snowfall,
which covered the ground to the depth of eight inches. The snow had been partially melted by the sun, but
was frozen hard during the ensuing night. Two days
later another snow storm occurred. The next morn-
ing found us on the ground, which is an ideal locality
for the nesting of this bird. The ground was then
covered with about five inches of crusted snow. In
company with several friends we immediately began
our search for the woodcock. We soon found the
tracks of a pair where they had been feeding or try-
ing to find food around partly frozen water holes. “We flushed the birds and began looking the snow-
covered ground over carefully for the nest. We found
none in that vicinity, but following the tracks of a
single bird, which evidently were made the previous
day, we came to the spot at least a quarter of a mile
distant where the bird had started on its walk to the
water holes. On examining the place nothing but a WOODCOCK 13 slight depression was
found, appearing somewhat
dirtier than the otherwise clean snow. “We were beginning to think that the bird had taken “We were beginning to think that the bird had taken
this place in a small clump of bushes as a refuge from
the snow storms, when it was suggested that perhaps
after all the nest might be under the hard frozen snow. This was no sooner suggested than we were down on
our knees, taking turns at melting the snow with our
breath. After a few minutes we were rewarded by
the sight of one egg, and, continuing our efforts, we
found a fine set of four eggs, the first lying directly
in the middle and on top of the three other eggs and
being separated from them by a layer of at least an
inch of snow. From this I take it that when the
first snow storm came, the bird had laid only three
eggs and found difficulty in keeping on them for any
great length of time. Philohela minor The woodcock lives on worms
from soft, marshy ground, and, consequently,
she
could not remain a long time on the eggs, or, possibly,
the large amount of falling snow made the bird con-
tinually shift her position until, by this constant mov-
ing, the eggs were covered with snow; then the next
day the other egg was deposited as before mentioned. After this the second snowfall occurred, which the
bird could not withstand, and so finally deserted nest
and eggs. We found several more nests in the same
way on that day, and in each case the eggs were found
as described.”
Mr. Reinecke records the finding in April, 1901, of Mr. Reinecke records the finding in April, 1901, of
five eggs in a woodcock’s nest on which the bird was AMERICAN GAME BIRD SHOOTING 14 sketched and from which she was afterward fright-
ened. The number is very unusual, if not previously
unexampled. As soon as the young emerge from the egg, they
leave the nest and follow the mother. Thencefor-
ward their development is rapid, and young birds have
been found well able to fly by April 10. Two broods
are usually reared in the Middle States. A curious
habit of the woodcock, which, though well attested, is
as yet but little understood, is its practice of carry-
ing its young from place to place, apparently to avoid
danger. Exactly how the mother bird does this is
not certainly known, but the weight of evidence seems
to show that she holds it clasped between her thighs,
as a rider does his horse, and does not carry it in
her weak and slender claws. She will sometimes
thus transport her young for a hundred yards or more,
and if pursued will even make a second flight with it. By the last of July, in favorable seasons, the young
of the second hatching are quite fit to look out for
themselves, and early in August the woodcock disap-
pear—that is to say, can no longer be found by those
who search for them. They retire to the dry hillsides
among the heavy undergrowth, and remain there until
the moult is complete. From such places—often among
thick growths of hazel or witch-hazel—they may some-
times be flushed by the ornithologist who is searching
for early migrants. Philohela minor In September they collect once
more in their accustomed haunts, and then are fat, in
good plumage, and fit for the gun. As soon as the young emerge from the egg, they
leave the nest and follow the mother. Thencefor-
ward their development is rapid, and young birds have
been found well able to fly by April 10. Two broods
are usually reared in the Middle States. A curious
habit of the woodcock, which, though well attested, is
as yet but little understood, is its practice of carry-
ing its young from place to place, apparently to avoid
danger. Exactly how the mother bird does this is
not certainly known, but the weight of evidence seems
to show that she holds it clasped between her thighs,
as a rider does his horse, and does not carry it in
her weak and slender claws. She will sometimes
thus transport her young for a hundred yards or more,
and if pursued will even make a second flight with it. By the last of July, in favorable seasons, the young By the last of July, in favorable seasons, the young
of the second hatching are quite fit to look out for
themselves, and early in August the woodcock disap-
pear—that is to say, can no longer be found by those
who search for them. They retire to the dry hillsides
among the heavy undergrowth, and remain there until
the moult is complete. From such places—often among
thick growths of hazel or witch-hazel—they may some-
times be flushed by the ornithologist who is searching
for early migrants. In September they collect once
more in their accustomed haunts, and then are fat, in
good plumage, and fit for the gun. *JS9U
UO
YIOIPOOM *J
UO
YIOIPOOM WOODCOCK Ti Twenty years ago there was much discussion as to
the manner in which the startled woodcock produces
the whistling sound usually heard as it springs from
the ground. The ranks of sportsmen were divided
into two factions, one of which held that the whistle
was vocal, while the other was as firmly convinced
that it was produced by the wings. Oddly enough, able ornithologists, who were also
sportsmen,
were divided on the question—and
are
probably still divided, for the matter has never been
satisfactorily
settled. Philohela minor Such
distinguished
men
as
William Brewster, of Cambridge, and the late Gur-
don Trumbull, of Hartford, whose “Names and Por-
traits of Birds Which Interest Gunners” will always
be remembered, took opposite sides on this question
and argued at length about it. The ever-increasing
scarcity of the woodcock and consequent inability to
observe it put an end to the discussion. Formerly it was legal all over the country to kill this Formerly it was legal all over the country to kill this
species during the month of July, at which time many
of the young were barely able to fly, and when, after
a late spring, some of the mother birds were even
still brooding the eggs of their second hatching. This
practice was most pernicious and is no longer per-
mitted in most States. The coming together in September of the birds
which have been mysteriously hidden away, no one
knows where, is often loosely spoken of as “‘the first
flight’”—in other words, is regarded as the beginning
of the southward migration. It is, however, nothing AMERICAN GAME BIRD SHOOTING 16 more than a collecting in favorite food localities of the
home-bred birds—those which have spent the summer,
or been reared, in the neighborhood. The first true migratory movement of the wood- The first true migratory movement of the wood-
cock usually follows a sharp frost early in October. The birds are not gregarious, and for the most part
move singly; though two, three, and even four have
been seen flying together, and sometimes six or eight
may be started in succession from a single small piece
of cover. The migration is performed during the
night, though in dull, cloudy weather there is some
movement in the daytime. Their flight is low over the
fields. This low flight is swift, and the birds are often
killed by flying against telegraph wires, and some-
times dash themselves against buildings. In New York and New Jersey the woodcock may
almost be considered as resident, for in mild winters
a few birds are to be found late in December and
early in February. The bird does not seem especially
to dread the cold, but the freezing up of the ground
cuts off the supply of food, and so obliges it to move
southward. Philohela minor Often, however, in the coldest weather,
an old fat bird may be found about some warm spring
hole, where the ground never freezes; and here, if
undisturbed, it may remain all through the winter. The principal food of woodcock is the earth-worm,
though they also devour many insects which are to be
found in the damp situations which they affect, and
have been
seen
to catch
butterflies. The “angle-
worm,” however, is the main reliance of this species, WOODCOCK. 17 and the number of these which a single bird will
devour in a given time is astonishing. Audubon says
that a woodcock will devour in a single night more
than its own weight in worms, and some experiments
on this point, made on a captive bird, entirely confirm
the observations of the great naturalist. This speci-
men was apparently a male, and weighed, at the time
of its capture, five ounces. His cage was two feet
long and one deep, and had been fitted up for him
by covering the bottom with long, dry moss, except
in one end,- where there was a box of wet earth, eight
inches square and three deep. The bird was fed alto-
gether on earth-worms, and these were buried, a few
at a time, in the mud. From the first this woodcock
manifested very little fear of man; and it was but a
short time before he so well understood what the open-
ing of his cage door meant, that at the approach of
his owner he would run to his ‘‘feeding-ground” in
anticipation of the meal. So eager was he that it
was necessary to push him away to the other end of
the cage while the worms were being buried. As
soon as he was permitted he would run to the mud
and “bore” for the worms. This was a very inter-
esting proceeding. He would push the point of his
bill into the earth at an angle of about sixty degrees,
and by two or three deliberate thrusts bury it to the
base. While doing this the left foot was slightly ad-
vanced and the body somewhat
inclined
forward. When the bill was wholly buried, he stood for a few
seconds perfectly still, as if listening. Philohela minor Perhaps he AMERICAN GAME BIRD SHOOTING 18 was doing so, but it seems more probable that he was
waiting to see if he could perceive any movement in
the earth near his bill. If none was felt, he would
withdraw his probe and thrust it in again a little
farther on. If, however, he detected any movement,
the beak was hastily withdrawn, rapidly plunged in
again in a slightly different direction, and the unfor-
tunate worm was brought to the surface and devoured
with evident satisfaction. When the supply of worms
was exhausted the bird carefully cleansed the mud
from his bill by means of his feet and, after giving
himself a shake or two, retired to the farthest corner
of his cage, buried his long beak among the feathers
of his back and settled himself for a quiet after-dinner
nap. Sometimes he would thrust his bill down among
the moss once or twice, and then walking to his water
dish would wash it by slowly moving his head from
side to side. After he had been confined for over a
month, the worms fed to the bird during twenty-four
consecutive hours were counted and weighed, and he
was found to have eaten two hundred worms, weigh-
ing five and one-quarter ounces. At the end of this
time he was
still eager for food. Somewhat later
he had increased one ounce in weight, and he now
ate in twenty-four hours no less than eight ounces of
worms. For nearly twenty years now the woodcock has
been growing more and more scarce, and it has been
generally accepted as true that it is on the way to ex-
tinction. Not many years ago so distinguished a WOODCOCK 19 naturalist as Dr. A. K. Fisher published in one of the
Year Books of the Department of Agriculture an ac-
count of Two Vanishing Birds, one of which was
the woodcock. Until within a few years summer shoot-
ing of woodcock has been permitted in a number of
States, a practice which resulted in the practical de-
struction of almost all the birds breeding and reared
in certain territories. So much shooting at a time
when the birds were but recently from the nest and
many of them able to fly only short distances, was
extremely destructive. Philohela minor The birds were so gentle and
at that season fly so easily that summer shooting was
really a sport for children rather than for men. If kept up long enough, this would have resulted in If kept up long enough, this would have resulted in
the extermination of the bird over considerable areas,
or in such a reduction in its numbers that some natural
change, which under ordinary circumstances might
be wholly harmless, might almost wipe the bird out
of existence. For many years far-seeing persons have
recognized this danger and have been laboring faith-
fully to have the practice of summer shooting stopped. It is only within a comparatively short time that their
efforts have been successful. Besides this danger, to which the woodcock were Besides this danger, to which the woodcock were
formerly exposed over much of the country, was an-
other which destroyed them by wholesale, although
such destruction came only at very long intervals. This was the occurrence of periods of severe cold in
their southern wintering grounds. Though such cold
periods do not come often, yet orange growers in AMERICAN GAME BIRD SHOOTING 20 Florida know to their cost that they do occur at in-
tervals. Frosts which merely chill the ground may
prevent the woodcock from feeding and so may force
them to congregate at a few open spots or warm spring
holes, where if found they can be readily killed, but
severe colds long continued are exceedingly destructive. In February, 1899, such a cold wave swept over In February, 1899, such a cold wave swept over
parts of the South, and its influence on bird life in
general was so interesting that I need not apologize
for quoting the gist of the observations made by Mr. Arthur T. Wayne, of Mt. Pleasant, S. C. I give this
account* of the great cold wave of February 13 and
14, 1899, chiefly in his own words, omitting only the
technical names of the various species: “The cold wave which struck the coast of South
Carolina was the severest recorded for two hundred
years. On Monday, February 13, the thermometer
registered 14° above zero, with the ground covered
with snow from four to five inches deep on a level,
while drifts were two feet deep. This is a remarkable
occurrence for the coast region and to be seen scarcely
in a lifetime. *Auk, Vol. XVI, 1800, p. 197. Philohela minor On Tuesday, at 6:55 a.m., the ther-
mometer registered 6° above zero. This exceedingly
cold weather came upon us very suddenly. It was
sleeting all day Sunday, February 12, but toward mid-
night grew
suddenly
colder,
and when
morning
dawned the whole country was covered with snow. The destruction of bird life caused by this cold wave WOODCOCK 2I can scarcely be conceived. To say that fox sparrows
and snow birds were frozen to death by the millions
is not an exaggerated statement, but a conservative
one. There was a tremendous migration of fox spar-
rows on Monday, February 13, following the coast
line of the mainland,
They apparently came from
the northeast
in a southwesterly direction. Thou-
sands tarried in my yard all day long and swarmed
on the piazza, fowl-yard and other places that would
afford protection. They would scratch away the snow
in order to find a bare place, singing—that is, the
stronger birds—the whole time, while their compan-
ions were freezing by the hundreds. While they were
numbed by the intense cold, boat-tailed grackles and
red-winged blackbirds would peck them at the base
of the skull, killing them and eating them. The
stronger fox sparrows would also eat their dead com-
panions. It was a most pathetic sight. I caught quan-
tities of fox sparrows, grass finches, snow birds and
chipping sparrows and put them into a large cage,
which I brought into the house and placed before a
large fire, with the hope of saving them from destruc-
tion ;
but, despite this, they all died. Very few of these
birds were emaciated, and the great majority were
fat. “The “The woodcock
arrived
in countless
thousands. Prior to their arrival I had seen but two birds the
entire winter. They were everywhere and were com-
pletely bewildered. Tens of thousands were killed by
would-be sportsmen, and thousands were frozen to AMERICAN GAME BIRD SHOOTING 22 death. The great majority were so emaciated that
they were practically feathers, and, of course, were
unable to withstand the cold. One man killed two
hundred pairs in a few hours. I shot a dozen birds. Late Tuesday afternoon I easily caught several birds
on the snow and put them into a thawed spot on the
edge of a swift-running stream
in order that they
would not perish, but upon going to the place next
morning I found one frozen. Philohela minor These were fearfully
emaciated and could scarcely fly. Two birds were
killed in Charleston, in Broad Street. It will be many
years before this fine bird can establish itself under
the most favorable conditions.” Mr. Wayne gives a list of sixteen species which he
found frozen to death, among them such hardy birds
as the meadow lark and hermit thrush. He goes on
to say:
“Bluebirds
and
pine warblers
were
decimated. “Bluebirds
and
pine warblers
were
decimated. Mocking birds, cardinals, Florida towhees, Carolina
wrens and all woodpeckers escaped.” It must take any species many years to recover from
a wholesale sweeping off of its individuals, such as took
place on this occasion, and if such a destruction of the
woodcock took place all along a section of its winter
home, as did in South Carolina, it is not strange that
this species should have been regarded by naturalists
as a vanishing bird. For many years in Louisiana, and possibly in other For many years in Louisiana, and possibly in other
portions of the Southern States where the conditions WOODCOCK 23 are favorable, the woodcock are killed during their
winter sojourn by firelighting. Audubon, in his “Ornithological Biographies,” says
that in Louisiana the negroes commonly killed wood-
cock at night by firelighting and striking the birds with
a pole or long stick. A resident of Louisiana described in Forest and
Stream one method of this destruction, by which enor-
mous numbers must be killed. He says: “This bird is migratory in this country and does not
breed here at all, yet our State has protected him. If
the States of his nativity were to protect him entirely
for a few years, in conjunction with the protection
afforded by the Southern States, no doubt he would
soon become abundant. “The birds are night feeders. Their favorite feed- “The birds are night feeders. Their favorite feed-
ing grounds are old sedge fields burned off clean, and
pasture lands that have been pastured closely, but they
can also be found in the cotton, corn and cane fields. They must be hunted on ground that is fairly clean
of weeds and grass, else they cannot be seen. “They are hunted with a torch. Philohela minor The primitive way
was a pine torch in an old-fashioned fire basket with a
long handle, extra fuel being carried in a sack, but the
torch has been superseded by the kerosene lamp. A
large lamp is placed in a tin frame with a glass front. The lamp has a strong reflector behind it and should
illuminate the ground for fifty feet. It is carried by a
man who does no shooting, suspended by a strap
around his shoulders and about waist high. Walking AMERICAN GAME BIRD SHOOTING 24 through the fields, as the light is veered around so as
to cover the ground within its range, the woodcock can
be seen squatting in his feeding place. “The darker the night the better; a drizzly night “The darker the night the better; a drizzly night
is the best of all. On starlight nights it is not easy
to get close enough to kill them with a long cane, which
the darkeys frequently use, or even to shoot them with
squib loads, but on dark, drizzly nights one can almost
catch them with the hands before they become accus-
tomed to the light, which temporarily dazzles them. The birds are usually found in pairs—unless one has
been killed—and squatting from three to six feet apart,
and not infrequently, if the night be very dark, the
hunter can kill both before flight. “A muzzleloader
is preferred on account of the “A muzzleloader
is preferred on account of the
cheapness of the ammunition. The birds are rarely
ever shot at more than thirty feet and frequently under
ten feet. For this reason squib loads are used. An or-
dinary charge of black powder is divided into two
loads, a wad run down on it, and the charge of No. 8
shot is about what a man can hold between his thumb
and fore finger, say fifteen or twenty pellets; more
would tear the birds. “The woodcock rarely spends over sixty to seventy “The woodcock rarely spends over sixty to seventy
days in Louisiana, but during this period many thou-
sands were killed before the enactment of prohibitory
laws, not only by the colored man, but by the whites
as well, in the manner mentioned. Philohela minor No doubt your
sportsmen critics will denounce the method, and, under
any circumstances other than those which actually exist WOODCOCK: 25 in Louisiana, their denunciation would be just, but
I opine if these same critics could be transported to
Louisiana in the woodcock season they would be sur-
prised to find in how short a time they would become
lovers of the night pothunt. “T know from personal experience. I was born a “T know from personal experience. I was born a
sportsman and I can recall, some thirty-five years ago,
when I scorned to shoot a woodcock on the ground,
but then I was new in the State. It did not take me
long to get broken in to the method. The great deli-
cacy of the bird and the almost impossibility of getting
him by daylight hunting begets the habit of night pot-
hunting, and, like other bad habits, it grows apace. “This section of the State is rather out of the wood- “This section of the State is rather out of the wood-
cock country, and I have not hunted them for twenty-
five years. They are here every year, but not plentiful
enough to warrant night hunting, but well do I re-
member a noted hunt of about thirty-five years ago. I was a visitor to Louisiana then. One dark, drizzly
night my brother-in-law was lamp-carrier for me, and
I killed seventy-two from 9 until I a.m. with a muzzle-
loading gun. No, it was seventy-one that I killed with
the gun, but when my ammunition became exhausted
on the way home we found a bird on the side of the
path. I drew the ramrod and killed it with a blow on
the head, making an even six dozen. “T cannot refrain from telling of my last woodcock
hunt. It was in January, 1885, just twenty-five years
ago. On a starlight night three of us started out for
a hunt, one gunner on each side of the light. The “T cannot refrain from telling of my last woodcock
hunt. It was in January, 1885, just twenty-five years
ago. On a starlight night three of us started out for
a hunt, one gunner on each side of the light. The AMERICAN GAME BIRD SHOOTING 26 birds were plentiful, but were so wild that we could not
get shots at half of those we found. Philohela minor The two of us
killed fourteen. “The next night was bright also, and I suggested a
new scheme. I, being a crack wing shot, was to load
my gun with the ordinary bird load, while my com-
panion was to use the squib; he to shoot them on the
ground and I to shoot those on the wing when flushed. “T instructed the light-carrier that when a bird “T instructed the light-carrier that when a bird
rose for flight he should keep the rays of light on
him as he flew. This was easily done by tilting the
lamp with the hands. The results of the hunt were
twenty-nine; my companion
killed fourteen and I
killed fifteen, all flying. I do not now recall that I
missed a single shot. Several times my companion,
shooting at too long range for his squib load, missed,
and I killed the bird in the air. But it was not re-
markable shooting at all, for when the light was
thrown on him he seemed practically to stop in the
air. I could almost have killed them with a rifle.” The vast alluvial region of southern Louisiana has
many large areas peculiarly favorable to woodcock life. Years ago, before the pothunter had effected so much
destruction, the woodcock congregated in those regions
in vast numbers in November and December. The
heavy tropical rains, of whose volume and persistency
the northern resident has little conception, softened
vast areas of land, cultivated and uncultivated, and
thus fitted them for the feeding grounds of the wood- “yYI0IP00 M JO JSON “yYI0IP00 M JO JSON WOODCOCK 27 cock. At the same season snipe abounded in enormous
numbers, and for the same reason. Another casual, but purely local, agent of destruc-
tion to the woodcock is the forest fires, which burn so
frequently in many of the Eastern States and which
run through groves or swamps where the woodcock
have their nests. In such States as New York, New
Jersey, Pennsylvania, Connecticut and Massachusetts,
small brush or forest fires started by careless railroad
engineers or thoughtless boys, occur in autumn and
spring, and these may travel over considerable stretches
of country, destroying great numbers of young seedling
trees, burning through the leaves, dry underbrush and
dead branches of swamps, and destroying the nests
of quail, woodcock and ruffed grouse, and sometimes
even injuring the birds themselves. Philohela minor These fires, while
doing harm here and there, are not of regular occur-
rence and work but little injury compared with those
prairie fires which in old times used to sweep over the
fertile States of the West, destroying the nests of the
prairie chickens, leaving the country bare of food for
them and often causing the farmer the loss of some of
his haystacks, or even of some of his buildings. Yet
the harm done by these eastern fires is a serious matter
in a region where game birds are few. An area so
burnt over is not likely to be occupied by woodcock
for several years. The birds will not breed there, nor,
in most cases, will they resort to such a burnt area for
food or rest. The years 1908 and 1909 seem to have shown dur- The years 1908 and 1909 seem to have shown dur- AMERICAN GAME BIRD SHOOTING 28 ing the fall migration a greater number of woodcock
than have been seen for many years. We have heard
of bags being made of fifteen or twenty birds to a
gun, and gunners have told us of starting thirty or
forty woodcock in a single day. One gunner of great
experience and knowledge of the habits of the birds and
the particular sections to which they delight to resort,
has told us that in the autumn of 1909 he started in
a single day by actual count not less than fifty birds. The number that he and a companion killed was some-
what less than one-third
this number. They kept
within the law of the State in which they were shoot-
ing, but the gunner confessed that the temptation to
overrun the limit was strong. The observations which have been carried on as to
the present abundance or scarcity of woodcock are not
sufficient to justify us in concluding that they are really
increasing, but two or three more favorable seasons
should make us hope that before long the birds will
be back with us again in something like their old-time
abundance. It is certain that of late years in parts of New Eng-
land birds have bred where they have not been seen
for many years before; and under present conditions,
when woodcock are killed only in the autumn, they are
usually fat, strong-winged birds, very different from
the little cheepers of mid-July. Philohela minor This improved condition of things is no doubt due This improved condition of things is no doubt due
to the adoption of proper game laws in the British
Provinces, New England and the States north of Mary- WOODCOCK 29 land. In this large area, which includes a considerable
portion of the summer range of the woodcock, shoot-
ing lasts for from one to two and a half months, begin-
ning from September 15 to November 1 in different
States, and ending November 15 to January 1. The
abolition of the barbarous practice of spring and sum-
mer shooting and the shortening of the open season
has resulted in this encouraging increase in the number
of the birds. Gallinago delicata. The snipe is a small bird, not much more than one-
half the weight of a woodcock. Its bill, which is dark
brown and considerably swollen at the end, is more
than twice as long as the head. The lower portion of
the tibia is naked; in other words, the feathers do not
come down to the tarsal joint, which they reach in
most
other
birds, and perhaps
in all land birds. The eyes are brown, and the feet are bluish or green-
ish gray. The upper parts are generally black or
blackish
brown,
divided
lengthwise
by streaks of
brown and whitish. The chin is whitish and the neck
reddish brown spotted with darker. The scapulars are
streaked with reddish brown, as are also the wing
coverts and secondaries. The tail feathers are blackish,
with a broad band of brownish red near the end, and
the tips white. The bird’s length is 10% inches, its
extent of wings from 16 to 17, and the bill is over 2%
inches long, and sometimes longer. As in the wood-
cock, so in the snipe, the bill is a sensitive organ of
touch. Allied to the woodcock, but wholly different from
it in appearance, habits and home, is the American
snipe, often wrongly called “English” snipe. Birds
30 30 30 AMERICAN SNIPE 31 of this genus are to be found all over the world, and
almost everywhere their habits are more or less alike. During their migrations they frequent fresh meadows
or wet ground, where the worms and insects which
constitute their food are to be found. They are swift
of wing, make long journeys, and, for the most part,
breed well to the North. It is not, however, the cold
which causes them to migrate, but rather the scarcity
of food which precedes the advent of cold weather. Many of the snipe along the Atlantic coast migrate
to a locality only just below the frost line. Often they
may be seen at any time in the winter on the fresh
meadows of Virginia and North Carolina, and if cold
weather should come and a freeze take ‘place, they
move on forty or fifty miles southward, to return again
as soon as milder weather has softened their feeding
grounds. The snipe goes southward to the Gulf of Mexico
and beyond. Gallinago delicata. As worked out by the American Ornith-
ologists’ Union, the range of the snipe is essentially as
follows:
North America and South America. Breeds
from northwestern Alaska, northern Mackenzie, cen-
tral Keewatin and northern Ungava, south to northern
California, southern Colorado, northern Iowa, northern
Hlinois, Pennsylvania and New Jersey. Winters from
northern California, New Mexico, Arkansas and North
Carolina, through Central America and West Indies
to Columbia and southern Brazil; remains in winter
casually and locally north to Washington and Mon-
tana, Nebraska, Illinois and Nova Scotia. Accidental AMERICAN GAME BIRD SHOOTING 32 in Hawaii, Bermuda and Great Britain. Cold affects
them little. My friend, Colonel W. D. Pickett, ob-
served snipe wintering in Wyoming, where the mercury
often went down to from twenty to thirty degrees
below zero. Here, in warm springs that were never
frozen, the birds remained and seemed to prosper all
through the winter. In the same way mallard ducks
winter in open water holes in the interior of Alaska. Usually the snipe reaches the middle and southern
New England States early in the month of April,
though this is a matter which depends largely on the
weather. Here they loiter for some time, the greater
part of them moving on farther to the North, where
they breed. Nests have been found in New York
State, and in the summer of 1908 I saw in August
on the banks of the Housatonic River, in Connecticut,
snipe which I believe had been hatched there. -Most of the snipe, however, go as far north as
Canada; and New Brunswick, Nova Scotia and suita-
ble localities just north of the boundary line between
the United States and the British possessions
are
favorite breeding grounds. The nest is usually a hollow in the open marsh, where The nest is usually a hollow in the open marsh, where
the moss or grass has been pushed aside and bent
down to make a concavity which will hold the four
eges. These, like those of many of this group, are
sharply pointed—pyriform—and always lie in the nest
with the four small ends together. They are grayish
green or olive in color, thickly dotted, blotched and
spotted with dark brown, the spots growing larger at s Snipe. ’
Nest of Wilson s Snipe. ’
Nest of Wilson AMERICAN SNIPE 33 the large end. Gallinago delicata. They measure more than an inch and
a half in length by a little over an inch in breadth. When hatched, the young leave the nest at once. They
are tiny little things covered with yellow and brown
down. At this age the bill is short, and the young
are unable to probe for food. Audubon says that at
first they seem to feed on minute insects found on
the surface of the mud, or amid grass and moss. It is
possible that they do so, but probably no one knows
very clearly just how they are nourished for the first
few days of their lives; but as they grow older and the
bill increases in length and strength, they begin to
feed as do the old ones, and probe the mire. On their return flight the snipe make their appear- On their return flight the snipe make their appear-
ance quite early and are often found on good ground
in small numbers in late August or early September. If the weather has been very dry, so that the area
soft enough to admit of their food being procured is
contracted, snipe may often be found in considerable
numbers on small wet places; but as the autumn flight
takes place gradually and slowly, the birds moving on
for short distances at a time, they are usually not found
in any such numbers as sometimes occur in spring
at the height of the migration, when some cold wave
checks the onward advance. It is commonly believed that snipe spend all their
time in wet and marshy places, and very likely they
do so, when undisturbed. Once, however, many years
ago, I visited a snipe ground near Vincennes, Ind.,
where the birds were astonishingly abundant. We AMERICAN GAME BIRD SHOOTING 34. killed many, and finally the snipe became wild and
flew away, not as if going to great distances, but as
if deserting this especial place. In the effort to learn
where the birds had gone I passed over a little rise of
ground on to a small, cultivated plateau of the prairie
running out between two arms of the marsh, and to my
great astonishment started a large number of snipe
from the dusty ground between the rows of potatoes. Gallinago delicata. A little later, entering a piece of timber, where the
ground was dry under foot, several snipe sprang up
in front of me and flew away. It was apparent that
these birds, being much harried on the marsh, had
taken refuge in this place, waiting until the cause of
the disturbance should disappear. I have never seen
snipe so abundant anywhere as they were in this place. The following morning, returning to the marsh, very
few were found, and those few extremely wild. The food of the snipe consists chiefly of earth-worms
and various insects which are found in the soft mire
of the marshes which they frequent.. Similar in its
food to the woodcock, its flesh is quite as toothsome,
and, indeed, it would be difficult by the taste alone to
tell which bird one was eating. The snipe is commonly called jack snipe, or English The snipe is commonly called jack snipe, or English
snipe, both misnomers, which should not be used by
sportsmen. It should properly be called snipe, common
snipe, or American snipe. In the South, according to
Audubon, the Creoles
call it cache-cache, no doubt
from its cry, while Mr. Nuttall gives “alewife bird,”
“from its arrival with the shoals of that fish.”
Years AMERICAN SNIPE 35 ago I called attention to a parallel name in Connecticut
and elsewhere in New England:
“As the bird arrives
about the same time as the shad and is found on the
meadows along the rivers where the nets are hauled,
the fishermen, when drawing their nets at night, often
start it from its moist resting places, and hear its sharp
cry as it flies away through the darkness. They do
not know the cause of the sound, and from the asso-
ciation they have dubbed its author the shad spirit.”
In the same way, Krider, in his sporting anecdotes, In the same way, Krider, in his sporting anecdotes,
speaks of the snipe as called shad-birds by many of
the fishermen along the Delaware. Until the time of Wilson, the American snipe was Until the time of Wilson, the American snipe was
regarded as identical with the English snipe, and it is,
of course, from this similarity of appearance that one
of its common names is derived. Gallinago delicata. Until within a comparatively short time, the snipe, Until within a comparatively short time, the snipe,
like many of our wild fowl, has been almost without
protection. To be sure, in the Northern States it was
not practicable to kill it in winter, but whenever to be
found it was fair game for the gunners of all the North-
ern States. On the other hand, it has always been
famous for the twisting flight, which renders it very
difficult
for inexperienced
gunners
to kill; yet at
times, when the birds are fat, tame and unsuspicious,
this difficulty is greatly reduced, and great numbers
may be killed. The coloring of the snipe is markedly protective in The coloring of the snipe is markedly protective in
character. White below, barred on sides with black,
with a breast more or less streaked, and upper parts AMERICAN GAME BIRD SHOOTING 26 black or gray, longitudinally striped with white, whitish
and cream color and tawny, it is extremely difficult to
see on the ground. Once, in Montana, riding near a
little mud flat, dotted with tussocks of yellow grass, I
saw at a little distance a snipe feeding in the mud. For some reason he had not noticed me until I got
quite close to him. Then he stopped, looked for a
moment, and took two deliberate steps which brought
him between me and a yellow tussock, the yellow of
which was, of course, constantly interrupted by shad-
ows
of darker—the
spaces between
the blades of
grass. Without taking my eye off him I looked at
the tussock, and after I had adjusted my field glasses,
could make out a snipe standing there in plain sight,
but invisible because of his background. After a mo-
ment or two the bird seemed to think that it had
been needlessly alarmed, and moved out again against
a dark background, where it was plainly seen; but
when I started my horse forward, it again became
alarmed and retreated to its position of shelter, which
again was in front of, and not behind, the tussock. It
seemed to understand that this background would ab-
solutely conceal it. This is, of course, only one of the
common devices of wild animals to escape the obser-
vation of their enemies. Gallinago delicata. The snipe is not likely to be taken for any other one
of our game birds, though the dowitcher, or brown-
back, one of its nearest relatives, resembles it rather
closely. Mr. Trumbull, in his admirable book, ““Names
and Portraits of Birds which Interest Gunners,” gives AMERICAN SNIPE 27 the derivation of the old word, dowitch,
as being
originally
“the Dutch
or German
snipe
(Duitsch,
Deutscher), probably employed to distinguish the red-
breasted snipe from the common ‘English’ snipe.” In spring and, to a less extent, in fall, the snipe often
practice a curious performance, called by some sports-
men booming and by others drumming. The birds,
of which there are likely to be a considerable number,
rise high in the air, circle more or less about overhead,
often uttering a twittering song, and then may sudden-
ly drop from a great height toward the ground, caus-
ing a humming or drumming sound, which is believed
to result from the air passing rapidly between the quill
feathers. An interesting account of this action and, in-
deed, of several habits of the snipe, was written by
Mr. Austen to Forest and Stream, years ago, and
commented on by Dr. Nicols, of Cookstown, both these
gentlemen residing in Canada. We quote Dr. Nicols’
remarks : “Tn your issue of April 23, Mr. H. Austen, of Hali-
fax, N. S., claims that the snipe does its booming (I
take it he means drumming) with his voice, and not, as
supposed, with his wings, and that this noise is only
made at dusk or long after dark. If Mr. Austen will
go out to any good snipe ground when these birds are
in, especially in the earty part of May, in the spring,
or September or October, in the fall of the year, on a
dull, overcast day, he will find numbers of snipe in the
air circling and soaring about and drumming to their
heart’s content, when he will be satisfied—or I am AMERICAN GAME BIRD SHOOTING 38 much mistaken—that the noise a snipe makes when
drumming or booming is made with his wings. “In the same letter Mr. Gallinago delicata. Austen writes:
‘All the
sound that I have ever heard in the daytime has been
their scaipe, except that on August 11, 1885, when
shooting on the marsh, I heard about and around me
who, who, who, only not in very loud tones, and for
quite a while was puzzled, until I found running about
me at my feet’ three tiny young snipe, which must have
been a very late, or, possibly second, brood.’
If Mr. Austen will go out where:these snipe breed, during the
mating season—that is, with us, in the month of May
—he will find that snipe sing, twitter and call. During
this season, snipe call one another pete, pete, pete. The
cock bird springs into the air, flying twenty or thirty
yards before lighting again, with his tail and head up,
singing, and twittering much like a bobolink. One
could hardly think that these tame, foolish birds were
the wild, swift-flying, hard-to-hit birds of the previous
month. “The snipe and woodcock both drum with their
wings. Woodcock drum about dusk, letting themselves
down from an elevated position plump on to the ground,
with wings set edgewise. Snipe generally drum on
dark and dull days, letting themselves down from a
high position with wings set edgewise, fifty or a hun-
dred feet, immediately soaring up again to circle around
as before. This they repeat for hours together. Years
ago, when snipe were plentiful in the Holland marshes,
a few miles from here, I have seen upon a dull day AMERICAN SNIPE 39 fifty or sixty snipe in the air at one time, circling and
drumming; in fact, every snipe on the marsh appeared
to be up in the air. I need hardly say that on such
occasions the bag was small. In those good old times
I remember in the month of October making a bag of
105 snipe in one day; the day was a bright, warm, still
day.” Galline. Among the gallinaceous birds, also called Galline—
cock family—and formerly Rasores, scratchers—are to
be found by far the most numerous and economically
the most important of the upland game birds. The
group includes the turkeys, guinea fowl, pheasants,
grouse, partridges, quail and some other groups, wild
and domesticated, which are especially used for food. On the one hand it is allied to the pigeons, and on the
other to the cranes, and through them to the limicoline
birds, which include the snipe, woodcock, plover and so
on. The great systematist, Huxley, divided the galli-
naceous birds into two groups, one of which he called
Alecteropodes, or fowl-footed, in which the hind toe is
small, and elevated above the others—as, for example,
in the domestic hen, the turkey and the grouse—while
to the other division he gave the name Peristeropodes,
or pigeon-footed, in which the hind toe is well devel-
oped and long, and all four toes are in the same plane
and rest equally upon a flat surface in walking—as the
pigeons, currassows and others. The English naturalists consider all the gallinaceous The English naturalists consider all the gallinaceous
game birds which we know here in northern North
40 40 40 GALLINACEOUS BIRDS 4I America, as well as a number of others, to belong to
the pheasant family (Phasianide) which in that view
includes the turkeys, the pheasants, the partridges, the
grouse and some others. Until recently American natu-
ralists were disposed to regard the pheasant family as
limited to the true pheasants, the turkeys, domestic fowl
and guinea fowl; and to place the grouse and the par-
tridges and quail in a family by themselves (Tetraont-
de), the grouse family. For the partridges of the Old
World the sub-family Perdicine was established, and
another sub-family (Odontophorine)
for the quails
of the New World. By American ornithologists these
two groups have recently been given family instead of
sub-family rank, and are called Perdicide and Odon-
tophoride. The turkeys have also been given family
rank, and are now called Meleagride. Although the various birds belonging to this group Although the various birds belonging to this group
may differ widely in many respects, all have certain
characteristics in common. In all the body is heavy
and round, owing to the great development of the pec-
toral muscles—what we call the breast in a bird—the
head is small and the neck rather long. Galline. The bill is short
and stout, much arched, and overhangs the lower
mandible. The digestive system has certain peculiarities corre-
lated with their habits of life. There is a large crop
capable of great distention; a strong gizzard—except in
the sage grouse—lined with a tough, almost horny,
coating, peculiarly adapted to grinding the hard sub- AMERICAN GAME BIRD SHOOTING 42 stances, as grain, nuts, and seeds, on which the birds
very largely feed. The wings are generally short and rounded, and the
flight, while often extremely swift, is more or less heavy
and labored and seldom greatly protracted. In some
species certain feathers of the wing are enormously
developed. The tail varies extremely. In some species it is very
long and pointed, in others extremely short; again nar-
row but long, or less long and very wide. In the pea-
cock, one of the large and showy pheasants, the coverts
of the tail greatly exceed the quills in length. In the
domestic fowl the tail develops a number of oddities. In the blackcock the quills on the outer sides are curi-
ously bent outward, whence one of its names, Lyrurus
—lyre-tailed. In the grouse and the partridges, the metatarsus, that
portion of the “leg” which is without the body, and
which in most birds is not covered with feathers but
scales, is short, whereas in such birds as the turkey or
the pheasant it is relatively much longer. This so-called
leg is really a part of the bird’s foot, and corresponds to
that part of the foot in man which lies between the
ankle and the toes, and in the horse to that portion
which lies between the hock and the pastern—the can-
non bone. In the pheasants, quails and partridges the feet are
naked, but in the grouse they are always more or less
protected by a covering of hairlike feathers, not un-
like those found on the feet of certain hawks and owls. GALLINACEOUS BIRDS 43 Sometimes these feathers descend only as far as the
toes, but in the ptarmigan (Lagopus—harefoot) the
whole foot is thickly covered with dense hairlike feath-
ers, which serve to protect it from cold and no doubt
to some extent facilitate progression over soft snow. Galline. In
the grouse, too, the toes are pectinated—provided with
a horny comblike structure—on either side, and these
are the only members of the group that are so pro-
vided. In some species, if not in all, these pectinations
are deciduous. Spurs with a bony core and a horny
sheath, thus resembling the horn of a cow—such as we
see in the turkey and the male of the domestic fowl—
are common to all the male pheasants. In some species
there are two or more spurs on each foot and in some,
spurs are present in the female. They are not found
in the grouse or the partridges. In the grouse family a little tuft of projecting feath-
ers runs forward on each side of the bill and covers the
nostrils. In the partridges the nostril is naked, but is
protected by a scale which overhangs it. In the gallinaceous birds the plumage is as varied In the gallinaceous birds the plumage is as varied
as it is possible to conceive, ranging from the most bril-
liant metallic sheens of all hues in some of the pheas-
ants, to the plainest, dullest clay color in some par-
tridges and grouse, as the sage grouse, or the white-
tailed ptarmigan in summer plumage. Many of the
American quails are singularly beautiful
birds, but
less by the brilliancy of their colors than by the fine con-
trasts or harmonies which their plumage exhibits. Of
all these birds in America, however, only the gorgeous AMERICAN GAME BIRD. SHOOTING 44 ocellated turkey of Yucatan and Guatemala can com-
pare in brilliancy with many of the pheasants of Asia. Grouse inhabiting arctic or alpine countries—called
ptarmigan—are notable for their seasonal changes of
plumage. They turn white in winter and are said to
be in a continuous state of moult at all seasons, except
for a brief period when they have assumed their sum-
mer dress, and again in winter after the completing of
the winter plumage. An exception to this rule is the
Scotch grouse, which does not turn white in winter,
presumably having lost that protective adaptation, be-
cause such a change of coloring is not required in the
region which it inhabits. Galline. The birds of this group are all formed for life upon The birds of this group are all formed for life upon
the ground, in this respect contrasting markedly with
that other division of gallinaceous birds, the so-called
pigeon-footed group, which shows a tendency toward
life in trees. This, of course, does not mean that fowl-
footed birds necessarily avoid trees, for as a matter of
fact many of them commonly roost in trees, and all re-
sort to them for refuge and often for feeding purposes. In many of the pheasants and in all the grouse there In many of the pheasants and in all the grouse there
are patches of naked skin on the body, which are usu-
ally particularly obvious during the breeding season. The comb and the wattles of the domestic hen offer ex-
amples of such naked patches. The turkey, of course,
has the head entirely naked, covered with roughnesses
and caruncles, with a notable process on the forehead. The grouse of North America have narrow, naked
patches over the eyes, which during the breeding season GALLINACEOUS BIRDS 45 become congested and large, and by popular writers
are sometimes called combs. Besides, several American
grouse, such as the sage hen, the pinnated grouse and
the dusky grouse, have on the sides of the neck naked
patches of skin, which in the breeding season are capa-
ble of being inflated, and when so inflated completely
change the appearance of the bird. In the prairie
chicken, these, when inflated, have been compared to
small oranges ; in the sage grouse they are much larger ;
while in the dusky grouse they appear smaller, and can
only just be seen through the white feathers which sur-
round them. The sharptail grouse has a naked place
in the same position on the neck, and so has the ruffed
grouse. All the grouse have peculiar and noisy methods of
ushering in the breeding season, of which the drumming
of the ruffed grouse, the booming of the prairie chicken,
the hooting of the blue grouse and the dancing of the
sharptail are familiar examples. These various actions
and sounds are not, however, exclusively confined to
the breeding season. Grouse are generally supposed to be polygamous, but Grouse are generally supposed to be polygamous, but
not all species are so. Galline. The quails, or American par-
tridges, are monogamous, the male and female remain-
ing together during the season of incubation and hatch-
ing. In fact, in case of accident to the female, the male
sometimes hatches out and rears the brood. The Ameri-
can quails possess shrill and rather pleasing voices,
while the calls of the grouse are often rough, hoarse
and disagreeable. AMERICAN GAME BIRD SHOOTING 46 In the north and in temperate regions America is
well provided with grouse, and in the south with quails. The American quails reach their greatest development
toward the tropics. Odontophoride. Of true partridges belonging to that family of galli-
naceous birds known as Perdicide there are none in
America, but their place is taken by another family,
known as Odontophoride, which has a wide distribu-
tion, with a number of species in the United States, and
a still greater expansion to the southward. While the grouse are all large birds, some of them While the grouse are all large birds, some of them
approaching the wild turkey in size, the American par-
tridges, or quails as they are commonly called, are all
small. The grouse have the lower legs and feet more
or less covered with hairlike feathers, and the nostrils
also covered with points of feathers reaching out on to
the bill, known as antiz. The grouse have over the
eye a naked strip of skin which in the breeding season
becomes to some extent enlarged and congested, so that
it is sometimes loosely spoken of as a comb, though it
is not a comb like that of the common hen. The Ameri-
can partridges on the other hand have the feet and
nostrils naked, lack the bare skin above the eye and
usually have short tails. The character which distin-
guishes them from the partridges of the Old World
(Perdicide) is found in the cutting edge of the mandi-
ble, which is toothed or notched. Sometimes this char- 47 47 AMERICAN GAME BIRD SHOOTING 48 acter is so faintly marked as hardly to be noticed, and
when this is the case it is not easy to point out any dis-
tinctive character separating them from the Old World
group. The division, however, is a convenient one, and
the
New World birds are sufficiently alike to be grouped
together. Though most numerous in tropical America, a num-
ber of beautiful species are found within the United
States. Such are the plumed quail of the California
mountains, the helmeted birds of the California low-
lands and of the interior, the scaled quail of the south-
west and the Massena, or Mearns, quail. In the United States and Mexico there are ten genera In the United States and Mexico there are ten genera
with forty species and sub-species belonging to the
Odontophoride. No members of the group range north
of the United States, except that the valley quail of
California has been introduced on Vancouver Island,
B. Odontophoride. C., and that formerly the Virginia quail was found
in small numbers in southwestern Ontario. QUAIL, BOBWHITE, PARTRIDGE. Colinus virgmianus,
Colinus virginianus floridanus,
Colinus virginianus texanus. Colinus virgmianus,
Colinus virginianus floridanus,
Colinus virginianus texanus. Of all these forms, by far the best known is the bob-
white—the partridge of the Southern States, and quail
of the north. It has been divided by naturalists into
several sub-species or geographical races inhabiting
southern New England and the Middle States, the
Southern States, Florida and Cuba, Texas and southern
Arizona; and in Mexico are found many other differ-
ent forms of the genus. All these, except those of
the extreme southwest—Mexico—differ
from each
other chiefly in size and in the varying intensity of the
blacks and browns which mark their plumage. Of those
of the United States the northern form, known as the
quail or Virginia partridge, is the largest; the southern
races, called the Florida bobwhite, the smallest and
blackest ;
while the Texas bobwhite is midway between
the two in size, and in color is paler and grayer than
either. . The general color of the typical bobwhite is reddish The general color of the typical bobwhite is reddish
brown, paler below and streaked, spotted or crossed
with black markings. The lower breast and belly be-
come whitish, and all the upper parts are crossed with
49 49 AMERICAN GAME BIRD SHOOTING 50 fine lines of blackish or dusky. In the male, the throat
and a broad line over the eye are white, the rest of the
head black or blackish streaked with brown. The sides
of the neck are spotted with triangular dots of black
and white, the back is reddish brown mottled with gray
and more or less streaked with tawny. The female is
paler than the male, and has the throat patch and the eet
OE EER
BOB WHITE QUAIL BOB WHITE QUAIL stripe over the eye buffy. Sometimes the young males
have this buff throat patch and superciliary stripe. The
tail is bluish gray, the under tail coverts white streaked
with reddish brown. The feathers of the flanks and
sides are reddish brown, striped with whitish. Length
about ten inches and wing about four and a half. Ex-
tent of wings fifteen to sixteen inches. Weight from
about six and a half to eight ounces. stripe over the eye buffy. Sometimes the young males
have this buff throat patch and superciliary stripe. QUAIL, BOBWHITE, PARTRIDGE. The
tail is bluish gray, the under tail coverts white streaked
with reddish brown. The feathers of the flanks and
sides are reddish brown, striped with whitish. Length
about ten inches and wing about four and a half. Ex-
tent of wings fifteen to sixteen inches. Weight from
about six and a half to eight ounces. QUAIL, BOBWHITE, PARTRIDGE SI The Florida bobwhite (C. virginianus floridanus)
averages somewhat smaller and considerably darker,
the black markings being wider and occupying relatively
more space on the feathers,
The Texan bobwh The Texan bobwhite (C. virginianus texanus) is
slightly smaller than the typical bobwhite, but larger
than that of Florida. Mr. Ridgway says an olive gray-
ish tint prevails over the whole back without conspicu-
ous black spots, and the general surface is usually dis-
tinctly barred with lighter; the black markings of the
lower parts are usually broad and nearly transverse, as
in C. virginianus floridanus. Bobwhite is the most widely distributed of our galli-
naceous birds except perhaps the turkey and the ruffed
grouse. In one form or another it is found from Mas-
sachusets south to Florida and the Gulf of Mexico, and
from southern Ontario, Canada, west and south through
eastern Minnesota, Southern Dakota, Nebraska, Kan-
sas, Oklahoma
and Texas,
into Mexico. Besides,
it has been introduced at various points west of its natu-
ral range and apparently has become thoroughly estab-
lished in Utah and Idaho. Jt is reported also to be
quite abundant in portions of California and in the
Willamette Valley, Oregon, as well as on certain islands
of Puget Sound, Washington. Though introduced and
doing well at various points—Denver, Fort Collins, etc. —in Colorado, some naturalists believe that it has of
itself advanced west with the farmers into eastern
Colorado. 52 AMERICAN GAME BIRD SHOOTING Being a bird that lives on the ground, bobwhite can-
not survive in a country of deep snows. Along the
northern border of its range it has often been almost ex-
terminated in certain regions, because a heavy snowfall
during the night has covered up the roosting birds, and
then a change in the weather melting the surface of the
snow has been followed by another change to freezing,
forming a hard crust through which the quail cannot
break, resulting in starvation and death. QUAIL, BOBWHITE, PARTRIDGE. There are
many records of bevies being found after the snow of
some severe storm has melted, close huddled together
precisely as they roosted. Long continued periods of
severe weather, when the ground is covered with snow
and ice, sometimes make food exceedingly hard to ob-
tain and at the same time render the birds more visible
to their many enemies. At the present time the quail may be said to be almost At the present time the quail may be said to be almost
extinct in Massachusetts, Connecticut and much of
New
York State, and in many sections they seem to be con-
stantly growing fewer in numbers. This is due largely
to over-shooting, but partly also to the destruction of
the covers which once afforded them so much more pro-
tection than at present. The reduction in their numbers
is a misfortune not merely to the sportsman, but to the
whole country, for investigations have shown the quail
to be a valuable ally to the farmer, destroying multi-
tudes of harmful insects, and also devouring vast quan-
tities of noxious weed seeds. The value of the services
which it performs is now coming to be understood by
the public at large, and it is reported that in certain QUAIL, BOBWHITE, PARTRIDGE 53 sections of the country—as in parts of Oklahoma—far-
mers refuse to permit gunners to venture on their prem-
ises in pursuit of bobwhite. The quail does not migrate and therefore is always
exposed to the dangers of winter, and where they have
once been exterminated efforts to replace them by other
quail imported from the south have seldom been success-
ful. The most hopeful indication for the protection of
the species is in the growing comprehension of its eco-
nomic value. Quail are extremely prolific, and under
ordinary conditions might survive the attacks of all
natural enemies, but when the climate sweeps off all the
birds in a particular region the progress of re-establish-
ment is slow. In old times in the south the practice of netting quail In old times in the south the practice of netting quail
was very generally indulged in. Powder and shot were
costly, while the net could be used over and over again. QUAIL, BOBWHITE, PARTRIDGE. This method of taking them, at first practiced only as a
means of procuring food, was carried on later for the
purpose of securing living birds to sell to persons who
desired to restock their covers with quail. Presumably
it has been in vogue up to within a short time, 1f not
now practiced in parts of Oklahoma and in Texas. A chief danger to the quail of the south is the non- A chief danger to the quail of the south is the non-
enforcement of the game laws, and the market shooting
by negroes, many of whom gun persistently almost the
whole year round and are excellent shots. The ordinary bobwhite is the northernmost and best The ordinary bobwhite is the northernmost and best
known species of this genus. Yet the smaller, darker
quail of the south and of Florida and the grayer form The ordinary bobwhite is the northernmost and best
known species of this genus. Yet the smaller, darker
quail of the south and of Florida and the grayer form AMERICAN GAME BIRD SHOOTING 54. of Texas are at once recognized by northern gunners as
essentially the same bird that they know at home in the
north. The uneducated birds of the southwest, how-
ever, do not afford the same sport furnished by birds
frequently pursued, but show the disposition to run be-
fore the dog exhibited by the other quail of the dry
country—Gambel’s, the scaled and the valley quails. It is said of bobwhite that years ago it was scarcely It is said of bobwhite that years ago it was scarcely
found west of the Missouri River, but that it has fol-
lowed the settlements north in Minnesota and west in
Nebraska. This may be true, but it is quite as likely
that it has always existed in this region, but was not
observed there until the country became more or less
thickly settled. Wherever found, the quail is resident and breeds. Al-
though occasionally large flocks occur, consisting of
twenty-five or thirty birds, it never packs, as do many
of the grouse of the open, and where such large flocks
are found it is probable that they consist of the first and
second broods of the same parents, or of the birds
hatched by two hen quail that have occupied the same
nest. QUAIL, BOBWHITE, PARTRIDGE. ,
It must be said, however, that there are at least two
records where packs of quail have been seen by good
sportsmen of great experience. One of these was
Edmund Orgill, who made the following interesting
report: “A curious experience occurred a short time ago to
a friend of mine who went on a hunt to north Missis-
sippi, where he had been earlier in the season and found QUAIL, BOBWHITE, PARTRIDGE 55 birds plentiful. He was accompanied by his brother,
and they had a brace of good dogs. They hunted for
hours without the sign of a bird, and could not account
for the apparent scarcity. At last their dogs made
game, but before they could establish a point the birds
commenced getting up all around and in front of them. They were confident that there were from 70 to 100
birds, and instead of their taking the usual quail flight,
they rose clear up in the air like a flock of pigeons, and
went away for half a mile. . They followed the line,
which was in an open country, and found a few scat-
tered birds in an old cotton field, fully the distance
named from where the birds took their flight. They
gave up finding more, and started again; and after a
prolonged tramp they had a duplicate of their first ex-
perience, finding a second flock or drove—not a bevy,
for they say there were nearly a hundred. Like the
former they made an immense flight. “Now, I know that it is common for the grouse in
Scotland to pack, occasionally the English partridges
do, and our prairie chickens; but I never saw or heard
of quail doing so before.”
On this report S. T. Hammond, referring to the On this report S. T. Hammond, referring to the
year 1852, commented as follows:
“In Iowa thirty-eight years
ago, when crossing “In Iowa thirty-eight years
ago, when crossing
through a piece of woods from one prairie to another,
we came upon a clearing of two or three acres of wheat
stubble, and a large drove of quail got up, at least 200
or 300, but they scattered in every direction. To this
day we can see that clearing and how the quail filled AMERICAN GAME BIRD SHOOTING 56 the air. QUAIL, BOBWHITE, PARTRIDGE. This is the only thing that has come under
our personal observation that looked like the Missis-
sippi case.”’ The quail is a bird of the open, building its nest in the
fields, in little patches of brush, or in fence corners, but
after the broods are grown, commonly taking refuge
when alarmed in woods or tangled brushy patches. Usually the winter home of the bevy is in some
swamp or woodland adjacent to fields where grain
or weed seeds
or other food
is accessible,
and a
few years ago their wanderings were limited to a
comparatively small area. The birds went to their
feeding grounds in the morning, walking from them
to some place where they sat in the sun and dusted
for two or three hours; toward night they fed again;
and then often walked in among high grass, where they
roosted. Of late years, since they have been so con-
stantly pursued, their habits appear to have undergone
some change, and they are believed often to roost in
the woods or swamps, but in old times it was common to
find these roosting places out among the stubbles or in
the open fields. Years ago, a gunner familiar with a
certain section of country, who had traveled over it
enough to learn where the different bevies of quail lived,
could almost certainly start each one of them and have
a couple of shots before they took refuge in swamp or
woods. In those days, however, a man who had such
knowledge—if he went shooting frequently—usually
contented himself with three or four birds a day. If his QUAIL, BOBWHITE, PARTRIDGE 57 outings came but seldom, he was likely to demand more
and to follow up the bevies. The broods of quail which have kept together all The broods of quail which have kept together all
through the winter usually break up as warm weather
approaches, and soon after this comes the mating. This
takes place earlier in the south, and in New England it
is often the first of June before the birds are mated and
the clear whistle of the male is heard. The nest is a
depression in the ground, lined to some extent with bits
of grass and weed stems and occasionally with a feather
or two dropped from the mother’s breast. Rarely the
parents build over the nest a dome-shaped roof. QUAIL, BOBWHITE, PARTRIDGE. Cap-
tain Bendire quotes Judge John M. Clark of Saybrook,
Conn., who saw a male bobwhite engaged in the work
of constructing a domed nest. Judge Clark says: “Tn May, 1887, while on a hill back of my house one
morning I heard a quail whistle, but the note, which
was
continually repeated, had a smothered
sound. Tracking the notes to their source I found a male bob-
white building a nest in a little patch of dewberry vines. He was busy carrying in the grasses and weaving a roof
as well as whistling at his work. The dome was very
expertly fashioned and fitted into its place without
changing the surroundings, so that I believe I would
never have observed it had he kept quiet.”
Captain Bendire also says that Mr. G. E. Beyer of Captain Bendire also says that Mr. G. E. Beyer of
New Orleans, La., found a nest constructed of pine nee-
dles, arched over and the entrance probably a foot or
more from the nest proper. Such constructions are very
unusual. The only concealment commonly found is that Captain Bendire also says that Mr. G. E. Beyer of
New Orleans, La., found a nest constructed of pine nee-
dles, arched over and the entrance probably a foot or
more from the nest proper. Such constructions are very
unusual. The only concealment commonly found is that AMERICAN GAME BIRD SHOOTING 58 offered by the weeds and grass that grow about the nest. Captain Bendire says that in the south, cotton rows are
a favorite nesting place. It rarely happens that for some reason or other a
second brood of quail may be very late. Such a case
was mentioned in Forest and Stream in February, 1879,
where a quail’s nest is stated to have been found in the
month of January. From twelve to eighteen white eggs are laid. Some- From twelve to eighteen white eggs are laid. Some-
times many more are found in a nest. In such cases
it is probable that two hens have shared a single nest. Captain Bendire tells of a nest of nine eggs taken in
Texas, all of which were more or less spotted and
streaked with reddish brown and lilac markings, es-
pecially about the larger end. QUAIL, BOBWHITE, PARTRIDGE. While the hen is sitting on her eggs, the male from
some elevated perch not far off whistles through much
of the day the cheerful notes which give him his name
and which are also interpreted as more wet. When
one is near the bird, a third note is heard preceding the
two most striking ones, and much lower pitched, mak-
ing the whole call seem more like ah bobwhite. It is
generally supposed that this call is peculiar to the male,
but the female also utters it, though not with the same
fullness and vigor as the male. On one occasion while
driving slowly along a narrow grassy lane I heard a
quail whistling at some distance ahead of the horse. We drew nearer and nearer, yet the sound seemed
hardly strong enough for that of a full-grown male,
and presently I distinctly saw whistling, a female quail, QUAIL, BOBWHITE, PARTRIDGE 59 which did not leave her perch on a fence post until the
horse had passed her and I was within four or five feet
of her. Then she dropped down into the grass close
to the fence. The rallying cry uttered by members of a scattered
bevy is a call of three notes, but entirely different from
the mating cry bobwhite. I believe the male bobwhite usually takes part in the I believe the male bobwhite usually takes part in the
work of incubation, and all writers are agreed that if
an accident happens to the female, the male incubates
the eggs and rears the young. When the eggs hatch,
the little ones, then scarcely larger than bumble-bees, at
once follow the parents, who look after them with every
manifestation of affection. Sometimes it happens that
the farmer while driving his mowing machine through
the tall grass, may see a male and a female quail rise
in front of him and flying but a few feet drop down
again. He knows then that somewhere in the grass
near his team are the tiny young whose lives are now
in jeopardy, and often he will turn his horses about
and go the other way, in order to give the parent birds
an opportunity to lead their young away from the dan-
ger spot. QUAIL, BOBWHITE, PARTRIDGE. Sometimes the little ones may be seen, a
dozen of them, hurrying after their parents across the
newly shorn grass, half hidden by its short stems. Oft-
en the parents strive to lure man or dog away from the
tender young by feigning injury, and on a signal from
the mother the young lie close hidden until the danger
is passed, 60 AMERICAN GAME BIRD SHOOTING During their earliest youth the young quail feed al-
most exclusively on soft insect food, but as they grow
larger they eat more and more seeds, and when autumn
comes, fruit of one kind and another and grain. In
New Jersey barrens, where grain fields are few and
far between, I have taken quail that seemed to have fed
exclusively on the acorns of the scrub oak. Most carnivorous reptiles, birds and mammals are
enemies to quail, and yet against the mammals and rep-
tiles they are fairly well able to protect themselves. Occasionally a snake devours the eggs in a nest or may
destroy a few of the very small young, but the depreda-
tions they commit are slight. Hawks are the enemies
from which the quail have most to fear, and of these
the goshawk, Cooper’s hawk and sharp-shinned hawk
are the most destructive. JI have more than once seen
a marsh hawk stoop at a quail, but I never saw this
hawk catch one. I have also seen a marsh hawk stoop
at a crow, and even at a great sage grouse that would
weigh many times what the hawk weighed. The grouse
seemed alarmed and ran away, but the crow merely
threatened the hawk with its beak and seemed not at all
disturbed. Colinus ridgwayi. The masked bobwhite is very little larger than the
Virginia bobwhite. It has the whole under side of the
head black and the white stripe over the eye is very
narrow or sometimes disappears entirely. The neck
and chest below the black throat is uniformly cinnamon
or reddish, like the other lower parts. The female is
almost exactly like the female of the Texas bob-
white, but usually has a strong band of cinnamon color
across the upper part of the chest. There are no special d There are no special differences in habit among the
various forms of bobwhite quail, except those which de-
pend on their surroundings—the character of the coun-
try which they inhabit. The masked bobwhite, or Ridg-
way’s quail, was described by Mr. Brewster from speci-
mens sent on from Mexico by F. Stevens. Previ-
ous to that, however,
Herbert Brown
of Tucson,
Arizona, had sent on specimens which were erroneously
identified as Grayson’s bobwhite, a Mexican species not
known to occur in the United States. In a paper entitled “Arizona In a paper entitled “Arizona Quail Notes,” published
in Forest and Stream in 1885, Mr. Brown writes about
this species in the following words:
61 In a paper entitled “Arizona Quail Notes,” published
in Forest and Stream in 1885, Mr. Brown writes about
this species in the following words:
61 61 61 62 AMERICAN GAME BIRD SHOOTING “The masked bobwhite is found in the country lying
between the Barboquivari Range in Arizona and the
Gulf coast in Sonora, more especially between Barbo-
quivari and the Plomoso, where this species is quite
abundant. ‘They are also found on the Sonoita Creek,
about 60 miles north of the Sonora line. From the
Sonoita Valley they range in a westerly direction fully
100 miles, and through a strip of country not less than
30 miles in width within Arizona Territory. Very
probably they may go beyond this, both to the east and
west. The habits of the masked bobwhite, so far as
we know them, appear to resemble very closely those
of the common quail, only slightly modified by the con-
ditions of their environment. They utter the character-
istic call of bobwhite with bold, full notes, and perch
on rocks or bushes while calling. Colinus ridgwayi. They do not appear
to be a mountain bird, but live on the mesas (table-
lands), in the valleys, and possibly in the foothills. “The masked bobwhite was, three years since, abun-
dant in the neighborhood of Bolle’s Well, a stage sta-
tion on the Quijotoa Road, near the northern end of
the Barboquivari Range, 29 miles southwest of Tuc-
son and about 40 miles north of the Mexican boundary
line. As the station was then comparatively new, the
grass thereabouts was high, and these quail could be
had for the taking; but now that the stock has eaten
away the grass, the birds have not for a year or more
been seen about the place. On the road from Bolle’s
Well, west to the Coyote Range (about 25 miles), these
quail were frequently to be met with, but the teamsters BOBWHITE IN MEXICO 63 and travelers have killed or frightened them off. One
of the former assured me that he had killed as many
as five at one shot. Ten miles south of Bolle’s, in the
Altar Valley, we came across a small covey, perhaps a
dozen inall. The bright, deep chestnut breast plumage
of the males looked red in the sun, and gave the birds
a most magnificent appearance. We secured but one,
a male, the rest secreting themselves in the tall sacaton
grass, which at this point was between 4 and 5 feet
high, and as we had no dog we did not follow them. Our next place to find them was on the mesa, southeast
of the peak, where we camped to hunt for them, but
they were scarce here, and we managed to secure but
few. “In addition to their bobwhite they have a second
call of hoo-we, articulated and as clean-cut as their
bobwhite. This
call of hoo-we
they used when
scattered, and more especially when separated toward
nightfall. At this hour I noted that although they oc-
casionally called bobwhite, they never repeated the
first syllable, as in the day time they now and then at-
tempted to do. In body they are plumpness itself; in
this respect, considering size, they overmatch the Ari-
zona quail (Callipepla gambeli), with which I com-
pared them. In actual size of body, however, the latter
is the larger. Colinus ridgwayi. Of three stomachs of this species exam-
ined, one contained a species of mustard seed, a few
chaparral berries, and some six or eight beetles and
other insects, ranging in length from a half inch down AMERICAN GAME BIRD SHOOTING 64 to the size of a pinhead. The second was similarly
provided, but contained, in lieu of mustard seed, a
grasshopper fully an inch in length. These two were
taken on the mesa. The third, from a bird taken in
the valley, contained about twenty medium-sized red
ants, several crescent-shaped seeds, and a large num-
ber of small, fleshy green leaves.” Mr. Brown pointed out, years ago, that the intro-
duction of live-stock into southern Arizona bade fair
to exterminate the masked bobwhite in that territory,
by the destruction of its nests and eggs by horses and
cattle, as well as by the eating of the cover among
which it lives. His prediction has been verified, and
as recently as June, 1909, he wrote, saying:
“Colinus
ridgwayt is a dead bird so far as Arizona is, as yet, con-
cerned, but it is again getting a good foothold in So-
nora, about 75 miles south of the line. I am almost
afraid to say anything about it, however, as I fear I
might send skin hunters into the country.” Other writers have noted in our common bobwhite
the same tendency to disappear in sections where gen-
eral farming has given place to stock-raising. The bobwhite family attains its greatest development
in Mexico. Though differing greatly in color, the pat-
tern of that color is somewhat similar in all the differ-
ent forms. Several are black, or black mottled with
white, on the breast and tail, where our bird is pale in
color, just freckled with black. All have the same
whistle with which we are so familiar in our own bird, 65 BOBWHITE IN MEXICO and generally their habits are much alike. They range
from the sea-level to an altitude of 7,500 feet. They have not the shyness or swiftness of wing of They have not the shyness or swiftness of wing of
the much hunted form of the East and South, and are
gentle, unsuspicious little birds, ready to run along on
the ground before the traveler or to dodge out of the
trail to one side. Colinus ridgwayi. Of the dozen forms described from Mexico, perhaps
only two, Ridgway’s bobwhite and the Texas bobwhite,
cross the boundary into United States territory. No
one is so familiar with this group in Mexico as Mr. FE. W. Nelson, the distinguished naturalist, whose work
in various branches of science, in Alaska, Mexico and
Central America, is so well known. In the Auk, for
April, 1898, he published a charming account of this
group, which is accompanied by an excellent plate of
Godman’s bobwhite, a species described by Mr. Nelson,
from the lowlands of southern Vera Cruz. Mr. Nelson
calls his sketch “With Bobwhite in Mexico,” and we
copy it here:
“While traveling in Mexico a few seasons ago, I “While traveling in Mexico a few seasons ago, I
arrived at a small town near the southern end of the
tableland in the State of Puebla. The first business
in hand was to secure suitable quarters for myself and
assistant. Having accomplished this, I was ready at
an early hour the following morning for a tramp into
the surrounding country. It chanced to be market
day, and passing the outskirts of the town I met a
straggling procession of Indians, in picturesque cos-
tumes, some driving heavily loaded donkeys, others car- AMERICAN GAME BIRD SHOOTING 66 rying on their own backs crates of fruits, vegetables,
hand-made pottery and other simple wares. All were
pushing forward, eager to take part in the keenly rel-
ished pleasures of petty chaffering, which would enable
them to return home with a few decimos knotted in
the ends of their sashes. Some of the men saluted me
with a polite ‘Buenos dias, sefior,’ but I noted that their
conversation was carried on in the Aztec tongue, as
spoken by their fathers centuries ago. “Once free of the last houses, a convenient opening “Once free of the last houses, a convenient opening
in the fence was soon found, and I crossed into a great
field, which reached for miles down the broad, open
valley. Areas covered with wheat and corn stubble
indicated the character of the last crops, while farther
away broad belts of brilliant green sugar-cane were in
vivid contrast to the dry browns and yellows of the
general surface. The sun was shining brightly, and
the fresh, balmy air seemed full of life-giving power. Colinus ridgwayi. The musical notes of meadow-larks were heard at in-
tervals, and on one side of the valley flocks of red-
winged blackbirds were swirling back and forth over
some small marshy spots grown up with tules. Through
the valley bottom flowed a little stream of clear, spark-
ling water, which, before reaching the distant shore of
the Pacific, runs a wild course through the mountain
gorges of Guerrero. Behind me arose the mysterious
pyramid of Cholula, crowned by a white-walled chapel,
which now occupies the place of ancient sacrifice. Over
to my right stood the gigantic form of the Smoking
Mountain—hoary old Popocatepetl—with the gleaming BOBWHITE IN MEXICO 67 robe of the White Lady—Iztaccihuatl—shining over
his shoulder. In front a sweeping plain descended for
many miles, through a district of great sugar estates,
to the far horizon, where it was walled in by the blue
front of distant mountains. “Turning to one side, I approached some scrubby
bushes which appeared to offer shelter for birds or
other game. Suddenly the familiar accents of my
mother tongue fell on my ear. I listened with bated
breath. Again arose in clear, round tones, the calls so
familiar in my boyhood days, bobwhite,
bobwhite. With eager steps I hastened forward to a small group
of acacias, and there, quietly perched on top of a bush,
was an old friend, the author of the notes. It is diffi-
cult to describe the mingled pleasure and exultation
caused by this unexpected meeting. It proved to be the
Puebla bobwhite (Colinus graysoni nigripectus, Nel-
son), and during the following days a number of oth-
ers were seen, and it became evident that my friend of
the first morning was one of a colony located in the
neighborhood. “Afterward, during my Mexican travels, I learned
that the bobwhites are widely spread in that country,
and although many of them have changed the color of
their dress more or less, yet their customs and tricks
of speech remain much the same as in their northern
home. “Ata later date during this same season, while work-
ing down the eastern slope of the Cordillera in Vera
Cruz, near the city of Orizaba, we found others of the AMERICAN GAME BIRD SHOOTING 68 family, known as the black-breasted bobwhite (Colinus
pectoralis, Gould). Colinus ridgwayi. They were living in brush-grown
and weedy old fields—sometimes straying about the
coffee plantations—and were on friendly terms with
most of their tropical neighbors. Fortunately, in these
parts guns and dogs are few, and mostly harmless, so
that Bob’s days were generally peaceful and contented. But even here life was not without its cares, for the
spotted tiger-cats and woolly-haired opossums, with
sad lack of consideration, were given to nocturnal raids
that filled them with terror and sometimes lessened
their numbers. “From Orizaba our wanderings led far away over
plains and mountains to the city of Tehuantepec, on the
hot lowlands bordering the Pacific coast. There we
found our friends again, but known as the Coyolcos
bobwhite (Colinus coyolcos, Miull.). They were com-
mon, and although their garb had changed consider-
ably, yet their voices and mode of life remained true
to the family traditions. Indeed, so fixed are old hab-
its among them that even long association with the
suave and politic Mexican has failed to cure Bob of one
custom that I often deplored during my youthful days,
when, gun in hand, I sought to make his acquaintance. I refer to that abruptness of manner which is shown in
such a disconcerting way when one comes upon him in
his favorite haunts. “Near Tehuantepec their home is on the partly wood- “Near Tehuantepec their home is on the partly wood-
ed and partly grassy plains. Old fields and grassy prai-
ries, that extend irregularly amid the scrubby forests BOBWHITE IN MEXICO 69 of that district, are their favorite haunts. Here the
mesquites, mimosas, acacias, cassias, Brazil-wood, eb-
ony, mahogany, Spanish cedar, and other tropical trees
and bushes, give the landscape quite a different aspect
from that which Bob is accustomed to see in his north-
ern home. Old cornfields and weedy indigo planta-
tions are popular resorts, and furnish an abundance of
food. Brush fences of thorny scrub are built about these
fields, and serve as fine places of shelter in times of dan-
ger. The quails do not penetrate heavily wooded bot-
toms along streams, where the moisture causes a vigor-
ous tropical forest growth, unless some farmer hews out
a clearing for his cornfields. In these forest belts the
motmots, trogons, red-and-yellow macaws, several spe-
cies of parrots and other tropical birds, abound, and a
little farther south troops of spider monkeys are en-
countered. Colinus ridgwayi. In many places it is but a few steps from
the dense shade of the bottoms, where the harsh screams
of the macaws dominate all other woodland notes, to
the borders of grassy prairies where our friends pass
their sedate lives, associated with meadow-larks and
sparrows. Throughout this region, where deer, pec-
caries, tree pheasants and other game is plentiful, small-
er birds are considered unworthy of powder and shot,
all of which conduces greatly to peace of mind among
the bobwhites. “While traveling down the coast from Tehuantepec
into Chiapas, we found them numerous most of the
way, and they were a constant source of interest and
pleasure. Their cheerful notes were frequently heard AMERICAN GAME BIRD SHOOTING 70 from the scrubby bushes near the trail, and the neat,
trimly-built little fellows carried on their small affairs
with little regard for our presence. While riding at
the head of the pack train, I frequently found them
scratching in the sandy trails, dusting themselves, or
searching for food. At such times it was amusing to
note the pretty air of doubt and hesitation with which
they awaited my approach before finally moving rather
deliberately a few yards to one side when I came too
near. Now and then the male could be heard uttering
little querulous notes, as if in subdued protest at being
disturbed. After entering Chiapas the coast was left
behind, and we passed into the interior through a se-
ries of beautiful open valleys ornamented with scat-
tered bushes and belts of trees. It was during the
rainy season, and the vegetation was growing luxuri-
antly ;
everywhere were myriads of flowers, and the in-
numerable plumelike heads of tall grasses nodded grace-
fully in the passing breezes. In these valleys the bob-
whites were very common. It generally rained during
the night, but the clouds broke away at dawn, leaving
a brilliantly clear sky. We were up and on our way at
sunrise, amid the invigorating freshness of early morn-
ing, when every leaf and twig bore a pendant water-
drop that sent out quivering rays of light with the first
touch of the sun. On every hand were new flowers and
strange birds. Colinus ridgwayi. Now and then the Central American
mockingbird, in full-throated ecstasy, poured out its
rich song, and over it all, at short intervals, the clear
call of bobwhite arose from a bush or low tree. At BOBWHITE IN MEXICO 71 an altitude of about 3,000 feet we passed out of their
range, and did not find them again until we reached
the valley of Comitan, on the Guatemalan border, where
their notes were heard. A few miles farther on, just
after entering Guatemala, a single female, which proved
to be quite different from those taken in Mexico, was
brought me by an Indian. This specimen served as
the type of the Guatemala bobwhite (Colinus insignis,
Nelson). Beyond this nothing was learned of them in
these remote parts. “From Comitan Valley we made a long circuit over
the Guatemalan highlands and reached the Pacific coast
again, on the border of Chiapas. There, on some grassy
prairies in the midst of the forested coast plain, a few
miles back from the sea, we found many bobwhites of
a previously unknown branch of the family (Colinus
salvini, Nelson). In this vicinity an attempt was
made, many years ago, to establish a large colony of
Americans. They came with great flourish of trum-
pets and large expectations, but the climate did its
silent work so effectually that two or three stranded
relics were all that remained. Over the desolate sun-
scorched
flats near by the same cheery call of the
quail sounded in the ears of the Mexican oxdrivers
and muleteers as they carried their cargoes of coffee
and cacao to the coast, that I heard from many a field
and thicket over thousands of miles of varied country
to the north. Among these sturdy little Americans
there appeared no sign of degeneration, and it was
pleasanter to meet them than some of my countrymen AMERICAN GAME BIRD SHOOTING 72 of a larger growth. So many failures at colonizing
people from the north in these hot southern lands had
come to my notice that I had become skeptical of its
successful accomplishment in any instance; yet here
in the tropics were the bobwhites, essentially a group
of the temperate regions, living as cheerfully as pos-
sible, and upsetting my preconceived ideas. Colinus ridgwayi. “After passing some time in this district we hired “After passing some time in this district we hired
an oxcart one evening, and were trundled across the
plains to the coast during the cool hours of the night. There, on the sandy shore, we waited ten days for a
steamer, which finally carried us back to Tehuantepec. From this place a railroad crosses the Isthmus to the
port of Coatzacoalcos, on the Gulf of Mexico, and we
took advantage of it to reach the eastern coast. Coat-
zacoalcos is a curious little town, destined to play an
important part in the development of southern Mexico
and western Guatemala. It is one of the few places
in Mexico where small frame houses are the prevail-
ing style, and reminds one more of some small mining
camp in the Far West than of a seaport on the Gulf
of Mexico. Here, where yellow fever, malaria and
other
ills stalk about, according to the season, we
heard of howling monkeys, jaguars, tapirs, and other
tropical creatures with which we still desired to be-
come more familiar. For this purpose we ascended
the Coatzacoalcos River about twenty miles, to the
town of Minatitlan, a place once noted for its enor-
mous trade in Spanish cedar and dyewoods. We re-
mained here some days, in the midst of the coast low- BOBWHITE IN MEXICO ie; lands, where the tropical forest is interrupted by grassy
prairies of considerable extent. In visiting these prai-
ries we were surprised and delighted to find another
of the bobwhites that had not been previously known
even to those most familiar with the ramifications of
this good old stock (Colinus godmani, Nelson. Aft-
erward we found them a few miles out of Coatzacoal-
cos, and they were also seen a little farther north, in
the open country about the shores of beautiful Lake
Catemaco. This latter point is probably near their
limit in gat direction. “The me ananen of the “The me ananen of the Mexican bobwhites is curi-
ous, and shows that the family has been long in the
land. They range over parts of the cool tableland, and
extend down to the tropical lowlands of both coasts,
but are unaccountably absent from many apparently
suitable places. Colinus ridgwayi. “Many “Many changes have taken place in their garb owing
to the influences and requirements peculiar to such va-
ried situations, but the general style is retained, so that
their relationship cannot be mistaken. “A representative of “A representative of this group lives in Yucatan,
which, it is said by some, belongs to the family proper;
but if this is so, there must be a bar sinister on its
escutcheon to account for some of its peculiarities. “At present eleven branches of bobwhites “At present eleven branches of bobwhites are known
to live in various parts of Mexico, and our work has
enabled us to introduce four of them to the friends of
the family. Wherever they were encountered over
this great area it was interesting to observe how closely AMERICAN GAME BIRD SHOOTING 74 they continue to resemble one another in notes and
habits. From the border of Canada to Guatemala they
hold true to a general style of speech and manners that
always betrays their connection, with the possible ex-
ception of the Yucatan branch, of which I am unable
to give any definite information. “For the charming qualities and pretty ways of these “For the charming qualities and pretty ways of these
little friends of the field, I trust their days may be
many and their numbers never grow less. “As it is quite possible that some of our mutual “As it is quite possible that some of our mutual
friends may have the opportunity to call upon these
Mexican connections of ‘our Bob,’ I have taken some
trouble to secure their names and addresses, which are
given below. The directory is complete, I believe, up
to date. “Tt. Colinus ridgwayi (Brewster). Ridgway’s bob-
white. Sonora; ranging south from the Arizona bor-
der. (Between 1,000 and 2,500 feet above sea-level. )
“2. Colinus virginianus texanus (Lawr.). Texas
bobwhite. Northeastern Mexico; Nuevo Leon and
Tamaulipas. (From near sea-level up to 2,500 feet.)
“3. Colinus graysoni (Lawr.). Grayson’s bobwhite. Southern part of tableland; from San Luis Potosi and
northern Jalisco to Valley of Mexico (3,000 to 7,500
reet)': “Tt. Colinus ridgwayi (Brewster). Ridgway’s bob-
white. Sonora; ranging south from the Arizona bor-
der. (Between 1,000 and 2,500 feet above sea-level. )
“2. Colinus virginianus texanus (Lawr.). Texas “2. Colinus virginianus texanus (Lawr.). Texas
bobwhite. Northeastern Mexico; Nuevo Leon and
Tamaulipas. (From near sea-level up to 2,500 feet.) “3. Very different
in appearance
from the bobwhite
group are the striking quails of the western and south-
western portions of the United States.
Of these birds
there are almost a dozen forms, some of them plumed,
others helmeted, others with full, soft crests, but all
very unlike the bobwhites.
These are among the most
beautiful of North American birds, and in many of
the regions which they inhabit are extremely abundant. Colinus ridgwayi. Colinus graysoni (Lawr.). Grayson’s bobwhite. Southern part of tableland; from San Luis Potosi and
northern Jalisco to Valley of Mexico (3,000 to 7,500
reet)': “4. Colinus graysoni nigripectus (Nelson). Puebla
bobwhite. Tableland of southern Puebla (3,000 to
6,000 feet). “cs. Colinus pectoralis (Gould). Black-breasted
bobwhite. Eastern base of Cordillera in Vera Cruz; BOBWHITE IN MEXICO 75 from Jalapa to Isthmus of Tehuantepec (500 to 5,000
feet). “6. Colinus godmani (Nelson). Godman’s bob-
white. Lowlands of southern Vera Cruz; probably also
ranging into Tabasco. (From sea-level to 1,500 feet. ) “7 Colinus coydlcos (Mull.). Coyolcos bobwhite. Pacific coast of Oaxaca and Chiapas; from city of
Tehuantepec to Tonala. (From sea-level to 3,000 feet. )
“8. Colinus atriceps (Ogilvie-Grant). Black-headed “8. Colinus atriceps (Ogilvie-Grant). Black-headed
bobwhite. Putla, western Oaxaca. (About 4,000 feet.)
“9. Colinus salvini (Nelson). Salvin’s bobwhite. “9. Colinus salvini (Nelson). Salvin’s bobwhite. Coast plains of southern Chiapas, near Guatemalan bor-
der. (Sea-level to 500 feet.) “to. Colinus insignis (Nelson). Guatemala bob-
white. Valley of Comitan in Chiapas, into adjacent
border of western Guatemala (3,000 to 6,000 feet). “T1. Colinus nigrogularis (Gould). Yucatan bob-
white. Yucatan. (Sea-level to 500 feet.”’) “T1. Colinus nigrogularis (Gould). Yucatan bob-
white. Yucatan. (Sea-level to 500 feet.”’) Very different
in appearance
from the bobwhite
group are the striking quails of the western and south-
western portions of the United States. Of these birds
there are almost a dozen forms, some of them plumed,
others helmeted, others with full, soft crests, but all
very unlike the bobwhites. These are among the most
beautiful of North American birds, and in many of
the regions which they inhabit are extremely abundant. MOUNTAIN QUAIL. Oreortyx pictus. Oreortyx pictus plumiferus. Oreortyx pictus confinis. This is one of the largest of our quails. Its upper
parts are brown or olive, or sometimes even bluish, the
inner webs of the tertiary feathers being buff, so that
when the wings are closed a distinct stripe is seen on
each side of the rump. The breast and head are in part
lead color; the long, backward directed crest of narrow
feathers growing from the top of the head is black; the
throat is chestnut, and from this a black stripe runs up
to the eye. The chin, front of cheeks, lower portion of
the lores, a line bordering the throat patch from the
posterior angle of the eye and forehead, are whitish. The effect of this is to surround the bill with a border
of whitish. The flanks are deep chestnut barred with
black and white. The thighs are reddish and the under
tail coverts black. In color, the female is very similar
to the male, but usually has the crest plumes smaller. The length is from 11 to 12 inches, and the wing from
514 to 514 inches. This is the typical mountain quail, ranging in the
California mountains from Santa Barbara, Cal., north
to Washington. In Oregon its range seems to extend
76 76 MOUNTAIN QUAIL 77 out to the eastward, where it has been found near Mt. Hood. It was first found north of the Columbia River
a good many years ago, and later was introduced near
Seattle, at Vancouver Barracks, and also on Whidbey
Island, in Puget Sound, where the birds have done
well. The plumed partridge (Oreortyx pictus plumiferus)
is a paler race, grayish olive above, but often with the
neck more or less lead-colored, like the breast, the edges =
ons: OS
a asp
eo Sta DP Gee
Ss
Cog (sees
See. aatses So YS
R
MOUNTAIN QUAIL MOUNTAIN QUAIL MOUNTAIN QUAIL of the tertiary feathers whitish, and the forehead white. It is found on both sides of the Sierra Nevada, from
eastern Oregon southward, and through California
south to northern Lower California. The San Pedro quail (Oreortyx pictus confinis) is a
still paler race, with a thicker bill, first found in the San
Pedro Mountains of Lower California. According to
Mr. Anthony, it ranges high up on both sides of the
mountains, to the highest peaks, estimated at about
12,500 feet, and is not common below 2,500 feet. AMERICAN GAME BIRD SHOOTING 78 The mountain quail is perhaps the most beautiful
American quail, though in such an attractive group it
is hard to say that one is handsomer than another. The
range of the species is limited to the mountains of the
Pacific coast, from Washington south through Oregon
and California, as stated. Captain Bendire quotes Prof. O. B. Johnson, of the Captain Bendire quotes Prof. O. B. Johnson, of the
University of Washington, to the effect that its north-
ern range has been extended by artificial means. He
says: “Twenty years ago this species was found but little
north of the Willamette Valley, Oregon, but they grad-
ually worked down the south side of the Columbia
River, toward Astoria, and in 1872 I was informed that
some of these birds, shot at Kalama, Washington, were
the first seen north of the Columbia. A crate of
trapped birds sent to the Seattle market were, some
time afterward, purchased by the Young Naturalists’
Society and set free. These have since multiplied nice-
ly, and others have been sent to Whidbey Island, forty
miles north of Seattle, where I understand they are
also doing well. A covey wintered in a barn lot with
the hens just at the outskirts of Seattle this winter.”
Quite a number were also liberated near Vancouver Quite a number were also liberated near Vancouver
Barracks, and did well. It is a moisture-loving species,
and delights in a country where the rainfall is heavy. The paler race of this species is found in the drier re-
gions of the Sierra and some of the desert ranges. This is quite an abundant species, found high up on
the mountains in summer, and also low down toward Mountain Quail. Mountain Quail. MOUNTAIN QUAIL 79 the desert in winter. MOUNTAIN QUAIL It is said that in spring many fol-
low up the snows as they melt, spending the summer
high up on the mountains, and then in the autumn re-
turn down the slopes, below the point where snow lies. The birds have a call not very unlike that of the bob-
white, a whistle of three syllables, and the attitude as-
sumed by the male while calling is not unlike that of
the common quail, the wings being drooped and the
point of the bill raised. The food consists of insects,
seeds, berries, and the buds and tops of tender plants. In the early spring they feed so much on the tops of
the wild garlic—one of the first green things to appear
—that the flesh sometimes tastes of the plant. This quail is a prolific This quail is a prolific breeder, the eggs usually vary-
ing in number from 9 to 15, though sometimes they
are much more numerous, a nest containing 19 eggs
being reported, as also a brood of 20 young birds. The
time of nesting varies from early April to late May. The eggs are white. In northe In northern Lower California Mr. Anthony found
that the San Pedro partridge does not differ greatly in
habit from the other forms. The race is confined tc
southern California and northern Lower California. The mountain quail is a bird only casu The mountain quail is a bird only casually pursued
by the gunner. Often they are flushed and shot, and
in some localities they are regularly pursued with dogs
and shot over points. In such places they are reported
to lie close, and to offer good opportunity for sport. The situations in which they are often found, rough
mountain slopes, overgrown with manzanita, chemisa! The mountain quail is a bird only casually pursued
by the gunner. Often they are flushed and shot, and
in some localities they are regularly pursued with dogs
and shot over points. In such places they are reported
to lie close, and to offer good opportunity for sport. The situations in which they are often found, rough
mountain slopes, overgrown with manzanita, chemisa! AMERICAN GAME BIRD SHOOTING 80 and other chaparral, will long protect them, and make
the work of shooting them very difficult for the dog,
and hardly less so for the gunner. MOUNTAIN QUAIL When the birds
take wing they are swift fliers, and soon out of sight. It has been suggested that these birds would do well It has been suggested that these birds would do well
in various localities in the southern Alleghany Moun-
tains, where they might take the place of the native
species, which seem to be growing scarcer. I do not
know, however, that any serious effort has been made
to acclimate them in such localities. Callipepla squamata.
Callipepla squamata castanogastris. Callipepla squamata. Callipepla squamata castanogastris. In size, not far from the Virginia quail; with a short,
thick crest, white at the tips. The rest of the head is
brownish or grayish, growing paler on the throat. The
back part of the neck, back and breast are bluish gray. A black border to each feather warrants the name
“scaled.””
The wings are pale brown, and the flanks
streaked with white. Other lower parts are buffy or
yellowish, the belly sometimes with a patch of chestnut
brown. Sexes alike. Length, g to 10 inches. Eggs
white or buff dotted with brown. Inhabits northwest-
ern Mexico, and the borders of the United States, from
western Texas to southern Arizona. The chestnut-bellied scaled partridge has the wings The chestnut-bellied scaled partridge has the wings
tipped with brown, tail bluish gray, the lower parts
behind deeper buff, sometimes yellowish, and a large
patch of rusty chestnut on the belly in the male and
sometimes in the female. This bird is found in east-
ern Mexico and in the lower Rio Grande Valley of
Texas. In northern Mexico, along the southwestern
border of the United
States, from western Texas
through southern New Mexico and Arizona, the scaled
partridge is abundant. 81 82 AMERICAN GAME BIRD SHOOTING The birds seem to shun the timber, and to be most
abundant on the high mesas, preferring the dry and
barren country. Mr. Herbert Brown, however, speaks
of having seen them immediately about water as well as
far away from it. Captain Bendire, however, intimates
that his experience leads him to think that the bird
scarcely requires water. Mr. E. W. Nelson tells us
that, while he has often found them far from water,
they nevertheless make regular visits to the watering SCALED QUAIL SCALED QUAIL places. All observers agree that they are exceedingly
shy and hard to approach, and that they are swift run-
ners, dodging in and out among the bushes with the
greatest ease, and soon out of sight. Even if flushed
they fly but a short distance, when they alight and
run again. Like many other gallinaceous birds, they
greatly enjoy taking dust and sand baths, and at such
times they act much like young chickens. In the eve-
ning they retire to roost, to ridges and knolls, and the
birds call to each other until the bevy has come to- Scaled Partridge. Scaled Partridge. SCALED PARTRIDGE; BLUE QUAIL 83 gether again. Sometimes the birds collect in consid-
erable numbers, 60 or 80 having been seen together. The breeding season begins in March, and the young
birds are usually hatched by the first of June, or earlier. There is some reason to think that two broods are
reared. The number of eggs ranges from 9 to 16, and
is commonly about 11 or 12. The eggs are sometimes
dead white, or again pale buff in color, and are dotted
with very small reddish brown spots scattered over
the entire egg. The scaled partridge is not a bird pursued for sport. The scaled partridge is not a bird pursued for sport. It is true that many of them are killed by gunners, but
merely for the food they afford, the birds being potted
on the ground at every opportunity. Of all the quail, this is the most difficult to make lie
to dog or man. In Arizona the army officers used to
hunt them on horseback, following up the birds and
shooting them whenever and however they could. The scaled quail, which is also sometimes called The scaled quail, which is also sometimes called
white topknot quail, or cottonhead, does not differ in
habits from
its relative,
the chestnut-bellied
scaled
partridge. Concerning the scaled partridge in southern Arizona,
Mr. Herbert Brown, of Tucson, writes me:
“The blue quail are less common than Gambel’s quail, “The blue quail are less common than Gambel’s quail,
and do not, as a rule, live on the desert proper, but in-
cline rather to the higher and rougher foothills. I saw
a dog tried on only one, but am inclined to believe in
their sprinting qualities. I have shot them in the foot-
hills of the Tucson Mountains, west of here, and as far AMERICAN GAME BIRD SHOOTING 84. east as the Graham Mountains, where 1 found them
quite plentiful. They are quick, active birds, and as
pretty as pictures. 1 take it for granted that they are
found much further east, but the eastern slope of the
Grahams is as far as my personal acquaintance goes.” California Quail. California Quail. CALIFORNIA QUAIL; VALLEY QUAIL. CALIFORNIA QUAIL; VALLEY QUAIL. Lophorty« californica. Lophortyx« californica vallicola. Two forms of this species are found on the Pacific
coast : a northern race, inhabiting the coast of Califor-
nia north of Monterey, and which has been introduced
in British Columbia, Washington and Oregon, and a
paler race found in the interior valleys and foothills
of California, south through Lower California to Cape
St. Lucas. In the California quail the flanks are streaked with
white on a brown or grayish ground. The feathers
of the belly, in the male, are edged with black, and have
a central patch of chestnut. The upper parts generally
are smoky brown, the inner edges of the tertiary feath-
ers buffy, the throat black, and the forehead whitish. The female has no black or white on the head, is plain
grayish or brownish, lacks the chestnut belly patch, and
has the scale-like markings of the under part less dis-
tinct. The crest is much shorter than in the male. The
length is 9% or Io inches, and the wing about 4%. This is the bird found in the coast valleys of Oregon
and Washington and California. The valley quail (L. californica vallicola) is paler, The valley quail (L. californica vallicola) is paler,
being grayish brown, the inner edges of the tertiary
85 85 86 AMERICAN GAME BIRD SHOOTING feathers whitish, and the flanks grayish brown. It is
a bird of the interior districts of California and Oregon,
south to Cape St. Lucas. The valley quail is an abundant species on the coasts
of California, Oregon and Washington, and has been
introduced on Vancouver Island and on a number of
the islands of Puget Sound. This, according to Dr. Suckley, was done by Governor Charles H. Mason
and Mr. Goldsborough, as.early as the year 1857. They {(iijpRwwMBbSes. daira,
cy
\,
soe
=
»s
SSO LT
CALIFORNIA QUAIL CALIFORNIA QUAIL have long been common on Whidbey Island and on
Vancouver Island, where they make, according to my
experience, far better shooting than they do in the dry
country of California. Of the two races, the California quail belongs espe- Of the two races, the California quail belongs espe-
cially to the coast, and the paler grayer form is found
in the drier interior, from western and southern Ore-
gon south, through western Nevada and interior Cali-
fornia, to, Cape St. CALIFORNIA QUAIL; VALLEY QUAIL. Lucas: owen 'Caltiornta:’)
Whis
species is still abundant, and many broods are found
on some California ranches. In some places they CALIFORNIA QUAIL; VALLEY QUAIL 87 are not popular, because believed to eat the grapes. Others more justly believe that the number of noxious
insects devoured by the valley quail far exceeds in
importance the small amount of fruit which they eat. In many places the too long open season and unrea- In many places the too long open season and unrea-
sonable bag limit have sadly depleted their numbers,
and unless complete protection or restocking is re-
sorted to this beautiful bird will become locally rare. The habits of this species differ very much in differ- The habits of this species differ very much in differ-
ent localities. They are tough, hardy birds, and some-
times surprise the ornithologists by turning up in places
where they would not at all be expected. On the other
hand, they sometimes suffer severely from cold and
snow. Mr. A. C. Lowell, writing from Ft. Bidwell, in
northeastern California, reported to Captain Bendire:
“These birds are unable to stand the severe cold of this
region, especially when accompanied by a heavy fall
of snow. In the winter of 1887-88 about two feet of
snow
fell, followed by three very severe nights in
which the thermometer reached 28° below zero. This
killed most of the birds. In the following fall I heard
of but three or four coveys of quail within a radius of
sixty miles, where thousands had been the year before. They ranged from the northern end of Warner Valley
south to Reno, Nevada, and were especially numerous
in Buffalo Canyon and along the western shore of
Pyramid Lake. They were very common up to the
summits of the Warner Mountains, which attain here
an altitude of about 6,000 feet. Though the canyons Mr. A. C. Lowell, writing from Ft. Bidwell, in
northeastern California, reported to Captain Bendire:
“These birds are unable to stand the severe cold of this
region, especially when accompanied by a heavy fall
of snow. In the winter of 1887-88 about two feet of
snow
fell, followed by three very severe nights in
which the thermometer reached 28° below zero. This
killed most of the birds. CALIFORNIA QUAIL; VALLEY QUAIL. In the following fall I heard
of but three or four coveys of quail within a radius of
sixty miles, where thousands had been the year before. They ranged from the northern end of Warner Valley
south to Reno, Nevada, and were especially numerous
in Buffalo Canyon and along the western shore of
Pyramid Lake. They were very common up to the
summits of the Warner Mountains, which attain here
an altitude of about 6,000 feet. Though the canyons 88 AMERICAN GAME BIRD SHOOTING and water courses
along those
slopes were
their
favorite resorts,
I have never seen nor heard of a
covey of these quail down in the cultivated fields of the
valleys. Here, at least, they prefer to live exclusively
on the brushcovered hillsides.” On the other hand, we have known of cases in Cali-
fornia where a brood of quail came regularly every
evening to drink from the fountain immediately in
front of a ranch house. In certain parts of southern
California the quail has found the orange trees safe
roosting places, and in the evening comes down from
the brush-covered hills to the orchards. In Lower California, Mr. A. W. Anthony found the
valley partridge very common in the mountains up to
an altitude of about 9,000 feet. He adds:
“Both in southern and Lower California
I was told by the Indians and native Mexicans that
during very dry seasons the valley quail do not nest,
but remain in flocks during the entire summer. This
statement I was able to verify by personal observation
during the summer of 1887. ‘These birds were seen
by me in large flocks throughout the spring and sum-
mer months, and only two or three broods of young
were noticed. Birds taken during April, May and
June showed but little development of the ovaries. Should the winter rains, however, be sufficient to in-
sure an abundance of seeds and grasses, the coveys
begin to break up early in March, and from every hill
in the land the loud challenge of the male is heard. The call notes of this sub-species are quite varied, fre- . Nest of California Quail . Nest of California Quail CALIFORNIA QUAIL; VALLEY QUAIL 89 quently the same bird changing his call six or seven
times within half an hour.” Mr. CALIFORNIA QUAIL; VALLEY QUAIL. William Proud wrote Captain Bendire from
Butte County, California, concerning this species, as
follows: “Hundreds of these birds roost every night in the
shrubbery around my house. Some of them are very
tame, feeding among the chickens and coming on
the verandah. They appear to know that they are pro-
tected. They mostly roost in thick brush, and on the
ground when the brush is not at hand. In early sea-
sons they begin to pair in the last week of February,
but the time varies somewhat according to the season. During this period there is considerable fighting among
the males for the favor of the coveted female. This
is kept up until they are suitably mated and the nest-
“ing season arrives. This usually begins here about
the last week in March, when the pairs scatter among
the shrubbery along the banks and creeks and in adja-
cent ravines, along hedgerows and brush fences, and
on the borders of cultivated fields. The earliest nest
I ever found was on March 15, and on April 15 I
met young birds probably a couple of days old. I
consider 14 eggs to be about the average number laid
by these birds, and have found as many as 24 in a
nest. The large sets I attribute to other hens laying
in the nest, probably young birds which have failed to
make preparation for their own eggs. On May 21
my dog pointed a valley partridge on her nest, which
contained 22 eggs, and every one hatched. AMERICAN GAME BIRD SHOOTING go “During incubation the male is very attentive and
watchful, usually taking an elevated position near the
nest, where, with crest erect and tail spread, he bids
defiance
to all intruders,
uttering an
oft repeated
whew-whew-whew. When the breeding hen leaves the
nest to feed, should he be absent from the post of duty,
her cry of tobacco-tobacco, very plainly given, brings
him wp at.once. ... “As soon as the young are hatched they immediately
leave the nest, keeping under cover as much as possi-
ble. Should the brood be disturbed, the old birds will
run and flutter along the ground to draw the attention
of the dog, or whatever may have frightened them, to
themselves and away from the young. In about ten
days these can fly a short distance. CALIFORNIA QUAIL; VALLEY QUAIL. The valley par-
tridge feeds on insects and the young and tender leaves
of clover and green peas; later, on grain and various
small seeds; in the fall they eat wild grapes, and are
also very partial to the seeds of the amaranth, alsc those
of Mentzclia levicaulis. Here only one brood is raised
in a season, and incubation, as nearly as I can ascertain,
lasts about twenty-eight days.”
The eggs of this species range from 12 to 16, nests The eggs of this species range from 12 to 16, nests
occasionally, as already stated, containing many more. The eggs are cream-white in color, spotted and blotched
with different shades of dark and light brown and drab
scattered over the whole egg. Lophortyx gambeli. Lophortyx gambeli. This is a richly-colored bird, and has the flanks
bright chestnut streaked with white. There are no
scalelike markings on the belly, and the central patch is
black, the forehead dark, and the back of the head red-
dish. The upper parts are much as in L. californica. The female resembles that of L. californica vallicola,
but has the flanks chestnut and the belly without any
trace of scalelike markings. It is, perhaps, a little
larger than the California partridge. Its range is north-
western Mexico and the neighboring portions of the
United States, from southern California and Arizona
to western Texas, and north as far as southern Utah. Besides this, other species of this genus are found Besides this, other species of this genus are found
in Mexico, and in one or two of these the sexes are
markedly unlike. Gambel’s quail is a dweller in the desert country of
the south, and ranges from eastern southern Califor-
nia through Arizona, and much of New Mexico, into
western Texas. It is also found in southern Nevada
and portions of Utah, and south into western Mexico. With a general similarity to the valley quail, it has With a general similarity to the valley quail, it has
gI gI AMERICAN GAME BIRD SHOOTING 9Q2 habits not unlike that species, except so far as these
habits are modified by its different surroundings. Like many gallinaceous birds, Gambel’s quail is very
social in habit, and at the proper season they get to-
gether in great flocks, and when alarmed and driven
to wing may get up all about one, only to disappear
almost at once among the thick cover or in the distance. Dr. Coues, in his article on this species published in Dr. Coues, in his article on this species published in
“The Birds of the Northwest,” designates the valleys
of the Gila and Colorado as its centers of abundance. This article is well worth quoting in part, as painting
charming pictures of a region little known to most
sportsmen, but one of extreme interest. He says:
“An interesting fact in the distribution He says:
“An interesting fact in the distribution
of this species is the effect of the Colorado desert in
shutting it off from the fertile portions of California. This dreary, sterile waste offers a barrier to its west-
ward extension that is only exceptionally overcome. Lophortyx gambeli. Although the birds enter the desert a little way, they
rarely reach far enough to mix with the representative
species of California (L. californicus). The strip of
country that mostly assists in their occasional passage
westward is along the Mojave River, a stream rising in
the San Bernardino Mountains, and flowing eastward
toward the Colorado, from which it is shut off by a
range of hills, and consequently sinks in the desert at
Soda Lake. Among other birds, the two kinds of
plumed
quail—Gambel’s
and
the California—meet
along this comparatively fertile thoroughfare upon neu-
tral ground, as Drs. Heermann and Cooper, as well as GAMBEL’S QUAIL 93 myself, have witnessed. Much further south Colonel
McCall found birds at Alamo Mucho, forty-four miles
west of the Colorado; but still the desert is in effect
the barrier I have represented, and the two quails,
speaking generally, do not meet. One wonders the
less at this who has any good idea of the Colorado
desert, such as may be gained, for example, from the
following passage from Colonel McCall’s article, which
remains associated in my mind with the plumed quails,
with all the freshness of first impressions. Speaking
of the Alamo, where he shot a pair, “Here is in truth
a desert!’ exclaims the colonel. ‘Figure to yourself,
if you can, a portion of this fair earth where for some
hundreds of miles the whole crust seems to have been
reduced to ashes by the action of internal fires; behold
a vast plain of desolation, surrounded, and at intervals
intersected, by abrupt mountain ranges which are little
better than gigantic heaps of scoria. Imagine this scen-
ery to be actually glowing under the direct rays of the
midsummer sun, and you may have some idea of the
prospect that meets the eye of the traveler who looks
out upon the desert from the well of the Alamo. You
may perceive in his rear a few stunted cottonwood trees,
scattered along the edge of a channel in which appar-
ently water once was, but now is not; while around
him, here and there, is a light-leaved mesquite that
stretches forth its slender arms and appears to invite
him to a shade that is but a mockery. Lophortyx gambeli. Here it was that
I first heard the plaintive voice of this bird as he strove
to cheer his mate while occupied in the tedious task AMERICAN GAME BIRD SHOOTING 94 of indubation.’
And singularly enough, the bird is al-
most equally hemmed in by desert to the eastward as
well; for according to the same writer’s accounts, ‘A
sandy desert between the Pecos and Devil’s River is
the barrier beyond which the species under considera-
tion has not extended its range.’
“Such a glimpse of the haunts of the plumed quail “Such a glimpse of the haunts of the plumed quail
makes one wonder how it ever became a game bird at
all; how sportsmen could be able to make game of it
without being themselves rather made game of. But
great as are the natural disadvantages of the surround-
ings, the bird’s attractions are still greater, and partly
so from this very fact ; but Arizona is a large place, and
one need not always endure desert in his quail-shoot-
ing. There is a much brighter side to the picture. We
have just seen only the darkest possible. Perhaps no
territory rivals Arizona in variety of climate, physical
geography and natural productions. Between rugged
mountains
that lift snow-capped peaks among the
clouds, lie hidden pleasant green valleys whose fresh
verdure contrasts with surrounding desolation. Vast
primeval forests stretch for leagues, or are only inter-
rupted by oak and cedar openings. Bright fringes of
cottonwoods and willows mark the devious courses of
streams, where walnut and cherry are scattered, and
grape vines cling to them and roses bloom beneath leafy
boughs. Here is plenty, at least, if not peace. Noth-
ing mars the pleasures of the chase but the chances of
being chased. Were it not for Indians, we should have
here the acme of quail-shooting. GAMBEL’S QUAIL 95 “Gambel’s quail may be looked for in every kind of
cover. Where they abound it is almost impossible to
miss them, and coveys may often be seen on exposed
sand heaps, along open roads, or in the cleared patches
around settlers’ cabins. If they have any aversion, it
is for thick, high pine woods without any undergrowth;
there they only casually stray. Lophortyx gambeli. They are particularly
fond of the low, tangled brush along creeks, the dense
groves of young willows that grow in similar places,
and the close-set chaparral of hillocks or mountain
ravines. “T have often found them also among huge granitic
boulders and masses of lava, where there was little or
no vegetation except some straggling leaves, and have
flushed them from the dryer knolls in the midst of a
reedy swamp. Along the Gila and Colorado they live
in such brakes as I have described in speaking of
Abert’s finch, and they frequent the groves of mes-
quite and mimosa that form so conspicuous a feature of
the scenery in those places. These scrubby trees form
dense interlacing copses, only to be penetrated with the
utmost difficulty ;
but beneath their spreading, scrawny
branches are open, intersecting ways, along which the
quail roams at will, enjoying the slight shade. In the
most sterile regions they are apt to come together in
numbers about the few water-holes or moist spots that
may be found, and remain in the vicinity, so that they
become almost as good indication of the presence of
water as the doves themselves. A noteworthy fact in
their history is their ability to bear, without apparent AMERICAN GAME BIRD SHOOTING 96 inconvenience, great extremes of temperature. They
are seemingly at ease among the burning sands of the
desert, where for months the thermometer daily marks
a hundred, and may reach a hundred and forty, ‘in the
best shade that could be procured,’ as Colonel McCall
says; and they are equally at home, the year round,
among the mountains, where snow lies on the ground
in winter. “The quail’s food is made up of various substances. Like the rest of its tribe, it is chiefly granivorous, eat-
ing seeds of every description; but fruits and insects
form a large portion of its fare. It devours insects of
such sorts as it can capture, and particularly those kinds
that infest plants. In the fall it gathers cherries and
grapes, and other ‘fruits,’ properly speaking, as well as
the various berries not usually so called. It visits patches
of the prickly pear (Opuntia), to feed upon the soft,
juicy tunas, that are eaten by everything in Arizona,
from men and bears to beetles. Lophortyx gambeli. In the spring it shows
fondness for the buds of different plants, particularly
mesquite and willow; birds shot at this time are fre-
quently found with sticky bits of the buds about their
bills. But though they thus feed so extensively upon
this substance containing salicine, I never noticed that
the flesh acquired a bitter taste. There is, as yet, little
cultivated grain in Arizona, but doubtless some future
historian will have to add our cereals to the bird’s list,
and speak of Gambel’s quail as frequenting old corn
and wheat-fields and the neighborhood of hayricks,
where a large share of its food is to be gleaned. Like GAMBEL’S QUAIL 97 other Galline, it swallows quantities of sand and gravel
to facilitate, it is supposed, the trituration in the gizzard
of the harder kinds of food. “T believe that the quail moults at least twice a year, “T believe that the quail moults at least twice a year,
but the spring change is apparently less complete, and
certainly more gradual, than that of the fall, the birds
seeming rather to furbish up a part of their plumage
than to furnish themselves with entirely new attire. By
the latter part of summer (at Fort Whipple) the plu-
mage is faded and worn with incubation and the care
of the young, and the renewal begins as soon as the
latest brood is reared. The process is a long one, and
the birds are rarely found at any season in such poor
condition as to be unfit for preservation, nor are they
ever deprived of flight. No crest is occasionally found
for a short time in early autumn, but new feathers gen-
erally sprout before all the old ones are dropped. I
think they are shed from behind forward, so that the
front ones are lost the last. The fully developed crest
is a striking and beautiful ornament, hardly to be sur-
passed in stylishness and jaunty effect. It averages an
inch and a half in length, and sometimes reaches two
inches in the most vigorous males; in the female it is
rarely over an inch. The male’s is a glossy jet black;
the female’s has a brownish cast. The number of feath-
ers composing it is variable; five or six is usual, but
there may be ten. Lophortyx gambeli. They all spring from a single point
on the top of the head—just behind the transverse white
line that crosses the crown from eye to eye. The feath-
ers are club-shaped, enlarged at the tip and curling over AMERICAN GAME BIRD SHOOTING 98 forward, together forming a helmet-shaped bundle. The webs are loose; they bend backward from the shaft
so that this forms the front border of the feather. Each
feather is thus folded or imbricated over the next suc-
ceeding, and the whole are packed into a single fascicle
in this manner. The crest is freely movable, and its
motions are subject to voluntary control. It is usually
carried erect, but sometimes drops forward, or oblique-
ly, over one eye, and occasionally is allowed to hang
backward, though it cannot be made to lie close over
the occiput. The crest sprouts when the chicks are
only a few days old, about the time that the first true
feathers appear upon the wings and tail... . “All quail are Precoces, as already explained, and “All quail are Precoces, as already explained, and
the chicks of this species are certainly precocious little
things, if we may judge by their actions when they are
disturbed. They run about as soon as they are hatched,
though probably not ‘with half shell on their backs,’
as some one has said. In a few days they become very
nimble, and so expert in hiding that it is difficult either
to see or catch them. When the mother bird is sur-
prised with her young brood, she gives a sharp warning
cry that is well understood to mean danger, and then
generally flies a little distance to some concealed spot,
where she crouches, anxiously watching. The fledg-
lings, by an instinct that seems strange when we con-
sider how short a time they have had any ideas at all,
instantly scatter in all directions, and squat to hide as
soon as they think they have found a safe place, remain-
ing motionless until the reassuring notes of the mother GAMBEL’S QUAIL 99 call them together again with an intimation that the
alarm is over. Then they huddle close around her, and
she carefully leads them off to some other spot where
she looks for greater security in the enjoyment of her
hopes and pleasing cares. Lophortyx gambeli. As long as they require the
parents’ attention they keep close together, and are
averse to flying. Even after becoming able to use their
wings well they prefer to run and hide, or squat where
they may be, when alarmed. If then forced up, the
young covey flies off, without spreading, to a little dis-
tance, often realighting on the lower limbs of trees or
in bushes, rather than on the ground. As they grow
older and stronger of wing they fly further, separate
more readily, and more rarely take to trees; and some
time before they are full grown they are found to have
already become wary and difficult of approach. As
one draws near where a covey is feeding, a quick, sharp
cry from the bird who first notices the approach, alarms
the whole, and is quickly repeated by the rest as they
start to run, betraying their course by the rustling of
dry leaves. Let him step nearer, and they rise with
a whirr, scattering in every direction. “Newly hatched birds may be found all summer, and “Newly hatched birds may be found all summer, and
incubation goes on from, say, early in May until the
middle of August. Not that any single pair are en-
gaged so long, but that different broods may be hatched
during all this time. A greater number of old birds
pair in April, and hatch their first brood some time
during the following month. Most of them doubtless
raise another. Others appear to defer incubation for AMERICAN GAME BIRD SHOOTING 100 a month or two, and have but one brood. The first
chicks that I saw in the summer of 1865 were hatched
in May, and I found others the same year only a few
days old in August; but by this month almost all the
birds of the year were well grown, and by September
were in condition to offer legitimate sport. In Octo-
ber a few are found not yet ready to be shot; but the
great majority are as large as the parents, and nearly
as strong of wing. Lophortyx gambeli. The season may, therefore, be said
to begin in October and continue into March; but birds
should not be pursued later than the middle of this
month, for although few birds, if any, actually mate
before April, it is cruel, as well as injudicious, to dis-
turb them while they are preparing to do so. The be-
ginning of the pairing season may be known to be
at hand when certain peculiar cries, different from any
usually emitted during the fall and winter, are heard. “These notes are a sign that the coveys are breaking
up and mating is about to commence. They are analo-
gous to the bobwhite of the eastern quail (Ortyx vir-
ginianus), and are uttered, as with that species, more
particularly in the breeding season. The note is a loud,
energetic, two-syllable whistle, delivered in a clear,
ringing tone. It is difficult to write down intelligently,
but, once heard, is not likely to be afterward mistaken,
except for one of the cries of the black-headed gros-
beak. It sounds to my ears something like the forcible
pronunciation of the syllables killink, killink, indefinite-
ly repeated, sometimes in a rising and sometimes with
a falling intonation. The old cocks, if they can be “AVAING
JeosopoIg
“SG
*AQ
9Y}
Jo
UOTsstuIOd
Ag
‘Tend
sjequreg “SG
*AQ
9Y}
Jo
UOTsstuIOd
‘Te
sjequreg GAMBEL’S QUAIL IOI seen so engaged, are found strutting along some fallen
log or gesticulating from the top of some broad rock
or stepping with dignified air along the pathway under
the bushes ; sometimes even perched in a tree or bush,
without other motion than the heaving of the chest;
but wherever they may be, they have one mind in com-
mon, their only thought to secure the admiration and
then the favor of birds more modest if not more fair. “A beautiful sight it is to see the enamored birds
pressing suit with all the pomp and circumstance of
their brilliant courtships—the firm and stately tread,
with body erect, of comely shape, displayed to best ad-
vantage ; the quivering wings, the motion of the plumes,
that wave like the standard of knights errant, the flash-
ing eyes—bespeak proud consciousness of masculine
vigor. The beautiful bird glances defiance, and chal-
lenges loudly, eager for a rival; but none disputes, and
he may retire, his rights proven. Lophortyx gambeli. Only a gentler bird
is near, hidden in a leafy bower, whence she watches,
admiring his bearing, fascinated by the courage she sees
displayed, hoping every moment that the next will
bring him, dreading lest it may. As their eyes meet
she trembles, and would turn to fly, but cannot; his
glow again, but with a different light, even more in-
tense than before. With an exultant cry he flies
straight to her, and, like the true knight he is, for just
one instant bends till his breast touches the ground, and
is then erect again. He leans toward her, half spreads
his wing over her, and pleads in an undertone. She AMERICAN GAME BIRD SHOOTING 102 listens, but draws away; she listens, but only stands
irresolute; she listens, and, listening, yields. “They must prepare for new duties. With deep “They must prepare for new duties. With deep
sense of responsibility and earnest solicitude the pair
now cast about for a suitable spot for their home. They
search through the tall, rank herbage alongside the
stream, through the willow copses, among fallen moss-
covered logs that are scattered around the glen, and
at length make up their minds. Little more is needed
than to fix upon the spot, for the nest is a simple affair,
the work of a few hours, perhaps, scratching a suitable
depression and lining it with a few dried grasses
pressed together. Day by day eggs are laid, till a dozen
or more fill the nest. They cannot be distinguished
from those of the California quail. They measure an
inch and a quarter in length by an inch in breadth,
and are almost pyramidal in shape, the larger end flat-
tish and very broad, the other narrow and pointed. The color is a buff, or rich cream, dotted and spotted
all over with bright brown, and splashed here and
there with large blotches of the same. When the fe-
male is not pressed to lay, the pair ramble about to-
gether in close company until the complement is fin-
ished. Then she gives up all recreation, grown already
quite sober and maternal, and resolutely sets about her
long tour of duty. But she is not forgotten because she
can no longer share the idle pleasures of her lord. Lophortyx gambeli. Mounted on a stump or bush near by, he stands watch,
and continually solaces her with the best music he can
make. It is not very harmonious, to be sure; in fact, GAMBEL’S QUAIL 103 his ditty at such times is a medley of odd notes, sound-
ing rather lugubrious than hilarious, but it is presum-
ably satisfactory to the one most concerned. So the
long days pass, for two weeks or more, till feeble cries
come from the nest; the mother dries and cuddles the
curious little things, and the delighted birds, brimful of
joy, lead their family off in search.of food. “From the number of eggs s “From the number of eggs sometimes found in a
nest it becomes a question whether birds, hard pressed,
may not occasionally deposit in nests not their own. We have no positive evidence that it may occur, but
observation has rendered it highly probable, and such
is the case with some other birds, as the rails andi, I
think, the Virginia quail. However this may be, it is
pretty certain that broods of young sometimes coalesce
at a varying time after hatching. I do not remember
to have myself seen a covey of more than twenty, but
it is currently reported upon good authority that troops
numbering as many as fifty partly grown birds, and
including several old ones, may be met with. This
raises, of course, the question of polygamy, so common
in birds of this order, and something may be said in
favor of the view. The same surmise has been made
in the case of L. californicus, but I believe it remains
to be proven. I am bound to observe that I have never
witnessed anything supporting this view. . . . “Man is, I suppose, the quail’s w “Man is, I suppose, the quail’s worst enemy; what
the White does with dog and gun the Red accomplishes
with ingenious snares. The Indians take great num-
bers alive in this way, for food or to trade with the AMERICAN GAME BIRD SHOOTING 104. whites along the Colorado, and they use the crests for
a variety of purposes that they consider ornamental. I saw a squaw once who had at least a hundred of them
strung on a piece of rope-yarn for a necklace. Lophortyx gambeli. But the
birds have other foes : the larger hawks prey upon them,
so also do the wolves, as I had good evidence upon one
occasion, when hunting in a precipitous, rocky place
near Fort Whipple. I heard a covey whispering about
me as they started to run off in the weeds, and fol-
lowed them up to get a shot. They passed around a
huge boulder that projected from the hillside, and then,
to my surprise, suddenly scattered on wing in every
direction, some flying almost in my face. At the same
instant a wolf leaped up from the grass where he had
been hiding, a few feet off, intending to waylay the
covey, and looking very much disappointed, not to say
disgusted, at the sudden flight. We had marked the
covey together, and were hunting it up from opposite
sides, and neither of us could account for their flushing
so unexpectedly. Then he caught sight of me, and it
was a question which of us was most surprised. How-
ever, I felt that I owed him a private grudge for get-
ting in the way of the birds and spoiling my shot, so
I fired both barrels in quick succession. With nothing
but mustard seed in my gun, I hardly expected to more
than frighten the beast, but he was so near that he
rolled over quite handsomely, his hindquarters para-
lyzed with the charge, which took effect in the small
of the back. I kept his skin as a trophy, and since that
time have had unlimited confidence in small shot.” GAMBEL’S QUAIL 105 This account was written, I believe, as long ago as
1872, or 1873, but it remains the best description of
Gambel’s partridge that has been writtten. The birds appear t The birds appear to be as numerous in their range
as ever they were, and with the increase of water
which goes with the progress of irrigation in Arizona
and New Mexico they are likely to increase still further. Captain Bendire cites an obse Captain Bendire cites an observation of Mr. Herbert
Brown, of Tucson, Arizona, which shows that the Gila
monster (fZeloderma) eats the eggs of this species. MASSENA QUAIL. Cyrtonyx montezume mearnst. Cyrtonyx montezume mearnst. This handsome and highly-colored bird is of the
southwest, and until very recently has never been pur-
sued for sport, though often killed for meat. In ap-
pearance it is unlike any of our United States par-
tridges, having large feet, a full, soft crest, and being
more or less dotted below with large round spots of
pure white. The sides of the head are marked with
black and white stripes; the chin, throat, and a band
across the fore neck, are all black ; the crest is brownish;
the upper parts are brown, barred and spotted with
black and streaked with white, buff or reddish. The
wing quills are spotted with white on their outer webs,
while the thighs, belly and under tail coverts are black. The flanks and sides are lead-color, marked with many
round spots of pure white, and the middle line is chest-
nut. The female is paler everywhere, but has the upper
parts streaked and barred as in the male. The head
lacks the distinctive white and black stripes, and the
sides are slightly barred with black. Length, 8%
to g inches; wing, about 5, or a little more. This quail
is found in western Texas, New Mexico, Arizona, and
well down into western and central Mexico. The typi-
106 106 106 MASSENA QUAIL 107 cal montezume and other species occur in Mexico. One
found in Guatemala and southern Mexico has the flanks
chestnut instead of lead-color. This bird was described as long ago as 1830, yet This bird was described as long ago as 1830, yet
for many years thereafter little or nothing was known
about it, and it was only in 1890 that the eggs were
found and described. MEARNS, OR MASSENA QUAIL MEARNS, OR MASSENA QUAIL The Massena quail, or, as it is now called, the Mon-
tezuma quail, is notable for its odd and beautiful col-
oring and for its extraordinary simplicity and lack of
suspicion, a quality which is reflected in the name “fool
quail,” given it in some sections. This title, commonly
enough applied to certain unsuspicious grouse and
quail, is an unintentional testimonial by man to his own
bloodthirstiness, declaring in effect that any bird or ani-
mal must be a fool that does not know that civilized AMERICAN GAME BIRD SHOOTING 108 man is a natural butcher. Cyrtonyx montezume mearnst. It takes wild things some
time to realize that there are no gentle men, and obvi-
ously this lesson is learned more slowly by birds or
animals living in places unfrequented by man than in
regions where he is more numerous. Even the ruffed
grouse, which, in thickly settled regions, is the wisest,
wildest and most wary of birds, is in some localities so
unafraid that the passer-by who will tie a noose of cord
on the end of a six-foot pole, may pass the loop over
the bird’s head and drag it from its perch. The Mearns quail is reported to be less graceful in The Mearns quail is reported to be less graceful in
carriage and less elegant in shape than some of its rela-
tives which inhabit the same region, but may not this
be only another way of stating that it is unsuspicious
-and not easily alarmed? The wariest of gallinaceous
birds, if ignorant of the presence of an enemy, carries
its feathers more or less loosely, walks with a short
neck, and has a rounded back, thus presenting an ap-
pearance very different from the same bird when it
is startled or alarmed and about to take to flight. Then the neck is stretched upward, the bird stands high
on its legs, all its feathers are pressed close to its body,
its crest is raised, and it stands there alert and pre-
pared to dart away at a second’s warning. In Texas this bird is known as the black quail, or In Texas this bird is known as the black quail, or
the black-bellied quail, while, as said, in Arizona, on
account of its gentle nature it is called fool quail. It
is said to frequent rocky ravines heading well up into
the mountains, but of recent years has come to the
ranches, and is found feeding in the green fields. Cap- Mearns’ Quail. Mearns’ Quail. MASSENA QUAIL 109 tain Bendire says that in Mexico he has several times
seen them living contentedly in cages. Capt. W. L. Carpenter saw the Massena partridge in Capt. W. L. Carpenter saw the Massena partridge in
the Rio Grande Valley, near Taos, Mexico, and on the
headwaters of the Black and White rivers, where he
believed it bred. Cyrtonyx montezume mearnst. In the breeding season, in Arizona,
they are said to frequent live-oak scrub and patches
of rank grass, at an altitude of seven to nine thousand
feet. “Here,” according to Mr. John Swinburne, writ-
ing to Captain Bendire, “they are summer residents,
only descending to much lower altitudes in winter. They lie very close at all times, allowing one to almost
step on them before they move. I have seen this species
on the White Mountains during the breeding season,
and saw young birds of the year shot there. Even
the adults seemed very stupid when suddenly flushed,
and after flying short distances would alight and at-
tempt to hide in most conspicuous places. I have seen
men follow and kill them by throwing stones.” Captain Bendire also quotes Mr. G. W. Todd, as
follows: “T first met with the Massena partridge in Bandera
County, Texas, in 1883, where they are very scarce,
and I learned but little of its habits for a long time. They are very simple and unsuspicious, and apparently
live so much in barren and waste places that they do
not see enough of man to make them afraid. On see-
ing a person they generally squat at once, or run a little
way and hide. They will hardly fly until one is almost
on them, but when they finally do fly they go much AMERICAN GAME BIRD SHOOTING T10 further than either the Texan bobwhite or the scaled
partridge, and on alighting they run rapidly for a lit-
tle distance and then squat again, generally flushing
easier the second time. It is rare to see more than
six together; two or three are more often met with. In the fall of 1886 I found a covey of five, on a wet
and misty day, and killed three of them with a Win-
chester rifle before the remaining two flew. I never
found their eggs nor met with small young until this
year. I saw but a single bird this season, and this
seemed to be entirely alone. They are not very abun-
dant here, and are always found in the most barren
places, among rocks and wastes where even prickly
pear is stunted and no bush grows over three feet
high. Cyrtonyx montezume mearnst. When scared they utter a kind of whistling
sound, a curious combination between a chuckle and
a whistle, and while flying they make a noise a good
deal like a prairie hen, though softer and less loud, like
chuc-chuc-chuc, rapidly repeated. “The only nest of this species I have ever seen was
situated under the edge of a big bunch of a coarse spe-
cies of grass, known as ‘hickory grass.’
This grass
grows out from the center, and hangs over on all sides
until the blades touch the ground. It is a round, hard-
stemmed grass, and only grows on the most sterile soil. According to my observations, the Massena partridge
is seldom seen in other localities than where this grass
grows. I was riding at a walk up the slope of a bar-
ren hill, when my horse almost stepped on a nest, touch-
ing just the rim of it. The bird gave a startled flut- MASSENA QUAIL II! ter, alighting again within three feet of the nest, and
not over six feet from me;
then she walked away with
her crest slightly erected, utering a low chuckling
whistle, until lost to view behind a Spanish bayonet
plant (Yucca) about thirty feet off. 1 was riding a
rather unruly horse, and had to return about thirty
yards, to tie him to a yucca, before I should examine
the nest. This was placed in a slight depression, pos-
sibly dug out by some animal, the top of the nest being
on a level with the earth around it. It was well lined
with fine stalks of wire-grass almost exclusively, the
cavity being about five inches in diameter and two
inches deep. At the back, next to the grass, it was
slightly arched over, and the overhanging blades of
grass hid it entirely from sight. The nest was more
carefully made than the average bobwhite’s nest, and
very nicely concealed.” The food of the Massena quail, in western Texas and
New Mexico, appears to consist of small bulbs, to se-
cure which the birds often dig or scratch out holes to
the depth of two inches. In the mountains they also
eat acorns, mountain laurel, tubers of nut grass, cedar
and other berries. Cyrtonyx montezume mearnst. The nest is likely to be built on a hillside, sometimes The nest is likely to be built on a hillside, sometimes
well hidden, and often sunk quite deeply in the ground. The eggs seem to run from 8 to 10 in number. They
are pure white in color, and, while often glossy, are
sometimes more or less rough and granulated. As already stated, the Massena quail is scarcely pur- As already stated, the Massena quail is scarcely pur- AMERICAN GAME BIRD SHOOTING
| 112 sued for sport, and on this point Mr. Herbert Brown,
of Tucson, Arizona, writes me as follows: “The Massena quail, or, as it is now called, Monte-
zuma quail, will lie well to a dog, but, practically speak-
ing, they are not hunted as a game bird; in fact, not
hunted at all. They are mountainous in their habits,
and are not commonly killed. “This
bird, among
the Mexicans,
is known
as
‘suinea,’ and also as ‘chacalaca.’
The latter word is
commonly applied to persons of garrulous or talkative
dispositions. As the male bird, when strutting among
the females, keeps up a continual clatter, the Mexican
people, with their readiness for characterization, call
them ‘chacalaca.’ “Tt is among the most peculiar of our game birds,
and I much regret that it is so little known. Of all
the quail it is the most easily domesticated. Some years
ago a young female was sent to me from Sonora. As
I then had no means of keeping it, I gave it to a friend. It was raised about the house, and was as tame as a
pet chicken. A swinging door led into the kitchen, and
it was always careful to await its opportunity, and make
its run through as the door swung open. Some years
ago I heard of a small bunch on the Sonoita that fed
regularly with the chickens at the ranch house. At a
ranch house on the Sopre two adult birds took up with
the chickens, and in the spring following raised a brood
of little ones, some four or five of which grew to ma-
turity. Eventually they fell into disfavor and were
driven away or killed. This was due to the pugnacity MASSENA QUAIL 113 of the male bird. They appeared to have no use for
the chickens, and were continually fighting them with-
out provocation. of the male bird.
They appeared to have no use for
the chickens, and were continually fighting them with-
out provocation.
The big ones could stand it, but the
little ones were the sufferers,” Cyrtonyx montezume mearnst. The big ones could stand it, but the
little ones were the sufferers,” Tetraonide. The differences between the American grouse and
quail have been pointed out in two earlier chapters,
but, nevertheless, may be repeated here. The feet of
all the grouse are covered with hair-like feathers. In
this group a tuft of small feathers runs forward from
the forehead along each side of the bill and covers the
nostrils, while in the quails the nostril is naked and is
protected by an overhanging scale. All the grouse
have on head over the eyes narrow patches of naked
skin, and most or all of them have on the sides of the
neck bare patches, which are often inflatable. The pectinations on the toes of the grouse are shed
-annually. Mr. Manly Hardy of Brewer, Me., informs
me that in the ruffed grouse the whole scaly envelope
of the foot is shed each year. The grouse are usually large birds, while the quail The grouse are usually large birds, while the quail
are small, and in the grouse the tail is usually long,
while in all the quail it is short. The outline drawings, showing the bill, foot, wing The outline drawings, showing the bill, foot, wing
and tail of the various grouse and quail will assist those
who desire to learn these characters. 114 114 114 DUSKY GROUSE. Dendragapus obscurus. Dendragapus obscurus fuliginosus. Dendragapus obscurus richardsom. Dendragapus obscurus sierre. The dusky grouse is the largest of the American
wood
grouse,
sometimes
weighing
four
pounds. Above, the male is dusky gray or dull black, usually
more or less waved with fine, blacker lines. Sometimes
there is a variety of light and dark color on the back
and wings, producing a mottled effect. The tail is
black with a gray band across the end, usually from
half an inch to an inch wide. The under parts are
slaty gray, somewhat streaked with white on the flanks. The bird is from twenty to twenty-three inches long,
and is stout and graceful. On the side of the neck there
is a patch of white surrounding a naked, inflatable
bare space which is hardly, or not at all, to be seen,
except during the breeding season. The throat is also
white or very pale. The much smaller female is grayish or brownish in The much smaller female is grayish or brownish in
color, marked with spots and bars of blackish. This is the typical dusky or blue grouse, and is This is the typical dusky or blue grouse, and is
found in the Rocky Mountains, west as far as they ex-
tend, east to the Black Hills, south to the Mogollon
115 115 AMERICAN GAME BIRD SHOOTING 116 Mountains of western New Mexico and the White
Mountains of middle eastern Arizona. The sub-species,
known as the sooty grouse, is found on the northwest
coast in the region of great precipitation, north to an
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DUSKY GROUSE DUSKY GROUSE southern Alaska and southern Yukon. It is darker
colored, with a narrower tail band, lacks the whitish
space on the side of the neck and has the throat darker. Often or usually the scapulars or shoulder feathers
are without distinct white streaks or spots. southern Alaska and southern Yukon. It is darker
colored, with a narrower tail band, lacks the whitish
space on the side of the neck and has the throat darker. Often or usually the scapulars or shoulder feathers
are without distinct white streaks or spots. DUSKY GROUSE. DUSKY GROUSE 17 The adult female is much darker than the typical
dusky grouse, and often has the upper parts distinctly
reddish, instead of being merely gray or buffy. Richardson’s grouse, found in the northern Rocky Richardson’s grouse, found in the northern Rocky
Mountains, has the tail without the terminal band of
gray; otherwise it is much like the dusky grouse, but
usually the tail feathers are broader and the tail more
even. There are certain places in the Rocky Moun-
tains—for example, in northwestern Montana—where
the dusky grouse and Richardson’s grouse intergrade,
and often it is impossible to say to which form a speci-
men belongs. In this latter region the female is often
gray, black mottled, with little or no tinge of buff. The sierra grouse, with a narrower tail band than The sierra grouse, with a narrower tail band than
the typical form, is found in the region from Fort
Klamath, Oregon, south, through the mountains of
California, to Mount Pifios, near old Fort Tejon. The dusky grouse, although not at all known by east- The dusky grouse, although not at all known by east-
ern sportsmen, is yet easily the finest of our American
species. It is exceeded in size only by the sage grouse,
but in beauty, in edible qualities and in the sport it
furnishes it far excels that species. The dusky grouse is found in all the mountain re- The dusky grouse is found in all the mountain re-
gions of the farther West, from Alaska south as far
as the White Mountains of Arizona. Naturalists sepa-
rate it into four forms, as given above; but in habits,
and in the qualities which interest the gunner, all are
much alike. It is not a migratory bird, but remains
essentially in one locality throughout the year, al-
though, like many mountain-dwelling birds and mam- AMERICAN GAME BIRD SHOOTING 118 mals, the altitude at which it lives changes with the
changing season. One of the earliest spring sounds in the country
where the dusky grouse are found is the hooting of
the male, a booming or blowing sound analogous to
the grating song of the sage grouse or the tooting of
the pinnated grouse. The call possesses a certain ven-
triloquial quality, which makes it exceedingly difficult
to follow, and it is often hard to locate the bird that is
uttering it. DUSKY GROUSE. A similar difficulty is usually found in
attempting to follow up the drumming
of the ruffed
grouse. This call and the accompanying mating ac-
tions were well described by a correspondent, “Stand-
stead,” in Forest and Stream twenty years ago. He
wrote: “While driving near the city. [ Victoria, B. C.]
with the veteran shot, R. Maynard, we saw a pair of
blue grouse quite near the trail, and the cock bird gave
us a most entertaining exhibition of the charms that he
displays in wooing his mate. Like a turkey cock he
strutted about with his wings trailing on the ground,
his tail feathers erect and spread out fart-like to their
fullest extent, his neck distended, and on each side of
his neck the feathers were turned out so as to resemble
a pair of round white rosettes, nearly three inches in
diameter, with an oblong red spot in their center, where
the skin of the neck was exposed. His head seemed
to be crowned with a fiery red comb. Excepting the
rosettes, he was in appearance a miniature turkey gob-
bler. Every few seconds he would strut up to his de-
mure but sleek-looking mate, puff out his neck and *& e
>
i
Be
em
a aL
Sierra Dusky Grouse and young. From group in American Museum of Natural History. Sierra Dusky Grouse and young. From group in American Museum of Natural History. DUSKY GROUSE 119 with a jerky movement of his head utter his boom,
or hoot, boom-boom-boom. As he grew more and
more demonstrative in his actions, his modest mate
flew up to an overhanging limb to escape his familiari-
ties, and we drove away, leaving him still strutting on
the ground underneath the tree where his mate sat
perched.” Another writer refers to the sound uttered at this
season by the male dusky grouse as “growling” or
“groaning,” and notes, as have many others, the pe-
culiarity that when near it often seems quite distant,
and when distant it sometimes seems near, appearing to
come from every direction but the true one. The female usually makes her nest in the open at the
foot
of the mountains,
quite
a
little way
from
the timber, perhaps under some little clump of brush at
the foot of a steep bluff, partly clad with pines, or
perhaps among the aspens in some mountain valley. DUSKY GROUSE. The eggs vary in number from seven to fifteen, and
perhaps are oftenest nearer the smaller number. They
are buffy in color and are’usually more or less thickly
spotted with fine dots of reddish brown or even choco-
late. These dots and spots are usually quite distinct
and seldom or never run into blotches and cloudings,
as is usually the case with the eggs of the ptarmigan. After the nest is constructed and the eggs are laid,
the male leaves his mate and by slow stages betakes
himself to the mountain-tops, where, in midsummer,
old cocks and barren hens are found in the extreme
edge of the timber, and very often on the alpine mea- AMERICAN GAME BIRD SHOOTING 120 dows and amid the rocks above timber line. The female
all through the summer,
leads her brood of little
ones about where insects, berries and seeds are most
accessible. Like many other grouse, if her young are
closely approached she feigns to be hurt, and runs off
with dragging wings and seemingly feeble footsteps
to lure the enemy to pursue her; but, after she has
been followed for a little distance, takes wing and
flies off up the mountain, to return on foot when the
danger is past. By the middle of July the young are
usually somewhat larger than quails, and at this time,
instead of immediately hiding when approached, they
take wing for a short flight, and, immediately on reach-
ing the ground, hide, lying as close as any birds that
I have ever seen. At such times, if the ground be
fairly open, it is not difficult to see them as they crouch
close to the earth, but as soon as they recognize that
your eye has caught them, they spring into the air for
another flight. I recall one morning when, traveling
along a little valley, with a companion, we started
several broods of well-grown young, of which we
killed seventeen by shooting their heads off with our
rifles. The birds were needed for food for the camp. As the young birds grow larger and more hardy,
the mother leads them higher up the mountain, and
by the end of August or first of September they are
feeding among the green timber, at which time their
principal food in some localities is a species of low-
growing red huckleberry. DUSKY GROUSE. By September or October
the birds are nearly as large as the parent, and long be- DUSKY GROUSE I21 fore this it is their practice when disturbed to fly up
into the branches of the pines, where they stand erect
with feathers pressed close to their bodies, each looking
for all the world like a broken-off dead branch. Often in August or September broods of these birds
may be found on the bare mountain-side, feeding on the
huckleberries, or among low sarvis trees. In such a
case splendid and very easy shooting may be had at
these great birds, but if a flock be scattered in the tim-
ber, or among thick alders, the shooting is quite as
difficult as that of the eastern ruffed grouse. Although
these birds can know little about the shotgun, they are
often quite as acute as the partridge in putting the
trunk of a tree or a clump of bushes between the gun-
ner and themselves. On the other hand, when they
take refuge in the branches of a tree, and stand there
watching you, they will often permit half-a dozen shots
to be fired at them without moving, unless hit. In Captain Bendire’s admirable account of the sooty In Captain Bendire’s admirable account of the sooty
grouse,
the northwestern form of D. obscurus, he
quotes a letter from Captain (now General) T. E. Wilcox, in which the writer says:
“I once caught a
mother grouse with a fish hook. I had my rod on my
shoulder and suddenly came upon a covey about the
size of quails and caught one with my hands. This
made the old bird frantic; she attacked me, and alight-
ing on my rod, the hook pierced her foot. I was
pulling her in when my leader broke and she flew off. Of course, I released her chicken. I killed a male in
the
Boise
Mountains,
December
2,
1879,
which AMERICAN GAME BIRD SHOOTING r22 weighed three pounds ten ounces; but some killed by
me in the Cascade Mountains seemed to be much larger. While on Lake Chelan in 1883, hunting white goats, I
flushed a covey of grouse and here heard for the first
time the call note of the female for her young. It
was low, but distinct, something like that made by the
bobwhite just before it flushes. DUSKY GROUSE. At this time, the last
of August, the birds were well grown. I have always
found these birds near water. In 1881, while going to
Indian Valley, Idaho, I rode past some, one being near
enough to touch with a switch I had in my hand, yet
they all walked out of the trail as quietly as domestic
fowls
would
have
done and
then resumed
their
dusting.” Richardson’s grouse, the form of the dusky grouse
found in the northern Rocky Mountains from southern
Montana,
north
far into
British
America,
resem-
bles in habits the other forms and is quite abundant. About the Continental Divide, specimens are some-
times seen which show approach to obscurus, or to the
sooty grouse. In many places, where seldom disturbed
by man, they are exceedingly gentle and unsuspicious,
and a brood will walk slowly along before one, feed-
ing and calling to one another in a particularly inno-
cent and attractive way. It is stated that the birds spend their winters largely It is stated that the birds spend their winters largely
among the branches of the pine trees, and that they feed
to a very great extent on the pine needles. Mrs. Florence Merriam Bailey says of the winter Mrs. Florence Merriam Bailey says of the winter
habits of the dusky grouse in New Mexico: ‘asnoig
Aysng ‘as
Aysng DUSKY GROUSE 123 “Near our camp by the foot of Pecos Baldy, Mr. Bailey discovered a winter roosting tree of the grouse. The tree was on a sheltered part of the wooded slope
and was so densely branched that after a prolonged
rain the ground beneath was perfectly dry. The earth
was strewn with winter droppings, composed entirely
of the leaves of conifers. Conifer needles had also
been eaten by three of the grouse that were taken... in July and August, but at this season the birds were
living principally on such fresh food as strawberries,
bearberries (Arctostaphylos uva-urst), sheperdia ber-
ries, flowers of the lupine and paint brush, seeds, green
leaves, grasshoppers, caterpillars, ants and other in-
sects. DUSKY GROUSE. But one crop contained twenty-seven strawber-
ries, twenty-eight bearberries
and twelve sheperdia
berries, besides flowers, leaves and insects, while the
accompanying gizzard was
filled with seeds, green
leaves and insects.”
In some sections of the West the dusky grouse are In some sections of the West the dusky grouse are
reported to pack in the autumn, forming very large
flocks, but I have never seen anything of the kind. Usually, in my experience, only the members of a
single brood, ranging in numbers from eight to twenty,
are found together. Sometimes, however, in summer,
on the high peaks, flocks of a dozen old males are
found associated together. It is said that in Colorado and parts of Montana, It is said that in Colorado and parts of Montana,
where grain is grown adjacent to the mountains or the
timber occupied by the dusky grouse, the birds are
frequently found feeding on the stubble fields. If this AMERICAN GAME BIRD SHOOTING 124 is the fact they must give great shooting in such
situations. | It is readily conceivable that this bird might be in-
troduced into the mountains of the Eastern States—
for example, in New England, Pennsylvania, Virginia
and North Carolina, and in these sections, if for a time
protected, might establish itself. In the West it is
usually found at altitudes much higher than these,
though, in summer, females and young broods of the
dusky grouse inhabit essentially the same territory as
the ruffed grouse. The dusky grouse has but three vernacular names The dusky grouse has but three vernacular names
commonly in use—blue grouse, pine grouse and gray
grouse. Dusky grouse and blue grouse are the two
most common. ‘CANADA GROUSE, SPRUCE PARTRIDGE. Canachites canadensis. Canachites canadensis osgoodi. Canachites canadensis canace. Two small wood grouse, notable for their extreme
simplicity and lack of fear of man, are found in the
evergreen country, Canadian life zone, of the east,
north and west. Of these the first is the Canada grouse,
a small, blackish bird, variously marked below with
spots of white and with the tail tipped with rusty red-
dish. The male above is black, waved with dark gray,
and beneath black; its throat bordered with white,
many of the feathers tipped with white; the sides and
the flanks marked by long white streaks. The female
is barred with black and rusty, but has the sides and
the scapulars or shoulder feathers streaked with white. The tips of the tail feathers are bright rusty. Length,
14% inches; wing about 7 inches and tail 5 inches. It
is found in northern New England, New York, Michi-
gan, Minnesota and through Canada to Alaska. The three forms of the Canada grouse are very The three forms of the Canada grouse are very
closely related, and, from the sportsman’s viewpoint,
hard to separate. True canadensis is restricted to the
northern part of the range—eastern
flanks of the
Rocky Mountains west of Edmonton, Alberta, easterly
to Labrador Peninsula, and Alaska from Bristol Bay
125 The three forms of the Canada grouse are very
closely related, and, from the sportsman’s viewpoint,
hard to separate. True canadensis is restricted to the
northern part of the range—eastern
flanks of the
Rocky Mountains west of Edmonton, Alberta, easterly
to Labrador Peninsula, and Alaska from Bristol Bay
125 125 AMERICAN GAME BIRD SHOOTING 126 to Cook Inlet and Prince William Sound; osgoodi
to Mount McKinley Range and Yukon region, east
to Great Slave and Athabaska Lakes; canace to Mani-
toba, southern Ontarie and New Brunswick, south to
northern Minnesota, Wisconsin, Michigan,
New York
and New England. Another race, atratus, has been de-
scribed from the coast region of Alaska. The Canada grouse is not a familiar bird to the TAIL OF CANADA GROUSE TAIL OF CANADA GROUSE sportsman, and is scarcely ever shot over dogs, but
the angler and traveler through the wilderness of the
North often meets it in the depths of the forest. There
is little to be said about shooting it, but in its life his-
tory there is much that is worth recording. ‘CANADA GROUSE, SPRUCE PARTRIDGE. Most of
this has already been written down in works on natural
history, but as many of these are not easily accessible
to sportsmen, it is well to repeat some of these facts. This is one of the most unsuspicious and least fear-
ful of man of all the grouse. Often it may be killed
by stones or sticks thrown at it, or a noose of twine sportsman, and is scarcely ever shot over dogs, but
the angler and traveler through the wilderness of the
North often meets it in the depths of the forest. There
is little to be said about shooting it, but in its life his-
tory there is much that is worth recording. Most of
this has already been written down in works on natural
history, but as many of these are not easily accessible
to sportsmen, it is well to repeat some of these facts. This is one of the most unsuspicious and least fear- This is one of the most unsuspicious and least fear-
ful of man of all the grouse. Often it may be killed
by stones or sticks thrown at it, or a noose of twine CANADA GROUSE, SPRUCE PARTRIDGE 127 tied to the end of a stick may be passed over its head
as it sits on a limb, and it may thus be dragged from
its perch. A bird of such confiding disposition would
not afford much sport, and besides this the deep forests
which this species inhabits are far from the usual
haunts of the gunner. The range of the Canada grouse extends from The range of the Canada grouse extends from
northern New England, north through Labrador, west
to northern Minnesota and northwestward to Alaska. We have seen them on the shores of Prince William
Sound and Cook Inlet in that territory. Its northern
limit, according to Swainson, extends to the parallel
of 67°. It is not migratory, and breeds wherever found,
and its favorite home is in the dense swamps and ever-
green thickets of the north land. It is upon the buds
and leaves of these evergreens that it chiefly feeds, and
from this food its flesh often takes a resinous taste that
is not agreeable. The breeding season is in May or June, according to
latitude. The mating actions of the male—his drum-
ming—differ much from those of other grouse. ‘CANADA GROUSE, SPRUCE PARTRIDGE. Major
Bendire quotes a correspondent as saying:
“After
strutting back and forth for a few minutes the male
flew straight up as high as the surrounding trees, about
fourteen feet; here he remained stationary an instant,
and while on suspended wing did the drumming with
the wings,
resembling
distant
thunder,
meanwhile
dropping down slowly to the spot from where he
started, to repeat the same thing over and over again.”
Mr. Everett Smith says in Forest and Stream:
‘The Mr. Everett Smith says in Forest and Stream:
‘The AMERICAN GAME BIRD SHOOTING 128 Canada grouse performs its drumming upon the trunk
of a standing tree of rather small size, preferably one
that is inclined from the perpendicular, and in the
following manner:
Commencing near the base of
the tree selected, the bird flutters upward with some-
what slow progress, but rapidly beating wings, which
produce the drumming sound. Having thus ascended
fifteen or twenty feet, it glides quietly on wing to the
ground and repeats the maneuver. . Favorite places
are resorted to habitually, and these drumming trees
are well known to observant woodsmen. I have seen
one that was so well worn upon the bark as to lead
to the belief that it had been used for this purpose
for many years. This tree was a spruce of six inches
diameter, with an inclination of about fifteen degrees
from the perpendicular, and was known to have been
used as a drumming tree for several seasons. The
upper surface and sides of the tree were so worn by
the feet and wings of the bird or birds using it for
drumming, that for a distance of twelve or fifteen feet
the bark had become quite smooth and red as if
rubbed.” Major Bendire quotes Manly Hardy as saying: “My
father, who had opportunities to see them drumming,
told me they drum in the air while descending from a
tree. They would fly up on a tree and then start off
and drum on their way to the ground like a Quaker
grasshopper. When on the ground they scratch a great
deal more than other grouse do.” CANADA GROUSE, SPRUCE PARTRIDGE 129 Thus there seems to be a wide individual variation
in the practice of this nuptial performance. ‘CANADA GROUSE, SPRUCE PARTRIDGE. The number of eggs laid by the Canada grouse varies The number of eggs laid by the Canada grouse varies
from nine to thirteen, though rarely two or three more
may be found ina nest. The ground color of the eggs is
buff, and they are irregularly spotted and blotched with
reddish brown, which sometimes is very dark. Speci-
mens are rarely seen with few or no markings. A number of years ago, Mr. Watson L. Bishop, of A number of years ago, Mr. Watson L. Bishop, of
Kentville, Nova Scotia, was successful in domesticat-
ing some Canada grouse, and he contributed to Forest
and Stream many interesting observations on the hab-
its of these birds in confinement. He says:
“As the
nesting season approaches I prepare suitable places for
them by placing spruce boughs in such a way as to
form cozy little shelters, where the birds will be pretty
well concealed from view. I then gather up some old
dry leaves and grass and scatter it about on the ground,
near where I have prepared a place for the nest. The
bird pays no attention to this until she wants to lay. She will then select one of these places, and after
scratching a deep cup-shaped place in the ground, de-
posit in it her eggs. When the hen is on the nest she
is continually making a kind of cooing sound, which
I have never heard them make on any other occasion. If there should be sufficient material within easy reach
of the nest, the bird will sometimes cover the eggs up,
but not in all cases. “No nesting material is taken to the nest until after “No nesting material is taken to the nest until after
three or four eggs are laid. After this number has AMERICAN GAME BIRD SHOOTING 130 been deposited, the hen, after laying an egg, and while
leaving the nest, will pick up straws, grass and leaves,
or whatever suitable material is at hand, and throw it
backward over her back as she leaves the nest, and by
the time the set is complete, quite a quantity of this
litter is collected about the nest. She will then sit on
her nest and reach out and gather in the nesting ma-
terial and place it about her, and, when completed, the
nest is very deep and nicely bordered with grass and
leaves. ‘CANADA GROUSE, SPRUCE PARTRIDGE. “So strong is the habit or instinct of throwing the
nesting materials over the back, that they will fre-
quently throw it away from the nest, instead of to-
ward it, as the hen will sometimes follow a trail of
material that will turn her right about, so that her
head is toward the nest, but all the time she will con-
tinue to throw what she picks up over her back. This, of
course, is throwing the material away from the nest. Discovering her mistake, she will then right about
face and pick up the same material that an instant
before was being thrown away, and throw it over her
back again, toward the nest. “The way they will steal eggs from one another
would do credit to
a London pickpocket. Two hens
had their nests near together, perhaps two feet apart,
and as each hen laid every other day, one nest would
be vacant while the other would be occupied. The
hen that laid last would not go away until she had
stolen the nest egg from the other nest and placed it
in her own. I once saw a hen attempt to steal an egg ‘aqe[q Ss uoqnpny wo1g
‘asnolg
epeurg ‘as
epe CANADA GROUSE, SPRUCE PARTRIDGE 131 from another nest that was twenty feet away. She
worked faithfully at it for half an hour or more, but
did not succeed in moving the coveted egg more than
about eight feet, it being uphill. The egg so fre-
quently got away from her, and rolled back a foot or
more each time, that she at last got disgusted and gave
up the task. “On going to the pen one evening I found one of “On going to the pen one evening I found one of
the hens on the nest, and I knew she was beginning to
set, as all the others had gone to roost. Slipping my
hand under her, I found three eggs—the nest egg, the
one just laid, and the one stolen from the other nest. ‘CANADA GROUSE, SPRUCE PARTRIDGE. I picked two of them up and held them before her,
when she all at once placed her bill over the one held
between my thumb and fore finger and tried to pull it
out of my hand; I did not let her have it, however, and
she immediately stepped upon the side of the nest, and
placing her bill over the remaining egg, drew it up out
of the nest and pushed it back out of sight, as much as
to say:
“You have two, and that is all you can have.’
I must confess that it was with great reluctance I
took these eggs from her, she pleaded so hard for
them. “The male bird begins to strut in March. I re-
member very well the first time I saw one strutting. I had obtained the bird in the fall, and he used to sit
about bunched up almost in a round ball, as the female
did, until one morning, when I went to feed them,
I found him strutting. His attitude was so different AMERICAN GAME BIRD SHOOTING 132 that one would scarcely have known it was the same
bird. “T will describe as nearly as I can his conduct and at-
titude while strutting. The tail stands almost erect,
the wings are slightly raised from the body and a little
drooped, the head is still well up, and the feathers of
the breast and throat are raised, and standing out in
regular rows, with the feathers of the nape and hind
neck well back, forming a smooth kind of cape on the
back of the neck. ‘This smooth cape contrasts beauti-
fully with the ruffled black and white feathers of the
throat and fore breast. The red comb over each eye is
enlarged, until the two nearly meet over the top of the
head. This comb the bird is able to enlarge or reduce
at will, and while he is strutting the expanded tail is
moved from side to side. The two center feathers
do not move, but each side expands and contracts al-
ternately with each step as the bird walks. This move-
ment of the tail produces a peculiar rustling like that
of silk. This attitude gives him a very dignified and
even conceited air. ‘CANADA GROUSE, SPRUCE PARTRIDGE. He tries to attract attention in
every possible way by flying from the ground up ona
perch, and back to the ground, making all the noise he
can in doing so. Then he will thump some hard sub-
stance with his bill. I have had him fly up on my
shoulder and thump my collar. At this season he is
very bold and will scarcely keep enough out of the
way to avoid being stepped on. He will sometimes sit
with his breast almost touching the earth, his feathers
erect as in strutting, and making peculiar nodding and CANADA GROUSE, SPRUCE PARTRIDGE 133 circular motions of the head from side to side; he
will remain in this position two or three minutes at
a time. He is a most beautiful bird, and shows by
his actions that he is perfectly aware of the fact. “As the spring and summer advance, the food given
these Canada grouse must be changed with the season,
and it is only with a perfect knowledge of their wants,
and with constant care, that they can be safely carried
through the heat of the summer and the moulting sea-
son. In the nesting season the females are very quar-
relsome, and at this time more than two or three can-
not be kept in the same pen, but in July they may be all
turned together again and they will agree very well
until the following March.” The winter food of the Canada grouse is chiefly
leaves and buds of spruce and tamarack. In spring,
when insects make their appearance, no doubt they feed
largely on them and on the leaves of various plants,
while at the ripening of the berries these are eaten. Little or nothing is said by writers generally about
the gathering together of these birds in great flocks,
yet, like many other species of our grouse, at certain
seasons of the year they seem to unite in great com-
panies. Major Bendire
quotes Manly Hardy,
of
Brewer, Maine, as saying:
“A Micmac Indian, whom
I consider reliable, tells me of having seen a pack of
many thousands, somewhere east of Halifax, Nova
Scotia, on which their whole village lived for weeks,
moving after them when they moved. The males
greatly preponderate over the females, at least two to AMERICAN GAME BIRD SHOOTING 134 one. ‘CANADA GROUSE, SPRUCE PARTRIDGE. They feed almost entirely on the needles of spruce
and fir, also hackmatack and berries in summer. They
show a preference for some fir trees over others, as
J have seen them return to the same tree until it was
nearly stripped. When disturbed they always take to
the trees, walking about in them.” Canachites franklini. Extremely similar to the foregoing, but with the
tail black to the end, or bordered with white; its cov-
erts white-tipped. The female has the tail feathers
tipped with white and the upper tail coverts streaked
with white, as also have the full-grown young of both
sexes which have not yet assumed the plumage of
the adult. It is slightly larger than the Canada grouse:
length, over 15 inches; wing, 7% inches;
tail, 5 inches
or over. Franklin’s grouse is found in southern Alaska, cen-
tral British Columbia and west central Alberta, south
to western
Montana,
central
Idaho
and
northern
Oregon east of the coast range. At many points in
northwestern Montana, northern and central Idaho it
is abundant. It is the bird commonly called “fool hen”
in the western mountains, though this, after all, is a
general term applied to any grouse or quail that has
not yet learned that man is an enemy to be dreaded. The
differences
in plumage between
Franklin’s The
differences
in plumage between
Franklin’s
grouse and the Canada grouse are so slight that they
are not likely to be noticed by the average gunner,
though at once obvious to the ornithologist. So far
135 135 135 AMERICAN GAME BIRD SHOOTING 136 as is known, their habits are closely alike, though about
the present species not very much has been written. Unless they should greatly change in habits, neither Unless they should greatly change in habits, neither
the Canada grouse of the East nor the Franklin’s
grouse of the West is likely ever to offer much sport
to the gunner. These little birds are so gentle and
confiding that they hardly take the trouble to move out
of the intruder’s way; they will stand on the ground TAIL OF FRANKLIN’S GROUSE TAIL OF FRANKLIN’S GROUSE or a limb to have a dozen shots fired at them, or may
be pelted with stones and sticks without manifesting
great alarm. I knew of one at which several rifle
shots were fired, the last of which cut off one of the
bird’s feet. It staggered on its perch, and then, re-
gaining its balance, stood there on one foot, apparently
wondering what had happened. It is often possible
to capture these birds by means of a noose tied to a
pole six or eight feet long, by means of which the
noose can be slipped over the head. Canachites franklini. It used to be not unusual, and no doubt in many lo- or a limb to have a dozen shots fired at them, or may
be pelted with stones and sticks without manifesting
great alarm. I knew of one at which several rifle
shots were fired, the last of which cut off one of the
bird’s feet. It staggered on its perch, and then, re-
gaining its balance, stood there on one foot, apparently
wondering what had happened. It is often possible
to capture these birds by means of a noose tied to a
pole six or eight feet long, by means of which the
noose can be slipped over the head. It used to be not unusual, and no doubt in many lo- It used to be not unusual, and no doubt in many lo- FRANKLIN’S
GROUSE 137 calities still is so, for a brood of these birds to fly into
a tree and sit there unalarmed until the heads of half or
all of their number have been cut off by the rifle balls of
some passing traveler. The flesh is excellent eating,
and in places where fresh meat is not easily to be had,
many of these birds are killed along the trails. On
the other hand, it is to be remembered that the country
in which they live is sparsely inhabited by man, and
that those which he kills constitute only a very small
fraction of the whole number in a district. On the
other hand, it is difficult to comprehend how these birds
escape their natural enemies, and heavy toll of them
must be taken by lynx, fox and wolf. With the destruction
of the forests, which now
threatens
us, the numbers of both these forms of
grouse must constantly diminish, but before the birds
are too greatly reduced in numbers some means should
be devised for their preservation. Franklin’s grouse is still more gentle and unsuspi-
cious than the Canada grouse, its eastern cousin. One
may frequently walk up to within fifteen or twenty feet
of these birds as they sit on the ground or on the low
limbs of trees without apparently being noticed by
them. When they discover the approach of man they
seem at first but little interested, but as one draws
nearer, they are likely to stretch up their heads and look
more carefully. Canachites franklini. If really alarmed or uneasy they stand
very erect, holding the neck outstretched, and often
raise the feathers on the sides of the neck just below
the head. This habit is common to this species and to AMERICAN GAME BIRD SHOOTING 138 the dusky grouse, and is seen to a greater or less extent
in other members of the family, as the ruffed, pinnated
and sharp-tailed grouse. Franklin’s grouse is commonly found in the summer Franklin’s grouse is commonly found in the summer
in timbered valleys, often close to the water, but some-
times ‘also on timbered plateaus much higher in the
mountains. I have not observed that it seems especially
to prefer swampy lands, as the Canada grouse is sup-
posed to, but rather a thickly timbered country, not far
from water. Even if alarmed and forced to take to wing, they do
nothing more than fly up into the branches of the tree
immediately above them, where they always either
huddle down on a branch, as if to rest again, or, if
seriously frightened, stand with head erect, stretching
the neck out, first in one direction and then in another,
until the cause of alarm has passed, or their suspicion
has become allayed. Contrary to what might be expected, the spruce
partridge,
found
in west
central
Alaska,
is not
Franklin’s grouse, but a form of the Canada grouse. RUFFED GROUSE—PARTRIDGE. Bonasa umbellus. Bonasa umbellus togata,
Bonasa umbellus umbelloides,
Bonasa umbellus sabini. The ruffed grouse is peculiar to North America, and
is found mainly in the Transition and Canadian life
zones. Ornithologists
recognize
four geographical
races in different sections of the continent. Of these
four forms, the typical species (Bonasa umbellus) in-
habits the eastern United States as far north as north-
ern Massachusetts, thence westward to and beyond
the Mississippi River; in southern Vermont, southern
New York, through Ohio, Michigan, Wisconsin and
southern Minnesota, touching the eastern portions of
North and South Dakota, eastern Nebraska, eastern
Kansas and Missouri, northern Arkansas, Tennessee,
Kentucky, and in the Alleghanies to Georgia. The
ruffed grouse of northern New England, northern New
York, northern Michigan and eastern Oregon, known
as B. umbellus togata, the Canada ruffed grouse, is
found also northward to Nova Scotia, Manitoba, cen-
tral Keewatin, southern Ungava and British Columbia. The gray ruffed grouse (Bonasa umbellus umbelloides)
occurs in the central Rocky Mountains, from Colorado,
139 139 AMERICAN GAME BIRD SHOOTING 140 Utah and western South Dakota to Alaska, along the
Yukon and Mackenzie rivers, throughout much of Brit-
ish North America, east as far as Manitoba. Another
darker race
(Bonasa umbellus sabini)
inhabits the
wooded country of the northwest coast region of south-
ern Alaska, to Humboldt County, California. 32312
<
sash
on /
RUFFED GROUSE RUFFED GROUSE All these forms are very similar in appearance, and
it is not unusual to find in one locality a bird which, in
color, may closely resemble those of some far distant
locality. The most that can be said for these races is
that they average lighter or darker—as the case may
be—than certain other relations, the center of whose
abundance may be far distant. RUFFED GROUSE—PARTRIDGE I4I Bonasa umibellus is above, reddish brown or grayish
brown, varied with black, brown and gray in different
shades, the scapulars and wing coverts streaked with
whitish or cream color, the rump and upper tail cov-
erts with long streaks or spots of grayish or yellowish. Tail long and wide, gray or reddish, more or less band-
ed with paler, each pale band bordered by a narrow,
irregular blackish edge. A broad sub-terminal band of
black or dark brown near the end, followed by a nar-
rower terminal band of grayish. RUFFED GROUSE—PARTRIDGE. Feathers of the tufts
on the side of the neck—the ruff—usually broadest
at the ends, and black in color, sometimes with green-
ish reflections. Occasionally the ruff, instead of being
black, is dark brown, or even pale chestnut. The throat
is pale buff, sometimes dotted with darker. The lower
parts are whitish or yellowish, barred with dull brown,
broadest and darkest on the flanks. The lower tail
coverts are white-tipped. The female is similar to the
male, but smaller, and with the plumage slightly paler. At the same time the plumage often fails to give any
suggestion of sex. There is a high-pointed crest on
the head. The lower portion of the tarsus—that is,
of the foot—is naked. This is a general description of the ruffed grouse. This is a general description of the ruffed grouse. The typical uwmbellus is described as mostly reddish
above. The sub-species, B. umbellus umbelloides, is
mostly gray, and has the tail always gray. It is re-
garded as the Rocky Mountain form. B. wmnbellus togata, the Canada ruffed grouse, is B. wmnbellus togata, the Canada ruffed grouse, is AMERICAN GAME BIRD SHOOTING 142 mostly grayish, and the tail is usually gray, but some-
times tinged with reddish. B. umbellus sabini is dark reddish, with little or no
gray. This is the northwest coast form, found in the
region of great precipitation, and by many is consid-
ered the most beautiful of the ruffed grouse. The descriptions of these sub-species do not always The descriptions of these sub-species do not always
give a very clear idea of the grouse of the regions the
various forms are supposed to inhabit. The center of
such a region, no doubt, will be occupied by birds of
the typical form. On the border lines, however, where
the sub-species tend to meet, individuals may some-
times be found that are typical of some taken thousands
of miles away. The length of the ruffed grouse varies from 16 to
19 inches. The wing measures from 7 to 7% inches,
and the tail from 514 to 7 inches. The extent of wing
varies from 22 to 25 inches, and the bird commonly
weighs from 21 or 22 up to about 30 ounces. Grouse
are often reported to weigh 2% or 2% pounds; but it
may be doubted if birds so heavy are ever found. RUFFED GROUSE—PARTRIDGE. The ruffed grouse is the best known and most highly The ruffed grouse is the best known and most highly
esteemed of the game birds of the North, and in Can-
ada, New England, northern New York, Pennsylvania,
Michigan and Wisconsin it 1s now the most important
of the upland birds. In the South the bird is called “pheasant,” and in
the North, especially in the Northeast, “partridge’”—
both misnomers, since it is neither. In parts of Canada RUFFED GROUSE—PARTRIDGE 143 it is called ‘birch partridge,” in contradistinction to
the Canada grouse, called “spruce partridge.” The grouse spend the winter in thick, deep swamps,
or else on steep hillsides in sections where evergreens
grow, which give protection from the snow, in dense
runs, and white birch and other thickets. Life is easy for the birds, which wander about over
their limited range, scratching, when the snow is not
too deep, for the fruit of the skunk cabbage, for the
fruit and leaves of wintergreen and partridge berry and
arbutus, for hibernating insects, for nuts overlooked
in autumn by themselves and the squirrels; or, if the
ground is deeply snow-covered and ice-bound, taking
to the tree-tops, where they glean a plenteous harvest
of buds, and usually come out in spring strong and well
nourished. At Lake George, New York, the grouse
feed extensively on the buds of ironwood, poplar, birch
and apple, from late October to early April, irrespective
of the snowfall. An hour or two before sunset they
fly to the trees and gorge on the buds and catkins. In New England and the Middle States the mating
time comes in early April, after the weather has grown
warm and the grass and flowers have begun to spring. By this time the grouse has begun to sound his drum-
ming call. In a country where grouse are plenty you
may hear the sound from a hundred hills, but you will
find it a difficult matter to trace it to its source, and
when you approach the place from which you thought
it came, you will find it is no longer heard there, and
when it next reaches your ear it seems a long way off. AMERICAN GAME BIRD SHOOTING 144 In the Rocky Mountains, in September, a few years
ago, I saw a grouse cross the trail along which I was
walking and disappear in the bushes to the left. RUFFED GROUSE—PARTRIDGE. The
clump of bushes was a small one, not more than twenty
yards across. I watched the bird while it was walking
perhaps ten yards, and then went on, but had hardly
come to where the bird had crossed the path when I
heard it drum. The sound was not loud and gave the
impression of being quite distant. I stood still for a
moment, trying to see the bird, but without success,
and after a little while the drumming began again, still
sounding as distant as before. I then very quietly made
my way into the bushes, advancing six or eight yards
toward the point where I had last seen the grouse, but
the drumming was not resumed, and after a few mo-
ments I withdrew, but had hardly reached the trail
when I again heard the sound. A second excursion
into the bushes was no more successful than the first
had been, but somewhat later, on my return, hearing
the drumming, I again went into the bushes, and just
beyond where I had seen the grouse disappear I saw
it walk away among the brush. This is mentioned only
to indicate how very faint the sound of the drumming
may be, although it is quite close at hand. Few subjects have been more discussed by American Few subjects have been more discussed by American
naturalists and sportsmen than this same drumming,
a sound so familiar to outdoor men as hardly to need
description. It is commonly thought to be a mating
call, though by no means sounded only at the pairing
season in spring; for the roll of the drumming, sound- ‘aspoyH
“Wa
‘<O
Aq
poeydeis0j0ydg
*Zur}jnIys ‘asnorn peyny ‘a
“Wa
‘<O
Aq
poeydeis0j0ydg
*Zur}jnIys ‘asnorn peyny RUFFED GROUSE—PARTRIDGE 145 ing like far-off thunder, is often heard in late summer
and autumn. It is possible that this autumnal drum-
ming is made by the young males, just as sometimes in
autumn a young male songbird may be heard to prac-
tice in low tones the lay which it will utter during the
mating season of the following spring. A great variety
of explanations have been given as to the method by
which the drumming is produced. RUFFED GROUSE—PARTRIDGE. It is made by the
wings, and the observations of ornithologists have es-
tablished the fact that the sound is caused by the out-
spread wings of the bird rapidly beating the air with-
out striking the bird’s body or any other object. Pho-
tographs taken by Professor Hodge show the wings
beating in front of the breast as the bird sits upright
during the act. The belief that the resonance of the
sound is caused in part because, while drumming, the
bird stands on a hollow log, was long ago abandoned. One of the best descriptions of the process of drum-
ming is that given by Mr. Manly Hardy, of Brewer,
Maine, who says:
“The cock grouse usually selects
a mossy log near some open hedge, clearing, or woods
road, and partly screened by bushes, where he can see
and not be seen. When about to drum he erects his
neck feathers, spreads his tail, and with drooping wings
steps with a jerky motion along a log for some distance
each way from his drumming place, walking back and
forth several times and looking sharply in every direc-
tion. Then standing crosswise, he stretches himself to
his fullest height and delivers the blows with his wings
fully upon his sides, his wings being several inches clear AMERICAN GAME BIRD SHOOTING 146 from the log. After drumming he settles quietly down
into a sitting posture and remains silently listening for
five or ten minutes, when, if no cause for alarm is dis-
covered, he repeats the process.” Mr. Ernest E. Thompson, in his “Birds of Mani-
toba,”’ describes the drumming as he saw it. It is said
that if, during the drumming, another male grouse
makes its appearance near the drumming place, the
birds fight with much spirit. An account of such a bat-
tle is given by a correspondent of Forest and Stream,
who writes from Schenectady, New York, and signs
himself ‘“Dorp.”
He says, in substance:
“T was walking along a country road, about the first “T was walking along a country road, about the first
of June, when I heard from the depths of an adjacent
wood an unusual rustling sound for which I could not
account. The sound
stopped, began again, again
stopped, and was repeated at intervals of a minute or
two. RUFFED GROUSE—PARTRIDGE. Not knowing what caused the sound, I crossed
the fence and cautiously approached the place. After
I had gone about fifty yards, screening myself as much
as possible behind trees,
I came up to within twenty
yards of a large dead log, upon which stood two ruffed
grouse in battle array. They stood something like six
feet from each other, with the black feathers around
their necks raised till they almost pointed toward their
heads, which were lowered, and which they were shak-
ing at each other in defiance. In a few moments they
gradually approached each other, and when about two
feet apart they rushed together, the momentum and
their wings carrying them into the air about a foot RUFFED GROUSE—PARTRIDGE 147 above the log. While there they struck repeated blows
and then fell back to the log, where a short tussle took
place, when they separated and moved back to a dis-
tance of about six feet from one another. “After an interval they advanced toward each other “After an interval they advanced toward each other
again and repeated the same performance as before. The desperate battle went on by ‘rounds’ till several
were fought. How long the fight had been going on
before I came upon the scene I had no means of know-
ing, but after they had come together several times in
my presence, the bird that seemed the smaller moved
backward, still presenting a defiant attitude, with his
head lowered, till he came to the end of the log, when
he jumped off and disappeared. “Upon this the victor raised himself and stretched “Upon this the victor raised himself and stretched
up until he nearly stood on his toes, expanding his
wings at the same time. It looked for a moment as if
he would crow. Then settling himself back, he brought
his wings down against his breast with a sound like
that produced by forcibly striking a bass drum. Again
he raised his wings and repeated the sound. Still
continuing to raise and lower his wings more and more
rapidly, the sound finally rolled away ‘in one continu-
ous murmur and was still.’
The sound was indescrib-
able. As soon as he had drummed he jumped off the
log and disappeared. RUFFED GROUSE—PARTRIDGE. Wishing to know if the female,
the probable cause of the conflict, was near by, I ad-
vanced rapidly and started her, together with the vic-
tor, and a short distance away the defeated bird.”
An interesting account of a case of drumming in An interesting account of a case of drumming in AMERICAN GAME BIRD SHOOTING 148 the autumn, with its possible cause, is given by this
same correspondent of Forest and Stream, who says: “On one occasion, while out shooting in Clifton
Park, I fired at a grouse, which fell to the ground and
fluttered over it for some distance, but before I could
reach him rose again and, flying high, disappeared
over the tops of the trees in the woods. Pursuing, I
looked the ground well over, but was unable to find
him. It so happened that two or three days after this
occurrence I was again on the same ground, and re-
membering the wounded grouse, went in search of him. I had not gone far when I heard a grouse drum, and
moving cautiously I finally got behind an old moss-
covered stone wall that ran through the woods. I had
often heard grouse drum, but I had never seen one per-
form the act, and I was very desirous to do so. After
a while he drummed again, and I moved along the
fence, nearer, having by this time got his bearings. Upon looking over the wall I saw the bird standing
on what I supposed to be a stone about five or six inches
in height. After he had drummed he pecked at the
supposed stone and then bobbed his head and tail up
alternately,
the latter being spread out. Then he
jumped off the object and walked in a wide circle
around it, after the manner of the turkey cock, with
wings lowered. After an interval he got on the stone
again and drummed and performed as before. About
this time something alarmed the drummer and he dis-
appeared into the covert. “Upon going to the spot where he had drummed I “Upon going to the spot where he had drummed I Faster
The roll
The drumming Ruffed Grouse. Photographed by C. F. Hodge. The roll Faster The drumming Ruffed Grouse. Photographed by C. F. Hodge. RUFFED GROUSE—PARTRIDGE. RUFFED GROUSE—PARTRIDGE 149 was astonished to find what I supposed a stone to be
a dead male grouse. Upon it the drummer had stood
and drummed, and about it he had paraded as described. The dead bird had one shot only, through his head. It was the same I had shot a couple of days before,
which had flown there and fallen dead. “T have read that the drumming of the ruffed grouse
is a demonstration of love and courtship, but from
this incident I conclude that it is also either one of
rivalry and triumph, or sounding a knell or requiem
of a departed mate or friend.” With this should be compared Audubon’s account of
the actions of a wild turkey cock after a fight in which
he has killed his rival. This same correspondent of Forest and Stream, an
observant man, has expressed the view that the grouse
drums to announce the approach of day at dawn, to
announce
any
approaching
unusual
atmospherical
change during the night or day, to make his where-
abouts known to the female, to celebrate her coming,
to announce a triumph over a rival, to sound a requiem
over a dead mate or friend, to amuse himself. Most
of these motives are precisely those which are supposed
to actuate the domestic cock in his crowing, or in the
flapping of his wings which commonly accompanies
the crowing. It is certainly true that the grouse drums
at night as well as by day, and that he frequently drums
just before daylight and often just before “a change of
weather.”
Although so excellent an observer as Captain Ben- Although so excellent an observer as Captain Ben- AMERICAN GAME BIRD SHOOTING 150 dire thinks that the drumming cannot be considered a
love note because, as he says, “It may be heard almost
every month in the year and sometimes in the night as
well as in the day time,”’ I am disposed to think that it
is really in part a mating call. Other grouse perform
certain operations usually thought to be connected with
the mating time in autumn as well as in spring. The
sharp-tailed grouse holds its dances in autumn, and I
have been told that the dusky grouse hoots in the
autumn as well as in spring, though by no means so
vigorously. RUFFED GROUSE—PARTRIDGE. Certainly we may believe that at the
proper season of the year it possesses an attraction for
the female, and S. T. Hammond, in his capital book,
“My Friend the Partridge,” gives some testimony to
this effect. The nest of the ruffed grouse is built almost any-
where, yet perhaps most often on a hillside more or
less steep, overlooking a swamp or a piece of woods. I have found nests among thick cedars on a hillside, or
perfectly open and exposed, at the foot of a cedar tree
in a mowing lot close to a fence, and again between two
cedars in an open piece of cedar wood, where there was
no apparent cover whatever. The eggs are laid usually
in April or early May, and by the middle of that month
the clutch of twelve to seventeen is usually completed. The eggs are cream color or buffy, rarely spotted with
brown at the larger end. If the female is startled from
her nest she leaves it with a roar of wings, whose rapid
beats often spread over the precious eggs the dry leaves
by which the nest is likely to be surrounded. On a num- ‘asno
payn
jo
ison ‘as
pay
jo
iso RUFFED GROUSE—PARTRIDGE I51 ber of occasions I have seen the eggs almost wholly
hidden by this simple means. Most sportsmen know how very difficult it is to Most sportsmen know how very difficult it is to
see game birds when they are not in motion. The
bird’s feathers harmonize so admirably with its sur-
roundings—whether these be the leaf-strewn ground
of October, the bare branches of December, or the yel-
lowing grass and weed stems of July—that it is often
almost impossible to detect the bird, even though one
knows precisely where it is. I recall an occasion when,
happening to cross a fence which separated a road from
a mowing lot, I almost stepped on a female grouse as
I sprang to the ground. She was sitting on a nest with
thirteen eggs, at the foot of a large cedar. Often after
that I used to go down to the lot and slowly approach
the place to look at the bird as she sat on her eggs. She soon became so accustomed to me that she mani-
fested no alarm, and I could approach quite close to
her. RUFFED GROUSE—PARTRIDGE. It always took me some little time to see the bird,
though she sat in plain sight, with only half a dozen
slender grass stems between her head and me. After
looking for some time at the spot where I knew she
sat, the shape of her head, her markings, and above all
her bright eye, would gradually grow out of the con-
fusion of the grass stems in front of her and the cedar
bark beyond, and I could see the whole bird plainly. Yet if I turned my eyes away it again took a little time
to find and recognize her. The eggs hatch from the first to the middle of June, The eggs hatch from the first to the middle of June,
and the young often number as many as a dozen or AMERICAN GAME BIRD SHOOTING 152 fifteen. They leave the nest at once and nimbly follow
the careful mother, as her chickens follow a domestic
hen. She leads them quietly through the woods, teach-
ing them the while how to live their lives and how to
keep themselves safe from their enemies. At her call
of alarm each chick sinks down on the ground and dis-
appears, looking like a leaf, a bit of stick or a pebble. No one can recognize them as living things, and their
only danger is that some clumsy person may step on
one of them. Meantime the mother, with feathers erect
and trailing wings, is limping in front of the intruder,
falling down, pushing herself along on her breast, pant-
ing as if in the very agony of death, often “growling”
or “whining” in the effort to lure the enemy away from
the brood. Usually she succeeds. No dog, and few
boys and men, can resist the temptation to catch a
partridge. The pursuer runs forward and almost
grasps her, but his approach seems to give her a little
strength and she flutters feebly forward. A few steps
more and she will be his; but still she evades him, and
presently, after having gone thirty or forty yards, she
rises on strong wing and, swift as a bullet, darts off
among the tree trunks. RUFFED GROUSE—PARTRIDGE. If the pursuer returns to the
place where he first saw her, sits down and remains
there quiet, after a time he will see her return on foot,
call together her little brood and start off again on her
travels. In Captain Bendire’s admirable work, so often cited, In Captain Bendire’s admirable work, so often cited,
Manly Hardy, after describing the actions of the
mother bird, says: RUFFED GROUSE—PARTRIDGE 153 “T have once seen the old cock with the brood, and
on this occasion he gallantly defended the rear until
the rest made good their escape. He stood with wings
raised and tail spread, ready to fight the intruder. I
have seen the young fly into a tree when still in the yel-
low down, and when not larger than a pine grosbeak
they will fly long distances, giving the alarm note of
‘quit, quit, just like an old bird. The young a few
days old are shyer than the wariest adult. The noise
made by the ruffed grouse in flying ‘is made on purpose’
to alarm others in the vicinity; they can fly as quietly
as any bird if they choose. “The males never congregate during the breeding “The males never congregate during the breeding
season or after, and I never but once saw two adult
males within one-fourth of a mile of each other between
April and September. I consider that the drumming
is not a call to the females, as they drum nearly or
quite as much in the fall as in the spring, and I have
heard them drumming every month in the year. I have
never seen the least evidence that the ruffed grouse is
polygamous.” With this last statement I quite agree. I know of
no evidence to justify the common assumption that this
grouse is polygamous. When the young birds are two or three weeks old When the young birds are two or three weeks old
their wing feathers have grown enough to enable them
to fly, and sometimes, if suddenly surprised, some of
them may leave the ground and fly up into low
branches, where they stand motionless with their necks AMERICAN GAME BIRD SHOOTING 154 stretched upward and may be so closely approached that
one may almost take them in the hand. RUFFED GROUSE—PARTRIDGE. By the middle of August the young are almost fully By the middle of August the young are almost fully
grown, and as cool weather approaches they begin to
separate, no longer keeping in a close flock, but wan-
dering off by twos, threes and fours. As the weather
grows colder they become still more solitary in their
ways, although up to midwinter two or three may
often be found together. This has been my experience; but other observers, This has been my experience; but other observers,
whose opinions are entitled to the greatest respect, be-
lieve that the broods keep together until midwinter,
unless broken up and scattered by the gunners. But
if this is true, how do the birds get together after the
“crazy season’? Dr. A. K. Fisher expresses the opinion that com-
paratively few grouse become “crazy” birds. He is
inclined to believe that the “‘crazy”’ birds are lost birds
—1t.é., individuals that for some cause or other have
been driven from their homes, and do not know how
to return. It is during late September and October that the
grouse for a short time wander about in a most eccen-
tric manner and indulge in such unusual performances
that this is sometimes called their ‘‘crazy season.”
Having separated, they seem to be searching for new
homes or perhaps for winter quarters, and wander
about in seemingly aimless fashion, so that they are
often found in most unusual places. Such are vacant
buildings, village streets, the lawn of a country place, RUFFED GROUSE—PARTRIDGE 155 or even within a covered country bridge. At this time,
too, they often fly against houses or fences, or through
windows, opened or closed, and in this way many birds
are killed. I have personal knowledge of three birds
flying against houses, one against a fence and two
through windows, opened or closed, on a single farm
in Connecticut. : A correspondent of Forest and Stream says:
“Once while walking along the street I saw a g “Once while walking along the street I saw a grouse
dusting in the roadway. At first I supposed it was a
hen, but as I approached, ittrose, flew straight down the
street and in at the opening of the large covered bridge
that spanned the Mohawk. The bird lit on a beam near
the roof. Getting up to the beam I took the bird off. RUFFED GROUSE—PARTRIDGE. He remained perfectly motionless, as is their habit
when approached by man. This grouse when he rose
in the roadway made comparatively little noise. . . .”
The same writer says:
“I recollect an incident that The same writer says:
“I recollect an incident that
occurred in our city where a grouse flew into a small
barn and perched on a beam. The owner of the prem-
ises having invited a gentleman into the barn on some
business, the guest said to the owner:
‘I see you keep
poultry.”
“No,’ said the owner, ‘I keep no poultry, but
my neighbors do, and I wish they would come and
take that
pullet away. She has been here now
three or four days and I am tired of feeding her.’
The
visitor looked a little more sharply and said:
‘That is
not a pullet; that is a partridge’; and so it was, to the
great surprise of the other.”
The food of the ruffed grouse is very varied. In The food of the ruffed grouse is very varied. In AMERICAN GAME BIRD SHOOTING 156 summer—and especially for the young birds—insects,
notably grasshoppers and crickets, make up a portion
of its diet. It is fond of berries of all sorts, and black-
berry patches and wild grape vines are often visited
by it when the fruit is ripe. Indeed, from midsum-
mer until early winter there are always berries for them
to feed on. In autumn also they feed on fallen apples,
and wild apple trees in the midst of woods are a fa-
vorite resort for them morning and evening. They eat
some grass and the leaves of many plants. They feed
on the fruit of the skunk cabbage (Symplocarpus) ;
early in the winter tearing away the pithy covering that
holds the seeds and picking them out from their spongy
bed, or later gathering them from the ground. Most of all they like nuts, such as chestnuts, acorns
and beechnuts. I have taken from the crops of grouse
two or three pignuts, a double handful of chestnuts and
as many beechnuts as I could hold in one hand. There
is a record of a small snake having been taken from a
grouse’s crop. The Biological Survey has shown that over Io per The Biological Survey has shown that over Io per
cent. RUFFED GROUSE—PARTRIDGE. of the food of the ruffed grouse is animal and
89 per cent. vegetable matter. The vegetable food is
seeds, more than 11 per cent.; fruit, more than 28 per
cent. ; leaves and buds, more than 48 per cent. Most of
the insects eaten are injurious; either those that prey
upon the growing crop or borers destructive to the
forest. Every ruffed grouse that is killed, if he had
lived out his time would have destroyed a great mul- RUFFED GROUSE—PARTRIDGE 157 titude of harmful insects and so would have done good
service to the farmer. Although grain has not been found in the crops of Although grain has not been found in the crops of
any of these grouse, there is no doubt that it is eaten
when obtainable. The birds are often seen scratching
in the roads, presumably for oats dropped by the horses,
and I have vivid recollection of a partridge which one
winter could be started two or three times a day at the
head of a small pond where corn had been scattered
along the edge of the water to attract the black ducks. This grouse frequented the place all through the winter
to feed on the scattered corn. In autumn, winter or spring it resorts to orchards to In autumn, winter or spring it resorts to orchards to
feed on the buds of the trees, but it probably does no
harm in this way. Buds and the leaves of poplar,
birch, ironwood and willow form a large portion of
the food of the grouse. It is said that the buds of the
black birch are sometimes eaten so largely that they
impart a taste to the bird’s flesh. In Alaska, E. W. Nelson found the grouse feeding on spruce buds and
declared that the flesh had become bitter from this diet. There was long a belief that the flesh of the ruffed
grouse was sometimes poisonous because it ate the buds
of the laurel. The statement was made by Alexander
Wilson, and cases are quoted by Mr. Judd to show
that there is a foundation for the belief. On the other
hand, grouse which had fed on laurel have been eaten
without any resulting harm. RUFFED GROUSE—PARTRIDGE. It is quite possible that
where injury has resulted, it came from leaving the
bird undrawn for a long time after death, when the AMERICAN GAME BIRD SHOOTING 158 poison from crop and intestines spread itself through
the flesh. Grass, flowers and especially red clover are
often found in the crop of the ruffed grouse, and many
other plants in small quantities. A grouse shot at
Lake George had its crop distended with the leaves of
the spearmint. Few birds are more local in their habits than the Few birds are more local in their habits than the
ruffed grouse, and one who is familiar with their ways
can usually visit the same spot in the woods at the
same time of the day and be quite sure of starting a
bird. A brood of birds started and scattered in all di-
rections will be found a few hours later within a few
hundred yards of the place from which they flew. I
believe that a circle with a diameter of half a mile will
cover the range of a group of ruffed grouse during the
summer or during the winter. In spring and autumn,
of course, there are marked changes of location, the
causes of which are not well understood. We merely
know that these changes take place, and that they shift
their ground with the seasons, usually being found in
different places in summer, autumn and winter. The
bird has a certain place for resting—basking in the sun
or wallowing in the dust—another for feeding and
another for drinking. Very often it passes between
these different places on foot, but not infrequently it
flies. It is perhaps during their flights in new and un-
familiar localities that they dash themselves against
houses or other obstacles. Occasionally they are killed
by flying against telegraph wires. When a grouse rises, it is likely to fly swiftly and When a grouse rises, it is likely to fly swiftly and When a grouse rises, it is likely to fly swiftly and RUFFED GROUSE—PARTRIDGE 159 straight at a distance of not more than from three to
thirty feet above the ground. As it approaches the
place where it wishes to alight it sets its wings and
sails straight for the place and there alights on the
eround. RUFFED GROUSE—PARTRIDGE. I do not think that it ever turns up in the
air as a quail does, but on the other hand I have never
seen a bird actually alight. In winter I have, however,
seen where they had come to the ground after a short
flight, and the impression of the bird’s breast in the
snow two or three inches deep. The breast
evi-
dently came down very close to the ground, sweeping
away the snow, and the grouse came to its feet at
once. I have seen a bird fly from the top of a high hill to
a piece of woods much lower down. It came straight
until it was almost over the woods, and then, making
a wide half circle, swiftly lowered and alighted among
the dry leaves not more than twenty steps from me. The various methods by which the grouse avoid ob- The various methods by which the grouse avoid ob-
servation are little known and are seldom observed by
those who are only occasionally in the woods and who,
when there, are possessed with the idea that they must
kill as many birds as possible in the time at their dis-
posal. The ones more likely to see such things are the
men who spend much time in the woods and have the
leisure to sit down and observe, watching the ways of
the wild creatures, which, after a time believing that
the intruder has gone away, emerge from their hiding
places and resume their usual pursuits. But if the ob- AMERICAN GAME BIRD SHOOTING 160 server makes a movement and the bird sees him, it will
stop for a moment, look carefully, and then, turning,
will seem to melt out of sight. It is most difficult to
tell when it disappears. Under such circumstances a
bird will sometimes fly, but more often will run for two
or three yards and disappear, and then springing from
the ground at a greater distance will fly off low, not
being seen after it takes to wing. Rarely one may see a frightened grouse on the Rarely one may see a frightened grouse on the
ground and may get up close to it, but if he does this
and wishes to observe the bird, let him avoid looking
directly at it. RUFFED GROUSE—PARTRIDGE. If it catches the observer’s eye, it is al-
most sure to fly, and when it flies the very fact that you
are so close to it may make the shot a difficult one. On
one occasion many years ago, after shooting two or
three times at a grouse, the dog pointed it at the foot
of a great oak tree. The other dog backed, and my
two brothers and myself, realizing where the bird must
be, surrounded the foot of the tree. Presently we saw
it crouched on the ground between two roots, looked
squarely at it and talked about it. That grouse must
have had an unhappy time, for it feared to fly. For
some little time we looked and talked of it, and then,
fearing that the pointing dog might try to seize the
bird, which was almost under his nose, one of us
stepped forward and grasped his collar. As this was
done, the grouse took wing, twisted around the root
of the tree, passing within two feet of one of my broth-
ers, dodged away and up and over a little rise of Daydreaming. Photographed by C. F. Hodge. Daydreaming. Photographed by C. F. Hodge. RUFFED GROUSE—PARTRIDGE 161 ground twenty yards from us, escaping untouched, not-
withstanding the fact that three shots were fired at it. The grouse sometimes takes refuge in the snow in
cold weather, and is credited with diving into drifts at
nightfall and passing the night there. A friend walk-
ing through the deep snow along a little swale, through
which passed a brook, saw sticking up out of the snow
what he supposed was an odd-looking stick. He de-
clared to himself that it looked just like a partridge’s
head, and when he had come within a few feet of the
place a bird rose out of the snow like a whirlwind and
flew away to a near-by swamp. No doubt these birds often inspect the gunner when
he is little aware of their observation. Dorp tells of a
case of that kind in the following words:
“T turned to resume my walk, and as I did so I cast “T turned to resume my walk, and as I did so I cast
my eyes upward, and there on a plateau covered with
moss and projecting nearly over me stood a ruffed
grouse looking intently down upon me. RUFFED GROUSE—PARTRIDGE. He was twelve
feet from me, as I afterward measured it. He was
partly turned sideways, with the neck stretched and
head bent down, and made a beautiful picture with
background of evergreen. “He remained motionless, as I did also, for about a
half minute, when he slowly turned and was quickly
lost to sight. This was the closest I remember ever to
have been to this wild bird in his native state when
seen. I have been closer, but then I did not know it
until he flew. “This bird had heard me walking and his curiosity AMERICAN GAME BIRD SHOOTING 162 had prompted him to come to the edge and look down
to discover the cause of the noise. Perceiving that my
back was toward him he knew that I could not see him
and so remained. Perhaps he had not seen enough of
me to satisfy him, and when I turned around he knew
I could not reach him. He had probably never been
shot at and knew nothing of the power of the gun which
I held in my hand, or of man’s ability to harm at a
distance.” In a country where it is much pursued by man or
dog, the ruffed grouse,
if unexpectedly approached
closely, is likely to walk or run away rather than to fly. A good grouse dog follows the trail of the bird slowly
and not much faster than the bird itself can run. Those
of us who have possessed good grouse dogs, which are
always scarce enough, have often seen them leave the
trail, make a wide circle and come up on the other side
of the bird to head it off and stop it. Unless some-
thing like this is done, the bird may readily enough
be followed to the edge of the cover, where it will
presumably take wing and disappear. If a dog is
alone—especially if it be a dog of red or yellow color,
suggesting a fox—the bird is likely merely to hop up
on a limb well out of reach and remain there looking
down at the enemy; but if a man follows the dog, the
bird, if he gets into a tree, is likely to stand close to
the tree trunk and remain absolutely motionless. RUFFED GROUSE—PARTRIDGE. Ruffed grouse are subject to more or less unex- Ruffed grouse are subject to more or less unex-
plained periods of scarcity. During one season the
woods will contain their usual number of the birds, and RUFFED GROUSE—PARTRIDGE 163 the following year very few may be found. After
that it may take some years for the birds to re-
cover themselves and again to become reasonably abun-
dant. Whenever such a period of scarcity occurs,
sportsmen very naturally endeavor to assign reasons
for the reduced numbers of the birds. Among the causes suggested are these: that they Among the causes suggested are these: that they
have been swept away by an epidemic disease, that they
have been destroyed by insect enemies, that they have
been killed by hawks, owls and foxes, that the breeding
season has been unfavorable, that the winter’s snow and
cold have killed them, while many men believe that
over-shooting furnishes the best reason of all. None of
these explanations appear to fit all cases. The birds
may succumb to disease, but there appears to be no evi-
dence that they do so. The young chicks in traveling
through the woods and swamps undoubtedly occasion-
ally pick up wood ticks which suck their blood, and
occasionally a young and weakly bird may perish from
this cause. Those who attribute the scarcity of grouse
at any time to hard winters—to their being covered up
and frozen in under the snow—cannot know much
about grouse nature. The bird is found far up in the
north, where it is exposed to weather far more rigorous
than it can ever experience in temperate climes, and if
it had been so tender as to be killed by the winter, it
would long ago have been exterminated in the moun-
tains of Alaska, along the Mackenzie River and south-
ern Ungava. It seems more probable that over-shoot-
ing must have much to do with these disappearances AMERICAN GAME BIRD SHOOTING 164 of the grouse, not directly, perhaps, but because by
constant attacks the breeding stock is kept down so low
that when conditions arise even slightly unfavorable to
the species, and a few birds are swept away, the breed-
ing stock is so reduced that not enough birds are reared
the next season to replenish the covers. RUFFED GROUSE—PARTRIDGE. The widest differences of opinion about this matter The widest differences of opinion about this matter
exist between sportsmen and naturalists of experience. So good a field naturalist as Nap. A. Comeau, in his
recent book, entitled “Life and Sport on the North
Shore,” says of the ruffed grouse:
“In some years they are abundant for a time and “In some years they are abundant for a time and
then disappear. I have noticed that heavy sleet in
winter will sometimes drive them away from certain
tracts of country. Since 1905 they have been pretty
scarce all over the country (the north shore of the Gulf
of St. Lawrence). I think this must be due to some
kind of contagious disease, something similar, proba-
bly, to the ‘grouse disease’ of Scotland. There is no
other way of explaining their scarcity over such an
immense extent of territory. Where the country has
opened up, and there are only patches of wood here and
there, it would be reasonable to suppose that they might
have been exterminated by over-shooting and snaring;
but where there are thousands of miles of forests, and
not one in a hundred shot over, it cannot be put down
to excessive shooting. As to natural enemies, they do
not seem to have been any more numerous here than
elsewhere. Last year (1908) I was over six weeks in
the woods with two of my boys, and we only saw six. *jSoU UO BsnoID payny
-
5. ple
,
ae
3
= *jSoU UO BsnoID payny RUFFED GROUSE—PARTRIDGE 165 From various points throughout the country, both in-
land and along the coast, I received the same reports—
no grouse.” Though constantly pursued by man during the open
season and exposed to the attacks of a multitude of
natural enemies, the ruffed grouse in many of our
covers seems still almost to hold its own. There are
seasons of abundance, when the birds are more numer-
ous than usual, and others of scarcity, when sportsmen
fear that they are about to disappear forever from par-
ticular localities; but they continue to exist, and will
long exist over much of the wooded country of the
Eastern United States. The cutting off of the forests
constitutes the gravest danger to which they are ex-
posed. RUFFED GROUSE—PARTRIDGE. Where this is done the birds disappear, but,
even after the heavy timber has been cut off, a period of
ten or twelve years often results in the reforestation of
the tract, at first only with underbrush and saplings,
but later with larger trees. Then the ruffed grouse tend
to come back again. For the ruffed grouse is a dweller in thickets. It
seldom frequents the open land, except that it may
venture out a little way from the edge of swamp or
forest to pick up the grain in a cultivated field, or to
eat the blackberries, huckleberries or wild grapes which
ripen in some opening at the edge of the woods. For
the most part, however, it is found in cover, sometimes
quite open, among tall tree trunks of great size, or
again in the most tangled swamp, among thickets of
alder, blackberry, catbrier and grape vines. Wherever AMERICAN GAME BIRD SHOOTING 166 found—in a country that has been much gunned—it is
well able to take care of itself. By much training it
has acquired a great variety of tricks and stratagems
which it practices to the utter discomfiture of many
gunners. It may rise far ahead of the dog and out
of shot and fly straight up a mountainside out of sight,
so that it is impossible to mark it down; or if for some
good reason of its own it continues to lie, it will very
likely let man and dog pass it, and then when the man
is tangled up in difficult brush and is trying to push
his way ahead, the partridge with thunderous roar will
rise behind him and disappear before he can free him-
self from his fetters and bring the gun to his shoulder. Very commonly the partridge runs rapidly ahead of the
dog, sometimes in a straightaway course, apparently
to make sure that it will be well out of gunshot before
it rises, or, again, it may run straight away, and then,
bending off to right or left, may come around nearly
to its trail again so that its pursuers will pass it. This
is the precise trick played by the moose and sometimes
by deer and bear when the conditions are favorable for
tracking them. RUFFED GROUSE—PARTRIDGE. A favorite device is to rise behind a
tree trunk, a clump of brush, a great rock or even a
stone wall, and to keep this barrier between itself and
the gunner until safely out of range. The flight of the grouse is very swift, and though The flight of the grouse is very swift, and though
when well under way usually flying straight, yet often
it rises on a curve, so that one may easily shoot behind
it. Although often rising from the ground with a
thunderous roar of wings, which may upset the nerves RUFFED GROUSE—PARTRIDGE 167 of its pursuer, the grouse does not always do this. Fre-
quently it takes wing as quietly as the smallest bird, so
that unless the shooter happens to be looking in the
bird’s direction he may not be aware that it has flown. Sometimes, too, it will merely hop up into a tree and
remain there, standing close to the trunk or to some
branch, stiff, straight and motionless, and looking like
a stub of wood. An old partridge may be counted on
to do some unexpected thing. It deals in surprises. Its
grace and beauty and the readiness with which it adapts
itself to changing conditions, as well as the difficult
places that it inhabits and the charm of its surround-
ings, unite to command the gunner’s admiration. Although where constantly shot at it practices a Although where constantly shot at it practices a
variety of such stratagems, yet in regions seldom pene-
trated by man and where it has not been pursued, it
is absolutely gentle and unafraid, and if startled from
the ground flies no farther than to a low branch of a
tree where it may sit with outstretched neck and erect
crest while half a dozen shots are fired at it from pistol
or rifle. If, however, a ball should strike the branch
on which it is resting, or if a twig cut from above it
drops down and touches the bird, it darts away with
the swift flight with which we are all so familiar. If
in a park or in private grounds the grouse are left un-
disturbed, they may often be seen walking about and
feeding, paying little attention to men who may pass
near them, recognizing that no danger is to be appre-
hended from them. WILLOW PTARMIGAN, Lagopus lagopus. Lagopus lagopus allent. Lagopus lagopus alexandre. In summer the male has the head and neck chest-
nut, often becoming darker below. On the neck and
chest this is often barred and flecked with black, as it
is also on the flanks and generally on the under parts. The belly is more or less slate color. The quills and
outer wing coverts are white and the rest of the upper
parts irregularly barred with tawny, brown and black. Many of the feathers are tipped with whitish. The
female is less deeply colored and is spotted with a
paler tawny or yellow. The length is 14 to 17 inches,
wing, 7 to 7/4 inches. The Newfoundland form, known as Allen’s ptarmi-
gan, is slightly different, and is described as having a
few of the secondaries, quills and wing coverts more
or less mottled with dusky, and the shafts black. But
this difference may be only seasonal. Mr. Austin Hobart Clark, who reported on the birds Mr. Austin Hobart Clark, who reported on the birds
collected and observed during the cruise of the ‘Alba-
tross in the North Pacific, found in southern Alaska a
new form of willow grouse, L. l. alexandre. It is
somewhat smaller than the willow grouse of the North,
168 168 WILLOW PTARMIGAN 169 and hasya smaller, weaker bill. In full summer plum-
age its upper parts are deep rich chestnut, thickly and
finely waved with black, the tip of each feather narrow-
ly white. The lower throat and fore neck are chestnut,
the breast becoming finely cross-lined with black, which
increases toward the belly. The sides and flanks are
very dark. The r The range of the willow ptarmigan, often called the
willow grouse, is chiefly confined to the Arctic regions
in North America, reaching from Alaska, over much
of the British Provinces, to the Atlantic, and rarely
straggling south into the United States—Minnesota,
New York and New England. Twenty-five or thirty
years ago it was not uncommon to find specimens of
this bird in the hands of taxidermists in New York,
the birds having presumably been shipped to that city
from southern Canada. In many parts In many parts of the North the birds are exceeding-
ly abundant and form no inconsiderable portion of the
winter food supply of the Indians, while many are
killed about the Hudson’s Bay posts. WILLOW PTARMIGAN, Much of what
we know about these birds comes from E. W. Nel-
son, who made his observations on the shores of the
Bering Sea, and from L. M. Turner, who studied them
in Labrador, with his headquarters at Ungava. From these observations we can From these observations we can get a good idea of
the life of the willow ptarmigan. In winter these birds seem to be In winter these birds seem to be to some extent
migratory, and, moving southward—partly, no doubt,
in search of food—are found in considerable numbers as AMERICAN GAME BIRD SHOOTING 170 far south as latitude 50°; yet great numbers remain
in the wooded districts of the North as far as latitude
670 Mr. E. W. Nelson, whose studies of the fauna of
the Northwest and whose ethnological work among the
Eskimos of Alaska are so well known, says that in the
northern portions of their respective ranges
these
grouse
are summer
residents—frequenting
the ex-
tensive open country and being most abundant along
the barren seacoast region of the Bering Sea and
Arctic coast, “but in autumn, the last of August and
during September, they unite in great flocks and mi-
grate south to the sheltered banks of the Kuskoquim
and Yukon rivers and their numerous tributaries. In
early spring, as the warmth of the returning sun begins
to be felt, they troop back to their breeding grounds
once more. “During a large portion of the year these birds form
one of the most characteristic accompaniments of the
scenery in the northern portion of Alaska. During the
winter season these birds extend their range south to
Sitka and Kadiak.... “Toward the end of March, as some bare spots
commence to show on the tundra, the Eskimos say this
will bring the ptarmigan from the shelter of the in-
terior valleys, and their observation proves true. At
St. Michael these birds commence their love-making
according to the character of the season—on some
years by the first of April their loud notes of challenge
are heard, but the recurrence of cold weather usually WILLOW
PTARMIGAN yg! puts a temporary stop to their proceedings. About
the fifth or fifteenth of this month the first dark feath-
ers commence to appear about the heads and necks of
the males. WILLOW PTARMIGAN, During some seasons
the males make
scarcely any progress in changing their plumage up
to the middle of May, when I have frequently seen
them with only a trace of dark about the head and
neck. In the spring of 1878 the first males were heard
calling on the 26th of April, and on April 27th, in
1879, the males were just commencing to moult, show-
ing a few dark feathers, but these seasons were un-
usually late. In autumn the change frequently com-
mences
the last of September and by the first of
October it is well under way, the winter moult being
completed toward the end of this month... . “At the Yukon mouth, on the evening of May 24th, “At the Yukon mouth, on the evening of May 24th,
these ptarmigan were heard uttering their hoarse notes
all about. As we were sitting by the tent my in-
terpreter took my rifle and, going off a short distance,
worked a lump of snow to about the size of one of
these birds. Fixing a bunch of dark-brown moss on
one end of the snow to represent the bird’s head, he
set his decoy upon a bare mossy knoll; then retiring a
short distance behind the knoll he began imitating the
call of the male until a bird came whirring along and
taking up the gauntlet lit close by this supposed rival
and fell a victim to the ruse. iv
At this time the males were continually
pursuing each other or holding possession of prominent
knolls, frequently rising thence five to ten yards in AMERICAN GAME BIRD SHOOTING 172 the air, with quick wing strokes and descending with
stiffened
wings,
with
the
tips curved
downward. While ascending they uttered a series of notes which
may be repeated by the syllables ku-ku-ku-ku, which
is changed as the bird descends to a hard rolling
kr-r-r-r-r, in a very deep, guttural tone, ending as
the bird reaches the ground. Frequently a pair would
fly at each other full tilt and a few feathers would be
knocked out, the weaker bird quickly taking flight
again, while the victor rises as just described and
utters his loud note of defiance and victory. On an-
other
occasion
when
the birds
are
more
evenly
matched they fight fiercely until the ground is strewn
with feathers. WILLOW PTARMIGAN, “On May 24th almost all these birds are paired, but
some did not complete their nuptials until the first
few days in June. This grouse takes but a single mate
in northern Alaska, and I am informed by the natives
of Unalaska that the same is the case with the rock
grouse found on the Aleutian Islands, nor have I ever
known of the ptarmigan assembling in numbers about
any special meeting place to carry on their love affairs;
they scatter about, as previously mentioned, being seen
singly here and there on prominent knolls over the
flat country. Early in June, rarely so early as the
last of May, the first eggs are laid; by June 20th and
25th the downy young are usually out, and when ap-
proached the female crouches close to the ground
among her brood. When she sees it is impossible to
escape notice she rolls and tumbles away as though WILLOW PTARMIGAN 173 mortally injured, and thus tries to lead one from her
chicks. The young at the same time try to escape by
running away in different directions through the grass. At this season the female and male both moult and
assume a plumage which differs considerably
The
young are fledged and on the wing at varying dates
through July and are nearly full grown by the first
to the tenth of August.”
Mr. R. Mac Mr. R. MacFarlane, long chief factor to the Hud-
son’s Bay Company, but now retired and resident in
Winnipeg, whose observations on the fauna of many
parts of the Far North are so valuable, gives some
interesting notes about the willow ptarmigan in one
of the regions which he has made famous. He says! “This species is exceedingl “This species is exceedingly abundant in the neigh-
borhood of Fort Anderson, on the lower Anderson
River, and in the wooded country to the eastward. It is not, however, common in the Barren Grounds,
especially from Horton River to Franklin Bay, where
it is replaced by L. rupestris. The nest is invariably
on the ground, and consists of a few withered leaves
placed in a shallow cavity or depression. The female
sometimes leaves it only when almost trodden under
foot, in fact, several were swooped upon and caught
thereon by hand. They usually begin to lay about
the end of May or the beginning of June. WILLOW PTARMIGAN, The process
of moulting, or the gradual assumption of their sum-
mer plumage, commences a week or two earlier. The
female lays from seven to ten, twelve, and, occasional-
ly, as many as thirteen eggs, which I find was the AMERICAN GAME BIRD SHOOTING 174 greatest number recorded; and we had reason to know
that some, at least, of the nests were used by ptarmigan
several seasons in succession. When very closely ap-
proached as stated, the female would frequently flut-
ter off, sometimes spreading her wings and ruffling
her feathers, as if to attack or frighten away intrud-
ers, and at other times calling out in distressed tones,
and acting as if she had been severely wounded. “In one instance, where an Indian collector had
found a nest which contained seven eggs, he placed a
snare thereon; but on returning to the spot a few hours
afterward, he was surprised to find that six of the
eggs had disappeared in the interim, and as no egg-
shells were left behind they were in all probability re-
moved by the parents to a safer place. The male bird
is generally not far away from the nest, and his pe-
culiar hoarse and prolonged note is frequently heard,
the more especially between the hours of 10 p.m. and
2 A.M. Both, however, displayed great courage and
devotion in protecting from capture their young, which
we often encountered on our return coast trips. “About the end of September, during October, and “About the end of September, during October, and
early in November
L. lagopus assembles
in great
flocks, but during the winter it was seldom that more
than two or three dozen were ever noticed in single
companies. They are, however, most winters very
numerous in the neighborhood of Fort Good Hope and
other Hudson’s Bay Company posts in the Mackenzie
River district; but as spring sets in, they begin to mi-
grate northward, and it is very doubtful if many breed WILLOW PTARMIGAN 175 to the south of latitude 68°, at least in the valley of
the Anderson.” The change of summer plumage to that of winter
begins on the abdomen and gradually extends over the
entire body, the head changing last. This change
occurs between September toth and the last of October,
the young assuming their winter garb a little in ad-
vance of the adults. WILLOW PTARMIGAN, In spring the change is reversed,
commencing about the head of the birds and thence
passing over the rest of the body. After the head has
assumed its summer plumage, the change on the rest
of the body goes on very slowly, and in many instances
is never perfected, the back and abdomen of the ma-
jority of summer specimens being never entirely free
from white feathers. The wings remain white through-
out the year. There is considerable individual varia-
tion in the plumage of adult specimens shot at the same
season. Mr. Charles Sheldon, who spent the winter of 1908
and 1909 on the shoulders of Mt. McKinley, saw there
the willow ptarmigan, the rock ptarmigan and the
white-tailed ptarmigan. These birds were all abun-
dant, and there was no difficulty at any time in starting
out and in the course of a short walk killing with
a .22 rifle a sufficient number to last for food during
several days. In spring, at the mating season, the willow ptarmi- In spring, at the mating season, the willow ptarmi-
gan was extremely noisy, and at that time was seen
much in the trees, often sitting on the topmost spray,
from which he uttered his crowing call, and often AMERICAN GAME BIRD SHOOTING 176 made short flights. Long before the snow disappeared
the birds had commenced to assume the dark plumage
of summer, the head and neck changing first. The
birds were now very noticeable against the snow. The
males fought with much energy. After the young
had been brought off the males disappeared. When startled in winter the birds often fly a short When startled in winter the birds often fly a short
distance, and then, alighting on the snow, remain
crouched there, perfectly motionless, so that they are
extremely hard to see. As I have often noticed with the white-tailed ptarmi- As I have often noticed with the white-tailed ptarmi-
gan, the willow ptarmigan, when the wind is blowing
fiercely and the snow driving, will often alight on a
drift and quickly scratching a hole will crouch in it,
thus protected from the wind and the drifting snow,
which, however, may sometimes quite cover them over. WILLOW PTARMIGAN, In the summer of 1899, while the Harriman Alaska In the summer of 1899, while the Harriman Alaska
expedition was in Yakutat Bay, some of the members
who were strolling up a ravine back of the sealing vil-
lage of the Indians, came upon a female ptarmigan
with half a dozen young. The young ones did not at
once hide, and one of them was caught in the hand. The mother was anxious and uneasy for her little ones
and walked about within three or four feet of us, and
when the young one was caught she flew close about the
captor. When walking on the ground she clucked
like a setting hen, but with a deeper note; and after
her young had been released she called to them, warn-
ing them to remain hidden, with the same note that a
domestic hen uses to warn her chickens that she sees “eySe[W Ul URsTUIe}qd MOTI “eySe[W Ul URsTUIe}qd MOTI WILLOW PTARMIGAN 177, a hawk in the sky. When at last we walked away,
leaving the family alone, she also walked off into the
alder bushes. The wings and a few breast feathers
of this mother bird were white, but the rest of her
plumage was black and tawny, much like that of a
female spruce grouse. Of this species at Ungava, in Labrador, Mr. L. M. Turner says: “In the spring these birds repair, as the snow melts,
to the lower grounds and prepare for the nuptial sea-
son. About the toth of April they may be heard
croaking or barking on all sides. A male selects a
favorable tract of territory for the location of the nest,
and endeavors to induce a female to resort to that
place. He usually selects the highest portion of the
tract, whence he launches into the air, uttering a bark-
ing sound of nearly a dozen separate notes, thence
sails or flutters in a circle to alight at the place whence
he started, or to alight on another high place, from
which he repeats the act while flying to his former
place. Immediately on alighting he utters a sound
similar to the Indian word, chu-xrwan (what is it?),
and repeats it several times, and in the course of a
few minutes again launches in the air. WILLOW PTARMIGAN, “T cannot speak accurately on the subject, but think “T cannot speak accurately on the subject, but think
that seventeen days are required to incubate the eggs. On the 2oth of June I obtained a young bird of this
species, which was less than forty-eight hours out of
the shell. This was the earliest record. Thousands
of these young must perish annually, either from the
cold rains or from their parents being killed for food. . . . After the middle of August the birds have ac-
quired a good size and are then feeding on berries of
various kinds. They are then quite tender, of nearly
white flesh, and when properly prepared form a pleas-
ant food for the table. The young birds of the year
attain their full growth by the first of November.”
It thus seems that this ptarmigan is monogamous, a “T cannot speak accurately on the subject, but think
that seventeen days are required to incubate the eggs. On the 2oth of June I obtained a young bird of this
species, which was less than forty-eight hours out of
the shell. This was the earliest record. Thousands
of these young must perish annually, either from the
cold rains or from their parents being killed for food. . . . After the middle of August the birds have ac- . . . After the middle of August the birds have ac-
quired a good size and are then feeding on berries of
various kinds. They are then quite tender, of nearly
white flesh, and when properly prepared form a pleas-
ant food for the table. The young birds of the year
attain their full growth by the first of November.”
It thus seems that this ptarmigan is monogamous, a It thus seems that this ptarmigan is monogamous, a
pair mating and remaining together through the breed-
ing season, the male taking part in the care of the
young, showing a strong attachment for them and
being devoted to the female. The food of this species consists chiefly of the buds The food of this species consists chiefly of the buds
and leaves of various willows and birches, as well as
berries of different sorts, which during the summer
are exceedingly abundant in that country. WILLOW PTARMIGAN, [Early in
the morning hundreds of these birds may be heard,
continuing until nearly eleven o’clock, when the bird
then becomes silent until after three o’clock, when he
again goes through the same performance, though
with less vigor than in the morning. In the course of
a few days a female may be found in the vicinity. The AMERICAN GAME BIRD SHOOTING 178 actions of the male are now redoubled, and woe be to
any bird of his kind which attempts to even cross his
chosen locality. Battles ensue which for fierceness are
seldom equaled by birds of larger size. “In the vicinity of Fort Chimo the nesting of this “In the vicinity of Fort Chimo the nesting of this
species begins during the latter part of May. The
nest is usually placed in a dry spot among the swamps,
or on the hillsides
where straggling bushes grow. The nest is merely a depression in the mosses and
contains a few blades and stalks of grass, together with
a few feathers from the parent bird, which is now
in the height of the moulting from the winter to the
summer plumage. “The first eggs obtained were two on June 1, 1884,
this being the earliest record at Fort Chimo. The
number laid for a set varies greatly in different locali-
ties. At Fort Chimo seven to nine is the usual num-
ber, although in exceptional instances
as many
as
eleven and rarely thirteen may be found. “While
I was
at St. Michael
(Norton
Sound, “While
I was
at St. Michael
(Norton
Sound,
Alaska) I frequently found nests containing as many
as fifteen, and several times found seventeen. I was
there informed that over twenty eggs had been taken
from a single nest. On neither side of the continent
did I hear that more than one female deposited eggs
in the same nest. I can affirm that a clutch of seven
eggs may be taken, and if the nest be not disturbed,
the female will deposit nearly the same number again. These may again be taken, and not over three eggs
will be deposited, and if disturbed a third time she will WILLOW
PTARMIGAN 179 lay no more unless she selects a new location, which,
of course, would be difficult to ascertain. WILLOW PTARMIGAN, The eggs,
like those of other ptarmigan, are cream-colored as to
groundwork and are marked with blotches, spots and
cloudings of reddish and dark brown, often entirely
covering the ground color. Captain Bendire tells us
that all these markings are readily removable from the
freshly laid egg. AMERICAN GAME BIRD SHOOTING 180 The form of willow ptarmigan, known as Allen’s
ptarmigan, was described from Newfoundland by Dr. Stejneger nearly twenty-five years ago. The differ-
ences between it and the bird of the mainland are
trifling, being chiefly confined to the color of the shafts
of primaries and secondaries, which are black. This
is still
a common bird of Newfoundland, where it is
often called “partridge” by the settlers, and where, in
more remote districts, it still offers very good shooting,
though nowhere nearly so abundant as formerly. It
lives on the barrens, feeding on seeds and berries,
and in winter on the buds of alders, willows and
birches. It breeds on the ground among the spruces. Perhaps no writer has lived longer in the region in-
habited
by the willow
grouse
than Napoleon
A. Comeau, who, in his recent work, “Life and Sport
on the North Shore of the Gulf of St. Lawrence,”
gives some interesting notes on the species. He says:
“Of all the above species [of grouse], the willow “Of all the above species [of grouse], the willow
ptarmigan is by far the most abundant, and of con-
siderable value as an article of food during its years
of passage. It is an irregular migrant, in this wise,
that it does not come regularly every season, like most
other birds, and sometimes we may be three or four
years without seeing any. I have gathered consider-
able data on this subject, and I find that about every
tenth year is one of great abundance. Here are some
of the dates:
1863 and 1864, extremely abundant;
1867, disappeared this year; 1871, a few were seen this
year, but none between 1867 and 1871; 1872 and 1873, WILLOW PTARMIGAN 181 very numerous again and total disappearance in 1876;
1882, a few observed; 1883, 1884 and 1885, great
abundance; 1887, disappeared entirely; 1891, a few
seen, gradually increasing each year till 1895, when
there were considerable quantities; 1897, none; 1903
and 1904, abundant, and a few seen every winter since
to date, 1909. WILLOW PTARMIGAN, “At one time it was supposed that these years of “At one time it was supposed that these years of
abundance on the coast were due to heavy sleet in the
interior, covering up all the buds and preventing the
birds from feeding, and thus forcing them to seek food
elsewhere. I have noticed that this will affect them
to some slight extent, but the dates given show too
much regularity for this to be the true cause. My be-
lief is that it is due to the food supply. Having ex-
amined thousands of the crops of these birds I found
that over ninety per cent. contained the buds of a
species of willow, popularly known here as pussy wil-
low, Salix arctica? The balance were buds of the
birch, poplar and the mountain ash and its berry. I
also noticed a few seeds that I could not identify. “After a year or two of great abundance, all the “After a year or two of great abundance, all the
willows are destroyed by the breaking of the tips and
the buds, and the shrub takes about two years to re-
cover, which is generally by fresh sprouts from the
roots. As the food fails the birds have to move else-
where. It would, therefore, appear from my data
that it takes about ten years to go around their circle
of migration. I say circle because their line of flight
seems
to indicate
this. They first appear on the AMERICAN GAME BIRD SHOOTING 182 Labrador coast line flying south, and continue so till
they reach our large rivers, like the Manicouagan,
Bersimis and the Saguenay, seldom going west of this
last. These large rivers are followed up in a west
and northwesterly direction, the birds scattering inland
over a tract that includes the Lake St. John and Lake
Mistassini region, then down to the shores of Hudson’s
Bay, where Dr. Milne and Mr. Peter McKenzie told
me they flew north all along the coast line to Ungava,
then south again to the Labrador, and so on. The
range of the flight on this side of Hudson’s Bay would
cover about ten degrees of latitude and in round fig-
ures form a circle around this big peninsula of about
two thousand miles. WILLOW PTARMIGAN, As this immense body of ptarmi-
gan moves on during a season of abundance, stragglers
are left behind, which breed, giving another and lesser
batch to migrate the second year, when fewer strag-
glers are left, till the third or fourth season, when no
more are seen for a time. Their total absence varies
from four to six years. They seldom or never breed
in the lowlands, always seeming to prefer high and
bare mountainous sections. In June, 1893, I saw a
pair several times. They probably had their nest with-
in a mile of our house (Godbout). The earliest ap-
pearance of any large migration was October 29, 1872,
but as a rule it begins here from the 15th of November
to December. “The regular flight along the seashore lasts about “The regular flight along the seashore lasts about
four to six weeks. After that the birds seem to scatter
inland and feed. When on the move they fly very WILLOW PTARMIGAN 183 early in the morning, sometimes so early that it is im-
possible to distinguish them unless there happens to
be some dark background. The morning flight, when
abundant, will last an hour or two. The size of the
flock varies, in ordinary seasons, from ten to fifteen or
twenty. In years of great abundance, flocks of a hun-
dred or more are common. On the 14th of December,
1885, I saw at Trinity Bay, six miles east of Pointe
des Monts, one flock which contained many thousands. It was a continuous mass of birds over half a mile
long and from sixty to a hundred yards wide. I have
never seen anything approaching this before, nor have
I since. When in large flocks they are, as a rule, shy,
especially if the weather is very cold or windy, and
will rise long before one gets within ordinary range. On the wing, however, they do not seem to mind any-
thing in their way, flying over and around one with-
out apparently noticing him. They are very strong
on the wing, not any faster than the ruffed grouse, but
capable of sustaining much longer flights, occasionally
going five or six miles without resting. WILLOW PTARMIGAN, “In small b “In small bunches and in snowy and mild weather
they are quite tame, and when feeding in the thick
willow bottoms it is hard work to get them to rise out of
them. After feeding they congregate around some
clump of willow, and scratching a small hollow in the
snow, will lie perfectly still for hours, the top of the
head just level with the snow, the black eye and beak
alone betraying their presence. The popular idea of
their diving in the snow to escape pursuit is most AMERICAN GAME BIRD SHOOTING 184 ridiculous. At night they will occasionally burrow
in the snow, but only during high winds or very cold
weather, the usual way of resting being the small
hollow mentioned. Another rather remarkable thing
in connection with ptarmigan is the apparent dispro-
portion of the sexes. Out of the many thousands that
I have killed and examined, only about twenty-five per
cent. were males. In winter they prefer low valleys and
the borders of rivers and lakes and dense willow
patches, but as the season advances they seek the
higher ranges, choosing those that face the midday
sun. When flying over water, as they often do in
crossing bays or large rivers, they keep very near the
surface, just about a foot or so above it. Over land
the reverse is the case, for sometimes they rise high
over the tops of the tallest trees. The most remarkable
thing about them, however, is their seasonal change of
plumage. In 1885 I had the pleasure of attending the
meetings of the American Ornithologists’ Union, in
New York. At one of the meetings a very interesting
paper was read by Dr. Stejneger on this subject. The
doctor exhibited two specimens which came from New-
foundland, and which, in his opinion, were a sub-
species confined to the island. This distinction was
based particularly on the coloration of the primaries. The birds shown had nearly all the tips of the pri-
maries black. Since my return I have taken special
pains to examine a great number of birds. On those
killed prior to 15th November I found the same
coloration, more or less, as on the species shown, but WILLOW PTARMIGAN 185 after that date there was a gradual whitening of the
primaries, and in many cases only the shafts were
white. WILLOW PTARMIGAN, “During the last two migrations, taking the best
years, 1895 and 1904, I took some trouble to try and
find out approximately how many birds were killed
between certain points. During the first year men-
tioned, between Mingan and Godbout, 175 miles of
coast, 30,000 were
killed. In the second
(1904),
14,000, but I am sure that during 1885 nearly 60,000
must have been shot or snared. When a flight begins,
every man, woman and boy able to handle a gun is out. To avoid accidents, which are very rare indeed, each
gun occupies a certain point or station, and shoots at
all the birds that pass or light in his vicinity. The
ladies keep watch of those that may light near the
houses. The bags vary, of course, according to the
skill of the shooter and his method of shooting. If he
is there for business he will take all the pot shots. He
can frequently get five or six in one shot. I have seen
fourteen killed in a single shot. A few will only shoot
on the wing, but there are many days when the wing
shooter comes out ahead. The biggest bag I ever
made (it was in 1885), shooting at flying birds, was
eighty-two brace in one morning. At Caribou Islands,
that winter, nets were tried, but they were not very
successful, more being got by shooting. Indians fre-
quently snare them by setting their snares around wil-
low clumps, where the birds feed. It is a very simple
arrangement. A twig is stuck in the snow, a twine AMERICAN GAME BIRD SHOOTING 186 snare is tied to it, a very light support placed under it
to hold it in position, and it is ready. In walking
around, the bird runs into it, then tries to rise on feel-
ing the snare, only to tighten the noose. There is a
little fluttering, and it is all over.” ROCK PTARMIGAN. Lagopus rupestris. Lagopus rupestris reinhardi. Lagopus rupestris welch. Lagopus rupestris nelsoni. Lagopus rupestris atkensis. Lagopus rupestris townsend. Lagopus rupestris evermannt. Lagopus rupestris chamberlaint. Lagopus rupestris kelloge. Lagopus rupestris dixont. The bill of the rock ptarmigan is much more slender
than that of the willow ptarmigan, and the bird is
somewhat smaller, measuring from 13 to 1434 inches. In winter the bird is white, with black tail; but a line
running from the eye to the bill is also black. It is
said that the female does not always have this black
spot covering the lores. In summer the male is grayish brown, the feathers In summer the male is grayish brown, the feathers
being crossed with fine black lines, which tend to form
irregular zigzag bars. The shoulder feathers, or scapu-
lars, are
chiefly
black, which
gives the
effect
of
large black spots on the back. The quills of the wing
—primaries and secondaries—and the outer tertiaries
187 187 187 AMERICAN GAME BIRD SHOOTING 188 are white. The lores and top of the head are black,
the ends of the feathers on the latter being tipped with
brown. Breast regularly barred with blackish and
light brown, and the sides the same, but with finer
black bars; beneath, white. The summer plumage of the female is brighter, yel- The summer plumage of the female is brighter, yel-
lowish or reddish, spotted and barred with black, and
having the quills and secondaries always white. The
lower plumage is somewhat paler and grayer; the dot-
tings and barrings are black, often quite fine; yet the
barrings show a tendency to form spots and heavier
bars. The head and neck are more yellowish and
barred with dusky. The rock ptarmigan, in its various forms, is scat-
tered over Arctic America in general, except the ex-
treme north, including Greenland and the Aleutian
Islands, and is found southeast as far as the Gulf of
St. Lawrence, occurring on Anticosti Island. Mr. Nelson reports it a common resident of the
mainland
of Alaska, where
it inhabits
the higher
ground during the summer, but is driven down by
winter to the lower levels. Its habits do not appear
to differ greatly from those of the common willow
ptarmigan. It breeds in large numbers on the Barren
Grounds, from which Mr. MacFarlane reported on its
breeding habits, nests and eggs. ROCK PTARMIGAN. It does not appear to
be so prolific a bird as the willow ptarmigan, its eggs
being fewer in number and apparently running from
nine down to four or five. The female sits closely on
her eggs, and her color harmonizes so well with that ROCK PTARMIGAN 189 of the surrounding vegetation that usually when dis-
covered she is seen only by accident. In Alaska the eggs are laid in May and in the Bar-
ren Grounds somewhat later, usually between the mid-
dle of June and July. The eggs are quite like those
of the willow ptarmigan, but are slightly smaller. The
ground color runs from cream color to yellowish, and
the spots and blotches vary from a dark brown to a
claret red. Sometimes these spots are so numerous as
almost to hide the ground color. Reinhardt’s ptarmigan, a sub-species of the rock Reinhardt’s ptarmigan, a sub-species of the rock
grouse, is found in Greenland and throughout Labra-
dor, and Mr. L. M. Turner, whose notes on the birds
of Labrador and Ungava are so familiar to ornitholo-
gists, tells us about all that is known of it. He says
that it prefers open ground, and rarely enters even the
skirts of the wooded tracts. “The mating season begins in May, and during this
period the male acts in the strangest manner to secure
the affection of its mate. He does not launch high
in the air and croak like the willow ptarmigan, but
runs around his prospective bride with tail spread,
wings dragging like those of the common turkey, or
else with head and neck stretched out and breast in
contact with the ground, pushing himself in this man-
ner by the feet, which are extended behind. The male
at this time ruffles every feather of his body, twists his
neck in various positions, and the supra-orbital pro-
cesses are swollen and erect. He utters a most peculiar
sound, something like a growling kurr-kurr, and as AMERICAN GAME BIRD SHOOTING 190 the passion of the display increases, the bird performs
the most astonishing antics, such as leaping in the air
without effort of wings, rolling over and over, acting
withal as if beside himself with ardor. The males
engage in most desperate battles; the engagement lasts
for hours, or until one is utterly exhausted, the feath-
ers of head, neck and breast strewing the ground. ROCK PTARMIGAN. A maneuver is for the pursuing bird to lead the other
off a great distance and suddenly fly back to the female,
who sits or feeds as unconcerned as it is possible for a
bird to do. She acts thoroughly the most heartless
coquette, while he is a most passionately devoted lover. He will die rather than forsake her side, and often
places himself between the hunter and her, uttering
notes of warning for her to escape, while attention
is drawn to him, who is the more conspicuous. “When the young are with the parents they rely
upon their color to hide themselves among the nearly
similar vegetation from which they procure their food. I am certain I have walked directly over young birds
which were well able to fly. If the parent birds are
first shot, the entire number of young may be secured,
as they will not fly until nearly trodden upon, and then
only for a few yards, while they may easily be seen. I have found on two occasions an adult female with a
brood of thirteen young. All of the flocks were se-
cured without trouble. At other times only three or
four young would be found with both parents. The
young are very tender when first hatched; no amount
of most careful attention will induce them to eat, and ROCK PTARMIGAN IQ! after only a few hours’ captivity they die. I never
could keep them alive above twelve hours. The change-
able weather, sudden squalls of snow or rain, must
be the death of scores of these delicate creatures. Their note is a soft piping pe-pe-pe, uttered several
times, and has the same sound as that of the young
of the bobwhite (Colinus virginianus).”
For the first few weeks of their life the young of For the first few weeks of their life the young of
this species cannot be distinguished from those of the
willow ptarmigan, but when a month old they may
be readily told apart by the bill. The food of this species consists of insects, berries,
leaves and buds of the birch and willow. Mr. Kumlien,
at Cumberland Sound, shot one whose crop was full
of moss. Welch’s ptarmigan, described by Mr. ROCK PTARMIGAN. Brewster from
Newfoundland, is reported as a highland form of the
rock ptarmigan, and confined to the sides and sum-
mits of rock hills and mountains in the interior of
Newfoundland. The rock ptarmigan is a very local
bird, and for the most part spends its life on or near
the hills where it was reared. It has been spoken of
as a truly alpine species, rarely found below the line
of stunted black spruces, except in the depth of winter,
when they descend to the lowlands and sometimes min-
gle with the willow grouse. The settlers call it moun-
tain partridge to distinguish it from the willow grouse,
known as partridge. Nelson’s ptarmigan is another form of the rock Nelson’s ptarmigan is another form of the rock
grouse, which is confined to the islands of Unalaska, AMERICAN GAME BiRD SHOOTING 192 Akutan and Unimak of the Aleutian group. It spends
most of the time on higher ground, but at the breed-
ing season comes down to the narrow valleys to rear
its young. The nest is usually placed in tall grass
in some valley or on the open tundra. Turner’s ptarmigan, another subspecies,
is found
at Atka, one of the islands of the Aleutian chain. It
occurs also on Amchitka Island. The bird is larger
than the mainland form, and is quite numerous on
these islands. Mr. Turner reports the eggs as from
eleven to seventeen in number. The habits of the
subspecies are not known to differ from those of the
other forms of this ptarmigan. Townsend’s ptarmigan from Kiska Island, Ever- Townsend’s ptarmigan from Kiska Island, Ever-
mann’s
ptarmigan from Attu Island, Chamberlain’s
ptarmigan from Adak Island, Dixon’s ptarmigan from
the Sitkan Islands, and Kellog’s ptarmigan from Mon-
tague Island, complete the list of closely related forms
from the Alaskan Islands, where this species seems to
have been very easily influenced by local conditions. Townsend’s ptarmigan from Kiska Island, Ever-
mann’s
ptarmigan from Attu Island, Chamberlain’s
ptarmigan from Adak Island, Dixon’s ptarmigan from
the Sitkan Islands, and Kellog’s ptarmigan from Mon-
tague Island, complete the list of closely related forms
from the Alaskan Islands, where this species seems to
have been very easily influenced by local conditions. WHITE-TAILED PTARMIGAN. Lagopus leucurus. Lagopus leucurus peninsularis. Lagopus leucurus. Lagopus leucurus peninsularis. Winter plumage snow-white throughout; the bill
and eyes being the only dark spots to be seen. The
summer plumage is buff, or, later in the season, clay
color, coarsely barred with black on a pale ground. There is little difference between the male and the
female. The tail is always white. In the young the tail feathers at first are not white, In the young the tail feathers at first are not white,
but are mottled with brownish. They become white
with moulting, however. The sub-species, peninsularis,
inhabits the alpine The sub-species, peninsularis,
inhabits the alpine
mountains of central Alaska, northern Yukon, N. W. Mackenzie, south to Cook Inlet, Kenai Peninsula and
southern Yukon. In all America, the especial home of the grouse,
there is no prettier member of this family than the
white-tailed ptarmigan. Like all its kind, it loves the
cold and snow, but, unlike the other American mem-
bers of the group, it substitutes altitude for latitude
and is an inhabitant of the lofty mountains of the
West, from central Alaska and northern Yukon south
to Washington and New Mexico. Here on the very
edge of perpetual snowfields, not far from some brawl-
193 193 AMERICAN GAME BIRD SHOOTING 194 ing ice-fed torrent, and within the sound of the roar of
avalanche and the thunder of glacier, the little ptarmi-
gan spends his life, seldom disturbed by enemies. In summer he wears a livery of black and clay color, In summer he wears a livery of black and clay color,
which harmonizes so well with the rocks among which WHITE-TAILED PTARMIGAN WHITE-TAILED PTARMIGAN his life is passed that it is most difficult to see him,
if he wishes to remain unseen and does not move;
while in winter, as he journeys over the frozen wastes,
his plumage is as white as the snows on which he
walks. His must be a contented, care-free life, for he
has few enemies to fear. The sneaking coyote and
the stealthy wild cat seldom visit his mountain soli- WHITE-TAILED PTARMIGAN 195 tudes. Rarely the eagle’s broad pinions cast their dark
shadow over snowfield and rock slope, but the eagle
is generally in search of larger game, the tender young
of the big horn or of the white goat. WHITE-TAILED PTARMIGAN. The great bears
that in early summer prowl over the range, looking for
the young grass or digging out mice, or later pick-
ing the luscious huckleberries, do not give a thought
to the ptarmigan, unless by chance they stumble on
her nest, when it suffers the fate of every thing eatable
that comes in Bruin’s way. Of all the animals of the mountains, the one that
the ptarmigan has most to fear is perhaps the pine
marten. He is always traveling about, alow and aloft;
equally at home among the trees of the forest and the
rocks of the peaks, always hungry, always searching
for food; and, while it may be doubted whether he
destroys many full-grown ptarmigan, we may feel sure
that he compasses the death of many young and pil-
lages many a nest. Although the white-tailed ptarmigan
is abundant
enough in the high mountains which it inhabits, it is
scarcely known at all to sportsmen. Only the hardy
spirit who climbs above timber line in search of sheep
or goats, or that other enthusiast whose highest pleas-
ure it is to reach the summit of the loftiest mountain
peaks, ever reaches the home of this bird; and as
neither of these ever burdens himself with a shotgun,
it is almost never killed for sport. In Colorado, where
many prospectors and miners carry on their operations
far above timber line, the ptarmigan is often killed for AMERICAN GAME BIRD SHOOTING 196 food, but in other parts of the West man interferes but
little with the abundance of this species. This ptarmigan is found in considerable numbers
above timber line on most of the mountains of western
America. It does not occur in the Sierra’ Nevada,
nor in the Cascades south of Washington. It is no-
where very abundant, though I have seen them, in
autumn, in flocks of twenty-five or thirty, and recall
an afternoon when, on Mount Jackson, in Montana,
two of the party secured twenty-two of the birds. Yet, as a rule, they are nowhere very common, a single
brood often seeming to occupy its own range of ter-
ritory, which is not encroached on by others. I have
not seen them in large flocks as mentioned below by
Mr. Trippe. WHITE-TAILED PTARMIGAN. The nest of the white-tailed ptarmigan is built high The nest of the white-tailed ptarmigan is built high
up on the mountain-side above timber line, and may be
located anywhere among the loose stones and rocks. A little depression is perhaps scratched out near a
patch of grass or weeds, and here the mother bird
deposits her eight or nine eggs, buff in ground color,
sometimes spotted with many small reddish or brown
dots, or, again, peculiarly blotched with larger mark-
ings of the same color. The nest is usually quite in
the open, but the grayish mother bird so closely resem-
bles the stones among which she nests as readily to
escape observation. Moreover, the confiding nature
of the bird gives her such courage that she will remain
on the nest, unscared even by the close approach of
some great danger. WHITE-TAILED PTARMIGAN 197 Mr. A. W. Anthony wrote to Captain Bendire:
“Twice have I escaped stepping upon a sitting ptarmi-
gan by only an inch or so, and once I reined in my
horse at a time when another step would have crushed
out the life of a brood of nine chicks, but an hour or
so from the egg. In this case the parent crouched at
the horse’s feet and, though in momentary danger of
being stepped on, made no attempt to escape until
I had dismounted and put out my hand to catch her. She then fluttered to the top of a rock a few feet dis-
tant and watched me as I handled the young, constant-
ly uttering low, anxious protests. The chicks were
still too young to escape; mere little wet bunches of
down, that stumbled and fell over one another when
they attempted to run. “Miners in whom I have confidence told me that they
have lifted sitting ptarmigan from the nest and han-
dled the eggs, while the bird stood but a few feet dis-
tant, watching her treasures and uttering an occasional
squeak like a sitting hen. One which had her nest
near the trail between the cabin and the mine was an-
noyed in this way so often that she would attempt to
regain the nest while the eggs were being handled, and
had to be frequently pushed aside. WHITE-TAILED PTARMIGAN. She never failed
to peck at the hand and utter her protesting ki-r-r-r-r
whenever one attempted to touch her, and made no
attempt to fly away.” All the evidence given by people who have found
the nests of this bird indicates that the sitting female AMERICAN GAME BIRD SHOOTING 198 is no wilder than the average domestic hen in the
same situation. The following very interesting account of this bird The following very interesting account of this bird
appears in Dr. Coues’ “Birds of the Northwest,” for
which it was prepared by Mr. Trippe; it relates en-
tirely to the species in Colorado:
‘The white-tailed
ptarmigan is a very: abundant bird on the main range,
living entirely above timber line the year round, ex-
cept during the severest part of the winter, when it
descends into the timber for shelter and food, occasion-
ally straggling as low as
10,000
feet. It begins
to change color about the middle of March, when a few
specks of blackish brown begin to appear in the plum-
age of the oldest males, but the change is very slow,
and it is late in April before there is much black visible,
and the close of May or early in June before the sum-
mer plumage is perfect. The ptarmigan builds
its
nest in the latter part of June and commences hatch-
ing toward the close of the month, or early in July. The nest—which is almost always placed on or near
the summit of the ridge, or spur, many hundred feet
above timber line-—is merely a depression in the ground,
lined with a few straws and white feathers from the
mother’s breast. The eggs are eight in number, of a
light buff brown, thickly sprinkled with spots of dark
chocolate brown, somewhat thicker at the larger end. While on her nest the bird is very tame. Once, while
walking near the summit of the range, I chanced to
look down and saw a ptarmigan in the grass at my very
feet ;
at the next step I should have trodden upon her. WHITE-TAILED PTARMIGAN. WHITE-TAILED PTARMIGAN 199 Seeing that she did not appear frightened, I sat down
gently, stroked her on the back, and, finally, putting
both hands beneath her, raised her gently off the nest
and set her down on the grass, while she scolded and
pecked my hands, like a setting hen, and, on being
released, merely flew off a few yards and settled on
a rock, from which she watched me till I had gone
away. Late in July I came across a brood of young
ones, apparently not more than four or five days old. They were striped with broad bands of white and
blackish brown, and looked precisely like little game
chickens. The mother flew in my face and hit me with
her wings, using all the little artifices that the quail
and partridge know so well how to employ, to draw me
away; while her brood, seven
or eight in number,
nimbly ran and hid themselves in the dense grass and
among the stones. On another excursion above timber
line, toward the close of August, I found most of the
young ones nearly grown, and strong on the wing;
but one brood was of the size of quails, showing that
some birds must begin breeding much later than others,
or that they: occasionally raise two broods. These
little ones were colored much like the older birds, hav-
ing blackish-brown bodies and pure white tails. About
the first of September the ptarmigan begins to change
color again, but, as in the spring, the process is very
gradual, white feathers appearing one by one and
taking the place of the dark ones. The white on the
lower parts enlarges first, then the white areas on
the wings, and next, white specks appear on the upper AMERICAN GAME BIRD SHOOTING 200 parts, becoming larger and more numerous as the sea-
son wears on; but so gradual is the change that a
month after it begins there is not much difference in
the plumage perceptible, the general aspect being that
of summer. There is much more of the light rufous,
however, and the appearance is lighter and grayer, as
though bleached. The dark areas predominate, how-
ever, throughout October, and, as I have been in-
formed by. WHITE-TAILED PTARMIGAN. persons who have killed them throughout
the year, it is late in December or January before they
become pure white, some few birds showing occasional
dark spots even throughout the latter month. “The ptarmigan feeds upon the leaves and stalks of
various alpine plants, being particularly fond of those
of a species of Cassia, the flowers of which I have
frequently taken from its crop. It also lives largely
upon insects, and in winter is said to subsist on the
buds and leaves of the pines and firs. Its flesh is light-
colored, though not as white as thateof the gray grouse,
to which it is usually considered inferior for the table. In localities where it is seldom molested, it is very
tame, and I have been informed by persons whose
word is worthy of belief, that they have frequently
killed it with sticks; but when persistently persecuted
it soon becomes wild and leaves the range of a shotgun
with surprising quickness. After hunting several large
flocks for three or four days, they grew so shy that
it was difficult to approach within gunshot, although
at first they had been comparatively tame. Nimble of
foot, the ptarmigan frequently prefers to run away on WHITE-TAILED PTARMIGAN 205. the approach of danger rather than take wing, run-
ning over the rocks and leaping from point to point
with great agility, stopping every little while to look at
the object of alarm. I have sometimes chased them half
a mile or more over the rocky, craggy ridges of the
main range, without being able to get within gunshot
or force them to take wing. The flight of the ptarmi-
gan is strong, rapid and, at times, sustained for a
considerable distance, though usually they fly but a
few hundred yards before alighting again. It re-
sembles that of the prairie hen, consisting of rapid
flappings of the wings, alternating with the sailing
flight of the latter bird. The note is a loud cackle,
somewhat like the prairie hen’s, yet quite different,
and when uttered by a large flock together, reminds
one of the confused murmur and gabble of a flock of
shore birds about to take wing. It is a gregarious
bird, associating in flocks throughout the year, except
in the breeding season. WHITE-TAILED PTARMIGAN. The different broods gather
together as soon as they are nearly grown, forming
large flocks, sometimes of a hundred or more. The
colors of this bird closely resemble those of sur-
rounding objects at all seasons of the year. In its
summer plumage of speckled black and gray it is very
difficult to detect while sitting motionless among the
gray and lichen-covered rocks. The ptarmigan is ap-
parently well aware of this, and often squats and re-
mains quiet while one is walking past, trusting to its
resemblance to the surrounding rocks to escape obser-
vation. So perfect is this resemblance that sometimes, AMERICAN GAME BIRD SHOOTING 202 on seeing one alight at a certain spot, and withdrawing
my eyes from it a moment, I have been unable to find
it again, although I knew the exact place where it
sat, until
a movement on the part of the bird betrayed
its position. In summer the white areas of the plum-
age are completely hidden while the bird is squatting,
although plainly visible while on the wing; in winter
the first appearing black specks are concealed beneath
the white feathers, and at this period, as I am in-
formed, it is almost indistinguishable from the snow. On being pursued, it will dive into the snow and re-
appear at a considerable distance.” My experience with this bird has been chiefly in
Wyoming, Montana and British Columbia, and usually
in the autumn. In the month of November some
change in the plumage will be noticed, though this
is chiefly in the reduction of the dark-colored areas,
rather than in the appearance in them of white feath-
ers. Where little disturbed by man, the birds pay but
slight regard to the presence of the hunter, and feed
along in a close flock without scrutinizing him. If
he ventures so near as to alarm them, and this some-
times may be within a few feet, they may rise on the
wing against the stiff breeze and sail along for a few
yards, looking much like a flock of domestic pigeons,
to alight after going a very short distance. WHITE-TAILED PTARMIGAN. If the
spot where they strike the ground is exposed, and the
wind is blowing, the birds crouch flat on the ground,
head to the wind, or those that have alighted near
stones or rocks projecting from the earth, run and ‘aseun
surids
“URsTUII
peyte}-a ‘aseu
surid
“URsTUI
peyte}-a WHITE-TAILED PTARMIGAN 203 crouch behind them; or, if they alight on a snow bank,
they quickly scratch out for themselves a little cavity
in the snow, large and deep enough to contain the
body, so that the wind blows over them. On the other hand, in Montana, among the high
mountains of the Saint Mary’s region, where hunters
and mountain climbers are often seen, these birds are
sometimes quite wild, rising at a considerable dis-
tance with a loud cackle, and flying a quarter of a
mile to alight again on some prominent rock, upon
which they run about with tail erect and head thrown
back, cackling in alarm, and ready at an instant’s no-
tice to take to wing again and fly still farther. It is
only in this region that I have pursued them with a
shotgun, and here they are as quick on the wing and
as hard to hit as any of the grouse. Sometimes, when
following scattered birds along the rough mountain-
side, they would pitch down past me from the rocks
on which they had perched, with a flight not less rapid
than that of the New England ruffed grouse, as he
darts down from the top of some pine tree in which
he has hidden himself. Two or three years ago, Joseph Kipp, of Montana,
while crossing from the west to the east side of the
range, through the Belly River Pass, in July, came
upon a brood of half-grown ptarmigan. The mother
attacked him vigorously, and was so persistent that
he caught her and several of the young, and carried
them with him for half a day, when he let them go
again, because he had nothing which they could eat. Two or three years ago, Joseph Kipp, of Montana,
while crossing from the west to the east side of the
range, through the Belly River Pass, in July, came
upon a brood of half-grown ptarmigan. WHITE-TAILED PTARMIGAN. The mother
attacked him vigorously, and was so persistent that
he caught her and several of the young, and carried
them with him for half a day, when he let them go
again, because he had nothing which they could eat. AMERICAN GAME BIRD SHOOTING 204 It is hardly to be supposed that these birds would do
well on food other than that to which they are accus-
tomed in their home among the high mountains, and
any attempt to domesticate them would be foredoomed
to failure. It is not improbable that if some one who
resided high up in the mountains should try to rear
the young he might succeed, but they could not be
taken away from the mountains where they belong. In summer or autumn single birds are often met In summer or autumn single birds are often met
with high up on the peaks, presumably the males whose
mates are then busy with their nests or young, and
these individual birds usually seem wild. They will
often stand and look until quite closely approached,
and then run swiftly fifteen or twenty yards, and then
stopping, stand erect and watch until again approached. While nowhere
very abundant,
the white-tailed While nowhere
very abundant,
the white-tailed
ptarmigan is yet numerous on all the higher moun-
tains which suit the requirements of its life. Half a
dozen broods may be found within a range of two
miles along the mountain-top, while the number of
eggs varies from five or six to fourteen or fifteen. Occasionally, at the approach of winter, a considerable
number of the birds will be found together, but I have
not seen more than twenty-five or thirty in a flock. Though, in summer, insects, flowers of the heather, Though, in summer, insects, flowers of the heather,
berries and seeds undoubtedly constitute a large part
of the white-tailed ptarmigan’s food, nevertheless I
believe that at all seasons they feed to a considerable
extent on the buds and tips of the willow, the largest
shrubs which grow near the tops of the mountains. On WHITE-TAILED PTARMIGAN 205 many occasions I have examined their craws and have
usually found them stuffed with these willow buds. None of these examinations, however, has been made
later than December. -tailed ptarmigan
is Of all our grouse the white-tailed ptarmigan
is
probably the one least exposed to persecution by man. WHITE-TAILED PTARMIGAN. No doubt some are killed by lynxes, martens and
weasels, but on the whole its enemies must be few in
number. PINNATED GROUSE. Tympanuchus cupido. Tympanuchus americanus. Tympanuchus americanus attwateri. Tympanuchus pallidicinctus. Tympanuchus cupido. Tympanuchus american The familiar prairie chicken or prairie hen of a
generation ago was
the pinnated
grouse,
once
so
abundant in Illinois, Indiana, Kentucky and to the
westward. It is remembered by older readers as abun-
dant in our markets, where it sold for seventy-five cents
per pair. In later years the term prairie chicken has
been applied equally to the sharp-tail grouse, a species
of more western distribution; and, generally, in the
western country, any grouse found in the open are
called: “chickens,”
The pinnated grouse include four forms, grouped The pinnated grouse include four forms, grouped
together under the genus Tympanuchus, a name which
refers to the inflatable sac on the neck of all these
grouse. All these forms are so similar as hardly to
be distinguished by any one save a practiced ornitholo-
gist. Birds of this group were formerly abundant on
the Atlantic coast, as well as throughout much of the
western
country
until
the
semi-arid
plains
were
reached. So far as we know, their western boundary,
roughly stated, was western Minnesota, eastern Ne-
206 PINNATED GROUSE 207 braska and Kansas, the Indian Territory and western
Texas. Birds of this genus are brownish or clay color, cross-
banded by dark brown or black. The tail is short and
rounded, and the sides of the neck are provided with wh
17044 %
=e Su rll ||
|
=~ G5
(
x
PINNATED GROUSE 17044 % PINNATED GROUSE conspicuous wing-shaped tufts of straight, stiff black
feathers, beneath which is a naked inflatable air sac. In greater detail the color above is pale brownish, In greater detail the color above is pale brownish,
barred with dusky and buff; beneath, paler, broadly
barred or banded with brown. The quills of the wing
are gray or brownish, with buff or whitish on the
outer webs. The chin, throat and cheeks are buff, AMERICAN GAME BIRD SHOOTING 208 the cheeks marked with brownish spots. A dark-
brown stripe runs from the corner of the mouth under
the eye and across the ear, and above this is a stripe
of buff. The black tufts of feathers on the side of
the neck are stiff and narrow, the longest ones being
two and a half inches. The tail feathers are black-
ish, tipped with white; the under tail coverts white-
tipped. PINNATED GROUSE. In the female the neck tufts are smaller,
usually less than two inches in length, and the tail
feathers are barred with light brown. In the ordinary prairie hen of the Mississippi Valley, In the ordinary prairie hen of the Mississippi Valley,
Tympanuchus americanus, the scapulars are without
white dots near the ends, and the neck tufts of the
male are composed of more than ten narrow feathers,
whose edges are parallel and whose ends are rounded,
or sometimes almost square. The feathered young are
more or less dotted with patches of white and black
and the top of the head is reddish brown. The bird’s
length is from eighteen to nineteen inches for the male,
and a little less for the female. The Martha’s Vineyard heath hen, T. cupido, is The Martha’s Vineyard heath hen, T. cupido, is
slightly smaller than the western prairie hen, has large
and noticeable spots of whitish at the end of the scapu-
lar feathers. The neck tufts of the male have not more
than ten of the narrow feathers, all of which are sharply
pointed. The lesser prairie hen, T. pallidicinctus,
is also
slightly smaller than the Mississippi Valley prairie
hen, and is recognizable because each dark bar across
the plumage consists of a continuous brown bar en- PINNATED GROUSE 209 closed between narrower black feathers; in other words,
the edges of this dark bar are black, and this is true all
over the bird. The lesser prairie hen is found in Kan-
sas,
southwestern
Missouri,
Oklahoma
and _ west
central Texas. Along the coast region of Texas and southwestern Along the coast region of Texas and southwestern
Louisiana is found a well-marked race of pinnated
grouse, which Major Bendire named in honor of Mr. H. P. Attwater, who brought the bird to the attention
of ornithologists. It is about the size of the lesser
prairie hen, but has the foot feathered only on the
upper two-thirds of the tarsus; the tips of the long
feathers of the wing-like neck tufts are square. THE HEATH HEN. The bird of the Atlantic coast now known as the
heath hen is almost extinct, the only existing colony
being
on
Martha’s
Vineyard,
an
island
off the
coast of Massachusetts. It was formerly found in
Massachusetts, Connecticut, Long Island, New Jersey,
Pennsylvania, Maryland and Delaware, but has long
been exterminated from most of these regions. Thus, long before any of the other so-called prairie Thus, long before any of the other so-called prairie
grouse had been discovered, the pinnated grouse was
well known. Indeed, it may very well be that when
the Pilgrim Fathers landed at Plymouth Rock, the first
flesh meat that they tasted in the new country was the
coast form of what in later times was called prairie hen
or prairie chicken, then known as heath cocke or heath AMERICAN GAME BIRD SHOOTING 210 henne, later changed in pronunciation to héth’n. The
bird was long abundant in Massachusetts, in the open,
brushy country around the seacoast, where, no doubt,
it fed, as do its descendants to-day, at Martha’s Vine-
yard, on acorns, berries, grass and insects. It was
well known in New England in the first quarter of the
nineteenth century, but disappeared soon after that. The old New England writers speak of the heath The old New England writers speak of the heath
cocke as common, so that, according to Wood, “Hee
that is a husband and will be stirring betime may kill
halfe a dozen in a morning.”
Mr. William Brewster, in his interesting and com- Mr. William Brewster, in his interesting and com-
plete paper,
entitled “The Birds of the Cambridge
Region of Massachusetts,” says:
“T have been permitted to quote the following in- “T have been permitted to quote the following in-
teresting passage from ‘Notes of conversations with
Eliza Cabot, written down by her son, J. E. C. (abot),’
and printed for private circulation in 1904:
‘T recol-
lect the western prairie grouse in this part of the coun-
try. I saw one once in Newton; and once after I was
married, your father went down to the cape fishing,
and in the woods there I saw a grouse very near me
and saw him puff up that orange they have on the
side of the neck.’
Eliza Cabot was born on April 17,
1791, and married about 1811. Her granddaughter,
Mrs. THE HEATH HEN. Charles Almy, thinks it probable that she saw
the grouse in Newton about the beginning of the nine-
teenth century, and the one on the ‘cape’ (Cape Cod,
no doubt) about 1812. That both birds were heath
hens can scarcely be doubted, for there is no evidence PINNATED GROUSE 211 that living western grouse of any kind were intro-
duced into Massachusetts at so early a period.”
From the evidence given by Mr. Brewster and other From the evidence given by Mr. Brewster and other
writers it may be assumed that the heath hen was
more or less abundant on the site of Boston at the time
that city was founded, and there is no reason why it
should not have been numerous in other favorable sit-
uations along the New England coast and to the south-
ward. Early writings
tell us that it was
so. It
was found along the seaboard south of New Jersey,
and the late C. S. Wescott, of Philadelphia, frequently
spoke of it as having occurred—according to tradition
—in Maryland and Delaware, on the shores of the
Chesapeake Bay and on the Peninsula of Maryland and
Virginia. Nuttall, as late as 1832, says of the heath hen:
“Along the Atlantic coast they are still met with on
the grouse
plains of New Jersey,
on
the brushy
plains of Long Island, in similar shrubby barrens in
Westford, Conn., in the islands of Martha’s Vineyard
on the south side of Massachusetts Bay, and formerly,
as probably in many other tracts, according to the
information
which
I have
received
from
Lueut.-
Governor Winthrop, they were so common on the
ancient bushy site of the city of Boston that laboring
people or servants stipulated with their employers not
to have the heath hen brought to table oftener than a
few times in a week!”
Linsley, in his list of Connecticut birds, eleven years Linsley, in his list of Connecticut birds, eleven years AMERICAN GAME BIRD SHOOTING 212 later than Nuttall, speaks of the birds as already ex-
tinct in that State. Giraud, writing about 1840, says that even then the
bird was practically extinct on Long Island, but that it
had been abundant thirty years before. Audubon quotes an interesting letter from a Mr. Audubon quotes an interesting letter from a Mr. David Eckley, of Boston, who was in the habit of
shooting prairie hens on Martha’s Vineyard. THE HEATH HEN. This
letter declares that “Nashawenna
is the only other
island of the group on which they are found,” and
further along adds:
“It would be difficult to assign
a reason why they are found upon the islands above
named, and not upon others, particularly Nashann,
which, being large, well wooded, and abounding in
feed, seems quite as favorable to the peculiar habits
of the birds.” Even at that time, according to this letter, the heath
hens on Martha’s Vineyard were scarce, for Mr. Eck-
ley says that the result of a few weeks’ residence of
a party of three is ten brace of birds. The same gentleman says:
“We frequently meet The same gentleman says:
“We frequently meet
with the remains of such as have been destroyed in vari-
ous ways, but more particularly by the domestic cat,
which prowls the woods in a wild state, and which
often receives a very unwelcome salute for the mis-
chief it does. Owls, hawks and skunks also do their
part toward the destruction of these valuable but de-
fenceless birds. In these ways they are thinned off much
more effectually than by the sportsman’s gun. They fre-
quent no particular soil, and, like all other hunting, PINNATED GROUSE 213 wherever the food is, there is the likeliest place for
the game. In addition to this rule as a guide, we look
for their fresh tracks among the sandy barberry hil-
locks and along the numerous patches which intersect
that remarkable part of the Vineyard called Tisbury
Plain. Into this, should the birds fly from the hedges,
as they sometimes do, it is almost impossible to start
them a second time, as there are no trees or large
objects to mark their flight. Being mostly covered with
scrub oak of a uniform height, with occasional mossy
hollows, it affords them a place of refuge, into which
they fly for protection, but from which they soon
emerge, when the danger is passed, to their more
favourite haunts.”
This letter was written in December, 1832. This letter was written in December, 1832. The ornithologists of the first half of the ninete The ornithologists of the first half of the nineteenth
century did not differentiate the pinnated grouse of the
Mississippi Valley from the eastern bird, and spoke
of the pinnated grouse as even then almost extermi-
nated from its old range on the Atlantic coast. THE HEATH HEN. PINNATED GROUSE 215 So numerous were they a short time since in the bar-
rens of Kentucky, and so contemptible were they as
game birds, that few huntsmen would deign to waste
powder and shot on them. In fact, they were held
in pretty much the same estimation, or, rather, abhor-
rence, that the crows are now in Pennsylvania or other
of the Middle and Southern States, as they perpetrated
quite as much mischief upon the tender buds and fruits
of the orchards, as well as the grain in the fields,
and were often
so
destructive
to the crops,
that
it was
absolutely
necessary
for
the
farmers
to
employ their young negroes
to drive them away
by shooting off guns and springing
loud
rattles
all around the plantations from morning till night. As
for eating them, such a thing was hardly dreamed of,
the negroes themselves preferring the coarsest food to
this now much admired bird; while the young sports-
man exercised his skill in rifle shooting upon them,
in anticipation of more exciting sport among the other
prized denizens of the plains and forest. Prairie chick-
ens have not only deserted Long Island, Martha’s Vine-
yard, Elizabeth Island, New Jersey, and their other
haunts to the eastward, but they have also removed
even farther west than the barrens of Kentucky. . . .” So numerous were they a short time since in the bar-
rens of Kentucky, and so contemptible were they as
game birds, that few huntsmen would deign to waste
powder and shot on them. In fact, they were held
in pretty much the same estimation, or, rather, abhor-
rence, that the crows are now in Pennsylvania or other
of the Middle and Southern States, as they perpetrated
quite as much mischief upon the tender buds and fruits
of the orchards, as well as the grain in the fields,
and were often
so
destructive
to the crops,
that
it was
absolutely
necessary
for
the
farmers
to
employ their young negroes
to drive them away
by shooting off guns and springing
loud
rattles
all around the plantations from morning till night. THE HEATH HEN. A con-
temporary statement of interest as to the heath hen is
that made by Elisha J. Lewis in “The American Sports-
man,” published in Pennsylvania, 1857. He says:
“The prairie hen was, no doubt, at one time widely “The prairie hen was, no doubt, at one time widely
disseminated over our whole country, more particularly
in those portions interspersed with dry, open plains sur-
rounded by thin shrubbery or scantily covered with
trees. Unlike the ruffed grouse, this bird delights in
the clear, open prairie grounds, and will desert those
districts entirely which in the lapse of time become AMERICAN GAME BIRD SHOOTING 214 covered with forest. These birds are very rare—
in fact, may almost be considered extinct in the North-
ern and Middle States. Within a few years they were
quite abundant on some portions of Long Island. They
were also to be found in Burlington County, N. J.,
and in some few other places. There are, however,
still a few to be found on the Jersey plains, and every
season we hear of some of our sporting acquaintances
exterminating a small pack. We know of ten braces
being killed this season (1848), and about the same
number last year by the same party; and, as usual, in
both instances these scarce and beautiful birds were
butchered long before the time sanctioned by the strong
—or, rather, the weak—arm of the law. “Thus it is that the destructive hand of the would- “Thus it is that the destructive hand of the would-
be respectable poacher, as well as the greedy gun of
the pothunter, hastens to seal the fate of the doomed
prairie hen in these eastern
regions, and we may
predict with great certainty that ere long not one will
be found, save upon the rich plains of the West; from
which also, in course of time, they will be driven and
ultimately perish, root and branch, from before the
unerring guns of their ruthless destroyers. We un-
derstand that there are still a few of these birds to be
found in Pennsylvania—we believe in Northampton
County—where the pine forests are thin and open and
the country about them such as prairie hens delight in. They have always been abundant in the barrens of
Kentucky and Tennessee, as also in the balmy plains
and fertile prairies of Louisiana, Indiana and Lllinois. THE HEATH HEN. As
for eating them, such a thing was hardly dreamed of,
the negroes themselves preferring the coarsest food to
this now much admired bird; while the young sports-
man exercised his skill in rifle shooting upon them,
in anticipation of more exciting sport among the other
prized denizens of the plains and forest. Prairie chick-
ens have not only deserted Long Island, Martha’s Vine-
yard, Elizabeth Island, New Jersey, and their other
haunts to the eastward, but they have also removed
even farther west than the barrens of Kentucky. . . .”
Lewis says also in the course of this article that Lewis says also in the course of this article that
the pinnated grouse are easily domesticated, and will
pair and hatch in captivity—all this from Audubon. The species has been extinct for more than forty The species has been extinct for more than forty
years in New Jersey and Pennsylvania. Mr. Witmer
Stone
says that up to
1868, and probably
later, AMERICAN GAME BIRD SHOOTING 216 a few were said to occur on the barren plains which
cover portions of Ocean and Burlington counties in
New Jersey. This is a part of the pine barren region,
an elevated, dry tract covered with dwarf pines, which
average not more than a foot and a half in height. Turnbull,
in his “Birds of Eastern Pennsylvania,”
states that in 1869 a few survived in Monroe and
Northampton counties in that State. The heath hen is very similar to the prairie hen, but The heath hen is very similar to the prairie hen, but
slightly smaller. The tufts of pointed neck feathers
are shorter and, as already said, the bird has slight
points of difference in color. Though called a wood-
land bird, it is much in the open or in the thick,
low scrub oak and pines which cover
an area of
forty miles square. There are thought to be not more
than 150 or 200 of these birds left alive, and they
are therefore more nearly extinct than the buffalo. They are already protected by law and should be still
more strongly protected by public opinion of the resi-
dents of Martha’s Vineyard, who ought to feel proud
of this bird and to do everything in their power to
preserve it. Not much had been written about the heath hen on
Martha’s Vineyard until the year 1885, when Mr. THE HEATH HEN. William Brewster visited the island for the special
purpose of studying the bird. He reported the re-
sults of this visit in the Awk, and in 1890 repeated the
trip and gained additional information, which was
printed in Forest and Stream. He said :
“Throughout Martha’s
Vineyard,
the heath hen “Throughout Martha’s
Vineyard,
the heath hen PINNATED GROUSE
* 217 (locally pronounced héth’n,
as this grouse
is uni-
versally called) is well known to almost every one. Even in such seaport towns
as Cottage City and
Edgarstown, most of the people have at least heard
of it, and in the thinly settled interior it is frequently
seen in the roads or along the edges of the cover by
the farmers, or started in the depths of the woods
by the hounds of the rabbit and fox hunters. “Its range
extends,
practically,
over
the entire “Its range
extends,
practically,
over
the entire
wooded portion of the island, but the bird is not found
regularly or at all numerously outside an area of about
forty square miles. This area comprises most of the
elevated central portions of the island, although it also
touches the sea at not a few points on the north and
south shores. In places it rolls into great rounded
hills and long, irregular ridges, over which are scat-
tered stretches of second-growth woods, often miles
in extent, and composed chiefly of scarlet, black, white
and post oaks, from fifteen to forty feet in height. Here
and there, where the valleys spread out broad and
level, are fields which were cleared by the early set-
tlers more than a hundred years ago, and which still
retain sufficient fertility to yield very good crops of
English hay, corn,
potatoes
and other vegetables. Again, this undulating surface gives way to wide, level,
sandy plains, covered with a growth of bear, chinqua-
pin and post-oak scrub, from knee to waist high,
so
stiff and matted as to be almost impenetrable; or to
rocky pastures, dotted with thickets of sweet fern, bay- AMERICAN GAME BIRD SHOOTING 218 berry, huckleberry, dwarf sumac and other low-grow-
ing shrubs. “Clear, rapid trout brooks wind their way to the
sea through open meadows, or long, narrow swamps,
wooded with red maples, black alders, high huckleberry
bushes, andromeda and poison dogwood, and overrun
with tangled skeins of green briars. THE HEATH HEN. “At all seasons the heath hens live almost exclusively
in the oak woods, where the acorns furnish them abun-
dant food, although, like our ruffed grouse, they occa-
sionally at early morning and just after sunset ven-
ture out a little way in the open to pick up scattered
grains of corn or to pluck a few clover leaves, of which
they are extremely fond. They also wander to some
extent over the scrub-oak plains, especially when blue-
berries are ripe and abundant. In winter, during long-
continued snows, they sometimes approach buildings,
to feed upon the grain which the farmers throw out to
them. A man living near West Tisbury told me that
last winter a flock visited his barn at about the same
hour each day. One cold, snowy morning he counted
sixteen perched in a row on the top rail of a fence
near the barnyard. It is unusual to see so many to-
gether now, the number in a covey rarely exceeding six
or eight, but in former times packs containing from
one to two hundred birds each were occasionally met
with late in the autumn. “Only one person of the many whom I questioned “Only one person of the many whom I questioned
on the subject had ever seen a heath hen’s nest. It was
in oak woods, among sprouts at the base of a large PINNATED GROUSE 219 stump, and contained either twelve or thirteen eggs. The date, he thought, was about June 10. This seemed
late, but I have a set of six eggs taken on the Vineyard
July 24, 1885, and on July 19, 1890, I met a blueberry
picker who only the day before had started a brood of
six young, less than half grown. These facts prove
that this bird is habitually a late breeder. “The farmers about Tisbury say that in spring the “The farmers about Tisbury say that in spring the
male heath hen makes a booming or tooting noise. This, according to their descriptions, must resemble
the love notes of the western pinnated grouse. About
sunrise, on warm, still mornings in May, several birds
may be sometimes heard at once, apparently answer-
ing one another. THE HEATH HEN. “During my stay at Martha’s Vineyard, I obtained “During my stay at Martha’s Vineyard, I obtained
as many estimates as possible of the number of heath
hens which are believed to exist there at the present
time. My most trustworthy informants were, credit-
ably, averse to what was apparently mere idle guessing ;
but when I questioned them, first as to the extent of
the region over which the birds ranged, and next as to
how many on the average could be found in a square
mile within this region, they answered readily enough,
and even with some positiveness. As already stated,
the total present range of the heath hen covers about
forty square miles. The estimates of the average
number of birds per mile varied from three to five,
giving from 120 to 200 birds for the total number. These estimates, it should be stated, relate to the num-
ber of birds believed to have been left over from last AMERICAN GAME BIRD SHOOTING 220 winter. If these breed freely and at all successfully,
there should be a total of fully 500, young and old to-
gether, at the beginning of the present autumn. When
one considers the limited area to which these birds are
confined, it is evident that within this area they must
be reasonably abundant. I was assured that with the
aid of a good dog it was not at all difficult to start
twenty-five or thirty in a day, and on one occasion eight
were killed by two guns. This, however, can be done
only by those familiar with the country and the habits
of the birds.” The fact that but a small remnant was left of this
once widely distributed species aroused much interest
in it, and after a time the Massachusetts authorities
began to consider measures for its preservation. Be-
fore any steps looking to its preservation had been
taken, the numbers of the birds had still further di-
minished, and observations made on the island from
October, 1906, to May, 1907, at a time when they were
collected in large flocks, seemed to justify the conclu-
sion that the number of individuals was less than one
hundred. The report of the commissioners on fisheries and
game for the year ending December 31, 1907, declares
that by actual count of the flocks located in various
sections of the range, seventy-seven individuals were
enumerated. THE HEATH HEN. In May, 1906, a destructive forest fire
swept practically the entire breeding grounds, and very
few birds were reared that season. The summer of
1907, however, was a favorable one. At least ten *AIOYSIP{
TeINJEN
Jo
wnasnyy
uvollowy
ul
dnois
wong
‘jsou
pue
9ssnoIg
poajeuulg PINNATED GROUSE 22% broods were successfully reared, and it was believed
that the number had more than doubled. The first protective law relating to the heath hen is The first protective law relating to the heath hen is
said to have been passed in 1831. This provided for a
closed season from March 1 to September 1. In 1837
there was established a closed season of four years,
which in the same year was extended for five years
more. These Acts, however, permitted any town to
suspend the law, so far as that town was concerned,
for such a period as might be deemed expedient, and
in 1842 the town of Tisbury did suspend the law for
a period of ten days on more than one occasion. In
fact, the law in behalf of these birds fell into desuetude
and no effort was made to enforce it up to about 1905. The inhabitants of Martha’s Vineyard felt a local pride
in having there a bird found nowhere else in the world,
but this local pride was not strong enough to protect
the species. An observer who visited the island in the spring of
1906 made to the commissioners on fisheries and game
of Massachusetts
a report which
gives much _ in-
teresting detail as to the habits of the heath hen at the
particular time when they are preparing to mate, and
is well worth quoting in full. He says: “At 6 p.m. we arrived at the point where we hoped
to find traces of the heath hen. Ina
cleared field, about
thirty rods from the road, we distinctly saw two large
birds. On our nearer approach they squatted close
and their protective coloration was so effective that
although we knew almost exactly the precise location AMERICAN GAME BIRD SHOOTING 222 of the birds, we could not distinguish them. We
crawled behind the nearest cover and remained mo-
tionless for perhaps ten minutes. At length the long
shadows from the descending sun enabled us to dis-
tinguish the birds as they crouched with head close
to the ground among the very scanty vegetation. THE HEATH HEN. After
another interval of motionless activity on our part,
one bird quickly arose and began feeding, apparently
without suspicion; soon two more birds arose as if by
magic from the ground. Then began a most inter-
esting series of antics. These birds were joined by
five others, coming in singly and on foot from the
scrub in various directions. The birds came frequently
within forty paces of our hiding place, and in one
instance alighted on a small oak tree twenty-three paces
from our camera. While not near enough for suc-
cessful photographing, we were well situated for using
our field-glasses. The birds were all actively feeding
in the open field, apparently on grasshoppers and other
insects, but nipping red clover leaves very freely. They
moved leisurely about. Frequently two birds, some-
times as much as a hundred to a hundred and fifty
yards apart, ran directly toward each other, dancing
and blowing on the way, with the so-called ‘neck wings’
pointed upward in a V form. On facing each other,
both squatted and remained motionless from one to
five minutes. We could see none of the nodding and
pecking motions of the head so commonly indulged in
by domestic fowls when fighting; rarely was there
sparring with the bill, or striking with the feet or PINNATED GROUSE 223 wings. In twelve or fifteen encounters, only three or
four times did they strike thus, and only once did we
see ‘feathers fly.’
Most of the energy seems to be
spent in posturing and blowing. Generally, one of the
combatants backs slowly away, suddenly stopping if
the opponent advances too rapidly. In all these fight-
ing tactics the similarity of habits with those of the
domestic fowl were very marked. From all directions
came the peculiar foot, like distant tugboats in a fog,
all having whistles of the same pitch. This call may
be well imitated by blowing gently into the neck of a
two-drachm homeopathic vial. Each call extends over
a period of two seconds, and is repeated at frequent
intervals. It is prefaced by a run of about one yard,
with very rapid, mincing steps. The strides, however,
are so short that the bird does not advance rapidly. The tail is spread and the wings dropped after the
manner of the strutting turkey-cock. THE HEATH HEN. When the tail
is spread, the white under-tail coverts are conspicuous
and remind one forcibly of the ‘white flag’ of the deer
and antelope, or of our gray rabbit. The head is then
depressed and the neck outstretched forward, until it
is parallel with the surface of the ground; the neck
tufts are elevated to a V shape. The bright, orange-
colored air-sacs on each side of the neck, directly be-
hind the tufts of feathers, are slowly inflated until
they reach apparently the size of a tennis ball, when
they appear like two small ripe oranges, one protruding
from either side of the neck. The duration of the call
appears to closely coincide with the period of infla- AMERICAN GAME BIRD SHOOTING 224 tion, and seems to be emitted as the air enters the sac,
rather than when the air is expelled. The collapse of
the sac is sudden. The sound is ventriloquial, and
it is very difficult to locate the direction or distance
whence it comes, unless the bird can be seen. A sec-
ond sort of call is much less frequent and closely re-
sembles a single syllable of the hoot of the barred owl. “Another characteristic antic was a peculiar combi- “Another characteristic antic was a peculiar combi-
nation of a short run, a sudden jump of three to five
feet into the air, and a rapid uncoordinated flop and
scramble in the air, the bird usually alighting within
ten or twenty feet of the starting point, but turn-
ing so as to face at least at right angles, or even in
the opposite direction from which it started. When in
the air it emits a peculiar cacophonous call or cackle,
which, when heard at a distance, gives the impression
of a hearty burst of laughter. The purpose of these
semi-somersault-like maneuvers appeared to be to at-
tract the attention of other birds, possibly even as a
challenge, for frequently they seemed to precede the
somewhat pacific duels described above. The effect
of these sounds, together with the tooting calls, in
the mists which so often obtain in their habitat before
sunrise, is weird in the extreme. At 4:15 A.M., on
May 2, these sounds were practically continuous, with-
out appreciable interval, apparently from all directions. At 4:45 A.M. six birds could be counted, all in sight
at once. THE HEATH HEN. They appeared to resort to a particular, clear
space, of about two acres in extent, where the antics
just described were carried on. All the birds, except PINNATED GROUSE 225 one, were observed to have the orange-colored air-
sacs. These were probably cocks. We saw only one
bird which we suspected might be a hen. The other
hens were probably nesting, or at least had secured
mates, and no longer resorted to the promenading
place. As the sun rose high the tooting became less
frequent; the birds became more restless, often flying
to the neighboring
low oaks, resting there
until
disturbed.” William
Hazen Gates,
of Williamstown,
Mass.,
worked that spring with the Massachusetts commis-
sioners, studying the habits of the heath hen, in order
to secure information which might be of use in arti-
ficially propagating the species. He says:
“On May 31, while wandering across the plains, “On May 31, while wandering across the plains,
three heath hens were started, and each taking wing
flew nearly out of sight before alighting. As I watched
the birds, a call, resembling to a slight degree that of
an ordinary barnyard cock calling to the hens, was
heard not far distant. The place was noted as nearly
as possible, and then cautiously I made my way there. When the place was reached I looked for birds, but
could see none. I then sat down and determined to
wait, in order to see if any birds could be heard. The
ground was covered with leaves, so the least stir would
have been heard. I listened and also looked for signs
of anything moving, but none appeared. I sat there
for fully twenty minutes and, hearing nothing, con-
cluded that either there were no birds or else they had
gone as IT approached. As I rosea bird flew up within AMERICAN GAME BIRD SHOOTING 226 twenty feet of where I had been watching. The bird
had been within sight all the while, but probably had
crouched
in the leaves and remained
invisible. It
would have been interesting to note how much longer
the bird would have stayed in this position without
moving. Another bird was started some fifty feet
from this one. “On this same day the foots of one or more heath “On this same day the foots of one or more heath
hens were heard between half-past four and five a.m. THE HEATH HEN. The birds are early risers and late bed-goers. Once
they were heard to toot at 3:30 A.M., or about an hour
before sunrise; and several times their call note was
heard as early as this. It is probable, though, that
they do not begin to stir quite so early, beginning their
breakfasting about sunrise or a little earlier. The mid-
dle of the day is generally spent in the shade, or in dust-
ing in the sun in the roads. Late in the afternoon, as
the air begins to cool, they take to feeding again, and
can be seen in the open fields. They will often feed
till nearly an hour after sundown. I do not know
whether they roost in the low shrubbery or on the
ground at night. Mother birds with young, however,
stay on the ground, but it is likely that this is done
only while the young are too small to roost and need
the shelter of the mother. “On June 29 a bevy of heath hens was found. The
mother bird took flight, cackling, and flew some fifty
feet or so. The young scudded in every direction, and
were entirely out of sight by the time I reached the
spot. I hunted around through the leaves some, but PINNATED GROUSE 227 fearing that I might accidentally step on one, did not
search very carefully, and so did not see any. Two
days later, what I think must have been the same bevy
was again seen, but about half a mile from the place
where they were first seen. This time they were in a
more or less cleared space, and six of the young were
counted. One or two squatted just where they were,
and it looked as if one might go right up to them
and pick them up. I did not, however, disturb them. These birds were apparently not over a week old. “On July 2 a mother heath hen and four young were “On July 2 a mother heath hen and four young were
seen dusting in a road about 11 A.M. Upon seeing me
the mother ran to the bushes and called to the young. As I went by I could hear the mother hen at the side
of the road in the bushes. The same day in the after-
noon, a mother hen and one young bird were seen. THE HEATH HEN. “On July 7, while walking through the brush near “On July 7, while walking through the brush near
the Cromwell cottage, soon after sundown, I heard
some peeping ahead. Getting on my hands and knees,
I crawled toward the sound. The peeping continued as
I approached, so I knew that I had not been per-
ceived. Finally, at a distance of some twenty or
twenty-five feet, I saw a mother hen with wings spread
under the thick foliage of a stunted oak. She was
more or less silent, only occasionally uttering a low
call, somewhat resembling that of a hen as she calls
her chicks at night under her wings. The young, how-
ever, peeped quite often as they stole in and out from
under the wings of the mother. I think they could
not have been much more than a day or two old. AMERICAN GAME BIRD SHOOTING 228 Like the chicks of other fowls, they could not seem to
get settled for the night, but would stray in and out. Then as they sought a place of shelter again they
would shove one of their fellows out from under the
mother’s wing. However, as darkness grew the rest-
lessness ceased, and by the time it was too dark to
see the group everything was silent. How many there
were in the bevy I could not tell, but it seemed that
there must have been at least six or eight.” Though the number of these birds is so pitifully
small, yet, as already suggested, they pack in the same
way as does the western pinnated grouse. The report
of the Massachusetts commissioners on fisheries and
game for 1907 states that on January 11, 1908, a flock
was counted which contained not less than fifty-five
nor more than sixty birds. The same report declares
that the birds remained in these flocks until late in
February and began to utter their calls on the approach
of warm weather, and when this takes place the flocks
break up and the season for mating begins. The elabo-
rate performances earlier described begin about April
I, and end about the middle of June, being at their
greatest height the last of April and early part of May. The chicks are hatched in June and in July. When
about the size of quails they make long flights when
alarmed. THE HEATH HEN. Among experiments carried on by the Massachusetts
commission was the taking of a set of nine heath hen
eggs, which were placed under a bantam hen. Only
one of these eggs hatched, and the chick was at once PINNATED GROUSE 229 killed by the hen. The hen was subsequently given
some pheasant eggs, and hatched them and reared the
chicks with all possible care. That this bird possesses a high interest, not only
for the sportsman, but because it is now on the very
point of extinction, is obvious. Much has been said
and written about its preservation, but the credit for
setting on foot a movement which it is hoped may pre-
vent the extermination of the species is due to Mr. J. E. Howland. He urged the importance of the situation
on the Massachusetts commission, and a permanent
guardian was located in the region inhabited by the
birds to study their habits and enforce the law. Some-
what later, Representative Mayhew introduced a bill
into the General Court, placing under the use and
control of the commission such lands as may be do-
nated, leased, purchased or otherwise placed under
temporary or permanent control, as a refuge and breed-
ing area for the heath hen. A number of individuals
have subscribed money to enable the commission to take
advantage of this law, and about sixteen hundred acres
have been placed under special protection. The Massa-
chusetts legislature has authorized the commissioners
to take such unimproved lands upon Martha’s Vine-
yard—not exceeding one thousand acres—as they may
deem necessary for the purpose of making fire stops
for the protection from fire of the feeding and breed-
ing grounds of the pinnated grouse, or of otherwise
securing the maintenance and increase of such pinnated
grouse or of any other species of wild birds upon said AMERICAN GAME BIRD SHOOTING 230 islands. Two thousand dollars was appropriated for
work incidental to these purposes and for an inves-
tigation and reports upon the best methods and possible
cost of protecting and increasing the colonies of birds
on the island. This appropriation, and the authority
to take the land, ought to protect the heath hen from
a danger which has been of late years the most im-
portant element in reducing its numbers—the bush and
forest fires. The pinnated grouse of the West have
suffered from the same cause. THE HEATH HEN. An investigation by the commissioners on fisheries
and game of the problems involved in the preservation
of the heath hen leads them to believe that one or more
extensive areas, whether they be called reserves, sanc-
tuaries or refuges, should be acquired by the common-
wealth and patrolled and maintained. Such refuges
should include the chief breeding and feeding grounds
of the birds, and on the land should be sown crops of
clover and such cultivated grains as they delight to
feed on; that suitable fire stops or breaks should be
maintained in order to reduce the danger of bush fires,
so destructive to the birds in the past; that every pre-
caution should be taken against the contagious diseases
which might be transmitted to these wild birds through
domestic fowls; that as soon as the number of birds
is sufficiently increased, systematic artificial incubation,
feeding and breeding should be begun for the purpose
of rearing annually an increased number of birds. The
commissioners believe that by artificial propagation the
number of eggs laid may be increased, while it is PINNATED GROUSE 220 obvious that this means will lessen the loss resulting
from the destruction of nests, eggs and young birds
by natural enemies, whether mammals or birds. They
believe that the expenditure involved would be prac-
tically that incidental to ordinary poultry raising, ex-
cept that on account of the hardiness and vigor of the
grouse it would need little or no winter shelter. The commissioners, in their report for the year 1907, The commissioners, in their report for the year 1907,
report contributions from private individuals of not
less than
$2,420 for the purpose of preserving these
birds. Of this sum, the town of Tisbury and the Mid-
dlesex Sportsmen’s Association made the largest con-
tributions of $200 each, but there are a large number
of subscribers, and the list ought to be largely added
to, since every sportsman and every naturalist in the
land should be ambitious to have some share in the
good work of re-establishing this splendid bird. THE PRAIRIE HEN. This is the form of pinnated grouse that has been
known to sportsmen. Its range was chiefly in the
Mississippi Valley, including Minnesota, Michigan,
western Ohio, Kentucky, but perhaps not Tennessee,
though it was found in Louisiana, southern Texas,
Indian Territory, Kansas, Nebraska and the eastern
parts of both Dakotas. It is now found in Manitoba,
southeastern Saskatchewan, to eastern Colorado, north-
eastern Texas, Arkansas, western Kentucky and In-
diana and intermediate regions, where it was at one AMERICAN GAME BIRD SHOOTING 232 time enormously abundant, and, being exceedingly
tame at certain seasons of the year, was very readily
destroyed. Throughout almost all this region the bird was resi-
dent, though in the northern portion it regularly made
seasonal changes of location, which, though commonly
called migratory, hardly deserve to be so character-
ized. On this point Mr. W. W. Cooke, in his report
on “Bird Migration in the Mississippi Valley,” says:
“The prairie chicken is commonly said to be a resi- “The prairie chicken is commonly said to be a resi-
dent bird, and so it is in a larger part of this range,
but in Iowa a regular though local migration takes
place. This has been mentioned by former writers, and
in the spring of 1884 a special study was made of the
matter. Many observers unite in testifying to the facts
in the case, and, what is still more important, there is
not a dissenting voice. One of the observers does not
exaggerate when he says: ‘Prairie chickens migrate as
regularly as the Canada goose.’
Summing up all the
information
received,
the facts of the case
are
as
follows:
In November and December large flocks of
prairie chickens come from northern Iowa and southern
Minnesota to settle for the winter in northern Missouri
and southern Iowa. This migration varies in bulk with
the severity of the winter. “During an early cold snap immense flocks come
from the northern prairies to southern Iowa, while in
mild, open winters the migration is much less pro-
nounced. During a cold, wet spring the northward
movement in March and April is largely arrested on “During an early cold snap immense flocks come
from the northern prairies to southern Iowa, while in
mild, open winters the migration is much less pro-
nounced. THE PRAIRIE HEN. During a cold, wet spring the northward
movement in March and April is largely arrested on PINNATED GROUSE 233 the arrival of the flocks in northern Iowa, but an
early spring with fair weather finds them abundant
in the
southern
tiers
of counties
in Minnesota,
and many
flocks
pass
still
farther
north. The
most remarkable feature of this movement is found
in the sex of the migrants. It is the females that
migrate, leaving the males to brave the winter’s cold. Mr. Miller,
of Heron Lake,
Minn.,
fairly
states
the case when he says:
‘The females in this latitude
migrate south in the fall and come back in the spring,
about one or two days after the first ducks; and they
keep coming in flocks of from ten to thirty for about
three days, all flying north. The grouse that stayed
all winter are males.’ ” Audubon noticed and spoke of these movements
nearly a hundred years ago, for in his account of this
species he says: “During the first years of my residence at Hender-
son (Kry.), in severe winters, the number of grouse of
this species was greatly augmented by large flocks of
them that evidently came from Indiana, Illinois and
even from the western side of the Mississippi. They
retired at the approach of spring.”
When John James Audubon first lived in Kentucky, When John James Audubon first lived in Kentucky,
the “Barrens”—by which is meant open stretches of
land without timber—swarmed with these birds, and
they were looked on more or less as a pest. They were
credited with committing much mischief among the
fruit trees of the orchards in winter, and in the spring
they fed on the grain in the new-sown fields. They AMERICAN GAME BIRD SHOOTING 234 were so abundant as to be caught in pens and traps,
and any one could kill as many as he wished. Indeed,
Audubon speaks of a friend who was fond of practicing
rifle shooting, who killed upward of forty in one morn-
ing and did not pick them up. Twenty-five years later Audubon speaks of them as Twenty-five years later Audubon speaks of them as
at that time not being found in any numbers east of
the State of Illinois, and says that there, too, they are
decreasing at a rapid rate. THE PRAIRIE HEN. At the approach of spring the large packs, which At the approach of spring the large packs, which
have held together during the winter, break up into
smaller companies of from twenty to fifty, and before
long—in March or April—the mating begins. This
has been spoken of by many writers as the booming
of the prairie chicken, or the dance of the prairie hen,
though this last term is more commonly applied to the
spring maneuvers of the sharp-tailed grouse. This
mating has been described in a somewhat spectacular
manner by Audubon, but recent observers have not seen
such fierce encounters as he describes. An excellent
account of this mating play was printed in Forest and
Stream many years ago by Judge John Dean Caton,
an early settler of Illinois, who had been familiar with
these birds for almost all his life. He says: “The spring of the year is the season of courtship
with them, and it does not last all the year round, as
it does with humans, and they do it in rather a loud
way, too; and instead of taking the evening, as many
people are inclined to do, they choose the early morn-
ing. Early in the morning you may see them assemble PINNATED GROUSE 235 in parties, from a dozen to fifty together, on some
high, dry knolls, where the grass is short, and their
goings on would make you laugh. The cock birds have
a loose patch of naked yellow skin on each side of their
neck just below the head, and above these on either
side, just where the head joins the neck, are a few long
black feathers, which ordinarily lie back on the neck,
but which, when excited, they can pitch straight for-
ward. These yellow naked patches on either side of the
neck cover sacs which they can blow up like a bladder
whenever they choose. These are their ornaments,
which they display to the best advantage before the
gentler sex at these love feasts. This they do by blow-
ing up these air-sacs till they look like two ripe oranges,
on each side of the neck, projecting their long, black
ears right forward, ruffling up all the feathers of the
body till they stand out straight, and dropping their
wings to the ground like a turkey cock. THE PRAIRIE HEN. Now they look
just lovely, as the coy, timid maidens seem to say, as
‘they cast side glances at them, full of admiration and
love. “Then it is that the proud cock, in order to complete
his triumph, will rush forward at his best speed for
two or three rods through the midst of the lovesick
damsels, pouring out as he goes a booming noise, al-
most a hoarse roar, only more subdued, which may be
heard for at least two miles in the still morning air. This heavy booming sound is by no means harsh or
unpleasant; on the contrary, it is soft and even har-
monious. When standing in the open prairie at early AMERICAN GAME BIRD SHOOTING 236 dawn, listening to hundreds of different voices, pitched
on different keys, coming from every direction and
from various distances, the listener is rather soothed
than excited. If this sound is heavier than the deep
keynotes of a large organ, it is much softer, though
vastly more powerful, and may be heard at a much
greater distance. One who has heard such a concert
can never after mistake or forget it. “Every few minutes this display is repeated. I
have seen not only one, but more than twenty cocks
going through this funny operation at once, but then
they seem careful not to run against each other, for
they have not yet got to the fighting point. After a
little while the lady birds begin to show an interest in
the proceedings by moving about quickly, a few yards
at a time, and then standing still a short time. When
these actions are continued by a large number of birds
at a time, it presents a funny sight, and you can easily
think they are moving to the measure of music. “The party breaks up when the sun is half an hour
high, to be repeated the next morning and every morn-
ing for a week or two before all make satisfactory
matches. It is toward the latter part of the love season
that the fighting takes place among the cocks, probably
by two who have fallen in love with the same sweet-
heart, whose modesty prevents her from selecting be-
tween them.” Audubon reports that he tried the experiment of
puncturing the inflatable air-sacs on the neck of a male
prairie chicken. THE PRAIRIE HEN. He caught one of the birds and passed PINNATED GROUSE 237 the point of a pin through each sac, and found that
thereafter it was unable to toot. He performed this
same experiment with another bird on one side only,
and found that the next morning it uttered the tooting
sound with the uninjured air-sac, but could not inflate
the one that had been punctured. He states that his
efforts to decoy this species by imitating its curious
sounds were unsuccessful, ‘‘although the ruffed grouse
is easily deceived in this manner.”
After the close of the mating operations the locations After the close of the mating operations the locations
of the nests are selected. Often they may be in hedges
and the margins of clumps of underbrush, in fence cor-
ners or along the borders of sloughs, but often, again,
in the middle of a field amid the tall grass. The eggs
number from eleven to fourteen, and sets of twenty
or even twenty-one eggs are not unknown. They vary
in color from cream to light olive or pale brown, and
are often regularly spotted with fine pin-points of
reddish brown. Captain Bendire regards the prairie
chicken as one of the most prolific of our game birds. Now, however, comes the season of danger; the eggs Now, however, comes the season of danger; the eggs
have been deposited in a slight depression, scratched
out among the weeds or grass, and the hen begins to
brood. If she has nested early and the season is late,
the streams may rise and flood her nest and destroy
the eggs or drown the tiny young, if they have already
hatched ; or early prairie fires, burning among the dead
grass and weeds of the preceding season, may destroy
mother and clutch alike; or, later still, the mowing
machine may kill the mother or the young, too small AMERICAN GAME BIRD SHOOTING 238 to fly and too inexperienced to force themselves through
the thick grass away from the approaching danger. In old times it used to be said that in wet seasons thou-
sands and thousands of prairie chickens’ nests were
ploughed under when the fields were being prepared
for grain. Certain it is that the combination of al! these dangers, together with the insatiate gunner, at
one time came very near exterminating the pinnated
grouse from the States of Illinois and Indiana. THE PRAIRIE HEN. If the mother bird is fortunate enough to bring off
her young, she leads them about much as do other
grouse, to the best feeding grounds. She is watchful
of danger for them, and at her warning cry the young
squat on the ground, which they so closely resemble
that it is almost impossible to find one of them. The
mother uses every art to lead the intruder away from
the brood. The birds grow rapidly, and by the middle
of August—the date at which up to within a few years
it has been legal to shoot them—are nearly two-thirds
grown. They are then very easily killed, and the
sport becomes mere butchery. When cold weather ap-
proaches, however, they grow stronger of wing, and
soon after this pack. Audubon was perhaps the first to announce that Audubon was perhaps the first to announce that
the pinnated grouse is easily tamed and easily kept. He declares also that they breed in confinement. A
number that he had while at Henderson were turned
loose in his garden and orchard, and within a week
became so tame as to allow him to approach them. They readily ate corn and vegetables, became so gentle PINNATED GROUSE 239 during the winter as to feed from the hand of his wife,
and altogether acted as domestic poultry might act. In spring they went through the operations of mating
just as did their wild brethren, and a number of them
hatched, but at last the birds proved so destructive in
the garden that they were ordered to be killed. nother page, birds sent to Eng- As will be seen on another page, birds sent to Eng-
land became quite tame, and many years ago I had a
dozen of these grouse in New York, which, when turned
out in the spring, so readily accustomed themselves to
their surroundings that they followed a man who was
spading the garden and scratched and crowded over the
freshly turned-up earth in search of insects. They were
less wild than so many domestic hens. its ways, the pinnated grouse suggests a In many of its ways, the pinnated grouse suggests a
domestic fowl. Though often carrying its tail droop-
ing toward the ground, it often carries it upright, as
a hen carries her tail. THE PRAIRIE HEN. The mother of a young brood
will fight for it, or at least will try to frighten away
an intruder. The young chicks constantly talk to each
other as they move along, and if one of them discovers
an insect and runs after it all those within sight join
in the pursuit. pson, in his “Birds of Manitoba,” Mr. E. E. Thompson, in his “Birds of Manitoba,”
points out that, while it was only in 1881 or 1882 that
the pinnated grouse was found in Manitoba, before
1890 it had become common at many points, such as
Winnipeg, Portage la Prairie and other localities. Pre-
vious to this, in 1872, Dr. Coues had written: AMERICAN GAME BIRD SHOOTING 240 “T have no reason to believe that it occurs at all in
northwestern Minnesota or North Dakota.” In Manitoba they seem more or less to associate with
the sharp-tailed grouse, so much so that sometimes birds
of both species will be started from before the dog. When winter comes, however, and the sharp-tailed
grouse go into the woods, the pinnated grouse stay out
on the prairie. LESSER PRAIRIE HEN. The range of the lesser prairie hen has already been
SIVED
iy" While riding over the prairie through the western
part of the Indian Territory in the month of March,
I have more than once driven for hours through flocks
of these birds, contentedly feeding on the prairie and
wholly disregarding the wagon, which passed close
to them. On these occasions we must have seen many
thousands of the birds, and it is probable that imme-
diately after this the packs broke up and the mating
season began. The pursuit of this form differs in no respect from
that of its slightly larger relative to the North and
Fast. The most southern range in Texas of Attwater’s
prairie hens is given as just north of Fort Brown, near
the coast, and it is very abundant south of San Antonio. 241 PINNATED GROUSE THE PINNATED GROUSE TO-DAY. An inquiry made in 1906 among the game commis-
sioners of States where prairie chickens were formerly
very abundant brought out a number of replies of
great interest. Mr. E. E. Earle, then chief deputy
warden of the State of Indiana, wrote to Forest and
Stream as follows:
“Our supply of pinnated grouse decreased rapidly “Our supply of pinnated grouse decreased rapidly
from year to year until 1901. Prior to that time the
open season had run from September 1 of any year to
February 1 of the succeeding year. Under this law
large numbers of these birds were slaughtered every
year, they being young, not gun shy, and easily found. “In 1901 a law was passed prohibiting the killing of “In 1901 a law was passed prohibiting the killing of
pinnated grouse, or possession of same, at any time
between January 1 and November 10, and prohibiting
export of such birds. Under the provisions of this
act, which was rigorously enforced, pinnated grouse
have increased in numbers, and may be found in great
droves on our prairies and marshes. “T was in Porter County last March, and one duck “T was in Porter County last March, and one duck
hunter told me of having seen in one flock what he
estimated to have been one hundred prairie chickens,
and such scenes are by no means rare. Wise laws and
strict enforcement of same will increase them in any
country that is suitable for their habitation.”
Illinois, in the youth of men who are now elderly, Illinois, in the youth of men who are now elderly,
was the great chicken ground of what used to be called AMERICAN GAME BIRD SHOOTING 242 the West, but the birds were so persecuted that a few
years ago it was supposed that this grouse was nearly
extinct there. Within the last few years, however, a
great change is reported. State Game Commissioner
John A. Wheeler wrote: “My deputy game wardens throughout the State re-
port prairie chickens, pinnated grouse, rapidly increas-
ing. Our deputy wardens in Wayne County report
3,000 birds in that county by actual count. In Sanga-
mon County, from personal observation and reports
from the deputy wardens, I am convinced that we
have nearly that many birds. THE PINNATED GROUSE TO-DAY. From all over the State
we are receiving encouraging reports of the increase of
prairie chickens.” Almost
the northwestern
limit
of the pinnated
grouse’s range is western Minnesota, and of this coun-
try Mr. S. F. Fullerton, then the executive agent of
Minnesota’s board of game and fish commissioners,
reported interestingly. It is obvious that unless the
cultivation of the land is such as to provide food for
the pinnated grouse they will not do well there, and the
character of Minnesota farming, which is largely dairy-
ing on small farms, is not such as to encourage occu-
pancy by the pinnated grouse. In two letters, Mr. Fullerton said:
“The pinnated grouse, or prairie chicken, is disap- “The pinnated grouse, or prairie chicken, is disap-
pearing from a large section of our State. It cannot
stand civilization, but it is very odd that in new por-
tions of the State that have been opened up they are PINNATED GROUSE 243 very plentiful, and we have sections in Minnesota where
the pinnated grouse are just as thick as they ever were. “The State game warden of Manitoba states that “The State game warden of Manitoba states that
their prairie chickens are not disappearing at all; in
fact, they are increasing under rigid protection and
stopping the sale. That is only across an imaginary
line of Minnesota and Dakota, where these birds are
plentiful, but in the southern part of our State, where
dairying has taken the place of grain-raising, there are
hardly any of the birds left. The sharp-tailed grouse,
however, are different, as they are found in the brush
country in great numbers where farms are opened up. “A very pleasing thing happened to me last March. “A very pleasing thing happened to me last March. I was up in the northern part of the State, and in a
drive of three hours I came across a stretch of land that
had been cleared of jack pine. The clearing was several
miles in extent, but it was surrounded by jack pine. The snow at the time was over two feet deep. The
land last year was cultivated, some corn grown on it
and some wheat and other coarse grain. In that clear-
ing, the man with me and I counted over two hundred
pinnated grouse. THE PINNATED GROUSE TO-DAY. They appeared to have wintered
finely and seemed in good condition.”
When we reach Nebraska, we approach the limit of When we reach Nebraska, we approach the limit of
the pinnated grouse and enter the region of the sharp-
tail just as we do in Minnesota. Both species were
formerly abundant in Nebraska, but were so overshot
by thoughtless gunners and by market shooters that
they became rare. Mr. G. L. Carter, then chief warden AMERICAN GAME BIRD SHOOTING 244 of Nebraska’s game and fish commission, wrote fully
about birds in Nebraska:
“We have both the pinnated and sharp-tailed grouse, “We have both the pinnated and sharp-tailed grouse,
and we are so proud of them that it is a pleasure for
me to tell you about them. The pinnated grouse are
found more abundantly in the central and eastern parts
of the State, while the sharp-tailed grouse are found in
the northwest portion. “A peculiar thing is that we seldom find a sharp- “A peculiar thing is that we seldom find a sharp-
tailed grouse south of the Platte River; when we do, it
is late in the season. The breeding and rearing grounds
are principally in the northern and western sections,
but a few are raised in other parts of the State. “During the winter months, on account of scarcity “During the winter months, on account of scarcity
of food in the north part of the State, which is prin-
cipally grazing country, the birds are driven farther
south, usually along the Platte River valley, some-
times going as far south as the northern counties of
Kansas. “A few days ago, while going from this city to
Omaha, I saw a bunch of perhaps 100 pinnated grouse
within eight miles of the Omaha city limits. It is
only through our effective game laws, passed during
the winter of 1901, that we have these birds to any
extent. Prior to that time they were being slaughtered
by the market hunters from everywhere and shipped
to the market. We were able to convince the Legis-
lature of 1901, that there had been shipped out of this
State 235,000 of these birds during the year 1goo. We have had this shipping stopped, and, as a result, PINNATED GROUSE 245 ranchmen and farmers throughout the State report
birds more plentiful than at any time during the past
fifteen years. THE PINNATED GROUSE TO-DAY. “Tf we take care of these birds, we will have them
for a great many years to come, as we have bound-
less acres of territory which will never be cultivated,
and which afford splendid breeding grounds for them.” Missouri is a State where in the past there have
been many pinnated grouse, and from this State also
we have a good report as to an abundant stock of birds
which might easily be increased by proper care. State
Game Warden J. H. Rodes’ account of things will be
read with interest: “Tn twenty-five, if not more, of the 114 counties in
this State, we have prairie chickens remaining in
greater or less quantities. Originally, as you know,
throughout the prairie districts of this State, these
birds abounded in very liberal supply, but having had
absolutely no protection up to and prior to the enact-
ment of the present game and fish laws, which went
into effect on June 15, 1905, they were industriously
hunted almost to the point of extermination, and were
wholly killed off in many counties where they had
formerly been found abundant. “Tt was a common practice—notwithstanding we “Tt was a common practice—notwithstanding we
had a statute prohibiting it—to begin shooting these
birds after July 1, clear on and through the hunting
season, when and wherever they could be found. Now
that they are protected during the entire year, except
from November 15 to December 15, we hope they will AMERICAN GAME BIRD SHOOTING 246 multiply and re-establish themselves in considerable
number. To illustrate:
This county, Pettis County,
which is little larger, perhaps, than the average size
county and fairly densely populated—Sedalia alone,
the county seat, having a population of something over
15,000
inhabitants,
and many
more
average-sized
towns—has yet remaining in it, I would say, five or
six hundred birds. “Of course, it is difficult to estimate even approxi-
mately the number of birds remaining, yet it is no un-
common sight to see flocks of ten to twenty-five of
these birds in the larger pastures and cornfields. A
very reliable person told me the other day that he saw
in the western part of our county a drove of about forty
birds. Doubtless this was an accumulation of several
flocks that were feeding together. THE PINNATED GROUSE TO-DAY. “While, as stated, this county is thickly populated,
there yet remain many large pastures on which the
virgin sod has never been broken, being used as pasture
lands, and perhaps some as large as a thousand acres
or more. They breed in these pastures and meadows
and feed in adjacent oat and corn fields during the sum-
mer season. They were seen in unusually large num-
bers this winter, which I can but attribute to the fact
that special effort was made to protect these birds dur-
ing their breeding season last year. “We were fortunate enough to secure one or two “We were fortunate enough to secure one or two
early convictions for hunting them out of season, and
the gunners took alarm and very few were killed. What is true in this county, is true in many other coun- PINNATED GROUSE 247 ties where like conditions exist. We intend to make
these birds the object of special care and supervision
in the hope that they may multiply and yet be seen in
large quantities in this State. They should be pro-
tected during every month of the year for a period of
five or six years, but the truth is that very few of
the birds can be killed during the thirty days of the
open season, as they are then full grown and are very
strong, and will not lie before a dog and are very
shy.” Kansas is another State where the prairie grouse was
formerly abundant, but here the pinnated grouse is
always the common bird, and the sharp-tail the ex-
ception. Mr. D. W. Travis, State fish and game war-
den, gave in brief but very telling form the history of
the wholesale destruction and rapid decrease of prairie
chickens until about ten years ago, and then of the
change of sentiment and an increase of the birds during
the year 1905. Mr. Travis says: “Up to the year 1885 pinnated grouse were very
plentiful in Kansas and especially so in the western
part. From that time they decreased very rapidly,
until about 1900. Between those dates a grouse was
seldom seen in the eastern half of the State, and but
very few in the western half. The decrease was caused
by the late burning of the prairies all over the western
half of the State. THE PINNATED GROUSE TO-DAY. Settlers were filling this section rap-
idly, and it seemed to be the general opinion that all
dead grass should be burned, which destroyed all food
and insects, starving the birds out and leaving no nest- AMERICAN GAME BIRD SHOOTING 248 ing grounds. Again, meat was scarce and high, and
the settlers killed grouse the year round in order to
supply their tables, and also killed thousands and sent
them to eastern markets. This, with the hundreds of
eastern market hunters, caused the almost complete ex-
termination of the grouse in Kansas. “About 1900, the people began to realize that the “About 1900, the people began to realize that the
grouse were nearly exterminated, and a crusade for
their protection started. Stringent laws were passed
in 1903, and still more severe ones in 1905, and I am
pleased to state that in the fall of 1905 in a number
of the central counties the birds have increased to num-
bers beyond expectations, and to-day the grouse can
be found in many of the eastern counties. With proper
protection, pinnated grouse will be abundant in all
parts of the State within five years. The day of the
game butcher, the pot and market hunter is past.” Texas is now and always has been a State where
pinnated grouse were abundant, and it is so large that
there are
still great numbers of birds there. The
growth of the game protective sentiment, which has
been so marked within the last year or two, promises
to protect these birds. There are other States, as Michigan and Wisconsin,
that have a few pinnated grouse, and the bird occurs
rarely in the Province of Ontario. Prof. Walter B. Barrows, of the Michigan Agri- Prof. Walter B. Barrows, of the Michigan Agri-
cultural College, whose writings on ornithological top-
ics are familiar to many of our readers, says:
“This bird was formerly fairly common over the “This bird was formerly fairly common over the PINNATED GROUSE 249 southern half of the Lower Peninsula, and up to twenty
years ago was abundant in many places in the prairie
regions of the three southernmost tiers of counties. Even ten years ago it was not uncommon in this county
(Ingham), and undoubtedly a few still exist here. THE PINNATED GROUSE TO-DAY. I
am not able to say positively where it is most abun-
dant now, but my latest reports, some two and three
years ago, indicated that there were still a good many
in Calhoun, Kalamazoo and Van Buren counties, and
it is fair to presume that they were locally common in
the three
counties
south
of those
and _ bordering
Indiana. “There are vague Michigan accounts of the presence
of this species along the Lake Michigan shore as far
north as Traverse City, fifty years ago, but I have no
reason to believe that the species has ever been common
north of the Saginaw Grand Valley in about latitude
43%°. The sharp-tailed grouse may at one time have
existed in the upper part of the Lower Peninsula and
in parts of the Upper Peninsula, but at present it is not
known to occur except in Isle Royale in Lake Superior,
where there appears to be a flourishing colony, but as
yet I have been unable to obtain specimens so as to
determine positively the sub-species.”’ It would seem perfectly practicable to reintroduce
this bird in eastern localities where once it was abun-
dant, but this of course could be done only in situations
where the bird would be sure to be protected; in other
words, in large preserves. It is conceivable that the
birds might live and do well on some of the large AMERICAN GAME BIRD SHOOTING 250 estates of Long Island, and even on the Shinnecock
Hills, but it is hardly to be hoped that they could ever
be brought back as a game bird. They would have to
be regarded as merely beautiful natural objects. SHARP-TAILED GROUSE. Pediecetes phasianellus. Pediacetes phasianellus columbianus. Pediecetes phasianeilus campestris. Pediecetes phasianellus. Pediacetes phasianellus columbianus. Pediecetes phasianeilus campestris. The sharp-tailed grouse, while about the same size
as the pinnated grouse, is a somewhat plumper and
stouter bird, and, from the fact that its tail is smaller,
does not seem so large. The tail of the sharp-tail is
graduated, the middle pair of feathers projecting much
beyond the rest; it has no tufts of feathers on the side
of the neck. It has a high crest, is brownish or rusty
above, varied with spottings and barrings of black and
darker brown. Large round white spots mark the
wing coverts, and the scapulars are somewhat streaked
with white, while the outer webs of the quills of the
wing are spotted with white. The lower parts are for
the most part white, varied with V-shaped marks of
dusky on ‘front and sides of breast. The female is
similar, but a little smaller. The differences between
the three forms consist chiefly in the shades of colors. The northern sharp-tailed grouse (Pediecetes pha-
sianellus—Linn.) is very dark-colored, with the dark
markings on the upper parts very heavy, while the
white marks on the wings show out strongly against
this dark color. The feathering on the legs is dark The northern sharp-tailed grouse (Pediecetes pha-
sianellus—Linn.) is very dark-colored, with the dark
markings on the upper parts very heavy, while the
white marks on the wings show out strongly against
this dark color. The feathering on the legs is dark
251 251 251 AMERICAN GAME BIRD SHOOTING 252 brownish gray. In length it varies from about 19
inches for the largest males to about 15 inches for the
smallest females, and the wing measures from 8 to 8}
inches. Most of the tail feathers are extremely stiff,
pointed and white-edged. This northern bird is found in central Alaska, and lf
7,
WETEIS Wy
eee Dy. oy;
Wee atneee eerie:
efi
SHARPTAILED
GROUSE SHARPTAILED
GROUSE in the interior of British Provinces, north as far as
Great Slave Lake, south as far as Moose Factory,
Lake Winnipeg, Temiscaming, the northern shore of
Lake Superior and the east side of James’ Bay, in
Labrador. It is never seen in the United States. The western
sharp-tailed
grouse,
or Columbian The western
sharp-tailed
grouse,
or Columbian SHARP-TAILED GROUSE 253 sharp-tailed
grouse
(P. p. SHARP-TAILED GROUSE. columbianus)
is very
much paler in color, being grayish or clay color and
marked with black, but with the black marks less sharp
and strong. It is found in central British Columbia
and central Alberta, south in the western United States
as far as northern California, Nevada and Utah, east
to the border of the plains in Colorado. Its range is
chiefly west of the Rocky Mountains. It is slightly
smaller than the northern form. The more familiar sharp-tail of the Middle West
(P. p. campestris)
is abundant on the plains from
southern Manitoba and southern Alberta, south through
the United States to Wyoming and Kansas, east as far
as Wisconsin and Illinois, and west to eastern Colo-
rado. It is bright rusty in color and its dark mark-
ings are much less conspicuous than in the northern
form. The sharp-tailed grouse, which of late years has
come to be known over much of the West as prairie
chicken, is thus—in one or other of its three forms—
a bird of wide distribution. It is found from Kansas,
on the south, to central Alaska on the north, and from
British Columbia, California and Nevada, on the west,
to James’ Bay on the east. It occurs, as said, sparsely
south of the Great Lakes, but in the United States—
except
in this locality—its
range
is chiefly west
of the Mississippi River. While during the greater
part of the year it seems to be a bird of the prairie, it is
yet often found high up in little mountain valleys, and
often in a country that is completely wooded. AMERICAN GAME BIRD SHOOTING 254 Like many others of our best game birds, the sharp-
tailed grouse has been so unremittingly pursued that
it is rapidly becoming more and more scarce, and
promises before long, in all regions where it is pursued
with dog and gun, to become as rare as its relative,
the pinnated grouse. In habits the birds are all closely alike, except that In habits the birds are all closely alike, except that
we may assume that the northern form has modified
its habits in accordance with its environment. Mr. Roderick MacFarlane found this species breeding in
1884 near Fort Providence. The two last-named forms
of the sharp-tailed grouse—which is also called white
belly, speckle belly, willow grouse and pin tail—are
common all through the northwestern United States. SHARP-TAILED GROUSE. They are birds of the open land, yet at certain seasons
of the year resort commonly to willows or brushy
ravines, from which sometimes they get up in a thick
flock, like a brood of gigantic quail. EK. E. Thompson, writing of the prairie sharp-
tailed grouse
in Manitoba,
describes
its prenuptial
dancing in the following language:
“After the dis-
appearance of the snow, and the coming of warmer
weather, the chickens meet every morning at gray dawn
in companies of from six to twenty on some selected
hillock or knoll, and indulge in what is called ‘the
dance.’
This performance I have often watched. It
presents the most amusing spectacle I have yet wit-
nessed in bird life. At first, the birds may be seen
standing about in ordinary attitudes, when suddenly
one of them lowers its head, spreads out its wings SHARP-TAILED
GROUSE 255 nearly horizontally and its tail perpendicularly, distends
its air-sacs and erects its feathers, then rushes across
the ‘floor,’ taking the shortest of steps, but stamping its
feet so hard and rapidly that the sound is like that of a
kettle drum; at the same time it utters a sort of bub-
ling crow, which seems to come from the air-sacs,
beats the air with its wings, and vibrates its tail, so
that it produces a low, rustling noise, and thus con-
trives at once to make as extraordinary a spectacle of
itself and as much noise as possible. “As soon as one commences
all join in, rattling, “As soon as one commences
all join in, rattling,
stamping, drumming, crowing and dancing together
furiously ; louder and louder the noise, faster and faster
the dance becomes, until at last, as they madly whirl
about, the birds are leaping over each other in their
excnement,
After a/\brief spell) the: enersy! of, the
dancers begins to abate, and shortly afterward they
cease and stand or move about very quietly, until they
are again started by one of their number ‘leading off.’ “The whole performance reminds one so strongly
of a Cree dance as to suggest the possibility of its
being the prototype of the Indian exercise. The space
occupied by the dancers is from 50 to 100 feet across. ... The dancing is indulged in at any time of the
morning or evening in May, but it is usually at its
height before sunrise. SHARP-TAILED GROUSE. Its erotic character can hardly
be questioned, but I cannot fix its place or value in the
nuptial ceremonies. The fact that I have several times
noticed the birds join for a brief set-to, in the late
fall, merely emphasizes its parallelism to the drumming AMERICAN GAME BIRD SHOOTING 250 and strutting of the partridge, as well as the singing
of small birds.” I have several times witnessed this dancing in No-
vember, just about the time that cold weather sets in,
and have seen it carried on for two hours, immediately
after sunrise, but have never been so fortunate as to get
close enough to the birds to hear their stamping sound
like a kettle drum. In the dances I have witnessed,
there was heard not only the crowing, of which Mr. Thompson speaks, but also a sharp, high-pitched cackle,
each note being separated from the other by a percep-
tible interval. Some years ago I sent Major Bendire some notes
on this species, which I quote here:
“This species is
partly migratory, and there is the very greatest dif-
ference in the habits of the birds in summer and winter. As soon as the first hard frosts come in the autumn, the
birds seem to take to the timber and begin to feed on
the buds of the willow and the quaking aspen. At this
time they spend a large portion of their time in the
trees, and are very wild. In the Shirley Basin, in
western Wyoming, a locality where I have never seen
any of these birds in summer, they are abundant in
winter. At this season they live in quaking aspen
thickets, along the mountains, and there I have seen
hundreds of them roosting on top of a big barn, which
stands just at the edge of a grove of quaking aspen
timber. It was always easy in the morning, just after
sunrise, to step out of the house, and with a .22 caliber Some years ago I sent Major Bendire some notes
on this species, which I quote here:
“This species is
partly migratory, and there is the very greatest dif-
ference in the habits of the birds in summer and winter. SHARP-TAILED GROUSE. As soon as the first hard frosts come in the autumn, the
birds seem to take to the timber and begin to feed on
the buds of the willow and the quaking aspen. At this
time they spend a large portion of their time in the
trees, and are very wild. In the Shirley Basin, in
western Wyoming, a locality where I have never seen
any of these birds in summer, they are abundant in
winter. At this season they live in quaking aspen
thickets, along the mountains, and there I have seen
hundreds of them roosting on top of a big barn, which
stands just at the edge of a grove of quaking aspen
timber. It was always easy in the morning, just after
sunrise, to step out of the house, and with a .22 caliber ‘aye]q
S,uoqnpny
wo1g
"asnoig pareydieys ‘ay
S,uoqnpn
wo1g
"asnoig pareydieys SHARP-TAILED
GROUSE 257 rifle shoot off the heads of as many of these birds as
were needed for eating for the next two or three days. “T have only one note on these birds which seems “T have only one note on these birds which seems
particularly worth mentioning, and of this I spoke in
my report to Colonel William Ludlow, on the birds
noticed during a reconnaissance to the Black Hills of
Dakota in 1874, which was published by the Engineer
Bureau of the War Department. The sharp-tailed
grouse has a cry which is unlike that of any other
grouse with which I am familiar, although something
very similar has been observed in the case, I think, of
one of the ptarmigans. On the plains of Dakota in
1874, having scattered a brood of sharp-tailed grouse,
consisting of a mother and a dozen well-grown young,
I sat down to wait for them to get together. The
mother had flown to the top of a hill not far off, where
she sat on the ground in plain sight, and after a few
moments began to call to the young, which immediately
answered her from the different points where they had
taken refuge. The call of the mother and the young
was a guttural, raucous croak, which quite closely re-
sembled the croaking of a raven at a little distance. SHARP-TAILED GROUSE. I plainly saw the old bird utter its note, and subse-
quently followed up the calls uttered by more than
one of the young ones, until I started them, and killed
one or two as they flew.”
In winter the food of the sharp-tailed grouse consists In winter the food of the sharp-tailed grouse consists
largely of rose-berries and the buds of willows, cotton-
woods and aspens. In summer and autumn, grass-
hoppers, insects and various berries, together with AMERICAN GAME BIRD SHOOTING 258 grass and the leaves of various plants, form their
chief food. I have seen them feeding by hundreds
in the alfalfa patches of the ranchmen, and have found
their crops and throats stuffed with the green leaves,
together with a few of the seeds. Almost everything
in the nature of berries, insects, seeds and green leaves
is devoured by this bird. In the winter and autumn, the sharp-tailed grouse In the winter and autumn, the sharp-tailed grouse
inhabiting a prairie country, spend most of their time
in the river bottoms, among or close to the willow and
cottonwood trees, on the buds of which they feed at
this season, and it is not uncommon to see large flocks
of them roosting among the branches of these trees
in the early morning, apparently too much chilled to
notice the approach of man. Mr. E. E. Thompson, in his “Birds of Manitoba,”
describes in some detail the habits of the sharp-tailed
grouse in winter. He says that it spends the winter
nights in the snow, which is always soft and penetrable
in the woods, though out on the plains it is beaten into
drifts of ice-like hardness. “As the winter wanes, it is not uncommon for a snow
storm to be accompanied by sleet. The storm always
drives the chickens into the drifts, and afterward levels
the holes they formed in entering. The freezing of the
sleet then forms a crust, which resists all attempts at
escape on the part of the birds, many of which, accord-
ing to the account of hunters, are starved and thus
perish miserably. I met with a single instance of this
myself. SHARP-TAILED GROUSE 259 “Before the winter is over many of the birds, by con-
tinuously pulling off frozen browse, have so worn their
bills that when closed there is a large opening right
through, immediately behind the hook. SHARP-TAILED GROUSE. Early in April
the few that have survived the rigors and perils of their
winter life spread over the prairie once more and soon
scatter to enter on their duties of reproduction.” No one has written about this bird more charmingly
than Dr. Elliott Coues, whose article on the prairie
form is well worth quoting. He first heard the call of
the sharp-tailed grouse in North Dakota, when he was
alone in camp, not far from Fort Randall—at the time
his home—where he had gone to shoot water fowl. He
says: “Awakened before it was light by the sonorous cries
of the wild fowl making for the reedy lake where I had
encamped, I arose—there was no need to dress—pushed
off into the expanse of reeds in a light canoe I had
brought with me, and with my gun across my knees
sat quietly waiting for light to come. The sense of
loneliness was oppressive in the stillness that preceded
morning, broken only by the quack or plash of the wild
duck, and the distant honking of a train of wild geese
winnowing their sinuous way afar. I felt desolate—
almost lost—and thought how utterly insignificant man
is in comparison with his self-assertion. The grand
bluffs of the Missouri, rising past each other intermina-
bly, were before me in shadowy outline, that seemed to
change and threaten
to roll upon me;
all around
stretched the waste of reeds, secret, treacherous, limit- AMERICAN GAME BIRD SHOOTING 260 less—unmoved, yet whispering to the water about their
roots with a strange trickling. But the light came on;
the distant hills took shape and settled in firm gray out-
line against the sky, and a breath of fresher, purer air,
messenger of morning, passed over the lake, dispelling
the vapors that hung reluctant, and causing the reeds to
sway in graceful salute to the coming sun. A sparrow
chirped from her perch with joy; a field lark rose from
her bed in the grass, tuning her limpid pipe to a song
of gladness; and the wild fowl plashed about right
heartily, when the highest hilltop was touched with
gold, and another and another, till the scene was il-
lumined to the very bosom of the lake. SHARP-TAILED GROUSE. The feathered
orchestra sounds never so impressive as when it ushers
in the day; never so fine and complete as when familiar
voices sing the higher notes to the strange deep bass of
the grouse; heard for the first time, as it was on this
occasion, the effect is indescribable. No one could say
whence the sound proceeded, nor how many birds, if
more than one, produced it; the hollow reverberations
filled the air, more like the lessening echoes of some
great instrument far away, than the voice of a bird at
hand. I listened to this grand concert, absorbed in
the reflections it stirred within me, no longer alone,
but in company I love, till the booming fell less fre-
quently upon my ear, and then ceased—it was broad
day; the various birds were about their homely avoca-
tions, and I must betake myself to practical concerns. “Thus, in no faltering accents of timid expectancy, “Thus, in no faltering accents of timid expectancy,
but in the bold tone of assured success, the grouse SHARP-TAILED
GROUSE 261 calls upon his intended mate to forget the shyness that
will no longer serve their purpose; nor does the invi-
tation lack defiance to a rival who may presume to dis-
pute his rights. At the rallying cry the birds assemble,
in numbers of both sexes, at some favorable spot, and a
singular scene
ensues
as the courtship
progresses. There is a regular ‘walk-around,’ as ludicrous, to the
disinterested observer, as some of the performances on
the comic stage. The birds run about in a circle, some
to the right, others to the left, crossing each other’s
path, passing and repassing in stilted attitudes, stop-
ping to bow and squat in extravagant postures, and
resuming their course, till one would think their heads
as well as their hearts were lost. But this is simply their
way, and they amuse themselves in such fashion till
the affair is settled. The cocks have bristled and
swelled, strutted and fought, till some have proven their
claims to first choice, and others have concluded to
take what they can get. Their subsequent history, I
am sorry to state, is neither particularly creditable to
themselves nor of absorbing interest to us. Leaving
them to go about their business in their usual humdrum
way, let us look to what now occupies their mates. SHARP-TAILED GROUSE. “A nest will soon be required for her eggs, and the
hen has to select suitable premises, though, being an
architect of only the humblest order, she has little build-
ing work to do; and, moreover, not being fastidious,
her choice is made without difficulty. I have found
the nests in such various locations that I can hardly
determine what her preference is, if, indeed, she have AMERICAN GAME BIRD SHOOTING 262 any. I suppose the site depends much upon circum-
stances. She will enter a tract overgrown with the
low, scrubby willow bushes, so abundant in our higher
latitudes, and settle beneath one of these; she will ram-
ble along the edge of a wooded stream and hide in a
patch of tall weeds; she will stroll out on the bound-
less, bare prairie, and take a tuft of grass at random. But wherever she makes down her bed she is solicitous
to conceal it, not only from the rude glances of men,
but from the equally cruel eye of her many four-footed
enemies. Her method of concealment is most artful—
perfected by its witlessness. With admirable instinct,
she will avoid a place that offers such chances of con-
cealment as to invite curious search; her willow bush
is the duplicate of a thousand others at hand; her tuft
of grass on the prairie is the counterpart of a million
others around; her nest will be found by accident
oftener than by design. And when, stooping over a
warm nest on the prairie, whence she has just fluttered
in dismay, we note how exposed it seems, now that it
is found, we wonder how the dozen blades of grass
that overarch the eggs, or the rank weed that shadows
them, could have hidden the home so effectually that we
nearly trod upon the bird before we saw her. She is
now but a few yards off, in plain view, amid the scrubby
prairie herbage, perhaps squatting, but more likely mov-
ing away with a swaying motion of the head at each
step. We will not combine murder with the robbery
we are about to commit, and let us hope she will be
consoled in time. SHARP-TAILED GROUSE. Lifting up the eggs carefully, one by SHARP-TAILED GROUSE 263 one, we find the nest to be merely a few spears of grass,
pressed down and somewhat circularly arranged with,
in all probability, a few feathers that appear to have
rather been mechanically detached from the mother
bird than laid down by design. If the place is near our
northern border, and early in June, we shall probably
find the eggs quite fresh; but by the third week of that
month they will be about hatching. At this period,
should we, for any sufficient reason, destroy the setting
bird, we should find her in sad plight—her plumage,
harsh and worn, entirely gone from a large space on
her belly; her flesh thin and flabby, and her crop con-
taining only a few buds of some weed that grows
close by her nest, with some grasshoppers or other
insects. “No bird is a more faithful mother than this grouse;
no one clings to her eggs more steadfastly, or guards
her young with more sedulous care. In proof of how
close she will set while incubating, let me mention two
instances that came under my observation. One poor
bird was actually trodden upon and killed, and some
of her eggs smashed. On another occasion, I drove
a large four-mule ambulance over a nest; the animals
shied as they stepped over it, when the bird fluttered
out from between their legs. Stopping instantly, I
discovered the nest just between the hinder wheels. ©
The grouse lies hard and close, never relinquishing hope
of escaping observation until the last moment. “The young, as usual among gall moment. “The young, as usual among gallinaceous birds, run
about almost as soon as they are hatched, and it is in- ment. “The young, as usual among gallinaceous birds, run
about almost as soon as they are hatched, and it is in- . “The young, as usual among gallinaceous birds, run
about almost as soon as they are hatched, and it is in- AMERICAN GAME BIRD SHOOTING 264 teresting to witness the watchful solicitude with which
they are cherished by the parent when she first leads
them from the nest in quest of food, glancing in every
direction, in her intense anxiety, lest harm befall them. SHARP-TAILED GROUSE. “Throughout the region of the Red, Pembina and
Souris or Mouse rivers, where I observed the birds
during the summer, I found them mostly in the under-
brush along the streams, which they seemed to seek
instinctively as affording the best shelter and protec-
tion, as well as plenty of food. Where they were most
abundant I frequently observed the ‘scratching holes’
in the bare earth among the bushes, where they resorted
to dust themselves, and, most probably, in the instances
of ungrown coveys, to roost. Late in the summer and
in September, those who cared to shoot the tender
young found them to lie well to a dog; in fact, to
lie so close that they were flushed with difficulty with-
out one. No game birds could be tamer or more readily
destroyed. Except when temporarily
scattered by
molestation, the coveys kept close together, and only
occasionally left the covert to stray on the adjoin-
ing prairie. They appeared to be feeding chiefly on
wild-rose seeds, and those of another kind of plant “Returning to the newly hatched chicks, we will note
their characteristics as they progress toward maturity. The down in which they are clothed when hatched
is rather dingy yellow, mottled on the crown, back and
wings with warm brown and black; it extends to the
toes, but leaves a bare strip along the hind edge of the
tarsus; the bill and feet are light brown. They are
about as large as bantam chickens of the same age,
and very pretty little things, indeed. They are very
quick in their movements, scrambling to squat and
hide on the least alarm, even at this early age. ... “Throughout the region of the Red, Pembina and
Souris or Mouse rivers, where I observed the birds
during the summer, I found them mostly in the under-
brush along the streams, which they seemed to seek
instinctively as affording the best shelter and protec-
tion, as well as plenty of food. Where they were most
abundant I frequently observed the ‘scratching holes’
in the bare earth among the bushes, where they resorted
to dust themselves, and, most probably, in the instances
of ungrown coveys, to roost. SHARP-TAILED GROUSE. She clucks matronly to bring them to brood under her
wings, or to call them together to scramble for a choice
morsel of food she has found. Should danger threaten,
a different note alarms them;
they scatter in every di-
rection, running, like little mice, through the grass till
each finds a hiding place; meanwhile, she exposes her-
self to attract attention, till, satisfied of the safety of
the brood, she whirrs away and awaits the time when
she may reassemble her family. In the region where
I observed the birds in June and July, they almost in-
variably betook themselves to the dense, resistant un-
derbrush,
which
extends
for
some
distance
out-
ward from the wooded streams, seeking safety in this
all but impenetrable cover, where it was nearly impossi-
ble to catch the young ones, or even to see them, until
they began to top the bushes in their early short flights. The wing and tail feathers sprout in a few days, and
are quite well grown before feathers appear among
the down of the body. The first coveys seen able to
rise on wing were noticed early in July; but by the
middle of this month most of them fly smartly for short
distances, being about as large as quails. Others, how-
ever, may be observed through August, little, if any,
larger than this, showing a wide range of time of
hatching, though scarcely warranting the inference of
two broods in a season. SHARP-TAILED GROUSE 265 “Returning to the newly hatched chicks, we will note
their characteristics as they progress toward maturity. The down in which they are clothed when hatched
is rather dingy yellow, mottled on the crown, back and
wings with warm brown and black; it extends to the
toes, but leaves a bare strip along the hind edge of the
tarsus; the bill and feet are light brown. They are
about as large as bantam chickens of the same age,
and very pretty little things, indeed. They are very
quick in their movements, scrambling to squat and
hide on the least alarm, even at this early age. ... SHARP-TAILED GROUSE. Late in the summer and
in September, those who cared to shoot the tender
young found them to lie well to a dog; in fact, to
lie so close that they were flushed with difficulty with-
out one. No game birds could be tamer or more readily
destroyed. Except when temporarily
scattered by
molestation, the coveys kept close together, and only
occasionally left the covert to stray on the adjoin-
ing prairie. They appeared to be feeding chiefly on
wild-rose seeds, and those of another kind of plant “Returning to the newly hatched chicks, we will note
their characteristics as they progress toward maturity. The down in which they are clothed when hatched
is rather dingy yellow, mottled on the crown, back and
wings with warm brown and black; it extends to the
toes, but leaves a bare strip along the hind edge of the
tarsus; the bill and feet are light brown. They are
about as large as bantam chickens of the same age,
and very pretty little things, indeed. They are very
quick in their movements, scrambling to squat and
hide on the least alarm, even at this early age. ... “Throughout the region of the Red, Pembina and “Throughout the region of the Red, Pembina and
Souris or Mouse rivers, where I observed the birds
during the summer, I found them mostly in the under-
brush along the streams, which they seemed to seek
instinctively as affording the best shelter and protec-
tion, as well as plenty of food. Where they were most
abundant I frequently observed the ‘scratching holes’
in the bare earth among the bushes, where they resorted
to dust themselves, and, most probably, in the instances
of ungrown coveys, to roost. Late in the summer and
in September, those who cared to shoot the tender
young found them to lie well to a dog; in fact, to
lie so close that they were flushed with difficulty with-
out one. No game birds could be tamer or more readily
destroyed. Except when temporarily
scattered by
molestation, the coveys kept close together, and only
occasionally left the covert to stray on the adjoin-
ing prairie. They appeared to be feeding chiefly on
wild-rose seeds, and those of another kind of plant AMERICAN GAME BIRD SHOOTING 266 equally abundant along the river bottoms. SHARP-TAILED GROUSE. The ma-
jority of these birds were ungrown up to September,
and scarcely any had at that date begun to assume their
new plumage. Up to this time I do not recollect that
I ever saw one alight in a tree; and they were still, for
the most part, under charge of the parent, as separate
families, rather than as the indiscriminate packs in
which they afterward associate. With the advance of
the month these family associations seemed to break
up, the change of plumage was finished, the birds grew
strong of wing, and able in all respects to look after
themselves. No longer solicitous
of shelter,
they
haunted the innumerable ravines that make down to
the streams, and strolled in company far out on the
prairie. In this region, at least, they showed little
wariness all through the month. I could generally
walk up to a covey in fair view on the bare prairie, even
to within a few feet, before they would fly, and they
seldom went far before realighting. Their appearance
when not obscured by the herbage is characteristically
peculiar. They seem to stand remarkably high on their
legs, and generally carry their short, pointed tail some-
what elevated; the singularity is increased when the
long neck is outstretched, as it generally is when they
are on the lookout. On alighting after being flushed,
if not much alarmed, they often stand motionless at
full height, but if badly scared, squat closely, and are
then difficult to find if not exactly marked down. If
without a dog, one may pass and repass among them
without finding one, unless he happen to stumble on SHARP-TAILED GROUSE 267 them; and often, going away after such want of suc-
cess, one may look back to find the heads of the whole
lot raised above the grass, intently regarding his re-
treat. It is astonishing how closely they can squat—
even laying the head flat upon the ground, and appear-
ing scarcely half their natural size. At this season
their food appears to be chiefly grasshoppers. I have
opened numbers to find their crops crammed with these
insects, only varied with a few flowers, weed-tops,
succulent leaves, and an occasional beetle or spider. SHARP-TAILED GROUSE. “By the first of October the sharp-tailed grouse have “By the first of October the sharp-tailed grouse have
mostly finished the renewal of their plumage, are all
full grown and strong of wing; their habits are con-
siderably modified. They grow wary and watchful,
flushing often at long distances to fly clear out of
sight, and running far on the ground. They also be-
gin to alight on trees, a habit, however, not confirmed
until somewhat later, when, with the advance of cold
weather and the failure of former supplies of food, they
assume the routine of their winter life. The close
coveys of the earlier season are for the most part broken
up, and the birds wander often alone in search of food. They haunt the interminable ravines along the Mis-
souri, making away from the river bottoms in search
of food, but mostly returning at evening to roost in
the trees. Early in the morning they may be seen
leaving their perches in straggling troops, flying high
and swiftly to other feeding grounds; and again in
the evening, if one loiter beneath the immense cotton-
woods, where, during the day, scarcely a chicken was AMERICAN GAME BIRD SHOOTING 268 to be seen, he will observe their return, till the trees
are almost covered, and the air resounds with the
hoarse kuk-kuk-k-k-k. Frequently, in very cold and
especially in falling weather, the grouse will not leave
their perches during the day, but may be seen at any
hour roosting quietly in the tops of the tallest cotton-
woods. They are decidedly not graceful objects under
these circumstances. They look very large, sharply
defined among the bare straggling branches against
the gray sky, and assume ungainly attitudes, particu-
larly when standing erect on their long legs, with out-
stretched necks and upturned tails. Their behavior
under these circumstances varies in a manner to me
inexplicable. Sometimes a group thus scattered among
the treetops will permit the closest approach desired,
and more than one may be brought down before the
rest are off in alarm; not seldom one may fire twice or
thrice at the same bird without dislodging it, or kill
several without stirring from his tracks. But ordi-
narily the chickens’ wits serve them to better purpose
than this. SHARP-TAILED GROUSE. As we approach, when just beyond range,
the crackling of the underbrush attracts the attention
of one of the birds, which before had been squatting
‘like a bump on a log’; he rises on his feet and twists
his neck around to have a look. The rest follow his
example. A moment more the warning kuk-kuk-k
sounds, and the nearest bird leaves his perch—the cry
is taken up by the rest, and the whole are off to settle
again a few hundred yards away, and tempt renewed
pursuit that is likely to end as unsuccessfully. From SHARP-TAILED GROUSE 269 the sportsman’s standpoint, the arrangement is wholly
unsatisfactory when the birds behave so; nor when they
are tame is it much more attractive; for, unless a supply
of meat be the only point, dropping chickens from the
trees is no more exciting than robbing a hen-roost. Killed under these circumstances, the food of the grouse
is readily ascertained; in the dead of winter it consists
chiefly of the berries of the cedar, and buds of the pop-
lar or cottonwood and willow, still closely sealed, await-
ing the coming of spring. I have taken from one
crop a double handful of such food, almost as dry as
when swallowed. This diet does not improve the
quality of the flesh; a chicken at this season is quite a
different thing from one killed earlier in the season. The rating of the grouse as an article of food neces-
sarily varies, not only with circumstances, but accord-
ing to individual preferences. 1, myself, do not esteem
it very highly. A tender young grouse, early in the
season, is not to be despised, but all such specially
flavored meat is likely to soon become distasteful, espe-
cially if, on one or two occasions, a person has been
forced upon a surfeit of it. Confined to grouse for a
few days, most persons, I should judge, would find
relief in mess-pork. “The mode of flight of this species is not peculiar ;
it rises with a startling whirr from the ground, till it
attains a certain elevation—its straight, steady course,
performed with great velocity by alternate sailing and
flapping, are points it shares with its relatives. SHARP-TAILED GROUSE. The
wing-beats are rapid and energetic, giving it an im- AMERICAN GAME BIRD SHOOTING 270 pulse that enables it to sail long distances, when the
wings are held stiffly expanded to their full extent,
somewhat decurved, and with the points of the quill
feathers separated. The bird’s voice is highly char-
acteristic. It is so almost invariably uttered during
flight, at particular moments with reference to the de-
livery of the wing strokes, that for some time after my
first acquaintance with the birds I was in doubt whether
the sound were mechanical or vocal; nor was the un-
certainty removed until I had heard it from the birds
at rest. The ordinary note of alarm is almost invaria-
bly sounded just before the bird takes wing, whether
from the ground or from a tree, and is usually repeated
with each succeeding set of wing-beats, seeming to be
jerked out of the bird by its muscular efforts. But we
hear it also when, the bird being at rest, it becomes
alarmed, yet not sufficiently to fly away; and when a
bird is passing at full speed, sufficiently near, we may
clearly distinguish the mechanical whirring sound of
its wings, as well as, sometimes, the creaking rustle of
its tail feathers as it turns its flight. When roosting
at ease among the trees, and probably at other times,
the grouse have a different set of notes—a sociable
cackling or clucking, with which they entertain each
other. “In conversation
with
Captain
Hartley,
of the
Twenty-second Regiment, an accomplished sportsman,
well acquainted with the ways of our game birds, I
was informed of an interesting point of difference in
the habits of this bird and the pinnated grouse. In SHARP-TAILED
GROUSE 271 entering a cultivated field the latter goes on foot, and
may consequently be readily trailed by a dog, while the
sharp-tail flies in, and is only likely to be overhauled
by the dog’s winding it, or coming accidentally upon
it. The same gentleman has noted the preference of
this species for the skirts of woods, brush and broken
places generally, in contrast to the entirely open places
which the pinnated grouse frequents.”
The eggs of the sharp-tail, while often plain cream- The eggs of the sharp-tail, while often plain cream-
colored, are at times greenish in color and are some-
times marked with small spots of reddish brown and
lavender. SAGE GROUSE. Centrocercus urophasianus. SHARP-TAILED GROUSE. They do not appear
to lay such large
clutches as the pinnated grouse, though, after all, the
difference in numbers is not marked. Centrocercus urophasianus. The sage grouse may always be recognized by its
great size and by the fact that its tail is longer than
the wings, and is graduated, with narrow
pointed
quills. The upper plumage is gray, variously marked
and streaked with black. Some of the tertiary feathers
are tipped with white or streaked with white; the chin,
cheeks and throat are spotted black and white, but there
is usually a distinct white streak on the side of the
head running a short way back of the eye. The neck
is black in front, while the fore breast is white or
grayish, and on either side of the lower neck in the
whitish areas are stiff, coarse hair-like feathers. The
belly is black, and the lower tail coverts black tipped
with white. The male is from 26 to 30 inches long,
with a wing of from 12 to 13 inches, and a tail about
the same length. The birds weigh from 4 to 8 pounds. The female is much smaller; has the chin and throat
white and the black patch on the front of the neck
speckled. Half-grown birds are browner than their
parents, with a warmer tone of rufous in their plumage,
more like the young of the dusky grouse. The length
of the female is from 20% to 23 inches. Excepting the wild turkey, the sage hen, as it is often Excepting the wild turkey, the sage hen, as it is often
272 SAGE GROUSE 273 called, is the largest North American gallinaceous
bird. Full-grown males sometimes almost equal the
female wild turkey in size, and have been reported to
weigh about eight pounds. SAGE GROUSE SAGE GROUSE The sage grouse is an inhabitant of the high, dry,
artemisia or sage-brush covered plains of the Western
States. It is found on the plains and flanks of the
Rocky Mountains, in the great central plateau, in the
northwest territories, and in parts of southern British AMERICAN GAME BIRD SHOOTING 274. Columbia. Its eastern range covers parts of North
Dakota, and extends thence westward through Mon-
tana, Wyoming and Utah to Oregon, California and
Nevada. The sage grouse is interesting from the fact that it
possesses a soft, membranous stomach; in this respect
differing from other gallinaceous birds. Its food varies
with the seasons, and no doubt in winter the leaves of
the so-called sage brush make up the most of this. Centrocercus urophasianus. The sage grouse takes its name from this plant, on The sage grouse takes its name from this plant, on
which, at certain seasons, it feeds extensively. Yet
it eats other food, including grain. In Forest and
Stream of August 29, 1889, Mr. George H. Wyman
says:
“The sage cock will eat the leaves from a sage
bush when it cannot get berries or grain, but it will go
farther for a morning’s feed from a wheat field than
any bird I know, except the wild geese. I have killed
sage fowls with stomachs filled with ripe wheat picked
up the same morning in places where none was to be
had nearer than eight miles, and in fact with no culti-
vation of any kind nearer in any direction. They fly
long distances in search of food, but return to roost in
the same place at night, generally on some steep hillside,
free from shrubs or high grass.”
This may have been
an error of observation. Very likely there was some
unknown source of grain supply nearer at hand. But
this, of course, cannot be proved. It is stated by some
observers that sage leaves are resorted to only when
other food is hard to obtain; but this is a mistake. The leaves and flowers of the sage have been found SAGE GROUSE 275 in the crops of adults and young during the season of
greatest food supply. The leaves, blossoms and the
pods of other plants, together with many seeds and
grasshoppers and crickets, are said by Captain W. L. Carpenter to constitute a large part of this food. Mr. Robert W. Williams is quoted by Captain Bendire as
having taken a bird, the crop of which was filled with
the blossoms of a species of goldenrod (Solidago). In past years controversies as to the edibility of the In past years controversies as to the edibility of the
flesh of the sage cock have often taken place, some
writers holding that the flesh is so strongly flavored
by the sage that it is unpleasant to the taste, while
others maintained with equal earnestness that the flesh
was delicate and without unpleasant flavor. Centrocercus urophasianus. Obviously,
the flavor of the flesh of any bird depends in great
measure on the food which it eats, and birds which
stuff themselves with a particular sort of vegetation,
whether it be buds of the birch, of the laurel, of the
spruce or of the wormwood, if left undrawn for a time
may taste of the plant on which they have been feeding. It is a safe plan to dress the sage grouse as soon as
may be after it has been killed, but even if this is not
done, young birds are not likely to have a strong taste,
though old ones may. Like other prairie grouse, the sage cock goes through Like other prairie grouse, the sage cock goes through
a courtship which is noteworthy. This is well de-
scribed by Captain Bendire in his ‘‘Life Histories of
North American Birds,” in which he says:
“Early one morning, in the first week of March, “Early one morning, in the first week of March,
1877, I had the long-wished-for opportunity to observe AMERICAN GAME BIRD SHOOTING 270 the actions of a single cock while paying court to sev-
eral females near him. ... His large, pale-yellow
air-sacs were fully inflated, and not only expanded for-
ward, but apparently upward as well, rising at least an
inch above his head, which, consequently, was scarcely
noticeable, giving the bird an exceedingly peculiar ap-
pearance. He looked decidedly top-heavy and ready to
topple over at the slightest provocation. The few long,
spiny feathers along the edges of the air-sacs stood
straight out, and the grayish white of the upper parts
showed in strong contrast with the black of the breast. His tail was spread out fan-like, at right angles to the
body, and was moved from side to side with a slow,
quivering movement. The wings were trailing on the
ground. While in this position he moved around with
short, stately and hesitating steps, slowly and gingerly,
evidently highly satistied with his performance, utter-
ing, at the same time, low, grunting, guttural sounds,
somewhat similar to the purring of a cat when pleased,
only louder. This was kept up for some ten minutes. After having regained his usual attitude it was hard
to believe that this was the same bird I had seen but a
few minutes before.” Mr. Centrocercus urophasianus. Frank Bond has described at some length a
hitherto unnoticed act of the male sage grouse, which
he witnessed in Wyoming. The bird, after inflating
its air-sacs, bent forward until its breast touched the
ground and pushed itself forward along the ground. This action, carried on for days, seems to explain the SAGE GROUSE 277, wearing away of the stiff feathers on the lower neck
and upper breast so often noticed in this species. The nest of the sage grou pecies. The nest of the sage grouse is usually placed at the
foot of some sage bush, or sometimes at the foot of a
bunch of rye grass, whose outer leaves, bending over,
may conceal the nest. Commonly it contains from
eight to eleven eggs, nearly as large as a hen’s egg,
greenish white or brownish in color, more or less
heavily spotted with round, but not large, dots of
brown and blackish. The period of incubation js given
as twenty-two days. There is little There is little or no nest, and the eggs lie in a mere
hollow scratched out in the bare ground. The sitting
bird harmonizes so wonderfully with the ground on
which she rests that one may pass within a foot or two
without seeing her. Major Ben Major Bendire quotes Captain William L. Car-
penter, U. S. A., who says:
“I found a nest at Fort
Bridger,
Wyoming—where this species is numerous—
June 1, with nine fresh eggs. I was standing alongside
a sage bush watching butterflies, several times looking
down
carelessly without
seeing anything
unusual,
when, happening again to glance at the foot of the
bush in the very place before observed, I saw the wink-
ing of an eye. Looking more intently, a grayish mass
was discerned, blending perfectly with the color of the
bush, which outlined itself into the form of a sage hen
not 2 feet from my foot. She certainly would have
been overlooked had not the movement of her eyelids
attracted my attention. I stood there fully five minutes AMERICAN GAME BIRD SHOOTING 278 admiring the beautiful bird, which could have been
caught in my butterfly net, then walked back and forth
and finally passed around the bush to observe it from
behind. Not until then did it become frightened and fly
away with a loud cackling. Centrocercus urophasianus. The nest was a depression
at the foot of a sage bush, lined with dead grass and
sage leaves. The spot was marked and visited several
times, always passing within
a few
feet without
alarming the bird.” While the mother bird is sitting, the males are scat-
tered over the prairie, two or three often being found
together, and when alarmed starting off with heavy,
lumbering flight to fly half a mile or a mile. When hatched, the young leave the nest and follow When hatched, the young leave the nest and follow
the mother. When quite young they are as expert at
hiding as are most of the grouse at this age. They are active and hardy, and for the first few
weeks of their life bear a certain resemblance to the
young turkey, less perhaps in color than in length of
neck and the active way in which they move about on
their long legs. The mother is devoted, and Captain
Bendire quotes Mr. Wm. G. Smith, who caught six
young sage chickens
one June in Carbon County,
Wyoming, as saying: “The female flew at my legs and followed me 200
yards to where my wagon was standing, constantly
making hostile demonstrations, while the young kept
calling.” The young families roost on the ground, on the
sides of shallow ravines or on the prairie above, and *AaAIng yeosojorg “S ‘Q jo uoTsstused Ag
"ISNOID a3BS *AaAIng yeosojorg “S ‘Q jo uoTsstused Ag
"ISNOID a3BS *AaAIng yeosojorg “S ‘Q jo uoTsstused Ag
"ISNOID a3BS SAGE GROUSE 279 the places where they have spent the night remind one
much of the roosting place of a brood of quail. When
full-feathered and half grown, they are very gentle
and unsuspicious, and it used to be common to see a
brood of them walk along feeding before
a man who
was following them up, trying to shoot their heads
off with a rifle or a six-shooter. They paid no attention
to the shots, but if one was wounded and fluttered
about, all flew without delay. At this age they seem
very social, constantly calling to each other with a
high-pitched, peeping note, and if one wanders off
from the flock or is left behind, he becomes very un-
easy and runs hard in his efforts to overtake the family. Centrocercus urophasianus. When full grown, and at the approach of cold weather,
the birds become more shy and fly more readily. On
flying they give a hoarse cackle, somewhat like that
of the sharp-tailed grouse, but deeper. The sage grouse always roosts on the ground, and The sage grouse always roosts on the ground, and
I have never seen them in what could fairly be called
a tree. Captain Bendire reports having seen them on
the horizontal limb of the juniper, about two feet from
the ground. A number of years ago, in response to an inquiry
from him, I wrote Major Bendire as follows:
“On a very few occasions I have seen the sage grouse “On a very few occasions I have seen the sage grouse
standing on the branches of a sage bush, sometimes 2
or 3 feet from the ground, but I imagine this is
quite an unusual position for the bird. This species
commonly, I think, goes to water twice a day, flying
down to the springs and creek bottoms to drink in the AMERICAN GAME BIRD SHOOTING 280 evening, then feeding away a short distance, but roost-
ing near at hand. In the morning they drink again and
spend the middle of the day on the upland. The young
birds, when feeding together, constantly call to one
another with a low, peeping cry, which is audible only
for a short distance. This habit I have noticed in
several other species of our grouse, notably in the
dusky grouse and sharp-tail. “In western Wyoming the sage grouse packs in Sep- “In western Wyoming the sage grouse packs in Sep-
tember and October. In October, 1886, when camped
just below a high bluff on the border of Bates’ Hole
in Wyoming, I saw great numbers of these birds just
after sunrise, flying over my camp to the little spring
which
oozed
out
of the
bluff,
200
yards away. Looking up from the tent at the edge of the bluff above
us we could see projecting over it the heads of hundreds
of the birds, and as those standing there took flight,
others stepped forward to occupy their places. The
number of grouse which flew over the camp reminded
me of the old-time flights of passenger pigeons that I
used to see when I was a boy. Centrocercus urophasianus. Before long the narrow
valley where the water was, was a moving mass of
gray. I have no means of estimating the number of
birds which I saw, but there must have been thousands
of them.” Although the sage hen is a large bird, rises slowly,
and lumbers off with a good deal of noise, nevertheless,
after they have attained their full growth, it is not
always easy to hit these birds when in full flight, espe-
cially if going with the wind. SAGE GROUSE 281 Except when in flight, the sage grouse is deliberate
in its movements, and I have seldom seen a bird on foot
that appeared to hurry. More often they walk delib-
erately along, with heads stretched high, watching the
intruder until the time comes for flight, when they
spring from the ground with the cackling cry already
mentioned and soon disappear over the next hill. The
flight is often very much extended. The only occasion when I recall seeing a sage grouse
run was once when a bird that I had started flew several
hundred yards and alighted in plain sight on a hillside
on the other side of a valley. A marsh hawk, which
was hunting near where the grouse alighted, flew to it
and several times stooped at it and appeared to reach
for it with its feet. The grouse at once started and
ran swiftly along the hillside until it reached some high
sage brush, the hawk following and now and then
making a clumsy dive at it. THE WiLp TURKEY: Meleagris gallopavo. Meleagris gallopavo silvestris. Meleagris gallopavo osceola. Meleagris gallopavo intermedia. Meleagris gallopavo merriamt. America has given to the world its largest game bird
and perhaps most important domestic fowl—the turkey. It is purely American, and its ancestry goes back a long
way, for it existed here in far-off Tertiary times, por-
tions of the skeleton of a turkey having been found in
the Miocene deposits of Colorado, and the bones of
other species in the post-Pliocene of New Jersey. Of
these last, one was about the size of the existing turkey,
but taller, while another was much smaller. At this
time, the mastodon lived along the Atlantic coast, while
the far older turkeys of Colorado had as associates the
huge Brontotherium and many other creatures long
extinct. When the white men came to these shores they found
turkeys in plenty. The flesh constituted a good share
of the food of the natives, who wore cloaks or robes
made of turkey feathers. Not very long after the
discovery of the New World the bird was taken to
282 282 283 THE WILD TURKEY Europe, and there received a variety of names in dif-
ferent countries, most of which referred to India, carry-
ing out the early idea that America was a part of the
Indies. Thus the bird was called by the English “cock
of India”; in French, poule d’Inde, contracted to dinde, WILD TURKEY
hen of India; in Spanish, gallo or gallina de India, cock
or hen of India; in German, Jndianische Henne or
Huhn, Indian hen, and also Calecutischer Hahn or WILD TURKEY hen of India; in Spanish, gallo or gallina de India, cock
or hen of India; in German, Jndianische Henne or
Huhn, Indian hen, and also Calecutischer Hahn or
Henne, cock or hen of Calcutta. It was also called by
the Spaniards the Moorish hen, referring to a supposed hen of India; in Spanish, gallo or gallina de India, cock
or hen of India; in German, Jndianische Henne or
Huhn, Indian hen, and also Calecutischer Hahn or
Henne, cock or hen of Calcutta. It was also called by
the Spaniards the Moorish hen, referring to a supposed AMERICAN GAME BIRD SHOOTING 284 African origin, while in Egypt the Arabic name is Dik
runu, fowl of Turkey. THE WiLp TURKEY: Precisely why it should have been called turkey by Precisely why it should have been called turkey by
the English it is hard to say, except that as Turkey was
a part of the Far East, it may have been supposed to
have some relation to India. It has been suggested
that the name by which we know the bird is a corrup-
tion of a Hebrew word tukki, said to mean peacock,
that this term was applied to the turkey, when it was
received in Spain, by the Jews, who then monopolized
the business of selling poultry, and that from this point
of first introduction the name spread with the bird over
a part of Europe. This bird, taken to Europe by the Spaniards soon This bird, taken to Europe by the Spaniards soon
after the conquest, was the Mexican turkey. The common wild turkey once found over most of The common wild turkey once found over most of
eastern North America was for a long time the only
form known in the United States, and this was thought
to be the progenitor of all the domesticated races of
turkeys. In 1856, however, the English naturalist,
Gould, described the Mexican turkey as a distinct
species, and much later other observers called attention
to a turkey from Florida differing slightly from the
ordinary wild turkey, and to yet another different
one from the Rio Grande. Later still, E. W. Nelson
found that the turkey of Arizona presented constant, if
slight, differences from the wild turkey of the plains
and that of Mexico, and described it as Merriam’s
turkey. To the untrained eye the differences between certain THE WILD TURKEY 285 of these subspecies are slight, and the sportsman will
do well to try to learn the geographical ranges of the
different forms, for in most cases the locality will be
to him a better guide in identifying the bird he kills
than will any color description. The Mexican turkey
is markedly different from the bird of the East, and
the two will readily be distinguished. The differences
between the various turkeys are found chiefly in the
coloring of the rectrices, or tail feathers, and of the
tail coverts and the primaries. THE WiLp TURKEY: The original wild turkey—to which the name Melea- The original wild turkey—to which the name Melea-
gris gallopavo was given—has been shown to be the
bird later described by Gould as the Mexican wild
turkey. It is notably different from the eastern form,
for its tail, tail coverts and the feathers of the lower
rump are tipped with white or whitish, while the
eastern and northern turkey has those feathers tipped
with deep rusty or even with rich dark chestnut. The
ordinary domestic turkey shows the whitish tippings
of the feathers of tail, tail coverts and lower rump—
characters derived from its ancestor, the turkey of
Mexico. The Mexican turkey occupies the wooded
mountain slopes bordering the Mexican tablelands on
the south and west, ranging north to Chihuahua, but
it does not reach the United States. Mr. Nelson has
shown where
it grades into Merriam’s turkey on
the north, while to the south in southeastern Mexico
and Central America, it is replaced by a strikingly dis-
tinct species, the.brilliantly hued ocellated turkey. The eastern wild turkey, which was long considered The eastern wild turkey, which was long considered AMERICAN GAME BIRD SHOOTING 286 the true Meleagris gallopavo, thus becomes a subspecies
of the Mexican turkey and is now known as Meleagris
gallopavo silvestris. It and the closely similar Florida
race (osceola) have the ends of the upper tail coverts
and tail feathers dark chestnut. The common northern
wild turkey has the primaries, or stiff quill feathers of
the wing white barred with black, while the Florida
wild turkey has the primaries black, with small white
bars, which are broken and usually do not reach the
shafts of the feathers. These differences are constant,
otherwise they would not be regarded as subspecific
characters. Down in parts of southern Texas and northeastern
Mexico
is found
another
subspecies
(intermedia)
known as the Rio Grande turkey, or Elliott’s turkey. It looks much like the common ‘wild turkey, but the
rump feathers have a coppery bronze bar across them
close to the ends, and are tipped with dark yellowish. The tail coverts are chestnut, with narrow black cross
bars, and are broadly tipped with buff. The tail
is mottled pale chestnut and black, has a black bar across
it close to the end, and is tipped with yellowish buff. THE WiLp TURKEY: The difference in appearance between Flliott’s turkey
and the common turkey is not easily expressed in
words, but any one who has an opportunity to compare
two specimens of the same age and sex will readily see
that they are not exactly alike. Moreover, the female
of the Rio Grande turkey has grayish tips to the feath-
ers on the upper part of the body, and thus differs
strikingly from any other species of turkey. 287 THE WILD TURKEY From the wild turkey of the East, Merriam’s turkey
may be distinguished by the whitish tips of the feath-
ers of the lower rump, tail coverts and tail. From the
Mexican turkey it may be known by its velvety black
rump and the greater amount of rusty rufous, succeed-
ing white tips on the tail coverts and tail, and by the
distinct black and chestnut barring of the middle tail
feathers. Merriam’s turkey thus appears to be about mid-
way between the eastern wild turkey and the wild tur-
key of Mexico, and in fact the eastern wild turkey
grades into Merriam’s turkey, as Merriam’s turkey
grades into the Mexican turkey, whose white-tipped
tail coverts and white-tipped tail, as seen in the do-
mestic turkey, are so characteristic. So in North America we have five forms of turkey,
the ranges of which are fairly well defined. The
Florida
wild
turkey
(Meleagris
gallopavo The
Florida
wild
turkey
(Meleagris
gallopavo
osceola), described by Scott about twenty years ago,
is confined to Florida, but the precise limits of its range
are not yet known. The type specimen came from
Tarpon Springs, Fla. The Rio Grande turkey (Meleagris gallopavo inter- The Rio Grande turkey (Meleagris gallopavo inter-
media) was described by the late George B. Sennett
in 1892. It is a striking bird, found in the lowlands
of southern Texas and also in northeastern Mexico,
where it ranges from the coast lowlands up to over
3,000 feet in the adjacent mountains. Merriam’s turkey (Meleagris gallopavo merriamt) Merriam’s turkey (Meleagris gallopavo merriamt)
was described by E. W. Nelson in 1900. It ranges AMERICAN GAME BIRD SHOOTING 288 from southern Colorado south through Arizona and
New Mexico, and grades into the Mexican turkey on
the south and into the eastern wild turkey on the east. THE WiLp TURKEY: On all streams flowing east from the Rocky Mountains
over the great plains, from the Niobrara, which is near
the northern boundary of Nebraska, south nearly to
the Rio Grande, turkeys were formerly common, and
these were the ordinary bird of the Mississippi Valley. They lived along these various rivers, many of which
have their heads in the mountains, and following up
these streams to the mountains, there intergrade with
the mountain bird. E. W. Nelson has shown where this
takes place. In these days, when the common wild turkey is ex-
tinct over much of its former range, it is very difficult
to define with exactness the former boundaries of that
range. We know that it was abundant in southern
New England and to the south. Audubon speaks of it
rather vaguely as found in southern Vermont, New
Hampshire and Maine, and it is certain that it was
once very abundant in Massachusetts, where many
years ago I saw a skin taken at Mt. Tom about 1848
or 1840. Wm. Brewster,
in his admirable volume on the
“Birds of the Cambridge Region of Massachusetts,”
published in 1906, as No. IV of the Memoirs of the
Nuttall Ornithological Club, has gathered much inter-
esting information concerning the turkey in eastern
Massachusetts and in Maine. He says: “Morton, referring, no doubt, to his experience at “urpesy
SAayINT
plIM “
SAayINT
plIM THE WILD TURKEY 289 Merrymount, now Wollaston, only a few miles south
of the Cambridge region, where he lived from 1625
to 1628, and again in 1629 and 30, says:
‘Great flocks
(of turkeys) have fallied by our doores;...I hada
Salvage who hath taken out his boy in a morning, and
they have brought home their loades about noone. I
have asked them what number they found in the woods,
who have answered, Neent Metawna, which is a tho-
sand that day.’
Wood confirms this by stating that
‘sometimes there will be forty, three score, and an hun-
dred of a flocke, sometimes more and sometimes lesse;
their feeding is Acornes, Hawes and Berries, some of
them get a haunt to frequent our English corne:
In
Winter when the Snow covers the ground they resort
to the Seashore to looke for Shrimps and such small
Fishes at low tides. THE WiLp TURKEY: Such as love Turkie hunting must
follow it in Winter after a new falne Snow, when he
may follow them by their tracts; some have killed ten
or a dozen in halfe a day; if they can be found towards
an evening and watched where they peirch, if one come
about ten or eleaven of the clocke he may shoote as
often as he will, they will sit unlesse they be slenderly
wounded. These Turkie remaine al the yeare long, the
price of a good Turkie cocke is foure shillings; and he
is well worth it for he may be in weight 40 pound; a
Hen two shillings.’
Josslyn mentions seeing, probably
at Black Point (now Scarborough), Maine, “‘threescore
broods of young Turkies on the side of a Marsh, sun-
ning of themselves in a morning betimes, but this was
thirty years since [in 1638 or 1639], the English and AMERICAN GAME BIRD SHOOTING 290 the Indian having now [1671] destroyed the breed, so
that ’tis very rare to meet with a wild Turkie in the
Woods.’ “That the species was formerly found throughout
the Cambridge region, there can be no reasonable
doubt. Turkey Hill in Arlington may well have de-
rived its name from the presence there of this noble
bird in early Colonial days. Indeed, Mr. Walter Faxon
writes me that an acquaintance of his has seen ‘in a
manuscript diary of the ancestor of an Arlington man
...anentry of killing some Wild Turkeys in the region
about Turkey Hill.’
At Concord, less than ten miles
further inland, the species had not become wholly ex-
tinct at the beginning of the past century. The late
Steadman Buttrick of that town, a keen lover of field
sports and a man of undoubted veracity, who died in
1874, used to delight in narrating how, when a boy,
he had made repeated but invariably fruitless expedi-
tions in pursuit of the last wild turkey that is known
to have lingered in the region about his home. He
often saw the bird, a fine old gobbler, but it was so very
wary that neither he nor any of the other Concord
gunners of that day ever succeeded in getting a fair
shot at it. THE WiLp TURKEY: It was in the habit of roosting in some tall
pines on Ball’s Hill, whence, when disturbed, it usually
flew for refuge into an extensive wooded swamp on
the opposite (Bedford) side of Concord River. Mr. Buttrick was born in 1796. As he was presumably at
least twelve or fifteen years of age before he began to
use a gun effectively, it is probable that his experience THE WILD TURKEY 291 with the wild turkey happened some time between 1808
and 1815.” The turkey was abundant in the southwestern por-
tions of the province of Ontario and occurred through
much of New York, in Pennsylvania and Ohio, from
which last State it has been exterminated in compara-
tively recent years. It is said that a few turkeys still
linger in Pennsylvania. Michigan, Wisconsin, south-
ern Minnesota and Iowa all once had turkeys enough. They were abundant in Nebraska, reaching beyond the
northern boundary of the State, for Captain W. L. Carpenter found turkeys on the Niobrara River, and
Dr. Coues speaks of good evidence of their occurrence
as far north as Yankton on the Missouri—about the
same latitude as the mouth of the Niobrara. It is well
understood that the turkey was fairly abundant on
many streams flowing into the Missouri or its tribu-
taries south of the Platte River, and undoubtedly they
worked up many of these streams into the mountains. Indians in whom I have confidence have told me of
killing turkeys on tributaries of the South Platte in the
mountains west of where Denver now stands. From
that point south Merriam’s turkey was undoubtedly
abundant in the mountains. The turkey found on the
plains to the south of the Platte, westward until the
mountains are reached, is presumably the eastern form
(Meleagris gallopavo silvestris). Captain L. H. North, who as a little boy moved with Captain L. H. North, who as a little boy moved with
his family into Nebraska in the year 1856, says of the Captain L. H. North, who as a little boy moved with
his family into Nebraska in the year 1856, says of the AMERICAN GAME BIRD SHOOTING 292 streams in eastern central Nebraska forty or fifty years
ago: “There were a good many wild turkeys here on the
Loup River, the Elkhorn and Shell Creek when we
came here. Ed. THE WiLp TURKEY: Chambers tells me they were often
seen on the Niobrara River in early days—say in 1877. I do not recall that any turkeys were seen when the
Pawnee scouts were out in 1867, guarding the track
layers on the plains toward the mountains, but at that
time turkeys were found on the Platte River near old
Ft. McPherson—not far below the forks of the Platte.” In August, 1909, Forest and Stream printed a letter
from me inquiring as to the western range of the wild
turkey. This inquiry brought out some extremely in-
teresting information which indicates that the former
range of the turkey extended
regularly
to
South
Dakota. In my letter I asked what the northern and
western range of the turkey was, and whether any of
Forest and Stream’s correspondents had ever known of
its being found in the Black Hills. In response to this
Sandy Griswold, of Omaha, Neb., sent to Forest and
Stream a letter, from which I quote the essential para-
graphs: “The query whether wild turkeys ever got as far
West as the Black Hills I am unable to answer; I do
know, however, that no longer ago than 1894 they had
found their way as far as the foothills this side of the
Black Hills in South Dakota. “T was camped on the Lake Creek marshes that fall, “T was camped on the Lake Creek marshes that fall,
duck shooting, and on the third of November Alfred THE WILD TURKEY 293 Reshaw, a young halfbreed Sioux, who was one of our
camp helpers and guides, killed a twenty-one-pound
black and tan turkey in the scraggy pine hills along
White River, twenty miles north of our camp and forty-
five or fifty miles this side of the Black Hills. He killed
the bird flying, from out of a bunch of five which he
had jumped from a patch of ground cherries on one
of the bluffs. He knew what the birds were, as he and
his brother had killed several the previous winter
in the same vicinity. “Two days later Alfred, the late George W. Scrib- “Two days later Alfred, the late George W. Scrib-
ner, of San Francisco, and I went to White River,
where the Sioux had killed his gobbler, and although
we hunted assiduously for hours up and down on
both sides of the river, we found no turkey. THE WiLp TURKEY: We
did find plenty of sign, however, in almost every rose
thicket and among the dried ground cherries from
which Alfred had flushed his birds. We found fresh
tracks and fresh droppings, showing that the birds had
been there after the day the Sioux made his kill. “Along the White River in this particular region “Along the White River in this particular region
are extensive fastnesses well adapted to the fancy of
wild turkeys, low scraggy acorn-bearing oaks, deep
arroyos, with numerous springs, thickets of plum, crab
and grape, rose fields, ground and choke cherry patches
and many vegetable growths on which the birds feed
in the fall and summer.” The Reshaws (Richard) are a well-known family of
Sioux mixed bloods, descendants of one or more French AMERICAN GAME BIRD SHOOTING 204 Creoles who served the American Fur Company about
1850 or earlier, and who married Sioux women. Through the kindness of Colonel Hugh L. Scott,
superintendent of the U. S. Military Academy, I am
enabled to give the most northerly definite record of
the wild turkey on the Missouri River of which I
have any knowledge. Colonel Scott, learning of my
interest in this subject, recalled that more than twenty
years ago General D. L. Magruder, U. S. A. (retired),
had told him of killing wild turkeys near Fort Ran-
dall, Dakota, in 1855. He therefore wrote to General
Magruder and received from him a letter dated Sep-
tember 6, 1909, as follows:
“From July, 1855, to October, 1860, I was stationed “From July, 1855, to October, 1860, I was stationed
at the different garrisons along that stream [the upper
Missouri River] from old Fort Pierre Chouteau to Fort
Randall. “From July, 1855, to October, 1860, I was stationed
at the different garrisons along that stream [the upper
Missouri River] from old Fort Pierre Chouteau to Fort
Randall. “On December
17, 1855, I accompanied General
Harney upon a hard winter’s march, from Fort Pierre
Chouteau to the mouth of the Niobrara River. The
march was by land as far as the present site of Fort
Randall, where we were compelled by heavy snowdrifts
in the ravines to abandon the prairie and take to the
ice upon the river, where the march was continued,
both going and returning, until our arrival back at
Fort Pierre, February 17, 1856. THE WiLp TURKEY: “During the trip, both going and returning, I killed “During the trip, both going and returning, I killed
deer, rabbits, grouse and turkeys to supply our mess,
finding each of the kinds of game in plenty and quite
fat in most of the heavily timbered points along both “During the trip, both going and returning, I killed
deer, rabbits, grouse and turkeys to supply our mess,
finding each of the kinds of game in plenty and quite
fat in most of the heavily timbered points along both THE WILD TURKEY 295 sides of the river. The turkeys were particularly fine-
flavored, their food being abundant, consisting mostly
of wild grapes, rose apples (the seed pod of the wild
rose), cottonwood buds and hackberries, the latter ap-
parently their favorite, at least to judge by the quantity
contained in their crops. “The last turkey killed by me was at a return camp “The last turkey killed by me was at a return camp
about thirty miles above Fort Randall. Beyond that
point I have no personal experience, but while stationed
at Fort Pierre I was told by the interpreter of the
fort that turkeys formerly were quite abundant in the
heavy timber about the mouth of the Big Cheyenne
River about thirty miles above.”
The statement made by the interpreter at Fort Pierre The statement made by the interpreter at Fort Pierre
furnishes quite satisfactory evidence that turkeys were
once found on the Missouri River as far north as the
mouth of the Cheyenne River. Colonel Scott has also called my attention to the diary
of Lieut. Rufus Saxton, printed in Vol. I, Pacific R. R. Reports, 1853-4, which says of Cedar Island, on the
Missouri River, below Ft. Pierre:
“Saw wild turkeys
for the first time. They are seldom seen above this
point, and have never, I believe, been found beyond the
Rocky Mountains.”
The reference, of course, is to
the northern Rocky Mountains. THE WiLp TURKEY: Alexander Henry, the Younger, states that in 1806 Alexander Henry, the Younger, states that in 1806
the Cheyenne Indians coming up from the south
brought with them the tails of turkeys which the Man-
dans and Minitari greatly desired for use as fans and
for which they traded, and from this we may infer that AMERICAN GAME BIRD SHOOTING 296 there were no turkeys found on the Missouri or its
tributaries as far north as the then location of those
tribes—Knife River. I suspect that in the Rocky Mountains turkeys sel-
dom or never crossed the divide between the north
and south forks of the Platte, and that they never got
as far west as the Black Hills. No bird is more gentle and unsuspicious than the
turkey until it has learned that man is an enemy, and
after that no bird is more wary and alert. Mr. Hen-
shaw speaks of the lack of suspicion on the part of these
birds, and Captain Carpenter tells how—when his com-
mand was preparing to establish Fort Niobrara—a wild
turkey came to the camp and ventured out of the under-
brush to feed on the grain spilled by the animals. When Florida first began to be a resort for northern
tourists, turkeys were very abundant and not at all
shy or suspicious. They often associated with the
domestic turkeys, and one wild gobbler became so tame
that he would feed unconcernedly within a few feet
of a man. On this point Dr. Ralph, writing about
1890, said to Captain Bendire:
“One can hardly believe that the wild turkeys of “One can hardly believe that the wild turkeys of
to-day are of the same species as those of fifteen or
twenty years ago [that is, 1872 to 1875]. Then they
were rather stupid birds which it did not require much
skill to shoot, but now I do not know of a game bird
or mammal more alert or more difficult to approach. THE WiLp TURKEY: Formerly I have often, as they were sitting in trees
on the banks of some stream, passed very near them, “suryynyys
‘AayIny,
pli “s
‘AayIny,
pli THE WILD TURKEY 297 both in our boats and in steamers, without causing
them to fly, and I once, with a party of friends, ran
a small steamer within twenty yards of a flock which
did not take wing until several shots had been fired
at them.” The turkey, while usually resident in a certain sec-
tion, is yet said to be prone to wander, and to be by
no means as local in its habits as the bobwhite or the
ruffed grouse. Sometimes they will remain in a desira-
ble location for a long time and then will leave it—for
no apparent reason. On the plains the birds used to
spend the night roosting in the trees of the bottoms,
and after drinking in the morning would wander up
on the prairie about the heads of ravines and there
feed on grasshoppers and other insects and on sand
cherries and tunas, returning in the heat of the day to
the shade of the underbrush or even of a cut bank. Turkeys feed chiefly on vegetable matter. In old
times the saying, that a good mast year was a good
turkey year, passed into a proverb. They eat beech-
nuts, chestnuts, various acorns, pecan nuts, persim-
mons, the fruit of the cactus, all sorts of wild berries or
seeds and grains and other vegetable matter, besides
all insects. In the central and southern Rocky Moun-
tains the fruit of the pifion forms a large part of their
subsistence. As determined by the Biological Survey,
the turkey’s food consists of 154 per cent. of animal
matter and nearly 84% per cent. of vegetable matter. Of the vegetable matter, buds and leaves constitute
nearly 25 per cent., fruit nearly 33, and other seeds AMERICAN GAME BIRD SHOOTING 2098 nearly 20 per cent. In the western country turkeys
are great eaters of grasshoppers. They also destroy
the tobacco worm and moth when they can get them. The breeding season for the turkey ranges from
February to May, according to the latitude which the
bird inhabits. At the breeding time, and, indeed,
throughout the year until mating in the early spring,
the hens and young birds associate together and apart
from the gobblers. THE WiLp TURKEY: At mating time the gobbler’s actions are those of the At mating time the gobbler’s actions are those of the
domestic turkey. He gobbles loudly, struts and spreads
his tail, drags his wings on the ground and puffs him-
self out until he has made the proper impression on
the hen. Often several birds are going through this
performance about a single hen, and fights between
the males are common, and, it is said, sometimes with
fatal results. The nest is a mere hollow scratched in the ground,
lined or not lined with straws, grass and a feather or
two. The eggs vary in number from eight to four-
teen. Captain Bendire reports a case where there were
twenty-six eggs in a nest, but two hens were at the nest,
one sitting on the eggs and one standing close by
them. It is likely, therefore, that occasionally two hen
turkeys share a nest, as two quail sometimes do. Like many ground-nesting birds, the turkey is ex- Like many ground-nesting birds, the turkey is ex-
ceedingly hard to see when on her nest, and of the
turkey, as of other birds, various instances of this
have been related. Captain B. F. Goss, writing May,
1882, in southern Texas, says: THE WILD) TURKEY 299 “We were encamped quite near the nest; one morn-
ing I noticed a hen turkey stealing through the bushes
and suspected she was going to her nest. We watched
her carefully for three mornings, and having pretty
nearly located the nest, commenced
a close search,
and examined, as we thought, every inch of ground. I
was about giving up, when looking down almost at my
feet, I saw the bird sitting on the nest. She at once
ran; she had allowed me to pass several times within
a foot of her without moving, and seemed to know at
once when she was seen. THE WiLp TURKEY: I have often noticed this
trait in birds of this genus; as long as unseen you
can tramp all around them, but they seem to know
at once when they are seen and lose no time in getting
away.” As long as the hiding bird, which sees and hears its
pursuer, is convinced that he is still ignorant of its
position, it feels safe, but the moment it recognizes by
the expression of the man’s eye that its hiding place has
been detected, it is off without delay. The eggs of the wild turkey are not at all unlike The eggs of the wild turkey are not at all unlike
those of the domestic bird. Usually they are cream-
colored, dotted with finer or larger spots of reddish
brown, chocolate and sometimes lavender. Captain
Bendire states that the spots are more often very
small and fine than large. The young follow the mother as soon as hatched, but The young follow the mother as soon as hatched, but
Audubon says, “As the hatching generally takes place
in the afternoon they frequently return to the nest to
spend the first night there.” AMERICAN GAME BIRD SHOOTING 300 The young are believed to be very tender and subject
to many dangers from dampness. Some writers de-
clare that the mother leads them on high ground for
the first week or two of their life in order that they
may escape the dangers of dew or rain from the grass. Audubon says:
“To prevent the disastrous effect of
rainy weather the mother, like the skilful physician,
plucks the buds of the spicewood bush and gives them
to her young”! The little birds are able to fly at about
two or three weeks old, and soon after that leave the
ground and roost on the low branch of a tree sheltered
under their mother’s wings. When danger threatens,
the mother turkey, like many other gallinaceous birds,
calls to her young, which at once crouch and hide and
cannot then be seen. It is said that if the male turkey finds a nest of eggs It is said that if the male turkey finds a nest of eggs
upon which the hen is sitting he will destroy them, and
that if he comes upon a brood of newly hatched young
he will kill them. THE WiLp TURKEY: It is certain that during the autumn
and winter the young birds and the females associate
together, while the old males keep by themselves and
do not begin to seek the society of their mates until the
approach of spring. In the Rocky Mountains the nests are built at an al- In the Rocky Mountains the nests are built at an al-
titude of from 3,000 to 5,000 feet, but as the weather
grows warmer and the snow disappears, the old hen
leads the young up to the higher mountains, so that
they finally summer at from eight to ten thousand feet. In the late autumn, when the weather grows cold and
snows come on the mountain ranges, the birds move THE WILD TURKEY 301 down again to sheltered cafions or timbered river val-
leys, where they spend the winter. In the southern States turkeys have always been In the southern States turkeys have always been
abundant and their stronghold is still there—parts of
Virginia, the Carolinas, Georgia, Alabama, Mississippi,
Louisiana, Arkansas, Missouri and Texas. Merriam’s
turkey is said to be almost extinct in Colorado, but is
still abundant in Arizona and New Mexico. That there
should be occasional outlying colonies of a few birds
in Iowa and Nebraska, such as Mr. Griswold is told
of, seems very surprising, but such colonies cannot last
long unless protected by the owners of the land on
which they live. The turkey, grandest of game birds, has been ex-
terminated over much of its former range. Great in
size, and valuable for food, he is an object of pursuit
wherever found. So, throughout the farming country
of the North and West the turkey is gone and gone for-
ever. As the country is settled up, is his complete ex-
termination to follow? Domesticated, he will always
survive, but should we not strive to retain the old wild
turkey of the eastern States in his untamed wild state,
self-dependent, one of the typical and interesting in-
habitants of our primitive forests and our far-stretch-
ing southern plains? PART. 3
UPLAND SHOOTING sash
hours Cassia Pertes.. AHOEN SCO
BALTIMORE hours Cassia Pertes.. From Bull. 21, Biological Survey,
Dept. of Agriculture,
By Permission. WOODCOCK SHOOTING. Many years ago I contributed to the Century Maga-
gine an article on this species, and from it, by the kind
permission of the Century Company, I extract a few
paragraphs on some details of the woodcock shooting
of those
days. Birds
were
plenty then,
as we
may hope that some day they will be again, if wise
measures are taken for their protection. The woodcock is not often seen and is quite con- The woodcock is not often seen and is quite con-
tented to be overlooked. He has no brilliant song to
catch the ear, no gaudy plumes to attract the eye, nor
does he perform graceful evolutions high in the air
in the broad glare of day. He is truly a modest fowl,
and except at night, or during the twilight of morn-
ing or evening, he does not willingly venture into sit-
uations where he can be viewed by the casual wan-
derer through field or wood. One who desires to make
his acquaintance must penetrate into the depths of the
most tangled swamps to find him at home. Even
here, during the day, he is usually half asleep. Not
so drowsy, however, as to be unaware of the approach
©
305 305 305 AMERICAN GAME BIRD SHOOTING 306 of an intruder. The soft rustling of the leaves, the
occasional snapping of a dry twig, and the sound of
the heavy footfall rouse him from his doze or his
day dream. He moves sideways beneath the spread-
ing leaves of a tuft of skunk cabbage, and with head
turned on one side, and great eyes spread to their wid-
est, watches for the approaching form. Once in a
while something may cause him to take the alarm
and dart away before it is within sight; but usually he
lies close, and when he rises it is near at hand. He
springs from the ground uttering a shrill, twittering
whistle and twists about in his upward flight to dodge
the branches which spread in a network above him,
until he has topped the undergrowth, and then darts
off in a straight line for fifty or a hundred yards, to
plunge once more into his beloved cover. The dog is a most important auxiliary in woodcock The dog is a most important auxiliary in woodcock
shooting. WOODCOCK SHOOTING. A very few sportsmen employ cocker or
field spaniels, which are trained to range close to the
gun, and to give tongue as soon as they strike the
scent, thus warning the shooter of the proximity of
the bird and preparing him for its possible rising. But most men use the setter or pointer. A good wood-
cock dog should work close; that 1s, within sight of
the gun. Often where the undergrowth is very thick,
it becomes necessary to attach a bell to the dog’s col-
lar, so that if it pass out of sight for a few moments
its whereabouts may still be known by the sound. Late in November you will still find a few birds,
and at this time they will be lusty and strong of wing, ‘spilg oures) BulysIUeA OMY ,, SJOUSIYy “MV Iq wor
“a
*SUIYINOIN YIOIPOOM ‘spilg oures) BulysIUeA OMY ,, SJOUSIYy “MV Iq wor
“a
*SUIYINOIN YIOIPOOM WOODCOCK
SHOOTING 307 and will test your skill. Cross the meadows then and
go down into the swamp, working along near the edge,
where the underbrush is not too thick, and the soil
under the leaves, as you can see in the cattle tracks,
is rich and black. Just beyond you, on the left, a steep
hillside rises sharply from the edge of the swamp, its
surface overgrown with low cedars, sumacs and bay-
berry bushes. The old dog comes out of the swamp
and turns toward the slope, and as he crosses before
you glances back inquiringly. He knows the hillside,
and understands as well as you do that a cock is usu-
ally to be found on that warm southern exposure at
this time of the year. No need to wave the hand or
use any elaborate signal to tell him to work up among
the cedars and through the brush. A little sidewise
movement of the head, and he is breasting the steep
ascent and rustling among
the twigs and the crisp
leaves, while you walk along a cowpath at the foot of
the slope. If there be a bird there it will be sure to
fly toward the swamp, and must therefore cross in
front of you. WOODCOCK SHOOTING. For a few moments you hear the dog
as he works above you; then the sound ceases, and
as you pause to listen for it there comes to the ear
that shrill whistle, so like the midsummer twitter of
the kingbird, that warns you to “mark cock.”
You
see a brown flash among the green cedars, and the
bird darts out to plunge into the swamp; but as he
sees you he turns sharply and flies down the path,
straight away. You have plenty of time; bring up
your gun deliberately, cover the bird, and when it is AMERICAN GAME BIRD SHOOTING 308 about thirty yards distant fire, and it is yours. At
the report of the gun your dog appears on the bank
above, pauses a moment until you have slipped an-
other cartridge into the gun, and then dashes off. to-
ward where the bird lies. A word steadies him as
he approaches it, and after quartering once or twice
the scent reaches his nostrils. He feels for it, then
pauses, and at command steps forward, gently takes
the bird in his mouth and trots slowly toward you,
expressing as much pride and satisfaction in his face
and in his slowly wagging tail as if he had captured
the prize without any assistance of yours. On again,
along the border of the swamp, sometimes stooping
low to pass beneath the tangled underbrush, or forc-
ing your way through the thick alders, making the
dead stems crack and fly, or passing through a spot
free from low shrubs, where the tall, gray trunks of
the hardwood trees stand apart, and the footfall is
scarcely heard on the damp, dead leaves. For some
time the dog works quietly ahead of you, manifesting
none of the signs which would lead you to suspect
that birds were near; but as you approach a little arm
of the swamp which runs up a narrow ravine, the
merry action of the setter’s tail warns you to be pre-
pared for a point. Yes, there, where the wind has
swept aside the leaves, exposing the black mud _be-
neath, you see in it dozens of little round holes, which
tell you that the long bill has been at work here. Sud-
denly he stops, and stands quite still, except that the
tip of his tail moves a little from side to side. WOODCOCK SHOOTING. As WOODCOCK
SHOOTING 309 you step up to him he moves again, very slowly and
cautiously, and then suddenly stops and remains mo-
tionless. It is a pretty picture, and one that the sports-
man never tires of watching and admiring. The dog’s
fore foot is raised in the act of stepping, his tail is
straight and rigid, head a little above the line of the
back and slightly turned to one side, ears a little
pricked. Walk up beside him and look at his face, and
you will see, what his attitude already indicates, that
he is laboring under strong excitement. His nose is
perhaps within a few inches of the bird and the scent
is strong. You will see his eyes roll as he looks over
the ground before him. His forehead is knotted into
a frown, which shows how thoroughly in earnest he
Tse The bird is likely to fly up from beneath the dog’s
nose, so close to you that you cannot shoot without run-
ning the risk of either missing altogether or else blow-
ing him to fragments, and will then, perhaps, dart be-
hind a thick cedar or twist into some alders through
which you can hardly see to shoot. The alder runs, so numerous through the New Eng- The alder runs, so numerous through the New Eng-
land States, are most satisfactory places to work for
woodcock. These are usually the channels of little
brooks a few feet below the general level of the open
meadows through which they pass. The ground is
too damp to be successfully cultivated and the farmer
gives it up to the black alder, which attains a height
of from fifteen to twenty feet. Beneath these in the
wet, springy soil, the skunk cabbage (Symplocarpus), AMERICAN GAME BIRD SHOOTING 310 a variety of ferns, and many other moisture-loving
plants, grow
in wild luxuriance. These
runs,
or
swales, are often so narrow that the best way to hunt
them, if two are shooting together, is for each to take
a side and let the dog work between them. The
birds, when started, will either show themselves above
the alders, or, what is more likely, will break out on
one side or the other, and fly forward along the edge
of the bushes, giving a perfectly open shot and one
which not even a tyro ought to miss. WOODCOCK SHOOTING. In working out
such places the bell should be put on the dog, for it
is often so dark beneath the thick growth that it is
difficult to see him. Should he come to a point, and
the bird decline to rise, a heavy stick or stone thrown
into the bushes just in front of him will often flush it. One of the most instructive articles on woodcock One of the most instructive articles on woodcock
shooting ever written is from the pen of Mr. B. Wa-
ters and was published in Forest and Stream in the year
1903. Iam glad to be able to reproduce it—with a few
minor changes—in these pages. Of all the kinds of shooting of field and forest the Of all the kinds of shooting of field and forest the
sport of woodcock shooting holds the warmest place
in the hearts of its devotees. The woodcock shooter
is an enthusiast of enthusiasts. He may take a keen
pleasure in bringing other game birds to bag, but when
woodcock shooting is under consideration comparison
ceases. And indeed this sport possesses many fas-
cinating features peculiar to itself. First of all, it can
at best be indulged in only in very small quantities. There is but little of it when compared with the abun- WOODCOCK
SHOOTING 311 dance of other kinds of shooting, for the woodcock is
comparatively a rare bird and its season is a short one;
therefore the keen edge of enjoyment of woodcock
shooting is never dulled by surfeit. The habitat of
the bird is distinctly different from the home of all
other game birds, and on the earth’s surface only tiny
spots here and there meet the wants of its nature,
and many vast tracts of fertile country have no wood-
cock ground at all. It, too, is a bird of mystery, of whose coming and It, too, is a bird of mystery, of whose coming and
going no one knows. It is nocturnal in its habits, and
its haunts have been such secluded and unused spots
—rarely invaded by man—that it is seldom seen. WOODCOCK SHOOTING. The
residents of sections wherein is the home of the wood-
cock may never see one from year’s end to year’s
end, and, indeed, may go through life with no more
knowledge of them than that derived from hearsay ;
or, seeing one, may still remain in ignorance of its
identity. While the quail, the partridge, the snipe,
and other game birds are not unfamiliar to country
residents and are readily identified by them, the wood-
cock and its doings are shrouded in mystery. The
large woodpecker in some sections is called woodcock
by the country folk, while in other sections any plover
which has a long bill is often called by the same name. So little is the bird known, that sometimes when killed
it is called snipe, and sometimes the snipe is called
woodcock, by those who have not given the bird spe-
cial study or attention. Its life being so entirely with-
out the sight of man and in general so little being AMERICAN GAME BIRD SHOOTING 312 known of it, it is not strange that the little accurate
knowledge is obscured by the air of much mystery,
and that those who seek the bird find a fascination in
it greater than that of any other form of game bird
shooting. The bird itself is of peculiar form and of
rare richness in its colorings, and its flesh is esteemed
a morsel of choice excellence fit for the palate of the
most fastidious epicure. Thus it affords great sport
in its capture and is pleasing to the eye and to the
palate. The scarcity of the bird, its beauty, and the delicate
flavor of its flesh, all serve to enhance its value, and
its mystic life adds a charm to its pursuit which is
distinct from all others. Its home is generally in densely shaded nooks in Its home is generally in densely shaded nooks in
out-of-the-way places where man rarely enters and
where the soil is soft and moist, for in such places is
its food obtained. The alder runs and slopes in the
birches and nooks in the woods where springs or rivu-
lets or excessive moisture makes the ground soft, are
its favorite haunts, and sometimes in certain sections
it finds spots in the cornfields which are desirable feed-
ing grounds, though haunts and feeding grounds are
never plentiful. WOODCOCK SHOOTING. In Mississippi it is occasionally found
in open sedge fields. Many places which to the eye
have every appearance of being a suitable home for it,
still have no birds in them. The difficulties of woodcock shooting have been The difficulties of woodcock shooting have been
greatly exaggerated, particularly as concerns the ex-
traordinary skill required by the shooter, and the still WOODCOCK
SHOOTING eiiie, more extraordinary labor, and consequent fatigue, im-
posed on the dogs, the latter being an indispensable
factor in the sport if any success worth considering is
sought. While intrinsically the sport possesses all the
requirements of the highest degree of wing shooting,
the writers on it have deemed it fitting that it be
dressed in a glamor of romance, presumably that a lit-
tle knowledge might be presented in an elaboration of
high colors which touched on the sky, the sunshine
as it glinted through the alders, the beautiful color of
the foliage, the balmy zephyrs laden with nature’s per-
fumes, ad infinitum, all of which are present in all
other kinds of shooting, or, indeed, present if there is
no shooting at all. The shooting of woodcock is diffi-
cult, it is true, but not so extraordinary in its diff-
culty as to be distinctly special, and far from being
so difficult as most shooters make it from injudicious
selection of guns, loads, etc. Woodcock shooting is close shooting, the closest of Woodcock shooting is close shooting, the closest of
any kind of shooting recognized as legitimate sport
with the shotgun. While the woodcock is called a
game bird, it is gentle and mild in its habits, with
none of the pugnacity or extraordinary vitality pos-
sessed by members of the grouse family. The small-
est of shot is sufficiently heavy to kill it, and the cylin-
der-bore gun is amply close enough for the ranges
which one must accept in shooting it. The choke bore
of any kind is out of place in such extremely short
ranges, and unfit to use on a bird so easily killed;
though, strange to say, the use of it is not uncommon, AMERICAN GAME BIRD SHOOTING 314 owing, no doubt, to the fact that many men, owning
but one gun, must use it for all kinds of shooting, and
in other instances to the further fact of thoughtless-
ness concerning the proper requirements of the sport. WOODCOCK SHOOTING. Short barrels, too, are desirable, the difference in the
handling of a 30-inch barrel and a 24-inch barrel in
cover being far away in favor of the latter. Woodcock shooting is largely a matter of snap Woodcock shooting is largely a matter of snap
shooting; therefore,
a wider range at a much shorter
distance is a requisite if one is cultivating success in-
stead of nursing a fad in respect to the use of choke-
bore guns for all kinds of shooting, whether the guns
be fitting or otherwise. In the shooting of quail or
chickens or ruffed grouse—to a lesser degree with the
latter—a certain degree of deliberation and quick aim
can be practiced, but in woodcock shooting the oppor-
tunities for deliberation are the rare exception; hence
the need of adopting an. open gun to meet the require-
ments of quicker work and short ranges. Light loads
and smaller shot can be used successfully, some noted
shooters using dust shot exclusively. With a short
cylinder-bore gun—a true cylinder bore, not the modi-
fied choke bores, which are often called cylinder—such
a pattern can be secured at 15 or 20 yards as will
insure fair success to the average shot and the best
of success to the good one. It might be said that
such a gun and load are too murderous, and indeed
they would be in the hands of a man who could shoot
with any degree of precision if he could exercise de-
liberation; but as in the greater number of instances WOODCOCK
SHOOTING 315 the shooter has but an instant in which to act, the
results are far from being so fatal as one might fancy
them to be. Often there is but a momentary glimpse
of a dusky shadow flitting through or across a small
vista in the dense growth, and the shooter must fire
then or not at all, unless he is pleased at a purpose-
less tumult, that being the sum total when he shoots
and trusts to luck for the execution of his purposes. WOODCOCK SHOOTING. As in all other shooting, experience enables the As in all other shooting, experience enables the
sportsman to recognize the promising nooks for wood-
cock and the signs which denote its presence, these
being the holes made by it in boring in the ground
for its food, and other indications well known to the
shooter, and which can only be recognized by experi-
ence. As to the labor and fatigue imposed on the dog As to the labor and fatigue imposed on the dog
while seeking for woodcock, they are largely an ex-
aggeration. Wilson, in his work on the birds of North
America,
specifically mentions the fatiguing efforts
which the dog encounters in woodcock shooting, and
mentions that relays of dogs are necessary. As a
matter of fact, the work of the dog in woodcock shoot-
ing is the easiest of all kinds of shooting. He must
range close to the shooter—at most not beyond a gun-
shot—if he is to serve the best purpose in that kind
of shooting and it is not at all essential or desirable
that he work at high speed. It is essential, however,
that he be intelligent, and know thoroughly the best
manner of working to the gun and assisting the shooter
to get his shots in a manner to insure success. He AMERICAN GAME BIRD SHOOTING 316 should work diligently, but not hurriedly, and it is
hardly necessary to add that the work should be done
as silently as possible, though this also is true of all
other kinds of shooting. The dog should be a good retriever, otherwise a The dog should be a good retriever, otherwise a
large percentage of the birds will be lost, for many
times it is as difficult to find the bird after it is killed
as it is before. Dogs which run riot in this shooting
can soon tire themselves out, particularly in summer
shooting, when dogs are out of condition, and the
weather warm, but the consequent fatigue from such
overexertion and unfit condition cannot be justly at-
tributed to the difficulty of the sport. It is rather hard
work for the shooter, particularly him of the North,
where the quest must be made afoot, and where the
footing is difficult and insecure, though after all it is
but little more difficult than any other shooting in
which the shooter walks. WOODCOCK SHOOTING. As the dog often comes to a point in thick cover As the dog often comes to a point in thick cover
out of sight of the shooter, even though the point may
be but a few steps away from him, a bell attached to
the dog’s collar has been found of great assistance in
determining his whereabouts, and its silence indicates
when he stops on point, a matter very essential in con-
ducting the sport. Not every dog is a good wood-
cock dog, even though he may be excellent on quail,
snipe, chickens, etc. Some dogs appear to dislike the
work intensely, others refusing to recognize the bird
at all. A few take to it very kindly, and work to the
gun from observation to a useful degree far above WOODCOCK
SHOOTING 317 what could be established by the most careful train-
ing. The spaniel is but little used in the United States
for woodcock shooting, or any other kind of shooting,
for that matter, though there is no doubt but they could
be made eminently useful in field sport. In Louisiana, and other sections of the South, where
the woodcock seeks a clime more genial than that of
a northern winter, the conditions of shooting change
almost entirely. In sections at certain times, gener-
ally in the last of December and the fore part of Janu-
ary, they may be found in great numbers, and a bag
of twenty, thirty or forty in a day is not then consid-
ered remarkable. They frequent the switch-cane bot-
toms, or woods in the timbered prairie, in which the
heavy fall rains have softened the ground, and where
abundance of food can be found. Their stay in the
South is very short, for they start North immediately
on the lessening of the winter cold—after a stay of not
more than a few weeks—their coming and going then
being quite as silent and secret as in the North. They
are then killed in great numbers, both day and night,
by market shooters, and shipped to the home and dis-
tant markets. They have their choice feeding grounds
even in that land of abundance, and skill, diligent ef-
fort and knowledge of habitat are quite as essential to
success in the southern winter shooting as in the less
bountiful shooting of the North in summer and fall. WOODCOCK SHOOTING. So scarce has the woodcock been for the last dozen
years that some young gunners have never seen one,
and know them only from books. Happily, for the So scarce has the woodcock been for the last dozen
years that some young gunners have never seen one,
and know them only from books. Happily, for the 318 AMERICAN GAME BIRD SHOOTING past two or three years they seem to have been rapidly
increasing, a result, no doubt, of their protection in
summer over most of the country. It is interesting to recall the days of woodcock shoot- It is interesting to recall the days of woodcock shoot-
ing a generation ago and to compare their results with
those of recent times. For the past few years many
a gunner who devoted a week or ten days to faithful
tramping and shooting in eastern covers has thought
himself fortunate if in that time he killed three or
four woodcock. An item published in Forest and
Stream in 1874 speaks of three gunners who went out
early in the summer season and in one day killed sixty-
four woodcock. Twenty, twenty-five and thirty a day
were ordinary records for a single gun, and in No-
vember, 1876, we recall an account by a friend of a
day’s shooting which yielded him twenty-six woodcock
besides a less number of partridges and quail. SNIPE SHOOTING The snipe is notorious as an uncertain bird, and snipe
shooting as a sport that can never be depended on. In old times one used to walk mile after mile in the
hope that some of the birds might be started from some
favorite bit of feeding ground, but too often only dis-
appointment and weary leg muscles rewarded the en-
thusiastic tramper. On the other hand, snipe were
occasionally
found
in great abundance,
and could
hardly be driven away. Recollections of occurrences of
both kinds are laid away in the memories of all of
the older gunners. Near Vincennes, Ind., many years ago, there was a
famous feeding ground for snipe to which, during mi-
gration, the birds resorted in great numbers. Thither
I went one season with a companion to whom the
grounds were well known, and there I saw more snipe
han at that time I supposed existed. We were driven
to the edge of the marsh, and there, as the team drew
up and the wagon cramped to let us jump out, a dozen
snipe rose almost under the horses’ hoofs, flew fifteen
or twenty yards and alighted. Disregarding the advice of my friend, I had brought Disregarding the advice of my friend, I had brought
a dog with me, the best on partridges that I had ever
seen, but he proved absolutely useless here. The birds
319 319 AMERICAN GAME BIRD SHOOTING 320 were so many, the scents so numerous and confused,
that the poor old dog entirely lost his head and gal-
loped about aimlessly until called in and made to fol-
low at heel. The birds got up, a dozen at a time, flew
a few yards ahead and alighted, and then a number
of others got up and did the same thing. The report
of the gun put a considerable number on the wing,
yet at first did not seem to make the birds wild. Mean-
time I could hear my companion, who had gone off
by himself, shooting very regularly, and felt that his
greater experience was now standing him in good stead,
and that he must be acquitting himself very much bet-
ter than I was. For the multitude of the birds, and
the way in which they were rising on every side, con-
fused and unnerved me almost as much as it did my
dog. SNIPE SHOOTING Although that day I killed more snipe than ever
before or since, yet I do not look upon it as one of
the shooting days especially worth remembering. The
birds were too many, and I was not in condition to
take advantage of my opportunities. I have had more
satisfaction from a single ruffed grouse, neatly killed
as he darted away through the thick underbrush of the
swamp, than I did in that heavy bag of snipe, where
I loaded and fired all the afternoon, until my ammu-
nition was exhausted. Some such feeling as this, I believe, animates most
sportsmen when they reach grounds where birds are
so numerous that all the uncertainty of shooting is
taken away. Few of us, I think, care greatly to catch SNIPE SHOOTING 321 brook trout—strong and gamey though they may be—
out of a preserved pond. Something of this sort Mr. Waters expresses in Something of this sort Mr. Waters expresses in
the admirable article on snipe shooting which follows
this. He has tramped the marshes where snipe were
scarce, and again where, as in Louisiana, they were
enormously abundant, and he, better than most gun-
ners, knows the oddities and eccentricities of this re-
markable bird. Mr. Waters’ article, with slight changes, says:
According to the writings of ornithologists, According to the writings of ornithologists, the
breeding grounds of the snipe begin on their south-
ern boundary, at about 42 degrees of latitude, a paral-
lel through the northern part of Nebraska, Iowa, etc. The grounds extend thence north to the Arctic Circle. The snipe migrate leisurely southward as the winter
season approaches, tarrying on the available feeding
grounds, ultimately going as far south as the West
Indies and northern South America. It is a bird of the wet lands, and as said of the
woodcock, the available area affording its food sup-
ply is small as compared with the earth’s surface. Relatively, the places which are soft enough to be bored
with its sensitive bill, which contain food to its liking,
and enough to supply its needs, are exceedingly lim-
ited in number and area. SNIPE SHOOTING Soft and wet land may also be gravelly or sandy or Soft and wet land may also be gravelly or sandy or
clayey, and therefore unfit to sustain the animal and
vegetable life on which the snipe subsists; or from its
refractory nature it may be impervious to the deli- AMERICAN GAME BIRD SHOOTING 322 cate weapon with which nature has provided the snipe
for the capturing of its food. It therefore is appar-
ent that of all the wet lands there are only certain
parts which contain snipe food. Of the places which afford snipe food some are good Of the places which afford snipe food some are good
throughout the whole season,
as, for instance,
the
sloughs and marshes and parts of river valleys of the
prairie country wherein it makes its summer home. Other places are but temporarily available, as lands
made soft and wet by heavy rains. Such places may
serve it well for many weeks, as in Louisiana and
Texas in the fall and winter months, during the rainy
season, which in those States is largely the equivalent
of winter. Again, the snipe may seek its food in places
which are quite wet, as in some of the large wet
marshes, and again, in some other sections, it may
make its haunts on upland so firm that the hunter may
walk thereon pleasantly and dry-shod. While the woodcock, its long-billed confrére, is a While the woodcock, its long-billed confrére, is a
bird of the covert, the snipe is a bird of the open. On
these birds nature lays a more severe restriction con-
cerning a late stay in the North than she does on any
other game bird, for a snipe or woodcock attempting
to gain a subsistence in a frozen country is in a pa-
thetic situation indeed. Its food is said to be larve, tender roots of plants,
and worms, which it secures by boring, and also such
insects and other edible food as it can secure on top
of the ground. To the local sportsman the snipe’s habits in the SNIPE
SHOOTING 323 shooting season—which is mostly the migratory sea-
son—seem erratic and unknowable, if its unstable char-
acteristics may be called habits at all. It is in one place
to-day, and to-morrow in another. To-day there may
be an abundance, to-morrow a dearth. SNIPE SHOOTING Or it may go
contrary to its erratic reputation and remain a num-
ber of days about the same grounds. Still, the shooter
is largely in ignorance of what the snipe will do next. The weather and food conditions may be the same,
so far as observation can determine them, and yet
the birds come and go in their own whimsical way,
regardless of conditions. Some subtle, mysterious
impulse seems to impel the birds of a certain locality
either to come or go, though not in the manner of
birds which flock. Snipe fly mostly in ones or twos or threes, some-
times more, but always in small numbers. Being inde-
pendent in flight, it is difficult to uaderstand how the
common impulse to seek other grounds is at the same
time felt and acted on by all the snipe of a certain
neighborhood, or at least by most of them. There
are many exceptions, as a matter of course; as, for
instance, in a section where there are snipe in abun-
dance on a certain day, a part only may leave at the
same time. Indeed, a few snipe may be found on cer-
tain grounds throughout the whole season. Yet, how-
ever much the exception may affect the rule, the greater
part of the birds are erratic and lawless most of the
time. No doubt that which seems whimsical and mysteri- No doubt that which seems whimsical and mysteri- AMERICAN GAME BIRD SHOOTING 324 ous in the life of the snipe is really in harmony with
the needs of its nature. As it is nocturnal in habit,
it is difficult to study, and it is specially difficult for
the resident of one locality to observe its general habits
with any degree of precision. Seeing it in but one
small corner of its habitat, the local sportsman can
gain, at best, but a fragmentary knowledge of its needs
and its habits. Being swift of wing and enduring of flight, the
snipe undoubtedly feeds over vast areas of ground
many miles apart, twenty or thirty miles of flight being
of no more effort to it when in search of food than
twenty or thirty rods are to the prairie chicken. SNIPE SHOOTING When
snipe invade feeding grounds in vast numbers, as is
frequently the case, the grounds are soon thoroughly
bored, and all the food within reach is consumed;
thus it may be a necessity for them to seek food else-
where till the exhausted grounds have time to replen-
ish themselves. Many writers lay great stress on the difficulties of Many writers lay great stress on the difficulties of
snipe shooting. They treat it as a bird of phenomenal
swiftness and erratic flight, and the shooting of it as
requiring something extraordinary in the matter of
skill. As a matter of fact, snipe shooting, at certain
times, is the easiest of shooting. On warm days, when
the birds are fat and lazy, flying slowly and tamely,
with pendulous bills, as is often the case in the fall, in
the South, no bird a-wing is more easily killed. They
are then disinclined to fly. They indolently lie to the
dog’s points till the shooter walks them up, ‘adiug surjurod 19339S
5
oe ‘adiug surjurod 19339S SNIPE SHOOTING 325 The books teach that the snipe rises with a zigzag
flight against the wind, darting to right and left with
such rapid flashes of speed that the best of skilful
sportsmen are puzzled, and consequently make many
a miss. The snipe, it is true, goes against the wind when
there is a wind, and zigzags a few times to rise up-
ward before taking a straight course. Many writers
on snipe shooting lay it down as correct that the
shooter, to take advantage of this peculiarity in ris-
ing, should walk down wind, or advance to the dog’s
point down wind, so that when the snipe is flushed
it will fly toward him. All such teachings savor of
the novice, or of a skill which needs nursing. All
the difficulties are greatly exaggerated, zigzag, swift
flight and all. The zigzag of the snipe a-wing is in
the beginning of its flight, and nothing is easier than
to wait
a moment till it straightens out on a straight
flight. Then the killing is a matter of shooting on
the wing, similar to other wing shooting. SNIPE SHOOTING As to walking down wind to secure a better shot,
the sportsman need not concern himself about it in
the least, excepting perhaps on such days as are cold,
and days when the birds are very wild and rise at the
extreme range of the gun. As with pigeon shooting,
the really good shot does not let his birds get far if
they rise within range. Whether they zigzag or not,
he snaps them as soon as they are on the wing; or
being well on the wing, he permits them to get into
steady flight, and then delivers his fire. There is on AMERICAN GAME BIRD SHOOTING 326 the part of the experienced shot no particular attempt
to reach the bird from a weak quarter. He takes the
shooting as it comes. On windy days, or when the weather is cold, the On windy days, or when the weather is cold, the
snipe may be very wild and rise at extreme ranges. Shooting then is quite as much a test of the gun as
it is a test of the shooter’s skill. Few writers, how-
ever, pay any heed to the distinction, and consider it
all, be the rise far or near, as a matter of skill alone. At best, walking down wind on snipe is an uncertain
advantage, for they can fly down or across wind with
a swiftness and ease which dispose very quickly of
any trifling advantage of a few yards taken up wind
for the start. The habits of snipe, as oftenest described, are their
habits when they are lean and wild, or wild from a
change from warm to cold or from calm to windy
weather. But to teach that such is their regular man-
ner of flight would be on a par with teaching that
quail live in the tree-tops because they sometimes take
refuge there. Even when lean and wild, on a calm day the snipe
does not strain the skill of a good shot. But on a
windy day it is a different proposition. The wild,
lean snipe can dart very swiftly across or down wind,
and if to this be added rise at long range, the shooting
becomes really difficult, though then, as mentioned be-
fore, it is also a test of the gun. SNIPE SHOOTING When wildest, the snipe is exceedingly restless and When wildest, the snipe is exceedingly restless and
moves fitfully from place to place. It then takes alarm SNIPE SHOOTING 327 quickly, flying high out of range, with its bill extended
straight ahead. It can pitch to the ground from its
highest flight, darting downward with stiffened wings
and alighting with the greatest ease. In the course of migration the birds stop in favorite In the course of migration the birds stop in favorite
places where food is abundant, and oftentimes there
remain till the weather becomes unpleasant. As a
rule, they arrive in the South in a lean condition. When lean they are also wilder, regardless of weather
conditions. Shooting then, if limited to times when they are
wild, is shooting in its most difficult phases. But as
mentioned before, such difficulties of snipe shooting are
not the average of snipe shooting. Snipe shooting as to possible quantity varies widely, Snipe shooting as to possible quantity varies widely,
one locality with another. One locality may contain
but a few snipe to reward the shooter’s efforts, while
in other nearby localities they may fairly swarm, as
in Louisiana and Texas in the fall and spring months,
when the birds are migrating. In those States they
generally remain several weeks to enjoy the food abun-
dance. Some scattered ones in the South may be
found all through the winter. The heavy rains of fall
and spring, frequently a downpour of days in the far
South, soften the fat alluvial prairie lands, thereby
fitting hundreds of square miles for the snipe’s habitat. In particularly favorable sections of the prairie, cotton,
corn and sugar fields, they may at times be found in
thousands. A dog in such shooting is an encumbrance,
except to act as a retriever. There is no woodcraft AMERICAN GAME BIRD SHOOTING 328 necessary in such shooting. The sportsman walks
along till the birds are walked up. So rapidly will
he sometimes flush them that at every step it is fire
and load, and fire and load again. At such times the
gun becomes too hot to hold, and the shooter must
perforce stop till it is cool enough to handle. SNIPE SHOOTING Enormous bags of snipe have been made, particu- Enormous bags of snipe have been made, particu-
larly in Louisiana and Texas, where the greater part
of the flight of North America congregates for a few
weeks in the period of snipe migration. One of the
greatest, and I believe that it is referred to now as
the greatest bag, was made many years ago by Mr. Pringle, a wealthy sugar planter, of Louisiana, who
had great fame as a sportsman of rare skill. He bagged
400 and some odd snipe in one day. This is a large
bag, indeed. It is but one of thousands of large bags
in that section, so common as to excite no special
comment. I have told of these matters to shooters in the North
whose ‘success was measured by a dozen snipe, more
or less, as the result of a day’s shooting. Such large
bags being outside of their personal experience, they
have been pleased to consider it an idle tale. They
seemed to think that their narrow experience in shoot-
ing a few birds over a few acres of ground each year
was the measure of the world over. In regard to the big bag made by Mr. Pringle, it In regard to the big bag made by Mr. Pringle, it
may be added, by way of explanation, that he had ne-
groes to assist him, some to carry the spare guns, oth-
ers to carry the ammunition and to retrieve the dead SNIPE
SHOOTING 329 birds. I have been told by men who have hunted with
him that he is a most indefatigable walker, and pos-
sesses extraordinary quickness and accuracy in the use
of the shotgun, snapping the birds almost on the instant
that they take wing. In that land of game abundance, at that day, it was
not considered unsportsmanlike to kill all that the
sportsman pleased to kill, for however great the bags,
there was no apparent diminution in the numbers of
the birds. If the sportsmen killed many, their neigh-
bors derived the benefit of it. The killing, too, was at
irregular intervals, differing from the steady drain
made on the bird supply day after day by those who
shoot for market. SNIPE SHOOTING This circumstance of the record bag was a happen-
ing of many years ago, when the sentiment concerning
game preservation was different everywhere, North and
South, from what it is to-day. As to snipe shooting, and the way of it, the proper As to snipe shooting, and the way of it, the proper
manner to shoot them is to go forth and shoot them. In other words, the set manner of doing this thing
and that thing, as taught by some writers, is all very
well if one can do no better. There is no rule whereby snipe shooting can be There is no rule whereby snipe shooting can be
made soft and easy, and there is no sportsman with
proper ambition who will care to have his skill less
than the best test that the bird can offer. If the sports-
man’s skill is equal to the test, practice will improve
it. In any event, there is at least the pleasure of try-
ing to cope with the conditions. The proper skill is AMERICAN GAME BIRD SHOOTING 339 that which takes the shooting as it comes, instead of
picking out the easy shots, or easy combinations to
secure them. The best snipe gun is moderately choked, or an im-
proved cylinder bore. As in all open shooting, good
work may be done with a full choke, since the shooter
can pick his distance to shoot his birds. However, it
is not every man who can wait on his bird, or who
can estimate distances at a glance; therefore, it is bet-
ter to have a scatter gun which will be available for
instant use when the bird rises. A 12-bore is most
commonly used, and as for the size of shot, No. 8s or
Qs or 10s are good, the latter being quite large enough
when the birds are fat and lazy. As a bird to shoot over dogs, the snipe is inferior. Sometimes it is in such abundance that a dog is un-
necessary. At other times it is so wild that it will not
lie to the dog at all. If fat and tame, it may lie too
well. Again, it will frequent marshes so wet, cold and
rank with marsh grasses that it is impossible for a dog
to work satisfactorily, however good his intentions and
ability may be. SNIPE SHOOTING Very few dogs have a natural fondness for work Very few dogs have a natural fondness for work
on snipe. It is acquired in most instances. Some
dogs, good on upland game birds, thoroughly detest the
snipe and refuse to recognize it. On the other hand,
some dogs like snipe as a bird to work on, though such
are exceptional. Considered strictly as a bird of the open, the snipe
affords excellent open shooting. Yet there is never SNIPE SHOOTING 331 the weird uncertainty about it that some writers have
discovered. No doubt an easy explanation of the diffi-
cult shooting is found in the manner in which the
shooter handles his gun rather than in the manner of
the snipe’s flight. When snipe are in great abundance, and can be When snipe are in great abundance, and can be
bagged with little effort by walking them up, the
sportsman soon tires of the sport. It is too easy then
to be considered sport. On the other hand, when they
are scarce, wild, and will not lie to a dog, it is too
difficult and uncertain. The dog is eliminated then
as a factor, and the shooting is largely a matter of
taking chances. When the happy medium is found,
the birds being neither too wild nor too tame, it 1s
excellent sport indeed, though in Louisiana I noted
that where there was an abundance of both snipe and
quail the sportsman quickly tired of snipe shooting and
gave quail the preference. QUAIL SHOOTING. The quails of America cover a greater territory than
any other of our game birds, and are more faithfully
pursued than any. They are found, interruptedly,
from points in Canada far down into Central America,
and beyond. Over the eastern half of the continent,
and far better known than any other species, bobwhite
stands alone. Moreover, by one means or another, he
has extended his range, first to the Rocky Moun-
tains and then beyond that to the Pacific coast, while
the Texas form, and some Mexican species, drift far to
the southward. Bobwhite has been called the game bird of America, Bobwhite has been called the game bird of America,
and from early days has furnished sport to north-
erner and to southerner alike. It was never abundant in
Canada, though the gunners of southwestern Ontario
used to get a few; but forty or fifty years ago it was
very abundant in central New York, from which coun-
try, we believe, it has long been exterminated. Many
years ago the late Prof. O. C. Marsh, of Yale Col-
lege, used to relate with gusto stories of the good
quail shooting that he had had near Lockport, N. Y.,
in the days before the Civil War. Hard winters and overshooting have long ago al- Hard winters and overshooting have long ago al-
most exterminated the quail in Massachusetts, Con-
necticut, New York, and portions of Pennsylvania, yet
332 332 332 332 QUAIL SHOOTING ga it cannot be doubted that self-control on the part of
gunners would result in the re-establishing of these
birds in goodly numbers through many sections of
their former range. In a certain Connecticut town,
where the quail had been practically exterminated, the
gunners, for several years, have had an understand-
ing that no one of them should disturb these birds. The result of this protection has been that in the spring
of 1910 quail might be heard calling from every side
in certain sections of this town. It is said by the
gunners there that there will be great quail shooting
this fall. This may mean that the gunners will again
kill off all the quail, and that again for six or seven
years they will have no shooting. QUAIL SHOOTING. it cannot be doubted that self-control on the part of
gunners would result in the re-establishing of these
birds in goodly numbers through many sections of
their former range. In a certain Connecticut town,
where the quail had been practically exterminated, the In the West are the beautiful plumed and helmeted
quails of the mountain, of the valley and of the desert
—and those other birds of curious plumage and soft,
thick crest, Mearns’ quail and the scaled quail, also
known, respectively, as “fool quail” and ‘“‘cotton-head.”
Except for Mearns’ quail, these western forms do Except for Mearns’ quail, these western forms do
not, we are usually told, offer good shooting. They run
like deer before the dogs, rise at long distances, make
long flights, and as soon as they reach the ground
start running again. Mearns’ quail is said to be
an exception to this rule, and lies close and hard. So
difficult are valley quail to shoot over points, that
many gunners of southern California do not attempt
to use a dog to find them, while others use dogs only
for retrieving, or to flush the birds while running. Few people in the United States have had greater AMERICAN GAME BIRD SHOOTING 334 experience in quail shooting than Mr. B. Waters, who
has written one of the very best articles on this sub-
ject that has ever been penned, which in substance
reads as follows: BOBWHITE SHOOTING. From the personal point of view, each one gener-
ally has his own preference in respect to the bird which
he prefers to shoot to secure the greatest pleasure, and
this preference in turn determines the shooter’s opin-
ion that such bird is therefore the best of all birds
for the purpose of sport. Thus, one prefers ducks, and
not considering that his own personal idiosyncrasies,
or greater success, or habit and long association, or
what not, may have much to do with his preference, he
solemnly affirms that duck shooting is the best of all
shooting. And so with him whose choice of sport is
the shooting of some other bird—that bird is sure to
be exalted above all others. But from the standpoint of the greatest good to
the greatest number, quail shooting, for many reasons,
is the best of all shooting. It is a kind which affords
such mixed shooting—open and cover, slow and swift
—that shots can be found to meet the skill and fancy
of all, be the former
little or great and the latter
fastidious. There is much of the open quail shooting which is
not so difficult as to dishearten him of moderate skill,
while, on the other hand, shooting in cover tests the QUAIL SHOOTING 335 skill of the most expert sportsman. And again, taken
all in all, whether in open or cover, the quail shooter
of good average skill can compass a good showing
in results, and thus enjoy the pleasure which comes
from reasonable success. In this connection it may not be amiss to maintain In this connection it may not be amiss to maintain
that a certain degree of success
is essential to the
shooter’s pleasure. Many writers deprecate the con-
sideration of the bag, treating it as an irrelevant, gross
incident, so dominated by the beauties of nature and
the ethics of shooting, in the abstract, that it should
be mentioned in hushed tones or viewed with eyes
askance. The beautiful and the useful of sport should
go hand in hand. Each is a part of the great whole,
and as such should be equal factors of sportsmanship. To the sentimental, which ennobles and adorns the
useful of life, there must be added the material and
the practical. To the shooter there must be a reward
for his efforts. BOBWHITE SHOOTING. It has often been said that it is not
all of shooting to shoot, nor all of fishing to fish, for-
getting the converse, that, all of shooting or fishing
being absent, there is no shooting nor fishing at all. As to quail shooting in respect to quantity, there As to quail shooting in respect to quantity, there
is more of it than there is of any other kind of shoot-
ing, hence each shooter can better satisfy his longings
for sport if it be measured by the possibilities of the
bag or the number of opportunities offered. And
there is also more of it when measured by the matter
of time, for it extends through a season of about five
months, taking it as it is in the North and South. AMERICAN GAME BIRD SHOOTING 336 Thus, the man whose business cares leave him but a
few days for shooting, and these at no definite time,
has more possibilities of sport on quail than on any
other bird. But the very abundance of the quail seems to have But the very abundance of the quail seems to have
checked the proper appreciation of it. Not that it is
treated with neglect, but there seems to be a lack of
the enthusiasm and lavish use of the superlative, so
often to be noted when writers are discoursing on
the ruffed grouse or the woodcock. Nevertheless, it
is not uncommon to have a keen relish for what is
rare, even if it be not of the best, while the good may
be so common as to escape notice. The quail is more uniformly and widely distributed The quail is more uniformly and widely distributed
throughout the United States than is any other game
bird. Its habitat generally comprises both open and
cover, though whole districts are exceptions, as will
be touched on later; thus, besides giving the sports-
man a mixed style of shooting, is added the charm of
constant variety, and testing of the sportsman’s skill
in woodcraft. It differs from the ruffed grouse, whose
home is in the woods, a much smaller section relatively. BOBWHITE SHOOTING. All works on natural history, so far as I know, teach All works on natural history, so far as I know, teach
that the quail’s home comprises conjointly both open
and cover; and while this is true in a general way,
there are important exceptions to it, so much so that
a work devoted to the habits and habitat of the quail
as they are in one locality might be distinctly erroneous
if applied to the quail of some other locality. In this
respect it differs from the prairie chicken and the ruffed QUAIL SHOOTING 337 grouse—for of the one it may be said, without quali-
fication, that it is a bird of the prairie; of the other,
that it is a bird of the woods. Such sayings of them
will be found to be true wherever those birds may be
found. The quail thrives wherever it can obtain a food sup-
ply, in open or in cover. It readily adjusts its habits
to the dominating circumstances of food and cover,
whether it be in prairie or woods, or a country com-
prising both open and cover. In the country north of the Ohio and east of the In the country north of the Ohio and east of the
Mississippi River, it frequents the open fields largely,
preferring such as have a good food supply, with
hedges or old walls and fences fringed with brush, or
nearby woods and thickets to which it can run or
fly for shelter or safety. In such sections it rarely goes
far into the woods, preferring to skirt around the outer
edges of them, merely for protection and shelter. The
hawks are its deadly enemies, and it needs ever to be
alert in avoiding them. The quail oftenest roosts in the open fields, where
there is at least a few inches growth of grass, stubble
or weeds for concealment, and it uses the same place
many times if not constantly disturbed. This is indi-
cated by the grass or other vegetation being beaten
down in the roost, a small circular opening, about
two feet in diameter, and the pile of droppings in the
center of it. BOBWHITE SHOOTING. The birds huddle on the ground, bunched up close The birds huddle on the ground, bunched up close
in a circular form, with their heads outside; thus all AMERICAN GAME BIRD SHOOTING 338 face toward the outer circumference of the circle, which
cannot be approached without coming to the view of
some bird. This arrangement is said to prove ad-
mirable for the safety of the whole. In theory it
seems a wise arrangement; in practice it works very
badly, since they often fly reluctantly when they have
comfortably adjusted themselves for a night’s rest. The pointer or setter may also draw very close to them
then, generally doing it with greater precision than
when they are more scattered about, the evening hours
being more favorable for strong scent and accurate
pursuit. Were not dogs trained to such stanchness
as is required in shooting, they could easily, at such
juncture, spring in and capture, as indeed some par-
tially trained dogs will do under the circumstances, In the States of greatest bird abundance, as in Ar-
kansas, Mississippi, etc., and where there are many
ragweed fields, very destructive shooting often takes
place near the twilight hours, when the birds have set-
tled themselves for their slumbers. When the dog
points the bevy the shooter places himself at the proper
distance from the roost to obtain the best scatter of
the shot. Then the huddled birds, being flushed, swarm
up loosely together for three or four feet, when the
shooter takes a snap shot at them, and often does
nearly as much damage as if he had potted them on
the ground. It is hardly necessary to add that this
practice is disapproved by all true sportsmen. In Minnesota, Dakota, Nebraska, Kansas, and other In Minnesota, Dakota, Nebraska, Kansas, and other
prairie States, the quail readily adapts itself to the QUAIL SHOOTING 339 local peculiarities of the different sections, utilizing
such slight advantages as may offer, as hedges, fences,
the cover with which most streams are fringed, or
high weeds. The quail sadly needs cover for its protection, its
destroyers being both of air and earth—hawks, foxes,
cats, dogs—and the eggs, too, fall a prey to the rapa-
cious appetites of some of its enemies. BOBWHITE SHOOTING. In the South
the cur dogs of the negroes—every family owning one
or more, all kept in a kind of half-famished condition—
prowl through the fields seeking for food; they are
the very worst of egg destroyers. Were the quail
not so hardy and prolific, its fate would be swift, and
extermination certain. The negroes’ dogs seem to be almost omnivorous. In the fall they may be seen making daily visits to
some persimmon tree, under which they eat the fallen
fruit with apparent relish. Those which have some
claim to hound blood are not averse to making a meal
in the corn field, on corn when it is in the milky stage. With such rapacious enemies to contend against, the
destruction of the quail must be great, but in addi-
tion to all that, many are trapped and netted, methods
which destroy whole bevies at a time. But to return to the matter of the quail’s habitat: But to return to the matter of the quail’s habitat:
in certain parts of the South, as in the oak woods in
sections of Alabama, Texas, Arkansas, or in the pine
woods of Louisiana, Mississippi, etc., the quail may
live wholly in the woods, food, always a first consid-
eration, being there secured in abundance. AMERICAN GAME BIRD SHOOTING 340 In cover, some of the shooting is easy and some
of it very difficult, though hardly ranking in difficulty
with ruffed grouse shooting. The quail is neither so
wary nor so wild as the ruffed grouse. Shooting in
some parts of the pine woods is almost as easy shoot-
ing as shooting in the open, the ground being bare
except for its covering of dry pine needles. The
smooth trunks of the pine trees, standing several yards
apart, and free from limbs for thirty or forty feet. offer
no serious obstacle to the shooting. In other sections
of the pine woods, where the growth of the trees is
more stunted, and the limbs grow from near the
ground up, the difficulty of the shooting is second to
none, and in some sections is almost prohibitive. BOBWHITE SHOOTING. Again, there are sections wherein the quails live
on the open prairie, as in parts of Arkansas, and, the
shooting being strictly open, it much resembles chicken
shooting, excepting the difference in the size and speed
of the two birds, the quail being much the quicker to
get away at the start. The quail makes its flight in
the open prairie, alighting near any little bit of shrub-
bery, be it no more than a bush or two of sumach,
which, by the way, grows here and there on the prairie
in Arkansas. In the woods, when pursued, it fre-
quently takes to the tree-tops for safety, where it is
hidden indeed. On warm days, or when there has
been a long spell of pleasant weather, it is far less
wild than when the weather has been stormy, or when
there has been a sudden change from warm to cold. Such changes add to the difficulty of the shooting. On
point. On
point QUAIL SHOOTING 341 In the North, the quail makes its home where some
buckwheat or other grain field is available for a food
supply. It so arranges its haunts that it has some
cover within easy flight, in the densest part of which
it seeks safety when flushed, not refusing the heavily
timbered swamps if too much persecuted by the shooter. In such places it has an excellent chance of escape
from pursuit, or may foil its pursuer by simply run-
ning away; or, if pressed to take flight, it has many
chances for safety owing to the difficulty of shooting
accurately in the dense cover. New England shooting is the most difficult of all New England shooting is the most difficult of all
quail shooting, excepting, perhaps, shooting in the
dense pines and cat briers of the South. Then, to
have any satisfactory success, the scattered birds must
be diligently followed and sought in the thickets, be
they ever so dense. In this respect it differs from
shooting in the sections of more abundance, where
such close attention to the scattered birds is unneces-
sary, either for sport or the interest of the bag. In the South, where there is an abundance of birds,
comparatively, the sportsman rarely tarries with a bevy
which gives him any special difficulty. It is much
easier, and more satisfactory, to go on and seek more
birds. BOBWHITE SHOOTING. For this reason, even under favorable oppor-
tunities, the scattered birds are never, as a rule, hunted
till the last one is flushed, and flushed again, when it
is possible, as in the North. In New England, buckwheat fields are the quails’ In New England, buckwheat fields are the quails’
choicest resorts for food, and any adjacent brush, or AMERICAN GAME BIRD SHOOTING 342 long grass of swamp or upland, or the skirts of woods,
afford the shelter and protection that they need or
seek. No doubt the birds become wilder in the North
than in the South, for, first of all, the inclement
weather of the North tends to make them so, and there
is a much more relentless pursuit of them by the
shooter. The birds being scarce, after the bevy is scat-
tered the search continues while there is a hope of
finding a single remaining one; and if success with
them has been unsatisfactory, the shooter may return
later to catch them, when they are whistling to each
other in the attempt to come together as a bevy. In the broad plantations of Mississippi, Alabama, In the broad plantations of Mississippi, Alabama,
Tennessee, Arkansas, etc., a dog of reasonably wide
range is necessary, much wider than would be either
desirable or useful in New England, or similar sec-
tions in respect to cover; for there is much of the
country in the South, open and cover, which every-
where affords a bountiful food supply, and, therefore,
the birds are to be found in the most unexpected places. The cover and cultivated fields of the South do not
aid the hunter’s judgment as to quail haunts to the
degree that they do in New England shooting. In
the latter place there are comparatively few areas in
which the birds can get both food and cover together,
or even food alone, and the sportsman soon learns to
distinguish the favorable places. In the South, in cover
and open, there is food in abundance everywhere. There are vast fields, some of which are overgrown
with sedge grass, others with weeds, with fields of QUAIL SHOOTING 343 cotton and corn interspersed, any part of which is a
fit habitat for the birds; thus, the dog working out
such ground in the South can, as a rule, beat out all
parts of it with probable success. BOBWHITE SHOOTING. In the more open
grounds of the South the dog can be seen at long dis-
tances, so that a wide range is not detrimental in itself,
providing that the dog is really working to the gun,
and not self-hunting or semi-hunting. As to the manner in which the dog should hunt, no As to the manner in which the dog should hunt, no
hard and fast rule can be laid down which would apply
to all sections. Whatever may be the quail’s abiding place, it learns Whatever may be the quail’s abiding place, it learns
to make the most of its surroundings in promoting its
own safety and interests. It learns whether it is better
to fly or run in evading its pursuer, and the best strat-
egy to attain that end. If good cover is conveniently
near, it may trust to its wings at once for safety, and
to its legs and wings if followed into the cover. If the country is open, or with narrow and insuffi- If the country is open, or with narrow and insuffi-
cient cover, as in parts of Louisiana, and other sec-
tions where the ground is thoroughly cultivated,
it
takes a great deal to its legs and cunning devices. In
working on such birds the dog must learn to govern
his work by the circumstances of it. He might be an
excellent performer on quail in the North, and a poor
one in the South, or he might be a good one in Mis-
sissippi and a poor one in Louisiana, though the pre-
sumption is that if he were good in one section he
would become so in any other section after the neces-
sary experience. AMERICAN GAME BIRD SHOOTING 344 Many of the plantations of Louisiana are drained
by open ditches running parallel at reasonably equal
distances from each other, though the distances may
vary greatly one field with another and may be 50
or 200 yards, more or less, apart, while other ditches
of like arrangement intersect them at right angles;
thus a plantation may be cut with more or less regu-
larity into small squares surrounded by ditches. Some
plantations may be irregularly ditched, while others
with a far greater watershed may not be ditched at
all. BOBWHITE SHOOTING. The heavy rains round out the banks of the ditches
and their bottoms, and a fringe of weeds and brush,
thick and thin in places, strings along the banks and
makes a fairly good shelter for the quail. On these
land squares are grown cotton, or corn, or nothing,
as the case may be, though, if not cultivated, there is
always certain to be a good crop of weeds, affording
plenty of quail food. When flushed in such places the
quail may fly a few yards to the first ditch, or may
cross over two or three ditches before finding a place
to its liking. As mentioned elsewhere, the state of the weather
may greatly affect the quail’s habits. The bevy having
gone to the ditches for safety, the dog, to be useful,
must have great superiority in roading if he pursues
satisfactorily. When in the ditch the birds run swiftly
along the bottom. It is almost impossible, at first, to
induce the green dog to go into the ditch, or, being in,
it is impossible to make him remain there, though it
may not be over a foot deep, and dry. The green dog ‘sourd
aq}
ur
3urjo0o4S ‘sour
aq}
ur
3urjo0o4S QUAIL SHOOTING 345 will promptly cross out from one side to the other,
missing the scent, and accomplishing nothing useful. He does not know what is required of him. But once
he catches the idea, he soon improves on it, following
carefully along the bottom of the ditch and pointing
the scattered birds here and there, every few yards
apart, in ones and twos, the shooter having a good
opportunity from his position on the outside to kill as
the birds fly out. The shooting along ditches is not so easy as one The shooting along ditches is not so easy as one
might imagine. Sometimes the birds run swiftly sev-
eral hundred yards or more in the ditch, and may then
run out and across to other ditches, giving a trail which
may try the most experienced dogs to follow. BOBWHITE SHOOTING. If the birds happen to be near a cotton or corn field,
where the ground is bare, and there are no ditches for
concealment, they may run so fast and far that the
dog may never approach near enough to them to secure
a point, and the shooter who is inexperienced in this
work will be likely to think that his dog is surely
deceiving him. When near the woods, or switch-cane, the birds
often take shelter therein, and when in the latter cover
it is well to abandon further pursuit of them. In the sugar country, where there may be corn fields In the sugar country, where there may be corn fields
here and there among the broad levels of the sugar-
cane, the character of the shooting again changes. Many birds will be found in and around the corn fields,
and then it is very pretty shooting. It may not be amiss to mention, for the benefit of It may not be amiss to mention, for the benefit of AMERICAN GAME BIRD SHOOTING 346 those who shoot in the sugar country for the first time,
that they should keep their dogs out of sugar-cane
fields as much as possible. The cane, in harvesting, is
cut diagonally across with a knife, thus leaving a
stump with an edge which will cut a dog’s foot almost
as a knife would. It is a common matter for a dog to
split his toes or heel on cane stumps, with the result-
ant crippled condition, and no more work from him
for a time. In Mississippi, in the midwinter season, the birds
stay mostly in the woods. Good shooting may be had
in the South from the middle of November to the
first of March. Many of the Southern States have a
longer legal open season, but the dense cover and warm
weather make a natural limitation to the sport. The
weather is mild, the birds are strong, and the sport is
at its best, in the winter months of the South. Quail shooting, in the main, is close shooting, as to
the ranges at which the birds are killed. Most birds
drop within twenty-five yards, some much nearer than
that. A gun weighing from 6% to 7% pounds is of ample
weight. The 12-bore is most commonly used, though
the 16 and 2o-bores are excellent, and are preferred
by many sportsmen. BOBWHITE SHOOTING. Some shooters use guns of 28
caliber, and are enthusiastic over their work. As a
matter of course, the smaller bores may be much lighter
than the 12-bore. The 16 and 20-bores being smaller, their killing The 16 and 20-bores being smaller, their killing
circle is less, though they shoot with good force in QUAIL SHOOTING 347 comparison with the 12-bore. Closer holding is re-
quired to shoot them well. Whichever bore is used,
it should not be closely choked. There is no need of
a choke bore in quail shooting. It is an extremely difficult matter to induce the It is an extremely difficult matter to induce the
average shooter to use an improved cylinder-bore gun. The recommendation to use an open bore seems to be
construed as reflecting on his ability to shoot a close
gun, instead of being accepted as sound information
concerning the gun fit for that particular kind of game. It requires time to effect a reformation concerning the
use of the choke bores in quail shooting. The sports-
man should go forth equipped for his sport according
to his needs, and not to the whimsicalities of senseless
fashion. He should not take a full-choked gun in
cover, nor a cylinder-bore gun to shoot ducks. There
should be intelligent adjustment of means to ends. In-
dustry and skill and woodcraft should not be balked
by visionary theories and inappropriate weapons. The foregoing is written of the quail as it refers to
man’s pleasure afield with dog and gun. Naturally it
is not fearful of man, and rather prefers to dwell near
his haunts, not from an affection for him, but from
the fact that near cultivated sections there is always
more food to be found than in the uncultivated. The
matter of providing food for itself and its young is
quite as constant and insistent in the life of the quail
as it is in the life of man. It often nests in or near the cultivated field. Its It often nests in or near the cultivated field. Its
cheery, ventriloquial whistle, reiterating its favorite AMERICAN GAME BIRD SHOOTING 348 utterance, bobwhite, may be heard about the farms,
particularly in the morning hours, and bobwhite has
come into use to designate the bird itself. BOBWHITE SHOOTING. Sometimes
the call is uttered with a short introductory note, and
these, with a few alarm calls, or calls of inquiry when
the birds have become scattered, seem to be about all
the vocal means of communication which these birds
have. In many parts of the South, where the quail are in
greater abundance, their sweet notes may be heard in
many directions, at many distances, in the early morn-
ing and evening hours. These calls work great harm
to it, for by them the shooter learns the whereabouts
of all the birds in his neighborhood, and locates the
haunts of every bevy. , In the fall, the rallying cry
often serves to inform the sportsman as to the best
course for the morning’s hunt. In the South the quail
is called partridge. In the fall, when the shooting opens, the quail soon In the fall, when the shooting opens, the quail soon
learns of the gunning dangers, and its habits thereupon
change quickly to conform to a life of greater safety,
though it will run many risks to be near an abundance
of food. However, when danger is impending,
it
avoids the open much more than when danger is not,
and is more alert, quicker to suspect mankind, and
quicker to take alarm. When spring returns, the quail seem to lose their When spring returns, the quail seem to lose their
fears of man, and they breed with little reference to
concealment from him. Their confidence is unimpaired QUAIL SHOOTING 349 till fall approaches, when there is a repetition of all
the fears and troubles and dangers of preceding years. He who can average three kills out of six shots, He who can average three kills out of six shots,
cover and open, is an excellent marksman. The shooter
may make a run of ten or twenty straight kills, but
soon there are sure to come misses if he does not pick
his shots. In winter the shooting is much more diffi-
cult than in the fall. Of course, the man who never
misses is of the parlor, not of the field. The home of the greater number of the American
quails of the extreme southwest or on the Pacific coast
is in a country of great aridity. BOBWHITE SHOOTING. Moreover, much of
the vegetation of that country consists of thorny and
spiny plants, of which many are cactus, yucca or
mesquite. It is evident that over much of this country
the use of dogs in shooting these birds would be diffi-
cult, if not impracticable. For much of the year the
ground is so dry that no scent would lie, and a dog,
unless trained for that particular work, would be at
a great disadvantage. Moreover, in the swift traveling
performed by hunting dogs, as they are trained in the
South for field trial work, a dog, in a very short time,
would inevitably become crippled by the spines of the
cactus, which he could not help constantly running
over or into. If to these difficulties we add the fact
that the quails seldom or never lie to a dog, but run as
hard as they can at the first intimation of danger,
endeavoring to get into the thick brush, through which
they can thread their way faster than any animal can AMERICAN GAME BIRD SHOOTING 350 follow them, it will be seen that many of these quail
cannot be pursued for sport, as sport is commonly un-
derstood; that is to say, the shooting of a game bird
over a pointing dog. This a priori conclusion is con-
firmed by statements made by sportsmen who have
lived for many years in Arizona, one of whom, Mr. Herbert Brown, not only a sportsman, but a field natur-
alist of great ability, tells me that he has never heard
of a dog being used on Gambel’s partridge in all the
time he has lived in the southwest. Yet Allen Kelly
says that in the irrigated districts of the Imperial
Valley, Cal., Gambel’s quail lies well where there is
cover, but on the bare ground runs like a deer. I have seen the valley quail in southern California I have seen the valley quail in southern California
and the mountain
quail in the Sierras,
but have
never yet seen either hunted with dogs. That the
mountain quail can be shot over dogs is hardly to be
doubted, but the case is different with the valley quail
living in the lowlands of dry California. On the other
hand, in Vancouver Island the introduced valley quail
sometimes lies to a dog among the thick undergrowth,
much as the eastern quail would lie. BOBWHITE SHOOTING. When startled
they get up in a thick coveys of fifteen or twenty birds
and scatter and sometimes lie well. VALLEY QUAIL. Of the California valley quail in the vicinity of
Pasadena, N. P. Leach says on this point:
“Up here
on the mesas and among the sage brush and grease- QUAIL SHOOTING 351 wood in the foothills we use dogs when hunting quail,
chiefly for flushing and retrieving, but down on the
plains, amid the cactus and low brush, some hunters
use both setters and pointers. There, when you flush
a covey of quail and shoot in among them or over
them, they will very often scatter and then hide under
the cactus and lie to point. It is a cruel place to work
a dog. The first day that I took my dog down there,
he filled his shoulders and breast full of cactus spines. We pulled out most of them, but even then he was
in no condition to work for some days. “Over one-half of the quail hunters here do not “Over one-half of the quail hunters here do not
use a dog, and ninety per cent. of the other half use
spaniels for retrieving. The other small minority use
either setters or pointers. “When my spaniel flushes a covey, some of them “When my spaniel flushes a covey, some of them
fly my way, so that I am very sure of a double shot,
and sometimes an extra double at some of the laggards. Then, as the dog can outrun the birds, he keeps them
moving, which often gives me shots at fast-flying
birds. I have him so trained that I can control him
with my whistle; that is, bring him into heel, where
he will remain until ‘I tell him to go.”
It is hardly to be doubted that if either the valley, It is hardly to be doubted that if either the valley,
the mountain, or, in fact, Gambel’s quail, were to be
introduced in any section where the climate was mild
enough, they would, if properly protected, do well and
increase. The beautiful valley quail readily adapts
itself to confinement, becomes not at all shy, and has
been frequently known to build its nest and lay its AMERICAN GAME BIRD SHOOTING 352 eges. We do not know that any have been reared,
but no doubt this might readily enough be done, were
proper care exercised. GAMBEL’S QUAIL Dr. Coues, in an article in the Jbis many years ago,
said of the shooting of this bird in its home:
“Compared with the eastern quail (O. virginianus), “Compared with the eastern quail (O. virginianus),
from the sportsman’s standpoint, Gambel’s plumed quail
is more difficult to kill. Not that it rises with more
startling suddenness, or flies faster, for I noticed no
material differences in these respects; but when a bevy
is flushed, and one, or at most two, birds secured, it
is exceedingly difficult, and usually only by chance, that
other shots are obtained; for except under certain cir-
cumstances, they lie very badly, and when they drop
after being for the first time started it is, usually, not
to squat and remain hidden, but to run as fast and far
as possible; so that, if found at all, it will be dozens of
yards from where they were marked down. This pro-
pensity to run, which is also a great obstacle to their
being flushed within proper distance, is exceedingly
troublesome both to the sportsman and his dog; so
much so, that the best trained dogs can often be of lit-
tle or no service. It is true that this habit of running
affords many shots on the ground, and often places
the whole bevy directly under fire, but no true sports-
man would thus ingloriously fill his bag by potting a
bevy of such noble game birds. Like all their tribe, QUAIL SHOOTING 353 their flight is exceedingly rapid and vigorous, but it
is always even and direct, so that it only requires a
very quick hand and eye, and the usual intuitive calcu-
lation for cross shots, to kill them readily. Notwith-
standing all I have heard to the contrary, I consider
them far from being tough birds, and No. 8 shot is
abundantly large enough for them. The fault in most
cases, I presume, is with the shooter rather than the
shot. I may add that many of the places in which
bevies are found would compare unfavorably with the
worst woodcock brake of the Eastern States as regards
facilities for obtaining a fair shot. I have had a bevy
flushed all around me and hardly caught a glimpse of
a feather. But these, and all other difficulties, should
only increase one’s ardor and confer additional value
on the lovely birds when obtained.’ MOUNTAIN QUAIL. In the California Mountains, near various hill towns,
the mountain quail are often abundant, and late in
October, or early in November, after rain, or snows
turning to rain, we have occasionally started coveys
which, flying for no great distance, would pitch down
and lie so close that they could be walked up by a
gunner and a few good shots had. In such situations,
and at a time when the ground was moist enough to
make the scent lie well, I should expect to have good
shooting with a careful dog. In the dry West, however, conditions for shooting In the dry West, however, conditions for shooting AMERICAN GAME BIRD SHOOTING 354 differ so widely from those which prevail in the more
humid East that it will probably take a long time
for gunners and dogs to work out a system, at once
agreeable and effective, for shooting these game birds. Dr. Elliot declares that many are shot over dogs, but Dr. Elliot declares that many are shot over dogs, but
that far more are trapped and netted. The situation is still more difficult when birds of The situation is still more difficult when birds of
the extremely dry country of Arizona and New Mexico
are in question, though, as will be seen further on,
Mearns’ quail appears in quite a different category. SCALED QUAIL. Of his attempts to shoot the scaled, or blue quail,
Dr. Coues says:
“This species is a bird of noticeably terrestrial hab- “This species is a bird of noticeably terrestrial hab-
its, rarely taking to trees or bushes unless hard pressed
in one of those extremities into which some people
are fond of forcing any birds large enough to be
worth a charge of shot, and wary enough to make it
exciting sport to penetrate their poor bodies with it. It generally trusts to its legs rather than its wings,
though these are not at all deficient in size or strength. On level ground it glides along with marvelous celerity,
and makes good progress over the most rocky and
difficult places. As a consequence, it is rather diffi-
cult to shoot fairly, though it may be ‘potted’ in great
style by one so disposed; and it will probably require
several generations in training before it can be taught
to lie well to a dog. I am inclined to think, indeed, QUAIL SHOOTING 355 that the lying of quail, an essential feature for the chase
in its perfection, is almost as much a result of education,
as the ‘pointing’ that the intelligent brute who helps us
kill them has learned. In a primitive and strictly
natural condition, quail, as a general rule, rather use
their legs to escape pursuit than squat and attempt
to hide. That the reverse is the case with the Virginia
quail, I am perfectly aware, but this proves nothing
to the contrary, and I am inclined to think its crouch-
ing, till almost trodden upon, to be an acquired trick. This would surely be a poor way of escape from any
of its natural enemies—any carnivorous bird or mam-
mal; yet they find it to succeed so well against their
chief persecutor, that he has had to call in the aid of
a sharper-sighted, sharper-nosed brute than himself,
else he might stumble over stubble-fields all day with-
out seeing a bird, except by accident. I presume that
Virginia quail in the days of Captain Smith and Poca-
hontas were very much in the social status of the
Arizonian to-day; and these certainly trust to their
legs and wings rather than to the artifice of thrusting
their heads in tufts of grass and then fancying they
are safe.” Dr. D. G. SCALED QUAIL. Elliott,
a sportsman of long experience,
has this to say about the habits of the blue quail when
pursued :
“This bird runs with great speed and seems to be “This bird runs with great speed and seems to be
able to keep it up for a long distance, and flies with
much reluctance, alighting almost immediately and be-
ginning to run at once. When compelled to take wing, AMERICAN GAME BIRD SHOOTING 356 it rises with the usual whir-r-r, and proceeds on a
slightly curved line, rather straight ahead, and if, on
alighting, it should stop for a moment, it is almost at
once under the cover of some cactus or other low bush,
which affords a place of concealment; and from which
it can watch its pursuer, before starting to run again. “A dog is practically
useless
for hunting
the “A dog is practically
useless
for hunting
the
scaled partridge, for if he is well broken and attempts
to point a covey, the birds will run several hundred
yards, while he is standing, and then will add several
hundred more while he is trailing them, and the poor
animal becomes bewildered and disgusted and is apt
to run also. I know nothing so trying to the patience
of a sportsman as the tactics of this species, unless it
be the similar habits of other crested quail. Asa
rule,
this species was not very much hunted in the localities
I met with it, and it always seemed to me rather
singular that they should be so wary, for that is an
attribute
that wild creatures
usually acquire,
after
having made the acquaintance of man and learned
that his presence always brought wounds and death,
and that safety to themselves was only to be obtained
by leaving his vicinity as soon as possible. But these
birds seem instinctively to have ascertained this fact
before they ever saw a human being, and decamp at
once whenever a man appears.” QUAIL SHOOTING 357 MEARNS’ QUAIL. With Mearns’ quail the case is quite different. This
bird, while not at all shy, possesses markedly swift
flight, and lies exceedingly close. Dr. L. C. Frick, who has had much experience in Dr. L. C. Frick, who has had much experience in
shooting them in New Mexico, considers them a better
game bird than bobwhite. In shooting them he uses
pointer dogs, which have proved very efficient. Dr. Frick believes that if this bird could be introduced
into the Mississippi Valley and the country east of it,
it would at once become enormously popular as a game
bird. He mentions as a great point in favor of this
species, that when the birds rise the sexes can always
be distinguished, and that therefore cocks may be se-
lected for killing and the hens preserved. He believes
that the males greatly exceed the females in number,
and that therefore it is practicable to do a good deal
of shooting without lessening the productive power
of the birds in a particular district. The eggs are given
by Captain Bendire as eight or ten in number, but, on
the other hand, Mr. Nelson, who has been much in
the range of these birds, says: “T have never seen the Massena partridge in coveys
larger than would be attributed to a pair of adults with
a small brood of young. Frequently a pair raise but
three or four, and I do not remember having ever seen
more than six or seven of these birds in a covey.” Mearns’ quail is odd in appearance and odd in habits,
but one of its chief peculiarities seems to be that in AMERICAN GAME BIRD SHOOTING 358 a country where all the quail take to their heels and
run like race-horses, it alone, when it pitches down,
lies hard, and will let the dogs get up close to it before
flushing. We do not understand that.the birds are
particularly hard to hit. Though they fly swiftly, the
shooting is usually open, and only reasonable quickness
is required to hit the bird. RUFFED GROUSE SHOOTING. Dwelling in many sorts of country, sometimes, as
in the East, where it is constantly pursued, cunning to
the last degree, and practicing every stratagem; or
again, in the wild regions, dreading only its natural
enemies, and thus
fearless and bold, and trusting
wholly to strength of wing, the ruffed grouse is killed
in many ways and under varying conditions. Where
it is ignorant of firearms or of the danger from man,
it permits half a dozen shots to be fired at it at close
range, or sometimes may even be noosed from its perch
on a limb by a bit of string tied to the end of a pole. A graphic account of ruffed grouse shooting in a A graphic account of ruffed grouse shooting in a
region where they had been little disturbed, yet were
not wholly tame, was contributed to Forest and Stream
many years ago by that charming writer and good
sportsman, Mr. T. S. Van Dyke. In those days ruffed
grouse were abundant in the region referred to, as,
in fact, they have been up to comparatively recent times
in that country, as well as in certain sections of Michi-
gan, where, within only a few years, we have heard
of from twenty-five to thirty birds being killed in a
day by two guns. We give the substance of Mr. Van
Dyke’s account as follows: 359 359 AMERICAN GAME BIRD SHOOTING 360 Who that has heard the somber shades of the dense
pine forest throb beneath the strokes of his hoarse re-
sounding wing, or in the autumn woods has seen him
flash for an instant amid the hues of crimson and gold,
or pierce like a shaft of light the dark green of the
cat-brier swamp, can ever forget the ruffed grouse? What sportsman can forget the feelings with which
he has heard his drum-beat echo from the dark moun-
tain side, or through the bursting woods of spring,
or in those soft, still autumn days when the leaves
are falling through the mellow haze of Indian summer,
or, as sometimes heard, in the noon of night, in the
depths of the forest primeval? RUFFED GROUSE SHOOTING. Few pictures hang
more bright in the inner chamber of the sportsman’s
soul than the broad fanlike tail spread along his path
as he treads the trail of the deer, or its dark bands
shining on the carpet of checkered leaves or sweeping
over the mossy carpet of wintergreen or vanishing in
the heavy green of the laurel brake. Not even the majestic woodcock, with his solemn Not even the majestic woodcock, with his solemn
dignity; not bobwhite, with his sweet, graceful ways
and artless beauty; not the brilliant but erratic little
genius of the boggy meadow; not the noble turkey,
with his beamy bronze and bearded breast, can raise
such tender memories as this grouse. For all these
must be sought, and often sought in vain, in their
native haunts. But the ruffed grouse is a more famil-
iar spirit, and many a time plays across the sports-
man’s path when wandering over the sapling-clad slope
where the autumn woodcock lies in the full bloom "yjNour Ur pIIg YIM Buyurod 19}39g "yjNour Ur pIIg YIM Buyurod 19}39g SHOOTING THE WOODS GROUSE 361 of life and
fatness,
or when
following bobwhite
through the hazel thicket, or when roaming from pond
to pond in search of ducks among the vine-clad arbors
of the river bottom. And often the hunter of the deer
sees him strut before him as he sits resting on a fallen
log, and often, when on the trail of the deer in winter,
sees him shake the snow from his lightning wings, as,
bursting from its cover, the bird goes whizzing away
amid the snow-draped trees. Few of those who most love this noble bird have Few of those who most love this noble bird have
ever seen him in the simplicity of youth, before he
has left his mother’s side and gone forth to roam alone
the spangled shades of the rugged mountain side or
the somber shrubbery of the tangled glen. For his
hearthstone is too often in the dense mass of sum-
mer’s wealth, and few are the eyes that can follow
him into the deep, dark brake or into the shaggy cov-
ering of the mountain’s breast, until autumn’s frosts
have tattered their gay banners and trailed their green
glory in the dust. RUFFED GROUSE SHOOTING. For certainty of finding this grouse at home in his For certainty of finding this grouse at home in his
early days, with comfort in hunting him, few places
have ever equaled the bluff regions of the upper Mis-
sissippi. Here this grouse lived and loved, and stayed
until long after the autumn leaves were scattered on
a thousand winds; and even after the deep snows of
winter fell upon his early playground many a one re-
mained instead of seeking the covers of the bottom
lands. Here he may be found while the trees stand
in the full green of summer and before any hue of AMERICAN GAME BIRD SHOOTING 362 death has touched their shining heads. Years ago
these bluffs were studded from base to crest with large
oak trees, scattered more or less along the slopes, and
more abundant and dense of foliage around the sides
and heads of the numerous ravines. Where they hap-
pened to fall, the rich verdure of the white birch gen-
erally filled their places, and in the bottoms of the ra-
vines and along the base of the slopes the crab-apple
and wild plum and scrub oak formed abundant cover. Everywhere along the hills the ground below the trees
was densely carpeted with green, upon which the sun-
light flittered in a thousand shades through the open-
ings in the leaves above. And yet the walking was
always good, and the view generally free in all direc-
tions beneath. It was one of the fairest days in 1867 that, with a
friend and two dogs, I first roamed these pleasant
shades and found my old friend in a new kind of
home. From nearly the foot of the bluffs, where the
outer guard of soft maple and white-oak saplings be-
gan to encroach on the black oak of the hills, to very
near the top, where the birch was flying its bright
green flag from its snowy staff, the dogs were soon
racing to and fro, while we were strolling along be-
hind them, half way up the hillside. We soon came to
a shallow ravine where the ferns and the prairie grass
that covered the ground were taller and greener, and
the shade of the black oak and maple was deeper and
cooler than on the rest of the hillside. RUFFED GROUSE SHOOTING. The elder dog,
named Jack, had hunted such ground before
and SHOOTING THE WOODS GROUSE 363 seemed to know all about such places, and at once
started up the leeward side of the ravine with slow
and cautious trot, while the younger, named Frank,
seemed to have an intuition that the other dog knew
more than he did, and slowed down his pace to about
the same. And soon Jack’s trot subsided to a walk
as his nose caught the faint breeze that played over
the shady side of the hollow, and his tail slackened
its lashing motion and settled down to a slow wavy
swing. Quietly he moved along, with nose upraised
just above the deep green of the ferns and prairie grass
and the bright golden hue of the lady-slippers and the
carmine of the wild peas, raising it from time to time
still higher, with inquiring sniff, and swinging steadily
off to the leeward so as to keep the breeze fairly in
his nose all the time. And soon the old dog’s tail began
to straighten and the joints of his legs to stiffen, and
he turned his head slowly from side to side, and snuffed
the air more cautiously as he moved, more and more
slowly, along. And all the time Frank coming up
the other slope, some hundred yards away, with eyes
fixed intently upon Jack, imitating all his movements,
even more strongly than if he had smelt something
himself, instead of taking Jack’s word for it. Suddenly Jack stops, and as suddenly Frank does
the same, and at the same instant a line of mingled
white, black and gray, with roaring wings enveloping
the whole in a haze of brown, bursts from the rank
ferns some ten yards ahead of the dog and darts like
an arrow through the green arcade. AMERICAN GAME BIRD SHOOTING 364 Bang-whang go two barrels of the guns almost to-
gether, a feather parts from the long, outspread fan
behind the booming wings, and in a second more the
brown streak fades among the distant trees. RUFFED GROUSE SHOOTING. B-b-b-b-b goes another from almost the same place,
almost before the first one is out of sight, and bang
goes one barrel of each gun exactly together, and a
cloud of feathers floats from the downward whirling
bird, while with boisterous b-b-b-b-b seven or eight
more birds rise, curling, flashing, darting and whizzing
from the ferns in all directions. But Jack seems to have no anxiety about the birds But Jack seems to have no anxiety about the birds
that have fallen, and after going cautiously a few steps
forward, stops again, with slowly waving tail. Care-
fully he moves along, sniffing daintily at the air on
high, and swinging off occasionally to one side so as
to catch the full breeze, then, as he advances a few
paces beyond where the other birds had risen, his limbs
and tail gradually stiffen, until he again becomes quite
rigid, with Frank, on the other side of the ravine, imi-
tating all his motions almost as accurately as if the
two were connected by an electric wire. As we come up to him he suddenly relaxes, moves As we come up to him he suddenly relaxes, moves
off a few yards to one side again, and then, with nose
high upraised and body sunk low in the grass, he
crawls forward a few feet, in shape more like an alli-
gator than a dog, and then comes again to a standstill. As we advance a little in front of the dog three grouse
burst roaring from the ferns some twenty feet ahead
of us and dart away in different directions. One SHOOTING THE WOODS GROUSE 265 whirls downward out of a cloud of feathers; another
changes his course at the report of another gun, and
mounts skyward through the tree-tops; the third, dash-
ing the sunshine from his glistening wings, scuds away
through an open place, with the guns belching flame
and smoke vainly at the place he had just left; while
the one that had mounted above the trees, poising for
a second aloft, closes his wings and descends with a
heavy thump to the earth. The fallen birds retrieved, we went on to find the
scattered birds. Some three hundred yards we wan-
dered along, and suddenly Frank began to dawdle in
his pace. RUFFED GROUSE SHOOTING. With gently oscillating tail, he sniffed in-
quisitively at the breeze that swept up the hillside from
the long ravine below. To our senses it was laden
with the fragrance of ferns and wild buckwheat and
wild peas and white clover, with wild rose and mint;
but the dog smelt something more, for he suddenly
stopped with the quickness of thought, and at the same
instant a grouse broke, with uproarious wing, from the
deep green cover, some fifteen feet from his nose. Two charges of shot shivered the blended white and
green of the birch behind which he disappeared, the
air throbbed no more beneath the beat of his hoarse
wings, and a faint nebula of fine feathers drifted into
sight on one side of the tree. Up and down the hill again both dogs were soon Up and down the hill again both dogs were soon
beating the ground. In about five minutes Jack, com-
ing down the hill on a gentle canter, dropped into the
grass as suddenly as if shot, and lay there with only AMERICAN GAME BIRD SHOOTING 366 the tip of his nose visible above the ferns. As we came
to him a bird rose like a rocket, only a yard from the
dog, and whizzed upward as if bound for the stars. My triend’s first barrel decimated the banded feathers
of its broad, outspread tail, and he caught it with his
second barrel as it was speeding its bobtailed career
high among the branches of the old oak trees. As it
fell, another bustled, with riotous hubbub, almost from
the same spot from which the last one rose, and wheel-
ing, with its breast, mottled with black and white, in
full view, cleft the breeze so fast that the shot from
my gun was held back by the resistance of the air
waves. At least that was my theory then, and it ought
to suffice at this lapse of time. Some ten minutes passed away, and we found Frank Some ten minutes passed away, and we found Frank
anchored apparently to a stump in a little ravine far
up the hillside, with Jack indorsing his draft on our
confidence with his most statuesque attitude, about
thirty yards behind him. RUFFED GROUSE SHOOTING. The birch was waving in
the breeze above him, and the ferns were swaying
gently below his nose, the raspberries and blackberries
were still bright on the bushes in the ravine, and the
young oaks were as green as in the spring, but other
signs of life there were none. We threw stones in
ahead of the dog, but nothing moved. We tried to
urge the dog to flush them, but he would not budge. At the risk of losing a shot I went in, for the ravine
was deep and steep-sided. A few feet ahead of the
dog I slipped and fell, and in a twinkling the sky
above me seemed alive with roaring wings and meteors SHOOTING THE WOODS GROUSE 307 of white and black and brown mixed in a whirl that
made the air tremble even more than the thunder of
my companion’s gun, which was spouting flame and
smoke above my head. When I recovered myself I
found that four birds had made all the uproar, and that
my friend had pacified two of them. The grouse were so scattered that it was better to The grouse were so scattered that it was better to
search for a new flock than to try to find the single
birds that had flown far up and down the hillsides. So
we moved along several hundred yards until we came
to a broad-bottomed
ravine. Along the hills near
its head the oaks stood larger and closer than before,
the ferns were brighter, longer and greener, the birches
were taller, and maples and aspens were jostling them
aside. A soft fragrance of wild honey and thyme
haunted the dark, cool shades, and everything hinted
strongly of the favorite home of the ruffed grouse. Old Jack at once took the hint, and with gingerly Old Jack at once took the hint, and with gingerly
tread went marching up the bottom of the ravine, with
nose aloft and slowly undulating tail. Though he had
yet smelt nothing, the spirit of the place whispered
grouse so strongly that his fancy kept him on a half
point from the start, just as many a good old dog’s
imagination makes him change his pace the instant he
enters a dark, damp swamp, where everything breathes
the magic word, woodcock. RUFFED GROUSE SHOOTING. And even Frank seemed
enthralled
by the cool,
green,
silent
shades,
and
threaded the birchen bowers and the beds of fern with
more than usual care. But Jack went far up the hill several times, and came AMERICAN GAME BIRD SHOOTING 368 trudging
back, looking somewhat
dispirited;
and
Frank, after making his way as often up and down
through the ferns, seemed as badly muddled; yet both
seemed to think there must be game there. We passed
around the head of the ravine, over ground that seemed
especially made for grouse to spend the day in, but
they seemed to have that provoking trait that game
often exhibits, of ignoring the fine places you pick out
for it and preferring to make its.own selection. Further
down the ravine, below where the scrub oaks and ma-
ples and aspens broke into the heavier black oak that
robed most of the hills, and where the bottom widened
out into a little valley, lay a long thicket of crab-apple
and wild plum, edged with black haw and hazel where it
broke into the oak and maple of the hills. Knowing
that the birds ranged low as well as high, along these
hills, we went to it. The dogs soon disappeared within
the dense green shrubbery, and naught was heard of
them in a minute or more but the light rustle of their
feet. And not another minute seemed to pass away
before that, too, ceased. Leaving my friend on the outside, where he would Leaving my friend on the outside, where he would
be able to get a shot at anything that came out, I went
into the thicket. There stood Jack, bent: like a bow,
with tail and jowl nearly parallel, as he had evidently
thrown himself with a sudden whirl, upon striking the
scent from one side. And a few yards behind him,
half hidden in the deep green, stood Frank, with the
solemnity of a tombstone on a winter night. As I
stopped behind Jack there was a bewildering burst of SHOOTING THE WOODS GROUSE 369 uproarious wings, and a dozen or more birds went
darkliing through the green, some wheeling out of the
top, some scudding straight away, some darting low
toward the edges. RUFFED GROUSE SHOOTING. Quick as a flash I dropped on one
knee and sent a charge through the leaves where one’s
fanlike tail was vanishing on a sharp curve as I raised
the gun. But by the time the shot reached there it
was gone, and by the time I discovered it was gone
the rest were all gone. But dimly through an opening
1 could see my friend on the hillside, with half a dozen
grouse swiftly driving toward him. One went past
him like an arrow feathered with white and brown, and
was gone before he could raise his gun. Another,
whirling into sight above the brush, with its full white
breast, broadly mottled with black, brightly flashing in
the sun, just a trifle too late for me to shoot at, went
spinning by him with unruffled feathers at the report
of his gun. And then five or six more went roaring
on past, and above and behind him, while he, in con-
fusion, shifting his gun from one side to the other,
and hardly knowing what to shoot at, let them all go
by, and stood as if looking for more to come. Few American sportsmen have had so much expe- Few American sportsmen have had so much expe-
rience in upland shooting as Mr. B. Waters, whose
excursions have covered the game fields of almost the
whole United States east of the Rocky Mountains. Famous as a handler of hunting dogs, and equally fa-
mous as a crack shot, both at the traps and in the field,
his views on the shooting of any game bird will receive
respectful attention, for he is past master of the art. AMERICAN GAME BIRD SHOOTING 370 This is what he has to say about ruffed grouse
shooting : From the time when the mind of man runneth not
to the contrary in matters of shooting for sport, the
ruffed grouse, by common consent, has been classed
with the most difficult of game birds which the sports-
man endeavors to bring to bag under the approved
conditions of sportsmanship, if indeed it be not the
most difficult of all. RUFFED GROUSE SHOOTING. For it taxes the sportsman’s
nerve, patience, skill, woodcraft and endurance as no
other bird can tax them; and all these requirements
are necessarily supplemented by a gun of good killing
powers, one selected with special reference to cover
shooting; and last, but not least,
a dog of more than
ordinary intelligence and good intent and good train-
ing, if the sport is to have any successful results and
pleasing finish in its action. If any element of the
sportsman’s ruffed grouse craft be missing success is
marred accordingly. The ruffed grouse, in every art and article, is a bird The ruffed grouse, in every art and article, is a bird
to fill the sportsman’s ideal. Its habitat is in nature’s
most picturesque setting; the bird is beautiful in its
delicate tracings and markings, and rich and varied
in its colorings; racy of form and faultless in sym-
metry; wild, dashing, daring, alert, and infinitely re-
sourceful in its crafty devices when pursued; exclu-
sive in its habits, and, withal, a bird of rare excellence
for the table, its flesh being of a delicate texture and
pleasing flavor—so palatable, indeed, that it is by many
epicures more highly prized than is the flesh of any SHOOTING THE WOODS GROUSE age other game bird. With those who may vaunt the ex-
cellence of the woodcock, the snipe, the prairie chicken,
the duck, the turkey, etc., it also holds a high place in
their esteem; and the exceptional man, whose fancy
for one particular kind of bird prejudices him against
all others, will not speak unkindly of it. And yet,
delicious as it is when properly prepared for the table,
it can easily be spoiled by ill cooking, and of bad cooks
there is no end. The art of cooking it properly is as
rare as is the skill of killing it properly. RUFFED GROUSE SHOOTING. If it be cooked
too much, or if it be cooked improperly, it loses much
of its rich delicacy of flavor and texture, and becomes
dry and unpalatable; and in that unfortunate condi-
tion it probably was when that eminent authority, Wil-
son, partook of it, and thereafter, in his “American
Ornithology,” wrote of it:
“At these inclement sea-
sons, however, they are generally lean and dry, and,
indeed, at all times their flesh is far inferior to that
of the quail or of the pinnated grouse.”
Yet, as tastes
are not all alike, the superlative will probably be placed
|
according to the individual fancy in matters of food,
as in all other matters, and it is well that it is so. If
all fancied alike, all would be monotony. Nevertheless,
a man who cannot have a culinary spell cast over him
by a skilfully cooked ruffed grouse, it having been kept
a proper length of time after killing—not too long—
has no music in his soul, and may not even be fit for
treason and spoils. The ruffed grouse chooses rough and timbered sec- The ruffed grouse chooses rough and timbered sec-
tions, for it is strictly a bird of the woods and thick- AMERICAN GAME BIRD SHOOTING 372 ets, preferring the roughest parts of a hilly or moun-
tainous country, and of these it many times selects the
densest recesses, or the timber of seamy and rocky
hillsides, or where ledges, fallen tree-trunks and tree-
tops in the woods, secluded from man, guard against
intrusion, and even the timbered swamps are not ob-
noxious to it. For man it has the most uncompromising aversion. It selects its home in the places least frequented by
him, though once the home is determined on it holds
to it with dauntless persistency, let the gunner disturb
it as often as he may. In choosing its habitat it prefers that it be near a In choosing its habitat it prefers that it be near a
supply of good water and an abundance of good food,
for it is a good feeder. Whortleberries, blackberries,
beechnuts, acorns, chestnuts, partridge berries and buds
are readily accepted as food in their proper season. The bud of the laurel is said to render the flesh
poisonous for food purposes, though the belief seems
to rest more on tradition than on any direct evidence. RUFFED GROUSE SHOOTING. Unlike the quail, which prefers to make its home near Unlike the quail, which prefers to make its home near
the homes of man, and the prairie chicken, which sticks
closely to the grain field, the ruffed grouse is ever in-
tent on choosing its home and haunts distinctly apart
from those of man. In the East it is called “par-
tridge’”’; in sections of Pennsylvania and the South,
“pheasant.”
In the breeding season, when it has been free from In the breeding season, when it has been free from
pursuit and harassing alarms, it sometimes strays a
short distance from cover into the adjacent
fields, SHOOTING THE WOODS GROUSE 373. where grow palatable huckleberries and blackberries,
though rarely venturing further than a short flight,
and often but a few yards from cover. Though always a wary bird, and ever avoiding man, Though always a wary bird, and ever avoiding man,
it is not so wild and quick to take wing before the frost
and unsettled weather of fall set in as it is afterward;
yet if the gunner disturb it once or twice, the full
wildness of its nature, and its constant alertness to
avoid man, are fully and permanently aroused. Then
man and the places he frequents are shunned as much
as possible. Indeed, it is not a social bird with its
own kind. After the young birds have matured they
separate, and in the fall the gunner will find them in
ones and twos, and at rare times in threes. Given to the sportsman the conditions of an open
field, and therein a ruffed grouse on the wing, within
range, then the difficulties of killing it are but little,
if any, greater than those which obtain in the killing of
a prairie chicken on the open prairie; though whether
in open or cover, the ruffed grouse is always swift
and decisive in its flight. But in the open, whether
it be on field or prairie, there is an even light and an
unobstructed view. Then, for safety, the bird can rely
only on its swiftness of wing, all too slow when pitted
against the sportsman who can, under those circum-
stances, with studied quickness and deliberation, com-
mand a large circle around him. RUFFED GROUSE SHOOTING. Thus the ruffed
grouse is at a fatal disadvantage when shot at in the
open field, as is also every other bird pursued under
the same conditions; but these conditions are rare in- AMERICAN GAME BIRD SHOOTING 374 deed in ruffed grouse shooting, for, as mentioned be-
fore, it ventures into the open only on such infrequent
eccasions as it is tempted to search therein for food,
and then only in places seldom invaded by man, where
it fancies there is freedom from pursuit. To all fixed
habits there seems to be an exception for a short pe-
riod in the fall, when it is subject to a crazy wayward-
ness. While in the open field it is strong and swift of
wing, in cover it is at its best. It will, on occasion,
dash through the densest thickets with apparent ease,
with no diminution of its swiftest speed, seemingly
having a charmed manner of flying through tree-tops
and thickets as if they were but phantom trees of the
woodland, or shadows offering no obstruction to its
onward flight. And in its favorite haunts it is a master of self- And in its favorite haunts it is a master of self-
defense. It can
utilize
thickets,
trees,
old fences,
ledges, stone walls, swift wings and endless cunning
to evade its pursuer. Be the position of the shooter
what it may in reference to this bird in cover, it, when
flushed, takes instant advantage of the nearest thicket
or the trunk of a tree or old fence, keeping one or the
other between itself and the gunner in its line of flight,
thus in a great measure blocking all opportunity to
shoot, or at least hampering the shooter greatly, and
oftentimes causing a miss. The bird, in most instances, times its rise so as. to The bird, in most instances, times its rise so as. to
have the advantage of some nearby object as a shield
to its flight. On occasion it will display a courage SHOOTING THE WOODS GROUSE 375 bordering on audacity, permitting the shooter to pass
close by, and flushing after he is some yards further
onward. This wile is oftenest practiced after it has
been flushed, marked down and pursued. Both man
and dog are apt to pass it then, though they may fol-
low in the exact line of flight. RUFFED GROUSE SHOOTING. The shooter may hear
the irritating roar of the bird’s wings behind him, on
ground but a moment before passed over, or catch a
shadowy glimpse as it dashes away from some tree-
top. Owing to its short flights and its proneness to take Owing to its short flights and its proneness to take
a straight, or nearly straight line, the persistent shooter
may be able to mark and flush the bird again and
again. It sometimes, in repeated flights, returns to
near the place where it was first found, and it always
takes the flights so that ground and cover are to its
advantage in avoiding danger. Once in a while a foolish bird will be found, which
will do the very thing it ought not to do, commonly
paying for the lapse with its life; so that if there is
anything in the theory of heredity, the ruffed grouse
should be uniformly of high capabilities, the fool birds
being killed promptly, and never breeding. By far the greater part of the shooting is at close By far the greater part of the shooting is at close
range, as it needs must be in thicket or woods, where
the longest views are short, and obstructed by trees or
ledges or the undergrowth or the hilly nature of the
ground, where in the early season the view may not
be greater than a few yards or feet, if the leaves have
not fallen. AMERICAN GAME BIRD SHOOTING 376 It then is not an infrequent occurrence that the
shooter will hear the startling whir of wings close by
him, and yet be unable to shoot or to mark the bird’s
course, from inability to see the bird at all. The light
of the woods, broken and broken again, as it is through
the irregular openings in the tree-tops, and branches
and leaves interposing, with here and there shafts of
clear light, and masses of shadows interspersed every-
where, tends to interfere with quick and clear vision,
and adds to the difficulty of accurate shooting, thus
differing widely from shooting in the open. The successful ruffed grouse shooter must be ever The successful ruffed grouse shooter must be ever
promptly ready to shoot, and further, must be quick
of eye and action. RUFFED GROUSE SHOOTING. He must instantly decide on the
manner of making the shot, taking advantage of all
the few opportunities offered, and avoiding the ob-
structions which interpose. No studied effort at aim-
ing is possible. Cover shooting of all kinds requires
quick action, but ruffed grouse shooting requires the
quickest. Of all snap shooting, ruffed grouse shoot-
ing is the snappiest, and the successful shooter of that
bird must exce! in that kind of shooting, since in most
cases he will have but a brief instant’s glimpse of the
bird in the unfavorable mixed lights of shadows and
cover. For this shooting the gun should be light, short of For this shooting the gun should be light, short of
barrel—26 to 28 inches—and a cylinder bore, for a full
choked barrel is entirely out of place in such cover
shooting, equally unsatisfactory when it does or does ‘aspoyH
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payny SHOOTING THE WOODS GROUSE 377 not kill, it being a miss in the first instance, and often a
badly mutilated bird in the second. The average shooter will find that he has success
far below his opportunities, even when equipped with
the gun most fitting for the work. In this shooting
there is no waiting for opportunities to fit the gun. The successful hunter must take the shots that are The successful hunter must take the shots that are
offered, and as they are offered; it matters not how
difficult they appear or how brief the opportunities may
be. He may catch but a momentary glimpse of the
bird as it crosses some diminutive opening, or he may
see it for an instant in a maze of leaves and branches,
or he may get a partial glimpse of it and some dis-
turbed leaves in the course of its flight, yet these are
the opportunities which are the most numerous, and
which must be relied on for the bulk of the shooting;
in short, that is ruffed grouse shooting. RUFFED GROUSE SHOOTING. If the shooter be too indolent or apathetic to be ever
ready to shoot, or if he is too slow to take advantage
of the opportunities, his success will be but meager
so far as material results are concerned, though he
may be greatly encouraged by the belief that his last
ill success was due to faults in the bird, and that if he
can have another opportunity he will acquit himself
nicely. The opportunity comes,
and failure again
evokes more excuses. Once in rare whiles the shooter
will have a good opportunity, catching the bird in some
corner so favorable that the advantage is with the
shooter; but such instances are rare indeed, and by
themselves would make but little sport. 378 AMERICAN GAME BIRD SHOOTING To be ready for the opportunities the sportsman must
be quiet, and never relax his vigilance, and his gun
must be so held that it can instantly be brought into
position to shoot. The nerves of the shooter must be
constantly at a high tension in readiness for the rise
of the bird and the instantaneous shot. Every faculty
must be at a high key. The very moment that the
shooter relaxes his attention will be the moment that
the bird will rise, and before the sportsman can get
ready the opportunity is gone. It will be seen that
the man who dawdles with his gun, who is slow in
the handling of it, or who is noisy, cannot hope for
any satisfactory success in shooting the bird of game
birds, the ruffed grouse. On the other hand, he can be
keyed up to too high a pitch, overready when the bird
rises. A nervous flurry does nearly as much to disar-
range the desired results as does the more indolent
dawdling. There are those, however, who can never
overcome the nervous start at the roar of the bird’s
wings, though they may be perfectly undisturbed in
any other bird shooting. And the skill of the shooter, be it ever so high in
degree, must be supplemented by the work of a quiet,
well
trained,
industrious,
intelligent
dog,
for the
shooter is much better off without any dog at all than
with one that is riotous or one that ranges too far or
that is heedless of his work. Loud orders to the dog
have no place in ruffed grouse shooting. RUFFED GROUSE SHOOTING. The sports-
man himself cannot observe too great a silence. The
human voice alarms and puts the birds to flight. SHOOTING THE WOODS GROUSE 379 The work required of the dog in this shooting is dis-
tinctly different from that required in any other kind
of bird shooting, except, perhaps, woodcock shooting,
which in a way it resembles, though a higher degree
of dog intelligence and obedience are required, as the
ruffed grouse is far more cunning and wary than the
woodcock. The “partridge dog” should not work far from the
gun in cover, and he should be silent and diligent in
his quest. Many experienced shooters highly com-
mend the use of a small bell tied to the dog’s collar, its
low tinkling constantly indicating the dog’s where-
abouts in the thick cover; and generally, when the bell
stops, it indicates that the dog is on point, thus in
a way keeping the shooter posted by ear as to his dog’s
doings and whereabouts. The rattle-headed, highly nervous dog, or the one The rattle-headed, highly nervous dog, or the one
which gallops swiftly and merrily about, is distinctly
out of place in this kind of shooting. The esthetic
shooter, whose dog must carry a high head and a tail
lashing his sides merrily as he gallops and bounds
about in the ecstasy of his enjoyment, as the dogs many
times do in the idealists’ tales of great work afield,
would better take his fiery dog into the open, where
he can better disport himself unhampered, and where
his pretty ways may be admired without any unpleas-
ant interposition of the ruffed grouse. Such manner
of the dog’s seeking is incompatible with ruffed grouse
shooting, for the shooting should be the dominant fea-
ture, not the joyousness of the dog. AMERICAN GAME BIRD SHOOTING 380 A dog of fair gait and persistent industry can easily
beat out the necessary range, and the one which makes
his quest patiently and soberly and quietly, working
with judgment and honesty to the gun, will bring the
shooter satisfactory success in the results, to say noth-
ing of the incomparable comfort and pleasure in shoot:
ing over him. RUFFED GROUSE SHOOTING. Nine out of every ten dogs which are running with
high head and merry actions are running because they
are in high spirits and for their own pleasure, with no
thought of the birds or of work to the gun. When
they come on birds this is often a matter of chance,
and their point work is marked by deplorable errors. This kind of dog leads his partial master to believe
that when he wears off the wildness and wire edge
he will steady down to a useful grade of work; but
often when such dog has worn off his exuberance he
has worn off all there is in him of field performance,
and he either loafs, or does his work in the same slov-
enly manner, though, loafing, he does less of it. In shooting for sport, the shooter takes his birds
on the wing. Of course, in shooting for market, the
market shooter has no thought of sport or its prac-
tices. His one object is to kill the bird and bring it
to bag. The manner of it is of the least importance. His theory and practice are founded on commercial
principles; therefore, he shoots his birds as he can,
whether they be on the limb of a tree, the ground, or
flying. Some hunters have dogs trained to seek for the Some hunters have dogs trained to seek for the SHOOTING THE WOODS GROUSE 381 birds, and, finding them, they flush and follow them. When flushed by the dog, the bird generally takes to
the trees, and the dog, barking, so engages their atten-
tion that they fall an easy prey to the hunter, who
often bags every bird in the covey under such circum-
stances. Often, when flushed by the dog, they fly to
the tree-tops immediately overhead, where, in fancied
security, they watch the dog. The shooter then drops
them one by one, taking the lowest birds first. The
falling of the lowest ones does not disturb those above,
though if a top bird is dropped the others fly away
forthwith. As to the number a shooter can kill in a day, so
much depends on the shooter’s skill, the bird supply,
and the local shooting conditions, that these things
alone determine it. RUFFED GROUSE SHOOTING. In some sections of New England, two or three In some sections of New England, two or three
birds at the end of a day of diligent effort is consid-
ered a highly successful result, and it is not an infre-
quent occurrence in that section that a diligent day
may have no birds at all at its ending. In certain sections of New York, Wisconsin, Minne-
sota, Dakota, etc., and in the mountain sections where
the ruffed grouse abound, such a bag would be consid-
ered an absurdity, if held forth as an index to good
shooting, industry and superior results. While in North Dakota I have heard of one bag While in North Dakota I have heard of one bag
of eighty birds, made to one gun in a day, something
extraordinary. They were shot at the air holes along
the banks of the Red River, after it had frozen over, AMERICAN GAME BIRD SHOOTING 382 the birds coming to those places for water. This was
not recounted to me as a matter of sport, nor is it so
set forth here, but will give an idea of the numbers
of the ruffed grouse in the sections where it is in the
greatest abundance. But the sportsman who seeks the ruffed grouse for But the sportsman who seeks the ruffed grouse for
the true sport of it has a more exalted pleasure than
comes from shooting any other game bird. First of
all, he must be skilful with the gun, and when he
shoots, be he ever so skilful, he can only apply such
skill as he can muster in a moment, the opportunities
of ruffed grouse shooting being but mere fragments
of the opportunities accorded to shooting in the open. When at length the bird is brought to bag it repre- When at length the bird is brought to bag it repre-
sents a toiling through brush and bramble, wooded hill
and dale, scrambling
over
ledges and floundering
through swamps, all colored by constant expectancy,
unavoidably lost opportunities, and seeking to circum-
vent the birds by cunning woodcraft, supplemented by
the powers of the dog—a degree of cunning, skill and
persistent effort greater than that required in the shoot-
ing of any other bird. RUFFED GROUSE SHOOTING. It is shooting pitched in the highest key, and that
is why I think the shooter can justly feel a greater
glow of pleasure when he makes a successful shot at
ruffed grouse, and why he loves this sport above all
others, since it tests to the utmost his skill, his wood-
craft, his patience, his endurance and his dog; and of
the dogs, if he owns a good one, he owns one of a
thousand. SHOOTING THE WOODS GROUSE 383 Many sportsmen will not agree with Mr. Waters’ be-
lief that a ruffed grouse in the open is as easily brought
to bag as a prairie chicken. They even declare that the
ruffed grouse flies three times as fast as his pinnated
cousin, and is at least twice as difficult to hit. In his recent volume on “Life and Sport on the
North Shore of the St. Lawrence River,’ Mr. Nap. A. Comeau says:
“Ruffed grouse shooting in this section is not sport, “Ruffed grouse shooting in this section is not sport,
and is not regarded as such by the residents, for the
reason that neither the people nor the birds have been
educated to it. I can count on less than the fingers
of one hand all the men I know on this shore who will
deliberately flush a grouse to shoot it on the wing. As
for the birds themselves, unless they happen to be in
an open spot, they will not fly any distance. In the
woods, which are pretty dense here, when flushed they
simply rise off the ground, perching in the nearest tree
and stretching their necks to see you walking under
them. If it happens that a covey is started they will
frequently be all killed without any of the others around
taking flight. Many a time when in the woods trap-
ping we would not waste a shot on them, but simply
go to work and cut down a small sapling, tie a noose
or string at one end, slip it over their head and pull
them off the branch. At other times, for amusement,
we would go out with a bow and a blunt-headed arrow
and whack them off the trees at twenty feet range,
which is about the usual one that they are shot at here. “What a contrast to the educated ones! RUFFED GROUSE SHOOTING. Some years AMERICAN GAME BIRD SHOOTING 384 ago I received an invitation from C. Beatty, of Platts-
burg, Lake Champlain, to go and have a few days of
mixed shooting with him. It was late in September,
but most of the leaves were still on the trees. The first
day we had a grand duck shoot on Missisquoi Bay,
and after that an outing for woodcock. The last day
had been reserved for partridge and gray squirrels. We
had breakfast at daylight and were off. We had not
far to go to reach our ground—patches of hardwood
trees with a good deal of underbrush. We soon heard
some whirring off at our approach, but could not even
get a glimpse of them. After a time I got a crossing
shot at one, over fifty yards away, which I bagged,
and that was the only bird we got. But we surely
heard a dozen or more rising. I was simply astonished
that such a bird could be so shy. When I came back
here and told the natives about my experience, they
thought I was pulling the long bow. I believe it will
be many years before our birds get so highly educated.” DUSKY GROUSE SHOOTING To my mind, the most splendid of the many Ameri-
can grouse is the dusky, or blue, grouse found in the
Rocky Mountains, north to Alaska, and west, at vari-
ous points, to the Pacific coast. To be sure, it is not
as large as the sage grouse, yet it is a big bird, some-
times weighing up to four pounds, and nearly two
feet long. Its tender and delicate flesh is always good
eating, and its habits of life, in underbrush and timber SHOOTING THE WOODS GROUSE 385 along the mountain side, make it seem much more a
bird for sport than the larger sage grouse, which is
found on the dry, hot, open prairie. In these days, when cities, towns, villages and farms In these days, when cities, towns, villages and farms
are scattered all over the range of the dusky grouse,
there must be a multitude of men who follow this bird
with dog and gun, and shoot it much as people in the
Eastern States and the Mississippi Valley shoot the
ruffed grouse; yet, curiously enough, we hear very little
of killing this bird in a sportsmanlike manner. Shooting the dusky grouse before the broods are Shooting the dusky grouse before the broods are
full grown, and when they are more or less scattered
out to feed, is but tame sport. The birds lie like stones,
and fly straight and easily, dropping at .a touch of the
shot. In September and October, when they are full
grown, it is quite different, however. Then they are
strong of wing and fairly well able to take care of
themselves; all the birds are large, and while they still
present a fairly easy mark, they fly with great swift-
ness, and from the situations in which they are often
found the shooting calls for readiness and care. In the old days, when no one in the West thought In the old days, when no one in the West thought
of carrying a shotgun, it was often necessary to kill
birds for food, and then the young broods of dusky
grouse often gave one a little practice as they stalked
ahead of one up the valley or stood on the branches
of the trees of the mountain side. DUSKY GROUSE SHOOTING At a distance of
twenty or thirty yards, provided one thoroughly knew
his rifle, it was considered fair shooting to knock the
heads off four out of five of these young birds. In- AMERICAN GAME BIRD SHOOTING 386 evitably there was a miss now and then, when a walk-
ing bird would unexpectedly stop, or would move its
head to one side just as the trigger was drawn; but
as I say, we used to be able to cut the heads off four
out of five. In the same way, when a brood of dusky grouse flew
into a tree, and stood there unfrightened by the report
of the rifle,
a number of them could be secured, pains
being taken always to shoot the lowest bird—in def-
erence to an aged tradition—in order that others might
not be alarmed by a fluttering body falling close to
them. One of the best morning shootings that I ever had
at dusky grouse was in northwestern Montana, on
one of the high benches that overlook the St. Mary’s
Lakes. It was a rounded knoll—an old lateral mo-
raine—a mile or two long, once overgrown by aspens,
which had been killed by fire and had now fallen and
rotted. A new growth of aspens, just starting, reached
only about up to the knee. Among these little aspens
grew huckleberries, and the ground was more or less
carpeted with the vines of the bearberry—the smoking
weed called “larbe,” perhaps a corruption of the trap-
per French word ’herbe. On these berries several
broods .of grouse were feeding, and after camp had
been made near the upper end of the knoll I took my
shotgun and walked back over the ground where sev-
eral birds had been started. It was not long before, with a thunderous roar, a It was not long before, with a thunderous roar, a
full-grown bird rose but a few yards before me, and, ‘UIe}JUNOM 8q} UO asnoIg AYsNG
=
-
,
od
sy ‘UIe}JUNOM 8q} UO asnoIg AYsNG SHOOTING THE WOODS GROUSE 387 scaling off on the fresh breeze that was blowing down
the lakes, was thirty or forty yards off before the heel-
plate touched my shoulder. Luckily, I held on the
bird, and the center of the load hit it so that it fell quite
dead. DUSKY GROUSE SHOOTING At the report two more rose near me, and this
time I brought my gun to bear a little more quickly,
and killed the second one. Two or three more rose at
this report and flew down the lake, but they did not
fly the whole length of the knoll, and I marked them
down. It was evident that if this lasted I was going to have
good shooting, and so it proved. I went on, more
slowly and carefully, and tried to pick my shots. As
I had no dog, only the birds immediately in my way
got up, except that sometimes, when a shot was fired,
several rose ahead or to the right and left, and most
of these pitched off down the hill, and, scaling off on
the wind, reached cover in the thick aspens of the
lower ground, where I knew it would be useless to
follow them. Before I had reached the lower end of the knoll I
had more birds than I could carry, and I was not sorry
to see my companion coming after me on horseback
to relieve mc of the load. The shooting that he had
heard had notified him of the sport that I was having,
and he rightly concluded that I would need help. I
was interested to notice, as he came toward me, that
he put up a number of grouse, though his route was
not far from the one I had followed. When we had tied our birds on his horse it was When we had tied our birds on his horse it was AMERICAN GAME BIRD SHOOTING 388 found that I had already ten, which I estimated to aver-
age not far from three pounds apiece in weight. Just
at the end of the knoll, and as we were about to turn
back to go to camp, a grouse jumped up before me,
at the foot of a clump of aspens, and dived into them,
precisely as a ruffed grouse would pitch into a piece
of underbrush. Just as I should have snapped at a
ruffed grouse, so I snapped at this bird, and a moment
later a loud splash in the water, and a muffled drum-
ming, told that the shot had reached him just as he
was about to cross the river. DUSKY GROUSE SHOOTING My companion went
down, and riding out into the water, picked up the
eleventh bird. A little later, on the way home, another grouse A little later, on the way home, another grouse
sprang from some low aspens at some distance in front
of me and pitched into a growth of pines, and this
one I snapped at, again, but not with the success of the
former shot, for the bird passed through the pines and
flew a long way to a little island, where he seemed to
go down. If I had had a dog and a good shooting pony I
could undoubtedly have killed forty or fifty birds in
this one place; but forty or fifty would have been in-
excusable slaughter, since there were but two of us
in the camp, and we could not have used anything like
that number of birds. As it was, those that I got that
day lasted us for quite a long time, and most delicious
food they were. The white, juicy flesh, sweet, and well
flavored from the diet of berries on which the bird
had been fed, was most delicate. Properly cooked, no SHOOTING THE WOODS GROUSE 389 bird is worthy of higher commendation than the dusky
grouse. This is especially true when the birds on
the lower plateaus are feeding on the tiny red huckle-
berry that grows in such profusion in the pine woods. While the females are down in the lower ground,
attending to nest building, hatching, and the rearing
of their young, the old males and the barren females
resort to the higher land, often being found on the
mountain sides far above timber line. From such
places they are often startled by the goat or sheep
hunter, and pitching down from these great heights
take long flights, at last bringing up down among the
timber, and flying so far that no one knows just exactly
where they go. Nowhere, so far as my limited experience goes, 1s Nowhere, so far as my limited experience goes, 1s
the dusky grouse pursued in so systematic and sports-
manlike a manner as on Vancouver Island, near the
beautiful city of Victoria. DUSKY GROUSE SHOOTING My shooting of them there
dates back many years, and it may be that in recent
years the sportsmen of other parts of the Pacific coast
have taken to shooting this splendid bird over dogs, as
in old times they did near Victoria. What good shooting there was at Victoria twenty- What good shooting there was at Victoria twenty-
five years ago, and how varied the bags used to be! There were the pheasants rising like an explosion of
fireworks, sometimes from under your very feet, and
seeming—after you had ineffectively fired both bar-
rels in the air in your fright—to wave at you in de-
rision long brown tails that you almost felt you could
grasp by reaching out the hand. There were blue AMERICAN GAME BIRD SHOOTING 390 grouse, big, and straight flying, getting up with a roar,
and almost at once plunging into the dense thicket. There were ruffed grouse, simple birds, that you some-
times saw walking about on the ground not far from
your feet, but ready enough, after they had been
pointed by the dog and kicked out from their hiding
place, to practice all the arts that their cousins use
three thousand miles away. Then, finally, there were
the California quail, big flocks of them, more often
heard running through the underbrush than seen, yet
sometimes rising in thick flocks and darting off like
little blue bullets through the timber. It was here that, in company with two or three Vic-
toria sportsmen, I first saw dogs used on the blue
grouse; not always with success, for two wild young
puppies, blundering excitedly through the underbrush
and the heavy green forest, flushed the birds, some of
which took refuge in the branches of the tall cedars
or Douglas firs, quite out of reach of the shotgun. There was one old white setter, however, which re-
garded the younger dogs not at all, but trotted method-
ically through the forest in businesslike fashion. To
him and to his owner I attached myself, and during
the day had the opportunity to see him point half a
dozen birds in most workmanlike style. The grouse
lay well, and did not run ahead of the dog, as an edu-
cated ruffed grouse would have done. DUSKY GROUSE SHOOTING At the same
time, when
flushed, the birds displayed wonderful
quickness in putting some object between themselves
and the gun; though in this case, as there were two SHOOTING THE WOODS GROUSE 391 guns, the stratagem was not always successful. No
great amount of wisdom was required to circumvent
these birds. They had not been subjected to the con-
stant pursuit suffered by the ever persecuted ruffed
grouse of our eastern covers, and did not resort to his
puzzling devices. They afforded great sport, but the
shooting was very destructive to the birds. On the
other hand, the thick cover which prevailed over much
of the forest did not permit following up the birds,
and if not secured on the first rise they were not seen
again. No doubt at the present time the dusky grouse are
frequently shot over dogs in the Rocky Mountains. Any dog which had been broken on ruffed grouse would
be serviceable also in finding its larger relative. Yet
as the dusky grouse has been, until recently, altogether
without education in this respect, the extreme caution
needed in a dog used for ruffed grouse shooting would
scarcely be required for the dusky grouse. It would be interesting to know whether, in modern It would be interesting to know whether, in modern
days, the ruffed grouse, or the dusky grouse of the
north Pacific coast, had been sufficiently pursued to
acquire a wisdom which men of the eastern part of the
continent usually expect the ruffed grouse to possess. PTARMIGAN SHOOTING. Ptarmigan are never shot for sport, but only for
food. Occasionally a few birds may be secured on the
wing, but usually the ptarmigan is not found in situa-
tions where the gunner is able to pursue it in a sports-
manlike manner. Commonly he is traveling, is with-
out a shotgun, and is thinking more of filling his pot
for the night’s meal than of giving the bird a chance
to escape. Practically the only exception to this rule
is Newfoundland, where, under the name of partridge,
the ptarmigan is commonly shot over dogs, and gives
excellent sport. Mr. Comeau, in his “Life and Sport on the North Mr. Comeau, in his “Life and Sport on the North
Shore,” speaks most interestingly of the migrations of
the willow ptarmigan, and tells of the extraordinary
numbers of them that are killed. He says:
“During the last two migrations, taking the best “During the last two migrations, taking the best
years, 1895 and 1904, I took some trouble to try and
find out, approximately, how many birds were killed
between certain points. During the first year men-
tioned, between Mingan and Godbout, 175 miles of
coast,
30,000 were
killed;
in the second
(1904),
14,000; and I am sure that during 1885 nearly 60,000
must have been shot or snared. When a flight begins,
every man, woman and boy able to handle a gun is
out. To avoid accidents, which are very rare, indeed,
392 392 Willo
Ptarm Wil
Pta PTARMIGAN SHOOTING 393 each gun occupies a certain point or station, and shoots
at all the birds that pass or alight in his vicinity. The
ladies keep watch for those that may alight near the
houses. The bags vary, of course, according to the
skill of the shooter and his method of shooting. If
he is there for business he will take all the pot shots. He can frequently get five or six in one shot. I have
seen fourteen killed in a single shot. A few will
only shoot on the wing, but there are many days when
the wing shooter comes out ahead. The biggest bag
I ever made (it was in 1885) shooting at flying birds
was eighty-two brace in one morning. At Caribou
Island, that winter, nets were tried, but they were not
very successful, more being got by shooting. PTARMIGAN SHOOTING. Indians
frequently snare them by setting their snares around
willow clumps where the birds feed. It is a very sim-
ple arrangement. A twig is stuck in the snow, a twine
snare is tied to it, a very light support placed under it
to hold it in position, and it is ready. In walking
around the bird runs into it, then tries to rise on feel-
ing the snare, only to tighten the noose. There is a
little fluttering, and it is all over.”
Such is the abundance of the Such is the abundance of the willow ptarmigan, and
sometimes of the rock ptarmigan as well, though this
last is not so numerous, and such is the way in which
they are killed for food. Rarely it happens that Rarely it happens that the earnest ornithologist or
the big-game hunter in search of a new sensation toils
laboriously up to the home of the white-tailed ptarmi-
gan, carrying his shotgun with him. When he reaches AMERICAN GAME BIRD SHOOTING 394 the haunt of these little birds he is very likely to be
disappointed. Either, on the one hand, they trot along
before him, refusing to rise on the wing, or else they
are so shy that long before he is within gunshot they
take wing, and fly off along the mountain side, only
to repeat this operation when again approached. Like all the grouse, the white-tailed ptarmigan, when Like all the grouse, the white-tailed ptarmigan, when
almost fully grown, are very gentle and unsuspicious,
and may be approached within a few feet; but as the
nights grow colder and the autumn storms begin, they
grow more and more shy. Finally, in September or
October, they are wild birds, often rising at good gun-
shot distance, flying thirty or forty yards, and, alight-
ing on the hillside or on some great rock, walk about
with head and tail held high in air, in an attitude of
great suspicion. If by chance they are closely ap-
proached when on a steep hillside they often throw
themselves into the air and scale downward with great
swiftness, sometimes continuing their flight across a
narrow valley, so that it is quite impossible to follow
them. PTARMIGAN SHOOTING. In the days of their youth, before they have grown
shy, many are killed in the mining districts of the high
mountains with stones or clubs, by miners going to
and from their work. In Newfoundland, the willow ptarmigan, and occa- In Newfoundland, the willow ptarmigan, and occa-
sionally the Newfoundland form of the rock ptarmi-
gan, used to afford superb shooting over dogs; but as
with
so many
other game
birds
in many
places,
they have been so overshot there that it is now reported PTARMIGAN SHOOTING 395 that they are comparatively few in number and that
good shooting can no longer be had. What it was in
old times may be inferred from an article long ago
contributed to Forest and Stream by M. Harvey, who,
formerly wrote so much about Newfoundland, and,
as one of the historians of the island, did much to
make it better known to the world at large. The sub-
stance of the article is as follows: The ptarmigan or partridge (“patridge” in our local
vernacular)
of Newfoundland is quite equal to the
Scotch grouse, and, indeed, resembles it so closely that
it is difficult to make out any specific difference be-
tween the red grouse, gorcock or moorcock of Scotland
and the ptarmigan of Terra Nova. They are a most
delicious article of food, whether roasted, stewed; or
in white soups. All visitors to our shores admit that
the flavor of a plump partridge, well cooked, is unsur-
passed in richness and delicacy. They are of respect-
able proportions, a brace of them, in season, weighing
from three pounds to three pounds and a half. When
the shooting season opens, on the first of September,
they are in prime condition after feeding on the wild
berries, the partridge berry and cranberry being their
favorite food. In certain localities they are very abun-
dant, and to the sportsman there can be nothing finer
than a day’s partridge shooting over our breezy bar-
rens during the fine autumn weather. The air is then
cool and exhilarating, and the skies bright. The weird
and charming scenery is varied by countless lakes or AMERICAN GAME BIRD SHOOTING 396 ponds and by low, rounded hills, covered to the sum-
mit with the tapering firs. PTARMIGAN SHOOTING. The lakelets are bright with
the white and yellow water-lilies ;
between the summits
of the bold headlands along the coast glimpses are had
of the restless Atlantic. All these natural beauties,
combined with the excitement of the sport, furnish to
the lover of nature a day of rapturous enjoyment. It is a thrilling moment to the sportsman when, gun It is a thrilling moment to the sportsman when, gun
in hand and dog at foot, he finds himself among the
partridge coverts. His faithful dog scents the game,
and every nerve in his frame quivers as, step by step,
he thoughtfully and cautiously advances toward the
unseen covey; then, suddenly pausing, the right fore
paw balanced lightly, and every limb and muscle tense
and rigid, the beautiful animal is at once transformed
into a marble statue. Presently a whirr is heard, and
with a loud ca, ca, ca, a magnificent old cock rises on
the wing. Crack goes the gun, and down tumbles the
great bird, the scarlet tips over his eyes glistening like
rubies, as with a thud that gladdens the sportsman’s
heart he strikes the earth. Or perhaps a whole family
—father, mother and children—trises at once, and the
double barrels bang at them right and left, bringing
down two or three brace. At times a late covey is started, the chickens of which
are only two or three weeks old, just able to run smartly
along the ground. It is a touching sight then to see
the cock fearlessly exposing his life to save the lives
of his offspring. He tumbles along the ground a few
yards in advance of the dogs, rolling there in order PTARMIGAN SHOOTING 397 to decoy the sportsman from the brood, which the hen
is anxiously calling into the thicket. No more touch-
ing instance of paternal affection could be witnessed ;
no more touching proof among the lower creation of
self-sacrifice prompted by love. The poor feeble bird
would almost attack dogs and men in his efforts to
save his children. No true sportsman would harm a
bird under such circumstances.. Only a brute would
fire upon it. The dogs are called off, and father and
mother ptarmigan are soon rejoicing over their res-
cued family. PTARMIGAN SHOOTING. At times, the ptarmigan here are
so tame that
they can be killed with a stick, and at others so wild
that they will not allow you to approach within gun-
shot; and this is generally the case in winter, when
the snow is hard and crusty, and the noise made in
approaching them alarms them. They are shot at all
times by our population in the distant districts, but
a close time is now fixed by law, which will have a
good effect when the law can be enforced. There is another species found in Newfoundland, There is another species found in Newfoundland,
but it is comparatively rare—the rock ptarmigan, L. rupestris (Gmelin). It is a truly alpine species, and
is seldom found below the line of stunted black spruce,
except in the depths of winter, when they descend to
the lowlands and feed on the buds of dwarf trees, some-
times in company with the willow grouse. Our settlers
call this the mountain partridge. SHOOTING THE PRAIRIE GROUSE The shooting of the prairie grouse differs widely
from the same sport on the woodland species. In the
one case the birds are. likely to dart into cover, dis-
appearing almost at once, and snap shooting is the rule. In the other the bird rises in the open, seems to fly
with deliberation, and the gunner has every opportu-
nity to make all his calculations, take careful aim, and
cut down the bird before he gets out of shot. Only
in the case of the sharp-tailed grouse, after they have
moved from the high uplands into brushy ravines or
wooded hillsides, is there any similarity between the
two styles of shooting. In September, after the sharp-
tailed grouse are grown, they spend the middle of the
day in wooded ravines or willowy bottoms, where cover
is often thick, and a single gunner, who has no dog,
and is trying to tramp the birds out of their places of
concealment, may have to do more or less snap shoot-
ing; but if he have a good dog that can be trusted to
work through the bottom, the gunner can usually so
place himself as to have clear and open shots. As the laws of most States now read, the prairie As the laws of most States now read, the prairie
chickens—of whatever kind—are reasonably well pro-
tected; but formerly, when shooting began, during the
month of August, at which time the birds lie very
close, it was not uncommon for two gunners to ex-
398 SHOOTING THE PRAIRIE GROUSE 399 terminate a brood, and in many places, a few weeks
after the shooting season had begun, there were prac-
tically no grouse left on the prairie. When, however,
the shooting season begins in October, or even No-
vember, as is now sometimes the case, the birds are
shy, scarcely lie to a dog, and are strong of wing. To kill them in great numbers is not an easy matter
under such conditions. PINNATED GROUSE SHOOTING To-day there are few places, except in Texas, where
much pinnated grouse shooting may be had, but thirty
or forty years ago things were very different. Then
men would start out on foot, and after two or three
hours of shooting would return to their homes bending
under a heavy backload of birds. Even later than
that, great shooting was to be had in Illinois, Iowa,
Minnesota and Nebraska. One scarcely dare say how
large the bags used to be, but they were unquestion-
ably large. Men hunted from wagons, carrying relays
of dogs, with abundant water to keep them fresh at
all times. Driving from farm to farm, and from vil-
lage to village, they covered an immense area of ter-
ritory and killed a great number of birds. In fact, at
one time, during the first days of amateur photography,
it was common to see photographs of such wagons
hung around with game, and with the well-armed gun-
ners and their dogs standing near. Happily, the taste
for such pictures is slowly changing, though the big- AMERICAN GAME BIRD SHOOTING 400 game hunter still delights to photograph his camp with
half a dozen deer hanging up in it, or to have his own
picture taken with his foot upon the body of the slain
bear or moose. Twenty years ago, when there were
still prairie
chickens in Minnesota, and plenty of them, it was well
for the shooter to be early abroad. Breakfast must
be eaten, the dogs looked after, a drive of perhaps five
or six miles made to reach the ground, and it was neces-
sary to be called long before daylight to reach the
shooting’ ground as soon as the birds began to move. It is hard to leave a comfortable bed in which it It is hard to leave a comfortable bed in which it
seems only a few moments since one settled himself
to sleep, but a strong heart and prompt action brings
the gunner out on the floor, and a few minutes of activ-
ity in putting on his clothes causes him to forget the
comforts of bed. Then, how good breakfast tastes,
and how eagerly he looks forward to the sport before
him. The drive in the dark may be cold and uncom-
fortable, but the growing light promises warmth, which
exercise certainly will bring. PINNATED GROUSE SHOOTING In those days chicken shooting was lawful on the In those days chicken shooting was lawful on the
15th of August, and the nights, though often damp
and chilly, were not cold. There was an opportunity
when there bred in Minnesota many ducks which have
long since been expelled by the farmer, to get an occa-
sional shot at teal, shoveller, mallards, and other home-
breeding birds; but, after all, the chickens were the
important thing. When the ground was reached the
horses were put in the stable, or perhaps were driven SHOOTING THE PRAIRIE GROUSE 401 through gate or bars into the extensive green stubble,
where the dogs were turned loose and eagerly com-
menced to range. On they go for some hundreds of
yards, crossing each other at high speed, with heads
high in air and merry tail action; but presently there
is a pause, one of them checks himself, and makes game,
quartering here and there with nose close to the ground,
and then making a cast, circling to try to pick up the
trail of the running birds. The other dog joins him,
and with a little rivalry they press forward, first one
and then the other in the lead, until at last, drawing
more and more slowly, one stops, and the other backs
him. The shooters hurry forward on foot, and presently
are close behind the dogs, which are standing staunchly. One man pushes forward and walks up even with the
leading dog, while his companion steps off a little to
one side; and then, suddenly, just before the pointing
dog, there is a tremendous hubbub, and three birds get
up and hurry away on swiftly beating wings. Alas! they stayed too long. Two of them fall to the right
and left of the man standing by the dogs, while his
companion, a little to one side, accounts for the third. The rapidly succeeding shots start the rest of the bevy,
which in a moment are on the wing, another falling to
the last barrel of the man to one side. The dogs have
sunk to their haunches, and are gazing eagerly over the
stubble. In a moment of such excitement it is too
much to expect them to drop flat. PINNATED GROUSE SHOOTING Presently the guns are recharged and the dogs called Presently the guns are recharged and the dogs called AMERICAN GAME BIRD SHOOTING 402 up and sent forward for the dead birds. They advance
very gingerly, and, picking them up, bring them neatly
to hand. One of the dogs brings his always by the
wing or neck, while the other grasps his bird about
the body, and seems to rejoice in the huge mouthful
that he has; yet he never bites nor apparently even
pinches the bird. Long before this both men had been watching the
disappearing bevy, which flew far, and at last seemed
to go down in a slough of high grass which inter-
rupted the stubble. Here the young birds, lying like
stones, were pointed by the dogs and walked up by
the gunners, one after another, until almost the whole
covey was down; and so, very likely, it went on all
through the day, until the heat of the midday sun be-
came too strong for walking or shooting or dogs, and
a couple of hours’ rest was taken. Late in the afternoon this was repeated, and some-
times a great bag was made; but, after all, while this
was killing, and was exciting enough, it was not what
in these days would be called sport. The young birds
lie very close, they present a large mark, and are too
easy to hit. The great destruction finally led to the almost total
extermination of the pinnated grouse of the West in
regions where it had always been most abundant, and
to-day the sportsmen of that region are bearing the
burden of the sins of their fathers. The following charming account of shooting prairie SHOOTING THE PRAIRIE GROUSE 403 chickens in Illinois, in the old days, is abbreviated from
a sketch from the pen of Wilmot Townsend, whose
charming contributions to Forest and Stream have
been well known for so many years:
The prairie has been likened to the ocean, and the The prairie has been likened to the ocean, and the
simile is not altogether wide of the mark; and as one
looks out over the boundless expanse of rolling grass
land that spreads on every side until land and sky
meet on the horizon line, it does not require a great
effort of the imagination
to see the resemblance. PINNATED GROUSE SHOOTING Though the waving grass gives life to the view, it
lacks the mighty heave of the ocean; that ceaseless
pulsing of the waters is wanting; the swelling waves
of prairie land seem to have stiffened and died, deserted
by the impulse which gave them birth. The mere turning of the sod reveals a rich dark The mere turning of the sod reveals a rich dark
loam, that, without fertilizing, will yield immense crops
of corn and produce a wonderful return in golden
grain. In late autumn the chickens resort to the fields
of standing corn to glean, and rich pickings they find. On a still, clear morning the scolding of the cocks On a still, clear morning the scolding of the cocks
begins at sunrise, and the air resounds with their kek-
kek! kek-kek! until long after the hoar frost has dis-
appeared. Then you will see flock after flock leave
the prairie and enter the corn, there to remain through-
out the day, unless disturbed. A short hour before
sunset they run out to the edge and spring into sturdy
flight for the night bivouac on the open plain. At
this season these camps of the prairie chicken often
contain thousands of individuals, representing hundreds AMERICAN GAME BIRD SHOOTING 404. of coveys, that arrive from all quarters, coming some-
times from a distance of miles to this favored spot. One may frequently see some belated party string
across his path as he tramps along just after sunset. Silent as owls, they flit past on outspread wings, and
vanish in the gloom,
The shooting among the standing corn is, to me, the The shooting among the standing corn is, to me, the
most exciting of any of the methods pursued in hunting
this\ fine bird. \2) 20;
It is early in the forenoon, and I will see if I can- It is early in the forenoon, and I will see if I can-
not find some of the birds I saw enter that eighty-acre
piece a while ago. We are presently in the thick of it, and save for the We are presently in the thick of it, and save for the
rustling leaves on the tall stalks which rise above our
heads it is very still. PINNATED GROUSE SHOOTING The chickens are running, as
I see by the trailing of my dog, and it is necessary to
step out briskly in order to keep him in view. There! He has struck a hot scent! Hear how his tail is beat-
ing a tattoo on the stalks as he worms his way along,
crouching close to the ground in his eagerness to locate
the birds. At last, with head almost at right angles
with his body, he stands rigid. There they go. A dozen chickens whizz up through There they go. A dozen chickens whizz up through
the corn about us. I catch a glimpse of one just as
he clears himself, and pull on him almost without
sighting. Crash! thud! Down he comes, and quickly
I have him in hand, a fine young bird, as I find by hold-
ing him up so that his whole weight falls on the under
mandible, which breaks under the strain. The differ-
ence in plumage of these full-grown birds is very slight, Kansa
ing
in
ken
shooti
irie Chic
irie
Pra Kan
ing
in
ken
shoo
irie Chic
irie
Pra SHOOTING THE PRAIRIE GROUSE 405 and it is by this simple test that we may distinguish
young from old. In an old bird the lower mandible
always sustains
the weight of the body without
breaking. The old dog is pointing again. A single bird rises
this time, and sails off uninjured, though I give him
the contents of both barrels. How easy it is to miss
them in this cover, in spite of their size. We move on. There has been a covey about here, as numerous There has been a covey about here, as numerous
tracks tend to show. It will take careful work to put
them up now, as they have doubtless taken the alarm
ere this, and are keeping well ahead of us. There they
go again, off to the left. If we flush another bunch
in this cover they will probably get up all together,
as is their habit after being so disturbed. Try to pick
your birds, for though they rise with a thunderous roar
of wings, and look very large, you will find, as in
quail shooting, that pot shots can seldom be made. Here is the edge of the field. I think we will work Here is the edge of the field. PINNATED GROUSE SHOOTING I think we will work
around to where we entered, and after trying that little
patch between here and the house I will wait until to-
ward sundown, when they come out, and perhaps I
may get a little flight shooting. Chickens feed quietly among the corn, rarely be- Chickens feed quietly among the corn, rarely be-
traying their presence, save to the keen nose of your
dog. Once flushed, they lie very close for some time
after alighting, and will not scatter until thoroughly
satisfied that danger is past. This, however, is only
in cover where they have been used to feeding undis-
turbed. Later in the season, when constant gunning AMERICAN GAME BIRD SHOOTING 406 has made them wary, they will make for the open at
first flight, and rarely return before next morning. Look here! See where they have been dusting in this
dry loam, just like so many old hens. At four o’clock I walk out on the prairie to an old At four o’clock I walk out on the prairie to an old
haystack, that seemed to be located near the center
of their flight as they left the corn last evening, and
lie down to await developments. High overhead a
flock of wild geese is moving south; travelers, I guess,
from the height at which they fly, and the silence and
regularity of their action. Blackbirds in dense flocks
are sweeping past, with incessant noise of harsh, rasp-
ing cries. Close by, a miniature whirlwind is sucking
up the stray heads of buffalo grass and careering with
them a short distance, where it leaves them awaiting
the next puff that shall waltz them off again. Keeping a sharp lookout in the direction of the corn- Keeping a sharp lookout in the direction of the corn-
fields, I presently see a covey of chickens rise. They
come down well to one side, far out of range, but af-
fording me a fine opportunity to observe their manner
of flight. Rising with a burst of strong wing strokes,
they attain an elevation of from 20 to 30 yards, and
scale off on extended pinions for long stretches; then
with a renewal of wing-beats they acquire fresh impe-
tus. In this manner they alternate, now scaling, now
flying along, until lost to sight over the prairie. Here comes another covey. PINNATED GROUSE SHOOTING Now they are close Here comes another covey. Now they are close
upon me. Bang! one—bang! again, as two cross each
other in flight, and down they come. There are num-
bers of chickens sailing by, but none venture in my SHOOTING THE PRAIRIE GROUSE 407 direction. It is nearly dark when I reach the house,
as night shuts in very quickly. How cosy the firelight
makes the room after the silence of the prairie. The luxury of field shooting is pointed out in the
following aecount of shooting in the Indian Territory. It presents a striking contrast to the sorrows of the
eastern gunner of old times, who spent his shooting
days in wallowing through swamps, squeezing among
alders and trying to break down cat-briars and grape-
vines : Prairie chicken shooting is, par excellence, the sport
of the lazy man. It is the easiest of all land shooting. lirst, because the field is always open, and if one is
too lazy to walk he can shoot from a horse or wagon;
secondly, because early in the season, before the birds
are matured or have been too often disturbed, they lie
in the tall grass as close and as long as one wishes;
and thirdly, because they make a good big mark, flying
true, and not too rapidly, and there is so much of them
that one need have no fear of blowing them all to
pieces, leaving nothing but feathers in the air. If
they happen to get up too near for a shot you can
measure your distance, knowing there is no bush or
tree for them to dodge behind. Thus, in all respects,
they make fine game for one not disposed to be in a
hurry; and for these same reasons the gentle things
are easy plunder for the unscrupulous market hunter. PINNATED GROUSE SHOOTING Later in the season, during the last of September, the
birds are fully matured, have become stronger flyers, AMERICAN GAME BIRD SHOOTING 408 and have been made a little more shy from an occa-
sional shot among them, even in this far-off locality, |
and will not always allow a dog to approach so near
them; and if a bird gets up twenty-five or thirty yards
away one has to wink his eye pretty quick in order
to stop him, for being strong, they will carry off quite
a weight of shot unless winged, or hit in a vital part. We shot mostly on the Omaha Indian reservation, We shot mostly on the Omaha Indian reservation,
which, here, is quite rolling, but excellent land, covered
with a heavy growth of prairie grass; and there being
no fences, we could drive at will in almost any direc-
tion. The Indians had here a few fields of corn and
other grains, but there were thousands of acres over
which we could ride and shoot at pleasure. Our wagon
was a comfortable two-seated spring vehicle, with a
park top, which would carry four to six persons and
our dogs, and we had a couple of ponies, somewhat
larger than jack rabbits, for a team, which would walk
or run all day, but manifested a most decided disin-
clination to trot. We had two dogs with us, an Irish
and an English setter, and our landlord had two Rus-
sian setters which were at our service. Our mode of proceeding was about this: We would Our mode of proceeding was about this: We would
get an early breakfast, load up our dogs, guns, ammu-
nition, lunch, a big jug of water for ourselves and
the dogs, and point the ponies for the Indian reserva-
tion, when a ride of a little more than a mile would
bring us to good.shooting grounds. We always drove
to the leeward of the field over which we designed
to shoot. Then we would get out, leave Miss S. to Our mode of proceeding was about this: We would
get an early breakfast, load up our dogs, guns, ammu-
nition, lunch, a big jug of water for ourselves and
the dogs, and point the ponies for the Indian reserva-
tion, when a ride of a little more than a mile would
bring us to good.shooting grounds. PINNATED GROUSE SHOOTING We always drove
to the leeward of the field over which we designed
to shoot. Then we would get out, leave Miss S. to SHOOTING THE PRAIRIE GROUSE 409 manage the team, following slowly in our wake, and
occasionally marking birds for us, which services she
rendered in an admirable manner and with a new and
delightful pleasure to herself. With the four dogs,
the three of us keeping about two hundred yards apart
and moving in line as nearly as practicable, each would
generally find birds enough for his individual shoot-
ing without disturbing the others or placing them in
danger; and when one’s pockets became too heavy for
comfort or convenience he would
fall back to the
wagon and deposit his load. Occasionally we met at the wagon, to water the dogs,
eat lunch, smoke a cigar, look over our birds, and when
we were thoroughly rested, started out for another
tramp. Thus we would put in time until about 11
o’clock, when it was
time to bundle
ourselves
and
dogs into the wagon and drive back to the hotel for
dinner, after which, about 3 o’clock, we would find
ourselves again seated in the wagon and on our way
for the evening shoot, which usually lasted far into
the “twilight soft and gray.”’
To me there is a rare
and indescribable delight in shooting on a still, quiet
evening, watching the last rays of the setting sun, and
the last faint glimmer of light as it quietly passes away
under the gauzy curtain of night. We always found supper awaiting us on our arrival We always found supper awaiting us on our arrival
home, when, after caring for the dogs and shedding
our hunting traps, and taking a good bath, we gathered
about the little round table, doing ample justice to
broiled chicken and other good things. 410 AMERICAN GAME, BIRD SHOOTING Should you ask me how many birds we bagged I
could not tell you. I kept tally till we got past 100,
and then quit. We did not forget our friends, nor
neglect ourselves, for we sent away a box each day, and
kept a string hanging under the little porch of the
hotel from which our table was supplied at each meal. PINNATED GROUSE SHOOTING Thus we passed the week, changing our route occa-
sionally, always getting birds enough to make it en-
joyable sport, never turning it into downright slaugh-
ter, and leaving birds enough and to spare. And with
it all we had a good time. At the approach of cold weather the birds gather At the approach of cold weather the birds gather
together in large flocks and pack, as the. term
is, re-
maining in these packs all through the winter. At this
time they are shy, will not lie for a dog, get up at a
considerable distance and fly a long way. An interesting account of the birds at this time was An interesting account of the birds at this time was
written by Latrobe in his “Rambler in America.”
La-
trobe accompanied Washington Irving and one or two
other friends on an expedition to what was then the
Far West—beyond the Missouri River, and like most
Englishmen did not let pass the opportunity to get
some shooting. In 1832 he wrote of his efforts to shoot some
prairie fowl. This was in Indian summer, at a time
when the squirrels were at work among the dead leaves
beneath the hickory and pecan trees near the Osage
Agency, and when countless bands of water fowls and
flights of pigeons, which had been constantly observed ‘sneyyjso
TH
“gq
Aq
3uyured
WOlT
‘SUdHIIYD
WO
yseq
siojuI0g
iirc
oncihoW
HS
iy ‘s
TH
“gq
Aq
3uyured
WOlT
‘S
WO
yseq
siojuI0g SHOOTING THE PRAIRIE GROUSE AIT passing to the southward during a season of cold, had
ceased to be a source of wonderment. He says:
“The prairie fowl “The prairie fowl had now completely thrown aside
their summer habits. Instead of keeping apart in dis-
tinct families, scattered over a vast extent of country,
like our grouse at an earlier season, they now appeared
congregated in immense flocks in the immediate vi-
cinity of the farms. I had plenty of opportunity of
studying their habits, but to shoot a few brace, as
they were extremely wild, required frequently hours
of patient and wary exertion; whereas at an earlier
season a sportsman, if aided by a dog, might bag any
quantity from the pertinacity with which they will lie
close until forced to fly. PINNATED GROUSE SHOOTING “Tt appeared that at this time of the year all the birds
within an area of three or four miles square congre-
gated together by consent at sundown on a given spot
in the rank dried grass of the unburnt prairie to
sleep. Many a time have I seen them coming at sun-
set from every point of the compass with their re-
markable level and even flight over the swells of the
prairie toward the place of rendezvous, which a few
days’ observation enabled me to determine upon within
a quarter of a mile, and twice I was on the prairie
early enough to hear and see them rise, and the sight
was such as might make an English sportsman’s mouth
water. The number must have amounted to many
thousands, and the sound of their wings might be
heard a very great distance. After rising for about
half an hour they crowd the scattered trees on the edge AMERICAN GAME BIRD SHOOTING 412 of the prairie by hundreds at a time, after which they
disperse. Their wariness at this time is extreme; and
the slightest indication of the approach of man, even
at a great distance, is noticed by the cock, who, perched
on the topmost twig, elongates his neck and peeps first
on one side and then on the other with the most pro-
voking caution. How often have I been foiled, when,
after the most cautious approach, either in serpent
fashion like the Indian, dragging myself through the
grass inch by inch, or in an upright position striving
to counterfeit a tree stump, never stirring till the sen-
tinel looked another way, and then, by imperceptible
approaches and five more feet in five more minutes,
would I approach the tree within range—the careful
bird began to grow more and more doubtful and rest-
less and finally set up that clear, tremulous crow which
said:
‘There’s a rogue with a gun almost within shot,’
as plainly as though he had spoken English. The in-
stant and complete dispersion of the whole covey to a
great distance would be the immediate consequence.” SHARP-TAILED GROUSE SHOOTING Except for the seasonal movements already mention-
ed, the sharp-tailed grouse is very local in habit. Half
a dozen pairs of these grouse will build their nests and
hatch their young along three or four miles of prairie
stream,
or the dry ravines which lead down to it. Mother and young will spend the summer on a range
of prairie covering less than a square mile, and there, SHOOTING THE PRAIRIE GROUSE 413 in September or early October, one may find these
broods of full-grown birds scattered over the hillsides
at feeding time, and in the middle of the day gathered
together in the shade of willows or box elders, or in
a clump of rose bushes, from which, if disturbed, they
rise with a startling roar and much cackling, to fly
from seventy-five to two or three hundred yards, and
drop in some convenient cover along the ravine or
on a hillside. I have found them in southern Mon-
tana under just these conditions, where, if one had had
a good dog and a shooting pony, it would not have
been difficult to exterminate the whole brood. It is soon after this, however, as the nights grow
cold and frosty, that the birds get together in larger
companies, several broods uniting, and in the early
mornings take to the tops of the cottonwoods and wil-
lows, where they keep that excellent lookout that Mr. Latrobe has so well described in another species. At
that time they are shy and quite impossible of approach
in these exposed situations. Though the sharp-tailed
grouse
rises
from the Though the sharp-tailed
grouse
rises
from the
ground with noise enough to disconcert the gunner of
slight experience, it is not a difficult bird to kill. The
mark is large and the bird’s flight steady and straight. There is time enough to shoot carefully. Ata straight-
away, low-flying bird one may shoot directly, but
crossing shots demand that the bird should be led a
little. After all, this is almost wholly a matter of
experience, and as almost all the shots are in the open,
one may fire deliberately, and can, usually, if he misses, AMERICAN GAME BIRD SHOOTING 414 explain to himself why he did so and at the next shot
correct his error. SHARP-TAILED GROUSE SHOOTING Like all the prairie grouse with which I am ac- Like all the prairie grouse with which I am ac-
quainted,
the meat
of the full-grown
sharp-tailed
grouse is dark, though the half-grown birds are white-
meated. The flesh is tender and well flavored, as might
be expected of birds that had passed the summer feed-
ing on prairie insects and a variety of berries. In
winter, when the character of the food largely changes,
and willow and cottonwood buds constitute a large
share of its dietary, its flesh is less toothsome, being
much drier. The sharp-tailed grouse, owing to the character of
country which it inhabits, brushy ravines, willowy bot-
toms, and, at certain seasons of the year, hillsides over-
grown with underbrush and some large trees, is much
less easily killed than the pinnated grouse, and will
probably remain with us much longer than that spe-
cies. If it could be introduced to eastern covers, and
protected until it had established itself, it would prove
a grand bird for Atlantic coast gunners. So intro-
duced, and so acclimated, it would, however, bear only
the very lightest shooting, perhaps not more than three
or four per gun in a season. More than that would
soon result in their extermination, and the efforts put
forth to introduce them would thus be wasted. In the early days of western field trials held on In the early days of western field trials held on
prairie chickens in this country, the birds sought for
were chiefly the pinnated grouse. These proved not
sufficiently numerous, and were so readily killed that SHOOTING THE PRAIRIE GROUSE 415 they did not give the dogs an opportunity to show
off the best that was in them. A little later, therefore,
a change was made, and grounds were sought where
the sharp-tailed grouse was abundant. This was in
northwestern Minnesota and in Manitoba, and here,
for a number of years, were held what used to be called
the chicken trials. SHARP-TAILED GROUSE SHOOTING Before they have been much shot at the young sharp- Before they have been much shot at the young sharp-
tailed grouse lie well, and offer easy shooting ;
but when
they
attain
their
full
growth,
and
the weather
grows colder, they become much more shy, and while
still spending most of their time on the upland prairies,
seek refuge, when disturbed, in the ravines or the wil-
lows of the stream bottoms. During the winter they get together in considerable During the winter they get together in considerable
packs, and live largely among the underbrush of the
river bottoms; yet often they may be seen sitting on
the roofs of buildings or willow trees, or fences im-
mediately about the ranch, and sometimes at this sea-
son of the year, while roosting in such places, may be
killed in large numbers with a rifle. When it will lie, the sharp-tail furnishes fine sport When it will lie, the sharp-tail furnishes fine sport
with a dog, but often it runs for a considerable dis-
tance, and so rapidly that it may rise far ahead of the
dog. As soon as the weather has become cool a quick
eye and arm and a strong lead to the gun are required
to bring the bird down. In many respects the sharptail suggests the bobwhite In many respects the sharptail suggests the bobwhite
quail, especially in the way in which it leaves the
ground in flight and the speed with which it moves on AMERICAN GAME BIRD SHOOTING 416 the wing. In the late summer or early autumn it may
be seen stalking about over the ground, walking quite
differently from some of our other grouse, holding
the head and tail erect and lifting its feet quite high,
as if stepping over some obstacle before it. It is not
easy to recognize in this seemingly absurd, affected bird
the ball of feathers that a month later will rise from
before your dog and speedily be out of range. In some parts of the country I have heard gunners In some parts of the country I have heard gunners
speak disparagingly of the sharp-tailed grouse, per-
haps for no better reason than that they fly more
swiftly and rise with more tumult than the pinnated
grouse; but I regard them as one of the finest of our
game birds. SAGE GROUSE SHOOTING The sage grouse, which has its home on the high,
dry plains, where grows the artemisia, harmonizes in
color with the gray soil and gray stems of the sage
brush to an extraordinary degree. When the sage
grows high, and, as is often the case in river bottoms,
is interrupted by frequent clumps of rye grass, one
may even walk into a brood of the birds without see-
ing them, and perhaps have them rise all about him;
or, on a sudden, his eye may catch a dozen heads, on
tall, straight necks, staring at him from every side. The sage hen, it must be confessed, does not convey
to the observer the idea of a game bird. When tame
it is so gentle and unsuspicious, and when shy so SHOOTING THE PRAIRIE GROUSE At. clumsy and lumbering in its efforts to escape, that one
hardly gives it credit for game qualities. It is a splen-
did bird, however, and its great size makes it a trophy
of the shotgun eagerly to be sought after. The range of the sage grouse is commonly believed The range of the sage grouse is commonly believed
to be coterminous with that of the plant whose name
it bears. Though often seen in localities where no
sage is actually visible, it never, we think, occurs far
from the region where fhe plant is found. It is at home
alike where the stunted shrubs of the wormwood grow
only two or three inches high, or in valleys of the great
central plateau where they are real trees, as high as
one’s head. In cover such as this the traveler may
often ride up on a great flock of the birds, which are
usually so unsuspicious that they will stand for some
time before taking alarm. When the birds do rise they get up heavily, with a When the birds do rise they get up heavily, with a
lumbering flight; but after they have acquired momen-
tum they go much faster than one would suppose, and
it is easy to shoot behind crossing birds, whose great
size often tends to make the shooter careless. SAGE GROUSE SHOOTING Inhabiting, as it does, a region of extreme aridity,
and because the shooting season comes at the dryest
period of the year, dogs would be at a great disad-
vantage in the pursuit of this bird, and I personally
have never seen them used in this shooting. The bird
is large, it inhabits the open, and is readily seen at
some little distance. Though often unsuspicious, yet
as the season advances, or in places where they have
been shot, or much disturbed, they will often rise at AMERICAN GAME BIRD SHOOTING 418 a distance of 125 or 150 yards, and then make such
long flights that it is useless to think of following
them. The adult sage grouse is tough and heavily feath-
ered, and possesses great vitality. Unless properly
hit it will carry away a heavy charge of shot. Young
birds, on the other hand, drop to a mere touch. I have
killed a great many on the wing with small charges
of dust shot. Many years ago a correspondent of Forest and
Stream declared that, when there is reasonable cover,
sage grouse will often lie to a dog better than the pin-
nated grouse. He adds what is now well known—
that the peculiarities of its home do not give the dog a
fair chance to work or to do himself or the game
justice, since the artemisia grows only upon barren
prairies
some 4,000
to 10,000 feet above sea
level,
where the soil is hard and dry, with little vegetation
save stunted shrubs, cactus and an occasional clump of
wild grass, where rain rarely occurs and there is little
moisture in the air or upon the ground. When thick,
the tough sprawling crooked evergreen, or rather ever-
gray, shrub is often difficult for man, horse or dog to
get through. The sage cock is a good skulker and
runner, and not easily flushed if it can hide. The same idea is suggested in Dr. Newberry’s ac-
count of his first meetings with this great bird during
one of the early explorations for a railroad route. It
is in- Volume V
lof Paciie R. R. Reports:
“Coming into camp at evening, I had been attracted “Coming into camp at evening, I had been attracted SHOOTING THE PRAIRIE GROUSE 419 by a white chalk-like bluff, some two miles to the right
of our trail, which I visited and examined. SAGE GROUSE SHOOTING Near it
was a warm spring, which came out of the hillside, and,
spreading over the prairie, kept a few acres green and
fresh, strongly contrasting with the universal brown of
the landscape. In this little oasis I found some, to me,
new flowers, many reptiles, and a considerable number
of sharp-tailed grouse, of which I killed several, the
whole presenting attractions sufficiently strong—as we
were to remain in camp one day—to take me over there
next morning. I had filled my plant-case with flowers,
had obtained frogs, and snakes, and chalky infusorial
earth enough to load down the boy who accompanied
me, and had enjoyed a fine morning’s sport, dropping
as many grouse on the prairies as we could conveniently
carry. Following up the little stream toward the spring
on the hillside, a dry, treeless surface, with patches of
sage brush
(Artemisia tridentata),
I was suddenly
startled by a great flutter and rush, and a dark bird,
that appeared to me as large as a turkey, rose from the
ground near me, and uttering a hoarse hek fiek, flew off
with an irregular but remarkably well-sustained course. “T was just then stooping to drink from the little “T was just then stooping to drink from the little
stream, and quite unprepared for game of any kind,
least of all for such a bird, evidently a grouse, but so
big and black, so far exceeding all reasonable dimen-
sions, that I did not think of shooting him, but stood
with open eyes, and, doubtless, open mouth, eagerly
watching his flight to mark him down. But stop he
did not—so long as I could see him, now flapping, AMERICAN GAME BIRD SHOOTING 420 now sailing, he kept on his course till he disappeared
behind a hill a mile away. “T was, of course, greatly chagrined by his escape, but
knowing that, given one grouse, it is usually not difficult
to find another, I commenced looking about for the mate
of the one I had lost. My search was not a long one;
almost immediately she rose from under a sage brush
with a noise like a whirlwind, not to fly a mile before
stopping to look around, as the cock had done, but, by a
fortunate shot, falling helpless to the ground. SAGE GROUSE SHOOTING No deer-
stalker ever felt more triumphant
enthusiasm
while
standing over the prostrate body of a buck, or fisher-
man when the silvery sides of a salmon sparkled in his
landing-net, than I felt as I picked up this great, and to
me, unknown bird. I afterward ranged the hillsides
for hours, with more or less success, waging a war on
these birds, which I found to be quite abundant, but
very strong-winged, and difficult to kill. I repeatedly
flushed them not ten yards from me, and, as they rose,
poured my whole charge, right and left, into them,
knocking out feathers, perhaps, but not killing the bird,
which, in defiance of all my hopes and expectations,
would carry off my shot to such a distance that I would
not follow him, even did I know he would never rise
again. Here, as elsewhere, I found these birds con-
fined to the vicinity of the sage bushes, from under
which they usually spring. “A few days later, on the shores of Wright and “A few days later, on the shores of Wright and
Rhett lakes, we found them very abundant, and killed
all we cared to. A very fine male which I killed there “A few days later, on the shores of Wright and
Rhett lakes, we found them very abundant, and killed
all we cared to. A very fine male which I killed there “A few days later, on the shores of Wright and
Rhett lakes, we found them very abundant, and killed
all we cared to. A very fine male which I killed there SHOOTING THE PRAIRIE GROUSE 421 was passed by nearly the whole party, within thirty
feet, in open ground. I noticed him as soon, per-
haps, as he saw us, and waited to watch his movements. As the train approached he sank down on the ground,
depressing his head, and lying as motionless as a stick
or root, which he greatly resembled. After the party
had passed I moved toward him, when he depressed
his head till it rested on the ground, and evidently
made himself as small as possible. He did not move
till I had approached within fifteen feet of him, when
he arose, and I shot him. He was in fine plumage, and
weighed over five pounds,” TURKEY SHOOTING. There are many methods of shooting the wild turkey,
yet, after all, it is probable that it is more often killed
by accident than by any one of the more approved
methods. In other words, men traveling through the
woods or fields stumble on the turkeys, flush them,
and so get a shot. In the South, “calling” is the approved and most
used means of killing these birds. The hunter con-
ceals himself in a blind, imitates the call of the hen, and
so draws to him the other members of the flock. This
is practiced with especial success either in the autumn,
when the young turkeys are still innocent, or in the
spring, at gobbling time, when the gobblers are likely
to respond readily to the supposed call of the female. In regions where turkeys are plentiful, it is common
for hunters to go out in the afternoon, and, finding
a brood, to flush and scatter it. If night is near, the
turkeys are likely to try to get together before roost-
ing time, and to begin calling almost at once. If, how-
ever, it is too late in the day for them to do this, the
birds roost in the trees, and in the morning are still
more anxious to get together. The hunter, therefore,
starts out long before daylight, repairs to the locality
where he started the turkeys the night before, and con-
cealing himself there, either by means of a blind con-
422 422 422 ye
bk
ee
CES Sear eT
TS
Wild
Turk
in
the
Pines Wil
Tur
in
the
Pine TURKEY SHOOTING 423 structed the night before, or in some thicket, listens
carefully for the first calling of the turkey, to which he
responds. In this way he may succeed in drawing
several turkeys toward him. The instrument used in callin The instrument used in calling turkeys is sometimes
made of the bone of the bird’s wing, sometimes of a
little box floored with a piece of slate, the cry being
caused by the stroke of another piece of slate on the
slate in the box. At all events, the noise must be a
good imitation of the turkey’s cry, for the great bird
is one of the most acute of fowls, and very little escapes
either its eye or its ear. TURKEY SHOOTING. If the imitation of the cry is
a poor one, answers will soon cease to come, and the
birds will retreat. Some Some years
ago a correspondent,
Tripod,
wrote
from Mississippi a brief but vivid account of the kill-
ing of a turkey by Miss Mary C. Breckenridge, whose
experience with a shotgun was limited, but who, when
the occasion arose, showed herself an apt pupil of her
skilled instructor. She was new to the woods, and
after traveling for some distance made up her mind
that turkeys were a myth. Nevertheless, it was de-
cided to call, and after a time, in the open woods, Miss
Mary selected a good log for an ambuscade, while Tri-
pod used the yelper, and at once received an answer
from a nearby bird. The turkey kept approaching,
ing, but before he had been seen a shot in the distance
frightened him and he ceased answering. The turkey
hunters sat quietly for a time, uttering now and then
a little yelp, or cluck, when a twig snapped, and as AMERICAN GAME BIRD SHOOTING 424 Tripod carefully peeped over the log, there, not twenty-
five paces from them, came a big gobbler, all fluffed
up and strutting. He was coming nearer, so that
prompt action was necessary. Tripod goes on: “ “Now, Miss Mary, don’t move a bit,’ I caution. “Her gun lay across the log, pointing in the direc- “ “Now, Miss Mary, don’t move a bit,’ I caution. “Her gun lay across the log, pointing in the direc- “Her gun lay across the log, pointing in the direc-
tion from which the first answer had come, with its
muzzle among some twigs, to free it from which the
gun had to be withdrawn entirely from over the log
and pushed forward again in a new place, as the tur-
key was approaching from a different quarter, very
much askew to the log’s position. This difficult move-
ment in the face of the enemy was successfully accom-
plished. “ “Now take the gun cautiously, aim carefully at his
neck, low down, and shoot. Don’t hurry!’
“She did it just that way, and over rolled that big “She did it just that way, and over rolled that big
bird. TURKEY SHOOTING. With great exultation we then bore down upon
him, and saw him flutter, then quiver, and then lie
still,
On the lower part of his neck was much evi-
dence of No. 5 shot. Miss Mary then looked much
prouder than the turkey had looked a few moments
before, and very justly so—and said turkeys were not
a myth any more. I realized this myself as I bore this
tangible specimen to where our horses had been left.”
A successful turkey hunter, writing from Missouri A successful turkey hunter, writing from Missouri
recently, expressed his views of turkey hunting, and
what he says, which I give in substance, will apply to
the pursuit of the bird over much of its present range: TURKEY SHOOTING 425 To be a successful turkey hunter one must know his
country and conditions, for unless the ground be cov-
ered with snow, and the pea crop has been a late one,
the birds keep to the woods, where they are not easily
seen in the tangle of hazel and blackberry. Plenty of turkeys means a good crop of mast, for Plenty of turkeys means a good crop of mast, for
in autumn the chief food of the bird is the acorn, and
the feeding grounds of the bird will be where the sup-
ply of acorns is greatest. If the crop fails in the
swamps, most of the birds move into the rough hill
country, for when the mast fails in the lowlands the
hills are likely to have a very good crop, and a
failure in the hills means an abundant harvest of acorns
in the swamp. To find where the birds feed, however,
is not to get your game, or even to see it. Often the
scattered birds may be easily called up, but on the other
hand, some flocks of young birds refuse to respond to
the call. To stalk the turkey requires more care and skill
than to stalk the deer, and he who is successful in this
description of hunting may congratulate himself. On
the other hand, one may often blunder on turkeys
without special effort, and may believe that he has
mastered the art of turkey hunting; while the very
next day he may not be able to get within long range
of his game. TURKEY SHOOTING. Sometimes it is possible to walk close
up to turkeys, so that one wonders if he has not fallen
upon a flock of domestic birds. As turkeys commonly roost over a water-course, or As turkeys commonly roost over a water-course, or
slough, as protection from danger, it is sometimes pos- AMERICAN GAME BIRD SHOOTING 426 sible, late in the evening or early in the morning, to
pass under their roosts in a boat. If birds can be found
in such a situation there is opportunity for good shoot-
ing with a rifle. A very successful method of shooting them in Mis-
souri is to ride through the timber on horseback, for
the birds are not as shy of the approach of a rider as
they are of a footman. On the other hand, turkeys
alarmed are likely to make for swamps or down tim-
ber, where a horse cannot go. If a flock of turkeys is scattered in the evening, at If a flock of turkeys is scattered in the evening, at
sundown, or frightened from its roost at night, the
hunter who can be at the place before sunrise is likely
to be able to get a number of shots by calling up the
birds. Shooting turkeys from the roost at night is a method
that does not present many features of sport. Track-
ing birds in the snow is good sport, and calls for work
and endurance. It is a very certain method of get-
ting turkeys, but is obviously to be practiced only where
snow falls. An excellent description of the country in which
turkeys are found in Arkansas, and of the opportuni-
ties which used to come to the turkey hunters there,
is found in an account written by J. E. London in the
spring of 1900. He says:
“While I was engaged in having some assessment “While I was engaged in having some assessment
work done on some mining lands in Newton County,
Ark., in December, 1899, I was informed by some boys TURKEY SHOOTING 427 who came to our camp that on the mountain north of
us I could find a large drove of wild turkeys. “The mountains in that section are very rugged, and “The mountains in that section are very rugged, and
heavily timbered, but the woods are rather open, the
undergrowth being sparse and small. TURKEY SHOOTING. The timber is
white, black and post oak, hickory, beech, walnut,
cherry, and a half dozen other kinds, and such timber! Walnut six feet in diameter, and white oak the same. In fact, nowhere in the South does such a wealth of
timber exist. “One bright afternoon, taking my shotgun, I climbed
the steep mountain for about one mile, to where a
beautiful spring bursts from the rocks. I stopped,
and was enjoying the solitude of the gloomy, grand
old forest, when I espied a yellowhammer on a small
tree, and shot him. This disturbed two gray squirrels,
that ran to the top of a large wild cherry tree, but two
loads of No. 6 put them in my game bag. “I then started north along a path, but I had only “I then started north along a path, but I had only
gone a short distance when I walked out of the path
to look down into a grove of post oaks, when I beheld
a sight that put my. heart to beating like a trip-hammer. Twenty-six as fine turkeys as I ever saw, not two hun-
dred yards distant, were feeding as though nothing
could harm them, and paying no attention to the noise
I had made killing the bird and squirrels. Armed only
with a shotgun and some No. 2 shot cartridges, I began
the task of getting in reach of those turkeys. “Down on my knees and hands I went, and after “Down on my knees and hands I went, and after
forty minutes of hard work I was within seventy-five AMERICAN GAME BIRD SHOOTING 428 yards of them. Selecting two big gobblers that were
close together, and in line, I gave them both barrels,
and maybe there wasn’t a racket on that hillside. One
of them began to flop and whirl around like a chicken
with its head cut off, and the other flew about two hun-
dred yards at right angles with the remainder of
the drove,
that had gone over
a high bluff into
a ravine about one-quarter of a mile from where they
rose. He came to the ground, staggered a few steps,
rose, and when he had gone perhaps one hundred yards
he let go and came tumbling down dead. TURKEY SHOOTING. I ran to the
first one, and hanging him up in a small tree, followed
the other, and did the same with him. “Just then I heard some dogs on the opposite side “Just then I heard some dogs on the opposite side
of the mountain begin to bark, and a boy encouraging
them. I knew this would turn the other turkeys back,
so I hurried across a small draw and around to the
north of where they came to the ground, and climbed
on some large rocks, where I had a good view of the
mountain side. In a minute or two I saw two hens
coming for me on a dead run, and waited till they were
within forty yards. I gave one of them the left-hand
barrel, and as the other rose I knocked her a clean
somersault. “T now had four as fine turkeys as I ever saw, and “T now had four as fine turkeys as I ever saw, and
not caring to hunt any more that day, I hung the two
hens over my back and carried them to the gobblers. In sections where turkeys still exist, and where snow
falls in winter, a favorite method of hunting these TURKEY SHOOTING 429 birds is to take a track and walk the bird down. Usu-
ally, for several hours the turkey keeps well ahead of
the hunter, the man steadily following the track and
obliterating the turkey’s trail as he goes. After a
time the turkey is likely to grow tired, and at length
to hide, when it can be flushed and shot on the wing. Sometimes, to be sure, if the ground was unfavorable
for hiding, the turkey might fly, but usually it did not
go far, and its track could be taken again. A short
but well told story of tracking turkeys in the snow
was printed in Forest and Stream, October 26, 1907,
from a correspondent signing himself “Brinkstine.”
It
took place in Missouri:
“We walked two or three hours, and not a sign of “We walked two or three hours, and not a sign of
aturkey. Another hour, and yet no sign. We rounded
a deserted field with no success, further than seeing
plenty of rabbits and a few squirrels, but we did not
want to set the hills quaking by shooting such game. “We were tired. TURKEY SHOOTING. Walking in a foot of snow four “We were tired. Walking in a foot of snow four
or five hours steadily, and as silently as possible, 1s
not conducive to a hilarious feeling, as one grows cold
and hungry. We built a fire, ate our lunch, rested and
warmed ourselves. We were almost discouraged, but
started on with some hope of success. We walked
two or three hundred yards, and found a turkey track,
and a huge one. We had gone a quarter of a mile
when the tracks of three other turkeys joined the one
we were tracking. One turkey would not satisfy us
both, so we were glad to know that there were four
turkeys in the woods. Just as we were ready to start AMERICAN GAME BIRD SHOOTING 430 again we were chagrined to see George coming down
the hill, trailing the three that had joined ours; but as
age deserved consideration, we let him go in advance. age deserved consideration, we let him go in advance. “Thus we came to a bottom covered with a growth
of hazel brush, grape-vines and weeds, an ideal hiding
place for game. A cautious approach, and George
looked over a brush pile and saw the turkeys sitting
seventy-five or eighty yards away. It was impossible
to approach closer, and George saluted them with both
barrels, but got nothing. We marked the direction
they took, and followed, George alone, and Riley and
I after a huge one I had spotted. We struck a lively
canter and ran quite a distance, as we knew he would
run a long way before he tired and tried to hide. His
tracks indicated that he was getting tired, so we slowed
up and proceeded cautiously. Every fallen tree-top
was closely scanned. The tracks led down hill toward
a monster tree blown down during the summer, and
which still had its leaves on, forming a splendid hiding
place. We both recognized this immediately, and
started to go one on each side of the tree-top. I
stooped to go under the body, which lay three or four
feet off the ground, when out burst the turkey. I
‘dropped to get a view of him, and fired one shot as he
pitched down hill. TURKEY SHOOTING. We ran across the ravine, and at
the top of the next bluff found tracks where a turkey
had alighted and departed in hot haste. A run of half
a mile down the backbone of the hill, and we found
the turkey had flown. “Sadly we retraced our steps, while I caressed a “Sadly we retraced our steps, while I caressed a “Sadly we retraced our steps, while I caressed a TURKEY SHOOTING 431 weeping eye, through which a hazel twig had dragged,
and we mused on what might have been. “ What on earth are you fellows doing, anyway ” “ What on earth are you fellows doing, anyway ”
“We looked up, and there stood Uncle Will, giving “We looked up, and there stood Uncle Will, giving
us a rather tantalizing smile, we thought, as we re-
membered his caution as we left the wagon. We were
not particularly glad to have him see us just then, as
we came back panting and hatless. The best we could
do was to tell him the straight of it, and as he was
an experienced hunter maybe he could help us out. At the risk of being guyed, we even hinted that the
turkey had limped as it ran away. “ “Are you sure you hit it?’ he asked. “ “Are you sure you hit it?’ he asked. “ “Think I did. I know that I tried aw “ “Think I did. I know that I tried awful hard.’
“ “Well, step here and see what this is, and tell m “ “Well, step here and see what this is, and tell me
how it got there,’ pointing to a big gobbler lying in
a hole which it had made in the snow when it fell. How quickly I grabbed it up, admired its beauty and
guessed at its weight. “ ‘Flere,’ said Riley, ‘you’ve got your turkey. Mine “ ‘Flere,’ said Riley, ‘you’ve got your turkey. Mine
has just gone over the hill, and is on the other bluff. Come along, and we will get him, too.’
“We separated, seventy-five or one hundred yards “We separated, seventy-five or one hundred yards
apart, in order to cover more ground. We followed
the tracks ten minutes, when we saw the turkey rise
in open ground one hundred and fifty yards away. TURKEY SHOOTING. We
marked the direction and hurried on, found the tracks,
and followed briskly along the top of a broad, flat
ridge. We had taken only a few steps through the tall
grass when the turkey rose, twenty-five yards away, AMERICAN GAME BIRD SHOOTING 432 but directly between me and the sun. Quicker than
thought my gun went up, a flame of fire leaped out,
and was answered by the thud of the turkey as it
fell. “*“Confound you!’ said Riley good-naturedly, walking
over and picking up the turkey, a young hen. He took
the gobbler and made me take the hen, weighing only
about one-half of his load, and we set out for the wagon,
four or five miles away. ‘I am glad you got them, if
I didn’t get any,’ said he. ‘You got them both, and
that is just as good.’ ““No, it isn’t,’ I answered. ‘You claim that you
killed one, and [ll stand by you.’
“No, you won’t. You killed them both, and you’ve “No, you won’t. You killed them both, and you’ve
got to have the credit of it.’
And he was as good as
his word.” The story of an oddly secured Thanksgiving turkey,
killed in 1893, in the Alleghany Mountains, was inter-
estingly told in Forest and Stream, some years ago, by
Mr. Edw. Banks, now of Wilmington, Del. A party
of three, of whom Mr. Banks was one, had been shoot-
ing ruffed grouse near Bellwood, Pa., but without any
great success. There was a little snow on the ground
and many tracks of turkeys in the snow. Presently the
party went to a cold spring and sat down to eat lunch. While doing so one of them remarked that he wished
he could get a Thanksgiving turkey. Mr. Banks says:
“Lunch was about over when we heard a gun go off,
with a regular old black-powder dr-ro-o-oom. It was
not more than three or four hundred yards away from TURKEY SHOOTING 433 us, judging by the sound. Somebody said turkeys. The rest of us agreed. By common consent we jumped
up and went to a pair of bars where an old woods road
came out into the orchard, and stood looking in the
direction of the sound. TURKEY SHOOTING. “All at once out into the open, over the little valley, “All at once out into the open, over the little valley,
away up above the trees, sailed a turkey. At first it
looked as if it were coming toward us, but almost in-
stantly we saw that it had turned down the valley and
was flying directly away from us, along the hillside. Our eyes followed it as it kept on its way for about a
quarter of a mile, when suddenly up went its head
and down it came, ‘deader’n a mackerel.’ “ “He got it, after all,’ said some one. ‘He did,’ said
Bill. “Did you ever see anything like it?’ said Jake. “Then we returned to our lunch, and talked over the
wonderful thing we had seen. “ “He got it, after all,’ said some one. ‘He did,’ said
Bill. “Did you ever see anything like it?’ said Jake. “Then we returned to our lunch, and talked over the “Then we returned to our lunch, and talked over the
wonderful thing we had seen. “Bill was quiet for a while. Presently he broke
the silence with, ‘Say, do you suppose that fellow who
shot at that turkey saw it fall? Do you think he had
any idea that he hit it?? That was quite a new idea
to us, and we caught at it at once. It would be easy
to follow the road in the woods and find out whether
the man with the gun had turned off to the right or
left anywhere. His tracks in the snow would tell us. We had marked where the turkey fell, right alongside
of a monster pine which had been left when its breth-
ren were taken by the lumbermen. Some three hundred
yards along the road we saw where a man had come
down into the road, and saw, also, that his tracks, as ’ “Bill was quiet for a while. Presently he broke
the silence with, ‘Say, do you suppose that fellow who
shot at that turkey saw it fall? Do you think he had
any idea that he hit it?? That was quite a new idea
to us, and we caught at it at once. It would be easy
to follow the road in the woods and find out whether
the man with the gun had turned off to the right or
left anywhere. TURKEY SHOOTING. His tracks in the snow would tell us. We had marked where the turkey fell, right alongside
of a monster pine which had been left when its breth-
ren were taken by the lumbermen. Some three hundred
yards along the road we saw where a man had come
down into the road, and saw, also, that his tracks, as ’ AMERICAN GAME BIRD SHOOTING 434 well as those of his dog, led along the road in the
direction the turkey had flown. “Following the tracks, we finally came to the big
pine, which was down hill, on the right of the road. The turkey had fallen to the right of the pine, and so
was further from the road than the pine was. Jake
and I turned off into the woods, which was open
enough for us to have a good view of the ground. Bill went down the road a bit, to make sure that the
man with the dog had not turned off further along,
then he, too, turned to the right into the brush, and
began to investigate. “To Bill belongs the honor of finding that bird, a “To Bill belongs the honor of finding that bird, a
gobbler, too, wattled, bearded and bronzed as only an
old gobbler can be. He was a beauty, with not a
feather damaged and not a drop of blood on the snow. ... Was it our turkey, or did it belong to the man
who had shot at it? By this time he was a mile or more
on his way, and that turkey certainly did look good. “At all events, we took the bird back with us, and
if I remember rightly, it weighed just a fraction under
twenty-two pounds at Bellwood. There was not a fresh
shot mark on the body when I removed the skin for
the taxidermist, who found, however, when he was
taking the head from the neck, that a single small pel-
let, hitting the bird in the head, had cut a large vein in
the throat, and the bird had bled internally.” In Kansas, the Indian Territory and Texas, where
the turkeys necessarily roost in the trees growing alone
the river bottoms, the practice of shooting them on TURKEY SHOOTING 435 the roost was long a common one. TURKEY SHOOTING. If a roost was
found, the hunters sometimes approached it during the
afternoon, and concealing themselves, awaited the re-
turn of the turkeys, and perhaps the rising of the moon,
so that the great birds might be seen against the sky. At all times, and especially in sections where turkeys
had not been much hunted, it was easy to ride one’s
horse beneath a roosting-tree, and there dismounting,
to fire into the birds above. Stories are told of hunt-
ing parties sent out from the forts in Texas to get
fresh meat for the men, who, after a few days, returned
with a wagon loaded with turkeys. On the far-stretching a On the far-stretching and often level plains of Texas
turkeys were frequently coursed with greyhounds. A
troop of riders, with a number of dogs, rode in a long
line over the prairie, beating up an extent of country
half a mile or more wide. Minally, from some little
hollow or clump of bushes a brood of turkeys was
started, which took wing and flew, fast and far, over
the prairie. The dogs, accustomed to the sport, fol-
lowed some particular bird, and all the horsemen,
plying whip and spur, followed the dogs at top speed. The turkey’s flight was swifter than the speed of the
dogs, and after it had gone half a mile or more it
slanted slowly down to the ground and then raced
away with great swiftness; but the eager dogs soon
overtook it, and forced it to wing again. This time
it once more gained distance on the dogs, but now its
flight was much shorter than before, and once more
it came to earth. The third time, tired by its previous AMERICAN GAME BIRD SHOOTING 430 flights, it rose with difficulty, and the dogs were almost
under it, looking up, and now and then one of them
springing high in the air in an endeavor to seize it,
which at last one of them did. A graphic account of this sport, written by the late A graphic account of this sport, written by the late
Elliot Roosevelt, is given by Theodore Roosevelt in
his very charming book, “Hunting Trips of a Ranch-
man.” In old times, when turkeys were plentiful, and espe-
cially in the South, they were trapped and slaughtered
in considerable numbers. TURKEY SHOOTING. The commonest form of
trap was one formed of rails piled up, crossing at the
corners so as to make an ordinary rail pen. From
this a deep trench was dug out, slanting up to the level
of the ground. This trench was deep enough and
wide enough
for a turkey easily to pass through. Within the trap a little brush was thrown over the
trench, close to the rails. Then through the trench,
and running out some distance into the woods, was
laid a trail of corn. Turkeys wandering through the
woods found the corn, began to pick it up, and fol-
lowed it to the trench, and down the trench into the
pen, where more corn was scattered. In this way half
a dozen turkeys, or even a whole brood, might enter
the pen. When they had eaten the corn, and wished
to go away, they walked around the walls of the pen
looking for a place to get out, walking or jumping
across the trench, but never seeming to think of going
out the way they had come in. In this manner large
numbers of turkeys used to be caught. 437 TURKEY SHOOTING Audubon says that sometimes, after people had be-
come surfeited with the flesh of turkeys, they failed
to visit the traps which they had baited for some time,
and so the poor birds were allowed to starve to death. Another means of slaughtering turkeys, of which
we read, is by laying a trail of corn in some place
where turkeys congregate; then, hiding near at hand,
the gunner prepares to shoot at their heads and necks
when all are busily engaged in picking up the corn. In April,
1900, E. W. Nelson, the distinguished
field naturalist of the Biological Survey, described a
new sub-species of wild turkey from Arizona, under
the name Meleagris gallopavo merriann. This bird is
found in the mountains of Arizona, western New Mex-
ico, and south as far as the Mexican border, extending
north into southwestern Colorado. Little has been written about this bird, which pre- Little has been written about this bird, which pre-
sumably does not differ in habits from its relatives. Because it is so little known, we are glad to quote an
account written by E. A. Goldman, who secured for the
Biological
Survey the specimens
from which
Mr. Nelson described the subspecies. TURKEY SHOOTING. He has been Mr. Nelson’s companion on many He has been Mr. Nelson’s companion on many
expeditions, and in the Auk, in 1902, described a trip he
made into the Mogollon Mountains of northern Ari-
zona to secure a series of wild turkeys. He started
in January,
1900, and was
very successful. The
weather was cold, and promised snow, which raised
the collector’s spirits, since, of course, with snow on
the ground turkeys would be more easily tracked and AMERICAN GAME BIRD SHOOTING 438 found than if the ground was bare. They camped
upon a ridge separating Clear Creek Canyon and some
of the upper branches of Canyon Diablo, and from
here we will let Mr. Goldman tell his own story:
“The first turkey tracks were seen in spots of soft “The first turkey tracks were seen in spots of soft
soil among the pinons, soon after noon, and others
we crossed at intervals along the road. Late in the
afternoon, when near the upper edge of the pinon
belt, we entered what was evidently the feeding ground
of a large flock, for many tracks were seen crossing
the wagon road in several places. This encouraged us
to camp near here, and when we came to a trail lead-
ing down into Clear Creek Canyon, and indicating ac-
cessible water, we did so. After a few hasty prepa-
rations for the night the camp man was sent to the
bottom of the canyon, over a mile away, to water the
horses and fill casks for camp use, while the hunter
and I started out in opposite directions to look for
turkeys. I walked slowly and as quietly as possible
through fairly open pine and pifion woods, following
a half-circular course in order to avoid going too far
from camp so late in the day. Many tracks were seen,
but none of them had been made that day. At the end
of an hour or so I came to the head of a small side
canyon, about a mile from camp. The sun had just
set, and all nature seemed to have gone to sleep. Not
a sound broke the silence, except the slight, unavoidable
rustling of my own footsteps among the dry leaves. TURKEY SHOOTING. I stopped a moment to listen, and had about decided
to cut across to camp, when suddenly, from down the TURKEY SHOOTING 439 canyon, came
faint but unmistakable turkey notes,
which started me on a run in that direction. A few
minutes later I cautiously approached the place, where
probably over a hundred and fifty turkeys, all females
and young of the year, were noisily trying to settle
themselves for the night. They occupied the tops of
tall pines for about two hundred yards along one of
the steep walls of the canyon. In many of the trees
there were only two or three turkeys, but some of the
larger ones, and especially those with many dead
branches, contained from five to ten birds. Many of
them sat as closely together as possible, and constantly
craned their necks about, squawking, crowding each
other, and struggling for places. They flew frequently
from tree to tree, and sometimes a bird alighting clum-
sily on a crowded branch would knock off one or two
others, which would fly noisily to other places. At
first the disorder seemed to be general, and most of
the birds were crowding, or being crowded, and were
uttering loud cries of quit, quit, quit, with many modu-
lations, depending apparently upon the degree of ex-
citement. They rapidly became quieter, however, until
by the time it was dark they were settled for the night. When all was still I rose from the cover where I had
been concealed, and carefully noting the locality, left
the birds undisturbed, and picked my way across sev-
eral small canyons into camp. The hunter came in
soon afterward, and a comparison of notes showed that
we had located the same roost, he having come up
he canyon, while I went down, and each had decided AMERICAN GAME BIRD SHOOTING 440 to watch until dark, and not to begin to fire without
the other. About nine o’clock we returned to the place. The first few shots, fired rapidly, created a great com-
motion, and the air seemed to be filled with turkeys,
flying heavily off in all directions; but there were no
outcries, and in a few minutes all was quiet and no
more birds could be found. TURKEY SHOOTING. I was satisfied, however,
that we had secured as many as necessary for speci-
mens, though we did not know the exact number, for
some of them went thumping down to the bottom of the
canyon and others fell at some distance. “At daylight next morning we were again on the “At daylight next morning we were again on the
ground, and found the flock broken up into small par-
ties, which soon left their roosts and went off in vari-
ous directions. On leaving the trees the turkeys usu-
ally flew two or three hundred yards and then ran
rapidly until out of sight. During the following days
it became evident that all the tracks seen for several
miles about our camp had been made by this flock. No more fresh ones were found in the vicinity, show-
ing that the frightened birds had left the locality at
once. “Our lucky night hunt having given us a fine series
of females and young of the year, we then devoted
our attention entirely to the old gobblers. We hunted
steadily day after day, covering the country for miles
in all directions, without seeing any of the old fellows;
but they were in the country, and it could only be a
question of time until we found them. From the tracks
of the different flocks it appeared that the old males 441 TURKEY SHOOTING were living apart from the females and young. Fe-
males and young were seen several times, but were not
molested. Evidently these birds wandered far and
wide, for tracks a day or two old were often found
along some ridge, and no fresh ones were seen in the
vicinity for days. It did not appear that the birds
returned regularly to the same roosting place. Several
old roosts were found, usually among tall pines, near
the head or along the walls of some side canyon, which
were evidently occupied occasionally. None appeared
to have been used very long, and at least two had only
been occupied once. The birds spent the day wander-
ing over the broad and gently sloping ridges between
the canyons, and as evening approached worked to-
ward one of the canyons and roosted wherever night
overtook them. TURKEY SHOOTING. “Finally, on the ninth day, soon after noon, I came “Finally, on the ninth day, soon after noon, I came
upon some big gobbler tracks, which were evidently
only two or three hours old, and decided at once to
follow them. There were about fifteen of the old fel-
lows, and in crossing patches of soft soil they left a
broad trail, which became very indistinct, or disap-
peared altogether, on rocky ground. I had gone only
a short distance when my hunter, whom I supposed far
away, came up. He had found my moccasin tracks
following the turkey trail, and quickly overtook me. I was very glad to see him, for it was difficult work,
and even with our combined skill at trailing we made
slow progress. Sometimes we had no trouble for sev-
eral hundred yards, then suddenly we came to places AMERICAN GAME BIRD SHOOTING 442 where the birds had paused to feed, and found they
had wandered about in all directions, spreading among
the leaves. At such places the trail became so in-
volved that it was difficult to find the direction taken
when the birds left. When we came to hard or stony
places a few misplaced leaves or an overturned stone
or stick were the only things to guide us. Where the
trail became dim one of us usually went a trifle to the
right and the other to the left, so that unless the flock
changed its course abruptly, one or the other was pretty
sure to find some signs every few yards. Occasionally
we lost the trail altogether and had to go ahead and
to ‘cut other tracks’ in softer ground. For a while the
course followed was very crooked, and several times
it even doubled back and crossed itself, but late in the
afternoon it became evident that the flock was working
toward a branch of Clear Creek Canyon. Shortly be-
fore sunset the trail became so fresh that we kept a
sharper lookout ahead, expecting to sight the flock at
any moment. It was still proceeding in a leisurely
manner, as was plainly shown*by the number of places
where birds had paused to scratch out deep pits in
search of food. At sunset we were quite close to the
canyon, and I began to fear they would be able to
roost before we could overtake them. TURKEY SHOOTING. With the idea
that I could hear for a considerable distance the heavy
wing strokes they would make in rising to the roost,
I decided to go ahead and listen, leaving my companion
to follow the trail as best he could. I had only ad-
vanced about two hundred yards, to some higher TURKEY SHOOTING 443 ground, when I suddenly saw the flock, only about
forty yards to my left. The birds had not seen me,
and were walking quietly along in single file, following
a course directly parallel to the one I had taken. They
presented a fine sight, and I was strongly tempted to
shoot, but on second thought decided to follow them
until they roosted. Moving quietly out of sight in a
small arroyo, I ran back a short distance and gave a
low whistle, when my companion soon overtook me. Together we followed the birds, using great care not
to show ourselves. Food had ceased to interest them,
and they were evidently looking for a place to roost. They continued in single file, pausing occasionally to
look warily about, until they reached some high ground
overlooking a small canyon, along the slopes of which
stood several tall dead pines. The leader, a fine old
fellow, of unusual size, stopped, and the rest of the
flock came trailing up, and gathered in a group facing
the canyon. Several
low, tremulous
single notes,
quir-r-r-rt, quir-r-1-rt, quir-r-r-rt, were uttered, and
suddenly they took wing together and flew almost hori-
zontally out to the branches of the dead trees. After
leaving the ground no sound was heard except the
heavy flapping of wings. A few moved into the tops
of live trees a short distance farther down, but most
of them remained in the dead ones. Their behavior
was in marked contrast to that of the females and
young. There was no crowding and no confusion,
and in a remarkably short time they were settled for
the night and all was quiet. From where I was lying AMERICAN GAME BIRD SHOOTING AAA their bodies appeared in the gathering darkness like
enormous black fruits outlined sharply against the
glowing western sky. “When it became thoroughly dark we cautiously ap- “When it became thoroughly dark we cautiously ap-
proached the trees and took a stand almost under one
containing several birds. TURKEY SHOOTING. They were perched two or
three feet apart, so that only one could be shot at a
time. “As we knew the old fellows were very wary, we
held our guns in readiness as soon as we came within
range, and prepared to shoot at the first sign of alarm. I sent my companion to the next tree, and told him
to give a low whistle when he was ready. I held my
10-bore gun leveled at one of the birds, and it began
to feel very heavy before the signal was given. When
it finally came I fired both barrels in quick succession,
and was much gratified an instant later to hear a crash-
ing noise among the branches as two fine old gobblers
came tumbling down, landing before me with a loud
thump. Instantly heavy wing strokes could be heard
in all directions as the frightened birds left their
perches. My companion had also made successful right
and left shots into his tree. After some search we
each located and killed another turkey, after which
no more could be found. We then gathered the big
birds together, swung them well out of reach of prowl-
ing coyotes or mountain lions, and started for camp. When about a quarter of a mile from the roost I
saw by the dim moonlight
a turkey form among
the branches of a big pine. A chance
shot was TURKEY SHOOTING 445 made at it, and I was more than half surprised when
another big turkey came crashing down. ‘To reach
camp we had several deep, dark canyons to cross, with-
out trails, and floundered about, finding the usual ob-
stacles, which are unconsciously avoided in daytime,
but are always encountered at night. We were in a
cheerful frame of mind, however, and above noticing
small bruises and other mishaps. “The next forenoon we spent taking pack horses to
the roost by a circuitous route and bringing our game
to camp. In the afternoon we started on our return
to the railroad, and camped in the pifions. The snow-
storm we had hoped for until it could no longer serve
us came during the night, and the morning found
everything white and cold outside our tent. We made
a hurried breakfast, and after a long drive reached
Winslow in the afternoon. TURKEY SHOOTING. “The result of the trip was a series of thirteen speci-
mens, including adults of both sexes and the young of
the year. When specimens reached Washington Mr. Nelson found they represented an undescribed sub-
species, which he named Meleagris gallopavo merriami. Merriam’s turkey ranges in summer over the higher Merriam’s turkey ranges in summer over the higher
slopes of the Mogollon Mountains. In winter, and
especially when the snow lies over the summits, the
birds move down to the pifion belt, where food is abun-
dant. I found them feeding largely on the nuts of
the pifion (Pinus edulis). According to some of the
old hunters, they also eat the berries of the cedar (Junt-
perus utahensis), but none were found in the stomachs AMERICAN GAME BIRD SHOOTING 446 examined, although the turkeys, just before being
killed, had been wandering through the upper edge
of the cedars, where the ripe berries were exceedingly
abundant.” AIDS TO SHOOTING Many excellent books have been written on the art
of shooting, and yet it will be obvious to any one who
takes the trouble to think a little, that no one can learn
to shoot merely by reading books about it. The act
of shooting is a complex operation, calling for close
attention and the coordination of a variety of func-
tions. Comparatively few people become really skilful,
because few practice shooting enough to bring about
this co-ordination. Most of us remain beginners all
our lives. Many parts of the body are called into action
in discharging a shotgun: hand, arm, frame, eye and
brain must all act together to make the operation per-
fect, or nearly so. If any one of these factors fails the
act is incomplete. The way to learn to efficiently perform a complex
operation is to repeat it many times. In other words,
we learn how to do things by doing them over and
over again. A player of instrumental music must prac-
tice many hours a day before he can play swiftly and
correctly. A person learning how to use a delicate tool
must work with it a long time before he can accomplish
satisfactory results. The professional shooters—men
hired by the great arms or ammunition companies to go
about to all the shooting tournaments and
use the
products manufactured by these companies—devote
447 447 447 AMERICAN GAME BIRD SHOOTING 448 their lives to shooting, and are as skilful as any men
can be; yet, in fact, it is the rarest possible exception
when these men make perfect scores. I suppose that there is no shooting practice so good I suppose that there is no shooting practice so good
as to take the gun, say twice a day, for five minutes
in the morning and five minutes in the evening, and
throw it up to the shoulder, aiming at different objects
near at hand, and trying to catch the sight as quickly
as possible; in other words, to try to throw the gun
up as nearly as possible in the line of sight. If this
is done often enough it will prove the greatest aid in
shooting, and will tend to make shooting more rapid
and more accurate. Upland shooting may be divided into two branches, Upland shooting may be divided into two branches,
open shooting and snap shooting. AIDS TO SHOOTING In the first you see
your mark clearly, and aim at it; in snap shooting you
use only the “eye of faith,” shooting at a shadow, a
sound, or a moving leaf, and trust to Providence that
your gun is so aimed that the bird which you know
is there shall fly into the circle of the shot. Obviously,
open shooting grades into snap shooting, and no hard
and fast line can be drawn between the two. Some
men, who are especially talented, practice snap shoot-
ing at all times, in the open as well as in cover. In open shooting, as, for example, where the prairie In open shooting, as, for example, where the prairie
grouse are found, or quail are scattered in the open
fields, there is almost always plenty of time to take de-
liberate aim and kill your bird before it is out of reach. In the brush, however, where snap shooting must be
practiced, because the game is hardly seen, and its AIDS TO SHOOTING AAQ whereabouts,
distance
or
direction
must
often
be
guessed at, there is no time at all. The writers on
shooting of many years ago laid undue stress on snap
shooting, and their books seem to urge the sportsman
just entering his novitiate to get his gun off first and
learn how to aim at his game last. This, of course,
makes a quick shot, but in these days, unless one shoots
constantly at the traps, the opportunities for practicing
shooting are so very few that a man might live beyond
the allotted three score years and ten without ever
shooting at birds enough to learn how to hit anything. I believe the way to learn the fundamentals of shoot- I believe the way to learn the fundamentals of shoot-
ing is to practice with an empty gun until you have
learned to throw the gun up in the line of sight; in
other words, make your body handle your gun mechan-
ically, so that when you put it to your shoulder it will
point in the direction in which you are looking. If
you can do that you will have advanced far on the road
toward making yourself a good shot. In open shooting there is usually abundant time to In open shooting there is usually abundant time to
take aim. AIDS TO SHOOTING If the bird gets up anywhere near you, you
have time enough to fire both barrels at him and then
to stand and watch him for a second or two before
he is out of range. Now, if you had given one-quarter
of a second more to the effort to secure a good aim
on each of these shots you probably would have killed
the bird with the first or second barrel, instead of en-
during the mortification of a miss. The operations of
throwing the gun to the shoulder and pulling the trig-
ger take very little time, and although the bird appears AMERICAN GAME BIRD SHOOTING 450 to be going very fast, and seems likely to get out of
shot before you can pull the trigger, this apparent
swiftness of flight is due partly to the nervousness of
the shooter. Where the shooting is open there is al-
ways, or almost always, abundant time to point your
gun just where you want it. Most of the shots that are
missed are due to overeagerness, which means undue
haste. The shooter may feel perfectly certain that he
will miss his bird unless the gun is pointed very nearly
straight at it. The shot spreads over a circle of three
or four feet in diameter, and sometimes, even if the
gun is not pointed straight at the bird, the edge of the
charge of shot will hit it and perhaps knock it down;
but this is a chance not worth considering—the bird
should be hit with the center of the charge. It was,
perhaps, Frank Forrester who spoke of “deliberate
promptitude,’ and this deliberate promptitude is one
of the secrets of being a successful and a good shot. Aiter all, this merely means coolness and training, the
keeping of one’s wits about him all the time, and never
being disturbed or flustered by any event of the field. Many of the best and most experienced shots never
reach the point where they can hear birds get up all
about them without quickened heart-beats and jumping
nerves, yet many of them never show by their shooting
that they are discomposed by the noise. AIDS TO SHOOTING It has been my experience, on a number of occasions, It has been my experience, on a number of occasions,
to go out with people who had done little or no field
shooting, and I have found that a word of caution, the
mere warning, “Take your time,” when a bird rose, AIDS TO SHOOTING 451 or was expected to rise, helped many a man to retain
his self-possession and to kill birds that otherwise he
would have missed, firing the gun when they were
so close to him that if the charge had hit them they
would have been torn to pieces. In ways such as this
the man of experience may greatly help the novice with-
out in the least hurting his feelings or in any way
offending him. In his adm In his admirable little book, entitled “Hitting vs. Missing,” Mr. Hammond recommends practice by the
novice at stones skipped over the smooth surface of
a body of water. The suggestion is an excellent one,
for the striking of the shot on the water enables the
shooter to see precisely where he is holding with rela-
tion to the mark at which he is shooting. This, of
course, is what each gunner wishes to know when he
shoots at any object. Why did he miss? Did the
charge go above, below, or to one side of the mark? The shooter who could tell where each charge of shot
went, and had intelligence enough to apply this knowl-
edge to each subsequent shot, would speedily attain a
high degree of skill. It is generally It is generally known that a person properly placed
can see a charge of shot flying through the air toward
a target. The charge of shot, as it flies through the
air, is said to look like a long shadow composed of
many lengthwise strips. It is a common practice for
a trap shooter, when he finds that he is frequently miss-
ing his targets, to ask some friend to stand near him
and watch where the shot is going with relation to the AMERICAN GAME BIRD SHOOTING 452 target, whether under or over, before or behind. Given
this information, the shooter corrects his aim accord-
ingly. AIDS TO SHOOTING The many books that have been written on shooting The many books that have been written on shooting
give hints and suggestions which are of great value
to the young gunner, explaining what he should do,
and, more important than all, cautioning him against
the mistakes he is likely to make. This is helpful, but,
after all, nothing can take the place of practice. Each
beginner must necessarily go through the school of
preliminary practice, finally coming to clay targets,
thrown from the trap, which afford the best practice
of all. He may feel sure that the more practice he
has the better he will learn to shoot, but I believe that
the best practice of all, if it be faithfully continued,
is that taken with an empty gun, for five minutes at
a time, two or three times a day, throwing up the gun
on different objects and pulling the trigger the instant
the sight is caught; or throwing up the gun and then
swiftly swinging it on to some mark and pulling the
trigger just as this mark is reached. A point that must be remembered, and that has much
to do with success in shooting, or the lack of it, is the
disparity in strength and skill which exists in the two
arms of all men. Most men are right-handed, and are
constantly performing various tasks with the right
hand and arm, which thus becomes far more efficient
and much stronger than the left. A few people are
ambidextrous, and can use either hand indifferently. A
few men are left-handed, and can shoot from the shoul- AIDS TO SHOOTING 453 der, drive a nail, or write a letter with the left hand,
but cannot use the right hand efficiently. Now, with
the average right-handed man, shooting from the right
shoulder, the gun is supported, and, more important
still, is guided by the left hand; yet, as the man is unac-
customed to performing tasks with that hand, he has
far less control over its movements than he has over
the right hand. If he swings his gun to try to point
it at a certain object, he is likely to swing it too far,
or not far enough, or above or below the object. AIDS TO SHOOTING It becomes important, then, for the man who is train- It becomes important, then, for the man who is train-
ing himself to shoot well to endeavor to strengthen
and to train the left arm so that he may have it con-
stantly more and more under control, and thus may
make it do more exactly the things which he wishes. Such strengthening and training may be begun by the
use of dumbbells or the swinging of Indian clubs,
which will give strength to the muscles, and then by
the performance of simple tasks with the left hand,
which will teach it to respond better to its owner’s
will. Professional shooters, who are among the very best
shots in the country, find it necessary to the perform-
ance of their work that they should keep themselves
in the very best physical condition. Many of them
practice constantly
at gymnasium
work, including
dumbbells and Indian clubs, are careful about their
eating and drinking, and are, in fact, trained athletes. In order to do the work for which they are paid they
must be in good physical condition, and if they are AMERICAN GAME BIRD SHOOTING 454 unable to continue to do this work their services are
no longer required by the people who hire them. In old times the best shots that we used to know
were the market shooters. Many of them were men
who worked at some trade when they were not shoot-
ing for a living, their muscles were hard, their health
good, and they were in the pink of physical condi-
tion. When they took up their guns in summer or
autumn they shot all the time, and their constant prac-
tice, together with their strength and hardiness, made
them most destructive of the game they followed. In
the same way to-day, we find that the best shots along
the south Atlantic coast are the gunners who shoot
ducks for the market. Farming during the spring and
summer, fishing in late summer and early autumn,
and gunning through the autumn and winter, they
are always in splendid condition, and always in good
practice. Very different is the case of the city or office man,
who perhaps spends fifty weeks of the year at his desk,
walking each day only a few blocks. AIDS TO SHOOTING When his vaca-
tion comes he sets out on his shooting trip, and is sur-
prised and disappointed to find that he can neither
tramp nor shoot. Why should he be able to do either ? It is not by doing a thing half a dozen times a year
that one acquires skill in it. The man who is to under-
take tasks requiring skill, muscular effort, long wind,
or endurance of any kind, must prepare himself for
the task which lies before him. As I wrote some years ago, in “American Duck As I wrote some years ago, in “American Duck AIDS TO SHOOTING A55 Shooting,” “Skill in shooting is not born in any one. Just like reading and writing, it must be learned; and,
like reading and writing, the more practice one has
the more easily and better it is done. Many a pro-
fessional gunner who is a wonderful shot would find
it labor of the hardest kind to sit down and write a
four-page letter; and many a business or professional
man, who goes gunning perhaps once in two or three
years, finds that killing the fowl that give him shots
is something that he cannot accomplish. Many men
have noticed that sometimes at the end of a season
they can shoot very well; and then, if for two or three
years they do not go shooting, they find that they can-
not hit anything, and have to begin at the beginning
and learn it all over again. They have, perhaps, for-
gotten how to hold on their birds, and besides, their
muscles, through disuse, refuse at first to act with the
brain as they formerly did. This reflex action, so
called, can only be regained by practice.”
From the books on shooting much may be learned From the books on shooting much may be learned
about how to hold on straightaway birds, incomers,
quartering birds, and cross shots. It is well to remem-
ber that at twenty-five or thirty yards the charge of
shot from a cylinder-bore gun covers a circle two or
three feet in diameter, and that on quartering birds,
and those flying across one at such a distance, a cer-
tain allowance may be made for the great area of the
charge. AIDS TO SHOOTING In other words, if the shooter fires one foot
ahead of a crossing bird it will be quite certain to fly
into the load. If the shooter fires a little behind the AMERICAN GAME BIRD SHOOTING 456 bird, very likely it will be missed altogether, or only the
edge of the load will prick it. Birds fly fast, but shot
flies much faster, and while almost every one is likely
to shoot behind wild ducks, there is very much less dan-
ger of doing so on cross shots at upland birds. In the brush shooting which prevails over many parts In the brush shooting which prevails over many parts
of this country the birds usually get up very near te
the gunner, and the shooting is quick and close. A
cylinder-bore gun and a charge of small shot will give
better results for this shooting than a choke-bore gun
and large shot. I usually begin the season by using
one ounce of No. 10 shot for woodcock, partridges and
quail, but as the weather grows colder and the leaves
fall, the birds are likely to be wilder and to be a little
more heavily feathered, and for the very last of the
season an ounce of No. 8 shot may be used for quails
and partridges alike. In prairie shooting, toward the
end of the season, when the chickens have become wild
and get up at long range, heavier shot and a choke-bore
gun may profitably be employed. Sage grouse, being
very large and—the old ones—very tough birds, may
be shot at with No. 6 shot all through the season. In all shooting there is really nothing more impor- In all shooting there is really nothing more impor-
tant than that the gunner should know just what his
arm can do. If he thoroughly understands this, if he
will practice as recommended, and if he has his nerves
and impulses under good control, he can very soon
make of himself a more than average shot. CLOTHING The hunter’s comfort and endurance are largely de-
pendent on being properly equipped in the matter of
footwear and clothing. The average novice is more
than likely to be ill furnished in these essentials, even
though he be expensively furnished. For instance,
clothing which is too tightly fitted, or too heavy in
weight, adds unnecessarily to the strain of walking or
riding, thereby inducing premature fatigue, and mean-
while cramping the activity and lessening the enjoy-
ment. The clothing should always be loose enough
to admit of the freest movements of the limbs, the
matter of style being a secondary consideration from
a common-sense viewpoint. Proper footwear is also of prime importance, for
while the walker may proceed passably well with a
hampering fit as to clothing, he is at a constant and
painful disadvantage if he wears ill-fitting shoes. The
latter are sure to strain, chafe or blister the feet, so
more or less crippling the wearer and marring his en-
joyment. When the feet are sore the walker, in the
attempt to save his feet, is thrown out of his usual
stride, and this lack of balance throws strain on weaker
muscles, bringing on a general soreness of the body,
with the result that what should be a recreative pleas-
ire becomes a painful labor. 458 AMERICAN GAME BIRD SHOOTING The hunting field is no place for the breaking in of
new footwear. ‘The latter should be purchased months
ahead of its actual use afield, and should be worn often
enough to be thoroughly broken in, so that it may be
worn with comfort in actual hunting. The footwear should be as light in material as is The footwear should be as light in material as is
consistent with practical use. If the going is easy and
dry, the hunter may advantageously use some of his
old cast-off shoes, assuming, of course, that they have
some wear left in them. About two dozen hobnails,
driven in proper distribution in the soles and heels,
add greatly to their efficiency in walking, and protect
them from the grinding and cutting effects of rocks
in rough going. It should be observed that the hob-
nails are an indispensable adjunct to any hunting foot-
wear. CLOTHING In dry weather the soles of the boots or shoes
take on an exceedingly slippery polish, so much so that
walking on dry leaves or grass in shoes without hob-
nails presents the same difficulities as walking on ice. The specially manufactured hunting boots and shoes The specially manufactured hunting boots and shoes
are made of various kinds of excellent leather, heavy
and light, and in styles to suit the fancy of the pur-
chaser. The best makes have tough, pliable uppers,
strongly reinforced on the sides, double-soled from toe
to heel, hobnailed; soft and elastic as a whole, yet so
closely constructed, withal, as to be nominally water-
proof, comfortable and durable. The boots, as a rule,
are simply the shoes with a leg added, and they weigh
more accordingly. After all, lightness of weight in foot gear is a most CLOTHING 459 important consideration, and many sportsmen of great
experience wear only low shoes with canvas leggings
of the cavalry type. Some men wear tennis shoes with
leggings, and rejoice greatly in the ease with which
they can get about, walking down their more heavily
shod companions. Of course, tennis shoes wear out,
but on the other hand they are much less expensive
than heavy boots or shoes. I have seen them used in
big game hunting in the Rocky Mountains. The footwear should be selected with a considera- The footwear should be selected with a considera-
tion of the surface features of the ground on which it
will be used. On comparatively smooth ground a light
shoe can be worn with advantage, but on ground where
rocks and ledges are numerous a heavier shoe is neces-
sary to withstand the increased rough usage. The only advantage of the boot is that the leg serves The only advantage of the boot is that the leg serves
as a legging; but it has the disadvantage, which the
regular legging has not, that in warm weather, owing
to the tightness, and absence of ventilation, it causes
overheating and much discomfort. It also hampers the
free movement of the ankle. The combination of shoe and legging is much lighter The combination of shoe and legging is much lighter
than the boot, is better ventilated, and does not hamper
the leg action in the least. CLOTHING The moccasin-shoe is an excellent article of foot- The moccasin-shoe is an excellent article of foot-
wear, particularly in such hunting as enjoins the most
silent activity. It is a kind of compromise between
the moccasin and the shoe, being the former with a
light but tough sole added thereto. The material, how- AMERICAN GAME BIRD SHOOTING 460 ever, as to uppers, is of the lighter and tougher leath-
ers, such as calfskin, etc. These, in the main, are the essentials of hunting foot-
wear, though presented in many varied styles and of
varied materials. A city dweller, accustomed to the use of light foot-
wear day after day, seriously handicaps himself if he
wears unnecessarily heavy footwear afield, particu-
larly if he has reached an age which can no longer be
recognized as young. An excellent leather-top rubber shoe serves admir- An excellent leather-top rubber shoe serves admir-
ably for still hunting, for hunting in wet or winter, and
are strictly waterproof, but they draw the skin on per-
spiring feet to an injurious extent, and therefore, for
certain persons, are not a proper article of footwear. As to clothing, there is even a greater variety as to
style and material from which the hunter may select. Of the materials offered, corduroy, moleskin and can-
vas have held conspicuous place in the garments used
in upland shooting, though severahother kinds of mate-
rial also have strong claims to excellence. However, in upland shooting, any material having However, in upland shooting, any material having
a woolen admixture is quickly picked to pieces by
brush, briars or heavy grass, and does not possess the
peculiar advantages
of the aforementioned
cloths,
which are of cotton material, hard in finish, and of
great durability. Khaki and Duxbak are very superior cloths for hunt- Khaki and Duxbak are very superior cloths for hunt-
ing garments. Advantages of canvas are that it dries 461 CLOTHING quickly, keeps out heat and cold, will not hold burs,
and is cheap and durable. made of various kinds of skins are excellent Jackets made of various kinds of skins are excellent
protection in cold weather. They are light, pliable,
impervious to wind, have a smart appearance, though,
as a rule, the upland shooter rarely needs overclothing
for the sake of warmth. His exercise keeps him warm. CLOTHING Still, at the end of the day, if the hunter rides home,
he needs protection from the chill of evening, and
should have an overcoat in reserve for his comfort. One weight of outer clothing will serve for all seasons,
hot or cold. all requirements as to weather The shooter can meet all requirements as to weather
by selecting his underwear, light or heavy, according
to the temperature. If the shooter desires to provide against stormy If the shooter desires to provide against stormy
weather, he can find a large variety of storm coats,
light and heavy in weight, made of rubber, oilskin and
Duxbak, the latter being a canvas partially waterproof. If the shooter desires a raincoat for use on horseback,
he should be careful to so specify, as one not divided
in the back to a proper height so that it may spread
over the rider’s legs, will not be a protection from rain. GUNS AND LOADS. The selection of a gun is a matter which requires
careful consideration. Weight, bore, and shooting
qualities are not to be determined arbitrarily. As a rule, the novice desires the closest shooting As a rule, the novice desires the closest shooting
gun obtainable, regardless of whether it is appropriate
to the shooting he expects to have, or whether his skill
is equal to the exactions of such gun, be the shoot-
ing what it may. Even superior marksmen are prone
to use guns of too close pattern and loads of unneces-
sary power. If the shooter cares to own but one gun,
and uses it for all-round shooting, a compromise as
to weights, loads, ete., is desirable, though for cover
and open shooting special guns are quite the best, a
modified cylinder for the one and a closer choke for the
other. Whatever the bore, the gun should be as light as is
consistent with safety. An extra pound or two of
gun counts heavily in added weariness toward the end
of a day’s shooting, and at all times in the way of
slower manipulation. Quickness of execution is one
of the prime essentials in the practical use of the gun
in wing shooting. In the closing years of the past century the popu-
larity of the 10-bore rapidly waned, till at last it be-
came obsolete as a gun for upland shooting. In weight
462 GUNS AND LOADS 463 it ranged from 8 to 12 pounds,
and, as a rule,
was chambered for heavy loads; that is, from 4 to
5 drams, sometimes more, of black powder and 1%
ounces
of shot. The advantages
of the
10-bore
were in its capability to handle heavy loads, and
it is an excellent gun
for duck shooting and for
prairie chicken shooting late in the season. A light
10-bore, shooting light loads, is opposed to the prin-
cipal reason—that is, the capacity for shooting heavy
loads—which justifies its use. It was demonstrated,
after a number of years, that the ro-bore had very
little advantage in ballistic properties over the
12-
bore, but had the very serious disadvantage of extra
weight and more expensive ammunition. At present At present, bores smaller than 12, as 16, 20 and even
28, have their warm advocates, who present many
cogent reasons for their faith. GUNS AND LOADS. However, for all round
shooting purposes, the 12-bore is still the popular gun. In weight it runs from 6 to 8 pounds, or more, with
barrels of 26, 28, 30, 32 and 34 inches, 30 inches being
the favorite length. It is chambered for from a 2%
to a 34-inch shell, the commonest measurements, how-
ever, being 25% and 2°/, inches. The lightweight gun uses The lightweight gun uses a lighter load, there being
always a proper relation of load to weight of gun. If
the gun is excessively light, then its advantages in the
matter of handling a heavier load are lost, and it is
better to get a gun of smaller bore. The load for the
12-bore ranges from 2 to 4 drams of bulk pow-
der, according to the weight of the gun and the AMERICAN GAME BIRD SHOOTING 464 kind of game bird on which it is employed. Four
drams, however, is an excessive load, used chiefly by
pigeon and duck shooters, and by very few of them. Besides the punishing recoil, the excessive load strains
a gun seriously and shortens its life. The most desirable all-round load for a 12-bore is 3 The most desirable all-round load for a 12-bore is 3
drams of powder and 1% ounces of No. 7% chilled
shot. This is a very killing load, and is in general use
by target shooters at the traps. However, for special
shooting, different sizes of shot are essential. Some shooters use 3% or 3% drams of powder, but
the recoil is much greater, relatively, than that of 3
drams, and punishes the average shooter. The recoil
may be kept within reasonable limits by reducing the
weight of shot as the load of powder is increased; thus,
3% drams with 1 ounce of shot gives a high velocity,
and is very effective in duck shooting, at the same time
being free from excessive recoil. The only advantages of bores smaller than the 12
are their lightness, and the consequent greater ease in
carrying them and greater quickness in aiming, because
of their greater lightness. The ammunition for them
is somewhat cheaper, but not enough so to be a mat-
ter for serious consideration. GUNS AND LOADS. While the smaller bores
will deliver the load of shot with as much velocity as
the larger bores, it should be noted that the killing
circle is less, and that, therefore, a higher degree of
skill in pointing is a necessary correlative. If the bore
is such as to make the killing circle equal, then the pat-
tern is thin. Thus there is reason, while giving con- GUNS AND LOADS 465 sideration to the powers of the gun, to consider there-
with the capability of the shooter. Some shooters have a false pride concerning the Some shooters have a false pride concerning the
ownership of a cylinder bore, which is often assumed to
indicate inferior shooting qualities in the gun or lack
of skill in the shooter; but it is far more sensible for
the shooter to use a gun in accord with his shooting
capabilities, and the conditions as to cover shooting,
etc., and thereby achieve reasonable success in the field,
than it is to make a ridiculous failure with a gun which
shoots too close. A full-choke gun puts about 70 per cent. of the shot
in a 30-inch circle at 40 yards, a half-choke about 60
per cent., a quarter-choke about 50 per cent., an im-
proved cylinder 45 per cent., a cylinder—so called; it
commonly has a trifle of choke in it—35 per cent. It is a safe assumption that by far the greater num- It is a safe assumption that by far the greater num-
ber of birds,
in upland
shooting,
fall within
25
yards of the shooter; therefore,
the pattern made
at 40 yards is not the criterion by which to judge
of the gun’s powers at 25 yards. In cover shoot-
ing, a full-choke gun mutilates many birds to a degree
which renders them worthless. Some shooters have a 16-bore set of barrels fitted Some shooters have a 16-bore set of barrels fitted
to a 12-gauge frame, the barrels being correspondingly
heavy, the greater weight necessarily being about the
chambers; thus this greater weight negatives all the
advantages of the 16-bore in the attempt to shoot heavy
loads. GUNS AND LOADS. Again, the 20-bore, mounted on a 16-gauge frame, 466 AMERICAN GAME BIRD SHOOTING is another freakish perversion of the smaller bore,
though the fads and fancies of shooters are of much
trade value to the gun makers and repairers,
The smaller bores can rarely be used to advantage The smaller bores can rarely be used to advantage
with less than a quarter choke, it being obvious that
three-quarters of an ounce of shot should be held closer
together than an ounce and a quarter to insure a proper
distribution in the pattern. The 28-bore is a wonderfully effective gun for up-
land game shooting, and its lightness makes for quick
execution; but the killing circle is relatively small, and
a higher degree of skill is a requisite in its success-
ful use. The 16-bore, 26, 28 or 30-inch barrels, may weigh
from 5
to 634 pounds, and the 20-bore may have
about the same range in weights, with 614 pounds, per-
haps, as the maximum. Two sets of barrels, one set
of modified cylinder bore, the other a close choke, add
immensely to the efficiency of the gun. There is no doubt
but that
in the matter of de-
structiveness the repeating shotgun, “pump” and auto-
matic far surpass the double gun. Fortunately, at
the rise of a bevy, in upland shooting, the average
sportsman using such gun is not sufficiently expert
to fire more than two shots with any degree of cer-
tainty. Yet, having extra loads at his command, he
can take advantage of any slothful or timid birds which
rise after the main part of the bevy has flown, hence is
hardly ever caught unprepared. An expert with the repeating shotgun is terribly An expert with the repeating shotgun is terribly GUNS AND LOADS 467 destructive when in action afield. He can fire with
wonderful quickness and precision, and may destroy
a small covey at the first rise. Except for that feature
it has no advantages over the double and single-bar-
reled guns in such sections as have a legal limitation
to the number of birds any one shooter may kill in
one day. However great the destructiveness of any gun,
it may not be properly employed to exceed the statu-
tory limit. GUNS AND LOADS. It is urged as a plea against the repeater
that, its capacity to kill being so great, there is always
a temptation to kill more than the legal limit; but the
same argument can be urged against any other kind
of gun in the hands of a skilful but lawless shooter. Men have broken the law many times while using guns
other than repeaters, therefore there is nothing inher-
ent in the kind of gun which makes for good citizen-
ship. That is a point of ethics which refers to the
mari. Factory-loaded ammunition has been so admirably
improved that it is practically perfect. The average
shooter, in the attempt to kill, is prone to attribute fail-
ure to faulty ammunition or to a gun which does not
shoot straight or does not fit. If the shooter could
calmly persuade himself to look upon his failures with
the same unprejudiced opinion common among his com-
panions in respect to himself, he could safely place the
blame for all failures without any risk of creating an
argument. In any event, when afield, it is not an un-
wise policy to keep all explanations of one’s failures
to one’s self. Nine men out of ten do not care to listen AMERICAN GAME BIRD SHOOTING 468 to hard-luck stories, which oftenest have their origin
in incompetency; and the other man, who might be
sympathetic, right or wrong, is more than likely to
have troubles enough of his own. There are shooters who have a belief that they can There are shooters who have a belief that they can
produce better cartridges loaded by hand than those
made by machinery. Such belief is fallacious. Given
like materials, the machine-loaded cartridge is likely
to be the better of the two. To a certainty it will have
more uniform loading, as the pressures on the wad-
ding will be automatically alike. This uniformity is
beyond the compass of the hand. The kind and quality
of wadding, however, is an important factor in the ef-
fectiveness of any load, hence a well wadded hand load
may surpass a poorly wadded machine load; but this
is a matter of material and not of methods. GUNS AND LOADS. For the 16 and 20 gauges the loads of shot should For the 16 and 20 gauges the loads of shot should
be comparatively light, from three-quarters of an ounce
to one ounce, with from two to two and a half drams
of bulk powder, or a corresponding quantity of dense
powder. There should be no attempt to arbitrarily
make the smaller gauges equal to the 12 in weight
of ammunition. As already suggested, the advan-
tage of the smaller bores is in their lightness, and
therefore in their fitness for upland shooting, which
does not exact such hard killing qualities in a gun as
does wild fowl shooting. The gun should be selected with a view to its fit- The gun should be selected with a view to its fit-
ness for the special shooting of the owner, his physical
strength and skilful capabilities, which, summed up, GUNS AND LOADS 469 are quite apart from the matter of reducing all the
factors to an arbitrary standard. Any gun that is
overloaded, be it light or heavy, will kick unpleasantly,
and sometimes harmfully, as shown by a bruised shoul-
der, a contused cheek, and perhaps a cut trigger finger. As a consequence of a punishing recoil, the shooter As a consequence of a punishing recoil, the shooter
is quite likely to flinch or fudge, which, in turn, with
continued punishment, is quite likely to become habit-
ual, thereby greatly impairing success. The lighter
the gun, the more violent the recoil from overloading. If the shooter is physically so hardy that he can If the shooter is physically so hardy that he can
withstand the punishment consequent on excessive re-
coil, he nevertheless is quite likely to lose control of the
kicking gun in delivering the second shot. No argu-
ment is required to show that a man in the act of firing
both barrels, one quickly after the other, can main-
tain a perfect poise and do quick, accurate execution
with a gun which does not kick, and that he cannot
do so equally well with a gun which kicks so hard that
it throws him off his poise. There is no arbitrary rule. Each
case
must
be considered
by itself. As
a
rule, a large man will withstand the recoil better than
a small man. GUNS AND LOADS. Again, some men have the ability to take
most of the recoil on the hands and arms, which ab-
sorb the shock as would a spring, thereby relieving the
body from the hard shock which is communicated when
the gun is held firmly against the shoulder. Apart
from all this, the average gun, when overloaded, does
not equal its performance with its proper load. DOGS. Setters and pointers are the breeds of hunting dogs
which are chiefly used for work afield by American
sportsmen. These breeds display especial usefulness
in the pursuit of the various kinds of game birds, such
as quail, grouse, snipe and woodcock. Sport of this
kind is known as upland shooting, in contradistinction
to wild fowl shooting, a sport of marsh and water. Spaniels also have appreciative recognition, in a rela- Spaniels also have appreciative recognition, in a rela-
tively limited way, by certain sportsmen whose domi-
cile happens to be situated in a locality favorable to
spaniels’ use. But even at that, this breed holds such
an insignificant place in relation to the gun, in America,
that it may be considered as a negligible factor. In this country the areas in which the spaniel can In this country the areas in which the spaniel can
be worked to advantage are so few and unimportant,
as compared with the vast areas favoring the work of
the setter and pointer, that it is obvious that these natu-
ral conditions dominate and determine the sportsman’s
choice to an important degree. As few sections favor
the spaniel’s use, few sportsmen own him. His physi-
cal powers, compared with those of the pointer and
setter, are exceedingly limited, and he is thus at a dis-
advantage in the comparison with them. Nevertheless,
in certain areas containing much cover and irregular
ground, in which the view is constantly obstructed, the
470 470 470 Setter
standi
Ruffed
Grous Sette
stan
Ruff
Grou DOGS 471 work of the spaniel fits in admirably, assuming that
game is present in reasonable abundance. The spaniel’s working metho The spaniel’s working methods, natural and educa-
tional, are quite distinct from those of the pointer and
setter. The two latter hunt mute, and, having found
the birds, indicate the find by pointing them, the flush
being left to the shooter’s choice of time and manner. The spaniel trails the birds to their place of conceal-
ment, and, the opportunity offering, presses them to
a flush without any attempt at pointing, if he acts
properly; hence the term “springer.”
Some spaniels whi Some spaniels whimper, babble, or give tongue mer-
rily while trailing; others work mute. Giving tongue
at all is an objectionable trait, as in upland shooting
man and dog should work as silently as possible. DOGS. This
is especially important when the birds are wild and
seek safety in flight at the slightest alarm. Thus the
need of silence in upland shooting would seem to be
quite obvious. Howe However, this is contrary to some of the traditions
and usages governing the use of the spaniel afield. Stonehenge (J. H. Walsh), in his work “Stonehenge
on the Dog” (1873), in respect to this matter speaks
as follows:
“A good and somewhat musical tongue
was, by the old school of sportsmen, considered a desid-
eratum, in order not only to give notice that the dog is
on game, but also the particular kind which he is
‘questing,’ and which many good spaniels enable their
masters to distinguish by a variation in their notes. Formerly this was thought so important that if a AMERICAN GAME BIRD SHOOTING 472 spaniel happened to be mute he was hunted with a bell
around his neck, as is sometimes done with the setter
when used in covert. In the present day a very fash-
ionable breed (the Clumber) is invariably mute; but as
these dogs are chiefly used in aid of the battue, there
is not the same necessity for them to give notice of
their approach as in the case of spaniels used either
in wild pheasant shooting or for cocks, hares or rab-
bits. It will, therefore, appear that for every kind of
covert shooting but the battue we require a strong, use-
ful spaniel, capable of bearing exposure to the weather,
and neither too large for the runs nor too small to
bear work. Added to these qualities we want an ex-
quisite nose, and a musical but not noisy tongue, which
is all the more valuable if it will distinguish by its note
the various kinds of game.”
While giving tongue might charm the ear of the While giving tongue might charm the ear of the
English sportsman, and indicate the kind of birds ahead
of the spaniel, in this country, with its rough, irreg-
ular coverts, much of which are in the condition that
nature’s hand shaped them, a noisy dog is a detriment
to the sport. As to the advantages of flushing, there are many
qualifications. Considered in its proper relation as a
spaniel trait, the spaniel must be trained to work well
within gunshot of the sportsman. DOGS. He also must con-
duct his efforts at all times with a reasonable degree
of steadiness, to the end that the gun shall constantly
have
favorable
opportunity. This
intelligent
co- DOGS 473 ordination of effort between dog and gun is absolutely
essential to success. When on a trail, the spaniel should time his speed to
that of the shooter. When pressing in close on the
birds he should refrain from flushing till the shooter
is properly placed and ready for the shot. If he is
specially intelligent, accomplished and helpful, he may
get the birds between himself and the shooter before
flushing them, so that they may be driven toward the
shooter, thus enhancing the chances for a successful
shot. It is self-evident that a lawless spaniel, ranging out
of gunshot, and flushing the birds regardless of the
shooter’s interests, is in no sense an aid to the gun. Such misbehavior, on the contrary, insures that even
if the game be plentiful the opportunities of the shooter
are constantly ruined, and he then is much worse off
than if he had no dog at all. As the spaniel is not required nor desired to point, As the spaniel is not required nor desired to point,
his training is correspondingly simpler than that of
the setter and pointer. When trained, he beats out a
very limited area of ground about the shooter, keeping
at all times well within gunshot, flushing the birds with-
out any preliminary point as an entirely proper act. In
thick cover, or in ground so rough that the dog is much
hidden from view, the advantages of this manner of
work are manifest. In the case of the pointer and setter, which fre- In the case of the pointer and setter, which fre-
quently find birds far out of gunshot, the advantages AMERICAN GAME BIRD SHOOTING 474: of pointing, and of stanchness to the point till the gun
arrives in position, are also self-evident. By feathering, and a general change of manner and By feathering, and a general change of manner and
intensity of action, the spaniel indicates his recogni-
tion of the foot scent or the whereabouts of the birds. By the observation of similar mannerisms, displayed
when he catches the body scent, the shooter discerns
when the spaniel is close to the birds or is about to
spring on them. DOGS. Thus, while his preparation for effi-
cient field work requires but a moderate degree of
training, as compared with the more complex educa-
tion required for the setter and pointer, it is impera-
tive that his education,
so far as it goes, shall be
complete. It is obvious that in the interest of good
sport he must conform to the gun’s requirements. He should promptly obey the commands, “Heel,”
*Go on,” ‘Drop, ‘Fetch,’ “Come uns? + Seekidead. or “Find,” and “Steady,” the latter command being
of special importance, since it comes into frequent use
in many relations, as when the spaniel is roading too
fast, or is intent on a premature flush, or when he is
disposed to become riotous and generally disobedient. A proper observance of this order makes in many ways
for the better success of the gun and the pleasure of
the shooter; therefore, the importance of establishing
thorough obedience to it. As to methods of finding the birds, the spaniel, as a
rule, follows the foot scent, differing in this respect
from the best working pointers and setters, which carry
a high nose to catch and follow the body scent direct to DOGS 475 the birds. However, as the spaniel works in a narrow
area, while setters and pointers work over considerable
territory, each method, in its proper relation, has its
special fitness and value. In the matter of speed the spaniel is at opposites In the matter of speed the spaniel is at opposites
with his pointing brothers. Huis range, necessarily so
limited in scope, requires but a moderate degree of
speed to meet the requirements of the gun. Even
with moderate effort he has ample time to search out
every nook and corner within gunshot of the shooter,
as the latter meanders along. With a team of spaniels
—a team may be any reasonable number which the
fancy or needs of the shooter requires—nothing is
overlooked by them. If up to the proper requirements, the spaniel works
merrily, diligently and intelligently. He delights in
the quest. A team of well broken spaniels, working
in a section adapted to their peculiar methods and limi-
tations, affords excellent sport. Unfortunately, in the
United States such sections are few and far between. DOGS. In England, before the battue
and
driving had In England, before the battue
and
driving had
largely ousted the pointing dog and spaniel, the latter,
as a worker afield, held an important place in upland
shooting and in the esteem of sportsmen. There were
many good reasons for this choice—good reasons as
they pertain to English shooting, but otherwise in re-
spect to shooting in this country. On the English
game preserves
the dog trainers,
the gamekeepers
and the owners know every cover, every range and
every runway. They know where the birds most fre- AMERICAN GAME BIRD SHOOTING 476 quent, and, therefore, know how to work out every inch
of ground to the best advantage. For cover shooting the spaniel is a superior assist-
ant. His shorter leg and smaller size enable him to
pass readily through or under cover which would offer
a real obstacle to setter and pointer,
even if it did
not deter them from attempting it. As he works within
gunshot he meets
an
essential of close cover shoot-
ing. Flushing the birds under such conditions is a
distinct advantage. Thus, then, the springer may be
considered as a better worker than the pointing dog
for rough and cover shooting exclusively. It is obvious that a setter or pointer, when standing
stanchly in dense cover, out of sight of the shooter, is,
as an assistant, inferior to the spaniel, which flushes
the birds at once. To overcome this disadvantage,
some shooters tie a small bell to the collar of the
pointer or setter, and when its tinkle ceases, the dog
being hidden from view, the inference is that he then
is pointing. To overcome the disadvantage of pointing stanchly To overcome the disadvantage of pointing stanchly
in dense cover, some trainers school their setters or
pointers to flush to order, but such a practice tends to
disorganize the dog’s training as a pointing dog, as
he is likely to become unsteady and unreliable in this
most valuable particular. As showing the great popularity of the spaniel in As showing the great popularity of the spaniel in
England, the following is taken from “British Dogs,”
written by the late Mr. DOGS. Hugh Dalziel, and published
about twenty years ago:
“The spaniel is not only DOGS 477 the oldest breed we have that has been kept to the
hunting of fur and feather, as a help to hawking, net-
ting and the gun, but he is still the most generally,
useful of our game dogs, as he is the most universal
favorite. In field or covert, no dog works so close
as a well bred and well broken spaniel; neither fur
nor feather can escape him; no hedgerow is too thick,
no brake too dense for him to penetrate and force
out to view of the sportsman the reluctant game; he
is a most active, ardent and merry worker. His ‘wan-
ton tail,’ ever in motion while he quests, increases in
rapidity of action with that tremulous whimper that
tells so truly that he is near his game, and says to his
master, in terms that never deceive, ‘Be ready—it is
inenesa Referring to the ancient history of the spaniel, the
same author remarks:
‘‘Spaniels were, in olden times,
also known by the name of the game they were kept
to, as ‘a dog for the partridge,’ ‘a dog for the duck,’
‘a dog for the pheasant,’ as in our own day we still
have the cocker, or dog for the woodcock; but at what
date the term ‘springer,’ or ‘springing spaniel,’ was
introduced, I do not know, but presume it must have
been when the qualities of the setter, or ‘setting span-
iel,’ became fully developed and permanently fixed by
breeding setters from known setting spaniels only, and
keeping the breed of questing spaniels distinct; the
term ‘springer’ was probably given to them on account
of their natural disposition to rush in and flush or
spring the game.” 478 AMERICAN GAME BIRD SHOOTING In view of his special aptitude for work in cover
and rough country, the spaniel should fit in usefully
in many sections of Canada, New England, Pennsyl-
vania, etc., for woodcock and ruffed grouse shooting. Yet, even in those sections, the stretches of open game
country are so numerous, though contiguous to cover,
that much of the best shooting ground is beyond the
capabilities of the spaniel. Thus the advantages and
disadvantages average up in such a way that in the
compromise as to methods the setter and pointer have
the preference. DOGS. In open country, where range is an essential to suc- In open country, where range is an essential to suc-
cess, the spaniel has no standing as a worker. As he
ranges close, from compulsion, and flushes his birds
when he finds them, it is quite obvious that in a prairie
or open country he could not meet the requirements. In bench show competition, and as a house dog, the In bench show competition, and as a house dog, the
spaniel shines as a favorite. Some years ago his ad-
mirers made a vigorous attempt to organize spaniel
field trials in the United States, but the effort resulted
in failure. However, in England, spaniel trials have
been held with more or less success. The foregoing remarks, in the main, apply to field The foregoing remarks, in the main, apply to field
and cocker spaniels, but there are several other breeds
used for field work, chief of which are the Clumber
spaniel, a dog of heavy physique, slow and mute in
his work, and easily broken to work to the gun. The
Sussex spaniel is a dog of hardy constitution, a good
all-round worker, much prized at one time in England
for his superior usefulness afield. The English springer DOGS 479 is said to be identical with the old Norfolk spaniel, a
compactly built dog, standing much higher on his legs
than do some of the other spaniel breeds, and quite
disposed to give tongue on occasion. There is also
the Welsh springer, of comparatively recent classifica-
tion, and there are several others as yet unnamed. Of setters, three breeds—English, Irish and Gordon Of setters, three breeds—English, Irish and Gordon
—are recognized in America. In England, some authorities affect to recognize the In England, some authorities affect to recognize the
Russian setter, though everything purporting to be de-
scriptive of that breed is meager, and seems to rest
largely on hearsay. DOGS. Edward Laverack, in his work,
“The Setter,’ describes them as white, lemon and
white, liver and white, and black and white in color,
yet naively remarks:
“I have never seen but one pure
specimen, which was in the possession of the late Lord
Grantley, at Rannoch Barracks, head of Loch Rannoch,
Perthshire.”
He also stated that “these dogs are but
little known in this country.”
As a matter of hearsay,
he relates that the head keeper to Her Majesty, many
years ago, had a beautiful breed of Russian setters,
and that ‘“‘they were good, but most determined, wilful
and cbstinate dogs, requiring an immense deal of break-
ing, and only kept in order and subjection by a large
quantity of work and whip; not particularly amiable
in temper, but very high-couraged and handsome; an
enormous quantity of long, silky white hair, and a lit-
tle weak lemon color about the head, ears and body;
and their eyes completely concealed by hair.” AMERICAN GAME BIRD SHOOTING 480 In “Stonehenge on the Dog” special attention is
devoted to this setter. By way of illustration, a pic-
ture of ‘a Russian setter slightly crossed with English
blood” is presented. In many points it resembles the
griffon. Stonehenge remarks:
“They are now very
scarce in this country, of pure blood, and even the cross
with the English setter is seldom seen.”
Many years
ago Mr. R. L. Purcell Llewellin, who was sceptical
concerning the existence of this breed, offered a prize
for any Russian setter shown at the Birmingham dog
show. None was shown in response to the induce-
ment. The breed, if it ever existed as a breed, was
thought to be extinct many years ago. More likely it
was merely a cross-bred strain. The probabilities are
that it never was known in Russia. The product of a cross between the setter and pointer
is called a dropper. It is considered a mongrel cross,
and therefore is not recognized as a breed. However,
this cross has produced useful working dogs, though
even at that far inferior to the best straight-bred point-
ers and setters in respect to high-class performance. In certain quarters, which are without any recognized
authority in matters pertaining to breeding, there is
a belief that the dropper averages well and pleasingly
with the setter and pointer as a worker. DOGS. Well in-
formed, practical sportsmen know that while there
may be a passably good dropper now and then, their
average as workers afield is not above the mediocre. There is an abundance of data concerning field work
and field-trial competition which establishes the fact DOGS 481 that the dogs of pure blood are far superior. In the
matter of beauty the dropper has no standing. In any event, it is admitted that after the first cross In any event, it is admitted that after the first cross
the dropper deteriorates physically and as a worker. In any generation he does not breed true to any fixed
type. Perhaps he may have all the externals of the
setter or of the pointer, or he may show modified char-
acteristics of both. For instance, a pointer coat may
be accompanied by a rudimentary flag on the tail, or
the latter may have a bushiness foreign to true pointer
type. This cross is never attempted or approved by
careful breeders, and is repugnant to all true sportsmen. As a rule, the dropper is the result of ignorance in
breeding matters, or of cheap pot-hunting proclivi-
ties, or he is a makeshift of indigence. The origin of the setter is unknown. As a matter
of conjecture, it is held by certain writers that his
origin is in a spaniel ancestry. There is much of
plausibility in the arguments advanced to sustain the
assumption. In many respects, some breeds of spaniels
and setters bear a striking physical resemblance. They
possess many instincts in common, though differing in
some important particulars as to methods of pursuit. Some spaniels at times display rudimentary attempts
to point, while on the other hand there are many set-
ters which display a natural inclination to flush birds
without pointing them at all, or with but a mere pre-
tense of pointing them. Nevertheless, the evidence ad-
duced in support of a spaniel origin is too fragmentary
and too speculative to furnish actual proof; and, more- AMERICAN GAME BIRD SHOOTING 482 over, in the ancient writings there are conflicting opin-
ions on this point. DOGS. In “The Dogs of the British Islands” (edition of In “The Dogs of the British Islands” (edition of
1867) Stonehenge, from such evidence as was available,
reached a vague conclusion, as follows:
“As some dif-
ference of opinion appears to exist with regard to
Setters, we have determined thoroughly to satisfy our-
selves as to their origin and best form, and we have
called the best authorities
to our assistance.”
His
ipse dixit is that the Romans, after the invasion, “‘in-
troduced the Land Spaniel, if not the Water Dog also,
into this country” (England). Hecontinues:
‘When
used either with hawks or for the net (especially in
the latter case), a far heavier dog answered the pur-
pose than what we call a ‘High-ranging Setter.’
The
net enveloped a whole covey in its meshes, and few
manors would allow of many coveys being taken in
a day; while the disentangling the birds and securing
them allowed time for a heavy dog to rest and regain
his wind.”
This is a labored, far-fetched attempt to
account for the origin of the setter. All experienced
trainers know that any kind of dog that will hunt can
be trained to drop to shot, wing, order or signal, and
teaching a dog to drop to scent or sight of the birds
is not a difficult educational feat to encompass. But
Stonehenge states that a far heavier and less enduring
dog than the modern setter was used for the net, thus
proving—so far as he proves anything at all—unlike-
ness, instead of likeness. Stonehenge candidly confessed that he personally did Stonehenge candidly confessed that he personally did DOGS 483 not know the origin of the setter, in which respect he
differed to an important degree from some other writ-
ers, who make greater pretensions without possessing
any greater knowledge. He continues:
“But in after
years, as we shall see, dogs were required to point,” in
proof of which he quotes in part as follows, from the
writings of Richard
Surflet, who wrote
in 1600:
“There is another sort of Land Spannyels which are
called Setters, and they differ nothing from the former
(the springer) but in instruction and obedience, for
these must neither hunt, range, nor retain, more or less,
than as the master appointeth, taking the whole limit
of whatsoever they do from the eie or hand of their
instructor. DOGS. ‘They must never quest at any time, what
occasion soever may happen, but as being dogs with-
out voices, so they must hunt close and mute. And
when they come upon the haunt of that they hunt, they
shall sodainely stop and fall down upon their bellies,
and so leisurely creep by degrees to the game till they
come within two or three yards thereof, or so neare
that they cannot press nearer without danger of re-
trieving. Then shall your setter stick, and by no per-
suasion go further till yourself come in and use your
pleasure. Now the dogs which are to be made for
this pleasure should be the most principall, best, and
lustiest Spannyels you can get, both of good scent and
good courage, yet young, and as little as may be made
acquainted with hunting.’
The late Rawdon B. Lee,
a much better authority than Stonehenge,
writing
about the origin of the setter, states:
‘However, other AMERICAN GAME BIRD SHOOTING 484 writers, and perhaps more
reliable
ones, including
Delabere Blane (1840), say that Robert, Duke of
Northumberland, as early as 1555, is said to have
trained a setter to the net; and that other authorities
of antecedent dates notice the sitter, or setter, as a
dog used for sporting purposes.”
Stonehenge,
referring
to the foregoing,
states: Stonehenge,
referring
to the foregoing,
states:
“Soon afterward the Setter was produced, either by
selection, or by crossing the Talbot Hound with the
Spaniel.””
The inconsistency of Stonehenge—that is,
asserting in one relation that the setter is of spaniel
origin, and in another relation that its origin may be
in a cross of hound and spaniel—is apparent. Lee quotes quite extensively from Dr. Johannes Lee quotes quite extensively from Dr. Johannes
Caius, a Doctor of Physic of the University of Cam-
bridge, who wrote about the year 1570, the following
being directly to the point:
‘The dog called the Set-
ter, in Latin Judex.—Another sort of dog there be
serviceable for fowling, making no noise with foot or
tongue whilst they follow the game. These attend
diligently upon their masters, and frame their condi-
tions to such becks, motions and gestures as it shall
please him to exhibit and make, either going forward,
drawing backward, inclining right hand or yielding
to the left. In making mention of fowl, my meaning
here is of partridge and quail. DOGS. When he hath found
the bird he keepeth sure and fast silence, and stayeth
his steps and will proceed no further, and with close,
covert, watching eye, layeth his belly to the ground and
so creepeth forward like a worm. When he approach- 485 DOGS eth near to the place where the bird is, he lays down
and with a mark of his paws betrayeth the place of
the bird’s last abode, whereby it is supposed that this
kind of dog is called Index-setter, being, indeed, a
name both consonant and agreeable with his quality.”
Numerous other ancient writers refer to the set- Numerous other ancient writers refer to the set-
ting dog as a land spaniel, but it appears that the term
spaniel was generic in its application, being applied
indiscriminately
to several
distinct
breeds, thereby
hopelessly beclouding the subject. The term, as used
at the present day, frequently may have a distinctly
different meaning from that of the same term as used
centuries ago, and confusion and inaccuracy are sure
to arise in any discussion which does not take cog-
nizance of that fact. On this point, the following,
taken from “The Gentleman’s Recreation” (1697), is
specially pertinent:
‘The dog which you select for
setting must have a perfect and good scent, and be
naturally addicted to the hunting of feathers; and the
dog may be either Land Spaniel, Water Spaniel, or
mongrel
of them both;
either
the Shallow-flewed
Hound, Tumbrel, Lurcher, or small bastard mastiff.”
To the ‘Field Book,” a sportsman’s encyclopedia, To the ‘Field Book,” a sportsman’s encyclopedia,
published in London in 1833, is ascribed the following
definition:
“The English setter (Canis index, Variety
B) is a breed between the Spanish pointer and the large
water spaniel, and was famous for his steadiness and
exquisite sense of smelling. The hair on his body was
much more curled than the present breed, which has
been lengthened by the additional cross of the springer. 486 AMERICAN GAME BIRD SHOOTING He was also much more steady than the improved va-
riety, but then he has not the same speed to recom-
mend him. . . . DOGS. He has an elegant figure, and is of a
very pleasing diversity of color; added to this, his
skin is covered with a coat of beautifully curled hair,
very rilious on the lower margin of the tail, being alto-
gether an extremely handsome dog, and quite unri-
valed by any of the canine species.”
These views, in a way, are a summation of the evi- These views, in a way, are a summation of the evi-
dence bearing on the setter’s origin. For him whose
fancy or prejudgments favor a spaniel origin there is
much evidence to humor them. In like manner, there
is evidence to console him who prefers a spaniel and
pointer origin. For him who can impartially consider
the pertinent facts, there also is ample evidence that
we do not know the origin of the setter. It is lost in
the mists of the ages. What are considered as finalities,
at best are merely shrewd speculations concerning it. While recognizing that the act of pointing, in a
more or less rudimentary form, is exhibited by span-
iels and several other breeds of dogs, and, indeed, by
cur dogs, under certain circumstances, it is never like
the intensity and efficiency of that displayed by setters
and pointers. Asa matter of speculation, it is much
more plausible that the spaniel was crossed on the old
Spanish pointer, which was introduced into England
centuries ago, and that the cross-bred product might
have been the setter. Here was the pointing instinct
ready to hand to breed into the spaniel, an exceedingly
simple matter as compared to the far-fetched theory d to hand. ir
b
ing
Deliver d to hand. ir
b
ing
Deliver DOGS DOGS 487 that it was implanted by education. This is sustained
by the admitted fact that while the setter has infinitely
more pointing instinct than has the spaniel, he in turn
has much less than has the pointer. It may be ob-
jected that the setter does not show any physical re-
semblance to the pointer. As a matter of fact, he
shows a great deal, the chief distinction being in the
coat. <A cross of setter and pointer—the dropper—
may appear to be pure setter or pointer. DOGS. By selection,
the physical attributes of the pointer could soon be
bred out, while the instinct to point would be added
to the many others which the two breeds, spaniel and
pointer, possessed in common. As before remarked, the As before remarked, the act of pointing is not the
exclusive attribute of pointers and setters. The point
seems to be a necessary factor in the successful pursuit
of birds, and is many times developed by the dog from
observation, from a realization of its helpfulness in
effecting a capture. That a dog will change his meth-
ods of pursuit to meet its conditions is well shown by
the differences of method employed in following the
trail of a rabbit and that of birds. In the one case he
gives tongue vociferously, in the other he is cautious
and silent. While the point no doubt has its origin
in an instinctive impulse, as, indeed, the impulse to
hunt is instinctive, a high degree of intelligence in
its practical application is an essential of its useful-
ness to man. The modern setter The modern setter, as owned by the majority of
sportsmen
in the United
States,
is the Llewellin AMERICAN GAME BIRD SHOOTING 488 setter,
a term which denotes merely a certain line of
breeding, of mixed English and Gordon setter blood. This family of setters originated in the ‘60s of the
last century, and was a product of Barclay Field’s
Duke and Thomas Statter’s Rhcoebe, or either of them
or their progeny crossed on any Laverack setter. The
Duke-Rhcebes had a large percentage of Gordon set-
ter blood, and so were cross-bred. However, the cross
produced some grand working dogs and field-trial win-
ners. So great was the success of the Duke-Rhcebes
and Duke-Rhcebe-Laveracks in field work and field-trial
competition, that the superiority of this family was
generally conceded. It was known for a time as “The
Field Trial Setter,” and was so known when the first
importations to this country took place, in 1874. Al-
though there are many good native setters, so called,
in this country, the Llewellin is far away the most
popular with sportsmen. After Mr. Llewellin took up
the field-trial setter, and bred it for a short time, it
was called the Llewellin setter. DOGS. The Laverack setter was a strain of dogs bred by The Laverack setter was a strain of dogs bred by
the late Edward Laverack, who averred that they were
descended exclusively from Ponto and Old Moll, pur-
chased by him of Rev. A. Harrison in 1825, and sup-
posed to have been bred pure during thirty-five years
prior to that time. However, the Laverack pedigrees
have been subjected to much hostile criticism. There
is much good evidence which shows that Laverack in-
troduced outside crosses into his kennel, and the pedi-
gree table, published in his work, “The Setter,” con- 489 DOGS tains internal evidence tending to show that it is spu-
rious; that is to say, there are only four and five gen-
erations given to cover a period of about forty years,
showing that much of the breeding was done when
the dogs and bitches were old, and ordinarily, in many
instances, past the years of reproduction. Neverthe-
less, the Laverack setter, when at his best, was beauti-
ful in physique and a grand worker. The pure Laverack setter, in the years near the end The pure Laverack setter, in the years near the end
of Laverack’s life, became very delicate and difficult
to rear. The mortality was extremely high. At the
time of Mr. Laverack’s death—he died in 1877—he
had nearly lost the breed. There probably is not a
pure Laverack setter in existence to-day, but its blood
predominates in the Llewellin setter. The colors of the English setter are orange and The colors of the English setter are orange and
white, liver and white, black and white with tan mark-
ings, roan, lemon and white, black and white ticked
with large black splashes, known as blue Belton—a des-
ignation first employed by Laverack, naming it after
a village in Northumberland County, England—and
solid colors, liver, black, white, orange, lemon, though,
as solid colors are difficult to distinguish when the dog
is working
afield,
parti-colors
are more
desirable. Dogs should weigh from forty-five to sixty-five pounds,
and bitches a few pounds less. A dog, when too light
in weight, finds great difficulty in heavy going, whether
mud or dense vegetation. The origin of the Irish setter is unknown. He un-
doubtedly originated very far back in the past. While The origin of the Irish setter is unknown. He un-
doubtedly originated very far back in the past. DOGS. While AMERICAN GAME BIRD SHOOTING 490 the Irish setter color, to conform to the standard,
should be a deep red, or, as the Irish Setter Club of
England has it, “a rich golden chestnut,” in ancient
times the color was red and white, and the red then
was not so deep and rich in color. The insistence on
color is a bench show qualification. However, a trifle
of white on toes, forehead, chest or face is not a dis-
qualification. In English field trials and field work
the Irish setter has succeeded far better than he has
in the same line of effort in this country, though this
applies to his status of some years ago. The Irish set-
ter is but little used by American sportsmen, and in
field-trial competition is rarely seen. The latter-day Irish setter seems to be lacking in The latter-day Irish setter seems to be lacking in
speed, range, endurance and general capabilities, as
compared with his more popular congeners, the pointer
and English setter. Physically, like the English set-
ter, which, in the main, he resembles closely, he is the
embodiment of grace and beauty. Many things point to the conclusion that the black Many things point to the conclusion that the black
and tan, or Gordon setter, is of comparatively recent
origin. Ancient writers are silent concerning him,
which would indicate that he was unknown to them. No one knows where the Duke of Gordon obtained his
dogs. It is supposed that this breed is a composite
of setter, collie and bloodhound, the latter showing
strongly in the generous dewlap, the coarser skeleton,
the larger, heavier head, the haw, and the tendency,
when at work, to use on the trail the methods of a
hound rather than those of the setter. The collie cross DOGS 4QI is shown by the tail and its carriage, and by the ten-
dency to head off birds in manner similar to that
employed in herding sheep. In color the Gordon set-
ter is supposed to be deep black and tan, though there
is good proof that the dogs at Gordon Castle were
black and tan, black, tan and white, and liver and
white. DOGS. Laverack, in “The Setter,’ wrote of them as
follows:
“As far as my observation goes, they lack
the endurance of the other breeds that I have named;
they are coarser and heavier made, and have not the
light and agile action of the blacks, the black and
whites, the black-grays, or blue and lemon and white
Beltons I have tested. . . . Black-tans, as a rule, have
sour, coarse heads; shoulders loaded, heavy, and too
upright; are heavy and thick-limbed; large feet, often
too straight and tilty in the hind quarters; tail thick
and ropy. Many of the black-tans have obstinate, stub-
born tempers, and are not particularly easy to break.”’
The modern pointer is supposed to be a descendant The modern pointer is supposed to be a descendant
of the old Spanish pointer which was introduced into
England in the early part of the eighteenth century. The Spanish pointer was a coarse, slow dog, sadly in-
efficient in the matter of speed and range, to improve
which a foxhound cross was introduced. The much
racier modern type is said to be the result. In the late
’70s and early ’80s of the last century many pointers—
dogs and bitches—of excellent blood, and fame as
workers, were imported from England;
but, strange to
relate, only an exceedingly small percentage of their
offspring proved to be noteworthy as workers afield, AMERICAN GAME BIRD SHOOTING 492 and quite as small a percentage of these imported dogs
and bitches showed the excellent field form with which
they were credited abroad. Many of them were first
class in bench show competition, but as a whole they
were lamentably inferior as workers. However, dur-
ing the past two decades there has been wonderful im-
provement in this breed, quite a number of pointers
showing ability equal to that of the best setters in field
work and field-trial competition. The comparative qualities of setter and pointer have
been long debated without any final conclusions being
reached. It is held that the pointer, having much the
shorter coat as a rule, suffers much less from excessive
heat than does his longer-haired brother; but that dic-
tum has so many exceptions that it is far from being
accepted. DOGS. The shorter coat of the pointer, in compari-
son with that of the setter, counts for very little; for
the setter, when properly conditioned by actual field
work, loses nearly all his feather, and his coat, also,
is much thinned and shortened from contact with the
cover. Unless fully conditioned and acclimated, both
suffer much distress in hot weather, and then, if set at
work, soon become exhausted. It is claimed for the
pointer that he can do without water better than the
setter. I myself have shot over both breeds exten-
sively when the weather was hot, and where water
was scarce, and I never observed this difference. As
a rule, both breeds require an abundance of water in
hot weather. Occasionally, one—either pointer or set-
ter, as the case may be—will show phenomenal en- DOGS 493 durance without the usual supply of water, but this is
the rare exception, and furnishes no data on which to
formulate any rule applicable to either breed. It is claimed as a point of superiority for the setter, It is claimed as a point of superiority for the setter,
that his longer, protecting coat enables him to take
punishing cover much better than the pointer can. The
claim is fallacious. Many setters are quite intent on
saving their precious skins when working where there
is brush or bramble; on the other hand, many pointers
will face punishing cover without flinching. This
phase of work is dependent on the dog’s individual
courage and his love of hunting, naturally possessed,
much more than upon the physical attribute of coat. Some dogs can go through cover without much suffer-
ing, while others are punished severely. Some dogs
are continually suffering injuries under any condi-
tions. It all resolves itself into whether the dog tries
to save his skin, or is so stupid and awkward that he
cannot do so. As to the general traits of the two breeds, the pointer As to the general traits of the two breeds, the pointer
is much the greater glutton on opportunity, is much
more likely to go stale, and so remain indefinitely, from
overwork, and to lose his dash and industry in middle
life. As a breed, the pointer does not display the fire,
endurance and sharp quickness in his bird work that
the setter does, and so is greatly handicapped in field-
trial competition. DOGS. However, he takes to pointing much
earlier in life, and more naturally, than the setter, and
therefore is much easier to train, and retains his train-
ing much better. AMERICAN GAME BIRD SHOOTING 494 Nevertheless, the pointing instinct, if exercised be-
yond useful limitations, may be a serious impairment
of a dog’s usefulness, as, while it fundamentally is an
instinct, its usefulness to man depends entirely on its
intelligent application in meeting special requirements. The pointing instinct of the pointer and setter, com- instinct, its usefulness to man depends entirely on its
intelligent application in meeting special requirements. The pointing instinct of the pointer and setter, com-
bined with their propensity to hunt birds diligently,
establishes their chief claim to excellence in upland
shooting. We do not know whence the pointing in-
stinct was derived. Even if we concede that it was
a natural evolution from education to dropping for
the convenience of the net, this in no wise accounts
for its origin in the old Spanish pointer. It is more
reasonable to assume that the instinct to point is pres-
ent in all breeds of dogs which will hunt birds. Of
course, it differs much in degree of intensity and man-
ner of application in breeds other than pointers and
setters, but it is there, nevertheless. Cur dogs which
have been used for all round pot-hunting purposes,
acquiring thereby a knowledge of the best methods in
the pursuit of birds, oftentimes learn to trail the latter
and make use of the point when the birds are located. As a matter of course, they are far inferior to setters
and pointers in every respect, in upland shooting, but
that is aside from the matter under consideration. When roading birds the cur dog observes the stealthy,
mute methods of the setter and pointer, although when
in pursuit of fur he may give tongue frantically, as
may also the setter and pointer. This seems to indicate
that it is an instinct common to all dogs, and exhibited DOGS 495 with more or less intensity under certain conditions,
as already mentioned. The exigencies of the pursuit of different animals The exigencies of the pursuit of different animals
determines in the main the choice of methods. DOGS. It is
quite obvious that a noisy, impetuous method, which
might be successful in the pursuit of an animal that
trusts for escape to its wits and legs, would wholly
fail if employed in the pursuit of an animal which can
fly swiftly away. If we assume that the point is an in-
telligent act, useful to the dog in securing a food sup-
ply, there then is no more difficulty in accounting for
it than there is in accounting for any other phenomena
of canine life. Foxes and wolves draw on their prey
and make the point, which is merely the pause to
accurately locate the exact position of the prey, pre-
paratory to the final spring to capture. The feathering
and bristling of the hair on the back are not exclusive
traits of bird dogs. All dogs, on the eve of a bloody
attack, exhibit it. Every one at all familiar with the
habits of dogs has seen even cur dogs, strangely met,
draw toward each other with hair bristling, stopping
betimes in the attitude of a point, all concluding with
a final rush which may end in battle or friendship,
accordingly as the principals are fighters or bluffers. Cats, in stalking their prey, exhibit many of the char-
acteristics of drawing and pointing. Whoever has
seen a setter or pointer make a mighty spring after
pausing a motnent—that is, pointing—in an effort to
capture birds, has noted the amazing swiftness of the
act, and that to make such a supreme effort the dog AMERICAN GAME BIRD SHOOTING 496 must set himself at the start, so that all his muscles
would co-ordinate to accomplish it. This pause, so
useful in the dog’s natural efforts to secure a food
supply, serves a double purpose in such attempt: it
enables him to locate the birds accurately before mak-
ing the final spring to capture, and it enables him to
spring powerfully and accurately. Sometimes, withal,
the dog makes a false estimate, and springs wide of
the mark, thus totally failing in his attempt. The point, however, does not always indicate the The point, however, does not always indicate the
presence of game. Under such conditions the act is
called a false point, and it may be a rarity in the field
work of a dog, or it may be of frequent occurrence,
or may happen irregularly. DOGS. A dog which habitually
false points
is next
to worthless
for field work. The fault is displayed by some dogs under unfavorable
weather conditions, which affect the scent. The ma-
jority of dogs will false point when fagged out or
when stale from overwork, day after day. There is
no cure for habitual false pointing. It probably is
the result of a faulty brain or nose, or both combined,
or, it may be, of overcaution. The standard of field work is precisely alike for The standard of field work is precisely alike for
pointers and setters. In their methods of work, natu-
ral or acquired, there are no particular differences
which can properly be classed as peculiar exclusively
to either breed. Yet each pointer or setter, in per-
forming identical tasks, may have his own individual
methods and mannerisms, no two dogs working or
planning alike, just as no two men think or act pre- DOGS 497 cisely alike. Thus, while
the main
essentials
of
field work, such as ranging, judgment, pointing, road-
ing, etc., are constants, there may be an infinity of
variable detail in accomplishing like results. cisely alike. Thus, while
the main
essentials
of
field work, such as ranging, judgment, pointing, road-
ing, etc., are constants, there may be an infinity of
variable detail in accomplishing like results. For the better comprehension of the terminology of For the better comprehension of the terminology of
upland shooting, some explanations will be of value. The terms pointing, standing, and setting, denote The terms pointing, standing, and setting, denote
the pause, generally very tense and spirited, which the
pointer or setter makes when either has located birds
definitely, or which, when trailing them, he makes in
an attempt to locate their direction or whereabouts. The dog, when pointing, may be quite close to the
birds or a long distance from them, variable circum-
stances entering into the consideration of this mat-
ter, such as whether the wind brings the scent directly
from the birds to the dog’s nose; whether the dog’s
nose is keen enough to locate the birds without press-
ing in close to them; whether at the time the birds are
in thick or thin cover, or no cover at all; or whether
it is at a season when they are either reluctant to take
wing, or will flush instantly at the slightest alarm. DOGS. All these different circumstances the wise, capable, ex-
perienced field dog recognizes and observes. The functional powers of the dogs’ noses, in respect The functional powers of the dogs’ noses, in respect
to pointing, vary greatly in the matter of efficiency. There is nothing remarkable in this if we consider
that there is much variability in sense organs—as
in the eyesight of man. Supplementary to a dog’s efficiency in locating and
pointing birds are his intelligence, as shown by the AMERICAN GAME BIRD SHOOTING 498 judgment he displays in conducting his efforts and his
honesty of purpose and cheerful effort in working to
the gun; these qualities, in sporting parlance, being
known by the comprehensive term “bird sense.”
But a dog which has a knowledge of approved meth- But a dog which has a knowledge of approved meth-
ods in work to the gun, and skill in combining them
successfully according to ever varying circumstances,
combined with endurance, cheerfulness and loyalty, is
a rarity indeed. He is the genius of his kind. Never-
theless, however excellent a dog may be naturally, un-
less he has an affection, or at least a tolerance, for the
shooter, he will not perform at his best, even if he will
perform at all. So much for the trained dog. Left to his own un-
hampered will, the natural proclivity of the untrained
dog is to impetuously follow his own impulses when
in pursuit. At first he trusts to his speed alone. After
a certain amount of experience he begins to use his
intelligence. When hunting for himself he displays
great enthusiasm and industry. This natural method,
called self-hunting, is not in accord with the best inter-
ests of the gun. The true theory of training is to preserve the dog’s
best natural methods, enforcing only such restraint as
the needs of the shooter require. No trainer can force
a dog to hunt if the latter is disinclined to do so. The dog’s natural
inclination,
after locating the The dog’s natural
inclination,
after locating the
birds, is to spring in to capture, but this being against
the interests of the shooter, the dog is trained to check
his impulse to spring while still encouraged to main- DOGS 499 tain the point. This is not always an easy task to
accomplish. DOGS. While the act of pointing is commonly
considered as a marvelous exhibition of the dog’s scent-
ing powers, it really is not any more so than is any
other organic function. Drawing and roading are terms used to denote the Drawing and roading are terms used to denote the
manner in which a dog approaches the birds by means
of their scent. Drawing has reference to the approach by the body
scent. The dog, when drawing, carries his nose well
up above the ground, and, if he performs in the best
manner, goes to the birds with quickness and precision. The dog which follows the birds by roading may The dog which follows the birds by roading may
do so with reasonable quickness and accuracy, or with
such awkwardness, slowness and inefficiency that he
is next to worthless. In respect to locating the birds, all that the trainer In respect to locating the birds, all that the trainer
can do to assist is to afford the dog proper opportuni-
ties for practice. Each dog selects his own methods,
and arbitrary methods cannot be forced on him. There is a vast difference in the functional powers There is a vast difference in the functional powers
of dogs’ noses and intelligence, therefore all dogs can-
not work up to the same standard. Drawing is far away the best method of locating Drawing is far away the best method of locating
birds, although in certain conditions of weather, when
the scent lies low, the dog which carries a high nose
on the trail encounters a difficult problem. But, un-
der favorable conditions, when he strikes a trail he
may follow it at top speed and locate the birds with
marvelous quickness and sharpness. Such a performer AMERICAN GAME BIRD SHOOTING 500 would have the birds located before the dog which
picks his way painfully and slowly along by the foot
scent had well begun. The advantage of drawing as
compared to roading is thus apparent, and it is of spe-
cial advantage in field trial competition. Some dogs have the ability to draw or road, adapting Some dogs have the ability to draw or road, adapting
their efforts to the scenting conditions, and acquitting
themselves creditably. However, every dog which car-
ries a high nose in the attempt to locate is not neces-
sarily a good performer. DOGS. ‘There are wide-ranging,
high-nosed dogs which are poor finders. The only test
by which a dog’s merit can be properly determined is
his actual work afield. Ranging is a term which denotes the casts taken Ranging is a term which denotes the casts taken
by the dog, to and fro, while beating out the ground
in the search for birds. It may be done in many quite
different but successful ways, or it may be done in a
way which at once demonstrates that the dog has
no hunting intelligence whatever so far as the needs
of the gun are concerned. Inability, due to stupidity,
cannot be overcome by training. Neither a foolish
man nor a foolish dog should be expected to surpass
his natural limitations. A good nose is an aid of inestimable value in sup- A good nose is an aid of inestimable value in sup-
plementing industry and good judgment. It com-
mands a great area, thus enabling its possessor to
search out much more territory with fewer casts than
is possible to his fellow with the poorer nose. The good ranger maintains
constant
observation The good ranger maintains
constant
observation
of the wind. He skirts promising stretches of cover DOGS 501 on the leeward side, so that, if there are therein any
birds in hiding, he will catch their scent. He recog-
nizes the likely places for birds, and, planning with
them in mind, he conducts his range after a well-laid
scheme, thereby economizing time and effort. On the
other hand, the dog possessing a poor nose is quite
likely to range here and there, hither and thither, hit
or miss, as it may happen, working the same ground
over repeatedly while leaving other places, perhaps the
most likely ones for game, unnoticed. A fast dog, if he uses his nose properly, is the best, A fast dog, if he uses his nose properly, is the best,
provided he can maintain his pace uniformly for a con-
siderable time. It is obvious that such a dog can cover
a certain area in a certain time much more thoroughly
than a slower worker can. Many dogs will start at high speed, slowing down
gradually thereafter, till they practically quit. A lot
of stamina and pluck are required to maintain a fast
pace while hunting during several hours in succession. DOGS. A moderate degree of speed, uniformly sustained, is
much to be preferred over high speed of uncertain
duration. Some slow dogs, of surpassing cunning in
their work, are excellent bird finders. Though a dog
may be slow he nevertheless may take wide casts, and
if his nose and judgment are good, combined with
honest work to the gun, he has the essentials of a hunt-
ing dog. As to methods of hunting out any game area, the
dog, if given the opportunity, learns them best when
left to his own devices, without interference from AMERICAN GAME BIRD SHOOTING 502 his trainer. The latter can hamper the dog’s progress
by interference, but he can do very little to assist. With age and experience, all intelligent dogs improve
in their hunting methods. Many of the best finding dogs are those which have Many of the best finding dogs are those which have
indulged in self-hunting, a term which denotes that
the dog hunts independently, as when he sneaks off
alone, or in the company of other self-hunting dogs,
or as he pleases when with his trainer. Under such
conditions he hunts till wearied or till satisfied, which
may be in a few hours or a few days, according to
the endurance, longing, and the food supply. Commonly, after a vagrant self-hunting trip, the
dog returns mere skin and bone, the exposure, pro-
longed exertion and short rations producing their natu-
ral results. The vagrant, under such circumstances,
regains his flesh as quickly as he lost it, if properly fed. Vagrant excursions should be strictly suppressed. Vagrant excursions should be strictly suppressed. They afford unlimited opportunity for the formation
of bad habits, such as harrying sheep, killing fowls,
robbing nests, etc. The self-hunting proclivity mani-
fests itself sometimes when working to the gun. The
self-hunters will apparently work under control for a
longer or shorter time, gradually ignoring the shooter,
till at last they go where and when they piease. This
insubordinate stage lasts according to the whim of the
dog. It may be for a brief while, or it may last a day
or two. Meanwhile the dog is learning a great deal
in respect to ways and means of finding birds, though
of no service whatever to the gun while so absent. DOGS. ‘sneuysO
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3u00ys DOGS 503 In ranging, the dog should adjust his efforts to fit
the circumstances of cover and open, casting closer
in a broken cover country than in an open country, and
he should come into view of the shooter at reasonable
intervals to note the proper course as a base line to
guide him in keeping within proper bounds. When
hunting in cover exclusively,
as in hunting ruffed
grouse and woodcock,
his proper range should not
much exceed that of the spaniel; that is, gunshot. Back, back s Back, back stand, or back set, is when one dog stands
stanchly at sight of a pointing dog, the attitude of the
backing dog being much like that which he assumes
on a point, though in many instances it is more or less
relaxed. In any case, as the dog advances in years,
he backs with less intensity. If the pointing dog has pointed If the pointing dog has pointed false a few times,
the backing dog, observing that the act is deceptive,
loses confidence in his fellow worker, disregards his
points, and goes on about his own affairs, or breaks
in to see what it is all about. Dogs quickly comprehend Dogs quickly comprehend the significance of each
other’s movements. Some deferential dogs will back
when they observe their bracemate drawing on game,
being satisfied to take a subordinate position at every
opportunity. Other dogs, on the contrary, may cast
in ahead at top speed when they see a dog roading or
drawing, being ambitious to secure the point. Some
will back well for a moment, then move forward a
few steps at a time, playing craftily to steal the point. Some dogs, again, will back or point honestly when AMERICAN GAME BIRD SHOOTING 504 under the eye of the shooter; out of sight, they are
lawless. Again, some dogs which point well refuse
to back at all. So far as work to the gun is concerned, the purpose
of the back, apart from its spectacular features, is to
guard against interference with the pointing dog. DOGS. If
a dog refuses to back he can be taught to drop instantly
to oral command or to signal or to his bracemate’s
point. This detracts from the spectacular beauties of
shooting, but a good dog should not be condemned
because of failure in this respect. Quartering denotes a system of beating out the Quartering denotes a system of beating out the
ground in substantially parallel casts at right angles
to the course of the shooter. This method has never
been esteemed in this country, although it was viewed
with favor in some sections of England. It fitted in
usefully in English shooting, as on the English pre-
serves the birds, in many fields, were quite as likely
to be in one place as another; therefore, it was an
essential that every bit of ground be searched out thor-
oughly. In this country, there being much unpromising
area in ratio to likely area, the really serviceable dog
uses his brains in hunting only in the likely places. These conditions are ever changing. The local con-
ditions must always be the guide of action; no arbitrary
rule is possible. Before attempting to shoot game over a dog’s points
with a serious purpose to secure enjoyable sport, it
is better to give him a thorough schooling in the dif-
ferent commands, and their significance, and therewith DOGS 505 sufficient discipline in obeying them, so that, when
afield, there will not be too many conflicting ideas in
his head. Added to all this, the dog should have suff-
cient experience in working on birds and working to
the gun, so that there will be sport instead of chaos,
Beyond a perfunctory feeding of his dog, the aver- Beyond a perfunctory feeding of his dog, the aver-
age dog owner, in times past, gave little thought to
the animal’s general well-being. If given an abundance
of food, without any consideration as to whether it
met the dog’s bodily needs, he was considered to have
received all the attention necessary. In recent years,
however, there has been a better understanding of the
need of promoting the comfort and general welfare of
the dog, and to this understanding bench shows and
field trials have contributed
incalculable missionary
work. Much valuable literature has become available
on all subjects of canine interest, and so a better gen-
eral knowledge of the dog’s requirements. DOGS. There are various kinds of excellent proprietary dog There are various kinds of excellent proprietary dog
foods on the market, so cheap as to price, though ex-
cellent as to quality, that they are within the reach of
the most slender purse. Table scraps, assuming that
they are other than potato skins and bare bones, make
a good dog food. Milk, sweet, sour, or buttermilk,
may be given to the dog in abundance. The latter is
a specially good summer drink for the dog, as it also
is for man. Contrary to a popular belief, milk does not
generate worms. If an owner does not care to purchase proprietary
foods, and the table scraps are insufficient for the needs AMERICAN GAME BIRD SHOOTING 506 of his kennel, scraps of mutton, beef, or goat meat,
stewed with cabbage, onions, beets, turnips, and other
vegetables—observing that all are strictly sound and
good—make an excellent food during the idle months. When a dog is used for field work the food should
be almost wholly meat. Sheep’s heads and tripe make
an excellent food, preferably in a stew. The former
should be skinned, and boiled till the bones can be
readily removed from the flesh. Corn meal, though
quite commonly used, is one of the poorest of dog
foods, much of it being undigested, as can readily be
known by an examination of the dog’s droppings. For working dogs, one feed after the day’s work
is ended is quite sufficient. They then should have all
that they will eat. If fed in the morning, digestion
is suspended while the dog is ranging about, and he
is carrying with him the equivalent of so much dead
weight, with no nutritive qualities for the time being. If fed on a meat diet when working, the dog is al- If fed on a meat diet when working, the dog is al-
most exempt from bowel troubles, while if fed much
corn meal such troubles are frequent. For the latter
there is no better or prompter remedy than lean beef,
cut into small pieces, and heated through in a hot oven,
thus being only blood rare. If a dog’s stomach is loaded when he is at work his
powers of scent are seriously impaired. It matters not
whether he is fed on meat or vegetables. DOGS. Nature has
wisely provided that while digestion is in progress the
dog’s sense of smell is dulled. Even man, whose sense
of smell is incomparably inferior to that of the dog, DOGS 507 can smell a dinner a long way off when he is hungry;
whereas, after dinner, the same gratifying fragrance is
unrecognized. Man’s own requirements are no data on which to Man’s own requirements are no data on which to
judge of the food needs of the dog. The latter, being
carnivorous, requires food far less frequently than
man, while the latter, in turn, feeds less frequently than
the ox, which is wholly herbivorous. Next to a good food supply, the dog needs a com- Next to a good food supply, the dog needs a com-
fortable, well-ventilated, sanitary place in which to
sleep. Any man who cannot afford such a place, and
neglects his dog’s comfort in this respect, cannot af-
ford to own a dog, consistently with humane require-
ments. With this, the dog should have plenty of exer-
cise. It is best that he should have entire freedom,
but as this is not always possible, particularly within
city limits, regular times each day should be allotted
for that purpose. PART
III
THE SHOOTING
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eIGMqog A LOOK BACKWARD When the white men came to America they found
wild birds and animals very abundant. The repro-
ductive energy of indigenous species more than made
up for their destruction by natural enemies. Human
beings were not numerous in proportion to the area of
the land, and took only what they needed. The balance
of nature was preserved. It was rudely disturbed by the arrival of civilized It was rudely disturbed by the arrival of civilized
man with his firearms, though for a time he made little
impression on the life of the great continent to which
he had come. Birds and animals continued abundant,
though close to the settlements they were soon driven
away or destroyed. Thus Josselyn, writing in 1671. tells, as already said, how thirty years before turkeys
were very abundant at Black Point, now Scarborough,
Maine, where many broods of young might be seen in
a morning, but that the English and the Indians have
destroyed them, “‘so that ‘tis very rare to meet with a
wild Turkie in the Woods.”
This was only about fifty
years after the landing of the Pilgrim Fathers. Gee Gee Gee 512 AMERICAN GAME BIRD SHOOTING In the early days of the settlements game was killed
for food, and not for sport. The arm carrying the
single ball comes first into a new country, and the shot-
gun follows at some distance of time. The musket or
rifle is a necessity; the shotgun a luxury. The rifle
protects life and property, supplies food for the family,
destroys the wild beasts that would prey upon the
settler’s stock. The shotgun is used in hours of leisure
and recreation. Food captured by its aid is a delicacy. It is the implement of sport. In the settlement of
America this has been everywhere the case. The hardy pioneer disdained to kill his game with The hardy pioneer disdained to kill his game with
more than a single ball, which it was his pride to plant
wherever he chose, and in his expert hands the old
crooked-stock pea-rifle was his dependence for support,
as it was the terror of the savage tribes into whose ter-
ritory he pushed his fearless way. A LOOK BACKWARD Many, many years
later, when the land had been cleared and waving fields
of corn and wheat had taken the place of the wild
grasses that once grew rank and thick along the valleys
and over the prairie, when the large game had almost
entirely disappeared, the children or the grandchildren
of the rifleman began to use the shotgun. Geese and
ducks, the noisy grouse, the brown quail, the whistling
woodcock and the twisting snipe became the objects of
pursuit to those whose fathers had killed the elk, the
moose and the buffalo. The fathers hunted for meat. With them it was a fight for life. Each ball and each
charge of powder was to be accounted for and must do
its work. The sons and grandsons inherited the hunt- A LOOK
BACKWARD 513 ing spirit from their sires, but no pressing need drove
them to the field. They shot—for pleasure—wild
fowl and game birds when these were to be had, but if
not these, too often they destroyed in their ignorance
tiny songbirds which were their best friends. Many a man has seen with his own eyes the expul- Many a man has seen with his own eyes the expul-
sion of the rifle by the shotgun from more than one
section of the land. Little more than a generation ago
the shotgun was scarcely known in central Nebraska. Then
it was
necessary
on
occasion
to
fight
off
Sioux or Cheyennes or Arapahoes, while the buffalo,
elk, deer and antelope were needed for food. The
years have rolled by; the riffle has disappeared. Either
it went westward to the mountains with its owner, or,
rusty and dirty, it stands to-day neglected in a corner
of the garret. All the young men and boys have shot-
guns, and in the season the geese that visit the river
bottoms, the ducks of the sloughs, the prairie chickens
on the upland and the quail in the tow-heads are con-
stantly pursued. So, in the settling up of a country the weapon adapts
itself to the game to be killed. As long as large mam-
mals are abundant the rifle holds its own, but when
these are gone it must give place to the weapon of
shorter range. A LOOK BACKWARD It was not until toward the middle of the nineteenth It was not until toward the middle of the nineteenth
century that the birds of the United States began to be
shot for sport. Before this the markets offered induce-
ments to the thoughtless or the greedy to destroy birds
with great recklessness. Game birds were taken in nets, AMERICAN GAME BIRD SHOOTING 514 snares and traps in vast numbers, and as early as 1855
and ’56 a writer said:
‘The havoc and slaughter, not
only by the mercenary and cruel, but by the thoughtless
and improvident, have consequently been very great,
both of birds and animals, thousands of buffaloes, deer
and hares, hundreds of wolves, panthers and bears, as
well as myriads of partridges and hecatombs of grouse,
have been slain and trapped. The markets of all our
principal cities have been most bountifully supplied with
every description of game—in fact, filled to repletion
with partridges and grouse; not, however, as many sup-
pose, with birds alone coming from the far, far West,
but by incredible numbers of half-starved victims snared
in our own and the neighboring States or mercilessly
shot down, whole coveys at a ‘blow,’ as they sit huddled
together on the treacherous snow, stupefied and be-
numbed with the chilling blasts of the north.”
Some of the sportsmen of that day were Englishmen, Some of the sportsmen of that day were Englishmen,
and of these Wm. Henry Herbert—Frank Forester—
was one of the most famous. Herbert wrote in charm-
ing style, and professed high ideals of sport. His writ-
ings were read by all who were fond of shooting and
fishing, and exercised a good influence. Birds at that
time were so extremely abundant—from the point of
view of to-day—that no one realized the importance of
moderation in killing, and the bags made were very
large. Herbert inculcated good views of sport, and his
teachings were far in advance of the practice of his day. Nevertheless, it was then a point of pride, as indeed it
still was at a very recent day, to do a little better—that A LOOK BACKWARD 515 is, to kill a few more birds—than your neighbor or your
rival. A LOOK BACKWARD Although all the larger game birds had been exter-
minated in the Eastern and Middle States by the time
of the Civil War, it was not until the advent of the
breech-loader in America, about 1868 or ’69, that bird
shooting began to be excessive in nearby localities. Yet,
by the middle of the following decade, complaints began
to be heard of the scarcity of game. People who a few
years before had had no difficulty in killing thirty birds
in a day, grumbled when they could get but fifteen, or
perhaps ten, and found vacant covers long known to
be depended on for a certain number of birds. These
were the first warnings of the scarcity of game birds,
which, since that time, we all have known. In those days, too, the gunners interested themselves
much in the question as to which was the most useful
arm, the breech-loader or the muzzle-loader. Those
who had adopted the new weapon vaunted its conven-
ience and general efficiency, while the more conservative
declared that the breech-loader did not throw shot with
the force of the muzzle-loader, and declared that they
would never change their weapons. It was about this time that the first periodicals de-
voted exclusively to shooting and fishing were estab-
lished. These were, first, the American Sportsman,
which lived but two or three years, and then Forest and
Stream, which subsequently absorbed the successor of
the American Sportsman, the Rod and Gun. These
journals were immediately successful, because of the AMERICAN GAME BIRD SHOOTING 516 interest felt in their subjects by the sportsmen and field
naturalists of that day. Sportsmen had much to say
on various topics, and naturalists wished to discuss
many matters which could hardly be brought up in pub-
lications devoted strictly to science. Many sportsmen
were careful and accurate observers, possessed of ex-
perience
and
education,
and
writing
delightfully. Later a large crop of periodicals devoted to these
sports sprang up, most of which, before very long,
perished for lack of support, their places being taken
by others. These earliest periodicals, being directed by men of
foresight and experience, urged upon their readers the
importance of preserving the natural resources of the
country. They preached game protection and forest
protection, and while converting not a few of their
readers, at first made little or no impression on the
general public. A LOOK BACKWARD Such educational work goes slowly at
first, especially when the teaching tends to limit the
recreation and pleasures of the public by urging them
to practice self-control. It was thus many years before any definite results
could be seen from these teachings, and in the mean-
time the works of destruction went on. Perhaps the
most radical and most important step ever taken in
game protection in this country was in the year 1894,
when Forest and Stream announced its platform plank,
“Forbid the Sale of Game.”
Meantime a constant,
though slow, improvement was taking place in the
game laws, and a few sportsmen, by earnest work with ‘trend
ueadoing ‘t
ueadoing A LOOK BACKWARD ig the legislatures, backed up the work which Forest and
Stream had set on foot. Meantime, sportsmen at large discussed with much
earnestness the question of what should be done to re-
stock the game covers. The first efforts took the direc-
tion of importing bobwhites from the South and turn-
ing them loose in covers that had been shot out. This
was done, but the birds were soon killed off, for no
one seemed to think of stopping shooting. Then came
the idea of importing foreign birds, and about 1878 or
*79 some one suggested the importation to America of
the Old World quail (Coturnix), a migratory bird
which breeds in Central and Northern Europe, goes
south
in autumn,
crosses
the Mediterranean
and
spends the winter months in Africa. These birds were
easily obtained and were inexpensive. Considerable
numbers of them were imported. They appeared to
take readily to their new home and many of them bred. Nevertheless, after a time they disappeared without
any apparent cause. It was reported that flocks of
them had flown out to sea and been drowned, and it is
quite possible that, migrating to the shores of the Gulf
of Mexico, they attempted to cross that body of water
and perished. At all events it is now many years since
one of these birds has been authentically reported as
taken in America. Attempts were made to acclimatize the English Attempts were made to acclimatize the English
pheasant in the United States a hundred years ago, but
the birds turned loose in New Jersey all disappeared. Another attempt was made early in the nineteenth cen- AMERICAN GAME BIRD SHOOTING 518 tury, near Belleville, N. A LOOK BACKWARD J., but again the birds disap-
peared. It is said that between 1820 and 1830 pheas-
ants were turned out near Baltimore, Md., but by 1830
the last of the birds seems to have vanished. In 1880, Hon. O. N. Denny, then U. S. Consul at In 1880, Hon. O. N. Denny, then U. S. Consul at
Tien-Tsin, China, shipped some ringnecked pheasants
to Oregon. Most of these died, but the following year,
according to the report of the Fish and Game Protector
of Oregon for 1895-96, another shipment was made
which did better, and these were set free on the ranch
of Mr. John Denny, in the Willamette Valley, in Lynn
County, Oregon, where they did well and increased
rapidly. In the late winter or early spring, 1885, an
important importation came to Portland. These were
again from Consul Denny, and were sent to the people
of Oregon in care of a sportsmen’s association of Port-
land. They included several species. Efforts were
made to induce the legislature to enact a law protecting
them and to make a small appropriation for their care
until they could become established. The legislature
laughed at these requests and treated them with so
much scorn as to create quite a little sympathy for the
sportsmen’s association, and incidentally for the pheas-
ants. The owner of Protection Island, in Puget Sound,
offered to give the birds a home and protect them if
desired, and they were turned out there. In 1882 two hundred pairs of English pheasants were In 1880, Hon. O. N. Denny, then U. S. Consul at
Tien-Tsin, China, shipped some ringnecked pheasants
to Oregon. Most of these died, but the following year,
according to the report of the Fish and Game Protector
of Oregon for 1895-96, another shipment was made
which did better, and these were set free on the ranch
of Mr. John Denny, in the Willamette Valley, in Lynn
County, Oregon, where they did well and increased
rapidly. In the late winter or early spring, 1885, an
important importation came to Portland. These were
again from Consul Denny, and were sent to the people
of Oregon in care of a sportsmen’s association of Port-
land. They included several species. Efforts were
made to induce the legislature to enact a law protecting
them and to make a small appropriation for their care
until they could become established. A LOOK BACKWARD The legislature
laughed at these requests and treated them with so
much scorn as to create quite a little sympathy for the
sportsmen’s association, and incidentally for the pheas-
ants. The owner of Protection Island, in Puget Sound,
offered to give the birds a home and protect them if
desired, and they were turned out there. In 1882 two hundred pairs of English pheasants were In 1882 two hundred pairs of English pheasants were
brought to New York from England to stock Pierre
Lorillard’s game preserve in Monmouth County, N. J. They did well there, and with others imported later by _ ‘yueseayd-asnorg
Aysng
pruqAy ‘y
Aysng
pruqAy A LOOK BACKWARD 519 Pierre Lorillard and Rutherford Stuyvesant and turned
out at various points in New Jersey and along the
border between that State and New York, stocked in a
limited way a considerable territory west of the Hudson
River. In the year 1881, nineteen Chinese pheasants were
imported to Victoria. Vancouver Island, and set free
there, while a law was enacted giving them absolute
protection for five years. They increased astonish-
ingly, so that when the closed time was ended there
was excellent shooting in the neighborhood of Victoria,
and it was estimated that during the first season not
less than 3,000 pheasants were killed. During the
winter the birds could be seen, sometimes along the
roadside and often in the cultivated fields, and were
evidently very abundant. The introduction of the pheasants in Oregon and The introduction of the pheasants in Oregon and
California was generally welcomed, and public feeling
protected the birds. It appeared that the climate was
well suited to them. They increased rapidly, and now
for many years have been abundant on the coast, but
chiefly in the region of great precipitation. They have
apparently not spread greatly to the south, into the dry
region of California, although in that State there are
believed to be some persons breeding pheasants in semi-
confinement in a small way. The birds are popular on the coast. Some fruit-rais- The birds are popular on the coast. Some fruit-rais-
ers, to be sure, declare that they injure the crops, but
on the whole they are regarded as a welcome addition AMERICAN GAME BIRD SHOOTING 520 to the fauna of the region. In a few instances they
have interbred with the dusky grouse. A LOOK BACKWARD During the last thirty years a number of different During the last thirty years a number of different
exotic species have been suggested as useful birds for
American shooters. Pheasants have done fairly well in
certain
eastern
localities
besides
those
mentioned. Major W. A. Wadsworth introduced them into the
Genesee Valley, and Dr. W. Seward Webb has a large
preserve in Vermont, where birds are hand-reared and
turned out to be shot in battues once or twice a year. The Blooming Grove Park Association in Pennsylvania
has successfully raised pheasants, and each year the
members are permitted to kill a certain number, while
many other parks and associations, to say nothing of
some
State farms. and a multitude of commercial
people, have been engaged in the work of rearing these
handsome birds. It has been demonstrated that the
pheasant will endure the rigorous climate of the north,
at least as far as central or northern New York. The pheasants are not without their opponents. It
has been alleged that they destroy or drive off our
native game birds; that they injure certain crops; and
that while they themselves are immune to certain dis-
eases, they yet may communicate these diseases to our
native birds. The latest candidate for public favor on the roll of
foreign game birds is the so-called Hungarian par-
tridge—the gray partridge of Europe. It is a bird
nearly as large as the ruffed grouse, and is hardy and
offers good shooting. It is only within three or four ‘aspiijizg
uvadoing ‘a
uvadoing A LOOK BACKWARD 521 years that it has been imported in considerable numbers,
the earliest importations having taken place in 1905 or
’06. It is reported to have done well, and by many is
believed to be the coming game bird. Besides those named, few Besides those named, few foreign game birds have
been introduced, and those in small numbers only. Mr. W. W. Thomas many years ago was instrumental in
importing and liberating in Maine some capercailzie
and black game. Black game was also imported and
turned loose on the Island of Newfoundland. So far
as known, nothing was ever heard of the birds. Much
later, some capercailzie were imported and turned out
in Algonquin Park, in Canada, whence for several
years reports have come of young broods seen. A LOOK BACKWARD Some
European grouse, including, we believe, ryper, black
game and capercailzie, were turned out on Grand Island
in Lake Superior, but these have all disappeared. Most of the efforts at r Most of the efforts at restocking our covers with
exotic birds have been carried on in an unintelligent and
aimless way. The belief seems to prevail that because
our native species have disappeared—while game birds
in Great Britain are still so abundant that good shooting
can generally be had—therefore these foreign species
are more prolific and better fitted to survive than our
native birds. People who reason in this way forget the
widely different conditions prevailing in the two coun-
tries—the great size of the land holdings in Great
Britain compared with those in this country, the fact
that trespass laws are there strictly enforced, and the
further fact that game is there regarded as the personal AMERICAN GAME BIRD SHOOTING 522 property of the owner of the land; while in the
United States up to within a few years the game has
been considered the property of whomsoever might re-
duce it to possession. On one side of the water are large estates to which
the public is not admitted, while game birds are arti-
ficially reared and carefully protected to prevent de-
struction by natural enemies, as well as by poachers. The result of this is that at the end of a season a crop
of game is harvested and sold. In the United States are
a series of small land holdings, over which until within
a very short time, everyone, man or boy, citizen or alien,
was at liberty to roam freely and to destroy at will; the
game is left to reproduce itself, is exposed to attacks by
its natural enemies as well as by all human beings who
may wish to take it, and is only protected by laws
which exist on the statute books, but are inefficiently
enforced. Here each gunner wishes to shoot, and in
practice may shoot, from daylight to dark, seven days
in the week. Add to this that a large portion of the
public here is accustomed to the use of firearms, and
that we have the best guns and ammunition in the
world, and it is not difficult to see why game is very
scarce in most sections of North America. A LOOK BACKWARD In the thickly settled districts of the Eastern and
Middle States matters have gone so far that it is diffi-
cult to suggest a means by which a stock of birds for
field shooting may again be obtained, except the one
means, which gunners will adopt last of all, the practice
of self-control in their shooting. One result of this A LOOK BACKWARD 523 state of things is that a considerable proportion of the
men who formerly followed the dog afield, and a very
large proportion of their descendants, have taken to
shooting targets at the trap, for the reason that within
the reach of most of us there are no birds to be had. I have never advocated the introduction into this I have never advocated the introduction into this
country of foreign species of game, believing that our
native species answer our requirements better than any
foreign birds, and believing also that there is great
danger in introducing exotic species into any country. I have believed that by the establishment of game
refuges in various sections of the country the native
game might be preserved and increased to such an ex-
tent that it would overflow into adjacent territory, and
that thus in each game refuge we should have a perma-
nent source of supply which would at least prevent the
extinction of species for any locality. I feel about the
introduction of exotic species much as does Mr. Brews-
ter, who, in his ‘Memoir on the Birds of the Cam-
bridge Region,” says, speaking of the pheasant:
“From the standpoint of the naturalist, the introduc- “From the standpoint of the naturalist, the introduc-
tion of most exotic forms of animal life must be a
matter of regret rather than of satisfaction, and these
pheasants, despite their undeniable beauty of form and
coloring, and reported value as game, seem deplorably
out of place ina New England landscape. A LOOK BACKWARD Even if they
did not crowd out our quail or ruffed grouse—as it
has been feared they may—or devastate our cultivated
crops—as they are already accused of doing—it would
have been much wiser to expend the time and money AMERICAN GAME BIRD SHOOTING 524 which have been devoted to their naturalization in fos-
tering and increasing our stock of native game birds.” The turning loose of foreign birds to take care of
themselves in a climate to which they are unaccustomed
and among conditions more or less different from those
in which their ancestors have lived, is not likely soon to
make much difference in our shooting. A few hundred
birds turned loose in a township or a county would
have to increase enormously before they would be suf-
ficiently numerous to make the shooting good. Every-
one killed would reduce the breeding stock, the process
of reproduction would be slow and the final results, even
if favorable, might not be important for a generation. Besides this, there are serious possible dangers in Besides this, there are serious possible dangers in
the turning loose of foreign birds. There are some
reasons for thinking that these foreign birds carry with
them the germs of certain diseases to which they them-
selves are immune, but which may be communicated to
our native birds with fatal results. It is believed by
some investigators that the domestic fowl carries with
it the germs of a disease which is fatal to the turkey,
and to our quail and grouse, although the young of the
hen do not suffer from it. Many examples might be cited of the danger of intro-
ducing into a new land an animal, harmless in its own
home, but which when transported to a country where
conditions are especially favorable to its existence and it
finds few or no enemies, has increased to such an ex-
tent as to become a nuisance, if not a public menace. The cases of the rabbit in Australia, of the English spar- A LOOK BACKWARD 525 row in North America, and of the mongoose in some
of the West Indian Islands, suggest themselves at once. ly, within the past two or three years, the ex- Happily, within the past two or three years, the ex-
periment of hand-rearing some of our native game birds
has apparently advanced well along the road to success. A LOOK BACKWARD If it cannot be said that any of the public establishments
for rearing such birds have been successful in any great
degree, it is yet true that they hatch and partly rear
many birds. Almost always it has happened that before
the birds reached maturity, certainly before they had
reached the breeding age, death in some form or other
has overtaken them. It was left to a private individual,
a professor in a New England university, to take up
this matter of rearing native birds as a hobby and to
succeed in it beyond the expectation of anyone, possibly
beyond his own hopes. Certain it is that Prof. C. F. Hodge, of Clark University, at Worcester, Mass., has
succeeded in rearing from the egg a considerable num-
ber of ruffed grouse and quail, which birds are no more
timid and fearful of the members of their owner’s
family than they are of their fellow birds. Not only are
they tame in this way, but they manifest no fear what-
ever of strangers. Within their enclosures they carry
on the operations of their daily life with the same un-
concern that they would manifest if they were hidden
in the depths of one of their native swamps, and this
whether human beings are
in the vicinity or not. Broods of quail which Professor Hodge has reared and
turned out to shift for themselves will come at his
whistle, flying from all directions, expecting to be fed. 526 AMERICAN GAME BIRD SHOOTING Gradually legislators are awakening to the increas-
ing scarcity of bird life about us, and to a compre-
hension of the invaluable services performed by the
birds for agriculture. It has taken the public a long
time to begin to comprehend what these services mean,
and it is yet, standing only at the threshold of this
knowledge. But having made this small beginning,
our faith may be strong that this interest will increase,
and that before long the birds will be generally recog-
nized as a natural resource of this country which should
be conserved. In a number
of the States
and provinces
the
law now provides that there shall be no spring shoot-
ing; that non-game birds shall not be killed nor their
nests and eggs disturbed, and that the shooting seasons
shall be short—not more than two, or at most three
months. A LOOK BACKWARD In some States the laws provide that only a
limited number of birds may be taken during one day
or during one season. This provision should obtain
everywhere, as well as the now very generally accepted
view that game birds shall not be sold. In many States
we have excellent laws, which, however, lack some-
thing of satisfactory enforcement. TRANSPLANTING AMERICAN GAME BIRDS. It was between the years 1885 and 1895 that the
greatest interest was felt in trying to restock the covers
of the Middle States with bobwhites imported from
the South. They were turned out in large numbers,
but, as I have said, did not increase the numbers of
local birds because it did not occur to sportsmen to stop
shooting. A few men paid for the birds in any locality,
and the general public turned out and killed them all,
very often without so much as saying “thank you” to
the men who had paid the bills. But bobwhite was not the only native bird which
sportsmen tried to introduce on the Atlantic Coast. Efforts have been made at various times to introduce
the pinnated grouse of the Mississippi Valley to the
old home of the heath hen. This might easily have
been done, and could be done to-day, provided only the
birds were introduced into game refuges where abso-
lute protection would be assured them. The birds for-
merly submitted readily to confinement, bore railway
journeys well and speedily adapted themselves to new
conditions. While most of these attempts have been forgotten. While most of these attempts have been forgotten. or remain only as tradition, we have fortunately a
record of one of them. This account is from the pen
of Mr. N. H. Bishop, well known a generation ago as
527 527 527 AMERICAN GAME BIRD SHOOTING 528 a traveler and canoeist, and was printed in Forest and
Stream in December, 1874. It tells of the introduction
of the pinnated grouse and two species of Pacific Coast
quail in eastern Maryland. The essential parts of
his letter are as follows: “About five years since a resident of Philadelphia
sent
to! Dr.) Py J) Purnell, near Berl) Worcester
County, Maryland, a few pairs of prairie chickens and
a covey of both the valley and mountain partridge, or
quail. Dr. Purnell has an estate of fifteen hundred
acres lying along the banks of Newport Creek, which
stream flows into Sinepuxent Bay, on the eastern shore
of Maryland. Since the war, this estate has been
worked for the Doctor by his tenants. Much of it is
woodland and salt meadows. The partridges were for
some time kept confined in the house and then were
set at liberty. TRANSPLANTING AMERICAN GAME BIRDS. They soon disappeared, except one pair,
which returned daily to the kitchen door to be fed. For
some cause the pair went to a neighbor’s house, on the
same estate, and for some weeks were fed from the
kitchen door. This pair of birds nested in the garden
near the house, and raised a brood of young birds. The
covey left their old quarters, and were heard from but
once after their departure. A person reported that he
saw the covey of ‘California quails on the other side of
the creek.’
This was two years since. It is now sup-
posed that these partridges have been shot by gunners
or have died from natural causes. “The prairie chickens adapted themselves to their “The prairie chickens adapted themselves to their
new home with but little trouble to the proprietor of the TRANSPLANTING AMERICAN GAME BIRDS 529 estate. Their nests filled with eggs were found along
the fences of the fields near the meadows. The birds
became tame, visiting the cattle yards, and feeding near
the buildings of the farm. They multiplied rapidly. A
law was passed by the Maryland legislature protecting
them from gunners. The birds seemed to like the large
salt meadows of the estate, and exhibited but little fear
of strangers. Unfortunately for the birds, a number of
terrapin hunters from New Jersey ascended the bay
and river in their small vessels. Seeing these tame
birds on the meadows, the Jerseymen commenced a
war of extermination upon them, which soon resulted
in the destruction of almost the entire lot. A work-
man on Dr. Purnell’s estate informed me that he had
seen eighteen prairie chickens in the cornfield, near the
house, in November of the present year. It was the
only covey left by the Jersey terrapin hunters, who
came up from Chincoteague Inlet. The same gentle-
man who sent these fine birds to Dr. Purnell is about
to send down from New Jersey the ruffed grouse,
called in that State and Pennsylvania, the pheasant. There are no ruffed grouse on the Peninsula.”
In Forest and Stream of Nov. 25, 1880, Mr. C. S. In Forest and Stream of Nov. 25, 1880, Mr. C. S. Wescott reported that a half-grown prairie chicken had
been brought to Mr. TRANSPLANTING AMERICAN GAME BIRDS. John Krider, of Philadelphia, for
mounting, the bird having been sent from lower Dela-
ware or Maryland with a lot of quail to a Philadelphia
game dealer. The bird was not preserved. Mr. Wes-
cott believed—and no doubt he was right about it—that
this was a descendant of the birds liberated in Mary- AMERICAN GAME BIRD SHOOTING 530 land about 1869 by Dr. Purnell. If this is true, it
would seem that these birds had survived and bred for
a period of ten years, indicating that the conditions
were then very favorable in that locality. About the year 1870 a number of birds are said to About the year 1870 a number of birds are said to
have been imported from the West and set free on
Long Island, where they did well, and reared broods,
but finally disappeared. Within two or three years
after that the late Shepard F. Knapp, of New York,
long a member of the Southside Club and an enthusi-
astic sportsman, told me of having seen on Long Island
two prairie chickens that had recently been killed some-
where near Islip. 1) 0. In the year 1874 a number of sportsmen strongly
urged the restocking of certain barren areas in New
Jersey with pinnated grouse, which had been extermi-
nated there not very long before. Their idea was to
import the birds from Illinois, where they were then
numerous. That same year there was talk and correspondence in That same year there was talk and correspondence in
England and in the United States as to the desirability
of importing the pinnated grouse to England and at-
tempting to establish them on the Scotch moors. The
price put upon the birds seems to have prevented any
action in this direction. Dealers asked £2 5s. per pair
for the birds, but later Mr. Chas. Hallock succeeded in
finding an animal dealer who agreed to furnish 100
pairs at £1 Ios. per pair. To-day many sportsmen
would be glad to close with an offer such as this. Mr. Brewster, in the work already quoted, tells of Mr. Brewster, in the work already quoted, tells of TRANSPLANTING AMERICAN GAME BIRDS 531 an importation of these birds turned out in Massachu-
setts. TRANSPLANTING AMERICAN GAME BIRDS. - He says:
arly spring of 1884 or 1885, six pairs of “Tn the early spring of 1884 or 1885, six pairs of
prairie hens brought from Iowa were liberated by Mr. Robert B. Nesbitt, of Cambridge, at various points
along Concord Avenue, between Belmont and Concord. He tells me that he was afterwards informed—on
somewhat questionable authority, however—that sev-
eral of these birds reared broods of young that season. I can vouch for the fact that a year or two later an adult
male spent most of the spring in a grain field near the
village of Carlisle, Massachusetts, where it was seen
by my friend, Mr. George H. Robbins, and several of
his neighbors. “Another bird of the same sex was met with by Mr. Walter Faxon in the Fresh Pine Swamps (on the Ar-
lington side of Little River) on May 14, 1892. The
latter instance may be taken to indicate that at least a
few of these grouse may have succeeded in maintain-
ing themselves for a number of years, but there are no
good reasons for believing that any of them are still
living or have left living descendants. In short, the at-
tempts to establish them permanently in the Cambridge
region, as well as in other certain parts of Massachu-
setts where they were liberated about the same time,
have evidently proved a complete failure.”
Efforts have been made to naturalize various Amer- Efforts have been made to naturalize various Amer-
ican game birds in England, but so far as known, with
no permanent success, except in the case of the turkey,
and this only as a domestic bird. In 1861 Mr. Grant- AMERICAN GAME BIRD SHOOTING 532 ley F. Berkeley,
a member of Parliament, wrote from
Winkton House, in Hampshire, to his friend, Capt. Geo. D. Bayard, saying: “My friend in America up to
this last week has been sending me over prairie grouse
and quail for naturalization for our society for that
purpose here, and I am charmed by being put in mind
of the plains, by having a male prairie grouse walking
about my garden, tamer than an English pheasant,
coming to my whistle for food and making the devil’s
own howls, with the skin blown out on either side of
his neck, strutting and running around and calling for
his mate, but I have no mate to give him. TRANSPLANTING AMERICAN GAME BIRDS. The four
prairie grouse that came over are all males. A friend
of mine, Lord Malmsbury, has imported some of your
wood grouse, and they are doing well. “T have sent out to different places a great number
of quails, some from Canada and some from New
York,’ More than twenty years later Mr. Frank Sturgis, of
Chicago, sent a large number of live pinnated grouse
to a friend in England, who acknowledged them in a
letter published in Forest and Stream in the following
letter from Neath, under date of March 22, 1883, as
follows: “T have unfortunately been upon the invalid list at
the seaside for a few days, or I would have written at
once to acknowledge Mr. Cheetham’s letter of the 3d
inst., and to thank you most heartily and most sincerely
for your princely present of prairie grouse. I am TRANSPLANTING AMERICAN GAME BIRDS 533 sure that I am quite right in stating you have altogether
excelled all previous attempts in this direction, and
the successful outcome of the shipment reflects the
greatest credit upon your excellent arrangements. The
birds being so rare, and the quantity so far in excess of
any private requirements, I hope you will be pleased
rather than offended at the distribution which has been
arranged. Acting in concert with Mr. Henry Nash,
who communicated with Mr. Lowell, we have pre-
sented twenty to her Majesty the Queen, for Balmoral,
and the Prince of Wales has been graciously pleased
to accept twenty for Sandringham. We have sent ten
to the Zodlogical Gardens in Regent’s Park, where they
are very much prized and valued, and we have turned
out sixteen to take their chances upon our Welsh
hills—jointly upon Lord Jersey’s property and upon
shooting land which is leased by the writer. Such of
these last birds as have since been seen were all doing
well, and I have a report to-day that those which were
sent to the Zoological Gardens are also well and getting
less shy than when first turned out. The birds which
were sent to the Zoological Gardens are the only ones
now in confinement, and it is understood that the so-
ciety will reserve half of any young birds which may
result in case those which are turned out should not in-
crease their numbers. I shall hope to report further
satisfactory progress very shortly. “PHILIP W. What became of these birds we do not know; that a TRANSPLANTING AMERICAN GAME BIRDS. FLOWER.” “PHILIP W. FLOWER.” What became of these birds we do not know; that a AMERICAN GAME BIRD SHOOTING 534 large number of them must have reached their destina-
tion in good condition seems obvious, since no less
than sixty-six are accounted for in the letter. Bobwhite was introduced into England in the earlier
half of the last century, and is reported to have done
well for a time. No serious attempt has been made to transplant No serious attempt has been made to transplant
from one part of America to another any others of the
grouse, except that one or more consignments of sharp-
tailed grouse are said to have been shipped to Massa-
chusetts, where they were turned out and disappeared. Sharp-tailed grouse were planted also on Grand Island
in Lake Superior, but this is in or close to the native
range of the species. What the fate of these Grand
Island sharp-tail grouse may have been we do not
know. Possibly they shared the fate of the exotic
grouse there introduced, all of which, we are told, have
disappeared. We read in the accounts of the early Spanish ex-
plorers that the wild turkey had been domesticated by
the Indians before the white man’s coming. A consid-
erable portion of the sustenance of certain Florida In-
dians was drawn from their turkeys, and we are told
that the carnivorous animals in the great zoological
gardens kept up by the Aztec, Montezuma, were fed on
the flesh of turkeys. The domestic turkey is a descendant of the wild The domestic turkey is a descendant of the wild
turkey of the Southwest. The bird was carried to
Spain and thence spread over Europe, finally reaching
England. That the common wild turkey of the East TRANSPLANTING AMERICAN GAME BIRDS 535 may also be domesticated has been shown by experi-
ments carried on, on a plantation near Farmville, Va.,
by Prof. Robert Lee Blanton, an account of which was
published in Forest and Stream, vol. 74, p. 450. When, if ever, game refuges shall be established in When, if ever, game refuges shall be established in
the various States—game refuges properly protected,
be it understood—they should be stocked with wild
turkeys. DESTRUCTION: AND PROTECTION. Where game birds are left to look out for themselves,
civilized man and the conditions he brings with him
constitute their most destructive enemy. It is not so
much the number of birds killed by man as the changes
which he works in the land that affect the birds and
often make life impossible for them. Of these changes,
the most important is the clearing up and cultivation of
woodland—the cutting down of forests and the drain-
ing of swamps. Most wild creatures require cover to
afford them shelter from their enemies, while the far-
mer wishes the ground to be free from all cover
except that which his crops afford, and these crops are
standing for a few months only. Without cover, the
quail, the wild turkey, the grouse and the woodcock
cannot exist. Wild birds depend for safety largely on
their protective coloring, which makes it easy for them
to escape notice in their chosen haunts, and on their
habit of flying—on the approach of danger—to refuges
where they may hide. But if forest and brush are cut
away the partridge and the quail have no places in
which to seek safety ; if the tall grass of prairie sloughs
and ravines is mowed and the soil ploughed up, the
prairie chicken is without place of concealment; if the
swamp is drained and the alders, birches and spice-
536 536 ‘aspoH
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y DESTRUCTION AND PROTECTION 537 wood that grow in it are cut down, the woodcock no
longer has a home. While all these changes are going on, gunners are While all these changes are going on, gunners are
becoming more numerous. With the settling up of the
country there is an increasing demand for the flesh of
game,
and, between
the attacks
of man,
the de-
struction of their old homes and the ravages of pred-
atory birds and mammals, in many regions the game
birds have been almost exterminated. Among their natural enemies are all the carnivorous
mammals, many of the hawks and owls, and not a few
domestic animals. DESTRUCTION: AND PROTECTION. In the old times, when birds were
less pursued by man and had a range far wider than at
present, their annual increase in numbers more than
made up the loss from the attacks of natural enemies,
but over much of the territory of the United States
that time has long passed. In thickly settled countries, the d In thickly settled countries, the domestic dog, self-
hunting through the spring and summer, destroys great
numbers of the nests and eggs of song and game birds,
while the house cat, the pet of the kitchen or the house
cat run wild, is very destructive. The harm done by
the farmer’s dog in his travels through the fields is not
generally understood. Two or three dogs from neigh-
boring farms may start out in company and be gone all
day—perhaps for several days—hunting. Among the
prey that they capture may, perhaps, be a woodchuck,
the remains of which the farmer finds later and speaks
of with pride as having been killed by his dogs. He
does not know that while hunting the dogs may have AMERICAN GAME BIRD SHOOTING 533 destroyed dozens of the nests of game birds and song-
birds, which are the farmer’s best friends, since they
are ever-working unpaid police officers, destroying all
through the summer days the insect enemies which
prey upon his crops. If the work of these insect-eating
birds were to be stopped for a week, or for a month,
the damage done to the crops of the United States
would be incalculably great. In certain sections of the South an extremely de-
structive enemy of ground-nesting birds of all sorts is
the wild hog, which roams the forest, literally seeking
what he may devour. The number of nests and eggs
of turkeys, ruffled grouse and quail that these animals
search out and destroy is very great. Some States have
laws providing that hogs shall not be allowed to run
at large, but such States are exceptions. On the other
hand, it must be said that in States so thinly settled
that hogs and cattle are permitted to run wild, there
are comparatively few dogs and cats that roam the
fields and woods. Mr. DESTRUCTION: AND PROTECTION. Herbert Brown reported a few years ago that
previous to the introduction of ranch cattle the masked
quail was quite common in southern Arizona, but that
the cattle eating off and trampling down the tall grass
had so destroyed the breeding and hiding places of this
bird that it had practically disappeared from the United
States side of the line, and at latest reports this was
still the condition, as is seen by what he says on page
64. Under such adverse conditions, it is not strange
that our stock of splendid game birds grows smaller Winter quarters of Dr. Hodge’s Quail. Photographed by C. F. Hodge. Winter quarters of Dr. Hodge’s Quail. Photographed by C. F. Hodge. DESTRUCTION AND PROTECTION 539 year by year. The problem as to what shall be done to
keep this stock from wholly disappearing gives cause
for much anxiety. For American sportsmen, American game birds are
the best, and there is no reason why we should not have
an abundance of these, provided only for a short time
we practice a little self-control. The wild turkey was
formerly found over the whole United States, east of
the Missouri River, south of the Niobrara and east of
the Rocky Mountains. Grouse of one sort and another
formerly abounded over almost the whole United States
and Canada, while in the southern portions of the
United States there were great numbers of quail. These birds sportsmen may bring back, in large meas-
ure, if they care to; but they cannot do this by killing
them at every opportunity. All these birds are prolific
and probably all can be hand-reared. If this can be
done, they may be turned out in numbers on game
refuges—if these shall be established—and if absolutely
protected there, will soon spread themselves over the
surrounding country. They must be protected, how-
ever, alike against the gunner and against the attacks
of their natural enemies. It is not yet fu It is not yet fully known what might be done with
our grouse and quail, but the experiments of Prof. C. F. Hodge, of Worcester, with the ruffed grouse and
bobwhite, justify the belief that all grouse may be
reared in confinement. A short period of experimen-
tation by men interested in the subject will probably
result in the semi-domestication of practically all the AMERICAN GAME BIRD SHOOTING 540 American grouse. DESTRUCTION: AND PROTECTION. These birds, so reared in their
natural ranges and then set free in refuges, would re-
stock covers now barren. The process of restocking
could be kept up indefinitely, and after a time at a very
modest cost. If one-half as much time and money
had been expended in attempts to domesticate or hand-
rear some of our native species as has been devoted to
the importation and rearing of exotic forms, the prob-
lem of our game supply would now be much simpler
than it is. With the Limicoline birds the case is somewhat dif-
ferent. No experience has been had with them, and
nothing is known about confining them or inducing
them to breed in confinement. On the other hand, as
migratory birds they are exposed to the gunner only
for a short time, and then pass on to sections less
thickly settled, and where, therefore, the dangers are
less. In the United States most of the efforts to rear game
birds have been devoted to foreign species, but to this
rule the State of Massachusetts is a notable exception. On its public game farm it has for several years con-
sistently tried to rear quail and ruffed grouse, and
though not altogether successful, much experience has
been gained. It was left to another Massachusetts man, Dr. Clif-
ton F. Hodge, working from pure love of his task, to
accomplish the greatest results. The work done by the Massachusetts Commissioners The work done by the Massachusetts Commissioners
of Fisheries and Game, and by Dr. Hodge, is set forth ‘aspoH Oa ES) Aq poydeis0joyud
*peyozey ysnf syoryo ssnoig poyny *peyozey ysnf syoryo ssnoig poyny DESTRUCTION AND PROTECTION 541 at some length in the Reports of the Massachusetts
Commissioners for the years 1907 and 1908. In 1907 the efforts of the Massachusetts Commis- In 1907 the efforts of the Massachusetts Commis-
sioners to rear ruffed grouse in confinement resulted in
little more than the acquisition of some experience as
to what not to do. It was learned, for one thing, that
the males are extremely disposed to fight. During the
winter, in a pen of three young birds one male killed
another with which it had grown up. Even during
the mating season there was need of great care lest
the male kill the female. DESTRUCTION: AND PROTECTION. His attacks on the females
caused them great alarm, since it was impossible for
them to escape from the cage, and they hurt them-
selves in their efforts to do so. In fact it was not
safe to put the male in the coop for mating without
closely watching him, and it was necessary to remove
him almost at once. The birds in the coops of the commission soon be-
came noticeably unafraid. They paid little attention
to what was going on outside, and did not display the
timidity and restlessness of the bobwhite. It was ob-
served that they ate with appetite and appeared to
enjoy their food. Nevertheless, the male grouse died
late in the summer, evidently from an infectious dis-
ease communicated to him probably through infected
ground. No one has approached the success which has at-
tended the work of Prof. C. F. Hodge, of Clark Uni-
versity, who for some years, as a rest and recreation
from his ordinary work, has been trying to rear ruffed AMERICAN GAME BIRD SHOOTING 542 grouse and quail in domestication. His experience is
extraordinarily suggestive, and we quote much of his
report
to the Massachusetts Commissioners. His
permits for the year allowed him to take 80 eggs of
the ruffed grouse and to keep three pairs of bobwhite
quail
for purposes
of propagation. He had
54
grouse eggs, from which 38 chicks were hatched, and
of these he says: “As will be seen, the eggs did not hatch as well
as in the previous years of experiment, possibly due
to cold weather in May. The eggs were all hatched
under Cochin bantam hens, as usual. Chicks are ac-
counted for as follows: “Fifteen chicks, June 6th, left with hen mother,
began dropping off after the first two weeks, and were
all dead by August 15th. “Fifteen chicks, June 6th, transferred on morning of “Fifteen chicks, June 6th, transferred on morning of
hatching to brooder; 2 escaped,
1 died, probably as
result of accidental injury, 2 died from swallowing
objects too large to pass into the gizzard (black cricket
and large spider), 10 are healthy, well-grown birds at
present. “Eight chicks, June roth, transferred to brooder, and
all did well, until, on sudden change of weather, tem-
perature ran too high and killed them all. DESTRUCTION: AND PROTECTION. “The experiment was conducted this season on my “The experiment was conducted this season on my
new place, on uncontaminated ground, and I thought
it desirable to try once more the simpler method of
rearing with the hen. The result is decisive against
this method. The fact that the hen carries parasites, DESTRUCTION AND PROTECTION 543 which are likely to prove fatal to young grouse, prob-
ably explains why our native species have not long
since been domesticated. All former
attempts
to
domesticate them have been made in the poultry yard
and its environment. The same is now being proved
to explain why the turkey, another native American
species, cannot be reared on ground contaminated by
domestic fowls. This is the fifth year in which the
experiment of rearing ruffed grouse with bantam hens
has been tried, and although the second year 6 birds
were reared out of a clutch of 12, for all the other
years fatalities by this method have amounted to 100
per cent. This season is particularly decisive because
the work was done on new ground under most favor-
able conditions, with the benefit of past experience, and
in clear competition with the brooder method.”
On the other hand his experience led him to believe On the other hand his experience led him to believe
that except
for accidents,
practically
every
ruffed
grouse chick hatched may be reared to maturity by the
brooder method. Brooders connected with yards in
which the chicks may run during warm days offer a
method which is not expensive. The young develop
and feather out rapidly, and except for the two first
weeks need no artificial heat, except during cold storms. Professor Hodge suggests the possibility of rearing Professor Hodge suggests the possibility of rearing
a strain of hens to be hatched in incubators and reared
on uncontaminated ground which possibly might be
safe for mothers and brooders. Professor Hodge found the bobwhite quail much Professor Hodge found the bobwhite quail much
easier to handle than the grouse, and experimented AMERICAN GAME BIRD SHOOTING 544 with a single pair kept as pets in the house. The hen
laid 18 eggs in a nest made by the pair in a window
cage, but since the birds did not seem inclined to in-
cubate, the eggs were placed under a bantam hen. DESTRUCTION: AND PROTECTION. Of
the 13 eggs first laid 6 hatched, but the chicks being
left with the hen all died within ten days. Of the
remaining 5, 3 hatched under a second hen and to
these 5 eggs were added 11 obtained from the State
Hatchery. Ten chicks were obtained from the 16
eggs. Of these, 3 died by accident and showed ne
trace of the disease of liver and ceca which has proved
fatal to so many of our young game birds as it has to
turkeys. The remaining 7 birds did well from Sep-
tember to the date of this report, November, 1907. Eleven quail chicks just out of the incubator were
obtained August 19th from the State Farm, were
reared in a brooder and to date are alive and well. One of the great difficulties attending these efforts One of the great difficulties attending these efforts
to rear native game birds has been the persistent attacks
of vermin, and this Professor Hodge was able to wholly
guard against during the summer of 1907. Rats were
troublesome, a skunk killed a hen quail, and a multi-
tude of skunks were about. Traps were kept con-
stantly set and the results were surprising—for it must
be remembered that Professor Hodge lives in the large
city of Worcester. These traps caught 17 skunks and
a great number of rats, and all these before any dam-
age had been done by the animals. Professor Hodge
is carrying on
this interesting experimental work
purely as a labor of love, though in 1909 a grant of ‘aspoy “yO Aq paydeisojoyd
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Ty ‘aspoy “yO Aq paydeisojoyd
“SHOTYO ayIYMQGog suIpsagq DESTRUCTION AND PROTECTION 545 $500 from the Carnegie Institute paid a portion of the
expenses. The The work of the Massachusetts State Farm started
in the year of 1906 with a flock of breeding quail
numbering 47. At the close of the season a partial
count indicated 30, and at the beginning of winter 21
were left, while at the opening of the breeding season
of 1907 there were but 12, or only about 25 per cent. of
the original number. A nu A number of chicks were hatched in incubators, and
this method seemed to Arthur Merrill, the superin-
tendent, more successful than the use of hens. DESTRUCTION: AND PROTECTION. The
loss from dead embryos was less than the usual break-
age of eggs, and the chicks were stronger. The in-
cubators were run in the same manner as for pheasants. The young quail were fed o pheasants. The young quail were fed on dried ants’ eggs and
maggots, live ants’ eges and insects being added when
practicable, with fruit, seeds and grain as the bird
grew older. Insect food is unquestionably most de-
sirable for young birds, but it is difficult to obtain. Berries, grasses and clover, or indeed almost any green
food, are essential, but difficult. to secure. On the
whole, up to the close of the year 1907 the experi-
menters had learned chiefly what was to be avoided,
and not what was to be done. In his report to the Commi In his report to the Commissioners on Fisheries and
Game of Massachusetts for the year ending Decem-
ber 31, 1908, Professor Hodge gives at length his
experience for the previous season, writing under date
of November 27th. Little could be done toward experi- AMERICAN GAME BIRD SHOOTING 546 menting with ruffed grouse, yet one clutch of 12 eggs
was secured,
10 of these hatched and 3 reared to
maturity. In the loss of 7 of these chicks a very im-
portant discovery was made. Insect food being essen-
tial to the young birds, a large number of striped plant
bugs were obtained by sweeping the grass where they
were abundant with insect nets. The young chicks
ate them eagerly, and went to sleep and died as if they
had been chloroformed. Professor Hodge points out
that these bugs give out a strong odor similar to that
of the squash bug, and Conradi found that when squash
bugs were fed to toads, these died as though poisoned
with chloroform. While Conradi found that 5 or 6
squash bugs might be sufficient to kill a toad, Miss
Morse has fed as many as II to bobwhite at a single
meal with no apparent ill effect. Toads ate the plant
bugs in large quantities without injury, but it is ap-
parent that strong-smelling bugs should not be fed to
young ruffed grouse. The experiments with bobwhite proved very success- The experiments with bobwhite proved very success-
ful. The season began with 5 pairs and several extra
cocks. DESTRUCTION: AND PROTECTION. The first egg was laid by May 6th, and 4
of the hens had begun laying by May roth. Not less
than 247 eggs were laid, an average of 49 to the
pair, but Mr. Merrill, of the State Game Farm, had
a hen quail which laid 100 eggs. Two of Professor
Hodge's hens died during the summer, but the 3 that
survived laid an average of 65 eggs each. In all 114
chicks were hatched and 75 reared. A most excellent
showing, but one that will unquestionably be greatly Hodge. C.F. ite reared by Dr. Bobwh Hodge. C.F. ite reared by Dr. Bobwh DESTRUCTION AND PROTECTION 547 improved on as time goes on. Professor Hodge’s letter
on his experiments shows extraordinary progress made
in learning of the lives of these birds,
There is a wide range of There is a wide range of difference in the actions of
different pairs of bobwhites, especially in respect to
breeding and care of young. One pair which occupied
a large cage with the ruffed grouse got along with
the larger birds without any trouble; the hen laid 58
eggs, making and filling several nests. On August
20th the cock began brooding a nest with 16 eggs, and
on September 13th hatched
15 chicks. Both birds
joined in the care of the brood. Another pair had
occupied a cage 6 by 12 feet and produced in all 69
eggs, but neither bird showed any signs of brood-
ing. The cock, however, took charge of and reared
a brood of chicks, hatched under a bantam, but while
he was doing this the hen quail refused to associate
with him. A pair was kept in a cage 3 by 6 feet and
produced 68 eggs, all laid in a single nest. The eggs
were taken away from time to time, because neither
bird showed signs of brooding. Nevertheless, on the
9 eggs last laid, the hen began to sit September 12th
and brought out 9 chicks October 6th. The cock was
attentive to his mate on the nest, but was seen sitting
on it only once for a short time, while the hen was
feeding. Both birds joined in the care of the young. DESTRUCTION: AND PROTECTION. Of the two unmated cocks one adopted and took care of
a brood of chicks hatched under a hen, while the other
refused to care for the chicks, pecked them savagely
and would probably have killed any that had been left AMERICAN GAME BIRD SHOOTING 548 in his cage. It is evident, therefore, that a cage 3 by 6
feet is not too small for quail to do well in; a fact
of interest, for it renders the work of experimenting
in this matter practicable for almost anyone. Some temperatures of these birds were taken, and Some temperatures of these birds were taken, and
it was found that the interior body temperature of a
brooding male bobwhite was no less than 111 degrees—
remarkably high. The brooding quail gives the eggs
rather long periods of cooling once or twice a day. The records thus seem to suggest for incubators a
temperature one or two degrees higher for quail than
for domestic fowls. It has been noted also that the
chicks of the ruffed grouse and the quail seem to thrive
much better when the brooders are kept much warmer
than is advised in the directions for domestic chickens. This is necessary, apparently, only for the first ten
days or two weeks, but for that time it seems well
to keep the temperature five degrees higher than re-
quired for domestic fowl. The conclusion thus reached
was confirmed by an experience in September when, of
68 chicks in brooders, 24 died in two days. Professor
Hodge’s explanation
of the ‘“brooder
troubles,” which have caused the death of so many
apparently healthy birds, seems wholly plausible. He
regards these troubles as acute indigestion and sug-
gests aremedy. He gives elaborate directions for feed-
ing, lays an especial stress on lining brooders, which
should have no chinks or cracks into which one of these
tiny birds may creep, on keeping them free from in-
fection from domestic fowls, and on giving the birds DESTRUCTION AND PROTECTION 549 sufficient water and a warm dust bath. These two
things, he believes, attach the birds strongly to a hom-
ing place. His birds were at liberty during the day. He says: “The little fellows have an affinity for brush patches,
corn, asparagus beds, or any sort of natural cover,
like that of ducks for water. DESTRUCTION: AND PROTECTION. Instead of proving the
nuisance that I at first feared, this affinity may be
turned to excellent account if properly indulged and
arranged for. I always keep a little brush-pile, with an
armful of hay or freshly cut weeds thrown over it,
in the home cage. This also attracts them to the
place. If they have located in a clump of bushes or
a weed patch, they are safe for the day. Should a
thunder storm come up, and the birds are as tame as
they should be, they can be whistled home, or an insect
net be put over the entire cluster and they can be car-
ried into the brooder. When they began to fly in the
second week, I expected to be obliged to clip their
wings. I did not do this, however, and, as the sequel
proved, their ability to fly is a great advantage in
rearing them. Within the past few days a wind storm
opened one of my cages containing adult and young
birds. Though able to fly for miles, I drove them
back into the cage as easily as if they had been a flock
of little chickens. During the summer
the flocks
would range out afoot to feed and fly home. In feed-
ing the birds I used from the first
a low whistle, in
imitation of the feeding note of the chicks themselves. On coming home at noon or night the flock might be AMERICAN GAME BIRD SHOOTING 550 nowhere in sight, but on giving the whistle I would
be assured by a chorus of eager replies, and in a
moment the air would be full of whirring wings, as the
flock flew in with a rush and lighted at the entrance of
the cage. This might be repeated as many as a dozen
times a day. “Wintering the bobwhite offers no difficulty. I set “Wintering the bobwhite offers no difficulty. I set
the cages with the shelter end open to the south, build
a brush wood-pile in the middle of the cage, see that
they are supplied with weed seed, grain mixture and
water when snow is lacking, and keep a cabbage, a
mangel wurzel or an apple where they can pick at it. DESTRUCTION: AND PROTECTION. It might be well to see that the cluster is not imprisoned
under the ice after a sleet storm, but the brush-pile
has afforded insurance against this so far. This brush
wood-pile is made by placing a few stout branches
on the ground in a sheltered sunny exposure, and on
these pile about two feet of weeds, cut before the seeds
fall—ragweed, lamb’s quarter, pigweed, smart-weed,
wild buckwheat (chaff straw or loft sweepings would
do if weeds are not at hand) ; then pile on a foot or two
of stout brush which cannot be crushed down by heavy
snows, and on top of this place a good thick covering
of weeds. This will give the flock scratching mate-
rial all winter, afford shelter from cold and storms, and
protect from vermin, especially hawks, owls and cats. For bobwhite in the open I think this simple winter
provision would insure against winter killing and ex-
tend the range of the species at least several hundred
miles to the north. The great value of the bird in Ruffed Grouse feeding from hand. Photographed by C. F. Hodge. Ruffed Grouse feeding from hand. Photographed by C. F. Hodge. DESTRUCTION AND PROTECTION 551 insect and weed seed destruction will amply ena farm-
ers for their work. “As spring comes on the cocks fight a good deal;
but still breeders generally advise keeping several
pairs in the same cage. I tried both methods this
season, keeping four pairs in the same cage (6 by 12
feet) through May and June. They laid well under
these conditions, but there was so much disturbance
and persecution that there was little hope of either
hens or cocks beginning to brood. Each pair was then
given a separate cage, and they did so much better
that this plan will be generally adopted in future.” Professor Hodge gives various suggestions replying
to many inquiries that have come to him, as to practical
methods of rearing bobwhite. What he has said, how-
ever, applies equally to the ruffed grouse and prob-
ably to the prairie chicken as well. The results which
he has obtained in six years of experiment will appear
to most people very gratifying and very encouraging
to further experimentation in this field. DESTRUCTION: AND PROTECTION. He very truly
says: “We now see clearly that we must bid farewell to
all our native game birds or work out methods of
propagating them.”
Besides the birds in question Professor Hodge ex- Besides the birds in question Professor Hodge ex-
perimented with a pair of sharp-tail grouse from the
Saskatchewan. The birds did very well and Professor
Hodge discovered that they were exceedingly fond of
clover. No doubt if he had given them alfalfa they
would have eaten it with still greater relish, since, in AMERICAN GAME BIRD SHOOTING 552 their natural range, whenever the alfalfa is green, these
birds resort to it, and for a part of the year it seems to
constitute one of their chief foods. The hen prairie chicken laid ten eggs and then died, The hen prairie chicken laid ten eggs and then died,
showing intense infection with coccidia, which appears
to be the bane of so many of our native birds, including
the domestic turkey. Five of the eggs were hatched
under a bantam hen, but all died in Professor Hodge’s
absence from home. He was promised eggs or breed-
ing stock in prairie chickens for the following year,
and hoped to experiment with them. In 1909, however, circumstances prevented his work
on the problem. He reared three ruffed grouse and
fourteen quail, and in 1910, owing to the pressure of
affairs he turned over his breeding stock to the Massa-
chusetts Commission. While Professor Hodge was doing this work at
Worcester, the superintendent of the Sutton Hatchery,
the Massachusetts Game Farm, was studying the same
problems. The results obtained were not great in the
number of birds, but much was learned as to the most
serious disease likely to be met with, and the adapt-
ability of quail and grouse to domestication. It was
found that disease rather than the method of feeding
is the controlling factor in rearing the young. One
hundred and twenty-two ruffed grouse were hatched,
but all except 2 birds were lost, though 11 lived to
the age of seven weeks. Many of the deaths were due
to coccidium, and others to what is called by the writer
“brooder pneumonia,” and the inference drawn from DESTRUCTION AND PROTECTION 553 the report is that. DESTRUCTION: AND PROTECTION. many of these young birds received
infection either from the hens which hatched them, or
from the ground on which they were reared, this
ground being a place where poultry had been kept. English pheasants appeared to be more resistant to dis-
ease than the grouse and quail. During this season most of the eggs laid by the quail During this season most of the eggs laid by the quail
were well fertilized, and no quail failed to lay. Four
hundred and seventy eggs were laid and 222 hatched. There seemed no advantage in penning the birds with
a greater number of females than males, and during
the season of 1907, when the birds were paired, results
were much more favorable than before. In a number
of cases the males were active in caring for the chicks,
and in one case a male adopted and reared a clutch of
young ones. While in earlier attempts at hatching,
much loss was experienced from breakage of eggs by
the bantam hens, and while the next year incubators
were used, hatching 80 per cent. of the eggs, during
the season
of 1908 incubators were unsatisfactory,
while hens did much better than before, the greater
number breaking no eggs at all. In other words, the
result of each season shows the importance of more
and more experiments on the part of those who have
these matters in charge, and as they acquire this experi-
ence their success will, of course, be greater. The food difficulty remains, but in this matter much The food difficulty remains, but in this matter much
also is being learned. Like Professor Hodge, Mr. Merrill concludes that Like Professor Hodge, Mr. Merrill concludes that
there appears to be no advantage of large pens over AMERICAN GAME BIRD SHOOTING 554 small ones. To be sure, the birds found some insect
food, but this was soon exhausted. Small pens have
given entire satisfaction for wintering quail and grouse,
the only difficulty being wet and dampness when snow
melts. The pen should be kept entirely dry, and should
be open to the fresh air. A better comprehension of the mating and nesting A better comprehension of the mating and nesting
processes of the quail and ruffed grouse is very nec-
essary. The breeding of the grouse is complicated by
the uncertain and quarrelsome temper of the male. DESTRUCTION: AND PROTECTION. The
problem of hatching, like that of food, requires further
study. Meantime, of course, the work of bacterio-
logical investigation must be kept up, for one of the
first problems in rearing these birds is to understand
the diseases to which they are subject under these new
and unnatural conditions. The success of the Sutton Farm with pheasants has The success of the Sutton Farm with pheasants has
been better than with our native game, but even so has
not been at all satisfactory. CONCLUSION. In view of all our experience it is clear that Amer-
ican sportsmen cannot continue to kill off their game
and still to have their covers full of birds. I believe
that a time will come when there will be good shooting
in many parts of the country, but this will not be until
gunners have been taught the lesson of self-control. Methods must be devised for restocking our fields and
woods, and the birds put out must have an opportunity
to live and to reproduce their kind. This means better
enforcement of law, shorter open-seasons and a bag
limit. As conditions precedent to good shooting, must
come also game refuges and the private preserve. If
sportsmen long ago had been wise enough to demand
from the legislatures the establishment of game refuges,
we should now have public preserves which might have
been large enough for anyone. Instead of demanding
this, gunners often grumbled bitterly when land own-
ers posted their fields and declined to allow everyone
to run over their land, tear down their fences and
shoot their domestic fowls, and even their animals. The private preserve has come to stay, and it is a
good thing for gunners at large that it has come. What the general public should now demand is game
refuges—public preserves—where the birds may in-
crease and from which they will certainly scatter out 550 AMERICAN GAME BIRD SHOOTING 550 for the benefit of the general public. By the time such
refuges have been generally established the problem of
rearing our game birds will probably have been solved,
and from that time on there will be little difficulty in
our finding each autumn a few birds on which to work
our dogs and exercise our skill in shooting. Only a few years ago the shooting seasons com-
monly began on September
Ist and closed Janu-
ary Ist. Now in most States—above all in those States
where birds are least abundant—the shooting season
lasts for two months only and often closes Decem-
ber 1st. Almost without our noticing it the length
of the season has been reduced one-half. In many
States bag limits have been established, and while too
often these limits are disregarded, the sentiment that
this
law
should
be obyed
is constantly
growing
stronger. CONCLUSION. Everywhere
sportsmen
might
accomplish
much
more toward increasing the supply of game if they
were organized and would work together for a common
object. In some States organizations have been ef-
fected, but sufficient interest has not yet been aroused
to make them ready to work together for the common
purpose. Sometitnes, too, the organization is brought
together more for the purpose of making a showing
or redounding to the credit of some one individual
than
it is for actual fish and game protective work,
and when this takes place, the organization becomes
a mere name, wholly inefficient for practical purposes. The State of Connecticut possesses an organization CONCLUSION Bez modeled on right lines. There are town gun clubs,
which are members of the County Game Protective As-
sociation, and these County Game Associations choose
delegates who are members of the executive council
of the central State Game Protective body. The town
eun clubs are active and especially interested in their
own especial territory. The occasional meetings held
where all the towns of the country are represented,
keep up this interest, and when the county delegates
attend the meetings of the State Association they are
able to report intelligently on what is taking place and
on what should be done. If every State in the Union
had such an organization, and if every State from time
to time chose delegates to a central association which
represented the English-speaking race on the conti-
nent, matters would go forward much more rapidly,
and much more would be accomplished than at present. Another thing that works against the sportsmen is Another thing that works against the sportsmen is
that they have no method of communication with each
other. The means are at their hand in the various
sportsmen’s journals, but only a small proportion of the
great number of gunners in thelandread
these journals. If they were read by most sportsmen, who thus kept
themselves informed as to what legislation is on foot
and what other sportsmen are doing, intelligent action
could be taken by a large body of men on the measures
which should be taken to increase our stock of game. Sportsmen would then be acting together as one body,
and many of the problems which now trouble us would
have been solved. CONCLUSION. 558 AMERICAN GAME BIRD SHOOTING It is worth the while of all who are actively inter-
ested in having our game supply increased to keep
themselves informed of the legislation that is taking
place, of the progress that is being made in the artificial
rearing of fish and game, and above all of the views and
opinions of other sportsmen. Unless those interested
in this subject are willing to work together and to
bring their united intelligence and influence to bear
on the public, the reforms will come only slowly. The objects which sportsmen must work for are,
the enforcement of the laws that we already have, and
the passage and enforcement of better laws. Among the better laws needed are, first, statutes
authorizing the establishment of game refuges; sec-
ond, the prohibition of the sale of game; third, short
shooting seasons and a reasonable bag limit. Many of the States now insist on a payment by gun- Many of the States now insist on a payment by gun-
ners of a shooting license fee, and in many States the
aggregate of these fees amounts to a great sum. It
is proper that a large share of this money should be
expended for the benefit of those who pay the tax, and
a proportion of that expenditure should be for intel-
ligent experimentation in rearing our native gamebirds. During the past twenty years great progress has During the past twenty years great progress has
been made in game protection. To-day more interest
is felt in this subject than ever before, and more money
is available for it. If sportsmen will work together
to a common end, the next ten or fifteen years should
see in many parts of the United States better shooting
than has been had there for a generation.
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Electroluminescent Organic and Quantum Dot LEDs: The State of the Art
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Journal of display technology
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I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS T
HE development of lighting solutions over the last 150
years has been driven by number of factors, namely lower
cost, increased brightness, longer lifetime, and more efficient
devices that use more environmentally-sustainable materials. T The tungsten light bulb was a revolutionary step up from
candles and oil lamps. Owing to its effectiveness at filling
rooms with white light at the flick of a switch, it quickly became
the lighting solution of choice and became a fixture in almost
all buildings with a source of electricity. But the tungsten lamp
was not perfect; grossly inefficient it created significant excess
heat. Nonetheless, its easy manufacturability and low cost
meant it would be some time until its use began to be rivalled by
the much more efficient compact fluorescent light bulb (CFL)
(50–60 lm/W cf. 8–10 lm/W) that was introduced in the 1970s. Besides using less energy, CFLs also lasted much longer:
in the region of 5000 hours cf. 800 hours. They have since
become ubiquitous in households and commercial buildings as
governments around the world introduced phase-out programs
of traditional (so-called incandescent) bulbs in favor of newer
generations of energy-efficient lighting. See Fig. 1. LED light bulbs make use of the principle of electrolumines-
cence (EL), although it is first appropriate to consider the me-
chanics of photoluminescence (PL) . PL is the term given to
describe the radiative decay process in a material; it is a more
general term than fluorescence or phosphorescence, which de-
scribe emission from specific states: either excited singlet or
triplet states, respectively. For organic molecules, fluorescence
usually occurs from the lowest vibrational level of the excited
singlet state. It is a so-called spin-allowed transition, so it oc-
curs quickly, typically on the nanosecond time scale. Phospho-
rescence, in contrast, occurs after intersystem crossing from an
excited singlet state to an excited triplet state; and as this process
is spin forbidden, it typically occurs over a longer period—in
the microsecond to second time frame. The process is gener-
ally described figuratively using a Jablonski diagram that shows
the absorption and deactivation processes of organic molecules
(Fig. 2). Many heavy metals, such as transition metals (e.g., Ir,
Pt) and lanthanide/rare earths are “phosphorescent” owing to
spin-orbit coupling, which weakens the rules around spin al-
lowed and spin forbidden transitions. As can be seen in Fig. 480 480 JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 Manuscript received January 08, 2015; revised March 12, 2015; accepted
March 19, 2015. Date of publication March 31, 2015; date of current version
May 04, 2015. 1Minamata Convention on Mercury. [Online] Available: http://www.mer-
curyconvention.org Poopathy Kathirgamanathan, Lisa M. Bushby, Muttulingam Kumaraverl, Seenivasagam Ravichandran, and
Sivagnanasundram Surendrakumar Poopathy Kathirgamanathan, Lisa M. Bushby, Muttulingam Kumaraverl, Seenivasa
Sivagnanasundram Surendrakumar Poopathy Kathirgamanathan, Lisa M. Bushby, Muttulingam Kumaraverl, Seenivasagam Ravichandran, and
Sivagnanasundram Surendrakumar Fig. 1. Historic development of more efficient lighting solutions. Abstract—A detailed background into the reasons for the
feverish interest and growing impact on commercial market share
of organic light-emitting diode (OLED) technology for lighting
and display applications is given. Following this, the increasing
discussion over whether electroluminescent devices based on
quantum dots will ever challenge that of organics in displays is
considered. The unique physics and chemistry of quantum dots
results in narrow emission bands and increased stability over
organic dyes meaning the potential for this is significant. The
current best efficiencies of quantum dot devices and associated
device structures from the literature are presented followed by
a comparison of devices fabricated with organic materials, in
particular those of polymers, metal complexes (fluorescent and
phosphorescent) and small molecules. Fig. 1. Historic development of more efficient lighting solutions. Although more energy efficient, the CFL bulb has its own
environmental concerns, containing as it does the hazardous
substance mercury, which means a number of controls must
be instituted during manufacture and disposal to prevent re-
lease into the environment and to protect human health. Parties
and signatories to the UN's Minamata Convention on Mercury1
have agreed to ban the production, export, and import of cer-
tain types of CFL by 2020 owing to their mercury content. As
a result, the non-mercury-containing third generation of light
bulb, the LED, which also provides further efficiency improve-
ments (60–80 lm/W) over the CFL bulb has taken over signifi-
cant swathes of market share and is continuing to do so. Index
Terms—Inorganic
light-emitting
diodes,
organic
light-emitting diodes (OLEDs), quantum dots. The authors are with the Institute of Materials and Manufacturing, College
of Engineering, Design and Physical Sciences, Wolfson Centre for Materials
Processing, Brunel University London, Uxbridge, UB8 3PH, U.K. (e-mail:
p.kathir@brunel.ac.uk). This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecommons.org/licenses/by/3.0/ This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecom I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS 2, the energy associated with emission
is less than that of absorption (excitation) meaning that emitted
photons have less energy than those that are absorbed and so Color versions of one or more of the figures are available online at http://
ieeexplore.ieee.org. Digital Object Identifier 10.1109/JDT.2015.2418279 Digital Object Identifier 10.1109/JDT.2015.2418279 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 481 Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro-
cesses of organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 5. Mechanics of electroluminescence in organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro-
cesses of organic molecules. Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro-
cesses of organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 2. Jablonski diagram showing absorption and emissive deactivation pro
cesses of organic molecules. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 4. Mechanism of thermally activated delayed fluorescence. Fig. 5. Mechanics of electroluminescence in organic molecules. Fig. 3. Absorption and emission spectra of Zrq . Fig. 3. Absorption and emission spectra of Zrq . the spectra are shifted to longer wavelengths. In addition, as ab-
sorption can occur from any one of several vibrational energy
levels in the ground state to any one of various excited vibra-
tional states, absorption and emission spectra of typical organic
molecules, which include the vibrational levels of many atoms,
are characteristically broad. See Fig. 3. Fig. 5. Mechanics of electroluminescence in organic molecules. Major breakthroughs in harnessing EL have been made pos-
sible through the development of wide bandgap light emitting
diodes (LEDs) in the 1960s, the development of evaporated or-
ganic films in the 1980s and most recently through the develop-
ment of solution-processable quantum dots. Where the excited triplet state is long lived and close in en-
ergy to the excited singlet state, an additional mechanism for
fluorescence may occur. Known as thermally activated delayed
fluorescence (TADF), the singlet state is populated through: 1)
excitation and 2) intersystem crossing then occurs to the triplet
state. I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS In the case of LCDs, a
white light source is used as the backlight and a liquid crystal,
that can filter the white light to a variable degree, is placed in
front of each subpixel in a device. The “filter” allows a range
of wavelengths appropriate to its color through at any one time
thus creating the multi-color capability. The brightness of the
subpixel is controllable as the liquid crystal is energized or not
to block or transmit light. It is the crystal filtering mechanism
that causes poor viewing angles on LCD displays. The first observations of EL in organic materials proved
problematic in terms of the potential for commercialization of
the technology, however. For instance, in 1965, Helfrich and
Schneider had to apply currents of 50–1000 V to anthracene
crystals to observe fluorescence [4], and progress was further
hampered by difficulties in crystal growth and short lifetimes. Around the same time, General Electric introduced the first
commercial LEDs based on the inorganic semiconductor
GaAsP [5]and over the next few decades, while research into
organic EL waned, that related to inorganic LEDs flourished. It is possible to use LEDs as the white light source in LCD
displays and these tend to produce better contrast and color co-
ordinates than fluorescent backlights. Plasma displays are gen-
erally not as efficient as so-called LED-LCDs. An inorganic (semiconductor) LED essentially consists of
several layers including an n-type layer with a surplus of neg-
ative electrons and a p-type layer with an insufficient amount
of electrons (also described as a layer with a surplus of positive
holes). An active layer is sandwiched between them. When an
electric voltage is applied, the electrons and holes flow towards
the layer they are attracted to. Their paths cross in the active
layer where they recombine radiatively and light is emitted. See
Fig. 6. In the first instance, LED technology would seem to tick
many of the boxes required for display applications. ZnS and
SrS are among the most common phosphor materials used in
thin-film electroluminescent (TFEL) displays, although to ob-
tain EL, the phosphor must be intentionally doped with either
transition metal [7]or lanthanide luminescent impurities [8]. Ex-
panding the applicability of semiconductor materials beyond
lighting and backlighting solutions continues to pose significant
challenges owing to the difficulties with the requirement to dis-
play more than one color. I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS At this point, rather than the triplet relaxing back to the
ground state: 3) a second intersystem crossing process back to
the excited singlet occurs, from where: 4) fluorescence takes
place, as shown in Fig. 4. One of the first observations of EL was in the 1930s by Des-
triau [1], who found that certain zinc sulphide phosphors could
be excited by low voltage, low frequency alternating current. Crucially, no heat was emitted during the process creating the
potential for highly-efficient lighting solutions. Destriau also
discovered that doping ZnS with “activators” such as Mn
,
Cu
or Ag , could lead to enhanced emission, although charge
compensation, i.e. the addition of “co-activators” such as Cl ,
Br
or I
is an important factor in this. EL is the phenomenon whereby light is emitted from a ma-
terial following the application of an electric field to it. The
process can be described by an analogous mechanism to that
of PL where rather than exciting the molecule through the ab-
sorption of radiation, the molecule is excited electrically. The
molecule then relaxes to its ground state radiatively and EL re-
sults (Fig. 5). Between 1953–1955, Bernanose published details of one of
the first observations of EL of organic compounds, namely acri-
dine derivatives [2], [3], in which brightness vs applied field
followed the same emission laws to those found for inorganic
phosphors. In particular, Bernanose studied acridine orange on
cellulose films from which EL was observed following the ap-
plication of high alternating voltages. His studies found that the
emission spectrum was independent of the applied field with the The energy levels of organic molecules are often described as
the lowest unoccupied molecular orbital (LUMO), or the excited
state, and the highest occupied molecular orbital (HOMO), or
the ground state, as can also be seen in Fig. 5, with the distance
between the two levels the energy bandgap (Eg). 482 JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 Fig. 6. Principle of electroluminescence in semiconducting (inorganic) and organic LEDs. Fig. 6. Principle of electroluminescence in semiconducting (inorganic) and organic LEDs. proposed mechanism being either direct excitation of the dye
molecules or excitation of electrons. another. CRTs, for example, have a much higher power usage
than LCDs and require bulky devices. I. HISTORIC DEVELOPMENT OF LIGHTING SOLUTIONS A succession of LEDs based on III–V semiconductors, such
as GaAs, GaP, AlGaAs, InGaP, AlInGaP, were all developed,
but devices tended to be red, orange, green and yellow as the
color of the emission is based on the energy gap of the semi-
conducting material, and blue-colored devices remained elu-
sive. It was some time before blue diodes based on materials
such as ZnSe or GaN were developed and these still exhib-
ited significantly lower efficiencies than other diodes [6]. It was
for the blue emitting GaN-based device that The Nobel Prize
in Physics 2014 was awarded. p-i-n junction inorganic LEDs
offer long lasting and more efficient alternatives to older lighting
technologies. Under active-matrix conditions, multi-color LEDs can be as-
sembled into an EL display as demonstrated by Barrow and
Tuenge of Planar Systems, Inc. [9]in a prototype multi-color dis-
play using ZnS:Mn and ZnS:Tb phophor layers. EL subpixels
can be arranged in two-dimensional arrays each with its own
row and column address and associated data value. To make a
full color display, one or more subpixels of different colors are
grouped together to form a pixel. Thus each pixel on an EL dis-
play includes one or more subpixels, e.g. red, green and blue. More than 30
years on, however, full-color TFEL displays
based on semiconductors are generally only available in small
format active matrix design as cost-effective full-color panels
remain problematic to commercialize and the devices are still
only used in backlighting applications. KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 483 Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 7. Multi-color OLED devices and the ability to generate white light. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 8. Simplicity of OLED design vs LEDs. Fig. 9. Selected commercial OLED displays. III. INTRODUCING OLED TECHNOLOGY TO THE MARKET:
TWO ASPECTS—LIGHTING AND DISPLAYS Interest in organic EL was revived when Tang and VanSlyke
fabricated devices using thin evaporated films ( 100 nm)
of aluminum 8-hydroxyquinoline
Alq
and low voltages
(
10 V) [10]. This breakthrough development made it clear
that, given improvements in efficiency and reliability, it would
be possible to manufacture low-cost, flexible, multi-color and
white displays based on organic EL. Further attention was
drawn to this possibility with the advent of similar devices
fabricated by Burroughes et al. [11] based on the conjugated
polymer polyparaphenylenevinylene (PPV) which could be
formed via the low-cost route of spin casting from solution
and subsequent thermal treatment. The development of a solu-
tion-processable precursor for PPV and poly(fluorenes) made
this a potentially attractive approach for simple light-emitting
diode fabrication. Fig. 9. Selected commercial OLED displays. OLEDs rely on a central layer of emissive organic material
that is electroluminescent, that is, it will emit light when ex-
cited by an electrical current. Electrons are injected from the
cathode and holes from the anode. These travel inwards into the
device where they recombine to form excitons in a singlet:triplet
ratio of 1:3. The spin-allowed emission from fluorescent exci-
tons may occur rapidly, otherwise the normally non-radiative
triplet excitons may emit if the active layer is phosphorescent. As there are often difficulties in injecting electrons and holes
(carriers) into the organic layer, the structure of an OLED usu-
ally includes additional layers, such as electron injecting and
transporting layers and hole injection and transport layers to
facilitate the injection of charge carriers. The layers are de-
posited on top of each other with the first deposited on a sub-
strate (Fig. 7). The anode must be transparent to allow the pho-
tons produced to be seen, and so it is usually transparent ITO,
although it could equally be polyethyleneterephathalate (PET)
coated with a transparent conductor for a flexible device or any
other transparent material. involves the deposition of additional layers of emissive material
onto the same substrate (Fig. 8). The inclusion of polymers and organic molecules as emissive
layers in devices of various structures have been the subject of
many thousand research papers and patent applications since the
1980s each demonstrating an improvement in efficiency, color
coordinates, lifetime or brightness over the last, with the result
that the efficiencies of the devices and thus the commercial vi-
ability of the technology improved exponentially. II. MULTI-COLOR DISPLAY TECHNOLOGIES Traditional technologies for multi-color displays utilizing
cathode ray tubes (CRT) or liquid crystal displays (LCD) have
significant limitations that prohibit their use in one way or IV. QUANTUM DOTS Quantum dots (QDs) have become the subject of intense aca-
demic and industrial (Nanoco, QD Vision, Nanosys, Samsung,
Dow Chemicals) research over the past 15 years owing to their
novel electronic, electrical, optical and catalytic properties with
wide ranging applications in displays, lighting, lasers, solar cells
and photoelectrochemical cells. Quantum dots are nanocrystals composed of III–V semicon-
ductor (e.g. GaN, GaP, GaAs, InP and InAs) and II-VI semicon-
ductor (e.g. ZnO, ZnS, CdS, CdSe, and CdTe) materials with
all three dimensions in the approximately 1–10 nm size range. Among these, CdSe/ZnS systems have been studied most as
far as the EL is concerned, although, Cd is environmentally re-
stricted owing to its toxicological properties and its viability as
a commercial material is consequently questionable. Fig. 11. White OLED vs white LED emission spectra. OLEDs can get much closer values of 90–95 to the ideal. The
CIE chart showing the relative positions of warm and cool light
is given in Fig. 10 and a typical emission spectra of a white
OLED and white LED is given in Fig. 11 where the significant
emission in the blue region of the spectrum can clearly be seen
for the LED. It is the size of the QD that imparts unique physical properties
on the material: in contrast to the bulk semiconductor, the elec-
trons in a nanocrystal exhibit quantum mechanical effects. The
so-called quantum confinement phenomenon occurs as the size
of the semiconductor becomes comparable to or smaller than
the exciton Bohr radius, and where the electron and hole are
confined by the boundaries of the material. It leads to discrete
energy levels, known as “confinement states”, as predicted by a
particle in a box (Schrödinger's) equation [see (1)]. While technology into OLED lighting and research is some-
what in its infancy, the same kind of improvements that were
seen in OLED displays enabling commercial devices over the
last few years are expected in lighting solutions in the next 3
years. For example, in 2009, Matsushita (National Panasonic)
Electric Works demonstrated a WOLED with an efficiency of
37 lm/W with a lifetime in excess of 10000 hours at an initial
luminance of 1000 cdm
and improved it to 150 lm/W with a
lifetime in excess of 20,000 hours at 3000 cdm
in 2014 [12]. III. INTRODUCING OLED TECHNOLOGY TO THE MARKET:
TWO ASPECTS—LIGHTING AND DISPLAYS OLEDs are now a commercial reality in many display appli-
cations (Fig. 9) and it is worthy of note that as they can be de-
posited onto virtually any substrate, e.g. glass, ceramic, metal or
plastic. It is also possible to manufacture flexible or curved de-
vices, which is not the case with semiconducting LEDs, LCDs
or plasma technologies. In addition to use in displays, OLEDs have the potential to be
used in lighting applications. A key advantage of using an OLED light over an LED light
source is its favorable color rendering properties as given by
its color rendering index (CRI). The CRI has been defined the
International Commission of Illumination (CIE) as the effect of
an illuminant on the color appearance of objects by conscious
or subconscious comparison with their color appearance under
a reference illuminant. Effectively, it is the quantitative measure
of the ability of a light source to reveal the colors of objects in
comparison with a natural light source. Devices are typically assessed through a number of character-
ization measurements that include color coordinates (perceived
color), current density (A cm ) versus voltage, luminance
(a measure of brightness in cd m ) versus voltage, current
efficiency (cd A) versus luminance, power efficiency (lm/W)
versus luminance and lifetime (a measure of the stability of the
device). Critically, the incremental cost and ease of producing multi-
color devices over single color or white devices is negligible as
the fabrication technique of making a multi-color device simply Semiconductor LED light bulbs have struggled to reach the
more desirable warmer whites with values of 80–85 compared
to the highest CRI achievable of 100 for a black body radiator. JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 484 Fig. 12. Projected market for OLED lighting, QDs and displays [IDtechEX,
Santa Clara, November 2014]. Fig. 10. CIE color coordinate chart showing warm and cool warm light. Fig. 10. CIE color coordinate chart showing warm and cool warm light. Fig. 12. Projected market for OLED lighting, QDs and displays [IDtechEX,
Santa Clara, November 2014]. Fig. 11. White OLED vs white LED emission spectra. IV. QUANTUM DOTS In addition to Matsushita, there are a number of large compa-
nies heavily involved in OLED lighting, such as Samsung, LG,
Pioneer, Hitachi, Osram, Philips, GE, and BOE. (1) (1) where
is the quantum number;
is Planck’s constant;
is the
electronic mass, and
is the width of the box. These
levels
correspond
to
bonding
(HOMO)
and
anti-bonding (LUMO) levels in the material (Fig. 13). While
bulk semiconductors have a set bandgap (Eg) between the
valence and conduction bands, an effect of quantum confine-
ment means that the bandgap energy of a QD is inversely
proportional to its size (smaller QDs emit higher energy than The market for both OLED lighting and displays has grown
steeply since 2000, with the market, particularly for OLED
lighting, predicted by IDTechEx to continue to show strong
growth through 2018 and beyond (Fig. 12). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART
485 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 485 Fig. 13. Energy bandgaps of bulk semiconductor and QD materials. Fig. 15. Cluster diameter versus energy gap. Fig. 15. Cluster diameter versus energy gap. Fig. 13. Energy bandgaps of bulk semiconductor and QD materials. QDs absorb all wavelengths higher in energy than their
bandgap and convert them into a single color, i.e. they have
broad absorption spectra, but narrow emission spectra. This
feature gives them advantages over organic fluorophores be-
cause the excitation wavelength can be anywhere within a
broad range [15]. The narrow emission spectra of QDs and the
tunability of such results in an extremely wide color gamut,
and thus QD displays have the potential for improved color
saturation over OLED displays. Fig. 14. Top image of PL of selected quantum dots in hexane [QD source:
Sigma Aldrich]. Schematic of tunability of QDs—effect of size distribution on
color of PL emission below. QLEDs are also solution processable (low cost) and have
theoretical performance limits that meet or exceed that of all
other display technologies [16]. As both the color coordinates
and luminous efficiencies of QLEDs are good they are there-
fore capable of being more power efficient. Furthermore, as with
OLED displays, they can be deposited on any substrate enabling
many exciting possibilities in terms of shape and design. It is
well-known that the absorption spectrum shifts to red as the size
of the nanocrystal increases. IV. QUANTUM DOTS Typical emission spectra show a
full-width at half maximum (FWHM) of 30–40 nm depending
on the degree of monodispersity achieved during synthesis [17]. QDs can be both electrically and optically excited, where in-
ternal EL quantum efficiencies as high as 90% have been ob-
tained for certain materials [18]. Fig. 14. Top image of PL of selected quantum dots in hexane [QD source:
Sigma Aldrich]. Schematic of tunability of QDs—effect of size distribution on
color of PL emission below. larger QDs), and this means that the emission from a QD is
color tunable (Fig. 14). Owing to colloidal stability and the ability to make thin films
without disrupting the physical integrity of the crystal, the use
of QDs in flexible EL displays becomes a possibility. Further,
solution processing enables low cost full color display man-
ufacturing, e.g., using spin processing [19] or inkjet printing
[20] techniques. Such techniques can be applied to additive pro-
cessing, such as vacuum deposition [21], and hence are more
affordable methods for display fabrication. The bandgap is also
dependent on the cluster diameter, and this also affects the dis-
tribution of spectral emission, i.e. the smaller the cluster, the
narrower the emission band. See Fig. 15. Although radiative, a QD that comprises of a single semi-
conducting material generally exhibits low quantum efficiencies
owing to non-radiative electron-hole recombinations that occur
at defects in the crystal and dangling bonds on the surface. For
example, Xie et al. [13] found that after 12 hours in air, the ab-
sorption spectrum of an InP QD sample shifted to the blue dra-
matically, which was attributed to the fast shrinkage of the in-
organic core by oxidation. As a result, shell materials, typically
with wider bandgap energies, such as CdS or ZnS are grown on
the surface of the core [14] in a process known as inorganic pas-
sivation. Confining charge carriers within the luminescent core
and away from the surface significantly reduces the effects of
surface defects caused by atoms on the surface of the crystal. In
this way, states that may function as centers for non-radiative
recombination are protected and there is a consequent increase
in photoluminescent quantum yield (PL QY). QDs are generally prepared by reacting inorganic precursors
in the presence of organic ligands, which eventually form a
molecular coating around the QD luminescent core and stabilise
the nanoparticles against aggregation (Fig. 16). IV. QUANTUM DOTS Factors that are important to optimize during inorganic pas-
sivation include the energy levels of the shell and core, the for-
mation of a defect-free and uniform coating, coherence strain
between with the shell and core lattice, and the thickness of
the shell layer. The maximum PL efficiency of the core/shell
QD is dependent on the thickness of the shell layer, which has Xie found that after growing a ZnS shell around the InP core,
the absorption properties of the nanocrystal were unchanged fol-
lowing exposure to air, indicating a much improved stability. Further, they found that the PL QY of the InP dot was low
(
1%), but that it reached
40% for the core/shell ones. 486 JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 Fig. 16. Example of a passivated core-shell quantum dot structure. Fig. 16. Example of a passivated core-shell quantum dot structure. Fig. 18. Typical synthetic route developed by Ludolph et al for the preparation
of TOPO-capped CdSe using a simple precursor [26]. Fig. 17. Energy bandgaps in Type I, Reverse Type I and Type II QDs. Fig. 19. Schematic of capping ligand replacement mechanism. Fig. 17. Energy bandgaps in Type I, Reverse Type I and Type II QDs. been found to be less than two monolayers thickness for op-
timum properties of a CdSe/CdS core/shell structured nanopar-
ticle. Thicker capping layers lead to the formation of misfit dis-
locations, which are also sites of non-radiative recombination,
leading to a decrease in the PL QY [22]. Fig. 19. Schematic of capping ligand replacement mechanism. The surface of the core/shell QD may still possess highly re-
active dangling bonds, and these can be used to further passivate
the nanocrystal from its surrounding environment through the
co-ordination of an organic ligand, such as tri-n-octylphosphine
oxide (TOPO), trioctylphosphine (TOP), thiols (e.g. -mer-
captoethanol), carboxylic acids (e.g. oleic acids), octadecene,
oleylamine or 3-(aminopropyl)trimethoxysilane (APTMS). The ligand chelates to the surface of the QD by donating lone
pair electrons to the surface metal atoms, resulting in a QD
that demonstrates reduced particle agglomeration, reduced
sensitivity to oxidation, improved electronic stability and may
be soluble in relatively non-polar media [24]. It also stabilises
the nanocrystals to the extent that it possible to extract them as
free-standing powders [25]. IV. QUANTUM DOTS KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART
487 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 487 Fig. 21. HOMO-LUMO levels of selected QD materials and hole and electron
transporting materials. Fig. 20. Conventional OLED device structure. Fig. 20. Conventional OLED device structure. Further, QDs can be anchored by polymers (in particular
light-emitting polymers) by producing a monomer with a phos-
phonyl group, carrying out the QD synthesis in this medium
and reacting the product with a polymerizable group (e.g.,
phenylvinylene) [27]. Fig. 21. HOMO-LUMO levels of selected QD materials and hole and electron
transporting materials. This ability for solution-based synthesis enables effective
control of the crystal size distribution. The phenomena of
“Ostwald ripening” is utilized whereby the higher free energy
of smaller QDs makes them lose mass to larger size QDs,
eventually disappearing. Using a co-ordinating solvent, such as
TOPO, stabilizes the QD dispersion, improves the passivation
of the surface and provides a steric barrier to slow the growth
of the QD. The final size of the QD is mainly controlled by the
reaction time and the temperature. As crystal size is directly
correlated to the HOMO-LUMO bandgap of the material, it
is possible to take aliquots during the reaction process, and
observe the corresponding absorption wavelength shift to stop
the reaction when the appropriate size of crystal is achieved. This method has been used extensively to synthesize II-VI and
III-V QDs [28]. The method provides sufficient thermal energy
to anneal defects resulting a monodispersed solution of QD. A
typical standard deviation of particle size of 5% is achieved. TABLE I
HOMO-LUMO LEVELS OF TYPICAL QDS
Fig. 22. Example OLED emissive layers with HTL/ETL energy levels. TABLE I
HOMO-LUMO LEVELS OF TYPICAL QDS IV. QUANTUM DOTS The quantum efficiency of the QD will be further increased by
a defect-free, and uniform shell coating to minimize the number
of non-radiative recombination sites within the QD. Further,
when the shell material coats the core surface the bonding lat-
tice, lattice parameters, such as coherency strains result and can
play an important role in the properties of these core/shell sys-
tems. For instance, strain may cause the absorption and emission
spectra of core/shell QDs to be red shifted [23]. Depending on the bandgaps and the relative position of the
electronic levels of the involved semiconductors, the shell can
have different functions in core/shell QDs. Fig. 17 gives an
overview of the band alignment of the bulk materials where
three types can be distinguished: Type I where the bandgap of
the shell is larger than that of the core, Reverse Type I where the
bandgap of the shell is smaller than that of the core and Type II
where either the valence band edge or the conduction band edge
of the shell material is located in the bandgap of the core. Ludolph et al. [26] has pioneered a route of synthesis for the
preparation of a TOPO (tri-n-octylphosphine oxide) capped de-
vice, as shown in Fig. 18. Organically-capped QDs may be photostable as the interface
between the capping molecules and surface atoms is generally
weak leading to the failure of passivation and the creation of
new surface states under UV radiation. Surface modification by
ligand replacement is possible to tailor the properties of the QD
to the use for which it is required, for example it can be made
hydrophobic or hyrophilic. Ligand replacement can be carried
by reacting with an excess of another ligand or a ligand that
has a higher complexing ability (high formation constant), for
example a bidentate ligand (thiols), as shown in Fig. 19. It is crucial to match the energy levels of the shell and core. For Type I QDs, passivation of the QD surface should be with
a material that has a larger bandgap energy to increase the ef-
ficiency of charge injection into the radiative core. A wider
bandgap shell material is desired to create a potential barrier
around the QD core to confine the excitons. Confinement of
charge carriers in the core region by the band offset potentials
results in efficient and photostable luminescence from QDs. V. OLED AND QLED DEVICES Of the different types of emissive layer in OLEDs, typically
small molecules, e.g., Alq
have been shown to demonstrate
good device lifetimes owing to the enhanced inherent stability
of the molecules over phosphorescent materials, e.g. Ir
,
phosphors offer high efficiency along with high internal
quantum efficiencies, and polymer materials, e.g., PPV, have
the advantage of being solution processable, but have lower EL
efficiency. y
Owing to the multiplicity of the excited states formed when
the charges meet in the active layer of an OLED, around 25%
of excitons are generated in the singlet state, with three times
more triplet excitons (approximately 75%) being formed. This
means that OLEDs based on fluorescent molecules have a
maximum internal efficiency of around 25%. The efficiency of
OLEDs can be dramatically improved by using phosphors, such
as organometallic complexes with iridium or platinum metal
ions that enhance spin-orbit coupling and enable emission from
the formally forbidden triplet state. Phosphorescent OLEDs
consequently have theoretical maximum internal efficiencies
of 100% as in addition to the 75% of triplet excitons generated,
relaxation of spin-orbit coupling rules means that the singlets
that are also generated may be converted to triplets through
intersystem crossing. Fig. 23. Historic current efficiency data for OLED and QLED red [A], green
[B], and blue [C] devices. Recent OLED activity utilizes thermally activated delayed
fluorescence (TADF), or upconversion to achieve near unity in-
ternal EL quantum yields from fluorphors by backfilling the sin-
glet state with triplet excitons (
reverse intersystem
crossing). The mechanism is described in Fig. 4, earlier. The
need for more costly phosphorescent materials is eliminated,
and the possibility of obtaining blue emitters that give very high
total singlet yields [30], [31], is introduced. Indeed, Uoyama et
al. [32] have reported OLEDs using EL metal-free molecules
in which the energy gap between the singlet and triplet state is
minimized by design allowing upconversion of excitons from
the non-radiative triplet to the radiative singlet state thereby
achieving efficiencies comparable to those in phosphorescence-
based OLEDs. Green, orange and sky-blue OLEDs were fab-
ricated with external EL quantum efficiencies of 19.3 1.5%
(equivalent to 64.3%–96.5% internal efficiency), 11.2
1% and
8.0
1%, respectively. be wet processed into devices such as by inkjet printing or die
coating much more cost effectively. Blue colored OLED devices remain a key challenge. V. OLED AND QLED DEVICES The basic structure of an OLED consists of one or more or-
ganic films deposited between two electrodes, one of which is
transparent (Fig. 20). Under electrical bias, electrons are in-
jected from the cathode and holes from the anode into the or-
ganic material where they travel in the applied field until they
meet and form an excited state that leads to radiative emission. Fig. 22. Example OLED emissive layers with HTL/ETL energy levels. Devices fabricated using films that are deposited by subli-
mation under vacuum are more costly than devices fabricated
with films that are deposited from solution, e.g., by spin casting. Film thicknesses range from 10 to 100 nm. The emissive layer
should have a high quantum yield and be an effective charge
transporter. Additional layers are usually added between the
anode/cathode and organic layer as hole or electron transporting
aids. Considering the energy alignment of the ground and ex-
cited electronic states of the emissive monolayers and the sur-
rounding organic thin films is critical to the fabrication of an
efficient device in order to reduce the energy level barriers be-
tween layers, i.e. the ETL and HTL should be optimized as a
well charge balanced device will have higher efficiency [29]. Fig. 21 shows energy levels of some common hole and electron transport materials used in OLEDs and HOMO-LUMO levels
of typical QDs are shown in Table I. In addition to QLEDs, we consider three types of OLED
(Fig. 22), i.e. those where the emissive layer is based on
polymers (PLED), fluorescent small molecules (e.g., Alq ,
OLEDs) or organo–metallic phosphorescent small molecules
(e.g., Ir ppy
, Ir ppy
acac , PHOLEDs). External quantum efficiency (EQE) can be expressed in anal-
ogous manner to that of an OLED by a well understood multi-
plication of four factors as described by (2) (2 (2) (2) JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 488 Fig. 23. Historic current efficiency data for OLED and QLED red [A], green
[B], and blue [C] devices. where where recombination efficiency of holes and electrons; recombination efficiency of holes and electrons; fraction of charge carrier recombination in the
emissive layer resulting in excitons with spin-allowed
optical transitions; photoluminescent efficiency of the emitter; fraction of emitted photons that are coupled out of
the device
; refractive index of the substrate (glass). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 489 TABLE II
SELECTED TYPICAL PERFORMANCE DATA OF OLED AND QLEDS AT 1000 CDM
Data given in the order: 1) CIE color coordinates (x,y); 2) lifetime; 3) current efficiency; 4) power efficiency TABLE II TABLE II
SELECTED TYPICAL PERFORMANCE DATA OF OLED AND QLEDS AT 1000 CDM Data given in the order: 1) CIE color coordinates (x,y); 2) lifetime; 3) current efficiency; 4) power efficiency Fig. 24. Conventional [A] and inverted [B] QLED device structures. towards improved efficiencies for red, green and blue devices,
both OLEDs and QLEDs. A comparison of typical device performance data based on
fl
t
h
h
t
l
d QD
i i
l
Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 26. Red device structure adapted from Kim and Jang [36]. Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 25. Mashford et al. red device structure adapted from [35]. Fig. 26. Red device structure adapted from Kim and Jang [36]. Fig. 24. Conventional [A] and inverted [B] QLED device structures. Fig. 24. Conventional [A] and inverted [B] QLED device structures. towards improved efficiencies for red, green and blue devices,
both OLEDs and QLEDs. A comparison of typical device performance data based on
fluorescent, phosphorescent, polymer and QD emissive layers
at 1000 cdm
is given in Table II. It demonstrates that the
performance of QD devices still lag behind those of OLEDs and
that progress on blue devices across the board is still required. QD device structures are largely similar to those of OLEDs
(Fig. 24). A comparison of typical device performance data based on
fluorescent, phosphorescent, polymer and QD emissive layers
at 1000 cdm
is given in Table II. It demonstrates that the
performance of QD devices still lag behind those of OLEDs and
that progress on blue devices across the board is still required. Fig. 26. Red device structure adapted from Kim and Jang [36]. has reported a red QD (CdSe/CdS) device that reaches a peak
efficiency of 19 cd/A and 25 lm/W with an optimum thickness
of 45 nm of QD and 0.68, 0.31 CIE coordinates. The lifetime
was estimated to be 4 hours at 1000 cdm
. The device config-
uration is shown in Fig. 25. p
g
q
QD device structures are largely similar to those of OLEDs
(Fig. 24). V. OLED AND QLED DEVICES Com-
pared to red (initial luminance 1000 cdm
, 500,000 hours) and
green (initial luminance 1000 cdm
, 200,000 hours, according
to Universal Displays at the OLED Summit 2013), the blue
devices typically have lifetimes in the range 10,000–20,000
hours). Color coordinates of fluorescent blue OLEDs (0.15,
0.15) are considered acceptable with lifetimes of 50,000 hours
at 1000 cdm
reported by Idemitsu Kosan and Merck. Blue
phosphorescent OLEDs have poorer color coordinates of
(0.20, 0.35) with a reported lifetime of 8000 hours at initial
1000 cd/m . Printed PHOLEDs have even poorer lifetimes
[33]. A further point of note is that OLEDs based on organic
molecules have been found to degrade with time during
prolonged excitation in a process known as photobleaching. Owing to the luminescent core being inorganic, which is then
passivated by a protective inorganic shell, QDs are often much
more resistant to photobleaching than organic dyes [34]. Strong intermolecular interactions usually result in the low
solubility of organic molecules, further they tend to form crys-
talline domains in the film state, which can act as carrier traps
and raise the operational voltage of a device. As such, organic
molecules in OLEDs are usually deposited onto a device by sub-
limation under vacuum, a costly technique, but polymer OLED
materials are readily soluble in ink solvents meaning they can Despite high theoretical performance levels of QLEDs in
practice, thus far efficiencies still lag behind those for OLEDs,
but as can be seen in Fig. 23, there is a marked general trend KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART It is frequently found that in the literature on QLED and
OLED devices that not all the relevant data (EQE, power ef-
ficiency, CIE co-ordinates and device lifetime) are reported for
each device. While the PL efficiencies of QDs are high, the main
reason for low EQEs in devices is largely attributed to poor
charge carrier injection into the QD layers. Mashford et al.[35] Kim
and
Jang
[14],
[36]
presented
devices
using
CdSe/CdS/ZnS. Their red device achieved 6.5 cd/A and
CIE (x,y)-coordinates of (0.70, 0.30), with a device structure
shown in Fig. 26 and their green device an impressive 28 cd/A
(0.16, 0.75), structure in Fig. 27. The lifetime was not reported. JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 490 Fig. 27. Green device structure adapted from Kim and Jang [36]. Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 29. Cd-free device structure (2011). (Lim et al. [32]) Fig. 30. Cd-free device structure (2013). (Lim et al. [38]) Fig. 27. Green device structure adapted from Kim and Jang [36]. Fig. 28. Yellow device structure adapted from Qasim et al. [37]. Fig. 30. Cd-free device structure (2013). (Lim et al. [38]) ITO, while holes are injected from Al. The HTL materials they
used possessed different HOMO energy levels in the range of
5.1–6.0 e.V. ITO, while holes are injected from Al. The HTL materials they
used possessed different HOMO energy levels in the range of
5.1–6.0 e.V. Fig. 28. Yellow device structure adapted from Qasim et al. [37]. HOMO-LUMO levels are easily determined from solution
electrochemistry (viz. cyclic voltammetry) and the absorption
edge from UV/VIS absorption spectroscopy of thin films [45]. Kathirgamanthan et al. [46] reported absorption, photolumines-
cence and cyclic voltammetry studies on red and green QDs
based on CdSe/ZnS provided by QD Vision. A yellow device has been reported by Qasim et al. [37] that
achieves 1.6 cd/A with peak EL emission at 575 nm. Again, life-
time data is not given. The device structure is given in Fig. 28. These devices show the potential of QDs in devices for dis-
play applications, but their commercial viability is negligible
owing to their reliance on the highly toxic and environmentally
restricted Cd. VI. CONCLUSION AND FUTURE DEVELOPMENTS VI. CONCLUSION AND FUTURE DEVELOPMENTS One of the highest priorities for the commercialization of
QLED technology will be to find suitable Cd-free quantum
dots. InP-based dots and CuInS [44] are already showing much
promise in this respect. We have also seen impressive results
from devices that use ZnO cores with a MgO shell, indicating
that ZnO is also a promising future Cd-free material. In terms
of improving performance of QLEDs, there will certainly be
much effort directed at optimizing the efficiency of devices. Research by Shen et al. [41], for example, has found that as the
shell thickness of the quantum dot affects the charge confine-
ment and charge injection in the QD. They recently reported a
conventional QD device structure with a power efficiency of
19.7 lm/W for green emission. g
As organic hole transporting materials with low HOMO
levels (deep HOMO levels) are not readily available (for
OLEDs, this is not so), the performance of QLEDs can be
further improved by enhancing energy transfer from the hole
transport layers into the QD layer through the careful selection
of transport materials that can efficiently transfer excitons to
QDs via Förster energy transfer [41]. Kim et al. [36] have
described high-efficiency inverted QLED devices, which are
useful for active matrix displays. They deposited two ETL
layers, the first Al doped ZnO, followed by a second ETL. A maximum current efficiency of 28.29 cd/A and a power
efficiency of 22.11 lm/W was achieved for their stacked ETL
inverted green device; an approximately three times improve-
ment on a QLED with a single ETL. Fig. 31. Absorption spectra of red and green QDs (upper chart) and PL of red
and green QDs thin films (lower chart). Fig. 32. Cyclic voltammogram showing redox behavior of red QD in CH CN
with CF SO Li as the supporting electrolyte. Using materials with high PLs in the red, green, and blue re-
gions of the visible spectrum, Anikeeva et al. [43] were able
to increase the efficiency of QLEDs four-fold for green devices
and by 30% for orange devices. However, they found that there
were still challenges in improving the efficiency of blue QLEDs,
presumably owing to incomplete energy transfer from organics
(weak spectral overlap between the blue QD and the electron
and hole transporting materials used). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 491 Fig. 31. Absorption spectra of red and green QDs (upper chart) and PL of red
and green QDs thin films (lower chart). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART As a result, efforts are now beginning to shift
towards Cd-free device structures. While Lim et al. [38] pre-
sented results from a Cd-free QD in 2011 using InP, the effi-
ciency was poor at 0.006 cd/A (Fig. 29). Two years later, his
group reported a 10.9 cd/A green device [39] that was also cad-
mium free. (Fig. 30). The red QD’s absorption spectra (Fig. 31) of thin films spin
coated from a hexane solution showed an edge, which we have
attributed to the emissive energy gap in the nanocrystal at
nm and which was at
nm as a thin film. The
green QDs showed clear absorption peaks at
nm
(in hexane); 509 nm (thin film). The PL spectra, also shown in
Fig. 31, indicate the typical normal distribution type shape. The
red QD showed PL emission peaks at
(thin
film);
nm (in hexane) and the green at
(thin film);
nm (in hexane). Kwak et al.[40] has reported bright and efficient inverted
structure red, green and blue QLEDs with maximum luminances
of 23,040, 218,800 and 2250 cd/m and external quantum ef-
ficiencies of 7.3%, 5.8%, and 1.7%, respectively. The devices
showed turn-on voltages as low as the bandgap energy of each
QD and long operational lifetime, which they attributed to direct
exciton recombination within QDs through the inverted device
structure. In their device, the electrons were injected from the Cyclic voltammograms (CV) were carried out in acetonitrile
(QDs in hexane diluted with acetonitrile) and lithium trifluo-
romethanesulphonate was used as the supporting electrolyte (Pt
foil: working electrode; Pt wire: counter electrode; Ag/AgCl:
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colloidal quantum dot LEDs,” ACS Nano, vol. 3, pp. 3581–3586, Nov. 2009. [2] A. Bernanose, M. Comte, and P. Vouaux, “Electroluminescence of or-
ganic compounds,” J. Chim. Phys., vol. 50, pp. 64–68, 1953. [30] D. Y. Kondakov, T. D. Pawlik, T. K. Hatwat, and J. P. Spindler,
“Triplet annihilation exceeding spin statistical limit in highly efficient
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thracene crystals,” Phys. Rev. Lett., vol. 14, pp. 229–231, Feb. 1965. [31] F. B. Dias et al., “Triplet harvesting with 100% efficiency by way
of thermally activated delayed fluorescence in charge transfer OLED
emitters,” Adv. Mater., vol. 25, pp. 3707–3714, May 2013. [5] N. Holonyak Jr and S. F. Bevacqua, “Coherent (visible) light emission
from Ga As
junctions,” Appl. Phys. Lett., vol. 1, pp. 82–83,
Dec. 1962. [6] M. G. Craford and F. M. Steranka, “Light emitting diodes,” in Ency-
lopaedia of Applied Physics, G. L. Trigg, Ed. Weinheim, Germany:
Wiley-VCH, 1994, vol. 8, pp. 485–514. [32] H. Uoyama, K. Goushi, K. Shizu, H. Nomura, and C. Adachi, “Highly
efficient organic light-emitting diodes from delayed fluorescence,” Na-
ture, vol. 492, pp. 234–238, Nov. 2012. [33] T. Tsujimura,
OLED
Displays:
Fundamentals
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Applica-
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thin-film electroluminescent devices with rare-earth-ion-activated SrS
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enhanced charge injection,” Nature Photon., vol. 7, pp. 407–412, Apr. 2013. JOURNAL OF DISPLAY TECHNOLOGY, VOL. 11, NO. 5, MAY 2015 492 if the saturated dark blue (CIE x,y (0.15, 0.03) can be harnessed
to give long life-time of at least 10000 hours at 1000 cdm
. [25] J. Gilbertson and T. Davis, “Colloidal CdSe quantum dot electrolumi-
nescence: Ligands and light-emitting diodes,” Microchim. Acta, vol. 160, pp. 345–350, Jun. 2007. [26] B. Ludolph, M. A. Malik, P. O'Brien, and N. Revaprasadu, “Novel
single molecule precursor routes for the direct synthesis of highly
monodispersed quantum dots of cadmium or zinc sulfide or selenide,”
Chem. Commun., vol. 17, pp. 1849–1850, 1998. ACKNOWLEDGMENT The authors thank QD Vision Inc., Lexington, MA, USA, for
the provision of red and green QDs. They also thank Profs J. Buckingham, G. Rodgers, and J. Silver for encouragement. [27] M. Green, “The Nature of Quantum Dot Capping Ligands,” J. Mater. Chem., vol. 20, pp. 5797–5809, Apr. 2010. [28] C. B. Murray, D. J. Norris, and M. G. Bawendi, “Synthesis and charac-
terisation of nearly monodisperse CdE E
S, Se, Te semiconductor
nanocrystallites,” J. Amer. Chem. Soc., vol. 115, pp. 8706–8715, Sep. 1993. VI. CONCLUSION AND FUTURE DEVELOPMENTS Direct charge injection
may also have a higher proportional contribution to the emis-
sion from the blue QLED, as manifested in its low EQE value
of 0.4%. They suggest this may be remedied by design and syn-
thesis of wide bandgap hole and electron transporting organic
materials for improved exciton energy transfer and direct charge
injection into blue QDs. Along with continued improvements to
luminance and lifetime results for OLEDs and QLEDs, we also
expect to see the emergence of “hybrid” devices that incorpo-
rate emissive layers using different types of emissive material,
for instance a device could contain a blue emitting TADF layer,
a green phosphorescent layer and a red QD layer. Fig. 32. Cyclic voltammogram showing redox behavior of red QD in CH CN
with CF SO Li as the supporting electrolyte. determined the HOMO level to be set at
5.6 e.V. and the
LUMO level at
3.4 e.V. for the red QD. Furthermore, we expect to see developments in the OLED
lighting arena in the coming years owing to the appealing warm
white light that can be created. The saturated colours from QDs,
hitherto unattainable from OLEDs, give a technical advantage
to QDs. However, the life-times of electroluminescent devices
(RGB) are all very short (typically, 100 hours at 1000 cdm
)
and need urgent attention. We have fabricated efficient red and
green devices with organic electron injectors and transporters
in inverted devices, the results of which will be published else-
where. We anticipate a great future for quantum dots particularly A clear CV for the green QD could not be obtained as it did
not form a stable colloid in the acetonitrile. This is supported
by Zeta potential measurements that we carried out. A value of
66.32 mV was obtained for the red QD in acetonitrile indicating
excellent stability in solution, but a value of
39.68 mV was ob-
tained for the green QD indicating very poor colloidal stability. We estimated a HOMO level of
5.72 e.V. for the green QD
from the CV and a value of
2.41 e.V for its LUMO level from
the absorption edge [46]. KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART 493 KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART Poopathy Kathirgamanathan received the Ph.D. degree from Exeter University, Devon, U.K., in
1980. Poopathy Kathirgamanathan received the Ph.D. degree from Exeter University, Devon, U.K., in
1980. Poopathy Kathirgamanathan received the Ph.D. degree from Exeter University, Devon, U.K., in
1980. in recognition of his achievements in developing electroactive and light emit-
ting materials and winner of awards for the best R&D in OLED's in 2006 and
2007 at the Organic Semiconductor Conference (Frankfurt, Germany). He is an
Associate Editor of the IEEE/OSA JOURNAL OF DISPLAY TECHNOLOGY since
2012. He was recently awarded GB £4 million by EC to develop flexible OLED
lighting. He has been a Fellow of the Royal Society of Chemistry since 1992,
and of the Institute of Physics since 1994 in recognition of his achievements in developing electroactive and light emit-
ting materials and winner of awards for the best R&D in OLED's in 2006 and
2007 at the Organic Semiconductor Conference (Frankfurt, Germany). He is an
Associate Editor of the IEEE/OSA JOURNAL OF DISPLAY TECHNOLOGY since
2012. He was recently awarded GB £4 million by EC to develop flexible OLED
lighting. He has been a Fellow of the Royal Society of Chemistry since 1992,
and of the Institute of Physics since 1994 1980. He is a Professor of Electronic Materials Engi-
neering, Wolfson Centre, Brunel University, U.K.,
and is one of the worlds leading experts in Organic
Electronics. Following the Ph.D. degree from Exeter
(Sir Arthur Reed Scholar), he pioneered research
and development in the area of Organic Electronics
at Newcastle University, Cookson Group plc., Uni-
versity College London, and London South Bank
University (Chair in Electronic Materials Engineering since 1993). In 2000,
he founded OLED-T, based on the technology that he developed at London
South Bank University and served as the CTO of OLED-T until the company
and most of the I.P. (48 patents) of OLED-T was sold to Merck Chemicals,
Germany, in August 2008. He moved to Brunel University, Uxbridge, U.K., in
March 2009, where he continues to develop new materials for OLEDs, QLEDs,
OPV, and OTFT, in addition to electrochemistry and nanotechnology. He is
passionate about the commercialization of OLEDs for displays and lighting
and OPV (Organic PV). KATHIRGAMANATHAN et al.: ELECTROLUMINESCENT ORGANIC AND QUANTUM DOT LEDs: THE STATE OF THE ART He has received a total of GB £2.1 million pounds
from Royal Society, TSB, EU, and industrial funding over the last three years. He has over 200 publications and patents, and over 150 conference papers. Prof. Kathirgamanathan is currently an Executive Committee Member of the
Materials Chemistry Group, Society of Chemical Industries, London, U.K., and
also served as the Chairman of the Group from 1999 to 2000. He is also a Com-
mittee Member of the Materials Chemistry Division (RSC) since July 2013,
and the Regional Vice President, Europe, Society for Information Displays,
since May 2013. He was a recipient of prestigious Sir Monty Finniston Award REFERENCES Surendrakumar, “Red and green quantum dot based LEDs demon-
strating excellent color coordinates,” in SID Conf. Proc., 2015. Lisa M. Bushby, photograph and biography not available at time of publication. Lisa M. Bushby, photograph and biography not available at time of publication. Muttulingam Kumaraverl, photograph and biography not available at time of
publication. Seenivasagam Ravichandran, photograph and biography not available at time
of publication. Prof. Kathirgamanathan is currently an Executive Committee Member of the
Materials Chemistry Group, Society of Chemical Industries, London, U.K., and
also served as the Chairman of the Group from 1999 to 2000. He is also a Com-
mittee Member of the Materials Chemistry Division (RSC) since July 2013,
and the Regional Vice President, Europe, Society for Information Displays,
since May 2013. He was a recipient of prestigious Sir Monty Finniston Award Prof. Kathirgamanathan is currently an Executive Committee Member of the
Materials Chemistry Group, Society of Chemical Industries, London, U.K., and
also served as the Chairman of the Group from 1999 to 2000. He is also a Com-
mittee Member of the Materials Chemistry Division (RSC) since July 2013,
and the Regional Vice President, Europe, Society for Information Displays,
since May 2013. He was a recipient of prestigious Sir Monty Finniston Award Sivagnanasundram Surendrakumar, photograph and biography not available
at time of publication.
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https://www.epj-conferences.org/10.1051/epjconf/201611402139/pdf
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English
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Numerical experiment about the arrangement determination of multiple bluff cylinders which obtain the most suitable new type stir
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EPJ web of conferences
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1 Introduction demerit in which carbon dioxide falls off from the liquid
by the centrifugal force produced in a motion of the
muddler will occur. The good method of raising the
efficiency of stirring should just make a muddler
reciprocating movement within the glass horizontally. However, the method is not efficiently mixed over the
overall fluid in the glass. In the restaurant in Japan, various alcoholic beverages are
offered like the countries of Europe. Beer, sake, white
distilled liquor, wine, whiskey, etc. are prepared there. Consumption of plum wine with soda is increasing in
such an alcoholic beverage. This is because plum wine
with soda passes along a throat smoothly. In women or
the younger age group, it is reported that the amount of
consumption of plum wine with soda is increasing [1]. In
response to this matter, alcoholic beverage brewing
industry companies vied in production increase-in-
quantity sale of a "soda cocktail" drink mutually, and
planned it. In order to attain differentiation on sale, the
improvement idea of packaging, addition of a sales
promotion article, etc. were planned. Development of a
new type "muddler" was desired as one of the plans of
differentiation on sale. Here, although the "muddler" is
"cocktail stir" in use, it is the popular name currently
called in usually in the food-and-drink industry of Japan. The popular name was used in this study. Generally, the
"muddler" of the stirring use of the cocktail drink is a
long and slender bar configuration. The method for use
agitates by making it circle horizontally in the liquid in a
glass. Since the liquid in a glass also rotates
synchronizing with a muddler, in the shape of an ordinary
muddler, stirring efficiency is low. Therefore, in order to
obtain enough stirring effect, it is necessary to continue
rotating a muddler over a long time. In a "soda cocktail",
if it continues rotating a muddler over a long time, the The author is observing that mix as the symmetrical
vortex street discharged from two circular cylinders
which are oscillating in the direction of a flow flows and
goes down through study of a lock-in phenomenon until
now, and rearrangement occurs as a vortex of another
kind [2, 3]. By using the lock-in phenomenon and
phenomenon of vortical rearrangement, the author
conjectured what can promote liquid stirring. 1 Introduction Then, the
author performed creation development of the muddler
(type shaken longitudinal) of the new shape based on
interference stirring by vortices. And the muddler was
used as the product and sent out to the market [1]. This paper reports the details which are the numerical
simulations performed in the early stage of the creation
development. Numerical experiment about the arrangement determination of multiple
bluff cylinders which obtain the most suitable new type stir Yoshifumi Yokoi1,2,a
1National Defense Academy, Department of Mechanical Engineering, 1-10-20 Hashirimizu, Yokosuka 239-8686, Japan
2Techniche Universität Berlin, Müller-Breslau-Str. 8, 10623 Berlin, Germany Yoshifumi Yokoi1,2,a Abstract. When developing the stirring rod for mixing two liquid with which the densities into which gas
melted differ in little stirring operation, verification of the stirring effect was performed in the numerical
simulation using a vortex method. Since it is calculation for specification examination of the preceding
paragraph which creates a producing model, it becomes important to obtain a calculation result early and to
feed back to the design. Two dimension calculation of the double file circular cylinder group which carried out
staggered arrangement of the nine circular cylinders was performed. The aspects of the flow which strayed off
from the design point or the design point were shown. The mixed degree was judged from the degree of the
complexity of the flow pattern. In a design point, the complicated flow by a vortex is formed with oscillating 1
period, and interference stirring occurs. Adaptation ranges other than a design point are that the ranges of the
value of P/L are 0.8-1.2. DOI: 10.1051/
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9 DOI: 10.1051/
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⃝Owned by the authors, published by EDP Sciences, 201
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9 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits
distribution, and reproduction in any medium, provided the original work is properly cited.
2.1 Test design of the muddler for numerical
simulations Mixture of ume liquor and carbonated water was assumed
with the cylinder glass with a capacity of 460 ml (the a Corresponding author: yokoi@nda.ac.jp a Corresponding author: yokoi@nda.ac.jp This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits
distribution, and reproduction in any medium, provided the original work is properly cited.
EPJ Web of Conferences this thing, the design of the coil centre distance L and the
design of a coil pitch P were performed. The value of P/d
was set to 3.5 in order to secure the space which has been
rolled before the circular cylinder by the side of
downstream by the separating shear layer discharged
from the circular cylinder by the side of the upstream. The value of space ratio P/L which is the ratio of a coil
pitch P and the coil centre distance L was decided to be
1.0 (design point). As a result, it was decided that it
would be P = L = 14 mm. The length of the coil section
was made into the 4 times roll (4P = 56 mm) in
consideration of the appearance as the muddler. The
conditions of oscillation were decided as follows. Half
oscillating amplitude a was set to 30 mm so that
oscillating amplitude might become equivalent to the
length of the coil section. Frequency f of oscillation was
set to 1 Hz in consideration of the cardiac beats rate of
the human body at the time of drinking. diameter of 70 mm, the depth of 120 mm) containing ice. In order to make it mix moderately in a little operation, a
motion of the muddler makes it reciprocating movement
in the perpendicular direction of the glass. In order to
enlarge the contact surface product when moving in one
operation in the inside of a liquid, the tip section of a
stick made shape of a muddler the shape rolled spirally. It
was considered as the shape of a coil. The shape is shown
in figure 1. The circular cylinder which became this coil
shape plans making a vortex, discharging and agitating
surrounding fluid by the mutual interference of the
discharged vortices. The muddler is inconvenient with the
metal which rusts on a health concept target in order to
touch a drink. It becomes a cost overrun although what is
necessary is just to use stainless steel for the material. a Corresponding author: yokoi@nda.ac.jp The cast made of resin which can be mass-produced at a
low price also from the field of manufacture expense was
assumed. In consideration of intensity and productivity,
the diameter d of a material round bar was determined as
4 mm. 2.2 The calculation method and apparatus As for a vortex shedding occurring independently
mutually, it is known for parallel two circular cylinder
that the ratio (G/d = (L - d)/d) of the circular cylinder
interval G and the circular cylinder diameter d is 1.0 or
more. In tandem two circular cylinders, it is known that
the separating shear layer discharged from the upstream
side circular cylinder at the time of about P/d (ratio of
circular cylinder center interval P and circular cylinder The numerical simulation was performed by commercial
fluid analysis tools and the commercial note type
computer. The fluid analysis tools of the marketing used
"Uzu Cruise 2D Ver.1.1.3 Rev.H" by College Master
Hands, Inc. The numerical computation technique of the
used fluid analysis tools is a vortex method which
introduced the surface of wall vortex element method
which is a type the vortex method application level 3
which used together the boundary element method and
the vortex method developed. The details as the
calculation technique of the vortex method are written to
reference [5] and reference [6]. The computer used the
commercial note type personal computer (NEC LaVie
LL850/L). Figure 1. Muddler tip section shape
0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
P/L
1.0
0.9
0.8
0.7
0.6
0.5
2.0
1.6
1.2
1.4
L/d
P/d
Independent vortex shedding in parallel case
Independent vortex shedding
in tandem case
0.4
0.3
0.2
Figure 2. The diagram of the calculation points
diameter d) = 2.5 will carry out a reattachment to the
downstream side circular cylinder [4]. In consideration of
which is a type the vortex method application level 3
which used together the boundary element method and
the vortex method developed. The details as the
calculation technique of the vortex method are written to
reference [5] and reference [6]. The computer used the
commercial note type personal computer (NEC LaVie
LL850/L). Moving
direction Figure 1. Muddler tip section shape
Moving
direction Figure 1. Muddler tip section shape
Movin
directio Moving
direction 0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
P/L
1.0
0.9
0.8
0.7
0.6
0.5
2.0
1.6
1.2
1.4
L/d
P/d
Independent vortex shedding in parallel case
Independent vortex shedding
in tandem case
0.4
0.3
0.2
Figure 2. 3.1 Aspect of the flow in a design point The aspect of the flow for every time progress in a design
point (P/L = 1.0, L/d = 3.5, P/d = 3.5) is shown in figure
3. Here, "Origin" is in the state of the calculation start. "T.D.C." and "B.D.C." mean the top dead center of
oscillation, and the bottom dead center of oscillation,
respectively. The "cycle" expresses one period and the
numeric character in a parenthesis shows the number of
calculation steps. Since preservation of calculated data
was performed every ten steps, the flow pattern of each
period, the top dead center, and the bottom dead center
used the thing of the step near it. It is imagined that
mixture of flow is promoted at the time of the turn to the
bottom dead center from the top dead center and the turn
to the top dead center from the bottom dead center. When
it sees through oscillating one period, it turns out that the
complicated
flow
by
vortices
was
formed
and
interference stirring has occurred. Furthermore, the flow
increased complexity by continuing oscillation. After In such the state, in order to investigate the
applicability of space ratio (P/L), the value of space ratio
(P/L) was varied and the simulation was performed. The
value of space ratio (P/L) was changed with 0.25, 0.5, 0.8,
1.0, 1.2, and 2.0. The total number of times of repetition
calculation is 1000 times, and this corresponds by 2.5
times the oscillating period. Figure 2 shows a design Figure 3. The time history of the flow pattern in a design point P/L = 1.0, L/d = 3.5, P/d = 3.5
(a) Origin (0)
(i) 2 cycle (750)
(b) T.D.C. (80)
(c) 0.5 cycle (170)
(d) B.D.C. (270)
(e) 1 cycle (360)
(f) T.D.C. (460)
(g) 1.5 cycle (560)
(h) B.D.C. (650)
(j) B.D.C. (850)
(k) 2.5 cycle (950)
(l) Final calculation (1000) (c) 0.5 cycle (170) (d) B.D.C. (270) (a) Origin (0) (b) T.D.C. (80) (d) B.D.C. (270) (c) 0.5 cycle (170) (a) Origin (0) (b) T.D.C. (80) (c) 0.5 cycle (170)
(g) 1.5 cycle (560) (a) Origin (0)
(e) 1 cycle (360) ( )
(
)
(f) T.D.C. (460) (h) B D C (650) (i) 2 cycle (750)
(e) 1 cycle (360) (e) 1 cycle (360) (h) B.D.C. (650) (f) T.D.C. (460) (g) 1.5 cycle (560) (h) B.D.C. (650)
(l) Final calculation (1000) (g) 1.5 cycle (560)
(k) 2.5 cycle (950) (f) T.D.C. 2.3 Calculation conditions point and each calculating point in each space ratio with
the aspect of the flow of parallel two circular cylinders,
and the aspect of the flow of tandem two circular
cylinders. An abscissa is the distance ratio in the case of
parallel two circular cylinders, and an ordinate is the
distance ratio in the case of tandem two circular cylinders. The straight lines in the figure mean the space ratio (P/L). This
calculation
is
calculation
for
specification
examination of the preceding paragraph which creates a
producing model. So, it is important to obtain the
calculation result early and to feed back to a design. In
this study, two dimensional calculation of the circular
cylinder group which treated the section including the
main axis of the coil, then carried out staggered
arrangement of the nine circular cylinders was performed. The mixed degree was judged from the degree of the
complexity of the flow pattern. Two kinds of liquids with
which density differs from specific gravity in fact are
mixed. However, in order to verify the process of mixture
by the vortex, it was considered as single fluid and the
physical-properties value of water was used for
calculation here. Here, the spatial restriction supposing a
glass, ice, etc. is not provided. The state of early fluid is
stationary. Half oscillating amplitude was set to a = 30
mm, and the oscillation frequency was set to f = 1 Hz. The Reynolds number (Re = umaxd/) based on the
maximum movement speed of body (umax = 2af) was
about 660. If the Strouhal number was calculated using
the body oscillation frequency and amplitude, the value
of the Strouhal number was about 0.02. 2.2 The calculation method and apparatus The diagram of the calculation points 0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
P/L
1.0
0.9
0.8
0.7
0.6
0.5
2.0
1.6
1.2
1.4
L/d
P/d
Independent vortex shedding in parallel case
Independent vortex shedding
in tandem case
0.4
0.3
0.2 Figure 2. The diagram of the calculation points Figure 1. Muddler tip section shape diameter d) = 2.5 will carry out a reattachment to the
downstream side circular cylinder [4]. In consideration of diameter d) = 2.5 will carry out a reattachment to the
downstream side circular cylinder [4]. In consideration of diameter d) = 2.5 will carry out a reattachment to the
downstream side circular cylinder [4]. In consideration of diameter d) = 2.5 will carry out a reattachment to the
downstream side circular cylinder [4]. In consideration of 02139-p.2 02139-p.2 EFM 2015 3.1 Aspect of the flow in a design point (460)
(j) B.D.C. (850) (k) 2.5 cycle (950) (l) Final calculation (1000) (j) B.D.C. (850) (i) 2 cycle (750) Figure 3. The time history of the flow pattern in a design point P/L = 1.0, L/d = 3.5, P/d = 3.5 02139-p.3 EPJ Web of Conferences Figure 4. The time history of the flow pattern when straying off ±20% from a design point (P/L = 0.8 and 1.2)
Origin T.D.C. 0.5 cycle B.D.C. 1 cycle
P/L = 1.0, L/ d= 3.5, P/d = 3.5
P/L = 0.8, L/d = 3.5, P/d = 2.8
P/L = 0.8, L/d = 4.375, P/d = 3.5
P/L = 1.2, L/d = 2.917, P/d = 3.5
P/L = 1.2, L/d = 3.5, P/d = 4.2 Origin T.D.C. 0.5 cycle B.D.C. 1 cycle
P/L = 1.0, L/ d= 3.5, P/d = 3.5 P/L = 1.0, L/ d= 3.5, P/d = 3.5 P/L = 0.8, L/d = 3.5, P/d = 2.8 P/L = 0.8, L/d = 4.375, P/d = 3.5 P/L = 1.2, L/d = 2.917, P/d = 3.5 Figure 4. The time history of the flow pattern when straying off ±20% from a design point (P/L = 0.8 and 1.2) e history of the flow pattern when straying off ±20% from a design point (P/L = 0.8 and 1.2) independent of each circular cylinder are discharged. Figure 4 shows the flow patterns of the conditions used as
P/L = 0.8 and P/L = 1.2 which strayed off from the space
ratio in a design point ±20 %. On every condition, the
complicated flow by a vortex was formed with oscillating
one period, and interference stirring has occurred, and it
can grasp so that it may scarcely change to the aspect of
the flow in a design point. On the other hand, figure 5
shows the flow pattern in the case of the space ratio
which strayed off from the design point further. When a
result is seen, it turns out that enough on any conditions
stirring is not obtained. These matters show that the
ranges of P/L of space ratio suitable for stirring are 0.8-
1.2. Here, when it is on the line of the same space ratio,
the one where the value of L/d or P/d is smaller is
considered to be a desirable thing on the size of the
product. passing over oscillating two periods, mixture by large-
scale vortices can be seen. 3.1 Aspect of the flow in a design point This thing suggests that "good
stirring" is obtained by little movement. EFM 2015 EFM 2015 Figure 5. The time history of the flow pattern when straying off further from a design point (P/L = 0.25, 0.5 and 2.0)
Origin T.D.C. 0.5 cycle B.D.C. 1 cycle
P/L = 1.0, L/d = 3.5, P/d = 3.5
P/L = 0.25, L/d = 7.0, P/d = 1.75
P/L = 0.5, L/d = 3.5, P/d = 1.75
P/L = 0.5, L/d = 7.0, P/d = 3.5
P/L = 2.0, L/d = 3.5, P/d = 7.0 Origin T.D.C. 0.5 cycle B.D.C. 1 cycle
P/L = 1.0, L/d = 3.5, P/d = 3.5 T.D.C. P/L = 1.0, L/d = 3.5, P/d = 3.5 P/L = 0.25, L/d = 7.0, P/d = 1.75 P/L = 0.5, L/d = 3.5, P/d = 1.75 P/L = 0.5, L/d = 7.0, P/d = 3.5 P/L = 2.0, L/d = 3.5, P/d = 7.0 P/L = 2.0, L/d = 3.5, P/d = 7.0 Figure 5. The time history of the flow pattern when straying off further from a design point (P/L = 0.25, 0.5 and 2.0 3.2 Examination of the space ratio suitable for
stirring The aspects of the flow of space ratios other than a design
point are shown in figure 4 and figure 5. The aspect of
the flow of the design point is also displayed on both
figures for comparison. In the design point, the
complicated developed flow is formed oscillating one
period. Therefore, the flow patterns of the "initial state",
the "top dead center", the "half cycle", the "bottom dead
center", and the "one period" are shown here, respectively. The conditions currently colored by yellow all over the
figure show the case where the vortices which became 02139-p.4 References 1. T. Monna, Y. Yokoi, Proc. 50th all Japan packing
technical research convention, 265-268 (2012) The "Kanpai-muddler" was the schedule of 20,000
piece creation in the first plan. However, since it was
reputable, additional creation of the " Kanpai-muddler "
was performed, and 80,000 totals were created. 2. Y. Yokoi, K. Hirao, Trans. Jpn Soc. Mech. Eng. 74-
748, 2466-2475 (2008) 3. Y. Yokoi, K. Hirao, Trans. Jpn Soc. Mech. Eng. 77-
775, 590-601 (2011) Figure 8. The surface of the covering letter attached to the
muddler e covering letter attached to the 4. JSME ed., JSME Standard S012, B78(1998) 5. K. Kamemoto, Computational Fluid Dynamics, 2-1,
20-29 (1993) 6. K. Kamemoto, Computational Fluid Dynamics, 2-2,
28-39 (1994) Acknowledgement In order to develop the stirring rod for mixing two liquid
with which densities called sodium carbonate and ume
liquor differ in little stirring operation, verification of the
stirring effect was performed in the numerical simulation
using the vortex method. The following conclusions were
obtained. An author expresses gratitude to Sapporo Breweries, Ltd.,
Daiko Advertising, Inc., and Avalanche, Inc. The
opportunity to which an author carries out research from
the staff of those companies was obtained. And they sent
out the product borne by this research to the market. An
author expresses deep gratitude to the staff members
anew. (1) In a design point, a complicated flow is made from
oscillation of the one period by vortices. And interference
stirring occurs. (2) Adaptation ranges other than the design point are
that the ranges of the value of P/L are 0.8-1.2. (3) The calculation using the vortex method is the
simple technique whose product development is possible
while making it feed back to the design. 02139-p.5 EPJ Web of Conferences is written. Here, the busy condition of the muddler was
also shown. Appendix The data of "the new type muddler" which the author
developed are attached. Figure 6 shows the blueprint and
its finished goods. The material of parts is plastic resin. This article is the goods developed for advertisement
sales promotion of ume liquor. Figure 7 shows the
muddler attached to the package of ume liquor. The sale
was performed by such packaging. Figure 8 shows the
covering letter attached to the muddler. Since the
campaign was performed in Japan, it is written in
Japanese. The muddler is shown in the beer jug which a
model woman (right-hand side) has in a hand. The author
(left-hand side) is also introduced as a designer. Since
"the cheers" was called "Kanpai" in Japan, this muddler
was named the "Kanpai-muddler." Figure 9 is the back of
the covering letter, and the method for use of the muddler Figure 6. The blueprint and finished goods of the muddler Figure 8. The surface of the covering letter attached to the
muddler Figure 9. The back of the covering letter attached to the
muddler and the busy condition of the muddler Figure 6. The blueprint and finished goods of the muddler Figure 7. Packaging of goods Figure 9. The back of the covering letter attached to the
muddler and the busy condition of the muddler Figure 7. Packaging of goods Figure 7. Packaging of goods 02139-p.6
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https://openalex.org/W2914789585
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https://europepmc.org/articles/pmc6433726?pdf=render
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English
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Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β‐cells
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Journal of Cellular and Molecular Medicine
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cc-by
| 8,601
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Correspondence Correspondence
Dusica Maysinger, Department of
Pharmacology and Therapeutics, McGill
University, Montreal, QC, Canada. Email: dusica.maysinger@mcgill.ca Funding information
Canadian Diabetes Society; Canadian
Institutes of Health Research; McGill
University K E Y W O R D S
autophagy, islets, LAMP2, PLIN2, TFEB Received: 27 July 2018 | Revised: 14 December 2018 | Accepted: 29 December 2018
DOI: 10.1111/jcmm.14172 Received: 27 July 2018 | Revised: 14 December 2018 | Accepted: 29 December 2018
DOI: 10.1111/jcmm.14172 Received: 27 July 2018 | Revised: 14 December 2018 | Accepted: 29 December 2018 DOI: 10.1111/jcmm.14172 Type 2 diabetes is associated with suppression of autophagy
and lipid accumulation in β‐cells Jeff Ji1 | Maria Petropavlovskaia2 | Armen Khatchadourian1 | Jason Patapas2 |
Julia Makhlin2 | Lawrence Rosenberg2 | Dusica Maysinger1 Jeff Ji1 | Maria Petropavlovskaia2 | Armen Khatchadourian1 | Jason Patapas2 |
Julia Makhlin2 | Lawrence Rosenberg2 | Dusica Maysinger1 1Department of Pharmacology and
Therapeutics, McGill University, Montreal,
QC, Canada 1Department of Pharmacology and
Therapeutics, McGill University, Montreal,
QC, Canada Abstract Abstract
Both type 2 diabetes (T2D) and obesity are characterized by excessive hyperlipidae‐
mia and subsequent lipid droplet (LD) accumulation in adipose tissue. To investigate
whether LDs also accumulate in β‐cells of T2D patients, we assessed the expression of
PLIN2, a LD‐associated protein, in non‐diabetic (ND) and T2D pancreata. We ob‐
served an up‐regulation of PLIN2 mRNA and protein in β‐cells of T2D patients, along
with significant changes in the expression of lipid metabolism, apoptosis and oxidative
stress genes. The increased LD buildup in T2D β‐cells was accompanied by inhibition
of nuclear translocation of TFEB, a master regulator of autophagy and by down‐regu‐
lation of lysosomal biomarker LAMP2. To investigate whether LD accumulation and
autophagy were influenced by diabetic conditions, we used rat INS‐1 cells to model
the effects of hyperglycaemia and hyperlipidaemia on autophagy and metabolic gene
expression. Consistent with human tissue, both LD formation and PLIN2 expression
were enhanced in INS‐1 cells under hyperglycaemia, whereas TFEB activation and
autophagy gene expression were significantly reduced. Collectively, these results sug‐
gest that lipid clearance and overall homeostasis is markedly disrupted in β‐cells under
hyperglycaemic conditions and interventions ameliorating lipid clearance could be
beneficial in reducing functional impairments in islets caused by glucolipotoxicity. 2Department of Surgery, McGill University,
Montreal, QC, Canada 2.5 | qRT‐PCR For RNA extraction, human pancreatic samples stored at −80°C
in RNAlater were homogenized in RLT buffer and processed in
QiacubeTM (Qiagen, Toronto, ON, Canada) using RNEasy mini kit
according to manufacturer protocol. Quality and integrity of RNA
was assessed by 1.5% agarose gel electrophoresis. For RNA ex‐
traction in cultured cells, INS‐1 were seeded at 2 000 000 cells in
150 mm plates (Sigma) and 48 hours after seeding were exposed to
5 mmol/L or 30 mmol/L [GLU] with or without 500 μmol/L OA, PA
or 250 μmol/L OA + 250 μmol/L PA for 24 hours. Cells were lysed in
RLT buffer and processed as above. Equal amounts of RNA, based
on OD260, were reverse transcribed using oligo‐dT primers and
Omniscript RT kit (Qiagen). One microlitre of cDNA was used for a
20 μL qPCR reaction performed with IQTM SYBR® Green Supermix
(Bio‐Rad, Mississauga, ON, Canada) in CFX96TM Real‐Time System 2.3 | Stable EGFP‐TFEB transfection of INS‐1 cells INS‐1 cells were seeded in 6‐well plates (Starstedt, Montreal, QC,
Canada) and transfected with pEGFP‐N1‐TFEB (CMV promoter,
neomycin resistance) using Lipofectamine 2000 (FischerScientific)
in culture medium for 48 hours. The medium was supplemented
with 400 μg/mL geneticin (Sigma, Oakville, ON, Canada) to select
for resistant cells and subsequently for single colonies by reseeding
into 96‐well plates. EGFP‐positive clones showing functional TFEB
translocation when starved in HBSS for 1 hour at 37°C were cul‐
tured with 200 μg/mL geneticin in the medium. Targeted inactivation or repression of autophagy in β‐cells has
been shown to cause diabetes in animal models. β‐cell–specific Atg7
knockout led to islet degeneration in mice, accumulation of protein
aggregates and decreased insulin production.20 Similarly, β‐cell–spe‐
cific Tsc‐2 knockout, which caused mTORC1 hyperactivation and
repression of autophagy, increased mitochondrial oxidation and ER
stress, resulting in β‐cell failure.21 mTORC1 is a central kinase re‐
sponsible for regulating many aspects of metabolism, energy utili‐
zation and cell growth in response to nutrient abundance within the
cell. A direct effect of mTORC1 activity on LD formation in rat islet
cells has been previously reported.22 mTORC1 inhibits autophagy
partly through phosphorylation of transcription factor EB (TFEB)
which prevents its nuclear translocation. During starvation, mTORC1
is suppressed and TFEB translocates to the nucleus and up‐regulates
genes involved in autophagic and lysosomal production.23 TFEB is
necessary for lipid degradation in the liver24 but its role in human
pancreatic islets in the context of T2D has not been reported. 2.2 | Cell culture INS‐1 rat insulinoma cells (AddexBio, San Diego, CA, USA) were cul‐
tured in RPMI‐1640 media containing 11.1 mmol/L [GLU], 2 mmol/L
L‐glutamine, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 2 g/L
sodium bicarbonate, 10% FBS, 50 μmol/L 2‐mercaptoethanol, 1%
penicillin‐streptomycin (Invitrogen, Waltham, MA, USA) and main‐
tained at 37°C with 5% CO2. 2.4 | FA/BSA complex preparation Oleic acid (OA) (Sigma) and palmitic acid (PA) (Sigma) were dis‐
solved in Krebs‐Ringer bicarbonate buffer complexed with 5%
fatty‐acid free BSA (Sigma) under gentle heating and stirring and
sterile‐filtered through a 0.22 μm filter. FA concentration was quan‐
tified using Wako HR series NEFA‐HR(2) according to manufacturer
instructions. The goal of this study was to investigate the impact of T2D on
LDs, autophagy and islet metabolism by assessing the expression
and localization of PLIN2, TFEB, lysosome‐associated membrane
protein‐2 (LAMP2) and genes associated with metabolism, oxida‐
tive stress, apoptosis and mitochondrial function in human pancre‐
atic tissue from normal and T2D subjects. We have suggested that
nutrient overload in diabetes causes LD accumulation due to de‐
creased TFEB activation and suppression of autophagy and tested
this hypothesis in vitro, using the rat insulinoma β‐cell line INS‐1. and as a platform for the recruitment of signalling molecules.6,7
Perilipin associated proteins (PAT) coat and stabilize the surface of
LD by reducing lipases access to the lipid core. Perilipin‐2 (PLIN2)
is the main housekeeping LD protein that is ubiquitously expressed
and used as a marker for LDs in many human and animal tissues.8
PLIN5 is highly expressed in oxidative tissues and plays multiple
metabolic roles such as fatty acid (FA) mobilization and has been
shown to regulate post‐prandial insulin secretion in β‐cells.9,10 High
cellular lipid uptake (eg, post‐prandial period) increases PLIN2 (and
other PAT proteins) expression and triggers their recruitment to
the LD surface.11 These events temporarily reduce the cytosolic FA
concentration. However, chronic lipid overload can lead to the for‐
mation of abnormally large and unstable LDs prone to FA spillover,
which could lead to endoplasmic reticulum (ER) stress, induction of
apoptosis and insulin resistance in β‐cells.12,13 The timely removal of
excessively ‘leaky’ LDs is therefore necessary to prevent lipotoxicity
in β‐cells. Aside from cytosolic lipase activity, lipophagy is emerging
as an important mechanism for LD degradation using the autophagic
pathway14,15 and is suggested as a general mechanism for degrading
LD in diverse cell populations.16-19 RNAlaterTM (Qiagen, Toronto, ON, Canada) for RNA extraction or
fixed in 10% formalin (Fisher Scientific, Ottawa, ON, Canada) and
paraffin‐embedded for immunolabelling (Pathology Unit, Montreal
General Hospital, Montreal, Quebec, Canada). Donor information is
summarized in Table S1. The study consisted of 22 ND and 17 type
2 diabetic patients. 1 | INTRODUCTION that excessive lipids and the ensuing lipotoxicity play a major role in
these processes by promoting insulin resistance and β‐cell dysfunc‐
tion.3 However, the exact impact of lipid overload on β‐cell dysfunc‐
tion remains poorly understood. T2D is characterized by β‐cell failure, insulin resistance, hypergly‐
caemia, and hyperlipidaemia. β‐cells are nutrient sensors that reg‐
ulate insulin secretion in response to elevated levels of glucose and
lipids.1 Nutrient overload is viewed as the main cause of insulin resis‐
tance that increases the demand on β‐cells to secrete more insulin. If β‐cells fail to adapt to insulin resistance and become dysfunctional,
glucose intolerance and overt diabetes develops.2 Evidence suggests LDs are dynamic organelles implicated in metabolic disorders
including obesity and T2D, which are marked by abnormal LD ac‐
cumulation.4,5 Structurally, LDs are natural micelles composed of a
hydrophobic core containing triglycerides, cholesterol esters and a
corona made of phospholipids that can serve as lipid storage sites J Cell Mol Med. 2019;23:2890–2900. J Cell Mol Med. 2019;23:2890–2900. JI et al. 2891 2.11 Cells were seeded into 24‐well plates (Starstedt) at 150 000 cells
per well for 24 hours treatment or 75 000 cells per well for 48 hours
treatment, 24 hours post‐seeding, cells were incubated in treatment
media for 24 hours/48 hours. Cell count analysis: Cells were incu‐
bated with 10 μmol/L Hoechst 33342 for 10 minutes followed by
imaging using a fluorescence microscope with a 4X objective. Four
images per well were taken to represent the average number of
cell in the well. The number of nuclei per field were counted using
ImageJ. MTT assay 500 ug/mL MTT (Sigma) was added to cells and
incubated for 40 minutes at 37°C. After incubation, the media were
aspirated and DMSO was added to each well to dissolve formazan
crystals. The absorbance was read at 595 nm using a plate reader. 2.12 | Microscopy Confocal imaging was performed with Zeiss LSM 510 NLO inverted
confocal microscope using a Plan Achromat 63X/1.4 Oil DIC objec‐
tive (Carl Zeiss Canada Ltd., Toronto, ON, Canada). All images were
acquired at a resolution of 1024 × 1024 pixels (x,y). Fluorescence mi‐
croscopy was performed with a Leica DMI4000 inverted microscope
(Leica Microsystems Inc, Concord, ON, Canada) using a 4×, 10× or
63× oil objective captured using a Leica DFC345 FX camera (Leica
Microsystems Inc). 2.7 | Lipid droplet staining and quantification INS‐1 cells were seeded onto poly‐d‐lysine coated coverslips and in‐
cubated for 48 hours before treatment with 500 μmol/L OA in nor‐
moglycaemic (5 mmol/L [GLU]) or hyperglycaemic (30 mmol/L [GLU])
medium for 24 hours. Torin‐1 (Tocris, Bristol, UK) was added 3 hours
before and with the 24 hours treatment. Cells were fixed in 2% PFA
in PBS+ (with 0.5 mmol/L MgCl2, 1 mmol/L CaCl2) for 10 minutes at
room temperature (RT). After washing with PBS+ cells were labelled
with 20 μmol/L BODIPY 493/503 and 10 μmol/L Hoechst 33342 for
10 minutes at RT and washed with PBS+. Coverslips were mounted
onto microscope slides with non‐hardening EverBrite Mounting me‐
dium (Biotium, Fremont, CA, USA) and the edges sealed with nail
polish. 2.10 After treatment, cells were fixed in 2% PFA in PBS+ (with 0.5 mmol/L
MgCl2, 1 mmol/L CaCl2) for 10 minutes at RT and permeabilized with
0.1% Triton X‐100 in PBS+. Cells were blocked with 10% goat serum
and incubated in primary antibody overnight at 4°C and incubated in
secondary antibody for 1 hour at RT. Coverslips were mounted onto
microscope slides using Aqua Poly/Mount. 2.1 | Human pancreatic tissue Adult human pancreata were obtained from Quebec Transplant with
prior consent for research use. Pancreatic tails were preserved in JI et al. JI et al. 2892 2.6 Tissue sections (5 μm) of pancreatic samples were deparaffinized in
xylene and rehydrated in ethanol‐water mixtures. Antigen retrieval
was performed in 95°C citrate buffer (10 mmol/L, 0.05% Tween‐20,
pH 6.0) for 40 minutes and was followed by blocking in 2% horse
serum/10% goat serum/1 mmol/L HEPES/0.1% sodium azide/0.3%
Triton X‐100 in HBSS for 2 hours. Incubation with primary an‐
tibodies (Table S3) at 4°C overnight was followed by secondary
antibodies, staining of nuclei in Hoechst 33342 or DAPI and mount‐
ing using Aqua‐Poly/Mount (Polysciences, Warrington, PA, USA). Quantification was performed in ImageJ by assessing the average
fluorescence intensity normalized to the area and expressed as ar‐
bitrary units. 2.8 | TFEB translocation test INS‐1‐TFEB‐EGFP cells seeded in 24‐well plates (Starstedt) were
treated in 5 mmol/L or 30 mmol/L [GLU] in the presence or ab‐
sence of 500 μmol/L OA, PA or 250 μmol/L OA + 250 μmol/L PA
for 48 hours. After treatment, cells were starved for 1 hour in HBSS,
fixed in 2% PFA for 10 minutes at RT and washed twice with PBS+. After labelling nuclei with 10 μmol/L Hoechst 33342, cells were
imaged by fluorescence microscopy and the images were analysed
using ImageJ. Four fields were randomly chosen per well and EGFP‐
positive cells were randomly chosen per field. TFEB‐EGFP nuclear
translocation was assessed as the ratio of the area of normalized
EGFP fluorescence intensity in the nucleus over the cytosol. A
higher ratio indicates greater nuclear TFEB‐EGFP translocation. 2.9 | Western blot After treatment, total cellular protein was extracted using RIPA
buffer containing protease inhibitors. Protein concentration was
quantified using the Pierce BCA Protein Assay (ThermoFisher,
Waltham, MA, USA). Proteins were subjected to SDS‐PAGE and
transferred onto PVDF membrane. Membranes were blocked with
5% milk/TBST and incubated with primary antibodies followed by
horseradish peroxidase‐conjugated secondary antibodies. Blots
were developed using ClarityTM Western ECL Substrate (BIO‐RAD,
Hercules, CA, USA) and imaged using Amersham Imager 600 (GE
Healthcare, Saint‐Laurent, QC, Canada). (Bio‐Rad) and primer pairs shown in Table S2. Multiple plates of ex‐
perimental data, run with an inter‐plate calibrator, were combined
into gene studies using glyceraldehyde 3‐phosphate dehydrogenase
(GAPDH), β‐actin and succinate dehydrogenase complex flavopro‐
tein (SDHA) as reference genes in human samples and β‐actin and
β‐tubulin in INS‐1 samples (all M < 0.5). Fold change in gene expres‐
sion (∆∆Ct) were obtained using the data analysis software CFX
Manager3.1. 2.13 Statistical significance was considered at P < 0.05. Statistical
analysis was done using R v3.2.2. 2.13 All data are expressed as means ± standard error of the mean (SEM). Statistical differences were analysed by either student's t test or
two‐way analysis of variance (ANOVA) followed by post‐hoc Tukey's JI et al. JI et al. 2893 at P < 0.05. Statistical
y increased in
(Figure 1C). This abnormal accumulation of PLIN2 and PLIN5 and, by
extension, of LDs in β‐cells is similar to that in hepatocytes in fatty liver
disease or macrophages in atherosclerosis, where it is usually linked
to the abnormal lipid catabolism.27 This prompted us to investigate
whether the observed LD accumulation in T2D is accompanied and
perhaps caused by an impaired processing of lipids in β‐cells. FI G U R E 1 Expression of Lipid Droplet
markers in the human pancreas. (A)
Gene expression analysis of lipid droplet
regulation genes from pancreatic tails
(CAV1 = caveolin 1, PLIN2 = perilipin 2,
FSP27 = fat specific protein 27). ND = 11
patients, T2D = 10 patients, graph
represents average fold change ± standard
error of the mean (SEM) in gene
expression over ND. (B) Pictomicrograph
of sections double immunolabelled with
PLIN2 (red), C‐peptide (green) and stained
with Hoechst 33342 (blue). Images are
representative from four different donors
per group. White arrows point to acinar
tissue (exocrine tissue) which did not up‐
regulate PLIN2. (C) Photomicrograph and
fluorescence quantification of sections
labelled with PLIN5 (red), C‐peptide
(green) and stained with Hoechst 33342
(blue). Quantified from >21 islets from
two different donors per group. Statistical
analysis was evaluated by Student's t
test and significance is indicated by *
(compared to ND), *P < 0.05 (Figure 1C). This abnormal accumulation of PLIN2 and PLIN5 and, by
extension, of LDs in β‐cells is similar to that in hepatocytes in fatty liver
disease or macrophages in atherosclerosis, where it is usually linked
to the abnormal lipid catabolism.27 This prompted us to investigate
whether the observed LD accumulation in T2D is accompanied and
perhaps caused by an impaired processing of lipids in β‐cells. (Figure 1C). This abnormal accumulation of PLIN2 and PLIN5 and, by
extension, of LDs in β‐cells is similar to that in hepatocytes in fatty liver
disease or macrophages in atherosclerosis, where it is usually linked
to the abnormal lipid catabolism.27 This prompted us to investigate
whether the observed LD accumulation in T2D is accompanied and
perhaps caused by an impaired processing of lipids in β‐cells. test. 3.2 | TFEB activation is suppressed and LAMP2
abundance is reduced in T2D (A) Representative images
of TFEB immunolabelling in ND, and
T2D patients and quantification of TFEB
nuclear fluorescence of cells within
islets. Islets regions (encircled) were
traced from bright‐field images based
on morphology and nuclei were stained
with Hoechst 33342. Quantifications
are from ND = 8 patients (471 cells
measured), T2D = 6 patients (416 cells
measured). (B) Representative images
of LAMP2 immunolabelling in ND and
T2D patients and quantification of
LAMP2 fluorescence within the islet. Quantifications are from ND = 6 patients
(48 islets measured), T2D = 6 patients
(61 islets measured). Statistical analysis
was evaluated by Student's t test and
significance is indicated by * (compared to
ND), *P < 0.05, error bars = SEM receptor‐alpha (PPAR‐α, 2.1 ± 0.3 ↑, P < 0.01) (Figure 3E), while
PPAR‐γ expression did not differ between T2D and ND. Together,
these changes in mRNA level in mitochondria, anti‐ROS defense and
apoptosis‐associated genes in the pancreata of T2D subjects, sug‐
gest the activation of compensatory mechanisms to withstand meta‐
bolic stress and that the effects of diabetes were likely still within
the adaptable range and have not reached the stage of irrevocable
damage. To assess the morphology and architecture of the islets, we
immunostained for glucagon (α‐cells), insulin (β‐cells) and somato‐
statin (δ‐cells) using sections from several patients in both ND and
T2D groups. Both ND and T2D islets contained similar distribution
of α‐, β‐ and δ‐cells (Figure S2), suggesting that the altered metabolic
profiles in T2D subjects were not caused by gross morphological
changes. 3.3 | Expression of islet metabolism, redox and
apoptosis related genes is altered in T2D Since T2D has a major impact on islet metabolism, we examined
the expression of pancreatic genes regulating mitochondrial func‐
tion, apoptosis and oxidative stress, as illustrated in Figure 3. Among
mitochondrial genes, there was a significant up‐regulation in the
expression of Carnitine palmitoyltransferase 1A (CPT1A), a crucial
FA transporter on the surface of mitochondria (3 ± 0.5 ↑, P < 0.01,
Figure 3B). This suggests an increase in β‐oxidation to combat the
increased lipid load. Elevated β‐oxidation would increase the trans‐
membrane potential of the mitochondria. Accordingly, Uncoupling
protein 2 (UCP2) was also up‐regulated in T2D (1.7 ± 0.2 ↑, P < 0.05)
suggesting an increase in non‐ATP coupled proton leak (Figure 3B). 3.2 | TFEB activation is suppressed and LAMP2
abundance is reduced in T2D We assessed whether T2D leads to changes in LD‐associated pro‐
teins: PLIN2, caveolin (CAV1) and fat specific protein 27 (FSP27) by
qRT‐PCR (Figure 1A). PLIN2 mRNA was significantly up‐regulated
in T2D (2.5 ± 0.3 ↑ fold increase, P < 0.001) (Figure 1A). However,
there was no significant difference in gene expression of other LD‐
associated proteins – CAV1, which plays an important role in cho‐
lesterol transport, lipogenesis and LD biogenesis,25 or FSP27, which
promotes LD growth by lipid exchange and fusion.26 We evaluated the homeostatic clearance of lipids by autophagy
by focusing on the activation of TFEB. TFEB nuclear transloca‐
tion gauged by immunofluorescence was significantly decreased
in islets from T2D donors compared to ND (3 ± 0.01 ↓, P < 0.001)
(Figure 2A), which suggested down‐regulation of autophagic genes. Since TFEB controls lysosome‐related genes, we quantified LAMP2
protein, a structural marker for lysosomes and found that it was sig‐
nificantly decreased in T2D compared to ND (3.3 ± 0.2 ↓, P < 0.001,
Figure 2B). These data support our hypothesis that T2D‐associated
reduction in islet TFEB and subsequently in lysosomal activity led to
suppression of autophagy. Immunostaining of pancreatic sections for the LD‐associated pro‐
teins showed a marked increase in PLIN2 protein in T2D islets, and
demonstrated its predominant localization in β‐cells based on co‐stain‐
ing with C‐peptide (Figure 1B), whereas it was undetectable in the
acinar tissue. PLIN5 was also up‐regulated in β‐cells from T2D donors JI et al. JI et al. 2894 2894 |
JI et al. FI G U R E 2 Autophagy markers in
human islets. (A) Representative images
of TFEB immunolabelling in ND, and
T2D patients and quantification of TFEB
nuclear fluorescence of cells within
islets. Islets regions (encircled) were
traced from bright‐field images based
on morphology and nuclei were stained
with Hoechst 33342. Quantifications
are from ND = 8 patients (471 cells
measured), T2D = 6 patients (416 cells
measured). (B) Representative images
of LAMP2 immunolabelling in ND and
T2D patients and quantification of
LAMP2 fluorescence within the islet. Quantifications are from ND = 6 patients
(48 islets measured), T2D = 6 patients
(61 islets measured). Statistical analysis
was evaluated by Student's t test and
significance is indicated by * (compared to
ND), *P < 0.05, error bars = SEM 2894 | FI G U R E 2 Autophagy markers in
human islets. 3.2 | TFEB activation is suppressed and LAMP2
abundance is reduced in T2D UCP2 up‐regulation was previously reported in response to oxi‐
dative stress in β‐cells.28 We next evaluated expression of several
genes related to anti‐oxidant defence and apoptosis and found sig‐
nificant up‐regulation of glutathione peroxidase 1 (GPX1, 3.8 ± 0.3
↑, P < 0.001) and heme oxygenase 1 (HMOX1, 2.2 ± 0.3 ↑, P < 0.01)
in T2D pancreata (Figure 3C), which correlated with an increased
immunofluorescence for Nrf2 (Figure S1), a transcription factor
regulating GPX1, HMOX1 and other genes involved in oxidant de‐
fense and redox signalling.29 One of these genes, p62 (SQSTM1),
is directly involved in autophagy and is used as a measure of au‐
tophagy flux,30 was also up‐regulated in T2D (Figure 3). Expression
of anti‐apoptotic gene BCL2 was strongly increased (11.3 ± 4.6 ↑,
P < 0.05) (Figure 3D), however pro‐apoptotic gene BAX was also up‐
regulated (2.3 ± 0.3 ↑, P < 0.001). Finally, we examined transcription
factors linked to metabolism. T2D leads to significant changes in the
expression of transcription factors forkhead box protein 1 (FOXO1,
1.7 ± 0.3 ↑, P < 0.05), and peroxisome proliferator‐activated 3.4 | Hyperglycaemia increases LD and Plin2 in
INS‐1 CPT1A = carnitine palmitoyltransferase 1A couples carnitine to acyl groups which enables fatty acids to enter the mitochondria and undergo
β‐oxidation. UCP2 = uncoupling protein 2 enables protons to flow back into the mitochondria matrix without oxidative phosphorylation
which generates heat instead of ATP, p62 = sequestome 1, delivers cargo to autophagosomes for degradation, regulates Nrf2 activity
by binding with Keap1. HMOX1 = heme oxygenase 1, converts denatured heme, oxygen and NADPH to Billiverdin, carbon monoxide
and iron. It is an anti‐oxidant gene up‐regulated by oxidative stress. GPX1 = glutathione peroxidase 1 is an anti‐oxidant enzyme which
scavenges hydrogen peroxide. BAX = BCL2 associated X, BCL2 = BCL2 are respectively pro‐apoptotic and anti‐apoptotic regulators. FOXO1 = forkhead box protein 1, PPAR‐α = peroxisome proliferator activated receptor‐α, PPAR‐γ = peroxisome proliferator activated
receptor‐γ are transcription factors which regulates many facets of metabolism. Gene expression analysis of (B) mitochondria, (C) oxidative
stress/oxidant defense, (D) apoptosis‐related, (E) metabolism regulation related genes from pancreatic tails. Graphs represent average fold
increase ± standard error of the mean (SEM) in gene expression between groups over ND. N = 11 patients for ND, N = 10 patients for T2D. Statistical analysis was evaluated by Student's t test and significance is indicated by * (compared to ND), *P < 0.05 translational level and that Plin2 alone is not sufficient for LD for‐
mation and growth. (Figure S3C,D). INS‐1 cells treated with OA under hyperglycae‐
mia showed a considerable increase in the number of LD (five‐
fold increase in LD area compared to normoglycaemia, P < 0.01,
Figure 4A,B). Furthermore, two‐way ANOVA analysis revealed sig‐
nificant synergistic effect (P < 0.05) between hyperglycaemia and
OA treatment on LD area per cell. The increased LD storage due
to hyperglycaemia was partially abolished by pre‐treatment with
the mTOR inhibitor/autophagy inducer Torin‐1, which suggests
that activation of autophagy could reduce lipid accumulation. In
addition, we show that hyperglycaemia alone was the main factor
leading to significant up‐regulation of Plin2 (P < 0.001, Figure 4C),
while FA alone (normoglycaemic conditions) only slightly elevated
Plin2 mRNA. Interestingly, FA treatment under hyperglycaemia
lowered the effect of hyperglycaemia on Plin2 expression. This
discrepancy between Plin2 mRNA and the LD number under
FA+30 mM [GLU] may indicate that Plin2 is also regulated at the 3.4 | Hyperglycaemia increases LD and Plin2 in
INS‐1 Although pancreatic dysfunction in T2D and obesity is multifac‐
torial and involves a host of metabolic and hormonal changes,
two major contributors are hyperglycaemia and hyperlipidaemia. To better understand the observed changes in LDs and in gene
expression in T2D pancreata, we examined the direct effects of
hyperglycaemia and hyperlipidaemia on LD and autophagy in vitro
using INS‐1 cells. 5 mmol/L glucose was taken as normoglycaemia
and 30 mmol/L glucose as hyperglycaemia. Hyperlipidaemia was
induced by 500 μmol/L OA, 500 μmol/L PA or 250 μmol/L oleic
acid +250 μmol/L palmitic acid (OA+PA), as used by others.22,31
This was the maximal dose which did not significantly reduce cell
number or induce apoptosis (Figure S3A,B,E) yet it did increase mi‐
tochondrial metabolic activity relative to 5 mmol/L [GLU] control 2895 JI et al. FI G U R E 3 Metabolism, redox and apoptosis related gene expression in human pancreata. (A) Schematic of the genes assayed below. CPT1A = carnitine palmitoyltransferase 1A couples carnitine to acyl groups which enables fatty acids to enter the mitochondria and undergo
β‐oxidation. UCP2 = uncoupling protein 2 enables protons to flow back into the mitochondria matrix without oxidative phosphorylation
which generates heat instead of ATP, p62 = sequestome 1, delivers cargo to autophagosomes for degradation, regulates Nrf2 activity
by binding with Keap1. HMOX1 = heme oxygenase 1, converts denatured heme, oxygen and NADPH to Billiverdin, carbon monoxide
and iron. It is an anti‐oxidant gene up‐regulated by oxidative stress. GPX1 = glutathione peroxidase 1 is an anti‐oxidant enzyme which
scavenges hydrogen peroxide. BAX = BCL2 associated X, BCL2 = BCL2 are respectively pro‐apoptotic and anti‐apoptotic regulators. FOXO1 = forkhead box protein 1, PPAR‐α = peroxisome proliferator activated receptor‐α, PPAR‐γ = peroxisome proliferator activated
receptor‐γ are transcription factors which regulates many facets of metabolism. Gene expression analysis of (B) mitochondria, (C) oxidative
stress/oxidant defense, (D) apoptosis‐related, (E) metabolism regulation related genes from pancreatic tails. Graphs represent average fold
increase ± standard error of the mean (SEM) in gene expression between groups over ND. N = 11 patients for ND, N = 10 patients for T2D. Statistical analysis was evaluated by Student's t test and significance is indicated by * (compared to ND), *P < 0.05 FI G U R E 3 Metabolism, redox and apoptosis related gene expression in human pancreata. (A) Schematic of the genes assayed below. 3.5 | Hyperglycaemia inhibits TFEB nuclear
translocation and down‐regulates autophagy Given that TFEB nuclear translocation and LAMP2 expression are
suppressed in the islets of T2D, we hypothesized that hyperglycae‐
mia and hyperlipidaemia may inhibit TFEB nuclear translocation and
autophagy. To test our hypothesis, INS‐1 cells stably transfected
with TFEB‐EGFP were exposed to 5 or 30 mmol/L [GLU] in the
presence and absence of FAs for 48 hours and then subjected to
starvation to measure TFEB nuclear translocation. Treatment with
OA and OA+PA under hyperglycaemic conditions significantly re‐
duced TFEB nuclear translocation (Figure 5A,B). These results
show that the combination of high glucose concentration with lipid 2896 | | 2896 JI et al. FI G U R E 4 Effect of hyperglycaemia and hyperlipidaemia on LD and LD‐associated proteins in INS‐1. (A) Representative image and (B)
quantification of INS‐1 pre‐treated with 1 μmol/L Torin‐1 for 3 h before treatment with 5 mM (normoglycaemic) [GLU] or 30 mM [GLU]
(hyperglycaemic) medium ±500 μM oleic acid (OA) for 24 h and stained for LD with BODIPY 493/503 (green) and Hoechst 33342 (blue). LD
was quantified as the total area of LD per cell and represents the mean from three independent experiments. (C) Gene expression analysis
of INS‐1 Plin2 under 5 mmol/L [GLU], 30 mmol/L [GLU]), treated with 500 μmol/LM OA, 500 μmol/L palmitic acid (PA) or 250 μmol/L
OA + 250 μmol/L PA. Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups from
three independent experiments. Statistical analysis was evaluated by two‐way ANOVA followed by Tukey's test. Dual arrows * indicate a
significant main effect due to hyperglycaemia (P < 0.05), * beside each point indicate significant difference (P < 0.05) compared to 5 mmol/L
[GLU] Ctrl FI G U R E 4 Effect of hyperglycaemia and hyperlipidaemia on LD and LD‐associated proteins in INS‐1. (A) Representative image and (B)
quantification of INS‐1 pre‐treated with 1 μmol/L Torin‐1 for 3 h before treatment with 5 mM (normoglycaemic) [GLU] or 30 mM [GLU]
(hyperglycaemic) medium ±500 μM oleic acid (OA) for 24 h and stained for LD with BODIPY 493/503 (green) and Hoechst 33342 (blue). LD
was quantified as the total area of LD per cell and represents the mean from three independent experiments. (C) Gene expression analysis
of INS‐1 Plin2 under 5 mmol/L [GLU], 30 mmol/L [GLU]), treated with 500 μmol/LM OA, 500 μmol/L palmitic acid (PA) or 250 μmol/L
OA + 250 μmol/L PA. 3.5 | Hyperglycaemia inhibits TFEB nuclear
translocation and down‐regulates autophagy Graphs represent average fold increase ± standard error of the mean (SEM) in gene expression between groups from
three independent experiments. Statistical analysis was evaluated by two‐way ANOVA followed by Tukey's test. Dual arrows * indicate a
significant main effect due to hyperglycaemia (P < 0.05), * beside each point indicate significant difference (P < 0.05) compared to 5 mmol/L
[GLU] Ctrl overload inhibits TFEB activation. Hyperglycaemia lowered the
expression of Tfeb, Lamp1, and Lc3 (Figure 5C), meaning that au‐
tophagy was down‐regulated at the transcriptional (TFEB), lysoso‐
mal (Lamp1) and autophagosome assembly (LC3) levels. Expression
of p62, a scaffold protein known to bind ubiquitin and LC3 and
degrade by autophagy,30 followed the same pattern as LC3 except
for PA+30 mmol/L [GLU] when no down‐regulation by high glucose
was observed. Since p62 protein accumulates when autophagy is
inhibited, we used p62 accumulation (seen as aggregated puncta)
as a measure of autophagic blockage. Chloroquine (inhibitor of
autophagy) was used as a positive control for autophagy blockage
(Figure S4A). PA+30 mmol/L [GLU] significantly increased p62 lev‐
els and aggregation (Figure S4A,B), while OA+PA+30 mmol/L [GLU]
had a lesser effect. Inhibition of autophagy was further confirmed
by a significant decrease in LC3BII/I ratio under hyperglycaemia
(Figure S4C). Given that down‐regulation of autophagy is known to
dysregulate insulin production and islet metabolism, we performed
qRT‐PCR for Ins1 and Ins2, Ppar‐α, Ppar‐γ, Ucp2 in INS‐1 cells ex‐
posed to 5 or 30 mmol/L [GLU] with or without OA, PA, OA+PA
(Figure S5). Under normoglycaemia, FA treatments stimulated a significant increase in insulin expression, whereas under hypergly‐
caemia FA had a smaller effect, suggesting that prolonged hypergly‐
caemia can reduce insulin production. This corroborates with the
recently reported significant reduction in insulin expression in INS‐1
cells after 14‐day exposure to 22.4 mmol/L [GLU] due to methyla‐
tion silencing of the insulin promoter.32 4 | DISCUSSION (C) Gene
expression analysis of INS‐1 Tfeb,
Lamp1, Sqstm1 and Lc3 under 5 mmol/L
[GLU]/30 mmol/L [GLU]), treated with
500 μmol/L OA, 500 μmol/L palmitic
acid (PA) or 250 μmol/L OA +250 μmol/L
PA. Graphs represent average fold
increase ± standard error of the mean
(SEM) in gene expression between groups
from three independent experiments. Statistical analysis was evaluated by two‐
way ANOVA followed by Tukey's test. Dual arrows * indicate significant main
effect due to hyperglycaemia (P < 0.05),
* beside each point indicate significant
difference (P < 0.05) compared to
5 mmol/L [GLU] Ctrl up‐regulated PLIN2 and substantially increased OA‐induced LD for‐
mation. The primary function of LDs is to store lipids for energy but
also to prevent acute lipotoxicity by sequestering otherwise toxic
FA.4 In isolated rat β‐cells, the accumulation of LD was inversely pro‐
portional to the cytotoxicity for a given FA, for example palmitate
lead to lower triglyceride accumulation and greater cell toxicity com‐
pared to oleate.34 However, nutrient‐induced LD accumulation is
also associated with β‐cell dysfunction.22 Reduction in LD by PLIN2
knockdown in β‐cells lead to decreased ER stress, enhanced autoph‐
agy and decreased β‐cell apoptosis in diabetic animal models.35This
is consistent with our observations of the changes in autophagy‐re‐
lated proteins TFEB and LAMP2 and in the gene expression in T2D
pancreata suggesting the activation of compensatory mechanisms
to withstand metabolic stress. For instance, to reduce the ATP/ADP
ratio under hyperglycaemic conditions, mitochondria can increase
proton leakage through uncoupling proteins. We report an increase
in both UCP2 and CPT1A expression in the pancreas of T2D patients which suggests an increase in both FA mitochondrial import and in
β‐oxidation. We also observed a significant increase in mitochondrial
metabolic activity in INS‐1 exposed to hyperglycaemic conditions
after 24 and 48 hours. In this study, PLIN2 protein was used as a LD marker to gauge the
size of the LD pool. 4 | DISCUSSION Results from this study show that hyperglycaemia and hyperlipi‐
daemia in humans and rat insulin‐producing cells (INS‐1) lead to: (1)
an up‐regulation of LD‐associated protein PLIN2, (2) a significant
decrease in TFEB activity and in lysosomal biomarker LAMP2, con‐
sistent with inhibition of autophagy and (3) dysregulation of genes
implicated in lipid metabolism, mitochondrial function and cell
survival. A schematic of the proposed mechanisms is illustrated in
Figure 6. Under physiological conditions, β‐cells preferentially use FA over
glucose through β‐oxidation.33 We showed that hyperglycaemia JI et al. JI et al. 2897 OA‐induced LD for‐
which suggests an increase in both FA mitochondrial import and in FI G U R E 5 TFEB translocation and
autophagy‐associated gene expression
in INS‐1. (A) INS‐1‐TFEB‐EGFP was
cultured for 48 h in normoglycaemia
(5 mmol/L [GLU]), hyperglycaemia
(30 mmol/L [GLU]) and treated with
500 μmol/L OA, 500 μmol/L PA, or
250 μmol/L OA + 250 μmol/L PA and
starved for 1 h in HBSS to induce TFEB
nuclear translocation. (A) Representative
images, (B) quantification of nuclear/
cytosolic TFEB fluorescence ratio. n > 50 cells from three independent
experiments. White arrows point to
TFEB nuclear translocation. (C) Gene
expression analysis of INS‐1 Tfeb,
Lamp1, Sqstm1 and Lc3 under 5 mmol/L
[GLU]/30 mmol/L [GLU]), treated with
500 μmol/L OA, 500 μmol/L palmitic
acid (PA) or 250 μmol/L OA +250 μmol/L
PA. Graphs represent average fold
increase ± standard error of the mean
(SEM) in gene expression between groups
from three independent experiments. Statistical analysis was evaluated by two‐
way ANOVA followed by Tukey's test. Dual arrows * indicate significant main
effect due to hyperglycaemia (P < 0.05),
* beside each point indicate significant
difference (P < 0.05) compared to
5 mmol/L [GLU] Ctrl FI G U R E 5 TFEB translocation and
autophagy‐associated gene expression
in INS‐1. (A) INS‐1‐TFEB‐EGFP was
cultured for 48 h in normoglycaemia
(5 mmol/L [GLU]), hyperglycaemia
(30 mmol/L [GLU]) and treated with
500 μmol/L OA, 500 μmol/L PA, or
250 μmol/L OA + 250 μmol/L PA and
starved for 1 h in HBSS to induce TFEB
nuclear translocation. (A) Representative
images, (B) quantification of nuclear/
cytosolic TFEB fluorescence ratio. n > 50 cells from three independent
experiments. White arrows point to
TFEB nuclear translocation. 4 | DISCUSSION PLIN2 is actively degraded in the cytosol when
it is not on the surface of LD.36 Interestingly, a number of studies re‐
port that PLIN2 has additional functions not directly related to lipid
accumulation, such as mediating Wnt/LiCl signalling,37 activation of
ER and unfolded protein response in β‐cells (UPR),35 and inhibition
of glucose uptake through interactions with SNAP23.38 Expression
of PLIN2 is up‐regulated by lipids,39 ROS,40 ER stress and UPR.35
We found that 30 mmol/L [GLU] alone was the strongest inducer
of PLIN2 expression in INS‐1, whereas FA alone did not have an ef‐
fect (Figure 4). We were surprised to see no effect of FA on PLIN2
mRNA in low glucose, contrary to the notion that PLIN2 expression
is up‐regulated by lipids.39 However, the differing culturing glucose 2898 | 2898 JI et al. FI G U R E 6 Schematic for dysregulated TFEB and lipid droplet accumulation during hyperglycaemia and hyperlipidaemia. Elevated glucose
and FFA increase mTORC1 activity which leads to greater suppression of TFEB through inhibitory phosphorylation. As a consequence,
LDs accumulate inside of β‐cells due to increased lipid load as well as decreased lipophagy due to the down‐regulation of autophagy and
lysosomal biogenesis FI G U R E 6 Schematic for dysregulated TFEB and lipid droplet accumulation during hyperglycaemia and hyperlipidaemia. Elevated glucose
and FFA increase mTORC1 activity which leads to greater suppression of TFEB through inhibitory phosphorylation. As a consequence,
LDs accumulate inside of β‐cells due to increased lipid load as well as decreased lipophagy due to the down‐regulation of autophagy and
lysosomal biogenesis both a mouse model of type 1 diabetes (Akita mice) and in obese
patients.47 concentration and different treatment times complicates the com‐
parison between our study and that of Faleck et al. 39 Active mTORC1 up‐regulates anabolic processes and suppresses
autophagy. Vernier et al have previously demonstrated that hyper‐
glycaemia and hyperlipidaemia lead to increased mTORC1 activ‐
ity.22 Our data on inhibition of LDs in 30 mmol/L [GLU]+OA treated
cells by Torin‐1 are consistent with those findings. As an extension,
we found a decreased rate of starvation‐induced TFEB nuclear
translocation in β‐cells pre‐treated with elevated glucose and FA. Hyperactivation of mTORC1 may be a general metabolic dysregu‐
lation due to nutrient overload. 4 | DISCUSSION Rapamycin inhibition of mTORC1
reversed insulin resistance in adipose tissue, skeletal muscle and
liver in hyperinsulinaemic rats.48 Importantly, rapamycin inhibition
of mTORC1 greatly diminished LD accumulation in rodent β‐cells.22
Although abnormally active mTORC1 leads to deleterious diabetic‐
like symptoms, normal mTORC1 function is still critical for β‐cell pro‐
liferation and metabolic function. Long‐term rapamycin treatment
leads to worsened glucose intolerance, lipid accumulation in the liver
and β‐cell loss in diabetic animals.49,50 Whether increased TFEB ac‐
tivation can rescue β‐cell loss and improve insulin sensitivity has yet
to be explored. In both genetic and diet‐induced models of obesity,
TFEB overexpressing mice had lower blood glucose levels, improved
glucose tolerance and decreased fat gain compared to WT, whereas
TFEB‐knockout mice experienced increased fat gain.24 Interestingly,
TFEB activity is increased during physical exercise and is necessary
for the beneficial effects of exercise such as increased FA oxidation,
insulin response and improved mitochondrial function in skeletal
muscle.51 Up‐regulation of PLIN2 in T2D patients is likely caused by a com‐
bination of hyperglycaemia, hyperlipidaemia, inflammation and oxi‐
dative stress, among others. As a chronic phenomenon, prolonged
accumulation of PLIN2 and LD can contribute to ER stress and β‐cell
loss. Consistent with this, we show an increase in pro‐apoptotic gene
BAX in T2D as well as up‐regulation of anti‐apoptotic gene BCL2 and
oxidant defense genes (GPX1, HMOX1, NRF2) to counteract ROS
induced by ER stress. The PLIN family of proteins are also known
to regulate the activity of a multitude of LD associated proteins,41
including lipases such as ATGL42 and HSL.43 Future studies should
examine the expression and activity of LD associated proteins such
as lipid synthesis enzymes, lipases and trafficking proteins to com‐
pletely characterize the regulation of lipid storage and breakdown
in T2D. Our data, from T2D samples and INS‐1 cells, are consistent
with inhibition of autophagy by hyperglycaemia and hyperlipidae‐
mia. Autophagy (and lipophagy) plays multiple roles in maintaining
cellular homeostasis.44 TFEB is a master regulator of autophagic
genes and lysosome biogenesis. A previous study has reported
that mice fed a high‐fat diet have reduced TFEB mRNA levels in
pancreatic islets.45 Our observation of reduced TFEB nuclear
translocation and LAMP2 protein levels in T2D islets is consistent
with previous findings of reduced LAMP2 and cathepsin‐B/D gene
expression in islets from T2D patients.46 Reduction in TFEB activ‐
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&lt;i&gt;Letter to the Editor:&lt;/i&gt; A comparison of F-region ion velocity observations from the EISCAT Svalbard and VHF radars with irregularity drift velocity measurements from the CUTLASS Finland HF radar
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Annales geophysicae
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A comparison of F-region ion velocity observations
from the EISCAT Svalbard and VHF radars with irregularity
drift velocity measurements from the CUTLASS Finland HF radar
J. A. Davies, T. K. Yeoman, M. Lester, S. E. Milan
Radio and Space Plasma Physics Group, Department of Physics and Astronomy, University of Leicester,
University Road, Leicester, LE1 7RH, UK
e-mail: jaq@ion.le.ac.uk Received: 24 September 1999 / Revised: 21 February 2000 / Accepted: 24 February 2000 Received: 24 September 1999 / Revised: 21 February 2000 / Accepted: 24 February 2000 and Ossakow, 1983) and observational (Villain et al.,
1985; Ruohoniemi et al., 1987; Baker et al., 1990;
Davies et al., 1999), suggests that the motion of
F-region irregularities measured by HF coherent scatter
radars, which, unlike their VHF or UHF counterparts,
are not limited to E-region observation at high-latitude
due to the refractive nature of radio propagation at HF,
is determined by the ambient plasma ¯ow. This has also
been found to be true of irregularities generated
arti®cially
in the F-region by
high-power heating
facilities (e.g. Eglitis et al., 1998). Abstract. During August 1998, the UK EISCAT special
programme SP-UK-CSUB, which combines operation
of both the mainland VHF and Svalbard UHF inco-
herent scatter radars, was run for several hours around
magnetic midnight on four consecutive days. The
CUTLASS Finland HF coherent scatter radar was, at
these times, operating in a discretionary mode, sounding
on all 16 beams, one at high-time resolution. This study
presents a comparison of the velocities measured by
coherent and incoherent techniques during the SP-UK-
CSUB experiments. Agreement, particularly between
the ion velocities measured by the EISCAT Svalbard
radar and irregularity drift measurements by the Fin-
land radar, is remarkable, thereby validating the scien-
ti®c integrity of both data sets. This work highlights the
substantive contribution to our understanding of the
solar-terrestrial environment which can be made by use
in concert of incoherent and HF coherent scatter radars. The studies by Villain et al. (1985), Ruohoniemi et al. (1987) and Baker et al. (1990) were based on data taken
over a restricted time interval and, consequently, on a
very limited number of velocity pairs. In contrast,
Davies et al. (1999) compared EISCAT UHF radar
observations from over 1000 hours of common pro-
gramme 1 (CP-1) and common programme 2 (CP-2)
experiments to the drift velocity of natural irregularities,
the latter measured by the CUTLASS Finland HF
radar. Despite their extensive database, Davies et al. (1999) still found themselves limited to a relatively small
number of velocity pairs, the EISCAT mainland site
being by no means optimally located within the Finland
radar ®eld-of-view from which to receive F-region
returns. A comparison of F-region ion velocity observations
from the EISCAT Svalbard and VHF radars with irregularity
drift velocity measurements from the CUTLASS Finland HF radar
J. A. Davies, T. K. Yeoman, M. Lester, S. E. Milan
Radio and Space Plasma Physics Group, Department of Physics and Astronomy, University of Leicester,
University Road, Leicester, LE1 7RH, UK
e-mail: jaq@ion.le.ac.uk Here, irregularity drift speeds measured by the
CUTLASS Finland radar are compared to ion velocity
observations by the EISCAT mainland VHF radar and
the EISCAT Svalbard radar (ESR). Key words: Ionosphere (ionospheric irregularities;
plasma convection; instruments and techniques) 1 Introduction Irregularity drift velocity measurements by coherent
scatter radars have been compared to bulk plasma
velocity measurements by other instrumentation, in-
cluding satellite borne probes and incoherent scatter
radars. Haldoupis and Schlegel (1990), and authors
referenced therein, con®rmed experimentally previous
theoretical evidence that the phase velocity of E-region
irregularities, measured with the STARE VHF coherent
scatter radar, is limited in magnitude to near the ion-
acoustic speed. Evidence, both theoretical (e.g. Keskinen Ann. Geophysicae 18, 589±594 (2000) Ó EGS ± Springer-Verlag 2000 Ann. Geophysicae 18, 589±594 (2000) Ó EGS ± Springer-Verlag 2000 LETTER Correspondence to: J. A. Davies 2 Experimental arrangement In SP-UK-CSUB, which is
identical to the VHF common programme CP-4, long
pulse and power pro®le codes are transmitted. The long
pulse, to which data this study is limited, provides line-
of-sight observations, on each beam, over 20 gates of
range resolution 65.3 km with the ®rst gate centred at a
range of 533.0 km. Like those from Svalbard, VHF
observations were analysed at 1 min temporal resolu-
tion. Beam 1 of the VHF radar is aligned in azimuth
along beam 7 of the Finland radar whereas beam 2 is
less convenient with regard the present study in this
respect, crossing as it does beams 8, 9 and 10 of the
Finland radar. Fig. 1. The geographic location of the ®eld-of-view of the CUTLASS
Finland radar, the ESR beam (solid line) and both the VHF radar
beams (dashed lines). Beams 7 and 9 of the CUTLASS radar are
highlighted explicitly. The locations of the radars are represented by
large solid circles. The smaller solid circle indicates the position of the
geomagnetic pole while the position of the geographic pole is
represented as a cross within a circle During the third run, ionospheric scatter is observed by
the Finland radar within the common viewing area only
between 2100 and 2200 UT on August 22. Over the
other two runs, there is a preponderance of coherent
scatter identi®ed as being of ionospheric origin within
that portion of the Finland ®eld-of-view accessible to
the incoherent scatter radars. During the third run, ionospheric scatter is observed by
the Finland radar within the common viewing area only
between 2100 and 2200 UT on August 22. Over the
other two runs, there is a preponderance of coherent
scatter identi®ed as being of ionospheric origin within
that portion of the Finland ®eld-of-view accessible to
the incoherent scatter radars. Figure 2 presents long pulse observations of line-of-
sight ion velocity from 2100 UT on 21 August to 0100
UT on 22 August 1998 from the EISCAT Svalbard
radar (Fig. 2a), and beams 1 and 2 of the mainland
VHF radar (Fig. 2b, c, respectively). Figure 2d, e
depicts line-of-sight irregularity drift velocity measure-
ments along beam 7 and beam 9, respectively, of the
CUTLASS Finland radar. 2 Experimental arrangement The UK EISCAT special programme SP-UK-CSUB,
which combines operation of both the VHF radar and
the ESR, was run over four four-hour intervals com-
mencing at 21 UT on 20, 21, 22 and 23 August 1998. During these times, the CUTLASS radar at Hankasalmi
in Finland (e.g. Davies et al., 1999 and references
therein) was operating in a discretionary mode in which
all 16 beams (numbered clockwise 0 to 15) were sounded
at a dwell time of 2 s, but with the radar returning to 590 J. A. Davies et al.: A Comparison of F-region ion velocity observations LETTER Fig. 1. The geographic location of the ®eld-of-view of the CUTLASS
Finland radar, the ESR beam (solid line) and both the VHF radar
beams (dashed lines). Beams 7 and 9 of the CUTLASS radar are
highlighted explicitly. The locations of the radars are represented by
large solid circles. The smaller solid circle indicates the position of the
geomagnetic pole while the position of the geographic pole is
represented as a cross within a circle sound on beam 9 between each; the scan is said to camp
on beam 9. In total 75 range gates were sampled along
each beam with a gate length of 45 km, and a range to
the ®rst gate of 180 km. The ESR, located at Longyearbyen (e.g. Wannberg
et al., 1997) was, during SP-UK-CSUB, directed south-
ward, with a geographic azimuth of 161.6° and an
elevation of 31.0°. The ESR beam is aligned, at least in
azimuth, along beam 9 of the CUTLASS Finland radar
®eld-of-view. The ESR was transmitting the GUP0
radar code, a multi-frequency long pulse. Received
signals were integrated over 10 s and the data subse-
quently analysed at a temporal resolution of 60 s. Analysis of ESR data is such that the user can vary
the range resolution, in this case with gating of 12 km
below a range of 330 km, which corresponds to an
altitude of around 175 km, and 36 km range gates above
330 km. In
SP-UK-CSUB,
the
EISCAT
VHF
radar at
Tromsù operated in a split beam mode with one beam
(beam 2) directed along the boresight which corresponds
to a geographic azimuth of 359.5° and the other (beam 1)
phased 14.5° west to an azimuth of 345.0°; both beams
are at a elevation of 30°. 2 Experimental arrangement Coherent returns identi®ed as
arising from ground scatter are excluded as indeed are
all Finland radar data with a line-of-sight irregularity
drift velocity of magnitude less than 50 m s)1; the latter
is an attempt to remove potentially misidenti®ed ground
scatter (e.g. Davies et al., 1999). Velocities are plotted as
a function of geomagnetic latitude. Velocities are
de®ned as being positive towards the radar, except in
the case of the Svalbard radar where positive velocities
represent ¯ow away from the radar in order to retain a
consistency between the general orientations of the
observations. An in-depth assessment of the geophysical
processes arising during this interval is not attempted
here, that being the subject of a forthcoming study. Figure 1 presents a map on which is depicted the
®eld-of-view of the CUTLASS Finland radar, beams 7
and 9 of which are illustrated explicitly. Moreover,
coverage by the ESR is represented by a solid line and
the VHF radar beams are indicated by dashed lines. The
large ®lled circles represent the locations of the radars
themselves and the cross within a circle, the geographic
pole; the magnetic pole is indicated by the small ®lled
circle. 3 Results at altitudes below some 140 km,
where ion-neutral collisions are signi®cant, will not be
representative of the component of the E ´ B velocity
along the radar line-of-sight. ments, being of F-region origin. The lowest range gate
for VHF observations corresponds to an altitude of
some 280 km while the lowest altitude at which the
Svalbard radar observes is 108 km. As such, it should be
borne in mind that ion velocities in the ®rst six or so
ESR range gates, i.e. at altitudes below some 140 km,
where ion-neutral collisions are signi®cant, will not be
representative of the component of the E ´ B velocity
along the radar line-of-sight. ignored. Although deviations from free-space propaga-
tion have been found to be small, less than the length of
a single range cell (e.g. Ruohoniemi et al., 1987), they
are, nevertheless, not insigni®cant, particularly in stud-
ies of this type. Despite these possible sources of discrepancy, many
small-scale ¯ow features observed by the Finland radar
appear to be mirrored in the incoherent scatter obser-
vations, for example the equatorward velocity burst
observed at around 2300 UT both in beam 9 of the
CUTLASS ®eld-of-view and by the Svalbard radar. The
relationship between F-region ion velocity and irregu-
larity drift velocity is investigated on a more quantita-
tive basis by employing appropriate observations from
all but the ®rst SP-UK-CSUB experiment. Even a cursory glance reveals good general agree-
ment between the observations from the coherent and
incoherent systems, and, indeed, between the two
incoherent scatter radars themselves. Exact agreement
is
not anticipated due to disparities
between the
sampling volumes of the systems (the beam width of
the incoherent scatter radars being almost an order of
magnitude smaller than that of the HF radar) and the
integration periods of the data sets; velocities from both
the ESR and VHF radars are integrated over 60 s,
whereas beam 9 of the Finland radar is integrated for 2
out of every 4 s and beam 7 for only 2 s out of a total
scan time of 64 s. Further discrepancies will be intro-
duced due to geometrical factors. Finland velocities can
be assumed orthogonal to the local magnetic ®eld, this
being the condition necessary for coherent scatter. This
need not be so for incoherent scatter, and, indeed, is not
so here. 3 Results It should also be noted that in the calculation of
the range from which coherent returns are attributed
assumes straight line propagation of HF waves, i.e. refraction eects are neglected and group retardation is Ion velocities measured by the ESR are compared
statistically to the irregularity drift velocities from beam
9 of the Finland radar in Fig. 3a while velocities from
beam 1 of the VHF radar are compared to drift speeds
from Finland Beam 7 in Fig. 3b; the sign convention of
the previous ®gure is maintained, with positive velocities
directed towards the Finland radar. As with the previ-
ous ®gure, all Finland radar data with a line-of-sight
irregularity drift speed less than 50 m s)1, in addition to
those returns conventionally identi®ed as arising from
ground scatter, are excluded. Moreover, a 3 dB thresh-
old has been applied to the CUTLASS data and a 2%
signal-to-noise ratio level to the EISCAT VHF obser-
vations. In the situation where there are multiple
estimates of irregularity drift velocity within a single Fig. 3a±c. Scattergram of irregularity drift velocity a measured by the
Finland radar versus F-region ion velocity measured by the ESR and
b along beam 1 of the VHF radar, during the SP-UK-CSUB
experiments. Both ion velocity and irregularity drift speed are binned
with a width of 50 m s)1, with the number of observations in each bin
represented by the colour scale to the right. c Reproduces the data
from the upper panel of Fig. 5 of Davies et al. (1999), in which ion
velocity from the UHF radar is plotted against F-region irregularity
drift velocity from the Finland radar, in an identical format to a and
b. The solid lines indicate a hypothetical equality of irregularity drift
and ion velocity from the upper panel of Fig. 5 of Davies et al. (1999), in which ion
velocity from the UHF radar is plotted against F-region irregularity
drift velocity from the Finland radar, in an identical format to a and
b. The solid lines indicate a hypothetical equality of irregularity drift
and ion velocity from the upper panel of Fig. 5 of Davies et al. (1999), in which ion
velocity from the UHF radar is plotted against F-region irregularity
drift velocity from the Finland radar, in an identical format to a and
b. The solid lines indicate a hypothetical equality of irregularity drift
and ion velocity Fig. 3a±c. 3 Results The four runs of SP-UK-CSUB are not equally useful
with regard a comparison of irregularity drift velocity
and F-region ion velocity. During the ®rst run, 20
August 2100 UT±21 August 0100 UT, there are a
limited number of coherent echoes observed by the
Finland radar within the viewing range of the ESR and
VHF radars, mainly in the lower CUTLASS range gates
of the common ®eld-of-view. The vast majority of these
returns are, however, characterised by very narrow
spectral widths and low velocities and, as such, have
been identi®ed as ground scatter. For the purpose of the
present study this interval is not considered further. The vagaries of propagation in the HF band is such
that the altitude from which scatter originates is not well
determined. Elevation angle-of-arrival measurements by
the Finland system are consistent with the ionospheric
scatter observed in the common viewing area of the
three instruments during the SP-UK-CSUB experi- 591 J. A. Davies et al.: A Comparison of F-region ion velocity observations Fig. 2a±e. Line-of-sight velocity measurements during the SP-UK-
CSUB experiment run from 2100 UT on 21 August to 0100 UT on 22
August, 1998. Successive panels illustrate data from a the EISCAT
Svalbard radar, b beam 1 of the VHF radar, c be
radar, d beam 7 of the CUTLASS Finland radar an
Finland radar, each as a function of geomagnetic l
J. A. Davies et al.: A Comparison of F-region ion velocity observations LETTER Svalbard radar, b beam 1 of the VHF radar, c beam 2 of the VHF
radar, d beam 7 of the CUTLASS Finland radar and e beam 9 of the
Finland radar, each as a function of geomagnetic latitude Fig. 2a±e. Line-of-sight velocity measurements during the SP-UK-
CSUB experiment run from 2100 UT on 21 August to 0100 UT on 22
August, 1998. Successive panels illustrate data from a the EISCAT J. A. Davies et al.: A Comparison of F-region ion velocity observations 592 LETTER ments, being of F-region origin. The lowest range gate
for VHF observations corresponds to an altitude of
some 280 km while the lowest altitude at which the
Svalbard radar observes is 108 km. As such, it should be
borne in mind that ion velocities in the ®rst six or so
ESR range gates, i.e. 3 Results Scattergram of irregularity drift velocity a measured by the
Finland radar versus F-region ion velocity measured by the ESR and
b along beam 1 of the VHF radar, during the SP-UK-CSUB
experiments. Both ion velocity and irregularity drift speed are binned
with a width of 50 m s)1, with the number of observations in each bin
represented by the colour scale to the right. c Reproduces the data 593 J. A. Davies et al.: A Comparison of F-region ion velocity observations LETTER radar, in an identical format to Fig. 3a, b. In this case
the ion velocity plotted is the component of the vector
velocity (the UHF being a tristatic system) resolved
along the direction of the appropriate Finland beam. Davies et al. (1999) dierentiated between E- and
F-region drift velocities on the basis of interferometric
evidence. Figure 3c, in common with Fig. 3a, b, reveals
a number of points corresponding to anomolously high
values of irregularity drift velocity, which are attributed
to uncorrelated spikes sometimes observed in CUT-
LASS data (Davies et al., 1999). EISCAT integration period, these have been averaged
together. Moreover, where there are more than one ion
velocity observation within a single CUTLASS range
gate, as is the case with the ESR, these too have been
averaged together. Although coherent returns of veloc-
ity less than 50 m s)1 have been excluded, the average of
the irregularity drift velocity over an EISCAT integra-
tion period may still be less than that value. ESR range
gates centred below 140 km altitude are excluded from
the analysis, the velocities therein not being representa-
tive of magnetised ¯ow. EISCAT integration period, these have been averaged
together. Moreover, where there are more than one ion
velocity observation within a single CUTLASS range
gate, as is the case with the ESR, these too have been
averaged together. Although coherent returns of veloc-
ity less than 50 m s)1 have been excluded, the average of
the irregularity drift velocity over an EISCAT integra-
tion period may still be less than that value. ESR range
gates centred below 140 km altitude are excluded from
the analysis, the velocities therein not being representa-
tive of magnetised ¯ow. g
The line-of-sight ion velocities measured by the ESR
and along beam 1 of the VHF radar must be corrected
for geometrical eects in order that direct comparison
be made with the Finland drift observations. 3 Results As noted
previously, coherent scatter requires the radar wave
vector to be near orthogonal to the magnetic ®eld. Although at approximately the same azimuths as beams
7 and 9 of the Finland radar, the ion velocities measured
by the incoherent scatter systems are not perpendicular
to the magnetic ®eld, rather at an angle determined by
the radar beam elevation and direction of the local
geomagnetic ®eld. The ion velocities are corrected such
that they represent the ®eld-perpendicular ¯ow, albeit
assuming that there is no ®eld-parallel motion of the ion
population, an assumption the validity of which is
discussed in more detail in the subsequent section, and
that the radar beam lies in the same plane as the
magnetic ®eld, the latter of which is reasonable under
these circumstances of a mainly northward directed
beam. The solid lines on Fig. 3 indicate a hypothetical
equality of irregularity drift and ion velocity for
comparative purposes. (
)
The correspondence, not inconsistent with the equiv-
alence of irregularity drift and bulk plasma motion in
the F-region, noted by Davies et al. (1999) is surpassed
in the present observations, especially with regard the
EISCAT Svalbard radar, corrected velocities from
which compare remarkably well with the line-of-sight
drift velocity measurements from the CUTLASS Fin-
land radar. There is signi®cant spread, however, which
may be ascribed to a number of potentially deleterious
eects such as discrepant temporal and spatial sampling. There does appear, however, to be a small yet systematic
oset, of some 50 to 100 m s)1, between the irregularity
drift measurements and the ion velocities determined by
the Svalbard radar. It is suggested that this is attribut-
able, at least in part, to the assumption that there is no
motion of the ion population along the magnetic ®eld
direction, used in correcting the incoherent scatter
velocity measurements from the Svalbard and VHF
radars such that they represent ®eld-perpendicular
¯ows. On the nightside, the general antisunward hori-
zontal motion of the neutral atmosphere, in response to
pressure gradients set up by solar heating, will drive ions
up the magnetic ®eld with a velocity of some several tens
of m s)1 (e.g. Rodger et al., 1992). Invoking an upward
®eld-aligned velocity of this order does indeed signi®-
cantly reduce the oset observed between the HF drift
and ESR velocity measurements. 4 Discussion These observations, made over a four day interval,
provide an exceptional opportunity for the comparison
of F-region ion velocity with the drift speed of F-region
®eld-aligned irregularities. This is especially noteworthy
given that in the work of Davies et al. (1999) coherent
returns from the Finland radar were observed in the
vicinity of the EISCAT site for a time totalling less than
4 h out of an entire database comprising more than
1000 h of measurements from the two most frequently
run UHF common programmes, taken over a three year
interval. Due to the geometrical arrangement of the SP-
UK-CSUB experiment, the present VHF and ESR
observations have the advantage over the UHF obser-
vations employed in the velocity comparison of Davies
et al. (1999) of being simultaneous over a number of
CUTLASS Finland range gates. Moreover, the present
study concentrates on latitudes poleward of those
considered by Davies et al. (1999) where the nature of
HF
propagation
limits
Finland
returns
from
the
F-region. y
Perhaps one of the most noteworthy features of this
work is the number of velocity pairs available for
comparison, over 3300 for the ESR and nearly 1500 for
the VHF radar compared to a mere 525 simultaneous
estimates of UHF ion velocity and F-region irregularity
drift speed in the study of Davies et al. (1999); this is
even more remarkable given that the latter was derived
from a data set more than 50 times as long as the
combined lengths of the four runs of SP-UK-CSUB. In
addition to the superior geometrical arrangement of the
SP-UK-CSUB experiment for a study of this type, with
regard the location and extended latitudinal range of the
common viewing area, this is possibly also attributable
to the conditions associated with the more active part of
the solar cycle. References operation of the CUTLASS Finland HF radar. Line-of-
sight velocity measurements from the three instruments
exhibit a consistency even with regard to small-scale
features in the convective ¯ow pattern. The geometrical
arrangement of the experiment permits, moreover, the
direct statistical comparison of line-of-sight F-region ion
velocities measured by the ESR and along one beam of
the VHF radar with irregularity drift velocity measure-
ments from the Finland radar. Baker, K. B., R. A. Greenwald, J. M. Ruohoniemi, J. R. Dudeney,
M. Pinnock, P. T. Newell, M. E. Greenspan, and C. I. Meng,
Simultaneous HF radar and DMSP observations of the cusp,
Geophys. Res. Lett., 17, 1869±1872, 1990. Davies, J. A., M. Lester, S. E. Milan, and T. K. Yeoman, A
comparison of velocity measurements from the CUTLASS
Finland radar and the EISCAT UHF system, Ann. Geophysicae,
17, 892±902, 1999. Eglitis, P., T. R. Robinson, M. T. Rietveld, D. M. Wright, and
G. E. Bond, The phase speed of arti®cial ®eld-aligned irregu-
larities observed by CUTLASS during HF modi®cation of the
auroral ionosphere, J. Geophys. Res., 103, 2253±2259, 1998. Statistical comparison of ion velocities measured by
the ESR and irregularity drift measurements by the
Finland radar is particularly satisfying, despite a num-
ber of potential sources of discrepancy, demonstrating
once again that the CUTLASS system provides an
invaluable spatial context in which to interpret small-
scale features in the high-altitude ionosphere identi®ed
by incoherent scatter techniques and, indeed, how
signi®cant a contribution to our understanding of
solar-terrestrial physics can be made by collaborative
use of observations from incoherent and HF coherent
scatter radars. Haldoupis, C., and K. Schlegel, Direct comparison of 1-m
irregularity phase velocities and ion acoustic speeds in the
auroral E-region ionosphere, J. Geophys. Res., 95, 18989±19000,
1990. Keskinen, M. J., and S. L. Ossakow, Theories of high-latitude
ionospheric irregularities ± A review, Radio Sci., 18, 1077±1091,
1983. Rodger, A. S., R. J. Moett, and S. Quegan, The role of ion drift in
the formation of ionisation troughs in the mid- and high-latitude
ionosphere ± A review, J. Atmos. Terr. Phys., 54, 1±30, 1992. Ruohoniemi, J. M., R. A. Greenwald, K. B. Baker, J.-P. Villain, and
M. A. McCready, Drift motions of small-scale irregularities in
the high-latitude F-region: an experimental comparison with
plasma drift motions, J. Geophys. Res., 92, 4553±4564, 1987. Acknowledgements. 5 Summary and conclusions During August 1998, the UK EISCAT special pro-
gramme SP-UK-CSUB, combining operation of both
the mainland VHF and Svalbard incoherent scatter
radars, was run in conjunction with discretionary mode g
Figure 3c reproduces the data from the upper panel
of Fig. 5 of Davies et al. (1999), in which ion velocity
from the UHF radar is plotted against the drift velocity
of F-region irregularities measured by the Finland J. A. Davies et al.: A Comparison of F-region ion velocity observations 594 LETTER The Editor-in-chief thanks C. Senior for her help in evaluating
this paper. Acknowledgements. The authors wish to acknowledge support
from the EISCAT group at the Rutherford Appleton Laboratory
and other members of the UK campaign team. Additional thanks
are due to N. M. Wade. EISCAT is funded collaboratively by the
research councils of Finland, France, the Federal Republic of
Germany, Japan, Norway, Sweden and the UK funding body,
PPARC. CUTLASS is funded jointly by PPARC, the Swedish
Institute for Space Physics and the Finnish Meteorological
Institute. References The authors wish to acknowledge support
from the EISCAT group at the Rutherford Appleton Laboratory
and other members of the UK campaign team. Additional thanks
are due to N. M. Wade. EISCAT is funded collaboratively by the
research councils of Finland, France, the Federal Republic of
Germany, Japan, Norway, Sweden and the UK funding body,
PPARC. CUTLASS is funded jointly by PPARC, the Swedish
Institute for Space Physics and the Finnish Meteorological
Institute. p
p y
Villain, J.-P., G. Caudal, and C. Hanuise, A SAFARI-EISCAT
comparison between the velocity of F-region small-scale irreg-
ularities and the ion drift, J. Geophys. Res., 90, 8433±8443, 1985. Wannberg
G.,
L. Wolf,
L.-G. Vanhainen,
K. Koskenniemi,
J. RoÈ ttger, M. Postila, J. Markkanen, R. Jacobsen, A. Stenberg,
R. Larsen, S. Eliassen, S. Heck, and A. Huuskonen, The
EISCAT Svalbard radar: A case study in modern incoherent
scatter radar system design, Radio Sci., 32, 2283±2307, 1997. The Editor-in-chief thanks C. Senior for her help in evaluating
this paper.
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Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection
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Virology journal
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cc-by
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BioMed Central BioMed Central Received: 13 June 2009
Accepted: 15 July 2009 Received: 13 June 2009
Accepted: 15 July 2009 © 2009 Sainz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2009 Sainz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research
Three-dimensional Huh7 cell culture system for the study of
Hepatitis C virus infection
Bruno Sainz Jr†1, Veronica TenCate†1 and Susan L Uprichard*1,2 ddress: 1Department of Medicine, The University of Illinois at Chicago, Chicago, IL 60612, USA and 2Department o
mmunology, The University of Illinois at Chicago, Chicago, IL 60612, USA Published: 15 July 2009
Virology Journal 2009, 6:103
doi:10.1186/1743-422X-6-103
Received: 13 June 2009
Accepted: 15 July 2009
This article is available from: http://www.virologyj.com/content/6/1/103
© 2009 Sainz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: In order to elucidate how Hepatitis C Virus (HCV) interacts with polarized
hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-
associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As
such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were
used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells. Results: When cultured in the RWV, Huh7 cells became morphologically and transcriptionally
distinct from more standard Huh7 two-dimensional (2-D) monolayers. Specifically, RWV-cultured
Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic
drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4α, Albumin, TTR and
α1AT), were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis
revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D
counterparts with the expression of HCV receptors, cell adhesion and tight junction markers
(CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, β-Catenin and E-
Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization. Conclusion: These findings show that when cultured in 3-D, Huh7 cells acquire a more
differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly
permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of
HCV infection on host cell function in a more physiologically relevant cell culture system. anese Fulminant Hepatitis, JFH-1) that can replicate and
produce infectious HCV in vitro in the Huh7 human
hepatoma-derived cell line [2-4], allowing for the study of
the entire viral life cycle. This system, however, is limited
in that it makes use of a non-differentiated cell line that
does not recapitulate the cellular conditions encountered
by HCV in vivo [5,6]. In particular, hepatocyte polarity is
likely relevant to HCV entry as growing evidence suggests
interplay between HCV and tight junction (TJ) proteins Virology Journal Open Access Page 1 of 8
(page number not for citation purposes) http://www.virologyj.com/content/6/1/103 http://www.virologyj.com/content/6/1/103 claudin-1 (CLDN1) [7] and occludin [8,9] is essential for
viral uptake. In fact, recent reports surprisingly suggests
that hepatocyte polarity may restricts HCV entry [10,11]. While an inverse relationship between viral entry and
hepatocyte polarity would potentially represent a unique
determinant of HCV entry, to date attempts to dissect this
relationship have been difficult and inconclusive due to
the inability of cell culture grown hepatocyte-derived cell
lines, such as Huh7 cells, to mimic the complex polarized
phenotype of hepatocytes in vivo. To circumvent these
restriction, studies investigating HCV entry into Caco-2
cells [10] and HepG2 cells [11] have been performed as
these cells can polarize to differing degrees in vitro, how-
ever, neither Caco-2 or HepG2 cells supports efficient
HCV infection limiting their utility. As such, a more phys-
iologically relevant hepatocyte tissue culture model is still
needed to assess if cell polarity negatively affects HCV
infection. lished using previously described techniques [13,14],
with minor modifications. Briefly, 5 × 106 Huh7 cells
were trypisinized, incubated with 250 mg Cytodex-3
microcarrier beads (Sigma, St. Louis, MO) for 30 minutes
at room temperature in a total volume of 30 ml complete
DMEM. Cell-bead complexes were introduced into the
RWV bioreactor (Synthecon, Inc., Houston, TX) at a ratio
of 20 cells/bead, transferred to 37°C, and vessel rotation
was initiated at 20 rotations per minute. Medium was
replenished every 24 h and rotation speed was increased
as aggregates developed to maintain cells in free-falling
suspension. lished using previously described techniques [13,14],
with minor modifications. Briefly, 5 × 106 Huh7 cells
were trypisinized, incubated with 250 mg Cytodex-3
microcarrier beads (Sigma, St. Louis, MO) for 30 minutes
at room temperature in a total volume of 30 ml complete
DMEM. Cell-bead complexes were introduced into the
RWV bioreactor (Synthecon, Inc., Houston, TX) at a ratio
of 20 cells/bead, transferred to 37°C, and vessel rotation
was initiated at 20 rotations per minute. Medium was
replenished every 24 h and rotation speed was increased
as aggregates developed to maintain cells in free-falling
suspension. Protocols for JFH-1 in vitro transcription and HCV RNA
electroporation have been described elsewhere [28]. RNA isolation and RTqPCR Total cellular RNA was isolated by the guanidine thiocy-
anate method using standard protocols [29]. One μg of
RNA was used for cDNA synthesis using TaqMan reverse
transcription reagents (Applied Biosystems, Foster City,
CA), followed by SYBR green real-time quantitative PCR
analysis (RTqPCR) using an Applied Biosystems 7300
real-time thermocycler as previously described [30]. Rela-
tive expression levels of hepatocyte-specific genes and
Phase I and Phase II metabolic genes were assessed using
the primers described in [30] and normalized to β-actin
levels. HCV JFH-1 and GAPDH transcript levels were
determined relative to a standard curve of serially diluted
plasmid containing the JFH-1 cDNA or the human
GAPDH gene, respectively, using primers described in
[28]. In the current study we demonstrate that RWV-cultured
Huh7 cells formed complex, multilayered, 3-D aggregates
that exhibited up-regulation of metabolic and hepatocyte-
specific transcripts as well as increased expression and re-
localization of tight junction, cell adhesion, and polarity
markers. Importantly, these aggregates remained highly
permissive for HCV infection suggesting that hepatic
polarity does not limit HCV entry in 3-D-cultured Huh7
cells. As such, RWV-cultured Huh7 cells may represent a
more appropriate physiologically relevant system for fur-
ther in vitro studies of HCV entry and infection dynamics. Immunofluorescence
f Immunofluorescence analysis of aggregates was per-
formed as previously described [14]. Briefly, Huh7 3-D
aggregates were fixed with 4% (v/v) paraformaldehyde
(Sigma, St. Louis, MO), free aldehydes quenched with 50
mM NH4Cl in DPBS for 10 minutes at room temperature
and cells permeabilized with 0.1% Triton-X 100 (Fisher). In parallel, Huh7 2-D monolayers were seeded in 8-well
chamber slides at 80% confluence and fixed 48 hours post
seeding. 3-D aggregates and 2-D monolayer cells were
stained with antibodies specific for scavenger receptor
class B member 1 (SR-BI) (BD Biosciences, Franklin Lakes,
NJ), CD81 (AbD Serotec, Raleigh, NC), CLDN1 (Abnova,
Taipei, Taiwan), CD26 (Abcam, Cambridge, UK), β-Cat-
enin (Zymed, San Francisco, CA), E-cadherin (Zymed),
zona occludens 1 (ZO-1) (Zymed), Occludin (Zymed) or
HCV E2 (C1 [31]) overnight at 4°C, followed by incuba- http://www.virologyj.com/content/6/1/103 JFH-
1 cell culture-propagated HCV (HCVcc) viral stocks were
obtained by infection of naïve Huh7-1 cells at a multiplic-
ity of infection (MOI) of 0.01 focus forming units (FFU)/
cell, using medium collected from Huh7 cells on day 18
post transfection with in vitro transcribed pJFH-1 RNA as
previously described [2]. The NASA-engineered RWV is a horizontally rotating
cylindrical culture vessel which reduces shear and turbu-
lence associated with conventional stirred bioreactors;
therefore, it simulates aspects of microgravity similar to
the environment encountered during fetal development
[12-14]. In contrast to conventional static tissue culture
systems, cells grown in the RWV are cultured in "sus-
pended animation" where they are continuously free-fall-
ing [12,15]. Thus, while the 2-D environment of plastic
substrates may alter gene expression and prevent cellular
differentiation [12,16-21], the fluid dynamics of the RWV
culture system allow cells to co-localize into three-dimen-
sional (3-D) aggregates, promoting in vivo-like exchange
of growth factors and efficient cell-to-cell interactions [12-
14,20,21]. This in vivo-like environment thus can pro-
mote transformed and primary cell lines to become more
structurally and functionally similar to their in vivo coun-
terparts [13,15,20-24]. Page 2 of 8
(page number not for citation purposes) Background g
Hepatitis C virus (HCV), a liver tropic positive-stranded
RNA flavivirus, infects ~170 million people worldwide,
causing acute and chronic hepatitis and hepatocellular
carcinoma [1]. However, since its discovery in 1989, a
major obstacle impeding HCV research has been the lack
of robust cell culture and small animal infection models. Notably significant advancement has been made with the
identification of a genotype 2a HCV consensus clone (Jap- Page 1 of 8
(page number not for citation purposes) Page 1 of 8
(page number not for citation purposes) Virology Journal 2009, 6:103 HCV infection kinetics Huh7 3-D aggregates were infected with JFH-1 HCVcc at
an MOI of 0.01 FFU/cell at day 1, 7 or 14 post RWV seed-
ing by injection of the viral inoculum directly into the
RWV. At indicated times post infection (p.i.), medium
was harvested for titration analysis and RNA was isolated
from ~0.5 ml of aggregates for reverse transcription fol-
lowed by RTqPCR as described above. High-fidelity 3-D Huh7 RWV aggregates
Figure 1
High-fidelity 3-D Huh7 RWV aggregates. (A) Phase
contrast micrograph of Huh7 3-D aggregates cultured in the
RWV for 14 days (400×). (B) Fluorescence micrograph of
Hoechst-stained OCT sections of 3-D Huh7 aggregates
(400×). (C-D) Light micrographs of H&E-stained paraffin sec-
tions of 3-D Huh7 aggregates [200× (C), 600× (D)]. (*) = 100
μm microcarrier bead. Cell culture and viruses Huh7 cells (also known as Huh7/scr cells [25,26] and
Huh7-1 cells [27]) were obtained from Dr. Chisari (The
Scripps Research Institute, La Jolla, CA) [2] and cultured
as previously described [2]. 3-D Huh7 cultures were estab- Page 2 of 8
(page number not for citation purposes) Page 2 of 8
(page number not for citation purposes) Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 High-fidelity 3-D Huh7 RWV aggregates
Figure 1
High-fidelity 3-D Huh7 RWV aggregates. (A) Phase
contrast micrograph of Huh7 3-D aggregates cultured in the
RWV for 14 days (400×). (B) Fluorescence micrograph of
Hoechst-stained OCT sections of 3-D Huh7 aggregates
(400×). (C-D) Light micrographs of H&E-stained paraffin sec-
tions of 3-D Huh7 aggregates [200× (C), 600× (D)]. (*) = 100
μm microcarrier bead. tion with a 1:1,000 dilution of an appropriate Alexa555-
conjugated secondary antibody (Molecular Probes,
Carlsbad, CA) for 1 h at room temperature. Cell nuclei
were stained by Hoechst dye. Bound antibodies were vis-
ualized via confocal microscopy (630×, Zeiss LSM 510,
Germany). Images were analyzed using Zeiss LSM Alpha
Imager Browser v4.0 software (Zeiss), and brightness and
contrast were adjusted using Adobe® Photoshop® (San
Jose, CA). Alternately, 3-D aggregates were embedded in
OCT freezing medium (TissueTek, Fisher) or paraffin, sec-
tioned and stained with Hoechst dye or Hematoxylin and
Eosin (H&E), respectively. Establishment of Huh7 3-D Aggregates gg
g
To assess the utility of the RWV as a culture method for
Huh7 cells, Huh7 cells were cultured on Cytodex-3 micro-
carrier beads in the RWV for 26 days. Morphological and
cytological examination of cultures demonstrated that
Huh7 cells efficiently adhered to the collagen-coated
microcarrier beads and that these individual beads then
assembled to form 3-D tissue-like aggregates containing
~10–20 beads per aggregate (Fig. 1A). To determine if
these aggregates consisted of multilayered cells, aggregates
were embedded in OCT freezing medium or paraffin, sec-
tioned, stained with either Hoechst stain (Fig. 1B) or H&E
(Fig. 1C–D) and examined by fluorescence or light micro-
scopy, respectively. Panels C and D highlight the multilay-
ered cellular infrastructure of the Huh7 3-D aggregates,
while Hoechst's staining in Panel B illustrates similar
infrastructure and confirms that the aggregates are devoid
of necrotic cores. Expression and Organization of Cellular Tight Junction
and Polarity Markers in 3-D Huh7 Aggregates
While the effect of HCV on cell polarity and TJs (and vice-
versa) cannot be accurately studied in 2-D monolayer
Huh7 cultures [10], these interactions are of particular Infectivity titration assay Culture supernatants were serially diluted 10-fold and
used to infect triplicate Huh7 cultures in 96-well plates. At
24 h p.i., cultures were overlayed with complete DMEM
containing 0.4% methylcellulose (Fluka BioChemika,
Switzerland) to give a final concentration of 0.25% meth-
ylcellulose. Seventy-two hours p.i., cells were fixed in 4%
paraformaldehyde (Sigma), and immunohistochemically
stained for HCV E2 using the anti-HCV E2 antibody C1
[31]. Viral titers are expressed as FFU/ml, determined by
the average E2-positive foci number detected at the high-
est HCV-positive dilution. Gene Expression within Huh7 3-D Aggregates p
gg
g
One measure of hepatocyte differentiation is up-regula-
tion of expression of transcription factors such as hepato-
cyte nuclear factors (HNF) [32,33], which regulate the
expression of liver secretory proteins [33] such as albumin
[34], alpha-1-antitrypsin (α1AT; [35]), and transthyretin
(TTR; [36]). Likewise, induction of enzymes and trans-
porters involved in Phase I and II xenobiotic metabolism
[37,38], which include cytochrome P450s (CYPs) and
UDP-glucuronosyltransferase (UGTs) enzymes, respec-
tively, is another hallmark of hepatocyte differentiation. Hence, to determine whether culturing Huh7 cells in the
RWV allows for cellular differentiation at the transcrip-
tional level, expression of hepatocyte-specific genes, CYPs,
and UGTs were analyzed. At indicated times post seeding,
total cellular RNA was extracted from 0.5 ml of 3-D Huh7
aggregates or 2-D Huh7 monolayers grown to confluence,
and relative gene expression was assessed by RTqPCR
analysis. As illustrated in Fig. 2, mRNA levels for the hepa-
tocyte-specific genes and the CYP and UGT enzymes were
significantly induced in 3-D Huh7 aggregates (relative to
2-D Huh7 monolayers) and increased in a time-depend-
ent manner while cultured in the RVW. Expression and Organization of Cellular Tight Junction
and Polarity Markers in 3-D Huh7 Aggregates Expression and Organization of Cellular Tight Junction
and Polarity Markers in 3-D Huh7 Aggregates While the effect of HCV on cell polarity and TJs (and vice-
versa) cannot be accurately studied in 2-D monolayer
Huh7 cultures [10], these interactions are of particular Page 3 of 8
(page number not for citation purposes) http://www.virologyj.com/content/6/1/103 Virology Journal 2009, 6:103 Increased differentiation-specific gene expression in 3-D
Huh7 RWV aggregates
Figure 2
Increased differentiation-specific gene expression
3-D Huh7 RWV aggregates. At indicated time points
post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5
104 cells) were removed from the RWV, pelleted at 1400
RPM for 5 minutes and total RNA extracted. Expression
(A) hepatocyte-specific genes, (B) Phase I (CYP) and (C)
Phase II (UGT) metabolic genes in Huh7 3-D aggregates w
assessed by RTqPCR. Expression of each transcript, relat interest as TJ proteins are involved in the entry of numer-
ous viruses [39-41] and the TJ proteins CLDN1 [7] and
occludin [8,9] have recently been shown to be involved in
HCV entry. Therefore, we assessed the expression and
organization of the HCV receptors (CD81 and SR-B1), cell
adhesion molecules (E-Cadherin and β-Catenin), cellular
TJ proteins (CLDN1, ZO-1 and Occuldin-1) and the
polarity marker (CD26) in 3-D Huh7 aggregates and their
2-D monolayer counterparts (Fig. 3). The expression of
known HCV receptors and polarity markers were
increased in 3-D Huh7 aggregates as compared 2-D Huh7
monolayers, similar to that observed by Mee et al in polar-
ized Caco-2 cells [10]. This was not a consequence of
increased mRNA levels, as normalized transcript levels for
all markers examined were similar between 3-D and 2-D
Huh7 cultures, as determined by RTqPCR (data not
shown). Increased differentiation-specific gene expression in 3-D
Huh7 RWV aggregates
Figure 2
Increased differentiation-specific gene expression in
3-D Huh7 RWV aggregates. At indicated time points
post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 ×
104 cells) were removed from the RWV, pelleted at 1400
RPM for 5 minutes and total RNA extracted. Expression of
(A) hepatocyte-specific genes, (B) Phase I (CYP) and (C)
Phase II (UGT) metabolic genes in Huh7 3-D aggregates was
assessed by RTqPCR. Expression of each transcript, relative
to 2-D Huh7 monolayer cultures, was determined using the
method [50], by normalizing to β-actin expression
and is graphed as fold induction compared to 2-D monolay-
ers. Increased d
Huh7 RWV
Figure 2 Increased differentiation-specific gene expression in
3-D Huh7 RWV aggregates. At indicated time points
post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 ×
104 cells) were removed from the RWV, pelleted at 1400
RPM for 5 minutes and total RNA extracted. Expression of
(A) hepatocyte-specific genes, (B) Phase I (CYP) and (C)
Phase II (UGT) metabolic genes in Huh7 3-D aggregates was
assessed by RTqPCR. Expression of each transcript, relative
to 2-D Huh7 monolayer cultures, was determined using the
method [50], by normalizing to β-actin expression
and is graphed as fold induction compared to 2-D monolay-
ers. 2-DDC T Page 4 of 8
(page number not for citation purposes) Expression and Organization of Cellular Tight Junction
and Polarity Markers in 3-D Huh7 Aggregates 2-DDC T As expected, the cell adhesion molecules E-Cadherin and
β-Catenin were membrane localized both in 2-D and 3-D
Huh7 cultures; however, there was a profound decrease in
the accumulation of nuclear β-Catenin-containing com-
plexes in the 3-D Huh7 aggregates. Because atypical
nuclear localization of β-Catenin in transformed cells has
been well documented [42], the loss of this cancer-specific
phenotype in the 3-D cultured Huh7 aggregates is consist-
ent with the loss of cancer-specific markers observed in
other continuous cell lines when cultured in the RWV
[14,23]. Additionally, in contrast to the 2-D Huh7 mon-
olayers, TJ markers localized to apicalateral and/or baso-
lateral planes in the 3-D Huh7 aggregates consistent with
localization patterns observed in primary hepatocytes
[6,43] and normal liver tissues [44]. Finally, CD26, a cell
surface ectopeptidase that localizes to the apical plane of
polarized cells [45], was non-detectable in 2-D Huh7
monolayers, while, apical staining of this marker was
apparent in distinct areas of 3-D Huh7 aggregates (Fig. 3). Taken together, these data demonstrate that the expres-
sion and distribution of cell adhesion and TJ proteins,
including known HCV entry receptors, is enhanced and
more polarized in 3-D Huh7 cultures than in 2-D monol-
ayers. Increased d
Huh7 RWV
Figure 2 HCVcc Infection of Huh7 3-D Aggregates f
f
gg
g
Because it has been suggested that hepatocyte polarization
is inversely related to the permissiveness of the cell for
HCVcc infection [10,11], we sought to determine if Huh7
3-D cultures were permissive for HCVcc infection. As
such, 3-D Huh7 cultures were inoculated with HCVcc
JFH-1 1, 7, or 14 days post RWV-seeding and culture
supernatant and cellular RNA were harvested at various
time points p.i. for titration of extracellular viral titers and
RTqPCR analysis of viral RNA, respectively. Fig. 4A illus-
trates that HCV not only infected the Huh7 3-D aggre-
gates, but that the kinetics of HCV RNA expansion and Page 4 of 8
(page number not for citation purposes) Page 4 of 8
(page number not for citation purposes) Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates
Figure 3
Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregat
Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodie
specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy
(630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sectio
which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which th
section was taken. The scale bar equals 20 μm. Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates
Figure 3
Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggrega
Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibod
specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy
(630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y secti
which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which t
section was taken. The scale bar equals 20 μm. Reorganiza
Figure 3 g
p
g
j
p
gg
g
g
Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates. HCVcc Infection of Huh7 3-D Aggregates Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodies
specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy
(630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sections,
which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z
section was taken. The scale bar equals 20 μm. HCVcc infection as cells infected 1 day post RWV seeding
(Fig. 4B-C), suggesting that differentiation and polariza-
tion does not negatively affect HCVcc infection in this 3-
D cell culture model. infectious virus production increased exponentially to lev-
els comparable to those reported using the robust 2-D
Huh7 system [2,46]. To determine the percentage of cells
expressing HCV proteins, indirect immunofluorescence
analysis of infected 3-D Huh7 aggregates was performed. Fig. 4B shows that the majority of Huh7 cells were positive
for the HCV E2 glycoprotein and that the entire aggregate
was permissive for HCV infection rather than just the cells
at the periphery, demonstrating that HCV can spread
throughout the aggregates. Importantly, Fig. 4D and 4E
illustrate that aggregates allowed to differentiate in the
RWV for 7 or 14 days were as equally permissive for Page 5 of 8
(page number not for citation purposes) http://www.virologyj.com/content/6/1/103 Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 Robust HCVcc infection in 3-D Huh7 RWV cultures
Figure 4
Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at
an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul-
ture supernatant and intracellular RNA were collected at the
indicated times p.i. Normalized intracellular HCV RNA copy
numbers, displayed as HCV RNA copies/μg total cellular
RNA (line), were determined by RTqPCR. Infectivity titers,
expressed as FFU/ml (bars), were determined by immunohis- aggregates relative to Huh7 monolayers (Fig. 2). Addition-
ally, we observed increased expression and organization
of cellular HCV receptors, cell adhesion, tight junction
and polarity-specific proteins, and the loss of cancer-asso-
ciated nuclear localization of β-Catenin, in RWV 3-D
Huh7 aggregates as compared to 2-D monolayers (Fig. 3). These data therefore suggest that the RWV environment
promotes differentiation of Huh7 cells down a more
hepatocyte-like route. Importantly, since these 3-D Huh7
cultures remain highly permissive for HCVcc infection,
this system represents a new in vitro cell culture system for
the study of HCV infection and antiviral drug studies in
more polarized, xenobiotically-competent cells. Relevant to the study of HCV, expression of the HCV
receptors CD81 and SR-B1 were both diffuse and poorly
organized in 2-D cultured Huh7s cells, while their expres-
sion was increased and localized to apical TJ regions and
basolateral-sinusoidal surfaces in 3-D aggregates. Like-
wise, TJ proteins, which typically localize to the apical sur-
face
in
polarized
hepatocytes
[43],
were
more
concentrated at the apical surface of 3-D Huh7 aggregates
as compared to monolayer cultures. Notably however, the
TJ protein CLDN1, a recently identified HCV receptor [7],
not only localized to TJs, but was also present at both api-
cal and basolateral surfaces in 3-D aggregates. This locali-
zation pattern is in agreement with other studies [47] and
the model proposed by Reynolds et al., describing tight-
junctional (apical) and nonjunctional (basolateral) forms
of CLDN1 in polarized hepatocytes [44]. As suggested by
Mee et al, it may be that these non-junctional pools of
CLDN1 have a direct role in HCV entry [11]. Interestingly,
Battle et al., have demonstrated a correlation between
HNF4α and cell adhesion and TJ molecules expression
and organization [48]. Whether this is also the case in the
3-D Huh7 aggregates, which have increased HNF4α
expression (Fig. 2A) remains to be determined. http://www.virologyj.com/content/6/1/103 Nonethe-
less, the ability of 3-D cultured Huh7 cells to better organ-
ize cell adhesion and TJ proteins is a phenotype consistent
with other RWV-cultured cell types [14,21,23]. As such,
RWV-cultured Huh7 cells provide an appropriate model
for investigating HCV entry, particularly the interaction,
organization, and stoichiometry of HCV receptors and TJ
proteins. Additional analyses to determine the extent of
differentiation and polarization of 3-D Huh7 aggregates is
still warranted and a focus of ongoing studies. Robust HCVcc infection in 3-D Huh7 RWV cultures
Figure 4
Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at
an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul-
ture supernatant and intracellular RNA were collected at the
indicated times p.i. Normalized intracellular HCV RNA copy
numbers, displayed as HCV RNA copies/μg total cellular
RNA (line), were determined by RTqPCR. Infectivity titers,
expressed as FFU/ml (bars), were determined by immunohis-
tochemical analysis of 10-fold serially diluted culture superna-
tants on naïve Huh7 cells. (B) Indirect immunofluorescence
analysis of HCV E2 expression in HCV-infected 3-D Huh7
aggregates 14 days p.i. Additional 3-D Huh7 cultures were
infected on day 1 (C), 7 (D) or 14 (E) post seeding in the
RWV. Aggregates were fixed 10 days p.i. and stained with a
human anti-E2 antibody (C1) and anti-human-Alexa 555 sec-
ondary antibody. Images were captured via confocal micros-
copy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha
Imager Browser v4.0 software (Zeiss). Image brightness and
contrast were adjusted using Adobe®Photoshop® (San Jose,
CA). (*) = 100 μm microcarrier bead. Small vertical panels
represent x-z sections of larger x-y sections, which were
compiled by taking 0.5 μm steps through corresponding x-y
sections. Red lines indicate the plane from which the z sec-
tion was taken. The scale bar equals 20 μm. Robust HCVcc infection in 3-D Huh7 RWV cultures
Figure 4
Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at
an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul-
ture supernatant and intracellular RNA were collected at the
indicated times p.i. Normalized intracellular HCV RNA copy
numbers, displayed as HCV RNA copies/μg total cellular
RNA (line), were determined by RTqPCR. Discussion Here we demonstrate that Huh7 cells cultured in RWV
bioreactors form multi-layered tissue-like aggregates that
are phenotypically distinct from traditional Huh7 2-D
monolayers (Fig. 1 and 2). Specifically, the RWV-environ-
ment promoted increases in hepatocyte-specific, as well as
Phase I and II metabolic gene transcripts in 3-D Huh7 Page 5 of 8
(page number not for citation purposes) Page 5 of 8
(page number not for citation purposes) Virology Journal 2009, 6:103
Robust HCVcc infection in 3-D Huh7 RWV cultures
Figure 4
Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at
an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Cul-
ture supernatant and intracellular RNA were collected at the
indicated times p.i. Normalized intracellular HCV RNA copy
numbers, displayed as HCV RNA copies/μg total cellular
RNA (line), were determined by RTqPCR. Infectivity titers,
expressed as FFU/ml (bars), were determined by immunohis-
tochemical analysis of 10-fold serially diluted culture superna-
tants on naïve Huh7 cells. (B) Indirect immunofluorescence
analysis of HCV E2 expression in HCV-infected 3-D Huh7
aggregates 14 days p.i. Additional 3-D Huh7 cultures were
infected on day 1 (C), 7 (D) or 14 (E) post seeding in the
RWV. Aggregates were fixed 10 days p.i. and stained with a
human anti-E2 antibody (C1) and anti-human-Alexa 555 sec-
ondary antibody. Images were captured via confocal micros-
copy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha
Imager Browser v4.0 software (Zeiss). Image brightness and
contrast were adjusted using Adobe®Photoshop® (San Jose,
CA). (*) = 100 μm microcarrier bead. Small vertical panels
represent x-z sections of larger x-y sections, which were
compiled by taking 0.5 μm steps through corresponding x-y
sections. Red lines indicate the plane from which the z sec-
tion was taken. The scale bar equals 20 μm. p
g
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors' contributions BS and VT participated in the design of the study, per-
formed the experiments and drafted the manuscript. SLU
designed the study and participated in drafting the manu-
script. All authors read and approved the final manu-
script. 14. Lamarca HL, Ott CM, Honer Zu Bentrup K, Leblanc CL, Pierson DL,
Nelson AB, Scandurro AB, Whitley GS, Nickerson CA, Morris CA:
Three-dimensional growth of extravillous cytotrophoblasts
promotes differentiation and invasion. Placenta 2005,
26:709-720. 15. Goodwin TJ, Prewett TL, Wolf DA, Spaulding GF: Reduced shear
stress: a major component in the ability of mammalian tis-
sues to form three-dimensional assemblies in simulated
microgravity. J Cell Biochem 1993, 51:301-311. http://www.virologyj.com/content/6/1/103 Infectivity titers,
expressed as FFU/ml (bars), were determined by immunohis-
tochemical analysis of 10-fold serially diluted culture superna-
tants on naïve Huh7 cells. (B) Indirect immunofluorescence
analysis of HCV E2 expression in HCV-infected 3-D Huh7
aggregates 14 days p.i. Additional 3-D Huh7 cultures were
infected on day 1 (C), 7 (D) or 14 (E) post seeding in the
RWV. Aggregates were fixed 10 days p.i. and stained with a
human anti-E2 antibody (C1) and anti-human-Alexa 555 sec-
ondary antibody. Images were captured via confocal micros-
copy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha
Imager Browser v4.0 software (Zeiss). Image brightness and
contrast were adjusted using Adobe®Photoshop® (San Jose,
CA). (*) = 100 μm microcarrier bead. Small vertical panels
represent x-z sections of larger x-y sections, which were
compiled by taking 0.5 μm steps through corresponding x-y
sections. Red lines indicate the plane from which the z sec-
tion was taken. The scale bar equals 20 μm. To date, attempts to study HCV in polarized cells have
been limited to colorectal adenocarcinoma Caco-2 cells
[10] or HepG2 cells [11], neither of which support robust
HCVcc infection. Although an inverse relationship
between cell polarization and HCV entry into polarized
Caco-2 [10] and HepG2 [11] cells has been observed no
such phenotype was observed in 3-D Huh7 aggregates. Specifically, 3-D Huh7 aggregates, infected at various Page 6 of 8
(page number not for citation purposes) Page 6 of 8
(page number not for citation purposes) http://www.virologyj.com/content/6/1/103 Virology Journal 2009, 6:103 http://www.virologyj.com/content/6/1/103 ice grant CA-133266 from the National Cancer Institute and the University
of Illinois Chicago Council to support Gastrointestinal and Liver Disease
(UIC GILD). VTC was supported by an Institutional Ruth L. Kirchstein
National Research Service Award (DK-007788-07) from the National Insti-
tute of Diabetes and Digestive and Kidney Diseases. ice grant CA-133266 from the National Cancer Institute and the University
of Illinois Chicago Council to support Gastrointestinal and Liver Disease
(UIC GILD). VTC was supported by an Institutional Ruth L. Kirchstein
National Research Service Award (DK-007788-07) from the National Insti-
tute of Diabetes and Digestive and Kidney Diseases. stages of differentiation (e.g. day 1, 7 or 14 post seeding),
were equally permissive for HCVcc infection (Fig. 4B–E). Abbreviations 8. Liu S, Yang W, Shen L, Turner JR, Coyne CB, Wang T: Tight junc-
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entry and are downregulated during infection to prevent
superinfection. J Virol 2009, 83:2011-2014. HCV: hepatitis C virus; JFH-1: Japanese Fulminant Hepa-
titis; RWV: rotating wall vessel; 3-D: three dimensional; 2-
D: two-dimensional; HCVcc: hepatitis C virus cell-cul-
tured produced; MOI: multiplicity of infection; FFU: focus
forming units; RTqPCR: real-time quantitative PCR; SR-
B1: scavenger receptor class B member 1; CLDN1: clau-
din-1; ZO-1: zona occludens 1; H&E: hematoxylin and
eosin; p.i.: post infection; HNF: hepatocyte nuclear fac-
tors; α1AT: alpha-1-antitrypisn; TTR: transthyretin; CYP:
cytochrome P450s; UGT: UDP-glucuronosyltransferase;
TJ: tight junction. p
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"BioMed Central will be the most significant development for
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Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
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BioMedcentral Publish with BioMed Central and every
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https://openalex.org/W2954380475
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https://www.openaccessrepository.it/record/24287/files/fulltext.pdf
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English
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Consequential LCA and LCC using linear programming: an illustrative example of biorefineries
|
The international journal of life cycle assessment
| 2,019
|
cc-by
| 10,425
| ERROR: type should be string, got "https://doi.org/10.1007/s11367-019-01650-6\nThe International Journal of Life Cycle Assessment (2019) 24:2191–2205 https://doi.org/10.1007/s11367-019-01650-6\nThe International Journal of Life Cycle Assessment (2019) 24:2191–2205 LCI METHODOLOGY AND DATABASES Abstract Purpose This paper aims to demonstrate how LCA can be improved by the use of linear programming (LP) (i) to determine the\noptimal choice between new technologies, (ii) to identify the optimal region for supplying the feedstock, and (iii) to deal with\nmultifunctional processes without specifying a certain main product. Furthermore, the contribution of LP in the context of\nconsequential LCA and LCC is illustrated. Methods We create a mixed integer linear program (MILP) for the environmental and economic assessment of new technologies. The model is applied in order to analyze two residual beech wood-based biorefinery concepts in Germany. In terms of the optimal\nconsequences for the system under study, the principle of the program is to find a scaling vector that minimizes the life cycle\nimpact indicator results of the system. We further transform the original linear program to extend the assessment by life cycle\ncosting (LCC). Thereby, two multi-objective programming methods are used, weighted goal programming and epsilon constraint\nmethod. Results and discussion The consequential case studies demonstrate the possibility to determine optimal locations of newly\ndeveloped technologies. A high number of potential system modifications can be studied simultaneously without matrix inver-\nsion. The criteria for optimal choices are represented by the objective functions and the additional constraints such as the available\nfeedstock in a region. By combining LCA and LCC targets within a multi-objective programming approach, it is possible to\naddress environmental and economic trade-offs in consequential decision-making. Conclusions This article shows that linear programming can be used to extend standard LCA in the field of technological choices. Additional consequential research questions can be addressed such as the determination of the optimal number of new production\nplants and the optimal regions for supplying the resources. The modifications of the program by additional profit requirements\n(LCC) into a goal program and Pareto optimization problem have been identified as promising steps toward a comprehensive\nmulti-objective LCSA. Keywords Eco-efficiency . Epsilon constraint . Goal programming . Mixed integer . Multi-objective optimization . Spatial LCA Keywords Eco-efficiency . Epsilon constraint . Goal programming . Mixed integer . Multi-objective opt Consequential LCA and LCC using linear programming: an illustrative\nexample of biorefineries Maik Budzinski1\n& Mattia Sisca1,2 & Daniela Thrän1,3 Received: 20 November 2017 /Accepted: 11 June 2019\n# The Author(s) 2019\n/Published online: 24 June 2019 * Maik Budzinski\nmaik.budzinski@ufz.de 1 Introduction In contrast,\nattributional LCA focuses on describing the life cycle of a\nproduct by attributing the environmental flows to the product\nunder study. The choice for either determining consequences or attribu-\ntional impacts also affects the way of modeling in terms of the\nmultifunctional problem. This problem arises when a process\nprovides more than one product that is not used within the\nsystem under study (Heijungs and Frischknecht 1998;\nHeijungs and Guinée 2007). In this case, the environmental\nburdens associated with the multifunctional process need to be\nallocated to different products. Several procedures have been\ndeveloped to solve the multifunctional problem (Heijungs and\nSuh 2002). Consequential LCA approaches prefer to use sub-\nstitution method to deal with the problem, whereas attribution-\nal LCA is a synonym for using the partitioning method\n(Ekvall and Weidema 2004; Thomassen et al. 2008; Schmidt\n2010). The ISO standard suggests the expansion of the system\nas one method to deal with multifunctional unit processes\n(ISO 2006). Also, partitioning method is recommended,\nwhere dividing unit processes and expanding the product sys-\ntem cannot be avoided. Several authors have identified that\nsystem expansion is conceptually equivalent to substitution\n(Heijungs and Guinée 2007; Tillman et al. 1994). Thereby,\nthe expansion of additional functions related to the co-\nproducts can either be carried out by subtracting the functions\nfrom the according process (substitution aka avoided burden\nmethod) or including the functions into the final demand vec-\ntor (system expansion by expanding the functional unit). In\nany way, dealing with multifunctional processes by using\npartitioning and/or substitution method depends on a model\nchoice (Majeau-Bettez et al. 2015). The choice of a certain\nmodel to deal with multifunctional processes is usually argued\nby the invertibility of the technology matrix (avoid rectangular\nmatrix) and the argumentation that the model represents real\nworld more closely than others would do (Suh et al. 2010). However, to solve systems with more products than processes\n(rectangular technology matrix), linear programming is a suit-\nable way (Heijungs and Suh 2002). The aim of this paper is, hence, to demonstrate how LCA\ncan be extended by the use of linear programming (i) to de-\ntermine the optimal choice between new technologies, (ii) to\nidentify the optimal region for supplying the feedstock, and\n(iii) to deal with multifunctional processes without specifying\na certain main product. 1 Introduction Responsible editor: Sangwon Suh Electronic supplementary material The online version of this article\n(https://doi.org/10.1007/s11367-019-01650-6) contains supplementary\nmaterial, which is available to authorized users. Electronic supplementary material The online version of this article\n(https://doi.org/10.1007/s11367-019-01650-6) contains supplementary\nmaterial, which is available to authorized users. Life cycle assessment (LCA), and in particular consequential\nLCA, is regarded as an appropriate tool for the assessment of\nenvironmental impacts of new bio-based technologies (e.g.,\nPawelzik et al. 2013). Despite the publication of various con-\nsequential modeling approaches, consequential LCA is still\nfar from a proper systematization (Zamagni et al. 2012). Although the use of consequential and attributional LCA for\ndecision-making is still being discussed (Ekvall et al. 2016;\nHertwich 2014; Plevin et al. 2014), all approaches are based\non the definition of consequential LCA that “is designed to\ngenerate information on the consequences of decisions”\n(Ekvall and Weidema 2004). Consequential LCA attempts to * Maik Budzinski\nmaik.budzinski@ufz.de 1\nHelmholtz Centre for Environmental Research – UFZ,\nPermoserstraße 15, 04318 Leipzig, Germany 2\nPolitecnico di Milano, Piazza Leonardo da Vinci 32,\n20133 Milan, Italy 3\nDeutsches Biomasseforschungszentrum (DBFZ) gemeinnützige\nGmbH, Torgauer Straße 116, 04347 Leipzig, Germany 2192 Int J Life Cycle Assess (2019) 24:2191–2205 of LCA with linear programming (LP) has a longer tradition. Azapagic and Clift (1995, 1998) introduced LP in the field of\nprocess engineering. Thereby, LP is used to allocate environ-\nmental burdens to multiple co-products by means of the mar-\nginal values at the solution of the LP model. Recent studies\nexist in the field of biomass utilization. Vadenbo et al. (2017)\nused a multi-objective optimization model to determine the\noptimal activity levels of a set of biomass process options. Kostin et al. (2012) carried out an assessment with a mixed-\ninteger linear programming approach for ethanol production\nchains. Based on a rectangular choice-of-technology model\n(Duchin and Levine 2011), Kätelhön et al. (2016) built a tech-\nnology choice model (TCM) for consequential LCA of rice\nproduction. Despite the attempts to incorporate LCA into a\nconsistent multi-objective framework combining linear pro-\ngramming and life cycle sustainability assessment (LCSA)\n(e.g., Gong and You 2017; Steubing et al. 2011; Liu et al. 2010), a conceptual discussion on how consequential LCA\nand LCC can be extended by LP is still scarce. determine how environmental flows will change due to the\nconsequences of a decision (Curran et al. 2005). 1 Introduction In doing so, we use the matrix-based\nnotation of Heijungs and Suh (2002) and create a rectangular\nmixed integer linear program and carry out a case study to\nillustrate its applicability. Thereby, two wood-based\nbiorefinery concepts are analyzed in terms of the optimal con-\nsequences for the total system under study. Furthermore, we\nmodify the original linear program into a weighted goal pro-\ngramming problem and a Pareto optimization model those\nextend the environmental focus on sustainability by adding\nthe economic perspective through a life cycle costing (LCC)\napproach. 3.1 Goal and scope Goal and scope\nstudy concerns two exemplar\nepts and data are based on\n6). The first biorefinery co\nnosolv lignin, and biomethan\nuces the same products as the\ns replaced by ethanol. The an\nmultifunctional biorefinery p\ne 1. The goal of the exempl\nly, we want to determine th\nopriate to reduce environmen\nnt production system. Second\nh site the biorefineries shoul\nly, to identify districts for\ntocks. 1\nAnnual input and output of b\nBi\nethane (m3)\n5.0\nene (kg)\n4. olysis lignin (MJ)\n1.9\nol (MJ)\n0 The study concerns two exemplary biorefinery concepts. The\nconcepts and data are based on Budzinski and Nitzsche\n(2016). The first biorefinery concept produces ethylene,\norganosolv lignin, and biomethane. The second configuration\nproduces the same products as the first one except for ethylene\nthat is replaced by ethanol. The annually produced amounts of\nthe multifunctional biorefinery processes are illustrated in\nTable 1. The goal of the exemplary case study is threefold. Firstly, we want to determine the biorefinery that is more\nappropriate to reduce environmental impacts in regard to the\ncurrent production system. Secondly, we want to determine at\nwhich site the biorefineries should be located (Fig. 1). And\nfinally, to identify districts for supplying woody biomass\nfeedstocks. An interesting but not often recognized characteristic of\nLCA is the existence of a price model beside the quantity\nmodel (As = f), which is similar to input-output models. This\nbecomes obvious when a price vector α is introduced (cf. Heijungs et al. 2013). Using this price vector, it is possible\nto compute the value added v per process. Table 1\nAnnual input and output of biorefinery processes\nBiorefinery 1\nBiorefinery 2\nBiomethane (m3)\n5.05·107\n5.05·107\nEthylene (kg)\n4.14·107\n0\nHydrolysis lignin (MJ)\n1.96·109\n1.96·109\nEthanol (MJ)\n0\n1.87·109\nOrganosolv lignin (kg)\n6.22·107\n6.22·107\nSodium hydroxide (kg)\n−5.04·105\n−5.04·105\nMagnesium sulfate (kg)\n−3.57·105\n−3.57·105\nPhosphorus (kg)\n−3.57·105\n−3.57·105\nSulfur (kg)\n−3.57·105\n−3.57·105\nSulfuric acid (kg)\n−3.76·106\n−3.76·106\nFodder yeast (kg)\n−2.32·106\n−2.32·106\nLime, packed (kg)\n−3.84·104\n−3.84·104\nRefrigerant (kg)\n−4.94·104\n0\nHeat, natural gas (MJ)\n−5.86·107\n0\nElectricity, natural gas (kWh)\n−1.08·109\n−1.35·109\nChemical factory (unit)\n−1.05·108\n−9.52·107\nTreated wastewater (m3)\n−3.33·10−2\n−3.33·10−2\nWater (kg)\n−2.84·106\n−3.16·106\nBeech wood (kg dm)\n−4.00·108\n−4.00·108 Table 1\nAnnual input and output of biorefinery processes v ¼ A0α\nð4Þ ð4Þ h ¼ Qg The so-called multifunctional problem arises if a process\ngenerates more than one product and if and only if the deliv-\nered functions are not used in the same proportion in the sys-\ntem (Heijungs and Frischknecht 1998). The most common\napproaches to solve the multifunction problem can be classi-\nfied into partitioning method and substitution method\n(Heijungs and Guinée 2007). By applying these procedures,\nthe technology matrix A becomes square, since an equal\namount of rows and columns exists. The partitioning method\ndivides the multifunctional process into processes that are\nmono-functional by the use of allocation factors. Several ways\nto gain these factors are possible, e.g., due to physical or\nmonetary characteristics of the products. The substitution\nmethod requires the definition of a mono-functional process,\nwhich provides the avoided product to the multi-functional\nprocess. Adding this process to the technology matrix A\nmakes its inversion possible. 2 Computational structure of LCA and LCC The computational structure of LCA is comprehensively de-\nscribed by Heijungs and Suh (2002). Mathematically, the first\nstep of life cycle inventory (LCI) analysis is the determination\nof the scaling vector s. The components of this vector scale\nprocesses of a system up or down such that the output of unit\nprocesses exactly matches the final demand. The final demand\nvector f includes the reference flows r, which are defined\nwithin the goal and scope of a LCA study. The scaling vector\ncan be determined by the multiplication of the inverse of tech-\nnology matrix A with the final demand vector. A major issue for standard life cycle assessment, which\naddresses one state of a particular product system, might be\nthe fact that no optimal production mixes can be determined. Usually, in LCA, this point is addressed by creating a small\nnumber of scenarios analyzing different product system alter-\nnatives iteratively (e.g., Budzinski and Nitzsche 2016; Renouf\net al. 2018). However, such procedure can be very time-con-\nsuming. Particularly in consequential LCA, this could be an\nissue. In many cases, not only the consequences of a potential\ndecision need to be described, but rather the optimal decision\nitself shall be identified. To overcome limitations in LCA,\namong other methods such as fuzzy programming (Tan et al. 2008) or graph methods (Vance et al. 2015), the combined use s ¼ A−1 f; with f i ¼\n> 0if i∈r\n0otherwise\n\u0001\nð1Þ s ¼ A−1 f; with f i ¼\n> 0if i∈r\n0otherwise\n\u0001\nð1Þ ð1Þ 2193 Int J Life Cycle Assess (2019) 24:2191–2205 g ¼ Bs ð3Þ 3 Exemplary case study The next step of the LCI analysis is to specify the inventory\nvector g according to the reference flows. This is done by\nmultiplying the intervention matrix B with the scaling vector. In the following sections, we demonstrate how the previously\nmentioned equations can be modified to create a rectangular\nchoice-of-technology model in regard to the goal and scope of\nthe study (Sect. 3.1). Therefore, we define a mixed integer\nlinear program (Sect. 3.2) and determine the results of an\nexemplary case study (Sect. 3.3). To include LCC aspects,\nwe transform the original problem into a weighted goal pro-\ngramming problem and a Pareto optimization model in Sects. 3.4 and 3.5, respectively. ð2Þ 1 http://www.cechemnet.de/Netzwerk/cechemnet_en v ¼ A0α To determine the value added in terms of a reference flow,\nthe elements of the scaling vector s can be multiplied by the\nvalue added of the corresponding processes, where 1 is the\nsummation operator (vector of ones). v0\nscaled ¼ 1 diag v\n0\n\u0003 \u0004\ns\n\u0003\n\u0004\nð5Þ ð5Þ ð5Þ This value added vector can then be used to determine life\ncycle costs (LCC). Heijungs et al. (2013) define life cycle cost\nof a composite final demand as l ¼ ∑j−vscaled\nj\n, where j runs\nover all case such that technical coefficient aij > 0, where ref-\nerence flows i is such that fi ≠0. In other words, the life cycle\ncosts of reference flows, which are specified in the final de-\nmand vector, are the negative sum of the corresponding entries\nin the scaled value added vector. 2194 Int J Life Cycle Assess (2019) 24:2191–2205 For simplicity reasons, we focus on four chemical parks of\nSchwarzheide. We assume that all chemical parks can provide\nFig. 1 Potential location for the two biorefinery types and annual availability of residual beech wood from forests within German districts (scenario\n100%) the two biorefinery types and annual availability of residual beech wood from forests within German districts (scenario Fig. 1 Potential location for the two biorefinery types and annual availability of residual beech wood from forests within German districts (scenario\n100%) Schwarzheide. We assume that all chemical parks can provide\nthe required infrastructure and utilities in the same manner. Furthermore, we suppose that either one or none of the\nbiorefinery concepts can be built at a location. Data on resid-\nual beech wood are estimated at the level of 37 administrative\ndistricts in Germany according to Polley and Kroiher (2006). For simplicity reasons, we focus on four chemical parks of\nthe Central European Chemical Network1 as potential plant\nlocations, which would in principal allow the building of the\ntwo biorefinery concepts: Leuna, Böhlen, Zeitz, and 2195 Int J Life Cycle Assess (2019) 24:2191–2205 This residual wood is currently not used and remains in forests\nafter chopping, which might be possibly used as feedstock for\nbiorefineries without inducing feedstock rivalry (Michels\n2009). Since the 100% use of available beech wood residuals\nis quite optimistic, we introduce a second scenario in which\nthe availability in each region is reduced by 50% (cf. Michels\n2009). v ¼ A0α The transport distances from the districts to the four\npotential plant locations can be covered by train and/or truck. In this study, we assume that up to 200 km, the wood is\ntransported only by truck. For distances greater than\n200 km, the wood is additionally transported by train. Thereby, we further assume that a minimal distance (between\n10 and 30 km depending on the availability of train network)\nis covered by truck. Varying transport distances for other pre-\nproducts of the biorefineries are neglected. multi-output processes are considered in the upstream system\nand, hence, is more general. The overall program can be summarized by the formulas 8\nto 11 considering that the scaling factors of biorefinery pro-\ncesses s1, s2, s3, s4, s5, s6, s7, and s8 are integers. Thereby, Eq. 8\nrepresents the objective function of minimizing environmental\nimpacts. Equation 9 illustrates the system under study includ-\ning all potential biorefineries, feedstock regions, and transpor-\ntation alternatives. Equation 10 includes the capacity con-\nstraints for feedstock availability in the regions. Equation 11\nconsiders the fact that only one of the two biorefinery options\ncan be built at a potential location. By using the linear pro-\ngramming model, we are interested in the reduction of the\ntotal environmental impacts of the current system. Hence,\nthe objective function results from Eqs. 2 and 3, and the target\nis to find a vector s that minimizes the total impacts h of the\nsystem, which is expressed by matrix A. Avisualization of the\nmodeling principle including the sub-matrices of A is given in\nFig. 2. The fundamental principle of the model is to check\nwhether environmental impacts can be reduced by biorefinery\n1 or 2 considering 4 potential locations (ABRS) and 37 regions\nof wood supply (AS). If not, the model would only choose\ncurrent processes (ARef) to provide the components of the final\ndemand. We assume that the new biorefinery products compete with\nexisting products of the current system. Hence, we define\nsubstitutable reference products for each biorefinery product:\nbiomethane vs. natural gas, bio-based ethylene vs. fossil eth-\nylene, hydrolysis lignin vs. lignite briquettes, ethanol vs. pet-\nrol, and organosolv lignin vs. polyol. The definition of these\navoided products is a long-discussed issue in LCA. Since the\ngeneral societal aim is to move towards a bio-based economy,\nwe use fossil-based references that provide the same\nfunctions. v ¼ A0α The amounts of reference flows are specified in regard to\nthe German demand of these products within the current sys-\ntem. Thereby, the reference flows need to be specified in the\nmanner that the products of current processes must provide the\nsame functions as of the biorefinery products. In the example,\nwe assume that this is guaranteed, e.g., by referring to MJ in\nthe case of ethanol and gasoline. Minimize h ¼ QBs\nð8Þ\nsubject to Minimize h ¼ QBs\nð8Þ ð8Þ e h\nQ s\nð8Þ\nsubject to subject to subject to subject to As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n:\nif i∈r\nif i∈T; S\notherwise\nð9Þ\n0≤s≤c\nð10Þ\ns1 þ s2 ≤1\ns3 þ s4 ≤1\ns5 þ s6 ≤1\ns7 þ s8 ≤1\nð11Þ As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n:\nif i∈r\nif i∈T; S\notherwise\nð9Þ As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n: ð9Þ For LCIA, we chose the category climate change taking\ninto account the three substances CO2, CH4, and N2O. ð10Þ ð11Þ As ¼ f A ¼\nABRS\n0\n0\n0\nARef\n−A*\nup\nAup\n−A**\nup\n−A***\nup\n0\n0\n0\nAS\n−A*\nS\n0\n−A*\nT\n0\n0\nAT\n0\n2\n664\n3\n775\nð12Þ by the inequation ð12Þ ð7Þ 3.2 The linear programming model According to Heijungs and Suh (2002), the principle of ex-\ntending LCA by LP is to relax the balance equations into\ninequations. Hence, we can replace the equation with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℕ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℕ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℕ The rectangular technology matrix Awith the dimension of\nproducts-by-processes consists of several sub-matrices. As ¼ f\nð6Þ\nby the inequation\nAs≥f:\nð7Þ ð6Þ As≥f: By using this formulation, the solution of such program\nwould allow flows that are greater than those specified in the\nfinal demand vector. Regarding the special case in which pro-\ncesses are defined to be strictly mono-functional, the formu-\nlation as As = f would also work for solving the LP. The for-\nmulation in Eq. (7) also provides solutions for cases in which These sub-matrices have the following dimensions: These sub-matrices have the following dimensions: ABRS\nir × jBRS (5 × 8)\nARef\nir × jRef (5 × 5)\nAup\niup × jup (17 ×\n17) ABRS\nir × jBRS (5 × 8)\nARef\nir × jRef (5 × 5)\nAup\niup × jup (17 ×\n17) −A*\nup\niup × jBRS (17 ×\n8)\n−A**\nup\niup × jS (17 × 37)\n−A***\nup\niup × jT (17 ×\n296)\n−A*\nT\niT × jBRS (8 × 8)\nAT\niT × jT. (8 × 296)\n−A*\nS\niS × jT. (37 × 296)\nAS\niS × jS.(37 × 37). with,\nir\nbiorefinery/reference product\niup\nproduct of upstream process\niT\ntransported wood\niS\nsupplied wood in a district\njBRS\nbiorefinery process\njRef\nsubstitutable reference process\njup\nupstream process\njS\nregion for supplying wood\njT\ntransport option\nThe columns of matrix ABRS represent the biorefinery op-\ntions jBRS of each type at each potential location. The rows of\nthis matrix represent the products ir which can also be pro-\nduced by current reference processes jRef. Matrix ARef\nincludes current available processes jRef that produce the same\nfunctions ir as the biorefinery processes. To address the choice for a certain district, which deliver\nthe residual beech wood, the model is extended by the matri-\nces AT, −A*\nT, AS and −A*\nS. Matrix AS is an identity matrix to\nconsider the different alternatives of beech wood supply from\nthe 37 districts. Matrices −A*\nS and ATare needed to include the\nalternatives of beech wood transport from each district to each\none of the 8 biorefinery alternatives. The linkage of the these\nalternatives to the biorefinery options is represented by matrix\n−A*\nT. Upstream processes in matrix Aup, which are needed to\nprovide the pre-products of the biorefineries, the cultivation,\nand transportation of wood and the reference products, com-\nplete the rectangular A matrix. As≥f: The program is designed as an LCA for a final demand fr\non reference flows r, which are characterized by the products\nof the biorefinery system and the corresponding reference\nproducts (biomethane/natural gas, bio-based ethylene/fossil\nethylene, hydrolysis lignin/lignite briquettes, ethanol/petrol,\norganosolv lignin/polyol). The amounts for the multiple com-\nponents of the functional unit are taken from statistical sources\n(e.g., VCI 2018; SDK 2017) and represent those of a current\nGerman demand. Other components of the final demand\nFig. 2 Simplified flowchart\nInt J Life Cycle Assess (2019) 24:2191–2205\n2196 Int J Life Cycle Assess (2019) 24:2191–2205 2196 Fig. 2 Simplified flowchart Fig. 2 Simplified flowchart includes current available processes jRef that produce the same\nfunctions ir as the biorefinery processes. To address the choice for a certain district, which deliver\nthe residual beech wood, the model is extended by the matri-\nces AT, −A*\nT, AS and −A*\nS. Matrix AS is an identity matrix to\nconsider the different alternatives of beech wood supply from\nthe 37 districts. Matrices −A*\nS and ATare needed to include the\nalternatives of beech wood transport from each district to each\none of the 8 biorefinery alternatives. The linkage of the these\nalternatives to the biorefinery options is represented by matrix\n−A*\nT. ir\nbiorefinery/reference product\niup\nproduct of upstream process\niT\ntransported wood\niS\nsupplied wood in a district\njBRS\nbiorefinery process\njRef\nsubstitutable reference process\njup\nupstream process\njS\nregion for supplying wood\njT\ntransport option Upstream processes in matrix Aup, which are needed to\nprovide the pre-products of the biorefineries, the cultivation,\nand transportation of wood and the reference products, com-\nplete the rectangular A matrix. The program is designed as an LCA for a final demand fr\non reference flows r, which are characterized by the products\nof the biorefinery system and the corresponding reference\nproducts (biomethane/natural gas, bio-based ethylene/fossil\nethylene, hydrolysis lignin/lignite briquettes, ethanol/petrol,\norganosolv lignin/polyol). The amounts for the multiple com-\nponents of the functional unit are taken from statistical sources\n(e.g., VCI 2018; SDK 2017) and represent those of a current\nGerman demand. Other components of the final demand The columns of matrix ABRS represent the biorefinery op-\ntions jBRS of each type at each potential location. The rows of\nthis matrix represent the products ir which can also be pro-\nduced by current reference processes jRef. f S;T ¼ 0 is introduced. We further define the first eight scaling factors for the\nbiorefinery processes to be integers. This choice is neces-\nsary, since the data collection for the biorefineries is based\non process simulation for a specific capacity. The inter-\nmediate flows and environmental interventions associated\nwith this capacity are not up- or downscalable in a linear\nmanner. The program is completed by introducing lower\nbounds (set to 0) and a vector of upper bounds c. In\nregard to the availability of residual beech wood in each\ndistrict, the data of Fig. 1 is considered as upper bounds\nfor sjS. The upper bounds c for the scaling factors of\nbiorefinery processes (sjBRS ) were set to 1, since we as-\nsume that only one biorefinery can be built at a chemical\npark. To study the origin of wood supply it is possible to analyze\nthe corresponding scaling factors of the transport processes jT. These factors represent the contribution to the overall wood\ndemand of the biorefineries. Figure 3a illustrates the solutions\nfor the 100% scenario. Thereby, the biorefinery in Zeitz is\ndelivered from regions in the middle west of Germany and\nfrom regions in the middle south. In contrast, the biorefinery\nin Schwarzheide is delivered by regions in the northeastern\npart of Germany. The LCI data for upstream biorefienry processes and\nfor the substitutable reference system is taken from the\necoinvent 3.2 database (cut-off model). The whole prob-\nlem and the corresponding data can be studied in more\ndetail in the Electronic Supplementary Material\n(MILP_LCA.xlsx). Due to the reduced wood availability in the 50% scenario,\nregions supplying the feedstock to biorefinery 1 in Leuna are\ndistributed throughout Germany (Fig. 3b). Hence, a large\ncatchment area is required to fulfill the demand of the\nbiorefinery in the scenario with reduced residual beech wood\navailability. 3.3 Results To solve the mixed integer linear program, the intlinprog\nsolver in Matlab was used. An additional scenario is an-\nalyzed in which the available amount of beech wood is\nreduced by 50%. The results for 100% scenario (1.14 Mt\ndm beech wood in Germany) indicate that two\nbiorefineries with a capacity of 0.4 Mt dm are built at\nthe considered chemical parks. Since the scaling factors\nfor biorefineries are defined as integers, not the total\navailable amount of beech wood is exhausted. The opti-\nmal locations to build the biorefineries are Zeitz and\nSchwarzheide. It is visible that for both scenarios, the\nethylene-producing biorefinery 1 is more preferable to\nreduce greenhouse gas emissions than the ethanol-\nproducing biorefinery 2 (Table 2). When reducing the\navailable residual wood for all districts by 50%, another\nlocation becomes more preferable than those of the 100%\nscenario. This is an interesting result, since one might\nassume that one of the locations identified in the 50% As≥f: Matrix ARef Int J Life Cycle Assess (2019) 24:2191–2205 2197 vector f are set to zero. The design of an LCA for a demand on\nthe functional unit does not take into account the linkage of\ndownstream processing with the modeled upstream process-\ning so that economy-wide impacts are cut off (Suh 2004). However, the modeling in regard to the functional unit is a\ngeneral principle of LCA (Peters and Hertwich 2006). To en-\nsure the balance between supply and demand of transported\nbeech wood, the constraint\n0\n0\nAS\n−A*\nS\n0\n−A*\nT\n0\n0\nAT\n0\n\u0005\n\u0006\ns ¼\nf S;T ¼ 0 is introduced. would be chosen. The total reduction of the impacts on\nclimate change compared to the impacts of the current\nsystem (hRef = QRef ∙BRef ∙ARef\n−1 ∙fr) is 4.76 Mt/a CO2\neq. in the 100% scenario and 2.38 Mt/a CO2 eq. in the\n50% scenario. The contribution analysis for the 100% scenario shows\nthat current substitutable processes still provide the ma-\njor part of the overall GHG emissions (lignite production\n88.48%, natural gas production 8.24%, petrol production\n1.95%, ethylene production 1.05%, polyol production\n0.25%) whereas the biorefinery system plays a minor\npart (biorefineries 0.12%, wood production −0.20%, oth-\ner upstream processes including transport 0.12%). Due to\nthe feedstock availability of German beech wood resid-\nuals, only a small ratio of current GHG emissions could\nbe reduced. Detailed information on contribution analy-\nses is provided in the Electronic Supplementary Material\n(MILP_LCA.xlsx). f S;T ¼ 0 is introduced. 3.4 Inclusion of LCC by multi-objective optimization For example, it might also reasonable to explicitly take\ninto account the profit for reference processes competing with\nbiorefineries, or to use discounted cash flow analysis taking\ninto account the time value of money (e.g., net present value). However, additional data must be collected. For simplicity\nreasons, we chose the standard approach of LCC in this study. Minimize whdþ\nh þ wzd−\nz\nð15Þ\nsubject to Minimize whdþ\nh þ wzd−\nz\nð15Þ ð15Þ subject to subject to subject to As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n:\nif i∈r\nif i∈t\notherwise\nð16Þ As\n≥f i > 0\n¼ f i ¼ 0\n≥f i ¼ 0\n8\n<\n: ð16Þ QBs þ d−−dþ ¼ h\nð17Þ\nps þ d−−dþ ¼ z\nð18Þ\n0≤s≤c\nð19Þ\ns1 þ s2 ≤1\ns3 þ s4 ≤1\ns5 þ s6 ≤1\ns7 þ s8 ≤1\nð20Þ QBs þ d−−dþ ¼ h\nð17Þ\nps þ d−−dþ ¼ z\nð18Þ\n0≤s≤c\nð19Þ\ns1 þ s2 ≤1\ns3 þ s4 ≤1\ns5 þ s6 ≤1\ns7 þ s8 ≤1\nð20Þ QBs þ d−−dþ ¼ h\nð17Þ\nps þ d−−dþ ¼ z\nð18Þ ð17Þ ð19Þ 0≤s≤c To deal with multiple objectives in linear programming,\nbasically, three groups can be distinguished: a priori, interac-\ntive, and a posteriori methods (Hwang and Masud 1979). The\nfirst class requires the definition of preferences between goals\nat the beginning of the solution process. The latter class re-\nquires the prioritization at the end after the presentation of all\nPareto optimal solutions. To illustrate the pros and cons of the\nbroad range of applications, we modify the linear program in\nthe next sections choosing weighted goal programming for an\na priori approach and epsilon-constraint method for an a\nposteriori approach. ð20Þ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℤ with s1, s2, s3, s4, s5, s6, s7, s8 ∈ℤ Table 3 shows the result of the GP model for both scenar-\nios. Contrary to the LP model, the optimal solution is the\nethanol concept (biorefinery 2) for both scenarios. The opti-\nmal chemical parks of the chosen biorefineries are identical to\nthose of the LP model. The regions that deliver the wood\nfeedstock to the plants are also identical. Therefore we omit\nto illustrate them again in the map. 3.4 Inclusion of LCC by multi-objective optimization For h,\nwe chose 0, since the aim of a sustainable economy can be\nregarded to be carbon neutral. For the profit target z, we\nchoose an arbitrarily large value (1.00E + 15 €) which is\nequivalent to maximize the objective function. The weighting\nfactors w result in a normalization and prioritization of the\ntarget deviations. For greenhouse gas emissions, we use the\nweighting factor wh = 0.025 which is the value of expected\ndamage costs in terms of €/kg CO2-eq (De Bruyn et al. 2010). The choice of this factor is concerned by uncertainty,\ncomparable to those related to the monetization of environ-\nmental impacts. Due to the illustrative character of this study,\nwe do not assess this in detail. However, robust optimization\nmay take uncertainty of parameters explicitly into consider-\nation (Wang and Work 2014). The weighting factor wz is set to\n1, since the unit of the profit target is €. Equation 13 consists of two major terms. The first term\nconsiders the cash-flows related directly to the biorefinery\nprocesses jBRS including revenues for biorefinery products processes jBRS including revenues for biorefinery products\n( ∑\niBRS¼1\nIBRS\nABRS\niBRS;jBRS \u0002 αiBRS ), payments for pre-products\n( ∑\niup¼1\nIup\nA*\niup;jBRS \u0002 αiup ), payments for wood ( ∑\niT¼1\nIT\nA*\niT;jBRS \u0002 αiT ),\nand other payments such as for personal and insurance\n( ∑\nK\nk¼1\nFk jBRS ). The second term represents the payments related\nto the transportation options jT of wood. This expression can\nbe simplified in matrix notation as Maximize z ¼ pËCs;\nð14Þ ð14Þ where the components of vector p specify the profit of pro-\ncesses j. This LCC approach is congruent to the proposed one by\nHeijungs et al. (2013), but differs in the way that only those\ncosts are taken into account, which are relevant for the invest-\nment decision for the biorefinery options. This choice is con-\ncerned with taking the view of potential investors maximizing\nthe profit of a biorefinery. Thereby, only the costs are consid-\nered that are relevant for the decision on the profitability of the\nbiorefinery (including costs for transportation and additional\ninputs). Besides to this simple LCC approach, however, there\nare further possibilities to consider cost information by the\nmodel. 3.4 Inclusion of LCC by multi-objective optimization Since we have not considered transportation and other costs so\nfar in this case study, we adopt the model by introducing an\nadditional objective for life cycle costing. In doing so, we\nintroduce for the specific biorefineries an additional input fac-\ntor matrix F with subcategories k (expressed in monetary\nunits): personal costs, taxes, insurance, and administration. As Heijungs et al. (2013) showed, the consideration of costs\nfor the inputs and outputs of the processes of the technology\nmatrix A can be carried out by using a price vector α. Since\nwe focus on the investor’s perspective, price information is\nonly required for products that are relevant for the profitability\nof the biorefineries. The additional objective function, which\nincludes costs for processed and produced commodities of the\nbiorefinery processes as well as the costs for transported beech\nwood, can be derived as Int J Life Cycle Assess (2019) 24:2191–2205 2198 Table 2\nResults of the mixed integer linear program, per annum Table 2\nResults of the mixed integer linear program, per annum\n100% scenario\n50% scenario\nBiorefinery 1, Leuna (pc.)\n0\n1\nBiorefinery 2, Leuna (pc.)\n0\n0\nBiorefinery 1, Zeitz (pc.)\n1\n0\nBiorefinery 2, Zeitz (pc.)\n0\n0\nBiorefinery 1, Böhlen (pc.)\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n0\nBiorefinery 1, Schwarzheide (pc.)\n1\n0\nBiorefinery 2, Schwarzheide (pc.)\n0\n0\nNatural gas (m3)\n9.580053·1010\n9.585027·1010\nFossil ethylene (kg)\n5.017197·109\n5.058598e·109\nLignite (MJ)\n3.214008·1013\n3.214204·1013\nPetrol (MJ)\n7.340000·1011\n7.340000·1011\nPolyol (kg)\n4.342704·108\n4.964352·108\nh (kg CO2-eq.)\n7.152422·1011\n7.156019·1011\nPotential savings h −hRef (kg CO2-eq.)\n−7.193181·108\n−3.596181·108 Maximize z ¼\n∑\njBRS¼1\nJ BRS\n∑\niBRS¼1\nIBRS\nABRS\niBRS;jBRS \u0002 αiBRS\n \n! −\n∑\niup¼1\nIup\nA*\niup;jBRS \u0002 αiup\n \n! −\n∑\niT ¼1\nIT\nA*\niT ;jBRS \u0002 αiT\n \n! −∑\nK\nk¼1\nFk jBRS\n \n! \u0002 sjBRS−∑\njT¼1\nJ T\n∑\niup¼1\nIup\nA***\niup;jT \u0002 αiup\n \n! \u0002 sjT\nð13Þ ð13Þ Fig. 3 Optimal regions to supply beech wood to the biorefineries Fig. 3 Optimal regions to supply beech wood to the biorefineries Fig. 3 Optimal regions to supply beech wood to the biorefineries Fig. 3 Optimal regions to supply beech wood to the biorefineries 2199 Int J Life Cycle Assess (2019) 24:2191–2205 respectively. The target function of the goal program mini-\nmizes the deviations in terms of the desired values. 3.4 Inclusion of LCC by multi-objective optimization The reason for choosing\nbiorefinery 2 is the higher priority to minimize the negative\ndistance to the economic target value (d−\nz ) compared to the\npriority of minimizing the positive distance to the environ-\nmental target value (dþ\nh ). Thus, the total annual amount of\nenvironmental savings is reduced from 0.72 Mt/a and 0.36 Mt/\na CO2-eq to 0.37 Mt/a and 0.18 Mt/a CO2-eq in the 100% 3.4.1 Goal programming In weighted goal programming, the objectives are taken ex-\nplicitly into account as constraints (Miller and Blair 2009). The principle of goal programming (GP) is to use slack vari-\nables dh and dz that measure the deviation from the desired\nenvironmental and economic target values h and z, Int J Life Cycle Assess (2019) 24:2191–2205 2200 Table 3\nResults of the goal program, per annum\n100% scenario\n50% scenario\nBiorefinery 1, Leuna (pc.)\n0\n0\nBiorefinery 2, Leuna (pc.)\n0\n1\nBiorefinery 1, Zeitz (pc.)\n0\n0\nBiorefinery 2, Zeitz (pc.)\n1\n0\nBiorefinery 1, Böhlen (pc.)\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n0\nBiorefinery 1, Schwarzheide (pc.)\n0\n0\nBiorefinery 2, Schwarzheide (pc.)\n1\n0\ndþ\nh (kg CO2-eq.)\n7.155941·1011\n7.157785·1011\nd−\nz (€)\n9.999991·1013\n9.999995·1013\nProfit z−d−\nz (€)\n9.174736·107\n4.509840·107\nPotential savings hRefS−dþ\nh (kg CO2-eq.)\n−3.674181·108\n−1.830181·108 Table 3\nResults of the goal program, per annum process-based LCC. However, a possible way is to implement\nan additional constraint in the LP model that ensures the ex-\nceeding of a minimal positive profit value. scenario and the 50% scenario, respectively. The annual\nprofits are 92 Mio € and 45 Mio € in the 100% scenario and\nthe 50% scenario, respectively. When introducing economic\naspects, this result is similar to the one of Budzinski and\nNitzsche (2016) who also concluded that the ethanol-\nproducing concept has a better economic performance than\nthe ethylene-producing concept. However, in Budzinski and\nNitzsche (2016), only the ethanol biorefinery is profitable,\nwhich is contrary to the results of this study. The reason for\nthat is the authors’ use of a dynamic cost calculation approach\nthat takes the time value of money into account. Thereby, the\ninternal rate of return on the investment must be at least equal\nthe minimum rate of return a decision-maker is willing to\naccept. The time value of money usually is not considered in 3.4.2 Epsilon-constraint method Comparing this difference with the\noverall GHG emissions of the system, one might assume that\nthe results are not reliable in terms of data inaccuracies. To\naddress this point, we have a look at the reference flows of the\nfinal demand vector. The chosen amounts represent an esti-\nmated total demand for Germany and, hence, are quite high. However, lower values would not lead to different results and\nwould not reduce uncertainty. In fact, the choice of values for\nthe considered reference flows is arbitrary. The determination\nof optimal locations for biorefineries is, hence, not affected by\nthis choice. Since we here assume identical conditions at the\npotential chemical parks, the identification of optimal loca-\ntions is only determined by the transport distances and the\ncorresponding impacts and costs. On the contrary, the choice\nfor the type of biorefinery in terms of environmental impacts\nis mainly dominated by its emissions and those of the refer-\nence processes. In terms of profit, the optimal solution is\nmainly dominated by the corresponding costs for biorefinery\n(pre)-products. In conclusion, the results for the optimal\nchoices of locations for biorefineries may be considered being\nrobust in the face of data uncertainties in other parts of the\nmodel. A problem that might result is numerical issues when\nsolving a problem with large discrepancies in order of Pareto optimal solutions are solutions that cannot be im-\nproved in any of the two objectives without degrading the\nother objective. The Pareto optimal solutions of the 100%\nscenario are illustrated in Fig. 4. In this example, three\nPareto optimal solutions are identified. The dominated solu-\ntions, which are not Pareto optimal, are below the blue line. However, more comprehensive problems can result in various\nnon-dominated solutions. Solution P1 suggests biorefinery 1\n(ethylene) in Zeitz and in Schwarzheide. In contrast to this\nenvironmentally most preferable solution, P3 contains the\nmost preferable solution in terms of profit (Table 4). Thereby, biorefinery 2 (ethanol) is located in Zeitz and\nSchwarzheide. The solution P2 can be interpreted as a com-\npromise between these extreme solutions of maximal profit\nand minimal impacts on climate change. Furthermore, the\nsolution may become worthwhile due to the fact that both\nbiorefinery concepts (ethylene and ethanol) would be built\nin Zeitz and Schwarzheide. This mixture of technologies\nmight be also interesting for decision-making. 3.4.2 Epsilon-constraint method A disadvantage of goal programming might be the require-\nment of defining of preferences at the beginning of the solu-\ntion process. Alternatively, a posteriori methods are available\nto prioritize between goals after the generation of all Pareto\nefficient solution. In doing so, epsilon-constraint method has\nbeen already used in the field of LCA (e.g., Azapagic and Clift\n1999). Here, in this study, we apply the epsilon-constraint\nmethod in GAMS (Mavrotas 2009a, b). Thereby, the target Fig. 4 Pareto front (100%\nscenario). h: environmental\nimpacts; z: profit Fig. 4 Pareto front (100%\nscenario). h: environmental\nimpacts; z: profit Fig. 4 Pareto front (100%\nscenario). h: environmental\nimpacts; z: profit 2201 Int J Life Cycle Assess (2019) 24:2191–2205 Table 4\nResults of the epsilon-constraint method (100% scenario), per annum\nP1\nP2\nP3\nh (kg CO2-eq.)\n7.154561·1011\n7.155251·1011\n7.155941·1011\nz (€)\n5.183191·107\n7.178963·107\n9.174736·107\na)\nb)\nBiorefinery 1, Leuna (pc.)\n0\n0\n0\n0\nBiorefinery 2, Leuna (pc.)\n0\n0\n0\n0\nBiorefinery 1, Zeitz (pc.)\n1\n0\n1\n0\nBiorefinery 2, Zeitz (pc.)\n0\n1\n0\n1\nBiorefinery 1, Böhlen (pc.)\n0\n0\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n0\n0\n0\nBiorefinery 1, Schwarzheide (pc.)\n1\n1\n0\n0\nBiorefinery 2, Schwarzheide (pc.)\n0\n0\n1\n1 Table 4\nResults of the epsilon-constraint method (100% scenario), per annum function of profit (Eq. 14) is additionally involved in the op-\ntimization problem as solution is nearly located to another such as P2 in the 50%\nscenario, clearly is an advantage of a posteriori approaches\nover a priori methods. Minimize h sð Þ; −z sð Þ\nð\nÞ:\nð21Þ ð21Þ Minimize h sð Þ; −z sð Þ\nð\nÞ: A special look requires the influence of transportation dis-\ntances on the overall result (profit and environmental im-\npacts). In this study the transportation of feedstock only has\na limited influence on the overall profit of the biorefinery. For\ninstance, the difference between biorefinery type 2 in Leuna\ncompared to the location in Böhlen is 177,280 € per year\n(Table 5). In regard to the potential reduction of GHG emis-\nsions, the difference is even less significant. Here, the\nbiorefinery 2 in Leuna has a reduction potential at 2000 kg\nCO2 eq. per year higher than in Böhlen (Pareto point 2 and 3\nof 50% scenario, Table 5). 3.4.2 Epsilon-constraint method In contrast to\ngoal programming in which only one solution is determined in\naccordance with the a priori defined priority order, Pareto\noptimization allows the decision-maker to deal with trade-\noffs after investigating the non-dominated solutions. The non-dominated solutions of the 50% scenario are illus-\ntrated in Fig. 5. The most beneficial solution P1 in terms of\nimpacts on climate change is given by the biorefinery 1 locat-\ned in Leuna. Biorefinery 2 in Leuna is the most profitable\nsolution P3. Contrary to the 100% scenario, the compromise\nsolution P2 is near to P3 suggesting biorefinery 2 in Böhlen. An interesting fact is that P3 (in both scenarios) is the solution\nof the goal programming approach (Sect. 3.4.1). However, the\nidentification of all non-dominated solutions, even if the Int J Life Cycle Assess (2019) 24:2191–2205 2202 extension of LCA is to provide a broader and systematic as-\nsessment of consequences. The implicit environmental com-\nparison of new bio-based technologies with fossil reference\ntechnologies can be regarded as a feature that has not been\nprovided by other optimization models within the field of\nLCA. The LCC formulation used four our purpose is congru-\nent to the suggestion of (Heijungs et al. 2013), since we only\nfocus on the processes that provide the reference flows. However, it differs in the way that we only need to collect\ndata for costs which are relevant for the decision-maker as a\nbiorefinery investor and neglecting the costs for current tech-\nnologies. This and the consideration of environmental targets\ncan be interpreted as an eco-efficiency approach. However,\ninstead of simply creating the fraction between an economic\nvalue and an environmental value (ISO 2012), the approach in\nthis study allow to assess a specific target achievement, i.e. being less pollutant than current available technologies. magnitude. Back substituting the solution vectors s into Eqs. 9\nand 16, however, identified the results´ reliability in terms of\naccuracy. magnitude. Back substituting the solution vectors s into Eqs. 9\nand 16, however, identified the results´ reliability in terms of\naccuracy. Fig. 5 Pareto front (50%\nscenario). h: environmental\nimpacts; z: profit 4 Discussion LCA has been developed for the assessment of environmental\nimpacts of a product. To broaden the scope of LCA, Udo de\nHaes et al. (2004) propose three general strategies: the use of\nLCA in a toolbox, hybrid analysis, and the extension of LCA. Using LCA in a toolbox, the limitations shall be overcome by\nadditional separate models that are used without a data link. The extension of LCA is considered with one consistent linear\nmodel. Thereby, LCA and the other tool are fully compatible. Hybrid modeling as a mixture of both approaches that com-\nbines LCAwith other models and linking these by data flows. The extension with linear programming (LP) leads to a con-\nsistent linear model that determines the optimal choice among\nothers for the total system under study. Interdependent choices\nin different regions can be studied simultaneously without\nmatrix inversion, since with LP even rectangular systems\ncan be solved. The criteria for choices are represented by the\nobjective function (minimizing impacts on climate change)\nand the additional constraints (e.g., available feedstock in a\nregion). It is shown that the modification of the program by\nadditional profit requirements (LCC) into a goal program and\na Pareto optimization approach also enables to incorporate\nmultiple objectives within the decision-making process. Thereby, regional biomass availability and transport logistic\noptions can be taken into consideration. The benefit of this Dealing with multiple objectives, GP needs an a priori\nweighting of the different goals. In contrast, epsilon constraint\nmethod uses the concept of Pareto efficiency in which the\nsolution is optimal in which an increase of a target would\nresult in a decrease of another target. The advantage of GP is\nthat one optimal solution can be determined whereas in the a\nposteriori method, the decision-maker is encouraged to decide\nfor a compromise. Besides these two methods for multiple\nobjective decision making, there exist further methods, which\ncannot be discussed here. A general overview including the\npros and cons is given by Hwang and Masud (1979). Rectangular LP models for choosing technologies have\nbeen used in input-output economics. Duchin and Levine\n(2011, 2012) introduced a rectangular model to study the Fig. 5 Pareto front (50%\nscenario). h: environmental\nimpacts; z: profit Fig. 5 Pareto front (50%\nscenario). 4 Discussion But instead of sectors as in input-output\nmodels, the columns of the technology matrix in LCA repre-\nsent processes, which are usually modeled in more detail. Furthermore, these processes can be multi-functional (e.g.,\nKätelhön et al. 2016), which is contrary to input-output\nmodeling in which the assumption of homogenous sector out-\nputs is implied by creating the technical coefficient matrix\n(Miller and Blair 2009). In our example, only the biorefinery\nprocesses are multi-functional. Solving the multifunctional\nproblem for these technologies can be interpreted as a system\nexpansion approach. Furthermore, this approach is equivalent\nto substitution method, since the implicit aim of the introduced\nmodels is the substitution of current processes by less pollut-\nant processes. To compare the biorefineries, the systems are\nexpanded by current substitutable processes which produce\nthe same type of products. Due to that a programming prob-\nlem results, in which we seek to find the optimal substitution\nof current processes by a set of biorefinery alternatives. Other\nprocesses in the example are mono-functional. However, if\nother multioutput processes would be considered within the\nupstream processes of the biorefinery or the reference system,\nthe multi-functionality can be solved by the program\ninterpreting it as a kind of surplus method (Heijungs and\nSuh 2002). When minimizing the total environmental impacts\nof the system under study, larger amounts of supplied products\nwould be allowed compared with those in the final demand. It\nis crucial to take this into consideration, since more products\nand hence more functions are possible in the final supply\nvector of the optimized system. On the other hand, if products\nof current technologies are not provided by the new technol-\nogies, the program would ensure that at least the amounts of\nthe current system are generated. In this case a study would\nonly be meaningful, if processes are modeled in a sufficient\ndetailed manner. In other words, it must be decided which Compared to common consequential LCA approaches\nwhich use substitution method to solve the multifunctional\nproblem, the definition of determining products wherein all\nother co-products are summarized into one avoided product\ngroup (e.g., Weidema 2001; Suh et al. 2010) is not necessary. Here, in contrast, all products of the new technologies are\nconsidered as determining products without distinguishing\nbetween the multiple products. For each biorefinery product,\nsubstitutable reference products are determined. 4 Discussion h: environmental\nimpacts; z: profit Int J Life Cycle Assess (2019) 24:2191–2205 2203 Table 5\nResults of the epsilon-constraint method (50% scenario), per annum\nP1\nP2\nP3\nh (kg CO2-eq.)\n7.15709438·1011\n7.15778471·1011\n7.15778469·1011\nz (€)\n2.514068·107\n4.492112·107\n4.509840·107\nBiorefinery 1, Leuna (pc.)\n1\n0\n0\nBiorefinery 2, Leuna (pc.)\n0\n0\n1\nBiorefinery 1, Zeitz (pc.)\n0\n0\n0\nBiorefinery 2, Zeitz (pc.)\n0\n0\n0\nBiorefinery 1, Böhlen (pc.)\n0\n0\n0\nBiorefinery 2, Böhlen (pc.)\n0\n1\n0\nBiorefinery 1, Schwarzheide (pc.)\n0\n0\n0\nBiorefinery 2, Schwarzheide (pc.)\n0\n0\n0 Table 5\nResults of the epsilon-constraint method (50% scenario), per annum process, e.g., a certain ethylene plant, is substitutable by the\nnew process. Even in LCA, which uses more disaggregated\nprocesses than input-output analysis does, this is usually not\nthe case. For instance, in our example only one process for\nethylene generation represents a bundle of ethylene plants. Furthermore, the ethylene production is mono-functional,\nwhich does not represent real-life complexity, since ethylene\nusually is produced with other co-products (e.g., propylene). Including those co-products, however, can lead to binding\nconstraints that are not achievable by the new technologies. In our example this becomes obvious if the bundled process of\nfossil ethylene production would additionally provide propyl-\nene. Since it seems not realistic to achieve a model that repre-\nsents all production processes at a plant level, the only viable\nway is to make a model choice. The question is thus, what\nimplicit assumption is appropriate in terms of the goal of the\nstudy (Zamagni et al. 2012). A general discussion is beyond\nthe scope of this article. However, in terms of the predominant\ngoal of the exemplary case studies, to identity the best alter-\nnative from a set of new biorefinery options, we argue that\nusing aggregated mono-functional processes with average da-\nta seems to be sufficient. To increase the reliability of future\ncase studies, however, the assessments should be enhanced by\nsensitivity analyses using different approaches for allocating\nthe environmental impacts to the substitutable reference prod-\nucts (e.g., partitioning by physical and monetary factors). optimal choice of technologies. Before that, Carter (1970)\napplied a square choice-of-technology model using linear pro-\ngramming in a similar manner. Our model works in principle\nthe same way. References Azapagic A, Clift R (1995) Life cycle assessment and linear program-\nming – optimization of product system. Comput Chem Eng 19:229–\n234 Azapagic A, Clift R (1998) Linear programming as a tool in life cycle\nassessment. Int J Life Cycle Assess 3:305–316 Azapagic A, Clift R (1999) Life cycle assessment and multiobjective\noptimisation. J Clean Prod 7(2):135–143 Budzinski M, Nitzsche R (2016) Comparative economic and environ-\nmental assessment of four beech wood based biorefinery concepts. Bioresour Technol 216:613–621. https://doi.org/10.1016/j.biortech. 2016.05.111 Carter A (1970) Old and new structures as alternatives: optimal combi-\nnation of 1947 and 1958 technologies. In: Carter a, structural change\nin the American economy. Harvard University Press, Cambridge Curran MA, Mann M, Norris G (2005) The international workshop on\nelectricity data for life cycle inventories. J Clean Prod 13:853–862 De Bruyn S, Korteland M, Markowska A, Davidson M, de Jong F, Bles\nM, Sevenster M (2010) Shadow Prices Handbook Valuation and\nweighting of emissions and environmental impacts. Delft, CE\nDelft, March 2010 4 Discussion In our opin-\nion, these choices for all products of the biorefineries seem\ncloser to reality than whether clustering several co-products 2204 Int J Life Cycle Assess (2019) 24:2191–2205 Acknowledgements This work has been carried out within the project\nSpitzencluster BioEconomy (031A078A). Acknowledgements This work has been carried out within the project\nSpitzencluster BioEconomy (031A078A). into one avoided product group or declaring the multi-\nfunctional processes to be mono-functional by using\npartitioning method. Funding information The German Federal Ministry of Education and\nResearch financially supported this project. Towards a comprehensive LCSA framework, some\npotential directions for further research shall be\nbroached. The characterization matrix Q can be easily\nmodified by corresponding characterization factors to\ntake into account additional environmental impact cate-\ngories (e.g., midpoint or endpoint categories). Targets for\nvarious impact categories can be considered separately\nwithin a multi-objective framework. On the other hand,\nnormalization and weighting could be introduced within\na single environmental target function. Thus the optimi-\nzation could be carried out in terms of a single environ-\nmental score which takes into account various normal-\nized and weighted impact indicator results. In the same\nmanner various social categories could be dealt with. By\nimposing a single score for each of the three pillars of\nsustainability (LCA, LCC, social LCA), the assessment\nof technological alternatives would be extended toward a\ncomprehensive LCSA within a multi-objective frame-\nwork. By identifying environmental, social, and econom-\nic benefits of new technologies (especially the compari-\nson with existing technologies those produce equivalent\nproducts), this framework would be also suitable to sup-\nport methods for estimating the maturity of technologies\nsuch as the technology readiness level (Hicks et al. 2009). Open Access This article is distributed under the terms of the Creative\nCommons Attribution 4.0 International License (http://\ncreativecommons.org/licenses/by/4.0/), which permits unrestricted use,\ndistribution, and reproduction in any medium, provided you give\nappropriate credit to the original author(s) and the source, provide a link\nto the Creative Commons license, and indicate if changes were made. 5 Conclusions Duchin F, Levine SH (2011) Sectors may use multiple technologies si-\nmultaneously: the rectangular choice-of-technology model with\nbinding factor constraints (revised). Rensselaer working papers in\neconomics number 1101 This article showed how (mixed integer) linear program-\nming can be used to extend standard LCA towards com-\nprehensive decision-making. Additional consequential re-\nsearch questions can be addressed such as the determi-\nnation of the optimal number of new production plants\nand the optimal region for supplying feedstocks while\nalso taking into consideration transport logistic options. The implicit environmental comparison of new bio-based\ntechnologies with fossil reference technologies can be\nregarded as a feature that has not been provided by other\noptimization models within the field of LCA. The exten-\nsion of LCA by linear programming remains a consistent\nlinear model, which is able to broaden the scope for\nconsequential assessments. The benefit of this extension\nof LCA is to provide a broader and systematic assess-\nment of consequences. The modifications of the program\nby additional profit requirements (LCC) into a goal pro-\ngram and Pareto optimization problem have been identi-\nfied as promising ways toward a comprehensive multi-\nobjective LCSA. Duchin F, Levine SH (2012) The rectangular sector-by-technology mod-\nel: not every economy produces every product and some products\nmay rely on several technologies simultaneously. J Econ Struct. https://doi.org/10.1186/2193-2409-1-3 Ekvall T, Weidema BP (2004) System boundaries and input data in con-\nsequential life cycle inventory analysis. Int J Life Cycle Assess 9:\n161–171 Ekvall T, Azapagic A, Finnveden G, Rydberg T, Weidema BP, Zamagni\nA (2016) Attributional and consequential LCA in the ILCD hand-\nbook. Int J Life Cycle Assess 21:293–296 Gong J, You F (2017) Consequential life cycle optimization: general\nconceptual framework and application to algal renewable diesel pro-\nduction. ACS Sustain Chem Eng 5(7):5887–5911 Haes HAU, Heijungs R, Suh S, Huppes G (2004) Three strategies to\novercome the limitations of life-cycle assessment. J Ind Ecol 8:19–\n32 Heijungs R, Frischknecht R (1998) A special view on the nature of the\nallocation problem. Int J Life Cycle Assess 3:321–332 Heijungs R, Guinée JB (2007) Allocation and “what-if” scenarios in life\ncycle assessment of waste management systems. Waste Manag 27:\n997–1005 Heijungs R, Suh S (2002) The computational structure of life cycle as-\nsessment. 5 Conclusions J Ind Ecol\n14:335–353 Kostin AM, Guillén-Gosálbez G, Mele FD, Jiménez L (2012) Identifying\nkey life cycle assessment metrics in the multiobjective Design of\nBioethanol Supply Chains Using a rigorous mixed-integer linear\nprogramming approach. Ind Eng Chem Res 51(14):5282–5291 Tan RR, Culaba AB, Aviso KB (2008) A fuzzy linear programming\nextension of the general matrix-based life cycle model. J Clean\nProd 16(13):1358–1367 Thomassen MA, Dalgaard R, Heijungs R, De Boer I (2008) Attributional\nand consequential LCA of milk production. Int J Life Cycle Assess\n13:339–349 Liu P, Pistikopoulos EN, Li Z (2010) An energy systems engineering\napproach to the optimal design of energy systems in commercial\nbuildings. Energy Policy 38:4224–4231 Tillman A-M, Ekvall T, Baumann H, Rydberg T (1994) Choice of system\nboundaries in life cycle assessment. J Clean Prod 2(1):21–29 Majeau-bettez G, Wood R, Hertwich EG (2015) When do allocations and\nconstructs respect material, energy, financial, and production bal-\nances in LCA and EEIO? J Ind Ecol 20:67–84 Vadenbo C, Tonini D, Astrup TF (2017) Environmental multiobjective\noptimization of the use of biomass resources for energy. Environ Sci\nTechnol 51:3575–3583 Mavrotas G (2009a) Effective implementation of the ε-constraint method\nin multi-objective mathematical programming problems. Appl Math\nComput 213(2):455–465 Vance L, Heckl I, Bertok B, Cabezas H, Friedler F (2015) Designing\nsustainable energy supply chains by the P-graph method for minimal\ncost, environmental burden, energy resources input. J Clean Prod\n94:144–154 Mavrotas G (2009b) Generation of efficient solutions in multiobjective\nmathematical programming problems using GAMS. Effective im-\nplementation of the ε-constraint method. Online: https://www.gams. com/modlib/adddocs/epscm.pdf. Accessed Nov 2017 VCI (2018) Chemiewirtschaft in Zahlen 2018. Verband der Chemisechen\nIndustrie e.V. Online: https://www.vci.de/vci/downloads-vci/\npublikation/chemiewirtschaft-in-zahlen-print.pdf. Accessed Nov\n2018 Michels J (2009) Pilotprojekt “Lignocellulose-Bioraffinerie“ –\nGemeinsamer Schlussbericht zu den wissenschaftlich-technischen\nErgebnissen aller Teilvorhaben [Pilot project “Lignocellulose\nBiorefinery“ - Final scientific and technical report of all project\npartners]. DECHEMA Gesellschaft für Chemische Technik und\nBiotechnologie. http://www.fnr-server.de/ftp/pdf/berichte/\n22001307.pdf. Accessed Nov 2017 Wang R, Work D (2014) Application of robust optimization in matrix-\nbased LCI for decision making under uncertainty. Int J Life Cycle\nAssess 19(5):1110–1118 Weidema B (2001) Avoiding co-product allocation in life-cycle assess-\nment. J Ind Ecol 4(3):11–33 Miller RE, Blair PE (2009) Input-output analysis. Foundations and ex-\ntensions. Cambridge University Press Zamagni A, Guinée J, Heijungs R, Masoni P, Raggi A (2012) Lights and\nshadows in consequential LCA. 5 Conclusions Kluwer Academic Publishers Int J Life Cycle Assess (2019) 24:2191–2205 2205 Polley H, Kroiher F (2006) Struktur und regionale Verteilung des\nHolzvorrates und des potenziellen Rohholzaufkommens in\nDeutschland im Rahmen der Clusterstudie Forst- und\nHolzwirtschaft. Arbeitsbericht des Institut für Waldökologie und\nWaldinventuren 2006 / 3 Eberswalde, November 2006 Heijungs R, Settanni E, Guinée J (2013) Toward a computational struc-\nture for life cycle sustainability analysis : unifying LCA and LCC. Int J Life Cycle Assess 18:1722–1733 Hertwich E (2014) Understanding the climate mitigation benefits of prod-\nuct systems: comment on “using Attributional life cycle assessment\nto estimate climate-change mitigation...”. J Ind Ecol 18:464–465 Renouf MA, Poggio M, Collier A, Price N, Schroeder BL, Allsopp PG\n(2018) Customised life cycle assessment tool for sugarcane\n(CaneLCA) - a development in the evaluation of alternative agricul-\ntural practices. Int J Life Cycle Assess 23:2150–2164 Hicks B, Larsson A, Culley S, Larsson T (2009) A methodology for\nevaluating technology readiness during product development. In:\nProceedings of international conference on engineering design,\nICED '09, 24–27 august 2009, Stanford University, Stanford, CA,\nUSA Schmidt JH (2010) Comparative life cycle assessment of rapeseed oil and\npalm oil. Int J Life Cycle Assess 15:183–197 Hwang CL, Masud ASM (1979) Multiple objective decision making —\nmethods and applications: a state-of-the-art survey. Lecture notes in\neconomics and mathematical systems, vol 164. Springer-Verlag,\nBerlin SDK (2017) Braunkohlenförderung in Deutschland. Statistik der\nKohlewirtschaft e.V. Online: https://kohlenstatistik.de/19-0-\nBraunkohle.html. Accessed Nov 2018 Steubing B, Ballmer I, Gassner M, Gerber L, Pampuri L, Bischof S,\nThees O, Zah R (2011) Identifying environmentally and economi-\ncally optimal bioenergy plant sizes and locations: a spatial model of\nwood-based SNG value chains. Renew Energy 61:57–68 ISO (2006) International standard ISO 14044. Environmental manage-\nment — life cycle assessment — requirements and guidelines ISO (2012) International standard ISO 14045. Environmental manage-\nment — Ecoefficiency assessment of product systems — principles,\nrequirements and guidelines Suh S (2004) Functions, commodities and environmental impacts in an\necological economic model. Ecol Econ 48:451–467 Kätelhön A, Bardow A, Suh S (2016) Stochastic technology choice mod-\nel for consequential life cycle assessment. Environ Sci Technol 50:\n12575–12583 Suh S, Weidema B, Schmidt JH, Heijungs R (2010) Generalized make\nand use framework for allocation in life cycle assessment. 5 Conclusions Int J Life Cycle Assess 17:904–918 Pawelzik P, Carus M, Hotchkiss J, Narayan R, Selke S, Wellisch M,\nWeiss M, Wicke B, Patel MK (2013) Critical aspects in the life cycle\nassessment (LCA) of bio-based materials - reviewing methodologies\nand deriving recommendations. Resour Conserv Recycl 73:211–\n228 Peters GP, Hertwich EG (2006) A comment on “functions, commodities\nand environmental impacts in an ecological-economic model”. Ecol\nEcon 59:1–6 Publisher’s note\nSpringer Nature remains neutral with regard to\njurisdictional claims in published maps and institutional affiliations. Plevin RJ, Delucchi MA, Creutzig F (2014) Using Attributional life cycle\nassessment to estimate climate-change mitigation benefits misleads\npolicy makers. J Ind Ecol 18:73–83"
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https://jurnalpapuaasia.unipa.ac.id/jurnalpapuasia/article/download/143/127
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POTENSI JASA LINGKUNGAN PADA KAWASAN TAMAN WISATA ALAM KLAMONO, KABUPATEN SORONG
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Jurnal Kehutanan Papuasia/Jurnal Kehutanan Papuasia (Journal of Papuasia Forestry)
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cc-by
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Kurey, dkk Kurey, dkk Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Abstrak Penelitian ini bertujuan untuk mengetahui jenis - jenis komoditi dan bentuk aktifitas
pemanfaatan jasa lingkungan pada kawasan Taman Wisata Alam Klamono. Metode yang
digunakan adalah metode deskriptif dengan teknik observasi dan wawancara. Observasi
dilakukan terhadap komoditi jasa lingkungan yang ada pada kawasan Taman Wisata Alam
Klamono dan wawancara dilakukan pada masyarakat di Kampung Klawana. Hasil
penelitian menunjukkan bahwa komoditi jasa lingkungan terdiri atas a). potensi sumber air
yang meliputi sungai Klagene dan sungai Klawana. b). Keanekaragaman hayati meliputi
potensi flora dan potensi fauna c). Penyerapan karbon meliputi tumbuhan berkayu dan
tumbuhan non kayu d). Panorama bentang alam. Aktivitas pemanfaatan jasa lingkungan
meliputi sumber pemanfaatan tata air, flora, fauna, tumbuhan berkayu, tumbuhan non kayu
dan panorama bentang alam. Potensi pengembangan jasa lingkungan dapat dinilai dengan
mengunakan kriteria penilaian daya tarik, aksesibilitas, kondisi sosial ekonomi masyarakat,
keamanan pengelolaan, dan sarana prasarana. Hasil skoring mengindikasikan bahwa Taman
Wisata Alam Klamono dapat dikembangkan tetapi perlu pertimbangan beberapa aspek
strategis oleh pemerintah setempat. Kata kunci: jasa lingkungan, wisata alam, keanekaragaman hayati, flora fauna, penyerapan
karbon (The Potency of Ecosystem Services in Klamono Nature Park, District of Sorong) Akwila Johanes Kurey1 Yohanes Y. Rahawarin1 dan Dominggas M.H. Renwarin1
Jurusan Kehutanan, Fakultas Kehutanan Universitas Papua Manokwari, Papua Barat,
98314. Tlp/Fax: +62986211065. Penulis Korespondensi: Email: jo.rahawarin@gmail.com
Diterima: 15 Mei 2019| Disetujui: 01 Juli 2019 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Abstract This study works toward varieties of commodity and types of ecosystem service usage
activity in Klamono nature park. The study has applied descriptive method through
observation and interview techniques. The observation is focusing on the ecosystem service
commodities in surrounding area and interview is carried out towards the local inhabitants
of Klawana village. The result pointed out that ecosystem services consist of a). water
resource potency that covered Klagene and Klawana rivers, b). natural resource, in
particular the potency of flora and fauna, c). carbon absorption from woody and non
woody plant species in the nature park, d). landscape panorama. The usage activities of
ecosystem services covered water use, flora, fauna, woody and non woody plants, as well
as ecosystem landscape. The prospect of ecosystem service development can be assessed by
applying criteria such as attractiveness, accessibility, socio-economy status of the
inhabitant community, management security, and facilities and infrastructure. Scoring 124 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Kurey, dkk Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) results indicated that Klamono nature park can be developed, however it required a
number of strategic considerations by the local government. Keywords: ecosystem service, natural tourism, natural resource, flora fauna, carbon
absorption PENDAHULUAN endemis di Sumatera dan 10 marga
endemis di pulau Jawa. Sejauh ini telah
diketahui 154 jenis mamalia, dimana 93
jenis merupakan endemis yang terdiri atas
41 jenis dari kalangan monotremata dan
marsupialia, serta 52 jenis dari kalangan
kelelawar dan binatang pengerat (Petocz
1987). Potensi
yang
begitu
tinggi
membutuhkan
upaya
perlindungan,
pengawetan dan pemanfaatan yang lestari
untuk menjaga keanekaragamannya. Hutan
merupakan
salah
satu
sumberdaya alam yang multifungsi dalam
menunjang kehidupan umat manusia yang
tidak hanya terbatas sebagai tempat
konservasi keanekaragaman hayati dan
pemeliharaan fungsi ekosistem, tetapi
juga menghasilkan barang dan jasa bagi
masyarakat (Rahawarin 2010). Hutan sangat penting bagi kehidupan
di muka bumi, terutama bagi kehidupan
generasi mendatang. Kesalahan dalam
pengelolaan
hutan
berarti
menyiksa
kehidupan generasi mendatang. Untuk
mencegah kesalahan dalam pengelolaan
hutan, maka fungsi hutan harus dipelajari
dan dimengerti secara holistik (utuh). Pengelolaan hutan bukan hanya sekedar
menetapkan hutan sebagai perlindungan
tanah, iklim, sumber daya air dan
pemenuhan kebutuhan akan kayu dan
produk lainya. Namun, pengelolaan hutan
harus ditujukan untuk mendayagunakan
semua lahan demi kepentingan negara,
bahkan dunia (Arief 2005). Kawasan konservasi selain berfungsi
sebagai kawasan yang dilindungi, juga
mempunyai fungsi lain sebagai sistem
penyangga kehidupan dan sebagai sarana
kegiatan pengembangan penelitian dan
ilmu pengetahuan, pendidikan, wisata
alam dan penunjang budidaya. Upaya
melestarikan sumberdaya alam hayati dan
ekosistemnya
bukan
hanya
menjadi
tanggung
jawab
pemerintah
tetapi
merupakan
tanggung
jawab
semua
lapisan masyarakat. Hal ini sesuai dengan
amanat Undang-Undang Nomor 41 tahun
1999 pasal 68 ayat 1 dan 2 serta pasal 29
ayat
1
yang
menyatakan
bahwa
masyarakat
selain
mempunyai
hak
menikmati kualitas lingkungan hidup
yang dihasilkan hutan, memanfaatkan
hutan dan hasil hutan sesuai dengan
peraturan yang berlaku, tetapi juga
berkewajiban ikut serta dalam menjaga
kawasan
hutan
dari
gangguan
dan
kerusakan (Pangesti 2005). Tujuan dari
penelitian
ini
antara
lain
untuk
mengetahui jenis-jenis komoditi jasa
lingkungan,
mengetahui
aktivitas
pemanfaatan komoditi jasa lingkungan Salah satu wilayah di Indonesia yang
memiliki sumber daya hutan yang paling
potensial adalah Papua. Formasi hutan
papua kaya akan jenis, general dan famili
khas yang tidak dijumpai pada formasi
lain dan dapat dijadikan tanaman berguna
bagi
manusia. Jumlah
flora
papua
diperkirakan 20.000-25.000 jenis dengan
keendemikan diperkirakan antara 60-90%
(Johns 1997; Lekitoo et al. 2017). Terdapat 124 marga tanaman berbunga
endemis, bila dibandingan dengan 59
marga endemis di Kalimantan; 17 marga 125 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk sebanyak 5 orang. Variabel Penelitian Variabel
yang
diamati
dalam
pelaksanaan penelitian ini meliputi: jenis
komoditi
jasa
lingkungan,
aktivitas
pemanfaatan
jasa
lingkungan,
dan
kemungkinan
pengembangan
pemanfaatan jasa lingkungan. Teknik Pengumpulan Data Data yang dikumpulkan terdiri atas
data primer dan data sekunder. Data
primer
dikumpulkan
dengan
teknik
observasi, dokumentasi dan wawancara. Data primer meliputi: jenis komoditi jasa
lingkungan, keberadaan jasa lingkungan,
lokasi timbulnya jasa lingkungan yang
dihasilkan, dan kegiatan pemanfaatan jasa
lingkungan. Data sekunder dikumpulkan
dengan cara pencatatan dan dokumentasi
dari instansi terkait serta studi pustaka
berupa laporan penelitian yang berkaitan
dengan topik penelitian. Metode dan Teknik Penelitian Penelitian
ini
dilakukan
dengan
menggunakan metode deskriptif dengan
teknik observasi lapang dan wawancara. Observasi dilakukan terhadap komoditi
jasa lingkungan yang ada pada kawasan
TWAK, sedangkan wawancara dilakukan
terhadap masyarakat yang memanfaatkan
jasa lingkungan. PENDAHULUAN Responden umum
adalah masyarakat
yang
tinggal
di
kampung Klawana. Jumlah responden
yang diwawancarai sebanyak 30% dari
total
jumlah
masyarakat
Kampung
Klawana sebanyak 110 KK yang berada
di sekitar kawasan TWAK. dan mengkaji kemungkinan pemanfaatan
jasa lingkungan di kawasan Taman
Wisata Alam Klamono (TWAK). Pelaksanaan Penelitian Kriteria yang dinilai adalah daya tarik,
aksesbilitas, kondisi
lingkungan dan
sosial
masyarakat,
keamanan
dan
pengelolaan, sarana dan prasarana. Pada
setiap kriteria memiliki nilai dan bobot. Nilai setiap kriteria (S) yang diperoleh
dari hasil akan dikalikan dengan bobot
(B)
kriteria
dengan
jumlah
nilai Pengelolaan dan Analisis Data Data yang dikumpulkan dianalisis
mengunakan kriteria penelitian potensi
obyek dan daya tarik alam yang telah
dimodifikasi
dari
pedoman
kriteria
penilaiaan
pengembangan
(Direktorat
Jendral
Perlindungan
Hutan
dan
Konservasi
Alam,
Departemen
Kehutanan, 2003/dan Direktorat Produk
Wisata
Direktorat
Jendral
Destinasi
Pariwisat Departemen Kebudayaan dan
Pariwisata 2007). Kemudian data yang
dianalisis diuraikan
dan
ditampilkan
dalam bentuk tabel dan gambar. Tempat dan Waktu Penelitian ini dilaksanakan di areal
Taman Wisata Alam Klamono Kabupaten
Sorong dengan lamanya waktu penelitian
sekitar 3 minggu, yaitu dari tanggal 26
Januari - 16 Februari tahun 2019. Yang
menjadi obyek dalam penelitian ini
adalah potensi jasa lingkungan pada
kawasan
TWAK,
sementara
subyek
penelitian adalah masyarakat yang berada
pada kampung disekitar kawasan TWAK. @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Penentuan Responden Penentuan responden dibagi menjadi
dua
yaitu
responden
kunci
(key
respondent)
dan
responden
umum
(common respondent). Responden kunci
terdiri dari aparat kampung sebanyak 2
orang dan tokoh masyarakat sebanyak 3
orang sehingga total responden kunci 126 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk N
= Jumlah nilai unsur unsur kriteria @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA N
= Jumlah nilai unsur unsur kriteria unsurunsur kriteria (N). jumlah nilai
unsur-unsur kriteria diperoleh dari nilai
yang sudah ada pada tabel penilaiaan
yang disesuaikan dengan kondisi dan
karakter lokasi obyek penelitian dan dapat
dihitung
mengunakan
rumus
seperti
berikut : Penilaian potensi jasa lingkungan
berdasarkan modifikasi dari Pedoman
Kriteria
Penilaiaan
Pengembangan
Direktorat Jendral Perlindungan Hutan
dan
Konservasi
Alam,
Departemen
Kehutanan (2003) dan Direktorat Produk
Wisata
Direktorat
Jendral
Destinasi
Pariwisat Departemen Kebudayaan dan
Pariwisata
(2007),
dengan
kriteria
penilaian seperti pada tabel 1. S = B × N S = B × N Keterangan : S
= Nilai kriteria S
= Nilai kriteria B
= Bobot nilai kriteria Tabel 1. Daftar kriteria penilaian jasa lingkungan yang telah dimodifikasi
No. Unsur yang dinilai
Bobot
1. Daya tarik
6
2. Aksesibilitas
5
3. Kondisi lingkungan sosial ekonomi masyarakat
5
4. Keamanan dan pengelolaan
3
5. Sarana dan prasarana
3 Nilai maximum akan diperoleh dari
jumlah total skor tertinggi, sedangkan
nilai minimum diperoleh dari jumlah total
skor
terendah. Hasil
perhitungannya
berupa kategori dan selang penilaian
potensi jasa lingkungan pada kawasan
TWAK. Nilai maximum akan diperoleh dari
jumlah total skor tertinggi, sedangkan
nilai minimum diperoleh dari jumlah total
skor
terendah. Hasil
perhitungannya
berupa kategori dan selang penilaian
potensi jasa lingkungan pada kawasan
TWAK. Selanjutnya
hasil
penelitian
akan
dikategorikan berdasarkan nilai selang,
dimana
selang
yang
ditetapkan
dikategorikan dalam 3 kategori, yaitu:
baik, sedang dan buruk. Nilai selang akan
dihitung dari rumus : Selang (S) = S max – S min/
Banyaknya kategori Selang (S) = S max – S min/ Selang (S) = S max – S min/
Banyaknya kategori Tabel 2. Kategori selang penilaiaan potensi jasa lingkungan kawasan TWAK
No. Kategori
Interval/Selang
1. Buruk
1090 – 1600
2. Sedang
1601 – 2110
3. Baik
2111 - 2620 obyek wisata alam. Kriteria penilaiaan
masuk dikategorikan sebagai berikut : Kategori dan interval/selang diambil
sebagai
acuan
standarisasi
untuk
menentukan layak dan tidaknya suatu
obyek yang akan dikembangkan sebagai g
g
a. Kategori Buruk berarti bahwa potensi
jasa lingkungan pada TWAK tidak
dapat dikembangkan. 127 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk raya Sorong - Klamono tepatnya di
Kilometer 37. b. Kategori Sedang berarti bahwa potensi
jasa lingkungan dapat dikembangkan
dan masih diperlukan pertimbangan
kembali dari pemerintah. Sama seperti sungai Klagene, sungai
Klawana juga merupakan sungai yg
mengalir melewati kawasan TWAK. Kondisi sungai yang masih alami dengan
banyaknya vegetasi berada di sepanjang
kiri dan kanan sungai. N
= Jumlah nilai unsur unsur kriteria Di sebut sungai
Klawana karena sungai ini adalah sungai
yang mengalir keluar dari TWAK ke
kampung Klawana. c. Kategori Baik berarti bahwa potensi
jasa lingkungan pada kawasan TWAK
harus dikembangkan sebagai nilai
keunggulan bagi kabupaten sorong. Data yang dikumpulkan diolah secara
tabulasi dan dianalisis secara deskriptif
dan
selanjutnya
ditampilkan
dalam
bentuk tabel dan gambar/foto. Jenis Komoditi Jasa Lingkungan Berdasarkanhasil observasi lapangan
pada kawasan TWAK jenis komoditi jasa
lingkungan meliputi yang ditemukan
berupa: 1). tata air, 2). keanekaragaman
hayati, 3). penyerapan karbon, dan 4). bentuk-bentuk pemanfaatan sumber daya
alam dan jasa lingkungan. Berikut ini
merupakan karakteristik dari masing
masing jenis komoditi jasa lingkungan: Tata air Berdasarkan hasil pengamatan terdapat
sumber air yang berada di dalam kawasan
TWAK,
diperoleh
informasi
berupa
keberadaan sungai Klagene dan sungai
Klawana yang merupakan sungai-sungai
yang mengalir di dalam kawasan TWAK. Sungai Klagene dan sungai Klawana
adalah merupakan anak sungai dari
sungai induk pada Distrik Klamono yaitu
sungai Klasofot. HASIL DAN PEMBAHASAN Kekayaan
keanekaragaman
hayati
merupakan salah satu potensi pada aspek
jasa
lingkungan
yang
berpontensi
dikembangkan sebagai parawisata dan
ekowisata. Selain itu, terdapat juga
kawasan
konservasi
yang
memiliki
potensi cukup tinggi yang tersebar di
berbagai tipe ekosisitem yang terdapat di
dalam
kawasan
TWAK. Potensi
lingkungan
yang
berada
di
dalam
kawasan TWAK berupa panorama alam,
berbagai jenis flora dan fauna serta
beberapa sungai yang mengalir di dalam
kawasan. Potensi tersebut merupakan
potensi untuk menunjang kegiatan wisata
alam. Jenis Komoditi Jasa Lingkungan Jenis Komoditi Jasa Lingkungan @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Fauna Terdapat potensi berbagai jenis satwa
khususnya
jenis-jenis
yang
telah
dilindungi
undang-
undang
seperti
Kakatua
Jambul
Kuning
(Cacatua
galerita
triton),
Kakatua
Raja
(Proboscinger aterrimus), Nuri Merah
Kepala
Hitam
(Lorius
lory),
Cenderawasih (Paradiseae spp.), Kasuari
(Casuarius
casuarius),
Rusa
(Rusa
timorensis)
dan
Kangguru
Pohon
(Dendrolagus sp.). Tumbuhan Berkayu (woody plant) kemudian diikuti oleh jenis Dillenia
indica dan Vileburna rubescent masing-
masing dengan nilai INP 10,841% dan
10,202%. Untuk tingkat pancang, jenis
yang memiliki INP tertinggi adalah
Cleytanthus papuana dengan 15,580%,
kemudian
disusul
oleh
jenis
Sphatiostemon javaensis dengan nilai INP
12,845% dan jenis Villebrunea rubescent
dengan nilai INP 12,674%. Sementara
untuk tingkat pohon, jenis yang memiliki
nilai INP tertinggi adalah Artocarpus
altilis dengan nilai 25,695%, disusul oleh
jenis Pometia pinnata dengan nilai INP
24,199% dan jenis Teijsmaniodendron
bogoriense dengan nilai INP 14,031%. y (
y p
)
Hasil pengamatan langsung yang dapat
di amati dalam Kawasan TWAK, terdapat
berbagai jenis tumbuhan berkayu, yang
sebagai penyerap karbon dan penyedia
oksigen. Terdiri dari 49 jenis tumbuhan
berkayu dari 26 family yang terdapat di
Kawasan TWAK Untuk menunjang jasa
linkungan dan perlindungan kawasan
Konsevasi dalam peningkatan tempat
wisata TWAK terdapat tiga jenis famili
tumbuhan
berkayu
yaitu,
Fabaceae,
Sapindaceae,
dan
Moraceae,
yang
dominan dalam kawasan TWAK. Jenis –
jenis tumbuhan berkayu yang ditemukan
memiliki potensi daya tarik yang sangat
menarik baik dari karakter morfologi
daun, batang, bunga dan buah. Pesona ini
menjadi suatu hal yang unik dan jarang
ditemukan ditempat lainnya. Hal ini di
tunjukan
dengan
kelangkaan
dan
endemiknya jenis-jenis yang ditemukan
pada kawasan tersebut. Jenis – jenis
tumbuhan
berkayu
yang
ditemukan
beserta daya tarik jasa linkungan antara
lain: Artocarpus altilis, Pometia pinnata,
Teijsmaniodendron
bogoriense,
Ficus
pungens, Melanolepis multiglandulosa,
Ficus trachypison, Cleytanthus papuana,
Artocarpus
odoratasimus,
Trichospermum javanicum, Macaranga
mapa
Spatiostemon
javensis Penyerapan Karbon Potensi
jasa
lingkungan
berupa
penyerapan karbon merupakan salah satu
jasa penting dalam kawasan konservasi
yang juga berperan menyediakan oksigen
da udara segar dari dalam kawasan
TWAK. Dengan hadirnya berbagai jenis
vegetasi hutan di Kawasan TWAK,
memberikan potensi ketersediaan oksigen
dan udara segar yang melimpah. Hasil
identifikasi di lapangan, terdapat dua
kelompok tubuhan yang petensial sebagai
penyerap karbondioksida dan selanjutnya
sebagai agen penyedia oksigen. Trichospermum javanicum, Macaranga
mapa,
Spatiostemon
javensis, Tumbuhan Bukan Kayu (non woody
plant) Flora Berbagai jenis tumbuhan di dalam
kawasan ini merupakan jenis-jenis yang
umumnya tumbuh di hutan dataran
rendah. Kegiatan
inventarisasi
telah
dilakukan di dalam kawasan TWAK
(Maturbongs 2011) dengan perolehan
data
berdasarkan
tingkatannya
yaitu
tingkat semai ditemukan sebanyak 102
jenis, pancang 102 jenis, dan pohon 102
jenis. Dari jenis data tersebut diperoleh
nilai INP pada masing-masing tingkat
vegetasi yang berbeda. Untuk tingkat
semai, Cleytanthus papuana memiliki
nilai INP tertinggi yakni 16,649%, Sungai Klagene berasal dari mata air
yang berada di luar kawansan TWAK
yang tepatnya di bawah kaki gunung
Mariat dan mengalir di dalam kawasan
sampai bermuara pada sungai induk
sungai Klasifot atau yang biasa di sebut
sungai
induk
Klamono. Keberadaan
sungai Klagene dapat di lihat dari jalan 128 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk Bird Watching g
Mengamati burung (bird watching)
secara
langsung
di
dalam
hutan
merupakan kegiatan wisata alam yang
dapat memberikan pengetahuan. Selain
itu pengunjung juga dapat melakukan
pengambilan gambar/foto dari obyek-
obyek satwa tersebut. Kegiatan bird
watching
di
dalam
TWAK
dapat
dilakukan dengan mengunjungi spot-spot
pengamatan burung. Terdapat berbagai
macam jenis burung yang dapat dijumpai
di dalam dan sekitar kawasan termasuk
jenis burung
endemik
seperti jenis
Kakatua
Jambul
Kuning
(Cacatua
galerita
triton),
Kakatua
Raja
(Proboscinger aterrimus) Nuri Merah
Kepala Hitam (Lorius lory). Pemanfaatan Tata Air Pemanafaatan tata air pada sungai
Klagene telah dilakukan oleh masyarakat
Kampung Klawana dan Wonosari. Sungai
Klagene hanya dimanfaatkan sebagai
patokan
atau
batas
alam,
yang
memisahkan hak ulayat pada kawasan
TWAK dari 2 marga suku Moy yaitu
marga
Mambringgofok
dan
Osok. Pemanfaatan
tata
air
pada
sungai
Klawana juga dapat dilakukan oleh
masyarakat
Kampung
Klawana
dan
Wonosari, namun sungai Klawana hanya
dimanfaatkan sebagai tapal batas kedua
kampung tersebut. Selain itu belum ada
pemanfaatan lain yang dilakukan oleh Tumbuhan Bukan Kayu (non woody
plant) Komposisi tumbuhan non kayu pada
kawasan
TWAK
Kabupaten
Sorong
antara lain: rotan, palem, pandan, bambu,
anggrek, paku-pakuan, herba dan liana
yang merupakan tumbuhan non potensial
sebagai peyerap karbon dan menyediakan
oksigen untuk menujang potensi jasa 129 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk lingkungan dan perlindungan kawasan
konservasi pada kawasan TWAK. Secara
detail, jenis-jenis tumbuhan bukan kayu
yang ditemukan antara lain dari jenis
rotan (Calamus aruensis Bacc., Calamus
kayensis Bacc., Korthalsia), dari jenis
palm (Arenga macrocalyx Ex., Aracea
sp., caryato rumpicna Mart., Licuala
telifera., Licuala sp., Metroxilon sagoo
Rotbb., Pigafeta filaris, Pinanga punicea,
Rhopaloblaste
sp.),
jenis
pandanus
(Pandanus
tectorius
Sol.,
Pandanus
furcatus Roxb., Pandanus conoides Lam.,
Pandanus
odoratissimus,
Freycinetia
sp.), jenis bambu (Bambusa vurgaris,
Schozostacchyum brachyclaudum Kurz.,
Schozostachyum lima, Schozostachyum
zollingeri, neolaleba atra) dan jenis
anggrek (Bullbophyllum sp., Calanthe sp.,
Colagyne
asperata,
Corimborkis
veratrifolia,
Dendrabium
bofalce,
Dendrobium
bracteosum,
Gramatophyllum
scriptum
BL.,
Gramatophyllum
speciosum
LINDL.,
Phalaenopsis
amabilis,
Pholidota
imbricatai,
Pomotocalpa
sp.,
dan
Spothoglottis plicata BL.). lingkungan dan perlindungan kawasan
konservasi pada kawasan TWAK. Secara
detail, jenis-jenis tumbuhan bukan kayu
yang ditemukan antara lain dari jenis
rotan (Calamus aruensis Bacc., Calamus
kayensis Bacc., Korthalsia), dari jenis
palm (Arenga macrocalyx Ex., Aracea
sp., caryato rumpicna Mart., Licuala
telifera., Licuala sp., Metroxilon sagoo
Rotbb., Pigafeta filaris, Pinanga punicea,
Rhopaloblaste
sp.),
jenis
pandanus
(Pandanus
tectorius
Sol.,
Pandanus
furcatus Roxb., Pandanus conoides Lam.,
Pandanus
odoratissimus,
Freycinetia
sp.), jenis bambu (Bambusa vurgaris,
Schozostacchyum brachyclaudum Kurz.,
Schozostachyum lima, Schozostachyum
zollingeri, neolaleba atra) dan jenis
anggrek (Bullbophyllum sp., Calanthe sp.,
Colagyne
asperata,
Corimborkis
veratrifolia,
Dendrabium
bofalce,
Dendrobium
bracteosum,
Gramatophyllum
scriptum
BL.,
Gramatophyllum
speciosum
LINDL.,
Phalaenopsis
amabilis,
Pholidota
imbricatai,
Pomotocalpa
sp.,
dan
Spothoglottis plicata BL.). masyarakat maupun Balai Konservasi
Sumberdaya Hutan (BKSDH). Sejauh ini
hanya dilakukan kegiatan pengawasan
dan perlindungan oleh pemerintah dan
pemegang hak ulayat. Jungle Tracking Kawasan TWAK menyediakan jalur
tracking
alam
yang
memikat. Di
sepanjang jalur track, pengunjung dapat
menikmati suasana alam dan udara yang
segar, selain itu disepanjang jalur track
bisa mengenal berbagai macam jenis
tumbuhan endemik TWAK serta dapat
berjumpa dengan satwa langka yang
menarik dari beberapa jenis burung paruh
bengkok yang ada di Papua. Fishing Terdapat beragam jenis ikan air tawar
yang berada di sungai Klagewe di
Kawasan
TWA
Klamono. Kegiatan
memancing (fishing) merupakan salah
satu kegiatan wisata alam yang dapat di
lakukan kepada pengunjung. Hal menarik
yang dapat
dilakukan yaitu
sambil 130 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk pusat kota kabupaten menuju kawasan
TWAK dengan waktu tempuh 2-4 jam
tergantung kendaraan darat apa
yg
digunakan. memancing pengunjung dapat menikmati
keindahan alam, Tetapi pada pelaksanaan
sendiri belum adanya pengelolaan yg baik
dari pemanku kepentingan (stakeholder)
dalam
menentuan
arah
kebijakan
pemanfaatan dan pengembangan Taman
Wisata Alam Klamono. Dari
kondisi
lingkungan
sosial
ekonomi masyarakat sekitar juga sangat
berperan penting dalam penyeimbangan
potensi jasa lingkungan . unsur – unsur
yang dinilai dari kondisi sosial ekonomi
masyarakat antara lain: tata ruang wilayah
objek,
mata
pencaharian,
tingkat
Pendidikan, dan tanggapan masyarakat
terhadap pengembangan TWAK. Kondisi
sosial
ekonomi
masyarakat
sekitar
berdasarkan hasil wawancara bahawa
masyarakat
pada
kampung
klawana
cukup mendukung dengan (nilai 375)
karena adanya sikap positif yang di
tujukan oleh masyarakat sekitar. @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Pengembangan Pemanfaatan Jasa
Lingkungan Kelayakan pengembangan dilakukan
melalui
beberapa
kriteria
untuk
mengambarkan
karakteristik
potensi
wisata alam yang layak dikembangkan
atau
sebeliknya
belum
dapat
di
kembangkan. Potensi wisata alam pada
kawasan Taman Wisata Alam Klamono
meliputi 5 (lima) kriteria, yaitu daya tarik,
aksesibilitas, kondisi lingkungan sosial
ekonomi masyarakat, keamanan dan
pengelolaan dan sarana dan prasarana. j
y
Keamanan dan pengelolaan kawasan
adalah salah satu hal penting dalam
melestarikan potensi jasa lingkungan
yang berada pada kawasan TWAK. Penilaian keamanan dan pengelolaan
dalam kawasan dapat dinilai dgn kriteria
penilaian sebagai berikut: keamanan
Kawasan
yang
meliputi
kebakaran,
penebangan liar, perambahan, keamanan
pengunjung dan pengolahan kawasan. Dari aspek keamanan dan pengelolaan di
beri (nilai 240) dari segi keamanan masih
kurang
baik,
karena
masih
adanya
aktifitas manusia pada kawasan yang
menyebapkan
kebakaran
akibat
pembukaan lahan, penebangan liar yang
di lakukan besar besaran dari dalam
maupun di luar kawasan TWAK, oleh
para pemilik hak ulaiyat maupun dari luar
dengan. Daya tarik merupakan nilai dari obyek
yang menjadi salah satu unsur utama
dalam menarik wisatawan datang ke
tempat tertentu. Penilaian terhadap unsur-
unsur daya tarik di lakukan untuk
mengetahui potensi daya tarik TWAK
yang di nilai meliputi unsur keindahan
alam, keunikan sumber daya alam,
banyaknya sumber daya alam yang
menojil, serta jenis kegiatan wisata alam. Dari skoring yang dilakukan, diperoleh
nilai 450 yang masuk kategori cukup
tinggi. Aksesibilitas merupakan salah satu
faktor penting yang menentukan mudah
atau tidaknya wisatawan datang ke daerah
tujuan wisata misalnya sarana trasportasi
dan jalan. Dari aksebilitas, TWAK di
tunjang
dengan
aksesibilitas
yang
memadai (725) k arena mudan di akses
mengunakan kendaraan mobil atau motor
dengan waktu tempuh yang relatif cepat. Kondisi jalan darat yang cukup baik dari Sarana
penunjang
yang
sangat
diperlukan dalam menjalankan kegiatan
wisata alam dengan tujuan memberikan
rasa aman dan nyaman bagi pengunjung. Unsur-unsur kriteria sarana dan prasarana 131 @ Asosiasi Peneliti Biodiversitas Papuasia - Fakultas Kehutanan UNIPA Jurnal Kehutanan Papuasia 5 (2): 124–133 (2019) Kurey, dkk umum yang berdomisili di Kampung
Klawana, sebagian besar menyetujui
apabila
TWAK
dikembangkan
dan
dilestarikan secara baik. Hal ini menjadi
modal
dasar
yang
tentunya
untuk
dikembangkan
dimasa
depan
karena
adanya
persetujuan
dan
dukungan
masyarakat setempat. Namun dimana saat
ini dalam hal keamanan dan pengelolaan
masih ada banyak terjadi pelanggaran
misalnya penebangan liar yang di sengaja
(ilegal logging), dan perambahan hutan
(pengunaan lahan). Pengelolaan kawasan
sendiripun belum ada misalnya pintu
gerbang masuk kawasan, rambu rambu
kawasan, pos penjagaan untuk keamanan
kawasan. Pengembangan Pemanfaatan Jasa
Lingkungan Selama
ini
dari
pihak
pemerintah dan balai besar konservasi
sumberdaya hutan masih belum ada
kesepakaan
tentang
status
kawasan
Taman Wisata Alam Klamono dengan
pemegang hak ulayat (Mambringgofok). Sejauh
ini
justru
baru
diadakanya
pendekatan
seperti
kegiatan
dari
BBKSDH
melalui
pembentukan
kelompok tani hutan, mitra polhut di
Kampung Klawana, Kampung Klamono
dan Wonosari. Selain itu ada juga bantuan
berupa ternak sapi pada tahun 2018,
penangkaran buaya pada tahun 2018,
pembibitan
pada
tahun
2018,
dan
pelatihan RHL, dalam bentuk uang tunai
ataupun secara lansung diberikan. Oleh
karena itu di harapkan kepada pemerintah
setempat dan Balai Besar Konservasi
Sumberdaya Hutan Provinsi Papua Barat
untuk
dapat
mengembangkan
dan
meningkatkan
aspek
pengelolaan
Kawasan terutama pengamanannya di
sekitar kawasan TWAK. yang dinilai yaitu unsur sarana berupa
rumah makan, sarana angkutan umum
dan prasarana berupa jalan, jembatan,
jaringan listrik, jaringan air minum dan
jaringan telepon. Hasil penilaian sarana
dan prasarana yg di nilai di sini adalah
sarana dan prasarana yang berada di
sekitar TWAK atau yang terdapat pada
kampung
klamono
sedangkan
pada
kawasan TWAK sendiri belum adanya
perhatian daan aktifitas aktifitas pada
kawasanTWAK. Sarana dan prasarana
(nilai 135) sarana rumah makan dan
sarana angkot umum dapat diakses
dengan mudah sebelum kita mengunjungi
TWAK. Sedangkan prasarana seperti
jalan, jembatan, jaringan listrik, jaringan
air minum, dan jaringan telepon dapat
membantu kita
untuk sampai pada
kawasan TWAK. umum yang berdomisili di Kampung
Klawana, sebagian besar menyetujui
apabila
TWAK
dikembangkan
dan
dilestarikan secara baik. Hal ini menjadi
modal
dasar
yang
tentunya
untuk
dikembangkan
dimasa
depan
karena
adanya
persetujuan
dan
dukungan
masyarakat setempat. Namun dimana saat
ini dalam hal keamanan dan pengelolaan
masih ada banyak terjadi pelanggaran
misalnya penebangan liar yang di sengaja
(ilegal logging), dan perambahan hutan
(pengunaan lahan). Pengelolaan kawasan
sendiripun belum ada misalnya pintu
gerbang masuk kawasan, rambu rambu
kawasan, pos penjagaan untuk keamanan
kawasan. Selama
ini
dari
pihak
pemerintah dan balai besar konservasi
sumberdaya hutan masih belum ada
kesepakaan
tentang
status
kawasan
Taman Wisata Alam Klamono dengan
pemegang hak ulayat (Mambringgofok). Sejauh
ini
justru
baru
diadakanya
pendekatan
seperti
kegiatan
dari
BBKSDH
melalui
pembentukan
kelompok tani hutan, mitra polhut di
Kampung Klawana, Kampung Klamono
dan Wonosari. Selain itu ada juga bantuan
berupa ternak sapi pada tahun 2018,
penangkaran buaya pada tahun 2018,
pembibitan
pada
tahun
2018,
dan
pelatihan RHL, dalam bentuk uang tunai
ataupun secara lansung diberikan. Pengembangan Pemanfaatan Jasa
Lingkungan Oleh
karena itu di harapkan kepada pemerintah
setempat dan Balai Besar Konservasi
Sumberdaya Hutan Provinsi Papua Barat
untuk
dapat
mengembangkan
dan
meningkatkan
aspek
pengelolaan
Kawasan terutama pengamanannya di
sekitar kawasan TWAK. Hasil rekapitulasi nilai terhadap 5
unsur penilaian yang terlah di modifikasi
modifikasi
dari
Pedoman
Kriteria
Penilaiaan
Pengembangan
Direktorat
Jendral
Perlindungan
Hutan
dan
Konservasi
Alam,
Departemen
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Prediction of Under-Detection of Paediatric Tuberculosis in the Democratic Republic of Congo: Experience of Six Years in the South-Kivu Province
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RESEARCH ARTICLE Editor: Nerges Mistry, Foundation for Medical
Research, INDIA Received: June 17, 2016
Accepted: December 9, 2016
Published: January 6, 2017 Methods Copyright: © 2017 Andre´ et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. We collected TB notification data from 2010 to 2015 and analysed the space-time variations
in notification for the different forms of TB among the 113 health facilities (HF) the South-
Kivu Province, a region with a low HIV incidence. The different forms of TB notified were:
smear positive pulmonary TB (SS+PTB), smear negative pulmonary TB (SS-PTB) and
extra-pulmonary TB (EPTB). We further analysed the distribution of these different forms of
the disease per age group and explored the possibility to predict the detection of PedTB. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This work was supported by TB REACH
grant (Stop TB Partnership) (http://www.stoptb. org/global/awards/tbreach/wave2.asp). Emmanuel Andre´1,2*, Yvette Lufungulo Bahati3, Eric Mulume Musafiri4, Olivier Bahati
Rusumba5, Dimitri Van der Linden2, Francis Zech1 1 Institut de Recherche Expe´rimentale et Clinique, Universite´ Catholique de Louvain, Brussels, Belgium,
2 Cliniques Universitaires Saint-Luc, Brussels, Belgium, 3 Hoˆpital Provincial Ge´ne´ral de Re´fe´rence de
Bukavu, Bukavu, Democratic Republic of Congo, 4 Coordination Provinciale Lèpre et Tuberculose, Bukavu,
Democratic Republic of Congo, 5 Ambassadeurs de Lutte contre la Tuberculose, Bukavu, Democratic
Republic of Congo a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * emmanuel.andre@uclouvain.be * emmanuel.andre@uclouvain.be Introduction Citation: Andre´ E, Lufungulo Bahati Y, Mulume
Musafiri E, Bahati Rusumba O, Van der Linden D,
Zech F (2017) Prediction of Under-Detection of
Paediatric Tuberculosis in the Democratic Republic
of Congo: Experience of Six Years in the South-
Kivu Province. PLoS ONE 12(1): e0169014. doi:10.1371/journal.pone.0169014 In the field of tuberculosis (TB), and particularly in regard to paediatric TB (PedTB), clinical
skills of health professionals play an important role in determining quality of care. In an era
where novel diagnostic technologies and efficient treatment regimens are being made avail-
able for the poorest, we must not divert our attention from the importance of clinical skills, as
this deliverable remains the cornerstone of individualized patient care and ultimately the
best assurance for optimal use of resources. The aim of our work was to study the epidemi-
ology of PedTB and the determinants of PedTB under-detection in the South-Kivu Province
of the Democratic Republic of Congo (DRC), a setting with nearly no technical resources
allowing to support the clinical diagnosis of PedTB, i.e. chest X-rays, rapid molecular tests
or culture laboratories. Editor: Nerges Mistry, Foundation for Medical
Research, INDIA Editor: Nerges Mistry, Foundation for Medical
Research, INDIA Conclusion Our observations strongly suggest that under-detection of PedTB is associated with insuffi-
cient clinical skills and technical resources at the HF level which similarly affects other forms
of the disease, in particular SS-TB. We demonstrated that, in the specific context of South-
Kivu, under-detection of PedTB can be predicted by a low SS-TB/SS+PTB ratio in the adult
population. In the context of severely under-resourced settings, this ratio could be used to
rapidly identify HF that should benefit in priority from deeper evaluation, and eventually tar-
geted interventions. Under-Detection of Tuberculosis among Children in DRC bacteriological confirmation (SS-TB) among adults, the number of TB cases notified by the
HF and the fact that the HF was supported or not by Me´decins Sans Frontières (MSF). The
proportion of SS-TB among adults was found to be a valid indicator for predicting the level of
detection of PedTB at the same HF. Abbreviations: DRC, Democratic Republic of
Congo; EPTB, Extra pulmonary tuberculosis; HZ,
Health Zone; HF, Health facility; INH, Isoniazid;
MSF, Me´decins Sans Frontières; MTB,
Mycobacterium tuberculosis complex; NGO,
Nongovernmental organization; PedTB, Paediatric
tuberculosis; RIF, Rifampicin; SS+PTB, Smear-
positive pulmonary tuberculosis; SS-PTB, Smear-
negative pulmonary tuberculosis; SS-TB, Smear
negative tuberculosis (SS-PTB and EPTB); TB,
Tuberculosis; WHO, World Health Organization. Results Significant differences were observed between HF in regard to the proportion of paediatric
TB and the proportion of SS-TB among adults. We found a strong correlation between the
proportion of PedTB and three major factors: the proportion of TB cases with no Competing Interests: The authors have declared
that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 1 / 9 Introduction The World Health Organisation (WHO) estimates that there were 9.6 million new patients
infected with tuberculosis (TB) in 2014. Globally, an estimated 10% of TB patients are children
less than 15 years, but two third of these are currently un-diagnosed or un-notified. The chal-
lenge of under-detection of TB is thus much more important among children than adults for
which approximately one third of TB patients are currently missed [1]. Paediatric TB (PedTB)
can rapidly evolve towards life-threatening manifestations such as miliary TB or meningitis
[2]. Improving the capacity of health care providers to detect TB among children will not only
save lives, but also have major repercussions in regard to the control of the epidemic. The Democratic Republic of Congo (DRC) lies among the few high TB burden countries
which did not meet any of the 2015 targets in regard to TB control: incidence is not falling and
neither prevalence nor mortality was reduced by 50% in comparison with 1990 figures. In
2015, DRC notified 111,683 TB cases (146.8/100,000), roughly half of the estimated numbers
of cases estimated for this country by WHO. In this low-income country, children aged less
than 15 years represent 46% of the population compared to 26% globally [3]. Despite the over-
representation of this age group, only 11.4% of TB patients were less than 15 years in 2015. The aim of our work was to develop a method to rapidly identify health facilities (HF)
which have a higher probability to be facing PedTB under-detection as a result of the inability
of health-care providers to recognise this clinical entity. Such method would allow the design
and implementation of targeted interventions, but also to evaluate their impact on PedTB
detection. Materials and Methods
Study setting The DRC presents a very large network of HF constituting the public health system. TB-related
services are well integrated within primary health care services, which offer free access to TB
diagnostics—including rapid molecular tests in a minority of HF—and treatment [4]. Clinical guidelines developed by the National Tuberculosis Programme of DRC (17)
include recommendations for the clinical diagnosis of PedTB. These recommendations are rel-
atively scanty, as they take into account the current situation of the vast majority of HF which 2 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Under-Detection of Tuberculosis among Children in DRC lack technical resources such as X-rays, molecular tests or tuberculin skin test (TST). There-
fore, the diagnosis of PedTB is principally based on aspecific clinical symptoms and the notion
of TB contact in the household. In practice, the use of rapid molecular diagnostic tests, which
are being progressively scaled-up in the country since 2012, is currently limited to patients
with presumptive drug resistant TB or HIV-TB coinfection. Due to this particular context, and
considering that no drug resistance survey has been performed in DRC to date, the incidence
of drug-resistant TB (and particularly among children) cannot be precisely estimated. Regular
supervision activities are performed by the national and provincial teams, although some areas
are inaccessible due to insecurity or the high costs associated with travelling in the most
remote areas. The South-Kivu province lies along the eastern border of DRC and presents a population of
over 6,6 million inhabitants, mainly living in rural areas. Health delivery is provided to the
population through over 500 HF, among which 78 (2010–2013) to 113 (2014–2015) provide
TB services: Ziehl-Nielsen microscopy and provision of first-line anti-TB drugs. Since 2012,
ten laboratories located in urban HF have been progressively equipped with GeneXpert
MTB/RIF technology. The province does not have a laboratory able to perform mycobacterial
cultures—the most sensitive diagnostic method—and diagnostic samples are usually not sent
to the National Reference Centre in Kinshasa unless drug resistance is suspected. g
The province is divided in 34 HZ, and each HZ has 1 to 6 HF. A HF is responsible for a
median of 51,500 habitants (percentiles 10 and 90: 17,800 and 97,300). The number of TB cases
notified by each HF is largely variable: 10% of these report less than 7 cases per year, while 10%
report more than 140 cases, the median being 32. Materials and Methods
Study setting Among the 34 health zones (HZ), five are
mainly urban (Bagira, Kadutu, Ibanda, Kamituga, and Uvira), while the remaining 29 HZ are
located in rural areas. Some of the latter are relatively well served in terms of infrastructure and
communications, while 11 are difficult to reach due to the absence of roads or on-going armed
conflicts. Most of these isolated HZ are located in the centre and west of the province. As a
result of the long-lasting humanitarian crisis in this region, the vast majority of the 34 HZ
receive external support from non-governmental organisations (NGOs) for the delivery of pri-
mary health care services. This support is generally integrated within the existing public system
and fully consistent with the national guidelines. Four HF located in the HZ Shabunda, Fizi
and Kimbi-Lulenge (since 2012), are nevertheless considered as exceptions: these are supported
by Me´decins Sans Frontières (MSF) which applies a more “independent” policy in regard to
the organisation of health services, including supplementation of the health structures with
qualified medical staff, clinical guidelines and technical resources including laboratory equip-
ment [5]. In regard to the diagnosis of PedTB, MSF recommends a documented growth assess-
ment of the children suspected or exposed to TB. Furthermore, MSF supports sample
transportation and provides technical resources such as X-rays, HIV testing, TST or investiga-
tions for EPTB. During the study period, there was no significant difference in the population
covered by each HF between rural, isolated (enclaved), or MSF-associated HF (p = 0.14). Diagnosis of smear negative and extra-pulmonary TB among adults Adults with SS-TB accounted for 21.8% of all cases notified and 28.5% of adult cases. With regard to the yearly notified cases of SS-TB for each HZ during the 6 years, the per-
centage of SS-TB among adult cases was highly variable from one HF to the other. Moreover,
we observed significant differences of the average percentages between rural HZ (15.7%), rural
enclaved (19.4%), urban (37.5%), or MSF-supported HZ (34,7%). Notified incidence of TB Between 2010 and 2015, the South-Kivu province reported 28,878 cases of TB. Among the
26,337 patients for which clinical outcome was available, 3.73% deceased during the year fol-
lowing diagnosis. This proportion was higher among SS-PTB (6.22%) and EPTB (4.89%) com-
pared to SS+PTB (2.79%). During this period, the mean incidence of TB per 100,000 people
per year was 84.5 new cases, among which 63.6% were adults with a pulmonary disease con-
firmed by microscopy. A transient increase in notification was observed due to the intensifica-
tion of active case-finding strategies supported by two TB REACH grants [7]. g
g
pp
y
g
The notified incidence was largely dependent on the notification of adults with SS+PTB,
accounting for up to 100% of all TB notified in some HZ. Data collection and statistical analysis We collected standard notification reports provided by the National TB Program. These
reports include the notification of the different forms of TB, including smear positive pulmo-
nary TB (SS+PTB), smear negative pulmonary TB (SS-PTB) and extra-pulmonary TB (EPTB). In this analysis, SS-PTB and EPTB among adults are considered jointly as smear-negative TB
(SS-TB), while PedTB includes all forms of the disease among children under 15 years. We analysed the changes in the incidence and proportion of different clinical TB presenta- We analysed the changes in the incidence and proportion of different clinical TB presenta-
tions for each HF over a period of 6 years from 2010 to 2015. Each HF and each year was 3 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Under-Detection of Tuberculosis among Children in DRC studied as a separate unit, with an internal correlation for the successive results of the same
HF. The relations between the percentages of PedTB and diverse factors were examined using
a generalised linear regression of a beta-binomial variable. Actually, the percentages of paediat-
ric case followed a beta-binomial distribution, due to hyperdispersion of results between health
areas. We used quasi-least squares, which is suitable for calculating internal correlations to
adjust the regression, and chose the hypothesis of constant between-year correlation matrix. To calculate statistical significance, we used the “sandwich variance matrix” augmented by the
bias correction proposed by Morel, Bokossa and Neerchal [6], which may be evaluated by the
normal distribution. To study the relation between the percentages of PedTB cases with other
factors, we first explored the curves of non-linear regressions and further selected the adequate
expression to include these factors in linear univariate and multivariate regressions. We used
the logarithm for the total number of TB cases reported for the populations surveyed by the
HFs and the percentages of SS-TB adults. We report here only the results of linear regressions. The data being anonymised, ethical approval was not required for this retrospective study. PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Diagnosis of paediatric TB The proportion of children less than 15 years accounted for 14.6% of all TB cases notified in
the South-Kivu province, compared to 11.4% at the national level (p<0.00001). In total, 4,219
PedTB cases were notified, among which 18.1% had a bacteriologically confirmed diagnosis
and only two children were diagnosed with MDR-TB. In total, 56 MDR-TB cases were
reported during the same period. The proportion of PedTB cases notified varied significantly between HZ, with a ratio vary-
ing between 0% and 40%. This important variation cannot be explained only by demographic
particularities, and rather suggests an important heterogeneity in the ability to diagnose
PedTB between HF. Looking at the yearly notified cases of PedTB for each HF during the 6 years, we could not
find any significant difference between urban, rural and isolated (enclaved) HZ (p = 0.074). The strongest association was found between the ratio of PedTB and the ratio of SS-PTB 4 / 9 Under-Detection of Tuberculosis among Children in DRC Fig 1. Correlation between the percentage of PedTB and the percentage of SS-TB among adults. Circles represent the number of
TB cases notified for each Health Facility (HF) during one year (6 years are represented). A strong correlation (p<0.000001) was found
between the proportion of SS-TB among adults and the proportion of PedTB. doi:10.1371/journal.pone.0169014.g001 Fig 1. Correlation between the percentage of PedTB and the percentage of SS-TB among adults. Circles represent the number of
TB cases notified for each Health Facility (HF) during one year (6 years are represented). A strong correlation (p<0.000001) was found
between the proportion of SS-TB among adults and the proportion of PedTB. doi:10.1371/journal.pone.0169014.g001 among adults (Fig 1) (p<0.000001). Furthermore, strong correlations were observed between
PedTB detection and the total number of TB cases diagnosed (p = 0.000016) and with the pres-
ence of MSF in the HF (p<0.00001). The size of the population covered by each HF has no sig-
nificant correlation with the ratio of PedTB. Development and validation of a model predicting under-detection of
paediatric TB For the final multivariate linear regression, we used the results as a model to predict the num-
ber of PedTB cases for each HF and each year (Fig 2). This model is based on 3 variables easily
available through the standard notification reports: the percentage of SS-TB among adults, the
total number of TB cases notified by the HF during the year and an external support received
by MSF. In the multivariate regression, these three factors remain independently significant:
being in a HF with a low or great percentage of adults with SS-TB (p<0.000001), being or not
in a MSF-supported HF (p<0.000001) and being in a HF with a low or great number of TB
cases notified (p = 0.0011). The validity of this model was further confirmed by the analysis of
the normalized Anscombe’s residues (data not shown). Under-Detection of Tuberculosis among Children in DRC Fig 2. Application of the model predicting under-detection of PedTB at the HF level based on 3 informations: percentage of
SS-TB among adults, external support received by MSF and the total number of TB cases notified by the HF during the year. doi:10.1371/journal.pone.0169014.g002 Fig 2. Application of the model predicting under-detection of PedTB at the HF level based on 3 informations: percentage of
SS-TB among adults, external support received by MSF and the total number of TB cases notified by the HF during the year. Fig 2. Application of the model predicting under-detection of PedTB at the HF level based on 3 informations: percentage of
SS-TB among adults, external support received by MSF and the total number of TB cases notified by the HF during the year. doi:10.1371/journal.pone.0169014.g002 the disease which are notified through quarterly reports. This observation suggests that there is
a high inequality between HF in regard to the ability to diagnose complex clinical presenta-
tions of TB, including PedTB and SS-TB among adults. We could demonstrate with a strong
level of evidence that the main factors which impact the detection of PedTB in South-Kivu, a
setting with scarce technical resources, are linked with the clinical skills and the clinical experi-
ence of the health professionals. Indeed, the HF which receive from MSF an important input
in terms of qualified health professionals tend to diagnose more PedTB and other complex
forms of the disease. Furthermore, HF which diagnose many cases of TB (all forms of the dis-
ease), tend to be better skilled to recognise children affected by the disease. Of note, this critical
importance of clinical skills may be exacerbated by the very poor infrastructure available in
most HF: overall, only a very small proportion of TB suspects have access to X-ray, rapid
molecular diagnostic or TST. Particularities of the study setting and implication for TB surveillance The South-Kivu province reports an incidence of 80.7/100,000 for all forms of TB, much lower
than the 325/100,000 national incidence estimated by WHO. We found that the reported inci-
dence is highly variable between HF, and particularly when looking at the different forms of 5 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Conclusion There is a critical lack of medical doctors in rural areas of Africa. In 2013, the South-Kivu
province counted 408 doctors (among which 14 had a specialization degree) and 2,862 nurses
for a population of over 6,6 million. In such context, nurses, laboratory technicians or the so-
called “Rapidly Trained Agents” form the front line of primary health-care delivery and are
inevitably faced to medical conditions that they have not been trained to recognize. Further-
more, these professionals and the patients have very limited access to complementary tests
such as X-Ray and accurate laboratory tests, which could eventually palliate part of these
deficiencies. The direct consequences of this lack of clinical skills are under-detection, increased mortal-
ity and continued transmission of the disease in the community [10]. As expected, SS-TB
among adults are similarly affected by these insufficient clinical skills. In the South-Kivu prov-
ince, these patients show an increased mortality during the first year after diagnosis. In such context, performing a continuous assessment of all provider’s level of “knowl-
edge, attitudes and practices (“KAP”) related to TB would be the best option, but is not
something that can be implementable in many difficult contexts such as the rural health
zones of the South-Kivu province. Our findings suggest that a first screening of the facilities
which would beneficiate the most from a complete assessment can be done based on the—
already collected—data. We propose to use the ratio between SS-TB and SS+TB notification
among adults as an indicator for insufficient clinical skills and under-detection of TB among
children. This indicator should enable TB control programs and their partners to precisely and
promptly identify those settings requiring additional support to tackle under-diagnosis of
PedTB and other complex clinical presentations of the disease. The interventions should focus
in parallel on the awareness of PedTB, active case finding strategies, strengthening of clinical
skills oriented towards PedTB and finally the implementation of adequate technical resources
such as X-rays, TST and rapid molecular laboratory assays. Improving clinical skills of health
professionals should not be considered as the only priority, but the importance of these human
skills should not be under-estimated as they remain the cornerstone of rational use of diagnos-
tic and treatment resources. In DRC, availability of X-rays, TST, rapid diagnostic tests are still
anecdotal when considering the thousands of health facilities located in rural areas. Conclusion Further-
more, a recent study performed by the National Tuberculosis Programme revealed that only
47% of HF had access to anti-TB treatments with adapted paediatric drug formulation [11]. Continued efforts focusing on the quality of clinical skills are a keystone for tackling the
dramatic burden of infectious diseases among children in low-resource settings [12–14]. We
propose an innovative approach to rapidly identify those HF facing alarming indicators and
which would beneficiate the most from additional human and technical resources. Importance of clinical skills The very strong association between under-detection of SS-TB among adults and under-detec-
tion of PedTB is therefore to be explained by the fact that recognizing these complex clinical
entities require (particularly in the absence of performant radiological investigations and labo-
ratory assays) higher clinical skills than the common smear positive pulmonary form of the
disease. Interestingly, four rural HF supported by MSF had the highest proportion of TB cases
diagnosed (mean 26.7%), and seemed to perform better than urban facilities in regard to
PedTB detection. The explanation of these high figures are probably the result of highly quali-
fied and adequately supported health staff and free access to X-rays or laboratory testing. Insufficient clinical skills among health professionals has been reported as the main reason
behind the frequent missed opportunities to diagnose and treat patients with TB that present
to health facilities [8]. A qualitative study performed in Tanzania [9] reports that “inadequate 6 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Under-Detection of Tuberculosis among Children in DRC awareness of the burden of childhood TB, limited knowledge of the wide spectrum of clinical
presentation and lack of clinical decision support strategies is detrimental to the health staff’s
central responsibility of suspecting and referring children with TB especially in the early dis-
ease stages”. The study authors stressed the importance of implementing strategies focusing on
awareness among health staff and clinical skills in order to improve diagnosis. S1 Fig. Validity of the model predicting under-detection of PedTB. Graphical representa-
tion of normalized Anscombe-s residues.
(TIF) Supporting Information S1 Fig. Validity of the model predicting under-detection of PedTB. Graphical representa-
tion of normalized Anscombe-s residues. (TIF) 7 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Under-Detection of Tuberculosis among Children in DRC S1 Dataset. Dataset. (XLS) S1 Dataset. Dataset. (XLS) Acknowledgments The authors would like to acknowledge the highly valuable comments made by Matthew Saun-
ders and Esto Bahizire during the redaction of this manuscript. Furthermore, the authors
would like to thank the staff of the Coordination Provinciale Lèpre et Tuberculose of the
South-Kivu province for the hard work of supervision of the health structures of the province,
which allowed to collect the information analysed in this manuscript. Finally, the authors
would like to thank the TB patients of the South-Kivu province, which are actively involved in
the implementation of sustainable solutions and working on a daily basis for ending the TB
epidemic. Writing – review & editing: EA EMM OBR YLB DVDL FZ. Writing – review & editing: EA EMM OBR YLB DVDL FZ. PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Author Contributions Conceptualization: EA FZ YLB DVDL. Resources: EA EMM OBR YLB DVDL FZ. Resources: EA EMM OBR YLB DVDL FZ. Supervision: FZ DVDL. Supervision: FZ DVDL. Validation: EA EMM OBR YLB DVDL FZ. Validation: EA EMM OBR YLB DVDL FZ. Visualization: EA FZ DVDL. PLOS ONE | DOI:10.1371/journal.pone.0169014
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tuberculosis/tuberculosis_en.pdf. 6. Morel JG, Bokossa MC, Neerchal NK. Small Sample Correction for the Variance of GEE Estimators. Biometrical Journal. 2003; 45(4):395–409. 8 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169014
January 6, 2017 Under-Detection of Tuberculosis among Children in DRC 7. Blok L, Sahu S, Creswell J, Alba S, Stevens R, Bakker MI. Comparative meta-analysis of tuberculosis
contact investigation interventions in eleven high burden countries. PLoS One. 2015; 10(3):e0119822. doi: 10.1371/journal.pone.0119822 PMID: 25812013 8. Claassens MM, Jacobs E, Cyster E, Jennings K, James A, Dunbar R, et al. Tuberculosis cases missed
in primary health care facilities: should we redefine case finding? Int J Tuberc Lung Dis. 2013; 17
(5):608–14. doi: 10.5588/ijtld.12.0506 PMID: 23575325 9. Bjerrum S, Rose MV, Bygbjerg IC, Mfinanga SG, Tersboel BP, Ravn P. Primary health care staff’s per-
ceptions of childhood tuberculosis: a qualitative study from Tanzania. BMC Health Serv Res. 2012;
12:6. doi: 10.1186/1472-6963-12-6 PMID: 22229965 10. Marais BJ. Strategies to improve tuberculosis case finding in children. Public Health Action. 2015; 5
(2):90–1. doi: 10.5588/pha.15.0028 PMID: 26393106 11. PNLT. Rapport e´pide´miologique de la Tuberculose2012. 12. References Farmer P, Robin S, Ramilus SL, Kim JY. Tuberculosis, poverty, and "compliance": lessons from rural
Haiti. Semin Respir Infect. 1991; 6(4):254–60. PMID: 1810004 13. Grange J, Zumla A. Tuberculosis and the poverty-disease cycle. J R Soc Med. 1999; 92(3):105–7. PMID: 10396250 14. Hansen C, Paintsil E. Infectious Diseases of Poverty in Children: A Tale of Two Worlds. Pediatr Clin
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Ttyh1 Protein is Expressed in Glia In Vitro and Shows Elevated Expression in Activated Astrocytes Following Status Epilepticus
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Ttyh1 Protein is Expressed in Glia In Vitro and Shows Elevated
Expression in Activated Astrocytes Following Status Epilepticus Elzbieta Wiernasz • Aleksandra Kaliszewska • Wojciech Brutkowski •
Joanna Bednarczyk • Malgorzata Gorniak • Beata Kaza •
Katarzyna Lukasiuk Received: 27 March 2014 / Revised: 26 September 2014 / Accepted: 8 October 2014 / Published online: 15 October 2014
The Author(s) 2014. This article is published with open access at Springerlink.com Abstract
In a previous study, we showed that Ttyh1
protein is expressed in neurons in vitro and in vivo in the
form of punctuate structures, which are localized to neu-
ropil and neuronal somata. Herein, we provide the first
description of Ttyh1 protein expression in astrocytes, oli-
godendrocytes and microglia in vitro. Moreover, using
double
immunofluorescence,
we
show
Ttyh1
protein
expression in activated astrocytes in the hippocampus fol-
lowing amygdala stimulation-induced status epilepticus. We demonstrate that in migrating astrocytes in in vitro
wound model Ttyh1 concentrates at the edges of extending
processes. These data suggest that Ttyh1 not only partici-
pates in shaping neuronal morphology, as previously
described, but may also play a role in the function of
activated glia in brain pathology. To localize Ttyh1
expression in the cellular compartments of neurons and
astrocytes, we performed in vitro double immunofluores-
cent staining using markers for the following subcellular
structures: endoplasmic reticulum (GRP78), Golgi appa-
ratus (GM130), clathrin-coated vehicles (clathrin), early endosomes (Rab5 and APPL2), recycling endosomes
(Rab11),
trans-Golgi
network
(TGN46),
endoplasmic
reticulum membrane (calnexin), late endosomes and lyso-
somes (LAMP1) and synaptic vesicles (synaptoporin and
synaptotagmin 1). We found that Ttyh1 is present in the
endoplasmic reticulum, Golgi apparatus and clathrin-
coated vesicles (clathrin) in both neurons and astrocytes
and also in late endosomes or lysosomes in astrocytes. The
presence of Ttyh1 was negligible in early endosomes,
recycling endosomes, trans-Golgi network, endoplasmic
reticulum membrane and synaptic vesicles. Keywords
Astrocytes Gliosis Microglia
Oligodendrocytes Status epilepticus Tweety Neurochem Res (2014) 39:2516–2526
DOI 10.1007/s11064-014-1455-3 Neurochem Res (2014) 39:2516–2526
DOI 10.1007/s11064-014-1455-3 ORIGINAL PAPER ORIGINAL PAPER Culture of Primary Astrocytes and Microglia Primary glial cell cultures were prepared from 1-day-old
Wistar rat pups, as previously described [13, 14]. Briefly,
the forebrains of the pups were aseptically removed and
homogenized through mechanical dissociation. The cells
were isolated from the cerebral cortices through trypsin-
ization
(0.025 %
trypsin
at
37 C
for
20 min)
and
mechanically dissociated into a single cell suspension. The
cells were plated onto poly-L-lysine-coated (#P4707,
Sigma) 75-cm2 culture flasks at a density of 3 9 105 cells/
cm2 in culture medium comprising DMEM with Gluta-
MAX and high-glucose at 4.5 g/l (#31966-021, Gibco),
supplemented with 10 % fetal bovine serum (FBS, #10500-
064,
Gibco),
penicillin
(100 U/ml)
and
streptomycin
(100 lg/ml) (#15140-148, Gibco). The cells were cultured
at 37 C, 95 % O2 and 5 % CO2. The culture medium was
changed after 3 days and then every 4–5 days. After
9–10 days in culture, the confluent glial cells were shaken
and centrifuged to recover microglia from astrocytes and
oligodendrocytes in culture. The loosely adhered microglia
were recovered through mild shaking and plated at a den-
sity of 2–3 9 105 cells/cm2 in 24-well plates or 60-mm
dishes. After changing the media to remove the nonad-
herent cells, the adherent microglia were incubated for
48 h. The expression of Ttyh1 mRNA is restricted to neural
tissue and the testis [8]. In the brain, Ttyh1 mRNA has
been detected in neurons [9, 10]. The expression of Ttyh1
protein is also neuronal and has been detected in neurons
in vivo and in vitro [6, 8, 11]. The overexpression of Ttyh1
protein in cell lines and neurons in vitro leads to excessive
filopodia formation [8, 11]. The involvement of Ttyh1 has
been implicated in aberrant neuronal structural plasticity
in vivo, as increased Ttyh1 protein expression was
observed in the molecular layer of the dentate gyrus during
epileptogenesis [11]. In a previous study, we have determined that Ttyh1
protein forms complexes with proteins localized in the
endoplasmic reticulum and Golgi [11]. Thus, the aim of the
present study is to verify these results using double
immunocytochemistry with markers of different cellular
compartments in vitro. In addition, we analyzed the
expression of Ttyh1 in glial cells in vitro and in vivo in the
normal and injured brain following status epilepticus. Culture of Primary Astrocytes and Microglia The
results show the ubiquitous expression of Ttyh1 protein in
astrocytes, oligodendrocytes and microglia in vitro and the
increased expression of Ttyh1 in astrocytes in the hippo-
campus following status epilepticus. We also demonstrate
that in migrating cultured astrocytes Ttyh1 is concentrated
at the margins of extending processes. To remove oligodendrocytes from astrocytes, primary
glial cells in 75-cm2 culture flasks were placed on a rotary
shaker and shaken at 37 C for 24 h (200 rpm). The culture
medium containing oligodendrocytes was removed, and the
astrocytes were plated onto 10-cm2 culture dishes. After
3 days, the astrocytes were trypsinized and plated onto poly-
L-lysine-coated glass coverslips (#1001/18, Glaswarenfab-
rik Karl Hecht GmbH & Co) in 12-well plates at a con-
centration of 75 9103 cells/ml and maintained in culture
for 72 h, followed by fixation and immunocytochemistry. Culture of Primary Hippocampal Neurons Culture of Primary Hippocampal Neurons Hippocampal neurons were cultured as previously descri-
bed [11, 12]. Briefly, the hippocampi were dissected from
E18 to E19 Wistar rat embryos and dissociated through
incubation in 0.04 % trypsin (#27250-0180, Gibco). The
cell suspension was plated onto poly-D-lysine-coated
(#P7280, Sigma) glass coverslips (#1001/18, Glaswaren-
fabrik Karl Hecht GmbH & Co) in 12-well plates. The cells
were cultured in Neurobasal G3 medium (#21103, Gibco),
supplemented with B27 (#17504, Gibco), 10 mg/l genta-
micin
(#15-750-037,
Gibco),
0.5 mM L-glutamine
(#G8540,
Sigma)
and
25 lM
L-glutamate
(#G1626, Introduction Campbell et al. [1] first described the tweety (twe, tty) gene
in the fli (flightless) genomic locus of Drosophila mela-
nogaster [1]. Mutations of tty in Drosophila result in the
loss of flight ability [1]. Homologs of the tweety (tty) gene
have also been identified in Caenorhabditis elegans and
mammals [2]. Three mammalian homologs, Ttyh1, Ttyh2
and Ttyh3, have been described [2–4]. Elzbieta Wiernasz and Aleksandra Kaliszewska have contributed
equally to this work. E. Wiernasz A. Kaliszewska J. Bednarczyk M. Gorniak
K. Lukasiuk (&)
Laboratory of Epileptogenesis, The Nencki Institute of
Experimental Biology, 3 Pasteur St, 02-093 Warsaw, Poland
e-mail: k.lukasiuk@nencki.gov.pl E. Wiernasz A. Kaliszewska J. Bednarczyk M. Gorniak
K. Lukasiuk (&)
Laboratory of Epileptogenesis, The Nencki Institute of
Experimental Biology, 3 Pasteur St, 02-093 Warsaw, Poland
e-mail: k.lukasiuk@nencki.gov.pl Human and mouse homologs of the D. melanogaster
tweety gene encode proteins predicted to be integral
membrane proteins that share a high degree of sequence
identity and common structural features with five or six
transmembrane regions positioned in a similar arrange-
ment. This arrangement comprises a pair of transmembrane
regions, followed by a hydrophobic region and an addi-
tional pair of transmembrane regions, which are followed
by more hydrophobic and transmembrane regions [2]. The
glycosylated N-terminus is located extracellularly, whereas W. Brutkowski
Laboratory of Imaging Tissue Structure and Function, The
Nencki Institute of Experimental Biology, Warsaw, Poland B. Kaza
Laboratory of Molecular Neurobiology, Neurobiology Center,
The Nencki Institute of Experimental Biology, Warsaw, Poland 12 3 Neurochem Res (2014) 39:2516–2526 2517 the involved in Ca2? binding, an acid-residue-rich C-ter-
minus is located cytoplasmically [5, 6]. The highest level
of sequence variation among Tweety-related proteins is
observed at the C-terminus, which likely specifies the
function of these proteins [3]. Sigma). After 5 days, the G3 medium was exchanged for
G2 medium (G3 medium without L-glutamate), and the
cells were maintained without changing the media until the
14th day of culture, when the cells were fixed and used for
immunocytochemistry. The human Tweety proteins are putative maxi-chloride
ion channels with differentially induced Cl- channel
activity [4, 7]. Tweety homolog 1 (Ttyh1) is a volume-
regulated Cl- channel, whose activity is regulated by cell
swelling, whereas the activity of Ttyh2 and Ttyh3 is reg-
ulated by ionomycin and calcium ions [4, 7]. Culture of Primary Astrocytes and Microglia Culture of Oligodendrocytes Rat oligodendrocyte precursor cells (OPCs) were prepared
according to a standard shaking method based on the dif-
ferential adherent properties of glia [15–17]. Briefly, the
forebrains from 1-day-old Wistar rat pups brains were
aseptically removed and homogenized through mechanical
dissociation. The cells were plated onto poly-L-lysine-
coated (#P4707, Sigma) flasks, maintained for 10 days in
DMEM/F12 (containing 10 % fetal calf serum, 2 mM
glutamine and antibiotics) and fed every 2–3 days by a
complete media change. At 10 days after plating, the cul-
ture flasks were placed onto a rotary shaker and pre-shaken
at 37 C for 6 h to remove the microglia cells. The medium
was changed, and the flasks were shaken at 37 C for an
additional 18–20 h. The cell suspension containing OPCs
was collected through centrifugation. The cell pellet was
suspended in OPC medium SATO medium containing
DMEM with GlutaMAX (#31966-021, Gibco) supple-
mented with 100 lg/ml BSA, 6.2 ng/ml progesterone,
16 lg/ml putrescine, 5 ng/ml sodium selenite, 400 ng/ml
T4, 400 ng/ml T3, 50 lg/ml holo-transferrin, 5 lg/ml
insulin, 10 ng/ml PDGF, 10 ng/ml FGF, penicillin (100
U/ml) and streptomycin (100 lg/ml)] and plated onto poly-
L-lysine-coated glass coverslips (#1001/18, Glaswarenfab-
rik Karl Hecht GmbH & Co) in 12-well plates at a con-
centration of 2 9 105 cells/ml. The cells were fed every
other day with 10 ng/ml PDGF and 10 ng/ml FGF until the
8th day of culture. To obtain differentiated oligodendro-
cytes, the OPCs were cultured for 7 additional days in
differentiation medium (DMEM
(#31966-021, Gibco)
containing 10 % fetal bovine serum (FBS, #10106-151,
Gibco), penicillin (100 U/ml) and streptomycin (100 lg/
ml) (#15140-148, Gibco)]. The medium was changed every
2–3 days. The differentiated oligodendrocytes were fixed
and used for immunocytochemical staining. Immunocytochemistry Primary hippocampal neurons, astrocytes, microglia and
oligodendrocytes in culture were fixed for 10 min in 4 or
2 % paraformaldehyde (#P6148, Sigma) in PBS containing
4 % sucrose (#S0389, Sigma), permeabilized with 0.5 %
Triton X-100 (#X100, Sigma) in PBS for 15 min, blocked
(60 min at RT) in 5 % bovine serum albumin (#A2153,
Sigma) (BSA) in 0.25 % Triton X-100 in PBS, and incu-
bated overnight at 4 C with primary antibodies diluted in
PBS containing 3 % BSA and 0.25 % Triton X-100. Sub-
sequently, the cells were washed in PBS and incubated
(2–2.5 h) with secondary antibodies diluted in PBS con-
taining 3 % BSA and 0.25 % Triton X-100. The cell nuclei
were counter-stained with 4,6-diamidino-2-phenylindole,
DAPI (1:1000, #D9542, Sigma). Each staining was per-
formed using 3–5 independent cultures. Amygdala Stimulation-Induced Status Epilepticus Amygdala Stimulation-Induced Status Epilepticus Scratch Assay Astrocytes (105 cells) were seeded onto 18 mmØ glass
coverslips pretreated with a solution of 100 lg/ml poly
poly-L-lysine (Sigma) and incubated in astrocyte culture
medium until reaching confluence. Confluent monolayers
were scraped with a 200 ll pipette tip. Cells were fixed in
4 % paraformaldehyde in PBS with 4 % sucrose 6, 24 or
48 h following wounding. Immunocytochemistry for Ttyh1
and GFAP was performed as described below. 12 3 2518 Neurochem Res (2014) 39:2516–2526 VA, USA, #E363-3-2WT-SPC) was implanted into the left
lateral nucleus of the amygdala under isoflurane anesthesia. For surface EEG recording, a stainless steel screw was
implanted contralaterally into the skull over the right
frontal cortex. After a 2-week recovery period, the animals
were electrically stimulated via the intra-amygdala elec-
trode to evoke SE. Stimulation consisted of a 100-ms train
of 1-ms biphasic square-wave pulses (400 lA peak to
peak) delivered at 60 Hz, every 0.5 s for 20 min (Master-8
Stimulator connected with an ISO-Flex stimulus isolation
unit, A.M.P.I., Israel). SE was terminated at 1.5–2 h after
stimulation through the intraperitoneal injection of diaze-
pam (20 mg/kg; Relanium, Polfa SA, Warsaw, Poland). The sham-operated control animals were implanted with
electrodes but did not receive electrical stimulation. Con-
trol (n = 7) and stimulated (n = 7) rats were sacrificed
4 days later. Animals were video-EEG monitored (Comet
EEG, Grass Technologies, West Warwick, RI) to confirm
development of SE lasting at least 90 min. VA, USA, #E363-3-2WT-SPC) was implanted into the left
lateral nucleus of the amygdala under isoflurane anesthesia. For surface EEG recording, a stainless steel screw was
implanted contralaterally into the skull over the right
frontal cortex. After a 2-week recovery period, the animals
were electrically stimulated via the intra-amygdala elec-
trode to evoke SE. Stimulation consisted of a 100-ms train
of 1-ms biphasic square-wave pulses (400 lA peak to
peak) delivered at 60 Hz, every 0.5 s for 20 min (Master-8
Stimulator connected with an ISO-Flex stimulus isolation
unit, A.M.P.I., Israel). SE was terminated at 1.5–2 h after
stimulation through the intraperitoneal injection of diaze-
pam (20 mg/kg; Relanium, Polfa SA, Warsaw, Poland). The sham-operated control animals were implanted with
electrodes but did not receive electrical stimulation. Con-
trol (n = 7) and stimulated (n = 7) rats were sacrificed
4 days later. Scratch Assay Animals were video-EEG monitored (Comet
EEG, Grass Technologies, West Warwick, RI) to confirm
development of SE lasting at least 90 min. Culture of Oligodendrocytes Image Acquisition and Analysis Image Acquisition and Analysis The Carl Zeiss LSM780 Spectral Confocal System inter-
faced with Axio Observer Z.1 inverted microscope equip-
ped with Plan Apochromat 409/1.4 Oil DIC, Plan
Apochromat 639/1.4 Oil DIC objectives, Ar ion (488 nm),
DPSS diode (561 nm), diode (405 nm CW/pulsed) laser
and Zen 2011 software were used for fluorescence image
acquisition. The images were processed using ImageJ software (Ras-
band, W.S., ImageJ, U. S. National Institutes of Health,
Bethesda, Maryland). The displayed images were not
manipulated beyond adjusting the histogram. Deconvolu-
tion was performed using Huygens Software (SVI, Hil-
versum, The Netherlands). Colocalization analysis was
performed with ImarisColoc (Bitplane, Zurich, Switzer-
land). Every time colocalization channel containing only
colocalized voxels was generated. Results are presented as
a merge of fluorescent channel and colocalization channel
for easier perception of colocalization sites. Pearson’s
correlation coefficient was calculated to quantify the
degree
of colocalization between
fluorophores. Ttyh1 Immunohistochemistry for western blot analysis using a standard protocol [11]. The experiment was repeated three times. Briefly, the cells
were lysed in lysis buffer containing 50 mM KCl, 50 mM
PIPES, 10 mM EGTA, 2 mM MgCl2, 0.5 % Triton X-100,
100 lM PMSF and 1 mM DTT. The brain tissue was
homogenized in the same buffer using a TissueRuptor
homogenizer (Qiagen). The lysates were centrifuged at
20,800g for 20 min at 4 C. The supernatants were col-
lected and stored at -20 C. The protein concentration was
determined using Protein Assay Dye Reagent (#500-0006,
BioRad). The samples were separated using SDS-PAGE
and transferred to nitrocellulose (#RPN303D, HybondTM-
ECL, Amersham). For immunodetection, the nonspecific
binding was blocked through incubation in 5 % non-fat
milk in TBST (0.5 M Tris, 0.9 % NaCl, 0.1 % Tween 20,
pH 8). Subsequently, the membrane was incubated in
a
solution
of
mouse
anti-Ttyh1
antibody
(1:500,
#WH0057348M4, Sigma) in TBST overnight at 4 C,
followed by incubation in a solution of secondary anti-
mouse horseradish peroxidase-linked antibody (1:5,000,
#NA931, Amersham Biosciences) in TBST for 2 h at RT. Chemiluminescent detection was performed using ECL
(#RPN2209, Amersham) according to the manufacturer’s
instructions. The protein molecular weights were estimated
using prestained protein markers. To verify that equal
amounts of protein were loaded, the membrane was strip-
ped and reprobed with mouse anti-GAPDH antibody
(1:300, #MAB374, Chemicon Millipore) in TBST over-
night at 4 C, followed by incubation with secondary anti-
mouse horseradish peroxidase-linked antibody. Immunohistochemistry All animal procedures were approved by the Ethical
Committee on Animal Research of the Nencki Institute,
and the experiments were conducted in accordance with the
guidelines of Directive 2010/63/EU of the European Par-
liament and the Council of the European Union (EU). Adult
male
Sprague–Dawley
rats
(290–320 g)
were
obtained from the The Mossakowski Medical Research
Centre (Polish Academy of Sciences, Warsaw, Poland) and
housed in a controlled environment (24 C, lights on
07:00–19:00) with free access to food and water. Status
epilepticus (SE) was triggered through electrical stimula-
tion of the amygdala as previously described [18], with
some
modifications
[19]. Briefly,
a
stimulating
and
recording bipolar electrode (Plastic One Inc., Roanoke, The rats were anesthetized with morbital (1.25 ml/kg, i.p.)
and perfused with 0.9 % NaCl (2 min, 30 ml/min), fol-
lowed by perfusion with 4 % paraformaldehyde in 0.1 %
phosphate buffer (PB; 0.1 M Na2HPO4, and 0.1 M NaH2
PO4, pH 7.4) for 20 min (30 ml/min) as previously
described [11]. Next, the brains were removed, postfixed
for 4 h in the same fixative, cryoprotected in 30 % sucrose
in 0.02 M PB buffer for 3–4 days, frozen on dry ice and
stored at -70 C. Coronal sections (30 lm) were cut and
stored in a cryoprotectant solution (30 % ethylene glycol,
25 % glycerol, 0.05 M PB) at -20 C. Immunohistochemistry was performed on free-floating
coronal brain sections using a standard procedure [11]. 123 2519 Neurochem Res (2014) 39:2516–2526 Briefly, for double immunofluorescence staining, after
blocking for unspecific binding for 2 h with PBS contain-
ing 3 % bovine serum albumin (BSA, Sigma) and 0.1 %
Triton X-100, a mixture of primary antibodies (diluted in
PBS containing 0.3 % BSA and 0.01 % Triton X-100) was
applied, and the cells were incubated overnight at 4 C. Subsequently, the sections were incubated for 2 h with the
appropriate secondary antibodies diluted in PBS (contain-
ing 0.3 % BSA and 0.01 % Triton X-100), and the nuclei
were counter-stained with 4,6-diamidino-2-phenylindole
(DAPI), mounted on gelatin-coated slides and coverslipped
in Vectashield (Vector Laboratories). For detection of
Ttyh1 with 3,30-diaminobenzidine (DAB) as a chromo-
phore, sections were incubated with anti-Ttyh1 antibody,
and then in a solution of appropriate biotinylated antibody. For signal detections, sections were incubated in avidin–
biotin solution (ABC kit Vectastain, #PK6100, Vector
Laboratories) and then in 0.05 % 3,30-diaminobenzidine
(DAB) solution containing 0.04 % H2O2. Antibodies The following primary antibodies were used in the present
study: mouse anti-Ttyh1 (1:200, #WH0057348M4, Sigma),
rabbit anti-Synaptotagmin 1 (1:100, #105102, Synaptic
Systems), rabbit anti-Synaptoporin (1:500, #102002, Syn-
aptic Systems), rabbit anti-Neuronal Class III b-Tubulin
(1:750, #PRB-435P, Covance), rabbit anti-LAMP1 (1:500,
#ab24170, Abcam), rabbit anti-GRP78 (1:200, #ab21685,
Abcam), rabbit anti-Calnexin (1:40, #ab22595, Abcam),
rabbit anti-GM130 (1:200, #ab52649, Abcam), rabbit anti-
TGN46 (1:500, #ab16059), rabbit anti-Clathrin (1:500,
#ab21679, Abcam), rabbit anti-Rab5 (1:500, #ab18211,
Abcam), rabbit anti-Rab11 (1:100, #71-5300, Invitrogen),
rabbit anti-APPL2 (home made, [20]), rabbit anti-Olig2
(1:500, #AB9610, Chemicon Millipore), rabbit anti-Iba1
(1:1,000,
#019-19741,
Wako), mouse
anti-GFAP-Cy3
(1:1,000,
#C9205,
Sigma). Anti-IgG
mouse
(1:200,
#553447,
BD
Pharmingen)
anti-IgG
rabbit
(1:200,
#ab46540, Abcam) were used as controls to test specificity
of immunostainings. The following secondary antibodies
were used in the present study: goat anti-mouse conjugated
with Alexa Fluor 488 (1:200, #A11001, Molecular Probes),
goat
anti-rabbit
conjugated
with
Texas
Red
(1:200,
#T2767, Molecular Probes), horse anti-mouse conjugated
with Texas Red (1:200, #TI2000, Vector Laboratories), and
anti-mouse biotinylated antibody (1:200, #BA2001, Vector
Laboratories). Western Blot Ttyh1-positive structures were present throughout the
entire cell, in the soma and in branches. The cell nuclei were counter-
stained with DAPI (blue). Scale bars 10 lm. The images represent
maximum intensity Z projections of confocal sections. DAPI, 4,6-
diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1,
Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte
lineage transcription factor 2 (Color figure online) oligodendrocyte immunostained with anti-Ttyh1 (green) and anti-
Olig2 (red). Ttyh1-positive structures were present throughout the
entire cell, in the soma and in branches. The cell nuclei were counter-
stained with DAPI (blue). Scale bars 10 lm. The images represent
maximum intensity Z projections of confocal sections. DAPI, 4,6-
diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1,
Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte
lineage transcription factor 2 (Color figure online) Fig. 2 Western blot analysis of Ttyh1 protein expression in protein
extracts isolated from microglia, cerebral astrocyte cultures and
hippocampal neuronal cultures. A single band represents Ttyh1
protein (predicted molecular weight of 37 kDa), which was detected
in all lysates. GAPDH (predicted molecular weight of 38 kDa) was
used as a protein loading control. GAPDH—Glyceraldehyde 3-phos-
phate dehydrogenase vesicle volume analysis was performed using Spots algo-
rithm based on absolute intensity included in Imaris soft-
ware. All the analysis were performed using at least five
images, which were obtained from 3 to 5 independent
experiments. Microscope pictures of DAB stained slices were taken
using a Nikon Eclipse 80i microscope equipped with a 109
and 209 objective and Wikom Evolution WF camera. Fig. 2 Western blot analysis of Ttyh1 protein expression in protein
extracts isolated from microglia, cerebral astrocyte cultures and
hippocampal neuronal cultures. A single band represents Ttyh1
protein (predicted molecular weight of 37 kDa), which was detected
in all lysates. GAPDH (predicted molecular weight of 38 kDa) was
used as a protein loading control. GAPDH—Glyceraldehyde 3-phos-
phate dehydrogenase Results Expression of Ttyh1 Protein in Neurons, Astrocytes,
Microglia and Oligodendrocytes In Vitro compared with oligodendrocytes. We have observed dif-
ferences in the size of the Ttyh1-immunopositive dots in all
cell types. Previous studies have shown the expression of Ttyh1
mRNA in rat cortical neurons and the widespread expres-
sion of Ttyh1 protein in neurons in vitro and in vivo [10,
11]. In the present study we evaluated the expression of
Ttyh1 protein in glial cells (astrocytes, oligodendrocytes
and microglia) cultured in vitro. Immunocytochemistry
with
the
anti-Ttyh1
antibody
revealed
the
abundant
expression of Ttyh1 in astrocytes, oligodendrocytes and
microglia in vitro. Pattern of expression was consistent
from culture to culture. Images of representative cells are
presented on Fig. 1. The western blot analysis confirmed the presence of
Ttyh1 protein in extracts of isolated hippocampal neurons,
cerebral astrocytes and microglia in vitro (Fig. 2). 123 Western Blot Proteins isolated from rat primary cultured neurons,
astrocytes, microglia and rat brain tissue were processed 12 3 123 Neurochem Res (2014) 39:2516–2526 2520 Fig. 1 Localization of Ttyh1 protein in rat astrocytes, microglia and
oligodendrocytes in vitro. a Double immunolabeling of primary
astrocytes for Ttyh1 and GFAP. Ttyh1-positive structures were
present in the entire cell, in astrocyte somata and in the branches
along the filament network stained with GFAP. b Ttyh1 immunore-
activity in microglia double-immunostained with anti-Ttyh1 (green)
and anti-Iba1 (red). Fine Ttyh1-positive, dot-like structures were
present throughout the entire cell. c Ttyh1 immunoreactivity in Fig. 1 Localization of Ttyh1 protein in rat astrocytes, microglia and
oligodendrocytes in vitro. a Double immunolabeling of primary
astrocytes for Ttyh1 and GFAP. Ttyh1-positive structures were
present in the entire cell, in astrocyte somata and in the branches
along the filament network stained with GFAP. b Ttyh1 immunore-
activity in microglia double-immunostained with anti-Ttyh1 (green)
and anti-Iba1 (red). Fine Ttyh1-positive, dot-like structures were
present throughout the entire cell. c Ttyh1 immunoreactivity in
oligodendrocyte immunostained with anti-Ttyh1 (green) and anti-
Olig2 (red). Ttyh1-positive structures were present throughout the
entire cell, in the soma and in branches. The cell nuclei were counter-
stained with DAPI (blue). Scale bars 10 lm. The images represent
maximum intensity Z projections of confocal sections. DAPI, 4,6-
diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1,
Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte
lineage transcription factor 2 (Color figure online) oligodendrocyte immunostained with anti-Ttyh1 (green) and anti-
Olig2 (red). Ttyh1-positive structures were present throughout the
entire cell, in the soma and in branches. The cell nuclei were counter-
stained with DAPI (blue). Scale bars 10 lm. The images represent
maximum intensity Z projections of confocal sections. DAPI, 4,6-
diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Iba1,
Ionized calcium binding adaptor molecule 1; Olig2, oligodendrocyte
lineage transcription factor 2 (Color figure online) Fig. 1 Localization of Ttyh1 protein in rat astrocytes, microglia and
oligodendrocytes in vitro. a Double immunolabeling of primary
astrocytes for Ttyh1 and GFAP. Ttyh1-positive structures were
present in the entire cell, in astrocyte somata and in the branches
along the filament network stained with GFAP. b Ttyh1 immunore-
activity in microglia double-immunostained with anti-Ttyh1 (green)
and anti-Iba1 (red). Fine Ttyh1-positive, dot-like structures were
present throughout the entire cell. c Ttyh1 immunoreactivity in oligodendrocyte immunostained with anti-Ttyh1 (green) and anti-
Olig2 (red). Expression of Ttyh1 in the Hippocampus in Control
Animals and After Amygdala Stimulation-Induced
Status Epilepticus We
detected
Ttyh1
immunoreactivity
in
astrocytes,
microglia and oligodendrocytes, and this result prompted
use to determine whether Ttyh1 is expressed in activated
glial cells in vivo. Following amygdala stimulation induced
status epilepticus, reactive astrocytes in hippocampus can
be observed as early as after 24 h and persist for at least In astrocytes and oligodendrocytes Ttyh1 was present in
dot-like structures in cell somata and in branches. Simi-
larly, in microglia Ttyh1-positive structures were evenly
distributed throughout the cytoplasm. Ttyh1 immunoreac-
tivity in astrocytes and microglia was weaker when 123 123 2521 Neurochem Res (2014) 39:2516–2526 Fig. 3 Ttyh1 expression in the normal rat brain and at 4 days after
amygdala stimulation-induced status epilepticus. DAB immunostain-
ing with anti-Ttyh1 antibody in the hippocampus of a sham control
brain (a) and at 4 days after status epilepticus (b). Fragments of
pyramidal layer and stratum oriens of CA3 region are framed and
enlarged in c (control brain) and d (stimulated brain). In the control
brain exclusively neuronal somata within pyramidal layer are stained
(indicated with arrowheads). At 4 days after status epilepticus
astrocyte-like profiles (arrows) and neuronal somata within pyramidal
layer (arrowheads) are revealed with immunostaining with anti-Ttyh1
antibody. Scale bars 500 lm for a and b, 50 lm for c and d brain exclusively neuronal somata within pyramidal layer are stained
(indicated with arrowheads). At 4 days after status epilepticus
astrocyte-like profiles (arrows) and neuronal somata within pyramidal
layer (arrowheads) are revealed with immunostaining with anti-Ttyh1
antibody. Scale bars 500 lm for a and b, 50 lm for c and d Fig. 3 Ttyh1 expression in the normal rat brain and at 4 days after
amygdala stimulation-induced status epilepticus. DAB immunostain-
ing with anti-Ttyh1 antibody in the hippocampus of a sham control
brain (a) and at 4 days after status epilepticus (b). Fragments of
pyramidal layer and stratum oriens of CA3 region are framed and
enlarged in c (control brain) and d (stimulated brain). In the control brain exclusively neuronal somata within pyramidal layer are stained
(indicated with arrowheads). At 4 days after status epilepticus
astrocyte-like profiles (arrows) and neuronal somata within pyramidal
layer (arrowheads) are revealed with immunostaining with anti-Ttyh1
antibody. Scale bars 500 lm for a and b, 50 lm for c and d Fig. 3 Ttyh1 expression in the normal rat brain and at 4 days after
amygdala stimulation-induced status epilepticus. Expression of Ttyh1 in the Hippocampus in Control
Animals and After Amygdala Stimulation-Induced
Status Epilepticus DAB immunostain-
ing with anti-Ttyh1 antibody in the hippocampus of a sham control
brain (a) and at 4 days after status epilepticus (b). Fragments of
pyramidal layer and stratum oriens of CA3 region are framed and
enlarged in c (control brain) and d (stimulated brain). In the control cells in the CA3 of animals subjected to amygdala stimu-
lation (Fig. 4). The increase in Ttyh1 immunoreactivity in
reactive astrocytes in the CA3 reflected the increased
intensity of immunofluorescence and the increase in the
number of Ttyh1-positive dots (Fig. 4d). 14 days [21]. We performed preliminary DAB immuno-
stainings to detect Ttyh1 protein in slices from rats 24 h, 4
and 14 days after amygdala stimulation-induced status
epilepticus and time-matched control animals. 4 days fol-
lowing status epilepticus majority of immunostained hip-
pocampal cells displayed star-like processes typical of
hypertrophic reactive astrocytes. They were more or less
evenly distributed among different layers (Fig. 3b, d). In
hippocampi of control animals only sparse neuron-shaped
somata were strongly labeled (Fig. 3a, c). They were found
mainly in the pyramidal layer. In other timepoints we did
not observe Ttyh1 expression in cells morphologically
resembling astrocytes (data not shown). To test possibility that status epilepticus induces Ttyh1
expression also in oligodendrocytes or microglia, we per-
formed double immunostainings using anti-Ttyh1 antibody
and anti-Iba1 (microglia marker) or anti-Olig2 (oligoden-
drocyte marker) antibody (data not shown). Ttyh1 immu-
noreactivity in microglia in vitro was negligible in the
control brain. Similarly, in the hippocampus of the stimu-
lated brain, Ttyh1 colocalization with Iba1 was only
occasionally observed. Ttyh1 immunostaining in oligo-
dendrocytes in vivo was difficult to interpret. Because the
oligodendrocytes insulating neuronal axons are thin and the
oligodendrocyte marker Olig2 is specifically localized
inside the nucleus, not in the branches, it is difficult to
distinguish immunoreactivity in oligodendrocytes. We To confirm Ttyh 1 astrocytic localization, we performed
double immunohistochemistry, using the anti-Ttyh1 and
anti-GFAP
antibody. Few
Ttyh1-positive
dots
were
observed in GFAP-positive astrocytes in the CA3 of the
sham control animals. In contrast, numerous Ttyh1-im-
munopositive elements were detected in GFAP-positive 12 3 3 2522 Neurochem Res (2014) 39:2516–2526 Fig. 4 Ttyh1 expression in astrocytes in the normal rat brain and at
4 days after amygdala stimulation-induced status epilepticus. a–
c Ttyh1 expression in the hippocampal CA3 of a sham control brain. Expression of Ttyh1 in the Hippocampus in Control
Animals and After Amygdala Stimulation-Induced
Status Epilepticus a Confocal image of immunofluorescence with anti-Ttyh1 (green) and
b the astrocyte marker anti-GFAP (red), c merged image of double
immunofluorescence staining demonstrating Ttyh1, GFAP and cell
nuclei counter-stained with DAPI (blue). d–f Ttyh1 expression in the
hippocampal CA3 pyramidal layer at 4 days after status epilepticus. d Confocal image of immunofluorescence with anti-Ttyh1 and e anti-
GFAP, f merged image of double immunofluorescence staining observed only few Ttyh1-positive dots in the close vicinity
of oligodendrocyte nuclei in the control brain and after
status epilepticus. deposited only in the part of the process most protruding
toward the scratch. In wider lamellipodia it was concen-
trated along the whole leading edge. Such uneven distri-
bution of Ttyh1 in astrocytes along the scratch at any
timepoint was observed only in fraction of cells. Expression of Ttyh1 in the Hippocampus in Control
Animals and After Amygdala Stimulation-Induced
Status Epilepticus a Confocal image of immunofluorescence with anti-Ttyh1 (green) and
b the astrocyte marker anti-GFAP (red), c merged image of double
immunofluorescence staining demonstrating Ttyh1, GFAP and cell
nuclei counter-stained with DAPI (blue). d–f Ttyh1 expression in the
hippocampal CA3 pyramidal layer at 4 days after status epilepticus. d Confocal image of immunofluorescence with anti-Ttyh1 and e anti-
GFAP, f merged image of double immunofluorescence staining
demonstrating Ttyh1, GFAP and cell nuclei counter-stained with
DAPI. The yellow arrows on green channel (a, d) are pointing single,
representative Ttyh1-positive structures. The same Ttyh1-positive
structure, marked by the yellow arrows on merge channel (c, f), show
the colocalization of Ttyh1 and GFAP. Note that in the control brain,
colocalization was negligible, while Ttyh1-positive punctuate staining
was observed throughout the astrocytes from the brain after status
epilepticus. Scale bar 5 lm. The images represent single confocal
sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary
acidic protein (Color figure online) rat brain and at
epilepticus. a–
am control brain. Ttyh1 (green) and
image of double
GFAP and cell
expression in the
tatus epilepticus. Ttyh1 and e anti-
escence staining
demonstrating Ttyh1, GFAP and cell nuclei counter-stained with
DAPI. The yellow arrows on green channel (a, d) are pointing single,
representative Ttyh1-positive structures. The same Ttyh1-positive
structure, marked by the yellow arrows on merge channel (c, f), show
the colocalization of Ttyh1 and GFAP. Note that in the control brain,
colocalization was negligible, while Ttyh1-positive punctuate staining
was observed throughout the astrocytes from the brain after status
epilepticus. Scale bar 5 lm. The images represent single confocal
sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary
acidic protein (Color figure online) demonstrating Ttyh1, GFAP and cell nuclei counter-stained with
DAPI. The yellow arrows on green channel (a, d) are pointing single,
representative Ttyh1-positive structures. The same Ttyh1-positive
structure, marked by the yellow arrows on merge channel (c, f), show
the colocalization of Ttyh1 and GFAP. Note that in the control brain,
colocalization was negligible, while Ttyh1-positive punctuate staining
was observed throughout the astrocytes from the brain after status
epilepticus. Scale bar 5 lm. The images represent single confocal
sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary
acidic protein (Color figure online) Fig. 4 Ttyh1 expression in astrocytes in the normal rat brain and at
4 days after amygdala stimulation-induced status epilepticus. a–
c Ttyh1 expression in the hippocampal CA3 of a sham control brain. Scratch-Induced Changes in Localization of Ttyh1
in Cultured Astrocytes Ttyh1 Immunopositive Puncta Volume and Identity
in Cultured Neurons and Astrocytes Ttyh1 Immunopositive Puncta Volume and Identity
in Cultured Neurons and Astrocytes Reactive astrogliosis, observed in many pathological brain
processes
including
epileptogenesis,
can
be
induced
in vitro by a scratch injury [22]. Astrocytes rapidly polarize
after lesion and extend their processes perpendicularly to
the scratch. We performed Ttyh1 immunofluorescent
staining on astrocytes fixed 6, 24 and 48 h after wounding. We observed gradual filling of the area of the scratch by
migrating astrocytes. In some migrating astrocytes we
observed concentration of Ttyh1 protein at the margin of
lamellipodia (Fig. 5). In narrow lamellipodia Ttyh1 was In neurons and in glial cells Ttyh1 is present in form of dot-
like structures of different sizes. Hypothetically this het-
erogeneity may reflect presence of Ttyh1 in different sub-
cellular structures. Neurons and astrocytes are particularly
interesting in terms of Ttyh1 expression, as in those types
of cells we were able to detect prominent Ttyh1 immuno-
reactivity in vivo. We asked whether in those two cell types
Ttyh1 is present in the same subcellular structures. Firstly, 123 123 2523 Neurochem Res (2014) 39:2516–2526 Fig. 5 Scratch-induced changes in localization of Ttyh1 in cultured
astrocytes. Cultured astrocytes were fixed 48 h after making a scratch
and labeled for Ttyh1 (green) and GFAP (red). Yellow dashed line
indicates the scratch. In presented astrocytes migrating toward the
scratch Ttyh1 was particularly concentrated at the margins of
lamellipodia. Ttyh1 formed deposits only in astrocyte processes most protruding toward the scratch (a) or along the leading edge (b). The
cell nuclei were counter-stained with DAPI (blue). Scale bar 25 lm. The images represent maximum intensity Z projections of confocal
sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary
acidic protein (Color figure online) Fig. 5 Scratch-induced changes in localization of Ttyh1 in cultured
astrocytes. Cultured astrocytes were fixed 48 h after making a scratch
and labeled for Ttyh1 (green) and GFAP (red). Yellow dashed line
indicates the scratch. In presented astrocytes migrating toward the
scratch Ttyh1 was particularly concentrated at the margins of
lamellipodia. Ttyh1 formed deposits only in astrocyte processes most protruding toward the scratch (a) or along the leading edge (b). The
cell nuclei were counter-stained with DAPI (blue). Scale bar 25 lm. The images represent maximum intensity Z projections of confocal
sections. DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary
acidic protein (Color figure online) Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured
neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was
determined using Imaris software. Ttyh1 Immunopositive Puncta Volume and Identity
in Cultured Neurons and Astrocytes Immunopositive puncta are
classified into groups depending on their volume. Percent distribution
of Ttyh1 vesicle volume in both neurons and astrocytes is similar,
with most puncta having volume 0.05–149 lm3 and only few percent
of puncta bigger than 0.35 lm3. Note that proportions of immuno-
positive puncta in different size groups vary between neurons and
astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05;
***p \ 0.001 We classified all Ttyh1 immunopositive puncta into one
of five groups depending on their volume (Fig. 6). Distri-
bution of Ttyh1 immunopositive puncta volume was
roughly the same for neurons and astrocytes, with rela-
tively small proportion of \0.05 lm3 puncta, highest pro-
portion
of
0.05–0.149 lm3
puncta
and
gradually
decreasing contribution of 0.15–0.249, 0.25–0.349 and
[0.35 lm3 puncta. However, percentages of puncta in
different size groups varied between neurons and astrocytes
as the puncta 0.05–0.149 lm3 were more abundant in
neurons than in astrocytes, while small puncta of
\0.05 lm3 and the large ones (0.25–0.349 and[0.35 lm3)
were more abundant in astrocytes. In a previous study, in which we performed a pull-down
assay using hippocampal protein lysates, we revealed that
Ttyh1 protein specifically binds to a number of proteins,
including a large group of proteins localized in the endo-
plasmic reticulum or Golgi apparatus [11]. To further
characterize the subcellular localization of Ttyh1 protein,
we performed double immunocytochemistry on primary
hippocampal neurons and primary cortical astrocyte cell
cultures using a number of antibodies against specific
subcellular markers. Ttyh1 protein has been detected in the
Golgi apparatus (Pearson’s correlation coefficient PCC,
neurons r = 0.30, astrocytes r = 0.32; Fig. 7a, e), endo-
plasmic reticulum (PCC, neurons r = 0.27, astrocytes
r = 0.47; Fig. 7b, f) and clathrin-coated vesicles (PCC,
neurons r = 0.27, astrocytes r = 0.35; Fig. 7c, g) in both
neurons and astrocytes. In astrocytes, Ttyh1 colocalized
with LAMP1, the marker of late endosomes and lysosomes
(PCC, r = 0.48; Fig. 7d). No colocalization of Ttyh1 with Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured
neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was
determined using Imaris software. Immunopositive puncta are
classified into groups depending on their volume. Percent distribution
of Ttyh1 vesicle volume in both neurons and astrocytes is similar,
with most puncta having volume 0.05–149 lm3 and only few percent
of puncta bigger than 0.35 lm3. Ttyh1 Immunopositive Puncta Volume and Identity
in Cultured Neurons and Astrocytes Note that proportions of immuno-
positive puncta in different size groups vary between neurons and
astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05;
***p \ 0.001 Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured
neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was
determined using Imaris software. Immunopositive puncta are
classified into groups depending on their volume. Percent distribution
of Ttyh1 vesicle volume in both neurons and astrocytes is similar,
with most puncta having volume 0.05–149 lm3 and only few percent
of puncta bigger than 0.35 lm3. Note that proportions of immuno-
positive puncta in different size groups vary between neurons and
astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05;
***p \ 0.001 Fig. 6 Analysis of distribution of Ttyh1 vesicle volume in cultured
neurons and astrocytes. Volume of Ttyh1 immunopositive puncta was
determined using Imaris software. Immunopositive puncta are
classified into groups depending on their volume. Percent distribution
of Ttyh1 vesicle volume in both neurons and astrocytes is similar,
with most puncta having volume 0.05–149 lm3 and only few percent
of puncta bigger than 0.35 lm3. Note that proportions of immuno-
positive puncta in different size groups vary between neurons and
astrocytes. Neurons, n = 5; astrocytes, n = 5; t test, *p \ 0.05;
***p \ 0.001 we addressed the issue indirectly. We compared distribu-
tion of Ttyh1 immunopositive puncta volume in cultured
neurons and astrocytes. Secondly, we tried to colocalize
Ttyh1 with different subcellular markers in neurons and
astrocytes. 12 3 2524 Neurochem Res (2014) 39:2516–2526 Fig. 7 Ttyh1 colocalization with subcellular organelle markers in
primary cultures of hippocampal neurons and cortical astrocytes. The
panels represent confocal images of immunofluorescent staining with
anti-Ttyh1 (green) and antibodies directed against different organelle
markers (red) and cell nuclei counter-stained with DAPI (blue). Intensity correlation-based analysis was performed on Imaris soft-
ware. The colocalized areas, representing the overlay of green and red
pixels, are shown as white dots (see yellow arrows that are pointing
representative examples of white dots). The colocalization of Ttyh1-
positive dot-like structures with the Golgi apparatus marker GM130
(a), endoplasmic reticulum marker, GRP78 (b), coated pits and
vesicles marker, clathrin (c) in neurons and astrocytes. Ttyh1
colocalizes with LAMP1 (late endosome-lysosome marker) in
astrocytes (d) and does not colocalize in neurons (h). Scale bar
5 lm. The images represent single confocal sections. LAMP1
was
detected
in
neurons
(PCC,
r = 0.021;
Fig. 7h). microgliosis occur in several brain areas, including hippo-
campus [21, 23]. In hippocampal slices from rats 4 days
after status epilepticus we observed Ttyh1 immunoreac-
tivity in cells possessing star-like processes characteristic
for hypertrophic astrocytes, while in control brain we
observed immunostaining only in neurons. We confirmed
astrocytic localization of Ttyh1 protein by collocalizing it
with GFAP. We conclude that in physiological conditions
Ttyh1 is expressed mainly in neurons, but in brain pathol-
ogy it may be also be expressed by astrocytes. Previously
we described Ttyh1 immunoreactivity in the hippocampi of
animals 14 days after status epilepticus [11]. At this time-
point we observed increased neuropilar Ttyh1 immunore-
activity in inner molecular layer of dentate gyrus, but we did
not detect immunostained astrocytic profiles despite the fact
that astrogliosis in epileptic hippocampus is persistent and
can be detected at this timepoint [21]. Therefore we
hypothesize that astrocytic expression of Ttyh1 is charac-
teristic for early phase of astrogliosis. Despite the lack of
Ttyh1 expression in astrocytes in control brains, we have
observed Ttyh1 expression in astrocytes in vitro which may
indicate that these cells do not maintain a complete resting
phenotype in dissociated culture. We did not observe We observed no colocalization of Ttyh1 protein with a
number
of
other
cell
compartment-specific
markers,
including markers of early endosomes (Rab5 and APPL2),
recycling
endosomes
(Rab11),
trans-Golgi
network
(TGN46), endoplasmic reticulum membrane (calnexin) and
synaptic vesicles (synaptoporin and synaptotagmin 1). Ttyh1 Immunopositive Puncta Volume and Identity
in Cultured Neurons and Astrocytes GM130,
130-kDa cis-Golgi matrix protein; GRP78, 78-kDa glucose-regulated
protein; LAMP1, lysosomal-associated membrane protein 1 (Color
figure online) positive dot-like structures with the Golgi apparatus marker GM130
(a), endoplasmic reticulum marker, GRP78 (b), coated pits and
vesicles marker, clathrin (c) in neurons and astrocytes. Ttyh1
colocalizes with LAMP1 (late endosome-lysosome marker) in
astrocytes (d) and does not colocalize in neurons (h). Scale bar
5 lm. The images represent single confocal sections. GM130,
130-kDa cis-Golgi matrix protein; GRP78, 78-kDa glucose-regulated
protein; LAMP1, lysosomal-associated membrane protein 1 (Color
figure online) Fig. 7 Ttyh1 colocalization with subcellular organelle markers in
primary cultures of hippocampal neurons and cortical astrocytes. The
panels represent confocal images of immunofluorescent staining with
anti-Ttyh1 (green) and antibodies directed against different organelle
markers (red) and cell nuclei counter-stained with DAPI (blue). Intensity correlation-based analysis was performed on Imaris soft-
ware. The colocalized areas, representing the overlay of green and red
pixels, are shown as white dots (see yellow arrows that are pointing
representative examples of white dots). The colocalization of Ttyh1- positive dot-like structures with the Golgi apparatus marker GM130
(a), endoplasmic reticulum marker, GRP78 (b), coated pits and
vesicles marker, clathrin (c) in neurons and astrocytes. Ttyh1
colocalizes with LAMP1 (late endosome-lysosome marker) in
astrocytes (d) and does not colocalize in neurons (h). Scale bar
5 lm. The images represent single confocal sections. GM130,
130-kDa cis-Golgi matrix protein; GRP78, 78-kDa glucose-regulated
protein; LAMP1, lysosomal-associated membrane protein 1 (Color
figure online) LAMP1
was
detected
in
neurons
(PCC,
r = 0.021;
Fig. 7h). Discussion Previous in vivo studies have indicated that in the brain,
Ttyh1 protein is abundantly expressed in punctuate, dot-like
structures localized in neuropil and neuronal somata [11],
and Ttyh1 mRNA in the normal brain is expressed in neu-
rons [10]. Therefore, we concluded that Ttyh1 is predomi-
nantly a neuronal protein. However, in the present study, we
detected Ttyh1 expression in astrocytes, microglia and ol-
igodendrocytes in vitro and this result prompted use to
determine whether Ttyh1 is expressed in activated glial
cells in vivo. In amygdala stimulation model of temporal
epilepsy neurodegeneration and consequent astrogliosis and 123 123 Neurochem Res (2014) 39:2516–2526 2525 is consistent with that of a previous study characterizing
proteins that form complexes with Ttyh1 in the brain tissue
[11]. The localization of Ttyh1 in the ER has also been
demonstrated in HEK293 and HEK293T cells and in the
mouse brain [6, 8]. The presence of Ttyh1 protein in the ER
and Golgi may reflect Ttyh1 protein synthesis and post-
translational modifications. It has been shown that glyco-
sylation occurs in the Golgi and is important for the proper
function of Tweety proteins [5]. A small population of
Ttyh1-positive structures has been detected in endocytic
vesicles and lysosomes. This interesting observation sug-
gests that Ttyh1 is present in transport vesicles and may be
involved in endocytosis and/or exocytosis. prominent Ttyh1 immunoreactivity in microglia and oli-
godendrocytes in vivo, either in slices from control rats or
from stimulated animals. However we can not exclude
possibility that in other pathological conditions microglia
and oligodendrocytes express Ttyh1. We performed a scratch injury on cultured astrocytes
monolayer to mimic reactive astrogliosis in vitro. In some
cells migrating into the scratched area we observed that
Ttyh1 is concentrated at the leading edge of lammelipodia. It has been shown in HEK 293 cell line, that Ttyh1 was
similarly concentrated at the peripheral membrane of cells,
where it induced formation of filopodia and co-localized
with a5 integrin subunit [8]. Integrins are crucial for cell
migration and its attachment to extracellular matrix. Dur-
ing the process of attachment of astrocytes, some integrin
subunits can be found at the margins of the cell on
lamellipodia [24, 25], which resembles pattern of Ttyh1
immunoreactivity in migrating astrocytes. Discussion Seizures acti-
vate integrin signaling and induce a turnover in adhesive
contacts [26, 27], which hypothetically may be linked to
increased Ttyh1 expression observed during epileptogene-
sis and epilepsy [10, 28]. We observed increased Ttyh1
immunoreactivity only in small proportion of migrating
astrocytes, so it is possible that Ttyh1 is involved only in
specific phase of cell migration and adhesion. Because Ttyh1 expression is primarily restricted to the
brain [8] and Ttyh1 is highly expressed in neurons, we
verified whether Ttyh1 is present in synaptic vesicles
involved in the storage and subsequent release of neuro-
transmitters at synapses. We were unable to detect colo-
calization with the synaptic vesicles markers synaptoporin
or synaptotagmin 1 in vitro. This observation is consistent
with a previous in vivo study showing the sparse colocal-
ization of Ttyh1 protein with presynaptic markers, and it is
reasonable to conclude that the majority of Ttyh1-positive
elements are not presynaptically localized in the brain [11]. This conclusion is not in agreement with the results of
Morciano et al. [9] who co-isolated Ttyh1 protein with the
docked synaptic vesicles. However, it is possible that the
immunofluorescence used in the present study was not
sensitive enough to detect minute amounts of synaptic
vesicle-bound Ttyh1 or that Ttyh1 is present at presynaptic
sites only under specific conditions. As we have showed in our previous paper [11], primary
hippocampal neurons after 8 days in culture show abundant
expression of Ttyh1 protein with characteristic differences
in size and distribution of Ttyh1-immunopositive dots. We
could distinguish two populations of Ttyh1-immunoposi-
tive dots: a population of large and a population of small
dots. The large Ttyh1-positive dots were localized to sites
adjacent to neuronal nuclei, and the small dots were local-
ized along neurites. Here, we used older neuronal cultures
(14DIV). In comparison to the younger culture (8DIV),
older neurons showed less abundant expression and more
homogenous distribution of Ttyh1 dots of different sizes. We conclude that the number and distribution of Ttyh1
immunopositive puncta in neurons in vitro depends on the
culture age. Such unequal distribution has not been
observed in glial cells in vitro. Percent distribution of Ttyh1
immunopositive puncta volume in neurons and astrocytes
was comparable. There were differences between neurons
and astrocytes in percentage of immunopositive puncta in
specific size groups, but it is difficult to asses whether these
differences have any functional meaning. References 13. Zawadzka M, Kaminska B (2005) A novel mechanism of FK506-
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trulation and muscle degeneration encodes gelsolin-like and
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elegans and humans. Proc Natl Acad Sci USA 90:11386–11390 14. Przanowski P, Dabrowski M, Ellert-Miklaszewska A, Kloss M,
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is little information on the potential molecular function of
Ttyh1, thus additional studies are required to determine the
precise role of this protein. Acknowledgments
This research was supported through funding
from Polish National Science Centre Grant 2011/03/NZ4/00302, the
Nencki Institute and the EU-funded grant BIO-IMAGINE: BIO-
IMAGing in research INnovation and Education (EU FP7 Grant
Agreement No. 264173). The authors would like to thank Prof. Marta
Miaczynska (Laboratory of Cell Biology, International Institute of
Molecular and Cell Biology) for anti-APPL2 antibody, Dr. Mal-
gorzata Zawadzka and Dr. Bartosz Wylot (Laboratory of Molecular
Neurobiology, Nencki Institute) for assistance with oligodendrocyte
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partments, including the lumen of the endoplasmic reticu-
lum, the cis network of the Golgi apparatus, clathrin-coated
pits and vesicles and late endosomes-lysosomes. This result Open Access
This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited. 12 3 2526 Neurochem Res (2014) 39:2516–2526 References References Gall CM, Lynch G (2004) Integrins, synaptic plasticity and epi-
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27. Wu X, Reddy DS (2012) Integrins as receptor targets for neu-
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pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
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pH Dependence of
Acrylate-Derivative
Polyelectrolyte Properties Thomas Swift Abstract There are many polymers formed of acrylate monomers in existence. Here we
interrogate four commonly-used examples and study how their solution properties
are pH dependent, or how their state of ionisation can affect their solution properties. Poly(acrylic acid) and poly(methacrylic acid) are both polyelectrolytes, with ioni-
sable functional groups that make them stimuli responsive, changing their hydro-
dynamic volume. Poly(acrylamide) is a mass-produced material used in a variety
of industrial applications, often with an anionic and cationic co-monomer, which
dictates both its efficacy and impact on the environment. Poly(N-isopropyl acryl-
amide) is a thermally responsive material with applications in smart bioengineering. In solution, these materials can interact with each other due to competing hydrogen
bonding interactions. However, this interpolymer complexation is dependent on both
the ionisation, and the conformational state, of the polymers involved. This review
focuses on the results from fluorescence tagging and turbidimetric techniques. Keywords: poly(acrylic acid), poly(methacrylic acid), poly(acrylamide),
poly(N-isopropylacrylamide), stimuli responsive, interpolymer complexation,
hydrodynamic volume, solution properties 1. Introduction The chemical structure of ionisation (or
dissociation/neutralisation) is thus: RCOOH ⇋ RCOO − + H +
(1) (1) and the dissociation constant (α) can be described by the Henderson-
Hasselbalch equation and the dissociation constant (α) can be described by the Henderson-
Hasselbalch equation α = ( [X] + [ H + ] − [ OH − ] ) / [RCOOH]
(2)
pH = p K a + log {α / (1 − α) }
(3) α = ( [X] + [ H + ] − [ OH − ] ) / [RCOOH]
(2) α = ( [X] + [ H + ] − [ OH − ] ) / [RCOOH] (2) pH = p K a + log {α / (1 − α) }
(3) (3) where X is the ionising (titrating) species and pKa the dissociation constant; the
pH at which 50% of the carboxylic groups have been ionised. However, for a poly-
acid, this is a more contentious issue than studying small molecules due to each acid
group is affected by the presence of neighbouring repeat units, which thus modify
their titration behaviour. In general, the first COOH group on a polymer backbone
shows a similar pKa to a small molecule analogue. However, as the polymer chain
becomes increasingly ionised, the building negative charge constrains further
deprotonation, and the pKa value alters with increasing pH. In this behaviour,
particularly polymeric electrolytes show divergent behaviour from small molecules,
and Katchalksy and Spitnik proposed a revision to the Henderson Hasselbalch
Equation [4]. pH = p K a + n log {α / (1 − α) }
(4) (4) pH = p K a + n log {α / (1 − α) } where n is a constant dependent on the ionic strength of the solution and the
strength of the polyacid. In a stationary solution, this plot should produce a straight
line (slope n, intercept pKa). However, this is rarely observed, particularly in aque-
ous solutions, and this was the first indication researchers had that many polymeric
macromolecules undergo a conformational rearrangement on the nanoscale in
response to chemical ionization [4, 5]. Over the years, this has proven fertile
ground for research, with poly(carboxylic acid)s receiving particular attention in
the literature as they are excellent, chemically distinct, model systems [3, 4, 5–13]. 1. Introduction A common feature of the many polymer systems formed from acrylate mono-
mers is their hydrophilicity; apparent either from their increased absorbency,
wettability or increased solubility. Whilst the latter is often overlooked in materials
science, it is of vital importance to a range of industries, as a multitude of polyacry-
lates form vital components in commercial products too varied to list, but including
dispersants, adhesives, emulsifiers, lubricants, flocculants, thickeners, surfactants,
sensors, delivery agents, coatings, chromatographic phases, grouting, passivation
and many more. As of 2018, the multi-million tonne polyacrylate global market
is still rising with an annual growth greater than 6% [1]. Research over the last
20 years into controlled radical polymerisation, and copolymerisation, has provided
increased insight into the distinct properties of these materials. However, even
50 years after the initial patenting of poly(acrylic acid) [2], new discoveries about
its fundamental properties are still being made [3]. In solution many, but not all, acrylate copolymers act as polyelectrolytes, con-
taining ionisable repeat units; and thus show some form of stimuli-response to pH. 1 Acrylate Polymers for Advanced Applications The solution forces that govern these properties are the same that give function to
biological macromolecules (i.e., peptides, proteins, DNA) and so many polyelectro-
lytes have been used as simple models for these more complex systems. However,
due to their applications are so widespread and varied, it is essential to any chemist
or engineer working with these sensitive materials to acquire some understanding
of the need to control their pH. Depending on the nature of these ionisable repeat units, a polymer can be clas-
sified as a ‘weak’ or ‘strong’ polyelectrolyte, governed by the pKa of the ionisable
groups. As samples containing carboxylic acid repeat units dissociate relatively
easily, they fall into the former category. 1. Introduction However, even non-responsive systems, such as poly(acrylamide), have been found
to demonstrate responsible macromolecular behaviour in the presence of corre-
sponding polymer systems via a process of interpolymer complex formation [14]. Many polyacrylates engage in hydrogen bond driven complex interactions. The field
has proven to be extremely complex due to the multitude of competing factors that
affect this often weak, almost always labile, interface. This chapter will discuss recent advances in the study of pH dependent poly-
acrylate solution behaviour, examining our improvements in understanding of 2 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 weak polyelectrolyte systems. Critically this review limits itself to studies of linear
polymer systems, as the properties of branched, or crosslinked, macromolecules are
fundamentally different [15, 16] and warrant further, separate discussion. Figure 1.
Polyacid chemical structures. g
Polyacid chemical structures. 2. Poly(carboxylic acids) 3 Acrylate Polymers for Advanced Applications macromolecular swelling results in dramatically changed properties between the
compact/swollen polymer. Compared to this, the equivalent deprotonation and
subsequent anionic charge drive PAA to adopt an extended state with a relatively
smooth transition, with only small changes to polymer physical properties save
additional anionic potential. These conformational responses to external stimuli
can be viewed as ‘smart behaviour’ and have led to the incorporation of acrylic
acid and methacrylic acid monomers being incorporated into a range of copolymer
systems to act as triggers and solvating groups in a range of applications. Due to the increased compaction, and hydrophobicity, of its globular state,
PMAA can solubilise low molar mass organic compounds in solution [12, 17, 32],
which is a property not shared by PAA [17, 32, 33]. This is particularly evidenced by
the fluorescence emission vibrational fine structure of the aromatic label pyrene. The pyrene excited state emits multiple emission bands, and the relative intensity of
bands 1 and 3 vary with different solvents, thus when dispersed in a solution it can
give an indication of system polarity [34, 35]. For example, the I3/I1 ratio is known
to vary between 0.55 (water) and 1.7 (n-pentane) [26]. This feature has been used
in the study of many polymer systems, and commonly used by spectroscopists to
study macromolecular aggregate structures such as colloids [36], microemulsions
[37], micelles [38] and microgels [39, 40]. For example when a 10−6 M solution of
pyrene was dispersed in an aqueous solution of PAA, the I3/I1 ratio did not shift
from ≈0.55 between pH 3 and 10, identical to the ratio seen for a dispersion in
water. This reflects the fact that any interaction between the fluorophore and the
polymer does not alter the microenvironment of the label, and confirms the exis-
tence of PAA in a water-swollen conformation across the entire pH range. In PMAA
at low pH, however, a I3/I1 ratio of 1.1 is commonly observed [12], indicating the
compact hypercoiled polymer provides hydrophobic shielding from the aqueous
solvent. When the pH of pyrene/PMAA solution is increased, this ratio begins to
decrease at pH 5, indicating the conformational rearrangement of the polymer,
until at pH 6 the probe is released into the solution, returning the fluorescence
emission ratio to the state seen in both pure water and PAA. 2. Poly(carboxylic acids) The two most comprehensively studied synthetic poly(carboxylic acid)s within
the literature are poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA)
respectively. Both contain a carboxylic acid repeat unit that dissociated to form a
negatively charged anion in low pH aqueous solutions. The additional methyl group
on the methacrylic acid functional group gives PMAA a degree of amphiphilic
behaviour [17] depending on the degree of ionisation (Figure 1). p
g
g
g
This additional hydrophobicity dominates the solution properties of PMAA,
leading to the aforementioned ‘anomalous’ Henderson Hasselbalch titration behav-
iour [4, 5, 7, 9], whilst PAA has long been considered a more ‘ideal’ system [18] as
it does not undergo as dramatic a macroscopic switch. As the carboxylic acid group
can only be classed as hydrophilic when the functional monomer is protonated,
PMAA undergoes a rapid swelling as the pH is increased, becoming an entirely
hydrophobic material with increasing anionic charge along the backbone. Extensive
investigations have been carried out into its behaviour using diverse methods
and techniques: pontentiometry [4, 5, 7, 10, 19], viscometry [8, 11], Raman spec-
troscopy [20], scattering methods [21–23] and fluorescence probe interrogation
techniques [3, 24–26]. The combined research has shown that PMAA undergoes
a dramatic conformational change between pH 4 and 6, (corresponding to an α
(degree of ionisation) between 0.1 and 0.3), whilst PAA adopts a relatively smooth
swelling process in the same pH range (initiating at the same degree of ionisation). In acidic media, due to the increased hydrophobicity, PMAA adopts a globular,
contracted structure designed to minimise unfavourable interactions between the
hydrophobic backbone and side chain and the aqueous solution, whilst PAA has
been described as a random, statistical coil [6, 7, 9]. The PMAA shows significantly
increased compaction due to the hydrophobic methyl backbone [8, 13, 22, 24–29],
that has been shown to induce hypercoiling [8]. This has two net effects—increased
hydrophobic density gives it both greater solubilisation potential but at the cost of
reduced solubility and mobility. As the degree of ionisation is increased from pH 4 to 6 the PMAA anionic units
begin populating the macromolecule backbone, resulting in a transition between
pH 5 and 6 where repulsive units between these charges initiate a macroscopic
switch from the compact to the water swollen (described in multiple places as
‘rod like’ [30, 31]) state. Due to the increased initial compaction in PMAA, this Figure 1. g
Polyacid chemical structures. 2. Poly(carboxylic acids) As such differences in behaviour between low and
high molar mass PAA materials have been observed, such as stark changes in the
polymer behaviour at oil-water interfaces [27, 48]. Figure 2. Conformation of polyacid with different degrees of ionisation. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569
Figure 2. Conformation of polyacid with different degrees of ionisation. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569
Figure 2. Conformation of polyacid with different degrees of ionisation. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 Figure 2. Conformation of polyacid with different degrees of ionisation. More recent data indicates that the length scale of the chain plays a role in this
transition. For instance, whilst in 0.1 M NaCl the hydrodynamic radii of PAA scales
with molar mass [46] the conformational rearrangement of the chai non ionisation
in low ionic strength liquids only occurs above a known molar mass lower limit [3]. Current results suggest this is a salt dependent phenomenon [41, 47] and has not
been observed in PMAA (although increasing polymer size does slow the kinetics of
polymer reconfiguration [27]). As such differences in behaviour between low and
high molar mass PAA materials have been observed, such as stark changes in the
polymer behaviour at oil-water interfaces [27, 48]. 2. Poly(carboxylic acids) This experiment
confirms both the increased solubilisation potential of PMAA over PAA and also the
fact that the transition occurs over a broad pH range. p
g
However, the electrostatic potential of these polyelectrolytes cannot be so
simply described as indicating that the swollen/collapsed state is neutral/charged
as there is an evident near neighbour effect present in polymers that is not seen in
comparative small molecule systems [41]. This has been evidenced by the different
acid dissociation titration behaviours seen in PMAA when comparing different
polymer tacticities [42]. In dilute solutions intrachain interactions across the
macromolecule tend to dominate its properties—the molecule can be considered
a single long chain surrounded by counter ions, and their solution properties are
thus governed by their corresponding electrostatic interactions, which are well
described by a range of mathematical theories [43, 44]. To summarise: due to
electrostatic repulsion ionisation of acrylate polyelectrolytes occurs over a much
wider pH range than observed in the equivalent small molecule, and at the ‘stated’
pKa only a fractional ionisation of repeat units will carry a negative charge. For
example, potentiometric titrations of PAA found that, at pH 4.5 (pKa of acrylic
acid and the point at which conformational change will occur) only 1/10th of the
acrylate repeat units in the polymer will carry this fractional charge [3, 45]. The
polymer will continue to ionise up to pH 11 with no further polymer swelling
observed despite increasing electrostatic potential of the system. Therefore, it is
inappropriate to suggest that the conformational change is driven purely by elec-
trostatic potential, as if this was solely the case further rearrangements at greater
degrees of ionisation would be observed (Figure 2). 4 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569
More recent data indicates that the length scale of the chain plays a role in this
transition. For instance, whilst in 0.1 M NaCl the hydrodynamic radii of PAA scales
with molar mass [46] the conformational rearrangement of the chai non ionisation
in low ionic strength liquids only occurs above a known molar mass lower limit [3]. Current results suggest this is a salt dependent phenomenon [41, 47] and has not
been observed in PMAA (although increasing polymer size does slow the kinetics of
polymer reconfiguration [27]). Figure 3. g
Polyacylamide chemical structures. Polyacylamide chemical structures. important factor in flocculation kinetics [53]. Following the adhesion of polymer
chain of sufficient length for a loop or tail to extend into the solvent, secondary
attachment to secondary particles can occur in a process known as ‘polymer bridg-
ing’. Polymer chains adsorbed on the particle surfaces via only a few points of
attachment leave the majority of the chain in solution whilst increasing adsorption
onto the particle to saturation reduces the flocculation potential of the polymer. Bridging can be impacted by both the charge density and the molecular weight of
the polymer [55] (Figure 4). important factor in flocculation kinetics [53]. Following the adhesion of polymer
chain of sufficient length for a loop or tail to extend into the solvent, secondary
attachment to secondary particles can occur in a process known as ‘polymer bridg-
ing’. Polymer chains adsorbed on the particle surfaces via only a few points of
attachment leave the majority of the chain in solution whilst increasing adsorption
onto the particle to saturation reduces the flocculation potential of the polymer. Bridging can be impacted by both the charge density and the molecular weight of
the polymer [55] (Figure 4). p y
g
Polyacrylamide is used in a range of other scenarios including erosion con-
trol [51, 57–59], medical implants [60–63], and reduction of water seepage via
increasing aqueous viscosity to both stabilise soil and dust prevention [51]. g q
y
p
Poly(acrylamide) was one of the first polymers used to reduce soil losses in furrow
irrigation [64] and the polymer has been sold commercially for this purpose since
at least 1995 [51]. Large quantities of this material are therefore escaping into the
environment [59, 65] and a body of research is being built up regarding its effect
on the ecosystem [66]. Generally, the polymer is considered non-toxic, with most
concerns around its use arising due to its close association from the potent neu-
rotoxin monomer from which it is formed. Since Swedish researchers discovered
that acrylamide can be found in heated foodstuffs [67–72], there has been low level
public concern about the use of polyacrylamides in a range of industries. However, studies of polyelectrolyte flocculants of all types have been carried
out and consistently poly(acrylamide) is identified as being the primary ‘toxicant’
[66]. 3. Poly(acrylamides) Not all polyacrylates demonstrate electrolyte properties, and one of the most
common non-ionisable acrylate materials produced today is acrylamide copolymers. This chapter concerns itself specifically with two specific materials of particular
interest with divergent properties, although there are a range of further examples. These polymers are poly(acrylamide) (PAM) and the hydrophobically modified
poly(N-isopropylacrylamide) (PNIPAM), whose properties are driven by the
additional hydrophobic groups along the polymer side chain. As such one is widely
used as an inexpensive, mass-market commodity whilst the other is a very heavily
investigated [40, 49], high value material with particular interest in its biomedical
applications [50] (Figure 3). pp
g
Random copolymers of acrylamide (both anionic, cationic and neutrally
charged) have been extensively used in the water industry for many years [51–54]. They are extensively employed to remove dissolved organic matter (DOM) for
water clarification purposes. Flocculation of fine particles can occur via several
mechanisms including polymer bridging, charge neutralisation, polymer-particle
complex formation and depletion flocculation; often a combination of several of
these processes [55]. Binding in poly(acrylamide) is primarily by hydrogen bonding
[56], although copolymerised sections may also assist with electrostatic interac-
tion or ion binding. In a sufficiently long polymer chain, there are many potential
binding sites, and once sufficient repeat units along a single polymer chain have
adhered to a particle surface, the adsorption is often considered irreversible despite
the fact each individual binding site is acting in an equilibrium [53]. Once a polymer
has adhered to a particle, it can be divided into three segments: trains (adhered to
the particle surface), loops (that extend from the surface) and tails (which project
into the solution). The speed by which the polymer shifts is difficult to assess but an 5 Acrylate Polymers for Advanced Applications Figure 3. Polyacylamide chemical structures. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 Testing of poly(acrylamide) interaction with the gill tissues of several aquatic
species including fish [66, 73, 75–77], crustaceans [66, 73, 77], algae [66, 77] and
insects [77] have been carried out. In many studies of adult fish, the anionic and
non-ionic form of the poly(acrylamide) cause only low levels of damage to the fish,
with effects increasing at higher concentrations [75]. However, sustained exposure
of organisms over a 40 day period has shown that low levels of these polymers
are intrinsically toxic to almost all aquatic fauna [73]. Environmental exposure is
unlikely to be sustained over long periods due to the polymer desorption to organic
matter but few studies have been undertaken into the metabolic rate at which
they are removed from living organisms. Even in tests where fish survival was
not impacted, the general activity and swimming behaviour of the fish were sub
normal [76]. Conversely the cationic form of the polymer is known to be far more
toxic, causing pathological issues at sub mg ml−1 concentrations, as the polymer
builds up on negatively charged gill surfaces [75, 77]. Reduced gill functional-
ity impairs oxygen uptake in the fish and results in death. Further studies have
shown that polyelectrolytes can cause adverse changes in fish organ cells (liver and
kidneys) [73], decrease animal locomotion and greatly increase respiratory rate. This suggests that the presence of dissolved flocculants may not be lethally toxic
but suggests it is capable of causing the fish elevated levels of distress. In inverte-
brates, their mechanical action was reduced as polyelectrolytes adsorbed onto their
body surfaces, reducing their vital functions [73], and again the cationic form of
the polymer is far more toxic than the anionic form. [77] In microcosms tests, it
has been shown that high polyelectrolyte concentration can reduce algal growth
[66, 77]. This in turn can increase the potential toxicity of the polymer as the algae
acts as a neutralising agent towards the polymer. To algae, even the anionic and
non-ionic polymer is toxic, negatively affecting both cell growth and O2 produc-
tion [73]. It has been observed that addition of combinations of both anionic and
cationic polymer can reduce toxicity [77] and several patents have been issued
suggesting that anionic polymers can be used to detoxify cationic polyelectrolytes
[78, 79]. Figure 3. Within much of the poly(acrylamide) literature, there has been a lot of
emphasis placed on the toxicity of the monomer, resulting in studies discounting
the effects of the polymer and only focusing on residual monomer spread [65]. However, some studies have shown that poly(acrylamide) is unlikely to degrade
into residual monomers, or any other toxic compounds [73], and this has only been
observed under specific harsh conditions [74]. Therefore, a complete study of the
environmental impact of these polymers should include the raw polymeric product. Figure 4. Increasing polymer adsorption to surfaces. Figure 4. g
4
Increasing polymer adsorption to surfaces. 6 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 Figure 5.
T
l h Figure 5. Typical thermoresponsive properties of polymers [83]: (temperature (T) vs. volume fraction (Ø)). The LCST of PNIPAM can be affected by the addition of hydrophobic or hydro-
philic end groups [84], or the molecular weight and concentration of the sample
[85]. Due to the LCST is reasonably close to body temperature, there has been much
work to manipulate PNIPAM to act as a drug-delivery agent or trigger or apply it in
other bio-engineering circumstances [86, 87]. pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 In conclusion, the discrepancy between anionic and cationic polymers in
regard to aquatic toxicity must be considered in the application of these polymers
[75, 77]. The cationic form of the polymer is regarded as generally more toxic but
the anionic form has also been shown to cause chronic, sub-lethal responses even at
low concentrations [66]. l h
h
h
l
l
d
d
l
d Although it has some larger applications, PNIPAM is not produced or utilised in
such great quantities. It is mainly of interest due to a thermally induced conforma-
tion the polymer exhibits at 32°C, caused by the hydrophobic isopropyl groups [49]. This ‘smart’ response has led to great interest in the polymer, both to understand
its properties and apply them in a range of fields, specifically in Bioengineering
[50]. In essence, the polymer has a lower critical solution temperature (LCST), a
conformational change that occurs via a two stage process. Firstly, the polymer has
an intramolecular collapse, where individual chains contract in upon themselves
as they break hydrogen bonds with the aqueous solution, followed by a second-
ary event of intramolecular aggregation of the collapsed coils [80]. This event is
triggered by the increasing entropic cost at high temperature of the restricted water
that solubilises the dissolved polymer chains below the LCST. The event has some
hysteresis between heating and cooling radii of gyration [81], governed by two
intermediate states that give PNIPAM four potential conformations: globule, molten
globule crumpled coil and coil [82]. During the collapse the globular state dispenses
approximately 34% of the water molecules [82], meaning that although this is a
desolvation event leading to an insoluble material, collapsed PNIPAM can never be
described as a hydrophobic system (Figure 5) [49]. 7 7 Acrylate Polymers for Advanced Applications Figure 5. Typical thermoresponsive properties of polymers [83]: (temperature (T) vs. volume fraction (Ø)). 4. Acrylate interpolymer complexes Interactions between multiple polymers in a formulation are almost inevitable,
and there has been plenty of studies of specific driving factors undertaken over
the last 50 years to build a strong picture of inter-polymer interactions. This phase
separation phenomena is observed in even the most dilute solutions, as it is driven
by a mixture of electrostatic, hydrogen bonding and hydrophobic interactions, all
dependent on pH, salt concentration and temperature [88, 89]. To our knowledge
this type of complex was first patented in 1966 [90], with much of the following
fundamental measurements carried out over the following decades [14, 91–94]. Since then, the system has been described as a laddered sequences of bonds between
the molecules, occasionally interrupted with loop defects [95], an evolution similar
to the model previously described of polymer adhesion to surfaces. This theory
originally posed that the polymers will form rigid, static structures due to repeated
hydrogen-bonding across molecules. More recent studies have put less emphasis
on the polymer rigidity and have given an alternative description of these repeated
labile interactions more as ribbons (i.e., two flexible materials that can slide over
each other). The interactions between PAA and PAM are one of the more studied systems of
interpolymer complex formation (IPC) [93, 96–101], and in both solution and solid
state the interaction has been shown to be pH dependant [14, 96]. Mixed solutions
of PAA and PAM form a turbid solution that precipitates when cooled [97]. This
phase separation follows the formation of complexes between PAA and PAM that
varies in structure depending on the concentration, medium and the ionisation
constant [97]. For complexes between PAA and a proton-acceptor polymer it has
been shown that IPCs will only form below a critical value of pH (pHcrit) [14, 99],
the structure dependent point above which any partial neutralisation of the poly-
acid inhibits complex formation [14, 100]. Early work within this field required high molecular weight materials to detect
complex formation [99, 102], however, modern instrumentation has facilitated
detection of smaller complexes down at the parts per million loading level [103]. 4. Acrylate interpolymer complexes The structure of the resultant IPC (whether in a gel or a compact solvated complex) 8 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 depends on the relative molecular weight of complexing partners, [104] but as this
is a multivalent effect of repeated binding sites, larger molecular weight materials
demonstrate stronger interactions. Furthermore, it has been indicated that very
large molecular weight polyacids have been seen to raise pHcrit [93]. g
g
p y
p
When dissolved in high ionisation solutions, both polymers have rapid seg-
mental motion, existing as random polymeric coils. If the solution ionisation is
decreased, this deprotonates the acidic polyelectrolytes and reduces its affinity for
inter-polymer complexation. This occurs as can now form both intramolecular
H-bonds internally across the chain backbone or intermolecularly forming H-bonds
with other polymers [105], leading to a rigid polymer mixture with restricted chain
motions. PAA forms stronger complexes to PAM than some other polymers
(i.e., poly(ethylene oxide) or poly(vinyl acetate)) due to additional ion-dipole inter-
action of the partially protonated amide groups and the C═O dipoles of PAA [106]. In ambient conditions the peak aqueous interaction between PAA and PAM occurs
≈ pH 2.69 [107] but this is affected by many environmental factors including tem-
perature [89, 101], ionic strength [93, 107, 108] and the addition of inorganic binders
[99]. The complex polymer/polymer/solvent ratio of interactions is temperature
sensitive causing PAM-PAA copolymers to become upper-critical solution tempera-
ture materials (an inversion of the LCST seen in PNIPAM where they become only
soluble above a specific temperature [87]). These combined external factors dete-
riorate the thermodynamic quality of the solvent, strengthening polymer-polymer
interactions by weakening polymer-water solvation [109]. However, below pHcrit,
only small portions of the PAM form into ‘multimacroion clusters’, indicating that
in an equivalent system with 1:1 acid/acrylamide repeat units, a large percentage of
acrylamide will be free in solution unbound to PAA [110]. This was the first result
of several which have cast doubt on the ladder model, and computational modelling
software of polymer/polymer ionic interactions has proposed a range of complexing
structures ranging from ladders to scrambled egg structures [111]. Figure 6. Figure 6. Three conformational states of PAA-PAM in mixed solutions. along the chain is deprotonated whilst more hydrophobically modified polymers
are more tolerant to partial ionisation when complexing with PAA. Computational
modelling of the solvation energy of each repeat unit shows a clear correlation
between solvation potential and pHcrit [14]. along the chain is deprotonated whilst more hydrophobically modified polymers
are more tolerant to partial ionisation when complexing with PAA. Computational
modelling of the solvation energy of each repeat unit shows a clear correlation
between solvation potential and pHcrit [14]. Clearly the polyacid dictates the potential of IPC with receptive polymers
[96], and in this mind, it is worth revisiting the PAA-PAM IPC structure. As the
pKa of the PAA is higher than pHcrit, it will be ‘non-swollen’ before it encounters a
complexing partner. Early literature in the subject, comparing the supposed ‘non-
response’ of PAA compared to PMAA cited, its only-slight alteration to solution
viscosity and inability to solubilise hydrophobic dyes as evidence it had no confor-
mational response. However, more recent studies with more sensitive techniques
have shown that this is not the case and PAA does indeed go through a lesser
swelling-contraction event. As such, it is proposed that the PAA can exist in three
potential conformations in the presence of a polymeric bonding partner (Figure 6). h
d
h
h l
l We suggest that compacted PAA has no entropic or enthalpic reason to uncoil
or swell prior to complexation. Given the combined evidence from two separate
sources that (1) most PAM chains are not binding to partners and (2) PAA chains do
not swell further apart on PAM binding (in fact there is some evidence of contrac-
tion), it seems reasonable to propose that the PAA-PAM complex is not amorphous
in nature, and certainly not an extended ribbon/ladder structure. 4. Acrylate interpolymer complexes Further experi-
mental evidence has shown that a PAA coil does not unwind or swell on addition
of a PAM polymer but potentially contracts into a smaller co-globule [112], and
an explanation for this can be found when considering the difference between the
pHcrit of IPC formation and pKa of PAA conformational change. p
p
g
Other acrylate materials, such as PNIPAM, demonstrate similar responses to
polyacids, and exhibit their own IPC potential [113]. Whilst PAM-PAA interactions
are dominant at lower temperatures, PAA-PNIPAM show increased interactions
at high temperatures, indicating that the complex formation is driven by hydro-
phobic interactions not seen in the base acrylamide structure [114]. Studies using
dissolved pyrene indicated these lead to stronger interactions between PAA and
PNIPAM than PAA-PAM [115, 116]. Furthermore, the hydrophobic isopropyl side
chain causes PNIPAM to alter its response to ionic strength. Whereas PAM-PAA
complexes are strengthened by increasing ionic strength, PNIPAM complexes show
decreased critical pH reducing their bond forming potential [109]. As the initial
critical pH for IPC formation was larger than 3, Khutoryanskiy theorised that the
increasing ionic strength partially dissociates the polyacid. As only non-ionised
carboxylic groups are able to form hydrogen bonds, this impedes IPC formation and
reduces the pKa. For PAA-PAM, pHcrit was found to be 2.7, whilst for increasing hydrophobic
additions to the acrylamide unit (poly(ethyl acrylamide), poly(dimethyl acryl-
amide), poly(diethyl acrylamide), the pHcrit was found to increase to 3, 4, 5 respec-
tively [14]. It is interesting to note that the most hydrophilic acrylamide polymers
(including a hydrophilic-functionalised poly(hydroxyl ethyl acrylamide)) show
lower pHcrit, indicating that their IPC with the polyacid are less tolerant of deprot-
onation. The PAA-PAM complex appears to separate when the first acid repeat unit 9 Acrylate Polymers for Advanced Applications Figure 6. Three conformational states of PAA-PAM in mixed solutions. 5. Conclusion This chapter reviews some of the recent developments in polyacrylate properties
and interactions, and delves deeply into their industrial applications to provide both
further context and understanding. During the early study many assumptions were
made due to the difficulty to analyse these large macromolecules, particularly in dilute
solutions, and our understanding of these systems has slowly evolved as more advanced
technology with greater sensitivity has facilitated deeper interrogation of these systems
[117]. This chapter only touches on a few choice themes of polymer-responsiveness and
ignored many of the more challenging aspects of the field. The state of ionisation of all
of these polymers has clearly been shown to have an effect on their solution properties,
and although the field is still under development after several decades of work, common
themes can be seen across the subject dictating macromolecular conformational changes. 10 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 pH Dependence of Acrylate-Derivative Polyelectrolyte Properties
DOI: http://dx.doi.org/10.5772/intechopen.82569 Acknowledgements This book chapter is both an update, summation of, and substantial revision
to my Ph.D., work area carried out at the University of Sheffield, originally under
Dr. Linda Swanson, who has published extensively on the photophysical analytical
techniques described in this paper. For further reference on the application of those
techniques in these systems, particularly in the study of polyelectrolytes, please see
her book chapter ‘Optical Properties of Polyelectrolytes’ [26]. Conflict of interest There are no conflicts of interest to declare. Abbreviations Abbreviations
IPC
interpolymer complex formation
DNA
deoxyribonucleic acid
DOM
dissolved organic matter
PAA
poly(acrylic acid)
PAM
poly(acrylamide)
PMAA
poly(methacrylic acid)
PNIPAM
poly(N-isopropylacrylamide) Thanks Written following the arrival of, and dedicated to, Jonathan Swift, born in 2017. Author details 11 Acrylate Polymers for Advanced Applications [17] Barone G, Crescenzi V, Liquori AM,
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Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity of Viking and Jakmania’s Supporters in the Indonesian Football Industry
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Suggested Citation: Wibowo, Andry. (2018). “Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity
of Viking and Jakmania’s Supporters in the Indonesian Football Industry” in MIMBAR PENDIDIKAN: Jurnal Indonesia untuk
Kajian Pendidikan, Volume 3(2), September, pp.149-168. Bandung, Indonesia: UPI [Indonesia University of Education] Press,
ISSN 2527-3868 (print) and 2503-457X (online). Pop Culture, Identity Conflict, and Chaos:
Studies on the Culture and Identity of Viking
and Jakmania’s Supporters in the Indonesian
Football Industry ABSTRACT: Football is most popular sport in the world community. Football is also one form of pop culture
itself, where football is no longer interpreted as a game or a way to exercise alone, but also become something
that can be interpreted as a culture that bind various communities in the world. This research, by using the
qualitative approach with case studies on conflict, will explore deeply about how social identity is present
in football, especially in the context of football supporters in Indonesia as part of the development of the
football industry in the world. This study has a focus on researching the phenomenon of cultural and local
identity in the “Viking” of PERSIB (Bandung Indonesian Football Association) and “Jakmania” of PERSIJA
(Jakarta Indonesian Football Association) supporter groups in football matches that are played in national
competitions. The formation of identity transforming become an identity conflict in a crowd of football
supporters to become the hallmark of football as part of the pop culture in Indonesia. KEY WORD: Pop Culture; Conflict; Identity; Crowds; Football in Indonesia. KEY WORD: Pop Culture; Conflict; Identity; Crowds; Football in Indonesia. ABSTRAKSI: “Budaya Pop, Konflik Identitas, dan Kerusuhan: Studi tentang Budaya dan Identitas Suporter
Viking dan Jakmania dalam Industri Sepakbola Indonesia”. Sepakbola merupakan olahraga yang sangat
populer pada masyarakat dunia. Sepakbola adalah juga salah satu bentuk dari budaya pop itu sendiri, dimana
sepakbola tidak lagi dimaknai sebagai sebauh permainan atau cara untuk berolahraga saja, melainkan menjadi
sesuatu yang dapat dimaknai sebagai sebuah kebudayaan yang mengikat beragam masyarakat di dunia. Penelitian ini, dengan menggunakan pendekatan kualitatif tentang studi kasus konflik, akan mengkaji secara
mendalam mengenai bagiamana pembentutakan identitas sosial hadir dalam sepakbola, terutama dalam konteks
suporter sepakbola di Indonesia sebagai bagian dari perkembangan industri sepakbola di dunia. Penelitian
ini memfokuskan kajian tentang fenomena identitas budaya dan lokalitas pada kelompok suporter “Viking”
dari PERSIB (Persatuan Sepakbola Indonesia Bandung) dan “Jakmania” dari PERSIJA (Persatuan Sepakbola
Indonesia Jakarta) dalam pertandingan sepakbola yang berlangsung melalui kompetisi nasional. Pembentukan
identitas hingga kemudian bertansformasi menjadi suatu konflik identitas dalam kerumunan berbentuk aksi
kerusuhan dan konflik menjadi ciri khas sepakbola sebagai bagian dari budaya pop di Indonesia. KATA KUNCI: Budaya Pop; Konflik; Identitas; Kerumunan; Sepakbola di Indonesia. About the Author: Andry Wibowo, M.H., M.Si. is a Student of Doctoral Program at the STIK-PTIK (Indonesian Police
Science Institute – Indonesian Police Science University), Jalan Tirtayasa Raya No.6, Kebayoran Baru, Jakarta Selatan, Jakarta,
Indonesia. About the Author: Andry Wibowo, M.H., M.Si. is a Student of Doctoral Program at the STIK-PTIK (Indonesian Police
Science Institute – Indonesian Police Science University), Jalan Tirtayasa Raya No.6, Kebayoran Baru, Jakarta Selatan, Jakarta,
Indonesia. E-mail: wibowoandry1993@gmail.coml MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
V l
3(2) S
b
2018 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
V l
3(2) S
b
2018 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 esia untuk Kajian Pendidikan,
Volume 3(2), September 2018 © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik Article Timeline: Accepted (July 1, 2018); Revised (August 17, 2018); and Published (September 30, 2018). INTRODUCTION part of the life in the world community. This condition encourages the form of
industrialization of football in the world,
which can be illustrated by an indication of
the massive amount of jersey sales (clothing
used in football) by sports equipment
industries, such as Nike and Adidas. This
example can indicate a massive football’s
industrialization by using an indication of
the role of media in viewing football as a
new culture that is regarded as something
that has a very high capital value. The
implications for the emergence of football
industry is like media, such as Bein Sport,
Fox Sport, and ESPN or Entertainment and
Sports Programming Network (Blair, 2011;
Clark, 2011; Billings & Ruihley, 2013;
Miles, 2015; and Eyong, 2018). The concept of pop culture is something
interesting to be studied since 1950s
(Rushkoff, 1994; Sifaki, 2003; and Loyo,
2012). The uniqueness in researching about
pop culture is driven by the emergence of
our awareness that the world has shown
the reality of social life that depends on the
cultures that are the way of life for the world
community (Swingewood, 1977 and 1998;
and Borg et al., 2005). The phenomenon
of cultural industrialization encourages
academics to look back on pop culture
as part of the life of modern society. It is
characterized by the inclusive nature of
culture in a social class, which transforming
into an exclusive one without distinction
of social class (Kellner, 2003; Adorno &
Bernstein, 2004; Borg et al., 2005; and
Ritzer, 2009). Football as a phenomenon of pop culture
in Indonesia can also be seen from the great
enthusiasm of society towards football in
Indonesia. Indonesia, as a country with
the fourth largest population in the world,
also ogled by the world football industry
as one of the countries that have the largest
market for football industry. The indication
can be seen from the large number of
football spectators in Indonesia, who watch
through the medium of technology, such
as television and internet; or who watch
directly in various stadiums to watch the
local or international competition (Clark,
2011; Jones, 2015; Gadiraju, 2016; and
Duerden, 2017). A simple example of the existence
of pop culture in today’s society can be
seen by the life of urban society today
is highly dependent on gadgets, such as
smartphones that no longer serve as a
means of communication but as part of a
lifestyle. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik
150 Pop Culture, Identity Conflict, and Chaos:
Studies on the Culture and Identity of Viking
and Jakmania’s Supporters in the Indonesian
Football Industry E-mail: wibowoandry1993@gmail.coml Suggested Citation: Wibowo, Andry. (2018). “Pop Culture, Identity Conflict, and Chaos: Studies on the Culture and Identity
of Viking and Jakmania’s Supporters in the Indonesian Football Industry” in MIMBAR PENDIDIKAN: Jurnal Indonesia untuk
Kajian Pendidikan, Volume 3(2), September, pp.149-168. Bandung, Indonesia: UPI [Indonesia University of Education] Press,
ISSN 2527-3868 (print) and 2503-457X (online). 149 149 149 © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos INTRODUCTION In the world of sports, there is also
a phenomenon similar to the phenomenon
of the smartphone in urban society. Football,
which at the beginning of the 20th century,
has a function not only as a game as well
and as a way to exercise for the community,
but football can be a part of bussiness and
entertainment instruments (Sullivan, 1979;
Blair, 2011; Billings & Ruihley, 2013;
and Jones, 2015). The meaning of football
has changed a lot in modern times, where
football has become part of lifestyle and
even a determinant value in society. Football in Indonesia is also used as a
pop culture industry. This is marked by the
presence of national competitions, such as Go-
Jek Traveloka Competition in 2017 ago. The
warm atmosphere of the local competition also
affects the enthusiasm of the public towards
the effort to support the football club based on
regional identity, for example Jakarta residents
tend to favor and support the PERSIJA
(Persatuan Sepakbola Indonesia Jakarta or
Jakarta Indonesian Football Association)’s
Club; West Java residents or Sundanese ethnic
groups tend to support the club of PERSIB Football is most popular sport in the
world community. Almost two-thirds of the
world’s population witnessed the World Cup
Championship’s event through the help of
technological developments, such as radio,
television, and internet. This illustrates that
football is no longer a way for doing body’s
exercise but also become an important 150 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 (Persatuan Sepakbola Indonesia Bandung or
Bandung Indonesian Football Association);
Minangkabau ethnic community or West
Sumatrans tend to support the SPFC (Semen
Padang Football Club); and so forth (Ayati,
2010; Hanifan & Herfiyana, 2014; Kristanto,
2016; Amirio, 2017; Sufiyanto, 2017; and
Wirayudha, 2018). also capitalizes on the crowd of football
supporters and their behavior inside and
outside the game. The identity of Vikings
and Jakmanias, for example, are two very
strong identity supporters in Indonesia, who
often clash these two groups in football
matches that have caused riots, security
disturbances, and public order disturbances. The uniqueness of the Viking and Jakmania
as a crowd identity is the identity of values,
which formed from the culture of football
and cultural identities which is produced
based on regional similarity, the value of
the struggle, and so forth (Berlonghi, 1995;
Kari, 2009; Conigliaro et al., 2015; Huda,
2016; Hadi, 2017; and Wirayudha, 2018). INTRODUCTION The combination of pop culture and
football industry in Indonesia is also
inseparable with the efforts to capitalize
on the issue of identity in the aspect of
football. Regional or ethnic distinctiveness
is attached to the football club itself. For
examples, fans club named themselves
to make self-identification of the crowds
with the same intention of supporting their
respective football clubs. Identification of
this identity can be exemplified by naming
Viking supporters for club supporters
of PERSIB; Jakmania for supporters of
PERSIJA; and so on (Brown, 1998; Dobson
& Goddard, 2001; Gibbons, 2012; Miles,
2015; Hadi, 2017; and Wirayudha, 2018). This study will look at the conditions
of Viking and Jakmania as an identity and
as a culture characterized by Viking and
Jakmania as a part of pop culture and as
a group that produces a “riot” identity in
Indonesia’s football industry. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik RESEARCH METHODS y
)
Identity is also highly visible when
football matches based on jersey use,
attribute use, and their own behavior. The
use of jersey as a symbol of identity has
always been a major determinant of self-
identification, which later became the
identity identification among supporters. Orange color, for example, to be the basic
color for Jakmania’s identity, blue color
for Viking, and so forth. The use of flag
attributes is also a form that symbolizes the
existence of the supporter group, especially
when the game is held. Behavior can also
be a symbol that depicts identity within a
crowd of football supporters. This can be
identified in the form of riots occurring in
the identity clashes between supporters in
football games (Ritzer, 2009; Fuller, 2017;
Hadi, 2017; and Wirayudha, 2018). Data collection process in this study is
using qualitative approach with case studies
on conflict between Viking and Jakmania
supporters (Creswell, 1998; Auerbach &
Silverstein, 2003; Yazid, 2015; and Wibowo,
2017). The conflict took place inside and
outside the PERSIB (Persatuan Sepakbola
Indonesia Bandung or Bandung Indonesian
Football Association) and PERSIJA
(Persatuan Sepakbola Indonesia Jakarta
or Jakarta Indonesian Football Association)
football matches that took place over the last
five years. The data collected are data generated
through in-depth interviews, direct
observation, focus group discussions,
expert panel discussions, and document
studies over the past five years. The data
generated will be analyzed with some
references, and information from Police
institution in Indonesia (Creswell, 1998;
Auerbach & Silverstein, 2003; Yazid, 2015;
and Wibowo, 2017). Riots in the football crowd became one
of the hallmarks of football in Indonesia. The football industry in Indonesia not
only shows football as a pop culture, but 151 151 ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos Jakmania and Viking, as a supporter
group, are a synthetic identity that have a
particular sub-culture. Sub-cultures of good
and bad supporters as a sub-culture identity
are found in Jakmania and Viking in their
daily behavior. The spirit of regionalism,
through the principles of conquering and
awakening, also marks as the sub-culture
of Jakmania and Viking. Both supporters
also use violent symbols on the club’s
symbolic status or supporters. FINDINGS AND DISCUSSION The Establishment of Viking and
Jakmania Identities as a Crowded
Football Supporters. As a synthetic identity
(Hoofnagle, 2007; ID Analytics, 2014; and
Voris, 2015), Viking is a metamorphosis of
Bobotoh, which became a common icon of
PERSIB (Persatuan Sepakbola Indonesia
Bandung or Bandung Indonesian Football
Association) supporters. Bobotoh is a
symbol of the continuity of the greatness
of the Padjadjaran kingdom in West Java
at this time (Ryza, 2011; Kristanto, 2016;
Hadi, 2017; and Kaka, 2017). The naming
of Viking is based on Ayi Beutik Suparman’s
imagination, on the values that existed in
the Vikings who once lived in Scandinavia
(Ryza, 2011; Hanifan & Herfiyana, 2014;
and Haryadi, 2014). Meanwhile, Jakmania is a popular
name used on the basis of intention to
attract as many members as the term “Jak”
symbolizes the identity of the names
“Jakarta” and “mania” as maniac. This
statement is supported by interviews
conducted on Gugun Gondrong and former
Governor of DKI (Daerah Khusus Ibukota
or Special Capital Region) Jakarta, Sutiyoso
(Ayati, 2010; Sufiyanto, 2017; Wirayudha,
2018; interview with Respondent C,
17/7/2017; and interview with Respondent
D, 24/7/2017). ;
,
)
Jakmania and Viking have an exploratory
and expansive character. This character
is formed naturally and fabricated in
times of growth Jakmania and Viking as a
community of football’s biggest supporters
in Indonesia. The growth of character in
Viking and Jakmania is also in line with
the development of football as an industry. This is coherent with the number of football
fans as the most popular sport in Indonesia. Family, peers, mass media, social media,
and club achievements have also been
influential as a trigger for the growing
number of Jakmania and Viking’s members. The expansion culture contained in the
Vikings and Jakmanias can be seen with
the pattern of expansion of members in one
region, and one ethnic group, which evolves
into different regions and to various ethnic
groups. In addition, its cultural expansion
is also influenced by the role of social
media as part of the information revolution,
which also enriches both positive and
negative impact in Jakmania and Viking’s
membership (Brown, 1998; Castells, 2000;
Ayati, 2010; and Eko, 2012).f Jakmania has a close relationship with
the character of poor people, who live in
Jakarta. RESEARCH METHODS PERSIJA
(Persatuan Sepakbola Indonesia Jakarta
or Jakarta Indonesian Football Association)
uses the meaning of Macan Kemayoran
(Kemayoran Tiger) as an imaginary value
to the supporters; and PERSIB uses the
Maung Bandung (Bandung Tiger) or Viking
imagination as an wild’s value on their
identity (Brown, 1998; Castells, 2000; Ayati,
2010; and Eko, 2012). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION Several factors that influencing
the dynamics of Jakmania’s identity are:
firstly, Jakarta as a city with multicultural
population, which causes the value of being
adopted would not could be in uniform. Secondly, the formation of impure Jakmania
comes from lower society as marked
by the intervention of the management
and Governor Sutiyoso in the Jakmania
organization (Brown, 1998; Huda, 2016;
Sufiyanto, 2017; and Wirayudha, 2018). The condition which influencing the
value in the organic process in the Viking
body is their condition in their community. The community will continue to hold
the value of culture and togetherness in
establishing various activities. Together,
PERSIB in West Java community put
together and form a new community entity. As a cultural binder, this condition is
also vulnerable to polluted inappropriate
notions, such as enmity with Jakmania that
continues to be produced without finding a
way out, and conflict without ending which
is characteristic of a cultural-based, and
conflict based on history and identity (Eko,
2012; Kristanto, 2016; and Wirayudha, 2018). In the perspective of the second relation,
the Vikings against Jakmanias, or otherwise,
have dual theoretical perspectives, namely:
Jakmania and Viking in the process of self-
looking can be regarded as an entity that has
solidity and solidarity, so transformed into
a big power (Canetti, 1973; Kennedy, 2014;
Kristanto, 2016; and Wirayudha, 2018). Jakmania in the process of viewing Viking
or reciprocally is regarded as a rivalry of
Jakmania and Viking, where they regard
each other as something monstrous (Le Bon,
2002; Kristanto, 2016; Fuller, 2017; and
Wirayudha, 2018). f Jakmania has a more dynamic group
identity. Several factors that influencing
the dynamics of Jakmania’s identity are:
firstly, Jakarta as a city with multicultural
population, which causes the value of being
adopted would not could be in uniform. Secondly, the formation of impure Jakmania
comes from lower society as marked
by the intervention of the management
and Governor Sutiyoso in the Jakmania
organization (Brown, 1998; Huda, 2016;
Sufiyanto, 2017; and Wirayudha, 2018). Jakmania and Viking have different
historical paths. Viking is a PERSIB
supporters who have been born first with a
background of fanaticism for PERSIB. The
love of the tribal base in West Java further
strengthens in the Viking itself, which
implies the emergence of a “hooliganistic”
infiltration that begins to enter into the
Viking self-worth. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION This identity is represented in the
form of Jakmania as illustrated by Clarke
McPhail (1997), which shows that members
of Jakmania tend to be lower class society
and people living in Jakarta (cf McPhail,
1997; Sufiyanto, 2017; and Wirayudha,
2018). This view is reinforced by
sociologists, such as George Ritzer (2009)
and Kenny M. John et al. (2001), who claim
that Jakmania is part of the symbol of the
lower middle class who living in Jakarta (cf
Ritzer, 2009; John et al., 2001; Fuller, 2017;
Sufiyanto, 2017; and Wirayudha, 2018). Theory of crowds offered by Elias Theory of crowds offered by Elias © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik
2 152 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 Canetti (1973); Gustav Le Bon (2002); and
others can look Jakmania and Viking as the
same groups, which have cultural properties
that are characterized by the character of
expansiveness in reaching the goal (Canetti,
1973; Le Bon, 2002; and Ritzer, 2009). Jakmania and Viking have similarities
in the value of irrational violence based
on criminogenic factors that are viewed
through the perspective of police officers in
Indonesia (Oliver ed., 2000; Le Bon, 2002;
Kari, 2009; Ayati, 2010; and Eko, 2012). conditions of Viking behavior until now. The
solidarity of the power of the mass grew. The implication of this is indirectly the
frequent politicians in West Java, who are
using Vikings in their campaign to attract
numbers of vote (Stott & Pearson, 2007;
Haryadi, 2014; Kristanto, 2016; Fuller,
2017; and Wirayudha, 2018).l 2017; and Wirayudha, 2018). The condition which influencing the
value in the organic process in the Viking
body is their condition in their community. The community will continue to hold
the value of culture and togetherness in
establishing various activities. Together,
PERSIB in West Java community put
together and form a new community entity. As a cultural binder, this condition is
also vulnerable to polluted inappropriate
notions, such as enmity with Jakmania that
continues to be produced without finding a
way out, and conflict without ending which
is characteristic of a cultural-based, and
conflict based on history and identity (Eko,
2012; Kristanto, 2016; and Wirayudha, 2018). Jakmania has a more dynamic group
identity. FINDINGS AND DISCUSSION This has greatly affected
their behavior, while in the stadium to
spectating matches (Conigliaro et al., 2015;
McLean, 2015; Kristanto, 2016; Rogers,
Woodhouse & Politowski, 2016; and
Wirayudha, 2018). Ethnic originality and birth places also
do not affect the internal segregation of
Jakmania’s membership identity. This
is shown by the existence of a Jakmania
named Bung Ferry, which is a figure
who admired Jakmania, but he was
born in Bandung, and originally was the
Commandos, a Pelita Jaya’s Fans Club in
West Java (Ismunanto, 2017; Sufiyanto,
2017; and Wirayudha, 2018). Hooliganistic conditions are always
in reproduction by a figure who admired
as a protector, i.e. Ayi Beutik Suparman. It further reinforces the value that is in
the Vikings with its hooliganism practices
that are considered as proud/cool/militant
people. The culture continues to shape the The value of militancy and holiganism 153 ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos adopted by Viking is more rooted than
Jakmania. In conflicts among supoorters,
Viking will be hostile to Jakmania with
more descending traits. This condition is
evidenced by the explanation of one of
the Vikings that use the nuanced attribute
of racist: “Let this Enmity Stay Eternal”
and “Jak is Dog” (Ayati, 2010; Eko, 2012;
Huda, 2016; and Rahayu, 2016). According
to a Viking’s informant, the use of the
attribute only applies what Ayi Beutik
Suparman used to say that the power of
Padjadjaran kingdom as a priority seed. Ayi Beutik Suparman is considered by
Viking’s members as a respected figure
and as a binding cultural identity other
than the Sundanese from the majority of
the population of West Java (Hanifan &
Herfiyana, 2014; Haryadi, 2014; Hadi,
2017; and interview with Respondent A,
3/7/2017). Executive of Jakmania stated, that is
not part of Jakmania. To anticipate the
widespread influence of Rojali, Chairman
of Jakmania formed various campaigns,
one of the campaigns is by campaigning 7
characteristics of Jak astray. The campaign
is to identify Rojali’s behavior that often
used undermines, vandalism, and anarchy
activities. Rojali became one of the
causes of the reproduction of the conflict,
especially with Viking. In the context of
a conflict involving Jakmania organizers,
“Rojali” is always placed as a scapegoat
without understanding the root cause of
the conflict itself (Huda, 2016; Wibowo,
2017; Wirayudha, 2018; and interview with
Respondent B, 10/7/2017). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik
154 FINDINGS AND DISCUSSION Rojali, according
to the claim of the Chairman of Central 154 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 driving factors, such as the decision of the
referee, and the provocation of the audience. This incident also resulted in one Policeman
from the Provost unit injured his nose hit by
a throw (cf Goldstein, 1993; Le Bon, 2002;
Chrysnanda, 2011; Rahayu, 2016; Fuller,
2017; and Ismunanto, 2017). at the venue of stadium (Abbot & Geddie,
2001; and Wibowo, 2017). Vikings that are
present dominated by young males and are
rarely seen by many children and women,
in addition to their difficult access to reach,
a great risk is also a consideration of the
infrequent presence of children and women
in every game (Ryza, 2011; Hadi, 2017; and
Kaka, 2017). The crowds, in the view of Gustav
Le Bon (2002), are (crowd’s) life is the
transformation of though (Le Bon, 2002). Another view represented by Elias Canetti
(1973), who sees crowds as transformation
of power. This shows that crowds have the
ability to influence and be influenced in
positions as objects or subjects (Canetti,
1973). In this context, the football audiences
can be said to be crowds which in popular
terms the football audiences are referred as
the twelfth player (Canetti, 1973; Le Bon,
2002; Kari, 2009; and Wibowo, 2017). Solo case in Central Java, in 2017,
indicated that the match between PERSIJA
versus PERSIB should be held in Bekasi,
West Java, but they do not get permission
and must be transferred to Solo by
PERSIJA’s management. The reasons are
based on security aspect; and because of
the fact that on the same day, there will
be a massive demonstration in Jakarta. The security forces use risky action by
presenting two large masses at a point that
are at risk of direct clashes. The transfer of
the venue became controversial, because
it happened in a fast tempo where within
three days the decision of the match in
Bekasi, West Java was canceled and had
to move to Solo in Central Java. Officials
did sudden preparations for the match
between PERSIJA dan PERSIB. FINDINGS AND DISCUSSION p
)
Characteristics of spectators from the
matches between PERSIB and PERSIJA can
be identified depending on where the game
is held. If the match held in Bandung, it
was seen that the Vikings, who attended the
game at the GBLA (Gelora [Gelanggang
Olahraga] Bandung Lautan Api or Bandung
a Sea of Flames’ Sports Arena) stadium,
were clustered irregularly. This is due to
a number of factors, firstly the factors of
crowd’s management and crowd’s control
(cf Berlonghi, 1995; Abbot & Geddie, 2001;
Ayati, 2010; and Duerden, 2017) that are not
working well; and secondly is the irregularity
of parking lot, the escape of the bottles that
enter the stadium, the dense access road to
and exit the stadium, to plotting officers as
ineffective security forces (McLean, 2015;
Ayati, 2010; and Duerden, 2017). In the phenomenon of Viking and
Jakmania, there are also other groups of
Viking’s border groups. Characters from
the border region that are part of Viking
members often looking for conflict with
Jakmania in areas such as Depok and Bekasi
in West Java. This group is also in the Viking
– Jakmania’s conflict, which always making
violence to the Jakmanias group. Viking’s
borders continue to reproduce existing
conflicts with Jakmania. The settlement of
prejudices arising from Viking, who think
on unfinished borders, will continue to be a
physical conflict even outside the stadium. The last case is the beating of Ricko (Viking)
by the Viking border that strikes Ricko as
Jakmania in the July 22, 2017’s match (Eko,
2012; Hanifan & Herfiyana, 2014; Huda,
2016; Hadi, 2017; and Wirayudha, 2018). The result of this irregularity is the
casualties in the Vikings. They are often
to be the victims of wrong targeting. KAPOLRESTABES (Kepala Kepolisian
Resort Kota Besar or Head of Big City
Police) Bandung has banned Jakmania
from attending the GBLA stadium, but
there are still beatings. This condition is
driven by Bobotoh (PERSIB supporters)’s
disappointment over the game, so the
Bobotoh acts irrational because of their In Jakmania, there is a group called the
Rojali group. This group is often to be the
main cause of Jakmania’s involvement in
conflicts, including Vikings. The central
Jakmania committee admitted the difficulty
in dealing with this group. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION The
selection of Solo as the venue is based
on the cooperation between officials of
PERSIJA with local officials from PERSIS
(Persatuan Sepakbola Indonesia Solo or
Solo Indonesian Football Association) in
Solo, Central Java (Rahayu, 2016; Saudale
& Ganesha, 2016; Hadi, 2017; Wibowo,
2017; and Wirayudha, 2018). On the other hand, the phenomenon of
conflict within the crowds also still uses
the concept of prejudice (Brown, 2011;
and Wibowo, 2017). It can be described in
the form of a Viking who always feels that
Jakmania will be a means of vengeance. This evident can be seen from the
statements of the perpetrators, who explain
the motive is hatred against Jakmania. If
match’s management can do well-organized
mechanism, the riots should be avoided
by using the crowd control concept that
focuses on crowds organizing crowd’s
action process (Berlonghi, 1994; Tamara
& Knutsson, 2011; Rahayu, 2016; Fuller,
2017; Ismunanto, 2017; and Wibowo, 2017). Police in the Solo case, in 2017,
used silahturahmi (good relationship)
approach before the game. The direction
of this gathering is done by KABAG OPS
POLRESTA (Kepala Bagian Operasi
Kepolisian Resort Kota or Head of City
Police Operation Division) Surakarta in
Central Java to minimize friction and asking
for cooperation between Pasoepati (PERSIS
supporters) with Jakmania (PERSIJA
supporters) in securing the match. Police The interesting thing about the
characteristics of Viking is the variety of
processes used in the assembling process;
see, for example, the presence of supporters
coming from different directions (cf John et
al., 2001; Redhead, 2009; and Hadi, 2017). Vikings, who go to the venue of the game,
are coordinated by their respective districts. the assembling process is accompanied by
a gathering process (gathering at one point)
that is associated with crowd’s management 155 ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos irregularities that will occur in Solo. The
small Solo area will have 17,250 people
coming from Jakarta; and if not done a good
crowds management process will be a big
potential riot (Berlonghi, 1994; Hadi, 2017;
Wibowo, 2017; and Wirayudha, 2018). It should also be strengthened by crowds
control conducted by Surakarta Police by
inspecting Jakmania entourage, who arrived
in Solo. This inspection resulted in a group
of supporters who were caught possessing
or consuming liquor. The perpetrator was
held in Surakarta Police until the match
was over (Hadi, 2017; Wibowo, 2017; and
Wirayudha, 2018). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik
156 FINDINGS AND DISCUSSION always maintaining security by calling for
a ban on the consumption of local liquor,
which is quite famous in Solo to the fans. It aims to strengthen the coordination and
security preparations that need to be done
(cf Parks, 1981; Braiden, 1992; Oliver
ed., 2000; Chrysnanda, 2011; Hadi, 2017;
Wibowo, 2017; and Wirayudha, 2018). Due to the displacement venue of this
match, the assembling process did not run
with massive force. Tickets sold for 17,250
tickets, which is the result of ticket sales for
4 days. Jakmania, in this context, succeeded
in mobilizing mass in large numbers in
preparation for a short time. This assembling
process (cf Berlonghi, 1994; John et al.,
2001; and Prpic et al., 2017) was escorted
by police to Jakmania’s entourage (Hadi,
2017; Wibowo, 2017; and Wirayudha, 2018). A similar phenomenon that occurs in
Bandung, West Java, and also occurs in
Solo, Central Java, is the action of raiding
the opposing supporters who are identified
as people who are not part of the supporters. Counted 6 times assault by Jakmania to
groups identified as Viking. This action is
not only triggered by prejudice as happened
in Bandung (Brown, 2011; Hadi, 2017;
Wibowo, 2017; and Wirayudha, 2018). However, it was also triggered by conditions
in the field of a match considered to be
detrimental to PERSIB, ranging from injury
to Kim Kurniawan, disallowed goal, until
a penalty decision to nourish the anger
and disappointment of the supporters (cf
Berlonghi, 1994; Brown, 2011; Hadi, 2017;
Wibowo, 2017; and Wirayudha, 2018). ;
,
;
y
,
)
This process is done with a form of
cooperation between the POLDA METRO
JAYA (Kepolisian Daerah Metropolititan
Jakarta Raya or the Great Jakarta
Metropolitan Regional Police); POLDA
JABAR (Kepolisian Daerah Jawa Barat or
West Java Regional Police); and POLDA
JATENG (Kepolisian Daerah Jawa Tengah
or Central Java Regional Police) who escort
a relay. To secure this process, Polices also
carried out the communication process
between the police and the community. Communications formed between the police
and the community are effective enough to
build mutual trust (Parks, 1981; Braiden,
1992; Goldstein, 1993; and Prpic et al.,
2017). A similar process is done when
traveling back from Solo to Jakarta or
dispersal process (Oliver ed., 2000; John et
al., 2001; Chrysnanda, 2011; Hadi, 2017;
Wibowo, 2017; and Wirayudha, 2018). FINDINGS AND DISCUSSION J k
i
fi
i
d i This process is done with a form of
cooperation between the POLDA METRO
JAYA (Kepolisian Daerah Metropolititan
Jakarta Raya or the Great Jakarta Chaos Tradition in Supporters as
Football Culture and Character in
Indonesia. Against the law and acting
aggressively to the Police becomes a
distinct culture in the lives of the hooligans
(Pearson, 1983; Poulton, 2008; and Wibowo,
2017). The culture of the crowd of football
supporters becomes a new culture that is
formed and produced in certain forms. Resistance to the Police, which seeks to
maintain security and order, is the hallmark
of the culture. The local ethnic and cultural
character is also a matter of encouraging
Viking and Jakmania groups to do things Jakmania entourage first arrived in
Solo at 02.00 AM (Ante Meridiem). They
were dominated by a group of 17-25
year-old boys, who will be channeled in
the velodrome area at Manahan Stadium
in Solo, Central Java, Indonesia. This
canalization aims to anticipate the MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 Indonesia, in addition to being shown in the
form of cultural identity and local identity, is
also indicated by the meaning of the value of
each victory achieved by each football club
in every game involving both clubs. The
club’s victory becomes an important marker
of the level of dominance possessed by
each of the crowd’s identities. This makes
it very important to look at such aspects as
the influence of the referee’s decision as that
is considered to affect the win or the defeat
of a club. The implication is that masculine
aggressiveness can be triggered by referee
decisions in the game. This is shown by
the many riot football supporters triggered
by goal scorer, yellow/red card given by
referee, and so forth (Williams & Dunning,
1990; Stone, 2007; Dick & Kernick, 2016;
Duerden, 2017; and Fuller, 2017).l that are contrary to the law, such as throwing
bottles and taking action against security in
certain ways (David et al., 2008; Hadi, 2017;
Wibowo, 2017; and Wirayudha, 2018). Culture supporters like this also happen
in other countries, like England for example. Eric Dunning (2006) shows how the football
industry has also spawned a violent industry. National characters within the context of
the nationalist issues of Wales, Ireland, and
Scotland are symbolized and applied in
support actions to their respective football
clubs. FINDINGS AND DISCUSSION The character of violence arises,
when these values are contained by football
matches that cause a pattern of masculine
aggressiveness to emerge as the root of
violence in football hooliganism (Dunning,
2006; Stott & Pearson, 2007; Rookwood &
Pearson, 2010; Lo, 2011; dos Reis, Lopes &
Martins, 2015; and Rogers, Woodhouse &
Politowski, 2016). Conflict in Matches as Cultural
Identity and Conflicts between Viking
and Jakmania. To understand the Vikings
and Jakmanias’ conflict, as a rivalry of
PERSIB (Persatuan Sepakbola Indonesia
Bandung or Bandung Indonesian Football
Association) and PERSIJA (Persatuan
Sepakbola Indonesia Jakarta or Jakarta
Indonesian Football Association), must
begin with the understanding that football
is a tool of Indonesian nation’s identity
struggle against the Dutch colonial. From
that history, football in Indonesia is a legacy
sport of colonial culture (transnational
sport) introduced by the Dutch. And, not yet
known the term football supporters. There
are clubs and players. Football also became
a portrait of social class (indigenous, non-
indigenous, and Dutch). It shows that since
the birth of football in Indonesia, the values
of social segregation, based on ethnicity
(Dutch, Chinese, and Native) have grown as
differentiators between clubs (Colombijn,
2000; Ayati, 2010; Kristanto, 2016; Rahayu,
2016; Sufiyanto, 2017; and Wirayudha, 2018). Violence became a concept that has a
relationship with the identity of football
supporters and became part of a popular
culture among certain fans in the national
football industry. This is as shown in the
Richard Guilianotti, Norman Bonney &
Mike Hepworth (1994)’s study which stated
that violence is capitalized with certain
interests such as political interests and
economic interests. Their research shows
that political interests in violence supporters
in Italy are used to distort aggression based
on reality, such as poverty and social welfare
that occurs in most supporter populations. Economic interests also affect chaos culture
reproduction in rioting crowds of football
supporters, where such influence could be
the destruction of trademark image and
capitalization of certain products that become
the main attribute in the identity symbol of
football supporters (Williams & Dunning,
1990; Guilianotti, Bonney & Hepworth,
1994; Dunning, 2006; dos Reis, Lopes &
Martins, 2015; and Wann et al., 2015). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION The Natives (Indonesia) raised their
identity through football clubs, such as
the BIVB (Bandoeng Inlandsche Voetbal The Natives (Indonesia) raised their
identity through football clubs, such as
the BIVB (Bandoeng Inlandsche Voetbal The mass culture element contained in
the phenomenon of football fanatical riots in 157 ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos of Sundanese moving widely (Utami, 2011;
Hanifan & Herfiyana, 2014; Kristanto,
2016; Hadi, 2017; and Wibowo, 2017). In
cyberspace, Bobotoh fans are also monitored,
there are settlers abroad such as Malaysia,
Singapore, Saudi Arabia, Britain, USA (United
States of America), and others (Kristanto,
2016; Bahri, 2017; and Putri, 2018). Bond) during the Dutch colonial period
as well as through the formation of other
club clubs based on regional names. It is a
manifestation of the Indonesian nationalist
attitude, to show a sense of coincidence
with the clubs in created by the colonialist. It
shows the character of the egalitarian concept,
which is shown by Indonesians or indigenous
(Colombijn, 2000; Hanifan & Herfiyana,
2014; Kristanto, 2016; and Hadi, 2017). The role of media, as identity formers,
also encourages in “how identity is formed”
(Croteau & Hoyness, 2003; and Effendy,
2003). As Manuel Castells (2000) states that
humans are passing through the industrial
age to the information age. With the era
of globalization and communication and
information technology, Manuel Castells
also stated that the information age will give
birth to a new social, industry, and cultural
network (Castells, 2000). The Japanese occupation government
(1942-1945) saw the important position
of football club formed by Indonesians
(indigenous) as a medium in showing
identity as Indonesian as well as a symbol
of resistance. Consequently, in the concept
of occupation (relations of domination),
colonial nation and colonized nation make
indigenous football club is considered very
dangerous for the Japanese nation. This
has implications for the banning policy of
Indonesian football club by the Japanese
government (Colombijn, 2000; Beck, 2015;
and Colombijn & Cote, 2015). (
,
)
This also happened to Jakmania
and Viking football supporter groups. Cyberspace becomes the arena of
communication, contestation (rivalitas),
as well as hostility supporters. This is
exacerbated by the capitalization of
Jakmania and Viking conflicts through
online and social media. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik
8 FINDINGS AND DISCUSSION This is shown by several things: firstly,
that Bobotoh as the parent of the synthetic
culture of delays has metamorphosed
naturally into several other support groups. The group’s naming is imaginary from
portraits of European supporters, such
as the Italian Ultras, the FCC (Football
Club Cincinnati) of Ohio, Vikings like
Scandinavia, and Orientalism that show
Asian identity. Secondly, social and class
segregation does not only happen to football
clubs in the Dutch colonial, but also happens
to football fans in the era of development. This means that social segregation is the
background of SARA (Suku, Agama, Ras,
dan Antargolongan or Ethnic, Religion,
Race, and Inter-Groups) that gave birth
to synthetic identity naturally or through
fabrication, which always grows in society
(cf Canetti, 1973; Voris, 2015; Kristanto,
2016; Hadi, 2017; Sufiyanto, 2017; and
Wirayudha, 2018). p
)
The emergence of fanatic football
supporters, in line with the era of national
football competition, is considered as part
of national development in the field of
sports during the New Order era (1966-
1998). Bobotoh grows in various regions in
West Java. In fact, its development grew in
other areas, inside and outside the country. The number was from dozens transformed
into hundreds of thousands and even
millions. Bobotoh is an imagine community
(Anderson, 1983), which is based on value
of ke-Sunda-an (Sundanese) in line with
PERSIB (Hanifan & Herfiyana, 2014;
Kristanto, 2016; Hadi, 2017). y
,
)
Vikings are symbols of conquering
values that are the product of Ayi Beutik
Suparman’s imagination of the legendary
name of the Scandinavian nation. Viking
identic with the values as a strong
nation, savage, brave, never give up,
and like to attack. Nonetheless, Viking
is a metamorphosis of Bobotoh, because
of its natural nature, the Vikings do not
recognize the semi-bureaucratic organizing
system (Haryadi, 2014; and interview with
Respondent A, 3/7/2017). Like the crowds,
Viking also develops naturally, through
the hereditary mechanisms of the family. Viking also develops to other regions and
is no longer confined to the Cibangkong
and Pasirluyu regions in West Java as the
Viking homebirth (cf Canetti, 1973; Hanifan
& Herfiyana, 2014; Kristanto, 2016; Hadi,
2017; and interview with Respondent A,
3/7/2017). According to John Voris (2015), Bobotoh
can be categorized as a synthetic identity
from ke-Sunda-an (Sundanese mattres). As part of Bobotoh, there is a group of
supporters called Viking PERSIB Fans
Club. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION From social media
network, the researchers can know that most
of Bobotoh or Viking comes from Cianjur
with 637,263 people; Garut with 627,240
people; Bandung in third place with 506,774
people; fourth is Jakarta with 260,056
people; and Bobotoh in Surabaya, East
Java is in fifth position with 93,965 people
(Hanifan & Herfiyana, 2014; Kristanto,
2016; Rahayu, 2016; Sufiyanto, 2017;
Wirayudha, 2018). From the historical description, PERSIB
as pride of Bandung’s citizens, especially
among Sundanese in the era of independence
is also a symbol of continuity or symbolic
metamorphosis of the Padjadjaran kingdom
in West Java. This spurred the formation
of a new sub-culture of identity of delays
or identities into Padjadjaran. PERSIB
is a symbol of a new identity formed in
Sundanese society that is identical with
Sundanese culture (Hanifan & Herfiyana,
2014; Kristanto, 2016; and Hadi, 2017). Bobotoh does not operate as limited in
Bandung or West Java. Although PERSIB
is headquartered in Bandung, but Bobotoh
supporters are widespread from Banjar,
Ciamis, Tasikmalaya, Bogor, Cirebon,
Kuningan, Karawang, and Bekasi in West
Java. Bobotoh also located outside the
Province of West Java, such as Jakarta,
Tangerang, and Serang in Banten Province. Widespread membership of Bobotoh in
accordance with the characteristic identity Based on the data of 5.3 million Bobotoh
in West Java, 26 percent are women. Approximately 1,378,000 this female Bobotoh
has demographic status with age ranging from
21-40 years old, married, and tend to like
PERSIB due to influence of husband, and
child (cited in Hanifan & Herfiyana, 2014;
Kristanto, 2016; and Hadi, 2017). In the match, Bobotoh will always meet
the stadium, either if PERSIB compete 158 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 in Bandung or Jakarta, with always bring
attributes. Bobotoh also performs watching
together in front of the television for those
who cannot go to another city if PERSIB
perform away games as a form of their
commitment to the club (Utami, 2011;
Hanifan & Herfiyana, 2014; Kristanto,
2016; and Hadi, 2017). The Bobotoh
generally have two major groups, namely:
Viking PERSIB Fans Club and Bobotoh
Maung Bandung Bersatu or the BOMBER. There are also Bobotoh type based on the
characteristics consist of: Bandung Ultras,
Flower City Casuals, Oriental Bobotoh, and
Singapore Bobotoh (Hanifan & Herfiyana,
2014; Kristanto, 2016; Hadi, 2017; and
interview with Respondent A, 3/7/2017). multiculturalism of football supporters. FINDINGS AND DISCUSSION Viking can be identified as a synthetic
identity of Bobotoh that indicates the
strength of transcultural or mix culture
as affecting the authentic culture of the
Sundanese. It also co-stars with football
as an imitation culture from European
football’s origin culture (cf Voris, 2015; and
Hadi, 2017). Vikings also remain familiar with group
values as the requirement of an identity, and
called Vikingnisme as a naturally formed From the naming of the Viking
and Bobotoh, it is true to show the 159 ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos society. The acculturation of values between
the Vikings as the imagine norm and the
delay as part of Bobotoh of PERSIB is seen
from value, loyalty (to the group), solidarity
(loyal friends), and values in urban society
(style) symbols, authentic identity brought
from the birth of each individual and social
environment, in which he lived to the
Viking group (Hanifan & Herfiyana, 2014;
Kristanto, 2016; and Hadi, 2017). is a determinant factor of the Viking
metamorphosis process. Even, if we
imagine the Vikings are a group of beliefs,
then, in the Vikings are also born rituals
that distinguish between members of
Viking with other Bobotoh. Ayi Beutik
Suparman becomes the object of a ritual
for Viking members, who are believed to
be able to give “strength” in the form of
courage (cf Canetti, 1973; Fathurrohman,
2014; Haryadi, 2014; and interview with
Respondent A, 3/7/2017). The growing number of Viking members
also boosts business growth related to
accessories and attributes of football
supporters. That supporters also have an
important role in an economic activity,
especially the football industry. In the
Viking group, there are symbolic figures
that influence the Viking development up
to now. The figure is Ayi Beutik Suparman,
who can be regarded as the central figure
in the Indonesian archipelago, like General
Soedirman for the military, Sukarno for
Marhaen nationalist, or R.A. (Raden Ajeng)
Kartini for women’s emancipation in
Indonesia (cf Stone, 2007; Fathurrohman,
2014; Hanifan & Herfiyana, 2014; Kennedy,
2014; Kristanto, 2016; Hadi, 2017; Kaka,
2017; and Eyong, 2018). Meanwhile, PERSIJA in Jakarta as a
club, that was born since the Dutch colonial
era, has a historical similarity with PERSIB
in Bandung. PERSIJA, previously named
VIJ (Voetbal Indonesische Jacatra), is a
symbol of the existence of indigenous people
of Betawi. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik
0 FINDINGS AND DISCUSSION In addition, Gugun
Gondrong as icon Jakmania is an artist who
uses his popularity as a magnet to attract
new members (Hamley, 2001; Sufiyanto,
2017; Wirayudha, 2018; and interview with
Respondent C, 17/7/2017). As a synthetic
identity of a new group, Jakmania members
are bound by the rules, which are mentioned
by AD/ART. From the beginning Jakmania
in form, in support of the development
of PERSIJA club from the social and
economic, such as ticket entry, merchandise,
etc. (Sufiyanto, 2017; and Wirayudha, 2018). Jakmania is a group of supporters,
whose development is heavily influenced by
bureaucratic policies. This is in line with the
common concern among Jakarta youngsters,
who love to watch PERSIJA with the
management and the Provincial Government
of DKI Jakarta under the leadership of
Governor Sutiyoso for the lack of audience
when the club Persija Jakarta compete. This
feeling of alienation and marginalization
is a major consideration of Jakmania’s
establishment as a new synthetic identity (cf
Colombijn, 2000; Voris, 2015; Sufiyanto,
2017; and Wirayudha, 2018). As the early history of football in
Indonesia as a means of identity struggle
against the Dutch colonial, Viking and
Jakmania relations as part of PERSIB
and PERSIJA competitiveness cannot
be separated from the form of harmony
metamorphosis relation to a relationship
that is conflict. In the history of relations,
they can be described as the crowd at the
Siliwangi Stadium was influenced by the
situation of the matches that took place in
the field, the emotions of the audience were
strongly influenced by the field conditions,
such as the outcome of the match (cf
Berlonghi, 1994; Colombijn, 2000; Abbot
& Geddie, 2001; Hanifan & Herfiyana,
2014; Conigliaro et al., 2015; Kristanto,
2016; Sufiyanto, 2017; Wibowo, 2017; and
Wirayudha, 2018). The pattern of Jakmania formation
differs from the pattern of formation that
occurs in Viking. If Viking is based on
natural, bottom-up, fluid, cultural values,
although familiar with simple organizational
systems, then Viking is a continuation of the
greatness of the Padjadjaran kingdom or
PERSIB. Jakmania is a tool or instrument
fabricated through the collaboration of the
provincial government and the will of the
Jakarta children, so that the underlying spirit
of Jakmania’s presence is the resurrection
values of the Jakarta community to host
its own region. FINDINGS AND DISCUSSION PERSIJA as a symbol of the spirit
of equality and resistance to colonial symbols
and class distinctions or VBO (Voetbalbond
Batavia Omstaken) as the Dutch formation. The difference with PERSIB is PERSIJA
originally named VIJ has had a group of fans
or fans known as VIJers (Sufiyanto, 2017;
and Wirayudha, 2018). In post-independence era, PERSIJA
and PERSIB are the founding initiators
of PSSI (Persatuan Sepakbola Seluruh
Indonesia or All Indonesian Football
Association). PERSIJA later named itself
as “Kemayoran’s Tiger”, which has the
meaning of a beast that in imagination as the
King of Forest, which has the characteristics
of ferocious, brave, in respect, and the ruler. This is reinforced by the use of red on club
uniforms (Kristanto, 2016; Sufiyanto, 2017;
and Wirayudha, 2018). Ayi Beutik Suparman is the founder and
patron of the entire sub-culture of the Viking
members. He was a charismatic figure, who
provided the first normative fundamentals of
the Vikings. His boldness became the most
basic reference value of Viking members. As a leader, from a group of natural and
cultural, willing to sacrifice for other
members has become the value that makes
him very much in love with the Viking groups
(Fathurrohman, 2014; and Haryadi, 2014). The existence of PERSIJA until 1994,
or the Indonesian league, is identified with
a club that does not have many supporters;
and this is indicated by the minimal number
of spectators. This affects the club’s internal
conditions. This situation prompted the role
of DKI (Daerah Khusus Ibukota or Special
Capital Region) Jakarta local government to
intervene to help PERSIJA as the club pride As a group that goes on naturally
and culturally, Ayi Beutik Suparman’s
leadership character becomes the main
magnet affecting the rapid growth of
Viking members, not only in Bandung but
also in other regions. If the analogy, with
the process of formation of butterflies,
it can be said that Ayi Beutik Suparman 160 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 of the capital. From it can be interpreted
that PERSIJA is a club that has a historical
value, in the dynamics of Indonesia, the
symbol of the pride of the capital, both
people and government (cf Berlonghi, 1995;
Colombijn, 2000; Sufiyanto, 2017; Wibowo,
2017; and Wirayudha, 2018). moves to invite the people of Jakarta to
join Jakmania as part of the exploration
process (Utami, 2011; Sufiyanto, 2017;
and Wirayudha, 2018). © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik FINDINGS AND DISCUSSION Jakmania was formed
based on the norms of the formation of
a social organization, one of which is by
arranging AD/ART (Anggaran Dasar/
Anggaran Rumah Tangga or Organizational
Constitution). Recruitment involves the
village, to socialize as well as encourage the
local people to watch the game conducted
by PERSIJA (Hanifan & Herfiyana, 2014;
Kristanto, 2016; Sufiyanto, 2017; and
Wirayudha, 2018). Crowds management failed to build in
Jakmania’s body, since the 2000s in the
process come to Bandung. The crowds
control operated by the POLDA METRO
JAYA (Kepolisian Daerah Metropolititan
Jakarta Raya or the Great Jakarta
Metropolitan Regional Police) on the road
trip agenda to the Siliwangi Stadium shows
a failure that can be identified through
the crowd’s stage based on Kenny M. John et al. (2001)’s research. At the stage
of assembling Jakmania, miscalculation Crystal crowds Jakmania, totaling
40 people into a community group that 161 ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos occurred which of the quota of 400 people
to 1,000 people (John, 2001; Wibowo, 2017;
and Wirayudha, 2018). f anytime and anywhere (Williams &
Dunning, 1990; Berlonghi, 1994; Hanifan
& Herfiyana, 2014; Kristanto, 2016; Fuller,
2017; Sufiyanto, 2017; Wibowo, 2017; and
Wirayudha, 2018). l This condition affects the next process
of gathering at the Siliwangi Stadium in
Bandung, the capacity of the stadium is not
large with the match allocation of the tickets
with the number of spectators in attendance
resulting in mass accumulation outside the
stadium. In this mass build up, there was a
clash between the Vikings and Jakmanias. In the process of dispersal shown by a group
of Jakmania, who will return to Jakarta,
involving clash activity with Vikings. From
the description, it can be interpreted that the
Police and match management is not ready
in the face of overcrowding situation, which
is always identic with Jakmania (Abbot &
Geddie, 2001; Kristanto, 2016; Fuller, 2017;
Wibowo, 2017; and Wirayudha, 2018). Conflicts are also exacerbated by the
interpretation of cases occurring, perpetrated
by both parties, both through interaction
with the direct victims/perpetrators as well
as the modified interpretations made by
Jakmania and Viking members. This extends
with the increasing number of Jakmania
and Viking members; and the use of social
media. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
ISSN 2527-3868 (print), 2503-457X (online), and http://ejournal.upi.edu/index.php/mimbardik
162 FINDINGS AND DISCUSSION The protracted conflict as well as the
modified interpretation of each of the sides
that resulted in the acute prejudice, not only
to individuals involved in the conflict but
extended to group members are generative
or hereditary (Blumer, 1968; Castells, 2000;
Sunarto, 2000; Brown, 2011; Hanifan &
Herfiyana, 2014; Kristanto, 2016; Fuller,
2017; Sufiyanto, 2017; Wibowo, 2017; and
Wirayudha, 2018). The prejudices held by Jakmania on
Viking came after the events that took place
at the Siliwangi Stadium since the 2000s. Jakmania feels like a loser, because they are
on the agenda to attend their away match,
which is not in line with expectations. This
action was responded by beatings to a group
of Vikings who attended a quiz show at one
of the TV stations in Jakarta. This conflict is
repeated and reproduced by the Vikings as
well as Jakmania as the basis for efforts of
conflict reproduction. This then produces a
prejudice that gave birth to a stereotype of
the Viking group’s rogue by the Jakmania
(cf Sears, Freedman & Peplau, 1994; CONCLUSION The characteristic of crowds in the
football industry in Indonesia is strongly
influenced by issues of cultural and local
identity. In addition, the character of
football supporters in Indonesia is strongly
influenced by social identity, such as socio-
economic level. This causes the level
of aggression in the football industry in
Indonesia seen in the form of riots and
competition of identity symbols, such as
jersey, colors, flags, and so forth. Then also
if drawn further, the formation of identity is
correlated with the phenomenon of football
as a pop culture, that is that football into a
culture that can be enjoyed universally, so it
can involve anyone and anywhere. ( f
p
Kristanto, 2016; Fuller, 2017; Wibowo,
2017; Wirayudha, 2018; and interview with
Respondent B, 10/7/2017). This illustrates the metamorphosis of
conflict between Jakmania and Viking. Conflict continues to occur involving
Vikings and Jakmanias, during matches and
beyond football matches. This illustrates
the metamorphosis of conflict with the
encouragement of precipitant factors, such
as bad crowd management to conflicts
related to group identity, which can happen The implications of looking at the
phenomenon of football in Indonesia as
a pop culture phenomenon also provide
help to illustrate that the level of masculine
aggression in riots between Viking in West
Java and Jakmania in Jakarta cannot be 162 MIMBAR PENDIDIKAN: Jurnal Indonesia untuk Kajian Pendidikan,
Volume 3(2), September 2018 released as a culture of its own produced
continuously to achieve certain interests. The phenomenon of behavior and attitude
of hooliganism in the body of Viking and
Jakmania as well as in the phenomenon of
hooliganism in England. l homogeneity of Sundanese culture. The
spirit of cultural homogeneity has been
acculturated with values and passion drawn
from the imagination of the “Vikings”
characterized by brave values, conquerors,
and loyalty; thus encouraging Bobotoh to
join the growing, growing, and growing
Viking supporters from the grassroots. The phenomenon of conflict involving
the two supporter groups has crystallized
into an identity conflict based on prejudice
between the two groups. Subsequent
forms of prejudice that also grew out of
the state and the public have given birth to
stereotypes that see Jakmania and Viking
as monstrous that have the potential to pose
a threat to security and order disorders,
especially if the group is present in the arena
of matches. CONCLUSION The root cause of the riots can be
summed up because the internalized
“prejudices” in both members of both
supporters’ camps are continuously
produced into a popular culture in the Viking
and Jakmania’s areas. The effort to build
trust in the level of awareness, peace, and
mutual respect is not easy, so it requires
a sustainable effort to build a culture of
sportsmanship in the football competition. However, cultural cultivation is not easy to
implement, because the riot culture is more
popular than the sprotif culture, peace, and
so forth. In accordance with the historical,
philosophical, cultural, and socio-economic
background, that inspired the formation
and founding of Jakmania in Jakarta and
Viking in Bandung, it can also be concluded
that initially both organizations were born
to provide support and love for PERSIJA
(Persatuan Sepakbola Indonesia Jakarta
or Jakarta Indonesian Football Association)
and PERSIB (Persatuan Sepakbola
Indonesia Bandung or Bandung Indonesian
Football Association)’s football clubs. But in
its journey, organization and management,
especially Jakmania, is more formal with
hierarchical organizational structure and
division of organizational tasks more
structured. Such forms and organizational
and management characteristics of course
have an influence on the behavior and
attitudes of members of Jakmania. The
youth empowerment of marginal and
highly heterogeneous community groups
in Jakarta has aroused the spirit of militant
Jakmania’s members coincidentally inspired
by community leaders, who are considered
to have high fighting spirit. Jakmania and Viking if they want to clean
up must carry out the members’ mental
reform that involves the reconstruction
of values. This is done considering the
unrest between the two members of the
organization, not only occurred in the arena
of matches between PERSIJA and PERSIB,
but also elsewhere such as Europe. The
acts of rioting between members of the
Jakmania and Viking support groups have
evolved into an identity crisis, requiring
the organization’s serious, and fundamental
guidance on mental, behavior, attitudes, and
action in accordance with the sportsmanship
values of football matches. The pattern
of guidance to members of Jakmania
and Viking should be done periodically,
structured, and professional so as to build
a sense of “trust” of both Jakmania and
Viking’s members. Chaotic atmosphere contained in football
matches in Indonesia are described through
the phenomenon of Viking and Jakmania
into an additional identity in the pop
culture in Indonesia. © 2018 by UPI (Indonesia University of Education) Press in Bandung, West Java, Indonesia
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is more informal and family spirit that is
driven by a figure who puts forward the
cultural and natural values based on the 163 ANDRY WIBOWO,
Pop Culture, Identity Conflict, and Chaos only can be considered as an effect, but as
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Developmental Trajectories of Internally and Externally Driven Temporal Prediction
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RESEARCH ARTICLE a11111 OPEN ACCESS OPEN ACCESS
Citation: Mento G, Tarantino V (2015)
Developmental Trajectories of Internally and
Externally Driven Temporal Prediction. PLoS ONE
10(8): e0135098. doi:10.1371/journal.pone.0135098 OPEN ACCESS
Citation: Mento G, Tarantino V (2015)
Developmental Trajectories of Internally and
Externally Driven Temporal Prediction. PLoS ONE
10(8): e0135098. doi:10.1371/journal.pone.0135098 OPEN ACCESS
Citation: Mento G, Tarantino V (2015)
Developmental Trajectories of Internally and
Externally Driven Temporal Prediction. PLoS ONE
10(8): e0135098. doi:10.1371/journal.pone.0135098 Editor: Hedderik van Rijn, University of Groningen,
NETHERLANDS Editor: Hedderik van Rijn, University of Groningen,
NETHERLANDS Received: February 25, 2015
Accepted: July 16, 2015
Published: August 11, 2015
Copyright: © 2015 Mento, Tarantino. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Received: February 25, 2015
Accepted: July 16, 2015
Published: August 11, 2015
Copyright: © 2015 Mento, Tarantino. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Received: February 25, 2015
Accepted: July 16, 2015
Published: August 11, 2015
Copyright: © 2015 Mento, Tarantino. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Copyright: © 2015 Mento, Tarantino. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Developmental Trajectories of Internally and
Externally Driven Temporal Prediction Giovanni Mento1*, Vincenza Tarantino1,2
1 Department of General Psychology, University of Padua, Via Venezia 8, 35131, Padua, Italy,
2 Department of Neurosciences: SNPSRR, University of Padua, Via Giustiniani 5, 35128, Padova (PD), Italy Giovanni Mento1*, Vincenza Tarantino1,2 1 Department of General Psychology, University of Padua, Via Venezia 8, 35131, Padua, Italy,
2 Department of Neurosciences: SNPSRR, University of Padua, Via Giustiniani 5, 35128, Padova (PD), Italy * giovanni.mento@unipd.it Abstract The ability to generate temporal prediction (TP) is fundamental to our survival since it allows
us to selectively orient our attention in time in order to prioritize relevant environmental infor-
mation. Studies on adult participants showed that externally and internally driven mecha-
nisms can be engaged to establish TP, both resulting in better behavioural performance. However, few studies on children have investigated the ability to engage internally and
externally driven TP, especially in relation to how these mechanisms change across devel-
opment. In this study, 111 participants (88 children between six and eleven years of age,
and 23 adults) were tested by means of a simple reaction time paradigm, in which temporal
cueing and neutral conditions were orthogonally manipulated to induce externally and inter-
nally driven TP mechanisms, as well as an interaction between the two. Sequential effects
(SEs) relative to both tasks were also investigated. Results showed that all children partici-
pating in the study were able to implement both external and internal TP in an independent
fashion. However, children younger than eight years were not able to combine both strate-
gies. Furthermore, in the temporal cueing blocks they did not show the typically-observed
asymmetric SE pattern. These results suggest that children can flexibly use both external
and internal TP mechanisms to optimise their behaviour, although their successful com-
bined use develops only after eight years of age. a1111 Introduction The ability to temporally predict relevant events (temporal prediction, or TP) is a fundamental
skill allowing us to reduce uncertainty about the future and to optimise our behaviour [1–3]. Crossing a busy street at the rush hour, or deciding whether to accelerate or to stop when our
car arrives at an amber traffic light are just two among thousands of daily situations in which a
bad prediction of the correct time to act may threaten our survival. In spite of this, the ontogen-
esis of the cognitive mechanisms enabling human beings to develop a ‘predictive brain’ is still a
challenging topic. In this study, we tried to address this issue by investigating the developmen-
tal trajectory of different types of TP. Data Availability Statement: All relevant data are
within the paper. Funding: This work was supported by the grant
‘Progetto Giovani Studiosi’ from the University of
Padua (prot. 1682/2012) to GM. http://www.unipd.it/
en/home-page. Competing Interests: The authors have declared
that no competing interests exist. 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction It has been demonstrated that TP can be externally generated by the temporal properties of
stimuli. More specifically, externally driven TP (E–TP) may be established by exploiting the
temporal information provided either by regular sensorial structures (i.e., auditory rhythms) or
discrete signals cueing the onset of task-relevant stimuli [4–15]. In both cases, the possibility of
using external information to predict the onset of upcoming events induces the orienting of
attention toward specific points in time. This ability is defined as temporal orienting (TO)
[1,3,4]. By contrast, in the absence of external cues providing information about incoming
events, TP must be deployed over time by using an internally driven process (I–TP). A classic
example of I–TP is the well-known variable foreperiod (FP) effect. When FPs of different dura-
tions are randomly presented in a block of trials with the same a priori probability of occur-
rence, the mean reaction time (RT) to targets decreases with increasing FPs [16–19]. The
specific benefit of long vs. short FP trials is mainly explained by the unidirectional flow of time
itself, which intrinsically and conditionally biases target predictability [6,16,20–23]. The proba-
bility of an event occurring at a given time is conditioned by the accumulating evidence that it
has not yet occurred, a phenomenon also known as hazard function [16,20–22,24]. An addi-
tional source of TP is reflected in the so-called sequential effects (SEs) [17,18,25–27]. Sequen-
tial effects rely on the observation that the temporal structure of trials itself can induce TP by
biasing the allocation of cognitive and motor resources over time. Specifically, a participant’s
ability to predict the onset of forthcoming events will depend on the temporal rule experienced
in the preceding trial. Behaviourally, this translates into slower RTs in the current trial, when
this is preceded by a longer FP in the previous trial, than when preceded by an equally long or
shorter one. Notably, SEs are asymmetric, as they are mainly present with the shortest FP in a
block of trials, but tend to vanish as FP increases. PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Participants One hundred and twenty participants were initially enrolled. Eight children were excluded
since they did not complete the experimental task. One subject was excluded because he
exceeded 2 SD from the age group RT average. The final sample was composed of 111 partici-
pants (88 children and 23 adults). Children were recruited from a primary school in the Vene-
tian Region of Italy and were allocated into one of three groups: 6–7, 8–9 and 10–11 years. Adults were the children’s parents or undergraduate psychology students at the University of
Padua. The demographic characteristics of the four groups are described in Table 1. Ethics statement. All children's parents and adult participants signed a written consent
form. The director of the school also signed an agreement that formally allowed the children to
be tested in the school. All experimental procedures were approved by the Ethics Committee of
the School of Psychology of the University of Padua (protocol n° 1362) and were conducted
according to the principles expressed in the Declaration of Helsinki. Development of Temporal Prediction on internal sources of information; this mechanism translates into behavioural advantage in
terms of motor preparation. Concerning the ability to use E–TP, there is evidence that preverbal infants are already able
to learn the temporal regularities as well as the durations associated with external sensorial
events [38–42]. This suggests that the ability to exogenously orient attention in time, making
use of external information, should also be operating at 6 years of age. The ability of using
external, discrete cues to endogenously orient attention in time has been much less investigated
in children. In a recent study, Johnson and colleagues [43] employed a cueing paradigm to dis-
entangle the automatic and voluntary mechanisms underlying spatial and temporal orienting
in children. Interestingly, they found that, although children were able to automatically orient
their attention in space and time, they showed voluntary orienting of attention in space but not
in time. More specifically, in the temporal cueing blocks children showed FP and SEs, but not
TO effects. As discussed by the authors, the lack of significant TO in their study may be
accounted for by the use of lateralised rather than central targets, which could have introduced
a spatial uncertainty bias that possibly reduced the behavioural benefit provided by the tempo-
ral cues. Moreover, Johnson and colleagues did not directly test the developmental trajectories
of cueing effects, given that they tested a single group of children ranging from six to sixteen
years of age. Hence, the questions are still open as to whether E–TP, generated by discrete tem-
poral cues, may emerge ontogenetically and whether and how it interacts with other forms of
implicit timing involving I–TP (i.e., FP and SEs) across development. To address the above questions, in this cross-sectional study we compared the developmen-
tal trajectories of E–TP and I–TP in six to eleven year-old children and adults according to a
paradigm in which a TO and a FP task were factorially combined to respectively induce E–TP
and I–TP; we also investigated their interplay. We adopted a child-friendly approach including
the presentation of central targets and a fixed 100% cue validity to facilitate children in the
deliberate use of cues to orient their attention in time, as well as to enhance the benefits of tem-
poral cueing by keeping fixed spatial target predictability. Sequential effects in both TP condi-
tions were also measured and analysed. Data Availability Statement: All relevant data are
within the paper. According to the ‘dual-process’ model pro-
posed by Vallesi and collaborators [28–30], while SEs per se are automatic in nature, their
observed asymmetry, depending on the relative durations, reflect a voluntary re-preparation
process triggered by the conditional probability of target appearance over time. Indeed, condi-
tional probability for target onset at the long FP is always equal to 1, irrespective of the previous
FP duration. While the cognitive mechanisms and the underlying neural bases of E–TP and I–TP in
adults are relatively known [1,3,6,10,11,17,18], how they emerge and interact ontogenetically is
less clear. In a pioneering study, Elliot [31] tested a large sample of children aged 5–13 years,
and adults performing a simple auditory FP task, including several preparatory intervals. Unlike many following studies (see below), he only reported a slight and non-significant FP
effect. However, this discrepancy may be attributed to methodological issues, including the
wide ranges of intervals used (1–16 s), making it harder for children to maintain sustained
attention over time. In a later study, Czudner and Rourke [32] tested children with brain-dam-
age, subdivided into two groups, ‘young’ (6 to 9 years) and ‘old’ (10 to 13 years), by using both
regular and irregular FPs. Interestingly, they found that young control, old control, and old
brain-damaged groups performed better than the young brain-damaged group, suggesting
that, with advancing years, brain-damaged children may adapt to and/or recover from deficit
(s) in their ability to develop and maintain a state of readiness to respond. A FP effect in chil-
dren between around 7 and 11 years was also reported by Ozmun et al. [33]. More recently,
Vallesi and Shallice [34] investigated both FP and sequential effects in 4 to 11 year-old children,
showing a developmental dissociation between the two effects; while SEs were already present
at 4–5 years of age, FP appeared gradually from 6–7 years on. These findings suggest that,
although being both implicit forms of temporal preparation, SEs reflect a more automatic pro-
cess than variable FP effects. This suggestion is supported by the observation that only the FP,
but not the SEs, are affected by dual-task interference [35–37]. In summary, the available stud-
ies provide consistent evidence that school-age children can implicitly generate TP by relying 2 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Stimuli and tasks Stimuli were presented on a 17–inch monitor at a resolution of 1,280 × 1,024 pixels. Partici-
pants were seated comfortably in a chair at a viewing distance of 60 cm from the monitor. All
participants performed two TP tasks within the same experimental session. The trial structure
was the same in both tasks. Each trial began with the display of a visual cue, followed by the presentation of a target
stimulus that stayed on the screen for a maximum of 3,000 ms. The visual cue consisted of a
picture of a black camera lens surrounded by a circle (total size of the stimulus: 840 × 840 pix-
els, 144 dpi, 10.62° × 10.54° of visual angle). The target stimulus consisted of a picture of an
animal, which was displayed centrally within the camera lens. The cue-target stimulus-onset-
asynchrony (SOA) was manipulated (either 600 or 1,400 ms). The inter-trial-interval (ITI) was
randomly manipulated between 600 and 1,500 ms. The task consisted of speeded target detec-
tion; participants were required to press the space bar with the index finger of the dominant
hand as quickly as possible at target occurrence. To encourage participants to produce a good
performance, they were given the following instruction: ‘Imagine that you are at the zoo and
you have a camera. Your task is to take a picture of the animals appearing within the camera
lens as soon as possible. To do that, you have to press the space bar with your index finger. Please, be careful to click as quickly as possible once you see the animals otherwise they will dis-
appear. Please, also be careful not to click before the animals appear!’ The experimental task included temporal and neutral cueing blocks, which were adminis-
tered block-wise rather than trial-by-trial in order to reduce top-down control required to
switch continuously from a predictive to a non-predictive setting [36]; this may have caused
additional difficulty for the children. As detailed below, children were also instructed block-by-
block that they could or not predict in advance the animals’ temporal onset. Temporal cueing blocks. In the temporal cueing blocks (Fig 1a) the visual cue provided
fixed temporal information concerning the SOA duration. In particular, the outer circle of the
camera lens was coloured either blue or orange. Each of the two colours was associated with a
SOA duration (600 or 1,400 ms). Development of Temporal Prediction collected all behavioural data. Children reported as having learning disabilities or neurological
or psychiatric disorders were excluded. All had normal or corrected-to-normal vision. Table 1. Experimental groups. Main demographic characteristics of the participants to the study. SD = Standard Deviation. Gender
Handedness
Group
Mean Age ± SD (range)
Female
Male
Left
Right
n
6–7
77 ± 4 months (69–84)
16
14
2
28
30
8–9
90 ± 4 months (84–96)
18
16
4
30
34
10–11
105 ± 4 months (97–127)
12
12
3
21
24
Adults
35.6 ± 15 years (18–59)
11
12
4
19
23
doi:10.1371/journal.pone.0135098.t001 Table 1. Experimental groups. Main demographic characteristics of the participants to the study. SD = Stand collected all behavioural data. Children reported as having learning disabilities or neurological
or psychiatric disorders were excluded. All had normal or corrected-to-normal vision. collected all behavioural data. Children reported as having learning disabilities or neurological
or psychiatric disorders were excluded. All had normal or corrected-to-normal vision. Experimental procedure The children underwent the experimental task individually in a quiet room at their school,
while the adults completed the experimental task at the University. The same experimenter 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Stimuli and tasks In other words, the colour of the visual cue provided fixed
information about the SOA duration, leading to a temporal short (T–Short) and a temporal
long (T–Long) SOA condition. The association between the colour of the outer circle and the
SOA duration was counterbalanced between subjects. In order to maximise the E–TP magni-
tude, the cue was 100% valid (the association between the colour and the SOA remained fixed
throughout the task), no catch trials were included (the target stimulus always appeared after
the SOA) and the target was always presented centrally. Neutral cueing blocks. In the neutral cueing blocks (Fig 1b), the outer circle surrounding
the camera lens was always black, providing no information about SOA duration; the cue 4 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction Fig 1. Experimental paradigm. The Temporal Cueing task (a) was purposely designed to induce externally
driven temporal prediction (E–TP). The visual cue provided fixed temporal information concerning the SOA
duration, which could be short (left panel) or long (right panel), according to the colour of the cue. By contrast,
the Neutral Cueing task (b) was specifically designed to induce internally driven temporal prediction (I–TP). In
this case participants never knew in advance the duration of the SOA, which could nevertheless have the
same short or long duration than in the Temporal Cueing task. Fig 1. Experimental paradigm. The Temporal Cueing task (a) was purposely designed to induce externally
driven temporal prediction (E–TP). The visual cue provided fixed temporal information concerning the SOA
duration, which could be short (left panel) or long (right panel), according to the colour of the cue. By contrast,
the Neutral Cueing task (b) was specifically designed to induce internally driven temporal prediction (I–TP). In
this case participants never knew in advance the duration of the SOA, which could nevertheless have the
same short or long duration than in the Temporal Cueing task. doi:10.1371/journal.pone.0135098.g001 doi:10.1371/journal.pone.0135098.g001 simply acted as a warning signal, which prepared participants for the forthcoming target with-
out furnishing temporal information about it. Nevertheless, regarding the temporal cueing
blocks, the SOA was also manipulated to create a neutral short (N–Short) and a neutral long
(N–Long) SOA condition (600 and 1,400 ms, respectively). To maximise the magnitude of the
I–TP effect, we used an ‘aging’ probability distribution [16,44], with an equal a priori odds
ratio for each SOA duration. Stimuli and tasks This probability distribution is known to obey a hazard function
[6,16,20]; that is, the subjective probability of target onset will increase over time, given that it
has not yet occurred. Experimental design As illustrated in Fig 2, the experimental manipulation yielded a factorial design in which the
CUE (temporal vs. neutral) and the SOA (short vs. long) factors were orthogonally contrasted
to calculate E–TP, I–TP and their combined effect (E/I–TP) on behavioural performance. Spe-
cifically, the E–TP effect was measured by comparing mean RTs between N–Short and T–
Short trials (TO–Short SOA) and between N–Long and T–Long trials (TO–Long SOA). The
effect of I–TP was measured by comparing mean RTs in N-Short trials to mean RTs in N–
Long trials (Uncued–FP Effect). Finally, the combined effect of E- and I–TP was measured by
comparing mean RTs in T–Short trials to mean RTs in T–Long trials (Cued–FP Effect). In the PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 5 / 18 Development of Temporal Prediction Fig 2. Experimental Design. The cue (Temporal vs. Neutral) and the SOA (Short vs. Long) were factorially
combined to obtain a measure of E–TP (revealed as the TO effect at short and long SOA), I–TP (revealed as
the Uncued–FP effect) and E/I–TP (revealed as the Cued–FP effect). doi:10.1371/journal.pone.0135098.g002 Fig 2. Experimental Design. The cue (Temporal vs. Neutral) and the SOA (Short vs. Long) were factorially
combined to obtain a measure of E–TP (revealed as the TO effect at short and long SOA), I–TP (revealed as
the Uncued–FP effect) and E/I–TP (revealed as the Cued–FP effect). doi:10.1371/journal.pone.0135098.g002 doi:10.1371/journal.pone.0135098.g002 doi:10.1371/journal.pone.0135098.g002 latter case, the performance of participants is supposed to be influenced by both the temporal
information of the cue (always valid) and the hazard function (i.e., the elapsing of time itself),
resulting in an interaction of external and internal TP (E/I–TP). Notably, all conditions were
matched for sensorimotor requirements, since the visual stimuli and the required response
were always the same across the experiment; the only difference between conditions was the
level of target predictability. Before starting the experimental session, participants were presented with a block of 20
training trials for each condition, to ensure they understood task instructions and had learnt
the temporal association between cue–type and SOA in the temporal cueing task. After train-
ing, the participants were asked to identify the temporal interval associated with each of the
cues to ensure understanding of the cues, and were reminded to detect the targets as quickly as
possible. All the children were able to correctly identify the cue–SOA association before start-
ing the experimental blocks. Experimental design After concluding the training, each child underwent a total of four
experimental blocks, including two temporal cueing and two neutral cueing blocks. Each block
lasted approximately four minutes and participants were able to take breaks between the
blocks. For all conditions, a total of 40 trials were delivered. The experiment was block-wise
randomised. The order of the tasks was counterbalanced between subjects. E-prime 2 software
(Psychology Software Tools, Pittsburgh, USA) was used to create and administer the stimuli. Development of Temporal Prediction were considered correct. Moreover, participants showing mean RTs deviating 2SD or more
from the group average were excluded. The Kolmogorov-Smirnov test revealed that accuracy
scores (percentages of correct responses) were not normally distributed. Hence, non–paramet-
ric tests were applied for accuracy analyses. Linear mixed models were conducted to test for
between– and within–subject effects on RTs since these were normally distributed. To reliably
control for the possible age–related effect on behavioural performance, RT raw data were loga-
rithmically transformed. As suggested by Puccioni and Vallesi [45], this allows the reduction of
intra– and inter–subject variability by partialling out the age-related effect across conditions. Hence, significant statistical effects resisting logarithmic transformations can be considered as
being due to reliable condition–specific effects rather than to the effects of general factors, such
as age–related motor response speed [46]. To explore the developmental trajectories of E–TP and I–TP and their combined effect (E/
I–TP) on behavioural performance, a 2 (Cue) × 2 (SOA) × 4 (Group) mixed ANOVA was per-
formed. Specifically, the behavioural benefit conferred by E–TP is revealed by the main effect
of Cue while its developmental trajectory was measured by the Cue × Group interaction. The
behavioural benefit conferred by I–TP is revealed by the main effect of SOA, and its develop-
mental trajectory by the SOA × Group interaction. Finally, E/I–TP was tested by the
Cue × SOA interaction, and its developmental trajectory by the 3-way Cue × SOA × Group
interaction. To evaluate the difference in the magnitude of the single TP effects across groups,
planned comparisons (one-way ANOVAs) were performed contrasting RTs on T–Short vs. N–
Short; T–Long vs. N–Long; N–Short vs. N–Long; and T–Short vs. T–Long. In addition, Pear-
son’s correlations were performed only on the children’s data to assess the relationships
between age and RTs for all TP effects (TO–Short, TO–Long, Uncued–FP and Cued–FP). Sequential effects were tested by comparing mean RTs in the current trial (SOAn) to those
obtained in the preceding trial (SOAn-1) across groups for both temporal and neutral cueing
tasks. More specifically, a 2 (Cue) × 2 (SOAn) × 2 (SOAn-1) × 4 (Group) mixed ANOVA was
performed. The overall behavioural benefit of SEs was revealed as the behavioural benefit con-
ferred by preceding trials on the current ones (main effect of SOAn-1), and its developmental
trajectory by the SOAn-1 × Group interaction. The Cue × SOAn-1 interaction tested SEs in rela-
tion to the task (temporal vs. neutral cueing), while its developmental trajectory was revealed
by the Cue × SOAn-1 × Group interaction. The SE asymmetry was globally tested by the SOAn
× SOAn-1 interaction, and its developmental trajectory by the 3–way SOAn × SOAn-1 × Group
interaction. Finally, the SE asymmetry in relation to the task (temporal vs. neutral cueing) was
revealed by the 3–way Cue × SOAn × SOAn-1 interaction, and how it is influenced by age by
the 4–way Cue × SOAn × SOAn-1 × Group interaction. For all analyses, an alpha level of 0.05
was used. Post-hoc Fisher's least significant difference (LSD) comparisons were performed to
evaluate pairwise differences among the means. The effect size was estimated by the partial eta
square measure (ηp
2). When appropriate, critical values were adjusted using the Greenhouse–
Geisser correction for violation of the assumption of sphericity. The raw response time data from the experiment are available in S1 Dataset. Data analysis As independent variables, age group (6–7 years vs. 8–9 vs. years vs. 10–11 years vs. adults), cue
(temporal vs. neutral), and SOA duration (short vs. long) were considered. Mean accuracy and
mean RTs were the dependent variables. Omissions, anticipated responses (within the cue and
100 ms after target onset), and delayed responses (1,500 ms after target onset) were considered
errors and excluded from analysis. Response times exceeding 2 SD from the individual average
for each dependent variable and for each participant were excluded. The remaining responses 6 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction Table 2. Behavioural measures. Mean and standard deviation (in parentheses) measures of accuracy (percentage of correct responses) and speed (reac-
tion times) for all groups and conditions. Accuracy (%)
Mean RTs (ms)
Group
T-Short
T-Long
N-Short
N-Long
T-Short
T-Long
N-Short
N-Long
6–7
93.4(1.1)
91.5(1.7)
95.7(1)
92.2(1.4)
490(12)
491(12)
562(15)
501(13)
8–9
94.5(1.8)
96.7(0.9)
98.7(2)
96.5(0.9)
433(13)
401(10)
481(12)
422(11)
10–11
95.2(0.9)
96.6(0.9)
98.6(0.5)
96.8(0.7)
390(11)
359(11)
433(13)
379(12)
Adults
95.7(1.1)
95.6(1.7)
99(0.5)
94.5(1.8)
313(12)
291(8)
355(10)
306(10)
Note that the analyses of variance reported in the text were performed using RT log-transformed data whereas the in the table we report raw RT data for
reasons of clarity. d i 10 1371/j
l
0135098 t002 Table 2. Behavioural measures. Mean and standard deviation (in parentheses) measures of accuracy (perce
tion times) for all groups and conditions. Note that the analyses of variance reported in the text were performed using RT log-transformed data whereas the in the table we report raw RT data for
reasons of clarity. Note that the analyses of variance reported in the text were performed using RT log-transformed data whereas th
reasons of clarity. doi:10.1371/journal.pone.0135098.t002 Mean RTs for each TP effect and Group are plotted in Fig 3. The 2 × 2 × 4 mixed ANOVA
revealed an expected Group effect (F(3,107) = 51.1, p < 0.0001, ηp
2 = 0.59). Post-hoc tests con-
firmed that RTs were progressively faster with age (6–7 years > 8–9 years > 10–11
years > adult RTs, all ps 0.04). The ANOVA also revealed the presence of the TO effect
(main effect of Cue; F(1, 107) = 92.33, p < 0.0001, ηp
2 = 0.46), with participants being faster in
detecting temporally than neutrally cued targets (Fig 3a and 3b), as well as the presence of the
FP effect (main effect of SOA; F(1, 107) = 60.89; p < 0.001, ηp
2 = 0.36), with RTs significantly
shorter in long than short SOA trials (Fig 3c). The Cue factor did not interact with Group ((F(3, 107) = 0.82; p > 0.95; ηp
2 = 0.02), while a
significant two-way Cue × SOA interaction emerged (F(1, 107) = 60.89; p < 0.001; ηp
2 = 0.36). Results Accuracy and RT scores are reported in Table 2. Overall, delayed and null responses decreased as a function of age; however, significant dif-
ferences emerged only in the T–Long condition (Kruskal-Wallis test H (3, N = 111) = 13.2,
p < 0.01), where the 6 to 7 year-old children showed a significantly lower accuracy level
(M = 91.5, SD = 1.34) compared to older children and adults. 7 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction Fig 3. Mean reaction times. Panels show the pair-wise comparison of the mean reaction times for the TO–Short (a), TO–Long (b), Uncued–FP (c) and
Cued–FP (d) effects for each age group. Error bars indicate standard errors of the mean. doi:10.1371/journal.pone.0135098.g003 Fig 3. Mean reaction times. Panels show the pair-wise comparison of the mean reaction times for the TO–Short (a), TO–Long (b), Uncued–FP (c) and
Cued–FP (d) effects for each age group. Error bars indicate standard errors of the mean. doi:10.1371/journal.pone.0135098.g003 Fig 3. Mean reaction times. Panels show the pair-wise comparison of the mean reaction times for the TO–Short (a), TO–Long (b), Uncued–FP (c) and
Cued–FP (d) effects for each age group. Error bars indicate standard errors of the mean. doi:10.1371/journal.pone.0135098.g003 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Post-hoc tests revealed all comparisons to be significant, although the behavioural benefit con-
ferred from temporal cues was greater in short (mean TO effect = 51 ms; Cohen’s d = 0.53)
than long trials (mean TO effect = 16 ms; Cohen’s d = 0.17) SOAs (p < 0.001). This asymmet-
ric effect was explained by the mean RTs in the T-Long trials being significantly faster than the
mean RTs in the T-Short trials (t(110) = -5.44; p < 0.001) and in N-Long (t(110) = -4.74;
p < 0.001). A significant interaction between Group and SOA was also found (F(3, 107) = 3.93;
p < 0.02, ηp
2 = 0.09). Planned comparisons confirmed that, when considering together tempo-
ral and neutral cueing conditions, the SOA effect (faster RTs for long than short SOAs)
emerged gradually, with the younger children showing a smaller effect (mean SOA effect = 29
ms; Cohen’s d = 0.22) than older children and adults (all mean SOA effects 35 ms; all
Cohen’s ds 0.37). Most importantly, we found a significant 3-way Cue × SOA × Group interaction (F(3, 107)
= 2.79; p < 0.05, ηp
2 = 0.07; Fig 3d). To better identify the source of this interaction, planned
comparisons across age groups (one-way ANOVAs) were performed. These allowed to statisti-
cally test for the presence of possible developmental dissociations for each TP effect. One-way
ANOVAs revealed no group effects on the TO–Short SOA, the TO–Long SOA or the Uncued–
FP effects (all Fs(3, 107) < 1.18; all ps > 0.32). By contrast, the Cued–FP effect was significantly
affected by age (F(3, 107) = 4.64; p < 0.005, ηp
2 = 0.14). The post-hoc analysis revealed that 6
to 7 year-old children significantly differed from older groups (all ps 0.02). More specifically,
as depicted in Fig 4, the Cued-FP effect was absent (around zero) in the youngest group, but
present and stable at later ages. In other words, the combined effect of internally and externally
driven TP (E/I–TP) emerged after 8 years of age. 8 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Sequential effects The 4 × 2 × 2 × 2 mixed ANOVA yielded a main SOAn-1 effect (F(1, 107) = 194.31; p < 0.0001,
ηp
2 = 0.64), confirming that RTs in the current trials were affected by the preceding ones (SEs). This effect was further influenced by development, as shown by the SOAn-1 × Group interac-
tion (F(3, 107) = 2.92; p < 0.05, ηp
2 = 0.07). This interaction was explained by the observation
that the advantage provided by the preceding trial on the current one diminished as age
increased, being greatest for the youngest group (43 ms on average) and smallest for the oldest
one (15 ms on average). We also found a SOAn × SOAn-1 interaction (F(1, 107) = 64.7; p < 0.001, ηp
2 = 0.38). This
interaction was explained by the fact that SEs were asymmetric, that is, RTs were overall We also found a SOAn × SOAn-1 interaction (F(1, 107) = 64.7; p < 0.001, ηp
2 = 0.38). This
interaction was explained by the fact that SEs were asymmetric, that is, RTs were overall
shorter on the current trials when these were preceded by short rather than long SOA trials. The ANOVA further revealed a 3–way Cue × SOAn × SOAn-1 interaction (F(3, 107) = 14.91; shorter on the current trials when these were preceded by short rather than long SOA trials. The ANOVA further revealed a 3–way Cue × SOAn × SOAn-1 interaction (F(3, 107) = 14.91; 9 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction Fig 4. Developmental trajectories of each TP effect. The upper panel shows the developmental
trajectories of each single TP effects derived by pair-wise comparisons. In particular, the upper panel shows
TO–Short (T–Short minus N–Short) and TO–Long (T–Long minus N–Long) effects, both measures of E–TP. The lower panel displays the developmental trajectories of the Uncued–FP (N–Long minus N–Short) effect
as a measure of I–TP and of the Cued–FP (T–Long minus T–Short) effect as a E/I–TP combined measure. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the text were
performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. doi:10 1371/journal pone 0135098 g004 Fig 4. Developmental trajectories of each TP effect. The upper panel shows the developmental
trajectories of each single TP effects derived by pair-wise comparisons. doi:10.1371/journal.pone.0135098.g004 Development of Temporal Prediction Fig 5. Sequential Effects. Mean RTs are plotted as a function of the current (SOAn; x axis) and preceding (SOAn-1; lines) cue-target intervals for each age
group and for Temporal (a) and Neutral (b) cueing Tasks. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the
text were performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. Fig 5. Sequential Effects. Mean RTs are plotted as a function of the current (SOAn; x axis) and preceding (SOAn-1; lines) cue-target intervals for each age
group and for Temporal (a) and Neutral (b) cueing Tasks. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the
text were performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. doi:10.1371/journal.pone.0135098.g005 shown by 6 to 7 year-olds in the temporal cueing task were symmetrical as compared to those
seen in the neutral cueing task and older children. Following Vallesi and Shallice [34] statistical approach, to corroborate this observation sub-
sequent 2 (Cue) × 2 (SOAn) × 2 (SOAn-1) repeated-measures ANOVAs were performed sepa-
rately on each group, with SOAn and SOAn-1 as the within-subjects variables. It emerged that
the Cue × SOAn × SOAn-1 interaction was significant only in the 6 to 7 year–old group (F(1,
29) = 4.72, p < 0.05) but not in the older ones (F(1, 33) = 2.26 for 8–9 year olds; F(1, 23) = 3.63
for 10–11 year olds; F(1, 22) = 3.44 for adults). Post-hoc tests conducted on the 6–7 year-old
group confirmed that, in the temporal cueing task RTs in the current trials were equally
affected when preceded by short or long SOAn-1 intervals (both p < 0.01). Whereas, in the neu-
tral cueing task the SE was present in short SOAn (p < 0.001) but not in long SOAn trials
(p > 0.07). In other words, they showed symmetrical SEs. By contrast, in the neutral cueing
task SEs were present only in short SOAn trials, with all participants being faster when these
were preceded by short rather than long SOAn-1 trials (symmetrical SEs). Sequential effects In particular, the upper panel shows
TO–Short (T–Short minus N–Short) and TO–Long (T–Long minus N–Long) effects, both measures of E–TP. The lower panel displays the developmental trajectories of the Uncued–FP (N–Long minus N–Short) effect
as a measure of I–TP and of the Cued–FP (T–Long minus T–Short) effect as a E/I–TP combined measure. Error bars indicate standard errors of the mean. Note that the analyses of variance reported in the text were
performed using log-transformed data whereas the the figure shows raw data for reasons of clarity. doi:10.1371/journal.pone.0135098.g004 p < 0.001, ηp
2 = 0.17). This interaction revealed that, in the temporal cueing condition, the SEs
were less asymmetric than in the neutral cueing condition. The 4–way Cue × SOAn × SOAn-1 × Group interaction was not significant (F(3, 107) = 0.13;
p > 0.94, ηp
2 = 0.01). It should be noted that a lack of significant interaction between SE asym-
metry and age group was previously reported by Vallesi and Shallice [34], who tested 4 to 12
year-old children with a variable FP paradigm. As discussed by these authors, this null interac-
tion effect may be accounted for by the high intra–and inter–subject variability of RTs in chil-
dren that could be only reduced, but not completely eliminated, by log-transformation [46]. Although the 4-way interaction was not significant, the visual inspection of the developmental
trajectory of each TP effect plotted separately for cueing conditions (Fig 5) suggests that SEs PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 10 / 18 Discussion The main aim of the present study was to investigate the developmental trajectories of different
types of temporal prediction (TP) mechanisms in typically developing school-aged children. To our knowledge, this study is the first purposely designed to delineate the developmental
trends of internal (i.e., the information provided by the passage of time itself) vs. external (i.e.,
the temporal information provided by discrete cues) TP mechanisms in such population. Over-
all, the results demonstrate that all children participating at the study (from six to eleven years)
were able to implement internally (E–TP) and externally (I–TP) driven TP mechanisms, show-
ing an efficient ability to orient attention in time and, consequently, to optimise their beha-
vioural performance. The ability to implement E–TP was demonstrated by the observation that
children benefitted from the information provided by external discrete temporal cues to
improve their performance in terms of target detection speed (i.e., TO effect). Furthermore,
findings highlighted two noteworthy issues. First, the TO effect did not interact with age, fol-
lowing a stable developmental trajectory. Indeed, while RTs showed a linear shortening with
increasing age, the RT difference between temporally vs. neutrally cued targets remained nearly
constant across age. Second, the presence of this effect was independent from the SOA dura-
tion, although its magnitude was larger for short than long SOAs. In other words, all partici-
pants exhibited faster RTs in temporal cueing as compared to neutral cueing blocks for both
short and long SOA durations, suggesting that the cognitive mechanisms underlying E-TP gen-
erated by discrete cues are fully established as early as six years of age and do not show signifi-
cant changes over time. A recent study by Johnson and colleagues [43] employed a cueing paradigm to test the
mechanisms underlying the ability to orient attention in space and time in children. Interest-
ingly, they found that children can voluntarily and automatically orient attention in space, as
revealed by the presence of both cueing and sequential effects on children’s behavioural perfor-
mance. However, in contrast to our findings, they found only automatic, but not voluntary,
temporal orienting in children. That is, children did not benefit from the temporal information
provided by symbolic cues (TO effect) although their performance was affected by the condi-
tional probability of target occurrence in time (FP effect) as well as the sequential temporal
structure of trials (SEs). Correlational analyses The correlational analyses conducted between the age and each single TP effect revealed that
the only index showing a significant correlation with age was the Cued–FP effect (r = 0.43, 95%
BCa CI [.27, .59], Bonferroni adjusted-p < 0.02), while the other effects were not affected by
age (all rs < 0.17; all ps > 0.07). That is, while the behavioural benefit conveyed separately by
E–TP and I–TP was developmentally stable, the effect conferred by their combined use
increased significantly with age. 11 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction event-related design including both temporal and neutral cues in the same block, children
failed to show TO effect, even if the target was centrally presented. We suggest that memory
demands associated with maintaining, retrieving and updating the cue meaning from trial to
trial, as well as the necessity to switch from a predictive to a non predictive context, may have
selectively impaired temporal, but not spatial, voluntary orienting. As suggested by the authors,
it should be taken into account that using cues to orient attention in time may be conceptually
more demanding than using spatial cues. Finally, we cannot completely exclude the possibility that the different kinds of stimuli
employed in the two studies may have further affected the possibility of eliciting the TO effect
in children. Indeed, in our study, we used child-friendly stimuli (i.e., coloured circles and pic-
tures of animals for the cue and the target, respectively) while Johnson and colleagues pre-
sented children with symbolic stimuli representing different parts of a clock-face for the cue
and the ‘+’ or ‘x’ signs for the target stimuli, which may be more difficult to discriminate. Most
importantly, Johnson and colleagues tested a single group of six to sixteen year-old children,
whereas in our study children were younger than eleven years. From eleven to sixteen years,
neurodevelopmental outcomes substantially change. In summary, in the light of the above con-
siderations it is difficult to draw reliable conclusions from the comparisons of the two studies. A second finding of our study was that children were able to use internal information (i.e.,
the passage of time itself) to predict target occurrence and consequently to orient attention in
time (I–TP). According to our experimental paradigm, the I–TP could be measured as the ben-
efit in RT speeding up, conferred by long vs. short SOAs in the context of either the temporal
(Cued–FP) or the neutral (Uncued–FP) cueing task. All participants showed a global shorten-
ing of RTs as well as a reduction of E–TP effect in long SOAs due to the variable FP effect [13–
17]. The FP effect is generally explained by the hazard function [6,16,26], which is defined as
the conditional probability of an event occurring at a given time, given that it has not occurred
yet. Discussion There are several reasons possibly accounting for the lack of consis-
tence between the results of Johnson and colleagues and those we report in the present study. Firstly, as discussed by the authors, the target presentation in their paradigm was lateralised
rather than central; this experimental choice may have disrupted the cueing effect in children. Indeed, it has been shown that the benefit of temporal cues is enhanced when the target posi-
tion is fixed and therefore spatially predictable [47]. Hence, it is possible that in our study the
central presentation of target stimuli and the consequent fixed spatial focus of attention made
it easier for children to benefit from temporal cues. A second possible explanation may rely on
the difference in the degree of temporal certainty experienced by children in the two studies. While Johnson and colleagues manipulated the cue validity (i.e., 80% of the cues provided
valid, and 20% invalid, information), we kept the validity of the cue constant to 100%. It follows
that, in order to optimise behaviour, in our study children could benefit both from the tempo-
ral cue and from the maximum temporal certainty that the target would have appeared always
at the time predicted by the cue. By contrast, the invalid conditions used by Johnson and col-
leagues may have introduced an additional difficulty given that, in such a case, children could
not completely trust the temporal information provided by the cue. It is also possible that the
mixing of temporal and neutral cueing conditions within the same experimental block in John-
son et al.’s study may have made the task too challenging for children (especially for the youn-
ger ones). In line with this, in a pilot study (unpublished data), we found that when using an PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 12 / 18 As suggested by Vallesi and Shallice
[34], the lack of significant interaction between age, task and the emerging of the typical SE
asymmetry can be accounted for by the high inter– and intra–subject variability, that can be
only reduced, but not completely eliminated, after applying a logarithmic transformation on
raw RTs [46]. Although the null 4-way interaction represents a limitation of the present study,
we nevertheless tried to provide further statistical evidence in support of the observed data. To
this aim, we conducted additional 3-way ANOVAs separately for the temporal and neutral cue-
ing task. These confirmed that, in the youngest children, the SE pattern was symmetrical only
in the temporal cueing task, given that their behavioural performance in SOAn trials was
equally affected irrespective of the duration of the preceding SOAn-1 trials. Remarkably, in the
neutral cueing task all groups, including the youngest one, exhibited the typical asymmetric
pattern described in the literature, with RTs in the current short, but not long, trials being
affected by SOAn-1 durations. The apparent presence of a developmental dissociation including either the Cued–FP effect
and the symmetrical SEs demonstrates that, while from six years of age onwards children can
successfully and independently make use of external and internal TP mechanisms to orient
attention in time, the ability to combine these two different sources of information emerges
only from eight years of age. A possible explanation for this is that, before eight years of age,
their limited available cognitive resources do not allow children to make a combined use of
external and internal strategies. From a theoretical perspective, according to the dual process
view [18] a controlled monitoring of the change of conditional probability would explain both
the FP effect and the asymmetry of SEs. In the first case, the subjective target predictability will
be highest in the longest SOAs due to the hazard function. In the second case, in any trial the
participant implicitly expects a repetition of the previous SOA, hence, the maximum prepared-
ness is biased toward the same SOA as that of the previous trial. If SOAn is shorter than SOAn-
1, then participants will be not fully prepared when the imperative stimulus occurs, resulting in
relatively slower RTs. In other words, the boost of expectation conditionally induced from the passage of time
heightened the probability of target occurrence, inducing faster responses in long cue–target
intervals. Similarly to TO, in the neutral cueing task the Uncued–FP effect did not show any
interaction with age, suggesting that the mechanisms underlying I–TP are already developed
and functioning at six years of age. This evidence is in line with most of previous studies report-
ing FP effects in children [32–34,43, 48–49]. As a third important finding, we found that, although even young children were able to
independently use internal and external mechanisms to generate TP, and consequently to ori-
ent their attention in time, their ability to combine these two sources of information emerged
only after eight years of age. According to our experimental design, the Cued–FP effect mea-
sures the combined use of external and internal TP processes. Namely, in the temporal cueing
blocks, the occurrence of targets is highly predictable based on the full validity of the cue (TO
effect) as well as on the hazard function that conditionally biases target predictability (FP
effect). Hence, the combined effect of both these sources of information on target occurrence
leads to the highest level of target expectancy, and therefore to the lowest RTs in T–Long trials. The evidence that in the temporal cueing blocks the FP effect (Cued–FP effect) emerged only
after eight years of age demonstrates that the ability to make a combined use of E–TP and I–TP
is developmentally constrained. We found a developmental dissociation also in SEs; in all age groups the behavioural perfor-
mance in the current trials (SOAn) was overall affected by the duration of the preceding ones
(SOAn-1). Yet, the magnitude of this effect depended on the SOAn duration; that is, the SEs
were greater in short rather than long SOAn, confirming the typical asymmetrical pattern
reported in the literature [17,18,26]. Critically, as suggested by the visual inspection of mean
RTs (Fig 5), the SE asymmetrical pattern seemed to be further affected by the interplay between 13 / 18 PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 Development of Temporal Prediction age group and task. Namely, in the temporal cueing task, the behavioural advantage conferred
by the preceding trials on the current ones appeared to be comparable for short and long SOAn
(i.e., SE symmetrical pattern) only in the six to seven-year old children, while this difference
became biased toward the short SOAn (i.e., asymmetrical SE pattern) at later ages. By contrast,
in the neutral cueing task, the magnitude of the SE asymmetry appeared to be constant across
all groups. Yet, we did not find an ominibus statistical validation of this effect, since the
ANOVA did not yield a significant 4-way interaction. As suggested by Vallesi and Shallice
[34], the lack of significant interaction between age, task and the emerging of the typical SE
asymmetry can be accounted for by the high inter– and intra–subject variability, that can be
only reduced, but not completely eliminated, after applying a logarithmic transformation on
raw RTs [46]. Although the null 4-way interaction represents a limitation of the present study,
we nevertheless tried to provide further statistical evidence in support of the observed data. To
this aim, we conducted additional 3-way ANOVAs separately for the temporal and neutral cue-
ing task. These confirmed that, in the youngest children, the SE pattern was symmetrical only
in the temporal cueing task, given that their behavioural performance in SOAn trials was
equally affected irrespective of the duration of the preceding SOAn-1 trials. Remarkably, in the
neutral cueing task all groups, including the youngest one, exhibited the typical asymmetric
pattern described in the literature, with RTs in the current short, but not long, trials being
affected by SOAn-1 durations. age group and task. Namely, in the temporal cueing task, the behavioural advantage conferred
by the preceding trials on the current ones appeared to be comparable for short and long SOAn
(i.e., SE symmetrical pattern) only in the six to seven-year old children, while this difference
became biased toward the short SOAn (i.e., asymmetrical SE pattern) at later ages. By contrast,
in the neutral cueing task, the magnitude of the SE asymmetry appeared to be constant across
all groups. Yet, we did not find an ominibus statistical validation of this effect, since the
ANOVA did not yield a significant 4-way interaction. However, when SOAn is longer than SOAn-1, it is assumed that partici-
pants can update and re-orient their attention over time and consequently re-prepare for opti-
mal preparation, resulting in SE abolishment for long intervals. Within this theoretical
framework, we speculated that, for young children, it may be difficult to efficiently monitor the
time–based conditional probability of target onset while concurrently using the temporal infor-
mation provided by the cues, which implies that the Cue–SOA associations has to be learnt,
maintained and retrieved trial-by-trial. This hypothesis is corroborated by the observation that
only the SE asymmetry, but not the presence of SEs per se (which is assumed to be more auto-
matic by the ‘dual process’ view), was disrupted in young children. In other words, the effort
made in using external, discrete cues to generate predictions over time may have disrupted the
monitoring of the conditional probability of target occurrence in young children. As an alternative explanation, we propose that six to seven year-old children may not yet have
developed the capacity to disengage attention from externally to internally driven information
when the two have to be simultaneously processed, requiring considerable effort to re-orient atten-
tion over time in long SOAn trials. In fact, younger children can effortlessly disengage and re-ori-
ent attention in the current trials (FP effect) as well as from the duration of the preceding trial (SE
asymmetry) when they only need to use I-TP. However, when they must make a combined use of PLOS ONE | DOI:10.1371/journal.pone.0135098
August 11, 2015 14 / 18 Development of Temporal Prediction I–TP and E–TP, the attentional disengagement may result more effortful for them, as revealed by
the absence of both the Cued–FP effect and by the SE asymmetry only in temporal cueing blocks. In support of this hypothesis, we also found that the Cued–FP effect, which is assumed to depend
on attentional resources more than other TP measures, is the only index showing a positive corre-
lation with age. That is, the older the child, the stronger the Cued–FP. A further, simpler hypothesis explaining why younger children did not show FP effect and SE
asymmetry in the temporal cueing task, is also that at this age children invest considerable atten-
tional resources on externally available cues, given that this is the first useful information always
providing valid target predictability. Acknowledgments The authors desire to thank all children and their families who participated to the study. A
thank goes to Federica De Osti for helping in collecting data as well as to Dr. Paola Bortoletto
and to all the staff of the ‘Istituto Comprensivo di Spresiano’, Treviso (Italy) for making possi-
ble this study. We would also like to thank Dr. Prematurata for inspiring the present study. Supporting Information S1 Dataset. Mean accuracy and response time for each participant. (XLSX) In other words, they can immediately and completely use
the cues to speed up their RTs to targets. By contrast, in the case of I–TP, children are only
required to perceive the elapsing of time, given that the cues do not provide useful information to
generate predictions. Then, they have to use the internal information to conditionally prepare for
the next time in which targets are likely to occur. Even if young children can effortlessly build up
a mental representation of durations, as indirectly shown by the presence of Uncued–FP effect,
this operation could be more demanding for them when it must be combined with the processing
of information externally provided by discrete cues, which do not require per se a dynamic and
metric temporal representation. Though, further studies are needed to better disentangle the
above different accounts. A possible way to better unravel the developmental mechanisms under-
lying dissociations in TP effects may come from neurophysiological studies depicting the timing
of the neural mechanisms underlying I–TP and E–TP across development. Although our present
data do not allow neurophysiological hypotheses to be tested directly, a possible hypothesis to be
addressed may be that while the single cortical networks underlying E–TP and I–TP are already
established at six years of age, they cannot be simultaneously recruited before the age of eight. It
may be also possible that their functional connectivity may not be yet developed until this time. In support of this view, structural neuroimaging studies using diffusion tensor imaging (DTI)
demonstrated a developmental increase in grey matter activation in frontal and parietal cortices
which would be in turn the direct consequence of the maturation of white matter connection
[50]. More specifically, the inferior parietal cortices, which have been shown to play a key role in
temporal orienting [3,4,23,51–54] and the lateral pre-frontal cortices, which are implied in the
monitoring of the hazard function [5,6,18,21] are among the last areas to myelinate, a change
that would result in more stable fronto-parietal activity. Although the neuroimaging approach
promises to provide a useful key for a deeper understanding of the developmental mechanisms
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Complementing the US Food and Drug Administration Adverse Event Reporting System With Adverse Drug Reaction Reporting From Social Media: Comparative Analysis (Preprint)
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Abstract Background: Adverse drug reactions (ADRs) can occur any time someone uses a medication. ADRs are systematically tracked
and cataloged, with varying degrees of success, in order to better understand their etiology and develop methods of prevention. The US Food and Drug Administration (FDA) has developed the FDA Adverse Event Reporting System (FAERS) for this purpose. FAERS collects information from myriad sources, but the primary reporters have traditionally been medical professionals and
pharmacovigilance data from manufacturers. Recent studies suggest that information shared publicly on social media platforms
related to medication use could be of benefit in complementing FAERS data in order to have a richer picture of how medications
are actually being used and the experiences people are having across large populations. Objective: The aim of this study is to validate the accuracy and precision of social media methodology and conduct evaluations
of Twitter ADR reporting for commonly used pharmaceutical agents. Methods: ADR data from the 10 most prescribed medications according to pharmacy claims data were collected from both
FAERS and Twitter. In order to obtain data from FAERS, the SafeRx database, a curated collection of FAERS data, was used to
collect data from March 1, 2016, to March 31, 2017. Twitter data were manually scraped during the same time period to extract
similar data using an algorithm designed to minimize noise and false signals in social media data. Results: A total of 40,539 FAERS ADR reports were obtained via SafeRx and more than 40,000 tweets containing the drug
names were obtained from Twitter’s Advanced Search engine. While the FAERS data were specific to ADRs, the Twitter data
were more limited. Only hydrocodone/acetaminophen, prednisone, amoxicillin, gabapentin, and metformin had a sufficient
volume of ADR content for review and comparison. For metformin, diarrhea was the side effect that resulted in no difference
between the two platforms (P=.30). For hydrocodone/acetaminophen, ineffectiveness as an ADR that resulted in no difference
(P=.60). For gabapentin, there were no differences in terms of the ADRs ineffectiveness and fatigue (P=.15 and P=.67, respectively). For amoxicillin, hypersensitivity, nausea, and rash shared similar profiles between platforms (P=.35, P=.05, and P=.31,
respectively). Conclusions: FAERS and Twitter shared similarities in types of data reported and a few unique items to each data set as well. Complementing the US Food and Drug Administration Adverse
Event Reporting System With Adverse Drug Reaction Reporting
From Social Media: Comparative Analysis Complementing the US Food and Drug Administration Adverse
Event Reporting System With Adverse Drug Reaction Reporting
From Social Media: Comparative Analysis Zeyun Zhou*, PharmD, MPH; Kyle Emerson Hultgren*, PharmD College of Pharmacy, Purdue University, West Lafayette, IN, United States
*all authors contributed equally Corresponding Author:
Kyle Emerson Hultgren, PharmD
College of Pharmacy
Purdue University
575 W Stadium Ave
West Lafayette, IN
United States
Phone: 1 765 494 5820
Email: khultgre@purdue.edu Corresponding Author:
Kyle Emerson Hultgren, PharmD
College of Pharmacy
Purdue University
575 W Stadium Ave
West Lafayette, IN
United States
Phone: 1 765 494 5820
Email: khultgre@purdue.edu Abstract The use of Twitter as an ADR pharmacovigilance platform should continue to be studied as a unique and complementary source
of information rather than a validation tool of existing ADR databases. (JMIR Public Health Surveill 2020;6(3):e19266) doi: 10.2196/19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 1
(page number not for citation purposes) JMIR PUBLIC HEALTH AND SURVEILLANCE JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren Original Paper KEYWORDS adverse drug reactions; FAERS; social media reporting; pharmacovigilance https://publichealth.jmir.org/2020/3/e19266 XSL•FO
RenderX XSL•FO
RenderX JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren social media and FAERS [10,11]. These studies have shed light
on the role of social media in ADR reporting. However, many
studies only examined one or two less commonly used
pharmaceutical agents, and some included more than 1000
drugs. While the inclusion tested a general scheme of social
media reporting, it overlooked the role of social media reporting
for common drugs. Background Adverse drug reactions (ADRs) are the unintended effect of
medicine at doses used for prophylaxis, diagnosis, or treatment
[1]. ADRs can occur anytime when a patient takes a medication. Factors including drug and food interactions, medication errors,
allergies, and metabolism contribute to the occurrence of ADRs. ADRs have been identified as one of the leading causes of death
in the United States. ADRs resulted in more deaths than the
pulmonary diseases, diabetes, HIV/AIDS, and pneumonia [2,3]. A systematic review on ADR-induced hospital admissions found
that 5.3% of hospital admissions were associated with ADRs
[4]. New drug therapies, the aging population, and polypharmacy
expose the population to increased risks of ADRs [5]. The
burden of ADRs necessitates appropriate detection and
assessment, and reporting is fundamental to successful
pharmacovigilance systems. The Center for Medication Safety Advancement (CMSA) at
Purdue University College of Pharmacy aims to adopt previous
research strategies and compare ADR reports in social media
and FAERS. Twitter was selected as the social media for
evaluation thanks to its simplicity and timeliness in information
sharing and access. Twitter users can report an ADR in one
tweet pursuant to the FDA guideline, which requires as a
minimum dataset to constitute a viable report an identifiable
patient, an identifiable reporter, a product exposure, and an
adverse event [12]. Additionally, the FDA does not require
reports to demonstrate causation or to be specific regarding the
type of error. All suspected medication errors, ADRs, or adverse
events are accepted as reports. Given the advantage of the
Twitter database, the objective of this study is to validate the
accuracy and precision of the research methodology and conduct
evaluations of social media ADR reporting via tweets for
commonly used pharmaceutical agents. The US Food and Drug Administration (FDA) Adverse Event
Reporting System (FAERS) is a database for reports of adverse
events, medication errors, and product quality complaints [6]. Although FAERS serves as a valuable data source for
postmarket pharmacovigilance, only drug manufacturers are
required to send reports received from health care professionals
and consumers to the FDA. Health care professionals and
consumers may voluntarily submit reports, which may lead to
incomplete data in FAERS. In order to obtain more
comprehensive information on drug products, multiple data
sources should be used to fill the information gap. Ethics Statement All social media data used in data collection and analysis were
extracted from public sources. Example tweets were paraphrased
and edited to prevent unmasking through a reverse search on
Twitter. FAERS reports on SafeRx were also anonymized. As
data used in this study were publicly available, no institutional
review board approval was sought. Social media has been proposed as a potential data source as it
allows an easily accessible information sharing platform with
almost no chronological and geographical constraints. A
systematic review of 51 studies compared ADR reports on social
media and other pharmacovigilance systems, and the review
noted that the prevalence of all ADR reports ranged from 0.2%
to 8% and social media contained more reports of mild ADRs
than severe ADRs [7]. Previous studies showed that ADRs were
underrepresented in clinical trial data, and less severe ADRs
were more frequently reported on social media. Social media
ADR reports reflected the ADRs reported on FAERS on average
11 months earlier [8,9]. Comparative studies suggested the
practicality of using social media as a complementary resource
and demonstrated a moderate agreement on ADR data between JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 2
(page number not for citation purposes) Overview This study was divided into 3 sections: drug selection, FAERS
data collection, and Twitter data collection. Collecting FAERS
data included searching for ADR reports of a pharmaceutical
agent and calculating relative frequencies of the 5 most
frequently reported ADRs, whereas Twitter data collection
required an additional step to identify relevant tweets according
to inclusion and exclusion criteria. Figure 1 demonstrates the
overall scheme for the methodology of this study. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 2
(page number not for citation purposes) https://publichealth.jmir.org/2020/3/e19266 XSL•FO
RenderX Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE cheme. ADR: adverse drug reaction; FAERS: FDA Adverse Event Reporting System. Figure 1. Methodology scheme. ADR: adverse drug reaction; FAERS: FDA Adverse Event Reporting System. brand names [15,16]. Accordingly, this study included common
brand names in the searching criteria as Twitter users could be
discussing ADRs using common brand names. For the data
collection purpose of this study, the most commonly used brand
name for each selected drug was identified according to
Micromedex: Norco for hydrocodone/acetaminophen, Synthroid
for levothyroxine, Deltasone for prednisone, Prinivil for
lisinopril, Amoxil for amoxicillin, Neurontin for gabapentin,
Glucophage for metformin, Lipitor for atorvastatin, Xanax for
alprazolam, and Norvasc for amlodipine. brand names [15,16]. Accordingly, this study included common
brand names in the searching criteria as Twitter users could be
discussing ADRs using common brand names. For the data
collection purpose of this study, the most commonly used brand
name for each selected drug was identified according to
Micromedex: Norco for hydrocodone/acetaminophen, Synthroid
for levothyroxine, Deltasone for prednisone, Prinivil for
lisinopril, Amoxil for amoxicillin, Neurontin for gabapentin,
Glucophage for metformin, Lipitor for atorvastatin, Xanax for
alprazolam, and Norvasc for amlodipine. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 3
(page number not for citation purposes) Pharmaceutical Agents To identify the 10 most popular prescribed medications,
prescription data were used from GoodRx, a health care
company that operates a telemedicine platform. GoodRx
generates a list of the top 10 drugs from monthly claims
submitted by pharmacies in the United States; in November
2017,
those
drugs
were
hydrocodone/acetaminophen,
levothyroxine, prednisone, lisinopril, amoxicillin, gabapentin,
metformin, atorvastatin, alprazolam, and amlodipine [13]. Previous studies included both brand and generic names in data
collection to expand the data that could be obtained [10,14]. Some studies further suggested that patients tended to use the
most common brand name in social media if a drug had multiple XSL•FO
RenderX JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren Statistical Analysis The analysis of ADR data from SafeRx and Twitter included
the following components: calculation of relative frequencies,
examination of ADR distribution, and test for association and
independence. A chi-square test was used to statistically quantify
the difference in ADRs between the FAERS data and Twitter
data. It was appropriate to use the chi-square test as no cell in
the cross-tabulation contained an expected value of 5 or below. The sample size required to achieve an a priori α<.01 was 96,
and samples from both sources exceeded the threshold. The null
hypothesis (H0) was “there is no significant difference between
FAERS data and Twitter data on common ADRs.” The failure aADR: adverse drug reaction. to reject H0 would signify that Twitter data were similar to and
independent from the FAERS data. The statistical analysis in
this study was conducted using SAS version 9.4 (SAS Institute
Inc). US Food and Drug Administration Adverse Event
Reporting System Data hydrocodone/acetaminophen, prednisone, levothyroxine could
be prescribed to individuals from all age groups regardless of
gender and geographic areas, Twitter’s population represented
a robust data source for generalizability. Purdue University College of Pharmacy’s CMSA designed and
maintained a searchable database for all published FAERS
reports since 2012 under SafeRx. SafeRx enables large-scale
studies to improve prescription medication safety as the database
contains a collection of 4,935,048 ADRs, representing 294,652
different drugs from the fourth quarter of 2012 through
December 2016. ADR reports were obtained via the FAERS
Data Explore function in SafeRx. The search criteria were set
to display data from March 1, 2016, to March 31, 2017, and the
data included both brand and generic names of selected drugs
as the primary suspect and the secondary suspect drug. After
obtaining all ADR reports from SafeRx, the 5 most reported
ADRs for each selected drug were recorded for data analysis. Tweets were obtained from the Advanced Search webpage on
Twitter’s website [18]. Both generic and brand names of the
selected medication were entered as keywords into the “any of
these words” field in the Advanced Search engine. To exclude
tweets containing advertisements, hyperlinks to external
webpages, and retweets, characters including “rt” for retweets,
“http,” and “.com” were entered into the “none of these words”
field. By eliminating tweets from pharmaceutical companies,
health care marketers, and agencies, Twitter data became more
comparative to the FAERS data. Table 1 describes additional
exclusion criteria in the selection of tweets. The “written in”
field was set so that only tweets in the English language would
be displayed. The time frame was chosen to be from March
2016 to March 2017 in order to correspond with the FAERS
data obtained from CMSA’s SafeRx database. All tweets
displayed were subsequently reviewed to include only those
that described ADRs after consuming the medication. Those
tweets served as the final source for data recording, which
included the username, offending medication, content of the
tweet, and types of ADRs. At the time of data collection, the
number of tweets was benchmarked at 100 for analysis. Twitter Data Searchability and generalizability were the main factors in
selecting Twitter as the social media platform. Twitter’s search
engine enabled keyword-based searching within a predetermined
time frame, and all public tweets containing the keyword could
be displayed. According to the Pew Research Center, Twitter
users were diverse in terms of age distribution and well balanced
in terms of gender and geographic areas at the time of study in
2016
[17]. As
medications
including Table 1. Additional exclusion criteria in the collection of tweets. aADR: adverse drug reaction. JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 4
(page number not for citation purposes) JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 5
(page number not for citation purposes) JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 5
https://publichealth.jmir.org/2020/3/e19266
(page number not for citation purposes)
L•FO US Food and Drug Administration Adverse Event
Reporting System Data Result A total of 40,539 FAERS ADR reports from March 1, 2016, to
March 31, 2017, were obtained via SafeRx. Table 2 summarizes
the 5 most reported ADRs for each of the 10 drugs. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 4
(page number not for citation purposes) XSL•FO
RenderX Zhou & Hultgren Drug and the top 5 adverse drug reactions Xanax resulted in 35 relevant tweets, lisinopril and Prinivil were
found in 33 relevant tweets, and only 3 relevant tweets were
found for atorvastatin and Lipitor. No relevant tweets were
found for keywords amlodipine and Norvasc. Due to the
insufficiency of relevant tweets to meet the benchmark, the final
Twitter data analysis did not include levothyroxine, alprazolam,
lisinopril, atorvastatin, and amlodipine. Table 3 presents the
ADRs reported for the remaining 5 drugs. JMIR PUBLIC HEALTH AND SURVEILLANCE JMIR PUBLIC HEALTH AND SURVEILLANCE Table 2. Five most frequently reported FDA Adverse Event Reporting System adverse drug reactions from March 1, 2016, to March 31, 2017, for ea
selected drug on SafeRx. n (%)
Drug and the top 5 adverse drug reactions
Hydrocodone/acetaminophen (Norco, n=1765)
429 (24.31)
Ineffectiveness
371 (21.02)
Nausea
353 (20.00)
Fatigue
345 (19.55)
Pain
267 (15.13)
Headache
Levothyroxine (Synthroid, n=3728)
881 (23.63)
Fatigue
828 (22.21)
Ineffectiveness
733 (19.66)
Nausea
664 (17.81)
Headache
622 (16.68)
Diarrhea
Prednisone (Deltasone, n=5689)
1423 (25.01)
Ineffectiveness
1332 (23.41)
Fatigue
1067 (18.76)
Dyspnea
976 (17.16)
Nausea
900 (15.82)
Diarrhea
Lisinopril (Prinivil, n=5386)
1243 (23.08)
Ineffectiveness
1172 (21.76)
Fatigue
1136 (21.09)
Diarrhea
1062 (19.72)
Nausea
773 (14.35)
Dyspnea
Amoxicillin (Amoxil, n=797)
328 (41.15)
Hypersensitivity
126 (15.81)
Fatigue
123 (15.43)
Diarrhea
121 (15.18)
Nausea
99 (12.42)
Rash
Gabapentin (Neurontin, n=5734)
1637 (28.55)
Ineffectiveness
1220 (21.28)
Fatigue
997 (17.40)
Nausea
966 (16.85)
Pain
914 (15.94)
Diarrhea
Metformin (Glucophage, n=5109)
1311 (25.66)
Hyperglycemia
1111 (21 75)
N
Zhou & Hultgre
JMIR PUBLIC HEALTH AND SURVEILLANCE n (%) Ineffectiveness
Nausea
Fatigue
Pain
Headache
Levothyroxine (Synthroid, n=3728)
Fatigue
Ineffectiveness
Nausea
Headache
Diarrhea
Prednisone (Deltasone, n=5689)
Ineffectiveness
Fatigue
Dyspnea
Nausea
Diarrhea
Lisinopril (Prinivil, n=5386)
Ineffectiveness
Fatigue
Diarrhea
Nausea
Dyspnea
Amoxicillin (Amoxil, n=797)
Hypersensitivity
Fatigue
Diarrhea
Nausea
Rash
Gabapentin (Neurontin, n=5734)
Ineffectiveness
Fatigue
Nausea
Pain
Diarrhea
Metformin (Glucophage, n=5109)
Hyperglycemia
Nausea
Ineffectiveness
Diarrhea https://publichealth.jmir.org/2020/3/e19266 n (%)
Drug and the top 5 adverse drug reactions
795 (15.56)
Fatigue
Atorvastatin (Lipitor, n=6588)
4601 (69.84)
Type 2 diabetes
586 (8.89)
Hypersensitivity
537 (8.15)
Fatigue
445 (6.75)
Ineffectiveness
419 (6.36)
Nausea
Alprazolam (Xanax, n=2551)
561 (21.99)
Ineffectiveness
548 (21.48)
Fatigue
547 (21.44)
Nausea
451 (17.68)
Anxiety
444 (17.40)
Headache
Amlodipine (Norvasc, n=3192)
696 (21.80)
Diarrhea
682 (21.37)
Fatigue
636 (19.92)
Ineffectiveness
611 (19.14)
Nausea
567 (17.76)
Dyspnea
Twitter Data Result
More than 40,000 tweets containing the drug names as keywords
from March 1, 2016, to March 31, 2017, were obtained from
Twitter’s Advanced Search engine. Although searching on
Twitter yielded an overall large quantity of tweets, ADRs of
some drugs were simply not mentioned in enough tweets. Within
the study period, searching keywords levothyroxine and
Synthroid yielded 50 relevant tweets, keywords alprazolam and
Xanax resulted in 35 relevant tweets, lisinopril and Prinivil were
found in 33 relevant tweets, and only 3 relevant tweets were
found for atorvastatin and Lipitor. No relevant tweets were
found for keywords amlodipine and Norvasc. JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 6
(page number not for citation purposes) JMIR PUBLIC HEALTH AND SURVEILLANCE Due to the
insufficiency of relevant tweets to meet the benchmark, the final
Twitter data analysis did not include levothyroxine, alprazolam,
lisinopril, atorvastatin, and amlodipine. Table 3 presents the
ADRs reported for the remaining 5 drugs. Zhou & Hultgren
JMIR PUBLIC HEALTH AND SURVEILLANCE n (%)
Drug and the top 5 adverse drug reactions
795 (15.56)
Fatigue
Atorvastatin (Lipitor, n=6588)
4601 (69.84)
Type 2 diabetes
586 (8.89)
Hypersensitivity
537 (8.15)
Fatigue
445 (6.75)
Ineffectiveness
419 (6.36)
Nausea
Alprazolam (Xanax, n=2551)
561 (21.99)
Ineffectiveness
548 (21.48)
Fatigue
547 (21.44)
Nausea
451 (17.68)
Anxiety
444 (17.40)
Headache
Amlodipine (Norvasc, n=3192)
696 (21.80)
Diarrhea
682 (21.37)
Fatigue
636 (19.92)
Ineffectiveness
611 (19.14)
Nausea
567 (17.76)
Dyspnea
Zhou & Hultgren
JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 7
(page number not for citation purposes) Twitter Data Result More than 40,000 tweets containing the drug names as keywords
from March 1, 2016, to March 31, 2017, were obtained from
Twitter’s Advanced Search engine. Although searching on
Twitter yielded an overall large quantity of tweets, ADRs of
some drugs were simply not mentioned in enough tweets. Within
the study period, searching keywords levothyroxine and
Synthroid yielded 50 relevant tweets, keywords alprazolam and https://publichealth.jmir.org/2020/3/e19266 XSL•FO
RenderX Table 3. Reported adverse drug reactions on Twitter from March 1, 2016, to March 31, 2017, for 5 drugs. Value %
Drugs and adverse drug reactions
Hydrocodone/acetaminophen
36
Fatigue
22
Ineffectiveness
10
Pruritus
9
Nausea
5
Mood changes
3
Vivid dreams
3
Insomnia
2
Headache
2
Constipation
2
Dizziness
1
Chest tightness
1
Delusion
1
Hallucination
1
Singultus
1
Inattention
1
Short-term amnesia
1
Sweating
1
Vomiting
Prednisone
25
Insomnia
23
Increased appetite
10
Mood changes
8
Moon face
8
Weight gain
5
Fatigue
4
Muscle weakness
3
Jitteriness
2
Diaphoresis
2
Tachycardia
2
Anxiety
1
Bradycardia
1
Cataracts
1
Xerostomia
1
Dyspnea
1
Heartburn
Zhou & Hultgren
JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Table 3. Reported adverse drug reactions on Twitter from March 1, 2016, to March 31, 2017, for 5 drugs. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 8
(page number not for citation purposes) JMIR PUBLIC HEALTH AND SURVEILLANCE Value %
Drugs and adverse drug reactions
16
Rash
15
Ineffectiveness
8
Nausea
5
Diarrhea
3
Fatigue
3
Pruritus
3
Vomiting
1
Stomachache
Gabapentin
31
Drowsiness
24
Fatigue
23
Ineffectiveness
8
Weight gain
5
Dizziness
2
Nausea
1
Blurred vision
1
Dysphasia
1
Confusion
1
Headache
1
Jitteriness
1
Mood changes
1
Vivid dreams
Metformin
57
Nausea
22
Diarrhea
5
Ineffectiveness
3
Fatigue
3
Renal dysfunction
2
Bloating
2
Headache
1
Hypersensitivity
1
Heartburn
1
Hypoglycemia
1
Mood changes
1
Vomiting
Drug and Adverse Drug Reaction Matching
The process was completed through consolidating the ADRs
reported in the Twitter dataset to match the top 5 ADRs from
frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile
with that of FAERS, one should fail to reject H0 according to
th P
l
f
th
hi
t
t T bl 4 h
t h d
Zhou & Hultgren
JMIR PUBLIC HEALTH AND SURVEILLANCE Value %
Drugs and adverse drug reactions
16
Rash
15
Ineffectiveness
8
Nausea
5
Diarrhea
3
Fatigue
3
Pruritus
3
Vomiting
1
Stomachache
Gabapentin
31
Drowsiness
24
Fatigue
23
Ineffectiveness
8
Weight gain
5
Dizziness
2
Nausea
1
Blurred vision
1
Dysphasia
1
Confusion
1
Headache
1
Jitteriness
1
Mood changes
1
Vivid dreams
Metformin
57
Nausea
22
Diarrhea
5
Ineffectiveness
3
Fatigue
3
Renal dysfunction
2
Bloating
2
Headache
1
Hypersensitivity
1
Heartburn
1
Hypoglycemia
1
Mood changes
1
Vomiting
Drug and Adverse Drug Reaction Matching
The process was completed through consolidating the ADRs
reported in the Twitter dataset to match the top 5 ADRs from
SafeRx. Following the matching, a chi-square test was
performed to test nonsignificant differences in the relative
frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile
with that of FAERS, one should fail to reject H0 according to
the P value from the chi-square test. Table 4 shows matched
ADRs between the two data sources, relative frequencies of
ADRs of each drug, and the results of chi-square test. Drugs and adverse drug reactions frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile
with that of FAERS, one should fail to reject H0 according to
the P value from the chi-square test. Table 4 shows matched
ADRs between the two data sources, relative frequencies of
ADRs of each drug, and the results of chi-square test. frequencies of an ADR between FAERS data and Twitter data. In order to demonstrate the similarity of Twitter’s ADR profile
with that of FAERS, one should fail to reject H0 according to
the P value from the chi-square test. Table 4 shows matched
ADRs between the two data sources, relative frequencies of
ADRs of each drug, and the results of chi-square test. Hydrocodone/acetaminophen 9
Nausea
5
Mood changes
3
Vivid dreams
3
Insomnia
2
Headache
2
Constipation
2
Dizziness
1
Chest tightness
1
Delusion
1
Hallucination
1
Singultus
1
Inattention
1
Short-term amnesia
1
Sweating
1
Vomiting
Prednisone
25
Insomnia
23
Increased appetite
10
Mood changes
8
Moon face
8
Weight gain
5
Fatigue
4
Muscle weakness
3
Jitteriness
2
Diaphoresis
2
Tachycardia
2
Anxiety
1
Bradycardia
1
Cataracts
1
Xerostomia
1
Dyspnea
1
Heartburn
1
Osteoporosis
1
Stomachache
1
Visual hallucination
1
Thirst
Amoxicillin
46
Hypersensitivity
JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 7
https://publichealth.jmir.org/2020/3/e19266
(page number not for citation purposes)
FO XSL•FO
RenderX Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE Drug and Adverse Drug Reaction Matching Drug and Adverse Drug Reaction Matching The process was completed through consolidating the ADRs
reported in the Twitter dataset to match the top 5 ADRs from
SafeRx. Following the matching, a chi-square test was
performed to test nonsignificant differences in the relative https://publichealth.jmir.org/2020/3/e19266 https://publichealth.jmir.org/2020/3/e19266 XSL•FO
RenderX XSL•FO
RenderX JMIR PUBLIC HEALTH AND SURVEILLANCE Zhou & Hultgren Zhou & Hultgren Table 4. Matched adverse drug reactions and chi-square test results for 5 drugs. P value
Chi-square
Relative frequencies,
Twitter data (%)
Relative frequencies, FAERSa data (%)
Drug and adverse drug events
Hydrocodone/acetaminophen
.60b
0.3
22.00
24.31
Ineffectiveness
.02
5.3
9.00
21.02
Nausea
<.001
14.7
36.00
20.00
Fatigue
<.001
13.2
2.00
15.13
Headache
Prednisone
<.001
18.8
5.00
23.41
Fatigue
<.001
47.0
1.00
18.76
Dyspnea
Amoxicillin
.35b
0.9
46.00
41.15
Hypersensitivity
.005
7.9
5.00
15.43
Diarrhea
.05b
3.8
8.00
15.18
Nausea
<.001
11.8
3.00
15.81
Fatigue
.31b
1.0
16.00
12.42
Rash
Gabapentin
.15b
2.1
22.00
28.55
Ineffectiveness
.68b
0.2
23.00
21.28
Fatigue
<.001
16.4
2.00
17.40
Nausea
Metformin
<.001
70.1
57.00
21.75
Nausea
<.001
12.7
5.00
19.04
Ineffectiveness
.30b
1.1
22.00
18.00
Diarrhea
<.001
11.9
3.00
15.56
Fatigue
aFAERS: US Food and Drug Administration Adverse Event Reporting System. bIndicates a P value above .05, leading to the failure of rejecting the null hypothesis and indicating that there is no difference in ADR frequency reported
Zhou & Hultgren
JMIR PUBLIC HEALTH AND SURVEILLANCE Table 4. Matched adverse drug reactions and chi-square test results for 5 drugs. adverse drug reactions and chi-square test results for 5 drugs. bIndicates a P value above .05, leading to the failure of rejecting the null hypothesis and indicating that there is no difference in ADR frequency reported
between FAERS and Twitter. bIndicates a P value above .05, leading to the failure of rejecting the null hypothesis and indicating that there is no difference
between FAERS and Twitter. ADRs remain one of the leading causes for preventable hospital
admissions, reduced quality of life, increased financial burdens
in the society, and mortality [19]. Prevention relies on adherence
to evidence-based medicine, monitoring, medication therapy
management, and pharmacogenomic testing [20]. Management
of ADRs should emphasize effective prevention and timely
detection, yet the current ADR reporting mechanism has shown
delays in detection [21]. The cause for delays is multifactorial. Consumers might not know about such a reporting system, and
the reporting steps could be troublesome. Drug and Adverse Drug Reaction Matching Further, as clinicians
and patients are not required to report ADRs, many could be
underreported. Social media and online resources have been
proposed as additional resources for pharmacovigilance. In
2017, MacKinlay et al [22] evaluated ADRs of 3055 drugs on
Twitter and found that Twitter had up to 72% precision of ADR
detection. By extracting ADRs of erlotinib, nivolumab, and
pembrolizumab through social health networks, Nikfarjam et
al [23] detected that social media ADRs were comparable and
7 months ahead of ADRs from literature reports. Along with JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 9
(page number not for citation purposes) Limitations This study does have two prominent limitations: sample size
and search methodology. Among multiple social media
platforms, only Twitter was selected as the data source. Despite
Twitter’s users being from multiple age groups, patients may
choose to share their ADR experiences on other sites such as
Facebook, Instagram, Reddit, and online forums, which
prevented this study from examining social media data across
different platforms. Additionally, due to Twitter’s privacy
setting, private tweets are not searchable, which can reduce the
number of tweets for data collection. The sample size of tweets
obtained for the drugs was relatively small compared with that
of FAERS reports from March 1, 2016, to March 31, 2017. The
sample size could be largely increased in future studies as
Twitter contains a large collection of tweets. During the search
process, the keywords hydrocodone/acetaminophen and Norco
yielded more than 100 tweets in the time period, which could
potentially improve the accuracy of Twitter ADR data. However,
there was a lack of relevant tweets for 4 of the 10 drugs, even
with the benchmark of 100 tweets. This situation could
potentially be resolved by extending the time frame to more
than 1 year; however, the extent of sample size improvement
might not be significant given the low number of social media
users when studying specific drugs such as atorvastatin and
amlodipine. Regarding the search mechanism, only one common brand name
per drug was used to search for tweets, yet many drugs have
multiple brand names. Lisinopril is sold under the brand names
Prinivil and Zestril, and levothyroxine has brand names
Synthroid, Levoxyl, and Thyrax. Using only one brand name
in the study could limit the number of tweets obtained in this
study, as patients might have shared their ADRs by using the
brand names that were not included in this study. Other
challenges to gathering all tweets through keywords include
typographical errors, abbreviations, and unstructured lexicons. Furthermore, social media intrinsically bears a limitation in
terms of patient follow-up. So far, research methodology
involving social media pharmacovigilance has yet to be capable
of investigating the causes of ADRs, the consequences of ADRs,
and the actions taken to resolve ADRs. Some challenges are
being tackled by computational technologies. For example, text
normalization and classification through machine learning have
been investigated by Sarker et al [27], and they offered insights
into processing text data on social media. Principal Findings Among the 5 drugs in the final analysis, a number of Twitter
ADR relative frequencies were not significantly different from
those of FAERS ADRs. For metformin, diarrhea was one of the
side effects. As no significant difference was detected between
FAERS and Twitter data on diarrhea (P=.30), it showed that
Twitter ADR reports could be further studied for their use as a
complementary
ADR
dataset. In
the
hydrocodone/acetaminophen group, there were no significant
differences in ineffectiveness between sources (P=.60). Gabapentin was shown to comparatively result in ineffectiveness
and fatigue according to FAERS and Twitter (P=.15 and P=.67,
respectively). Three ADRs of amoxicillin, hypersensitivity,
nausea, and rash, shared similar profiles on FAERS and Twitter
(P=.35, P=.05, and P=.31, respectively). https://publichealth.jmir.org/2020/3/e19266 Zhou & Hultgren JMIR PUBLIC HEALTH AND SURVEILLANCE the medication and seeing its effect. This study result should
also prompt patient education on regular monitoring and diet
adjustment when managing diabetes, as ineffectiveness for an
antidiabetic drug, metformin, was 19.04% and 5.00% of all
ADRs on FAERS and Twitter, respectively. Data mining to
track ineffectiveness for hydrocodone/acetaminophen may offer
a potential avenue for regulatory bodies in examining opioid
use patterns. the medication and seeing its effect. This study result should
also prompt patient education on regular monitoring and diet
adjustment when managing diabetes, as ineffectiveness for an
antidiabetic drug, metformin, was 19.04% and 5.00% of all
ADRs on FAERS and Twitter, respectively. Data mining to
track ineffectiveness for hydrocodone/acetaminophen may offer
a potential avenue for regulatory bodies in examining opioid
use patterns. numerous major publications on validating ADR reports across
different social media platforms, Hoang et al [24] took a step
further and incorporated content authenticity and user credibility
to improve ADR detection on Twitter. With more advanced
technology for data mining and ADR detection, social media
can serve as an additional channel for monitoring ADRs. In this study, 10 drugs were identified, and ADR reports of these
drugs on Twitter were retrospectively obtained by searching for
tweets containing the drug names that mentioned ADR
experiences. While adopting comparative methods used in
previous studies, this study specifically focused on the 10 most
commonly prescribed drugs to investigate if discrepancies
existed pursuant to different drugs. Based on the results of this
study, FAERS data and Twitter data showed some similar ADR
profiles
for
hydrocodone/acetaminophen,
amoxicillin,
gabapentin, and metformin. Principal Findings In the data collection process,
levothyroxine, alprazolam, lisinopril, and atorvastatin did not
appear as keywords in sufficient tweets from March 1, 2016,
to March 31, 2017. A possible explanation of the low number
of tweets is the demographics of patients taking these
medications. Atorvastatin, a lipid-lowering agent, is usually
initiated for elderly patients, as are the antihypertensive agents
lisinopril and amlodipine. Individuals aged 50 to 64 years and
those older than 65 years represented 21% and 10% of all
Twitter users, respectively [16]. Fewer Twitter users in these
age ranges could potentially explain the low number of tweets
for those drugs. The number of reports of these 3 drugs on
FAERS further demonstrates that the lack of tweets was due to
fewer users, as atorvastatin, lisinopril, and amlodipine had 6588,
5386, and 3192 reports on FAERS. Other social media–based
studies have also experienced this challenge and achieved
opposite conclusions due to inactivity for most of the drugs
studied on social media [25,26]. Nevertheless, data from the
remaining drugs indicates the potential role of Twitter as a
complementary source of ADR reporting to FAERS. https://publichealth.jmir.org/2020/3/e19266 JMIR Public Health Surveill 2020 | vol. 6 | iss. 3 | e19266 | p. 10
(page number not for citation purposes) Conflicts of Interest None declared. Acknowledgments We acknowledge the team that developed the SafeRx database and the CMSA staff for administrative support for this study. Publication of this article was funded in part by Purdue University Libraries Open Access Publishing Fund. We acknowledge the team that developed the SafeRx database and the CMSA staff for administrative support for this study. Publication of this article was funded in part by Purdue University Libraries Open Access Publishing Fund. Conclusion early detection and a reporting system for mild symptoms. To
demonstrate the accuracy and usability of social media ADR
data in complementing FAERS, future studies may benefit by
using a larger sample of data, including specific drugs, and
assessing multiple social media platforms. It is also important
to apply technology, along with structured reporting systems,
to avoid arbitrary entries to better provide health care
professionals, regulatory bodies, patients, and pharmaceutical
companies with robust ADR data. While the use of Twitter as an ADR reporting platform has
limitations, should be considered as a unique and complementary
source of information rather than a validation tool of an existing
ADR database. Future research should focus on validating
Twitter and other social media platforms using involving larger
sample sizes and different medications. Additionally, evaluating
the types of ADRs on social media that share the most similarity
with those on FAERS would be helpful to promote effective
use of this source of information. Limitations Other challenges of
social media ADR reporting may continue to be barriers for
taking full advantage of this data source. The similarities observed for some ADRs between Twitter and
FAERS data were disparate across the individual drugs studied. This variability further suggests that patients’actual experiences
with medications are not being shared with their providers or
that providers have not reported these experiences to national
ADR repositories at a similar rate. Moreover, the insufficiency
of tweets for some drugs may indicate that social media ADR
reporting should consider drug classes and the demographics
of patients taking them. One recommendation is to further
investigate social media ADR reporting for drugs that are
consumed by a population that represents a large share of social
media users and drugs that require early ADR detection. In addition to being a supplementary data source for
pharmacovigilance services, social media can also serve as a
resource for pharmaceutical companies, regulatory bodies,
researchers,
health
care
professionals,
patients,
and
policymakers. In this study, ineffectiveness appeared as an ADR
for hydrocodone/acetaminophen, gabapentin, and metformin
on both data sources. Gabapentin, for example, takes time to
exert its full effect in controlling neurological pain. As 23.00%
of Twitter ADRs and 28.55% of FAERS ADRs for gabapentin
were ineffectiveness, it should encourage prescribers and
pharmacists to consult patients on the time lag between taking Although social media cannot replace professional reporting
systems such as FAERS at this stage, studies including this
analysis have indicated the role of social media as a tool for XSL•FO
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media in pharmacovigilance: lessons from IMI WEB-RADR. Drug Saf 2019 Dec;42(12):1393-1407 [FREE Full text] [doi:
10.1007/s40264-019-00858-7] [Medline: 31446567] 26. van Stekelenborg J, Ellenius J, Maskell S, Bergvall T, Caster O, Dasgupta N, et al. Recommendations for the use of social
media in pharmacovigilance: lessons from IMI WEB-RADR. References Opioid epidemic in the United States. Pain
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30272184] 27. Sarker A, Belousov M, Friedrichs J, Hakala K, Kiritchenko S, Mehryary F, et al. Data and systems for medication-related
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30272184] Edited by G Eysenbach; submitted 10.04.20; peer-reviewed by D Degnan, KNB Nor Aripin; comments to author 04.05.20; revised
version received 09.06.20; accepted 25.06.20; published 30.09.20 Please cite as:
Zhou Z, Hultgren KE
Complementing the US Food and Drug Administration Adverse Event Reporting System With Adverse Drug Reaction Reporting From
Social Media: Comparative Analysis
JMIR Public Health Surveill 2020;6(3):e19266
URL: https://publichealth.jmir.org/2020/3/e19266
doi: 10.2196/19266
PMID: 32996889 ©Zeyun Zhou, Kyle Emerson Hultgren. Originally published in JMIR Public Health and Surveillance (http://publichealth.jmir.org),
30.09.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work, first published in JMIR Public Health and Surveillance, is properly cited. The complete bibliographic
information, a link to the original publication on http://publichealth.jmir.org, as well as this copyright and license information
must be included. https://publichealth.jmir.org/2020/3/e19266 XSL•FO
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Organizational and technological cycle of life of a construction object
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* Corresponding author: aljurgaitis@gmail.com, fayzdim@mail.ru Organizational and technological cycle of life of
a construction object
Muhammet Fakhratov1,*, Vitaly Chulkov1, Dmitry Fayzullin1, and Salavat Zaidullin1
1Moscow State University of Civil Engineering, 129337, 26, Yaroslavskoye Shosse, Moscow, Russia Abstract. During the life cycle, the state of an object is modified. The
information for stepwise and phased study of innovation processes is
characterized as local and torn in time, while the life cycle approach
regards the process of creating and developing technological innovations
as a dynamically synchronized system. The development of organizational
and technological systems is being implemented in two directions: the
improvement of basic and the creation of fundamentally new technologies. The life cycles of all objects, processes and systems are built on one info
graphic model: any life cycle begins with the birth, passes through the
stages of growth, maturity, decay and decline. Therefore, it is advisable to
consider the innovative investment and construction life cycle of an object
as a combination of a series of successive stages (cycles). They are sub-
cycles during the period from the beginning of the idea of the initial design
and construction of an object until the demolition, complete disassembly,
disposal of construction and demolition waste (CDW) and non-recyclable
parts of them. https://doi.org/10.1051/e3sconf /201911002161 https://doi.org/10.1051/e3sconf /201911002161 , 0
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2161 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative
Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2 Methods The development of organizational and technological systems is being implemented in two
directions: the improvement of basic and the creation of fundamentally new technologies. With the improvement and modernization of technologies, their transition to the stage of
maturity and saturation of the market with this commodity, further technological develop-
ment within the framework of the old paradigm becomes unprofitable, volume of sales and
profits fall. In the depths of the existing technological structures, fundamentally new solu-
tions, "breakthrough" technologies emerge, which lays the foundation for new technologi-
cal structures, industries and branches [2]. The life cycles of all objects, processes and systems are built according to a single info-
graphic model: any life cycle begins with the birth, passes through the growth, maturity,
wilt and decline stages (Figure 1). Fig. 1. The life cycle of an object, process or system. I - the stage of origin and the beginning of
growth; II - rapid growth; III - growth retardation, maturity; IV - stabilization; V - wilting, aging,
decay, end of life cycle. Fig. 1. The life cycle of an object, process or system. I - the stage of origin and the beginning of
growth; II - rapid growth; III - growth retardation, maturity; IV - stabilization; V - wilting, aging,
decay, end of life cycle. Fig. 1. The life cycle of an object, process or system. I - the stage of origin and the beginning of
growth; II - rapid growth; III - growth retardation, maturity; IV - stabilization; V - wilting, aging,
decay, end of life cycle. The infographic model indicated in Figure 1 adequately sets the dynamics and structure
of the life cycle of numerous objects of innovation management: innovative enterprises,
new equipment and technology, new market goods (norms, products and services). The
concept of the life cycle and its model (Fig. 1) provides: accounting for the use of various
resources (including the time factor, material, information and financial flows); identifica-
tion of the central trend of the process; visualization of the dynamics of transformations;
process deployment logic; the possibility of using alternative forecasting methods; identifi-
cation of the relationship of different objects. The concept of the life cycle requires the study of its constituent objects and systems
from the standpoint of their self-development and improvement. It is closely connected to
the system analysis that introduces regularity, completeness and complexity in the process
of innovation management. 1 Introduction Temporary, resource and organizational synchronization of all processes and production
stages is possible on the basis of life cycles of innovation processes, products and systems. Local information torn in time is characteristic for stepwise and phased study of innovation
processes, while the life cycle approach regards the process of creating and developing
technological innovations as a dynamically synchronized system. Innovation activity consists of a series of events combined into one logical chain. Each
component of this chain (the innovation cycle stage) has its own development logic, its own
laws and features. Connecting together, research, development and technological develop-
ment, investment and financial, marketing activities, production facilities and organization-
al structures are subordinated to one main goal - the creation of innovation [1]. Originating in the depths of marketing research, the life cycles of market goods (norms,
products and services), demand and technology in a short time have occupied a dominant
position in the study of economic objects, processes and systems. The concepts of the life
cycles of organizations, branches of industrial production, construction sites and structures,
machines and mechanisms, processes and procedures have gained significant development. , 0
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2161 https://doi.org/10.1051/e3sconf /201911002161 For the study of innovation, the life cycles of a new product, a new technique, technolo-
gy and innovative organizations as open systems are most important. The most productive modern idea of life cycles is the concept of large technological
systems, including the evolution and transformation of technologies as economic objects. The study of the life cycles of large technological systems leads to the theory of generations
of technics and technology, developing in the framework of both the traditional and the new
technological paradigm. 2 Methods Even if several innovations bring the same production effect, they have unequal eco-
nomic efficiency; differently affect changes in the production apparatus and the materials
used, and the organization of production. The urgency of the problem of choosing the object of innovation is that the correct ini-
tial choice predetermines the entire course of the subsequent innovation activity and makes
the development of production irreversible. 2 Methods In other words, innovation processes of different scale and level form the basis for the
development of activity systems. Studying the features of innovation processes in industri-
al, scientific, technical and organizational systems is the basis for increasing the effective-
ness of innovation management. 2 2 , 0
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2161 https://doi.org/10.1051/e3sconf /201911002161 In the dynamic system of the innovation process, subsystems (stages) of the creation,
production, and consumption of innovation are distinguished. The stage of creating innova-
tion requires the emergence of a new idea, new knowledge, the use of new equipment, new
technology, new materials to produce the idea into a prototype model [3]. The stage of sci-
entific and technical implementation of innovations is the result of engineering develop-
ments, laboratory tests, creation of design documentation, production of experimental and
prototypes, and technological preparation of production. Distribution, diffusion and com-
mercialization of innovations in the market conditions allow obtaining of the expected ef-
fect (Fig. 2). Fig. 2. Generalized scheme of life cycle of innovation. Stimulating the use of innovations in production is associated with the problem of their
correct choice. The variety of possible impact of each of the innovative solutions offered by
science and technology creates the complexity of their selection for manufacturing applica-
tion. Even if several innovations bring the same production effect, they have unequal eco-
nomic efficiency; differently affect changes in the production apparatus and the materials
used and the organization of production Fig. 2. Generalized scheme of life cycle of innovation. Stimulating the use of innovations in production is associated with the problem of their
correct choice. The variety of possible impact of each of the innovative solutions offered by
science and technology creates the complexity of their selection for manufacturing applica-
tion. Even if several innovations bring the same production effect, they have unequal eco-
nomic efficiency; differently affect changes in the production apparatus and the materials
used, and the organization of production. Stimulating the use of innovations in production is associated with the problem of their
correct choice. The variety of possible impact of each of the innovative solutions offered by
science and technology creates the complexity of their selection for manufacturing applica-
tion. 3 Results Innovations are an essential integral part of construction as a branch of business. This situa-
tion exists because of the relative duration of the construction of buildings and structures, as
well as due to the fact that construction projects, as a rule, are being built and rebuilt with
the prospect of operating them for decades (and sometimes centuries). Recently, the primary erection of buildings and structures in urban areas (when con-
struction is carried out on previously unbuilt territories) has become rare: the price for land,
newly erected or reconstructed utilities and transport routes and roads has risen sharply. More often, they carry out construction of buildings and their reorganization on the already
built-up territories (redevelopment, often with the demolition of existing buildings) [4, 5]. 3 3 , 0
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2161 https://doi.org/10.1051/e3sconf /201911002161 This allows us to consider the redevelopment, in relation to the previously built-up are-
as, as an innovation (urban planning, technical, technological, economic, etc.). Therefore, it is reasonable to consider the innovative investment and construction life
cycle of an object (Pic. 3.) as a combination of a series of successive stages (cycles) and
their subcycles during the period from the inception of the idea of the initial construction of
a building or structure until its demolition, complete disassembly, disposal of construction
and demolition waste (CDW) and non-recyclable part of them. Fig. 3. Infographic model of the innovation life cycle (ILC) of the construction project. Fig. 3. Infographic model of the innovation life cycle (ILC) of the construction project. Erection of the construction object (and the cycle of erection of the object, realized at this
stage); the content and sequence of implementation of enlarged sub-stages of stage I (from
I.1 to I.10 inclusive) are shown in Figure 4; I.1
I.2
I.3
I.4
I.5
I.6
I.7
I.8
I.9
I.10 I.1
I.2
I.3
I.4
I.5
I.6
I.7
I.8
I.9
I.10 I.1
I.2
I.3
I.4
I.5
I.6
I.7
I.8
I.9
I.10 Fig. 4. Notation conventions: Fig. 4. 3 Results Notation conventions: I.1 - development of technical specifications (TS) for the erection of the object (building or structure);
I.2 - development of a business plan; I.1 - development of technical specifications (TS) for the erection of the object (building or structur
I.2 - development of a business plan; I.1 - development of technical specifications (TS) for the erection of the object (building or structure);
I.2 - development of a business plan; I.2 - development of a business plan; I.3 - development of a constructive project for the future facility; I.4 - development of the project of the organization of construction (POC); I.5 - tendering and auctions (tender), objectification of the general contractor (developer); I.6 - development of working documentation for the construction object; I.7 - development of the project of production of works (PPW); I.8 - development of a work organization project (WOP); I.9 - construction and installation works (the actual construction of the object); I.10 - acceptance of the object and (if necessary) auction for the sale of the object. - Stage II: Reorganization of the construction object (and the cycle of reorganization of the
object, implemented at this stage Creorganization); - Stage III: Demolition and disposal of the construction object (and the cycle of demolition
of the object, implemented at this stage Cdemolition). of the object, implemented at this stage Cdemolition). 4 4 , 0
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2161 https://doi.org/10.1051/e3sconf /201911002161 The basis of the innovation life cycle (ILC) is a calendar organizational and technological
design and planning (COTDP) of construction and assembly processes, supported by timely
investment. The basis of the innovation life cycle (ILC) is a calendar organizational and technological
design and planning (COTDP) of construction and assembly processes, supported by timely
investment. COTDP is hierarchical functional database accompanying the construction object
throughout its innovation life cycle, which reflects all changes in the construction object
without any exception, i.e. local adjustments to the constructive project of the construction
object of organizational and technological projects: project of the organization of construc-
tion (POC), project of production of works (PPW), and work organization project (WOP). 4 Discussion and conclusions During the life cycle, the state of the object is modified. The flexibility of modern building
production is a natural result of the variety of types of construction products, natural, social
and technical factors influencing the choice of constructive and technological solutions. g
g
The efficiency of the construction industry is due to the desire to obtain high-quality
construction products with the lowest consumption of material, financial, labor, time re-
sources. Hierarchical functional database COTDP should be a market product inalienable from
the construction project (subject to continuous organizational and technological, software
and computer maintenance and support), with market value and being transferred from
owner to owner of the construction object as the object is being sold and purchased up to
the stage of its demolition and recycling. Only with this approach, each new owner (or operator) of a construction object must
treat it consciously and make decisions, possessing all the necessary information about its
history and current state of engineering systems of a building or structure, as well as its
premises and redevelopment. And only after the completion of the stage of demolition and disposal of the construc-
tion object, the hierarchical functional database COTDP can be officially discarded. Alt-
hough in this case, it is of archival and historical town-planning value. References 1. A. Komar, A. Kalgin, M. Fakhratov, O. Kikawa, V. Baev, V. Tsyro, Design and
reconstruction of enterprises of precast concrete, (Triada Publishing House
LLC, Tver, 2012) 2. A. Kalgin, M. Fakhratov, O. Kikawa, Advanced technologies in the production of
building materials. (Publishing House International Concrete Manufacturing
LLC, Tver, 2009) 3. A. Kalgin, M. Fakhratov, V. Chulkov, Production and use of building materials,
products and systems: Volume 1. Finishing materials, products and systems. 2nd edn. SVR-ARGUS LLC, (Moscow, 2009) 4. V. Chulkov, Reconstruction: Organizational and anthropotechnical reliability of
construction. A series of “Infographic basis of functional systems”. SVR-ARGUS
LLC, (Moscow, 2012) 5. A. Kalgin, V. Sohryakov, M. Fakhratov, V. Chulkov, Production and use of
building materials, products and systems: Volume 4. Production of building
structures of precast concrete. Design and reconstruction of enterprises. 2nd
edn. SVR-ARGUS LLC, (Moscow, 2012). 5
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Dental Post National Cancer Institute National Cancer Institute Qeios ID: F765NH · https://doi.org/10.32388/F765NH Source National Cancer Institute. Dental Post. NCI Thesaurus. Code C66863. A rod that a dentist has positioned and then cemented in the canal space in a tooth's
root. Qeios ID: F765NH · https://doi.org/10.32388/F765NH 1/1
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Three-dimensional label-free observation of individual bacteria upon antibiotic treatment using optical diffraction tomography
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1. Introduction Understanding how antibiotics induce bacterial cell death is essential in the study of microbiology and also to general healthcare
in order to help develop, improve, and apply treatment to patients [1]. Over the past years, the mechanism of antibiotics has
been studied intensively along with the use of new multiple classes of antibiotics that have succeeded in treating infectious
diseases [2]. However, the widespread use of antibiotics has resulted in emergence of drug-resistant bacteria, evoking a
significant challenge to the use of antibiotics [3, 4]. Since the mainstream approach to overcome this crisis primarily relies on
the discovery and development of newer, more efficient antibiotics, understanding how bacteria respond to antibiotics has
become more critical. A basic approach to understanding the mechanism of antibiotics is to observe the phenotypic or physical response of bacteria
to antibiotics. Phenotypic imaging has been one of the most popular diagnostic methods to achieve rapid antimicrobial
susceptibility testing [5-10], as it provides an easy, low cost, real-time single-cell level examination. Furthermore, phenotypic
analysis of bacteria has been exploited to extract useful information in various fields such as epidemiology and systems biology
[11]. Although the phenotypic response of bacteria to β-lactam antibiotics has been studied [12, 13], detailed phenotypic aspects
have been less explored with little available research on the changes in physical quantities such as cell mass, density, and
volume. These parameters are direct indicators of cell survival and growth [14, 15], which can provide fundamental insights
into the physical and chemical mechanism of bacterial response to antibiotics. p y
p
To revisit these unattended but physiologically relevant quantities of bacteria, we need an imaging modality that quantifies
the parameters mentioned above. However, conventional two-dimensional (2D) microscopic techniques, such as phase contrast
microscopy or differential interference contrast (DIC) microscopy, are not suitable for this purpose, because they are unable to
measure three-dimensional (3D) properties. Fluorescence microscopy, such as scanning confocal fluorescence microscopy,
allows 3D molecular imaging of cells [16], but the use of fluorescent dyes or proteins have limitations for studying the dynamic
alterations in bacteria over a long time due to issues of photo-bleaching and photo-toxicity [17]. Scanning electron microscopy
or atomic force microscopy is also not suitable for assessing the dynamics of live bacteria with high throughput [18, 19]. JEONGHUN OH,1,2 JEA SUNG RYU,3 MOOSUNG LEE,1,2 JAEHWANG JUNG, 1,2,† SEUNGYUN HAN, 1,2
HYUN JUNG CHUNG,3,4 AND YONGKEUN PARK1,2,5,* JEONGHUN OH,1,2 JEA SUNG RYU,3 MOOSUNG LEE,1,2 JAEHWANG JUNG, 1,2,† SEUNGYUN HAN, 1,2
HYUN JUNG CHUNG,3,4 AND YONGKEUN PARK1,2,5,* 1Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea
2KAIST Institute for Health Science and Technology, KAIST, Daejeon 34141, South Korea
3Graduate School of Nanoscience and Technology, KAIST, Daejeon 34141, South Korea
4Department of Biological Sciences, KAIST, Daejeon 34141, South Korea
5Tomocube Inc., Daejeon 34051, South Korea
†Current Affiliation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea
*yk.park@kaist.ac.kr 1Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, South Korea
2KAIST Institute for Health Science and Technology, KAIST, Daejeon 34141, South Korea
3Graduate School of Nanoscience and Technology, KAIST, Daejeon 34141, South Korea
4Department of Biological Sciences, KAIST, Daejeon 34141, South Korea
5Tomocube Inc., Daejeon 34051, South Korea
†Current Affiliation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea
*yk.park@kaist.ac.kr j
liation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea
i
k Current Affiliation: Mechatronics R&D Center, Samsung Electronics, Hwasung 18448, South Korea
yk park@kaist ac kr Abstract: Measuring alterations in bacteria upon antibiotic application is important for basic studies in microbiology, drug
discovery, and clinical diagnosis, and disease treatment. However, imaging and 3D time-lapse response analysis of individual
bacteria upon antibiotic application remain largely unexplored mainly due to limitations in imaging techniques. Here, we
present a method to systematically investigate the alterations in individual bacteria in 3D and quantitatively analyze the effects
of antibiotics. Using optical diffraction tomography, in-situ responses of Escherichia coli and Bacillus subtilis to various
concentrations of ampicillin were investigated in a label-free and quantitative manner. The presented method reconstructs the
dynamic changes in the 3D refractive-index distributions of living bacteria in response to antibiotics at sub-micrometer spatial
resolution. .
CC-BY 4.0 International license
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not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was
this version posted September 19, 2019.
;
https://doi.org/10.1101/775346
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was
this version posted September 19, 2019. ;
https://doi.org/10.1101/775346
doi:
bioRxiv preprint . CC-BY 4.0 International license
under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which was
this version posted September 19, 2019. ;
https://doi.org/10.1101/775346
doi:
bioRxiv preprint Three-dimensional label-free observation of individual
bacteria upon antibiotic treatment using optical diffraction
tomography JEONGHUN OH,1,2 JEA SUNG RYU,3 MOOSUNG LEE,1,2 JAEHWANG JUNG, 1,2,† S
HYUN JUNG CHUNG,3,4 AND YONGKEUN PARK1,2,5,* 2.1 Optical diffraction tomography To measure the 3D RI tomogram of individual bacteria, we employed optical diffraction tomography based on a Mach-Zehnder
nterferometer equipped with a digital micromirror device (DMD) [Fig. 1(a)]. To measure the 3D RI tomogram of individual bacteria, we employed optical diffraction tomography based on a Mach-Zehnder
interferometer equipped with a digital micromirror device (DMD) [Fig. 1(a)]. Fig. 1. Measurements of the 3D RI distribution. (a) Experimental setup. M1−M4, mirror; L1−L5, lens; FC, fiber coupler; DMD, digital
micromirror device. (b) Sequential illumination scanning for bacteria at 71 illumination angles and the corresponding 71 holograms. (c)
Images of the retrieved amplitudes and phases corresponding to the holograms. (d) Three-section view of the reconstructed RI
distribution. (e) 3D rendered image of bacteria. The color map shows various colors depending on the value of RI and its gradient value. Fig. 1. Measurements of the 3D RI distribution. (a) Experimental setup. M1−M4, mirror; L1−L5, lens; FC, fiber coupler; DMD, digital
micromirror device. (b) Sequential illumination scanning for bacteria at 71 illumination angles and the corresponding 71 holograms. (c)
Images of the retrieved amplitudes and phases corresponding to the holograms. (d) Three-section view of the reconstructed RI
distribution. (e) 3D rendered image of bacteria. The color map shows various colors depending on the value of RI and its gradient value. A coherent laser ( = 532 nm in a vacuum; SambaTM, Cobolt Inc., Sweden) beam was split into a reference beam and sample
beam using a 1 × 2 fiber coupler. The sample beam passed through 4-f relay lenses (L1−3), and was diffracted by a DMD
(DLPLCR6500EVM, Texas Instruments Inc., USA). A DMD controlled the angle of the sample beam impinging onto a sample,
by displaying digital grating patterns [32]. The time-multiplexing illumination technique was utilized to eliminate the unwanted
diffractions of a DMD [33]. The first-order diffraction beam illuminated a sample with a specific incident angle controlled by
the pattern projected onto the DMD. The beam diffracted from the DMD passes through a lens (L4) and a condenser lens (NA
= 1.2, water immersion, UPLSAPO 60XW, Olympus Inc.) in order to magnify the incident angle. The scattered light from the
sample was collected by an objective lens (NA = 1.2, water immersion, UPLSAPO 60XW, Olympus Inc.), and projected to the
image plane. 2.1 Optical diffraction tomography At the image plane, the sample beam and reference beam interfere and generate a spatially modulated hologram
pattern, which is recorded using a camera (FL3-U3–13Y3M-C, Point Grey Research Inc.). To combine two beams and generate
the interference pattern with the maximum contrast, a beam splitter and a linear polarizer were used, respectively. The beam
impinging onto the sample was controlled to have the maximum elevation angle limited by the NA of the condenser lens and
was scanned along a full azimuthal angle with 71 steps [Fig. 1(b)]. The total acquisition time for 71 holograms was
approximately 1 s (15 ms per hologram). 1. Introduction py
g
y
g
g p
[
]
To investigate the aforementioned parameters, optical diffraction tomography (ODT), one of the 3D quantitative phase
imaging (QPI) techniques, has several potentials [20]. ODT provides a non-invasive and non-contact 3D optical imaging [21]. ODT reconstructs the 3D refractive index (RI) maps of optically transparent samples based on interferometric imaging at
multiple illumination angles. Because RI distributions serve as both intrinsic cell markers and indicators of protein densities,
ODT meets the demands of quantitative phenotypic imaging of unlabeled live cells. Thus, its applications have been rapidly
expanded for investigations on hematology [22], microalgae [23], immunology [24], infectious diseases [25, 26], plant biology . CC-BY 4.0 International license
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bioRxiv preprint [27], cell biology [27, 28], neuroscience [29], and drug discovery [30]. Some bacterial studies have also used ODT [31], but
its application for bacterial microbiology is in a nascent stage. pp
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g
In this study, we investigated the 3D bacterial response to antibiotics by exploiting ODT. Our method provides a label-free,
real-time analysis of the phenotypic changes in bacterial cells at given antibiotic conditions. The quantitative analysis of 3D RI
maps enables examination of the physical changes in cells induced by antibiotic agents. We demonstrate the capability of ODT
for imaging the antibiotic response of bacteria in 3D by investigating the response of Escherichia coli and Bacillus subtilis to
a β-lactam antibiotic agent, ampicillin. The dynamics of bacteria were observed by mapping the 3D RI distribution in real time,
and the changes in morphological and biochemical parameters were analyzed. With its non-invasive, label-free, and quantitative
analysis, our method provides a useful tool for studying and developing antibiotic agents against clinical pathogens that threaten
public health. 2.3 Analysis of cell parameters and visualization methods To measure cell volumes, the voxels were selected which have an RI higher than a specific value determined by masking the
sample region at the initial time for each cell and averaging the RI values therein. Because the RI value of cell cytoplasm is
linearly related to its protein concentration, local protein concentration of a cell can be retrieved from the measured 3D RI
tomogram [46, 47]. The concentration at each point of the bacteria was determined from RI increment (RII), an increment of
an RI in a solution per an increment of concentration in a solute. We assumed that the value of RII is constant inside bacteria,
i.e. n(r) - nmedium = (RII) × C(r), where n is an RI inside the bacteria, nmedium is the RI of surrounding medium, and C is the
protein concentration. Therefore, the concentration distribution inside bacteria can be acquired using an RI distribution. In
addition, the RII of 0.185 mL/g is known as a typical value for proteins [42, 46]. Because many bacteria have proteinaceous
organelles [48], it is reasonable that the typical value of RII for proteins could be used in our analysis. The temporal changes
in the cellular dry mass are calculated as the total sum of the concentration at each voxel multiplied by its volume. Cytoplasm
concentration was calculated by averaging the concentrations at the internal voxels of bacteria. y
g g
The 3D rendered image was obtained using commercial software (TomoStudioTM, Tomocube Inc., Republic of Korea),
which delineates the overall RI distributions of bacteria more clearly [Fig. 1(e)]. The different colors indicate the presence of
subcellular organelles in bacteria with different RI values, suggesting that our technique provides sub-micrometer spatial
resolution to investigate individual bacterial cells. 2.2 3D RI tomogram reconstruction The 3D RI distribution of a sample is reconstructed from the measured multiple 2D holograms using the principle of optical
diffraction tomography [34, 35]. For each hologram pattern, amplitude and phase delay maps were retrieved using the field
retrieval algorithm [Fig. 1(c)] [36, 37]. Then, from these retrieved optical field maps with various angles, the 3D RI distribution
was determined [Fig. 1(d)]. ODT solves an inverse scattering problem for monochromatic light propagating in a linear, isotropic,
and nonmagnetic medium, which satisfies the 3D inhomogeneous Helmholtz equation [34]. For a slowly varying scattered
complex phase, the Rytov approximation was applied, which has shown better reconstruction performances than the Born . CC-BY 4.0 International license
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bioRxiv preprint approximation [38, 39]. According to the Fourier diffraction theorem, each optical field is mapped onto a sphere in the 3D
Fourier space [40]. Then, the inverse Fourier transform of the 3D Fourier space provides the RI distribution of the sample [41]. The theoretically calculated lateral and axial resolution of the used optical imaging system is 124 nm and 397 nm, respectively
[32, 42]. Due to the limited NAs of both the condenser and the objective lens, side scattering signals are not collected, which
is known as the missing cone problem. To resolve this missing cone problem, the regularization algorithm based on non-
negativity criteria was used [43-45]. approximation [38, 39]. According to the Fourier diffraction theorem, each optical field is mapped onto a sphere in the 3D
Fourier space [40]. Then, the inverse Fourier transform of the 3D Fourier space provides the RI distribution of the sample [41]. The theoretically calculated lateral and axial resolution of the used optical imaging system is 124 nm and 397 nm, respectively
[32, 42]. 2.4 Sample preparation Bacterial strains Escherichia coli (E. coli; #1682) and Bacillus subtilis (B. subtilis; #1021) were purchased from Korean
Collection for Type Cultures. E. coli cells were cultured in Tryptic soy broth (TSB) and B. subtilis were cultured in Nutrient
broth (NB). Both bacteria were cultured to the concentration of 1.0−5.0 × 108 cells/mL to obtain activated bacteria using a
nanophotometer (Implen). Centrifugation to wash the cells was repeated twice under the general condition of 8,000 × g for 5
minutes at 4℃ in 200 μL of PBS solution. Cells were then resuspended in TSB and NB to conduct imaging for the bacteria. Ten microliters of the resuspended solution were placed between two coverslips and then measured using the optical imaging
system. All the measurements were performed at room temperature (25℃). 2.2 3D RI tomogram reconstruction Due to the limited NAs of both the condenser and the objective lens, side scattering signals are not collected, which
is known as the missing cone problem. To resolve this missing cone problem, the regularization algorithm based on non-
negativity criteria was used [43-45]. 3. Results In order to systematically investigate the alterations in individual bacteria upon treatment with antibiotics, we measured the 3D
RI tomograms of E. coli and B. subtilis using ODT, and analyzed the measured images. 3.1 Changes in a bacterial cell revealed by time-lapse 3D imaging We first validated the 3D dynamic imaging capability of ODT by visualizing bacterial growth in the absence of antibiotics. We
imaged the dynamics of E. coli in growth medium with a time interval of 30 minutes up to 6 hours. Both the cross-sections and
rendered images of the measured 3D RI tomograms clarify the growth and division of individual E. coli [Figs. 2(a) and 2(b)]. Prominent cell division was observed and the increase in the number of E. coli over time is presented in Fig. 2(c). The number
of E. coli doubled constantly at intervals of 56 min, which is consistent with the generation time of E. coli at room temperature
[49]. These results clearly validate the label-free imaging capability of ODT for studying the dynamics of individual bacteria. . CC-BY 4.0 International license
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oRxiv preprint Fig. 2. (a) Three-section views of the reconstructed RI distributions of E. coli without antibiotics over time (at 0, 120, and 240 minutes). (b) 3D rendered images of bacteria corresponding to (a). The color map shows various colors depending on the value of the RI. (c) The
number of E. coli for the control group over time and the fitted curve. R-square = 0.9741. Fig. 2. (a) Three-section views of the reconstructed RI distributions of E. coli without antibiotics over time (at 0, 120, and 240 minutes). (b) 3D rendered images of bacteria corresponding to (a). The color map shows various colors depending on the value of the RI. (c) The
number of E. coli for the control group over time and the fitted curve. R-square = 0.9741. 3.2 Antibiotic responses of Escherichia coli and Bacillus subtilis 3.2 Antibiotic responses of Escherichia coli and Bacillus subtilis Next, we investigated the growth dynamics of individual bacteria under antibiotic treatments. As Gram-positive and Gram-
negative models, E. coli and B. subtilis were selected, respectively. The bacteria were treated with ampicillin at various
concentrations of 20, 100, and 300 g/mL and imaged at time intervals of 30 minutes after drug treatment. As a normal control
group, untreated bacterial samples were also measured at the same time intervals. Next, we investigated the growth dynamics of individual bacteria under antibiotic treatments. As Gram-positive and Gram-
negative models, E. coli and B. subtilis were selected, respectively. The bacteria were treated with ampicillin at various
concentrations of 20, 100, and 300 g/mL and imaged at time intervals of 30 minutes after drug treatment. As a normal control
group, untreated bacterial samples were also measured at the same time intervals. The results are shown in Fig. 3. In the absence of antibiotic treatment, both E. coli and B. subtilis exhibited active growth
and division. When the bacteria were treated with antibiotics, each cell showed significant morphological alterations, and also
presented a decrease in RI values, suggesting cell lysis. To effectively visualize these morphological alterations, the maximum
RI projection of each 3D RI tomograms is presented for each group at various time points in Fig. 3. Fig. 3. Maximum RI projection images with ampicillin over time. The red arrows indicate a bulge formed in E. coli. The entry with a
red border is presumed to be near the point at which the bacteria die. (a) and (b) are E. coli and B. subtilis, respectively. Fig. 3. Maximum RI projection images with ampicillin over time. The red arrows indicate a bulge formed in E. coli. The entry with a
red border is presumed to be near the point at which the bacteria die. (a) and (b) are E. coli and B. subtilis, respectively. . CC-BY 4.0 International license
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bioRxiv preprint Figure 3(a) shows the morphological changes in E. coli, due to the ampicillin treatment. As the concentration of ampicillin
increased, the bacteria underwent earlier cell lysis. 3.2 Antibiotic responses of Escherichia coli and Bacillus subtilis From the reconstructed 3D RI tomograms, the point where the RI value
drops suddenly could serve as the time point when the bacterial lysis initiated, because the decrease in the RI values of bacteria
indicates the efflux of cell cytoplasm. When the concentration of the applied ampicillin was 20, 100, and 300 g/mL, the
bacterial lysis occurred right after 180, 150, and 30 minutes, respectively. In the control group, E. coli showed stable growth
over time, whereas the bacteria treated with ampicillin did not divide. p
In addition, the ampicillin-treated bacteria exhibited distinct elongation of cell shape; the cell length increased without cell
division until the point when the RI value increased. Interestingly, the cells exposed to the ampicillin concentration of 20 g/mL
appeared like filaments, exhibiting a fiber-like cell shape. Notably, formation of bulges could be clearly detected in the
measured tomograms [the red arrows in Fig. 3(a)]. Bulge formation was observed in groups treated with ampicillin
concentrations of 20 or 100 g/mL. However, a bulge was not formed at 300 g/mL, which is a significantly high concentration. Furthermore, the rate of cell lysis increased noticeably as the concentration of ampicillin increased to 300 g/mL. Compared to the results with E. coli, B. subtilis exhibited significantly different responses to ampicillin [Fig. 3(b)]. First,
the bacterial cell lysis time was earlier than that of E. coli treated with low ampicillin concentration. When the ampicillin
concentration was 20, 100, and 300 g/mL, the cell lysis points were conjectured to be right after 90, 90, and 40 minutes,
respectively. At an even lower concentration, B. subtilis showed faster cell lysis than E. coli. In addition, B. subtilis exposed to
the low concentration of ampicillin (20 g/mL) did not exhibit morphological alteration into filament-like shapes as observed
with E. coli. Notably, bulge formation was not observed in B. subtilis. 3.3 Quantitative analysis results for morphological and biochemical parameters To investigate the morphological and biochemical characteristics of bacterial growth under antibiotic treatment, the following
cellular parameters were retrieved and analyzed from the measured 3D RI tomograms: cell volume, cellular dry mass,
cytoplasm concentration (See Methods). These cellular morphological and biochemical parameters were systematically
addressed as a function of time and at various antibiotic concentrations [Fig. 4]. Fig. 4. Cell volume, cellular dry mass, and cytoplasm concentration depending on the concentrations of ampicillin (0, 20, 100, and 300
g/mL) and time at the maximum volume. (a−d) and (e−h) are E. coli and B. subtilis, respectively. Fig. 4. Cell volume, cellular dry mass, and cytoplasm concentration depending on the concentrations of ampicillin (0, 20, 100, and 300
g/mL) and time at the maximum volume. (a−d) and (e−h) are E. coli and B. subtilis, respectively. Figure 4(a) shows the cellular volume of E. coli over time for various concentrations of ampicillin. Note that this analysi
was conducted at the single cell level, and each line in Fig. 4 indicates individual cells. For untreated E. coli, cells continued
growth and division, and thus the volumes of each cell remained within a certain range (0.5−1.4 fL). Furthermore, the volume
of untreated E. coli oscillated over time, implying continuous growth and division. Figure 4(a) shows the cellular volume of E. coli over time for various concentrations of ampicillin. Note that this analysis
was conducted at the single cell level, and each line in Fig. 4 indicates individual cells. For untreated E. coli, cells continued
growth and division, and thus the volumes of each cell remained within a certain range (0.5−1.4 fL). Furthermore, the volumes
of untreated E. coli oscillated over time, implying continuous growth and division. Figure 4(a) shows the cellular volume of E. coli over time for various concentrations of ampicillin. Note that this analysis
was conducted at the single cell level, and each line in Fig. 4 indicates individual cells. For untreated E. coli, cells continued
growth and division, and thus the volumes of each cell remained within a certain range (0.5−1.4 fL). Furthermore, the volumes
of untreated E. coli oscillated over time, implying continuous growth and division. Upon ampicillin treatment, E. coli did not exhibit cell division, as shown in Fig. 3. The volumes of treated E. coli showed
monotonic increases, followed by sudden plummets [Fig. 3(a)]. These time points are consistent with the cell lysis observed in
the 3D RI tomograms. 3.3 Quantitative analysis results for morphological and biochemical parameters agree with our observation that when ampicillin is applied, cell division stops and abnormal elongation and bulge formation
occurs. This abnormal growth occurred for a shorter time at high ampicillin concentration, indicating that E. coli burst to death
faster at higher antibiotic concentrations [Fig. 4(b)]. The cellular dry mass of E. coli is shown in Fig. 4(c) (See Methods). The graphs for the dry mass are similar to those of
cell volume [Fig. 4(a)], indicating that the cytoplasmic concentrations did not significantly change over time. This result can
also be confirmed from the measured 3D RI tomograms [Fig. 4(d)]. The cytoplasm concentration increased slightly as the
bacteria grew. After bacterial lysis, the internal contents escaped beyond the cell wall, and so, the cytoplasm concentration
dropped suddenly. Comparison between the control and ampicillin-treated groups clearly indicates that the sudden drop in
cytoplasm concentration was only notable in the latter case. Compared to E. coli, B. subtilis did not show noticeable increases in either cell volume or dry mass upon ampicillin
treatment [Figs. 4(e)-4(g)]. Rather, the bacteria rapidly reached a much smaller cell volume than the initial volume. The most
discernible change was observed for the temporal graphs of cytoplasm concentration; a decrease in cytoplasm concentration
caused by ampicillin was relatively well observed [Fig. 4(h)]. To clarify the different lysis behaviors between E. coli and B. subtilis, we reorganized and compared the species-dependent
changes in quantitative parameters. Among the various parameters, we chose to compare cell volumes because they showed
the most notable difference [Fig. 5]. At ampicillin concentrations of 20 and 100 g/mL, the difference in cell volume between
E. coli and B. subtilis was remarkable [Figs. 5(a) and 5(b)]. This is because abnormal growth was observed in E. coli treated
with ampicillin, but no such growth was observed in B. subtilis. Moreover, at ampicillin concentrations of 20 and 100 g/mL,
the time to reach a minimal cell volume was shorter for B. subtilis than for E. coli. In other words, B. subtilis appeared to
undergo lysis faster than E. coli. However, at the concentration of 300 g/mL [Fig. 5(c)], this tendency was lost. These
observations can be seen more clearly in Fig. 5(d), which represents the maximum volume of the bacteria at each concentration
as a relative value. These data sufficiently represent how ODT can quantitatively address the dynamics of the antibiotic response
of bacteria of bacteria. Fig. 5. 3.3 Quantitative analysis results for morphological and biochemical parameters Comparing the graphs for ampicillin concentrations of 20, 100, and 300 g/mL, as the concentration of
ampicillin increased, the bacteria showed earlier lysis, and the maximum volume of bacteria decreased. In addition, the
maximum volume of E. coli with ampicillin treatments was significantly larger than that of the control group. These results Upon ampicillin treatment, E. coli did not exhibit cell division, as shown in Fig. 3. The volumes of treated E. coli showed
monotonic increases, followed by sudden plummets [Fig. 3(a)]. These time points are consistent with the cell lysis observed in
the 3D RI tomograms. Comparing the graphs for ampicillin concentrations of 20, 100, and 300 g/mL, as the concentration of
ampicillin increased, the bacteria showed earlier lysis, and the maximum volume of bacteria decreased. In addition, the
maximum volume of E. coli with ampicillin treatments was significantly larger than that of the control group. These results Upon ampicillin treatment, E. coli did not exhibit cell division, as shown in Fig. 3. The volumes of treated E. coli showed
monotonic increases, followed by sudden plummets [Fig. 3(a)]. These time points are consistent with the cell lysis observed in
the 3D RI tomograms. Comparing the graphs for ampicillin concentrations of 20, 100, and 300 g/mL, as the concentration of
ampicillin increased, the bacteria showed earlier lysis, and the maximum volume of bacteria decreased. In addition, the
maximum volume of E. coli with ampicillin treatments was significantly larger than that of the control group. These results . CC-BY 4.0 International license
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bioRxiv preprint agree with our observation that when ampicillin is applied, cell division stops and abnormal elongation and bulge formation
occurs. This abnormal growth occurred for a shorter time at high ampicillin concentration, indicating that E. coli burst to death
faster at higher antibiotic concentrations [Fig. 4(b)]. 3.3 Quantitative analysis results for morphological and biochemical parameters (a−c) The relative value of cell volume for E. coli and B. subtilis with the ampicillin concentration of (a) 20 g/mL (b) 100 g/mL
(c) 300 g/mL. (d) The relative value of the maximum volume for E. coli and B. subtilis. Fig. 5. (a−c) The relative value of cell volume for E. coli and B. subtilis with the ampicillin concentration of (a) 20 g/mL (b) 100 g/mL
(c) 300 g/mL. (d) The relative value of the maximum volume for E. coli and B. subtilis. 4. Discussions and Summary In summary, we present a method to systematically investigate the alterations in individual bacteria upon antibiotic treatment. Employing ODT, the 3D RI of antibiotic-treated bacteria was measured over time in a label-free and non-invasive manner. The
3D RI reconstruction of E. coli and B. subtilis bacteria provides the morphological and biochemical parameters related to their
real-time alterations depending on the concentration of ampicillin, which were characterized by the inhibition of cell division,
bulge formation, abnormal elongation, and cell lysis. Because the RI inside the bacteria is directly related to the cytoplasm
concentration, dynamics of cytoplasmic concentrations and cellular dry mass were also analyzed quantitatively. Moreover, we
quantitatively deduced the bacterial lysis time through a sudden change in cell volume or cellular dry mass. This can also be
used effectively to study the cell cycle of bacteria [50]. Our measurements and observations with bacteria are consistent with those in the existing literature. Ampicillin from the
β-lactam class inhibits cell wall synthesis, leading to various changes in bacterial cell shape and size, and finally resulting in
bacterial cell death [51]. It is also intriguing to observe the changes in bacterial appearance depending on the ampicillin
concentration. Spratt explains that cell division of E. coli is inhibited even at low concentrations of ampicillin, resulting in
filamentation of E. coli; further, at higher concentrations, a bulge is formed in the middle of E. coli, and cell lysis occurs at
significantly high ampicillin concentrations [13]. In our experiments, the corresponding concentrations can be considered as
20, 100, and 300 g/mL, respectively. Comparing the cells with 20 g/mL of ampicillin to the control group, cell division was
inhibited, and abnormal growth, i.e. filamentation, was observed. As the concentration increased to 100 g/mL, the
filamentation phenomenon decreased, and the bulge became more pronounced. This is also consistent with the statement that
only lysis was observed at the concentration of 300 g/mL. . CC-BY 4.0 International license
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bioRxiv preprint The different responses of E. coli and B. subtilis to ampicillin are also noteworthy for discussion. Funding This work was supported by KAIST, BK21+ program, Tomocube, and National Research Foundation of Korea
(2017M3C1A3013923, 2015R1A3A2066550, 2018K000396). Disclosures Prof. Park and Mr. Moosung Lee have financial interests in Tomocube Inc., a company that commercializes optical diffraction
tomography and quantitative phase imaging instruments and is one of the sponsors of the work. 4. Discussions and Summary Ampicillin hinders
transpeptidation by combining with penicillin-binding proteins on maturing peptidoglycan strands, and the resulting reduction
in peptidoglycan synthesis causes cell lysis [13, 52]. In the case of Gram-positive bacteria including B. subtilis, the majority of
the cell wall is composed of peptidoglycan; thus, the effect on ampicillin is more pronounced [52], resulting in faster cell lysis. This explains the contrast in the E. coli and B. subtilis at ampicillin concentrations of 20 and 100 g/mL. In addition, there is
a difference in the reactivity of Gram-positive and Gram-negative bacteria to ampicillin, but ampicillin eventually leads to cell
death in both types of bacteria [53]. Our experiments confirmed that both E. coli and B. subtilis reached cell lysis in the presence
of ampicillin. Indeed, our results were based on small datasets but were sufficient to epitomize the antibiotic response in a
label-free, quantitative, and three-dimensional manner. Previously, 2D QPI techniques have been applied for the study of individual bacteria [54], [55, 56]. However, 2D QPI only
measures the optical phase delay maps, an integration of RI values along an optical path, and does not provide tomographic
measurements of individual bacteria. As demonstrated in this work, ODT enables the systematic investigation of various
volumetric information of individual bacteria. We emphasize that the 3D imaging technique exploited here is not limited to the
objects targeted in this paper. The species of bacteria and the type of antibiotics can be expanded as needed. In addition, using
this method, it is possible to conduct statistical analysis by increasing the number of microbes regardless of single cells or
multiple cells. It is also possible to infer the physical functions and biological characteristics of bacteria from their interior
structural features [57, 58]. Moreover, quantitative analysis for other morphological and biochemical parameters can be
attempted. For instance, when a bulge is formed in E. coli, each volume or cytoplasm concentration can be quantified by
separating the bulge and non-bulge portions. The lifetime of the bulge can also be measured. In this case, combination with the
segmentation method will serve as the key to analysis [24]. We believe that the methods presented in this paper will provide
microbiologists with broader insights into the various responses of bacteria to antibiotics. Acknowledgments The authors thank Dr. Weisun Park for helpful discussions. References Kohanski, M.A., D.J. Dwyer, and J.J. Collins, How antibiotics kill bacteria: from targets to network
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Marine deforestation leads to widespread loss of ecosystem function
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PLOS ONE RESEARCH ARTICLE Marine deforestation leads to widespread
loss of ecosystem function Matthew EdwardsID1*, Brenda Konar2, Ju-Hyoung KimID3, Scott Gabara1,4,
Genoa Sullaway1, Tristin McHugh1, Michael SpectorID1, Sadie SmallID1 1 Department of Biology, San Diego State University, San Diego, CA, United States of America, 2 College of
Fisheries and Ocean Sciences, University of Alaska Fairbanks, Fairbanks, Alaska, United States of America,
3 Marine Applied Biosciences, Kunsan National University, Gunsan, South Korea, 4 Department of
Environmental Science and Policy, University of California, Davis, California, United States of America a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * medwards@sdsu.edu Editor: Maura (Gee) Geraldine Chapman, University
of Sydney, AUSTRALIA Editor: Maura (Gee) Geraldine Chapman, University
of Sydney, AUSTRALIA Editor: Maura (Gee) Geraldine Chapman, University
of Sydney, AUSTRALIA Received: November 20, 2019
Accepted: February 13, 2020
Published: March 4, 2020 Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0226173 Copyright: © 2020 Edwards et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Abstract Trophic interactions can result in changes to the abundance and distribution of habitat-form-
ing species that dramatically reduce ecosystem functioning. In the coastal zone of the Aleu-
tian Archipelago, overgrazing by herbivorous sea urchins that began in the 1990s resulted
in widespread deforestation of the region’s kelp forests, which led to lower macroalgal abun-
dances and higher benthic irradiances. We examined how this deforestation impacted eco-
system function by comparing patterns of net ecosystem production (NEP), gross primary
production (GPP), ecosystem respiration (Re), and the range between GPP and Re in rem-
nant kelp forests, urchin barrens, and habitats that were in transition between the two habitat
types at nine islands that spanned more than 1000 kilometers of the archipelago. Our results
show that deforestation, on average, resulted in a 24% reduction in GPP, a 26% reduction in
Re, and a 24% reduction in the range between GPP and Re. Further, the transition habitats
were intermediate to the kelp forests and urchin barrens for these metrics. These opposing
metabolic processes remained in balance; however, which resulted in little-to-no changes to
NEP. These effects of deforestation on ecosystem productivity, however, were highly vari-
able between years and among the study islands. In light of the worldwide declines in kelp
forests observed in recent decades, our findings suggest that marine deforestation pro-
foundly affects how coastal ecosystems function. OPEN ACCESS Citation: Edwards M, Konar B, Kim J-H, Gabara S,
Sullaway G, McHugh T, et al. (2020) Marine
deforestation leads to widespread loss of
ecosystem function. PLoS ONE 15(3): e0226173. https://doi.org/10.1371/journal.pone.0226173 * medwards@sdsu.edu PLOS ONE PLOS ONE Introduction Currently, sea otters are largely absent
from or are in very low abundances on many of the islands and most of the kelp forests have
either disappeared from the archipelago or are in the process of disappearing, although some
small forests remain in their ‘historical state’ at scattered locations on most of the islands
[14,15] (Fig 1). These remnant forests provide a valuable benchmark against which we evalu-
ated the effects of widespread deforestation on an important metric of ecosystem function,
namely primary productivity. ME and (OCE1435205) to BK, and the National
Research Foundation (NRF-2018R1C1B6008523
and NRF-2015R1C1A1A01054831) to JHK. ME and (OCE1435205) to BK, and the National
Research Foundation (NRF-2018R1C1B6008523
and NRF-2015R1C1A1A01054831) to JHK. Competing interests: NO - Authors have no
competing interests. Characterizing patterns of biodiversity and primary productivity is essential to fully under-
standing ecosystem function [16,17]. The latter includes three basic metrics: gross primary
production (GPP), which describes all the CO2 fixed by the autotrophs during photosynthesis,
total ecosystem respiration (Re), which describes the release of CO2 during the production of
energy by autotrophs, heterotrophs, decomposers and microbes, and net ecosystem produc-
tion (NEP), which is the difference between GPP and Re and describes net changes in the total
amount of organic carbon in an ecosystem available for consumption, storage and export to
adjacent ecosystems, or nonbiological oxidation to carbon dioxide [18–21]. In general, ecosys-
tems with high rates of GPP also exhibit high rates of Re, with the central tendency being that
GPP and Re are in balance (i.e., similar in magnitude) and therefore have median GPP / Re
ratios close to 1.0, and NEP values near zero [21,22]. Indeed, a review of five decades (1950 to
1990) of studies in aquatic ecosystems demonstrated that these two opposing processes are
generally in balance, although unproductive ecosystems tend towards net heterotrophy with
GPP / Re < 1.0 and NEP < 0, while productive ecosystems tend towards net autotrophy with
GPP / Re > 1.0 and NEP > 0 [21,22]. Further, the amount of Re associated with any given GPP
in shallow coastal ecosystems tends to be greater when the complete benthic communities are
considered [22]. This may be especially true if microbial metabolism, which is an important
component of Re, is large compared to GPP [20–22]. This is important for coastal kelp forests,
which host a higher diversity of microbes relative to the adjacent ocean waters [23–27]. Introduction Consumers fundamentally affect ecosystems through trophic interactions [1]. These interac-
tions are especially important if they result in changes to the abundance or distribution of eco-
system engineers, such as forest-forming trees, which can lead to changes in microclimates,
biodiversity, primary production, nutrient cycling, and energy flow [2]. For example, the rein-
troduction of gray wolves (Canis lupus) into Yellowstone National Park, USA in the 1990s
resulted in increased predation on elk (Cervus elaphus) and subsequently reduced herbivory
on canopy-forming trees such as aspens (Populus tremuloides), willows (Salix spp.), and cot-
tonwoods (Populus spp.) [3]. This ultimately led to changes in the morphology and hydrology Data Availability Statement: Data are available on
our NSF bco-dmo data page at https://www.bco-
dmo.org/dataset/755658 Funding: This research was funded by grants from
the National Science Foundation (OCE1435194) to PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 1 / 21 PLOS ONE Deforestation and loss of ecosystem function of the region’s river systems and its riparian plant communities [4,5]. Similarly, large marine
algae, such as kelps, can form subtidal forests whose biogenic structures alter hydrodynamic,
nutrient and light conditions, modify patterns of biodiversity, enhance primary production
and carbon sequestration, and provide food and habitat for numerous other species [6–9]. Consequently, the loss of these forest-forming kelps and the benthic communities they support
can have dramatic impacts to how nearshore ecosystems function, especially if they occur over
large geographic areas. Indeed, kelp deforestation has occurred in numerous areas worldwide
in recent decades due to a variety of forcing factors [10,11], and the subtidal rocky reefs of the
Aleutian Archipelago serve as a model system to investigate the broader impacts of such defor-
estation. These forests have historically been dominated by dense populations of the surface
canopy-forming kelp Eualaria fistulosa, several species of understory kelps such as Laminaria
spp. and Agarum spp., the brown alga Desmarestia spp., and numerous species of fleshy read
algae. However, the collapse of sea otter (Enhydra lutris) populations led to large increases in
their primary prey, herbivorous sea urchins (Strongylocentrotus polyacanthus), which subse-
quently resulted in overgrazing and widespread losses of the region’s kelp forests [12]. This
collapse began in the late 1990s, likely in response to a dietary shift by killer whales toward sea
otters, and by 2000 sea otter densities had declined throughout the archipelago to around
5–10% of their estimated equilibrium density [13]. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 Introduction Conse-
quently, loss of these forests may lead to complex patterns of GPP, Re, and NEP within coastal
ecosystems. On one hand, reductions in primary producer biomass should result in lowered
GPP and thus reduced NEP. Alternately, deforestation may result in lowered abundances of
invertebrates, fishes and microbes, which may lead to reduced Re and thus enhanced NEP. At
the same time, loss of the habitat-forming kelps also results in elevated benthic irradiances
(measured as photosynthetically active radiation (PAR) [18] and thus potentially to enhanced PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 2 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 1. Three habitat types. Photographs of each habitat type showing (A) high abundance of benthic macroalgae and
canopy-forming kelps in the kelp forests, (B) lack of benthic macroalgae but remaining canopy-forming kelps and high
abundances of sea urchins in the transition habitats, and (C) lack of benthic macroalgae and canopy-forming kelps, but
high abundances of sea urchins in the urchin barrens. Fig 1. Three habitat types. Photographs of each habitat type showing (A) high abundance of benthic macroalgae and
canopy-forming kelps in the kelp forests, (B) lack of benthic macroalgae but remaining canopy-forming kelps and high
abundances of sea urchins in the transition habitats, and (C) lack of benthic macroalgae and canopy-forming kelps, but
high abundances of sea urchins in the urchin barrens. Fig 1. Three habitat types. Photographs of each habitat type showing (A) high abundance of benthic macroalgae and
canopy-forming kelps in the kelp forests, (B) lack of benthic macroalgae but remaining canopy-forming kelps and high
abundances of sea urchins in the transition habitats, and (C) lack of benthic macroalgae and canopy-forming kelps, but
high abundances of sea urchins in the urchin barrens. https://doi.org/10.1371/journal.pone.0226173.g001 https://doi.org/10.1371/journal.pone.0226173.g001 https://doi.org/10.1371/journal.pone.0226173.g001 compensatory production by any remaining fleshy macroalgae, encrusting coralline algae, and
microalgae [28–30], which can result in greater NEP. Thus, understanding how GPP, Re, and
NEP change with kelp forest change can be instrumental in discerning the broader impacts of
deforestation on ecosystem productivity. This may be especially relevant for the Aleutian
Archipelago where widespread kelp deforestation has resulted in significant reductions in
fishes, invertebrates and fleshy macroalgae, increases in the exposure of encrusting coralline
algae [12,31], and elevated benthic irradiances [14]. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 Results We used benthic chambers to study patterns of GPP, Re, and NEP within remnant kelp forests,
urchin barrens, and habitats that were in transition to becoming urchin barrens (i.e., they had
lost all benthic fleshy macroalgae but still had abundant stands of the canopy-forming Eualaria
fistulosa; Fig 1) at nine islands spanning more than 1000 kilometers of the Aleutian Archipel-
ago (Fig 2, Table 1). Kelp forests and urchin barrens occur as alternate stable states of one
another, often with sharply delineated boundaries between them, and exhibit little-to-no over-
lap in community assemblages [15,33] (Fig 1). Indeed, the benthic communities within our
chambers reflected these assemblages, with the chambers deployed in the kelp forests having
more than a 10-fold greater biomass of fleshy macroalgae, which were predominantly stipitate
kelps, than those deployed in the urchin barrens, and the chambers deployed in the urchin bar-
rens having a nearly 3-fold greater biomass of urchins than those deployed in the kelp forests
(Figs 3 and 4). The chambers deployed within transition habitats contained high abundances
of urchins and little-to-no fleshy macroalgae, except for the canopy-forming E. fistulosa. The
chambers within all three habitats had high bottom covers of encrusting coralline algae below
the fleshy macroalgae, which became exposed following deforestation. Benthic irradiances,
measured as photosynthetically active radiation (PAR), varied among the three habitat types
(ANOVA: F2,14 = 4.826, p = 0.025), but this was variable among the nine islands and two study
years (HabitatIsland(Year) interaction: F14,33, = 4.426, p <0.001; Table 2). Generally, PAR
was greatest in the urchin barrens, lowest in the kelp forests, and intermediate in the transition
habitats (Fig 5). We examined how GPP, Re and NEP, and the balance between GPP and Re differed among
the habitat types by measuring changes in seawater oxygen concentrations within replicate
(n = 3) chambers (collapsible benthic incubation tents; hereafter cBITs) that were placed on
the benthos over representative assemblages within each habitat type at each island. We pre-
dicted that NEP at the benthos would be reduced in the urchin barrens due to the loss of pho-
tosynthetic macroalgae. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 Results Instead, we found that NEP did not differ between the habitat types
ANOVA: F2,14 = 0.530, p = 0.600), nor did it differ from zero (i.e., GPP = Re) in any of the PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 3 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity
(NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset
version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity
(NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset
version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity
(NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset
version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. https://doi.org/10.1371/journal.pone.0226173.g002 Fig 2. Map of the Aleutian Archipelago. Map of the Aleutian Archipelago showing locations of the nine islands (denoted by red circles) where ecosystem productivity
(NEP, GPP and Re) was measured in the cBITs. Shoreline data was obtained from the Global Self-Consistent Hierarchical High-resolution Shoreline (GSHHG) dataset
version 2.3.4 (www.soest.hawaii.edu/wessel/gshhg/) [32]. https://doi.org/10.1371/journal.pone.0226173.g002 https://doi.org/10.1371/journal.pone.0226173.g002 habitat types (Table 2, Figs 6 and 7). The effects of habitat type on NEP, however, varied
among the nine islands visited during the two study years (HabitatIsland(Year) interaction:
F14,41, = 4.541, p < 0.001; Table 2). Specifically, NEP was greater in the urchin barrens than in
the kelp forests on five of the islands and lower in the urchin barrens on three of the islands,
with the average difference being 25.58 ± 373.26 mg O2 m-2 day-1 lower in the urchin barrens
(i.e., the deforested habitats) (Tables 3 and 4). The change on one island (Attu) was not deter-
mined due to lost replication (Tables 1 and 4). Results However, when averaged across all nine islands,
NEP was generally lowest (-239.73 ± 425.16 mg O2 m-2 day-1, mean ± SE) in the kelp forests,
highest (-59.60 ± 145.32 mg O2 m-2 day-1) in the transition habitats, and intermediate
(-120.08 ± 338.07 mg O2 m-2 day-1) in the urchin barrens (Table 3, Fig 4). Benthic GPP also
did not vary among the habitat types (ANOVA: F2, 14 = 0.234, p = 0.794), but when averaged
across islands, GPP was highest in the kelp forests (1,806.14 ± 521.75 mg O2 m-2 day-1;
mean ± SE), lowest in the urchin barrens (1,367.77 ± 483.99 mg O2 m-2 day-1), and intermedi-
ate in the transition habitats (1,494.22 ± 452.41 mg O2 m-2 day-1) (Fig 4; Table 3). Like NEP,
the effects of habitat type varied among the nine islands visited in the two study years (Habi-
tatIsland(Year) interactions: F14,41 = 2.166, p = 0.028; Table 2). Specifically, GPP was lower in habitat types (Table 2, Figs 6 and 7). The effects of habitat type on NEP, however, varied
among the nine islands visited during the two study years (HabitatIsland(Year) interaction:
F14,41, = 4.541, p < 0.001; Table 2). Specifically, NEP was greater in the urchin barrens than in
the kelp forests on five of the islands and lower in the urchin barrens on three of the islands,
with the average difference being 25.58 ± 373.26 mg O2 m-2 day-1 lower in the urchin barrens
(i.e., the deforested habitats) (Tables 3 and 4). The change on one island (Attu) was not deter-
mined due to lost replication (Tables 1 and 4). However, when averaged across all nine islands,
NEP was generally lowest (-239.73 ± 425.16 mg O2 m-2 day-1, mean ± SE) in the kelp forests,
highest (-59.60 ± 145.32 mg O2 m-2 day-1) in the transition habitats, and intermediate
(-120.08 ± 338.07 mg O2 m-2 day-1) in the urchin barrens (Table 3, Fig 4). Benthic GPP also
did not vary among the habitat types (ANOVA: F2, 14 = 0.234, p = 0.794), but when averaged
across islands, GPP was highest in the kelp forests (1,806.14 ± 521.75 mg O2 m-2 day-1;
mean ± SE), lowest in the urchin barrens (1,367.77 ± 483.99 mg O2 m-2 day-1), and intermedi-
ate in the transition habitats (1,494.22 ± 452.41 mg O2 m-2 day-1) (Fig 4; Table 3). Results Like NEP,
the effects of habitat type varied among the nine islands visited in the two study years (Habi-
tatIsland(Year) interactions: F14,41 = 2.166, p = 0.028; Table 2). Specifically, GPP was lower in habitat types (Table 2, Figs 6 and 7). The effects of habitat type on NEP, however, varied
among the nine islands visited during the two study years (HabitatIsland(Year) interaction:
F14,41, = 4.541, p < 0.001; Table 2). Specifically, NEP was greater in the urchin barrens than in
the kelp forests on five of the islands and lower in the urchin barrens on three of the islands,
with the average difference being 25.58 ± 373.26 mg O2 m-2 day-1 lower in the urchin barrens
(i.e., the deforested habitats) (Tables 3 and 4). The change on one island (Attu) was not deter-
mined due to lost replication (Tables 1 and 4). However, when averaged across all nine islands,
NEP was generally lowest (-239.73 ± 425.16 mg O2 m-2 day-1, mean ± SE) in the kelp forests,
highest (-59.60 ± 145.32 mg O2 m-2 day-1) in the transition habitats, and intermediate
(-120.08 ± 338.07 mg O2 m-2 day-1) in the urchin barrens (Table 3, Fig 4). Benthic GPP also
did not vary among the habitat types (ANOVA: F2, 14 = 0.234, p = 0.794), but when averaged
across islands, GPP was highest in the kelp forests (1,806.14 ± 521.75 mg O2 m-2 day-1;
mean ± SE), lowest in the urchin barrens (1,367.77 ± 483.99 mg O2 m-2 day-1), and intermedi-
ate in the transition habitats (1,494.22 ± 452.41 mg O2 m-2 day-1) (Fig 4; Table 3). Like NEP,
the effects of habitat type varied among the nine islands visited in the two study years (Habi-
tatIsland(Year) interactions: F14,41 = 2.166, p = 0.028; Table 2). Specifically, GPP was lower in PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 4 / 21 PLOS ONE Deforestation and loss of ecosystem function the urchin barrens than in the kelp forests on all but two of the islands, by an average of
461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Results Re also did not vary among the habitat
types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in
the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ±
546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2
Table 1. List of the nine islands in the Aleutian Archipelago where cBITs were deployed to measure NEP, GPP and Re during 2016 and 2017, and the six islands
where all macroalgae and invertebrates were collected from within the cBITs to estimate their biomass during 2016. The number cBITs deployed, the deployment
year, and whether macroalgae and invertebrates were collected from within the cBITs at each island are noted. Island
Year
No. cBITs deployed
Collections made? Kelp
Transition
Urchin
forests
habitats
barrens
Adak
2016
3
2
3
No
Amchitka
2017
3
2
3
Yes
Atka
2017
3
3
2
Yes
Attu
2017
3
3
1
Yes
Chuginadak
2016
2
2
3
Yes
Kiska
2017
3
3
2
Yes
Nizki
2017
3
3
2
Yes
Tanaga
2016
2
1
2
No
Yunaska
2017
3
3
3
No
Totals
25
22
21
6
https://doi.org/10.1371/journal.pone.0226173.t001 Table 1. List of the nine islands in the Aleutian Archipelago where cBITs were deployed to measure NEP, GPP and Re during 2016 and 2017, and the six islands
where all macroalgae and invertebrates were collected from within the cBITs to estimate their biomass during 2016. The number cBITs deployed, the deployment
year, and whether macroalgae and invertebrates were collected from within the cBITs at each island are noted. the urchin barrens than in the kelp forests on all but two of the islands, by an average of
461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Re also did not vary among the habitat
types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in
the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ±
546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2
Table 1. Results List of the nine islands in the Aleutian Archipelago where cBITs were deployed to measure NEP, GPP and Re during 2016 and 2017, and the six islands
where all macroalgae and invertebrates were collected from within the cBITs to estimate their biomass during 2016. The number cBITs deployed, the deployment
year, and whether macroalgae and invertebrates were collected from within the cBITs at each island are noted. Island
Year
No. cBITs deployed
Collections made? Kelp
Transition
Urchin
forests
habitats
barrens
Adak
2016
3
2
3
No
Amchitka
2017
3
2
3
Yes
Atka
2017
3
3
2
Yes
Attu
2017
3
3
1
Yes
Chuginadak
2016
2
2
3
Yes
Kiska
2017
3
3
2
Yes
Nizki
2017
3
3
2
Yes
Tanaga
2016
2
1
2
No
Yunaska
2017
3
3
3
No
Totals
25
22
21
6
https://doi.org/10.1371/journal.pone.0226173.t001
Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass
measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during
2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi.org/10.1371/journal.pone.0226173.g003
PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020
5 / 21 the urchin barrens than in the kelp forests on all but two of the islands, by an average of
461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Re also did not vary among the habitat
types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in
the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ±
546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2 the urchin barrens than in the kelp forests on all but two of the islands, by an average of
461.60 ± 578.69 mg O2 m-2 day-1 (mean ± SE) (Table 4). Re also did not vary among the habitat
types (F2,14 = 0.390, p = 0.684), but when averaged across all nine islands, Re was again highest in
the kelp forests (1,994.91 ± 574.11 mg O2 m-2 day-1), lowest in the urchin barrens (1,474.51 ±
546.83 mg O2 m-2 day-1), and intermediate in the transition habitats (1,553.84 ± 469.81 mg O2 m-2 Fig 3. Algae and invertebrate biomass. https://doi.org/10.1371/journal.pone.0226173.t001 Deforestation and loss of ecosystem function Fig 4. Algae and invertebrate biomass. Mean biomass (± SE) of (A) all kelps, and red, brown and green macroalgae, and (B) the most abundant
taxonomic groups of invertebrates collected from within the cBITs in each habitat type at six of the islands where the cBITs were deployed in 2017
(Table 1). Fig 5B is divided into two panels, with abundant taxa on the left panel, and rarer taxa on the right panel. https://doi org/10 1371/journal pone 0226173 g004 Fig 4. Algae and invertebrate biomass. Mean biomass (± SE) of (A) all kelps, and red, brown and green macroalgae, and (B) the most abundant
taxonomic groups of invertebrates collected from within the cBITs in each habitat type at six of the islands where the cBITs were deployed in 2017
(Table 1). Fig 5B is divided into two panels, with abundant taxa on the left panel, and rarer taxa on the right panel. Table 2. Results of separate three-way nested analyses of variance testing for differences in A) net ecosystem production (NEP), B) gross primary production
(GPP), C) ecosystem respiration (Re), D) the range between GPP and Re, and E) irradiance (PAR) among the two sample years, nine islands, and three habitat types
(kelp forests, transition habitats, and urchin barrens). For each analysis, year and habitat type were fixed factors, and island nested within year was a random factor. The model r2 is given for each analysis. (
)
)
y
p
(
)
)
g
)
(
)
g
p
y
yp
(kelp forests, transition habitats, and urchin barrens). For each analysis, year and habitat type were fixed factors, and island nested within year was a random factor. The model r2 is given for each analysis. https://doi.org/10.1371/journal.pone.0226173.t002 Results Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass
measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during
2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi org/10 1371/journal pone 0226173 g003 Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass
measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during
2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi.org/10.1371/journal.pone.0226173.g003 Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass
measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during
2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. Fig 3. Algae and invertebrate biomass. Box plots showing (A) Macroalgae (gray bars) and invertebrate (white bars) biomass
measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at six islands during
2017 (Table 1). Red diamonds represent mean values, and horizontal lines represent median values. https://doi.org/10.1371/journal.pone.0226173.g003 https://doi.org/10.1371/journal.pone.0226173.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 5 / 21 PLOS ONE Deforestation and loss of ecosystem function 6 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 PLOS ONE Deforestation and loss of ecosystem function Deforestation and loss of ecosystem function PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 Deforestation and loss of ecosystem function A) NEP (r2 = 0.74)
Source
Type III SS
df
Mean Squares
F-ratio
p-value
Year
146.911
1
146.911
8.319
0.006
Habitat
85.076
2
42.538
0.53
0.6
HabitatYear
365.04
2
182.52
10.336
<0.001
Island(Year)
610.045
7
87.149
4.935
<0.001
HabitatIsland(Year)
1122.729
14
80.195
4.541
<0.001
Error
724.009
41
17.659
B) GPP (r2 = 0.72)
Source
Type III SS
df
Mean Squares
F-ratio
p-value
Year
1060.514
1
1060.514
15
<0.001
Habitat
71.658
2
35.829
0.234
0.794
HabitatYear
416.847
2
208.424
2.948
0.064
Island(Year)
3497.967
7
499.71
7.068
<0.001
HabitatIsland(Year)
2144.29
14
153.164
2.166
0.028
Error
2898.811
41
70.703
C) Re (r2 = 0.78)
Source
Type III SS
df
Mean Squares
F-ratio
p-value
Year
1456.821
1
1456.821
24.825
<0.001
Habitat
125.51
2
62.755
0.39
0.684
HabitatYear
946.319
2
473.16
8.063
0.001
Island(Year)
3081.525
7
440.218
7.501
<0.001
HabitatIsland(Year)
2254.327
14
161.023
2.744
0.006
Error
2406.048
41
58.684
D) Range (r2 = 0.75)
Source
Type III SS
df
Mean Squares
F-ratio
p-value
Year
2503.758
1
2503.758
20.237
<0.001
Habitat
184.788
2
92.394
0.318
0.733
HabitatYear
1267.372
2
633.686
5.122
0.01
Island(Year)
6204.846
7
886.407
7.164
<0.001
HabitatIsland(Year)
4064.628
14
290.331
2.347
0.017
Error
5072.698
41
123.724
E) PAR (r2 = 0.83)
Source
Type III SS
df
Mean Squares
F-ratio
p-value
Year
0.898
1
0.898
8.503
0.006
Habitat
4.507
2
2.254
4.826
0.025
HabitatYear
1.231
2
0.616
5.832
0.007
Island(Year)
2.066
7
0.295
2.795
0.021
HabitatIsland(Year)
6.542
14
0.467
4.426
<0.001
https://doi.org/10.1371/journal.pone.0226173.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 7 / 21 PLOS ONE Deforestation and loss of ecosystem function Deforestation and loss of ecosystem PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 8 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 5. Production metrics. Box plots showing (A) Net Ecosystem Production (NEP), (B) Gross Primary Production
(GPP), (C) Ecosystem Respiration (Re), (D) the range between GPP and Re (Range), and (E) Irradiance (PAR), as
measured in the cBITs deployed within each habitat type (kelp forests, transition habitats, and urchin barrens) at nine
islands during 2016 and 2017 (Fig 2, Table 1). Red diamonds represent mean values, and horizontal lines represent
median values. Boxes within each graph that do not share letters represent significant differences between habitat pairs. https://doi.org/10.1371/journal.pone.0226173.g005 day-1) (Fig 4; Table 3). Deforestation and loss of ecosystem function As with NEP and GPP, the effects of habitat type varied among the nine
islands visited in the two study years (HabitatIsland(Year) interactions: F2,14 = 2.744, p = 0.006;
Table 2). Specifically, Re was lower in the urchin barrens than in the kelp forests on four of the
islands and greater in the urchin barrens on four of the islands, with the average difference being
472.09 ± 734.70 mg O2 m-2 day-1 lower in the urchin barrens (Table 4). Lastly, the range between
GPP and Re, which we believe to be a better measure of ecosystem function regarding productivity
than NEP alone, did not differ among the habitat types (ANOVA: F2,14 = 0.318, p = 0.733), but was
again greatest in the kelp forests (3,750 ± 1,069.01 mg O2 m-2 day-1), lowest in the urchin barrens
(2,860.94 ± 994.44 mg O2 m-2 day-1), and intermediate in the transition habitats (3,047.98 ± 910.36
mg O2 m-2 day-1) (Table 3, Fig 4). This again varied among the study islands visited in the two
study years (HabitatIsland(Year) interactions: F14,41 = 2.347, p = 0.017; Table 2). Specifically, the
range between GPP and Re was lower in the urchin barrens than in the kelp forests on five of the
islands and greater in the urchin barrens on two of the islands, with the average difference being
933.69 ± 1,262.65 mg O2 m-2 day-1 lower in the urchin barrens (Table 4). Although the effects of deforestation on all three metrics of productivity varied among the
islands visited in the two study years, some general patterns were evident. When considered
across all nine islands, GPP, Re and the range between GPP and Re were each greatest in the
kelp forests, intermediate in the transition habitats, and lowest in the urchin barrens. Specifi-
cally, GPP was 24% higher, on average, in the kelp forests than in the urchin barrens, and 17%
higher, on average, in the kelp forests than in the transition habitats, but it differed by only 7%
between the transition habitats and urchin barrens (Table 3, Fig 4). Benthic Re was 26% higher,
on average, in the kelp forests than in the urchin barrens, and 22% higher in the kelp forest
than the transition habitats, but it differed by less than 1% between the transition habitats and Fig 6. GPP versus Re. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 Deforestation and loss of ecosystem function Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. https://doi.org/10.1371/journal.pone.0226173.g007 https://doi.org/10.1371/journal.pone.0226173.g007 https://doi.org/10.1371/journal.pone.0226173.g007 the urchin barrens. The range was between GPP and Re was 24% greater, on average, in the
kelp forests than in the urchin barrens, and 19% greater in the kelp forests than in the transi-
tion habitats, but it varied by less than 6% between the transition habitats and the urchin bar-
rens. In contrast, GPP was, on average, greatest in the urchin barrens, intermediate in the
transition habitats, and lowest in the kelp forests in contrast, but it did not differ from zero (i.e. GPP = Re) in any of the habitats. These patterns, however, were highly variable among the dif-
ferent islands visited in the two study years for each of the production metrics. Altogether, this
indicated deforestation resulted in widespread but geographically variable losses to primary
production and respiration by the ecosystem. As with previous studies in aquatic ecosystems, we found that GPP and Re are generally in
balance, resulting in exhibit GPP / Re ratios near 1.0, and NEP values near zero [21,22]. When
examined within each cBIT separately, GPP and Re were consistently similar in magnitude
with no differences in GPP / Re ratios among habitat types (ANCOVA: F2,62 = 0.16, p = 0.852)
(Table 5, Fig 6). Further, the distribution of these ratios was symmetrical around 1.0 in each
habitat (Fig 7). Interestingly, the highest individual values of NEP were not observed in the
kelp forests but rather in the urchin barrens, which we believe was due to higher irradiances in
the urchin barrens than the other two habitats (Fig 5) combined with compensatory produc-
tion by the encrusting coralline algae and benthic diatoms [30]. However, those few observa-
tions aside, it is clear that all three benthic habitats remain in balance following deforestation,
with GPP Re, GPP / Re ratios 1, and median NEP values 0. Thus, although NEP may
help differentiate between productive and unproductive ecosystems [22], it poorly describes
changes in primary productivity following large-scale habitat change in the Aleutian Archipel-
ago. Deforestation and loss of ecosystem function Instead, it is clear that deforestation results in significant changes to the region’s benthic
communities, and these led to geographically variable reductions in GPP, Re and the range
between them, which better reflect a reduction in ecosystem functioning. Further, it appears
that even partial deforestation, where the benthic macroalgae and invertebrates have been lost
but the canopy-forming kelps remain, results in lower GPP and Re at the benthos that is simi-
lar to trends found in urchin barrens. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 Deforestation and loss of ecosystem function Relationship between gross primary production (GPP) and ecosystem respiration (Re) for each
habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each point represents
measurements from a single cBIT. Gray shading denoted 95% confidence intervals. https://doi.org/10.1371/journal.pone.0226173.g006 Fig 6. GPP versus Re. Relationship between gross primary production (GPP) and ecosystem respiration (Re) for each
habitat type across all nine islands where cBITs were deployed in 2016 and 2017 (Table 1). Each point represents
measurements from a single cBIT. Gray shading denoted 95% confidence intervals. https://doi.org/10.1371/journal.pone.0226173.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 9 / 21 PLOS ONE Deforestation and loss of ecosystem function ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 10 / 21 Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands
where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. https://doi.org/10.1371/journal.pone.0226173.g007
Deforestation and loss of ecosystem function PLOS ONE Deforestation and loss of ecosystem function Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands
where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands
where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Note the urchin barrens have the highest ratios observed, and the kelp forests have the largest number of low values. The vertical dashed line represents the 1:1 ratio. Fig 7. GPP and Re ratios. Frequency distribution of GPP / Re ratios within each habitat type across all nine islands
where cBITs were deployed in 2016 and 2017 (Table 1). Each data point represents measurements from a single cBIT. Discussion Trophic interactions can lead to changes in the distribution and abundance of habitat-forming
species, which can have profound impacts on ecosystem function [2,9]. Deforestation, in par-
ticular, can result in changes to biodiversity and energy flow [2], altered regional and global cli-
mates [34], and even lead to species extinctions [35]. Coastal kelps are a pertinent example of
such ecosystem engineers in nearshore habitats that have suffered declines in some locations
over the past few decades due to both biological and physical stressors [10,11,36–39]. Conse-
quently, while kelp populations remain stable in many of the world’s ecoregions [10,40,41], or
may even be expanding in some high latitude regions in response to ocean warming [39,42],
our study is relevant to many areas of the world where kelp forests have exhibited local to
broad scale declines [10,43–47]. Indeed, recent estimates suggest that global declines in kelp
abundances may be as high as 2% per year [11], which can negatively affect numerous other
species that depend on them for food and habitat. Certainly, the kelp forests of the Aleutian
Archipelago are in critical condition in the face of widespread overgrazing by urchins, and this PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 11 / 21 PLOS ONE Deforestation and loss of ecosystem function Table 3. Community productivity values (measured as mg O2 m-2 day-1) for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem
respiration (Re), and D) the Range between GPP and Re (Range) estimated for each habitat on each island. Data reflect the means (SD) of the replicate chambers in
each habitat (kelp Forests, Urchin Barrens and Transition Habitats). Positive values for NEP reflect net oxygen production and negative values reflect oxygen consumption
(net respiration). Negative values for Re reflect oxygen consumption (i.e. more negative values reflect greater respiration by the ecosystem). “NA” denotes not available due
to lack of replication (i.e. data are based on only one chamber at that island; see Table 1). Table 3. Community productivity values (measured as mg O2 m-2 day-1) for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem
respiration (Re), and D) the Range between GPP and Re (Range) estimated for each habitat on each island. Data reflect the means (SD) of the replicate chambers in
each habitat (kelp Forests, Urchin Barrens and Transition Habitats). PLOS ONE (Continued)
Chuginadak
1797.40 (188.72)
3576.43 (462.80)
2057.03 (15.29)
Kiska
2606.00 (1597.22)
1589.48 (613.66)
1665.17 (653.88)
Nizki
6135.68 (1014.08)
2281.17 (1026.88)
4293.65 (1143.33)
Tanaga
5710.56 (1538.90)
5682.77 (515.01)
5201.03 (NA)
Yunkasa
890.72 (121.60)
1115.60 (424.16)
3042.63 (1262.13)
Average
3750.03
2860.94
3047.98
SE
1069.01
994.44
910.94
https://doi.org/10.1371/journal.pone.0226173.t003 has had profound effects on the region’s benthic communities and on patterns of gross pri-
mary production and ecosystem respiration. Whether these forests will recover and return to
prior ecosystem functioning regarding these metrics is unknown, but observations of kelp for-
ests from other areas of the world suggest it is possible. For example, Laminaria longicruris for-
ests recovered from overgrazing following localized disease outbreaks that decimated sea
urchin populations in Nova Scotia [48], while L. hyperborea forests recovered in mid-Norway
due to low sea urchin recruitment [49]. Ecklonia maxima expanded its range eastward in
South Africa, coincident with cooling of the local ocean waters [50]. Likewise, Macrocystis pyri-
fera recovered along a ~100 km stretch of the Pacific coast of Baja California, Mexico following
nearly two decades of absence after the strong 1997–98 El Niño Southern Oscillation [51]. Recovery of the Eualaria fistulosa forests throughout the Aleutian Archipelago, however,
would likely require widespread mortality in the urchin populations, which today seems
unlikely. One potential contributing factor for this may lie in the low abundance of other
urchin predators, such as the urchin eating sea star, Pycnopodia helianthoides [38,52,53],
which historically has not been found in high abundances in the central or western Aleutians. Therefore, until predation on the urchins recovers or the urchin populations suffer widespread
disease that reduces their numbers, benthic algal abundances, GPP and Re will likely remain
generally lower in areas of kelp forest loss because the high abundance of urchins limits
regrowth of macroalgae and maintains the urchin barrens [15]. Thus, we present a benchmark
against which we can evaluate this recovery if it occurs, and understand the effects of further
deforestation in this ecosystem. Although we have learned much about the effects of the otter-urchin-kelp trophic cascade
in the Aleutian Archipelago, this study offers new insights into the consequences of such wide-
spread deforestation on the region’s benthic primary productivity. Certainly, benthic GPP, Re
and the range between them are generally greatest in the kelp forests where macroalgae, fish,
invertebrates, and microbial communities are all most abundant [15,23–26,33], while they are
lowest in the urchin barrens. PLOS ONE PLOS ONE Deforestation and loss of ecosystem function has had profound effects on the region’s benthic communities and on patterns of gross pri-
mary production and ecosystem respiration. Whether these forests will recover and return to
prior ecosystem functioning regarding these metrics is unknown, but observations of kelp for-
ests from other areas of the world suggest it is possible. For example, Laminaria longicruris for-
ests recovered from overgrazing following localized disease outbreaks that decimated sea
urchin populations in Nova Scotia [48], while L. hyperborea forests recovered in mid-Norway
due to low sea urchin recruitment [49]. Ecklonia maxima expanded its range eastward in
South Africa, coincident with cooling of the local ocean waters [50]. Likewise, Macrocystis pyri-
fera recovered along a ~100 km stretch of the Pacific coast of Baja California, Mexico following
nearly two decades of absence after the strong 1997–98 El Niño Southern Oscillation [51]. Recovery of the Eualaria fistulosa forests throughout the Aleutian Archipelago, however,
would likely require widespread mortality in the urchin populations, which today seems
unlikely. One potential contributing factor for this may lie in the low abundance of other
urchin predators, such as the urchin eating sea star, Pycnopodia helianthoides [38,52,53],
which historically has not been found in high abundances in the central or western Aleutians. Therefore, until predation on the urchins recovers or the urchin populations suffer widespread
disease that reduces their numbers, benthic algal abundances, GPP and Re will likely remain
generally lower in areas of kelp forest loss because the high abundance of urchins limits
regrowth of macroalgae and maintains the urchin barrens [15]. Thus, we present a benchmark
against which we can evaluate this recovery if it occurs, and understand the effects of further
deforestation in this ecosystem. Al h
h
h
l
d
h b
h
ff
f h
hi
k l
hi
d
Table 3. (Continued)
Chuginadak
1797.40 (188.72)
3576.43 (462.80)
2057.03 (15.29)
Kiska
2606.00 (1597.22)
1589.48 (613.66)
1665.17 (653.88)
Nizki
6135.68 (1014.08)
2281.17 (1026.88)
4293.65 (1143.33)
Tanaga
5710.56 (1538.90)
5682.77 (515.01)
5201.03 (NA)
Yunkasa
890.72 (121.60)
1115.60 (424.16)
3042.63 (1262.13)
Average
3750.03
2860.94
3047.98
SE
1069.01
994.44
910.94
https://doi.org/10.1371/journal.pone.0226173.t003 Table 3. Discussion Positive values for NEP reflect net oxygen production and negative values reflect oxygen consumption
(net respiration). Negative values for Re reflect oxygen consumption (i.e. more negative values reflect greater respiration by the ecosystem). “NA” denotes not available due
to lack of replication (i.e. data are based on only one chamber at that island; see Table 1). (net respiration). Negative values for Re reflect oxygen consumption (i.e. more negative values reflect greater respiration by the ecosystem). “NA” denotes not available due
to lack of replication (i.e. data are based on only one chamber at that island; see Table 1). NEP
Island
Kelp
Barren
Transition
Adak
-851.56 (409.34)
-431.10 (184.56)
-580.85 (89.91)
Amchitka
-694.81 (217.86)
-166.91 (141.38)
-151.16 (86.17)
Atka
-434.43 (72.69)
-231.05 (137.95)
-208.92 (143.52)
Attu
-176.96 (167.98)
1104.59 (NA)
98.52 (151.30)
Chuginadak
37.74 (15.07)
-537.29 (80.57)
105.85 (9.02)
Kiska
193.70 (93.03)
418.16 (58.16)
160.13 (63.01)
Nizki
-608.20 (177.58)
-20.29 (102.59)
-84.82 (78.95)
Tanaga
355.08 (361.67)
-909.49 (854.06)
254 (NA)
Yunkasa
21.82 (21.50)
-307.36 (96.49)
-129.13 (132.22)
Average
-239.74
-120.08
-59.6
SE
245.47
338.07
145.32
GPP
Island
Kelp
Barren
Transition
Adak
2018.08 (934.63)
2296.04 (1164.46)
2246.55 (184.47)
Amchitka
977.21 (552.29)
807.74 (304.87)
416.49 (188.14)
Atka
2450.84 (1340.11)
315.90 (60.74)
675.59 (266.57)
Attu
2238.97 (479.65)
2445.60(NA)
1826.29 (1482.52)
Chuginadak
917.57 (86.83)
1519.57 (199.70)
1081.44 (3.13)
Kiska
1399.85 (823.96)
1003.82 (335.91)
912.65 (347.63)
Nizki
2763.69 (420.47)
1130.44 (564.73)
2104.32 (610.25)
Tanaga
3032.82 (588.61)
2386.64 (684.53)
2727.91 (NA)
Yunkasa
456.27 (71.52)
404.17 (169.92)
1456.75 (627.00)
Average
1806.14
1367.77
1494.22
SE
521.75
483.99
452.41
Re
Island
Kelp
Barren
Transition
Adak
-2410.85 (763.16)
-2439.14(1273.10)
-2827.40 (94.57)
Amchitka
-1672.02 (334.43)
-974.65 (184.45)
-567.65 (101.96)
Atka
-2885.26 (1268.85)
-546.95 (77.21)
-884.52 (406.-6)
Attu
-2415.93 (564.65)
-1509.01 (NA)
-1727.77 (1313.82)
Chuginadak
-879.83 (101.90)
-2056.86 (265.44)
-975.60 (12.15)
Kiska
-1206.15 (775.20)
-585.66 (277.75)
-753.52 (308.09)
Nizki
-3371.99 (594.26)
-1150.73 (462.14)
-2189.14 (533.21)
Tanaga
-2677.74 (950.28)
-3296.13 (169.53)
-2473.12 (NA)
Yunkasa
-434.45 (50.10)
-711.43 (256.39)
-1585.88 (641.96)
Average
1994.91
1474.51
1553.84
SE
574.11
546.83
469.81
Range
Island
Kelp
Barren
Transition
Adak
3969.73 (1770.87)
4735.18 (2432.99)
5073.94 (279.04)
Amchitka
2649.23 (886.72)
1782.39 (483.68)
984.15 (290.10)
Atka
5336.10 (2608.91)
862.85 (16.48)
1560.11 (671.78)
Attu
4654.89 (1033.53)
4122.62 (NA)
3554.07 (2864.05)
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 12 / 21 PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 PLOS ONE Deforestation thus resulted in overall reductions in each of these
metrics, identifying a general loss of ecosystem function. This, however, was geographically
variable, with some islands showing elevated primary production following deforestation,
which we believe is due to higher irradiances combined with compensatory production by
microalgae (e.g. diatoms) and the coralline algal crusts. Indeed, we observed some of the high-
est production values in a few of the barrens cBITs where diatom mats formed within the
chambers during the deployments. These cBITs also tended to have low numbers of urchins
within them, and the chambers therefore appeared to exclude urchins from grazing the micro-
algae. In contrast, benthic primary productivity and respiration by the ecosystem are all similar
in the urchin barrens and transition habitats, which have similarly high abundances of urchins
and low biomasses of macroalgae [15,33], suggesting that the transition habitats have already
suffered reduced ecosystem functioning. This, of course, reflects patterns at the benthos and PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 13 / 21 PLOS ONE Deforestation and loss of ecosystem function Table 4. The effects of habitat change on patterns of productivity for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem respi-
ration (Re), and D the Range between GPP and Re (i.e. Range) estimated for reach island. “Change” reflects absolute differences in each metric (measured as mg O2 m-
2 day-1) as the habitat transitions from Kelp forests to Transition Habitats, Transition Habitats to Urchin Barrens, Kelp Forests to Urchin Barrens (i.e. the total change due
to deforestation). Positive values denote greater values for that metric and negative values denote lower values for that metric. NA denotes comparison “not available” due
to loss of replicates in one habitat that precluded reliable estimates of the change (see Table 1). At the bottom of each table are the average values and standard errors. Table 4. The effects of habitat change on patterns of productivity for A) Net ecosystem production (NEP), B) gross primary production (GPP), C) ecosystem respi-
ration (Re), and D the Range between GPP and Re (i.e. Range) estimated for reach island. “Change” reflects absolute differences in each metric (measured as mg O2 m-
2 day-1) as the habitat transitions from Kelp forests to Transition Habitats, Transition Habitats to Urchin Barrens, Kelp Forests to Urchin Barrens (i.e. the total change due
to deforestation). PLOS ONE Combining these with estimates of
macroalgal and invertebrate diversity and abundance revealed that the Aleutian Archipelago
suffered geographically variable losses to ecosystem function following widespread
deforestation. PLOS ONE PLOS ONE Deforestation and loss of ecosystem function Table 4. (Continued)
Chuginadak
259.63
1519.4
1779.03
Kiska
-940.83
-75.69
-1016.52
Nizki
-1842.03
-2012.48
-3854.51
Tanaga
NA
NA
-27.79
Yunkasa
2151.91
-1927.03
224.88
Average
-726.13
-390.51
-933.69
SE
1073.93
754.56
1262.65
htt
//d i
/10 1371/j
l
0226173 t004 not in the mid-water or at the surface where the canopy-forming Eualaria fistulosa remains
abundant in the transition habitats. It is likely that these canopy-forming macroalgae would
enhance GPP and perhaps result in positive values of NEP in the mid-water and at the surface
in both the kelp forests and transition habitats. However, at the benthos, GPP and Re remain
in balance following deforestation, leading to similar, near-zero NEP in all three habitats. We
believe this reflects balance between the autotrophic and heterotrophic components of the eco-
system. Specifically, the macroalgae exhibit positive GPP as they photosynthesize, grow and
increase in abundance, but this results in a concomitant increase in heterotrophic metabolism,
which enhances Re. In the face of deforestation, both GPP and Re are reduced, resulting in little
to no changes in NEP. Thus, we propose that, GPP, Re and the range between them are better
measures of changes to primary productivity than NEP. Combining these with estimates of
macroalgal and invertebrate diversity and abundance revealed that the Aleutian Archipelago
suffered geographically variable losses to ecosystem function following widespread
deforestation. not in the mid-water or at the surface where the canopy-forming Eualaria fistulosa remains
abundant in the transition habitats. It is likely that these canopy-forming macroalgae would
enhance GPP and perhaps result in positive values of NEP in the mid-water and at the surface
in both the kelp forests and transition habitats. However, at the benthos, GPP and Re remain
in balance following deforestation, leading to similar, near-zero NEP in all three habitats. We
believe this reflects balance between the autotrophic and heterotrophic components of the eco-
system. Specifically, the macroalgae exhibit positive GPP as they photosynthesize, grow and
increase in abundance, but this results in a concomitant increase in heterotrophic metabolism,
which enhances Re. In the face of deforestation, both GPP and Re are reduced, resulting in little
to no changes in NEP. Thus, we propose that, GPP, Re and the range between them are better
measures of changes to primary productivity than NEP. PLOS ONE Positive values denote greater values for that metric and negative values denote lower values for that metric. NA denotes comparison “not available” due
to loss of replicates in one habitat that precluded reliable estimates of the change (see Table 1). At the bottom of each table are the average values and standard errors. to loss of replicates in one habitat that precluded reliable estimates of the change (see Table 1). At the bottom of each table are the average values and standard errors. A) NEP
Kelp to Transition
Transition to Barren
Kelp to Barren
Island
Change
Change
Change
Adak
270.71
808.46
420.46
Amchitka
845.97
-318.07
527.9
Atka
225.51
-22.13
203.38
Attu
275.48
NA
NA
Chuginadak
68.11
-643.14
-575.03
Kiska
-33.57
258.03
224.46
Nizki
523.38
64.53
587.91
Tanaga
NA
NA
-1264.57
Yunkasa
-150.95
-178.23
-329.18
Average
253.08
-4.36
-25.58
SE
183.33
265.76
373.26
B) GPP
Kelp to Transition
Transition to Barren
Kelp to Barren
Island
Change
Change
Change
Adak
687.67
49.49
737.16
Amchitka
-560.72
391.25
-169.47
Atka
-1775.25
-359.69
-2134.94
Attu
-412.68
NA
NA
Chuginadak
163.87
438.13
602
Kiska
-487.2
91.17
-396.03
Nizki
-659.37
-973.88
-1633.25
Tanaga
NA
NA
-646.18
Yunkasa
1000.48
-1052.58
-52.1
Average
-255.4
-202.3
-461.6
SE
501.77
353.95
578.69
C) Re
Kelp to Transition
Transition to Barren
Kelp to Barren
Island
Change
Change
Change
Adak
416.55
-388.26
28.29
Amchitka
-1104.37
407
-697.37
Atka
-2000.74
-337.57
-2338.31
Attu
-688.16
NA
NA
Chuginadak
95.77
1081.26
1177.03
Kiska
-452.63
-167.86
-620.49
Nizki
-1182.85
-1038.41
-2221.26
Tanaga
NA
NA
618.39
Yunkasa
1151.43
-874.45
276.98
Average
-470.63
-188.33
-472.09
SE
578.86
423.11
734.7
D) Range
Kelp to Transition
Transition to Barren
Kelp to Barren
Island
Change
Change
Change
Adak
1104.21
-338.76
765.45
Amchitka
-1665.08
798.24
-866.84
Atka
-3775.99
-697.26
-4473.25
Attu
-1100.82
NA
NA
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 14 / 21 PLOS ONE Experimental design Our cBITs were made from 0.106 cm polycarbonate plastic triangle sheets glued to fiberglass-
reinforced vinyl panels (Fig 8). The frames were reinforced using stainless steel tubes with
stainless steel cable to facilitate handling and to ensure they held their pyramidal shape with an
internal volume of 192 L and a basal area of (0.64 m2). The cBITs each had 26” skirts around
the perimeter, upon which chain was laid to hold them to the benthos and prevent water
exchange with the surrounding environment. This was verified by injecting fluorescein dye
into the chambers and examining the perimeters for leaks. The polycarbonate walls were thin
and flexible to allow hydrodynamic energy transfer into the cBITs, thereby reducing boundary
layer formation around the macroalgal thalli. We verified this energy transfer using dissolving
plaster blocks placed within cBITs, and by using video analysis of internal seaweed and fluores-
cein dye movements within the chambers relative to seaweeds outside them [60]. Sensor arrays
that included a Photosynthetic Active Radiation (PAR) sensor (Odyssey Dataflow Systems
Ltd), and a Dissolved Oxygen (DO mg/L) and Temperature (˚C) sensor (MiniDOT Logger,
PME) were placed at the center of each cBIT (Fig 8). During two cruises aboard the R/V Oceanus in 2016 and 2017, we deployed cBITs in each
of the three habitats (kelp forest, urchin barrens, transition habitats) on each of nine islands
(Table 1; Figs 1,2 and 8) for 36-hour periods to measure both day and night patterns of NEP
and Re, and to ensure we captured a complete diurnal cycle. These islands span more than
1000 km and therefore experience differences in temperature, salinity, wave exposure and
other biotic factors [62]. Consequently, all cBITs deployments were done in the summer (i.e. July) of each year, in similar depths (i.e. 6–8 m), and under similar wave exposures (i.e. pro-
tected from ocean swells) in order to standardize factors that could affect productivity mea-
surements. The three habitat types were selected based on non-overlapping community
assemblages (i.e., kelp forests were chosen based on abundant E. fistulosa and dense assem-
blages of understory macroalgae; transition habitats were chosen based on abundant E. fistu-
losa, little-to-no understory macroalgae, and high abundances of urchins; urchin barrens were
chosen based on no E. fistulosa, little-to-no understory macroalgae and abundant urchins). These were then grouped in each island to reduce the effects within-island spatial heterogene-
ity in other environmental factors. Materials and methods While many past experiments examining primary production by autotrophic communities
have relied on laboratory experiments that do not incorporate natural fluctuations in abiotic
conditions, recent studies have identified techniques that measure primary production in situ,
thereby increasing the ecological realism of their experiments [54–57]. For example, in situ
chamber designs have been developed for estimating primary production by individual species
[55,56] and whole benthic communities [29,56,57]. In general, estimates of net ecosystem pro-
duction (NEP) on the benthos can be made by measuring changes in dissolved oxygen within
chambers that are placed in situ over macroalgae and invertebrate communities. In this study,
we deployed collapsible benthic isolation tents (cBITs) modelled after those described by Haas
et al. [58] and Calhoun et al. [59] that directly measured in situ benthic oxygen production and
allowed us to estimate gross primary production (GPP), ecosystem respiration (Re) and net
ecosystem production NEP by the benthic communities [28,29,55]. These cBITs were the same
ones used by Sullaway and Edwards [60] to measure loss of primary productivity following the Table 5. Analysis of covariance testing the effect of GPP and habitat on Re. Note the non-significant HabitatGPP interaction hat shows no differences in the slopes
(i.e. relationships) between GPP and Re among the three habitat types. See Fig 5 for graphical representation. Source
Type III SS
df
MS
F-ratio
p-value
GPP
8.460E+03
1
8.50E+03
3.20E+02
0.001
HABITAT
20.791443
2
1.00E+01
3.90E-01
0.68
HABITATGPP
8.6140845
2
4.30E+00
1.60E-01
0.852
Error
1.66E+03
62
2.70E+01
https://doi.org/10.1371/journal.pone.0226173.t005 Table 5. Analysis of covariance testing the effect of GPP and habitat on Re. Note the non-significant HabitatGPP interac
(i.e. relationships) between GPP and Re among the three habitat types. See Fig 5 for graphical representation. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 15 / 21 PLOS ONE Deforestation and loss of ecosystem function displacement of native giant kelp, Macrocystis pyrifera, by the invasive Sargassum horneri on
Catalina Island, CA. Further, because our cBITs encompassed whole benthic communities,
species interactions (e.g., shading), and invertebrate and microbial respiration were incorpo-
rated into production measurements. These interactions are often not captured in laboratory
experiments but are pertinent to understanding GPP, Re, and NEP [61]. PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 Experimental design For each deployment, three replicate cBITs were placed on
the benthos over targeted assemblages within each habitat type. However, occasionally, repli-
cates were lost due to logistical difficulties associated with the chamber-benthos seals
(Table 1). The water within each cBIT was replaced once per day by opening the side of the
chamber and completely replacing the water with new ambient seawater to reduce “chamber
effects” (i.e., the build-up of oxygen and depletion of inorganic carbon and nutrients). After
each deployment, the chambers and sensors were retrieved. At six of the islands (Table 1), all
organisms within each of the chambers’ benthic footprints were collected, brought back to the
ship, enumerated and weighed during our 2017 cruise. We measured NEP over the whole
diurnal cycle, Re during the nighttime hours, and calculated GPP during the day for each cBIT
during each incubation period separately according to Olive´ et al. [57]. Specifically, measure-
ments made during the night (the dark) were used to infer rates of Re, which were then com-
bined with measurements of NEP to estimate GPP by the autotrophs [18–20]. Ethical PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 16 / 21 PLOS ONE Deforestation and loss of ecosystem function Fig 8. Photograph of cBIT. Photograph of (A) cBIT before deployment showing 26” skirt around perimeter, flexible
polycarbonate walls, steel framing, anchor chain used to hold skirt and cBIT to the benthos, and (B) cBIT deployed in
kelp forest showing PAR and oxygen sensors placed both inside and outside the chamber. https://doi.org/10.1371/journal.pone.0226173.g008 Fig 8. Photograph of cBIT. Photograph of (A) cBIT before deployment showing 26” skirt around perimeter, flexible
polycarbonate walls, steel framing, anchor chain used to hold skirt and cBIT to the benthos, and (B) cBIT deployed in
kelp forest showing PAR and oxygen sensors placed both inside and outside the chamber. https://doi.org/10.1371/journal.pone.0226173.g008 https://doi.org/10.1371/journal.pone.0226173.g008 Approval: All procedures performed in studies involving fishes were in accordance with the
ethical standards of the institution or practice at which the studies were conducted (University
of Alaska Fairbanks Institutional Animal Care and Use Committee; Permit Number: 899401–
4). Acknowledgments We thank S. Lamerdin, and the captain and crew of the R/V Oceanus for excellent ship sup-
port. We thank J. Estes for offering historical perspectives on the Aleutian kelp ecosystem, and
M. Hatay for designing the cBITs. We are grateful to M. Good, S. Traiger, J. Metzger, A. Bland,
A. Ravelo, and B. Weitzman for assistance with field operations. We also thank the Alaska
Maritime National Wildlife Refuge for logistical support. Author Contributions Conceptualization: Matthew Edwards, Brenda Konar. Conceptualization: Matthew Edwards, Brenda Konar. Data curation: Matthew Edwards, Michael Spector. Data curation: Matthew Edwards, Michael Spector. Formal analysis: Matthew Edwards, Scott Gabara. Formal analysis: Matthew Edwards, Scott Gabara. Formal analysis: Matthew Edwards, Scott Gabara. Funding acquisition: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim. Investigation: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim, Scott Gabara, Genoa Sull-
away, Tristin McHugh, Michael Spector, Sadie Small. Methodology: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim. Project administration: Matthew Edwards, Brenda Konar. Supervision: Matthew Edwards. Supervision: Matthew Edwards. Validation: Ju-Hyoung Kim. Validation: Ju-Hyoung Kim. Visualization: Scott Gabara. Visualization: Scott Gabara. Writing – original draft: Matthew Edwards. Writing – original draft: Matthew Edwards. Writing – review & editing: Matthew Edwards, Brenda Konar, Ju-Hyoung Kim, Scott Gabara,
Genoa Sullaway, Tristin McHugh, Michael Spector, Sadie Small. Statistical analyses All analyses were done in either Systat ver. 12 or Primer ver 6. Prior to analyses, all data were
evaluated for normality by graphical examination of the residuals, which suggested they were
slightly non-normal. Data for NPP, GPP, Re and the Range between GPP and Re were then
square-root transformed and re-graphed, which suggested the problems were corrected. Data
for PAR were log transformed, which corrected the problem. The transformed data were then PLOS ONE | https://doi.org/10.1371/journal.pone.0226173
March 4, 2020 17 / 21 PLOS ONE Deforestation and loss of ecosystem function examined for equality of variances using Bartlette’s tests, which indicated they were homosce-
dastic. We then evaluated if urchin biomass, PAR, GPP, Re, NEP and the range between GPP
and Re varied among the three habitats (kelp forests, urchin barrens, and transition habitats),
the nine islands, and between the two study years using separate three-way Model III Nested
ANOVAs, with year and habitat type as fixed factors, island nested within year as a random
factor. We evaluated if the relationship between GPP and Re varied among habitats using
ANCOVA, with Re as the response variable, GPP as the covariate, and habitat type as the cate-
gorical independent variable. We evaluated if the ratios in any of the habitats differed from 1.0
(i.e. GPP = Re) by assessing if the value 1.0 occurred within the 95% confidence intervals
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Getting clean and harm reduction: adversarial or complementary issues for injection drug users
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Cadernos de Saúde Pública
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1 Bloomberg School of Public
Health, Johns Hopkins
University, Baltimore, U.S.A.
2 Friends Social Research
Center, Baltimore, U.S.A.
3 Ethknoworks,
Santa Fe, U.S.A. 733
ARTIGO ARTICLE 733
ARTIGO ARTICLE 733
ARTIGO ARTICLE J. Peterson
Department of Health Policy
and Management, Bloomberg
School of Public Health,
Johns Hopkins University.
Hampton House, 624 North
Broadway, Baltimore MD,
21205, Suite 492, U.S.A.
jpeterso@jhsph.edu Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Getting clean and harm reduction:
adversarial or complementary issues
for injection drug users Abstinência versus redução de danos:
questões conflitantes ou complementares
entre usuários de drogas injetáveis James Peterson 1
Shannon Gwin Mitchell 1
Yan Hong 1
Michael Agar 2,3
Carl Latkin 1 Abstract Traditionally, in the United States, the predom-
inant approaches to drug policy and drug treat-
ment have been characterized by such phrases
as “zero tolerance” and the “war on drugs”. This
abstinence-based political and ideological view
emerges from the substance use treatment ap-
proach embedded within the principals of the
moral model 1. This approach supports the be-
lief that abstinence is a prerequisite for sub-
stance users to modify their behavior. Many contemporary HIV prevention interven-
tions targeting injection drug users (IDUs) have
been implemented using Harm Reduction as a
theoretical framework. Among drug-using indi-
viduals, however, the abstinence-based “getting
clean” models espoused by Narcotics Anony-
mous and other widely adopted approaches to
drug treatment are often more readily accepted. This paper describes an ethnographic examina-
tion of the ideological dichotomy between Harm
Reduction and abstinence-based “getting clean”
treatment model which emerged during the pi-
loting phase of an HIV prevention intervention
in Baltimore City, Maryland, USA. This paper
describes how the conflict was identified and
what changes were made to the intervention to
help resolve the participants’ dichotomous think-
ing concerning their substance abuse issues. Street Drugs; Drug Abuse; Harm Reduction Piloting “STEP”: an HIV prevention
intervention The “STEP” study, the Harm Reduction pro-
gram in question, is a network and peer-orient-
ed HIV prevention intervention seeking to train
individuals engaged in the injection-drug-us-
ing community in Baltimore City. The study’s
short-term goal is the promotion of HIV pre-
vention behaviors among the program’s partici-
pants and their drug and sexual network mem-
bers. Based on our previous intervention effi-
cacy research 4,5,6,7, STEP utilizes social influ-
ence theory to promote the philosophy of Harm
Reduction among social network members. The study design is a controlled phase II effica-
cy trial with an equal attention control condi-
tion. The STEP program consists of four group
sessions (peer mentor training sessions), one
individual session, one drug (or sex) dyad ses-
sion, and periodic booster sessions up to six
months after completion of the intervention. The STEP intervention was tested and revised
through five waves of piloting, which occurred
over the eight-month period proceeding formal
program implementation. The process evalua-
tion (upon which this paper was based) utilized
ethnographic methods and was implemented
during the eight-month piloting phase. For many substance users, however, an un-
derstanding of recovery from substance use is
derived from the philosophy of self-help orga-
nizations such as Narcotics Anonymous (NA)
and Alcoholics Anonymous (AA) which pro-
mote an all-or-nothing belief concerning drug
use. The NA philosophy posits that complete
abstinence from all mood-changing chemicals
is the mark of success and anything less than
that is tantamount to failure; in a way, it can be
thought of as an individual level of the “zero
tolerance” policy. Method The STEP HIV prevention intervention pilot
sessions were conducted from July 11, 2003
through March 10, 2004. The STEP HIV prevention intervention pilot
sessions were conducted from July 11, 2003
through March 10, 2004. In this article we present salient lessons
learned while conducting an ethnographically
informed process evaluation during the pilot
phase of an HIV prevention intervention trial
aimed at injection drug users (IDUs) in Balti-
more City, Maryland. We will discuss three key
points which emerged during the course of our
process evaluation: (1) the program partici-
pants’ abstinence orientation, which dominat-
ed their views of drug use and initially created
tension with the program’s Harm Reduction
orientation; (2) how the contradictions between
these two orientations manifested themselves
in group interactions; and (3) how the facilita-
tors and program planners, along with the par-
ticipants, augmented the intervention’s lan-
guage, highlighting the similarities between
the two approaches and reducing the tensions
within group sessions. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Correspondence
J. Peterson On the other hand, Harm Reduction, and its
application within the HIV prevention and sub-
stance-use treatment arena, has met with a broad
range of acceptance throughout the world 2,
particularly among contemporary researchers. Harm Reduction is defined as a set of practical
strategies that are used to reduce the negative
consequences of substance use. Harm Reduc-
tion involves incorporating a continuous spec-
trum of strategies, ranging from safer use, to
managed use, to complete abstinence from
drugs. Harm Reduction tailors behavior change
to the unique conditions of substance use
along with the substance users, themselves
(Ptah A. Speech presented to the Community
Planning Leadership Summit by Director of
Policy Harm Reduction Coalition. March 2003). Street Drugs; Drug Abuse; Harm Reduction Street Drugs; Drug Abuse; Harm Reduction Proponents of Harm Reduction have been
accused of supporting continued drug use,
sabotaging efforts toward achieving abstinence,
undermining recovery, contradicting current
social policy, and selling out or giving in to the Peterson J et al. 734 Piloting “STEP”: an HIV prevention
intervention presence of illicit drug use in the society. How-
ever, those who adopt the principles of Harm
Reduction do so with the understanding that
drug use is a complex problem related to other
quality of life issues such as poverty, class,
racism, social isolation, and discrimination. They espouse that an approach to drug use
that attempts to mitigate its negative effects on
the drug-using individuals, their families, and
the communities where they live, should in-
clude non-coercive intervention strategies 3
and should not paint an overly simplistic pic-
ture of the problem. In addition, Harm Reduc-
tion allows for the creation of a non-adversari-
al and supportive milieu that engages drug-us-
ing individuals in a process where rewards and
continued supports are not just the consequence
of fundamental change, but are the incentives
for incremental gains. Based on a vast body of
empirical research, harm reduction is widely
accepted among public health professionals as
the cornerstone of HIV prevention among sub-
stance abusers. Ethnography The use of qualitative methods in conducting
process evaluations has demonstrated utility
and success in the evaluation realm. Patton 8
elucidates four characteristics that make quali-
tative inquiry “highly appropriate” for process
evaluation: (1) depicting process requires de-
tailed descriptions of how people engage with
each other; (2) the experience of process typi-
cally varies for different people, so their experi-
ence needs to be captured in their own words;
(3) process is fluid and dynamic so it can not be
fairly summarized on a single rating scale at
one point in time; and (4) participants’ percep-
tions are a key process which should be taken
into account. During the field observations, ethnographers
also identified information-rich opinion lead-
ers and key informants. In some cases, further
interviews were scheduled to elicit more feed-
back concerning the participants’ perspectives
on relevant social and cultural factors. These
interviews were also recorded. Specific to this study, LeCompte & Schensul
9 provide a detailed dialogue describing the use
of ethnography in the process of developing in-
tervention programs. Consistent with their un-
derstanding, ethnographic methods were used
in the STEP HIV prevention intervention devel-
opment process to (1) examine the acceptabili-
ty of the intervention to the staff as well as the
intervention participants; (2) assess the pro-
grammatic integrity of the intervention; (3)
monitor the usefulness of the intervention; and
(4) inform the design and development of the
intervention outcome evaluation. Participants Eligibility criteria for STEP participants includ-
ed: (1) at least 18 years of age, (2) willing to in-
vite at least one sex or drug network member,
and (3) self-reported unprotected sex or injection
drug use within previous 3 months. Pilot par-
ticipants were recruited using word-of-mouth,
advertising in community newspaper, flyers
posted throughout the neighborhoods, and
street outreach. The participants were primari-
ly African Americans from low-income, high-
crime, urban neighborhoods and all resided
within Baltimore City. A total of 29 individuals
were recruited and participated in the piloting
phase of the intervention. Among these partic-
ipants 21 were male, 8 were female. Twenty-
four of the participants were African American
and 5 were White. The intervention utilized a team of two co-fa-
cilitators (one Caucasian male and one African- ADVERSARIAL OR COMPLEMENTARY ISSUES FOR INJECTION DRUG USERS
735 American female). The research team consist-
ed of 4 ethnographers, the primary investigator
of the intervention, the project’s director, the
two facilitators, and several staff members. community. The procedure was thoroughly ex-
plained to each participant, and additional in-
formed consent was received prior to any field
observation work. Field work took place, by definition, out in
the community. The purpose of the communi-
ty-based field work was for the ethnographer to
observe the STEP participant actually imple-
menting the risk reduction skills and dissemi-
nating HIV prevention information learned in
the intervention sessions. The participants were
asked to conduct the sessions with either their
sex partner or a drug-using network member. Detailed field notes were taken by the ethnog-
rapher and recorded immediately after leaving
the field and entered into an ethnographic
database. Relevant activities and conversations
were audio-taped, when appropriate. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Data analysis Several types of ethnographic data were in-
cluded in the analysis: (1) recordings and de-
tailed notes from training sessions; (2) written
notes of the session observations; (3) record-
ings and written field notes from the fieldwork;
and (4) recordings and written notes from in-
terviews with key informants. The group notes, along with the field obser-
vation notes, were uploaded to an ethnograph-
ic database so that the ethnography team and
the STEP intervention project planners could
have easy access to the notes, track the progress
of the evaluation, and make adjustments to the
pilot program as necessary. Ethnographers
coded the field notes using themes that paral-
leled the domains and/or content areas identi-
fied as “session objectives” as well as identify-
ing new and emerging themes 9. Detailed sum-
maries were made with substantial retention of
original quotes and notes so as minimize the
“summary” bias and reproduce the true situa-
tions. Ethnography was utilized to inform the
process evaluation during the project’s piloting
phase, from August 2003 to March 2004. Data
were collected through a variety of methods,
including group observations, field observa-
tions, and the content analysis of session tapes
and interviews with STEP participants. In each
of the piloting waves, at least one ethnograph-
er was present during all of the classroom ses-
sions. An ethnographer conducted observa-
tions concerning the pilot sessions’ content
and the interactions among the facilitators and
the participants. Detailed notes of the observa-
tion were recorded. During session breaks an ethnographer con-
ducted informal interviews and participated in
casual conversations with the participants in
order to elicit feedback about the sessions, as
well as to establish and build rapport. After
earning the trust of the participants, an ethno-
grapher would individually query participants
as to whether they were willing to demon-
strate skills during observation activities in the Regular meetings were scheduled with
ethnographic team members in order to dis-
cuss and cross-validate preliminary insights
and observations. These insights were then
shared with the larger project research team
and the facilitators. These discussions provid-
ed opportunities to examine preliminary find-
ings from the perspectives of both the ethnog- Peterson J et al. 736 the idea of getting clean than on the Harm Re-
duction information presented during the pilot
sessions. Longer drug use history among par-
ticipants also created barriers to conducting
outreach. The use of AA/NA language
by program participants The participants’ conversations during the ses-
sions invariably focused on their drug use and
their desire to get clean or help their network
member get clean. For instance, in the early pi-
loting sessions, participants were asked about
their individual goals through their participa-
tion in the STEP project. Many of the partici-
pants responded by making “not using” their
primary goal rather than simply reducing HIV
risks associated with their drug use. In the conversations during group sessions, as
well as in their outreach activities, the language
of the participants was liberally peppered with
clichés and language often heard in AA and NA
meetings. Comments such as “I’m gonna keep
coming back”, “sick and tired of being sick and
tired”, and “I have to change people, places, and
things”, among others. Conversations with par-
ticipants and their network members suggested
that AA/NA significantly influenced their think-
ing and dialogue about their substance use and
concomitant issues. In many cases, the partici-
pants incorporated AA/NA language into their
risk reduction conversations with their network
members and it seemed as if participants had
internalized the “all-or-nothing” attitude un-
derlying the AA/NA model of drug dependence. The following field note includes the response
from one ethnographer’s observation of an out-
reach episode that a participant had coordinat-
ed in which several of his drug-using associates
were participants: “Well, I thought I was at an
NA meeting and I also thought the participant
planned it that way. The order that it [the con-
versation] fell in and the way everyone shared
behind each other. Then, when they started cry-
ing right behind each other. It was strange, but
then I thought maybe these people really needed
to talk to someone, and they just got emotional”. Th “ ll
hi
”
i
d
h
b There are some plausible explanations for
this finding. The ethnographic team hypothe-
sized that many of the participants may have
talked about getting clean because they thought
it was socially acceptable and what they need-
ed to say in order to obtain personal influence
within the program structure. Also, it is likely
that some participants actually wanted to get
clean, but may have had little or no other re-
sources to achieve this goal. In response, they
decided to utilize the Harm Reduction-based
research project as if it were a treatment pro-
gram. The project’s focus on Harm Reduction The STEP philosophy can be summed up in the
following sentence from session one, “we believe
that any positive change you are willing to make
is an important step towards Harm Reduction”. Session content centered upon various methods
and ways to reduce the participants’ potential
exposure to HIV by engaging in safer injection
practices and using condoms. All of the sessions
(group, individual, and dyad) were focused on
reducing injection and sex risks for contracting
HIV. This emphasis was clearly identified in the
facilitators’ statements and the discussions they
led during the sessions. Data analysis For instance, a 50-year-old African
American male participant who reported a 20-
year drug-use history discussed, in detail, the
resistance to his attempt at outreach that he
had to deal with as he initiated a conversation
with his drug-using partner, who he also iden-
tified as his sex partner. The conversation, as
he reported, was about him articulating the in-
formation he learned while participating in the
sessions. The participant was emphatic when
he mentioned the argumentative and con-
frontational behavior he met with from his
partner. The resistance the participant was ex-
periencing may have been the result of his ap-
proach being flavored with his subjective and
unrelenting understanding of getting clean, an
idea that had the potential to greatly overshad-
ow any “incremental change” notions garnered
from STEP’s Harm Reduction-based sessions. raphy team members and the project planning
staff and to make recommendations regarding
the intervention session content. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Blending divergent viewpoints At first glance the views of many of the partici-
pants appeared to be diametrically opposed to
the views espoused by the STEP project. How-
ever, once the “conflict” was identified, an ef-
fort was made to emphasize during the pro-
gram’s sessions that getting clean was just one
step along the path to a Harm Reduction plan. Facilitators began to emphasize that complete
abstinence was at the far end of the Harm Re-
duction continuum and that each incremental
step towards reducing exposure to HIV was al-
so a step towards their ultimate goal of getting
clean. Other key factors added to the sessions
which assisted the participants in the integra-
tion of the two perspectives were: The intervention sessions were conceived
with a flexible design that allowed for tailoring
the prevention intervention to the needs and
satisfaction of the participants. Thus, one of
the sessions in the STEP program was designed
to convey that to “talk the talk” you need to
“walk the walk” or you may be seen as hypo-
critical and less effective in your outreach ac-
tivities. Such an accomplishment required the
intervention planning group to reach a delicate
balance between the HIV prevention practices
espoused by the Harm Reduction arena and
the goals of the targeted group. Thus, the STEP
program emphasized that walking the walk
could be about harm reduction, that is, prac-
tice and preach safer injection practices. •
They were taught that the Harm Reduction
information and incremental changes they
made could help them get clean; The group facilitators mentioned that they
initially experienced some tension introducing
the Harm Reduction concept to the partici-
pants. The participants, while not being able to
clearly articulate their lack of understanding of
the concept of Harm Reduction, demonstrated
this fact in a variety of ways. During the group
sessions several of the participants articulated
a sense of pride when a participant announced
that he or she had accumulated any amount of
“clean-time” or amassed one day or more with-
out using drugs. However, while accruing “clean-
time” was a worthwhile goal, many of the par-
ticipants found that maintaining these efforts
at staying clean seemed insurmountable for
any length of time. The use of AA/NA language
by program participants It is also likely that as drug-dependent
individuals tended to focus their behavior on
acquiring and using drugs, they also viewed
drug use as the cause of the major problems in
their lives. From this standpoint, stopping drug
use would alleviate all or most of their other
problems, such as poor family relationships,
unemployment, and depression. Results from observations of the group ses-
sions suggest that many participants with a
long history of drug use were more focused on The “all-or-nothing” attitude may have been
a consequence of the AA/NA mindset that one ADVERSARIAL OR COMPLEMENTARY ISSUES FOR INJECTION DRUG USERS
737 has to “hit a bottom” before asking for and, in
many cases, receiving help. Several of the par-
ticipants articulated discussions and responses
throughout the piloting of the intervention ses-
sions which echoed the NA/AA language and
seemed to further convey the getting clean phi-
losophy rather than the program’s main focus
of Harm Reduction. with the concept of getting clean in the AA/NA
abstinence model. An example of this type of
behavior is inferred from the following note,
which describes the participants’ focus on
their own behavior during conversation in the
group sessions. An ethnographer wrote: “In
this session, the most prominent problem is the
tension between ‘partner’ and ‘myself’. Partici-
pants could not get out of the notion of ‘getting
clean’. Many times the discussion moved from
‘helping others’ to ‘helping myself’”. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Blending divergent viewpoints •
That helping others was a way to help them-
selves; •
That there were other goals in their lives be-
sides getting clean and that reaching those
goals might assist them in getting clean; and •
That getting clean is a process, not just an
end result, and that it requires positive steps. The group facilitators took special care to
celebrate any small gains toward risk and harm
reduction reported by participants. Discussion cated, interconnected components of the in-
tervention, the relationship of those compo-
nents to the program as a whole, and the im-
mediate reactions of the participants. Using
ethnographic methods to collect data, which
informed a process evaluation of the imple-
mentation of an intervention program, can fos-
ter a reflexive exercise, which enables immedi-
ate and responsive feedback for program de-
velopment. In the current study an ethnograph-
ically informed process evaluation was used to
obtain information during the pilot sessions
and assist with program development and de-
cision-making 12. The design and implementation of an HIV pre-
vention intervention is an arduous and com-
plex undertaking. Weeks et al. 10 suggest that in
communities such as the locales from where
the participants of this study were recruited, is-
sues of poverty, gender, ethnic identity, and lev-
el of acculturation come together to create a
unique set of high-risk conditions for the trans-
mission of drug-related epidemics. Strategies
to reduce the high risk of disease transmission
among the drug-using population have to be
culturally relevant to the participants and im-
plemented within multiple contexts 11. The par-
ticipants engaged in the pilot sessions of the
STEP HIV prevention intervention show that
“cultural relevance” means taking seriously the
issue of getting clean and the influence of tra-
ditional abstinence models like AA/NA. Traditionally, evaluation strategies have pri-
marily been used to determine cost effective-
ness and the impact of the prevention efforts
on the targeted group. Thus, outcome evalua-
tions provide a systematic method to deter-
mine (1) whether the program achieved the stat-
ed objectives and (2) whether the intervention
was a cost-effective activity. Obtaining an un-
derstanding of the aforementioned aspects and
characteristics of an intervention may have
some usefulness for evaluating whether a pro-
gram has achieved the intended objectives. However, describing key aspects of the pro-
gram’s dynamics has significant utility for the
design, development, proper implementation,
and facilitation of interventions that are tai-
lored to meet the specific needs of its targeted
group. Within the context of the public health are-
na, the relationship between the philosophical
principles underpinning the STEP approach to
Harm Reduction can be seen as challenging
the traditional abstinence-only models 2. Discussion Re-
searchers attempting to design and implement
a Harm Reduction-based intervention for IDUs
must ask the question, “How many drug users
‘just say no’ to Harm Reduction and why do they
do so?”. Injection drug users who have the tradi-
tional “getting clean” ideology may initially
struggle with the messages of an HIV preven-
tion intervention that has Harm Reduction as
its philosophical base. This preliminary and ex-
ploratory study shows that many of the IDUs
who participated in this pilot study focused on
abstinence as their only goal and not abstinence
as the end result of a process, as it is viewed
from the Harm Reduction perspective. Designing and implementing culturally rel-
evant HIV prevention interventions is a com-
plex and arduous task. Contemporary preven-
tion interventions designed to address the
transmission of HIV and other diseases among
specific populations are intricate and typically
are implemented within multiple contexts. Consequentially, evaluating HIV prevention in-
terventions is an indispensable component of
the overall effort. The IDU participants recruited to the STEP
program were at various stages in their drug-
using careers and brought a variety of needs
and experiences to the intervention that im-
pacted their receptivity to the project’s Harm
Reduction message. Through the use of ethnog-
raphy in the process evaluation phase of the
HIV prevention intervention, these needs and
prior experiences became clear to the research
team and provided both a need and an oppor-
tunity to include strategies merging the absti-
nence and Harm Reduction models. It is important for researchers and program
planners to know the extent to which all com-
ponents of the intervention are actually imple-
mented as planned. The use of ethnographic
methods within the process evaluation offered
a strategy, which allowed for systematically
documenting the step-by-step procedures in-
volved in the implementation of the interven-
tion, disentangling the factors that may con-
tribute to successful outcomes, and explicating
the components of the intervention that are in-
herent to the effectiveness and success of the
intervention effort. Reduction setting The AA/NA abstinence model advocates that if
a person does not remain clean or drug free,
then a negative label is attached to their efforts. Thus the participants’ perception was that they
must “walk the walk” in order to “talk the talk”. The participants consistently articulated their
discomforts with disseminating the recently
learned HIV prevention information if they
were not “clean” or they continued to use drugs
while participating with the outreach compo-
nent of the intervention. The idea, from their
perspective, was that they thought of them-
selves as being hypocritical if they conducted
outreach work while still using drugs. This no-
tion of hypocrisy appeared to be directly linked As the intervention developed, the facilita-
tors became more and more adept at eliciting
Harm Reduction-based “positive steps” while
acknowledging the heroic efforts that the par-
ticipants were making to reduce their risks of
contracting HIV and other infectious diseases
and, ultimately, get clean. The conflict resolved
with the concept of any positive change, which
was flexible and did not focus on either the me-
chanics of risk reduction or the ultimate goal of
getting clean. Peterson J et al. 738 Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Contributors Muitas intervenções para prevenir a infecção pelo HIV
entre usuários de drogas injetáveis adotam a técnica
de redução de danos como referencial teórico. Entre-
tanto, os próprios usuários tendem a preferir modelos
baseados na abstinência, defendidos pelos Narcóticos
Anônimos, além de outras abordagens adotadas am-
plamente para o tratamento da dependência química. O artigo descreve uma avaliação etnográfica da dico-
tomia ideológica entre redução de danos e o modelo
terapêutico baseado na abstinência (getting clean) du-
rante a fase piloto de uma intervenção para a pre-
venção de HIV na Cidade de Baltimore, Maryland, Es-
tados Unidos. O artigo descreve como o conflito foi
identificado e que mudanças foram introduzidas na
intervenção para ajudar a resolver as idéias dicotômi-
cas dos participantes em relação às questões de de-
pendência química. J. Peterson is the primary author of the manuscript. S. G. Mitchell assisted with manuscript development
and Y. Hong with consultation, proofreading, and
writing. M. Agar provided conceptualization, consul-
tation, proofreading, and scientific editing. C. Latkin
performed proofreading and scientific editing. Implications for research
and program development Delineating the particular group norms,
such as language use and the means by which
specific group participants get their needs met, Process evaluations that include ethnographic
methods are useful for explicating the compli- Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 ADVERSARIAL OR COMPLEMENTARY ISSUES FOR INJECTION DRUG USERS
739 is vital to determining the intervention strate-
gies and structure. The course of integrated ac-
tions that are included in achieving a specific
planned or stated outcome has value in dupli-
cating those program activities that have proven
to be effective, determining the integrity of the
program, assessing the acceptability of the
program by the targeted group, and identifying
key factors of the intervention that are being
carried out as planned. volved with deciphering the interactions be-
tween the intervention components, the par-
ticipants, and the facilitators, however ethno-
graphic methods were indispensable in obtain-
ing information needed to mitigate these chal-
lenges and were critical in furthering our un-
derstanding of the change process within the
HIV prevention intervention. Ethnographic
methods not only assisted with addressing the
specific questions related to obtaining an un-
derstanding of the overall project, but this ap-
proach facilitated efforts to bring those ques-
tions to light, one step at a time. Gaining an understanding of the process
involved in carrying out change efforts, such as
the STEP HIV prevention intervention, can
prove challenging. There are complexities in- Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 References 7. Dickson-Gomez J, Knowlton A, Latkin C. Hoppers
and oldheads: qualitative evaluation of a volun-
teer AIDS outreach intervention. AIDS Behav
2003; 7:303-15. 1. Brickman P, Rabinowitz VC, Karuza K, Coates D,
Cohn E, Kidder L. Models of helping and coping. Am Psychol 1982; 37:368-84. 2. Heller D, McCoy C, Cunningham C. An invisible
barrier to integrating HIV primary care with Harm
Reduction services: philosophical clashes between
the Harm Reduction and medical models. Public
Health Rep 2004; 119:32-9. 2. Heller D, McCoy C, Cunningham C. An invisible
barrier to integrating HIV primary care with Harm
Reduction services: philosophical clashes between
the Harm Reduction and medical models. Public
Health Rep 2004; 119:32-9. 8. Patton MQ. Qualitative research & evaluation
methods. Thousand Oaks: Sage Publications; 2002. 9. LeCompte M, Schensul J. Analyzing and inter-
preting ethnographic data in ethnographer’s tool-
kit. v. 5. Walnut Creek: AltaMira Press; 1999. 3. Harm Reduction Coalition. Principles of harm re-
duction. Newsline People AIDS Coalition N Y
1998 Jun; 7-8. 3. Harm Reduction Coalition. Principles of harm re-
duction. Newsline People AIDS Coalition N Y
1998 Jun; 7-8. 10. Weeks MR, Singer M, Himmelgreen DA, Rich-
mond P, Grier M, Radda K. Drug use patterns of
substance abusing women: gender and ethnic dif-
ferences in an AIDS prevention program. Drugs
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among drug users: outcome of a network-orient-
ed peer outreach intervention. Health Psychology
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among drug users: outcome of a network-orient-
ed peer outreach intervention. Health Psychology
2003; 22:332-9. 11. Steckler AB, Linnan L. Process evaluation for
public health interventions and research. San
Francisco: John Wiley and Sons; 2002. 5. Hopson RK, Peterson JA, Lucas KJ. Tales from the
‘hood’: framing HIV/AIDS prevention through in-
tervention ethnography in the inner city. Addic-
tion Research & Theory 2001; 9:339-63. 12. Hong Y, Mitchell S, Peterson J, Latkin CA, Tobin K,
Gann D. Ethnographic process evaluation: pilot-
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nal of Qualitative Methods 2005; 4(1). http://
www.ualberta.ca/~iiqm/backissues/4_1/html/ho
ng.htm. 6. Hopson RK, Lucas KJ, Peterson JA. HIV/AIDS talk:
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ers; 2000. p. 29-42. 6. Hopson RK, Lucas KJ, Peterson JA. Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Submitted on 23/May/2005
Approved on 30/Jun/2005 Acknowledgements This work was funded by the National Institute on
Drug Abuse, grant R01DA016555. Drogas Ilícitas; Abuso de Drogas; Redução de Danos Drogas Ilícitas; Abuso de Drogas; Redução de Danos Cad. Saúde Pública, Rio de Janeiro, 22(4):733-740, abr, 2006 Peterson J et al. 740 References References HIV/AIDS talk:
implications for prevention intervention and eval-
uation. In: Hopson RK, editor. How and why lan-
guage matters in evaluation: new directions for
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ers; 2000. p. 29-42. Submitted on 23/May/2005
Approved on 30/Jun/2005
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Decolonisation and the “Federal Moment”
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Diplomacy and statecraft
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© Michael Collins.
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. The moral rights of the named
author(s) have been asserted. © Michael Collins. Diplomacy & Statecraft, 24:21–40, 2013
Published with license by Taylor & Francis
ISSN: 0959-2296 print/1557-301X online
DOI: 10.1080/09592296.2013.762881 Decolonisation and the “Federal Moment” Federations involved alternative ways of thinking
about sovereignty, territoriality and political economy: a re-examination of
the rationale for their creation and the reasons for their failure may be of
some significance for historians looking back at the successes and failures
of the post-colonial nation-state. This article is an early attempt to sketch
some of the possible contours of what is a wide area of historical research. The article proceeds on the basis that the intersection between international
and imperial history constitutes a key pathway for making sense of some
of the most important trans-national and world historical developments in
twentieth century history.3 Downloaded by [University College London] at 08:18 08 September 2014 Specifically, the paper looks at the British Labour governments of
1945–51, and some of the imperial challenges faced by Britain in south-
ern and central Africa immediately following the South African election
in 1948 and up to the creation of the Central African Federation (CAF)
under the Conservatives in 1953. These two events were interconnected,
with fears of Afrikaner territorial, economic and political expansion influenc-
ing British policy making in the region.4 The CAF—a federal realm of the
British crown formed in 1953 by combining northern and southern Rhodesia
with Nyasaland—involved a denial of the principle of nationality. It thus ran
counter to a broad, if loosely defined acceptance of the emergence of African
nationalism by senior Labour figures. The abstract idea of national self-
determination gave way to the practical developmentalist imperialism that
came naturally to many senior Labour politicians and policy-makers. In addi-
tion, acute and multiple problems of race and identity arose in the southern
and central African region in the late 1940s, which impacted upon both
policy-making and wider, less tangible perceptions of Britain’s imperial and
national self. British policy makers redefined the terms of collaboration with
both white colonists and black nationalists, allowing them to secure on-going
control of political power and resources. Federation was thus ambivalent, a
move towards decolonisation as well as a means of securing imperial con-
trol. But insofar as federation can be read as a form of the “imperialism
of decolonisation,”5 the politics of Britain’s imperial identity were causal as
the British re-articulated justifications for the control of African peoples and
resources. Decolonisation and the “Federal Moment” MICHAEL COLLINS Post-1945 decolonisation involved the universal acceptance of
nation-statehood as the alternative to imperialism. Nationalism
vanquished its transnational competitors, notably imperialism and
Marxism. Alternatives to imperial rule that avoided sovereign states
on national lines, such as federations in the later 1940s and
1950s, have received less attention from historians. Federations
involved alternative ways of thinking about sovereignty, territorial-
ity, and political economy. British interest in creating federations,
for example the Central African Federation (CAF) in 1953, offers
some new perspectives on the strength of imperial ideology and
the determination to continue a missionary imperialism after the
Second World War. Federal thinking and practice was prominent
at this time in other European empires too, notably the French and
Dutch ones. The federal idea was also an aspect of the emerg-
ing European community. This is suggestive of a wider “federal
moment” that points to the importance of linking international,
trans-national, imperial, and world historical approaches. Downloaded by [University College London] at 08:18 08 September 2014 Downloaded by [University College Londo Post-1945 decolonisation involved the universal acceptance of nation-
statehood as the alternative to imperialism.1 Nationalism vanquished its
transnational competitors, notably imperialism and Marxism. Alternatives to
imperial rule that resisted the logic of sovereign states on national lines,
such as the federations created by the British in the later 1940s and 1950s,
as well as post-imperial federations created by decolonising political elites,
have arguably received less attention from historians than they might.2 From
a British imperial perspective, the interest in creating federations offers
an opportunity to re-examine—within the context of the “decline, revival
and fall” of the British Empire—the role of imperial ideology and Britain’s 21 22 M. Collins determination to continue a missionary imperialism after World War II. But
federal thinking and practice was also prominent at this time in other
European empires too, notably the French and Dutch ones. Equally, the
federal idea was an aspect of the emerging European community. This is
suggestive of a wider post-1945 “federal moment” that is linked, in con-
trast to the longer history of thinking about federations or forms of “closer
union” in British imperial circles, to the failures of the inter-war nation-state
and the specific challenges posed by decolonisation and the creation of
national states after 1945. Decolonisation and the “Federal Moment” It is significant—especially for those who are interested in lega-
cies of empire and decolonisation in the metrople—that this took place at a
relatively late stage, within the era of decolonisation itself. The events and Decolonisation and the “Federal Moment” 23 debates leading up to the creation of the CAF in 1953 reveal a conjuncture
of problems relating to imperial identity, imperial politics and international
pressures. Underlying these is a wider historical space in which the very idea
of sovereignty and nation-statehood was being contested. Variations of the federal idea have a long history in British imperial
thinking. The motivations for federal plans were most often a mixture of
practical, administrative, and cost-saving schemes, and sometimes sugared
with romantic notions of imperial unity. As early as the seventeenth century,
a federal model was floated as a solution to reducing the financial burden of
defending the West Indian plantations.6 In the late eighteenth century, figures
including Adam Smith and Lord Shelburne met the crisis in the American
colonies with calls for federation.7 But it was the end of the nineteenth
and beginning of the twentieth century that saw the emergence of a more
concerted and widespread movement for imperial federation and greater
imperial unity. Downloaded by [University College London] at 08:18 08 September 2014 The “colonial question” had come to the fore increasingly from the
1860s with “empire unity” occasionally demanded, and numerous schemes
“for federation, confederation, union, a colonial council, council represen-
tatives in Parliament, or occasional colonial conferences” advanced. “Little
emerged from the talk,” and yet “many of these ideas were to crop up again
and again for nearly a century.”8 One notable though short-lived venture
was the Imperial Federation League in 1884. Looking to Canada by way of
example, the League advocated an imperial parliament for Britain and the
self-governing territories gaining support from familiar figures in the “Greater
Britain” debate such as J. R. Seeley and J. A. Froude.9 The British branch of
the League was formally dissolved in 1894, undermined by divisions over
the necessity of putting forward formal proposals for federation versus the
development of a forum for an ongoing conversation about imperial ties and
closer association. The increasingly fraught debate over free trade and tariff
protection also split enthusiasts for federation at this time. Decolonisation and the “Federal Moment” p
p
Even so, the theme was given renewed impetus by the creation of the
Round Table Movement in 1909, with the journal of the same name founded
in 1910. The Movement initially pressed for organic union, but ultimately
for a Commonwealth of independent nation states, ironically the antithesis
of such a union. The Round Table Movement consisted largely of acolytes
of Lord Milner, his so-called “kindergarten” men such as Lionel Curtis. The
union in South Africa prompted thinking about a wider conception of union. In his more lofty and idealistic moments Curtis envisaged “an organic union
to be brought about by some establishment of an Imperial government,
constitutionally responsible to all the electors of the Empire, and with power
to act directly on individual citizens.”10 More concretely, even in light of the
recent Entente with France and Agreement with Russia, Curtis argued that a
“business arrangement for the support and control of Imperial defence and
foreign policy of the Empire” was necessary, or the empire “must break up.”11 24 M. Collins The impact of World War I on this kind of thinking was ambiguous. On the
one hand, there was a well-documented increase in Dominion assertiveness,
nationalism even. On the other, it also led to the development of greater
imperial participation in decision making at the centre. When, in December
1916, Lloyd George became Prime Minister, with the Milnerite and founding
member of the Round Table Philip Kerr as his Private Secretary, his call for a
special imperial war conference in 1917 and the creation of an Imperial War
C bi
“
l
b
i
h fi
i
f
h
h l
i
”12 p
p
p
Cabinet “came close to becoming the first executive for the whole empire.”1 What is noticeable is that calls for federation or closer union between
Britain and its empire, by which its advocates almost always meant the
colonies of white settlement, tended to be more vigorous and achieve greater
resonance at times of perceived weakness or vulnerability. If the Balfour
Declaration of 1926 and the Statute of Westminster constituted shifts towards
greater independence for the Dominions, the Great Depression forced a
shift in the opposition direction, towards greater economic inter-dependence. Here practical necessity tended to crowd out the loftier rhetoric of an earlier
period. Decolonisation and the “Federal Moment” This is interesting14 and per-
tinent to the federal moment of the 1950s in which will, desire and idea are
indeed stressed over and above means. The post war “federal moment” is
thus explicitly associated with decolonisation and the challenges of colonial
nationalism, and yet the desire and imagination to maintain Britain’s world
role were arguably of an earlier vintage. In his work on Sir Andrew Cohen, Ronald Robinson attributed a degree
of progressive foresight to the British Colonial Office in terms of its post-
1945 attitude towards decolonisation, specifically in Africa.15 With India
liberated, Palestine abandoned, and Malaya under threat, active steps were
taken to reorient British imperialism towards the strategically important site
of the Middle East, and to re-think the British role in Africa. In fact this pro-
cess and the ideas that pushed it forward had been underway before the
war, partially embodied in the progressive figure of Malcolm MacDonald,
Secretary of State for the Colonies from May 1938 to May 1940, and the
personal zeal that he brought to the Colonial Development and Welfare Act
of 1940. The shift in colonial policy towards Africa should not be seen as
a direct consequence of the war and the loss of imperial assets or prestige
that the British subsequently endured. Lord Hailey’s influential Africa Survey,
published in 1938, was an important moment in the steady re-evaluation of
the Lugardian idea of “trusteeship” that had framed African societies and cul-
tures as static, essentially ahistorical entities, with the British as benevolent
guardians, and in this regard at least primus inter pares when compared to
rival colonial powers.16 Downloaded by [University College London] at 08:18 08 September 2014 The flattering and deeply misleading self-image embodied in famous
Together wartime propaganda poster was indicative of British attempts
to portray their diverse “multi-racial” empire as the nemesis of Fascism’s
attempted racial holocaust. Their efforts were complicated, amongst other
things, by the obvious on-going economic exploitation underpinning the
empire, the brutal suppression of Gandhi’s 1942 “Quit India” campaign
and the awkward colour bar between black and white American G.I.s that
drew so much attention in wartime London, and in the British West Indies. Decolonisation and the “Federal Moment” Even so, the shift towards a more clearly defined and articulated
national identity on the part of the Dominions pointed toward a future
Commonwealth rather than any previously imagined imperial federation. Downloaded by [University College London] at 08:18 08 September 2014 The significance of this for our understanding of post-1945 fed-
eral experiments is twofold. The problem of colonial nationalism would
inevitably grow beyond the Dominions and India and would thus threaten
British control and influence in colonial territories. At the same time, the
strength of the association between Britain and its empire clearly remained
fundamental to the maintenance of a British world role. Commonwealth
would be central to this, and this would shape the British approach to nation-
alism. Federation would no longer be conceived as an imperial endeavour
formally constituting the relations between metropole and colony, but rather
as a way of maintaining British influence in particular parts of the empire,
a way of reconfiguring the politics of collaboration so as to defy the logic
of nationalism with its fetishisation of sovereign territoriality and hence to
maintain key British spheres of influence. What is surprising, perhaps, is the
enthusiasm with which some key British officials in the later 1940s pursued
this objective, for example Sir Andrew Cohen at the Colonial Office. In this sense, although the formal structure of the relationship may have
changed, the central objective of maintaining Britain’s world role and the
ideological belief in a liberal civilising mission is suggestive of a longer run
connection between the post-war period and the earlier liberal imperialists
such as Milner. As Jack Gallagher put it some time ago, “between the wars
British statesmen had been fearful for their empire. It had seemed so fragile
that another great war might well knock it to pieces. That did not hap-
pen. Whatever caused the end of empire, it was not the Second World War,
although this conclusion will not please those who think that the world came
to an end in 1945, or those who think that the world’s great age began anew Decolonisation and the “Federal Moment” 25 in 1945.”13 Gordon Martel, in his critique of what he saw as Paul Kennedy’s
economic determinist view of decline, stressed the importance of will and
ideas in this process of imperial reconfiguration. Decolonisation and the “Federal Moment” The Atlantic Charter of 1941, which Churchill had done so much to resist,
changed the tone of international public opinion.17 With the 1945 Charter of
the United Nations declaring “the respect for the principle of equal rights
and self-determination of peoples” as one of its basic purposes, a sec-
ond and, from the perspective of nationalists, more promising “Wilsonian
moment” was well under-way.18 Amongst its many foreign policy challenges,
the Labour government would have to respond to this new environment. Colonial Office policy shifts towards Africa certainly accelerated in
the later 1940s. It was not only domestic and international public opin-
ion that was changing, but also the politics of collaboration in the colonies 26 M. Collins themselves. In the face of rising African nationalism, which would increase
in the foreseeable future, the Colonial Office deemed it necessary that the
aforementioned “trusteeship model” of indirect rule—whereby the British
struck deals with African chiefs in order to maintain control of territory—
needed revision. Increasing urbanisation was underway in Britain’s African
colonies, and labour unrest was taking on surprisingly violent forms, notably
in Accra in 1948.19 Urgent action was required to improve Britain’s position. 9
g
q
p
p
A major component of this was to be the so-called “new approach”
to local government in Africa advanced by Arthur Creech Jones, Secretary
of State for the Colonies, and Andrew Cohen, Assistant Under-Secretary of
State, and Creech Jones’ “alter ego.”20 The purpose was to bring greater
African participation into the affairs of government. Robinson, in one of
the only significant scholarly works on Cohen, claimed that the May 1947
“Cohen Report” which outlined four stages of constitutional advance to incor-
porate Africans into local democracy, foresaw most African dependencies
being self-governing within a generation. Cohen’s “revolutionary manifesto”
was “inspired by the belief that if the dependencies were to be devel-
oped economically, their administration would have to be democratised and
nationalised.”21 But the general political radicalism of Labour’s approach and
support for nationalism and decolonisation has been grossly overstated.22 If
we are to understand why Labour begins to acquiesce in the creation of the
CAF by the late 1940s this misperception needs to be cleared up. In fact, in
the 1947 report only the Gold Coast was viewed as being capable of self-
government “in a generation.” Elsewhere, internal self-government would
take substantially longer. Decolonisation and the “Federal Moment” Independence is not even discussed.23 Downloaded by [University College London] at 08:18 08 September 2014 Cohen is a key figure here, whom Robinson presents as embodying the
spirit of the new policy, labelling him the “proconsul of African national-
ism,” rather misleadingly suggesting that his “constitution mongering” was
awakening the “slumbering genius” of national politics.24 Early in his career
at the Colonial Office, Cohen had gravitated towards the Fabian wing of the
Labour Party. The Fabian Colonial Bureau (FCB), founded in 1940, might be
seen as a spiritual home, and along with Creech Jones, Cohen corresponded
with, and highly regarded the opinion of, key figures in the FCB such as Rita
Hinden and Marjory Perham. In true Fabian spirit, the new approach to Africa
was not primarily constitutional but socio-economic and socio-political.25 In some senses, Fabians and democratic socialists from the British
Labour Party could have their cake and eat it too. They could make efforts at
colonial development which sought to increase dollar earning exports and
thus benefit the metropole in a period of desperate dollar shortage, whilst
simultaneously (and arguably sincerely) believing that in the longer run
this new economic development would create the socio-economic under-
pinnings of “de-tribalised” societies with a functioning civil society: trade
unions, political parties, news media. Modernised indigenous elites would
make excellent future collaborators—not least for Labour governments—in Decolonisation and the “Federal Moment” 27 a post-imperial world where formal constitutional decolonisation gave way
to looser but still substantial economic and political cooperation through
Commonwealth. Rita Hinden, looking back from the perspective of 1959 on the FCB and
its role in post-war imperial policy, put Labour’s predicament thus: To this massive legacy, the socialists were heirs; they had the duty to
decide on its use. Enjoy it? No, that would have been a violation of social-
ist principle. Reject it outright, and so remain true to the anti-imperialism
which socialists had always preached? Or, better still perhaps, accept
the heritage, but with the determination to nurse and develop it for the
advantage of its rightful owners till they themselves should have come of
age?26 Downloaded by [University College London] at 08:18 08 September 2014 Hinden recognised the “poverty and backwardness” of the colonies had
been deepened by imperialism, by “extorting the . . . wealth and alienating
. . . Decolonisation and the “Federal Moment” land from the people, by taxation and forced labour, by the despoliation
of the soil.” But, she believed that “even before the entry of imperialist
powers, the colonial territories were poor and economically backward” and
that poverty might “be sooner cured by prolonging imperial rule” than by
moving too quickly toward independence.27 Getting Labour’s approach to
empire straight is thus crucial in understanding not simply the acceptance of
federation in central Africa, but ultimately its advocacy. Neither nationalism
nor democracy was of paramount importance. What mattered was steady
and consistent social and economic development, and inter alia, continued
British control. British missionary imperialism was in rude health under
Labour. Margaret Joy Tibbets, a sharp-witted political officer at the United States
Embassy in London, saw the point early on. In a memorandum on “British
Colonial Development and Welfare Programs” she wrote that “it is the opin-
ion of the United States Embassy at London, that [Labour’s approach to
colonial development] is intended primarily to provide increased foodstuffs
and other primary products for the British people.”28 This was undoubtedly
true, but the realties of the Cold War, the apparent unpreparedness of African
peoples to govern themselves and the predatory threat of Communist infiltra-
tion were already softening the United States government’s attitude towards
the British Empire.29 The early Cold War made the southern African region
a strategic asset first and foremost because of South Africa’s rich source of
uranium, and instability in the region caused by political and racial ten-
sions offered opportunities to the Soviet Union to foment unrest, threatening
this rich and vital source of nuclear material.30 Hence the extent to which
“London retained a good deal of its old determination to shape the terms of
collaboration and make things happen on the ground”31 became increas-
ingly important to the United States, and opened up new opportunities
for the Labour government to reinvigorate colonial policy. In fact, Labour’s 28 M. Decolonisation and the “Federal Moment” Collins redeployment of imperial power in Africa after World War II, with hun-
dreds of new bureaucrats and the burgeoning of a technocratic, imperialist
developmentalism, has been called a “second colonial occupation”.32 The
United States would not stand in the way of the British if this meant Britain’s
empire would remain a bulwark against the encroachments of communism
in Africa, and to this end Marshall Aid was allowed to be spent within the
empire.33 p
Despite some recent corrections from Cooper and Hyam on the Labour
Party and Africa c. 1945–51, Robinson’s triumphalist account of the Colonial
Office—particularly his version of Cohen’s role—continues to deflect our
attention from an important moment in the decolonisation process in which
both trusteeship and a new civilising mission make a return. The creation of
the CAF is an important case in point. Some kind of amalgamation or feder-
ation of Britain’s central African territories had been mooted for some time,
an idea largely pushed forward by the white settler community for its own
purposes. Yet in early 1948 Creech Jones had declared to Roy Welensky,
Prime Minister of Northern Rhodesia, that the Labour Government, indeed
“no government” would “abandon” millions of black Africans in Nyasaland
and the Rhodesias to rule by a minority of white settlers.34 But in September
of the same year Cohen’s private meetings with United States diplomats
in London led them to surmise that the British government believed the
interests of Africans in southern and central would suffer “if Whitehall’s pro-
tecting hand were withdrawn.”35 Federation was looking increasingly likely. By September 1950 American diplomats produced a secret Memorandum
of Conversation for Washington’s consumption in which they directly quote
Cohen as saying that “it would obviously be advantageous to have a strong
federated Rhodesia as a counterweight to the Union [of South Africa].”36 In
the context of this particular federal project, the “race problem” and the
South African bogeyman were crucial drivers of British policy, allowing a
paternalistic imperialism to side-line black African nationalism (specifically in
Nyasaland) in favour of a new British dominion in central Africa. The spectre
of a pan-Afrikaner alliance that had prompted Milner and Chamberlain to go
to war in 1899 reared its head again.37 More broadly, the denial of national
sentiment in favour of non-national geo-political reform begs the question
of whether a more pervasive strategy is underway. Decolonisation and the “Federal Moment” Downloaded by [University College London] at 08:18 08 September 2014 The problems Britain faced on its southern African imperial periphery
after 1945 were manifold. Sympathy for newly defeated German fascism
in some quarters of the Afrikaner community re-opened and accentuated
old divisions between the Afrikaner and Anglo worlds. From London’s per-
spective, the growing political power of the Nationalist Party under Daniel
Malan did not augur well. The South African election of May 1948 brought
the (Afrikaner) National Party to power. The Nationalists did not con-
ceal their commitment to the most rigid separation of the races. In July
1949 the Prohibition of Mixed Marriages Act became law in South Africa, Decolonisation and the “Federal Moment” 29 making a marriage between a “European” and a “non-European” a criminal
offence. Further efforts to increase segregation followed. The long-standing
discrimination against the South African Indian community, which had
equally been a feature of Smuts’ United Party rule, was now internation-
alised via Jawaharal Nehru’s vigorous efforts to censure South Africa through
the United Nations and its new status as a Dominion within the British
Commonwealth. South Africa’s desire to incorporate the British protectorates
of Swaziland, Basutoland and Bechuanaland existed since the Union was
created and Dominion status conferred in 1910. Under Nationalist Party
government from 1948 incorporation was pursued with renewed vigour. In addition, South African ambitions to annex South West Africa (mod-
ern day Namibia) and the underlying possibility that Britain’s dependent
white settler colonies in southern and northern Rhodesia may gravitate—by
desire, design, or accident—towards South African-style apartheid was an
omnipresent concern in Whitehall. South African sensitivities over all these
matters caused chronic anxiety in London about the possibility of South
Africa declaring itself a republic and leaving the Commonwealth (as it would
eventually do in May 1961). Downloaded by [University College London] at 08:18 08 September 2014 y
y
All these issues, falling as they do under the umbrella of South African
expansionism, were further exacerbated by indigenous black political mobil-
isation. In Nyasaland, a new sense of black African political resistance gained
ground. Northern Rhodesia contained a large indigenous population, which
suffered exploitation in the copper mines and other extractive industries that
dominated its economy. Decolonisation and the “Federal Moment” Moreover, the way in which the Seretse affair taps into
pre-existing beliefs about the superiority of British civilisation helps us to
understand the turn towards federation in central Africa. Soon after Malan’s election, Seretse Khama and Ruth Williams were
declared prohibited immigrants in the Union of South Africa. This may not
have come as a surprise to Labour ministers, nor would it, in and of itself,
have been particularly destabilising of British policy in the region. The com-
plicating factor was the calls of successive South African governments to
have the three High Commission Territories—Seretse’s native Bechuanaland,
as well as Swaziland and Basutoland—incorporated into the Union of South
Africa.39 Under these circumstances, Labour ministers were advised that to
give the title of “Native Authority” to an African who had married a white
woman would be regarded as a deliberate provocation in South Africa. Malan
would use the opportunity to renew calls for the transfer of the Territories,
and to do so with the cross-party backing of the whites in the Union, as a
way of setting his own back yard in order, as Pretoria saw it. He would also
have the support of Sir Godfrey Huggins, the Prime Minister of Southern
Rhodesia, which was made clear in a statement to the all white Legislative
Assembly in Salisbury (Harare) on 7 July 1949.40 Downloaded by [University College London] at 08:18 08 September 2014 On the same day, after a meeting with Seretse Khama in Mafeking,
Evelyn Baring, High Commissioner in Bechuanaland, had, in his own words,
“his first opportunity of seeking help” from Douglas Forsyth, the Secretary
of South Africa’s Department of External Affairs. A top secret and per-
sonal letter was then sent to the Permanent Under-Secretary of State in
the Commonwealth Relations Office (CRO London), Sir Percival Leisching. Baring made it clear to Leisching that Forsyth had been very clear with him. Forsyth, Baring reported, had discussed the whole matter with Malan, who
had in turn discussed the question with his ministers and was greatly worried
and distressed. Decolonisation and the “Federal Moment” The struggles of indigenous peoples in South West
Africa were brought to the attention of international public opinion through
the United Nations and the political activism of the English Anglican reverend
Michael Scott, considered a “communist” by the United States.38 Amidst these
myriad complications, the event that captured the British domestic mood
most strongly, and in turn re-shaped the terrain upon which Labour had to
make its southern African policy, is the marriage of Seretse Khama to Ruth
Williams. It was September 1948 when Seretse Khama, a black African man from
the Bechuanaland Protectorate, studying law in England, married a white
English girl, Ruth Williams, at the Kensington Registry Office in London. Unfortunately for the newly married couple, Seretse was not just any old law
student from Africa. He was the grandson of Khama the Great, destined to
become the Kgosi (Chief) of the Bangwato, the largest and politically most
significant tribe in the Bechuanaland Protectorate. Seretse’s uncle, Tshekedi,
was the current Regent, recognised by the British Administration as the
Native Authority pending Seretse’s accession, once he was of the right age. The marriage of Seretse Khama and Ruth Williams caused problems
for successive British governments, first under Attlee’s Labour Party and then
under the Conservatives. Most importantly for our purposes, in the late 1940s
these fears fed into the on-going discussions about the CAF. In short, the 30 M. Collins “white settler problem” and the advent of Pretoria’s Nationalist apartheid
policy were key drivers of British policy towards southern Africa and the
CAF. The extent to which the Malan government in Pretoria was dictating
British policy in southern Africa reveals the link between race politics and
Britain’s self-perception as a benign power still capable of a progressive
liberal imperialism. Moreover, the way in which the Seretse affair taps into
pre-existing beliefs about the superiority of British civilisation helps us to
understand the turn towards federation in central Africa. “white settler problem” and the advent of Pretoria’s Nationalist apartheid
policy were key drivers of British policy towards southern Africa and the
CAF. The extent to which the Malan government in Pretoria was dictating
British policy in southern Africa reveals the link between race politics and
Britain’s self-perception as a benign power still capable of a progressive
liberal imperialism. Decolonisation and the “Federal Moment” Baring told Leisching that two points had emerged, and they
warrant quoting in full: First, official recognition of Seretse as chief, so long as it implies the resi-
dence in Serowe [Bechuanaland] of his English wife and the performance
by her of the duties of the first wife of a chief is what really matters to
the members of the government and probably to most South Africans. The mere residence of Seretse and his wife in Serowe without official
recognition is objectionable to them, but in Forsyth’s view of subsidiary
importance. He hopes that it might be avoided, but it is the recognition
of Seretse as chief which will be the match to set off the gunpowder. Huggins has also written to me taking exactly the same point of view.41 Decolonisation and the “Federal Moment” 31 Secondly, Baring added: Secondly, Baring added: [t]he political consequences in the Union of recognition would be far
more serious than I had realised. . . . The more extreme Nationalists will
use the Seretse incident to add fuel to those flames. They will argue that
our action demonstrates the folly of allowing the existence side by side
in southern Africa of two systems of native administration diametrically
opposed to one another. They will go on to say that South Africa should
not and cannot remain associated with a country which recognises offi-
cially an African chief married to a white woman, and they will make
Seretse’s recognition the occasion of an appeal of the country for the
establishment of a republic, and not only of a republic but a republic
outside the Commonwealth. Decolonisation and the “Federal Moment” Collins it added, uncomfortably for the Labour government, that Seretse’s “prospects
of success as a Chief are as bright as those of any native in Africa. . . . He
is admittedly the lawful and legitimate heir and, save for his unfortunate
marriage, would be in our opinion, a fit and proper person to assume the
chieftainship.”45 In light of this Noel-Baker warned Attlee that the Harragin
Report was “an inflammable document.”46 Attlee was suitably alarmed. “The
document is most disturbing,” he said. “In effect we are invited to go contrary
to the desires of the great majority of the Bangwato tribe, solely because of
the attitude of the governments of the Union of South Africa and Southern
Rhodesia. It is as if we had been obliged to agree to Edward VIII’s abdication
so as not to annoy the Irish Free State and the United States of America.47
Attlee decided that the Report should be referred to the Cabinet but then
accepted the advice of Gordon Walker that it should not be published. The
Harrigan Report was suppressed for 30 years. Downloaded by [University College London] at 08:18 08 September 2014 Following the February 1950 General Election Gordon Walker was
promoted to Secretary of State for Commonwealth Relations, replacing Noel-
Baker. With Leisching now his Permanent Under-Secretary, Gordon Walker
took the matter to the Cabinet with a new set of proposals. Seretse and Ruth
would be banished from the Bechuanaland Protectorate. His proposals were
accepted and a statement seeking approval was to be made to Parliament.48
Gordon Walker faced concern about the course of action proposed from
all sides of the House of Commons. The integrity of the government was
in question. The Seretse affair had become a cause celebre amongst the
anti-colonial left in Britain, and a propaganda coup for the Soviet Union.49
Churchill called the whole affair a “very disreputable transaction.”50 Following the February 1950 General Election Gordon Walker was
promoted to Secretary of State for Commonwealth Relations, replacing Noel-
Baker. With Leisching now his Permanent Under-Secretary, Gordon Walker
took the matter to the Cabinet with a new set of proposals. Seretse and Ruth
would be banished from the Bechuanaland Protectorate. Decolonisation and the “Federal Moment” Malan is desperately worried and feels he
could not successfully oppose an extremist offensive on these lines.42 Downloaded by [University College London] at 08:18 08 September 2014 In
a
minute
to
Philip
Noel-Baker—the
Secretary
of
State
for
Commonwealth Relations—Leisching informed him that in addition to the
communication from Baring, he had been separately told by General Byers,
Chief of the South African General Staff, that the British government’s recog-
nition of Seretse as chief of the Bangwato would “light a fire through all
the British colonial territories in Africa which would not soon be quenched.”
Leisching went on to tell Noel-Baker that “the very existence of white settle-
ment in these territories depended, in light of the numerical inferiority and
defencelessness of the white population, upon the principle that the native
mind regarded the white woman as inviolable.” He himself, he added, had
been unable to accept the “ultimate logical consequences of this principle
of non-discrimination when it takes practical forms affecting oneself or one’s
family in terms of miscegenation.” He did not “believe that many who hold
to their antipathy to the colour bar would, if confronted with this matter
in personal terms, view with equanimity, or indeed without revulsion, the
prospect of their son or daughter marrying a member of the Negro race.”43
The most senior civil servant at the CRO clearly viewed the problem through
the prism of both race and gender. Gordon Walker responded with his sup-
port for Baring’s proposal: “I would not put out of court the possibility of
declaring that a chief cannot have a white wife. There is a lot to be said for
this argument, and we should consider facing the uproar that would result. We must all think about this carefully.”44 Baring’s encounter with Forsyth appears to have decisively influenced
his thinking, and in turn influenced the position of the British govern-
ment. Having previously been minded to recognise Seretse as Chief, Baring
changed his position, saying the right course was now to “play for time” via
a commission of enquiry, which was subsequently set up under the chair-
manship of Lord Harrigan, a course of action that Noel-Baker was willing to
follow. For political reasons, the Harrigan Report came to the required con-
clusion that Seretse should not be recognised as Kgosi of the Bangwato. But 32 M. Decolonisation and the “Federal Moment” His proposals were
accepted and a statement seeking approval was to be made to Parliament.48
G
d
lk
f
d
b
h
f
i
d f In our understanding of the extent to which the race issue shaped
British government policy, it is important to note that during the House of
Commons debate on Seretse, Reginald Sorenson, a Labour member, asked
Gordon Walker whether South Africa had had any influence on the gov-
ernment’s decision. Gordon Walker replied in unambiguous language: “we
have had no communication from the government of the Union nor have we
made any communication to them. There have been no representations and
no consultation in this matter.”51 Gordon Walker’s statement to Parliament
was followed by a White Paper which said: “His Majesty’s Government were
of course aware that a strong body of European opinion in South Africa
would be opposed to recognition; but as stated in the House of Commons
on the 8 March, no representations on this matter have been received from
the government of the Union of South Africa or Southern Rhodesia.” Hyam
has referred explicitly to this incident and suggested Gordon Walker was
“perilously close” to lying.52 In fact, it is almost certainly clear that this was
indeed a lie to the House. On 30 June 1949, prior to Baring being persuaded
by Pretoria, Lief Egeland, South African High Commissioner in London, had
a meeting with Gordon Walker’s predecessor, Noel Baker, in which he made
the very same case about the impact of recognising Seretse’s marriage on Decolonisation and the “Federal Moment” 33 white opinion in the Union and beyond. He had made clear to the Secretary
of State that his visit was on the instructions of his Prime Minister, Malan,
and Gordon Walker could not reasonably claim he, or his civil servants,
were unaware of this.53 Either way, senior Labour ministers were paranoid about the race issue
and the impact it could have on Britain’s strategic position in Africa. A paper
presented by Gordon Walker to Cabinet in September 1950 claimed, fear-
ing South American expansion, that “the policies we detest in the Union,”
could be “established far to the North, and in the heart of this part of our
Colonial Empire.” He envisaged “terrible wars . . . Decolonisation and the “Federal Moment” Collins Robinson’s so-called proconsul of African nationalism was taking a different
stance. Cohen is perhaps better understood as a defender of a particular
vision of British liberalism, one with a long pedigree, and for historical pur-
poses this may be more interesting than the anodyne picture presented by
Robinson. It is important to recognise the ways in which this vision was
redeployed after World War II, indicating as it does the on-going vitality
of the British imperial mission long after 1945, the scepticism about the
nation-state that cut across party lines, and divisions between the civil service
and politicians. p
In August 1952, Henry Hopkinson, now Minister of State at the Colonial
Office under Winston Churchill’s Conservative Government, held sixty-eight
meetings in Northern Rhodesia and Nyasaland, coming to the conclusion
that any further delay would merely encourage “nascent nationalism.”58 By
the time the CAF came into being in 1953 it was accepted that Southern
Rhodesia would benefit economically from northern raw materials, and that
Nyasaland would provide a large pool of black labour. The issue of the
colour bar in the northern copper belt was fudged. With this in mind,
the suggestion that it was only after the Conservatives came to power
in 1951 that we see the return of “old-fashioned imperialism” is surely
misleading.59 There were some cases of consistency. Attlee, for example,
stuck to his belief that federation in central Africa ran counter to “the entire
premise of Labour’s African policy” because it set back the cause of African
nationalism.60 But we have seen how, in this instance, earlier talk in Labour
circles about political reform and the advance of black African nationalism
came to be superseded by a renewed vision of British imperial mission, a
vision that was accompanied by the implementation, eventually under the
Conservatives, of a tremendously ambitious, ultimately foolhardy project of
imperial state-building, a civilising mission, familiar in content but new in
form. Decolonisation and the “Federal Moment” between a white ruled East
Africa and a black ruled Western Africa,” and warned his colleagues that “our
whole work in Africa would be undone.”54 Fred Cooper has suggested that
after World War II, “[t]he old claims to colonial authority based on superior-
ity of race and civilisation were thoroughly discredited.”55 This, as Cooper
expertly shows, required Labour to justify its developmental imperialism in
technocratic terms. And yet arguments about race and civilisation do remain
of great importance. Downloaded by [University College London] at 08:18 08 September 2014 The conjuncture of race alongside geo-strategic considerations trumped
the idea that amalgamation and federation in Central Africa should be
postponed in order to protect the native African from white supremacist
ideologies, both from within the Rhodesias and spreading out of the Union. Now, members of the Labour Party took heed of the warnings out of Pretoria,
because they fed into pre-existing beliefs about the superiority of British
values and the on-going need for Britain to nurture and protect the black
African. They therefore acted against Seretse’s interests, persuaded that a
new dominion north of the Limpopo (the river separating the Union of
South Africa from Southern Rhodesia) was a higher goal. The fact that this
British bloc was supposed to stand for British “multi-racial” values against
the alien impact of the Afrikaner merely adds to the irony of Seretse’s treat-
ment. Cohen’s conversations with American diplomats in September 1950, in
which he reveals the radical shift from the position he held in 1949—now
believing that “it would obviously be advantageous” to have a federated
central Africa as a counterweight to South Africa—was contemporaneous to
Gordon Walker’s apocalyptic vision of race war in Africa, the Cabinet dis-
cussions about Seretse, the suppression of the Harrigan Report and Gordon
Walker’s deceitful statement to the House of Commons. By April 1951, in a
confidential minute, Cohen warned of the “Afrikaner danger” and Britain’s
“duty to the welfare of Africans.” In the context of Northern Rhodesia and
Nyasaland, Cohen stated that the native problem was one of bringing them
round to a “true realisation of their own interests.”56 In these matters it is
difficult to establish a forensic causal link, but the connection here seems to
be irresistible. By the end of 1951 Cohen felt that it was now crucial not to abandon the
field to “black nationalists,” which would have “disastrous consequences.”57 34 M. Decolonisation and the “Federal Moment” Downloaded by [University College London] at 08:18 08 September 2014 For the black Africans of Nyasaland and northern Rhodesia, talk of
socio-economic advancement and preparation for self-government had been
replaced by a new trusteeship and the re-mergence of pre-Word War II
approaches to collaboration on the British imperial periphery, embodied
in Hopkinson’s Lugardian conclusion that the black Africans of the CAF
would accept federation “taking their lead from the chiefs.”61 The history of
British imperial policy in southern Africa under Labour suggests that the great
changes apparently signalled by the Cohen Report of 1947 stalled in the face
of Afrikaner nationalism, white settler politics, and the complexities of race
relations. The missionary imperialism of key Fabian influences on the Labour
Party had always been pronounced. The turn to federation in central Africa,
what Hyam has called “the most controversial large-scale imperial exercise
in constructive state-building ever undertaken by the British government,”62
enabled the continuation of British control in central Africa and constituted a
late flowering of a deep-rooted, cross-party and inter-governmental belief in Decolonisation and the “Federal Moment” 35 Britain’s liberal imperial mission. It is suggestive that will and ambition could
still imagine their triumph over a paucity of resources.63 The CAF is but one instance of federation, and the story has only been
told from the perspective of high politics in the metropole. If there are links
between the missionary imperialism of an earlier age, which animated ideas
about federation, union, and commonwealth prior to 1945, the post-1945 fed-
eral moment is different, being explicitly a problem of decolonisation. The
nascent Commonwealth would be a commonwealth of independent nation
states. The federal option as deployed by the British meant resisting the
logic of nationalism as a response to the challenges of decolonisation, and
in the case of the CAF the pressures of white settler politics. Having acted as bridge between Labour and Conservative administra-
tions in the negotiations over the CAF, Cohen went on to become Governor
of Uganda in 1952. His time there is remembered rather well by many
Ugandans, especially for his liberal and tolerant views, his focus on social
and economic development, and his racial inclusivity. But it was not with-
out controversy. Decolonisation and the “Federal Moment” Yet the compression of the historical space
between empire and nation-state has arguably prevented us from asking how
it was that for the purposes of decolonisation, national states became the only
alternative to empires.68 In the post-war period federal questions arose in
regard to other European empires.69 The United States was supportive of the
idea of European federation.70 Questions of federal and “world government”
continued to coalesce around debates about the United Nations.71 These may
not be incidental connections, but may point to a historical moment in which
decolonisation and the relationships between empire, nation, sovereignty,
identity, and political economy were being questioned. This comes at a time
when, as Charles Maier has suggested, the territorial basis of nation-statehood
was already weakening.72 Downloaded by [University College London] at 08:18 08 September 2014 Raising our sights a little we realise that the CAF was not isolated. There were numerous federations or mooted federations across the British
Empire during this period, including the Malay Federation, 1948–1963; the
East African High Commission, 1948–1961; the previously discussed Central
African Federation, 1953–1963; the West Indies Federation, 1958–1962; and
the Federation of South Arabia, 1962–1967. These were not always British ini-
tiatives, with nationalists and other agents in the colonies themselves thinking
through the possibilities of federation for their own ends. But the sheer
breadth of federal experiments is suggestive of a deeper historical connec-
tion to ideologies of power and structures of identity, the significance of
which may be lost if we understand these manoeuvres strictly in terms of
a peripheral theory of collaboration and control. The idea of a federated
empire has long and deep roots in British imperial ideology. Clearly the
Federation of Rhodesia and Nyasaland is distinct from the dream of a fed-
erated white Empire-Commonwealth imagined by the advocates of Greater
Britain in the late nineteenth century, or the protagonists of the Round Table
Movement which pushed the idea forward throughout the twentieth cen-
tury. The British had already imposed a federal constitution on India. But
exactly what is the relationship between idea of federation in the late 1940s
and 1950s and that previous tradition? How did questions of empire, nation-
hood, sovereignty, and territoriality play themselves out across the canvas of
a decolonising British Empire? Moreover, that canvas was in fact far bigger than the British Empire. Decolonisation and the “Federal Moment” Cohen exiled the Kabaka of Buganda in 1953, essentially
because of the Kabaka’s attempt to steer the course of Ugandan decoloni-
sation toward a state based on Buganda, rather than the whole protectorate
established by the British in 1894. In fact, Cohen was also very interested
in an East African Federation to match the Central African one. The idea
of an east African federation prompted vigorous political debate, support
and opposition, in London and in east Africa itself. It would be picked
up by local political leaders and newly independent states as a possible
way to realise greater cooperation and put pan-African ideals into practice. As Julius Nyerere put it in 1964, “there is one way in East Africa that the
present unity of opposition should become a unity of construction. The
unity and freedom movements should be combined, and the East African
territories achieve independence as one unit . . . this means Federation of
the Territories now administered separately.”64 The story of the failure of
east African federation needs to be told, both from the perspective of colo-
nial authorities and, crucially, from the perspective of indigenous political
elites.65 Why did federation seem like a good idea? In asking this ques-
tion, the possible flaws of the nation-state model itself would be central
to the discussion of the failure of the post-colonial state, alongside the
neo-colonialism of former imperial powers or the inequities of the world
economy.66 Downloaded by [University College London] at 08:18 08 September 2014 Answering these kinds of historical questions would take us into a wider
field of inquiry, one in which the historical constitution of the nation-state in
the post-war period could be re-examined. Whereas the Atlantic Charter and
the Charter of the United Nations had envisaged the triumph of national self-
determination, the 1950s was also a moment of deep aporia surrounding the
viability of the nation-state. As Ernest Gellner articulated so precisely, nation-
alism as an ideology holds that “the political and the national unit should be 36 M. Collins congruent.”67 The application of that principle had been one of the key
drivers of two world wars. Paradoxically, empire explicitly resists national-
ist logic. So too does federation. Decolonisation and the “Federal Moment” Decolonisation and the “Federal Moment” 37 NOTES 1. Versions of this article were presented at the International History Conference in Honour of
Kathy Burk, UCL, 14 June 2012 and the Brady–Johnson Colloquium on Grand Strategy, International
Security Studies, Yale University, 4 December 2012. I am grateful for the comments I received at both
events. I am also incredibly thankful to all participants and convenors at the July 2012 International
Seminar on Decolonization, National History Center, Washington DC, for their careful and sharp reading
of an earlier draft. 2. For a near-contemporary assessment, see R. L. Watts, New Federations: Experiments in the
Commonwealth (Oxford, 1966). My thanks to Jason C. Parker at Texas A&M for alerting me to this
source. 2. For a near-contemporary assessment, see R. L. Watts, New Federations: Experiments in th
Commonwealth (Oxford, 1966). My thanks to Jason C. Parker at Texas A&M for alerting me to th
source. 2. For a near-contemporary assessment, see R. L. Watts, New Federations: Experiments in the
Commonwealth (Oxford, 1966). My thanks to Jason C. Parker at Texas A&M for alerting me to this
source. 3. For example, in the context of Anglo–American and imperial histories, see K. Burk, Old World,
New World: The Story of Britain and America (New York, 2007); B. J. C. McKercher, Transition of Power:
Britain’s Loss of Global Preeminence to the United States, 1930–1945 (Cambridge, 1999); or, more broadly,
the Adventures With Britannia series, beginning with W. R. Louis, ed., Adventures With Britannia:
Personalities, Politics and Culture in Britain (Austin Texas, 1996). 4. See R. Hyam, “The Political Consequences of Seretse Khama: Britain, the Bangwato and South
Africa, 1948–1952,” Historical Journal, 29 (1986), pp. 921–47; and R. Hyam, “The Geopolitical Origins
of the Central African Federation: Britain, Rhodesia and South Africa, 1948–1953,” Historical Journal,
30(1987), pp. 145–72. Both are reprinted in R. Hyam and P. Henshaw, The Lion and the Springbok:
Britain and South Africa Since the Boer War (Cambridge, 2003). 5. W.R. Louis & R. Robinson, “The Imperialism of Decolonization,” Journal of Imperial an
Commonwealth History, 22(1994), pp. 462–511. 6. E. Wallace, The British Caribbean: From the Decline of Colonialism to the End of Federation
(Toronto, 1977), p. 86. This reference is attributable to Jason C. Parker’s as-yet-unpublished manuscript
on federation in the West Indies. 7. Smith called for a “federal union with America.” See E. C. Mossner, I. Simpson Ross, R.H. Campbell, D. D. Raphael, and A. S. 18. On the first “Wilsonian moment” see E. Manela, The Wilsonian Moment: Self-Determination
and the International Origins of Anticolonial Nationalism (New York, 2009). On the second one, see S. 15. R.E. Robinson, “Andrew Cohen and the Transfer of Power in Tropical Africa, 1940–1951,”
in W.H. Morris-Jones & G. Fischer, eds, Decolonisation and Africa: The British and French Experience
(London, 1980), pp. 50–72. Since Robinson himself was at the Colonial Office in the late 1940s working
alongside men such as Andrew Cohen, this raises questions about how the historian is to historicise peo-
ple and institutions with which he was so intimately involved. For another positive interpretation of the
Labour government’s approach to Africa see R. Pearce, “The Colonial Office and Planned Decolonisation
in Africa,” African Affairs, 83 (1984), pp. 77–93. Decolonisation and the “Federal Moment” The decolonising federal moment was a genuinely trans-national, world his-
torical one, which deserves deeper, comparative historical analysis.73 This
will hopefully reveal the complex ways in which historical actors at both
the core and periphery of empires imagined alternative forms of sovereignty
and political organisation, forms which it my yet serve us well to re-examine
from our postcolonial present. NOTES Collins Moyn, “Imperialism, Self-Determination, and the Rise of Human Rights,” in Akira Iriye et al., eds., Th
Human Rights Revolution: An International History (New York, 2012), pp. 159–78 19. For a comparative and comprehensive approach to labour questions in British and French
Africa, see F. Cooper, Decolonization and African Society: The Labor Question in French and British
Africa (Cambridge, 1996). On British East Africa, see P. Kelemen, “Modernising Colonialism: the British
Labour Movement and Africa,” Journal of Imperial and Commonwealth History, 34(2006), pp. 223–244. 20. R.E. Robinson, “Cohen, Sir Andrew Benjamin (1909–1968),” in H.C.G. Matthew and Brian
Harrison, eds, Oxford Dictionary of National Biography (Oxford, 2004), online edition, L. Goldman,
ed., January 2011, http://www.oxforddnb.com/view/article/32478. 21. Robinson, “Transfer of Power in Tropical Africa,” p. 62. 22. Pearce called the 1947 report “a mature and systematic exposition of Colonial Office thinking, a
consistent and conscious strategy of decolonisation.” R. Pearce, “Planned Decolonisation in Africa,” p. 86. Hyam has criticised the “wilder misinterpretations” that have been placed upon the pronouncements of
Cohen and others in 1947 but cites D. Williams’ Cambridge History of Africa, Vol. VII: From c. 1940 to
c. 1975 (Cambridge, 1984), 341 as an example, not mentioning Robinson. See R. Hyam, “Africa and
the Labour Government, 1945–1951,” in R. Hyam, Understanding the British Empire (Cambridge, 2010),
p. 242. Downloaded by [University College London] at 08:18 08 September 2014 23. See Cooper, Decolonization and African Society, pp. 202–215; R. Hyam, “Africa and the Labour
Government,” p. 242. 24. Robinson, “Transfer of Power in Tropical Africa,” passim. 25. The possible convergence of Fabian thinking about empire and the slightly later American
modernisation theory, for example Seymour Martin Lipset’s ideas about the socio-economic underpin-
nings of democracy, is perhaps worth further investigation. See S.M. Lipset, “Some Social Requisites
of Democracy: Economic Development and Political Legitimacy,” American Political Science Review,
53(1959), pp. 69–105; S.M. Lipset, Political Man: The Social Bases of Politics (New York, 1960). 26. R. Hinden, “Socialism and the Colonial World,” in A.C. Jones, ed., New Fabian Colonial Essays
(London, 1959), p. 9. I owe this quote to Charlotte Riley, UCL History Department. 27. Ibid., p. 13. 28. United States Embassy, London, memorandum on “British Colonial Development and Welfare
Programs,” p. 37. As Gallagher puts it with regard to the Second Anglo–Boer War, “indeed a good deal of what
is commonly described as imperialist aggression during this period should rather be seen as imperialist
counter-punching; or, to put it more demurely, as reactions by British policy-making to developments
outside Europe.” J. Gallagher, Decline, pp. 81–82. The parallels with southern Africa in 1948–1953 are
intriguing. NOTES Skinner, eds, The Correspondence of Adam Smith (Oxford, 1987),
p. 383. On Shelburne, see H. M. Scott, British Foreign Policy in the Age of the American Revolution
(O f
d 1990)
224 I
b
h
f h
f
S
h
C
UCL (Oxford, 1990), p. 224. I owe both of these references to Stephen Conway, UCL. 8. W.D. McIntyre, The Commonwealth of Nations: Origins and Impact, 1869–1971 (Minneapolis,
1977), p. 30 9. D. Bell, The Idea of Greater Britain: Empire and the Future of World Order, 1860–1900
(Princeton NJ, 2007), pp. 6–62. 10. J. Kendle, Round Table Movement and Imperial Union (Toronto, 1975), pp. 70–71 10. J. Kendle, Round Table Movement an 11. Curtis to Milner, 1908, in Ibid., p. 56. 12. Macintyre, Commonwealth of Nations, p. 172. 13. J. Gallagher, The Decline, Revival and Fall of the British Empire (Cambridge, 1982), pp. 141–142. 13. J. Gallagher, The Decline, Revival and Fall of the B 14. G. Martel, “The Meaning of Power: Rethinking the Decline and Fall of Great Britain,”
International History Review, 13(1991), passim; P. Kennedy, The Realties Behind Diplomacy: Background
Influences on British External Policy, 1865–1980 (London, 1983); idem., The Rise and Fall of Great Powers
(New York, 1987). 15. R.E. Robinson, “Andrew Cohen and the Transfer of Power in Tropical Africa, 1940–1951,”
in W.H. Morris-Jones & G. Fischer, eds, Decolonisation and Africa: The British and French Experience
(London, 1980), pp. 50–72. Since Robinson himself was at the Colonial Office in the late 1940s working
alongside men such as Andrew Cohen, this raises questions about how the historian is to historicise peo-
ple and institutions with which he was so intimately involved. For another positive interpretation of the
Labour government’s approach to Africa see R. Pearce, “The Colonial Office and Planned Decolonisation
in Africa,” African Affairs, 83 (1984), pp. 77–93. 16. J. Flint, “Planned Decolonization and Its Failure in British Africa,” African Affairs, 82(1983),
pp. 389–411; R. Pearce, The Turning Point in Africa: British Colonial Policy in Africa, 1938–1948
(London, 1982), p. 43. 17. E. Borgwardt, A New Deal for the World: America’s Vision for Human Rights (Cambridge MA
2007). 18. On the first “Wilsonian moment” see E. Manela, The Wilsonian Moment: Self-Determination
and the International Origins of Anticolonial Nationalism (New York, 2009). On the second one, see S. 38 M. 35. United States Embassy, London, “The Proposed Federation of Rhodesia and Nyasaland
28 October 1948, RG84, Classified General Records, Box 158, File 350 Britain-Commonwealth. 36. United States Embassy, London, “British Colonial Policies in Africa,” 20 September 1950, Ibid 33. This money has proved difficult to trace but the figure usually used is £98 million. See R.
Schreurs, “A Marshall Plan for Africa? The Overseas Territories Committee and the Origin of European
Cooperation in Africa,” in R.T. Griffiths, ed., Explorations in OEEC History (Paris, 1997). 31. J. Darwin, The End of the British Empire, p. 104
32. See F. Cooper, “Modernising Bureaucrats, Backward Africans and the Development Concept
in F. Cooper and R.M. Packard, eds, International Development and the Social Sciences: Essays on th
History and Politics of Knowledge (Los Angeles, 1997) pp. 64–92. 34. R. Welensky, Welensky’s 4000 Days (London, 1964). 31. J. Darwin, The End of the British Empire, p. 104 34. R. Welensky, Welensky’s 4000 Days (London, 1964). 38. C. Anderson, “International Conscience, the Cold War, and Apartheid: The NAACP’s Alliance
with the Reverend Michael Scott for South West Africa’s Liberation, 1946–1952,” Journal of World History,
19 (2008), pp. 297–326; R. Skinner, “Christian Reconstruction, Secular Politics: Michael Scott and the
Campaign for Right and Justice, 1943–45” in S. Dubow and A. Jeeves, eds., South Africa’s 1940s: Worlds
of Possibilities (Cape Town, 2005). Decolonisation and the “Federal Moment” 39 Decolonisation and the “Federal Moment” 38. C. Anderson, “International Conscience, the Cold War, and Apartheid: The NAACP’s Alliance
with the Reverend Michael Scott for South West Africa’s Liberation, 1946–1952,” Journal of World History,
19 (2008), pp. 297–326; R. Skinner, “Christian Reconstruction, Secular Politics: Michael Scott and the
Campaign for Right and Justice, 1943–45” in S. Dubow and A. Jeeves, eds., South Africa’s 1940s: Worlds
of Possibilities (Cape Town, 2005). 39. These calls found their impetus in the 1909 South Africa Act, which included consideration of
the possibility of handing over the three territories to South Africa at some undefined point in the future. 40 “Th
i
d
b ”
i
ld h
“ h
h
ib
’ d
i i
[
S 39. These calls found their impetus in the 1909 South Africa Act, which included consideration of
the possibility of handing over the three territories to South Africa at some undefined point in the future. 40. “There is no doubt,” Huggins told the House, “that the tribesmen’s decision [to support Seretse
as Kgosi] is a disastrous one. First it shows lack of racial pride in Bechuanaland; secondly, it is disastrous
from the effect it will have on neighbouring territories.” He told members that he had already written to
the High Commissioner, but would write again “informing him of the opinion of this House and how
disastrous it would be if this fellow is allowed to become Chief of Khama’s people.” S. Williams, Colour
Bar: The Triumph of Sereste Khama and His Nation (London, 2006), pp. 69–70. 40. “There is no doubt,” Huggins told the House, “that the tribesmen’s decision [to support Seretse
as Kgosi] is a disastrous one. First it shows lack of racial pride in Bechuanaland; secondly, it is disastrous
from the effect it will have on neighbouring territories.” He told members that he had already written to
the High Commissioner, but would write again “informing him of the opinion of this House and how
disastrous it would be if this fellow is allowed to become Chief of Khama’s people.” S. Williams, Colour
Bar: The Triumph of Sereste Khama and His Nation (London, 2006), pp. 69–70. 41. Baring to Leisching, 11 July 1949, DO (Dominions Office Archives, The National Archives, Kew,
London) 119/1283. 42. Ibid. 43. Minute by Leisching, 14 July 1949, DO119/1283, Williams, Colour Bar, pp. 72–3. 45. Decolonisation and the “Federal Moment” Harrigan Report, 1 December 1949, PREM (Prime Minister’s Office Archives, Archives, The
National Archives, Kew, London) 8/1308, Part 1, Ibid., p. 107. 45. Harrigan Report, 1 December 1949, PREM (Prime Minister’s Office Archives, Archives, The
National Archives, Kew, London) 8/1308, Part 1, Ibid., p. 107. 46. Noel Baker to Attlee, 21 December 1949, Ibid.; Williams, Colour Bar, p. 110. 47. Attlee to Gordon Walker, 22 January 1950, PREM 8/1308, Williams, Colour Bar, p. 113 48. Gordon Walker would lose the Smethwick constituency seat at the General Election of 1964 to
a Conservative Party candidate who ran an anti-immigration campaign with the slogan: “If you want a
nigger neighbour, vote Labour.” 48. Gordon Walker would lose the Smethwick constituency seat at the General Election of 1964 to
a Conservative Party candidate who ran an anti-immigration campaign with the slogan: “If you want a
nigger neighbour, vote Labour.” 49. R. Skinner, “The Moral Foundations of British Anti-Apartheid Activism 1946–60,” Journal of
Southern African Studies, 35 (2009), pp. 399–416. Cf. Hyam, “Political Consequences of Seretse Khama.” . R. Skinner, “The Moral Foundations of British Anti-Apartheid Activism 1946–60,” Journal of
n African Studies, 35 (2009), pp. 399–416. Cf. Hyam, “Political Consequences of Seretse Khama.” . J. Redfern, Ruth and Seretse: A Very Disreputable Transaction (London, 1955), p. 7. 50. J. Redfern, Ruth and Seretse: A Very Disreputable Transaction (London, 1955), p. 7. 51. Hansard, 8 March 1950, House of Commons Debates, Series 5, Vol. 472, p. 288 m, “Political Consequences of Seretse Khama,” p. 939 53. Egeland sent a telegram to Malan, copied to the CRO, and then followed up his telegram with
a private letter to Malan, telling him that at the very start of his meeting with Noel Baker in June he had
made it clear that his visit to the Secretary of State was made “on your [Malan’s] instructions.” See Egeland
to Malan, 24 March 1950, PM (National Archives of South Africa) Vol. 1/4/21, 1/15. / /
/
54. R. Hyam, ed., British Documents on the End of Empire, Vol. 2: The Labour Government and the
End of Empire 1945–1951 (London, 1992), p. 311. 54. R. Hyam, ed., British Documents on the End of Empire, Vol. 2: The Labour Government and the
End of Empire 1945–1951 (London, 1992), p. 311. 55. Cooper, Decolonization, p. 173. 56. Hyam, “Geopolitical Origins,” p. 163. 57. Quoted in R. NOTES 9, circulated to Certain American Diplomatic and Consular Officers by the State Department,
Washington D.C., 7 July 7 1948, RG 84 (National Archives and Records Administration, College Park, MD)
Classified General Records, Box 158, File 350 Britain-Colonies. 29. Cohen did not miss the opportunity to remind the American Embassy in London that whilst in
his view (and he was thought by American diplomats to be “probably the best-informed man in London
on the subject”) communism had not yet penetrated deeply into Africa, keeping it out would be possible
in large part due to the presence of British administration and control. See W. Stratton Anderson, United
States Embassy, London, “Communism in Africa: Cohen’s Comments,” 26 October, 1948, Ibid. 30. On United States policy towards South Africa see T. Borstelmann, Apartheid’s Reluctant Uncle:
The United States and Southern Africa in the Early Cold War (New York, 1993); and A. Thomson, U.S. Foreign Policy Towards Apartheid South Africa, 1948–1994 (New York, 2008). 30. On United States policy towards South Africa see T. Borstelmann, Apartheid’s Reluctant Uncle:
The United States and Southern Africa in the Early Cold War (New York, 1993); and A. Thomson, U.S. Foreign Policy Towards Apartheid South Africa, 1948–1994 (New York, 2008). 31. J. Darwin, The End of the British Empire, p. 104
32. See F. Cooper, “Modernising Bureaucrats, Backward Africans and the Development Concept,”
in F. Cooper and R.M. Packard, eds, International Development and the Social Sciences: Essays on the
History and Politics of Knowledge (Los Angeles, 1997) pp. 64–92. 33. This money has proved difficult to trace but the figure usually used is £98 million. See R. Schreurs, “A Marshall Plan for Africa? The Overseas Territories Committee and the Origin of European
Cooperation in Africa,” in R.T. Griffiths, ed., Explorations in OEEC History (Paris, 1997). 47. Attlee to Gordon Walker, 22 January 1950, PREM 8/1308, Williams, Colour Bar, p. 113. 68. Partha Chatterjee argued persuasively that anti-colonial nationalism was a “derivative discourse.”
atterjee, Nationalist Thought and the Colonial World: A Derivative Discourse (London, 1986). Critics 72. C. S. Maier, “Consigning the Twentieth Century to History: Alternative Narratives for the Modern
Era,” in American Historical Review, 105 (2000), pp. 807–31. Maier highlights “the spatially anchored
structures for politics and economics that were taken for granted from about 1860,” and puts in a “plea
for historians to envisage a historical era that took shape in the second half of the nineteenth century
and, just as important, effectively unraveled in the two or three decades before the [twentieth] century
formally ended.” Maier, “Consigning the Twentieth Century to History,” p. 808. Decolonisation and the “Federal Moment” Hyam, “Containing Afrikanerdom: The Geopolitical Origins of the Central Africa 57. Quoted in R. Hyam, “Containing Afrikanerdom: The Geopolitical Origins of the Central African
Federation, 1948–1953,” in Hyam & Henshaw, Lion and the Springbok, p. 219. 58. Ibid., p. 165. Federation, 1948–1953,” in Hyam & Henshaw, Lion and the Springbok, p. 219. 58. Ibid., p. 165. Federation, 1948–1953,” in Hyam & Henshaw, Lion and the Springbok, p. 219. 58. Ibid., p. 165. 59. Pearce, “Planned Decolonization,” p. 88. 60. Hyam, “The Labour Government and Africa,” note 54, pp. 266. Hyam contrasts this with Cohen’s
“determination to bring federation into being,” note 54, pp. 266. 61. Hyam, “Geopolitical Origins,” pp. 165. 61. Hyam, “Geopolitical Origins,” pp. 165. 62 Ibid 63. Cf. Martel, ‘The Meaning of Power’, passim. 63. Cf. Martel, ‘The Meaning of Power’, passim. 64
d
d
4 (
64)
4 64. J. Nyerere, “Freedom and Unity,” Transition, 14 (1964), p. 40. 65. There seems to be relatively little work done in this area, although east African federation did
attract the attention of some prominent political scientists in the 1960s. See J.S. Nye, Pan–Africanism and
East African Integration (Cambridge MA, 1965); C. Leys and P. Robson, eds, Federation in East Africa
(Oxford, 1965). 66. For an idiosyncratic and stimulating discussion of this problem see B. Davidson, The Black
Man’s Burden: Africa and the Curse of the Nation-State (New York, 1992). 66. For an idiosyncratic and stimulating discussion of this problem see B. Davidson, The Blac
Man’s Burden: Africa and the Curse of the Nation-State (New York, 1992). 67. E. Gellner, Nations and Nationalism (Oxford, 1983), p. 1. 68. Partha Chatterjee argued persuasively that anti-colonial nationalism was a “derivative discourse.”
P. Chatterjee, Nationalist Thought and the Colonial World: A Derivative Discourse (London, 1986). Critics 68. Partha Chatterjee argued persuasively that anti-colonial nationalism was a “derivative discourse.”
atterjee, Nationalist Thought and the Colonial World: A Derivative Discourse (London, 1986). Critics M. Collins 40 of empire who resisted nationalism were few and far between. Rabindranath Tagore had denounced both
imperialism and nationalism in the early twentieth century, but his lone voice was drowned out by the
rising tide of anti-colonial nationalism, the seductive political power of which not even Gandhi could
deny. See M. Collins, Empire, Nationalism and the Postcolonial World: Rabindranath Tagore’s Writings
on History, Politics and Society (London, 2012). 69. On the Malay federation see A.J. 73. Cf. F. Cooper and J. Burbank, Empires in Word History: Power and the Politics of Differenc
(Princeton, 2010), p. 10. 71. M. Mazower, Governing the World: The History of an Idea (London, 2012). 70. P. Winand, Eisenhower, Kennedy and the United States of Europe (Basingstoke, 1996). Decolonisation and the “Federal Moment” Stockwell, “Malaysia: The Making of a Neo-Colony?,” Journal of
Imperial and Commonwealth History, 26 (1998), pp. 138–156; and M. Jones, Conflict and Confrontation
in South East Asia, 1961–1965: Britain, the United States and the Creation of Malaysia (Cambridge,
2002). On federal experiments in French West Africa see F. Cooper, “Alternatives to Empire: France and
Africa After World War II,” in D. Howland and L. White, eds, The State of Sovereignty: Territories, Laws,
Populations (Bloomington, 2009). On the Dutch East Indies see J. Foray, “A Unified Empire of Equal
Parts: The Dutch Commonwealth Schemes of the 1920s–1940s,” forthcoming in the Journal of Imperial
and Commonwealth History. Downloaded by [University College London] at 08:18 08 September 20
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Seven naphtho-γ-pyrones from the marine-derived fungus Alternaria alternata: structure elucidation and biological properties
|
Organic and medicinal chemistry letters
| 2,012
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cc-by
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Seven naphtho-g-pyrones from the marine-
derived fungus Alternaria alternata: structure
elucidation and biological properties Mohamed Shaaban1,2*†, Khaled A Shaaban2† and Mohamed S Abdel-Aziz3 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Open Access © 2012 Shaaban et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. * Correspondence: mshaaba_99@yahoo.com
† Contributed equally
1Chemistry of Natural Compounds Department, Pharmaceutical Industries
Division, National Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt
Full list of author information is available at the end of the article Abstract Eight bioactive pyrone derivatives were identified from the culture of Alternaria alternata strain D2006, isolated
from the marine soft coral Denderonephthya hemprichi, which was selected as its profound antimicrobial activities. The compounds were assigned as pyrophen (1), rubrofusarin B (2), fonsecin (3), and fonsecin B (5) beside to the
four dimeric naphtho-g-pyrones; aurasperone A (6), aurasperone B (7), aurasperone C (8), and aurasperone F (9). Structures of the isolated compounds were identified on the basis of 1D and 2D NMR spectroscopy and mass (EI,
ESI, HRESI) data, and by comparison with the literature. Configuration of the four dimeric naphtho-g-pyrones 6-9
was analyzed by CD spectra, exhibiting an identical stereochemistry. Keywords: pyrone derivatives, Alternaria alternata, marine fungi, biological activity at Safaga coasts, Egypt) was selected due to its distinc-
tive features in the chemical and biological assays. We
therefore performed a bioassay-guided fractionation. 1. Background g
Infectional diseases and drug resistance phenomena are
the most effective reasons for the death of ca. 20 mil-
lions yearly. For example, tuberculosis (TB) was the
leading cause of ca. two million deaths due to a bacterial
pathogen, Mycobacterium tuberculosis, among them
more than 80% of TB patients living in sub-Africa and
Asia [1-4]. Thus, new and more-powerful drugs are
necessary to solve these problems. Marine microorgan-
isms, especially fungi, are still a less investigated
resource of bioactive substances [5,6]; recent investiga-
tions indicated their tremendous potential as source of
new drugs [7-13]. y g
The crude extract possessed in the agar diffusion test
potent activity against Pseudomonas aeruginosa, Staphy-
lococcus aureus and Candida albicans. For isolation of
the bioactive constituents, A. alternata D2006 was up-
scaled as a shaker-culture using GYMP medium [15]
(100% seawater) for 10 days. Thereafter, the obtained
black broth was worked up [16] and separated by a ser-
ies of chromatographic steps, yielding colourless semiso-
lid of pyrophen (1) and seven naphtho-g-pyrones (2, 3,
5-9) as yellow solids, among them four dimeric analo-
gues (6-9). Herein, we describe their separation, struc-
ture elucidation (using 1D and 2D NMR and MS (EI,
ESI, HRESI) data and antimicrobial properties. In this article, a report on the antimicrobial activity of
naphtho-g-pyrones (naphthopyran-4-ones) attracted our
interest [14]. During the investigation of fungal strains
for the production of structurally novel active com-
pounds from marine microorganisms, we found that the
EtOAc extract of the marine-derived fungal strain Alter-
naria alternata D2006 (isolated from a red soft coral,
Denderonephthya hemprichi, collected from the Red Sea * Correspondence: mshaaba_99@yahoo.com
† Contributed equally
1Chemistry of Natural Compounds Department, Pharmaceutical Industries
Division, National Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt
Full list of author information is available at the end of the article © 2012 Shaaban et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. 2. Taxonomy and characterization The fungal isolate was identified as A. alternata (Dema-
tiaceae) according to Barnett [17]. Microscopically, the
conidiophores were dark, simple, rather short or elon-
gate and contained simple or branched chains of coni-
dia. Conidia were dark, typically with both cross and
longitudinal septa, with various shapes, obclavate to
elliptical or ovoid. The fungal spores were multicellular,
dark and having thick cell walls. Page 2 of 8 Page 2 of 8 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 for 1 using the 2D NMR experiments for the first time
(Figure 1 and Table 1 [see Additional file 1]). for 1 using the 2D NMR experiments for the first time
(Figure 1 and Table 1 [see Additional file 1]). Compound 2 showed a molecular weight of m/z
287.09137 (HRESI MS), corresponding to the molecular
formula C16H15O5 [M+H]+. The
1H NMR spectra
(Table 2) displayed a chelated hydroxyl group (δ 14.96),
two m-coupled doublets (δ 6.56, 6.38) and two singlets
(δ 6.94 and 5.98), along with two methoxy signals (δ
3.99, 3.91) and an sp2 linked methyl (δ 2.35). The 13C/
HMQC spectra (Table 2) indicated the presence of 16
carbon signals, including 4 sp2 methines (δ 107.3-97.2),
3 sp2-oxy carbons (δ 162.6-160.6), 1 carbonyl of g -lac-
tone (δ 184.2) [20], 5 non-oxygenated sp2, 2 aromatic-
attached methyl ethers (δ 56.0, 55.4) and 1 sp2-attached
methyl (δ 20.6). Full assignment of the 2D NMR experi-
ments (Figure 2 and Table 2) established the structure
of 2 as rubrofusarin B, and excluded the structure of
the isomeric asperxanthon (11) in the same way [21]. Structure of 2 was not fully assigned using 2D NMR
before, which we report her to first time (see Additional
file 2). The closely related compound 3 afforded a molecular
weight of 290 Da (C15H12O5 by HRESI MS); EI MS gave
easily an ion peak at m/z 272 by expulsion of water
molecule. The 1H NMR spectrum exhibited aromatic
m-coupled doublets (δ 6.47, 6.31, J ~ 1.1 Hz) and a
methine singlet (δ 6.41), but in contrast to 2, two phe-
nolic hydroxy signals (δ 14.19, 10.18), and only one
methoxy signal (δ 3.84). In addition, an AB signal of dia-
stereotopic methylene protons (δ 3.14, 2.72, J ~ 16.8)
and a methyl singlet (δ 1.60) were visible. Based on 13C/
HMQC spectra (Table 2) and HMBC experiment (as it
was not fully assigned before using 2D NMR) (Figure 2), 3. Results and discussion The expectation of a dimeric rubrofusarin B (
was confirmed by 1H NMR spectra, where six s
Table 1 13C and 1H NMR data of pyrophen (1) in CDCl3
in [Hz])
Number
δc
δh
Number
δc
δh
2
164.7
-
7-NHCOCH3
170.3
-
3
88.0
5.43 (d, 2.2)
7-NHCOCH3
22.3
1.95 (s
4
171.0
-
8
38.1
3.09 (m
4-OCH3
55.7
3.73 (s)
1’
136.0
-
5
100.6
5.90 (d, 2.2)
2’,6’
128.6
7.16 (m
6
161.9
-
3’,5’
128.2
7.25 (m
7
52.3
4.98 (q, 7.8)
4’
126.5
7.21 (m
7-NHCOCH3
-
7.79 (d, 8.4)
Table 2 13C and 1H NMR data of rubrofusarin B (2) and
fonsecin (3) in CDCl3 (J in [Hz])
Number
2
3
δc
δH
δc
δH
2
167.4
-
100.0
-
2-CH3
20.6
2.35 (s)
27.6
1.60 (s)
2-OH
-
-
-
6.95 (brs)
3
107.3
5.98 (s)
47.6
3.14 (d, 16.8), 2.72 (d, 16. 4
184.2
-
197.5
-
4a
104.3
-
102.5
-
5
162.6
-
164.2
-
5-OH
-
14.96 (s)
-
14.19 (s)
5a
108.4
-
105.2
-
6
160.6
-
161.4
-
6-OCH3
56.0
3.99 (s)
55.6
3.84 (s)
7
97.2
6.38 (d, 2.2)
96.6
6.31 (brd, 1.1)
8
161.5
-
160.7
-
8-OH
-
-
-
10.18 (brs)
8-OCH3
55.4
3.91 (s)
-
-
9
97.8
6.56 (d, 2.2)
101.5
6.47 (s)
9a
141.0
-
142.9
-
10
101 0
6 94 (s)
101 0
6 41 (s) Table 1 13C and 1H NMR data of pyrophen (1) in CDCl3 (J
in [Hz])
Number
δc
δh
Number
δc
δh
2
164.7
-
7-NHCOCH3
170.3
-
3
88.0
5.43 (d, 2.2)
7-NHCOCH3
22.3
1.95 (s)
4
171.0
-
8
38.1
3.09 (m)
4-OCH3
55.7
3.73 (s)
1’
136.0
-
5
100.6
5.90 (d, 2.2)
2’,6’
128.6
7.16 (m)
6
161.9
-
3’,5’
128.2
7.25 (m)
7
52.3
4.98 (q, 7.8)
4’
126.5
7.21 (m)
7-NHCOCH3
-
7.79 (d, 8.4) Table 1 13C and 1H NMR data of pyrophen (1) in CDCl3 (J
in [Hz]) methine protons were visible, which were classified into
two m-coupled protons, two a-methines of the conse-
quent g -pyrones (δ 6.15, 6.08) and two singlet methines
(δ 7.35 and 7.24), together with six methyls, among
them four methoxy signals. 3. Results and discussion Based on these data and
search in literature, compound 6 was identified as auras-
perone A [22] (see Additional file 5) Compound 7 exhibited a close structural similarity
with fonsecin B (5); the molecular weight was determined
as 606 Da, corresponding to the molecular formula
C32H30O12 (HRESI MS). EI MS of 7 displayed an ion sig-
nal at m/z 570 as base peak, resulting from the expulsion
of two water molecules, affording the molecular weight
of aurasperone A (6). The 1H NMR spectrum established
a dimeric pattern of fonsecin B (5), where four sp2
methines protons being of two m-coupled protons and
two singlet methines; two methylene signals (δ 3.02 and
2.89) instead of the two a-methines of the consequent-g-
pyrones shown in 6, along with six methyls, among them
four methoxy signals and two sp3-bounded methyl signals
(δ 1.79, 1.46). In accordance, structure of 7 was assigned
as aurasperone B (7) [22]. (see Additional file 6) compound 3 was finally established as fonsecin (3) (see
Additional file 3). The facile loss of water by EI MS cor-
responded to the formation of TMC-256 A1 (4). Compound 5 displayed similar chromatographic prop-
erties and the same 1H NMR pattern as 3. The molecu-
lar weight of 5 was deduced as 304 Da, which is 14 amu
higher than that of 3, attributing to the methylation of
the phenolic hydroxyl group at 8-position, hence com-
pound 5 was identified as fonsecin B [22] (see Addi-
tional file 4) A third dimer 8 had a molecular weight of 592 Da
and a corresponding molecular formula C31H28O12. Three consecutive fragment ions (m/z 574, 556 and 525)
on EI MS corresponded to the expulsion of one H2O
molecule (to afford aurasperone F, 9), two H2O (dianhy-
droaurasperone C, 10) and 2H2O + OCH3, respectively. The 1H NMR spectrum displayed the same pattern as in
aurasperone B (7), except that the methoxy signal (δ
3.78) of 8-OCH3 in 7 was replaced by a phenolic hydro-
xyl group, pointing to aurasperone C (8) [23]. (see Addi-
tional file 7) 3. Results and discussion The expectation of a dimeric
was confirmed by 1H NMR spec
Table 1 13C and 1H NMR data of pyro
in [Hz])
Number
δc
δh
Numbe
2
164.7
-
7-NHCO
3
88.0
5.43 (d, 2.2)
7-NHCO
4
171.0
-
8
4-OCH3
55.7
3.73 (s)
1’
5
100.6
5.90 (d, 2.2)
2’,6’
6
161.9
-
3’,5’
7
52.3
4.98 (q, 7.8)
4’
7-NHCOCH3
-
7.79 (d, 8.4)
Table 2 13C and 1H NMR data of rubr
fonsecin (3) in CDCl3 (J in [Hz])
Number
2
3
δc
δH
δc
δH
2
167.4
-
100.0
-
2-CH3
20.6
2.35 (s)
27.6
1.6
2-OH
-
-
-
6.9
3
107.3
5.98 (s)
47.6
3.1
4
184.2
-
197.5
-
4a
104.3
-
102.5
-
5
162.6
-
164.2
-
5-OH
-
14.96 (s)
-
14
5a
108.4
-
105.2
-
6
160.6
-
161.4
-
6-OCH3
56.0
3.99 (s)
55.6
3.8
7
97.2
6.38 (d, 2.2)
96.6
6.3
8
161.5
-
160.7
-
8-OH
-
-
-
10
8-OCH3
55.4
3.91 (s)
-
-
9
97.8
6.56 (d, 2.2)
101.5
6.4
9a
141.0
-
142.9
-
10
101.0
6.94 (s)
101.0
6.4
10a
153.3
-
153.4
- compound 3 was finally established as fonsecin (3) (s
Additional file 3). The facile loss of water by EI MS co
responded to the formation of TMC-256 A1 (4). Compound 5 displayed similar chromatographic pro
erties and the same 1H NMR pattern as 3. The molec
lar weight of 5 was deduced as 304 Da, which is 14 am
higher than that of 3, attributing to the methylation
the phenolic hydroxyl group at 8-position, hence com
pound 5 was identified as fonsecin B [22] (see Add
tional file 4)
3.1. Aurasperones A-C and F
Compound 6 was obtained from fraction II as midd
polar yellow solid, displaying a molecular weight at m
570. 3. Results and discussion The fungal extract showed several UV absorbing (254
nm) yellow bands, exhibiting yellowish-green UV fluor-
escence at 366 nm. On spraying with anisaldehyde/sul-
phuric acid and heating they turned orange to dark red,
but showed no colour change with sodium hydroxide,
thus excluding peri-hydroxyquinones. O
NH
O
C
H3
H
O
C
H3
O
1
3
5
7
8
1'
4'
Figure 1 Selected HMBC (®) and H, H-COSY (bold lines)
correlations of pyrophen (1). The molecular formula of compound 1 was deter-
mined by HRMS as C16H17NO4; the 1H NMR spectrum
revealed signals for a phenyl residue, an amino NH
doublet, and two m-coupled methines (δ 5.90, 5.43). Further signals were a methine quartet, a methylene 2H
multiplet and two methyl singlets. The 13C NMR/
HMQC spectra indicated the existence of 16 carbons
corresponding to a phenyl residue, 2 up-field sp2
methines (δ100.6, 88.0), 4 quaternary sp2 atoms (δ171.0-
161.9), representing carbonyls or phenolic carbons, and
4 sp3 carbon signals (δ55.7-22.3). According to these
data, compound 1 was identified as pyrophen (1) [5],
which was isolated and reported previously from Asper-
gillus niger [18,19] and elucidated by crystal structure
analysis. Here, we report the full NMR assignments data Figure 1 Selected HMBC (®) and H, H-COSY (bold lines)
correlations of pyrophen (1). Figure 1 Selected HMBC (®) and H, H-COSY (bold lines)
correlations of pyrophen (1). Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Page 3 of 8 Page 3 of 8 compound 3 was finally established
Additional file 3). The facile loss of w
responded to the formation of TMC
Compound 5 displayed similar chr
erties and the same 1H NMR pattern
lar weight of 5 was deduced as 304 D
higher than that of 3, attributing to
the phenolic hydroxyl group at 8-p
pound 5 was identified as fonsecin
tional file 4)
3.1. Aurasperones A-C and F
Compound 6 was obtained from fr
polar yellow solid, displaying a mole
570. 3.1. Aurasperones A-C and F Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Page 4 of 8 O
O
OH
O
C
H3
O
CH3
C
H3
1
3
5
7
9
10
O
O
OH
O
C
H3
O
H
CH3
OH
1
3
5
7
9
10
Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). O
O
OH
O
C
H3
O
CH3
C
H3
1
3
5
7
9
10
Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). O
O
OH
O
C
H3
O
CH3
C
H3
1
3
5
7
9
10
Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). Figure 2 HMBC couplings in Rubrofusarin B (2) and Fonsecin (3). The optical rotations of the dimers had the same
negative sign and similar values indicating that the opti-
cal rotation value was dominated by the chiral axes
between the two naphthopyranone moieties (atropi-
somerism). The absolute configurations of dimeric
naphtho-g-pyrones have been determined by circular-
dichroism (CD). According to the literature [25], (S)-
configured dimeric naphtho-g-pyrones exhibit a first
positive Cotton Effect in the long-wavelength region, a
negative Cotton Effect at middle wavelength and then a
positive Cotton Effect at shorter one. In our experimen-
tal data, the CD spectra for three representative dimeric
naphtho-g-pyrones (6-8) showed closely related values
with pronounced Cotton Effects, recognizing them to
have the same patterns. In accordance, the ellipticity of
aurasperones A-C (6-8) showed three Cotton Effects,
one peak was shown firstly in the region of [θ]284-285
+359274-22843.4, then one trough between [θ]270-267
-151670-339938 and the last elliptical peak was shown
at [θ]227-219 +107899-5629. As the dimer 6 has no
further chiral elements, the chiral axis is dominating the
absolute configuration. Based on the revealed features
from the CD spectroscopic data, the four dimeric com-
pounds (6-9) have identical (S)-configurations around
their corresponding axis between C-10’ and C-7 (see
Additional file 9) studied compounds here showed weak cytotoxicity
(approx. 4-11%). 4.1. Sampling and isolation of the fungal strain 4.1. Sampling and isolation of the fungal strain
The reddish soft coral D. hemprichi was collected from
the Red Sea; approx. 30 km offshore from Safaga (east
Egypt) at a depth of approx. 30 m. Pieces of the coral
were rinsed three times with sterile seawater and then
aseptically cut into smaller pieces and shaken for 2 h. The aqueous supernatant was serially diluted, and each
200 μL were inoculated onto 15-cm Petri dishes, each
containing 50 mL of yeast extract/starch agar (yeast
extract 0.2 g/L, soluble starch 1.0 g/L, agar 20 g/L,
chloramphenicol 50 mg/L natural seawater at pH 6.0)
[7]. The black single colonies were picked from the
plates after inoculation for 25 days at 30°C and sub-
cultured on the same medium without chlorampheni-
col. The strain is deposited in the culture collection of
the Department of Microbial Chemistry, NRC, Cairo,
Egypt. 3.1. Aurasperones A-C and F Compound 6 was obtained from fraction II as middle
polar yellow solid, displaying a molecular weight at m/z
570. The expectation of a dimeric rubrofusarin B (2)
was confirmed by 1H NMR spectra, where six sp2 Table 2 13C and 1H NMR data of rubrofusarin B (2) and
fonsecin (3) in CDCl3 (J in [Hz])
Number
2
3
δc
δH
δc
δH
2
167.4
-
100.0
-
2-CH3
20.6
2.35 (s)
27.6
1.60 (s)
2-OH
-
-
-
6.95 (brs)
3
107.3
5.98 (s)
47.6
3.14 (d, 16.8), 2.72 (d, 16.8)
4
184.2
-
197.5
-
4a
104.3
-
102.5
-
5
162.6
-
164.2
-
5-OH
-
14.96 (s)
-
14.19 (s)
5a
108.4
-
105.2
-
6
160.6
-
161.4
-
6-OCH3
56.0
3.99 (s)
55.6
3.84 (s)
7
97.2
6.38 (d, 2.2)
96.6
6.31 (brd, 1.1)
8
161.5
-
160.7
-
8-OH
-
-
-
10.18 (brs)
8-OCH3
55.4
3.91 (s)
-
-
9
97.8
6.56 (d, 2.2)
101.5
6.47 (s)
9a
141.0
-
142.9
-
10
101.0
6.94 (s)
101.0
6.41 (s)
10a
153.3
-
153.4
- Table 2 13C and 1H NMR data of rubrofusarin B (2) and
fonsecin (3) in CDCl3 (J in [Hz]) Compound 9 was a fourth dimer with a molecular for-
mula C31H26O11; on EI MS, it displayed a fragment ion
at m/z 556 corresponding to an aromatized structural
analogue (dianhydroaurasperone C, 10), and a further
fragment at m/z 286 corresponded to rubrofusarin B
(2). The 1H NMR spectra displayed five sp2 methines (δ
6.87-6.08), one less than in 6, replaced by an AB signal
of a methylene group (δ 3.35-3.25). Accordingly, one of
the b-bounded methyls of the lactones was up-field
shifted (δ 1.65), while the other one was retained at δ
2.16 as in 6. In contrast to 6, only three methoxy signals
(δ 3.95-3.43) were visible, while the fourth one was
replaced by a phenolic OH. Based on these spectro-
scopic features, structure 9 was confirmed as aurasper-
one F [24] (see Additional file 8) The four dimeric naphtho-g-pyrones (6-9) were pre-
sently constructed from two naphtho-g-pyrone units,
which are not symmetrically linked; i.e. the first pyrone
(above) unit is linked via a middle aromatic moiety (10’-
position) to a terminal aromatic residue (7-position) of
the second pyrone (down) unit. Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. 4. Experimental The NMR spectra were measured on a Bruker AMX
300 (300.135 MHz), a Varian Unity 300 (300.145
MHz) and Varian Inova 500 (499.876 MHz) spectro-
meters. EI mass spectra were recorded on a Finnigan
MAT 95 spectrometer (70 eV). ESI MS was recorded
on a Finnigan LCQ with quaternary pump Rheos 4000
(Flux Instrument). HRMS were recorded by ESI MS on
an Apex IV 7 Tesla Fourier-Transform Ion Cyclotron
Resonance Mass Spectrometer (Bruker Daltonics, Bill-
erica, MA, USA). Optical rotation was measured on a
Perkin-Elmer Polarimeter, model 343. Flash chromato-
graphy was carried out on silica gel (230-400 mesh). Rf
values were measured on Polygram SIL G/UV254
(Macherey-Nagel & Co., Düren, Germany). Size exclu-
sion chromatography was done on Sephadex LH-20
(Lipophilic Sephadex, Amersham Biosciences Ltd.; pur-
chased from Sigma-Aldrich
Chemie, Steinheim,
Germany). Pyrophen (1)
l
l Colourless semisolid, UV-absorbing, no colour reaction
on spraying with anisaldehyde/sulphuric acid; Rf = 0.86
(CH2Cl2/5% MeOH); 1H NMR (300 MHz, CDCl3) and
13C NMR (125 MHz, CDCl3) see Table 1; EI MS m/z
(%) = 287.2 ([M]+, 28), 228.1 (8), 196.1 (40), 154.2 (100),
125.1 (16), 111.1 (6), 91.1 (12), 43.1 (11); (+)-ESI MS
m/z (%) = 596.9 ([2M+Na]+, 85), 310 [M+Na]+, 36), 288
([M+H]+, 100); (-)-ESI MS m/z 286 [M+H]-; (+)-HRESI
MS m/z 288.12301 ([M+H]+, calcd: 288.12303 for
C16H18NO4); 310.10490 ([M+Na]+, calcd: 310.10497 for
C16H17NO4Na). Table 4 Antimicrobial activities of the fugal extract (60
μg/disc (5-mm diameter)
Test organism
Extract activity (mm)
P. aeruginosa
17
S. aureus
23
C. albicans
20
A. niger
ND
ND, not detected. Table 4 Antimicrobial activities of the fugal extract (60
μg/disc (5-mm diameter)
Test organism
Extract activity (mm)
P. aeruginosa
17
S. aureus
23
C. albicans
20
A. niger
ND
ND, not detected. 3.2. Biological activities The antibiotic activity of compounds 1-8 was exam-
ined against 11 microbial test organisms using the agar
diffusion method (40 μg/disc) (Table 3). According to
the antimicrobial assay, the crude extract of the fungal
strain exhibited high activity against bacteria and
yeasts (Table 4). Nevertheless, only three of the iso-
lated metabolites were found to exhibited activity: pyr-
ophen (1) and rubrofusarin B (2) displayed high (28
mm) and moderate (12 mm) activity against C. albi-
cans, respectively, while aurosperone A (6) was active
(13 mm) against the plant pathogenic fungi, Rhizocto-
nia solani. In the brine shrimp assay (10 μg/mL), all Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Page 5 of 8 Page 5 of 8 Table 3 Antimicrobial (40 μg/disc (∅9 mm; [mm]) and cytotoxic (10 μg/mL) activities of compounds 1-8
Compound number
BSa
SAb
SVc
ECd
CAe
MMf
CVg
CSh
SSi
PSj
PUk
Brine shrimp
1
ND
ND
ND
ND
28
ND
ND
ND
ND
ND
ND
4.2%
2
ND
ND
ND
ND
12
ND
ND
ND
ND
ND
ND
11%
3
ND
ND
ND
ND
ND
ND
ND
ND
ND
13
ND
Nt
4
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8.8%
5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
5.0%
6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
9.7%
7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6.4%
8
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
9.7%
aBacillus subtilis, bS. aureus, cStreptomyces viridochromogenes (Tü 57), dEscherichia coli, eC. albicans, fMucor miehi, gChlorella vulgaris, hChlorella sorokiniana,
iScenedesmus subspicatus, jR. solani; kPythium ultimum
ND, not detected. microbial (40 μg/disc (∅9 mm; [mm]) and cytotoxic (10 μg/mL) activities of compounds 1-8
b
d
f
h
i
j
k /disc (∅9 mm; [mm]) and cytotoxic (10 μg/mL) activities of compounds 1-8 aBacillus subtilis, bS. aureus, cStreptomyces viridochromogenes (Tü 57), dEscherichia coli, eC. albicans, fMucor miehi, gChlorella vulgaris, hChlorella sorokiniana,
iScenedesmus subspicatus, jR. solani; kPythium ultimum
ND, not detected. and they were combined therefore, and concentrated in
vacuo, affording 5.5 g as black crude extract. 4.3. Isolation of the active constituents The obtained extract was applied to column chromato-
graphy on silica gel eluted by CH2Cl2-MeOH gradient
and monitored by TLC to afford five fractions: I (0.62 g),
II (1.21 g), III (0.71 g), IV (1.52 g) and V (0.22 g). Fraction
I was re-purified on silica gel column (DCM) followed by
Sephadex LH-20 (DCM/40% MeOH) to afford a colour-
less semisolid of phyrophen (1) (468.0 mg). Application
of Fraction II to PTLC (DCM/3% MeOH) followed by
purification on Sephadex LH-20 (DCM/40% MeOH) lead
to isolation of two yellow solids of rubrofusarin B (2, 11.0
mg) and aurosperone A (6, 13.0 mg), respectively. Frac-
tion III was purified using a silica gel column (DCM-
MeOH) followed by Sephadex LH-20 (DCM/40%
MeOH) to give a yellow solid of aurasperone F (9, 15.0
mg). Purification of the middle polar fraction IV via
PTLC (DCM/5% MeOH) followed by Sephadex LH-20
(MeOH) yielded three yellow solids of aurasperone B (7,
8.0 mg), aurasperone C (8, 14.0 mg) and fonsecin (3, 11.5
mg). As the same for IV, the polar fraction V afforded
three yellow solids of fonsecin B (5, 12.0 mg), aurasper-
one B (7, 3.4 mg) and aurasperone C (8, 4.1 mg). The well-grown agar slants of the fungal strain D2006
were served to inoculate 60 of 1-L Erlenmeyer flasks,
each containing 300-mL of GYMP medium (g/L): malt
extract (3), yeast extract (3), peptone (5), glucose (10),
agar (20) and 1000 mL of 100% seawater at pH approx. 6.5. The inoculated media was applied to additional cul-
tivation using a rotary shaker (150 rpm) for 10 days. After harvesting, the obtained black culture broth was
mixed with celite (approx. 1.5 kg) and then filtered in
vacuo. The afforded two phases, filtrate and mycelium,
were applied to exhaustive extraction by ethyl acetate. TLC of both organic extracts recognized their unique, 4.2. Fermentation and working up The well-grown single colonies of A. alternata were
inoculated in subculture agar slants containing malt
extract medium: malt extract (30 g/L), peptone 5 g/L),
agar (20 g/L), natural sea water (1000 mL); at pH
approx. 5.5 for 7 days at 30°C). The obtained grown
agar slants were served to inoculate 500-mL Erlenmeyer
flasks, each containing 100 mL of GYMP medium (g/L):
malt extract (3), yeast extract (3), peptone (5), glucose
(10) and 1000 mL natural seawater at pH approx. 6.5 at
30°C. The culture media was in turn applied to cultiva-
tion on a rotary shaker (10 days). After harvesting, the
afforded black broth was centrifuged (7,000 rpm for 15
min), and the obtained two phases, mycelial cake and
supernatant, were individually extracted with ethyl acet-
ate. The obtained unique black organic extracts were
applied to biological and chemical screenings. Aurasperone A (6) Yellow solid, UV-green fluorescence (365 nm), turned
orange with anisaldehyde/sulphuric acid; Rf = 0.82
(CH2Cl2/5% MeOH); [a]D
20 = -18.9 (c = 0.19, MeOH);
CD (c 1.1929 × 10-5 mol/L [c 6.8 μg/mL], MeOH) [θ]400
0 [θ]284 +22843, [θ]270 -36396, [θ]219 +5629; 1H NMR
(300 MHz, CD3OD) δ = 7.35 (s, 1H, H-10), 7.25 (s, 1H,
H-9), 6.51 (brd, 1H, J ~ 1.1 Hz, H-7’), 6.23 (brd, 1H, J =
1.1 Hz, H-7), 6.15 (s, 1H, H-3), 6.08 (s, 1H, H-3’), 3.95
(s, 3H, 6’-OCH3), 3.79 (s, 3H, 8-OCH3), 3.59 (s, 3H, 8’-
OCH3), 3.46 (s, 3H, 6-OCH3), 2.42 (s, 3H, 2-CH3), 2.13
(s, 3H, 2’-CH3); EI MS m/z (%) = 570.5 ([M]+, 44),
539.5 ([M-OCH3]+, 10), 513.4 (5), 286 (7), 167.2 (7),
145.2 (44), 116.2 (100), 85.1 (39), 55.1 (22), 43.1 (24). Aurasperone B (7) Aurasperone F (9) Yellow solid, UV-green fluorescence (365 nm), turned
orange with anisaldehyde/sulphuric acid; Rf = 0.55
(CH2Cl2/5% MeOH); 1H NMR (300 MHz, CD3OD) δ =
6.87 (s, 1H, H-10), 6.55 (s, 1H, H-9), 6.51 (d, 1H, J ~ 1.1
Hz, H-9’), 6.36 (brd, 1H, J ~ 1.1 Hz, H-7’), 6.08 (s, 1H,
H-3’), 3.95 (s, 3H, 6’-OCH3), 3.63 (s, 3H, 8’-OCH3), 3.43
(s, 3H, 6-OCH3), 3.35-3.25, (m, 2H, 3-H2), 2.16 (s, 3H,
2’-CH3), 1.65 (s, 3H, 2-CH3); EI MS m/z (%) = 556.5
([M-H2O]+, 5), 286.3 ([rubrofusarin B (2)]+, 8), 84.1
(12), 57.2 (10), 44.1 (100); (+)-ESI MS m/z (%) = 1172
([2M+Na+H]+, 19), 575 ([M+H]+, 100); (-)-ESI MS m/z
(%) = 1721 ([3M-H]-, 31), 1147 ([2M-H]-, 22), 573 ([M-
H]-, 100). 16
15
6)
Fonsecin B (5) Yellow solid, UV-green fluorescence (365 nm), turned
dark red with anisaldehyde/sulphuric acid; Rf = 0.44
(CH2Cl2/5% MeOH); 1H NMR (300 MHz, DMSO-d6) δ
= 14.09 (brs, 1H, 5-OH), 7.00 (brs, 1H, 2-OH), 6.68
(brd, 1H, J ~ 1.1 Hz, H-9), 6.55 (s, 1H, H-10), 6.38 (brd,
1H, J ~ 1.1 Hz, H-9), 3.84 (s, 6H, 6,8-OCH3), 3.14 (d,
1H, J ~ 16.8 Hz, H-3a), 2.72 (d, 1H, J ~ 16.8 Hz, H-3b),
1.61 (s, 3H, 2-CH3); EI MS m/z (%) = 304.3 ([M]+, 56),
286.3 ([M-H2O]+, 8), 262.3 (8), 247.2 (28), 246.2 (60),
220.2 (20), 218.2 (10), 149.2 (20), 145.2 (34), 127.2 (12),
116.2 (64), 101.2 (48), 84.1 (36), 66.1 (24), 59.1 (63),
43.1 (100). Fonsecin (3) Yellow solid, UV-green fluorescence (365 nm), turned
dark red with anisaldehyde/sulphuric acid; Rf = 0.38
(CH2Cl2/5% MeOH); 1H NMR (300 MHz, CDCl3) and
13C NMR (125 MHz, CDCl3) see Table 2; EI MS m/z
(%) = 290.2 ([M]+, 24), 272.2 ([M-H2O]+, 16), 243.2 (8),
232.1 (21), 189.1 (7), 175.1 (16), 101.1 (15), 85.1 (22),
59.1 (36), 43.1 (100); (+)-ESI MS m/z (%) = 291 ([M+H]
+); (-)-ESI MS m/z (%) = 289 ([M-H]-); (+)-HRESI MS
m/z
291.08631
([M+H]+,
calcd:
291.08631
for
C16H15O6). Yellow solid, UV-green fluorescence (365 nm), turned
orange with anisaldehyde/sulphuric acid; Rf = 0.26
(CH2Cl2/5% MeOH); [a]D
20 = -33.5 (c = 0.17, MeOH);
CD (c 4.29 × 10-5 mol/L [c 24 μg/mL], MeOH) [θ]400 0,
[θ]285 +359273, [θ]268 -339938, [θ]226 +107899.87; 1H
NMR (300 MHz, CD3OD): δ = 6.84 (s, 1H, H-10), 6.57
(s, 1H, H-9), 6.38 (d, 1H, J ~ 1.2 Hz, H-9’), 6.20 (d, 1H,
J ~ 1.2 Hz, H-7’), 3.93 (s, 3H, 6’-OCH3), 3.60 (s, 3H, 8’-
OCH3), 3.50 (s, 3H, 6-OCH3), 3.30-3.29 (m, 4H, 3,3’-
H2), 1.69 (s, 3H, 2-CH3), 1.49 (s, 3H, 2’-CH3); EI MS m/
z (%) = 574.3 ([M-H2O]+, 6), 556.3 ([M-2H2O]+, 42),
525.3 ([M-(2H2O+OCH3)]+, 32), 264.2 (7) 58.2 (28), 43.1
(100); (+)-HRESI MS m/z 615.14779 ([M+Na]+, calcd:
615.14729 for C31H28O12Na), m/z 593.16570 ([M+H]+,
calcd: 593.16534 for C31H29O12). Rubrofusarin B (2) Yellow solid, UV-green fluorescence (365 nm), orange
with anisaldehyde/sulphuric acid; Rf = 0.78 (CH2Cl2/5% Page 6 of 8 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 Hz, H-9’), 3.99 (s, 3H, 6’-OCH3), 3.78 (s, 3H, 8-OCH3),
3.63 (s, 3H, 8’-OCH3), 3.39 (s, 3H, 6-OCH3), 3.02, (d,
2H, J ~ 16.3 Hz, 3-H2), 2.89 (m, 2H, 3’-H2), 1.79 (s, 3H,
2-CH3), 1.46 (s, 3H, 2’-CH3); EI MS m/z (%) = 570.3
([M-2H2O]+, 100), 539.4 ([M-(2H2O+OCH3)]+, 74),
524.3 (5), 299.2 (12), 272.2 (13), 269.7 (24), 230.2 (18),
193.1 (12), 154.2 (14), 149.1 (19), 130.1 (48), 91.1 (54),
57.1 (30), 43.1 (57); (+)-HRESI MS m/z 607.18100 ([M
+H]+, calcd: 607.18100 for C32H31O12), m/z 629.16294
([M+Na]+, calcd: 629.16295 for C32H30O12Na). Aurasperone C (8) MeOH); 1H NMR (300 MHz, CDCl3) and 13C NMR
(125 MHz, CDCl3) see Table 2; EI MS m/z (%) = 286.2
([M]+., 100), 268.1 ([M-H2O]+, 12), 257.2 ([M-CHO]+,
44), 240.2 (8) 213.2 (5), 43.1 (7); (+)-ESI MS m/z (%) =
594.9 ([2M+Na]+, 14), 287 ([M+H]+, 100); (+)-HRESI
MS m/z 287.09137 ([M+H]+, calcd: 287.09139 for
C16H15O5). References 1. DWHO Report (2006) Global tuberculosis control: surveillance, planning,
financing E. World Health Organization, Geneva p 1 1. DWHO Report (2006) Global tuberculosis control: surveillance, planning,
financing E. World Health Organization, Geneva p 1 2. Alland D, Kalkut GE, Moss AR, McAdam RA, Hahn JA, Bosworth W,
Drucker E, Bloom BR (1994) Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic
methods. N Engl J Med 330(24):1710–1716. doi:10.1056/
NEJM199406163302403. 3. Whalen C, Horsburgh CR, Hom D, Lahart C, Simberkoff M, Ellner J (1995)
Accelerated course of human immunodeficiency virus infection after
tuberculosis. Am J Respir Crit Care Med 151(1):129–135 Acknowledgements g
The authors are deeply thankful to Prof. H. Laatsch for his Lab facilities and
unlimited support. We are appreciated greatly R. Machinek for the NMR
spectra, Dr. H. Frauendorf for the mass measurements, F. Lissy for biological
activity tests and A. Kohl for technical assistance. Dr. Mohamed Shaaban is
deeply thankful to the DAAD offices in Cairo and Bonn for kindly financing
of the project during the visiting period in Germany. Brine shrimp microwell cytotoxicity assay Supplementary Information accompanying this paper includes MS, NMR and
CD spectra. The cytotoxic assay was performed according to Taka-
hashi et al. [27] and Sajid et al. [28]. Author details
1 1Chemistry of Natural Compounds Department, Pharmaceutical Industries
Division, National Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt
2Institute of Organic and Biomolecular Chemistry, University of Göttingen,
Tammannstrasse 2, D-37077 Göttingen, Germany 3Department of Microbial
Chemistry, Genetic Engineering and Biotechnology Division, National
Research Centre, El-Behoos St., Dokki-Cairo 12622, Egypt 4.4. Biological activities
Antimicrobial activity Compounds 1-8 were dissolved in CH2Cl2/10% MeOH
at a concentration of 1 mg/mL. Aliquots of 40 μL were
soaked on filter paper discs (9 mm ∅, no. 2668, Schlei-
cher & Schüll GmbH, Germany) and dried for 1 h at
room temperature under sterilized conditions. The
paper discs were placed on inoculated agar plats and
incubated for 24 h at 38°C for bacterial and 48 h (30°C)
for the fungal isolates, while the algal test strains were
incubated at room temperature in day light. Yellow solid, UV-green fluorescence (365 nm), turned
orange with anisaldehyde/sulphuric acid; Rf = 0.48
(CH2Cl2/5% MeOH); [a]D
20 = -18.3 (c = 0.12, MeOH);
CD (c 2.83 × 10-5 mol/L [c 17.2 μg/mL], MeOH) [θ]400
0, [θ]284 +143232, [θ]267 -151670, [θ]227 +46610; 1H
NMR (300 MHz, CDCl3) δ = 14.51 (brs, 1H, 5’-OH),
14.08 (brs, 1H, 5-OH), 6.84 (s, 1H, H-9), 6.72 (s, 1H, H-
9), 6.37 (d, 1H, J ~ 1.1 Hz, H-7’), 6.14 (d, 1H, J ~ 1.1 For the fungal extract examination, representative test
microbes; P.aeruginosa, S. aureus, C. albicans and A. Page 7 of 8 Page 7 of 8 Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 niger were used. Both bacterial and yeast strains were
grown on nutrient agar medium (g/L): Beef extract 3;
peptone, 10; and agar, 20. The pH was adjusted to 7.2. The fungal strain was grown on Czapek-Dox medium
(g/l): Sucrose, 30; NaNO3, 3; MgSO4.7H2O, 0.5l; KCl,
0.5; FeSO4, 0.01; K2HPO4, 1; and agar, 20. The pH was
maintained at 6.0. The disc diffusion test has been done
according to Collins and Lyne [26]. Filter paper discs (5
mm diameter) were saturated with 200 μg from the cul-
ture extract, and located on the surface of the agar
plates (150 mm diameter containing 50 mL of solidified
media). The paper discs were placed on inoculated agar
plats and incubated for 24 h at 38°C (bacteria and yeast)
and 48 h at 30°C (fungi). Additional file 7: Spectral data of Aurasperone C (8). Four charts
(chart 40-43) containing the mass (ESI, HRESI MS) and NMR (1HNMR)
spectral data of Aurasperone C (8) Additional file 7: Spectral data of Aurasperone C (8). Four charts
(chart 40-43) containing the mass (ESI, HRESI MS) and NMR (1HNMR)
spectral data of Aurasperone C (8) Additional file 8: Spectral data of Aurasperone F (9). 5. Conclusions In this research article, eight bioactive pyrone derivatives
were identified from the culture of A. alternata strain
D2006, isolated from the marine soft coral D. hemprichi. Selection of the strain was based on its profound anti-
biotic and antimicrobial activities. Structures of the iso-
lated compounds were identified on the basis of 1D and
2D NMR spectroscopy and mass (EI, ESI, HRESI) data,
and by comparison with the literature. Configuration of
the four dimeric naphtha-g-pyrones 6-9 was analyzed by
CD spectra, exhibiting an identical stereochemistry. The
biological activity (antimicrobial and cytotoxicity) of the
fungal extract and its corresponding isolated compounds
were comparatively studied. This is as a trial to find out
new leading drugs to overcome some of the recently dis-
covered diseases. 4.4. Biological activities
Antimicrobial activity Three charts
(chart 44-46) containing the mass (ESI, EI MS) and NMR (1HNMR) spectral
data of Aurasperone F (9) Additional file 9: CD Spectra of Aurasperones A-C (6-8). Three charts
(chart 47-49) containing the CD spectral data of Aurasperones A-C (6-8). Additional file 9: CD Spectra of Aurasperones A-C (6-8). Three charts
(chart 47-49) containing the CD spectral data of Aurasperones A-C (6-8). Additional file 9: CD Spectra of Aurasperones A-C (6-8). Three charts
(chart 47-49) containing the CD spectral data of Aurasperones A-C (6-8). Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 21 October 2011 Accepted: 29 February 2012
Published: 29 February 2012 Additional material p
4. Zumla A, Grange J (1998) Tuberculosis. BMJ 316(719):1962–1964 5. Laatsch H AntiBase, a data base for rapid structural determination of
microbial natural products, and annual updates, chemical concepts,
Weinheim, Germany.http://wwwuser.gwdg.de/~hlaatsc/antibase.htm Additional file 1: Spectral data of Pyrophen (1). Ten charts (chart 1-
10) containing the mass (ESI, HRESI, EI MS) and NMR (1HNMR, 13CNMR, H,
H COSY, HMQC, HSQC, HMBC) spectral data of Pyrophen (1). 6. Laatsch H (2006) Marine bacterial metabolites. In: Proksch P, Müller WEG
(ed) Frontiers in marine biotechnology. Horizon Bioscience, Norfolk, UK pp
225–288. ISBN 1-904933-18-1 Additional file 2: Spectral data of Rubrofusarin B (2). Thirteen charts
(chart 11-23) containing the mass (ESI, EI, HRESI MS) and NMR (1HNMR,
13CNMR, H, H COSY, HMQC, HSQC, HMBC) spectral data of Rubrofusarin B
(2). 7. Shiono Y, Tsuchinari M, Shimanuki K, Miyajima T, Murayama T, Koseki T,
Laatsch H, Takanami K, Suzuki K (2007) Fusaristatins A and B, two new
cyclic lipopeptides from an endophytic Fusarium sp. J Antibiot
60(5):309–316. doi:10.1038/ja.2007.39. Additional file 3: Spectral data of Fonsecin (3). Nine charts (chart 24-
32) containing the mass (ESI, EI MS) and NMR (1HNMR, 13CNMR, H, H
COSY, HMQC, HSQC, HMBC) spectral data of Fonsecin (3). 8. Al-Zereini W, Schuhmann I, Laatsch H, Helmke E, Anke H (2007) New
aromatic nitro compounds from Salegentibacter sp. T436, an Arctic sea ice
bacterium. Taxonomy, fermentation, isolation and biological activities. J
Antibiot 60(5):301–308. doi:10.1038/ja.2007.38. 8. Al-Zereini W, Schuhmann I, Laatsch H, Helmke E, Anke H (2007) New
aromatic nitro compounds from Salegentibacter sp. T436, an Arctic sea ice
bacterium. Taxonomy, fermentation, isolation and biological activities. J
Antibiot 60(5):301–308. doi:10.1038/ja.2007.38. Additional file 4: Spectral data of Fonsecin B (5). Two charts (chart
33-34) containing the mass (EI MS) and NMR (1HNMR) spectral data of
Fonsecin B (5). 9. Namikoshi M, Akano K, Kobayashi H, Koike Y, Kitazawa A, Rondonuwu AB,
Pratasik SB (2002) Distribution of marine filamentous fungi associated with
marine sponges in coral reefs of Palau and Bunaken Island, Indonesia. J
Tokyo Univ Fish 88:15–20 Additional file 5: Spectral data of Aurasperone A (6). Two charts
(chart 35-36) containing the mass (EI MS) and NMR (1HNMR) spectral
data of Aurasperone A (6) 10. Lange L (1996) Microbial metabolites–an infinite source of novel chemistry. Pure Appl Chem 68(2):745–748 Additional file 6: Spectral data of Aurasperone B (7). Shaaban et al. Organic and Medicinal Chemistry Letters 2012, 2:6
http://www.orgmedchemlett.com/content/2/1/6 12. Abdelazim A (2004) Secondary metabolites of marine-derived fungi: natural
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University, Bonn, Germany 12. Abdelazim A (2004) Secondary metabolites of marine-derived fungi: natural
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University, Bonn, Germany 12. Abdelazim A (2004) Secondary metabolites of marine-derived fungi: natural
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University, Bonn, Germany 13. Feofilova EP (2001) The kingdom fungi: heterogeneity of physiological and
biochemical properties and relationships with plants, animals, and
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prokaryotes (Review). Appl Biochem Microbiol 37(2):124–137. (Translated
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and antibacterial activities of indigenous Streptomyces isolates from saline
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journal and benefi t from:
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7 Rigorous peer review
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7 Open access: articles freely available online
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7 Retaining the copyright to your article
Submit your next manuscript at 7 springeropen.com doi:10.1186/2191-2858-2-6
Cite this article as: Shaaban et al.: Seven naphtho-g-pyrones from the
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biological properties. Organic and Medicinal Chemistry Letters 2012 2:6. Submit your manuscript to a
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https://link.springer.com/content/pdf/10.1007/JHEP10(2018)047.pdf
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English
| null |
Angular analysis of B0 d → K∗μ+μ− decays in pp collisions at $$ \sqrt{s}=8 $$ TeV with the ATLAS detector
|
The Journal of high energy physics/The journal of high energy physics
| 2,018
|
cc-by
| 27,311
|
Received: May 11, 2018
Revised: September 5, 2018
Accepted: September 27, 2018
Published: October 8, 2018 Received: May 11, 2018
Revised: September 5, 2018
Accepted: September 27, 2018
Published: October 8, 2018 JHEP10(2018)047 The ATLAS collaboration Abstract: An angular analysis of the decay B0
d →K∗µ+µ−is presented, based on proton-
proton collision data recorded by the ATLAS experiment at the LHC. The study is us-
ing 20.3 fb−1 of integrated luminosity collected during 2012 at centre-of-mass energy of
√s = 8 TeV. Measurements of the K∗longitudinal polarisation fraction and a set of angu-
lar parameters obtained for this decay are presented. The results are compatible with the
Standard Model predictions. Keywords: Hadron-Hadron scattering (experiments) ArXiv ePrint: 1805.04000 https://doi.org/10.1007/JHEP10(2018)047 Contents
1
Introduction
1
2
Analysis method
2
3
The ATLAS detector, data, and Monte Carlo samples
4
4
Event selection
4
5
Maximum-likelihood fit
6
5.1
Signal model
7
5.2
Background modes
8
5.3
K∗cc control sample fits
10
5.4
Fitting procedure and validation
11
6
Results
11
7
Systematic uncertainties
18
8
Comparison with theoretical computations
21
9
Conclusion
23
A Correlation matrices
24
The ATLAS collaboration
30 Contents
1
Introduction
1
2
Analysis method
2
3
The ATLAS detector, data, and Monte Carlo samples
4
4
Event selection
4
5
Maximum-likelihood fit
6
5.1
Signal model
7
5.2
Background modes
8
5.3
K∗cc control sample fits
10
5.4
Fitting procedure and validation
11
6
Results
11
7
Systematic uncertainties
18
8
Comparison with theoretical computations
21
9
Conclusion
23
A Correlation matrices
24
The ATLAS collaboration
30 JHEP10(2018)047 1
Introduction Flavour-changing neutral currents (FCNC) have played a significant role in the construction
of the Standard Model of particle physics (SM). These processes are forbidden at tree level
and can proceed only via loops, hence are rare. An important set of FCNC processes involve
the transition of a b-quark to an sµ+µ−final state mediated by electroweak box and penguin
diagrams. If heavy new particles exist, they may contribute to FCNC decay amplitudes,
affecting the measurement of observables related to the decay under study. Hence FCNC
processes allow searches for contributions from sources of physics beyond the SM (hereafter
referred to as new physics). This analysis focuses on the decay B0
d →K∗0(892)µ+µ−, where
K∗0(892) →K+π−. Hereafter, the K∗0(892) is referred to as K∗and charge conjugation
is implied throughout, unless stated otherwise. In addition to angular observables such – 1 – as the forward-backward asymmetry AFB,1 there is considerable interest in measurements
of the charge asymmetry, differential branching fraction, isospin asymmetry, and ratio of
rates of decay into dimuon and dielectron final states, all as a function of the invariant
mass squared of the dilepton system q2. All of these observable sets can be sensitive to
different types of new physics that allow for FCNCs at tree or loop level. The BaBar, Belle,
CDF, CMS, and LHCb collaborations have published the results of studies of the angular
distributions for B0
d →K∗µ+µ−[1–8]. The LHCb Collaboration has reported a potential
hint, at the level of 3.4 standard deviations, of a deviation from SM calculations [3, 4]
in this decay mode when using a parameterization of the angular distribution designed
to minimise uncertainties from hadronic form factors. Measurements using this approach
were also reported by the Belle and CMS Collaborations [6, 8] and they are consistent
with the LHCb experiment’s results and with the SM calculations. This paper presents
results following the methodology outlined in ref. [3] and the convention adopted by the
LHCb Collaboration for the definition of angular observables described in ref. [9]. The
results obtained here are compared with theoretical predictions that use the form factors
computed in ref. [10]. JHEP10(2018)047 This article presents the results of an angular analysis of the decay B0
d →K∗µ+µ−
with the ATLAS detector, using 20.3 fb−1 of pp collision data at a centre-of-mass energy
√s = 8 TeV delivered by the Large Hadron Collider (LHC) [11] during 2012. 1The forward-backward asymmetry is given by the normalised difference between the number of positive
muons going in the forward and backward directions with respect to the direction opposite to B0
d momentum
in the dimuon rest frame. 1
Introduction Results are
presented in six different bins of q2 in the range 0.04 to 6.0 GeV2, where three of these bins
overlap. Backgrounds, including a radiative tail from B0
d →K∗J/ψ events, increase for q2
above 6.0 GeV2, and for this reason, data above this value are not studied. The operator product expansion used to describe the decay B0
d →K∗µ+µ−encodes
short-distance contributions in terms of Wilson coefficients and long-distance contributions
in terms of operators [12]. Global fits for Wilson coefficients have been performed using
measurements of B0
d →K∗µ+µ−and other rare processes. Such studies aim to connect
deviations from the SM predictions in several processes to identify a consistent pattern
hinting at the structure of a potential underlying new-physics Lagrangian, see refs. [13–15]. The parameters presented in this article can be used as inputs to these global fits. 2This equation neglects possible Kπ S-wave contributions.
The effect of an S-wave contribution is
considered following the method used by LHCb in ref. [3]. 2
Analysis method Three angular variables describing the decay are defined according to convention described
by the LHCb Collaboration in ref. [9]: the angle between the K+ and the direction opposite
to the B0
d in the K∗centre-of-mass frame (θK); the angle between the µ+ and the direction
opposite to the B0
d in the dimuon centre-of-mass frame (θL); and the angle between the two
decay planes formed by the Kπ and the dimuon systems in the B0
d rest frame (φ). For B
0
d
mesons the definitions are given with respect to the negatively charged particles. Figure 1
illustrates the angles used. 1The forward-backward asymmetry is given by the normalised difference between the number of positive
muons going in the forward and backward directions with respect to the direction opposite to B0
d momentum
in the dimuon rest frame. – 2 – φ
B0
d
µ+
µ−
K+
π−
θL
θK
Figure 1. An illustration of the B0
d →K∗µ+µ−decay showing the angles θK, θL and φ defined
in the text. Angles are computed in the rest frame of the K∗, dimuon system and B0
d meson,
respectively. Figure 1. An illustration of the B0
d →K∗µ+µ−decay showing the angles θK, θL and φ defined
in the text. Angles are computed in the rest frame of the K∗, dimuon system and B0
d meson,
respectively. Figure 1. An illustration of the B0
d →K∗µ+µ−decay showing the angles θK, θL and φ defined
in the text. Angles are computed in the rest frame of the K∗, dimuon system and B0
d meson,
respectively. JHEP10(2018)047 The angular differential decay rate for B0
d →K∗µ+µ−is a function of q2, cos θK, cos θL
and φ, and can be written in several ways [16]. The form to express the differential decay
amplitude as a function of the angular parameters uses coefficients that may be represented
by the helicity or transversity amplitudes [17] and is written as2 1
dΓ/dq2
d4Γ
dcosθLdcosθKdφdq2 =
9
32π
"
3(1−FL)
4
sin2 θK +FL cos2 θK + 1−FL
4
sin2 θK cos2θL
−FL cos2 θK cos2θL+S3 sin2 θK sin2 θL cos2φ
+S4 sin2θK sin2θL cosφ+S5 sin2θK sinθL cosφ
+S6 sin2 θK cosθL+S7 sin2θK sinθL sinφ
+S8 sin2θK sin2θL sinφ+S9 sin2 θK sin2 θL sin2φ
#
. 3ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in
the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre
of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r, Φ) are used in the transverse
plane, Φ being the azimuthal angle around the z-axis. The pseudorapidity is defined in terms of the polar
angle θ as η = −ln tan(θ/2). 3
The ATLAS detector, data, and Monte Carlo samples The ATLAS experiment at the LHC is a general-purpose detector with a cylindrical ge-
ometry and nearly 4π coverage in solid angle [19]. It consists of an inner detector (ID)
for tracking, a calorimeter system and a muon spectrometer (MS). The ID consists of
silicon pixel and strip detectors, with a straw-tube transition radiation tracker providing
additional information for tracks passing through the central region of the detector.3 The
ID has a coverage of |η| < 2.5, and is immersed in a 2T axial magnetic field generated
by a superconducting solenoid. The calorimeter system, consisting of liquid argon and
scintillator-tile sampling calorimeter subsystems, surrounds the ID. The outermost part of
the detector is the MS, which employs several detector technologies in order to provide
muon identification and a muon trigger. A toroidal magnet system is embedded in the MS. The ID, calorimeter system and MS have full azimuthal coverage. JHEP10(2018)047 The data analysed here were recorded in 2012 during Run 1 of the LHC. The centre-
of-mass energy of the pp system was √s = 8 TeV. After applying data-quality criteria, the
data sample analysed corresponds to an integrated luminosity of 20.3 fb−1. A number of
Monte Carlo (MC) signal and background event samples were generated, with b-hadron
production in pp collisions simulated with Pythia 8.186 [20, 21]. The AU2 set of tuned
parameters [22] is used together with the CTEQ6L1 PDF set [23]. The EvtGen 1.2.0
program [24] is used for the properties of b- and c-hadron decays. The simulation included
modelling of multiple interactions per pp bunch crossing in the LHC with Pythia soft
QCD processes. The simulated events were then passed through the full ATLAS detector
simulation program based on Geant 4 [25, 26] and reconstructed in the same way as data. The samples of MC generated events are described further in section 5. 2
Analysis method (2.1) −FL cos2 θK cos2θL+S3 sin2 θK sin2 θL cos2φ
+S4 sin2θK sin2θL cosφ+S5 sin2θK sinθL cosφ
+S6 sin2 θK cosθL+S7 sin2θK sinθL sinφ +S8 sin2θK sin2θL sinφ+S9 sin2 θK sin2 θL sin2φ
#
. (2.1) (2.1) Here FL is the fraction of longitudinally polarised K∗mesons and the Si are angular
coefficients. These angular parameters are functions of the real and imaginary parts of the
transversity amplitudes of B0
d decays into K∗µ+µ−. The forward-backward asymmetry is
given by AFB = 3S6/4. The predictions for the S parameters depend on hadronic form
factors which have significant uncertainties at leading order. It is possible to reduce the
theoretical uncertainty in these predictions by transforming the Si using ratios constructed
to cancel form factor uncertainties at leading order. These ratios are given by refs. [17, 18] as P1 =
2S3
1 −FL
(2.2)
P2 = 2
3
AFB
1 −FL
(2.3)
P3 = −
S9
1 −FL
(2.4)
P ′
j=4,5,6,8 =
Si=4,5,7,8
p
FL(1 −FL)
. (2.5) (2.2) (2.5) 2This equation neglects possible Kπ S-wave contributions. The effect of an S-wave contribution is
considered following the method used by LHCb in ref. [3]. – 3 – All of the parameters introduced, FL, Si and P (′)
j , may vary with q2 and the data are
analysed in q2 bins to obtain an average value for a given parameter in that bin. 4
Event selection Several trigger signatures constructed from the MS and ID inputs are selected based on
availability during the data-taking period, prescale factor and efficiency for signal iden-
tification. Data are combined from 19 trigger chains where 21%, 89% or 5% of selected
events pass one or more triggers with one, two, or at least three muons identified online
in the MS, respectively. Of the events passing the requirement of at least two muons, the
largest contribution comes from the chain requiring one muon with a transverse momentum
pT > 4 GeV and the other muon with pT > 6 GeV. This combination of triggers ensures
that the analysis remains sensitive to events down to the kinematic threshold of q2 = 4m2
µ, – 4 – where mµ is the muon mass. The effective average trigger efficiency for selected signal
events is about 29%, determined from signal MC simulation. Muon track candidates are formed offline by combining information from both the ID
and MS [27]. Tracks are required to satisfy |η| < 2.5. Candidate muon (kaon and pion)
tracks in the ID are required to satisfy pT > 3.5 (0.5) GeV. Pairs of oppositely charged
muons are required to originate from a common vertex with a fit quality χ2/NDF < 10. Candidate K∗mesons are formed using pairs of oppositely charged kaon and
pion candidates reconstructed from hits in the ID. Candidates are required to satisfy
pT(K∗) > 3.0 GeV. As the ATLAS detector does not have a dedicated charged-particle
identification system, candidates are reconstructed with both possible Kπ mass hypothe-
ses. The selection implicitly relies on the kinematics of the reconstructed K∗meson to
determine which of the two tracks corresponds to the kaon. If both candidates in an
event satisfy selection criteria, they are retained and one of them is selected in the next
step following a procedure described below. The Kπ invariant mass is required to lie in
a window of twice the natural width around the nominal mass of 896 MeV, i.e. in the
range [846, 946] MeV. The charge of the kaon candidate is used to assign the flavour of the
reconstructed B0
d candidate. JHEP10(2018)047 The B0
d candidates are reconstructed from a K∗candidate and a pair of oppositely
charged muons. The four-track vertex is fitted and required to satisfy χ2/NDF < 2 to
suppress background. 4
Event selection A significant amount of combinatorial, B0
d, B+, B0
s and Λb back-
ground contamination remains at this stage. Combinatorial background is suppressed by
requiring a B0
d candidate lifetime significance τ/στ > 12.5, where the decay time uncer-
tainty στ is calculated from the covariance matrices associated with the four-track vertex
fit and with the primary vertex fit. Background from final states partially reconstructed
as B →µ+µ−X accumulates at invariant mass below the B0
d mass and contributes to the
signal region. It is suppressed by imposing an asymmetric mass cut around the nominal
B0
d mass, 5150 MeV < mKπµµ < 5700 MeV. The high-mass sideband is retained, as the
parameter values for the combinatorial background shapes are extracted from the fit to
data described in section 5. To further suppress background, it is required that the angle
Θ, defined between the vector from the primary vertex to the B0
d candidate decay vertex
and the B0
d candidate momentum, satisfies cos Θ > 0.999. Resolution effects on cos θK,
cos θL and φ were found to have a negligible effect on the ATLAS B0
s →J/ψφ analysis [28]. It is assumed to also be the case for B0
d →K∗µ+µ−. On average 12% of selected events in the data have more than one reconstructed B0
d
candidate. The fraction is 17% for signal MC samples and 2–10% for exclusive background
MC samples. A two-step selection process is used for such events. For 4% of these events it
is possible to select a candidate with the smallest value of the B0
d vertex χ2/NDF. However,
the majority, about 96%, of multiple candidates arise from four-track combinations where
the kaon and pion assignments are ambiguous. As these candidates have degenerate values
for the B0
d candidate vertex χ2/NDF, a second selection step is required. The B0
d candidate
reconstructed with the smallest value of |mKπ −mK∗|/σ(mKπ) is retained for analysis,
where mKπ is the K∗candidate mass, σ(mKπ) is the per-event uncertainty in this quantity,
and mK∗is the world average value of the K∗mass. – 5 – The selection procedure results in an incorrect flavour tag (mistag) for some signal
events. The mistag probability of a B0
d (B
0
d) meson is denoted by ω (ω) and is determined
from MC simulated events to be 0.1088 ± 0.0005 (0.1086 ± 0.0005). 4
Event selection The mistag probability
varies slightly with q2 such that the difference ω −ω remains consistent with zero. Hence
the average mistag rate ⟨ω⟩in a given q2 bin is used to account for this effect. If a candidate
is mistagged, the values of cos θL, cos θK and φ change sign, while the latter two are also
slightly shaped by the swapped hadron track mass hypothesis. Sign changes in these angles
affect the overall sign of the terms multiplied by the coefficients S5, S6, S8 and S9 (similarly
for the corresponding P (′) parameters) in equation (2.1). The corollary is that mistagged
events result in a dilution factor of (1 −2⟨ω⟩) for the affected coefficients. JHEP10(2018)047 The region q2 ∈[0.98, 1.1] GeV2 is vetoed to remove any potential contamination from
the φ(1020) resonance. The remaining data with q2 ∈[0.04, 6.0] GeV2 are analysed in
order to extract the signal parameters of interest. Two K∗cc control regions are defined for
B0
d decays into K∗J/ψ and K∗ψ(2S), respectively as q2 ∈[8, 11] and [12, 15] GeV2. The
control samples are used to extract values for nuisance parameters describing the signal
probability density function (pdf) from data as discussed in section 5.3. For q2 < 6 GeV2 the selected data sample consists of 787 events and is composed of
signal B0
d →K∗µ+µ−decay events as well as background that is dominated by a combina-
torial component that does not peak in mKπµµ and does not exhibit a resonant structure in
q2. Other background contributions are considered when estimating systematic uncertain-
ties. Above 6 GeV2 the background contribution increases significantly, including events
coming from B0
d →K∗J/ψ with a radiative J/ψ →µ+µ−γ decay. Scalar Kπ contributions
are neglected in the nominal fit and considered only when addressing systematic uncertain-
ties. The data are analysed in the q2 bins [0.04, 2.0], [2.0, 4.0] and [4.0, 6.0] GeV2, where
the bin width is chosen to provide a sample of signal events sufficient to perform an angular
analysis. The width is much larger than the q2 resolution obtained from MC simulated
signal events and observed in data for B0
d decays into K∗J/ψ and K∗ψ(2S). Additional
overlapping bins [0.04, 4.0], [1.1, 6.0] and [0.04, 6.0] GeV2 are analysed in order to facilitate
comparison with results of other experiments and with theoretical predictions. 5
Maximum-likelihood fit Extended unbinned maximum-likelihood fits of the angular distributions of the signal decay
are performed on the data for each q2 bin. The discriminating variables used in the fit are
mKπµµ, the cosines of the helicity angles (cos θK and cos θL), and φ. The likelihood L for
a given q2 bin is L = e−n
N! N
Y
k=1
X
l
nlPkl(mKπµµ, cos θK, cos θL, φ; bp, bθ),
(5.1) (5.1) where N is the total number of events, the sum runs over signal and background compo-
nents, nl is the fitted yield for the lth component, n is the sum over nl, and Pkl is the pdf
evaluated for event k and component l. In the nominal fit, l iterates only over one signal – 6 – and one background component. The bp are parameters of interest (FL, Si) and bθ are nui-
sance parameters. The remainder of this section discusses the signal model (section 5.1),
treatment of background (section 5.2), use of K∗cc decay control samples (section 5.3),
fitting procedure and validation (section 5.4). 5.1
Signal model The signal mass distribution is modelled by a Gaussian distribution with the width given
by the per-event uncertainty in the Kπµµ mass, σ(mKπµµ), as estimated from the track
fit, multiplied by a unit-less scale factor ξ, i.e. the width given by ξ · σ(mKπµµ). The mean
values of the B0
d candidate mass (m0) and ξ of the signal Gaussian pdf are determined from
fits to data in the control regions as described in section 5.3. The simultaneous extraction
of all coefficients using the full angular distribution of equation (2.1) requires a certain
minimum signal yield and signal purity to avoid a pathological fit behaviour. A significant
fraction of fits to ensembles of simulated pseudo-experiments do not converge using the
full distribution. This is mitigated using trigonometric transformations to fold certain
angular distributions and thereby simplify equation (2.1) such that only three parameters
are extracted in one fit: FL, S3 and one of the other S parameters. For these folding schemes
the angular parameters of interest, denoted by bp in equation (5.1), are (FL, S3, Si) where
i = 4, 5, 7, 8. These translate into (FL, P1, P ′
j), where j = 4, 5, 6, 8, using equation (2.5). Following ref. [3], the transformations listed below are used: JHEP10(2018)047 FL, S3, S4, P
′
4 :
φ →−φ
for φ < 0
φ →π −φ
for θL > π
2
θL →π −θL
for θL > π
2 ,
(5.2)
FL, S3, S5, P
′
5 :
(
φ →−φ
for φ < 0
θL →π −θL
for θL > π
2 ,
(5.3)
FL, S3, S7, P
′
6 :
φ →π −φ
for φ > π
2
φ →−π −φ
for φ < −π
2
θL →π −θL
for θL > π
2 ,
(5.4)
FL, S3, S8, P
′
8 :
φ →π −φ
for φ > π
2
φ →−π −φ
for φ < −π
2
θL →π −θL
for θL > π
2
θK →π −θK
for θL > π
2 . 5.1
Signal model (5.5) FL, S3, S4, P
′
4 :
φ →−φ
for φ < 0
φ →π −φ
for θL > π
2
θL →π −θL
for θL > π
2 ,
(5.2) (5.2) (5.3) (
θL →π −θL
for θL > π
2 ,
FL, S3, S7, P
′
6 :
φ →π −φ
for φ > π
2
φ →−π −φ
for φ < −π
2
θL →π −θL
for θL > π
2 ,
(5.4)
FL, S3, S8, P
′
8 :
φ →π −φ
for φ > π
2
φ →−π −φ
for φ < −π
2
θL →π −θL
for θL > π
2
θK →π −θK
for θL > π
2 . (5.5) (5.4) (5.5) On applying transformation (5.2), (5.3), (5.4), and (5.5), the angular variable ranges
become cos θL ∈[0, 1],
cos θK ∈[−1, 1]
and
φ ∈[0, π],
cos θL ∈[0, 1],
cos θK ∈[−1, 1]
and
φ ∈[0, π],
cos θL ∈[0, 1],
cos θK ∈[−1, 1]
and
φ ∈[−π/2, π/2],
cos θL ∈[0, 1],
cos θK ∈[−1, 1]
and
φ ∈[−π/2, π/2], – 7 – – 7 – respectively. A consequence of using the folding schemes is that S6 (AFB) and S9 cannot
be extracted from the data. The values and uncertainties of FL and S3 obtained from the
four fits are consistent with each other and the results reported are those found to have
the smallest systematic uncertainty. Three MC samples are used to study the signal reconstruction and acceptance. Two of
them follow the SM prediction for the decay angle distributions taken from ref. [29], with
separate samples generated for B0
d and B0
d decays. The third MC sample has FL = 1/3
and the angular distributions are generated uniformly in cos θK, cos θL and φ. The samples
are used to study the effect of potential mistagging and reconstruction differences between
particle and antiparticle decays and for determination of the acceptance. The acceptance
function is defined as the ratio of reconstructed and generated distributions of cos θK,
cos θL, φ, i.e. it is compensating for the bias in the angular distributions resulting from
triggering, reconstruction and selection of events. Pkl = ε(cos θK)ε(cos θL)ε(φ)g(cos θK, cos θL, φ) · G(mKπµµ), Pkl = ε(cos θK)ε(cos θL)ε(φ)g(cos θK, cos θL, φ) · G(mKπµµ), where g(cos θK, cos θL, φ) is an angular differential decay rate resulting from one of the four
folding schemes applied to equation (2.1) and G(mKπµµ) is the signal mass distribution. The MC sample generated with uniform cos θK, cos θL and φ distributions is used to
determine the nominal acceptance functions for each of the transformed variables defined
in equations (5.2)–(5.5). The other samples are used to estimate the related systematic
uncertainty. Among the angular variables the cos θL distribution is the most affected by
the acceptance. This is a result of the minimum transverse momentum requirements on
the muons in the trigger and the larger inefficiency to reconstruct low-momentum muons,
such that large values of | cos θL| are inaccessible at low q2. As q2 increases, the acceptance
effects become less severe. The cos θK distribution is affected by the ability to reconstruct
the Kπ system, but that effect shows no significant variation with q2. There is no significant
acceptance effect for φ. Figure 2 shows the acceptance functions used for cos θK and cos θL
for two different q2 ranges for the nominal angular distribution given in equation (2.1). where g(cos θK, cos θL, φ) is an angular differential decay rate resulting from one of the four
folding schemes applied to equation (2.1) and G(mKπµµ) is the signal mass distribution. The MC sample generated with uniform cos θK, cos θL and φ distributions is used to
determine the nominal acceptance functions for each of the transformed variables defined
in equations (5.2)–(5.5). The other samples are used to estimate the related systematic
uncertainty. Among the angular variables the cos θL distribution is the most affected by
the acceptance. This is a result of the minimum transverse momentum requirements on
the muons in the trigger and the larger inefficiency to reconstruct low-momentum muons,
such that large values of | cos θL| are inaccessible at low q2. As q2 increases, the acceptance
effects become less severe. The cos θK distribution is affected by the ability to reconstruct
the Kπ system, but that effect shows no significant variation with q2. There is no significant
acceptance effect for φ. Figure 2 shows the acceptance functions used for cos θK and cos θL
for two different q2 ranges for the nominal angular distribution given in equation (2.1). 5.1
Signal model It is described by sixth-order (second-
order) polynomial distributions for cos θK and cos θL (φ) and is assumed to factorise for each
angular distribution, i.e. using ε(cos θK, cos θL, φ) = ε(cos θK)ε(cos θL)ε(φ). A systematic
uncertainty is assessed in order to account for this assumption. The acceptance function
multiplies the angular distribution in the fit, i.e. the signal pdf is JHEP10(2018)047 5.2
Background modes The fit to data includes a combinatorial background component that does not peak in the
mKπµµ distribution. It is assumed that the background pdf factorises into a product of one-
dimensional terms. The mass distribution of this component is described by an exponential
function and second-order Chebychev polynomials are used to model the cos θK, cos θL and
φ distributions. The values of the nuisance parameters describing these shapes are obtained
from fits to the data independently for each q2 bin. Inclusive samples of bb →µ+µ−X and cc →µ+µ−X decays and eleven exclusive B0
d,
B0
s, B+ and Λb background samples are studied in order to identify contributions of interest – 8 – K
θ
cos
1
−
0.8
−
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
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1
Probability Density
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
2
[0.04, 2.0] GeV
∈
2
q
2
[4.0, 6.0] GeV
∈
2
q
ATLAS
Simulation
L
θ
cos
1
−
0.8
−
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
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1
Probability Density
0
0.2
0.4
0.6
0.8
1
1.2
2
[0.04, 2.0] GeV
∈
2
q
2
[4.0, 6.0] GeV
∈
2
q
ATLAS
Simulation
Figure 2. The acceptance functions for (left) cos θK and (right) cos θL for (solid) q2 ∈
[0.04, 2.0] GeV2 and (dashed) q2 ∈[4.0, 6.0] GeV2, that shape the angular decay rate of equa-
tion (2.1). K
θ
cos
1
−
0.8
−
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
Probability Density
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
2
[0.04, 2.0] GeV
∈
2
q
2
[4.0, 6.0] GeV
∈
2
q
ATLAS
Simulation L
θ
cos
1
−
0.8
−
0.6
−
0.4
−
0.2
−
0
0.2
0.4
0.6
0.8
1
Probability Density
0
0.2
0.4
0.6
0.8
1
1.2
2
[0.04, 2.0] GeV
∈
2
q
2
[4.0, 6.0] GeV
∈
2
q
ATLAS
Simulation JHEP10(2018)047 Figure 2. The acceptance functions for (left) cos θK and (right) cos θL for (solid) q2 ∈
[0.04, 2.0] GeV2 and (dashed) q2 ∈[4.0, 6.0] GeV2, that shape the angular decay rate of equa-
tion (2.1). to be included in the fit model, or to be considered when estimating systematic uncertain-
ties. The relevant exclusive modes found to be of interest are discussed below. 5.2
Background modes Events with
Bc decays are suppressed by excluding the q2 range containing the J/ψ and ψ(2S), and by
charm meson vetoes discussed in section 7. The exclusive background decays considered
for the signal mode are Λb →Λ(1520)µ+µ−, Λb →pK−µ+µ−, B+ →K(∗)+µ+µ−and
B0
s →φµ+µ−. These background contributions are accounted for as systematic uncertain-
ties estimated as described in section 7. Two distinct background contributions not considered above are observed in the cos θK
and cos θL distributions. They are not accounted for in the nominal fit to data, and are
treated as systematic effects. A peak is found in the cos θK distribution near 1.0 and
appears to have contributions from at least two distinct sources. One of these arises from
misreconstructed B+ decays, such as B+ →K+µµ and B+ →π+µµ. These decays
can be reconstructed as signal if another track is combined with the hadron to form a
K∗candidate in such a way that the event passes the reconstruction and selection. The
second contribution comes from combinations of two charged tracks that pass the selection
and are reconstructed as a K∗candidate. These fake K∗candidates accumulate around
cos θK of 1.0 and are observed in the Kπ mass sidebands away from the K∗meson. They
are distinct from the structure of expected S-, P- and D-wave Kπ decays resulting from
a signal B0
d →Kπµµ transition. The origin of this source of background is not fully
understood. The observed excess may arise from a statistical fluctuation, an unknown
background process, or a combination of both. Systematic uncertainties are assigned to
evaluate the effect of these two background contributions, as described in section 7. Another peak is found in the cos θL distribution near values of ±0.7. It is associated
with partially reconstructed B decays into final states with a charm meson. This is studied
using Monte Carlo simulated events for the decays D0 →K−π+, D+ →K−π+π+ and
D+
s →K+K−π+. 5.2
Background modes Events with a B meson decaying via an intermediate charm meson
D0, D+ or D+
s are found to pass the selection and are reconstructed in such a way that – 9 – [MeV]
µ
µ
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3000
4000
5000
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s
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Signal
Combinatorial
Λ
(2S)
ψ
→
b
Λ
+
(2S) K
ψ
→
+
B
(2S) K*
ψ
→
s
B [MeV]
µ
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0
10000
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30000
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Combinatorial
Λ
ψ
J/
→
b
Λ
+
K
ψ
J/
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+
B
K*
ψ
J/
→
s
B
[MeV]
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Λ
(2S)
ψ
→
b
Λ
+
(2S) K
ψ
→
+
B
(2S) K*
ψ
→
s
B
Figure 3. Fits to the Kπµµ invariant mass distributions for the (left) K∗J/ψ and (right) K∗ψ(2S)
control region samples. The data are shown as points and the total fit model as the solid lines. The dashed lines represent (black) signal, (red) combinatorial background, (green) Λb background,
(blue) B+ background and (magenta) B0
s background components. [MeV]
µ
µ
π
K
m
5000
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5600
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0
10000
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30000
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50000
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Total Fit Model
Signal
Combinatorial
Λ
ψ
J/
→
b
Λ
+
K
ψ
J/
→
+
B
K*
ψ
J/
→
s
B JHEP10(2018)047 Figure 3. Fits to the Kπµµ invariant mass distributions for the (left) K∗J/ψ and (right) K∗ψ(2S)
control region samples. The data are shown as points and the total fit model as the solid lines. The dashed lines represent (black) signal, (red) combinatorial background, (green) Λb background,
(blue) B+ background and (magenta) B0
s background components. they accumulate around 0.7 in | cos θL|. These are removed from the data sample when
estimating systematic uncertainties, as described in section 7. 5.4
Fitting procedure and validation A two-step fit process is performed for the different signal bins in q2. The first step is a fit
to the Kπµ+µ−invariant mass distribution, using the event-by-event uncertainty in the
reconstructed mass as a conditional variable. For this fit, the parameters m0 and ξ are
fixed to the values obtained from fits to data control samples as described in section 5.3. A
second step adds the (transformed) cos θK, cos θL and φ variables to the likelihood in order
to extract FL and the S parameters along with the values for the nuisance parameters
related to the combinatorial background shapes. Some nuisance parameters, namely m0,
ξ, signal and background yields, and the exponential shape parameter for the background
mass pdf, are fixed to the results obtained from the first step. JHEP10(2018)047 The fit procedure is validated using ensembles of simulated pseudo-experiments gen-
erated with the FL and S parameters corresponding to those obtained from the data. The
purpose of these experiments is to measure the intrinsic fit bias resulting from the likeli-
hood estimator used to extract signal parameters. These ensembles are also used to check
that the uncertainties extracted from the fit are consistent with expectations. Ensembles
of simulated pseudo-experiments are performed in which signal MC events are injected into
samples of background events generated from the likelihood. The signal yield determined
from the first step in the fit process is found to be unbiased. The angular parameters ex-
tracted from the nominal fits have biases with magnitudes ranging between 0.01 and 0.04,
depending on the fit variation and q2 bin. A similar procedure is used to estimate the effect
of neglecting S-wave contamination in the data sample. Neglecting the S-wave component
in the fit model results in a bias between 0.00 and 0.02 in the angular parameters. Simi-
larly, neglecting exclusive background contributions from Λb, B+ and B0
s decays that peak
in mKπµµ near the B0
d mass results in a bias of less than 0.01 on the angular parameters. All these effects are included in the systematic uncertainties described in section 7. The
P (′) parameters are obtained using the fit results and covariance matrices from the second
fit along with equations (2.2)–(2.5). 5.3
K∗cc control sample fits The mass distribution obtained from the simulated samples for K∗cc decays, respectively
as q2 ∈[8, 11] and [12, 15] GeV2, and the signal mode, in different bins of q2, are found to
be consistent with each other. Values of m0 and ξ for B0
d →K∗J/ψ and B0
d →K∗ψ(2S)
events are used for the signal pdf and extracted from fits to the data. An extended unbinned
maximum-likelihood fit is performed in the two K∗cc control region samples. There are
three exclusive backgrounds included: Λb →Λcc, B+ →K+cc and B0
s →K∗cc. The
K∗cc pdf has the same form as the signal model, combinatorial background is described
by an exponential distribution, and double and triple Gaussian pdfs determined from MC
simulated events are used to describe the exclusive background contributions. A systematic
uncertainty is evaluated by allowing for 0, 1, 2 and 3 exclusive background components. The control sample fit projections for the variant of the fit including all three exclusive
backgrounds can be found in figure 3. The impact of the used exclusive background model
on the peak position and scale factor of the signal pdf is negligible. From these fits the
statistical and systematic uncertainties in the values of m0 and ξ are extracted for the B0
d
component in order to be used in the B0
d →K∗µ+µ−fits. From the J/ψ control data
it is determined that the values for the nuisance parameters describing the signal model
pdf in the Kπµµ mass are m0 = 5276.6 ± 0.3 ± 0.4 MeV and ξ = 1.210 ± 0.004 ± 0.002,
where the uncertainties are statistical and systematic, respectively. The ψ(2S) sample
yields compatible results albeit with larger uncertainties. These results are similar to those
obtained from the MC simulated samples, and the numbers derived from the K∗J/ψ data
are used for the signal region fits. – 10 – 6
Results The event yields obtained from the fits are summarised in table 1 where only statistical un-
certainties are reported. Figures 4 through 9 show for the different q2 bins the distributions
of the variables used in the fit for the S5 folding scheme (corresponding to the transfor-
mation of equation (5.3)) with the total, signal and background fitted pdfs superimposed. Similar sets of distributions are obtained for the three other folding schemes: S4, S7 and
S8. The results of the angular fits to the data in terms of the Si and P (′)
j
can be found
in tables 2 and 3. Statistical and systematic uncertainties are quoted in the tables. The
distributions of FL and the Si parameters as a function of q2 are shown in figure 10 and
those for P (′)
j
are shown in figure 11. The correlations between FL and the Si parameters
and between FL and the P (′)
j
are given in appendix A. – 11 – q2 [GeV2]
nsignal
nbackground
[0.04, 2.0]
128 ± 22
122 ± 22
[2.0, 4.0]
106 ± 23
113 ± 23
[4.0, 6.0]
114 ± 24
204 ± 26
[0.04, 4.0]
236 ± 31
233 ± 32
[1.1, 6.0]
275 ± 35
363 ± 36
[0.04, 6.0]
342 ± 39
445 ± 40
Table 1. The values of fitted signal, nsignal, and background, nbackground, yields obtained for
different bins in q2. The uncertainties indicated are statistical. JHEP10(2018)047 Table 1. The values of fitted signal, nsignal, and background, nbackground, yields obtained for
different bins in q2. The uncertainties indicated are statistical. Table 1. The values of fitted signal, nsignal, and background, nbackground, yields obtained for
different bins in q2. The uncertainties indicated are statistical. 6
Results [MeV]
µ
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∈
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Background
Figure 4. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[0.04, 2.0] GeV2. The (blue) solid line is a projection of the total
pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents
the signal component. These plots are obtained from a fit using the S5 folding scheme. [rad]
φ
0
1
2
3
π
Events / 0.04
0
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30
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∈
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Total Fit Model
Signal
Background Events / 25 MeV π
Events / 0.04 Events / 0.08 Events / 0.04 Figure 4. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[0.04, 2.0] GeV2. 6
Results The (blue) solid line is a projection of the total
pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents
the signal component. These plots are obtained from a fit using the S5 folding scheme. – 12 – [MeV]
µ
µ
π
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Figure 5. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[2.0, 4.0] GeV2. The (blue) solid line is a projection of the total pdf,
the (red) dot-dashed line represents the background, and the (black) dashed line represents the
signal component. These plots are obtained from a fit using the S5 folding scheme. [rad]
φ
0
1
2
3
π
Events / 0.04
0
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20
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∈
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Total Fit Model
Signal
Background Events / 25 MeV π
Events / 0.04 JHEP10(2018)047 Events / 0.08 Events / 0.04 Figure 5. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[2.0, 4.0] GeV2. 6
Results The (blue) solid line is a projection of the total pdf,
the (red) dot-dashed line represents the background, and the (black) dashed line represents the
signal component. These plots are obtained from a fit using the S5 folding scheme. – 13 – [MeV]
µ
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π
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Figure 6. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[4.0, 6.0] GeV2. The (blue) solid line is a projection of the total pdf,
the (red) dot-dashed line represents the background, and the (black) dashed line represents the
signal component. These plots are obtained from a fit using the S5 folding scheme. [rad]
φ
0
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3
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Events / 0.04
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Background π
Events / 0.04 JHEP10(2018)047 Events / 0.08 Events / 0.04 Figure 6. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[4.0, 6.0] GeV2. 6
Results The (blue) solid line is a projection of the total pdf,
the (red) dot-dashed line represents the background, and the (black) dashed line represents the
signal component. These plots are obtained from a fit using the S5 folding scheme. – 14 – [MeV]
µ
µ
π
K
m
5200
5400
5600
Events / 25 MeV
0
20
40
60
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∈
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Figure 7. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[0.04, 4.0] GeV2. The (blue) solid line is a projection of the total
pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents
the signal component. These plots are obtained from a fit using the S5 folding scheme. 6
Results [rad]
φ
0
1
2
3
π
Events / 0.04
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10
20
30
40
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Background [MeV]
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Events / 25 MeV
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∈
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Data
Total Fit Model
Signal
Background π
Events / 0.04 JHEP10(2018)047 Events / 0.04 Events / 0.08 Figure 7. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[0.04, 4.0] GeV2. The (blue) solid line is a projection of the total
pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents
the signal component. These plots are obtained from a fit using the S5 folding scheme. – 15 – [MeV]
µ
µ
π
K
m
5200
5400
5600
Events / 25 MeV
0
20
40
60
80
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= 8 TeV, 20.3 fb
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∈
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Figure 8. 6
Results The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[1.1, 6.0] GeV2. The (blue) solid line is a projection of the total pdf,
the (red) dot-dashed line represents the background, and the (black) dashed line represents the
signal component. These plots are obtained from a fit using the S5 folding scheme. [rad]
φ
0
1
2
3
π
Events / 0.04
0
20
40
60
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= 8 TeV, 20.3 fb
s
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∈
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cos
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∈
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Data
Total Fit Model
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Background π
Events / 0.04 [MeV]
µ
µ
π
K
m
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5600
Events / 25 MeV
0
20
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80
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∈
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Data
Total Fit Model
Signal
Background JHEP10(2018)047 Events / 0.08 Events / 0.04 Figure 8. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[1.1, 6.0] GeV2. The (blue) solid line is a projection of the total pdf,
the (red) dot-dashed line represents the background, and the (black) dashed line represents the
signal component. These plots are obtained from a fit using the S5 folding scheme. 6
Results – 16 – [MeV]
µ
µ
π
K
m
5200
5400
5600
Events / 25 MeV
0
50
100
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= 8 TeV, 20.3 fb
s
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∈
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Data
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[rad]
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Events / 0.04
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Data
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Background
Figure 9. The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[0.04, 6.0] GeV2. The (blue) solid line is a projection of the total
pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents
the signal component. These plots are obtained from a fit using the S5 folding scheme. [rad]
φ
0
1
2
3
π
Events / 0.04
0
20
40
60
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= 8 TeV, 20.3 fb
s
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Background [MeV]
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Events / 25 MeV
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∈
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Data
Total Fit Model
Signal
Background Events / 25 MeV π
Events / 0.04 JHEP10(2018)047 Events / 0.04 Events / 0.08 Figure 9. 6
Results The distributions of (top left) mKπµµ, (top right) φ, (bottom left) cos θK, and (bottom
right) cos θL obtained for q2 ∈[0.04, 6.0] GeV2. The (blue) solid line is a projection of the total
pdf, the (red) dot-dashed line represents the background, and the (black) dashed line represents
the signal component. These plots are obtained from a fit using the S5 folding scheme. – 17 – q2 [GeV2]
FL
S3
S4
S5
S7
S8
[0.04,2.0] 0.44±0.08±0.07 −0.02±0.09±0.02
0.15±0.20±0.10
0.33±0.13±0.08 −0.09±0.10±0.02 −0.14±0.24±0.09
[2.0,4.0] 0.64±0.11±0.05 −0.15±0.10±0.07 −0.37±0.15±0.10 −0.16±0.15±0.06
0.15±0.14±0.09
0.52±0.20±0.19
[4.0,6.0] 0.42±0.13±0.12
0.00±0.12±0.07
0.32±0.16±0.09
0.13±0.18±0.09
0.03±0.13±0.07 −0.12±0.21±0.05
[0.04,4.0] 0.52±0.07±0.06 −0.05±0.06±0.04 −0.15±0.12±0.09
0.16±0.10±0.05
0.01±0.08±0.05
0.19±0.16±0.12
[1.1,6.0] 0.56±0.07±0.06 −0.04±0.07±0.03
0.03±0.11±0.07
0.00±0.10±0.04
0.02±0.08±0.06
0.11±0.14±0.10
[0.04,6.0] 0.50±0.06±0.04 −0.04±0.06±0.03
0.03±0.10±0.07
0.14±0.09±0.03
0.02±0.07±0.05
0.07±0.13±0.09
Table 2. The values of FL, and S3, S4, S5, S7 and S8 parameters obtained for different bins in q2. The uncertainties indicated are statistical and systematic, respectively. q2 [GeV2]
P1
P ′
4
P ′
5
P ′
6
P ′
8
[0.04,2.0] −0.05±0.30±0.08
0.31±0.40±0.20
0.67±0.26±0.16 −0.18±0.21±0.04 −0.29±0.48±0.18
[2.0,4.0] −0.78±0.51±0.34 −0.76±0.31±0.21 −0.33±0.31±0.13
0.31±0.28±0.19
1.07±0.41±0.39
[4.0,6.0]
0.14±0.43±0.26
0.64±0.33±0.18
0.26±0.35±0.18
0.06±0.27±0.13 −0.24±0.42±0.09
[0.04,4.0] −0.22±0.26±0.16 −0.30±0.24±0.17
0.32±0.21±0.11
0.01±0.17±0.10
0.38±0.33±0.24
[1.1,6.0] −0.17±0.31±0.13
0.05±0.22±0.14
0.01±0.21±0.08
0.03±0.17±0.12
0.23±0.28±0.20
[0.04,6.0] −0.15±0.23±0.10
0.05±0.20±0.14
0.27±0.19±0.06
0.03±0.15±0.10
0.14±0.27±0.17
Table 3. The values of P1, P ′
4, P ′
5, P ′
6 and P ′
8 parameters obtained for different bins in q2. The
uncertainties indicated are statistical and systematic, respectively. q2 [GeV2]
FL
S3
S4
S5
S7
S8
[0.04,2.0] 0.44±0.08±0.07 −0.02±0.09±0.02
0.15±0.20±0.10
0.33±0.13±0.08 −0.09±0.10±0.02 −0.14±0.24±0.09
[2.0,4.0] 0.64±0.11±0.05 −0.15±0.10±0.07 −0.37±0.15±0.10 −0.16±0.15±0.06
0.15±0.14±0.09
0.52±0.20±0.19
[4.0,6.0] 0.42±0.13±0.12
0.00±0.12±0.07
0.32±0.16±0.09
0.13±0.18±0.09
0.03±0.13±0.07 −0.12±0.21±0.05
[0.04,4.0] 0.52±0.07±0.06 −0.05±0.06±0.04 −0.15±0.12±0.09
0.16±0.10±0.05
0.01±0.08±0.05
0.19±0.16±0.12
[1.1,6.0] 0.56±0.07±0.06 −0.04±0.07±0.03
0.03±0.11±0.07
0.00±0.10±0.04
0.02±0.08±0.06
0.11±0.14±0.10
[0.04,6.0] 0.50±0.06±0.04 −0.04±0.06±0.03
0.03±0.10±0.07
0.14±0.09±0.03
0.02±0.07±0.05
0.07±0.13±0.09 Table 2. The values of FL, and S3, S4, S5, S7 and S8 parameters obtained for different bins in q
The uncertainties indicated are statistical and systematic, respectively. JHEP10(2018)047 Table 3. The values of P1, P ′
4, P ′
5, P ′
6 and P ′
8 parameters obtained for different bins in q2. The
uncertainties indicated are statistical and systematic, respectively. 7
Systematic uncertainties Systematic uncertainties in the parameter values obtained from the angular analysis come
from several sources. The methods for determining these uncertainties are based either
on a comparison of nominal and modified fit results, or on observed fit biases in modified
pseudo-experiments. The systematic uncertainties are symmetrised. The most significant
ones are described in the following, in decreasing order of importance. • A systematic uncertainty is assigned for the combinatorial Kπ (fake K∗) background
peaking at cos θK values around 1.0 obtained by comparing results of the nominal fit
to that where data above cos θK = 0.9 are excluded from the fit. • A systematic uncertainty is assigned for the combinatorial Kπ (fake K∗) background
peaking at cos θK values around 1.0 obtained by comparing results of the nominal fit
to that where data above cos θK = 0.9 are excluded from the fit. • A systematic uncertainty is derived to account for background arising from partially
reconstructed B →D0/D+/D+
s X decays, that manifest in an accumulation of events
at | cos θL| values around 0.7. Two-track or three-track combinations are formed from
the signal candidate tracks, and are reconstructed assuming the pion or kaon mass
hypothesis. A veto is then applied for events in which a track combination has a mass
in a window of 30 MeV around the D0, D+ or D+
s meson mass. Similarly, a veto is
implemented to reject B+ →K+µ+µ−and B+ →π+µ+µ−events that pass the event
selection. Here B+ candidates are reconstructed from one of the hadrons from the K∗
candidate and the muons in the signal candidate. Signal candidates that have a three-
track mass within 50 MeV of the B+ mass are excluded from the fit. A few percent
of signal events are removed when applying these vetoes, with a corresponding effect
on the acceptance distributions. The fit results obtained from the data samples with – 18 – vetoes applied are compared to those obtained from the nominal fit and the change
in each result is taken as the systematic uncertainty from these backgrounds. This
systematic uncertainty dominates the measurement of FL at higher values of q2. vetoes applied are compared to those obtained from the nominal fit and the change
in each result is taken as the systematic uncertainty from these backgrounds. This
systematic uncertainty dominates the measurement of FL at higher values of q2. 7
Systematic uncertainties • The combinatorial background pdf shape has an uncertainty arising from the choice
of the model. For the mass distribution it is assumed that an exponential function
model is adequate; however, for the angular variables the data are re-fitted using
third-order Chebychev polynomials. The change from the nominal result is taken as
the uncertainty from this source. JHEP10(2018)047 • The acceptance function is assumed to factorise into three separate components, for
cos θK, cos θL and φ. To validate this assumption, the signal simulated events are
fitted with the acceptance function obtained from that same MC sample. Differences
in the fit results from expectation are small and taken as the uncertainty resulting
from this assumption. • A systematic uncertainty is assigned for the angular pdf model for the background
by comparing the nominal result to that with a reduced fit range of mKπµµ ∈
[5200, 5700] MeV, in particular to account for possible residues of the partially re-
constructed B-decays. • A correction is applied to the data by shifting the track pT according to the uncer-
tainties arising from biases in rapidity and momentum scale. The change in results
obtained is ascribed to the uncertainty in the ID alignment and knowledge of the
magnetic field. • The maximum-likelihood estimator used is intrinsically biased. Ensembles of MC
simulated events are used in order to ascertain the bias in the extracted values of the
parameters of interest. The bias is assigned as a systematic uncertainty. • The pT spectrum of B0
d candidates observed in data is not accurately reproduced by
the MC simulation. This difference in the kinematics results in a slight modification
of the acceptance functions. This is accounted for by reweighting signal MC simulated
events to resemble the pT spectrum found in data. The change in fitted parameter
values obtained due to the reweighting is taken as the systematic uncertainty resulting
from this difference. • The signal decay mode is resonant K∗→Kπ decay, but scalar contributions from
non-resonant Kπ transitions may also exist. The LHCb Collaboration reported an S-
wave contribution at the level of 5% of the signal [4, 30]. Ensembles of MC simulated
events are fitted with 5% of the signal being drawn from an S-wave sample of events
and the remaining 95% from signal. The observed change in fit bias is assigned as
the systematic uncertainty from this source. 7
Systematic uncertainties Any variation in S-wave content as a
function of q2 would not significantly affect the results reported here. – 19 – • The values of the nuisance parameters of the fit model obtained from MC control
samples and fits to the data mass distribution have associated uncertainties. These
parameters include m0, ξ, the signal and background yields, the shape parameter of
the combinatorial background mass distribution, and the parameters of the signal
acceptance functions. The uncertainty in the value of each of these parameters is
varied independently in order to assess the effect on parameters of interest. This
source of uncertainty has a small effect on the measurements reported here. • Background from exclusive modes peaking in mKπµµ is neglected in the nominal
fit. This may affect the fitted results and is accounted for by computing the
fit bias obtained when embedding MC simulated samples of Λb →Λ(1520)µ+µ−,
Λb →pK−µ+µ−, B+ →K(∗)+µ+µ−and B0
s →φµ+µ−into ensembles of pseudo-
data generated from the fit model containing only combinatorial background and sig-
nal components. The change in fit bias observed when adding exclusive backgrounds
is taken as the systematic error arising from neglecting those modes in the fit. JHEP10(2018)047 • The difference from nominal results obtained when fitting the B0
d signal MC events
with the acceptance function for B0
d is taken as an upper limit of the systematic error
resulting from event migration due to mistagging the B0
d flavour. • The parameters S5 and S8, as well as the respective P (′)
j
parameters are affected by
dilution and thus have a multiplicative scaling applied to them. This dilution factor
depends on the kinematics of the K∗decay and has a systematic uncertainty associ-
ated with it. The effect of data/MC differences in the pT spectrum of B0
d candidates
on the mistag probability was studied and found to be negligible. The uncertainty due
to the limited number of MC events is used to compute the statistical uncertainty of ω
and ω. Studies of MC simulated events indicate that there is no significant difference
between the mistag probability for B0
d and B0
d events and the analysis assumes that
the average mistag probability provides an adequate description of this effect. The
magnitude of the mistag probability difference, |ω −ω|, is included as a systematic
uncertainty resulting from this assumption. 7
Systematic uncertainties The total systematic uncertainties of the fitted Si and P (′)
j
parameter values are presented
in tables 2 and 3, where the dominant contributions for FL come from the modelling of
the angular distributions of the combinatorial background and the partially reconstructed
decays peaking in cos θK and cos θL. These contributions and in addition also ID alignment
and magnetic field calibration affect S3 (P1). The largest systematic uncertainty contribu-
tion to S3 (P1) comes from partially reconstructed decays entering the signal region. This
also affects the measurement of S5 (P ′
5) and S7 (P ′
6). The partially reconstructed decays
peaking in cos θL affect the measurement of S4 (P ′
4) and S8 (P ′
8), whereas the fake K∗back-
ground in cos θK affects S4 (P ′
4), S5 (P ′
5), and S8 (P ′
8). The parameterization of the signal
acceptance is another significant systematic uncertainty source for S4 (P ′
4). The systematic
uncertainties are smaller than the statistical uncertainties for all parameters measured. – 20 – 0
2
4
6
8
10
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2
[GeV
2
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0
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theory DHMV
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theory DHMV
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Figure 10. 7
Systematic uncertainties The measured values of FL, S3, S4, S5, S7, S8 compared with predictions from the
theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are
shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar
the total uncertainty. 0
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s JHEP10(2018)047 0
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theory DHMV
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CFFMPSV fit
theory DHMV
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0.4
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S
Data
CFFMPSV fit
theory DHMV
ATLAS
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= 8 TeV, 20.3 fb
s Figure 10. The measured values of FL, S3, S4, S5, S7, S8 compared with predictions from the
theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are
shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar
the total uncertainty. 4This result uses the experimental convention of equations (2.2)–(2.5) following the LHCb Collaboration’s
notation in ref. [3]. In the DHMV calculation, a different convention is used as explained by equation (16)
in ref. [15]. 8
Comparison with theoretical computations The results of theoretical approaches of Ciuchini et al. (CFFMPSV) [31], Descotes-Genon
et al. (DHMV) [32], and J¨ager and Camalich (JC) [33, 34] are shown in figure 10 for the S
parameters, and in figure 11 for the P (′) parameters, along with the results presented here.4 4This result uses the experimental convention of equations (2.2)–(2.5) following the LHCb Collaboration’s
notation in ref. [3]. In the DHMV calculation, a different convention is used as explained by equation (16)
in ref. [15]. – 21 – 0
2
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theory DHMV
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theory JC
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P'
Data
theory DHMV
theory JC
ATLAS
-1
= 8 TeV, 20.3 fb
s
Figure 11. The measured values of P1, P ′
4, P ′
5, P ′
6, P ′
8 compared with predictions from the
theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are
shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar
the total uncertainty. 8
Comparison with theoretical computations 0
2
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10
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theory DHMV
theory JC
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theory DHMV
theory JC
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[
q
0
2
4
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8
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]
2
[GeV
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q
1
−
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−
0
0.5
1
1.5
2
8
P'
Data
theory DHMV
theory JC
ATLAS
-1
= 8 TeV, 20.3 fb
s Figure 11. The measured values of P1, P ′
4, P ′
5, P ′
6, P ′
8 compared with predictions from the
theoretical calculations discussed in the text (section 8). Statistical and total uncertainties are
shown for the data, i.e. the inner mark indicates the statistical uncertainty and the total error bar
the total uncertainty. QCD factorisation is used by DHMV and JC, where the latter focus on the impact
of long-distance corrections using a helicity amplitude approach. The CFFMPSV group
takes a different approach, using the QCD factorisation framework to perform compatibility
checks of the LHCb data with theoretical predictions. This approach also allows informa-
tion from a given experimentally measured parameter of interest to be excluded in order to
make a fit-based prediction of the expected value of that parameter from the rest of the data. – 22 – With the exception of the P ′
4 and P ′
5 measurements in q2 ∈[4.0, 6.0] GeV2 and P ′
8 in
q2 ∈[2.0, 4.0] GeV2 there is good agreement between theory and measurement. The P ′
4 and
P ′
5 parameters have statistical correlation of 0.37 in the q2 ∈[4.0, 6.0] GeV2 bin. The ob-
served deviation from the SM prediction of P ′
4 and P ′
5 is for both parameters approximately
2.7 standard deviations (local) away from the calculation of DHMV for this bin. 8
Comparison with theoretical computations The devi-
ations are less significant for the other calculation and the fit approach. All measurements
are found to be within three standard deviations of the range covered by the different pre-
dictions. Hence, including experimental and theoretical uncertainties, the measurements
presented here are found to agree with the predicted SM contributions to this decay. JHEP10(2018)047 9
Conclusion The results of an angular analysis of the rare decay B0
d →K∗µ+µ−are presented. This
flavour-changing neutral current process is sensitive to potential new-physics contributions. The B0
d →K∗µ+µ−analysis presented here uses a total of 20.3 fb−1 of √s = 8 TeV pp col-
lision data collected by the ATLAS experiment at the LHC in 2012. An extended unbinned
maximum-likelihood fit of the angular distribution of the signal decay is performed in order
to extract the parameters FL, Si and P (′)
j
in six bins of q2. Three of these bins overlap in
order to report results in ranges compatible with other experiments and phenomenology
studies. All measurements are found to be within three standard deviation of the range
covered by the different predictions. The results are also compatible with the results of the
LHCb, CMS and Belle collaborations. A
Correlation matrices Four folding schemes are applied to the data in order to extract FL, S3, S4, S5, S7 and S8
from four separate fits. The P (′) parameters are subsequently derived from the fit results
using equations (2.2)–(2.5). It is not possible to extract a full correlation matrix between
fitted parameters obtained from different fits. In order to reconstruct the correlation ma-
trix, ensembles of pseudo-experiments are simulated using the pdf corresponding to the
nominal angular distributions. Each simulated ensemble has the four folding schemes ap-
plied to it and four fits are performed on the resulting samples. The distributions obtained
for pairs of parameters obtained from fits to these ensembles are used to compute Pearson
correlation coefficients for those pairs. Correlation matrices for FL and the S parameters
are reconstructed from all possible pairings for a given q2 bin. A similar method is used
to extract the correlation matrices for the P (′) parameters. This procedure is repeated for
each q2 bin studied in order to obtain correlation matrices given in the remainder of this
appendix. The correlation matrices are statistical only. Contributions from systematic
uncertainties are not included, since the measurement precision is statistically limited. • Table 4 (5) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [0.04, 2.0] GeV2. • Table 6 (7) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [2.0, 4.0] GeV2. • Table 8 (9) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [4.0, 6.0] GeV2. • Table 10 (11) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [0.04, 4.0] GeV2. • Table 10 (11) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [0.04, 4.0] GeV2. • Table 12 (13) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [1.1, 6.0] GeV2. • Table 12 (13) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [1.1, 6.0] GeV2. • Table 14 (15) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [0.04, 6.0] GeV2. Acknowledgments We thank CERN for the very successful operation of the LHC, as well as the support staff
from our institutions without whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Aus-
tralia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and
FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST
and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR,
Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DRF/IRFU, France;
SRNSFG, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong
SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS,
Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland;
FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation;
JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF, South
Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and
Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United
Kingdom; DOE and NSF, United States of America. In addition, individual groups and
members have received support from BCKDF, the Canada Council, CANARIE, CRC,
Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC,
ERDF, FP7, Horizon 2020 and Marie Sk lodowska-Curie Actions, European Union; In-
vestissements d’Avenir Labex and Idex, ANR, R´egion Auvergne and Fondation Partager – 23 – le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia
programmes co-financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel;
BRF, Norway; CERCA Programme Generalitat de Catalunya, Generalitat Valenciana,
Spain; the Royal Society and Leverhulme Trust, United Kingdom. The crucial computing support from all WLCG partners is acknowledged gratefully,
in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF
(Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF
(Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (U.K.) and BNL
(U.S.A.), the Tier-2 facilities worldwide and large non-WLCG resource providers. Ma-
jor contributors of computing resources are listed in ref. [35]. JHEP10(2018)047 Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2 A
Correlation matrices • Table 14 (15) shows the statistical correlation matrix for FL and S (P (′)) parameters
for the q2 bin [0.04, 6.0] GeV2. – 24 – FL
S3
S4
S5
S7
S8
FL
1.00
0.11
−0.13
0.03
0.16
0.24
S3
1.00
0.31
0.28
0.73
0.45
S4
1.00
0.58
0.19
0.22
S5
1.00
0.14
0.28
S7
1.00
0.59
S8
1.00
Table 4. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[0.04, 2.0] GeV2. FL
S3
S4
S5
S7
S8
FL
1.00
0.11
−0.13
0.03
0.16
0.24
S3
1.00
0.31
0.28
0.73
0.45
S4
1.00
0.58
0.19
0.22
S5
1.00
0.14
0.28
S7
1.00
0.59
S8
1.00
Table 4. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[0.04, 2.0] GeV2. Table 4. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[0.04, 2.0] GeV2. JHEP10(2018)047 P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
0.04
0.05
0.62
0.32
P ′
4
1.00
0.53
−0.08
−0.06
P ′
5
1.00
0.00
0.22
P ′
6
1.00
0.55
P ′
8
1.00
Table 5. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 2.0] GeV2. able 5. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 2.0] GeV2. Table 5. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 2.0] GeV2. FL
S3
S4
S5
S7
S8
FL
1.00
0.27
0.35
−0.04
−0.15
−0.37
S3
1.00
−0.08
−0.44
−0.09
−0.20
S4
1.00
0.60
−0.02
−0.12
S5
1.00
−0.11
−0.20
S7
1.00
0.63
S8
1.00
Table 6. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[2.0, 4.0] GeV2. Table 6. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[2.0, 4.0] GeV2. Table 6. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[2.0, 4.0] GeV2. P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
−0.12
−0.21
0.05
0.05
P ′
4
1.00
0.51
0.08
0.03
P ′
5
1.00
−0.23
0.22
P ′
6
1.00
0.66
P ′
8
1.00
Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. Table 7. A
Correlation matrices Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. Table 7. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[2.0, 4.0] GeV2. – 25 – FL
S3
S4
S5
S7
S8
FL
1.00
0.33
−0.18
0.04
0.22
0.28
S3
1.00
0.15
0.23
0.60
0.05
S4
1.00
0.52
0.03
0.01
S5
1.00
0.28
0.27
S7
1.00
0.60
S8
1.00
Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[4.0, 6.0] GeV2. FL
S3
S4
S5
S7
S8
FL
1.00
0.33
−0.18
0.04
0.22
0.28
S3
1.00
0.15
0.23
0.60
0.05
S4
1.00
0.52
0.03
0.01
S5
1.00
0.28
0.27
S7
1.00
0.60
S8
1.00
Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[4.0, 6.0] GeV2. Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[4.0, 6.0] GeV2. Table 8. Statistical correlation matrix for the FL and S parameters obtained for q2
∈
[4.0, 6.0] GeV2. JHEP10(2018)047 P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
0.11
0.34
0.41
0.16
P ′
4
1.00
0.37
0.06
0.04
P ′
5
1.00
0.39
0.33
P ′
6
1.00
0.62
P ′
8
1.00
Table 9. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[4.0, 6.0] GeV2. P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
0.11
0.34
0.41
0.16
P ′
4
1.00
0.37
0.06
0.04
P ′
5
1.00
0.39
0.33
P ′
6
1.00
0.62
P ′
8
1.00
Table 9. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[4.0, 6.0] GeV2. Table 9. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[4.0, 6.0] GeV2. FL
S3
S4
S5
S7
S8
FL
1.00
0.08
0.05
0.01
0.18
0.14
S3
1.00
−0.04
0.03
0.29
−0.16
S4
1.00
0.79
0.08
0.03
S5
1.00
0.03
−0.02
S7
1.00
0.60
S8
1.00
Table 10. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈
[0.04, 4.0] GeV2. Table 10. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈
[0.04, 4.0] GeV2. Table 10. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈
[0.04, 4.0] GeV2. A
Correlation matrices P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
−0.07
0.00
0.21
0.12
P ′
4
1.00
0.78
0.08
0.02
P ′
5
1.00
0.03
−0.04
P ′
6
1.00
0.59
P ′
8
1.00
Table 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
−0.07
0.00
0.21
0.12
P ′
4
1.00
0.78
0.08
0.02
P ′
5
1.00
0.03
−0.04
P ′
6
1.00
0.59
P ′
8
1.00
Table 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. able 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. Table 11. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 4.0] GeV2. – 26 – FL
S3
S4
S5
S7
S8
FL
1.00
0.05
0.00
0.07
0.18
−0.03
S3
1.00
0.41
0.46
0.32
−0.01
S4
1.00
0.60
0.09
0.03
S5
1.00
0.17
0.24
S7
1.00
0.67
S8
1.00
Table 12. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈
[1.1, 6.0] GeV2. Table 12. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈
[1.1, 6.0] GeV2. JHEP10(2018)047 P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
0.23
−0.09
0.08
−0.07
P ′
4
1.00
0.53
0.15
0.08
P ′
5
1.00
0.28
0.24
P ′
6
1.00
0.67
P ′
8
1.00
Table 13. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[1.1, 6.0] GeV2. able 13. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[1.1, 6.0] GeV2. Table 13. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[1.1, 6.0] GeV2. FL
S3
S4
S5
S7
S8
FL
1.00
0.03
0.01
−0.10
0.13
0.06
S3
1.00
−0.02
−0.09
0.32
−0.01
S4
1.00
0.68
0.00
0.04
S5
1.00
−0.05
0.03
S7
1.00
0.65
S8
1.00
Table 14. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈
[0.04, 6.0] GeV2. Table 14. Statistical correlation matrix for the FL and S parameters obtained for q2
[0.04, 6.0] GeV2. Table 14. Statistical correlation matrix for the FL and S parameters obtained for q2 ∈
[0.04, 6.0] GeV2. Table 15. Statistical correlation matrix for the P (′) parameters obtained for q2 ∈[0.04, 6.0] GeV References [1] Belle collaboration, J.T. Wei et al., Measurement of the Differential Branching Fraction
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Correlation matrices P1
P ′
4
P ′
5
P ′
6
P ′
8
P1
1.00
−0.02
−0.14
0.17
0.04
P ′
4
1.00
0.65
0.00
0.05
P ′
5
1.00
−0.06
0.09
P ′
6
1.00
0.61
P ′
8
1.00
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Girolamo35, B. Di Girolamo35, B. Di Micco72a,72b, R. Di Nardo35, K.F. Di Petrillo57, A. Di Simone50, R. Di Sipio165, D. Di Valentino33, C. Diaconu99, M. Diamond165, F.A. Dias3 A. Di Simone50, R. Di Sipio165, D. Di Valentino33, C. Diaconu99, M. Diamond165, F.A. Dias39 Dita27b, S. Dita27b, F. Dittus35, F. Djama99, T. Djobava157b, J.I. Djuvsland59a, P. Dita27b, S. Dita27b, F. Dittus35, F. Djama99, T. Djobava157b, J.I. Djuvsland59a, M.A.B. Do Vale78c, D. Dobos35, M. Dobre27b, C. Doglioni94, J. Dolejsi139, Z. Dolezal139, M. Donadelli78d, S. Donati69a,69b, P. Dondero68a,68b, J. Donini37, M. D’Onofrio88, J. Dopke A. Doria67a, M.T. Dova86, A.T. Doyle55, E. Drechsler51, M. Dris10, Y. Du58b, arte-Campderros159, A. Dubreuil52, E. Duchovni178, G. Duckeck112, A. Ducourthial132, J. Duarte-Campderros159, A. Dubreuil52, E. Duchovni178, G. Duckeck112, A. Ducourthial132 O.A. Ducu107,x, D. Duda118, A. Dudarev35, A.C. Dudder97, E.M. Duffield18, L. Duflot128, M. D¨uhrssen35, M. Dumancic178, A.E. Dumitriu27b,e, A.K. The ATLAS collaboration Ch
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M. Dano Hoffmann142, V. Dao152, G. Darbo53b, S. Darmora8, J. Dassoulas3, A. Dattagupta127, M. Dano Hoffmann142, V. Dao152, G. Darbo53b, S. Darmora8, J. Dassoulas3, A. Dattagupta127, T. Daubney44, S. D’Auria55, W. Davey24, C. David44, T. Davidek139, D.R. Davis47, JHEP10(2018)047 JHEP10(2018)047 P. Davison92, E. Dawe102, I. Dawson146, K. De8, R. De Asmundis67a, A. De Benedetti124, P. Davison92, E. Dawe102, I. Dawson146, K. De8, R. De Asmundis67a, A. De Benedetti124, S. De Castro23b,23a, S. De Cecco132, N. De Groot117, P. de Jong118, H. De la Torre104, F. De Lorenzi76, A. De Maria51,u, D. De Pedis70a, A. De Salvo70a, U. The ATLAS collaboration Duncan55, M. Dunford59a, H. Duran Yildiz4a, M. D¨uren54, A. Durglishvili157b, D. Duschinger46, B. Dutta44, D. Duvn M. Dyndal44, B.S. Dziedzic82, C. Eckardt44, K.M. Ecker113, R.C. Edgar103, T. Eifert35, G. Eigen17, K. Einsweiler18, T. Ekelof170, M. El Kacimi34c, R. El Kosseifi99, V. Ellajosyula99 M. Ellert170, S. Elles5, F. Ellinghaus180, A.A. Elliot174, N. Ellis35, J. Elmsheuser29, M. Elsing35 M. Ellert 70, S. Elles5, F. Ellinghaus 80, A.A. Elliot 7 , N. Ellis35, J. Elmsheuser 9, M. Elsing3
D. Emeliyanov141, Y. Enari161, O.C. Endner97, J.S. Ennis176, J. Erdmann45, A. Ereditato20, D. Emeliyanov141, Y. Enari161, O.C. Endner97, J.S. Ennis176, J. Erdmann45, A. Ereditato20, M. Ernst29, S. Errede171, M. Escalier128, C. Escobar172, B. Esposito49, O. Estrada Pastor172, A.I. Etienvre142, E. Etzion159, H. Evans63, A. Ezhilov134, M. Ezzi34e, F. Fabbri23b,23a, J. Faltova35, Y. Fang15a, M. Fanti66a,66b, A. Farbin8, A. Farilla72a, C. Farina135, M. Farina68a,68b, T. Farooque104, S. Farrell18, S.M. Farrington176, P. Farthouat35, F. Fassi34e, P. Fassnacht35, D. Fassouliotis9, M. Faucci Giannelli91, A. Favareto53b,53a, W.J. Fawcett131, L. Fayard128, O.L. Fedin134,q, W. Fedorko173, S. Feigl130, L. Feligioni99, C. Feng58b, E.J. Feng3 L. Fayard
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P. Fleischmann103, R.R.M. Fletcher133, T. Flick180, B.M. Flierl112, L.R. Flores Castillo61a, M.J. Flowerdew113, G.T. Forcolin98, A. Formica142, F.A. F¨orster14, A.C. Forti98, A.G. Foster21 D. Fournier128, H. Fox87, S. Fracchia146, P. Francavilla132, M. Franchini23b,23a, S. Franchino59a D. Francis35, L. Franconi130, M. Franklin57, M. Frate169, M. Fraternali68a,68b, D. Freeborn92, S.M. Fressard-Batraneanu35, B. Freund107, D. Froidevaux35, J.A. Frost131, C. Fukunaga162, T. Fusayasu114, J. Fuster172, C. Gabaldon56, O. Gabizon158, A. Gabrielli23b,23a, A. Gabrielli18, B. Galhardo136a,136c, E.J. Gallas131, B.J. Gallop141, P. Gallus138, G. Galster39, K.K. Gan122, S. Ganguly37, Y. Gao88, Y.S. Gao150,m, C. Garc´ıa172, J.E. Garc´ıa Navarro172, JHEP10(2018)047 JHEP10(2018)047 J.A. Garc´ıa Pascual15a, M. Garcia-Sciveres18, R.W. Gardner36, N. Garelli150, V. Garonne130, A. Gascon Bravo44, K. Gasnikova44, C. Gatti49, A. Gaudiello53b,53a, G. Gaudio68a, I.L. The ATLAS collaboration Gavrilenko108, C. Gay173, G. Gaycken24, E.N. Gazis10, C.N.P. Gee141, J. Geisen51 M. Geisen97, M.P. Geisler59a, K. Gellerstedt43a,43b, C. Gemme53b, M.H. Genest56, C. Geng103 S. Gentile70a,70b, C. Gentsos160, S. George91, D. Gerbaudo14, A. Gershon159, G. Gessner45, S. Gentile70a,70b, C. Gentsos160, S. George91, D. Gerbaudo14, A. Gershon159, G. Gessner45,
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L. Han58a, S. Han15d, K. Hanagaki79,w, K. Hanawa161, M. Hance143, B. Haney133, P. Hanke59 J.B. Hansen39, J.D. Hansen39, M.C. Hansen24, P.H. Hansen39, K. Hara167, A.S. Hard17 T. Harenberg180, F. Hariri128, S. Harkusha105, R.D. Harrington48, P.F. Harrison176, N.M. Hartmann112, M. Hasegawa80, Y. Hasegawa147, A. Hasib48, S. Hassani142, S. Haug20, R. Hauser104, L. Hauswald46, L.B. Havener38, M. Havranek138, C.M. Hawkes21, R.J. Hawkings g
D. Hayakawa163, D. Hayden104, C.P. Hays131, J.M. Hays90, H.S. Hayward88, S.J. Haywood141, S.J. Head21, T. Heck97, V. Hedberg94, L. Heelan8, S. Heer24, K.K. Heidegger50, S. Heim4 T. Heim18, B. Heinemann44,ar, J.J. Heinrich112, L. Heinrich121, C. Heinz54, J. Hejbal137, – 33 – L. Helary35, A. Held173, S. Hellman43a,43b, C. Helsens35, R.C.W. Henderson87, Y. Heng179, S. Henkelmann173, A.M. Henriques Correia35, S. Henrot-Versille128, G.H. Herbert19, H. Herde2 V. Herget175, Y. Hern´andez Jim´enez32c, H. Herr97, G. Herten50, R. Hertenberger112, L. Hervas g
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T.C. Herwig133, G.G. Hesketh92, N.P. Hessey166a, J.W. Hetherly41, S. Higashino79, E. Hig´on-Rodriguez172, K. Hildebrand36, E. Hill174, J.C. Hill31, K.H. Hiller44, S.J. Hillier21, M. Hils46, I. Hinchliffe18, M. Hirose50, D. Hirschbuehl180, B. Hiti89, O. Hladik137, X. Hoad48, J. Hobbs152, N. Hod166a, M.C. Hodgkinson146, P. Hodgson146, A. Hoecker35, M.R. Hoeferkamp116, F. Hoenig112, D. Hohn24, T.R. Holmes36, M. Homann45, S. Honda167, T. Honda79, T.M. Hong135, B.H. Hooberman171, W.H. Hopkins127, Y. Horii115, A.J. Horton149
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awamura51, E.F. Kay88, V.F. Kazanin120b,120a, R. Keeler174, R. Kehoe41, J.S. Keller33, G. Kawamura51, E.F. Kay88, V.F. Kazanin120b,120a, R. Keeler174, R. Kehoe41, J.S. Keller33 J.J. Kempster91, J. Kendrick21, H. Keoshkerian165, O. Kepka137, S. Kersten180, B.P. Kerˇsev Kempster91, J. Kendrick21, H. Keoshkerian165, O. Kepka137, S. Kersten180, B.P. Kerˇsevan89, R.A. Keyes101, M. Khader171, F. Khalil-Zada13, A. Khanov125, A.G. Kharlamov120b,120a, Keyes101, M. Khader171, F. Khalil-Zada13, A. Khanov125, A.G. Kharlamov120b,120a, harlamova120b,120a, A. Khodinov164, T.J. Khoo52, V. Khovanskiy109,*, E. Khramov77, J. Khubua157b, S. Kido80, C.R. Kilby91, H.Y. Kim8, S.H. Kim167, Y.K. Kim36, N. Kimura160, ubua157b, S. Kido80, C.R. Kilby91, H.Y. Kim8, S.H. Kim167, Y.K. Kim36, N. Kimura160, O.M. Kind19, B.T. King88, D. Kirchmeier46, J. Kirk141, A.E. Kiryunin113, T. Kishimoto161, M. Kind19, B.T. King88, D. Kirchmeier46, J. Kirk141, A.E. Kiryunin113, T. Kishimoto161,
* D. Kisielewska81a, V. Kitali44, K. Kiuchi167, O. Kivernyk5, E. Kladiva28b,*, D. Kisielewska81a, V. Kitali44, K. Kiuchi167, O. Kivernyk5, E. Kladiva28b,*, g
P. Klimek119, A. Klimentov29, R. Klingenberg45,*, T. Klingl24, T. Klioutchnikova35, P. The ATLAS collaboration Kluit118, P. Klimek119, A. Klimentov29, R. Klingenberg45,*, T. Klingl24, T. Klioutchnikova35, P. Kluit118, S. Kluth113, E. Kneringer74, E.B.F.G. Knoops99, A. Knue113, A. Kobayashi161, D. Kobayashi16 T. Kobayashi161, M. Kobel46, M. Kocian150, P. Kodys139, T. Koffas33, E. Koffeman118, M.K. K¨ohler178, N.M. K¨ohler113, T. Koi150, M. Kolb59b, I. Koletsou5, A.A. Komar108,*, Y. Komori161, T. Kondo79, N. Kondrashova58c, K. K¨oneke50, A.C. K¨onig117, T. Kono79,aq R. Konoplich121,am, N. Konstantinidis92, R. Kopeliansky63, S. Koperny81a, A.K. Kopp50, K. Korcyl82, K. Kordas160, A. Korn92, A.A. Korol120b,120a,ap, I. Korolkov14, E.V. Korolkova146 – 34 – O. Kortner113, S. Kortner113, T. Kosek139, V.V. Kostyukhin24, A. Kotwal47, A. Koulouris10, ,
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L I McClymont92 E F McDonald102 J A Mcfayden92 G Mchedlidze51 S J McMahon141 P.C. McNamara102, R.A. McPherson174,af, S. Meehan145, T.M. Megy50, S. Mehlha A. Mehta88, T. Meideck56, B. Meirose42, D. Melini172,h, B.R. Mellado Garcia32c, JHEP10(2018)047 JHEP10(2018)047 J.D. Mellenthin51, M. Melo28a, F. Meloni20, A. Melzer24, S.B. Menary98, L. Meng88, X.T. Meng103, A. Mengarelli23b,23a, S. Menke113, E. Meoni40b,40a, S. Mergelmeyer19, P. Mermod52, L. Merola67a,67b, C. Meroni66a, F.S. Merritt36, A. Messina70a,70b, J. Metc A.S. Mete169, C. Meyer133, J. Meyer118, J-P. Meyer142, H. Meyer Zu Theenhausen59a, ,
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P Reznicek139 R Rezvani107 R Richter113 S Richter92 E Richter Was81b O Ricken2 P. Reznicek139, R. Rezvani107, R. Richter113, S. Richter92, E. Richter-Was81b, O. Ricken24, M. Ridel132, P. Rieck113, C.J. Riegel180, J. Rieger51, O. Rifki124, M. Rijssenbeek152, A. Rimoldi68a,68b, M. Rimoldi20, L. Rinaldi23b, G. Ripellino151, B. Risti´c35, E. Ritsch35, I. Riu14,
f F. Rizatdinova125, E. Rizvi90, C. Rizzi14, R.T. Roberts98, S.H. Robertson101,af, A. Robichaud-Veronneau101, D. Robinson31, J.E.M. Robinson44, A. Robson55, E. Rocco97, C. Roda69a,69b, Y. Rodina99,aa, S. Rodriguez Bosca172, A. Rodriguez Perez14, D. Rodriguez Rodriguez172, S. Roe35, C.S. Rogan57, O. Røhne130, J. Roloff57, A. Romaniouk11
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(a)Department of Physics, Ankara University, Ankara; (b)Istanbul Aydin University, Istanbul;
(c)Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 4
(a)Department of Physics, Ankara University, Ankara; (b)Istanbul Aydin University, Istanbul;
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(a)Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul; (b)Istanbul Bilgi
University, Faculty of Engineering and Natural Sciences, Istanbul; (c)Department of Physics,
Bogazici University, Istanbul; (d)Department of Physics Engineering, Gaziantep University,
Gaziantep, Turkey 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan
14 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14 Institut de F´ısica d’Altes Energies (IFAE), Barcelona Institute of Science and Technology,
Barcelona, Spain 15
(a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b)Physics Department,
Tsinghua University, Beijing; (c)Department of Physics, Nanjing University, Nanjing; (d)University
of Chinese Academy of Science (UCAS), Beijing, China 15
(a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b)Physics Department,
Tsinghua University, Beijing; (c)Department of Physics, Nanjing University, Nanjing; (d)University
of Chinese Academy of Science (UCAS), Beijing, China – 40 – 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 17 Department for Physics and Technology, University of Bergen, Bergen, Norway 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley
CA, United States of America 19 Institut f¨ur Physik, Humboldt Universit¨at zu Berlin, Berlin, Germany Institut f¨ur Physik, Humboldt Universit¨at zu Berlin 20 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics,
University of Bern, Bern, Switzerland 21 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 22 Centro de Investigaci´ones, Universidad Antonio Nari˜no, Bogota, Colombia
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(a)Dipartimento di Fisica e Astronomia, Universit`a di Bologna, Bologna; (b)INFN Sezione di
Bologna, Italy 24 Physikalisches Institut, Universit¨at Bonn, Bonn, Germany JHEP10(2018)047 JHEP10(2018)047 25 Department of Physics, Boston University, Boston MA, United States of America 27
(a)Transilvania University of Brasov, Brasov; (b)Horia Hulubei National Institute of Physics and
Nuclear Engineering, Bucharest; (c)Department of Physics, Alexandru Ioan Cuza University of Iasi,
Iasi; (d)National Institute for Research and Development of Isotopic and Molecular Technologies,
Physics Department, Cluj-Napoca; (e)University Politehnica Bucharest, Bucharest; (f)West
University in Timisoara, Timisoara, Romania 27
(a)Transilvania University of Brasov, Brasov; (b)Horia Hulubei National Institute of Physics and
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(a)Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava;
(b)Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of
Sciences, Kosice, Slovak Republic 29 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 30 Departamento de F´ısica, Universidad de Buenos Aires, Buenos Aires, Argentina 31 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 32
(a)Department of Physics, University of Cape Town, Cape Town; (b)Department of Mechanical
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(a)Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies -
Universit´e Hassan II, Casablanca; (b)Centre National de l’Energie des Sciences Techniques
Nucleaires (CNESTEN), Rabat; (c)Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad,
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(e)Facult´e des sciences, Universit´e Mohammed V, Rabat, Morocco 34
(a)Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca; (b)Centre National de l’Energie des Sciences Techniques LPHEA-Marrakech; (d)Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda;
( ) 35 CERN, Geneva, Switzerland 36 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 36 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America Nevis Laboratory, Columbia University, Irvington NY, United States of America 38 Nevis Laboratory, Columbia University, Irvington NY, United States of America 39 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark
( )
( ) 40
(a)Dipartimento di Fisica, Universit`a della Calabria, Rende; (b)INFN Gruppo Collegato di Cosenz
Laboratori Nazionali di Frascati, Italy 41 Physics Department, Southern Methodist University, Dallas TX, United States of America 42 Physics Department, University of Texas at Dallas, Richardson TX, United States of America
43
( )
(b) 44 Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 45 Lehrstuhl f¨ur Experimentelle Physik IV, Technische Universit¨at Dortmund, Dor 45 Lehrstuhl fur Experimentelle Physik IV, Technische Universitat Dortmund, Dortmund, Germany
46 Institut f¨ur Kern- und Teilchenphysik, Technische Universit¨at Dresden, Dresden, Germany 46 Institut f¨ur Kern- und Teilchenphysik, Technische Universit¨at Dresden, Dresden, Germany 47 Department of Physics, Duke University, Durham NC, United States of America 48 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, Un 48 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom
49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy SUPA
School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom
49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 50 Physikalisches Institut, Albert-Ludwigs-Universit¨at Freiburg, Freiburg, Germany 51 II. The ATLAS collaboration Physikalisches Institut, Georg-August-Universit¨at G¨ottingen, G¨ottingen, Germany – 41 – 52 D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland 52 D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland
53
( )
(b) 53
(a)Dipartimento di Fisica, Universit`a di Genova, Genova; (b)INFN Sezione di Genova, Italy 54 II. The ATLAS collaboration Physikalisches Institut, Justus-Liebig-Universit¨at Giessen, Giessen, Germany 55 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56 LPSC, Universit´e Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge MA, United States
of America 58
(a)Department of Modern Physics and State Key Laboratory of Particle Detection and Electronics,
University of Science and Technology of China, Hefei; (b)Institute of Frontier and Interdisciplinary
Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong
University, Qingdao; (c)School of Physics and Astronomy, Shanghai Jiao Tong University,
KLPPAC-MoE, SKLPPC, Shanghai; (d)Tsung-Dao Lee Institute, Shanghai, China Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao; (c)School of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai; (d)Tsung-Dao Lee Institute, Shanghai, China JHEP10(2018)047 JHEP10(2018)047 59
(a)Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg;
(b)Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 59
(a)Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg;
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(b)Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany 60 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Jap 61
(a)Department of Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong;
(b)Department of Physics, University of Hong Kong, Hong Kong; (c)Department of Physics and
Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay
Kowloon, Hong Kong, China 62 Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 64
(a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b)ICTP, Trieste; (c)Dipartim
di Chimica Fisica e Ambiente Universit`a di Udine Udine Italy 64
(a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b)ICTP, Trieste; (c)Dipartimento 64
(a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b)
di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy 65
(a)INFN Sezione di Lecce; (b)Dipartimento di Matematica e Fisica, Universit`a del Salento, Lecce,
Italy 66
(a)INFN Sezione di Milano; (b)Dipartimento di Fisica, Universit`a di Milano, Milano, Italy 67
(a)INFN Sezione di Napoli; (b)Dipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 68
(a)INFN Sezione di Pavia; (b)Dipartimento di Fisica, Universit`a di Pavia, Pavia, Italy N Sezione di Pisa; (b)Dipartimento di Fisica E. The ATLAS collaboration Ferm 69
(a)INFN Sezione di Pisa; (b)Dipartimento di Fisica E. The ATLAS collaboration Fermi, Universit`a di Pisa, Pisa, Italy 70
(a)INFN Sezione di Roma; (b)Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 71
(a)INFN Sezione di Roma Tor Vergata; (b)Dipartimento di Fisica, Universit`a di Roma Tor Verga
Roma, Italy 72
(a)INFN Sezione di Roma Tre; (b)Dipartimento di Matematica e Fisica, Universit`a Roma Tre,
Roma, Italy 73
(a)INFN-TIFPA; (b)Universit`a degli Studi di Trento, Trento, Italy 74 Institut f¨ur Astro- und Teilchenphysik, Leopold-Franzens-Universit¨at, Innsbruck, Austria versity of Iowa, Iowa City IA, United States of Ame 76 Department of Physics and Astronomy, Iowa State University, Ames IA 76 Department of Physics and Astronomy, Iowa State University, Ames IA, United States of America epartment of Physics and Astronomy, Iowa State Un 77 Joint Institute for Nuclear Research, Dubna, Russia 78
(a)Departamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de
Fora; (b)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (c)Universidade
Federal de S˜ao Jo˜ao del Rei (UFSJ), S˜ao Jo˜ao del Rei; (d)Instituto de F´ısica, Universidade de S˜ao
Paulo, S˜ao Paulo, Brazil 79 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 80 Graduate School of Science, Kobe University, Kobe, Japan uate School of Science, Kobe University, Kobe, Japan 81
(a)AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, 81
(a)AGH University of Science and Technology, Faculty of Physics and Applied Computer Science,
Krakow; (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland AGH University of Science and Technology, Faculty of Physics and Applied Computer Science,
Krakow; (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland
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Fukuoka, Japan – 42 – 86 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 86 Instituto de F´ısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina
87 Physics Department, Lancaster University, Lancaster, United Kingdom Instituto de Fısica La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina
87 Physics Department Lancaster University Lancaster United Kingdom 87 Physics Department, Lancaster University, Lancaster, United Kingdom 88 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 89 Department of Experimental Particle Physics, Joˇzef Stefan Institute and Department of Physics,
University of Ljubljana, Ljubljana, Slovenia University of Ljubljana, Ljubljana, Slovenia 90 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom f
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(IN2P3), Villeurbanne, France JHEP10(2018)047 JHEP10(2018)047 96 Departamento de F´ısica Teorica C-15 and CIAFF, Universidad Aut´onoma de Madrid, Madrid,
Spain 97 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 97 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 98 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 98 School of Physics and Astronomy, University of Manchester, Manc 99 CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 100 Department of Physics, University of Massachusetts, Amherst MA, United States of America 100 Department of Physics, University of Massachusetts, Amherst MA, U 101 Department of Physics, McGill University, Montreal QC, Canada 102 School of Physics, University of Melbourne, Victoria, Australia 103 Department of Physics, University of Michigan, Ann Arbor MI, United States of America
4 104 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States
of America 105 B.I. The ATLAS collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus
106 Research Institute for Nuclear Problems of Byelorussian State University Minsk Belarus 105 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 106 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 107 Group of Particle Physics, University of Montreal, Montreal QC, Canada 108 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 109 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 110 National Research Nuclear University MEPhI, Moscow, Russia Research Nuclear University MEPhI, Moscow, Russi 111 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow,
Russia 112 Fakult¨at f¨ur Physik, Ludwig-Maximilians-Universit¨at M¨unchen, M¨unchen, Germany 113 Max-Planck-Institut f¨ur Physik (Werner-Heisenberg-Institut), M¨unchen, Germany 114 Nagasaki Institute of Applied Science, Nagasaki, Japan 115 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
116 116 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States
of America 117 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University
Nijmegen/Nikhef, Nijmegen, Netherlands 118 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam,
Netherlands 119 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 119 Department of Physics, Northern Illinois University, DeKalb IL, United States of America
120
(a)Budker Institute of Nuclear Physics, SB RAS, Novosibirsk; (b)Novosibirsk State University
Novosibirsk, Russia 120
(a)Budker Institute of Nuclear Physics, SB RAS, Novosibirsk; (b)Novosibirsk State University
Novosibirsk, Russia 121 Department of Physics, New York University, New York NY, United States of America
122 121 Department of Physics, New York University, New York NY, United State 122 Ohio State University, Columbus OH, United States of America te University, Columbus OH, United States of Ameri 123 Faculty of Science, Okayama University, Okayama, Japan 124 Homer L. The ATLAS collaboration Petersburg, Russia 135 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of
America 136
(a)Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas - LIP; (b)Departamento de
F´ısica, Faculdade de Ciˆencias, Universidade de Lisboa, Lisboa; (c)Departamento de F´ısica,
Universidade de Coimbra, Coimbra; (d)Centro de F´ısica Nuclear da Universidade de Lisboa, Lisboa;
(e)Departamento de F´ısica, Universidade do Minho, Braga; (f)Departamento de F´ısica Teorica y del
Cosmos, Universidad de Granada, Granada (Spain); (g)Dep F´ısica and CEFITEC of Faculdade de
Ciˆencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal JHEP10(2018)047 JHEP10(2018)047 (e)Departamento de F´ısica, Universidade do Minho, Braga; (f)Departamento de F´ısica Teorica y del
Cosmos, Universidad de Granada, Granada (Spain); (g)Dep F´ısica and CEFITEC of Faculdade de Ciˆencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 137 Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 138 Czech Technical University in Prague, Prague, Czech Republic es University, Faculty of Mathematics and Physics, P 140 State Research Center Institute for High Energy Physics, NRC KI, Protvino, Russia 141 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 142 IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 143 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA,
United States of America 144
(a)Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b)Departamento
F´ısica, Universidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Chile 144
(a)Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b)Departamento de
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152 152 Departments of Physics and Astronomy, Stony Brook University, Stony Brook NY, United States
America 153 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 154 School of Physics, University of Sydney, Sydney, Australia 155 Institute of Physics, Academia Sinica, Taipei, Taiwan 156 Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan 157
(a)E. The ATLAS collaboration Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK,
United States of America 125 Department of Physics, Oklahoma State University, Stillwater OK, United States of America 126 Palack´y University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Republic 127 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America – 43 – 128 LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France 129 Graduate School of Science, Osaka University, Osaka, Japan 130 Department of Physics, University of Oslo, Oslo, Norway 131 Department of Physics, Oxford University, Oxford, United Kingdom 132 LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France
133 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America ,
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133 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 133 Department of Physics, University of Pennsylvania, Philadelphia PA, Unite 134 Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNP
St. The ATLAS collaboration Javakhishvili Tbilisi
Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia ics Institute, Tbilisi State University, Tbilisi, Georgi 158 Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 159 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv,
Israel 160 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 161 International Center for Elementary Particle Physics and Department of Physics, University of
Tokyo, Tokyo, Japan 162 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 163 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 164 Tomsk State University, Tomsk, Russia 164 Tomsk State University, Tomsk, Russia 165 Department of Physics, University of Toronto, Toronto ON, Canada 166
(a)TRIUMF, Vancouver BC; (b)Department of Physics and Astronomy, York University, Toronto
ON, Canada – 44 – 167 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and
Applied Sciences, University of Tsukuba, Tsukuba, Japan 168 Department of Physics and Astronomy, Tufts University, Medford MA, United States of America f
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America 170 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 171 Department of Physics, University of Illinois, Urbana IL, United States of America
172 171 Department of Physics, University of Illinois, Urbana IL, United States of America
172 Instituto de F´ısica Corpuscular (IFIC), Centro Mixto Universidad de Valencia - CSIC, Valencia,
Spain 172 Instituto de F´ısica Corpuscular (IFIC), Centro Mixto Universidad de Valencia - CSIC, Valencia,
Spain 173 Department of Physics, University of British Columbia, Vancouver BC, Canada 174 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada JHEP10(2018)047 JHEP10(2018)047 175 Fakult¨at f¨ur Physik und Astronomie, Julius-Maximilians-Universit¨at W¨urzburg, W¨urzburg,
Germany 176 Department of Physics, University of Warwick, Coventry, United Kingdom 176 Department of Physics, University of Warwick, Coventry, United Kingdom
177 Waseda University, Tokyo, Japan 177 Waseda University, Tokyo, Japan 177 Waseda University, Tokyo, Japan 178 Department of Particle Physics, Weizmann Institute of Science, Rehovot, Israel 179 Department of Physics, University of Wisconsin, Madison WI, United States of America
180 180 Fakult¨at f¨ur Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universit¨at
Wuppertal, Wuppertal, Germany 181 Department of Physics, Yale University, New Haven CT, United States of America 182 Yerevan Physics Institute, Yerevan, Armenia a Also at Department of Physics, University of Malaya, Kuala Lumpur, Malaysia b Also at Borough of Manhattan Community College, City University of New York, NY, United
States of America c Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South
Africa d Also at CERN, Geneva, Switzerland e Also at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France f Also at D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve,
Switzerland g Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain
h Also at Departamento de F´ısica Teorica y del Cosmos, Universidad de Granada, Granada (Spain),
Spain g Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain
h Also at Departamento de F´ısica Teorica y del Cosmos, Universidad de Granada, Granada (Spain)
Spain i Also at Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago, Chile j Also at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United
Arab Emirates j Also at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United
Arab Emirates k Also at Department of Financial and Management Engineering, University of the Aegean, Chios,
Greece k Also at Department of Financial and Management Engineering, University of the Aegean, Chios,
Greece l Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY, United
States of America l Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY, United
States of America m Also at Department of Physics, California State University, Fresno CA, United States of America n Also at Department of Physics, California State University, Sacramento CA, United States of
America o Also at Department of Physics, King’s College London, London, United Kingdom p Also at Department of Physics, Nanjing University, Nanjing, China q Also at Department of Physics, St. The ATLAS collaboration Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbil
Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 157
(a)E. Andronikashvili Institute of Physics, Iv. The ATLAS collaboration Petersburg State Polytechnical University, St. Petersburg,
Russia r Also at Department of Physics, Stanford University, United States of America s Also at Department of Physics, University of Fribourg, Fribourg, Switzerland t Also at Department of Physics, University of Michigan, Ann Arbor MI, United States of America Also at Department of Physics, University of Michigan, Ann Arbor MI, United States of America
u Also at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy u Also at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy u Also at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy – 45 – v Also at Giresun University, Faculty of Engineering, Giresun, Turkey w Also at Graduate School of Science, Osaka University, Osaka, Japan x Also at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania y Also at II. The ATLAS collaboration Physikalisches Institut, Georg-August-Universit¨at G¨ottingen, G¨ottingen, Germany z Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain ucio Catalana de Recerca i Estudis Avancats, ICREA aa Also at Institut de F´ısica d’Altes Energies (IFAE), Barcelona Institute of Science and Technology,
Barcelona, Spain ab Also at Institut f¨ur Experimentalphysik, Universit¨at Hamburg, Hamburg, Germany ac Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University
Nijmegen/Nikhef, Nijmegen, Netherlands ad Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest,
Hungary JHEP10(2018)047 JHEP10(2018)047 ae Also at Institute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics
and Particle Irradiation (MOE), Shandong University, Qingdao, China af Also at Institute of Particle Physics (IPP), Canada ag Also at Institute of Physics, Academia Sinica, Taipei, Taiwan ah Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan ai Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia aj Also at LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France ak Also at Louisiana Tech University, Ruston LA, United States of America al Also at LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris,
France am Also at Manhattan College, New York NY, United States of America an Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia an Also at Moscow Institute of Physics and Technology State University, Dolgopru ao Also at National Research Nuclear University MEPhI, Moscow, Russia at National Research Nuclear University MEPhI, Mo ap Also at Novosibirsk State University, Novosibirsk, Russia aq Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan as Also at School of Physics, Sun Yat-sen University, Guangzhou, China ol of Physics, Sun Yat-sen University, Guangzhou, C at Also at The City College of New York, New York NY, United States of America au Also at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China au Also at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China
av Also at Tomsk State University Tomsk and Moscow Institute of Physics and Technology State av Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State
University, Dolgoprudny, Russia aw Also at TRIUMF, Vancouver BC, Canada ax Also at Universita di Napoli Parthenope, Napoli, Italy – 46 –
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English
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The Influencing Factors of Taxpayer Compliance: Risk Preferences as a Moderating Variable
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International journal of academic research in accounting, finance and management sciences
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cc-by
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Abstract This research was conducted to examine and determine the factors that influence taxpayer compliance with
risk preference as a moderating variable. The independent variable used in this study is understanding of
taxation regulations, tax sanctions, and tax rates. The population in this study was 1,613,317 taxpayers
registered at SAMSAT Bekasi City. Sampling was done using the Accidental Sampling method on taxpayers
registered at SAMSAT Bekasi City which produced 99 samples. The data analysis technique used is
Structural Equation Modeling (SEM) using the Partial Least Square application with a significance level of
5%. The results of this study indicate that understanding of tax regulations, tax sanctions, and tax rates has
a significant effect on the level of compliance of taxpayers, risk preference does not significantly influence
the relationship between understanding of tax regulations on tax compliance levels and risk preference has
a significant effect on the relationship between tax sanctions with taxpayer compliance level. Key words
Understanding of Tax Regulations, Tax Sanctions, Tax Rates, Risk Preferences, Taxpayer Compliance
Received:
26 Jan 2020
© The Authors 2019
Revised:
30 Jan 2020
Published by Human Resource Management Academic Research Society (www.hrmars.com)
This article is published under the Creative Commons Attribution (CC BY 4.0) license. Anyone may
reproduce, distribute, translate and create derivative works of this article (for both commercial and
non-commercial purposes), subject to full attribution to the original publication and authors. The full
terms of this license may be seen at: http://creativecommons.org/licences/by/4.0/legalcode
Accepted:
04 Feb 2020
Published Online:
07 Feb 2020 y words
Understanding of Tax Regulations, Tax Sanctions, Tax Rates, Risk Preferences, Taxpayer Compliance http://dx.doi.org/10.6007/IJARAFMS/v9-i4/6845 (DOI: 10.6007/IJARAFMS/v9-i4/6845) The Influencing Factors of Taxpayer Compliance: Risk Preferences as a
Moderating Variable The Influencing Factors of Taxpayer Compliance: Risk Preferences as a
Moderating Variable
Tresia Risda Saragih1, Khoirul Aswar2
Faculty of Economics and Business, Universitas Pembangunan Nasional Veteran Jakarta, Jl. RS Fatmawati, Pondok
Labu, Jakarta, 1E-mail: tresiarisda1106@gmail.com, 2E-mail: aswar_law@yahoo.com
Abstract
This research was conducted to examine and determine the factors that influence taxpayer compliance with
risk preference as a moderating variable. The independent variable used in this study is understanding of
taxation regulations, tax sanctions, and tax rates. The population in this study was 1,613,317 taxpayers
registered at SAMSAT Bekasi City. Sampling was done using the Accidental Sampling method on taxpayers
registered at SAMSAT Bekasi City which produced 99 samples. The data analysis technique used is
Structural Equation Modeling (SEM) using the Partial Least Square application with a significance level of
5%. The results of this study indicate that understanding of tax regulations, tax sanctions, and tax rates has
a significant effect on the level of compliance of taxpayers, risk preference does not significantly influence
the relationship between understanding of tax regulations on tax compliance levels and risk preference has
a significant effect on the relationship between tax sanctions with taxpayer compliance level. Key words
Understanding of Tax Regulations, Tax Sanctions, Tax Rates, Risk Preferences, Taxpayer Compliance
Received:
26 Jan 2020
© The Authors 2019
Revised:
30 Jan 2020
Published by Human Resource Management Academic Research Society (www.hrmars.com)
This article is published under the Creative Commons Attribution (CC BY 4.0) license. Anyone may
reproduce, distribute, translate and create derivative works of this article (for both commercial and
non-commercial purposes), subject to full attribution to the original publication and authors. The full
terms of this license may be seen at: http://creativecommons.org/licences/by/4.0/legalcode
Accepted:
04 Feb 2020
blished Online:
07 Feb 2020
1. Introduction Tresia Risda Saragih1, Khoirul Aswar2 Tresia Risda Saragih1, Khoirul Aswar2
1,2Faculty of Economics and Business, Universitas Pembangunan Nasional Veteran Jakarta, Jl. RS Fatmawati, Pondok
Labu, Jakarta, 1E-mail: tresiarisda1106@gmail.com, 2E-mail: aswar_law@yahoo.com 1,2Faculty of Economics and Business, Universitas Pembangunan Nasional Veteran Jakarta, Jl. RS Fatmawati, Pondok
Labu, Jakarta, 1E-mail: tresiarisda1106@gmail.com, 2E-mail: aswar_law@yahoo.com Abstract 1. Introduction According to research results in the
field the reason Bekasi residents are in arrears in tax payments is because lack of awareness of paying taxes
and his financial condition (Tempo, 2019). Based on the above, the researcher wants to conduct a research related to tax compliance. One
reason for the low morale of a tax is a tax rate that is too high so it is burdensome for the mandatory tax. In
the study of Atawodi and Ojeka (2012) high tax rates is a factor the most important thing that causes non-
compliance with taxes. Tax rate motorized vehicles are regulated in Law No. 28 of 2009 article 6, namely
for first motor vehicle ownership is at least 1% (one percent) and the highest of 2% (two percent). There
are a number of studies related to the effect of tax rates on taxpayer compliance such as research
conducted by Cahyani and Naniek (2019), Tawas et al. (2016), Helhel and Helhel (2014), Atawodi and Ojeka
(2012) where the results of the study stated that Tax rates have a positive effect on taxpayer compliance. Results of the study inversely proportional to the research conducted by Mas'ud et al. (2014) which the
results of the study tax rates have a negative effect on taxpayer compliance, and research conducted by
Mustofa et al. (2016), Yusro and Kriswanto (2014) which stated that the tax rate is not affect the
compliance of taxpayers. In addition, an understanding of tax regulations is also a factor which affects taxpayer compliance. If
taxpayers understand more about taxation regulations, the more taxpayers will understand the sanctions
that will be accepted if they neglect their tax obligations (Ananda et al., 2015). There are a number of
studies related to understanding regulations taxation of taxpayer compliance which is the result of the
research understanding of tax regulations has a positive effect on mandatory compliance taxes (Cahyani
and Naniek 2019, Mareti and Dwimulyani 2019, Asrianti, 2018, Utari and Setiawan 2019, Ananda et al. 2015, Adiasa 2013). The results of the study are inversely proportional to research by Syah and
Krisidiyawati (2017), Andinata (2015), Mas'ud et al. (2014) in which the understanding of tax regulations
has no effect taxpayer compliance. Another factor that is considered to affect tax compliance is tax sanctions. 1. Introduction In general, tax is compulsory contributions paid by the people to a country which useful for the
benefit of government and society because tax is state revenue that will be used for the construction of
public facilities. Therefore, taxes are important for state income. National development is one of the
ongoing government activities continuously. State revenue in 2018 amounted to Rp1,942.3 trillion or
102.5% with the tax ratio reaches 11.5% of the increased Gross Domestic Product (GDP) by 0.8% from 2017
(Kemenkeu, 2019). Even so, the acceptance ratio tax in Indonesia is the lowest among the G-20 countries
and countries emerging markets. To be equal to developing countries, Indonesia needs to have a tax ratio
of 20% of GDP. To achieve this target the government seeks to reform taxation in various ways, one of
which is building tax compliance and awareness. level of compliance Indonesian people in paying taxes are
still very low compared to other countries, as a comparison of the tax ratio in Malaysia of 16%, the
Philippines 14%, Thailand 17%, South Korea 25%, South Africa 27%, and Brazil 34% (Detik Finance, 2018). One of the local taxes levied by the local government from the people is tax Motorized Vehicles in
2018, the tax target in Bekasi will reach Rp. 928,833,000,000, but the realization was 35.13% or Rp. 326,292,255,850, with a total of 1,459,933 units of motorized vehicles. In April 2018, 546,922 units of
vehicles have not been paid annual taxes motorized vehicles or 37.46% (Tribun, 2018). In January 2019, the
number of motorized vehicles was at Bekasi as many as 1,613,317 units, while the number of motorized 269 International Journal of Academic Research in Accounting, Finance and Management Sciences
Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) vehicles is as much 620,073 units are still in arrears on motor vehicle tax with the potential for mandatory
receivables the tax is estimated to be more than IDR 186,000,000,000. According to research results in the
field the reason Bekasi residents are in arrears in tax payments is because lack of awareness of paying taxes
and his financial condition (Tempo, 2019). vehicles is as much 620,073 units are still in arrears on motor vehicle tax with the potential for mandatory
receivables the tax is estimated to be more than IDR 186,000,000,000. 1. Introduction There are a number of
studies related to the effect of tax sanctions on taxpayer compliance conducted by Cahyani and Naniek
(2019), Mareti and Dwimulyani (2019), Asrianti (2018), Syah and Krisdiyawati (2017), Setiawan and Meliana
(2017), Tawas et al. (2016), Helhel and Helhel (2014), Ardyanto and Utaminingsih (2014) where the results
of the study stated that tax sanctions have a positive effect on taxpayer compliance. The results of this
study are inversely proportional to that of the study conducted by Rahmawati and Yulianto (2018),
Andinata (2015), Pranata et al. (2015) which states that tax sanctions do not affect the tax taxpayer
compliance. According to Alabede et al. (2011) things that must be considered in taxpayers making tax
payments is a risk that taxpayers must consider before making tax payments. Torgler (2003) conveys that
decision a taxpayer can be influenced by his behavior towards the risks faced. Based on the description that
has been stated above, the main problems are will be examined as follows: a) Does understanding of tax regulations affect on taxpayer compliance? b) Does the tax sanction affect on taxpayer compliance? c) Does the tax rate affect on taxpayer compliance? d) Does risk preference affect on taxpayer compliance? e) Does risk preference can moderate the relationship between understanding of tax regulations
with taxpayer compliance? e) Does risk preference can moderate the relationship between understanding of tax regulations
with taxpayer compliance? f) Does risk preference can moderate the relationship between tax sanctions with taxpayer
compliance? 2.1. Prospect theory Prospect theory is a theory that explains how one takes decisions in uncertain conditions. Prospect
theory was first developed by Daniel Kahneman and Amos Tversky in the early 1980s, where this theory is
includes two scientific disciplines namely psychology and economics which is an analysis a person's
behavior in making decisions between two choices. Related research uses prospect theory as a theoretical
basis for examines taxpayer compliance with risk preferences as moderation, which is Mareti and 270 International Journal of Academic Research in Accounting, Finance and Management Sciences
Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) Dwimulyani (2019), Irawati and Sari (2019), Tambun and Witriyanto (2018), Setiawan and Meliana (2017),
Ardyanto and Utaminingsih (2014), Adustom (2013), Aryobimo and Cahyonowati (2012), Alabede et al. (2011), and Torgler (2003). The relationship of this research to prospect theory is to explain preferences
risks can affect taxpayer compliance. If a taxpayer has high risk, the taxpayer will not necessarily pay the
obligation the tax because if the taxpayer has the risk seeking nature though taxpayers have a high risk
then it will not affect taxpayers to keep paying taxes, whereas taxpayers who have the risk aversion if
taxpayers have low risk, the taxpayers will avoid it tax liability. 3.1. Effects of Understanding of Taxation Regulations on Taxpayer Compliance An understanding of taxation is needed so that taxpayers understand, understand, to be able to
apply what is contained in the regulations listed. In research Cahyani and Naniek (2019), Mareti and
Dwimulyani (2019), Utari and Setiawan (2019), Asrianti (2018), Mustofa (2016), Ananda et al. (2015), dan
Adiasa (2013) examine the influence of understanding of regulations taxation of taxpayer compliance. The
results of previous studies stated that understanding of tax regulations has a significant effect on taxpayer
compliance. Based on the description above, a hypothesis can be formulated as follows: H1 = Understanding tax regulations has a significant on rates taxpayer compliance. 2.2. Theory Planned of Behavior Theory of planned behavior is a theory that emphasizes the rationality of human behavior is also in
the belief that the target behavior is below control of individual consciousness. According to Ajzen (1991),
individual behavior is influenced by individual's intention (behavioral intention) towards that particular
behavior. There are three Components in Theory Planned Behavior, include: Behavioral beliefs, individual
beliefs will result from a behavior and evaluation of these results (beliefs strength and outcome
evaluation), such as taxpayer awareness. In addition, normative beliefs, namely beliefs about the normative
expectations of others and motivation to meet these expectations (normative beliefs and motivation to
comply), like understanding tax regulations. Next, Control beliefs about the existence of things that support
or inhibit that behavior will be displayed (control beliefs), such as tax penalties and tax rates and
perceptions about how strong the things that support and hinder the behavior (perceived power). Barriers
that may arise when the behavior is displayed can come from within oneself and from the environment. This theory explains that taxpayers will comply or not in their obligations in the field of taxation by
considering the benefits and effects of his actions in paying taxes. In addition the taxpayer will consider the
information obtained from outside. Thus, taxpayers will be influenced by certain factors in His actions are
control of trust. Research that uses theory of planned behavior as a theoretical basis for examining
taxpayer compliance, Widanaputra and Ratnadi (2019) using the independent variable awareness of
taxpayers, obligations moral, and environment, Novianti and Dewi (2017) with independent variables which
used is tax amnesty, and Nugraheni and Purwanto (2015) with variables independent awareness of
taxpayers, tax knowledge and understanding, sanctions tax, the tax authority, the quality of tax services,
attributable justice, procedural justice, and interactional justice. 3. Development of Hypotheses and Research Models The development of hypotheses in this study is based on factors that are allegedly having an
influence on the level of tax compliance, so that it can formulate as follows: 3.6. Effects of Risk Preference on the Relationship between Tax Sanctions with taxpayer com One of the goals of tax sanctions is to create compliance with payments tax. According to Elin and
Susi (2019) tax sanctions are a deterrent to compulsory tax does not violate existing rules. When the
taxpayer does not pay tax the vehicle will risk being netted in zebra operations carried out by the
authorities the police, administrative fines. So with these risks it will affect taxpayer compliance. When
taxpayers have jobs that are not still, taxpayers are aware of existing sanctions if they do not comply with
existing regulations and will add to the burden because many risks will be faced. This is in line with
prospect theory which the risk of financial condition is able to influence tax compliance that will result in
tax penalties if you do not pay the tax. In the research Asrianti (2018) risk preferences affect the
relationship of tax sanctions with taxpayer compliance. Therefore, researchers are interested re-test the
hypothesis to find empirical evidence so that the hypothesis which was formulated in this research,
include: H6 = Risk preference has a significant effect on the relationship between tax sanctions with taxpayer
compliance H2 = Tax sanction has a significant effect on taxpayer compliance.
3.3. Effect of Tax Rates on Taxpayer Compliance The tax rate is the amount of value that must be paid by taxpayers to country through the fiscus. In
the study of Atawodi and Ojeka (2012) stated that exorbitant tax rate cause taxpayer non-compliance. In
the research of Cahyani and Naniek (2019), Tawas et al. (2016), Helhel and Helhel (2014), and Atawodi and
Ojeka (2012) examine the tax rate on taxpayer compliance. The results of previous studies stated that the
tax rate has significant effect taxpayer compliance. Based on the description above, it can be formulated
hypothesis as follows: H3 = Tax rates has a significant effect on taxpayer compliance. 3.2. Effects of Tax Sanctions on Taxpayer Compliance Tax sanctions must be given as a form of punishment for taxpayers who are breaking the rules will be
a deterrent so as not to repeat it again so creating an increase in tax compliance. In Cahyani and Naniek's
research (2019), Mareti and Dwimulyani (2019), Asrianti (2018), Mustofa et al. (2016), Syah and
Krisdiyawati (2017), Setiawan and Meliana (2017), Tawas et al. (2016), Helhel and Helhel (2014), Ardyanto
and Utaminingsih (2014) tested tax sanctions for taxpayer compliance. The results of previous studies
stated that tax sanctions have a significant effect on taxpayer compliance. Based on description above,
then the hypothesis can be formulated as follows: 271 International Journal of Academic Research in Accounting, Finance and Management Sciences
Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) 3.4. Effect of Risk Preference on Taxpayer Compliance According to Torgler (2003) Risk preferences can affect a person's decision taxpayers against the risks
faced. The risk of being the one considered taxpayers in paying taxes (Alabede et al., 2011). When risk
preferences have a strong relationship with taxpayer compliance, then the level of taxpayer compliance will
be low. In the research of Dwi et al. (2019) as well Aryobimo and Cahyonowati (2012) risk preferences have
a positive effect on taxpayer compliance. On the other hand in the research of Irawati and Sari (2019),
Setiawan and Meliana (2017), Adiasa (2013) risk preference has no effect on compliance taxpayer. Based
on the description, the hypothesis can be formulated as following: H4 = Risk preference has a significant effect on taxpayer compliance. 3.5. Effects of Risk Preference on Relationships Between Understanding of Tax Regulations with
taxpayer compliance Risk preference as a moderating variable which means risk preferences can be strengthen or weaken
the relationship between understanding of tax regulations with tax compliance. In dealing with risks that
are happens every taxpayer must have a decision to face a risk. When the taxpayer has problems in his
social life, such as lack information about taxation or changes in policy will affect taxpayer compliance. In
the research of Dwi et al. (2019), Utari and Setiawan (2019), Asrianti (2018) Risk preferences can
moderated understanding about tax regulations with taxpayer compliance. Therefore, researchers are
interested re-test the hypothesis to find empirical evidence so that the hypothesis which was formulated in
this research, include: H5 = Risk preference has a significant effect on the relationship between understanding of tax
regulations with taxpayer compliance. 4. Methodology of research The method in this study used a questionnaire survey to taxpayers registered in SAMSAT Bekasi. A
total of 99 questionnaires were returned. The hypothesis in this study was tested using Structural Equation 272 International Journal of Academic Research in Accounting, Finance and Management Sciences
Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) Modeling (SEM) by the Smart Partial Least Square (PLS) application. The dependent variable in this study is
taxpayer compliance measured using indicators adopted from research conducted by Rahmawati and
Yulianto (2018). The independent variable in this study was to understanding of tax regulations measured using
indicators adopted from Adiasa (2013), tax sanctions from Ardyanto and Utaminingsih (2014), and tax rates
from Cahyani and Naniek (2019). In addition, the moderating variable in this study is risk preference
measured using indicators adopted from Adiasa (2013). This study was measured using a Likert scale with 5
levels (1 = Strongly Disagree, 2 = Disagree, 3 = Neutral, 4 = Agree, 5 = Strongly Agree). Figure 1. Conceptual Framework
H4 Figure 1. Conceptual Framework 5. Results and discussions Results Hypothesis Test Path & Bootstrapping Algorithm The results of testing this hypothesis use the structural equation modeling (SEM) model using smart
PLS software version 3.0. Table 2 shows that understanding tax regulations (PPP, t = 2.230 and p-value =
0.026), tax penalties (SP, t = 5.119 and p-value = 0,000), and tax rates (TP, t = 2,346 and p- value = 0.019)
affects taxpayer compliance (KP), which means H1, H2, and H3 are accepted. Meanwhile, risk preference
(PR, t = 1,947 and p-value = 0.052), which means H4 is rejected. The risk preference in moderating the
understanding of tax regulations (PR. PPP, t = 0.157 and p-value = 0.875) does not affect taxpayer
compliance, which means H4 is rejected. Finally, risk preference in moderating tax sanctions (PR. SP, t =
2.045 and p-value = 0.041) affects taxpayer compliance. Understanding tax regulations has a significant
effect on taxpayer compliance. Understanding tax regulations has a significant effect on the level of tax compliance. This shows that
the higher the taxpayer understands the tax regulations that have been set; the taxpayer tends to be
obedient in paying his obligations. The results of this study are supported by research of Cahyani and
Naniek (2019), Elin and Susi (2019), Asrianti (2018), Mustofa et al. (2016), Post et al. (2015), and Adiasa
(2013) who state that understanding of regulations taxation has a significant effect on taxpayer
compliance. The results of this study are also in accordance with the theory of planned behavior whereby
understanding tax regulations as normative beliefs that mean beliefs about the normative expectations of
others and motivation to fulfill these expectations (normative beliefs and motivation to comply). In the relationship of tax sanctions to taxpayer compliance shows that tax sanctions significantly
influence tax compliance. This shows that the application of sanctions imposed on violating taxpayers will
cause a deterrent effect so as to increase taxpayer compliance. The results of this study are in line with
research by Cahyani and Naniek (2019), Elin and Susi (2019), Asrianti (2018), Mustofa et al. (2016), Asrofi
and Krisdiyawati (2017), Tawas et al. (2016), Helhel and Ahmed (2018) 2014), and Arif and Nanik (2014)
which state that tax sanctions have a significant effect on taxpayer compliance The results of this study are
in accordance with the theory of planned behavior where tax sanctions are linked as control beliefs. 5. Results and discussions Validity and reability tests are done before testing hypothesis. Test the validity by looking at the
extracted average variance (AVE) with suggested values above 0.5 and reability test with see the value of
Composite Reliability and Cronbach's Alpha. Composite reliability value for all constructs above 0.7 which
means all constructs in this study, it is estimated that it meets the criteria and values suggested in
Cronbach's Alpha is above 0.6 for all constructs (Ghozali, 2014). Besides that, in assessing a model with PLS
will begin by looking at R- Square (R2) for each dependent latent variable (Ghozali, 2014). Test results
validity and Reliability can be seen in the following table: Table 1. Validity and Reliability Test Results Table 1. Validity and Reliability Test Results Table 1. Validity and Reliability Test Results
R2 = 0,612
Variabel
AVE
Composite Reliability
Cronbach’s Alpha
KP
0,575
0,841
0,743
PPP
0,534
0,821
0,709
PR
0,502
0,922
0,909
SP
0,631
0,872
0,803
TP
0,715
0,834
0,607 In the table above, the lowest AVE value is 0.502 at moderating variable is risk preference. Then it
can be concluded that throughout variable is declared valid. Then, the lowest value in the Composite
Reliability value is 0.821 for the variable understanding of tax regulations so it can be concluded that all
variables in this study have good reliability of each construct and Cronbach’s value Alpha for the lowest is
0.607 at the tax rate so it can concluded that all variables in this study had a reliability good for each
construct. R2 test results tax compliance is equal to 0.612 thus indicating that the variable is latent
independent namely understanding tax regulations, tax sanctions, tax rates, and moderation variables is
risk preferences, can explain latent variables dependent: taxpayer compliance of 61.2% and the remaining
38.8% explained by other factors outside this research such as socialization taxation, taxpayer awareness,
and others. 273 International Journal of Academic Research in Accounting, Finance and Management Sciences
Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) Table 2. Results Hypothesis Test Path & Bootstrapping Algorithm
Hypothesis
Variable
Path Coefficient
T Statistics
P Values
H1
PPP -> KP
0,206
2,230
0,026
H2
SP -> KP
0,458
5,119
0,000
H3
TP -> KP
0,222
2,346
0,019
H4
PR -> KP
-0,119
1,947
0,052
H5
PR. PPP -> KP
-0,015
0,157
0,875
H6
PR. SP -> KP
0,193
2,045
0,041 Table 2. 5. Results and discussions In this
study, tax sanctions support the level of taxpayer compliance, effective and efficient tax sanctions will
increase taxpayer compliance in meeting obligations because it can provide a deterrent effect to any non-
compliant taxpayers. Implementation of strict sanctions will harm taxpayers so that taxpayers would prefer
to carry out their obligations. In addition, the relationship of tax rates to taxpayer compliance shows that
tax rates has a significant effect on taxpayer compliance. This shows that the application of tax rates in
accordance with established regulations will increase taxpayer compliance. The results of this study were
supported by Cahyani and Naniek (2019), Tawas et al. (2016), Helhel and Ahmed (2014), and Atawodi and
Ojeka (2012) who stated that tax rates had a significant effect on tax compliance. The results of this study
are consistent with the theory of planned behavior whereby the tax rate as a perception of behavioral
control. Variable risk preference for taxpayer compliance shows that risk preference has no significant effect
on taxpayer compliance. This shows that the taxpayers in the study sample accept the existing risks so that
risk preferences do not affect taxpayer compliance. The results of this study are not in line with the
research of Dwi et al. (2019) and Aryobimo and Cahyonowati (2012) risk preferences have a positive effect
on taxpayer compliance. On the other hand, in the research of Irawati and Sari (2019), Setiawan and
Meliana (2017), Adiasa (2013) risk preferences do not affect tax compliance. This is not in line with the
prospect theory that even though the taxpayer has a high risk, it will not affect the taxpayer to continue
paying taxes, while the taxpayer who has a risk aversion if the taxpayer has a low risk, the taxpayer will 274 International Journal of Academic Research in Accounting, Finance and Management Sciences
Vol. 9 (4), pp. 269–278, © 2019 HRMARS (www.hrmars.com) actually avoid his tax obligations. Then, the risk preference variable in moderating the relationship of
understanding of tax regulations to taxpayer compliance shows that risk preferences cannot moderate the
relationship of understanding of tax regulations towards taxpayer compliance with taxpayer compliance. This shows that the taxpayers sampled in this study understand the tax regulations but the existing risk
cannot influence a taxpayer's decision to pay his taxes so that the risk preference cannot moderate the
relationship of understanding the tax regulations to the level of tax compliance. 5. Results and discussions The results of this study
are not in line with the research of Dwi et al. (2019), Utari and Setiawan (2019), and Asrianti (2018). Risk
preferences can strengthen the relationship between understanding tax regulations and taxpayer
compliance. However, this study is in line with the study of Adiasa (2013) which states that risk preferences
cannot moderate the relationship of understanding tax regulations to taxpayer compliance. The results of
this study are not in accordance with prospect theory, where taxpayers will make their own decisions in
dealing with existing risks. So, risk preference has no effect on the relationship between understanding tax
regulations and the level of tax compliance. Finally, risk preference in moderating the relationship of tax sanctions to taxpayer compliance shows
that risk preferences have a significant effect in moderating the relationship of tax sanctions to taxpayer
compliance with taxpayer compliance. This shows that taxpayers who have a high risk will affect the
relationship of tax sanctions to the level of tax compliance. The public will comply with the regulations if
there are sanctions that are applied because sanctions as a regulatory tool so that taxpayers become more
orderly because if they violate the rules set then the risk of being sanctioned will be higher. The results of
this study are in line with research by Asrianti (2018) which states that risk preference influences the
relationship of tax sanctions with taxpayer compliance. The results of this study are in accordance with
prospect theory, where taxpayers will make their own decisions in dealing with existing risks depending on
the perspective of the taxpayer to see whether the risk preferences will affect positive or negative. 6. Conclusions This research begins with the problem of taxpayer compliance in Indonesia which is still low and
requires improvements to taxpayer compliance. Problems with taxpayer compliance also occur with motor
vehicle tax at SAMSAT Bekasi. This study aims to examine and determine the factors that influence
taxpayer compliance with risk preference as a moderating variable. The results of this study indicate that
understanding of tax regulations, tax sanctions, and tax rates has a significant effect on tax compliance. Meanwhile, risk preference has no effect on taxpayer compliance. Then, risk preference has no effect on
the relationship between understanding tax regulations and taxpayer compliance. However, risk
preference influences the relationship of tax sanctions with taxpayer compliance. This study has a
limitation that this study only uses taxpayer research objects that is registered in SAMSAT Bekasi and
cannot be generalized because of the limited time and busyness of taxpayers. In addition, the majority of
the sample in this study came from respondents with a senior high school education or less. Meanwhile,
the sample with master degree (S2) or doctoral degree (S3) is very small. Suggestions for this research for SAMSAT Bekasi are expected to be more active in providing
information to the whole community through tax socialization to improve tax knowledge and
understanding both directly and through the mass media regarding the importance of paying taxes on time,
increasing supervision with zebra operations, and improving services to minimize the existence of brokers. Then, for further research it is expected to be able to add other variables such as awareness of taxpayers,
taxation socialization, service quality of focus and use the taxation understanding variable as a moderating
variable and expand the object of research not only to one SAMSAT but to all SAMSAT in the Bekasi area so
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Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation
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PloS one
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Rationally Engineered Synthetic Coculture
for Improved Biomass and Product
Formation Suvi Santala*, Matti Karp, Ville Santala Suvi Santala*, Matti Karp, Ville Santala
Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland OPEN ACCESS Citation: Santala S, Karp M, Santala
V (2014) Rationally Engineered Synthetic
Coculture for Improved Biomass and Product
Formation. PLoS ONE 9(12): e113786. doi:10. 1371/journal.pone.0113786 Editor: Guo-Qiang Chen, Tsinghua University,
China Received: September 12, 2014
Accepted: October 30, 2014
Published: December 3, 2014 Copyright: 2014 Santala et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited. Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper
and its Supporting Information files. Funding: The project was funded by Academy of
Finland (project ProChassis 272602, URL: http://
www.aka.fi/fi/A/), and by Suomen Kulttuurirahasto
(URL: http://www.skr.fi/) to MK. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manu-
script. RESEARCH ARTICLE Abstract In microbial ecosystems, bacteria are dependent on dynamic interspecific
interactions related to carbon and energy flow. Substrates and end-metabolites are
rapidly converted to other compounds, which protects the community from high
concentrations of inhibitory molecules. In biotechnological applications, pure
cultures are preferred because of the more straight-forward metabolic engineering
and bioprocess control. However, the accumulation of unwanted side products can
limit the cell growth and process efficiency. In this study, a rationally engineered
coculture with a carbon channeling system was constructed using two well-
characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture
showed symbiotic nature in terms of substrate utilization and growth. Recombinant
protein production was used as a proof-of-principle example to demonstrate the
coculture utility and the effects on product formation. As a result, the biomass and
recombinant protein titers of E. coli were enhanced in both minimal and rich
medium simple batch cocultures. Finally, harnessing both the strains to the
production resulted in enhanced recombinant protein titers. The study
demonstrates the potential of rationally engineered cocultures for synthetic biology
applications. Engineered Synthetic Coculture for Improved Performance concentrations of substrates, intermediates, or other potentially harmful
compounds. Common end-metabolites, such as organic acids, are rapidly
converted to other compounds. Thus, tolerance to high concentrations of
inhibitory compounds is not essential for individual members of a community. In
biotechnological applications, however, engineered pure cultures are typically
utilized as production platforms because of the easier bioprocess control and
more straight-forward genetic engineering. Nevertheless, establishing a sustainable
and economical bioprocess is a challenge, for which metabolic engineering and
synthetic biology are seeking solutions [1–4]. For example, accumulation of
unwanted side products and inefficient carbon and energy fluxes are common
issues. Since Escherichia coli is a widely used host in bioprocesses, considerable
effort is dedicated to controlling the overflow metabolism. In this phenomenon,
carbon is excessively transported into the cells, and the capacity of tricarboxylic
acid (TCA) cycle is exceeded. As a result, the metabolism is shifted to produce
acetate from acetyl coenzyme A (CoA) [5]. The accumulation of acetate results in
the induction of the stress response, growth inhibition, significant carbon loss,
and eventually reduced productivity [6, 7]. Previous strategies to neglect the negative effects of acetate include both genetic
engineering of E. coli to circumvent the acetate production and comprehensive
optimization of fed-batch bioprocesses [8–10]. These approaches, however,
potentially decrease the growth rate or require a substantial amount of
optimization and engineering work. ‘‘Pure cocultures’’ typically consisting of two
known strains are a more recent approach to improve the production platforms
[11, 12], and a trend towards more controllable and tunable cocultures involving
systems biology and synthetic biology solutions is growing stronger [13, 14]. For
example, Eiteman et al. [15] have described the co-fermentation of hexoses and
pentoses by a community of engineered E. coli. Tsai et al. [16] established a system
for ethanol production from cellulose by a synthetic yeast community. Although
the obtained ethanol yields are low, the study is an elegant example of the
utilization of an engineered community for bioenergy production. Previous strategies to neglect the negative effects of acetate include both genetic
engineering of E. coli to circumvent the acetate production and comprehensive
optimization of fed-batch bioprocesses [8–10]. These approaches, however,
ll d
h
h
b
l
f The culture systems described above are based on exploiting strains of single
species, but they represent the first steps towards consolidated processing of
sustainable substrate to products by an engineered consortium. PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Introduction In a microbial ecosystem, bacterial species cooperate by producing metabolites
that serve as substrates for other species of a community. Dynamic and symbiotic
interactions between different species enable an efficient carbon and energy flow. Furthermore, the interactions balance rapid environmental changes and high Competing Interests: The authors have declared
that no competing interests exist. 1 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Strains In the initial coculture, E. coli K12 BW25113 (from Yale E. coli Genetic Stock
Center CGSC, Connecticut, USA) was used. The single gene knock-out strain of
A. baylyi ADP1 (DSM 24193) (gene deletion high-affinity gluconate permease,
gntT, ACIAD0544) was employed in the study (kindly provided by Dr. Veronique
de Berardinis, Genoscope, France). In the single gene knock-out mutant, the
target gene is replaced with a gene cassette containing a kanamycin resistance gene
(Kanr) [18]. The strain ADP1Dgnt::Kanr/tdk was transformed with a construct
pVKK81-T-lux, and designated as ABlux. In the first stage of GFP production, E. coli K12 BW25113 was transformed with sfGFP/pAK400c and designated as ECsf. In the second stage, both E. coli K12 BW25113 and A. baylyi ADP1Dgnt::Kanr/tdk
were transformed with a plasmid pBAV1C-T5-GFP, resulting in strains designated
as ECg and ABg, respectively. For monitoring the individual GFP production of E. coli, a control strain ADP1Dgnt::Kanr/tdk transformed with a plasmid pBAV1C-
ara without the insert was constructed, and the strain was designated as ABc. Genetic constructs are described in the section ‘‘plasmid construction and
transformations’’. The same strains without the plasmids were used as
background control strains for fluorescence measurement. Engineered Synthetic Coculture for Improved Performance engineered to produce recombinant protein, and batch cocultures with different
substrate concentrations were conducted to demonstrate the effects of accelerated
growth to recombinant protein production. Finally, both strains were engineered
to produce the same recombinant protein, and cocultivations in a bioreactor were
carried out. Employing
different strains of a single species broadens the engineering possibilities; however,
the strains often possess very similar carbon utilization patterns, thus leaving
unresolved the issues related to by-product accumulation and the inefficient
carbon flow. On the other hand, genetic engineering is very challenging for mixed
populations which efficiently degrade complex substrates and mixed sugars [17]. In the present study, the advantages of both mixed populations and readily
engineered model strains are exploited; the well-characterized model organisms E. coli K12 and Acinetobacter baylyi ADP1 were employed for the construction of a
synthetic and symbiotic microecosystem with directed carbon flow for improved
culture performance. In the first stage of the study, the strains were cultured in a
minimal medium batch cultivation to monitor the coculture compatibility,
performance of individual strains, and carbon flow. Secondly, E. coli strain was PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 2 / 16 Engineered Synthetic Coculture for Improved Performance [19] using primers 59-AATAGCTAGCCTGTAGAAAAGAGGAAGGAAATAA-
TAAATGGAGAAAAAAATCACTGGATATAC-39 and 59-TAATAGTGCACT-
TACGCCCCGCCCTGCCAC-39 with restriction sites NheI and ApaLI. For
expression of bacterial luciferase operon, a plasmid pVKK81-T-lux (V. Santala
and M. Karp, unpublished data) containing the bacterial luciferase genes
luxCDABE from Photorhabdus luminescens and tetracycline resistance gene (T)
was used. For ADP1, the plasmids pVKK81-T-lux, pBAV1C-T5-GFP, and
pBAV1C-ara without an insert [22] were naturally transformed by the method
described previously [23], and the colonies were selected from Luria-Agar (LA)
plates (1 g/l NaCl, 5 g/l yeast extract, 10 g/l tryptone, 15 g/l agar) containing
tetracycline (10 mg/ml) and chloramphenicol (25 mg/ml), respectively. For E. coli,
the plasmids sfGFP/pAK400c and pBAV1C-T5-GFP were transformed by
electroporation and the colonies were selected from LA plates containing
chloramphenicol (25 mg/ml). The constructs were verified by restriction analysis,
fluorescence, or luminescence determination. Medium composition and cultivations For the initial coculture, minimal salts medium MA/9 described previously (9)
was used. Glucose (,50 mM) was used as the initial carbon and energy source. The strains were cultivated in batch bottles (50 ml medium/250 ml Erlenmeyer
flasks) at 37˚C and 300 rpm both individually and in cocultures for 12 h. Two
parallel bottles were used for each culture. For study of the GFP production of E. coli both in monoculture and in
coculture at variable substrate concentrations, the strains ECg and ABc were
cultivated in 5 ml of a MA/9 medium supplemented with 50 mM, 100 mM, or
250 mM glucose and 100 mM IPTG at 30˚C and 300 rpm. The cultivations were
performed both individually and in cocultures in two parallel tubes for 24 h. For 0.5-litre batch cultivations in a bioreactor, the monoculture (ECg) and the
cocultures (ECg and ABg or ECg and ABc) were cultivated in a modified Luria-
Bertani medium (1 g/l NaCl, 5 g/l yeast extract, 10 g/l tryptone) supplemented
with 2 mM MgSO4, 0.5 mM CaCl2, 3 mM FeCl3 and 100 mM glucose, and 25 mg/
ml chloramphenicol. The cultivation was carried out in a 1-litre vessel (Sartorius
Biostat B plus Twin System, Germany) with an online pH monitoring system at
30˚C for 10 h. The cultures were aerated by stirring (350 rpm) and supply of
oxygen (O2 partial pressure set to 20%). Equal inoculum sizes for each strain were
used. Control cultivations without glucose supplementation were conducted in
250 ml batch bottles in otherwise similar conditions. Luminescence, fluorescence, and biomass determination Plasmid construction and transformations For digestions and ligations, the enzymes and buffers were provided by Fermentas
(Lithuania) and used according to the manufacturer’s instructions. The PCR
reagents were provided by Finnzymes (Finland) (DNA polymerase PhusionTM
and buffer) and Fermentas (nucleotides). The primers were obtained from
ThermoFisher Scientific (Germany) with appropriate restriction sites. The superfolder GFP variant (from BBa_I746909) was amplified with primers
59-GAGTTCTAGAGAAGGAGATATACATATGCGTAAAGGCGAAGAGCTGTT-
C-39 and 59-CTAGCAAGCTTAGTGGTGATGGTGATGATGTTTGTATAG-39
with restriction sites XbaI and HindIII, respectively, and cloned to pAK400c vector
[19, 20] under the IPTG-inducible lactose promoter, resulting in construct sfGFP/
pAK400c. To construct the plasmid pBAV1C-T5-GFP with chloramphenicol
resistance gene cat, the original plasmid pBAV1K-T5-GFP [21] was partially
amplified with PCR using primers 59-TAATAGCTAGCTATTTAAAGATACC-
CCAAGAAGCTAATTATAAC-39 and 59-TAATAGTGCACTCGCTTGGACTCC-
TGTTGATAG-39. The product was ligated with cat gene amplified from pAK400c For digestions and ligations, the enzymes and buffers were provided by Fermentas
(Lithuania) and used according to the manufacturer’s instructions. The PCR
reagents were provided by Finnzymes (Finland) (DNA polymerase PhusionTM
and buffer) and Fermentas (nucleotides). The primers were obtained from
ThermoFisher Scientific (Germany) with appropriate restriction sites. The superfolder GFP variant (from BBa_I746909) was amplified with primers
59-GAGTTCTAGAGAAGGAGATATACATATGCGTAAAGGCGAAGAGCTGTT-
C-39 and 59-CTAGCAAGCTTAGTGGTGATGGTGATGATGTTTGTATAG-39
with restriction sites XbaI and HindIII, respectively, and cloned to pAK400c vector
[19, 20] under the IPTG-inducible lactose promoter, resulting in construct sfGFP/
pAK400c. To construct the plasmid pBAV1C-T5-GFP with chloramphenicol
resistance gene cat, the original plasmid pBAV1K-T5-GFP [21] was partially
amplified with PCR using primers 59-TAATAGCTAGCTATTTAAAGATACC-
CCAAGAAGCTAATTATAAC-39 and 59-TAATAGTGCACTCGCTTGGACTCC-
TGTTGATAG-39. The product was ligated with cat gene amplified from pAK400c 3 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Analysis for glucose and end-metabolite concentrations Glucose consumption and acetate accumulation/consumption in the cultivations
were determined with high performance liquid chromatography (HPLC) as
described previously [24]. Identification and quantification of glucose and end-
metabolites were based on co-chromatography using external standards. The
mediums were used as controls. Engineered Synthetic Coculture for Improved Performance measured immediately with a Victor 2 plate reader (Perkin Elmer Life Sciences,
Finland). Finland). The total biomass in the bottles and in the bioreactor was determined by optical
density measurement at the wavelength 600 nm every hour (OD600). Also, in the
initial coculture and in the bioreactor experiments, the cells were plated every two
hours (starting from 2 or 5 h) on LA plates with and without kanamycin (30 mg/
ml) in order to determine the individual cell numbers of E. coli and A. baylyi
according to CFU (colony forming units). For bioreactor batch cultures, 40 ml
biomass samples were taken at the end of the cultivation, centrifuged at 30,000 g
for 30 min and freeze-dried for gravimetric dry cell weight (CDW) determination. For fluorescence determination, 1 ml samples were taken from all cultivations,
centrifuged for 1 min at 10,000 g, and suspended in 1 ml 10 mM Tris-HCl buffer
(pH 8.2) containing 150 mM NaCl. The samples for fluorescence measurements
were diluted to linear range and measured as four 10–50 ml parallel samples with
Fluoroskan Ascent FL (Thermo Labsystems, Finland) using the wavelengths
485 nm (excitation) and 538 nm (emission). The total biomass in the bottles and in the bioreactor was determined by optical
density measurement at the wavelength 600 nm every hour (OD600). Also, in the
initial coculture and in the bioreactor experiments, the cells were plated every two
hours (starting from 2 or 5 h) on LA plates with and without kanamycin (30 mg/
ml) in order to determine the individual cell numbers of E. coli and A. baylyi
according to CFU (colony forming units). For bioreactor batch cultures, 40 ml
biomass samples were taken at the end of the cultivation, centrifuged at 30,000 g
for 30 min and freeze-dried for gravimetric dry cell weight (CDW) determination. For fluorescence determination, 1 ml samples were taken from all cultivations,
centrifuged for 1 min at 10,000 g, and suspended in 1 ml 10 mM Tris-HCl buffer
(pH 8.2) containing 150 mM NaCl. The samples for fluorescence measurements
were diluted to linear range and measured as four 10–50 ml parallel samples with
Fluoroskan Ascent FL (Thermo Labsystems, Finland) using the wavelengths
485 nm (excitation) and 538 nm (emission). Luminescence, fluorescence, and biomass determination For luminescence measurements, samples were taken from the batch cultivations
every hour and 200 ml of a sample (diluted when necessary) was applied on white
microtiter well plates in three parallel samples. The luminescent signal was PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 4 / 16 Engineered Synthetic Coculture for Improved Performance tools are widely available for the strain [21, 30, 31]. A. baylyi ADP1 exhibits a
relatively wide substrate range and is known to efficiently utilize organic acids
such as acetate as a sole carbon and energy source [32], although the detailed
mechanism for the uptake and utilization of acetate has not yet been
characterized. Moreover, our recent studies suggest that ADP1 neither produces
harmful overflow metabolites nor exhibits substrate inhibition (S. Santala and V. Santala, unpublished data). In order to establish a functional symbiotic coculture system with directed
carbon flow, and to produce unambiguous data for analyzing the coculture
performance, a mutant strain of A. baylyi ADP1 made deficient of utilizing
glucose was employed; according to the metabolic model of ADP1, the disruption
of a high-affinity gluconate permease (gntT, ACIAD0544) blocks the glucose
pathway. Due to the exceptional glucose utilization pathway of ADP1, a glucose
molecule is oxidized to gluconate on the outer surface of the inner membrane by
an electron carrier associated to glucose dehydrogenase, pyrroloquinoline quinine
(PQQ). Subsequently, the high-affinity gluconate permease GntT transports
gluconate into cells [32]. Here, a knock-out mutant strain A. baylyi
ADP1DgntT::Kanr/tdk [18] was employed. When glucose is sufficiently available
in the coculture, E. coli cells uptake glucose into the cells in excess and return a
significant amount of the carbon into the medium in the form of acetate. Thus, in
a coculture, the glucose negative mutant strain of ADP1 is dependent on the end-
metabolites of E. coli, enabling an experimental validation of the carbon flow and
fate. A schematic illustration of the proposed carbon flow is presented in Figure 1. In order to establish a functional symbiotic coculture system with directed
carbon flow, and to produce unambiguous data for analyzing the coculture
performance, a mutant strain of A. baylyi ADP1 made deficient of utilizing
glucose was employed; according to the metabolic model of ADP1, the disruption
of a high-affinity gluconate permease (gntT, ACIAD0544) blocks the glucose
pathway. Due to the exceptional glucose utilization pathway of ADP1, a glucose
molecule is oxidized to gluconate on the outer surface of the inner membrane by
an electron carrier associated to glucose dehydrogenase, pyrroloquinoline quinine
(PQQ). Subsequently, the high-affinity gluconate permease GntT transports
gluconate into cells [32]. Here, a knock-out mutant strain A. baylyi ADP1DgntT::Kanr/tdk [18] was employed. When glucose is sufficiently available
in the coculture, E. Establishing a synthetic coculture In natural ecosystems, different species interact through diverse forms of
cooperation, such as mutualism, parasitism, or symbiosis, creating a complex
network of integrated metabolic pathways. From a synthetic biology point of view,
cocultures can provide several advantages over monocultures by performing
multi-step tasks and being more catabolically versatile [25–27]. In this study, a synthetic coculture was constructed using two strains of
different species, E. coli K12 and A. baylyi ADP1, and the effects of connecting the
carbon metabolism of these strains were investigated. For the construction of a
functional, synthetic coculture, the bacterial species for the community were
chosen based on characteristics supporting one another. To promote straight-
forward system design and engineering, the availability of readily applicable
engineering tools and well-characterized metabolic networks was considered
essential here [28, 29]. In microbiological terms, the strains also share similar
preferences for growth conditions. A. baylyi ADP1 has been recently introduced as a competitive model organism
for genetic studies [18] and metabolic engineering purposes [22, 23], and genetic A. baylyi ADP1 has been recently introduced as a competitive model organism
for genetic studies [18] and metabolic engineering purposes [22, 23], and genetic 5 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 coli cells uptake glucose into the cells in excess and return a
significant amount of the carbon into the medium in the form of acetate. Thus, in
a coculture, the glucose negative mutant strain of ADP1 is dependent on the end-
metabolites of E. coli, enabling an experimental validation of the carbon flow and
fate. A schematic illustration of the proposed carbon flow is presented in Figure 1. For a functional community, the compatibility of the two strains is evidently
essential. In order to monitor the performance and growth of ADP1DgntT::Kanr/
tdk in the coculture, pVKK81-T-lux containing a bacterial luciferase genes
luxCDABE (from P. luminescens [33]) were transformed into the strain by natural
transformation. A luminescent colony was selected from a plate containing 10 mg/
ml tetracycline. The resulting strain ADP1DgntT::Kanr/tdk[lux_tetr] was desig-
nated as ABlux. In order to monitor the growth dynamics of E. coli and ABlux, the
strains were cultivated both separately and in cocultures for 12 h in batch
cultures. A minimal salts medium MA/9 supplemented with 50 mM glucose was
used in the experiment. Biomass and luminescence were determined from the
cultivations hourly. Selective plate count was also applied in order to determine
the individual cell numbers of E. coli and ABlux. The total biomass of the
coculture and the proportion of ABlux of the total biomass are presented in
Figure 2. The use of luminescent construct enabled specific real-time monitoring
of ABlux performance during the cocultivation (Figure 2, the inlet), and allowed
the observation of potential fluctuations and problem situations in the cultures,
not readily detectable by other means. According to the luminescence signal data,
ABlux starts to grow in the coculture after a short lag phase, which was
subsequently confirmed by the 5-hour timepoint plate count. After 11 h of
cultivation, the strain reached an approximately 6% proportion of the total
biomass. 6 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Engineered Synthetic Coculture for Improved Performance Figure 1. The proposed carbon flow in the wild type Escherichia coli culture and in the coculture of
engineered Acinetobacter baylyi ADP1 and E. coli. A) E. coli culture supplied with excess glucose readily
shifts to an overflow metabolism (*), producing large amounts of acetate into the culture medium. Acetate
inhibits growth and reduces product formation, and carbon flow is directed off the product route. B) In a
coculture involving directed carbon flow, the strain A. PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 baylyi ADP1 is made deficient in glucose utilization by a
knock-out of gluconate permease gntT and is solely dependent on the end-metabolites (acetate) of E. coli. Carbon is further metabolized and can be directed to biomass and the product of interest. Metabolic pathways
in the figure are simplified and only the main products are shown. Figure 1. The proposed carbon flow in the wild type Escherichia coli culture and in the coculture of
engineered Acinetobacter baylyi ADP1 and E. coli. A) E. coli culture supplied with excess glucose readily
shifts to an overflow metabolism (*), producing large amounts of acetate into the culture medium. Acetate
inhibits growth and reduces product formation, and carbon flow is directed off the product route. B) In a
coculture involving directed carbon flow, the strain A. baylyi ADP1 is made deficient in glucose utilization by a
knock-out of gluconate permease gntT and is solely dependent on the end-metabolites (acetate) of E. coli. Carbon is further metabolized and can be directed to biomass and the product of interest. Metabolic pathways
in the figure are simplified and only the main products are shown. doi:10.1371/journal.pone.0113786.g001 Figure 2. The total biomass of the coculture of E. coli and ADP1DgntT::Kanr/tdk[lux_tetr] (ABlux), and
the proportion of ABlux in the total biomass. The strain ABlux is deficient in utilizing glucose. During the
cultivation, the growth of ABlux was monitored in real-time via a luminescence reporter luxCDABE (the inlet). The cells were cultivated in a minimal salts medium supplied with glucose for 12 h. The mean and standard
deviation of the two independent cultures are shown. Note the logarithmic scale in biomass and luminescence
y-axes. Total biomass – line with squares; proportion of ABlux cells in total biomass – columns. doi:10.1371/journal.pone.0113786.g002 Figure 2. The total biomass of the coculture of E. coli and ADP1DgntT::Kanr/tdk[lux_tetr] (ABlux), and
the proportion of ABlux in the total biomass. The strain ABlux is deficient in utilizing glucose. During the
cultivation, the growth of ABlux was monitored in real-time via a luminescence reporter luxCDABE (the inlet). The cells were cultivated in a minimal salts medium supplied with glucose for 12 h. The mean and standard
deviation of the two independent cultures are shown. Note the logarithmic scale in biomass and luminescence
y-axes. Total biomass – line with squares; proportion of ABlux cells in total biomass – columns. doi:10.1371/journal.pone.0113786.g002 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 7 / 16 Engineered Synthetic Coculture for Improved Performance with the total decrease in glucose concentration being 9 mM. Detectable amounts
of acetate could not be observed in the coculture until the 11 h timepoint,
indicating efficient acetate utilization by ABlux. The acetate concentration in the
end of the cultivation (2 mM) was lower compared to that of the monoculture of
E. coli. The strain ABlux did not exhibit growth in the monoculture due to the
lack of suitable carbon source, and neither consumption of glucose nor
accumulation of end-metabolites was detected (data not shown). We discovered that the coculture was beneficial for E. coli, as the cells grew
faster in the cocultivation compared to the monoculture in the studied
conditions. As expected, the proportion of ADP1 cells in the coculture increased
as a result of E. coli acetate production. According to the E. coli growth curve, the
consumption of acetate by ABlux positively affected the E. coli growth, which
could be observed as a shorter lag phase and more sufficient growth. Even though
the concentration of acetate did not reach the detection limit until the 8–10 h
timepoint the cultures already showed differential performance after the 5 h
timepoint, indicating that acetate already hinders growth at very low concentra-
tions. Eventually, the coculture resulted in more efficient glucose utilization by E. coli and a lower overall acetate concentration in the culture. Thus, it could be
concluded that the constructed carbon channeling system diminished the negative
effects of acetate and favorably affected the E. coli growth, and a relatively low
proportion of the supporting strain ABlux in the community was able to benefit
the growth. Production of recombinant protein by E. coli in the synthetic
coculture Engineered Synthetic Coculture for Improved Performance Figure 3. E. coli biomasses and substrate/end-metabolite concentrations. The cells were cultivated in a
minimal salts medium supplied with glucose for 12 h. A) E. coli cell number in monoculture (empty squares)
and in coculture (filled squares). B) Glucose (squares) and acetate (circles) concentrations in monoculture
(empty symbols) and in coculture (filled symbols). The mean and standard deviation of two independent
culture samples are shown. Note the logarithmic scale in biomass y-axis. Figure 3. E. coli biomasses and substrate/end-metabolite concentrations. The cells were cultivated in a
minimal salts medium supplied with glucose for 12 h. A) E. coli cell number in monoculture (empty squares)
and in coculture (filled squares). B) Glucose (squares) and acetate (circles) concentrations in monoculture
(empty symbols) and in coculture (filled symbols). The mean and standard deviation of two independent
culture samples are shown. Note the logarithmic scale in biomass y-axis. doi:10.1371/journal.pone.0113786.g003 The biomass of E. coli in the monoculture and in the coculture was calculated
according to optical densities and plate counts: the data is presented in Figure 3A. In the E. coli monoculture, the growth shifted to an exponential phase after 5–
6 hours of lag phase and reached a total cell number of ,6.15?108 ml21 after 12 h
of cultivation. In the coculture, the lag phase for E. coli was shorter, 4–5 h, and the
culture reached a slightly higher cell number of 6.63?108 ml21 at 12 h timepoint. The consumption of glucose and accumulation of end-metabolites in E. coli
monoculture and coculture were analyzed by HPLC (Figure 3B). The
consumption of glucose in E. coli monoculture was nearly linear after 7 h of
cultivation, with a total decrease of 6 mM in the glucose concentration. Acetate
could be reliably detected after an 8 h timepoint, the concentration reaching
4 mM at the end of the cultivation. End-metabolites other than acetate were not
detected. In the coculture, the glucose was utilized more rapidly and efficiently, PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 8 / 16 Engineered Synthetic Coculture for Improved Performance Figure 4. Biomasses and fluorescence of E. coli expressing sfGFP/pAK400c (ECsf) and A. baylyi
ADP1DgntT::Kanr/tdk expressing empty plasmid pBAV1C-ara (ABc) in mono and cocultures. Fluorescence is produced solely by E. coli expressing GFP. The strains were cultivated in a minimal salts
medium supplemented with 50; 100; or 250; mM glucose for 24 h. A) Biomasses (optical density, OD600) of
ECsf monocultures and cocultures (ECsfABc). B) Fluorescence signals measured for monocultures and
cocultures demonstrating the production of recombinant protein in cultures. The mean and standard deviation
of two independent cultures are shown. Figure 4. Biomasses and fluorescence of E. coli expressing sfGFP/pAK400c (ECsf) and A. baylyi
ADP1DgntT::Kanr/tdk expressing empty plasmid pBAV1C-ara (ABc) in mono and cocultures. Fluorescence is produced solely by E. coli expressing GFP. The strains were cultivated in a minimal salts
medium supplemented with 50; 100; or 250; mM glucose for 24 h. A) Biomasses (optical density, OD600) of
ECsf monocultures and cocultures (ECsfABc). B) Fluorescence signals measured for monocultures and
cocultures demonstrating the production of recombinant protein in cultures. The mean and standard deviation
of two independent cultures are shown. Fluorescence is produced solely by E. coli expressing GFP. The strains were cultivated in a minimal salts
medium supplemented with 50; 100; or 250; mM glucose for 24 h. A) Biomasses (optical density, OD600) of
ECsf monocultures and cocultures (ECsfABc). B) Fluorescence signals measured for monocultures and
cocultures demonstrating the production of recombinant protein in cultures. The mean and standard deviation
of two independent cultures are shown. doi:10.1371/journal.pone.0113786.g004 doi:10.1371/journal.pone.0113786.g004 cocultures are presented in Figure 4. The cocultures grew to higher optical
densities and produced more fluorescence with all studied glucose concentrations
compared to the respective monocultures, and the highest optical density and
fluorescence signal were obtained in the culture containing 50 mM glucose. Interestingly, the more glucose is present in the medium, the more significant is
the difference between the mono- and cocultures. This indicates that high
substrate concentrations may hinder E. coli growth by direct substrate inhibition
and by accelerated overflow metabolism. In the cultures containing 250 mM
glucose, a fluorescence signal of approximately 2-fold and an optical density of 3-
fold higher were obtained for the coculture compared to the monoculture. Thus,
the batch experiments further emphasized the coculture relevance for improved
culture performance and productivity in variable substrate concentrations. cocultures are presented in Figure 4. Production of recombinant protein by E. coli in the synthetic
coculture It was demonstrated that the coculture is beneficial for E. coli growth and biomass
production. The next step was to study the effects of the coculture on production
of the heterologous product that competes with the cell building blocks,
demonstrated here by recombinant protein production. Green fluorescent protein
(GFP) was chosen for the proof-of-principle production platform in order to
enable real-time monitoring of production dynamics and comparability between
the mono and cocultures. In addition, GFP has previously been successfully
expressed both in E. coli and A. baylyi ADP1 [21]. A plasmid containing a
superfolder variant of GFP (BBa_I746909) under inducible lactose promoter was
constructed. The resulting plasmid sfGFP/pAK400c was transformed to E. coli, the
clones were selected from chloramphenicol plates, and the strain was designated as
ECsf. To enable A. baylyi ADP1 growth in the coculture, plasmid pBAV1C-ara
(described elsewhere [22]) without an insert was transformed to the mutant strain
ADP1DgntT::Kanr/tdk and selected from the chloramphenicol plates, resulting in a
strain designated as ABc. The potential support of ABc for ECsf growth and
protein production in a coculture was studied in minimal medium batch cultures
supplied with different glucose concentrations: 50 mM, 100 mM, and 250 mM. The amount of GFP produced was estimated by fluorescence determination. The
end-point biomasses (OD600) and fluorescence signals for monocultures and 9 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Engineered Synthetic Coculture for Improved Performance Production of recombinant protein by the synthetic coculture in a
bioreactor PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Production of recombinant protein by the synthetic coculture in a
bioreactor The previous experiments demonstrated that ABc efficiently supports ECsf growth
in cocultures, being especially beneficial in cultures containing high substrate
concentrations by neglecting the negative effects of acetate. In an ideal case, the
secreted acetate could be directed to the product by the supportive strain. For
such an approach to be practical, a single expression construct that is functional in
both strains should be used. Indeed, an expression vector exploitable both in E. coli and A. baylyi ADP1 have been described [21]. Derived from the original
vector, a plasmid pBAV1C-T5-GFP was constructed and transformed to E. coli
and A. baylyi ADP1DgntT::Kanr/tdk. The vector contains the gene gfp under a
constitutive strong promoter (T5), and a chloramphenicol resistance gene (cat). The resulting strains were designated as ECg and ABg, respectively. To provide sufficient supply of nutrients and building blocks for the strains
under strong over-expression, and to study the coculture functionality in simple
unoptimized batch conditions without pH buffering or control, cultivations were
performed in a rich medium supplied with glucose. The batch cultivations for the
ECg and ABg coculture and ECg monoculture were carried out in a rich medium
supplied with 100 mM glucose and aeration. The cultivations were conducted in a
bioreactor in order to reveal more information about the culture characteristics
and dynamics; oxygen partial pressure, pH, biomass production (OD), To provide sufficient supply of nutrients and building blocks for the strains
under strong over-expression, and to study the coculture functionality in simple
unoptimized batch conditions without pH buffering or control, cultivations were
performed in a rich medium supplied with glucose. The batch cultivations for the
ECg and ABg coculture and ECg monoculture were carried out in a rich medium
supplied with 100 mM glucose and aeration. The cultivations were conducted in a
bioreactor in order to reveal more information about the culture characteristics
and dynamics; oxygen partial pressure, pH, biomass production (OD),
proportions of the coculture strains (%, CFU), substrate and end-metabolite
concentrations, and fluorescence signals were monitored throughout the 10-hour
cultivations (Figure 5). The coculture started to grow clearly faster after four
hours of cultivation, reaching a final optical density of 13, which was ,3-fold
higher compared to the biomass of ECg monoculture (OD 4.3). The cocultures grew to higher optical
densities and produced more fluorescence with all studied glucose concentrations
compared to the respective monocultures, and the highest optical density and
fluorescence signal were obtained in the culture containing 50 mM glucose. Interestingly, the more glucose is present in the medium, the more significant is
the difference between the mono- and cocultures. This indicates that high
substrate concentrations may hinder E. coli growth by direct substrate inhibition
and by accelerated overflow metabolism. In the cultures containing 250 mM
glucose, a fluorescence signal of approximately 2-fold and an optical density of 3-
fold higher were obtained for the coculture compared to the monoculture. Thus,
the batch experiments further emphasized the coculture relevance for improved
culture performance and productivity in variable substrate concentrations. 10 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Engineered Synthetic Coculture for Improved Performance Figure 5. Monitored bioreactor cultivations of the monoculture ECg and the coculture ECgABg. Both strains produce GFP by expressing the same
genetic construct pBAV1C-T5-GFP. The cultivations were performed in a bioreactor in a rich medium supplied with 100 mM glucose and aeration. A) Culture
parameters; pH (large circles) and oxygen partial pressure (pO2 %, small circles) for ECg monoculture (empty circles) and ECgABg coculture (filled circles
B) Total biomasses (OD600) of the cultures and proportion of ABg cells (%, CFU) in the coculture. C) Fluorescence signals (cts) for the monoculture ECg and
coculture ECgABg demonstrating the production of recombinant protein in the cultures. The mean and standard deviation of 2–4 independent culture
samples are shown. ECg - E. coli expressing pBAV1C-T5-GFP, ABg - A. baylyi ADP1DgntT::Kanr/tdk expressing pBAV1C-T5-GFP. Figure 5. Monitored bioreactor cultivations of the monoculture ECg and the coculture ECgABg. Both strains produce GFP by expressing the same
genetic construct pBAV1C-T5-GFP. The cultivations were performed in a bioreactor in a rich medium supplied with 100 mM glucose and aeration. A) Culture
parameters; pH (large circles) and oxygen partial pressure (pO2 %, small circles) for ECg monoculture (empty circles) and ECgABg coculture (filled circles)
B) Total biomasses (OD600) of the cultures and proportion of ABg cells (%, CFU) in the coculture. C) Fluorescence signals (cts) for the monoculture ECg and
coculture ECgABg demonstrating the production of recombinant protein in the cultures. The mean and standard deviation of 2–4 independent culture
samples are shown. ECg - E. coli expressing pBAV1C-T5-GFP, ABg - A. baylyi ADP1DgntT::Kanr/tdk expressing pBAV1C-T5-GFP. doi:10.1371/journal.pone.0113786.g005 the culture. This implies an efficient internal buffering system in the coculture,
which retains favorable growth conditions. Acetate concentrations started to differentiate after the 6 h timepoint. At the
end of the cultivation, the acetate concentration in the ECg monoculture was
13 mM, which probably affected the growth and cell performance negatively. In
the coculture, the acetate concentration was only 3 mM, indicating successful
recycle of acetate from the medium to the biomass and the product. The final
glucose concentrations were 65 mM in the coculture and 90 mM in the
monoculture. For the biomass samples collected at the end of the cultivations, the
obtained CDWs were 2.1 g/l and 5.1 g/l for ECg and ECgABg cultures,
respectively. Control cultivations without glucose supplementation were carried
out in batch bottles. Production of recombinant protein by the synthetic coculture in a
bioreactor The initial
proportions of the two strains were equal but the proportion of ABg seemed to
increase fastest between the 6–8 h timepoints, assumingly in parallel with the
exponential growth phase of ECg. It was noted that when cultured in a rich
medium, the proportion of ABg cells (20–30%) was much higher compared to the
minimal medium coculture where the proportion of ABlux was approximately 6%
by the end of the cultivation. The relatively high biomass of ABg did not, however,
negatively affect ECg growth, which further supports the hypothesis of ADP1 not
producing compounds inhibiting E. coli growth. proportions of the coculture strains (%, CFU), substrate and end-metabolite
concentrations, and fluorescence signals were monitored throughout the 10-hour
cultivations (Figure 5). The coculture started to grow clearly faster after four
hours of cultivation, reaching a final optical density of 13, which was ,3-fold
higher compared to the biomass of ECg monoculture (OD 4.3). The initial
proportions of the two strains were equal but the proportion of ABg seemed to
increase fastest between the 6–8 h timepoints, assumingly in parallel with the
exponential growth phase of ECg. It was noted that when cultured in a rich
medium, the proportion of ABg cells (20–30%) was much higher compared to the
minimal medium coculture where the proportion of ABlux was approximately 6%
by the end of the cultivation. The relatively high biomass of ABg did not, however,
negatively affect ECg growth, which further supports the hypothesis of ADP1 not
producing compounds inhibiting E. coli growth. The fluorescence signals differentiated after 4 hours of cultivation, the final
fluorescence signals being approximately 4-fold higher in the coculture compared
to the ECg monoculture. The higher biomass and consequently the higher
fluorescence signal of the coculture can be explained by the more propitious
growth conditions; in both cultures, pH started to drop after 3–4 hours of
cultivation, but the decline of pH in ECg monoculture was faster compared to the
coculture. In the monoculture, the final pH of 5.4 was reached at 8–9 h timepoint. In the coculture, pH decreased more moderately, but increased rapidly after the
8 h timepoint, reaching a final pH of 6.5, which is very close to the initial pH of PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 11 / 16 doi:10.1371/journal.pone.0113786.g005 Engineered Synthetic Coculture for Improved Performance such conditions (data not shown), suggesting that the relevance of the coculture
lies for the most part in the interactive glucose-acetate metabolism. The previous minimal medium cultivations constituted of ECsf and ABc strains
demonstrated that both biomass and product titers could be improved, even
though ABc was not contributing to the protein production. In a minimal
medium, ABc is completely dependent on the carbon provided by ECsf growth,
and the proportion of ABc cells is low, whereas rich medium enables more rapid
and independent growth of ABc (as for ABg), resulting in significantly higher cell
proportions consuming valuable building blocks from protein production. Thus,
we were interested to see how the increased number of ABc cells affects the protein
production in a rich medium when only ECg contributes to protein production
(Figure S1). In short, it was observed that the bioprocess trends, acetate
production, glucose consumption, and biomass production were very similar
compared to the ECgABg bioreactor coculture. According to the fluorescence
signals, the recombinant protein titer was 50% higher in the ECgABc coculture
compared to the ECg monoculture, which is expectedly less than improvements
gained in ECgABg coculture. It can be concluded that as protein overexpression is
highly dependent on the available biocomponents consumed by both the strains,
it is not energetically affordable to maintain the system in a rich medium with
high proportions of the supportive strain, without it participating in the
production. Thus, the availability of genetic tools and the possibility to rationally
engineer and commit both the strains to the production are crucial to take full
advantage of the carbon redirection, as demonstrated here. Furthermore, the
product example (recombinant protein) is constituted of amino acids instead of
carbon based molecules, and thus the benefits of carbon rerouting could be
potentially better exploited in production of hydrocarbons, such as fatty acid
derived compounds for bioenergy [22, 23]. Our findings suggest it is possible to
tune the coculture balance, nature, and dynamics by altering the medium,
substrate concentration, and genetic constructs. g
The accumulation of acetate is a widely recognized problem in bioprocessing of
E. coli. This study demonstrates that by exploiting a coculture with optimally
chosen strains, the growth, carbon utilization, and product formation can be
improved through symbiotic interactions, and the negative effects of acetate can
be diminished. It was found that the coculture does not grow sufficiently in PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 12 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 Conclusions Synthetic biology broadens the possibilities to tune sophisticated production
platforms, and coculturing is seen as a promising new frontier for taking the
bioproduction to the next level [35, 36]. This study demonstrates that rationally
engineered synthetic cocultures can improve biomass production, culture
viability, and product formation in simple unoptimized batch conditions. The
study extends applications of novel type of bioprocess optimization, and provides
clues for the development of functional, readily engineered, and dynamic
cocultures for synthetic biology applications. Engineered Synthetic Coculture for Improved Performance bioprocesses have demanded decades of optimization work to build up a cost-
effective bioproduction platform [34]. Thus, improvement of the utility,
profitability, and viability of cocultures requires significant research efforts. It is
impossible to exceed the production rates and yields of highly tuned and
optimized systems at a single cell level, but the power of cocultures most probably
lie in processes that cannot be readily optimized (e.g. processes exploiting diverse
industrial streams) or in processes involving variable conditions. Supporting Information Figure S1. Bioreactor data of ECg and ABc coculture. doi:10.1371/journal.pone.0113786.s001 (PDF) Author Contributions Conceived and designed the experiments: SS VS. Performed the experiments: SS
VS. Analyzed the data: SS VS. Contributed reagents/materials/analysis tools: SS. Wrote the paper: SS. Supervised and coordinated the study: MK VS. Acknowledgments We want to thank Dr. Anniina Kivisto¨ and Rahul Mangayil for their assistance in
HPLC analyses, as well as Antti Larjo for the computational metabolic analysis. 4.
Rabinovitch-Deere CA, Oliver JW, Rodriguez GM, Atsumi S (2013) Synthetic Biology and Metabolic
Engineering Approaches To Produce Biofuels. Chemical reviews. PLOS ONE | DOI:10.1371/journal.pone.0113786
December 3, 2014 3.
Purnick PE, Weiss R (2009) The second wave of synthetic biology: from modules to systems. Nature
reviews Molecular cell biology 10: 410–422. 2.
Lee SK, Chou H, Ham TS, Lee TS, Keasling JD (2008) Metabolic engineering of microorganisms for
biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 19: 556–563. Employing the coculture enabled the use of a high substrate
concentration in a simple batch culture, and the over-flow metabolism of E. coli
was successfully exploited in producing the protein of interest. Fluctuations in
sugar concentrations in industrial feedstocks can impose restrictions for single
strain cultures (6), whereas high substrate concentration is not an issue for the
coculture system presented here. Thus, with regards to sustainable and
economical carbon sources, exploiting a well-designed coculture may broaden the
possibilities to exploit challenging liquors with high concentrations of
carbohydrates. Nevertheless, cocultures are complex systems involving numerous interactions. Despite the possibility to reduce these interactions, it is very difficult to identify all
the mechanisms that are potentially beneficial for the culture. E. coli based 13 / 16 PLOS ONE | DOI:10.1371/journal.pone.0113786
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Separable States on Density Matrices and Emergent Systems
| null | 2,022
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cc-by
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Yasmín Navarrete 1,*,†
and Sergio Davis 2,3,† Yasmín Navarrete 1,*,†
and Sergio Davis 2,3,† 1
Instituto de Filosofía y Ciencias de la Complejidad, Los Alerces 3024 Ñuñoa, Santiago 7780192, Chile Research Center in the Intersection of Plasma Physics, Matter and Complexity (P mc), Comisión Chilena de
Energía Nuclear, Casilla 188-D, Santiago 8320000, Chile g
g
3
Departamento de Física, Facultad de Ciencias Exactas, Universidad Andres Bello, Sazié 2212 piso 7,
Santiago 8370136, Chile 3
Departamento de Física, Facultad de Ciencias Exactas, Universidad Andres Bello, Sazié 2212 piso 7,
S
i
8370136 Chil 3
Departamento de Física, Facultad de Ciencias Exactas, Universidad Andres Bello, Sazié 2212 piso 7,
Santiago 8370136, Chile g
*
Correspondence: yasmin.navarrete@gmail.com †
These authors contributed equally to this work. Abstract: In this paper, we present an analytical description of emergence from the density matrix
framework as a state of knowledge of the system, and its generalized probability formulation. This description is based on the idea of fragile systems, wherein the observer modifies the system
by the measurement (i.e., the observer effect) in order to detect possible emergent behavior. We
propose the use of a descriptor, based on quantum mutual information, to calculate if subsystems of
systems have inner correlations. This may contribute to a definition of emergent systems in terms of
emergent information. Keywords: emergence; quantum theory; density matrix; subadditivity 1. Introduction From quantum mechanics, it is well known that any separable state belongs to a
composite space that can be factored into individual states from separate subspaces. One
state is said to be correlated if it is not separable. As a matter of fact, to determine if a state
is separable or not is not trivial and the problem is classed as NP-hard from the theory of
complex systems [1]. Citation: Navarrete, Y.; Davis, S. Quantum Mutual Information,
Fragile Systems and Emergence. Entropy 2022, 24, 1676. https://
doi.org/10.3390/e24111676
Academic Editor: Masahito Hayashi
Received: 12 September 2022
Accepted: 11 November 2022
Published: 17 November 2022 Citation: Navarrete, Y.; Davis, S. Quantum Mutual Information,
Fragile Systems and Emergence. Entropy 2022, 24, 1676. https://
doi.org/10.3390/e24111676
Academic Editor: Masahito Hayashi
Received: 12 September 2022
Accepted: 11 November 2022
Published: 17 November 2022 entropy entropy entropy 2. Emergent Systems Several definitions of emergence exist, taking into account different aspects of their
origin or behavior. For instance, Peter Checkland [7] defines emergence as “the principle
that entities exhibit properties which are meaningful only when attributed to the whole,
not to its parts”. Emergent systems are structured in such a way that their components
interact, allowing for the structure of global patterns, depicted as a consequence of interre-
lations/correlations between subsystem elements, them being the result of complex and
self-organizing processes. This process may be triggered by an external stimulus. There are basically three types of emergence: simple, weak and strong ones, described
as follows: Simple emergence is composed by the combination of certain properties and relation-
ships between elements in a non-linear behavior. For instance, in order to achieve the
flight of an airplane, we cannot consider the motors, the propulsion system and their
wings separately; all of these properties must be considered together because they are
interconnected and they have interrelations through which flight emerges. This type of
emergence can be predicted from the functioning of its parts and it is referred to as the
concept of synergy, which means interacting or working together [8]. Weak emergence describes the emergence of properties of systems that may be pre-
dictable (not completely) and also reducible. They can be reduced to basic rules at an initial
time. After a while, the behavior can be unpredictable, as is mentioned in chaos theory [9];
nevertheless, it is possible to make computational simulations about such systems because
of the knowledge of the basic rules. Weak emergence is the product of complex system
dynamics (i.e., non-linear behavior, spontaneous order and adaptation); an example of the
latter is cellular automaton, known as Conway’s Game of Life [10–12]. Strong emergence is a case of non-expected emergence, as well as weak emergence. The difference lies in its non-reducible behavior, which appears just when the system is
running. As it is systematically determined by low-level attributes, it is not possible to
deduce it from the components at lower levels. The consciousness phenomenon is one
example of this type of emergence, and appears as a construction process. There is likely
no algorithm from the bottom up because it is a dynamical process evolving along time at
the highest level, with non-linear relations at the lower ones. Citation: Navarrete, Y.; Davis, S. Quantum Mutual Information,
Fragile Systems and Emergence. Entropy 2022, 24, 1676. https://
doi.org/10.3390/e24111676 A density operator, in quantum mechanics, is used to describe the statistical state of
a quantum system. The usual meaning of it is that the eigenvalues are the probabilities
of finding a system in one state corresponding to the eigenvectors. In a physical sense,
we can see its elements as relative frequencies corresponding to an appropriate ensemble
of N identical copies of the system that are in several possible states under a certain
setup or preparation protocol. Thus, we have a superposition of quantum states |vi⟩with
probabilities pi (real numbers) satisfying ∑pi = 1. However, it can be shown that the
density operator formalism can be recovered in a Bayesian formalism for noncommutative
expectations, wherein the system depends on the order of the measurements. This is
not restricted just to a quantum mechanical system, and can be understood under the
framework of what we have called a fragile system [2]. This operator provides a convenient
mean for describing quantum systems whose state is not completely known, it being
mathematically equivalent to a state vector approach [3,4]. Academic Editor: Masahito Hayashi Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. By applying the entropy to the density matrix, we can obtain the degree of disinfor-
mation of the state of the system. The systems can be composed of subsystems and, using
the subadditivity property (the probability of the whole is less than that of its parts) [5],
it is possible to quantify if the entropy of the whole is less than that of its parts. Holzer
and De Meer [6] make a comparison between the information at the system level with the
information at a lower level. As they state, “this measure gives a high value of emergence
for systems with many dependencies (interdependent components) and a low value of
emergence for systems with few dependencies (independent components)”; therefore, the Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/entropy Entropy 2022, 24, 1676. https://doi.org/10.3390/e24111676 Entropy 2022, 24, 1676 2 of 10 information of the whole is more than the information of the parts. In that sense, the
entropy can be a good parameter to measure a type of emergence in systems. This paper is organized as follows. In Section 2, we talk about emergent systems and
its current definitions. In Section 3, we define fragile systems as ones that are modified
by the act of measurement because of the change in internal variables. In Section 4, we
introduce the density matrix formalism. In Section 5, we depict a mathematical formulation
of emergent systems within the density matrix formalism. In Section 6, we show a concrete
example of a subadditive system. Finally, we provide some concluding remarks in Section 7. 2. Emergent Systems g
Another way of conceptualizing emergence is the separation of levels of complexity of
the system at different spatial or temporal scales [13]. y
p
p
Some main characteristics of strong emergence to consider are the following: Non-reducible phenomenon: the global state of the emergent system cannot be explain-
able, and neither is it reducible to its sub-system components. Downward causation: emergent high-level properties appear from a non-obvious con-
sequence of low-level properties, but, at the same time, all processes at the lower
level of hierarchy are constrained by and act in coherence with the laws of the higher
level [14]. Wholeness: a phenomenon wherein a complex, interesting high-level function appears as
a result of combining low-level mechanisms in straightforward ways. Entropy 2022, 24, 1676 3 of 10 3 of 10 Radical novelty emergence: a phenomenon wherein a system is designed according to
certain principles. Interesting unexpected properties arise from the behavior of
sub-system elements [15]. Radical novelty emergence: a phenomenon wherein a system is designed according to
certain principles. Interesting unexpected properties arise from the behavior of
sub-system elements [15]. In general, emergent systems are common in nature and technology. One example of
the latter is the speed of a vehicle affected by the center of gravity, the driver skills, weather
and friction, among other attributes (in this case, we can predict the emergent property of
the speed from the relation between components, it being the case of weak emergence). It is
possible to find strong emergent properties in the consciousness phenomenon [16], human
body and social phenomena, among others. One important example is ’self-awareness’,
which is a result of the interconnection of neurons in the brain [17]. In the case of biological systems (as well as in the case of social phenomena), emergent
models are appropriate for describing those situations. We can observe the characteristics
mentioned regarding strong emergence: non-reducibility, radical novelty, wholeness and
downward causation. The main aspect of biological emergent systems is that they may be observed from
inside them by the system (“from internal control of a system which might be fully con-
trolled by an observer/controller architecture that is part of the system” [18]); this is in
concordance with the autopoiesis theory proposed by the Chilean biologists H. Maturana
and F. 3. Fragile Systems In simple terms, a fragile system is one that is affected by the measurement [21]. This
distinguishes it from a non-fragile (classical) system, which is not modified upon observation. Because any system (being fragile or not) possesses information, we will think of
a system as a “black box” that can be found in different internal states, which is to be
denoted by λ. In general, λ contains many degrees of freedom, but we will not make use of
that inner structure here. The internal state λ contains all of the information necessary to
describe any aspect of the system. The crucial difference between a fragile system and a non-fragile one is that, in a fragile
system, access to the internal state λ is impossible, because it is precisely this internal state
that is modified by the measurement. As the modification of the state λ depends on the
details of the environment when carrying out the measurement (which we do not know
or control with accuracy), the outcome of a measurement is unavoidably stochastic, and a
mathematical formulation requires probability theory [22]. 2. Emergent Systems Varela [19] to define the self-maintaining chemistry of any living cell, which is under
the perspective of chemical organization theory used to formalize autopoietic structures,
“providing a basis to operationalize goals as an emergent process” [20]. When the measure-
ment is affected by the observer, it is the case of what we will call a fragile system. In the
next section, we will go deeper into the concept. 4. The Density Operator In this section, we formulate the density matrix operator, as a previous concept, in
order to define emergent systems using the calculation of entropies. The density operator is a positive semi-definite Hermitian operator of trace one. If A
is the matrix representation of an arbitrary observable ˆA, we can write A
=
a1
a2
... aN
a1a2 · · · aN
. (1) (1) Entropy 2022, 24, 1676 4 of 10 Hence, we have Hence, we have Hence, we have A
=
a1a1
· · ·
a1aN
a2a1
· · ·
a2aN
... ... ... aNa1
· · ·
aNaN
where ai is the conjugate of the element ai, and, in the case of real numbers, the same
element. We can take the average of different measurements represented by different
matrices A of the same observable, and then normalize and diagonalize it; finally, we obtain
the density matrix of a mixture as below: ˆρ =
N
∑
i
pi|v⟩⟨v|
(2) (2) where pi = P(λi|I) and λi is obtained from the eigen-value problem λi|vi⟩= λi ˆA [21]. where pi = P(λi|I) and λi is obtained from the eigen-value problem λi|vi⟩= λi ˆA [21]. We can formulate the density matrix operator by the use of a complex Hilbert space
just as in Von Neumann’s formulation of quantum theory [23]. For this, we consider an
arbitrary orthonormal basis set |n⟩(n = 1, . . . , N) with ⟨i|j⟩= δ(i, j), and define the density
operator [24] ˆρ as ˆρ :=
N
∑
i=1
N
∑
j=1
ρij|i⟩⟨j|,
(3) (3) with ρij complex numbers. Imposing that ˆρ is Hermitian, we see that the diagonal elements
ρii must be real and ρij = ρji. It is always possible to make a choice of such complex matrix
elements ρij so that they are proportional to the elements pij; these are given by ρij = ⟨ϵi| ˆρ
ϵj
= 1
2η
[pij + pji] + i[pij −pji]
,
(4) (4) where η = ∑i pii is a normalization factor that imposes Tr( ˆρ) = 1. Pure and Mixed States Consider an ensemble of measurements {|vi⟩}N
i=1. If the state vector is known [25],
the ensemble represents a pure state. Assuming that the system is in the state |v⟩, we can
expand it with respect to the eigenvector of a Hermitian operator ˆA as follows: |v⟩
= ∑
n
λn|vn⟩,
(5)
ˆA|v⟩
=
λn|v⟩. (6) (5) (6) Finally, we can define a pure state by the following term [25]: Finally, we can define a pure state by the following term [25]: ˆρ = |v⟩⟨v|. (7) (7) Because ˆρ2 = (|v⟩⟨v|)(|v⟩⟨v|) = |v⟩(⟨v|v⟩)⟨v| = ˆρ, we can distinguish a density
operator of a pure state by tracing; then, we have Because ˆρ2 = (|v⟩⟨v|)(|v⟩⟨v|) = |v⟩(⟨v|v⟩)⟨v| = ˆρ, we can distinguish a density
operator of a pure state by tracing; then, we have Tr( ˆρ) = Tr
ˆρ2
= 1. (8) (8) When we cannot repeat exactly the same initial condition, because of the noise of the
system, we represent this situation in a mathematical formulation in terms of an operator
called a density matrix for mixed states. This is a superposition of pure states [26]: ˆρ = ∑
i
pi|v⟩⟨v|,
(9) (9) Entropy 2022, 24, 1676 5 of 10 where the weights of each measurement satisfy the normalization condition ∑i pi = 1 . Each pi is the probability of finding a system in a given pure state. where the weights of each measurement satisfy the normalization condition ∑i pi = 1 . Each pi is the probability of finding a system in a given pure state. p
p
y
g
y
g
p
In contrast to a pure state, when we have a mixed-density matrix, the trace of the
square density matrix is given by the inequality Tr
ˆρ2
< 1. (10) (10) Hence, Tr
ˆρ2
, known as the degree of purity [25], can be used to distinguish between
pure and mixed states in a basis-independent manner. 5. Density Operator Formalism and Emergent Systems Earlier work by Prokopenko et al. [13] has set the basis for a discussion of complexity,
self-organization and the emergence of classical systems in information-theoretical terms,
particularly in terms of Shannon entropy and mutual information. Taking all of this into account, we will rephrase the earlier arguments in terms of the
(von Neumann) entropy of density matrices. However, first, let us review the behavior of
the information entropy (or Shannon entropy) for classical, correlated systems. We adopt the Bayesian view by Jaynes [27] and others of the Shannon entropy related
to the information content of a model based on, in principle, subjective probabilities, but
consistent with known facts. Shannon entropy is then a measure of missing information in
a probabilistic model about some aspect of reality, and is therefore dependent of the state
of knowledge used to construct said model. For a state of knowledge I, where we ask an
arbitrary question with N possible answers, denoted by the proposition A1, A2, . . . , AN,
the Shannon entropy is defined as S(I) := −
N
∑
i=1
pi ln pi
(11) (11) where pi is the probability P(Ai|I) of the answer Ai being true under I. Please note that,
for two ‘observers’ with different states of knowledge I1 and I2, the Shannon entropies
S(I1) and S(I2) that they assign to an unknown question will, in general, be different. For instance, if the first observer knows that A1 is true, whereas the second only knows
that either A1 or A2 is true, then S(I1) = 0 because P(Ak|I1) = δ1k, but S(I2) > 0 because
0 < P(A1|I2) < 1 and P(A2|I2) = 1 −P(A1|I2) > 0. In the case where the question involves the unknown value of one or more variable,
the information entropy directly translates in terms of the probability distribution. For in-
stance, for the joint probability distribution P(X, Y|I) of the variables X and Y under the
state of knowledge I, we have SXY(I) := −∑
x ∑
y
P(x, y|I) ln P(x, y|I). (12) (12) Using the product rule of probability, Using the product rule of probability, Using the product rule of probability, P(x, y|I) = P(y|x, I)P(x|I),
(13) (13) this entropy can always be separated into two terms [28], SXY(I) = SX(I) +
D
SY|X
E
I,
(14) (14) where the first term is the entropy of the variable X, and the second term, D
SY|X
E
I := −∑
x
P(x|I)∑
y
P(y|x, I) ln P(y|x, I)
(15) (15) Entropy 2022, 24, 1676 6 of 10 is the expected value of the conditional entropy of Y given X. This conditional entropy
cannot be negative, it being the expected value of a non-negative quantity. is the expected value of the conditional entropy of Y given X. This conditional entropy
cannot be negative, it being the expected value of a non-negative quantity. It is possible to extend the Bayesian idea of probabilities as degrees of belief constrained
on the available information to quantum systems [29,30]. Let ρ12 = ρ1 ⊗ρ2. We can obtain
the eigenvalues of ρ1 ai = {ρ1}ii and ρ2 bj = {ρ2}ii. Hence, the diagonal elements of ρ12
are given by all products of the form aibj, where i = 1, ..., n1 and j = 1, ..., n2. Here, n1 and
n2 are the dimensions of ρ1 and ρ2, respectively; then: S(ρ12)
=
−Tr(ρ12 ln ρ12)
(16)
=
−
n1
∑
i=1
n2
∑
j=1
(aibj) ln
aibj
. (16) On the other hand, the sum of the entropy of each system is given by On the other hand, the sum of the entropy of each system is given by S(ρ1) + S(ρ2) = −
n1
∑
i=1
ai ln(ai) −
n2
∑
j=1
bj ln
bj
. (17) (17) Thus, the entropy for an ensemble (ρ12) for which the subsystems are uncorrelated is
just equal to the sum of the entropies of the reduced ensembles for the subsystems. When
there are correlations, we should expect an inequality instead (called the subadditivity
property of entropy [5,31,32]), since, in this case, ρ12 contains additional information
concerning the correlations, which is not present in ρ1 and in ρ2 (those are the partial traces
of ρ12, respectively), as in (18) S(ρ12) ≤S(ρ1) + S(ρ2). (18) Given this inequality, we can use the mutual information formulation Given this inequality, we can use the mutual information formulation I := S(ρ1) + S(ρ2) −S(ρ12),
(19) (19) such that I > 0 whenever there is subadditive behavior and I = 0 for additivity. Using the product rule of probability, From Equation (19), we obtain a descriptor of weak emergency in systems that are
correlated. In these types of systems, new information emerges from the relation of its parts
in terms of correlations. 6. An Example of a Subadditive System (25) Γ1 : a = 1, b = 1,
Γ2 : a = 1, b = 3,
Γ3 : a = 3, b = 1,
Γ4 : a = 3, b = 3,
Γ5 : a = 2, b = 2,
Γ6 : a = 2, b = 4,
Γ7 : a = 4, b = 2,
Γ8 : a = 4, b = 4. (25) (25) Let us now define the measurement M(a, b) by M(a, b) :=
1 if a = b,
2, if a > b,
3 if a < b. (26) (26) such that the system undergoes the following transitions: Γ1 ↔Γ4, Γ5 ↔Γ8, Γ2 ↔Γ6 and
Γ3 ↔Γ7. In this way, if the system has a = 3 and b = 1, that is, it is in the state Γ3, and we
perform the measurement M, we will obtain a value M = 2 and the state will change to a′
= 4 and b′ = 2, that is, to Γ7. This makes the system fragile regarding the measurement M,
with the integers a and b being the hidden variables of the system. Lastly, we will define the
parity of each integer as 0 if the number is odd, and 1 if it is even. In this way, there are
only four observable states, namely s1 = (0, 0),
s2 = (0, 1),
s3 = (1, 0),
s4 = (1, 1),
(27) (27) where the first and second position of the tuple represent the parity bit of a and b, respec-
tively. Of these states, only s1 and s4 are consistent with the constraint I. Considering the
allowed transitions, we have that where the first and second position of the tuple represent the parity bit of a and b, respec-
tively. Of these states, only s1 and s4 are consistent with the constraint I. Considering the
allowed transitions, we have that P(s, s′|I) =
1
4 if s ∈{s1, s4} and s′ ∈{s1, s4},
0 otherwise. (28) (28) The matrix elements pij = P(s = si, s′ = sj|I) are given in Table 1, together with the
density matrix elements ρij. The latter are simply ρij = 1
2η
pij + pji + i[pij −pji]
= pij
η = 2pij,
(29) (29) because η = ∑4
i=1 pii = 1
2. 6. An Example of a Subadditive System As is depicted in Equation (4), we can write the density matrix as follows: ρij = ⟨ϵi| ˆρ
ϵj
= 1
2η
[pij + pji] + i[pij −pji]
. (20) (20) but where we now interpret pij as pij = P(ϵi, ϵ′
j|I),
(21)
η = ∑
i
pii
(22) (21)
(22) (22) where primed states are states after a measurement. f h
l
l
h ld h where primed states are states after a measurement. B
f th
i
li
ti
l
it
t h ld th t where primed states are states after a measurement. Because of the marginalization rule, it must hold that ∑
i
pij = P(ϵ′
j|I)
for all j = 1, . . . , N, ∑
i
pij = P(ϵ′
j|I)
for all j = 1, . . . , N,
(23) (23) while, simultaneously, while, simultaneously, ∑
j
pij = P(ϵi|I)
for all i = 1, . . . , N
(24) ∑
j
pij = P(ϵi|I)
for all i = 1, . . . , N (24) Entropy 2022, 24, 1676 7 of 10 7 of 10 must be true. As an example, consider an abstract system composed of two integers, a and
b, such that a ∈{1, 2, 3, 4} and b ∈{1, 2, 3, 4}. Let us set the constraint I that a and b are
either both even or both odd; then, there are eight allowed states, namely must be true. As an example, consider an abstract system composed of two integers, a and
b, such that a ∈{1, 2, 3, 4} and b ∈{1, 2, 3, 4}. Let us set the constraint I that a and b are
either both even or both odd; then, there are eight allowed states, namely Γ1 : a = 1, b = 1,
Γ2 : a = 1, b = 3,
Γ3 : a = 3, b = 1,
Γ4 : a = 3, b = 3,
Γ5 : a = 2, b = 2,
Γ6 : a = 2, b = 4,
Γ7 : a = 4, b = 2,
Γ8 : a = 4, b = 4. 6. An Example of a Subadditive System The states given by Equation (27) are equivalent to the ket
notation (|00⟩, |11⟩, |01⟩, |10⟩). The density matrix elements ρij correspond to the density
operator of a pure, but entangled state, ˆρAB = 1
2
|00⟩⟨00| + |00⟩⟨11| + |11⟩⟨00| + |11⟩⟨11|
=
Φ+
Φ+
(30) (30) Entropy 2022, 24, 1676 8 of 10 with |Φ+⟩as one of the Bell states, with |Φ+⟩as one of the Bell states, Φ+ :=
1
√
2
|00⟩+ ⟨11|
. (31) (31) Table 1. The set of joint probabilities pij = P(s = si, s′ = sj|I) (third column) for the fragile system
arising from the two-integer example described in Section 6. The fourth column shows the values of
the elements of the corresponding density matrix. i
j
pij
ρij
1
1
1⁄4
1⁄2
1
2
0
0
1
3
0
0
1
4
1⁄4
1⁄2
2
1
0
0
2
2
0
0
2
3
0
0
2
4
0
0
3
1
0
0
3
2
0
0
3
3
0
0
3
4
0
0
4
1
1⁄4
1⁄2
4
2
0
0
4
3
0
0
4
4
1⁄4
1⁄2 The reduced density matrices are The reduced density matrices are ρA = 1
2
1
0
0
1
,
ρB = 1
2
1
0
0
1
. Hence, ˆρAB ̸= ˆρA ⊗ˆρB, and the operator ˆρAB is not separable. The values of the von
Neumann entropy are SA = SB = ln 2 and SAB = 0, the latter being a pure state; therefore,
the system is subadditive. With this example, we have found a density operator that is not
only correlated but could describe an emergent system, since the entropy of the whole is
less than that of its parts. 7. Concluding Remarks In this work, we reviewed the idea of emergence in the context of quantum mechanics
and fragile systems in order to detect emergent behavior in systems under the framework
of the density matrix theory. We proposed the mutual information as a descriptor of
emergence in fragile systems [21]. When the parameter I > 0, there is more information in
the whole than its parts; therefore, new information emerges as a result of the interaction of
the subsystems as correlations. We have presented a concrete example of an operator that
could describe a subadditive system. We believe that this formulation may give some insights about adaptative, self-
sustainable and interconnected models. In particular, we aim to describe how, in an
entangled system, the observer is part of the observation by being an inseparable element
of it; for instance, by considering the side effect that produces the act of measurement (i.e.,
the observer effect [33–35]). Entropy 2022, 24, 1676 9 of 10 9 of 10 Author Contributions: Conceptualization, Y.N.; methodology, Y.N. and S.D.; software, S.D.; vali-
dation, Y.N. and S.D.; formal analysis, Y.N. and S.D.; writing—original draft preparation, Y.N. and
S.D.; writing—review and editing, Y.N. and S.D. All authors have read and agreed to the published
version of the manuscript. Funding: This research was funded by ANID FONDECYT 1220651 grant. Funding: This research was funded by ANID FONDECYT 1220651 grant. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References g
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English
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Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum
|
EvoDevo
| 2,013
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cc-by
| 12,711
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Developmental coupling of larval and adult stages
in a complex life cycle insights from limb Developmental coupling of larval and adult stages
in a complex life cycle insights from limb © 2013 Lee et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. * Correspondence: ysuzuki@wellesley.edu
†Equal contributors
Department of Biological Sciences, Wellesley College, 106 Central St.,
Wellesley, MA 02481, USA Developmental coupling of larval and adult stages
in a complex life cycle: insights from limb
regeneration in the flour beetle, Tribolium
castaneum Alison K Lee†, Christie C Sze†, Elaine R Kim and Yuichiro Suzuki* Alison K Lee†, Christie C Sze†, Elaine R Kim and Yuichiro Suzuki* Developmental coupling of larval and adult stages
in a complex life cycle: insights from limb
regeneration in the flour beetle, Tribolium
castaneum
L
l Developmental coupling of larval and adult stages
in a complex life cycle: insights from limb
regeneration in the flour beetle, Tribolium
castaneum Developmental coupling of larval and adult stages
in a complex life cycle: insights from limb
regeneration in the flour beetle, Tribolium
castaneum Lee et al. Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 RESEARCH
Open Access
Developmental coupling of larval and adult stages
in a complex life cycle: insights from limb
regeneration in the flour beetle, Tribolium
castaneum
Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 Background side-effects caused by gene silencing, it is difficult to
know how alterations of larval limb morphologies during
embryogenesis affect the adult morphology. Thus, in order
to assess the continuity between larval leg and adult leg
patterning, the larval appendage morphology was altered
by molecular perturbations during regeneration, to assess
the potential impact on the regenerated adult morphology. Tribolium is well suited for genetically perturbing regener-
ation, given its short life cycle, fully sequenced genome
[12], and amenability to functional analyses using RNA
interference (RNAi) [13]. Importantly, it develops external
larval appendages, such as legs and antennae, which are
capable of complete regeneration in a relatively short
amount of time [14]. The larvae are capable of regenerating
their appendages, and within two molts, much of the
larval appendage morphology is restored. Once they
become a pupa or an adult, Tribolium do not regenerate
their appendages. By disrupting larval leg re-patterning,
we can begin to address how larval leg patterning and
adult leg patterning might be linked. Complex life cycles, which contain two or more morpho-
logically discrete postembryonic phases, are found ubiqui-
tously among many metazoan taxa [1]. Such complex life
cycles can serve as adaptive strategies that allow different
developmental stages to evolve independently to occupy
larger ecological niches [1]. For example, holometabolous
insects, or insects that undergo complete metamorphosis,
produce distinct larval and adult morphologies that are
capable of freely adapting to different ecological habitats. The evolution of two morphologically distinct stages is
thought to be a key innovation that greatly aided the
diversification of these insects [1,2]. However, whether or
not the larval morphology is completely free to evolve
without influencing the adult phenotype remains unclear. The molecular mechanisms underlying the transition
from larval to adult stages are well-understood, yet the
cellular origins of the adult body remain an enigma. In
higher dipterans and a number of other insect species,
clusters of cells form a distinct tissue called an imaginal
disc, which develops inside the larva. In fruit flies, much
of the adult body forms from these imaginal discs, and
the larval cells make limited contributions to the adult
body [3]. However, in most other insects, adult structures
do not arise from free-floating imaginal discs, but rather
develop from cells associated with larval structures. In
lepidopterans, for example, imaginal cells embedded
within the larval tissues replace much of the larval-specific
cells to generate the adult structures [4,5]. Background In other insects,
such as beetles, polymorphic larval cells are thought to
make direct contribution to the developing adult body
[5,6], although more studies are needed before definitive
conclusions can be drawn. In this study, we knocked down the expression of four
transcription factors that have been previously identified as
key regulators of normal appendage patterning: dachshund
(dac), Distal-less (Dll), abrupt (ab) and spineless (ss). dac
codes for a transcription factor required for development
of the mid-limb segment along the proximal-distal (PD)
axis in Drosophila and Tribolium [15,16]. In Manduca, its
embryonic expression likewise suggests a conserved role in
mid-limb segment patterning [15-17]. In Tribolium, Dac
has been determined to play a critical role in both adult
mid-leg segment patterning [9,18] and adult antennal
segmentation [8]. dac expression in Tribolium has been
analyzed in the embryonic stage [15], but whether it has a
major function in patterning larval legs or antennae during
this stage is not known. Given that most insects do not make their entire adult
body from set-aside imaginal discs, the larval morphology
likely imposes some constraint on the development of the
adult morphology. To determine the extent to which
various stages of a complex life cycle can independently
evolve, an understanding of the developmental relation-
ships among the different stages is essential. In this study,
we examined the consequence of genetic perturbations
during larval appendage regeneration, to examine the degree
of developmental continuity that might exist between
larval and adult morphologies. Dll is a limb-patterning gene encoding a homeodomain
transcription factor required for distal limb development
in insects [8,9,18-20]. In Tribolium, Dll is required for
proper leg patterning along the PD axis during embryonic
development [20] and metamorphosis [9,18]. Dll has also
been established to be important for antenna segmentation
in the developing Tribolium pupa [8] and embryo [20]. In addition to Dac and Dll, several additional transcrip-
tion factors have also been identified to be important for
patterning limbs during metamorphosis [8,9]. We chose
to investigate the role of two of these transcription factors
during regeneration: the Bric-a-brac Tramtrack Broad
complex (BTB) protein, Ab, and the basic-helix-loop-
helix-PAS transcription factor, Ss. Ab is a BTB-zinc finger
transcription factor necessary for antennal and tarsal
segmentation in Tribolium. Knockdown of ab in Tribolium
larvae results in a reduction of the number of adult anten-
nal segments [8] and fusion of tarsomeres of the adult legs
[9]. Abstract Background: A complex life cycle, such as complete metamorphosis, is a key innovation that can promote
diversification of species. The evolution of a morphologically distinct larval stage is thought to have enabled insects
to occupy broader ecological niches and become the most diverse metazoan taxon, yet the extent to which larval
and adult morphologies can evolve independently remains unknown. Perturbation of larval limb regeneration
allows us to generate larval legs and antennae with altered limb morphologies, which may be used to explore the
developmental continuity that might exist between larval and adult appendages. In this study, we determined the
roles of several appendage patterning transcription factors, abrupt (ab), dachshund (dac), Distal-less (Dll), and
spineless (ss), in the red flour beetle, Tribolium castaneum, during larval appendage regeneration. The functions of
these genes in regenerating and non-regenerating limbs were compared using RNA interference. Results: During limb regeneration, dac and ss were necessary to re-pattern the same larval structures as those
patterned during embryogenesis. Removal of these two genes led to larval appendage patterning defects that
were carried over to the adult legs. Surprisingly, even though maternal knockdown of ab had minimal effects on
limb allocation and patterning in the embryo, it was necessary for blastema growth, an earlier phase of
regeneration. Finally, knockdown of Dll prevented the blastema-like bumps from re-differentiating into appendages. Conclusions: Our results suggest that, similar to vertebrates, the re-patterning phase of Tribolium larval limb
regeneration relies on the same genes that are used during embryonic limb patterning. Thus, the re-patterning
phase of regeneration is likely to be regulated by taxon-specific patterning mechanisms. Furthermore, Ab and Dll
appear to play important roles during blastema proliferation and re-differentiation, respectively. Finally, our results
show that continuity exists between larval and adult limb patterning, and that larval and adult leg morphologies
may be developmentally coupled. Thus, the evolution of imaginal discs may have been a key step towards
completely removing any developmental constraints that existed between larval and adult phenotypes. Keywords: Limb regeneration, Distal-less, Dachshund, Spineless, Abrupt, Tribolium castaneum, Patte * Correspondence: ysuzuki@wellesley.edu
†Equal contributors
Department of Biological Sciences, Wellesley College, 106 Central St.,
Wellesley, MA 02481, USA Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Page 2 of 16 Background During postembryonic development of Drosophila, We chose to work with a model holometabolous insect,
Tribolium castaneum, whose legs and antennae undergo
two major morphogenetic transitions during the life cycle:
1) embryonic to larval appendages during embryogenesis,
and 2) larval to adult appendages during metamorphosis
[7]. Knockdown studies in Tribolium have shown that the
embryonic patterning of larval legs and the metamorphic
patterning of adult legs rely on overlapping but distinct
sets of genes [8-11]. Because embryonic gene knockdown
often leads to lethality of the animal due to pleiotropic Page 3 of 16 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 mRNA isolation and cDNA synthesis
Larvae were dissected in 1X-PBS (0.02 M phosphate,
0.15 M NaCl, 0.0038 M NaH2PO4, 0.0162 M Na2HPO4;
pH 7.4) to remove the gut and the fat body. The
remaining tissue was homogenized in Trizol (Invitrogen,
Carlsbad, CA, USA), and RNA was subsequently extracted
using chloroform, treated with DNase (Promega, Fitchburg,
WI, USA) and precipitated in isopropyl alcohol. cDNA was
synthesized from 1 μg of total RNA using the cDNA syn-
thesis kit (Thermo Fisher Scientific, Pittsburgh PA, USA)
following the manufacturer’s instructions. mRNA isolation and cDNA synthesis
Larvae were dissected in 1X-PBS (0.02 M phosphate,
0.15 M NaCl, 0.0038 M NaH2PO4, 0.0162 M Na2HPO4;
pH 7.4) to remove the gut and the fat body. The
remaining tissue was homogenized in Trizol (Invitrogen,
Carlsbad, CA, USA), and RNA was subsequently extracted
using chloroform, treated with DNase (Promega, Fitchburg,
WI, USA) and precipitated in isopropyl alcohol. cDNA was
synthesized from 1 μg of total RNA using the cDNA syn-
thesis kit (Thermo Fisher Scientific, Pittsburgh PA, USA)
following the manufacturer’s instructions. Ab is involved in establishing and maintaining muscle
attachments and morphogenesis of adult appendages:
viable ab mutants have deformed antennae and bristles,
gnarled legs and wing venation defects [21-23]. Its role
during embryonic limb development is not known although
it has been identified as a regulator of neuromuscular
junction development in Drosophila, where it is expressed
in muscle cells but not neurons [21,24]. Ss is also required for leg segmentation in Tribolium
and Drosophila during metamorphosis [9,25]. Knockdown
of ss in Tribolium results in the development of adult legs
with fused tarsomeres. Background In addition, homeotic transforma-
tions of the distal portions of the adult antennae into a
tibia-like segment with a terminal claw result from ss
knockdown during the larval stage [8,10]. Similarly, in
Drosophila, Ss is required for specifying antennal identity,
and ss mutants exhibit antenna-to-leg transformations
[25-28]. During Tribolium embryonic antennal development,
Ss plays a critical role in establishing larval antennal
identity [10,29]. Silencing of ss during embryogenesis
results in the formation of newly hatched larvae with
legs instead of antennae [10,29]. However, its removal
does not appear to influence embryonic leg development;
thus, embryos develop larval legs normally even when ss is
silenced, while ss knockdown during metamorphosis
results in adults with fused tarsomeres. Cloning and double-stranded RNA (dsRNA) synthesis
Sequences of Dll [AF317551], dac [XM_964678], ab
[XM_969854] and ss [EU912437] were obtained from
confirmed sequences deposited in GenBank [8,10,18],
and primers were designed outside of highly conserved
regions to avoid off-target knockdown of gene expression. The list of primers used for cDNA amplification is given
in Table 1. The amplified cDNA product was isolated and
subsequently cloned into a TOPO TA vector (Invitrogen,
Carlsbad, CA, USA). Following plasmid identity confirm-
ation by sequencing, plasmid DNA was linearized via
restriction digestion. Each of the strands of the dsRNA was synthesized with
the T3 and T7 MEGAscript Kits (Ambion) following the
manufacturer’s instructions. Single-stranded RNA (ssRNA)
was combined and annealed to form dsRNA [30]. The
annealed product was analyzed by gel electrophoresis to
confirm annealing, and stored at −80°C until use. The final
concentrations of the dsRNA were 2 μg/μL for ab, dac, Dll
and
ampr
(plasmid
gift
from
Dr
Takashi
Koyama,
Gulbenkian Institute, Lisbon), and 2 μg/μL or 4 μg/μL
for ss. We hypothesized that the removal of patterning genes re-
quired for larval limb development would also be required
for larval limb regeneration. We also hypothesized that
disrupting the larval limb patterning during regeneration
would influence the adult limb patterning and illustrate
the continuity that exists between larval and adult limb
patterning. Thus, we determined the phenotypes of em-
bryonic appendages resulting from silencing Dac and Ab,
and the effects of dac, Dll, ab and ss knockdowns during
larval limb regeneration in Tribolium. We found that both
Ab and Dll are necessary for limb regeneration prior to
patterning, suggesting regeneration-specific functions of
these genes. Background We also found that perturbed patterning of
larval limbs leads to corresponding alterations in the adult
limb morphology, indicating the continuity of patterning
across larval and adult structures. DsRNA injection
A
l Approximately 0.5 μg (0.25 μL) of dsRNA was injected
into the dorsal side of the day-0 fifth or sixth instar
larvae (the final instar in our colony is typically the
seventh or eighth instar), between the first and second
abdominal segments, with a pulled 10-μL glass capillary
needle connected to a syringe. Control larvae were
injected with the same amount of bacterial ampr dsRNA. To visualize the effects of dsRNA on developing embryos,
parental RNAi was conducted on female Tribolium adults
[20]. Approximately 0.5 μL of dsRNA was injected into
the abdominal body cavity of the adult female insects. The
eggs from the dsRNA-injected female adults were separated
out from the flour every 4 to 5 days, starting one week
after dsRNA injection. The eggs were maintained at 29°C
for another 4 to 5 days to allow the eggs to develop. The
developing unhatched embryos or early hatched larvae
(first instar) were then processed with lactic acid to clear
the egg shell and the hard cuticle for fluorescent Results Embryonic functions of dac and ab We first report the effects of maternal dac and ab
dsRNA injection on embryonic appendage development,
since knockdown embryonic phenotypes for these genes
have not yet been reported. The results of maternal
RNAi are summarized in Table 2. Dll and ss knockdown
produced phenotypes similar to those reported previously
(see Additional file 1 and Tables 2 and 3) [10,20,29,33]. Leg and antennal ablations All of the dac knockdown embryos (n = 47) (Table 3)
developed normal antennal morphology (Figure 1E),
similar to antennae of the control ampr dsRNA-treated
embryos (Figure 1B). In contrast, 49% of the dac
dsRNA-injected embryos (Table 3) had partially fused
femur and tibiotarsus (Figure 1D and 1F), which were
not seen in the ampr dsRNA-injected control embryos
(Figure 1A and 1C). The coxa and trochanter, however,
were unaffected in size or segmentation. Thus, dac knock-
down caused a reduction in size of the femur-tibiotarsal
leg segments, whereas the antennae remained unaffected
(Figure 1E and 1F). Two days after dsRNA injections, the larval mid- and
hindlegs were ablated at the coxa on one side [14]. The
larvae were placed on double-sided tape after anesthesia,
and the legs were ablated using a pair of microscissors. The forelegs and the contralateral mid- and hindlegs
served as internal controls for analyzing the effect of
each dsRNA on regeneration of larval and adult legs. The larval antenna from the left side of the head was
ablated using a razor blade two days after dsRNA injection. Anesthetized larvae were placed ventral side uppermost on
double-sided tape, and the antennae were sliced off using
the razor blade. The contralateral antenna served as the
internal control. Larvae were stored in a 70% ethanol: 15%
glycerol solution until imaged. The legs and heads were
mounted in 80% glycerol and imaged using light and
fluorescence microscopy, respectively. The majority of ab knockdown embryos produced
through maternal RNAi exhibited an abnormal scrunched
phenotype (Table 3; Figure 1G-I), where the body and
appendages all appeared broader and compressed. The
antenna (n = 57) and legs (n = 58) of the ab-knockdown
embryos both exhibited this characteristic scrunched
phenotype, where the cuticle failed to be fully extended. However, these appendages had the typical number of
segments in the right proportions. This suggests that Ab
does not affect the structural formation or patterning of
these appendages in the embryo. Beetle husbandry Tribolium casteneum strain GA-1 was obtained from
Dr Richard Beeman (USDA ARS Biological Research
Unit, Grain Marketing & Production Research Center,
Manhattan, KS, USA). Beetles were raised on organic
wheat flour containing 5% nutritional yeast, and stocks
were maintained in plastic containers in a 29°C incubator
with approximately 50% relative humidity. No ethical
approval was necessary to work with Tribolium as they
are common household insect pests. Page 4 of 16 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Table 1 Primers used for ab, dac, Dll and ss dsRNA synthesis and knockdown verification
Gene
Application
Forward primer (5′->3′)
Reverse primer (5′->3′)
ab
RNAi
ACTCACAAAGGAGAAGGGAAA
CGTTGGTATTGAAAGGATGG
Knockdown verification
GCCTGTGATGGATGTTCGT
GTCTTGGGTCTGTCGCTCT
ss
RNAi
ATTACTCAAAACTGGCGCTTC
TGTTGTGTTAGTGGGAGGAGTT
Knockdown verification
GGAGATACCGCAGAAGGAAG
TTGTAGTCAGGGGCGATGT
dac
RNAi
CAGCATCGCATCTTCAAC
CTCCTCCCTCAGCCTTTCT
Knockdown verification
ACTGCACTACGGCCAGTTC
TCGTCCATGTCTTGATCCTT
Dll
RNAi
GGATAACAAACCCTTCACGAC
GCCTCTCCAACGATAAACAC
Knockdown verification
GGTGTGTTCGTAGTGCTTCC
CGCCTTCATCATCTTCTTGT
RNAi, RNA interference; ab, abrupt; ss, spineless; dac, dachshund; Dll, Distal-less. microscopy as previously described [31,32]: unhatched
embryos were incubated in 75% lactic acid, while hatched
larvae were incubated overnight at 60°C in 10% ethanol:
90% lactic acid solution. These were then stored in 70%
ethanol: 15% glycerol solution, or mounted in 80% glycerol. Embryos were imaged and scored using fluorescence
microscopy. microscopy as previously described [31,32]: unhatched
embryos were incubated in 75% lactic acid, while hatched
larvae were incubated overnight at 60°C in 10% ethanol:
90% lactic acid solution. These were then stored in 70%
ethanol: 15% glycerol solution, or mounted in 80% glycerol. Embryos were imaged and scored using fluorescence
microscopy. Knockdown verification
d
h h To determine whether gene expression was knocked
down in response to dsRNA injection, gene expression was
analyzed using semiquantitative PCR. Day-0 sixth-instar
larvae were injected with dac, Dll, ab, ss or control ampr
dsRNA. For each treatment, two days after molting into
the seventh instar, three larvae were pooled in Trizol and
homogenized to isolate their RNA. After DNAse treat-
ment, 1 μg of RNA was converted to cDNA and amplified
using semiquantitative PCR. The PCR was repeated until
the optimum cycle numbers were obtained, such that the
bands were unsaturated. The following cycle numbers were
used: dac = 42; Dll = 39; ab = 38; ss = 40; rp49 = 34. Besides the scrunched phenotype, ab knockdown
dramatically decreased the number of bristles that were
present on the surface of the embryonic cuticle. Compared
to the ampr dsRNA-injected embryos (Figure 1J), there
were fewer bristles present on the dorsal tergites of ab
knockdown embryos (Figure 1K). This suggests that
Ab is important for embryonic bristle formation and
cuticular expansion, but not for appendage segmentation
or patterning. Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Page 5 of 16 Page 5 of 16 Table 2 Summary of the knockdown effects observed in this study
Appendage
Gene
No ablation
Ablation
Maternal RNAi
Larval RNAi
Larval RNAi
Larva
Larva
Pupa/adult
Larva
Leg
Dll
Loss of distal leg segments
Loss of tissue identity
and regression
Loss of tissue identity and regression
Bump formation and
no outgrowths
dac
Partial fusion of femur and tibiotarsus
No effect
Loss of distal femur and tibia and
fusion of proximal tarsomeres
Partial fusion of femur
and tibiotarsus
ab
Slightly compressed but segment
number is unaffected
No effect
Partial loss of tarsomeres
Small bump formation
and no outgrowths
ss
No effect
No effect
Fusion of tarsomeres
No effect
Antenna
Dll
Loss of distal antennal segments
Loss of tissue identity
Partial transformation into a leg
Bump formation and
no outgrowths
dac
No effect
No effect
Partial loss of antennal segments
No effect
ab
Slightly compressed, but
segment number is unaffected
No effect
Partial loss of antennal segments
No outgrowths in some
larvae; others no effect
ss
Partial transformation into a leg
No effect
Partial transformation into a leg
Partial transformation into
a leg
The leg and antennal phenotypes observed after maternal RNA interference (RNAi) or larval RNAi of ab, dac, Dll and ss expression with and without ablation are
summarized. Knockdown of dac expression prevents regeneration of
the medial segments of legs When smaller larvae were injected, the larvae molted three
times, allowing us to see the effect of dac knockdown on
the regenerating larval leg. When ampr dsRNA-injected
larvae molted three times, the morphology of all segments
was restored (Figure 3A). In contrast, when dac expression
was knocked down, the femur and tibiotarsus of the
regenerated legs were partially fused (Figure 3B). This
leg phenotype was similar to the embryonic legs of
dac-knockdown larvae (compare Figures 1F and 3B). We next examined the effects of larval RNAi during
regeneration. All of the phenotypic effects observed in
this study are summarized in Table 2. To examine the role
of Dac during Tribolium leg regeneration, dac dsRNA was
injected into day-0 fifth- or sixth-instar larvae. Two days
later, the mid- and hindlegs on one side of these larvae
were ablated, and the regeneration of the ablated append-
age after every molt was recorded (Table 4). After one
molt, the site of ablation was completely healed in all
larvae (n = 18) (Table 4; Figure 2A and C). After two
molts, all larvae (n = 16) began to regenerate truncated
legs (Table 4). The regenerating mid- and hindlegs of the
dac dsRNA-injected animals had regenerated the coxa,
trochanter and distal claw, but the femur and tibiotarsal
segment appeared to be shortened (Figure 2D) compared
to the ampr dsRNA-injected regenerating limbs (Figure 2B). As reported previously, adults that developed after dac
dsRNA-injection in the larval phase lacked the distal
femur and tibia and had fused proximal tarsomeres [9,18]. When legs were ablated in these dac dsRNA-injected
animals, the regenerated legs in adults that underwent
two molts prior to metamorphosis had smaller femurs
and fused tarsomeres, unlike those of the uncut contralat-
eral legs (n = 12) (Table 4; Figure 4C and D). Knockdown verification
d
h h Dll, Distal-less; dac, dachshund; ab, abrupt; ss, spineless. Table 2 Summary of the knockdown effects observed in this study Table 2 Summary of the knockdown effects observed in this study
Appendage
Gene
No ablation Knockdown of dac expression prevents regeneration of
the medial segments of legs Eggs and newly hatched larvae from female
beetles injected with of 2 μg/μL ampicillin-resistance (ampr) (A-C), dachshund (dac) (D-F) and abrupt (ab) (G-I) double-stranded (ds)RNA were
collected and processed to compare the phenotypes of resulting antennae and legs. Newly hatched ampr dsRNA-treated larvae were used as a
control. The antennae (B,E,H) and legs (C,F,I) were visualized under fluorescence microscopy and are marked with arrowheads. Right panel shows
a color-level inversion image with the leg segments highlighted in yellow. (J) Lateral view of the abdomen of a newly hatched larva obtained
from female adults injected with ampr dsRNA. (K) Lateral view of the abdomen of the late embryo obtained from female adults injected with ab
dsRNA. Scale bars represent 0.1 mm. Figure 1 Effects of patterning gene knockdown on embryonic appendage development. Eggs and newly hatched larvae from female
beetles injected with of 2 μg/μL ampicillin-resistance (ampr) (A-C), dachshund (dac) (D-F) and abrupt (ab) (G-I) double-stranded (ds)RNA were Figure 1 Effects of patterning gene knockdown on embryonic appendage development. Eggs and newly hatched larvae from female
beetles injected with of 2 μg/μL ampicillin-resistance (ampr) (A-C), dachshund (dac) (D-F) and abrupt (ab) (G-I) double-stranded (ds)RNA were
collected and processed to compare the phenotypes of resulting antennae and legs. Newly hatched ampr dsRNA-treated larvae were used as a
control. The antennae (B,E,H) and legs (C,F,I) were visualized under fluorescence microscopy and are marked with arrowheads. Right panel shows
a color-level inversion image with the leg segments highlighted in yellow. (J) Lateral view of the abdomen of a newly hatched larva obtained
from female adults injected with ampr dsRNA. (K) Lateral view of the abdomen of the late embryo obtained from female adults injected with ab
dsRNA. Scale bars represent 0.1 mm. Knockdown of dac expression prevents regeneration of
the medial segments of legs The femur
and the segment corresponding to fused tarsomeres were Table 3 Summary of the effects of ab, dac, Dll and ss dsRNA injection on embryonic appendage development
Treatment (dsRNA)
Total number
of embryos
Antenna
Leg
Unaffected
F/Sc/T*
Short
Unaffected
F/Sc/T*
Short
ampr
49
49 (100%)
0 (0%)
0 (0%)
49 (100%)
0 (0%)
0 (0%)
dac
47
47 (100%)
0 (0%)
0 (0%)
23 (49%)
24 (F)(51%)
0 (0%)
Dll
53
4 (8%)
0 (0%)
49 (92%)
4 (8%)
0 (0%)
49 (92%)
ss
58
0 (0%)
58 (T)(100%)
0 (0%)
58 (100%)
0 (0%)
0 (0%)
ab
65
7 (11%)
57 (Sc)(88%)
1 (1%)
7 (11%)
58 (Sc)(89%)
0 (0%)
*F/Sc/T denotes embryos that are fused (F), scrunched (Sc) or transformed (T). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. Eggs collected from female beetles injected with ab, dac, Dll and ss dsRNA (2 μg/μL) of were collected and processed to determine the phenotypes of the
developing embryonic appendages, the antennae and legs. The ampr-treated embryos were used as the control. The antenna and legs were scored according to
the following categories: unaffected, F/Sc/T (fused, scrunched, or transformed compared to the control), or short (lengths of segments are different from the
control). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. mary of the effects of ab, dac, Dll and ss dsRNA injection on embryonic appendage development Table 3 Summary of the effects of ab, dac, Dll and ss dsRNA injection on embryonic appendage d *F/Sc/T denotes embryos that are fused (F), scrunched (Sc) or transformed (T). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. Eggs collected from female beetles injected with ab, dac, Dll and ss dsRNA (2 μg/μL) of were collected and processed to determine the phenotypes of the
developing embryonic appendages, the antennae and legs. The ampr-treated embryos were used as the control. The antenna and legs were scored according to
the following categories: unaffected, F/Sc/T (fused, scrunched, or transformed compared to the control), or short (lengths of segments are different from the
control). ampr, ampicillin-resistance; dac, dachshund; Dll, Distal-less; ss, spineless; ab, abrupt. Page 6 of 16 Lee et al. EvoDevo 2013, 4:20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Figure 1 Effects of patterning gene knockdown on embryonic appendage development. The fifth- or sixth-instar larvae were injected with ampr, dac, Dll, ab and ss dsRNA, and their legs were ablated. ampr dsRNA injections served as controls.
*Regeneration in the dac dsRNA-injected insects was different from that in the control ampr dsRNA-injected insects. ampr, ampicillin-resistance; dac, dachshund; Dll,
Distal-less; ab, abrupt; ss, spineless. Knockdown of dac expression prevents regeneration of
the medial segments of legs Table 4 Summary of effects of patterning gene knockdowns on larval leg regeneration in Tribolium
Larvae after 2 molts
Pupae after 1 molt
Pupae after 2 molts
dsRNA
injected
Concentration
Total
number
Number with
regenerated
legs
Number with no
regenerated
legs
Died before
pupation after
2 larval molts
Number
with leg
regeneration
Number
with no leg
regeneration
Number
with leg
regeneration
Number
with no leg
regeneration
ampr
2 μg/μL
23
10
0
3
13
0
7
0
dac
2μg/μL
18
16*
0
4
2
0
12*
0
Dll
2μg/μL
18
0
18
16
0
0
0
2
ab
2 μg/μL
28
0
19
13
0
9
0
6
ss
4 μg/μL
18
11
0
3
7
0
8
0
2 μg/μL
10
7
0
1
3
0
6
0
The fifth- or sixth-instar larvae were injected with ampr, dac, Dll, ab and ss dsRNA, and their legs were ablated. ampr dsRNA injections served as controls. *Regeneration in the dac dsRNA-injected insects was different from that in the control ampr dsRNA-injected insects. ampr, ampicillin-resistance; dac, dachshund; Dll,
Distal-less; ab, abrupt; ss, spineless. ummary of effects of patterning gene knockdowns on larval leg regeneration in Tribolium Table 4 Summary of effects of patterning gene knockdowns on larval leg regene The fifth- or sixth-instar larvae were injected with ampr, dac, Dll, ab and ss dsRNA, and their legs were ablated. ampr dsRNA injections served as controls. *Regeneration in the dac dsRNA-injected insects was different from that in the control ampr dsRNA-injected insects. ampr, ampicillin-resistance; dac, dachshund; Dll,
Distal-less; ab, abrupt; ss, spineless. Page 7 of 16 Page 7 of 16 Lee et al. EvoDevo 2013, 4:20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 http://www.evodevojournal.com/content/4/1/20 Figure 2 The effect of patterning gene knockdown on Tribolium larval leg regeneration one or two molts after leg ablation. (A,C,E,G,I,K)
Regenerating larval legs after one molt in larvae injected with ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA
(C), Distal-less (Dll) dsRNA (E), abrupt (ab) dsRNA (G), spineless (ss) dsRNA (2 μg/μl) (I) and ss dsRNA (4 μg/μl) (K). (B,D,F,H,J,L) Regenerating larval legs
after two molts in larvae injected with ampr dsRNA (B), dac dsRNA (D), Dll dsRNA (F), ab dsRNA (H), ss dsRNA (2 μg/μl) (J) and ss dsRNA (4 μg/μl)
(L). Arrowheads indicate the bases of the ablated legs. Middle panel shows the close-up image of the hindleg. Knockdown of dac expression prevents regeneration of
the medial segments of legs Right panel shows a color-level inversion
image with the regenerating leg segments highlighted in yellow. Cx, coxa; tr, trochanter; f, femur; tt, tibiotarsus. Scale bars correspond to 0.1 mm. Figure 2 The effect of patterning gene knockdown on Tribolium larval leg regeneration one or two molts after leg ablation. (A,C,E,G,I,K)
Regenerating larval legs after one molt in larvae injected with ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA
(C), Distal-less (Dll) dsRNA (E), abrupt (ab) dsRNA (G), spineless (ss) dsRNA (2 μg/μl) (I) and ss dsRNA (4 μg/μl) (K). (B,D,F,H,J,L) Regenerating larval legs
after two molts in larvae injected with ampr dsRNA (B), dac dsRNA (D), Dll dsRNA (F), ab dsRNA (H), ss dsRNA (2 μg/μl) (J) and ss dsRNA (4 μg/μl)
(L). Arrowheads indicate the bases of the ablated legs. Middle panel shows the close-up image of the hindleg. Right panel shows a color-level inversion
image with the regenerating leg segments highlighted in yellow. Cx, coxa; tr, trochanter; f, femur; tt, tibiotarsus. Scale bars correspond to 0.1 mm. (Table 5; Figure 5B). After two molts, all of the dac
dsRNA-injected larvae (n = 14) regenerated their cut
antennae. They either partially developed a new segment
distal to the site of ablation, or fully regenerated their
larval antennae, as observed in the ampr dsRNA-injected
animals (Figure 5B). All regenerated antennae were indis-
tinguishable from the unablated contralateral antennae reduced in length, and in some cases, were missing. These
data indicate that Dac plays a role in re-patterning the
medial segments during leg regeneration and that the
alteration in larval leg patterning is carried over to the adult
leg patterning. The silencing of dac expression during the larval stage
did not appear to affect the regeneration of antennae Page 8 of 16 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20 http://www.evodevojournal.com/content/4/1/20 Figure 3 Regenerated legs of larvae after three molts post gene knockdown and leg ablation. (A-C) Regenerating larval legs injected with
ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA (B) and spineless (ss) dsRNA (4 μg/μl) (C). The mid- and hindlegs
(marked with arrowheads) were removed. A close-up of the hindleg is shown in the center, and right panels show a color-level inversion image
with the regenerated leg segments highlighted in yellow. Fourth-instar larvae were injected on day 0, and legs were removed on day 2. Dll is essential for regeneration of appendages Dll is essential for regeneration of appendages
Injection of Dll dsRNA into day-0 sixth-instar larvae
followed by leg ablation resulted in no regeneration of
limbs. After one molt, all of the larvae (n = 18) were able
to heal the wound around the area of leg ablation
(Figure 2E; Table 4), similar to the ampr dsRNA-treated
animals. After two or more molts (Table 4), however, none
of the Dll dsRNA-injected larvae were able to regenerate
their ablated limbs (n = 18), even though the ampr
dsRNA-treated animals regenerated their limbs. All of the
ablated leg segments, such as the femur, tibiotarsus and
claw, were missing (Figure 2F). In the absence of Dll, only
a round blastema-like structure was present on the
ablated legs after two molts (Figure 2F), whereas in the
control ampr dsRNA-injected animals, all of the leg
segments were re-established after two molts (Figure 2B). In addition, the morphology of the contralateral, unablated
legs gradually became distorted with increasing number of Similarly, day-0 sixth-instar larvae (n = 10) injected with
2 μg/μL Dll dsRNA followed by antennal ablation did not
regenerate any structures after two molts (Table 5;
Figure 5C). They formed rounded large blastema-like
bumps that failed to grow back any of the antennal seg-
ments even after multiple supernumerary molts. Thus, Dll
appears to be necessary for re-differentiation during larval
appendage regeneration. Knockdown of dac expression prevents regeneration of
the medial segments of legs Figure 3 Regenerated legs of larvae after three molts post gene knockdown and leg ablation. (A-C) Regenerating larval legs injected with
ampicillin-resistance (ampr) double-stranded (ds)RNA (A), dachshund (dac) dsRNA (B) and spineless (ss) dsRNA (4 μg/μl) (C). The mid- and hindlegs
(marked with arrowheads) were removed. A close-up of the hindleg is shown in the center, and right panels show a color-level inversion image
with the regenerated leg segments highlighted in yellow. Fourth-instar larvae were injected on day 0, and legs were removed on day 2. molts, indicating that Dll is required to maintain leg
identity, as previously reported [18]. Most larvae continued
to molt as larvae and never metamorphosed. This
inability to metamorphose was also observed previously
in unablated larvae, so it was likely independent of
wounding. The two adults that metamorphosized after
more than two molts post Dll dsRNA injection did not
regenerate any structures on their ablated side (Table 4;
Figure 4I). Given that the blastema-like bump in these
Dll dsRNA-injected larvae were prominent, Dll appears
to be required for re-differentiation of the blastema-like
bump into a functional leg, but not for the formation of
the bump. (Figure 6B) in all adults that pupated after two molts
(n = 12), and both ablated and unablated antennae had
reduced numbers of segments as reported previously
[8,18]. This indicates that Dac does not play a major
role during antennal regeneration, whereas it is necessary
for proper antennal development during metamorphosis. Ab is required for blastema development The identity of the observed
structure and the claw-like structures could not be
determined, but the abnormal structure showed that
Ab strongly influences growth/patterning during regener-
ation, in addition to regulating the growth of blastemas. This was in contrast to the unablated legs of the adults
obtained from ab dsRNA-injected larvae, which were
affected such that the tarsomeres were reduced in number
(Figure 4F), but the femur and the claws at the tip of
the tarsus remained unaffected, as reported previously
[8,9]. Thus, although silencing of ab only affects tarsal
segmentation during metamorphosis, it prevents legs from
re-forming during regeneration. The effect of removing larval Ab expression on the
regeneration of antennae was variable. As with the ampr
dsRNA-injected control animals, sixth-instar larvae injected
with 2 μg/μL ab dsRNA after one molt were able to heal
their wounds and sometimes form blastema-like structures. After two molts, a few larvae (n = 4) were able to regener-
ate a new segment on the site of antennal ablation,
whereas the rest (n = 7) did not regenerate any new struc-
tures (Figure 5D; Table 5). However, the majority of the
animals that were injected with ab dsRNA and underwent
antennal ablation died as larvae, as they got trapped in the
old cuticle during the larval-larval molts. The head often
became trapped in the old cuticle, and the larvae eventually
died, presumably from starvation. Ab is required for blastema development Similarly, the
failed to show any signs of leg regeneratio
structures were visible at the ablated area
mass of unidentifiable tissue was observed w
femur nor tibia regenerated (Figure 4E, arro
particular adult that had developed a large
the second larval molt post injection, a
appendage developed (Figure 4F). The str
sclerotized and exhibited normal pigmentatio
development. However, this appendage had o
with several abnormal protrusions, as well
claw-like protrusions (Figure 4F) at the bound
the coxa and the segment. The identity of t
structure and the claw-like structures co
determined, but the abnormal structure s
Ab strongly influences growth/patterning dur
ation, in addition to regulating the growth o
Table 5 Summary of the effects following kno
Larvae after 2
dsRNA
injected
Concentration
Total
number
Number with
regenerated
antennae
Nu
no r
a
ampr
2 μg/μL
14
10
dac
2 μg/μL
27
14
Dll
2 μg/μL
10
0
ab
2 μg/μL
14
4
0.5 μg/μL
9
2
ss
2 μg/μL
22
21*
The left antennae of fifth- and sixth-instar larvae treated with
concentrations were ablated. The numbers of Tribolium treated
regeneration was different from the control ampr dsRNA-inject all cases, after one molt, the wound healed and a
smooth, rounded covering formed (Figure 2G). After the
second molt, the legs had still not regenerated in the ab
dsRNA-injected larvae (n = 19) (Table 4), and a small
bump was observed (Figure 2H), indicating that little cell
proliferation had taken place. Beyond the coxa, none of
the larval leg segments were distinguishable. Although
many larvae died prior to metamorphosis (n = 13), none
of the animals that pupated after two larval molts
regenerated pupal legs (n = 6). Similarly, the adults also
failed to show any signs of leg regeneration: often no
structures were visible at the ablated areas, or a tiny
mass of unidentifiable tissue was observed with neither
femur nor tibia regenerated (Figure 4E, arrows). In one
particular adult that had developed a large bump after
the second larval molt post injection, a malformed
appendage developed (Figure 4F). The structure was
sclerotized and exhibited normal pigmentation and bristle
development. However, this appendage had one segment
with several abnormal protrusions, as well as a pair of
claw-like protrusions (Figure 4F) at the boundary between
the coxa and the segment. Ab is required for blastema development We next examined the effect of silencing ab during the
larval stage on the regeneration of ablated larval legs. In Page 9 of 16 Lee et al. EvoDevo 2013, 4:20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 http://www.evodevojournal.com/content/4/1/20 Figure 4 (See legend on next page.) Page 10 of 16 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 (See figure on previous page.)
Figure 4 The effects of appendage patterning gene knockdowns on adult leg regeneration in Tribolium. (A,B) Adult leg phenotype of
larvae injected with 2 μg/μL ampicillin-resistance (ampr) double-stranded (ds)RNA. Adults formed from pupae that experienced one molt (A) or
two molts (B) after dsRNA injection. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. (C,D) Adults that formed after larvae were injected with 2 μg/μL dachshund (dac) dsRNA and underwent one molt (C) and two molts (D) before
pupation. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. Arrowheads indicate the segment
on the unablated leg that is missing in the ablated leg. (E,F) Adults that formed after larvae were injected with 2 μg/μL abrupt (ab) dsRNA and
underwent two molts before pupation. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. (E) Arrows indicate the site where the legs should have regenerated. (F) Arrow indicates the claw-like structure that developed at the base of the
leg. (G,H) Adults that formed after larvae were injected with 4 μg/μL ss dsRNA and underwent one molt (G) and two molts (H) before pupation. Close-up of the unablated (middle) and ablated (right) mid- (top) and hindlegs (bottom) are shown. (I) Adults that formed after larvae were
injected with 2 μg/μL Distal-less (Dll) dsRNA and underwent three molts before pupation. Arrows indicate the sites where the legs should
have regenerated. all cases, after one molt, the wound he
smooth, rounded covering formed (Figure 2G
second molt, the legs had still not regenerat
dsRNA-injected larvae (n = 19) (Table 4),
bump was observed (Figure 2H), indicating th
proliferation had taken place. Beyond the co
the larval leg segments were distinguishabl
many larvae died prior to metamorphosis (n
of the animals that pupated after two l
regenerated pupal legs (n = 6). Ab is required for blastema development Only two animals
survived to metamorphose after one molt: one of them
regenerated an antenna that resembled the unablated
contralateral antenna, and the other became a pharate adult
that did not exhibit any signs of antennal regeneration Table 5 Summary of the effects following knockdown of patterning genes on larval antennal regeneration in Tribolium
Larvae after 2 molts
Pupae after 1 molt
Pupae after 2 molts
dsRNA
injected
Concentration
Total
number
Number with
regenerated
antennae
Number with
no regenerated
antennae
Died before
pupation after
2 larval molts
Number
with antennal
regeneration
Number with
no antennal
regeneration
Number with
antennal
regeneration
Number with
no antennal
regeneration
ampr
2 μg/μL
14
10
0
0
4
0
10
0
dac
2 μg/μL
27
14
0
2
10
3
12
0
Dll
2 μg/μL
10
0
7
7
0
3
0
0
ab
2 μg/μL
14
4
7
11
2
1
0
0
0.5 μg/μL
9
2
7
8
0
0
1
0
ss
2 μg/μL
22
21*
0
9
0
1
12*
0
The left antennae of fifth- and sixth-instar larvae treated with abrupt (ab), dachshund (dac), Distal-less (Dll) and spineless (ss) double-stranded (ds)RNA at various
concentrations were ablated. The numbers of Tribolium treated with the ampicillin-resistance (ampr) dsRNA are also included as a control. *For ss, the pattern of
regeneration was different from the control ampr dsRNA-injected regeneration; the regenerating antennae transformed into a leg. Table 5 Summary of the effects following knockdown of patterning genes on larval a 5 Summary of the effects following knockdown of patterning genes on larval antennal regenerati cts following knockdown of patterning genes on larval antennal regeneration in Tribolium The left antennae of fifth- and sixth-instar larvae treated with abrupt (ab), dachshund (dac), Distal-less (Dll) and spineless (ss) double-stranded (ds)RNA at various
concentrations were ablated. The numbers of Tribolium treated with the ampicillin-resistance (ampr) dsRNA are also included as a control. *For ss, the pattern of
regeneration was different from the control ampr dsRNA-injected regeneration; the regenerating antennae transformed into a leg. Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Page 11 of 16 Figure 5 Effects of patterning gene knockdown on larval antennal regeneration. Larvae were treated with 2 μg/μL ampicillin-resistance
(ampr) (A), dachshund (dac) (B), Distal-less (Dll) (C), abrupt (ab) (D) and spineless (ss) (E) double-stranded (dsRNA). Left panels, the head of the
treated larvae; middle panels, unablated antennae; right panels, ablated antennae. Ab is required for blastema development (E) Arrow indicates the claw-like structure in the regenerated
antenna of ss dsRNA-injected larva. The arrowhead indicates the ectopic bristle on the transformed antenna. Figure 5 Effects of patterning gene knockdown on larval antennal regeneration. Larvae were treated with 2 μg/μL ampicillin-resistance
(ampr) (A), dachshund (dac) (B), Distal-less (Dll) (C), abrupt (ab) (D) and spineless (ss) (E) double-stranded (dsRNA). Left panels, the head of the
treated larvae; middle panels, unablated antennae; right panels, ablated antennae. (E) Arrow indicates the claw-like structure in the regenerated
antenna of ss dsRNA-injected larva. The arrowhead indicates the ectopic bristle on the transformed antenna. dsRNA (Table 5). There were two larvae with regenerating
antennae (developing new structures on the site of
ablation) after two molts; one of these larvae molted
successfully into an adult with a perfectly regenerated
antenna that resembled the uncut control antenna. However, the remainder of the larvae (n = 7) did not
show signs of antennal regeneration after two molts. In addition, decreasing the concentration to 0.5 μg/μL
did not decrease the rate of larval mortality (8 larvae
died after two molts). Thus, at either concentration, (not shown). The remaining larvae that survived past two
molts (n = 11) were unable to properly eclose or meta-
morphose, and ultimately died as larvae. The observed
deaths were not confined to larvae with ablated limbs:
many unablated Ab-knockdown larvae also died prior to
metamorphosis (not shown). To see if we could obtain higher survival rates, we
injected fifth-instar larvae (n = 9) with 0.5 μg/μL ab
dsRNA. The degree of antennal regeneration was variable,
as with the larvae injected with a higher concentration of Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Page 12 of 16 Lee et al. EvoDevo 2013, 4:20 Figure 6 Effects of patterning gene knockdown on adult antennal regeneration. Adults that formed after larvae were treated with 2 μg/μL
ampicillin-resistance (ampr) (A), dachshund (dac) (B) and spineless (ss) (C) double-stranded (ds)RNA. All injected animals underwent two molts prior
to the prepupal stage. Comparable pictures were taken under the same magnification. Figure 6 Effects of patterning gene knockdown on adult antennal regeneration. Adults that formed after larvae were treated with 2 μg/μL
ampicillin-resistance (ampr) (A), dachshund (dac) (B) and spineless (ss) (C) double-stranded (ds)RNA. All injected animals underwent two molts prior
to the prepupal stage. Comparable pictures were taken under the same magnification. Ss is not required for leg regeneration When ss was silenced prior to leg ablation during the
larval stage, both ablated legs began to regenerate by the
second molt (n = 11 for 4 μg/μL and n = 7 for 2 μg/μL;
Table 4; Figure 2J). These legs were similar to the regen-
erating
legs
of
ampr
dsRNA-injected
larvae
and
regenerated all of the larval leg segments. When smaller
larvae were injected, larvae that molted three times had
restored the morphology of leg segments, similar to
ampr dsRNA-injected larvae (compare Figure 3A and C). ss-knockdown without leg ablations resulted in the
formation of adults with phenotypes similar to those
obtained by Shippy et al. (2009) and Angelini et al. (2012) (Figure 4G and H) [9,10]. As described previously,
the tarsomeres were fused and were shorter than those of
the control ampr dsRNA-injected adults. In the adults
obtained from ss dsRNA-injected larvae followed by
leg ablations, the regenerated legs were morphologically
indistinguishable from the contralateral unablated legs
(Table 4; Figure 4G and H). From these results, we infer
that Ss is not necessary for larval leg regeneration. In the unablated antenna of ss-knockdown larvae, the
antennae transformed partially into a leg-like structure
during metamorphosis (n = 14). These animals have one
large structure that appears to comprise a fusion of the
femur, tibia and tarsomeres (Figure 6C; [10]). The adult
structure on the ablated side had distinct leg-like struc-
tures with clearly distinguishable tibia and tarsomeres, in
contrast to the contralateral unablated antennae, where the
tibia and tarsomeres appeared to be fused. Presumably, the
earlier transformation of the regenerating antenna into
leg-like phenotype accounts for the more enhanced leg-
like morphology observed in the ss-knockdown adult. Thus, similar to the effect of dac knockdown in regenerating
legs, the antennal patterning defects in ss dsRNA-injected
larvae were reflected in the adult antennal morphology. Taken together, our results indicate that Ss plays an im-
portant role in maintaining the identity of the antennae
during larval antennal regeneration and metamorphosis. When ss was knocked down in day-0 fifth- or sixth-
instar larvae, all of the ablated antennae (n = 22) (Table 5)
healed and formed rounded structures as seen in the
control ampr-knockdown animals after the first molt
(Figure 5A). After the second molt, all of the 2 μg/μL ss Ab is required for blastema development most ab dsRNA-injected larvae were unable to regen-
erate their ablated antennae after two molts (n = 7/11
for 2 μg/μL and n = 7/9 for 0.5 μg/μL; Table 5). Combined, these results indicate that Ab is required
for blastema development during regeneration of both
legs and antennae. dsRNA-injected larvae (n = 21) developed leg-like struc-
tures in the place of a regenerated antenna (Figure 5E;
Table 5). The regenerated antennae developed a claw and
a bristle pattern resembling those seen in larval legs
(Figure 5E, arrow and arrowhead). The unablated antennae
remained unaffected and did not transform, indicating that
Ss is only required when the larval antenna is regenerating. Such heteromorphic transformation of regenerating
appendages was never seen with the knockdowns of any
of the other transcription factors investigated here. Discussion
I
hi
d In this study, we examined the roles of limb-patterning
genes during embryonic development and regeneration,
and the degree of developmental coupling between larval
and adult limb-patterning (Table 2). We found that dac-
knockdown embryos developed legs with abnormally
shortened medial segments, while the antennae developed
normally. In addition, silencing of dac resulted in loss of
the medial segments of the regenerating legs; this altered
phenotype was carried over to the adult leg morphology. However, no major effects were detected in the regener-
ating antennae. Knockdown of Dll completely inhibited
regeneration of the ablated appendages, although a promin-
ent blastema-like structure formed. ab-knockdown embryos
exhibited a unique scrunched phenotype throughout their
entire body, including their antennae and legs; however,
each of the segments of these appendages was distin-
guishable. These embryos also displayed abnormal bris-
tle patterning on the dorsal tergites. Ab was found to be
essential for blastema growth/development. Ss-knock-
down larvae were able to regenerate their limbs, but a
heteromorphic transformation of the larval antenna to a
leg was observed when the antennae were removed. This
larval heteromorphic effect was also carried over to the
adult stage. Legs regenerated normally when the expression of ss
was knocked down. However, knockdown of ss in the
fifth or sixth instars affected the regeneration of the
antennae. The ablated antenna underwent a heteromorphic
transformation and developed into a structure that resem-
bled a leg. A similar transformation of an antenna to a leg
was also observed in embryos [10,29]. The embryonic
phenotypes resulting from dsRNA treatment suggest that
Ss is not involved in the embryonic patterning of legs but
plays a key role in specifying antennal identity. In contrast,
during metamorphosis, Ss is required for patterning the
distal segments of the developing adult legs [8-10,25]. Taken together, the functions of Ss and Dac during regen-
eration are similar to those seen during embryonic, rather
than metamorphic, development (Table 2). Dac and Ss function similarly during regeneration and
embryonic development Although dac dsRNA-injected Tribolium were able to
regenerate both the larval legs and antennae after two
molts (Tables 4 and 5), knockdown of Dac expression Knockdown verification We performed semiquantitative PCR to determine the
gene expressions of dac, Dll, ab and ss in response to
dsRNA injections. We confirmed that gene expression Page 13 of 16 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Page 13 of 16 disrupted the patterning of the medial segments in the re-
generating legs (Table 2; Figure 3B). Our results indicate
that Dac acts specifically in the legs to pattern the medial
segments during regeneration, which is consistent with
past studies of Dac in cricket leg regeneration [34]. In the
antennae, Dac seems to have no apparent role during larval
regeneration, and the regenerated adult antennae were
indistinguishable from the unablated antennae (Figures 5B
and 6B). Similar effects of patterning were observed in the
dac-knockdown embryos: in embryos affected by dac
dsRNA, the antennae developed normally, but the legs
had a shortened femur-tibiotarsus segment, akin to the
regenerated larval legs of dac-knockdown larvae. These
findings are consistent with previous studies, which have
determined that dac is most strongly expressed in the legs
and less so in the antennae [15,35], suggesting that the
role of Dac in patterning the medial segments of the
antenna is largely confined to metamorphosis [8], and not
regeneration or embryonic development. Thus, Dac func-
tions similarly during larval appendage regeneration and
embryonic development (Table 2). was knocked down through dsRNA injection by comparing
the expression in ampr dsRNA-injected animals with that
in the dsRNA-injected animals (Figure 7). Compared to
the control larvae, the target gene expression was reduced
in larvae injected with dsRNA of the respective genes
(Figure 7). Thus, we established that the phenotypes
shown in this study were the effects resulting from the
knockdown of each gene. Developmental uncoupling between larval and adult
morphologies An unresolved issue in holometabolous insect development
and evolution is the developmental continuity between
larval legs and adult legs. At the cellular level, there
appears to be a smooth transition between the larval
appendages and adult appendages in most insects except
in the case of lepidopterans, where the adult appendages
actually arise from set-aside imaginal cells that reside
within the larval legs and proliferate during the final instar
[4]. What is less clear is how patterning of larval append-
ages affects adult appendages. Our study shows that larval
appendage patterning is inherited by the adult appendages. For example, when Dac is silenced in the regenerating
larval legs, the medial segments are deleted, and this
patterning alteration is also observed in the adult stage. While unablated adult legs are also affected, the regenerated
leg is more severely affected. This implies that either the
fates of the presumptive adult cells are already specified
by the time they pupate or the number of cells present in
the larval appendage constrains the adult leg morphology. Regardless of the mechanism, our study indicates that
larval appendage patterning might pose a developmental
constraint on the adult appendages. Thus, while the evolu-
tion of metamorphosis allowed the generation of two
distinct morphs, the larva and the adult, which are each
adapted to a different habitat, the two life-history stages
may constrain each other, at least in some holometabolous
insects, such as beetles. Unlike the effect of silencing ab on regeneration, Ab was
not required for the initial allocation and subsequent for-
mation of either antennae or legs during embryonic devel-
opment (Table 2). During metamorphosis, the tarsomeres
became fused in ab-knockdown animals; however, the rest
of the leg was unaffected. Thus, while Ab is involved in the
distal patterning of adult legs during normal development,
Ab appears to play a distinct role during regeneration by
affecting the development and growth of the blastema
(Table 2). Together, these data indicate that the role of Ab
during regeneration is separate from its embryonic and
metamorphic function in normal development. The ab-knockdown phenotype is similar to that obtained
when Wnt-1 expression is silenced. When Wnt-1 is
silenced, the blastema fails to grow, and the legs are never
able to regenerate, whereas the amount of antennal regen-
eration is variable between individuals [14]. Thus, Ab and
the Wnt signaling pathway may influence the same process
during regeneration and may potentially interact. Developmental uncoupling between larval and adult
morphologies It is
interesting that both Wnt-1 and Ab knockdown animals
form rudimentary wings during metamorphosis (unpub-
lished observations and [14]). The heteromorphic transformations seen in ss knock-
down animals have interesting implications for under-
standing the developmental basis for naturally occurring
heteromorphic transformations. The theory of homeotic
transformation was first developed by Bateson (1894) to
describe the replacement of one structure, such as the
antennae, with a completely different one, such as the
tarsus of a leg [53]. Though at first believed to be isolated
examples of developmental anomalies, various studies
have demonstrated that heteromorphic transformations
could be experimentally induced by physical mutilation
during late development [54,55]. For example, injuring the
antennae of a sawfly, Cimbex axillaris, can cause the
heteromorphic transformation of the adult antenna into a
leg [55]. However, the molecular basis of postembryonic
heteromorphic transformation of an appendage has not
been well-characterized. Our study demonstrates that
given the continuity of larval and adult stages, alteration
of a single transcription factor, such as Ss, during a spe-
cific time period of larval life can induce the homeotic Ab is required for initiation of regeneration specific embryonic patterning genes are used to re-pattern
the appendages. Whether or not the first steps of limb re-
generation, such as wound healing and blastema formation,
are evolutionarily conserved processes remains to be seen. Our study shows that Ab plays an essential role in limb
regeneration. Beyond the formation of a small bump,
appendage regeneration appeared to be stunted in ab-
knockdown animals. This indicates that Ab may be
involved in cell proliferation of blastema cells during leg
regeneration. The effects of ab knockdown on antenna
regeneration, though less definitive given that the antennae
of a few larvae regenerated after two molts (Table 5), also
indicated that Ab is required for the initiation of antennal
regeneration. Developmental uncoupling between larval and adult
morphologies Dll is required for initiation of leg outgrowth during
regeneration None of the animals injected with Dll dsRNA were able
to regenerate any structure distal to the site of ablation
in either the legs (Tables 2 and 4; Figure 2E, 2F and 4I)
or the antennae (Tables 2 and 5; Figure 5C). For all of
the Dll-knockdown animals, a large bump formed on
the ablated sites and remained throughout the molts, indi-
cating that the bump is able to grow. However, no append-
ages ever differentiated from this bump, and the legs
were absent from the adult (Figure 4I). At this point, we
do not have any blastema markers so we are unable to
determine the nature of this bump. However, given the
previous finding that Dll is required to maintain the
appendage identity [18], Dll may be required for re-
differentiation of cells inside this bump. Figure 7 Knockdown verification of double-stranded RNA
(dsRNA)-injected larvae using semiquantitative polymerase
chain reaction. Day-0 sixth-instar larvae were injected with 2 μg/μL
ampicillin-resistance (ampr), dachshund (dac), Distal-less (Dll), abrupt
(ab) and spineless (ss) dsRNA, and total RNA was isolated from
day-2 seventh-instar larvae. This RNA was converted to cDNA, and
gene-specific primers were used to amplify the gene products. ampr
dsRNA-injected larvae served as controls. The cycle numbers were as
follows: ab = 38; dac = 42; Dll = 39; ss = 40; rp49 = 34. Figure 7 Knockdown verification of double-stranded RNA
(dsRNA)-injected larvae using semiquantitative polymerase
chain reaction. Day-0 sixth-instar larvae were injected with 2 μg/
ll
(
r) d
h h
d (d
)
l l
( ll)
b Figure 7 Knockdown verification of double-stranded RNA
(dsRNA)-injected larvae using semiquantitative polymerase
chain reaction. Day-0 sixth-instar larvae were injected with 2 μg/μL
ampicillin-resistance (ampr), dachshund (dac), Distal-less (Dll), abrupt
(ab) and spineless (ss) dsRNA, and total RNA was isolated from
day-2 seventh-instar larvae. This RNA was converted to cDNA, and
gene-specific primers were used to amplify the gene products. ampr
dsRNA-injected larvae served as controls. The cycle numbers were as
follows: ab = 38; dac = 42; Dll = 39; ss = 40; rp49 = 34. Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Page 14 of 16 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Conclusions 8. Angelini DR, Kikuchi M, Jockusch EL: Genetic patterning in the adult
capitate antenna of the beetle Tribolium castaneum. Dev Biol 2009,
327:240–251. 8. Angelini DR, Kikuchi M, Jockusch EL: Genetic patterning in the adult
capitate antenna of the beetle Tribolium castaneum. Dev Biol 2009,
327:240–251. Our study indicates that larval limb morphology is
linked to the adult limb patterning in Tribolium. We
propose that the evolution of free-floating imaginal discs
may have been a key step towards to completely removing
the developmental constraint that existed between the
larval and adult phenotypes. Thus, the evolution of insects
is characterized by a gradual loss of developmental coupling
between the juvenile and adult stages. 9. Angelini DR, Smith FW, Jockusch EL: Extent with modification: leg
patterning in the beetle Tribolium castaneum and the evolution of seri
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patterning in the beetle Tribolium castaneum and the evolution of serial
homologs. G3 (Bethesda) 2012, 2:235–248. g
10. Shippy TD, Yeager SJ, Denell RE: The Tribolium spineless ortholog specifies
both larval and adult antennal identity. Dev Genes Evol 2009, 219:45–51. 10. Shippy TD, Yeager SJ, Denell RE: The Tribolium spineless ortholog specifies
both larval and adult antennal identity. Dev Genes Evol 2009, 219:45–51. 10. Shippy TD, Yeager SJ, Denell RE: The Tribolium spineless ortholog specifies
both larval and adult antennal identity. Dev Genes Evol 2009, 219:45–51. 11. Namigai EKO, Suzuki Y: Functional conservation and divergence of BMP
ligands in limb development and lipid homeostasis of holometabolous
insects. Evol Dev 2012, 14:296–310. y
11. Namigai EKO, Suzuki Y: Functional conservation and divergence of BMP
ligands in limb development and lipid homeostasis of holometabolous
insects. Evol Dev 2012, 14:296–310. 11. Namigai EKO, Suzuki Y: Functional conservation and divergence of BMP
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required for normal eye and leg development in Drosophila. Development
1994, 120:3473–3486. Ab: Abrupt; ampr: Ampicillin-resistance gene; BTB: Bric-a-brac tramtrack broad
complex; dac: Dachshund; Dll: Distal-less; dsRNA: Double-stranded RNA;
FGF: Fibroblast growth factor; PBS: Phosphate-buffered saline; Ab: Abrupt; ampr: Ampicillin-resistance gene; BTB: Bric-a-brac tramtrack broad
complex; dac: Dachshund; Dll: Distal-less; dsRNA: Double-stranded RNA;
FGF: Fibroblast growth factor; PBS: Phosphate-buffered saline;
PCR: Polymerase chain reaction; PD: Proximal-distal; RNAi: RNA interference;
ss: Spineless; ssRNA: Single-stranded RNA. 17. Tanaka K, Truman JW: Molecular patterning mechanism underlying
metamorphosis of the thoracic leg in Manduca sexta. Dev Biol 2007,
305:539–550. g
p
PCR: Polymerase chain reaction; PD: Proximal-distal; RNAi: RNA interference;
ss: Spineless; ssRNA: Single-stranded RNA. 18. Suzuki Y, Squires DC, Riddiford LM: Larval leg integrity is maintained by
Distal-less and is required for proper timing of metamorphosis in the
flour beetle, Tribolium castaneum. Dev Biol 2009, 326:60–67. Received: 20 March 2013 Accepted: 4 June 2013
Published: 4 July 2013 transformation of the regenerating larval antennae and
subsequently influence the adult antennal identity. Received: 20 March 2013 Accepted: 4 June 2013
Published: 4 July 2013 Compartmentalization of developmental processes has
been proposed to be a major source of evolvability for
different stages of complex life cycles [1]. In insects with
larval limbs that transform into adult limbs, developmental
coupling between larval and adult stages might constrain
evolutionary change. Many derived insect species have
independently evolved imaginal discs that have enabled
complete separation of larval and adult life stages [1,2,56]. Thus, in addition to promoting rapid growth in an organ-
ism with a short life cycle, the evolution of imaginal discs
may serve as a final step towards complete dissociation of
larval and adult structures. Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Page 15 of 16 Page 15 of 16 Re-patterning phase of limb regeneration relies on taxon-
specific regulation Limb regeneration in most metazoans, including Tribolium,
can be classified into four general stages: wound healing,
blastema formation, blastema cell proliferation, and re-
patterning [14,36-38]. Although we have found that both
Dac and Ss are necessary for the re-patterning of certain
appendages in Tribolium, these genes most likely are not
conserved throughout the Metazoa, because they are spe-
cific to the patterning of arthropod appendage segments. Similarly, genes that are necessary for normal limb
patterning and re-patterning in vertebrates, like fibroblast
growth factor (FGF) [39-46], appear not to play significant
roles, at least during the earliest stages of Tribolium limb
re-patterning [47]. Thus, while certain factors appear
to play roles in regeneration across all regenerating
metazoans [14,47-52], genes involved in re-patterning are
unlikely to be a part of any conserved gene regulatory
networks underlying limb regeneration. Rather, it appears
that once the regeneration pathway is activated, taxon- Acknowledgements Okada TS: A brief history of regeneration research - For admiring
Professor Niazi’s discovery of the effect of vitamin A on regeneration. J Biosci 1996, 21:261–271. 30. Hughes C, Kaufman TC: RNAi analysis of Deformed, proboscipedia and sex
combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for
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(Phasmida). J Neurobiol 1989, 20:101–114. 31. Bolognesi R, Farzana L, Fischer TD, Brown SJ: Multiple Wnt genes are
required for segmentation in the short-germ embryo of Tribolium
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maxillopedia expression pattern and larval cuticular phenotype in wild-
type and mutant Tribolium. Genetics 2000, 155:721–731. doi:10.1186/2041-9139-4-20
Cite this article as: Lee et al.: Developmental coupling of larval and adult
stages in a complex life cycle: insights from limb regeneration in the flour
beetle, Tribolium castaneum. EvoDevo 2013 4:20. 33. Beermann A, Jay DG, Beeman RW, Hulskamp M, Tautz D, Jurgens G: The
short antennae gene of Tribolium is required for limb development and
encodes the orthologue of the Drosophila Distal-less protein. Development 2001, 128:287–297. 34. Nakamura T, Mito T, Bando T, Ohuchi H, Noji S: Dissecting insect leg
regeneration through RNA interference. Cell Mol Life Sci 2008, 65:64–72. 35. Dong PDS, Chu J, Panganiban G: Proximodistal domain specification and
interactions in developing Drosophila appendages. Development 2001,
128:2365–2372. 36. Bergantinos C, Corominas M, Serras F: Cell death-induced regeneration in
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disc cells precedes multipotency. Cell 2005, 120:383–393. 39. D’Jamoos CA, McMahon G, Tsonis PA: Fibroblast growth factor receptors
regulate the ability for hindlimb regeneration in Xenopus laevis. Wound
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dependency of regeneration: the role of distal-less and FGF signaling in
amphibian limb regeneration. Development 1996, 122:3487–3497. 41. Acknowledgements 23. Biehs B, Sturtevant MA, Bier E: Boundaries in the Drosophila wing imaginal
disc organize vein-specific genetic programs. Development 1998,
125:4245–4257. We thank Drs John Cameron, Jennifer Hood-DeGrenier, Kaye Peterman, Michelle
LaBonte and Kimberly O’Donnell, and the members of the Suzuki lab, for their
support and critical reading of the manuscript, and Dr Elizabeth Jockusch for
helpful discussions. We thank Dr Richard Beeman and Dr Yoshinori Tomoyasu for
the Tribolium beetles. This study was supported in part by Wellesley College and
by the National Science Foundation Grant number IOS-1027453 to YS. 24. Caygill EE, Johnston LA: Temporal regulation of metamorphic processes
in Drosophila by the let-7 and miR-125 heterochronic microRNAs. Curr Biol 2008, 18:943–950. Page 16 of 16 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 Lee et al. EvoDevo 2013, 4:20
http://www.evodevojournal.com/content/4/1/20 25. Duncan DM, Burgess EA, Duncan I: Control of distal antennal identity and
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the mammalian dioxin receptor. Genes Dev 1998, 12:1290–1303. 49. Yokoyama H, Ogino H, Stoick-Cooper CL, Grainger RM, Moon RT:
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regenerative capacity. Develop Growth Differ 2008, 50:13–22. 26. Emerald BS, Curtiss J, Mlodzik M, Cohen SM: Distal antenna and distal
antenna related encode nuclear proteins containing pipsqueak motifs
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130:1171–1180. 51. Vinarsky V, Atkinson DL, Stevenson TJ, Keating MT, Odelberg SJ: Normal
newt limb regeneration requires matrix metalloproteinase function. Dev Biol 2005, 279:86–98. 27. Struhl G: Spineless-aristapedia: a homeotic gene that does not control the
development of specific compartments in Drosophila. Genetics 1982,
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Epithelial morphogenesis in hydra requires de novo expression of
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antennal determinant spineless. Dev Biol 2007, 302:412–426. 53. Bateson W: Materials for the Study of Variation Treated with Especial Regard
to Discontinuity in the Origin of Species. London: Macmillan; 1894. 29. Toegel JP, Wimmer EA, Prpic NM: Loss of spineless function transforms
the Tribolium antenna into a thoracic leg with pretarsal, tibiotarsal, and
femoral identity. Dev Genes Evol 2009, 219:53–58. y
54. Acknowledgements Lee Y, Grill S, Sanchez A, Murphy-Ryan M, Poss KD: Fgf signaling instructs
position-dependent growth rate during zebrafish fin regeneration. Development 2005, 132:5173–5183. 42. Poss FD, Shen JX, Nechiporuk A, McMahon G, Thisse B, Thisse C, Keating
MT: Roles for Fgf signaling during zebrafish fin regeneration. Dev Biol
2000, 222:347–358. 43. Whitehead GG, Makino S, Lien CL, Keating MT: fgf20 is essential for
initiating zebrafish fin regeneration. Science 2005, 310:1957–1960. 44. Giampaoli S, Bucci S, Ragghianti M, Mancino G, Zhang F, Ferretti P:
Expression of FGF2 in the limb blastema of two Salamandridae
correlates with their regenerative capability. Proc R Soc Lond B Biol Sci
2003, 270(1530):2197–2205. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 45. Poulin ML, Patrie KM, Botelho MJ, Tassava RA, Chiu IM: Heterogeneity in the
expression of fibroblast growth-factor receptors during limb regeneration
in newts (Notophthalmus-viridescens). Development 1993, 119:353–361. • Convenient online submission • Thorough peer review 46. Han MJ, An JY, Kim WS: Expression patterns of Fgf-8 during development
and limb regeneration of the axolotl. Dev Dyn 2001, 220:40–48. 47. Mitten EK, Jing D, Suzuki Y: Matrix metalloproteinases (MMPs) are required
for wound closure and healing during larval leg regeneration in the flour
beetle, Tribolium castaneum. Insect Biochem Mol Biol 2012, 42:854–864. 48. Acknowledgements Nakamura T, Mito T, Tanaka Y, Bando T, Ohuchi H, Noji S: Involvement of
canonical Wnt/Wingless signaling in the determination of the positional
values within the leg segment of the cricket Gryllus bimaculatus. Develop
Growth Differ 2007, 49:79–88. 48. Nakamura T, Mito T, Tanaka Y, Bando T, Ohuchi H, Noji S: Involvement of
canonical Wnt/Wingless signaling in the determination of the positional
values within the leg segment of the cricket Gryllus bimaculatus. Develop
Growth Differ 2007, 49:79–88.
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https://openalex.org/W4237429783
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https://zenodo.org/record/1281304/files/article.pdf
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English
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The effect of the ionosphere on remote sensing of sea surface salinity from space: absorption and emission at L band
|
IEEE transactions on geoscience and remote sensing
| 2,002
|
public-domain
| 7,022
|
Manuscript received June 1, 2001; revised December 13, 2001.
D. M. Le Vine is with the Microwave Sensors Branch/Code 975, Labora-
tory for Hydrospheric Processes, Goddard Space Flight Center, Greenbelt, MD
20771 USA (e-mail: dmlevine@priam.gsfc.nasa.gov).
S. Abraham is with the Science System and Applications, Inc., Microwave
Sensors Branch/Code 975, Goddard Space Flight Center, Greenbelt, MD 20771
USA (e-mail: sabraham@synth.gsfc.nasa.gov).
Publisher Item Identifier S 0196-2892(02)04604-1. 771 771 IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 I. INTRODUCTION T T
HE SALINITY of the open ocean is important for under-
standing ocean circulation and for modeling energy ex-
change with the atmosphere. For example, salinity gradients
affect mixed layer processes, which influence fluxes of heat
near the surface [1]. Salinity and temperature determine water
density and are important factors in large-scale ocean circu-
lation [2]. Also, changes in salinity are primarily caused by
changes in freshwater (evaporation, precipitation, melting ice,
or river input). These changes are manifestations of elements of
the water cycle, which are poorly known over the ocean [3]. The Effect of the Ionosphere on Remote Sensing of
Sea Surface Salinity From Space: Absorption and
Emission at L Band David M. Le Vine, Fellow, IEEE, and Saji Abraham, Member, IEEE However, measurement of sea surface salinity (SSS) in the
open ocean presents a special challenge. This is so because the
dynamic range of SSS in the open ocean is relatively small
(about 5 K) and the requirements for a scientifically useful mea-
surement (about 0.05 K) put a severe constraint on radiometric
performance. A change of 0.05 K at L-band corresponds to a
change of about 0.1 psu [7] where “psu” denotes changes mea-
sured on the practical salinity scale [11]. To put the challenge of
this measurement in perspective, the dynamic range associated
with changes in soil moisture is on the order of 100 K [12] and
measurement requirements are 1–4 K [13]. Abstract—The purpose of this work is to examine the effects
of Faraday rotation and attenuation/emission in the ionosphere in
the context of a future remote sensing system in space to mea-
sure salinity. Sea surface salinity is important for understanding
ocean circulation and for modeling energy exchange with the at-
mosphere. A passive microwave sensor in space operating near 1.4
GHz (L-band) could provide global coverage and complement in
situ arrays being planned to provide subsurface profiles. However,
the salinity signal is relatively small and changes along the prop-
agation path can be important sources of error. It is shown that
errors due to the ionosphere can be as large as several psu. The
dominant source of error is Faraday rotation but emission can be
important. Among the important potential sources of error at L-band is
the ionosphere. The ionosphere causes a change in the direction
of polarization (Faraday rotation) and because the ionosphere is
lossy at L-band, there is both attenuation and emission along the
signal path. As will be shown here, both phenomena can cause
errors that are important for remote sensing of salinity at the
0.1-psu level of accuracy. A brief background is given in Ap-
pendices A and B to define Faraday rotation and attenuation in
the ionosphere. In Section II, the magnitude of Faraday rotation
and its effect on observed brightness temperature are presented. In Sections III–IV, attenuation and its effect on brightness tem-
perature (emission) are discussed. Index Terms—Ionospheric electromagnetic propagation, mi-
crowave radiometry, ocean salinity, remote sensing. U.S. Government work not protected by U.S. copyright. Manuscript received June 1, 2001; revised December 13, 2001. A. Magnitude Top: variation of vertical total electron content (VTEC) with solar activity. Bottom: Faraday rotation at the location 30 N, 220 E during high solar activity
(June 1989). The data are for a sensor altitude of 675 km and looking perpendicular to the satellite heading to the right. The solid and dashed lines are for local
time noon and 6 am, respectively. Rz. (Rz is a 12-month, running mean centered on the middle
of the month of interest.) The data are for June and the solid
line is for a local time of noon and the dashed line for 6 am. The lower panel shows Faraday rotation at this location for
several incidence angles,
. This example illustrates some
general characteristics: VTEC increases with solar zenith angle
and solar activity, and for a fixed sensor orientation Faraday
rotation increases with VTEC and incidence angle. Faraday
rotation varies as
VTEC ; hence, orientation of
the sensor with respect to the magnetic field affects the rate of
change with incidence angle and VTEC. These approximations are reasonable because
is slowly
varying with altitude and because there is little curvature
of the ray path. In (2),
is in tesla,
is in degrees,
and VTEC
dz is the vertical total electron con-
tent at the sub-satellite point in total electron content units
(10
electrons/m ). Rz. (Rz is a 12-month, running mean centered on the middle
of the month of interest.) The data are for June and the solid
line is for a local time of noon and the dashed line for 6 am. The lower panel shows Faraday rotation at this location for
several incidence angles,
. This example illustrates some
general characteristics: VTEC increases with solar zenith angle
and solar activity, and for a fixed sensor orientation Faraday
rotation increases with VTEC and incidence angle. Faraday
rotation varies as
VTEC ; hence, orientation of
the sensor with respect to the magnetic field affects the rate of
change with incidence angle and VTEC. (
)
It is clear from (1) and (2) that Faraday rotation depends on
electron density and magnetic field and also on the orientation
of the sensor with respect to the local magnetic field (i.e., on
). Examples of the effect of sensor orientation (scan pattern
and look angle) can be found in [16]. A. Magnitude Microwave remote sensing from space could provide the
necessary temporal and spatial sampling needed to understand
the role of salinity in these ocean processes [2], [4]. Changes
in salinity modulate the emissivity of the surface and cause
changes in emission that are sufficiently strong in the low
frequency portion of the microwave spectrum to be detected
with passive sensors [5], [6]. Measurements from space have
been proposed [7] and salinity differences were observed
from space with the L- band radiometer on SKYLAB 25 [8]. Recently, experiments with L-band radiometers on aircraft
have demonstrated that salinity can be retrieved with accuracy
useful for studying processes in coastal regions [9], [10]. The rotation of the polarization vector in the ionosphere is
due to the change along the propagation path from surface to
sensor of the phase,
, in (B3). Substituting (A6) into (B4) and
integrating along the propagation path, , one obtains ds
(1) (1) where
is the Faraday rotation in radians and
is the angle
between the direction of propagation and the Earth magnetic
field [14], [15]. To simplify the many calculations needed to
make a global map of
, (1) has been approximated by making
the change of variables ds
dz where
is the normal
to the surface at the subsatellite point nadir and
is the polar
angle between nadir and the line of sight to the surface (inci-
dence angle). Substituting for the plasma frequency,
and elec-
tron gyro frequency,
, from (A2)–(A3) and replacing
by its
value at an altitude of 400 km [16], one obtains VTEC (2) VTEC IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 772 Top: variation of vertical total electron content (VTEC) with solar activity. Bottom: Faraday rotation at the location 30 N, 220 E during high solar activity
89). The data are for a sensor altitude of 675 km and looking perpendicular to the satellite heading to the right. The solid and dashed lines are for local
n and 6 am, respectively. Fig. 1. Top: variation of vertical total electron content (VTEC) with solar activity. Bottom: Faraday rotation at the location 30 N, 220 E during high solar activity
(June 1989) The data are for a sensor altitude of 675 km and looking perpendicular to the satellite heading to the right The solid and dashed lines are for local Fig. 1. A. Magnitude For purposes of this paper,
it will be assumed that the sensor looks to the right (i.e., across
track in the plane perpendicular to the satellite heading). The
sensor will be assumed to be in a sun-synchronous orbit with an
altitude of 675 km, which is representative of orbits proposed
for microwave remote sensing at L-band [4]. Faraday rotation
will be computed using the International Reference Ionosphere
(IRI-95) [17] to generate the necessary electron density profiles
and the International Geomagnetic Reference Field (IGRF) [18]
for the magnetic field. Fig. 2 shows the global distribution of Faraday rotation for a
sensor with the orientation and altitude given above. The data are
for a local time of 6 am (left) and noon (right) for incidence an-
glesof20,30,40,and50degrees.ThesedataareforJune,1989,a
very active Sun near solar maximum with sunspot number, Rz
. Notice the north–south bias in these maps, reflecting that
the computation is for summer in the northern hemisphere. Also
notice the east–west symmetry, which reflects that the computa-
tion is for constant local time (6 am or noon). However, there are
significant deviations from these simple generalizations. For ex-
ample, notice at noon (right) the peaks in Faraday rotation south
of Australia and also over northern Asia. These patterns reflect
the complex variations of VTEC with latitude and longitude and Fig. 1 shows the Faraday rotation at a fixed location (30
N, 220 E) over the Pacific Ocean just off the coast of North
America. The top panel shows VTEC at this location as a
function of solar activity given in terms of the sunspot number LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 773 obal distribution of Faraday rotation for local time of 6 am (left) and noon (right). The data are for high solar activity (June 1989), an altitu
di
l
h
lli
h
di
h
i h
i
id
l
i di
d (20 30 40
d 50 d
) distribution of Faraday rotation for local time of 6 am (left) and noon (right). The data are for high solar activity (June 1989), an altitude of 675 km,
pendicular to the satellite heading to the right at incidence angles as indicated (20, 30, 40, and 50 degrees). ce of Faraday rotation on orientation with respect to
gnetic field (i.e.,
). trating the dependence on solar activity, local time and sensor
orientation can be found in [16] and [19]. A. Magnitude Additional examples illus-
trating the dependence on solar activity, local time and sensor
orientation can be found in [16] and [19]. Fig. 2. Global distribution of Faraday rotation for local time of 6 am (left) and noon (right). The data are for high solar activity (June 1989), an altitude of 675 km,
and looking perpendicular to the satellite heading to the right at incidence angles as indicated (20, 30, 40, and 50 degrees). the dependence of Faraday rotation on orientation with respect to
the local magnetic field (i.e.,
). Additional examples illus- the dependence of Faraday rotation on orientation with respect to
the local magnetic field (i.e.,
). Additional examples illus- the dependence of Faraday rotation on orientation with respect to
the local magnetic field (i.e.,
). Additional examples illus-
trating the dependence on solar activity, local time and sensor
orientation can be found in [16] and [19]. trating the dependence on solar activity, local time and sensor
orientation can be found in [16] and [19]. trating the dependence on solar activity, local time and sensor
orientation can be found in [16] and [19]. IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 774 Fig. 3. Error in brightness temperature due to neglecting Faraday rotation at noon (top) and 6 am (bottom) at 30 N, 220 E for high solar activity (June 1989). The
data are for a sensor at altitude of 675 km and looking perpendicular to the satellite heading to the right. The surface is ocean with S = 35 psu and T = 20 C. Fig. 3. Error in brightness temperature due to neglecting Faraday rotation at noon (top) and 6 am (bottom) at 30 N, 220 E for high solar activity (June 1989). The
data are for a sensor at altitude of 675 km and looking perpendicular to the satellite heading to the right. The surface is ocean with S = 35 psu and T = 20 C. B. Effect on Brightness Temperature where the “ ” and “ ”signs are for H- and V-polarization, re-
spectively. To compute
, it has been assumed that the surface
is flat and without roughness (waves or foam). In this case, the
emissivity is Faraday rotation is a potential source of error for remote
sensing because radiation of given polarization at the surface
will have mixed polarization when it arrives at the sensor. In
particular, given the physical temperature of the surface
and the emissivity
for horizontal and vertical polarization,
respectively, one obtains the following expression for the
brightness temperatures,
, at the sensor: (5) (5) where
are the Fresnel reflection coefficients (6a)
(6b) (6a) (3a)
(3b) (6b) Inverting (3) without correcting for Faraday rotation
can re-
sult in error because
and
are different functions of salinity
[6]. and
is the relative dielectric constant of sea water and is
a function of salinity and temperature. The relationship for
L-band reported by [6] has been used here. For purposes of deciding when the error might be significant,
it is convenient to examine the change in brightness temperature,
, due to Faraday rotation. From (3),
one obtains Fig. 3 shows
as a function of solar activity at the
fixed location (30 N, 220 E). The top panel is for local time of
noon and the bottom panel is for 6 am. The data are for the month
of June, a sensor at 675 km altitude, and looking to the right with
incidence angles of 10, 30, and 50 degrees. The Faraday rotation
and VTEC are given in Fig. 1. The calculations were made for (4) LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 775 global distribution of the error in brightness temperature as a function of incidence angle due to neglecting Faraday rotation at 6 am (le
ata are for high solar activity (June 1989; Rz = 158) and for a sensor at altitude of 675 km and looking perpendicular to the satellite h
face is ocean with S = 35 psu and T
= 20 C. urface with
psu and
C. Notice that
incidence angle and solar activity as one would expec Fig. 4. The global distribution of the error in brightness temperature as a function of incidence angle due to neglecting Faraday rotation at 6 am (left) and noon
(right). B. Effect on Brightness Temperature The data are for high solar activity (June 1989; Rz = 158) and for a sensor at altitude of 675 km and looking perpendicular to the satellite heading to the
right. The surface is ocean with S = 35 psu and T
= 20 C. an ocean surface with
psu and
C. Notice that
the error incurred by ignoring Faraday rotation increases with
incidence angle and solar activity as one would expect (because
Faraday rotation increases with these parameters) and the error IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 776 Fig. 5. Profiles of ionosphere parameters (electron density, collision frequency, temperature, and attenuation) at 30 N, 330 E for local times of noon (solid) and
6 am (dashed) and high solar activity (June 1989). Fig. 5. Profiles of ionosphere parameters (electron density, collision frequency, temperature, and attenuation) at 30 N, 330 E for local times of noon (solid) and
6 am (dashed) and high solar activity (June 1989). be neglected. Neglecting terms of the order
compared to
unity in (A6) and (B4), one obtains be neglected. Neglecting terms of the order
compared to
unity in (A6) and (B4), one obtains is much worse at noon than at 6 am. For incidence angles less
than 40 degrees, the error at 6 am is less than 0.3 K even at max-
imum solar activity. At noon, the error is much larger (Fig. 3, top
panel). (7)
(8) (7) Fig. 4 shows the global distribution of brightness temperature
error,
, over the oceans. These calculations were made
for an ocean surface with
psu,
C and for a
period of solar maximum (June 1989). The panel on the left is
for a local time of 6 am and on the right for noon. Notice that, at
6 am, mid-latitudes and moderate incidence angles the error in
brightness temperature is less than 0.3 K. However, much larger
values are possible at local noon, even at moderate incidence
angles. (To approximate the error in the estimate of salinity, the
ratio 0.5 K/psu is useful.) (8) (8) In order to compute
, both the electron density
and the
collision frequency
are needed. To estimate the collision fre-
quency, we use the “effective” collision frequency defined by
[20] (9) (9) where where (10) LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 777 TABLE I
INTEGRATED PARAMETERS (NADIR AND LOCATION: 30 N, 330 E) TABLE I
INTEGRATED PARAMETERS (NADIR AND LOCATION: 30 N, 330 E) TABLE I the signal that would arrive in the absence of attenuation (and
Faraday rotation). Calling this difference
, one obtains the signal that would arrive in the absence of attenuation (and
Faraday rotation). Calling this difference
, one obtains is the temperature of all constituents. The concentrations
are
in units of cm
and the collision frequencies are in Hz. Computations of
have been made here using the IRI-95
[17] to obtain electron density profiles and using the Mass-
Spectrometer-Incoherent-Scatter (MSIS) model [21] to gen-
erate the profiles of temperature and neutral particle density
(
and
) needed in (10). Values of
were computed as a
function of altitude in steps of 1 km and then integrated along
the ray path to obtain an optical depth:
dz. The
optical depth gives the attenuation of power (i.e., brightness
temperature). (11a) Since
is small, this is approximately Since
is small, this is approximately (11b) (11b) Values of
at (30 N, 330 E) are given in Table I. These
values are for nadir (vertical and horizontal polarization are
equal) and it is obvious from (11) that the attenuation increases
with incidence angle approximately as
. The values given
in Table I assume a water surface with a salinity of 35 psu and
temperature of 20
C. Values at selected locations around the
globe (shown in Fig. 6) are given in Table II for a local time of 6
am and in Table III for a local time of noon. The same values of
salinity and temperature are used at each location (35 psu and
20
C). The only variations are with parameters of the iono-
sphere. Notice that, at nadir, in the case of high solar activity
and noon, it is possible to have a loss of 0.01 K. However, the
loss is much less at 6 am, even at high solar activity. Fig. 5, shows vertical profiles at (30 N, 330 E). Profiles are
shown for electron density
, collision frequency
, tem-
perature
, and attenuation coefficient
for local times of 6 am
(dashed) and noon (solid). The calculations are for a period of
high solar activity (June 1989). Notice the peak in the profile
of attenuation coefficient at low altitude (near 100 km). IV. IONOSPHERE EMISSION Loss in the ionosphere also means that there will be emission
(additional signal which is not related to surface properties). There are two components of this emission, a signal emitted up-
ward toward the radiometer from the column of ionosphere be-
tween the sensor and surface and signal emitted downward that
is reflected at the surface into the sensor’s field-of-view. Using
a conventional radiative transfer model for a uniform layer, one
can write these terms as follows [7], [22]: LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE This
occurs because the collision frequency increases rapidly in low
altitudes. Hence, even though the electron density has decreased
substantially from its peak near 300 km (F-region), attenuation
is not negligibly small in the lower ionosphere. Integrated values
of these parameters are shown in Table I. The table shows values
for high and also low solar activity. Values for Faraday rotation
and the ellipticity factor
are included for reference. Notice
how much smaller
is than
. All of the integrated values are
for an altitude of 675 km and a vertical path. The values scale
roughly as
for incidence angles less than 50 degrees. A. Magnitude of Absorption (10) As shown in Appendix B, the dominant loss due to absorption
in the ionosphere is determined by the coefficient
[cf. (B3)
and (B4)]. As will be shown below, the term
(attenuation due
to ellipticity) in (B3) is very much smaller at L-band and can In these expressions, the subscripts , , and
denote elec-
trons, ions, and neutral particles, respectively. is the molec-
ular weight of ions and neutral particles (assumed equal) and B. Loss of Brightness Temperature The signal emitted from the surface is
and
it is attenuated as it propagates through the ionosphere to the
sensor. For purposes of estimating the effect of this attenuation,
define the loss in brightness temperature to be the difference
between the temperature that actually arrives at the sensor and (12) (12) (12) IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 778 Fig. 6. Location where the calculation of ionospheric absorption and emission were made. Fig. 6. Location where the calculation of ionospheric absorption and emission were made. where term is polarization-dependent because reflection at the surface
depends on polarization. (13a)
dx
dz
(13b)
dx
dz
(13c) (13a) Examples of the upwelling and downwelling terms in (12)
are given at a fixed location (30 N, 330 E) in Fig. 7. The radi-
ation is shown for nadir and an incidence angle of 50 degrees
as a function of local time. The data are for a period of high
solar activity (June 1989) and assume a surface with salinity of
35 psu and temperature of 20 C and a sensor at 675 km. The
upwelling radiation (top) is unpolarized. The downwelling ra-
diation is the net term,
, in
(12). This term is different for vertical polarization (middle) and
horizontal polarization (bottom) because
is different for
the two polarizations. (13a) (13a) (13a)
dx
dz
(13b) (13b) (13b) (13b)
dx
dz
(13c) Tables II and III list examples for the several locations
around the globe shown in Fig. 6. The data are for the month
of June and for high and low solar activity (Rz
and 8.5) corresponding to solar maximum and minimum,
respectively. The data in Table II are for local time of 6 am
and the data in Table III are for noon. Values are presented
for optical depth
, for the loss in brightness temperature due
to attenuation
, and for the upwelling radiation
and
downwelling radiation
. The results are for nadir (vertical
and horizontal polarization are the same). Notice that the total
radiation at the sensor (upwelling plus downwelling) ranges
from 0.001 to 0.055 K at 6 am depending on location and
solar activity. To put this in perspective, the salinity signal is
approximately 0.5 K/psu. Thus, a change of 0.05 K is an error
of about 0.1 psu. LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 779 TABLE II
IONOSPHERIC ATTENUATION AND EMISSION (6 AM) TABLE II
IONOSPHERIC ATTENUATION AND EMISSION (6 AM) TABLE III
IONOSPHERIC ATTENUATION AND EMISSION (NOON) V. COMMENTS B. Loss of Brightness Temperature This is important for monitoring salinity in
the open ocean but probably not important for applications in
the coastal ocean [23]. (13c) (13c) In these expressions, (13a) is the nominal emission from the
surface,
is the altitude of the sensor (675 km in the numer-
ical examples),
is the attenuation coefficient for
power at altitude
as given by (7),
is the emissivity of
the surface and
is the Fresnel reflection coefficient of
the surface as defined by (6), and
is the temperature of the
ionosphere and
is the temperature of the surface. is the
signal emitted by the ionosphere along the line-of-sight between
the surface and sensor. This term is due to thermal emission and
is the same for both polarizations. is the radiation emitted
by the entire column of ionosphere downward to the surface. This radiation is reflected at the surface and then attenuated as it
propagates from the surface toward the sensor. The net contribu-
tion is
as given in (12). This In these expressions, (13a) is the nominal emission from the
surface,
is the altitude of the sensor (675 km in the numer-
ical examples),
is the attenuation coefficient for
power at altitude
as given by (7),
is the emissivity of
the surface and
is the Fresnel reflection coefficient of
the surface as defined by (6), and
is the temperature of the
ionosphere and
is the temperature of the surface. is the
signal emitted by the ionosphere along the line-of-sight between
the surface and sensor. This term is due to thermal emission and
is the same for both polarizations. is the radiation emitted
by the entire column of ionosphere downward to the surface. This radiation is reflected at the surface and then attenuated as it
propagates from the surface toward the sensor. The net contribu-
tion is
as given in (12). This V. COMMENTS 3 (error due to Faraday rotation) and Tables II and III (error
due to attenuation and emission) that corrections for these
phenomena will have to be made to achieve salinity retrievals
accurate to 0.1–0.2 psu. Calculations have been presented here illustrating the effect
of Faraday rotation and attenuation/emission from the iono-
sphere on passive microwave remote sensing at L-band (1.4
GHz). The motivation for this work is current interest in remote
sensing of ocean salinity from space. To put the effects of the
ionosphere into context, the change of brightness temperature
with salinity is about 0.5 K/psu. It is clear from Figs. 2 and The data also indicate some obvious choices for remote
sensing. Clearly an optimum choice as far as minimizing errors
is 6 am local time and as close to a minimum of solar activity
as possible. Local time of 6 am is near the minimum in the
daily cycle of the ionosphere, (Fig. 7 is an example of the IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 780 Fig. 7. Diurnal variation of upwelling (top) and downwelling emissions
(vertical polarization, middle; horizontal polarization, bottom). The calculations
are at 30 N, 330 E during high solar activity (June 1989). The surface is ocean
with S = 35 psu and T
= 20 C. IRI-95 model [17] is a good representation of mean characteris-
tics of the ionosphere but is not a particularly good predictor of
current (instantaneous) behavior [24], [25]. It may be possible
to improve its ability to predict current behavior given input of
local, measured parameters [26], [27]. However, without such
input corrections for Faraday rotation and emission will likely
have to be based on other models or techniques. (One possibility
is to measure the third Stokes parameter [28].) Attenuation is proportional to
as in (7) which follows from
the Appleton–Hartree equation and simplifications for L-band. This is a “cold” plasma approximation. In the present study,
the collision frequency is taken as
(see (9)–(10)). It has been suggested that, when
, as is the case at
L-band, a better approximation is to use
[29]. Hence, it is likely that the values for emission and loss given
here are underestimated. Also, the results presented here employ
approximations that are reasonable at low and middle latitudes. Near the poles, additional care must be taken in the calculation
of attenuation [30]. APPENDIX A
REFRACTIVE INDEX AT L BAND The (A4) Sin
Sin
Cos
(A1) (A1) LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE
781 LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE LE VINE AND ABRAHAM: EFFECT OF THE IONOSPHERE ON REMOTE SENSING OF SSS FROM SPACE 781 tionalizing the denominator and only keeping terms of lowest
der in
and
and
in the numerator, one may
ite this as
Cos
(A5)
nally, using the definitions
where
(B4) nalizing the denominator and only keeping terms of lowest
in
and
and
in the numerator, one may
this as
Cos
(A5)
ly, using the definitions
where
(B4) alizing the denominator and only keeping terms of lowest
n
and
and
in the numerator, one may
his as
Cos
(A5)
, using the definitions
where
(B4) Rationalizing the denominator and only keeping terms of lowest
order in
and
and
in the numerator, one may
write this as
where Cos
(A5) Finally, using the definitions Finally, using the definitions (B4) Cos
(A6) Because the two modes propagate with different phase velocity,
the polarization vector changes and because the index of refrac-
tion
is complex, there is attenuation. For example, in the spe-
cial case
, both modes in (B3) are attenuated by the factor
and the polarization vector rotates through an angle
. The latter is Faraday rotation. The wave emerges linearly
polarized but with reduced amplitude and with the direction of
the polarization vector changed. When
the two modes
do not entirely add in phase and the wave has a slight ellipti-
cally polarized component. At L-band
is very small (Table I)
and will be neglected in this paper. The two dominant effects at
L-band are Faraday rotation (determined by
) and attenuation
(determined by
). (A6) (A6) and taking the square root of (A5), one obtains (A7) (A7) (A7) APPENDIX B
PROPAGATION OF A PLANE WAVE IN THE IONOSPHERE To obtain an expression for the complete path, one needs to
sum over all the layers. Assuming that the changes are suffi-
ciently gradual that we can neglect reflections as the boundaries
and that the layers are sufficiently thin and numerous that sums
are approximated by integrals, one obtains Assume a plane (flat) layered ionosphere divided into many
layers sufficiently thin that the index of refraction is constant
within each layer. That is, neglect curvature; assume that the
only variations are vertical and that the vertical variations are
slowly varying. We approximate the solution for propagation in
this model ionosphere by solving for plane-wave propagation
within each layer assuming constant index of refraction
and
later summing the solutions over all the layers. (B5) To begin, consider a linearly polarized wave propagating in
the -direction within the th layer. It has the following form: (B1) (B1) where where dz
(B6a)
dz
(B6b) It is possible to write this linearly polarized wave as the sum of
the two circularly polarized waves. Doing so and recognizing
that in the ionosphere the two modes propagate with different
velocities (i.e., index of refraction,
, in (A7)), one obtains (B6a) (B2) ACKNOWLEDGMENT The authors wish to thank the Dr. D. Bilitza (National Space
Science Data Center, Greenbelt) for providing atmosphere and
ionosphere models. where
and
denote unit vectors along the
and
axes, respec-
tively, and the subscript “ ” on
has been dropped to make the
notation easier. Substituting for
from (A7), one obtains APPENDIX A
REFRACTIVE INDEX AT L BAND The effective refractive index for electromagnetic wave
propagation in the ionosphere is given by the Appleton–Hartree
equation [31], [32] (A1), shown at the bottom of the page,
where
is frequency and
is the angle between the direction
of propagation and the geomagnetic field and where In these expressions,
is the plasma frequency (Hz) In these expressions,
is the plasma frequency (Hz) (A2) (A2) (A2) where
is the number of electrons per m and
is the electron
gyro frequency (Hz) Fig. 7. Diurnal variation of upwelling (top) and downwelling emissions
(vertical polarization, middle; horizontal polarization, bottom). The calculations
are at 30 N, 330 E during high solar activity (June 1989). The surface is ocean
with S = 35 psu and T
= 20 C. (A3) (A3) At L-band (1.4 GHz) the frequency is sufficiently larger than the
maximum plasma frequency and the gyro frequency that simpli-
fying assumptions can be made to the classical magneto-ionic
theory. In particular, the terms in (A1) proportional to Sin
can be neglected as long as
. This is called the quasi-
longitudinal approximations, and one obtains [14] diurnal variation). Remote sensing from a sun-synchronous
orbit with an equatorial crossing time of 6am/6pm would
provide observations near this minimum. Fig. 1 and Tables I–III
illustrate the dependence on solar activity. For a fixed local
time and location, Faraday rotation and emission/absorption
increase roughly linearly with the solar activity (IG) index, Rz. Cos
(A4) The studies presented here use the IRI-95 and are therefore
representative of climatalogical data for the ionosphere. IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 782 IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 [5] H.-J. C. Blume, B. M. Kendall, and J. C. Fedors, “Measurements of
ocean temperature and salinity via microwave radiometry,” Bound. Layer Met., vol. 13, pp. 295–308, 1978. [28] S. H. Yueh, “Estimates of Faraday rotation with passive microwave po-
larimetry for microwave remote sensing of earth surfaces,” IEEE Trans. Geosci. Remote Sensing, vol. 38, pp. 2434–2438, Sept. 2000. y
pp
[6] L. A. Klein and C. T. Swift, “An improved model for the dielectric con-
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sensing of ocean-surface salinity,” IEEE Trans. Geosci. Remote Sensing,
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[31] J. A. Ratcliffe, Magneto Ionic Theory and its Applications to the Iono-
sphere. Cambridge, U.K.: Cambridge Univ. Press, 1959. [8] R. M. Lerner and J. P. Hollinger, “Analysis of 1.4 GHz radiometric mea-
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mapper makes debut in coastal zone,” EOS, Trans. AGU, vol. 79, no. 173, pp. 176–177, 1998. [10] D. M. Le Vine, M. Kao, R. Garvine, and T. Sanders, “Remote sensing of
ocean salinity: Results from the Delaware coastal current experiment,”
J. Tech., vol. 15, pp. 1478–1484, 1998. David M. Le Vine (M’70–SM’79–F’95) received the
B.S. and M.S. degrees in electrical engineering, the
M.S. degree in physics, and the Ph.D. degree in elec-
trical engineering from the University of Michigan,
Ann Arbor. [11] E. L. Lewis, “The practical salinity scale 1978 and its antecedents,”
IEEE J. Oceanic Eng., vol. OE-5, pp. 3–8, 1980. [12] P. E. O’Neill, A. Hsu, T. Jackson, and C. Swift, “Ground-based
microwave radiometer measurements during the Southern Great Plains
’97 Experiment,” in Proc. IGARSS-98, Seattle, WA, July 1998, pp. 1843–1845. IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 Space Res., vol. 27, no. 1, pp. 153–156, 2001. [20] H. Rishbeth
and
O. K. Garriott,
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Working Group (SSIWG),”, http://www.esr.org, 1998. Saji Abraham (M’01) received the B.Sc. degree in
physics from Kerala University, India, and the M.Sc. degree in space physics from Andhra University,
India, and the Ph.D. degree in space physics from
the University of Delhi, India. He has been supporting NASA projects since
1997 and is currently employed with Science System
and Applications, Inc. (SSAI), Greenbelt, MD. His
current research includes developing forward models
to simulate brightness temperature at microwaves
for space-born sensors and sea surface salinity
retrieval algorithms. He has worked in research areas related to ionosphere
radio wave propagation techniques and has published scientific articles on
lower ionosphere dynamics and its coupling with the lower atmosphere through
planetary waves and Quasi-biennial Oscillations (QBO). Dr. Abraham is a member of the American Geophyical Union and the IEEE
Geoscience and Remote Sensing Society. Saji Abraham (M’01) received the B.Sc. degree in
physics from Kerala University, India, and the M.Sc. degree in space physics from Andhra University,
India, and the Ph.D. degree in space physics from
the University of Delhi, India. [24] A. J. Preble, D. N. Anderson, B. G. Fejer, and P. H. Doherty, “Compar-
ison between calculated and observed F region electron density profiles
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and IRI-95 using JPL-derived GIM’s for single frequency satellite
altimetry,” in Ionospheric Determination and Specification for Ocean
Altimetry and GPS Surface Reflection Workshop. Pasadena, CA: Jet
Propulsion Lab., 1997. [27] A. Komjathy, R. B. Langley, and D. IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 He has been employed as an Electronics Engineer
at the Harry Diamond Laboratory, Department
of the Army, and as a Research Engineer at the
Radiation Laboratory, University of Michigan. After
leaving the University of Michigan, he became
Assistant Professor in the Department of Electrical
Engineering at the University of Maryland, College Park. In 1973, he joined
NASA’s Goddard Space Flight Center, Greenbelt, MD, where he is currently
a member of the Microwave Sensors Branch. His current research has focused
on the development of techniques for microwave remote sensing of the
environment from space. [13] T. J. Schmugge, P. E. O’Neill, and J. Wang, “Passive microwave soil
moisture research,” IEEE Trans. Geosci. Remote Sensing, vol. GE-24,
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heights,” J. Geophys. Res., vol. 64, no. 7, p. 717, 1959. [16] D. M. Le Vine and S. Abraham, “Faraday rotation and passive
microwave remote sensing of soil moisture from space,” in Microw. Radiomet. Remote Sens. Earth’s Surf. Atmosphere, P. Pampaloni and S. Paloscia, Eds: VSP, 2000, pp. 89–96. Dr. Le Vine is a member of the International Union of Radio Science (URSI,
Commisions B, E and F) and the American Geophysical Union. Within the IEEE
he is affiliated with the Antennas and Propagation Society and the Geoscience
and Remote Sensing Society. He was president of the local (Washington, DC)
section of the Antennas and Propagation Society and has been active in en-
gineering accreditation. He is currently a member of the IEEE Committee on
Engineering Accreditation Activities (CEAA) and is one of the IEEE represen-
tatives on the Engineering Accreditation Commission (EAC) of ABET. He is a
member of Eta Kappa Nu, Tau Beta Pi, and Phi Kappa Phi. He was a Univer-
sity of Michigan Fellow and NASA/ASEE Faculty Fellow and is the recipient
of several service awards from the Goddard Space Flight Center. pp
[17] D. Bilitza, “International Reference Ionosphere—Status 1995/1996,”
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generation,” J. Geomagn. Geoelectr., vol. 49, pp. 123–148, 1997. [19] S. Abraham and D. M. Le Vine, “Evaluation of IRI-95 to correct errors
caused by Faraday rotation in passive microwave remote sensing from
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M. Goodberlet, “Sea surface salinity: Toward and operational remote
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Ed. Amsterdam, The Netherlands: Elsevier, 2000, pp. 321–335. sensing system,” in Satellites, Oceanography and Society, D. Halpern,
Ed. Amsterdam, The Netherlands: Elsevier, 2000, pp. 321–335. (B3) Ed. Amsterdam, The Netherlands: Elsevier, 2000, pp. 321–3 IEEE TRANSACTIONS ON GEOSCIENCE AND REMOTE SENSING, VOL. 40, NO. 4, APRIL 2002 Bilitza, “Ingesting GPS-derived
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English
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Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection
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BMC plant biology
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cc-by
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RESEARCH ARTICLE Open Access © 2011 Lang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Abstract Background: Among the various biochemical markers, fatty acids or lipid profiles represent a chemically relatively
inert class of compounds that is easy to isolate from biological material. Fatty acid (FA) profiles are considered as
chemotaxonomic markers to define groups of various taxonomic ranks in flowering plants, trees and other
embryophytes. Results: The fatty acid profiles of 2076 microalgal strains from the culture collection of algae of Göttingen
University (SAG) were determined in the stationary phase. Overall 76 different fatty acids and 10 other lipophilic
substances were identified and quantified. The obtained FA profiles were added into a database providing
information about fatty acid composition. Using this database we tested whether FA profiles are suitable as
chemotaxonomic markers. FA distribution patterns were found to reflect phylogenetic relationships at the level of
phyla and classes. In contrast, at lower taxonomic levels, e.g. between closely related species and even among
multiple isolates of the same species, FA contents may be rather variable. Conclusion: FA distribution patterns are suitable chemotaxonomic markers to define taxa of higher rank in algae. However, due to their extensive variation at the species level it is difficult to make predictions about the FA profile
in a novel isolate. Lang et al. BMC Plant Biology 2011, 11:124 Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Fatty acid profiles and their distribution patterns
in microalgae: a comprehensive analysis of more
than 2000 strains from the SAG culture collection Imke Lang1,2, Ladislav Hodac3, Thomas Friedl3 and Ivo Feussner1* Imke Lang1,2, Ladislav Hodac3, Thomas Friedl3 and Ivo Feussner1* * Correspondence: ifeussn@gwdg.de
1Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences,
Department of Plant Biochemistry, Göttingen, Germany
Full list of author information is available at the end of the article Background saturated and mono unsaturated FAs as well as trace
amounts of PUFAs, mostly linoleic acid (LA, 18:2(9Z,
12Z): where x:y(z) is a fatty acid containing X carbons
and y double bonds in position z counting from the car-
boxyl end)). On the other hand Chromalveolate algae
contain significant amounts of PUFAs [6]. The analysis of the overall fatty acid profiles as well as the
occurrence of fatty acids (FAs) in different lipid classes in
microalgae is an emerging field which is expected to reveal
the identification of novel FAs with a variety of new func-
tional groups [1]. Despite a number of reports has been
carried out and published, describing the contents as well
as the composition of polyunsaturated fatty acids (PUFAs)
in mostly marine microalgae [2-4], systematic approaches
that include different or even many genera of microalgae
and particularly those from freshwaters or terrestrial habi-
tats are still missing [5]. Among the various biochemical markers, FA or lipid
profiles represent a chemically relatively inert class of
compounds that is easy to isolate from biological material
and FA profiles are considered as chemotaxonomic mar-
kers to define groups of various taxonomic ranks in flow-
ering plants, trees and other Embryophytes [7,8]. Beside the identification of novel FAs, some recent stu-
dies report on the use of FAs and lipid profiles of algae as
biomarkers [1,9-11]. Viso et al. determined profiles of FAs
of nine different marine algal groups and they were able to
define even species-specific lipid compositions [4]. More-
over they found a roughly taxon specific profile when the
cells were cultured under identical growth conditions. Various strains and species of the cyanobacterium Nostoc Based on current knowledge, FA composition divides
microalgae roughly into two groups, i.e. on one hand the
cyanobacteria and green algae (Chlorophyta and Strepto-
phyta) which contain low amounts of FAs, predominantly * Correspondence: ifeussn@gwdg.de
1Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences,
Department of Plant Biochemistry, Göttingen, Germany
Full list of author information is available at the end of the article Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 2 of 16 identified by comparison to mass spectra with highest
similarity to the proposed substance in the MS-library
(Nist02 or Wiley98). By this some methyl esters of
branched FAs were detected, for example 12-methyl-14:0
or 3, 7, 11, 15-tetramethyl-16:0. Background Whereas for most of the
FAMEs, authentic standards or MS references were avail-
able, for some other substances only “best hit” identifica-
tion was possible. The DMOX derivatives enabled the
identification of the remaining 12 FAMEs. Unidentified
substances have yet to be verified with authentic stan-
dards, which are not available at this time point. The com-
plete database is shown as additional file 1. were screened for their FA content and the application of
a FA-based cluster analysis has been described for their
identification [12]. FA and lipid composition have also been used as bio-
markers to distinguish closely related microalgae at the
species and the generic levels [11,13]. Hitherto no sys-
tematic analysis has been carried out on a large scale basis
on either the profiles of lipids or FAs in microalgae. Therefore, we determined the FA profiles of all available
microalgal strains of the SAG culture collection of micro-
algae http://www.epsag.uni-goettingen.de which is one of
the most diverse and comprehensive resources of microal-
gae. At present (March 2011) 2291 strains of mainly
microscopic algae including a considerable variety of cya-
nobacteria is available. They comprise almost all phyla and
classes of eukaryotic algae, but an emphasis is put on algae
from freshwaters and terrestrial habitats. Bacteria in algal cultures (as contaminations or some-
times even through symbiosis) are well known and can be
found in culture strains of almost any algal culture collec-
tion. Only a small fraction (about 20%) of the studied SAG
strains may be in axenic state. Therefore, also the FA con-
tent of the contaminating bacteria may have contributed
to the obtained FA profile. To test this, we measured
methyl-15:0 and methyl 17:0 that are regarded as markers
for bacterial contaminations [4]. Only 34 strains out of the
2076 analyzed strains contained small amounts methyl-
15:0. This observed low rate of contaminating bacteria was
supported by microscopic controls which are routine in
the perpetual maintenance of algal strains (data not
shown). In summary, we conclude that only 1-2% of the
strains may have been contaminated and that there is only
a minor influence of bacterial contaminations on the
observed algal culture FA profiles. Distribution patterns of FAs may be valuable also as a
proxy to identify certain groups, species and strains of
microalgae of particular interest for applied research, i.e. Background due to the presence of certain FAs and/or high percen-
tages of total FA content. We also tested whether the
detected FA distribution patterns are meaningful in a phy-
logenetic context at various taxonomic levels, i.e. to define
taxonomic groups of microalgae by their FA patterns. It
would assist predicting FA content and/or presence of
other valuable compounds if the phylogenetic relation-
ships of algae were reflected in their FA distribution
patterns. In addition we compared the measured major FA pro-
files of 10 randomly chosen strains from different classes
with published data (Table 2), and it should be noted that
only one out of the 10 strains that were chosen from the
published data originated from the SAG collection. For 6
strains the FA profiles were very similar. In case of the 4
remaining strains major differences were observed in the
degree of desaturation of the FAs with different chain
lengths, which may be explained by the different cultiva-
tion conditions used in the different studies. Here the focus was set on esterified long chain FAs (C-
14 - C-24), which were analysed via Gas chromatography
(GC) with or without mass spectrometry (MS). The large
number of data obtained, were added into a database to
document the FA profiles of the studied microalgal strains. 1. A database of FA profiles from diverse microalgae 1. A database of FA profiles from diverse microalgae
The characterisation of FA profiles of the SAG microalgal
strains was performed by screening long chain FAs (C-14
- C-24) esterified within lipids. A total of 2076 culture
strains from the SAG (equal 91% of the SAG’s holding)
were screened. A database was established which con-
tained all identified FAs and some other hydrophobic
metabolites. An overview of all substances identified in the
algal strains screened is shown in Table 1. A total of 86
different substances were identified by mass spectrometry,
76 of which represent methyl esters of FAs. Out of the 76
fatty acids, 36 substances were identified by their mass
spectrum and by retention time according to a standard
substance, and the other 40 fatty acids were identified by
their mass spectra only. The remaining 10 substances
were identified by their mass spectra only as well. In com-
parisons with a standard substance, the compound was Results and Discussion 1. A database of FA profiles from diverse microalgae 2. Patterns of fatty acid composition 2. Patterns of fatty acid composition
FAME profiles were rather different among strains. As an
example, FAME profiles from four different genera, i.e. Chroococcus (Cyanobacteria), Closteriopsis (Chlorophyta,
Trebouxiophyceae), Pseudochantransia (Rhodophyta)
and Prymnesium (Chromalveolates, Haptophyta) are pre-
sented in Figure 1. Therefore it was anticipated to
recover certain different FA distribution patterns
between phyla, classes and genera of microalgae. In addi-
tion, it was tested whether differences in FA patterns can
also be found for groups at lower taxonomic rank, i.e. between species of the same genus or even among multi-
ples isolates of the same species. Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 3 of 16 Table 1 Overview of the FAMEs identified and other substances found in the analysed SAG microalgal strains
86 substances, 76 methyl esters of FAs
methyl esters of saturated straight-chain FAs
methyl esters of branched chain FAs
methyl esters of monoenoic FAs
14:0
12-methyl-14:0
14:1 (7Z)
16:0
13-methyl-14:0
14:1 (9Z)
17:0
14-methyl-15:0
15:1 (10Z)
18:0
14-methyl-16:0
16:1 (5Z)
19:0
methyl-3, 7, 11, 15-tetramethyl-16:0
16:1 (7Z)
20:0
16- o. 2. Patterns of fatty acid composition 15-methyl-17:0
16:1 (9Z)
21:0
17-methyl-18:0
16:1 (11Z)
22:0
6, 10, 14 trimethyl-2-pentadecanone
17:1 (8Z)
23:0
17:1 (9Z)
24:0
17:1 (10Z)
18:1 (9E)
methyl esters of dienoic FAs
methyl esters of trienoic FAs
18:1 (9Z)
15:2
16:3 (4Z, 7Z, 10Z)
18:1 (11Z)
16:2 (7Z, 10Z)
16:3 (6Z, 9Z, 12Z)
19:1 (11Z)
16:2 (9Z, 12Z)
16:3 (7Z, 10Z, 13Z)
20:1 (11Z)
17:2 (7Z, 10Z)
17:3
22:1 (13Z)
17:2 (9Z, 12Z)
18:3 (5Z, 9Z, 12Z)
24:1 (15Z)
18:2 (6Z, 9Z)
18:3 (6Z, 9Z, 12Z)
18:2 (8Z, xZ)*
18:3 (8Z, 11Z, 14Z)
18:2 (9E, 12E)
18:3 (9Z, 12Z, 15Z)
18:2 (9Z, 12Z)
19:3
18:2 (9Z, 14Z)
19:3
18:2 (11Z, 14Z)
20:3 (7Z, 10Z, 13Z)
19:2 (9Z, 12Z)
20:3 (8Z, 11Z, 14Z)
20:2 (11Z, 14Z)
20:3 (11Z, 14Z, 17Z)
22:2 (13Z, 16Z)
22:3
methyl esters of tetra-, penta-, and hexaenoic FAs
other substances
16:4 (4Z, 7Z, 10Z, 13Z)
(8Z, 11Z)-heptadeca-8, 11-dienal
16:4 (6Z, 9Z, 12Z, 15Z)
3-(3, 5-ditertbutyl-4-hydroxyphenyl) propionate
18:4 (5Z, 9Z, 12Z, 15Z)
3, 7, 11, 15-tetramethyl-2-hexadecen-1-ol
18:4 (6Z, 9Z, 12Z, 15Z)
8-(2-octylcyclopropyl) octadecanoate
19:4
2, 3, 4, 5- tetramethyl-3-hexen
20:4 (5Z, 8Z, 11Z, 14Z)
(5Z, 8Z, 11Z)-15, 16 epoxy 5, 8, 11-octadecadienoate
20:4 (8Z, 11Z, 14Z, 17Z)
Tetradecanamide
22:4 (7Z, 10Z, 13Z, 16Z)
Hexadecanamide
18:5 (3Z, 6Z, 9Z, 12Z, 15Z)
(9Z)-Octadecenamide
20:5 (5Z, 8Z, 11Z, 14Z, 17Z)
9, 10-methylene tetradecanoate
22:5 (4Z, 7Z, 10Z, 13Z, 16Z)
22:5 (7Z, 10Z, 13Z, 16Z, 19Z)
22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)
h
k d (*)
h
d
bl
b
d
l
l
d Table 1 Overview of the FAMEs identified and other substances found in the analysed SAG microalgal strains Table 1 Overview of the FAMEs identified and other substances found in the analysed SAG microalgal strains
86 substances, 76 methyl esters of FAs For the marked (*) FAMEs the double bond positions were only tentatively assigned. occurrence of these four PUFAs in a certain group of
microalgae is given as the percentage of strains with a
certain FA from all examined strains in Table 3.
Because the SAG culture collection focuses on micro-
scopic algae from terrestrial habitats, the Haptophyta,
Dinophyta and Phaeophyceae were just poorly repre-
sented. Therefore, the recovered distribution patterns in 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection occurrence of these four PUFAs in a certain group of
microalgae is given as the percentage of strains with a
certain FA from all examined strains in Table 3. Because the SAG culture collection focuses on micro-
scopic algae from terrestrial habitats, the Haptophyta,
Dinophyta and Phaeophyceae were just poorly repre-
sented. Therefore, the recovered distribution patterns in occurrence of these four PUFAs in a certain group of
microalgae is given as the percentage of strains with a
certain FA from all examined strains in Table 3. The distribution patterns of FAs among and within the
17 groups (phyla or classes) of microalgae and the cya-
nobacteria comprised by the examined strains was
investigated in more detail for four PUFAs which are of
high nutritional interest (Table 3). The frequency of Because the SAG culture collection focuses on micro-
scopic algae from terrestrial habitats, the Haptophyta,
Dinophyta and Phaeophyceae were just poorly repre-
sented. Therefore, the recovered distribution patterns in Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 4 of 16 Table 2 Comparison of the major FA composition of algae observed in this study against data published previously
Species
FA
(%
of
total)
Ref
14:0
16:0
16:1
16:2
16:3
16:4
18:0
18:1
18:2
18:3
18:4
20:4
20:5
22:6
Bacillariophyceae
Phaeodactylum
9.2
26.8
45.4
-
-
-
0.7
4.6
-
-
-
-
12.3
1.1
a
tricornutum
9.4
23.7
35.8
-
-
-
6.0
3.3
4.4
3.2
0.2
-
13.3
0.9
b
6.7
14.7
43.6
2.0
-
-
-
15.8
0.5
0.4
1.1
-
14.4
0.7
e
Thalassiosira weissflogii
25.9
28.8
28.7
-
-
7.4
1.5
3.3
-
0.3
-
-
4.0
0.1
b
8.8
36.6
40.5
-
-
-
-
14.0
-
-
-
-
-
-
e
Chlorophyceae
Dunaliella primolecta
0.4
21.8
4.5
0.9
2.5
12.3
0.8
6.4
6.2
41.1
4.1
-
-
-
b
0.6
26.0
0.9
-
-
-
1.6
16.3
7.0
38.7
0.6
-
-
-
e
Nannochloris sp. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection 1.8
15.1
16.6
-
0.2
-
1.0
57.7
0.6
0.8
0.3
5.9
-
-
b
13.3
17.8
-
-
-
-
-
23.9
10.8
28.2
6.1
-
-
-
e
Parietochloris incisa
-
10.0
2.0
1.0
1.0
-
3.0
16.0
17.0
3.0
-
46.0
1.0
-
c
0
19.8
-
5.2
-
-
18.2
10.2
14.3
14.3
-
14.0
4.3
-
e
Cyanophyceae
Nostoc commune
0.3
43.5
11.3
0.4
-
-
1.5
6.9
19.3
16.3
-
-
-
-
d
-
25.3
24.1
-
-
-
-
-
12.5
38.1
-
-
-
-
e
Synechocystis sp. 13.4
26.5
43.6
-
-
-
3.5
8.0
0.2
4.7
-
-
-
-
b
42.5
18.8
30.1
-
-
-
-
-
-
14.2
-
-
-
-
e
Haptophyceae
Pavlova lutheri
11.8
23.6
28.3
-
-
-
2.0
12.4
-
-
-
-
12.1
9.7
a
10.1
11.1
26.3
-
-
-
-
5.2
0.6
0.5
9.1
0.3
18.0
9.7
e
Prymnesiophyceae
Emiliana huxleyi
41.7
17.7
5.5
-
-
-
2.1
21.7
0.9
5.5
5.0
-
-
-
b
18.8
10.3
-
-
-
-
10.8
42.2
-
-
8.7
-
-
9.2
e
Raphidophyceae
Heterosigma akashiwo
6.2
46.3
21.3
-
-
0.4
0.5
2.7
1.6
4.2
7.3
-
8.7
0.7
b
6.6
40.0
12.7
4.0
-
-
-
-
4.5
6.7
5.2
3.5
14.8
-
e
a [3] most frequent FA, present in 15 out of 20 examined
groups (Table 3). In the Dinophyta, Haptophyta and
Euglenoids DHA-containing strains were particularly fre-
quent and DHA was found there in relatively high per-
centages of total FA content, i.e. in 60% or more of these
strains the DHA proportion was higher than 5%. In the
single studied dinophyte strain of Ceratium horridum the
DHA proportion was even 29.3%. In the other groups
DHA was found in rather low frequencies and also
mostly in rather small proportions, i.e. less than 1% of
total FA content. Although DHA was found in the Cryp-
tophyta and Bacillariophyceae in about every fifth strain,
its percentage of total FA content was less than 5% there,
except in Cryptomonas baltica SAG 18.80 (Cryptophyta)
where it is was 13.7%. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection Despite DHA was found in rather
low frequencies in the green algae (Chlorophyta), the sec-
ond highest DHA content of all SAG strains, 18.9% of
total FA, was found in the chlorophyte Chlorococcum these and other poorly represented groups may not be
representative for the whole group. For instance, for
Phaeophyceae mainly microscopic forms (e.g., Ectocar-
pus and the freshwater genus Bodanella) were available
and the examined Rhodophyta strains covered mostly
freshwater forms or those from terrestrial habitats (e.g.,
Porphyridium). Although diatoms are very diverse in
terrestrial habitats, the examined small sample of avail-
able diatom strains (18) does by far not adequately
represent this group which is probably the most species-
rich algal group. Also, for each of the two classes of
Stramenopiles (heterokont algae), Phaeothamniophyceae
and Raphidophyceae, just two strains are maintained at
the SAG and, therefore, are not further discussed here. Similarly, there is only a single strain of Chlorarachnio-
phyta (Rhizaria supergroup) in the SAG. The very long chain PUFA docosahexaenoic acid
(DHA, 22:6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)) was the third Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 5 of 16 Figure 1 Representative gas chromatograms of fatty acid
methyl esters from four species belonging to different algal
groups. a) Cyanobacteria, Chroococcus minutus SAG 41.79; b)
Chlorophyta, Closteriopsis acicularis SAG 11.86; c) Rhodophyta,
Pseudochantransia spec. SAG 14.96; d) Chromalveolates
(Haptophyta), Prymnesium parvum SAG 127.79. Fatty acid methyl
esters: a) 14:0, b) 14:1n-5, c) 16:0, d) 16:1n-9, e) 16:1n-7, f) 16:2n-6, g)
16:4n-3, h) 18:0, i) 18:1n-9, j) 18:1n-7, k) 18:2n-6, l) 18:3n-6, m) 18:3n-
3, n) 18:4n-3, o) 18:5n-3, p) 20:3n-6, q) 20:4n-6, r) 20:5n-3, s) 22:5n-3,
t) 22:6n-3. novae-angliae SAG 5.85, followed by the trebouxiophyte
Prototheca zopfii SAG 263-8 with 14.2%. Together these
findings are in accordance with DHA amounts described
before for specific groups of alga [3,4,14,15]. p
g
p
g
Eicosapentaenoic acid (EPA, 20:5(5Z, 8Z, 11Z, 14Z,
17Z)) was one of the most common PUFAs, found in all
of the 17 groups covered by our study (Table 3). EPA-
containing strains were particularly frequent in the
Eustigmatophyceae, Glaucophyta, Xanthophyceae and
Rhodophyta. The highest EPA proportions of total FA
content were in the Rhodophyta, with about 81% of the
strains exhibiting more than 10% EPA. The highest
values were 52.4% in Compsopogonopsis leptoclados
SAG 106.79 and 44.9% in Acrochaetium virgatulum
SAG 1.81. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection Also strains of three species of Porphyridium
contained high amounts of EPA (31.2% in P. sordidum
SAG O 500, 27.5% in P. aerugineum SAG 110.79, 26.7%
in P. purpureum SAG 1380-1a). This is in agreement
with a report on P. cruentum suggesting that red algae
are a rich source of EPA [16]. Despite EPA was rather
frequently found in the Glaucophyta, only about half of
all strains had EPA proportions greater than 10% (maxi-
mum 31.1% in Glaucocystis nostochinearum SAG 28.80). This is in agreement with another study which showed
high amounts of EPA (besides ARA) in the glaucophyte
Cyanophora paradoxa [17]. The highest percentage
(87%) of strains with an EPA proportion of greater than
10% was in the Dinophyta, but with a maximum of just
24.3% in Pyrocystis lunula SAG 2014. In the Euglenoids,
Xanthophyceae and Eustigmatophyceae about 67% of all
strains had an EPA proportion of greater than 10% with
maximum values of about 31% (31.4% in Heterococcus
fuornensis SAG 835-5, 31.6% in Euglena proxima SAG
1224-11a) and 34.6% in Goniochloris sculpta SAG 29.96. EPA was rarely found and mostly in insignificant
amounts (< 5%) in most green algae, but three strains
had an exceptionally higher content of about 20% of
total FAs (24.2%, Chlorella sp. SAG 242.80; 24.0%, Chla-
mydomonas allensworthii SAG 28.98; 22.3%, Cylindro-
capsa involuta SAG 314-1). EPA was the only FA
recovered from Chlorarachnion repens SAG 26.97
(Chlorarachniophyta). That Xanthophyceae and Eustig-
matophyceae contain EPA in relatively high proportions
while green algae rarely accumulate EPA supports pre-
vious studies [3,4,14,15,18]. EPA was rarely found and mostly in insignificant
amounts (< 5%) in most green algae, but three strains
had an exceptionally higher content of about 20% of
total FAs (24.2%, Chlorella sp. SAG 242.80; 24.0%, Chla-
mydomonas allensworthii SAG 28.98; 22.3%, Cylindro-
capsa involuta SAG 314-1). EPA was the only FA
recovered from Chlorarachnion repens SAG 26.97
(Chlorarachniophyta). That Xanthophyceae and Eustig-
matophyceae contain EPA in relatively high proportions
while green algae rarely accumulate EPA supports pre-
vious studies [3,4,14,15,18]. Figure 1 Representative gas chromatograms of fatty acid
methyl esters from four species belonging to different algal
groups a) Cyanobacteria Chroococcus minutus SAG 41 79; b) Figure 1 Representative gas chromatograms of fatty acid
methyl esters from four species belonging to different algal
groups. a) Cyanobacteria, Chroococcus minutus SAG 41.79; b)
Chlorophyta, Closteriopsis acicularis SAG 11.86; c) Rhodophyta,
Pseudochantransia spec. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection Table 3 Frequency of four selected PUFAs in 17 taxonomic groups of microalgae on which the examined 2071 strains
of the SAG culture collection were distributed, and the size of each group (in total number of strains) The frequency of PUFAs is shown as the percentage of the total number of strains examined per group. more than 5% and in the Eustigmatophyceae even no
strain reached 5%. ARA was rarely found in the green
algae, i.e. with an average frequency of about 14% in the
phyla Chlorophyta and Streptophyta, except for prasino-
phyte green algae where ARA was present in 42.9% of
all strains (Table 3). However, there were a few single
green algal examples with extraordinarily high ARA
contents, i.e. 73.8% (corresponding to 102 μg/mg of dry
weight, the highest ARA content detected in all investi-
gated SAG strains) in the chlorophyte Palmodictyon var-
ium SAG 3.92, followed by 52.9% in the chlorophyte
Trochisciopsis tetraspora SAG 19.95 and 51.8% in the
trebouxiophyte Myrmecia bisecta SAG 2043. That a
high ARA content was found in the latter strain is in
agreement with that it has been found a close relative
with Parietochloris incisa (syn. Lobosphaeropsis incisa,
Myrmecia incisa) [19]. P. incisa has been assigned an
“oleaginous microalga” and the richest plant source of
ARA known so far due to its capability to accumulate
high amounts of ARA (up to 59% of its total FA con-
tent) [20]. Interestingly, the SAG strain of P. incisa
(Lobosphaera incisa SAG 2007) had with 13.2% a much
lower ARA content (Table 2). more than 5% and in the Eustigmatophyceae even no
strain reached 5%. ARA was rarely found in the green
algae, i.e. with an average frequency of about 14% in the
phyla Chlorophyta and Streptophyta, except for prasino-
phyte green algae where ARA was present in 42.9% of
all strains (Table 3). However, there were a few single
green algal examples with extraordinarily high ARA
contents, i.e. 73.8% (corresponding to 102 μg/mg of dry
weight, the highest ARA content detected in all investi-
gated SAG strains) in the chlorophyte Palmodictyon var-
ium SAG 3.92, followed by 52.9% in the chlorophyte
Trochisciopsis tetraspora SAG 19.95 and 51.8% in the
trebouxiophyte Myrmecia bisecta SAG 2043. That a
high ARA content was found in the latter strain is in
agreement with that it has been found a close relative
with Parietochloris incisa (syn. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection SAG 14.96; d) Chromalveolates
(Haptophyta), Prymnesium parvum SAG 127.79. Fatty acid methyl
esters: a) 14:0, b) 14:1n-5, c) 16:0, d) 16:1n-9, e) 16:1n-7, f) 16:2n-6, g)
16:4n-3, h) 18:0, i) 18:1n-9, j) 18:1n-7, k) 18:2n-6, l) 18:3n-6, m) 18:3n-
3, n) 18:4n-3, o) 18:5n-3, p) 20:3n-6, q) 20:4n-6, r) 20:5n-3, s) 22:5n-3,
t) 22:6n-3. Figure 1 Representative gas chromatograms of fatty acid
methyl esters from four species belonging to different algal
groups. a) Cyanobacteria, Chroococcus minutus SAG 41.79; b)
Chlorophyta, Closteriopsis acicularis SAG 11.86; c) Rhodophyta,
Pseudochantransia spec. SAG 14.96; d) Chromalveolates
(Haptophyta), Prymnesium parvum SAG 127.79. Fatty acid methyl
esters: a) 14:0, b) 14:1n-5, c) 16:0, d) 16:1n-9, e) 16:1n-7, f) 16:2n-6, g)
16:4n-3, h) 18:0, i) 18:1n-9, j) 18:1n-7, k) 18:2n-6, l) 18:3n-6, m) 18:3n-
3, n) 18:4n-3, o) 18:5n-3, p) 20:3n-6, q) 20:4n-6, r) 20:5n-3, s) 22:5n-3,
t) 22:6n-3. Arachidonic acid (ARA, 20:4(5Z, 8Z, 11Z, 14Z)) was
most frequently found in the Phaeophyceae where it
was present in all strains except one investigated strain
(Table 3); in about 54% of all Phaeophyceae strains the
proportion of ARA was higher than 10%, but with a
maximum of just 17.7% in Halopteris filicina SAG Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 6 of 16 Table 3 Frequency of four selected PUFAs in 17 taxonomic groups of microalgae on which the examined 2071 strains
of the SAG culture collection were distributed, and the size of each group (in total number of strains)
no. of strains
DHA
EPA
ARA
GLA
Cyanobacteria
223
1.3
0.9
0.4
12.1
Plantae
Glaucophyta
15
80.0
46.7
6.7
Chlorophyta
Chlorophyceae
927
5.1
6.9
5.7
26.2
Trebouxiophyceae
253
4.3
16.6
22.9
6.3
Ulvophyceae
70
4.3
22.9
12.9
7.1
prasinophytes
21
14.3
33.3
42.9
57.1
Charophyta
159
1.3
17.6
13.8
31.4
Rhodophyta
78
70.5
67.9
3.8
Excavates
Euglenoids
131
42.7
44.3
51.1
Chromalveolates
Stramenopiles
Bacillariophyceae
18
22.2
44.4
11.1
11.1
Xanthophyceae
81
4.9
75.3
49.4
16.1
Eustigmatophyceae
17
88.2
41.2
5.9
Phaeophyceae
12
58.3
91.7
16.7
Chryso-/Synurophyceae
12
16.7
33.3
8.3
16.7
Haptophyta
13
84.6
61.5
7.7
Cryptophyta
Cryptophyta
27
22.2
66.7
3.7
3.7
Alveolates
Dinophyta
14
64.3
57.1
14.3
2071
The frequency of PUFAs is shown as the percentage of the total number of strains examined per group. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection Rela-
tively high percentages of GLA were found in species/
strains of Closterium (16.5% in C. baillyanum SAG 50.89,
8% in C. lunula SAG 7.84), but GLA was not found in
the other 12 strains of that genus. Similarly, in the many
strains available for Cosmarium (25) and Micrasterias
(16), GLA was found in only 11 and 2 strains, respec-
tively. The highest percentages of GLA were found in the
green algal class Chlorophyceae (29.9% in Deasonia mul-
tinucleata SAG 25.95, 28.5% in Desmodesmus multifor-
mis SAG 26.91) and in Cyanobacteria (24.8% in Spirulina
maxima SAG 84.79). In about one third (32%) of all
chlorophyte GLA strains this FA had precentages of 5%
and higher. Distribution of GLA in the cyanobacteria was
rather patchy, i.e. the 27 cyanobacteria strains with GLA
were mainly restricted to three genera, Calothrix (8
strains), Microcystis (7 strains) and Spirulina (6 strains). Also within each of these genera the GLA percentages
were quite variable, e.g. in Spirulina it varied from 4.6%
to 24.8%, and three strains where without GLA. FA com-
position has previously been used to discriminate cyano-
bacteria in isolates and natural samples at the generic
level [21,22]. To discriminate species of cyanobacteria, as
an additional marker the hydrocarbon composition was
used in an earlier study, but in our study we failed to
detect any substance out of this group [23]. Interestingly,
GLA was the only FA that was detected in more than
three out of the 223 examined strains. Therefore, the
SAG cyanobacteria strains may be roughly divided into
those with GLA present (few genera) and those where
almost no PUFAs were present. This corresponds to the
earlier findings that described a bipartition of cyanobac-
teria, independent of their taxonomic position, into gen-
era producing C-18 PUFA and those which do not
[24,25]. The prasinophyte genus Tetraselmis presented an y
Higher taxonomic levels analyses It was tested whether
distribution patterns of FA composition on the investi-
gated strains delineate the three “super groups” of
eukaryotic algae, Plantae, Chromalveolates and Exca-
vates (Euglenoids), and the cyanobacteria from each
other. The Plantae super group comprises exclusively
eukaryotes with plastids derived from primary endocyto-
biosis, i.e. a cyanobacterium was transformed into an
organelle through uptake and retention by the host cell
followed by the loss of much of its genome [26]. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 7 of 16 whether certain patterns of FA distribution among the
various investigated algal groups are present that may
correspond to their phylogenetic relationships. In a first
set of three analyses (higher taxonomic levels) it was
tested 1) whether FA distribution patterns may reflect
differences among algal phyla derived from primary
(Plantae supergroup) or secondary endocytobiosis (Chro-
malveolates, Euglenoids) compared to cyanobacteria
representing the plastid origin, 2) the distinction of phyla
within the Plantae supergroup (Chlorophyta, Strepto-
phyta, Rhodophyta/Glaucophyta) and 3) major evolution-
ary lineages (classes) within the Chlorophyta. A second
set of analyses focused at the generic level, i.e.it was
tested whether separation of genera as based on previous
18S rDNA sequence analyses suggested for Chlamydo-
monas s.l., Chlorella s.l. and Scenedesmus s.l. are reflected
in the FA distribution patterns. For the first set of ana-
lyses the many species (266) which were represented as
multiple strains (e.g., Chlamydomonas moewusii, 28) had
to be reduced to only a single strain per species to avoid
biases. This included also the multiple strains unidenti-
fied at the species level, i.e. labelled with “sp.” instead a
species name (e.g., Chlorogonium sp., 26). The SAG’s
Chlorophyta strains were particularly rich in such multi-
ple strains. Also excluded were those strains where only a
single FA was detected. This reduced the total number of
strains considered in our calculations to 1193. The
strains were then divided into eleven groups roughly cor-
responding to phyla or classes (Additional file 2). Strains
belonging to the Chlorophyta (61% of all investigated
strains) were further subdivided into the three classes,
Chlorophyceae, Trebouxiophyceae, and Ulvophyceae,
whereas the prasinophyte SAG green algal strains (1.7%
of all considered Chlorophyta strains) were excluded
from the analyses because they comprised only very few
species (10). The strains of Glaucophyta (15) and Rhodo-
phyta (81) were collectively treated as one composite
unit. The Rhizaria - Chlorarachniophyta, was represented
just by a single strain and, thus, was omitted from the
statistical analyses. group, Tetraselmis, and there in 12 out of the 17 available
strains and with variable proportions, i.e. 0.5 - 7.3% of
total FA content. In the Streptophyta, GLA was more
widely distributed, i.e. it was detected in 17 out of 41
examined genera. GLA distribution was rather variable
within strains and species of a certain streptophyte
genus, similar to findings of ARA in other genera. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection Lobosphaeropsis incisa,
Myrmecia incisa) [19]. P. incisa has been assigned an
“oleaginous microalga” and the richest plant source of
ARA known so far due to its capability to accumulate
high amounts of ARA (up to 59% of its total FA con-
tent) [20]. Interestingly, the SAG strain of P. incisa
(Lobosphaera incisa SAG 2007) had with 13.2% a much
lower ARA content (Table 2). 10.96. ARA had the highest proportion of total FA in
the Rhodophyta; there even about 77% of all strains had
an ARA content of more than 10% with a maximum of
68.3% in Pseudochantransia sp. SAG 19.96. Interestingly,
the ARA content was rather high but variable among
the eight examined multiple isolates of the rhodophyte
Porphyridium purpureum. While the average ARA pro-
portion was about 31% in six strains, it was just 3.8% in
SAG 1380-1d, but 44.5% in SAG 1380-1e. We have no
explanation for this variation yet; both strains were iso-
lated from marine habitats and are kept under the same
culture conditions. High proportions of ARA (as well as
EPA) were already found characteristic of another spe-
cies of Porphyridium cruentum [16]. ARA was present
in about half of all investigated Euglenoid strains and
with relatively high proportions of total FA content, i.e. about one third of the strains exhibited more than 5%
ARA with extraordinarily high values of 41.3% and
34.3% in Rhabdomonas incurva SAG 1271-8 and Khaw-
kinea quartana SAG 1204-9. Interestingly, another
strain of the same species K. quartana, SAG 1204-9,
had less than half (13.3%) of ARA content and in five
other species of Rhabdomonas no ARA was detected. This demonstrates that FA contents may be rather vari-
able between species of the same genus and even among
multiple isolates of the same species. Although about
half of all examined strains for the Xanthophyceae and
Eustigmatophyceae contained ARA (Table 3), they had
this FA in relatively low proportions. Only one fourth of
the ARA-containing Xanthophyceae strains exhibited g-Linolenic acid (GLA, 18:3(6Z, 9Z, 12Z)) was the third
most common FA in the studied sample of SAG microal-
gal strains, missing only in the Haptophyta, Dinophyta
and Euglenoids (Table 3). It was most frequently detected
in two lineages of green algae, the prasinophytes and the
Streptophyta. In prasinophytes, however, GLA was pre-
sent only in one out of five genera available for that Lang et al. 2.1 Distribution of four important PUFAs among strains of
the SAG algal culture collection Chro-
malveolate algae as well as the Euglenoids (the only
algal lineage of Excavates) acquired their plastids
through secondary endocytobiosis from rhodophyte and
a green alga, respectively [26,27]. To consider almost The prasinophyte genus Tetraselmis presented an
interesting example to test for FA variation among clo-
sely related isolates. Nine strains assigned to that genus
have been isolated from the same (marine) locality and
regarded as the same species by the isolator (U.G. Schlösser, pers. comm.). Only in two strains DHA was
present, but in very small traces (0.3% and 0.4%). In
contrast, ARA and GLA were found in all isolates with
percentages varying from 0.8% to 2.7% and 0.5% to
7.3%, respectively. y
2.2 Analysis of FA distribution patterns y
2.2 Analysis of FA distribution patterns The detected fatty acid (FA) composition of the 2076
investigated strains was statistically analyzed to test Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 8 of 16 equal numbers of strains for all four groups, 100 strains
of Plantae, Chromalveolates and Cyanobacteria were
randomly selected which closely amounts the total num-
ber of considered euglenoid strains (73). The ordination
which resulted from CVA (Canonical Variates Analysis,
multigroup discriminant analysis) pointed out a strong
difference between cyanobacteria/primary endocytobiosis
(Plantae) and the two groups representing secondary
endocytobiosis (Chromalveolates/Euglenoids) (Figure 2). The
observed
difference
was
without
exception supported by non-parametric significance tests for mul-
tidimensional data (NP-MANOVA and ANOSIM). Fol-
lowing SIMPER, the lowest observed dissimilarity
(63.55%) was between Cyanobacteria and Plantae, while
the highest (77.29%) was between Plantae and Chromal-
veolates. The first canonical variate (CV1) involved
99.99% of all possible differences among the four
groups, hence we examined for possible correlations
between this axis and FAs. Four FAs were significantly
and exclusively correlated with the first canonical variate supported by non-parametric significance tests for mul-
tidimensional data (NP-MANOVA and ANOSIM). Fol-
lowing SIMPER, the lowest observed dissimilarity
(63.55%) was between Cyanobacteria and Plantae, while
the highest (77.29%) was between Plantae and Chromal-
veolates. The first canonical variate (CV1) involved
99.99% of all possible differences among the four
groups, hence we examined for possible correlations
between this axis and FAs. Four FAs were significantly
and exclusively correlated with the first canonical variate Figure 2 Discrimination of cyanobacteria and three algal eukaryotic supergroups (Plantae, Chromalveolates, Excavates/Euglenoids) as
based on fatty acid distribution patterns of 373 investigated cyanobacterial and algal strains using Canonical Variates Analysis. The
two vectors shown indicate FAs significantly correlated with canonical axis 1. Lines encircle 95% of members of a particular group. Circles,
Cyanobacteria; crosses, Plantae; arrowheads, Excavates/Euglenoids; diamonds, Chromalveolates. Figure 2 Discrimination of cyanobacteria and three algal eukaryotic supergroups (Plantae, Chromalveolates, Excavates/Euglenoids) as
based on fatty acid distribution patterns of 373 investigated cyanobacterial and algal strains using Canonical Variates Analysis. The
two vectors shown indicate FAs significantly correlated with canonical axis 1. Lines encircle 95% of members of a particular group. Circles,
Cyanobacteria; crosses, Plantae; arrowheads, Excavates/Euglenoids; diamonds, Chromalveolates. Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 9 of 16 Page 9 of 16 (CV1), i.e. y
2.2 Analysis of FA distribution patterns 16:0 (rCV1 = -0.61/p < 0.001), 18:2(9Z, 12Z)
(rCV1 = -0.46/p < 0.001), 9-octadecanamid (rCV1 = 0.41/
p < 0.001), and 18:1(9Z) (rCV1 = -0.17/p = 0.001). In a
second analysis it was tested whether FA distribution
patterns distinguish phyla of the Plantae super group,
i.e. the two lineages of green algae, Chlorophyta and
Streptophyta [28,29], and the composite Rhodophyta/
Glaucophyta group. Because the latter was with 54
strains the smallest group, it was compared with equally large random samples from each the Chlorophyta and
Streptophyta (Table 3). The ordination diagram from a
CVA of the total of 162 investigated strains clearly sepa-
rated the Rhodophyta/Glaucophyta group from both
green algal phyla (Figure 3). CV1 involved 79% of all pos-
sible differences and even CV2 was with 21% not negligi-
ble. The significance tests, NP-MANOVA and ANOSIM,
supported the distinction of all three groups. SIMPER
showed the Rhodophyta/Glaucophyta composite group large random samples from each the Chlorophyta and
Streptophyta (Table 3). The ordination diagram from a
CVA of the total of 162 investigated strains clearly sepa-
rated the Rhodophyta/Glaucophyta group from both
green algal phyla (Figure 3). CV1 involved 79% of all pos-
sible differences and even CV2 was with 21% not negligi-
ble. The significance tests, NP-MANOVA and ANOSIM,
supported the distinction of all three groups. SIMPER
showed the Rhodophyta/Glaucophyta composite group Figure 3 Discrimination of 162 algal strains of the Plantae supergroup into three subgroups representing the Rhodophyta/
Glaucophyta composite group (arrowheads) and both green algal phyla, Chlorophyta (diamonds) and Streptophyta (circles) as based
on their fatty acid distribution patterns using Canonical Variates Analysis. The vectors shown indicate FAs significantly correlated with CV1
and CV2 Lines encircle 95% of members of a particular group Figure 3 Discrimination of 162 algal strains of the Plantae supergroup into three subgroups representing the Rhodophyta/
Glaucophyta composite group (arrowheads) and both green algal phyla, Chlorophyta (diamonds) and Streptophyta (circles) as based
on their fatty acid distribution patterns using Canonical Variates Analysis. The vectors shown indicate FAs significantly correlated with CV1
and CV2. Lines encircle 95% of members of a particular group. Figure 3 Discrimination of 162 algal strains of the Plantae supergroup into three subgroups representing the Rhodophyta/
Glaucophyta composite group (arrowheads) and both green algal phyla, Chlorophyta (diamonds) and Streptophyta (circles) as based
on their fatty acid distribution patterns using Canonical Variates Analysis. y
2.2 Analysis of FA distribution patterns Also in
contrast to the rDNA phylogenies, the FA analyses split
the genus Lobochlamys, i.e. L. culleus was part of the
“Chloromonas“ group while L. segnis belonged to the
“Reinhardtii“ group. Strains of Oogamochlamys were also
separated on both FA groups, in contrast to their species
assignments as based on the 18S rDNA analyses. rather dissimilar from both green algal phyla, i.e. there
were dissimilarities of 70.55% and 71.53% with the Chlor-
ophyta and Streptophyta, respectively. The lowest dissim-
ilarity (55.41%) among the three tested groups was
between Chlorophyta and Streptophyta. There were five
FAs significantly and exclusively correlated with CV1, i.e. 18:3(9Z, 12Z, 15Z) (rCV1 = 0.77/p < 0.001), 20:4 (rCV1 =
-0.49/p < 0.001), 20:5(5Z, 8Z, 11Z, 14Z, 17Z) (rCV1 =
-0.59/p < 0.001), 18:1(9Z) (rCV1 = 0.30/p = 0.001) and
16:0 (rCV1 = -0.56/p = 0, 001). Two FAs were correlated
exclusively with CV2, i.e. they discriminated Chlorophyta
and Streptophyta, 18:1(9Z) (rCV2 = -0.4477/p < 0.001)
and 9-octadecanamid (rCV2 = 0.34/p < 0.001). The by far
largest fraction of all considered strains (60.3%) were
from the Chlorophyta which made it interesting to test
whether FA distribution patterns can discriminate
between the three classes of Chlorophyta, the Chlorophy-
ceae, Trebouxiophyceae and Ulvophyceae. Ulvophyceae
was the smallest of the three with just 49 strains and,
therefore, random samples of almost the same size (54)
from each of the other two classes were used for the sta-
tistical analyses. The CVA did not reveal any distinct
groups, i.e. the analyzed strains tended to form three
groups corresponding to the three green algal classes, but
with a considerable overlap among them (Figure 4). However, the three classes were found significantly dis-
tinct from each other in both employed significance tests
and SIMPER. The latter and correlation analyses allowed
to consider 9-octadecanamid (rCV1 = -0.58/p < 0.001;
rCV2 = -0.22/p < 0.010) and the FA 18:2(9Z, 12Z) (rCV1 =
-0.44/p < 0.001; rCV2 = -0.53/p < 0.001) as the only vari-
ables to discriminate well Ulvophyceae from Chlorophy-
ceae/Trebouxiophyceae and Trebouxiophyceae from
Ulvophyceae/Chlorophyceae, respectively. Species and strains formerly assigned to a single genus
Scenedesmus were shown to be actually distributed on sev-
eral genera by rRNA gene sequence analyses. For example,
the genus Acutodesmus has been segregated from Scene-
desmus [31,32]. y
2.2 Analysis of FA distribution patterns A NMDS ordination plot of FA distribu-
tion patterns revealed a tendency among the studied
strains to be distributed on two clusters, i.e. one cluster of
8 strains of Acutodesmus (mainly including multiple
strains of A. obliquus) was clearly separated from another
cluster containing mainly strains of Scenedesmus s.str. (Figure 6). The multiple strains of S. vacuolatus were
grouped together with four other strains of the genus,
except for SAG 211-11n which was close to the Acutodes-
mus cluster. The multiple strains of A. obliquus, however,
were distributed on both clusters (Figure 6). Seven strains
of A. obliquus mainly formed up the Acutodesmus cluster,
whereas five other A. obliquus strains grouped together
with strains of Scenedesmus s.str. This means that within
the same green algal species, A. obliquus, two distinct FA
patterns exist. AFLP fingerprints already showed extensive
genetic variation among the multiple strains of A. obliquus
while ITS2 rDNA sequence comparisons demonstrated
conspecificity of the multiple strains, except for SAG 276-
20 (T. Friedl, unpubl. observation). Therefore, the finding
of A. obliquus strains being separated in two FA pattern
groups favours the view that genetic differences resolved
by AFLPs may correspond to different phenotypic proper-
ties. Consequently, it may be crucial to carefully record
which strain has been used in any application [33]. Though strain SAG 276-20 was found not to belong to
the same species, A. obliquus, its FA pattern suggests that
it may still be a member of Acutodesmus because it was
grouped in the Acutodesmus cluster (Figure 6). Generic level analyses The three previous analyses
showed that phylogenetic relationships at the level of
phyla and classes among algal groups were reflected in FA
distribution patterns using a large sample of strains. Therefore, in a second group of analyses, we tested
whether differences in FA distribution patterns may
resolve the same distinction of genera as in rRNA gene
sequence analyses. To test this, we selected three genera
which are widely used in biotechnological applications and
well represented by SAG strains, i.e. Chlorella s.l., Scene-
desmus s.l. and Chlamydomonas s.l.. Recent18S rRNA
gene sequence analyses revealed each of the three as para-
or polyphyletic assemblages encompassing several distinct
genera. For Chlamydomonas we selected 17 species
(53 strains), out of which 9 were represented by multiple
strains (e.g., C. reinhardtii, 16), which were distributed on
five independent lineages/clades (= genera) in the 18S
rDNA phylogeny [30]. y
2.2 Analysis of FA distribution patterns The vectors shown indicate FAs significantly correlated with CV1
and CV2. Lines encircle 95% of members of a particular group. Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 10 of 16 rather dissimilar from both green algal phyla, i.e. there
were dissimilarities of 70.55% and 71.53% with the Chlor-
ophyta and Streptophyta, respectively. The lowest dissim-
ilarity (55.41%) among the three tested groups was
between Chlorophyta and Streptophyta. There were five
FAs significantly and exclusively correlated with CV1, i.e. 18:3(9Z, 12Z, 15Z) (rCV1 = 0.77/p < 0.001), 20:4 (rCV1 =
-0.49/p < 0.001), 20:5(5Z, 8Z, 11Z, 14Z, 17Z) (rCV1 =
-0.59/p < 0.001), 18:1(9Z) (rCV1 = 0.30/p = 0.001) and
16:0 (rCV1 = -0.56/p = 0, 001). Two FAs were correlated
exclusively with CV2, i.e. they discriminated Chlorophyta
and Streptophyta, 18:1(9Z) (rCV2 = -0.4477/p < 0.001)
and 9-octadecanamid (rCV2 = 0.34/p < 0.001). The by far
largest fraction of all considered strains (60.3%) were
from the Chlorophyta which made it interesting to test
whether FA distribution patterns can discriminate
between the three classes of Chlorophyta, the Chlorophy-
ceae, Trebouxiophyceae and Ulvophyceae. Ulvophyceae
was the smallest of the three with just 49 strains and,
therefore, random samples of almost the same size (54)
from each of the other two classes were used for the sta-
tistical analyses. The CVA did not reveal any distinct
groups, i.e. the analyzed strains tended to form three
groups corresponding to the three green algal classes, but
with a considerable overlap among them (Figure 4). However, the three classes were found significantly dis-
tinct from each other in both employed significance tests
and SIMPER. The latter and correlation analyses allowed
to consider 9-octadecanamid (rCV1 = -0.58/p < 0.001;
rCV2 = -0.22/p < 0.010) and the FA 18:2(9Z, 12Z) (rCV1 =
-0.44/p < 0.001; rCV2 = -0.53/p < 0.001) as the only vari-
ables to discriminate well Ulvophyceae from Chlorophy-
ceae/Trebouxiophyceae and Trebouxiophyceae from
Ulvophyceae/Chlorophyceae, respectively. (2213, 1753) were included. The NMDS ordination clearly
separated the members of the “Reinhardtii“ clade (upper
right in Figure 5), except for three strains, from those of
the “Chloromonas“ clade (lower left in Figure 5). However,
the “Chloromonas“ group as revealed by the FA patterns
also included the three investigated strains of the “Moewu-
sii“ and four of the “Oogamochlamys“ clades which was in
contrast to the 18S rDNA phylogenies of [30]. y
2.2 Analysis of FA distribution patterns To better represent the “Oogamo-
chlamys“ clade also two strains from the UTEX collection Chlorella vulgaris forms another example where exten-
sive genetic variation among multiple strains of the same
species has been detected by AFLP analyses [33]. The 15
multiple SAG strains of C. vulgaris were compared to 19
other Chlorella and Chlorella-like strains, i.e. their closest
relatives as seen in 18S rDNA phylogenies, C. sorokiniana Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 11 of 16 Figure 4 Discrimination of 162 algal strains of the Chlorophyta into three subgroups representing the three green algal classes
Chlorophyceae (diamonds), Trebouxiophyceae (arrowheads) and Ulvophyceae (circles) as based on their fatty acid distribution
patterns using Canonical Variates Analysis. Both vectors correspond to variables (fatty acids) correlated with both canonical axes. Lines Figure 4 Discrimination of 162 algal strains of the Chlorophyta into three subgroups representing the three green algal classes
Chlorophyceae (diamonds), Trebouxiophyceae (arrowheads) and Ulvophyceae (circles) as based on their fatty acid distribution
patterns using Canonical Variates Analysis. Both vectors correspond to variables (fatty acids) correlated with both canonical axes. Lines
encircle 65% of members of a particular group. Figure 4 Discrimination of 162 algal strains of the Chlorophyta into three subgroups representing the three green algal classes
Chlorophyceae (diamonds), Trebouxiophyceae (arrowheads) and Ulvophyceae (circles) as based on their fatty acid distribution
patterns using Canonical Variates Analysis. Both vectors correspond to variables (fatty acids) correlated with both canonical axes. Lines
encircle 65% of members of a particular group. and C. lobophora, members of the Parachlorella clade
sensu [34] as well as more distantly related strains, i.e. from the Watanabea and Prasiola clades sensu [35]. NMDS ordination based on FA distribution pattern
showed almost no variation within the multiple strains of
C. vulgaris and clustered them together, except for strain
SAG 211-1e (Figure 7). Another cluster distant from C. vulgaris was formed by members of the Watanabea-
clade, whereas Chlorella-like algae of the Prasiola-clade
were not clustered together. Conclusion The algae collection at the SAG represents a valuable
resource of natural products as shown in the present
study for FAs and other hydrophobic metabolites. Sev-
eral general trends in FA distribution reflect phyloge-
netic relationships among phyla and classes as seen in
genomic and molecular phylogenies and this makes FA
distribution patterns an additional feature to define taxa
of higher rank in algae. However the FA profile alone
may be no useful marker to distinguish among different Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 12 of 16 Figure 5 Distinction of 54 strains previously assigned to Chlamydomonas s.l. (Chlorophyceae), into the “Reinhardtii“ (upper right) and
“Chloromonas“ (lower left) groups as based on fatty acid distribution patterns (non-metric multidimensional scaling, NMDS;
Manhattan distance, Kruskal’s stress = 0.17). Symbols indicate the lineages and genera as resolved in the rDNA analyses of Pröschold et al. (2001); circles, “Reinhardtii“ clade; empty arrowheads, Lobochlamys; filled circles, Oogamochlamys; filled arrowheads, Chloromonas; filled arrowhead
down, “Moewusii“ clade. Figure 5 Distinction of 54 strains previously assigned to Chlamydomonas s.l. (Chlorophyceae), into the “Reinhardtii“ (upper right) and
“Chloromonas“ (lower left) groups as based on fatty acid distribution patterns (non-metric multidimensional scaling, NMDS;
Manhattan distance, Kruskal’s stress = 0.17). Symbols indicate the lineages and genera as resolved in the rDNA analyses of Pröschold et al. (2001); circles, “Reinhardtii“ clade; empty arrowheads, Lobochlamys; filled circles, Oogamochlamys; filled arrowheads, Chloromonas; filled arrowhead
down, “Moewusii“ clade. Figure 5 Distinction of 54 strains previously assigned to Chlamydomonas s.l. (Chlorophyceae), into the “Reinhardtii“ (upper right) and
“Chloromonas“ (lower left) groups as based on fatty acid distribution patterns (non-metric multidimensional scaling, NMDS;
Manhattan distance, Kruskal’s stress = 0.17). Symbols indicate the lineages and genera as resolved in the rDNA analyses of Pröschold et al. (2001); circles, “Reinhardtii“ clade; empty arrowheads, Lobochlamys; filled circles, Oogamochlamys; filled arrowheads, Chloromonas; filled arrowhead
down, “Moewusii“ clade. Methods genera and species. For this, the comparison of further
metabolites, like sterols, entire lipids and hydrocarbons
should be considered. Thus, PUFA contents in microal-
gae are rather difficult to predict at the levels of genera
and species, making it difficult to select appropriate
strains for biotechnological research/applications which
aim at yielding high lipid contents. Therefore, each addi-
tional or novel isolate will be worth of examination for
its PUFA content. Preparation of microalgal cultures Preparation of microalgal cultures The microalgal cells were harvested from cultures at the
stationary phase and stored at -20°C. Stationary phase
was reached after different periods of culturing ranging
from three months to about one year, depending on the
strain-specific SAG’s standard maintenance protocols. Before FA extraction the algal material was lyophilised
for two days until the cell pellets were totally dry. Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 13 of 16 Alkaline hydrolysis, transesterification and extraction of
FA methyl esters (FAMEs)
corresponding FAMEs was accomplished by adding
150 μl sodium methoxide [36]. After 20 min shaking a
Figure 6 Separation of Acutodesmus (empty circles) from Scenedesmus s.str. strains (filled arrowheads) as seen in FA pattern
distribution. Multiple strains of A. obliquus are indicated by abbreviation “Aobl”, those of S. vacuolatus by “Svac”. E, P, T, strains of the genera
Enallax, Pectinodesmus and Tetradesmus (Non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.16). Figure 6 Separation of Acutodesmus (empty circles) from Scenedesmus s str strains (filled arrowheads) as seen in FA pattern Figure 6 Separation of Acutodesmus (empty circles) from Scenedesmus s.str. strains (filled arrowheads) as seen in FA pattern
distribution. Multiple strains of A. obliquus are indicated by abbreviation “Aobl”, those of S. vacuolatus by “Svac”. E, P, T, strains of the genera
Enallax, Pectinodesmus and Tetradesmus (Non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.16). Alkaline hydrolysis, transesterification and extraction of
FA methyl esters (FAMEs) Alkaline hydrolysis, transesterification and extraction of
FA methyl esters (FAMEs) Alkaline hydrolysis, transesterification and extraction of
FA methyl esters (FAMEs) corresponding FAMEs was accomplished by adding
150 μl sodium methoxide [36]. After 20 min shaking at
RT the FAMEs were extracted two times with 500 μl n-
hexane and 500 μl 1 M NaCl. The hexane phases were
transferred into a 1.5 ml tube and dried under streaming
nitrogen. Finally the FAMEs were redissolved in 10 μl
acetonitrile and analysed by GC. Prior to FAME extraction the dry weight of lyophilised
algal material was determined and then the samples were
transferred into a 2 ml tube. The samples were extracted
by adding 405 μl of methanol/toluol 2:1 (v/v) followed by
homogenisation of the cells with a potter (Heidolph RZR
2020, Schwabach) for 30 s. To avoid autoxidation, the
samples were overlaid with argon. As internal standard,
10 μg of tripentadecanoate (diluted in 10 μl toluol) was
added. Transesterification of lipid bound FAs to their Preparation of 4, 4-dimethyloxaline (DMOX) derivatives
The position of double bonds of unknown FAME iso-
mers was determined by analysing the corresponding DMOX derivatives to allow identification by MS [37]. FAMEs were prepared as described, but the hexane
phases were transferred into a 1.5 ml glass tube. Sam-
ples were dried under streaming nitrogen and 200 μl 2
redissolved with 50 μl chloroform. The DMOX deriva-
tives were separated on a 20 cm × 20 cm silica gel 60
TLC plate (Merck) with petrol ether/diethyl ether 2:1
(v/v) as a developing solvent The plate was sprayed
Figure 7 Comparison of FA patterns of multiple strains of Chlorella vulgaris (arrowheads) and their closer relatives (filled circles) with
more distantly related Chlorella-like green algae of the Watanabea- (empty circles) and Prasiola-clades (diamonds) sensu Darienko et
al., 2010 (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.12). Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124
Page 14 of 16 Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 14 of 16 Figure 7 Comparison of FA patterns of multiple strains of Chlorella vulgaris (arrowheads) and their closer relatives (filled circles) with
more distantly related Chlorella-like green algae of the Watanabea- (empty circles) and Prasiola-clades (diamonds) sensu Darienko et
al., 2010 (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.12). Alkaline hydrolysis, transesterification and extraction of
FA methyl esters (FAMEs) Figure 7 Comparison of FA patterns of multiple strains of Chlorella vulgaris (arrowheads) and their closer relatives (filled circles) with
more distantly related Chlorella-like green algae of the Watanabea- (empty circles) and Prasiola-clades (diamonds) sensu Darienko et
al., 2010 (non-metric multidimensional scaling, NMDS; Manhattan distance, Kruskal’s stress = 0.12). DMOX derivatives to allow identification by MS [37]. FAMEs were prepared as described, but the hexane
phases were transferred into a 1.5 ml glass tube. Sam-
ples were dried under streaming nitrogen and 200 μl 2-
alkenyl-4, 4-dimethyloxazoline (Sigma, München) were
added. After incubation at 180°C over night in a heating
block, the samples were cooled to RT and transferred
with 2 ml dichloromethane into a 12 ml glass tube and
reextracted with 5 ml hexane and 2 ml water. The hex-
ane phase was dried under streaming nitrogen and DMOX derivatives to allow identification by MS [37]. FAMEs were prepared as described, but the hexane
phases were transferred into a 1.5 ml glass tube. Sam-
ples were dried under streaming nitrogen and 200 μl 2-
alkenyl-4, 4-dimethyloxazoline (Sigma, München) were
added. After incubation at 180°C over night in a heating
block, the samples were cooled to RT and transferred
with 2 ml dichloromethane into a 12 ml glass tube and
reextracted with 5 ml hexane and 2 ml water. The hex-
ane phase was dried under streaming nitrogen and redissolved with 50 μl chloroform. The DMOX deriva-
tives were separated on a 20 cm × 20 cm silica gel 60
TLC plate (Merck) with petrol ether/diethyl ether 2:1
(v/v) as a developing solvent. The plate was sprayed
with 0.2% 8-anilino-1-naphthalene-sulfonic acid to
visualize the DMOX derivatives under UV-light. The
blue/yellow band of the DMOX derivatives was scraped
out and the derivatives extracted by consecutive addition
of 0.4 ml water, 2 ml methanol, 2 ml chloroform and 2
ml saturated NaCl solution. Between each step the Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Page 15 of 16 Page 15 of 16 Metric Multidimensional Scaling (NMDS). The signifi-
cance of the differences among a priori predefined algal
groups were tested using non-parametric multidimen-
sional significance tests (Non-Parametric Multivariate
Analysis of Variance, Analysis of Similarity) and visua-
lised as ordinations from multigroup discriminant analy-
sis (Canonical Variates Analysis). The percentages of
dissimilarity between group pairs were investigated con-
ducting SIMPER analysis. Alkaline hydrolysis, transesterification and extraction of
FA methyl esters (FAMEs) To link the significant differ-
ences among algal groups with particular variables/fatty
acids possibly contributing to the observed difference,
correlation analyses were conducted (Spearman’s rank
correlation coefficient, r/rho), permutation significance
tests). All statistical analyses and graphical visualisations
have been conducted in PAST version 2.07 software
package. Final graphical attributes required for publica-
tion were adapted in vector graphics editor Inkscape ver-
sion 4.7 and CorelDraw X3 Graphic suite. sample was vigorously mixed and finally centrifuged for
5 min at 3220 × g to separate phases. The lower phase
was transferred into a new glass tube and dried under
nitrogen stream, redissolved in 10 μl acetonitrile and
analysed with GC/MS. Statistical analyses of FA distribution patterns For each detected fatty acid (FA) its percentage of the
total FA content of a strain was used as variable. For the
investigation of the general structure of the data sets,
common indirect ordination techniques were used, i.e. Principal Components Analysis (PCA), Correspondence
and Detrended Correspondence Analysis, and Non- Identification of FAMEs by GC with flame ionisation
detection (FID) The prepared FAMEs were analysed by GC/FID using a
capillary DB-23 column (30 m × 0.25 mm, 0.25 μm coat-
ing thickness, J&W, Scientific, Agilent, Waldbronn)
according to Hornung et al. (2002). Helium was used as
carrier gas with a flow of 0.1 ml/min. The temperature
gradient was 150°C for 1 min, 150-200°C at 4 K/min, 200-
250°C at 5 K/min and 250°C for 6 min. Tripentadecanoate
was added to each sample for quantification and the
FAMEs were identified according to the retention time of
the corresponding peaks in the standard “F.A.M.E. Mix
C4-C24” (Sigma, München), which was injected before
every 50th run. The injection volume depended on the
concentration of FAMEs within in the sample. Identification of FAMEs by GC/MS FID signals which were not identified by their retention
times on GC and either represented FAMEs or other
unpolar substances were further analysed by their mass
spectra using a 6890 Gas Chromatograph/5973 Mass
Selective Detector system (Agilent, Waldbronn). The
GC/MS conditions were the same as for GC-analysis. The electron energy was 70 eV, the ion source tempera-
ture 230°C, and the temperature for the transfer line
added up to 260°C. The identification of unknown sub-
stances was done by comparison of the obtained mass
spectra with the mass spectra library NIST98 and the
“Lipid Library” of the Scottish Crop Science research
Institute http://www.lipidlibrary.co.uk/index.html. Additional file 2: Reduced FAME database for statistical analyses. The database contains information about clade, phylum, class, genus and
species identification (1st to 5th column) as well as SAG strain number
(6th column) and the amount of the different substances given as
relative proportion (following columns). Acknowledgements and Funding All chromatograms of the microalgal samples were ana-
lysed by using the ChemStation software version 9.03
(Agilent, Waldbronn). All peaks spanning a peak area of
more than 50 units were integrated. The amount of each
FAME was calculated using a defined amount (1 μg) of
the internal standard tripentadecanoate and the dry weight
(DW) of each sample: area of peak × 1 μg/area of tripenta-
decanoate/mg d.w = μg FAME/mg DW The authors are grateful to Dr. Fredi Brühlmann (Geneva) and Dr. Cornelia
Göbel (Göttingen) for their continuous support with analytical methods and
interpretation of data, Dr. Maike Lorenz (Göttingen) for continuous support
with microalgal handling, database work and interpretation of data, Prof. Dr. Rüdiger Hardeland (Göttingen) for providing with dinophyte strains. This
work was supported by Firmenich SA, Geneva. List of abbreviations
ALA
li
l
i
id AR ALA: α-linolenic acid; ARA: Arachidonic acid; CVA: canonical variance analysis;
DHA: docosahexaenoic acid; DMOX: 4, 4-dimethyloxaline; EPA:
Eicosapentaenoic acid; FA: fatty acid; FAME: fatty acid methyl ester; GC: gas
chromatography; GLA: γ-Linolenic acid; MS: mass spectrometry; NMDS: non-
metric multidimensional scaling; PA: palmitic acid; PUFAs: polyunsaturated fatty
acids; SAG: culture collection of microalgae in Göttingen; SDA: stearidonic acid. ALA: α-linolenic acid; ARA: Arachidonic acid; CVA: canonical variance analysis;
DHA: docosahexaenoic acid; DMOX: 4, 4-dimethyloxaline; EPA: Additional material Additional file 1: FAME database established of all SAG microalgal
strains screened. The database contains information about clade,
phylum, class, genus and species identification (1st to 5th column) as well
as SAG strain number (6th column) and the amount of the different
substances given as relative proportion (following columns). Additional file 2: Reduced FAME database for statistical analyses. The database contains information about clade, phylum, class, genus and
species identification (1st to 5th column) as well as SAG strain number
(6th column) and the amount of the different substances given as
relative proportion (following columns). Additional file 1: FAME database established of all SAG microalgal
strains screened. The database contains information about clade,
phylum, class, genus and species identification (1st to 5th column) as well
as SAG strain number (6th column) and the amount of the different
substances given as relative proportion (following columns). Additional file 2: Reduced FAME database for statistical analyses. The database contains information about clade, phylum, class, genus and
species identification (1st to 5th column) as well as SAG strain number
(6th column) and the amount of the different substances given as
relative proportion (following columns). Additional file 1: FAME database established of all SAG microalgal
strains screened. The database contains information about clade,
phylum, class, genus and species identification (1st to 5th column) as well
as SAG strain number (6th column) and the amount of the different
substances given as relative proportion (following columns). Author details
1 1Georg-August-University, Albrecht-von-Haller-Institute for Plant Sciences,
Department of Plant Biochemistry, Göttingen, Germany. 2Cyano-Biofuels
GmbH, Magnussstrasse 11, 12489 Berlin, Germany. 3Georg-August-University,
Albrecht-von-Haller-Institute for Plant Sciences, Department of Experimental
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Cite this article as: Lang et al.: Fatty acid profiles and their distribution
patterns in microalgae: a comprehensive analysis of more than 2000
strains from the SAG culture collection. BMC Plant Biology 2011 11:124. doi:10.1186/1471-2229-11-124
Cite this article as: Lang et al.: Fatty acid profiles and their distribution
patterns in microalgae: a comprehensive analysis of more than 2000
strains from the SAG culture collection. BMC Plant Biology 2011 11:124. 15. Authors’ contributions IL carried out the fatty acid analysis of all algal strains and drafted the
manuscript. LH performed the statistical analysis. IF and TF conceived of the
study, and participated in its design and coordination and helped to draft
the manuscript. All authors read and approved the final manuscript. Page 16 of 16 Lang et al. BMC Plant Biology 2011, 11:124
http://www.biomedcentral.com/1471-2229/11/124 Received: 6 May 2011 Accepted: 6 September 2011
Published: 6 September 2011 Received: 6 May 2011 Accepted: 6 September 2011
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Y Acad Sci 1987, 503:17-54. Submit your next manuscript to BioMed Central
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• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
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• Research which is freely available for redistribution
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irregularis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel
subaerial coccoid green alga. Phycologia 2011, 50:57-66. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
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A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability
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Digital Object Identifier (DOI):
10.1016/j.chom.2014.01.006 Published In:
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A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is
Required to Subvert Autophagy and Maintain Virion Stability Edinburgh Research Explorer Citation for published version:
Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the
Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host &
Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 Citation for published version:
Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the
Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host &
Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 Citation for published version:
Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the
Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host &
Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 Citation for published version: Citation for published version:
Beale, R, Wise, H, Stuart, A, Ravenhill, BJ, Digard, P & Randow, F 2014, 'A LC3-Interacting Motif in the
Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability', Cell Host &
Microbe, vol. 15, no. 2, pp. 239-47. https://doi.org/10.1016/j.chom.2014.01.006 SUMMARY et al., 2009; 2012; Wild et al., 2011; Zheng et al., 2009). Binding
partners of LC3/ATG8 family members typically contain an
LC3-interacting region (LIR). LIRs form intermolecular b sheets
with LC3/ATG8 family members by virtue of a consensus
W/FxxI/L motif, often preceded by acidic residues (Johansen
and Lamark, 2011). et al., 2009; 2012; Wild et al., 2011; Zheng et al., 2009). Binding
partners of LC3/ATG8 family members typically contain an
LC3-interacting region (LIR). LIRs form intermolecular b sheets
with LC3/ATG8 family members by virtue of a consensus
W/FxxI/L motif, often preceded by acidic residues (Johansen
and Lamark, 2011). Autophagy recycles cellular components and de-
fends cells against intracellular pathogens. While
viruses must evade autophagocytic destruction,
some viruses can also subvert autophagy for their
own benefit. The ability of influenza A virus (IAV) to
evade autophagy depends on the Matrix 2 (M2)
ion-channel protein. We show that the cytoplasmic
tail of IAV M2 interacts directly with the essential
autophagy protein LC3 and promotes LC3 relocali-
zation to the unexpected destination of the plasma
membrane. LC3 binding is mediated by a highly
conserved LC3-interacting region (LIR) in M2. The
M2 LIR is required for LC3 redistribution to the
plasma membrane in virus-infected cells. Mutations
in M2 that abolish LC3 binding interfere with fila-
mentous budding and reduce virion stability. IAV
therefore subverts autophagy by mimicking a host
short linear protein-protein interaction motif. This
strategy may facilitate transmission of infection be-
tween organisms by enhancing the stability of viral
progeny. While autophagy restricts the growth of pathogens poorly
adapted to life in the cytosol (e.g., Salmonella enterica), profes-
sional cytosol-dwelling bacteria, such as Shigella flexneri, evade
restriction by autophagy (Boyle and Randow, 2013; Mostowy
and Cossart, 2012). Other pathogenic microbes have evolved
mechanisms to actively benefit from host autophagy. Multiple
viruses deploy such subversion strategies, but the molecular
mechanisms involved are unclear (Feng et al., 2013; Heaton
and Randall, 2010; Klein and Jackson, 2011; Reggiori et al.,
2010). The sizes of RNA virus genomes are evolutionarily constrained
(Belshaw et al., 2008). Their need to hijack cellular machinery
while evading host immune responses requires complex coding
capacities and multifunctional viral proteins. Multiple distinct
peptide products are often produced from the same gene
(e.g., Firth and Brierley, 2012; Thomas et al., 1988; Wise et al.,
2012). A LC3-Interacting Motif in the
Influenza A Virus M2 Protein Is Required to
Subvert Autophagy and Maintain Virion Stability Rupert Beale,1,2,5 Helen Wise,3,4,5 Amanda Stuart,4 Benjamin J. Ravenhill,1 Paul Digard,3,4,* and Felix Randow1,2,*
1MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
2University of Cambridge, Department of Medicine, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK
3Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, UK
4Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 0QQ, UK
5These authors contributed equally to this work
C
@
(
)
@
(
) *Correspondence: paul.digard@roslin.ed.ac.uk (P.D.), randow@mrc-lmb.cam.ac.uk (F.R.)
http://dx.doi.org/10.1016/j.chom.2014.01.006 SUMMARY In addition, viral proteins subvert host physiology by
encoding short linear motifs (SLiMs) that mimic host protein
interaction interfaces, but which require little coding capacity
(Davey et al., 2011). No viral SLiMs have yet been described to
subvert autophagy. Take down policy Download date: 24. Oct. 2024 Cell Host & Microbe
Short Article Cell Host & Microbe
Sh
A Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc.
2 INTRODUCTION One important human pathogen affecting host cell autophagy
is influenza A virus (IAV). This enveloped, segmented negative
sense RNA virus infects a wide range of vertebrate species
and causes seasonal epidemics and sporadic pandemics in hu-
mans as well as outbreaks in domestic animals. Epidemic IAV
causes significant annual global mortality and in pandemic years
can result in millions of deaths (Taubenberger and Kash, 2010). One important human pathogen affecting host cell autophagy
is influenza A virus (IAV). This enveloped, segmented negative
sense RNA virus infects a wide range of vertebrate species
and causes seasonal epidemics and sporadic pandemics in hu-
mans as well as outbreaks in domestic animals. Epidemic IAV
causes significant annual global mortality and in pandemic years
can result in millions of deaths (Taubenberger and Kash, 2010). The ability of IAV to subvert autophagy is dependent on the
Matrix 2 (M2) ion-channel protein, which blocks fusion of auto-
phagosomes with lysosomes (Gannage´ et al., 2009). M2 is a
tetrameric integral membrane protein, made from a spliced tran-
script (Holsinger et al., 1994; Lamb et al., 1985). M2 comprises a
25 amino acid N-terminal ectodomain, a transmembrane a helix
and a 50 amino acid cytoplasmic domain. This cytoplasmic
tail contains a membrane-proximal amphipathic a helix and a Macroautophagy
(hereafter
autophagy)
can
generate
raw
materials at times of cellular stress by degrading cytoplasmic
contents (Mizushima and Komatsu, 2011). During starvation-
induced autophagy, cytoplasm is nonselectively engulfed into
double-membrane vesicles (autophagosomes), whose content
is digested upon fusion with lysosomes. This process has
been adapted for host defense, where invading pathogens are
selectively targeted by autophagy (Deretic, 2012; Levine et al.,
2011; Randow et al., 2013). Selective antimicrobial autophagy
relies on cargo receptors that simultaneously detect path-
ogen-associated ‘‘eat me’’ signals and bind to members of the
ATG8/LC3 family of ubiquitin-related proteins residing on auto-
phagosomal membranes (Boyle and Randow, 2013; Thurston The ability of IAV to subvert autophagy is dependent on the
Matrix 2 (M2) ion-channel protein, which blocks fusion of auto-
phagosomes with lysosomes (Gannage´ et al., 2009). M2 is a
tetrameric integral membrane protein, made from a spliced tran-
script (Holsinger et al., 1994; Lamb et al., 1985). M2 comprises a
25 amino acid N-terminal ectodomain, a transmembrane a helix
and a 50 amino acid cytoplasmic domain. Plasma Membrane To observe how IAV subverts the cellular autophagocytic ma-
chinery, we investigated the distribution of GFP-tagged LC3
upon virus infection. Human cell lines, stably transduced with
GFP-LC3, were infected with IAV strain A/PR/8/34 (H1N1) (Fig-
ure 1A and Figure S1 available online). In uninfected cells,
GFP-LC3 was distributed diffusely throughout the cytoplasm. Following infection, LC3 accumulated in the perinuclear region,
as previously described (Gannage´ et al., 2009). In addition,
GFP-LC3 strikingly relocalized to the cell periphery. To investi-
gate the subcellular localization of LC3 in infected cells, we
stained nonpermeabilized cells with wheat-germ agglutinin
(WGA) to label extracellular glycans. In uninfected cells, there
was little colocalization of GFP-LC3 and WGA, while in infected
cells GFP-LC3 and WGA colocalized at the plasma membrane,
but not on perinuclear autophagosomes (Figure 1B). To verify
the presence of LC3 at the plasma membrane and examine the
subcellular distribution of autophagosomes in IAV-infected cells,
we performed immunoelectron microscopy (Kabeya et al.,
2000). As expected, in both mock and IAV-infected cells, LC3
was present in double-membrane vesicles corresponding to au-
tophagosomes (Figure 1C). Infected but not uninfected cells also
demonstrated staining of the plasma membrane, both at sites of
active viral budding and in areas where less budding was taking
place (Figure 1C). LC3 G120A, a mutant that cannot be lipidated,
did not localize to autophagosomes or the plasma membrane. Taken together, these data demonstrate that IAV directs
LC3 to the plasma membrane, an unusual and unexpected
destination. Strong conservation of motifs frequently indicates biological
importance. We therefore examined conservation of the putative
M2 LIR motif (Squires et al., 2012). Of 2,685 unique M2 se-
quences, 2,661 have an FVSI or FVNI motif (Figure 3C). This
near-absolute conservation of the hydrophobic residues renders
the predicted b strand a strong candidate LIR motif. To test if IAV M2 binds LC3 via its putative LIR motif, we per-
formed LUMIER binding assays (Barrios-Rodiles et al., 2005;
von Muhlinen et al., 2013) with purified GST-LC3 and the cyto-
solic domain of M2 fused to luciferase. WT M2 and the naturally
occurring variant S93N bound LC3, whereas M2DLIR, M2
F91S, M2 V92S, and M2 I94S failed to bind (Figure 3D). M2
and the LIR-containing p62 compete for binding to LC3, indi-
cating that M2 binds the same site as the classical p62 LIR
(Figure S2A). Plasma Membrane To determine the monomeric binding affinity of
the interaction, a fluorescently labeled peptide corresponding
to the last 13 amino acids of M2 and purified LC3 protein
were used in fluorescence anisotropy equilibrium measure-
ments. A dissociation constant (KD) of 9.5 ± 1.2 mM was deter-
mined (Figure 3E). In vivo this interaction would be enhanced
by the tetrameric nature of M2, and reduced dimensionality
imposed by the presence of both proteins in membranes. To
test whether M2 bound LC3 in vivo in a LIR-dependent manner,
we performed GFP-trap pull-down experiments in GFP-LC3-
expressing cells infected with WT, DM2, or M2 F91S IAV. WT
M2, but not the M2 LIR mutant F91S, coprecipitated with
LC3 (Figure 3F). We conclude that the M2 cytosolic tail con-
tains a conserved LIR motif capable of directly interacting
with LC3 in infected cells. Influenza A Virus M2 Protein Drives Plasma Membrane
Localization of LC3 Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability membrane-distal region of unknown structure (Schnell and
Chou, 2008; Sharma et al., 2010; Stouffer et al., 2008). M2 has
multiple important roles in the virus life cycle. During viral entry
its ion channel activity is required to trigger disassembly in
response to lowered endosomal pH, and its cytoplasmic tail con-
tributes to virus assembly, budding, and morphogenesis (Ross-
man and Lamb, 2011). here DM2) due to mutation of the M2 splice donor site (Hutch-
inson et al., 2008; Wise et al., 2012) did not relocalize LC3 to
the plasma membrane or induce its perinuclear accumulation
(Figure 2B). GFP-LC3 G120A failed to relocalize in cells in-
fected with WT virus (Figure 2B), confirming the electron
microscopy data (Figure 1C) and indicating that the autophagy
machinery is required for IAV to redistribute LC3 in infected
cells. here DM2) due to mutation of the M2 splice donor site (Hutch-
inson et al., 2008; Wise et al., 2012) did not relocalize LC3 to
the plasma membrane or induce its perinuclear accumulation
(Figure 2B). GFP-LC3 G120A failed to relocalize in cells in-
fected with WT virus (Figure 2B), confirming the electron
microscopy data (Figure 1C) and indicating that the autophagy
machinery is required for IAV to redistribute LC3 in infected
cells. membrane-distal region of unknown structure (Schnell and
Chou, 2008; Sharma et al., 2010; Stouffer et al., 2008). M2 has
multiple important roles in the virus life cycle. During viral entry
its ion channel activity is required to trigger disassembly in
response to lowered endosomal pH, and its cytoplasmic tail con-
tributes to virus assembly, budding, and morphogenesis (Ross-
man and Lamb, 2011). Here, we report a LIR motif in the cytoplasmic tail of M2. The
M2 LIR motif causes the relocalization of LC3 to the plasma
membrane in IAV-infected cells at the time of virus budding,
and is essential to generate stable viral progeny. We propose
that in addition to blocking autophagosome maturation (Gan-
nage´ et al., 2009), IAV hijacks the autophagy machinery via the
M2 LIR motif to provide suitable resources for viral budding
and to enhance virion stability. Influenza M2 Contains a Conserved LIR Motif in Its
Cytoplasmic Tail Because IAV M2 is required for redistribution of LC3 in infected
cells, we speculated that it binds LC3 directly. LIR motifs are b
strands that form an intermolecular b sheet on binding to LC3
(Johansen and Lamark, 2011). To identify potential LIRs in M2,
we used JPRED to predict secondary structures in its cytosolic
tail. One a-helix and one b strand were predicted (Figure 3A). The predicted b strand contains a FVSI motif that matches the
consensus LIR motif (Johansen and Lamark, 2011). Among bio-
chemically proven LIR motifs, the M2 FVSI motif most closely
corresponds to those found in optineurin and ATG13, which
both contain FVxI as their hydrophobic core (Alemu et al.,
2012; Wild et al., 2011) (Figure 3B). Preceding the putative M2
LIR motif are three acidic residues, another typical feature of
LC3/ATG8 binding proteins. INTRODUCTION This cytoplasmic
tail contains a membrane-proximal amphipathic a helix and a 239 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 239 Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability Influenza A Virus M2 Protein Drives Plasma Membrane
Localization of LC3 The pattern of LC3 immunogold staining (Figure 1C) parallels
the known distribution of M2, which, though abundant at the
plasma membrane during budding, is incorporated into virions
at low levels (Leser and Lamb, 2005). Furthermore, GFP-LC3
and M2 closely colocalized at the plasma membrane (Figures
2A and S1). This finding prompted us to test whether M2 is
required for the relocalization of LC3. In contrast to wild-type
(WT) IAV, infection with a virus deficient in M2 (V7-T9; named Plasma Membrane Targeting of LC3 Depends on the M2
LIR Motif Plasma Membrane Targeting of LC3 Depends on the M2
LIR Motif To test whether relocalization of LC3 in infected cells depends
on the M2 LIR motif, cells expressing GFP-LC3 were infected 240
Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Cell Host & Microbe Cell Host & Microbe M2 LIR Motif Required for Influenza A Stability A
B
C
10µm
10µm
IAV, LC3
IAV, LC3 G120A
mock, LC3
GFP-LC3 DAPI NP merge
GFP-LC3 DAPI WGA merge
IAV
mock
IAV
e 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells
49 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. CT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. CT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with gold-labeled anti-GFP anti
arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. so Figure S1. A
GFP-LC3 DAPI NP merge
IAV NP B
10µm
10µm
GFP-LC3 DAPI WGA merge
IAV
mock
IAV B IAV C
IAV, LC3
IAV, LC3 G120A
mock, LC3
e 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells
49 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. T116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. C
IAV, LC3
IAV, LC3 G120A
mock, LC3 Figure 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells C3 to the Plasma Membrane in Influenza-Infected Cells Figure 1. Relocalization of LC3 to the Plasma Membrane in Influenza Infected Cells
(A) A549 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm. (B) HCT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. (C) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with gold-labeled anti-GFP antibody. Black arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. Figure 1. Relocalization of LC3 to the Plasma Membrane in Influenza-Infected Cells
(A) A549 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-NP. Scale bar, 10 mm.
(B) HCT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm.
(C) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with gold-labeled anti-GFP antibody.
Black arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability A
B A A A A Figure
2. Relocalization
of
LC3
to
the
Plasma Membrane Requires Influenza M2
(A) HCT116 cells expressing GFP-LC3 were in-
fected with IAV, fixed at 16 hr p.i., and stained with
anti-M2. Scale bar, 10 mm. (B) HCT116 cells expressing the indicated LC3
alleles fused to GFP were infected with WT or DM2
mutant IAV, fixed at 16 hr p.i., and stained with
anti-NP. Scale bar, 10 mm. See also Figure S1. with IAV encoding WT or mutant M2 polypeptides (Figures 3G
and S2B) and scored for the accumulation of LC3 at the plasma
membrane and in perinuclear autophagosomes (Figures 3H
and 3I). Both phenomena depended on M2. However, plasma
membrane relocalization of LC3 occurred efficiently only in cells
infected with viruses encoding functional M2 LIR motifs (WT or
M2 S93N), but not in those infected with viruses encoding LIR
motifs unable to bind LC3 (M2 F91S and I94S) (Figure 3H). This phenomenon was not due to a delay in replication of
mutant viruses (Figure S2C). In contrast, the proportion of cells
accumulating perinuclear autophagosomes was not signifi-
cantly different between WT and LIR mutants (Figure 3I). LC3
unable to become lipidated (LC3 G120A) did not redistribute
to either the perinuclear region or plasma membrane in IAV-
infected cells (Figures 3H and 3I). Thus, while IAV M2 causes
both the accumulation of autophagosomes and the relocaliza-
tion of LC3 to the plasma membrane, these functions are sepa-
B
core autophagy prot
and Komatsu, 2011
decreased the numb
However, upon infec
for the accumulatio
translocation of LC3
gest that influenza st
in a manner distinct
Taken together, o
lates the autophagy
the accumulation of
shown (Gannage´ et
of LC3 to the plas
in an ATG16L1-depe
contrast, the reloca
but not its perinuclea
interaction of LC3 w rable, since the M2 LIR motif is required
for the relocalization of LC3 to the
plasma membrane, but not its perinu-
clear accumulation. B B B Further
evidence
for
M2-mediated
but
LIR-independent
manipulation
of
the autophagy machinery came from
the analysis of LC3 lipidation in infected
cells (Figure 3J; also evident in Figure 3F). LC3
undergoes
ATG5/ATG12/ATG16-
dependent lipidation with phosphatidyl
ethanolamine, which provides a mem-
brane anchor to LC3 and is a critical
step in autophagosome biogenesis (Ichi-
mura et al., 2000; Kabeya et al., 2000). Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability Lipidated LC3 (LC3-II) migrates faster
in SDS-PAGE. Cells infected with WT
IAV or the control M2 mutant S93N con-
tained more LC3-II than mock-infected
cells, consistent with the virus triggering
autophagy (Figure 3J). Enhanced LC3-II
levels were dependent on M2 but only
partially dependent on the functionality
of its LIR motif, as infection with M2
F91S produced intermediate amounts
of LC3-II. To understand how M2 subverts auto-
phagy, cells expressing GFP-LC3 were
depleted of FIP200, a component of
the ULK kinase complex, or ATG16L1, a
core autophagy protein required for LC3 conjugation (Mizushima
and Komatsu, 2011) (Figure S3). Depletion of either protein
decreased the number of starvation-induced autophagosomes. However, upon infection with IAV, only ATG16L1 was required
for the accumulation of perinuclear autophagosomes and the
translocation of LC3 to the plasma membrane. These data sug-
gest that influenza stimulates autophagy downstream of FIP200,
in a manner distinct from starvation. with IAV encoding WT or mutant M2 polypeptides (Figures 3G
and S2B) and scored for the accumulation of LC3 at the plasma
membrane and in perinuclear autophagosomes (Figures 3H
and 3I). Both phenomena depended on M2. However, plasma
membrane relocalization of LC3 occurred efficiently only in cells
infected with viruses encoding functional M2 LIR motifs (WT or
M2 S93N), but not in those infected with viruses encoding LIR
motifs unable to bind LC3 (M2 F91S and I94S) (Figure 3H). This phenomenon was not due to a delay in replication of
mutant viruses (Figure S2C). In contrast, the proportion of cells
accumulating perinuclear autophagosomes was not signifi-
cantly different between WT and LIR mutants (Figure 3I). LC3
unable to become lipidated (LC3 G120A) did not redistribute
to either the perinuclear region or plasma membrane in IAV-
infected cells (Figures 3H and 3I). Thus, while IAV M2 causes
both the accumulation of autophagosomes and the relocaliza-
tion of LC3 to the plasma membrane, these functions are sepa- Taken together, our data demonstrate that IAV M2 manipu-
lates the autophagy machinery in two separate ways: by causing
the accumulation of perinuclear autophagosomes, as previously
shown (Gannage´ et al., 2009), and by inducing the translocation
of LC3 to the plasma membrane. Both phenomena occur
in an ATG16L1-dependent and FIP200-independent manner. In
contrast, the relocalization of LC3 to the plasma membrane,
but not its perinuclear accumulation, requires the LIR-dependent
interaction of LC3 with M2. Figure
2. Relocalization
of
LC3
to
the
Plasma Membrane Requires Influenza M2
(A) HCT116 cells expressing GFP-LC3 were in-
fected with IAV, fixed at 16 hr p.i., and stained with
anti-M2. Scale bar, 10 mm.
(B) HCT116 cells expressing the indicated LC3
alleles fused to GFP were infected with WT or DM2
mutant IAV, fixed at 16 hr p.i., and stained with
anti-NP. Scale bar, 10 mm.
See also Figure S1. Plasma Membrane Targeting of LC3 Depends on the M2
LIR Motif See also Figure S1 g
) A549 cells expressing GFP-LC3 were infected with PR8 strain IAV at an moi of 0.2, fixed at 16 hr p.i., and stained with anti-N
) HCT116 cells expressing GFP-LC3 were infected as above and stained with WGA. Scale bar, 10 mm. ) HCT116 cells expressing the indicated LC3 alleles fused to GFP were infected with IAV, fixed at 16 hr p.i., and stained with go
ack arrows, autophagosomes or autophagolysosomes; green arrows, labeling of plasma membrane. Scale bars, 500 nm. Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 241 Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability 242
Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability A
B
C
D
G
E
F
H
I
J
3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization
cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. parison of M2 LIR motif with established LIRs. o derived from 2,685 unique IAV M2 sequences. A
B
C
D E E
F E D G
H
I
J
Figure 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization
(A) M2 cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. (B) Comparison of M2 LIR motif with established LIRs. (C) Logo derived from 2,685 unique IAV M2 sequences. (D) LUMIER binding assay. Binding of luciferase-tagged cytoplasmic regions of the indicated IAV M2 variants expressed in 293ET cells to beads coate
purified GST or GST-LC3. (E) Fluorescence anisotropy of hydroxycoumarin-labeled M2 C terminus (DADDGHFVSIELE) against purified LC3. (F) Lysates of HCT116 cells expressing GFP-LC3 and infected with the indicated IAVs were immunoprecipitated with GFP-TRAP resin. Input and bound fra
were blotted with the indicated antibodies. Dotted line, irrelevant lanes removed. (G) HCT116 cells expressing GFP-LC3 infected with the indicated virus, fixed at 16 hr p.i., and stained with anti-HA. (H and I) Plasma membrane localization (H) or perinuclear accumulation (I) of GFP-LC3 in HCT116 cells expressing the indicated LC3 alleles and infected w
indicated viruses. Coverslips were assessed blindly in triplicate at 16 hr p.i. Mean and SD. **p < 0.01 by ANOVA. (J) Lysates of HCT116 cells expressing GFP-LC3 were blotted for indicated antigens at 16 hr p.i. with indicated IAV. See also Figures S2 and S3. G
H
I
J
e 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization H H G I Figure 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization
(A) M2 cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. (B) Comparison of M2 LIR motif with established LIRs. (C) Logo derived from 2,685 unique IAV M2 sequences. (D) LUMIER binding assay. Figure 4. M2 LIR Motif Required for Fila-
mentous Budding and Virus Stability Consis
a role for autophagy in filamentous budding, both dep
ATG16L1 and LIR motif mutation reduced the formatio
ment bundles (Figure S4). We conclude that the M2 L
is required for normal filamentous budding of IAV. 244
Cell Host & Microbe 15 239 247 February 12 2014 ª2014 Elsevier Inc (B) MDCK cells were infected with the indicated
PR8 MUd viruses, fixed at 10 hr p.i., and stained
without permeabilization with antiserum against
PR8 virus. (Top) Maximum intensity z stack pro-
jection of confocal images. (Bottom) Side-on view
of z stacks. Scale bar, 10 mm. (C) A549 cells infected with WT or mutant PR8 or
PR8 MUd viruses at an moi of 1. Cell-free super-
natants (16 hr p.i.) were harvested and frozen either
immediately or after having been left at room
temperature for 1 or 2 days. Experiments were
performed in parallel. Titers were determined in
triplicate after thawing by plaque assay; mean
and standard deviation are shown. **p < 0.01 by
ANOVA. S93N I94S See also Figure S4. in both a spherical and a filamentous
fashion, depending on the genetic back-
ground of the virus and the host-cell
type
(Bourmakina
and
Garcı´a-Sastre,
2003; Elleman and Barclay, 2004; Roberts
and Compans, 1998). Most human-path-
ogenic isolates of IAV form up to 10 mm
long filamentous particles that require
extensive membrane resources. Since
M2 influences filament formation (Iwat-
suki-Horimoto et al., 2006; Roberts and
Compans, 1998; Rossman et al., 2010),
we hypothesized that accumulation of
LC3 at the plasma membrane might
represent the virus mobilizing lipid re-
sources for budding. Accordingly, we
introduced the M2 LIR mutations into
a filamentous strain of IAV, PR8-MUd
(Noton et al., 2007). PR8-MUd produces
a profusion of micrometer-length fila-
ments arrayed in parallel bundles. While
the M2 S93N LIR variant resembled WT
virus, cells infected with the M2 F91S or
I94S virus encoding nonfunctional LIRs
produced fewer filaments with a lower
tendency to form bundles (Figure 4A). Confocal microscopy, which biases to-
ward detection of long filament bundles
(Simpson-Holley et al., 2002), revealed a
large difference in the propensity of cells
infected with WT or F91S PR8 MUd to
project filament bundles away from the
cell surface (Figure 4B). Consistent with
in filamentous budding, both depletion of
otif mutation reduced the formation of fila-
e S4). We conclude that the M2 LIR motif
filamentous budding of IAV. Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability Binding of luciferase-tagged cytoplasmic regions of the indicated IAV M2 variants expressed in 293ET cells to beads coated with
purified GST or GST-LC3. (E) Fluorescence anisotropy of hydroxycoumarin-labeled M2 C terminus (DADDGHFVSIELE) against purified LC3. (F) Lysates of HCT116 cells expressing GFP-LC3 and infected with the indicated IAVs were immunoprecipitated with GFP-TRAP resin. Input and bound fractions
were blotted with the indicated antibodies. Dotted line, irrelevant lanes removed. (G) HCT116 cells expressing GFP-LC3 infected with the indicated virus, fixed at 16 hr p.i., and stained with anti-HA. (H and I) Plasma membrane localization (H) or perinuclear accumulation (I) of GFP-LC3 in HCT116 cells expressing the indicated LC3 alleles and infected with the
indicated viruses. Coverslips were assessed blindly in triplicate at 16 hr p.i. Mean and SD. **p < 0.01 by ANOVA. (J) Lysates of HCT116 cells expressing GFP-LC3 were blotted for indicated antigens at 16 hr p.i. with indicated IAV. See also Fig res S2 and S3 Figure 3. A LIR Motif in Influenza M2 Is Required for LC3 Membrane Localization
(A) M2 cytoplasmic tail (from amino acid 65 to C terminus) with predicted a helix and b strand. FVSI motif is underlined. (B) Comparison of M2 LIR motif with established LIRs. (C) Logo derived from 2,685 unique IAV M2 sequences. (D) LUMIER binding assay. Binding of luciferase-tagged cytoplasmic regions of the indicated IAV M2 variants expressed in 293ET cells to beads coated with
purified GST or GST-LC3. (E) Fluorescence anisotropy of hydroxycoumarin-labeled M2 C terminus (DADDGHFVSIELE) against purified LC3. (F) Lysates of HCT116 cells expressing GFP-LC3 and infected with the indicated IAVs were immunoprecipitated with GFP-TRAP resin. Input and bound fractions
were blotted with the indicated antibodies. Dotted line, irrelevant lanes removed. (G) HCT116 cells expressing GFP-LC3 infected with the indicated virus, fixed at 16 hr p.i., and stained with anti-HA. (H and I) Plasma membrane localization (H) or perinuclear accumulation (I) of GFP-LC3 in HCT116 cells expressing the indicated LC3 alleles and infected with the
indicated viruses. Coverslips were assessed blindly in triplicate at 16 hr p.i. Mean and SD. **p < 0.01 by ANOVA. (J) Lysates of HCT116 cells expressing GFP-LC3 were blotted for indicated antigens at 16 hr p.i. with indicated IAV. See also Figures S2 and S3 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 243 Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 243 Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability Formation of Filamentous Virions Depends on the M2 LIR
The strong conservation of the M2 LIR motif and its requirement
for the accumulation of LC3 at the plasma membrane suggest
that LC3 relocalization benefits the viral life cycle. IAV can bud
A
B
0
1
2
5
6
7
days
0
1
2
4
5
6
days
IAV
S93N
I94S **
F91S **
log10 PFU/ml
log10 PFU/ml
IAV
S93N
I94S **
F91S **
F91S
IAV
IAV
I94S
S93N
F91S
C
PR8
PR8 MUd
a role for autophag
ATG16L1 and LIR m
ment bundles (Figu
is required for norm Figure 4. M2 LIR Motif Required for Fila-
mentous Budding and Virus Stability Figure 4. M2 LIR Motif Required for Fila-
mentous Budding and Virus Stability
(A) MDCK cells were infected with the indicated
PR8 MUd viruses at an moi of 5, fixed at 16 hr p.i.,
and visualized by scanning electron microscopy. Scale bar 10 mm Figure 4. M2 LIR Motif Required for Fila-
mentous Budding and Virus Stability A A
IAV
S93N I94S
F91S F91S IAV (A) MDCK cells were infected with the indicated
PR8 MUd viruses at an moi of 5, fixed at 16 hr p.i.,
and visualized by scanning electron microscopy. Scale bar, 10 mm. Formation of Filamentous Virions Depends on the M2 LIR
The strong conservation of the M2 LIR motif and its requirement
for the accumulation of LC3 at the plasma membrane suggest
that LC3 relocalization benefits the viral life cycle. IAV can bud
B
0
1
2
5
6
7
days
0
1
2
4
5
6
days
IAV
S93N
I94S **
F91S **
log10 PFU/ml
log10 PFU/ml
IAV
S93N
I94S **
F91S **
F91S
IAV
I94S
S93N
C
PR8
PR8 MUd
Scale bar, 10 mm. (B) MDCK cells were infected with the
PR8 MUd viruses, fixed at 10 hr p.i., a
without permeabilization with antiseru
PR8 virus. (Top) Maximum intensity z
jection of confocal images. (Bottom) Sid
of z stacks. Scale bar, 10 mm. (C) A549 cells infected with WT or mut
PR8 MUd viruses at an moi of 1. Cell-f
natants (16 hr p.i.) were harvested and fr
immediately or after having been lef
temperature for 1 or 2 days. Experim
performed in parallel. Titers were det
triplicate after thawing by plaque as
and standard deviation are shown. **p
ANOVA. See also Figure S4. in both a spherical and a fila
fashion, depending on the gene
ground of the virus and the
type
(Bourmakina
and
Garcı´a
2003; Elleman and Barclay, 2004
and Compans, 1998). Most hum
ogenic isolates of IAV form up t
long filamentous particles that
extensive membrane resource
M2 influences filament formatio
suki-Horimoto et al., 2006; Rob
Compans, 1998; Rossman et a
we hypothesized that accumu
LC3 at the plasma membran
represent the virus mobilizing
sources for budding. Accordin
introduced the M2 LIR mutati
a filamentous strain of IAV, P
(Noton et al., 2007). PR8-MUd p
a profusion of micrometer-len
ments arrayed in parallel bundle
the M2 S93N LIR variant resem
virus, cells infected with the M2
I94S virus encoding nonfunctio
produced fewer filaments with
tendency to form bundles (Fig
Confocal microscopy, which b
ward detection of long filament
(Simpson-Holley et al., 2002), re
large difference in the propensit
infected with WT or F91S PR8
project filament bundles away
cell surface (Figure 4B). DISCUSSION We describe a functional, highly conserved LC3-binding/LIR
motif in the C terminus of IAV M2. This LIR is required for the
generation of stable virions, which correlates with the M2 LIR-
dependent recruitment of the autophagosomal marker LC3 to
the plasma membrane of infected cells. In contrast, the M2
LIR is not required for the induction of autophagy per se, since
viruses bearing a mutant M2 LIR enhanced the proportion of
lipidated LC3 in infected cells similar to WT IAV. In contrast,
viruses lacking M2 scarcely stimulated LC3-II accumulation. We therefore propose a model whereby M2 subverts autophagy
by two different means: (1) via a LIR-independent mechanism
that, in accordance with a previous report (Gannage´ et al.,
2009), is not dependent on the M2 cytosolic domain containing
the LIR motif, and (2) via the LIR motif identified here, which
redirects LC3 to the plasma membrane. The two activities
may not be independent, since M2 may stimulate autophagy
via beclin-1 (Gannage´ et al., 2010) and utilize its LIR motif to
transport lipids to the plasma membrane. This mobilization of
resources via the autophagy machinery may be important to
prevent the depletion of the plasma membrane due to budding
virus particles. Transmission of infection between individuals and species
depends on survival of virions outside the host. The relative
contribution of respiratory droplets, fomite, and aerosol trans-
mission for IAV is still under debate, but the fomite route in
particular is a time-demanding process (Brankston et al.,
2007; Hall, 2013). Significantly, IAV with mutations in its LIR is
less stable. We have demonstrated that filament formation is
affected by LIR mutations. Ultrastructural studies of viruses
lacking the M2 C terminus (and thus the LIR motif) showed
that even spherical virions had decreased structural integrity
(Sugita et al., 2011). We conclude that the LIR motif is involved
in budding and is required for morphologically normal and sta-
ble viral progeny. Whereas decreased stability is detrimental to the spread of the
virus, it may have a technological use. There are significant con-
cerns surrounding the manipulation of highly pathogenic strains
of IAV, especially in potential gain-of-function experiments (Lan-
glois et al., 2013; Patterson et al., 2013). LIR mutations could
potentially provide enhanced biosafety by ensuring decreased
environmental stability. Motif To test whether the M2 LIR influences virus replication, we
examined growth of the PR8 and PR8 MUd viruses. As ex-
pected (Simpson-Holley et al., 2002), the nonfilamentous PR8
virus replicated to higher titers than the filamentous PR8 MUd
strain (Figure 4C). Within the two backgrounds, viruses encod-
ing functional and nonfunctional M2 LIR motifs replicated indis-
tinguishably, suggesting the M2 LIR does not directly affect the
production of infectious particles (Figures 4C and S2C). How-
ever, when viral supernatants were left at room temperature
for 24 or 48 hr, viruses encoding WT M2 or M2 S93N remained
stable, while viruses encoding nonfunctional LIRs (M2 I94S or
F91S) lost infectivity. Filamentous virus was especially sensitive
to LIR mutations; PR8 MUd encoding nonbinding M2 LIR
mutants suffered a 10-fold drop in titer after just 24 hr at room
temperature. M2 is largely excluded from mature virus particles (Rossman
and Lamb, 2011), and immunogold staining for LC3 was
observed only rarely in virions. This is consistent with the LIR
motif predominantly restoring the depleted plasma membrane
of host cells as virus particles bud, rather than contributing
directly to the viral membrane. Cells produce up to 104 IAV par-
ticles (Mo¨ hler et al., 2005). Even if only 1,000 spherical virus par-
ticles were produced, their surface corresponds to a substantial
fraction (20%) of the apical plasma membrane. The far greater
amount of membrane required to produce a filamentous virion
suggests that, as seen here, LIR function is of particular impor-
tance in supporting filament formation. Given that many other
RNA viruses (especially respiratory pathogens) produce filamen-
tous particles (Ba¨ chi and Howe, 1973; von Magnus, 1953; Yao
and Compans, 2000), it may be that other enveloped viruses
have also evolved mechanisms to mobilize lipid resources from
host cells via autophagy. We therefore conclude that the LIR motif in M2 is of functional
importance to IAV, as indicated by its high degree of conserva-
tion and its essential role in producing stable virions, a prerequi-
site for the transmissibility of infection. Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Figure 4. M2 LIR Motif Required for Fila-
mentous Budding and Virus Stability B
IAV B B F91S F91S 0
1
2
5
6
7
days
0
1
2
4
5
6
days
IAV
S93N
I94S **
F91S **
log10 PFU/ml
log10 PFU/ml
IAV
S93N
I94S **
F91S **
C
PR8
PR8 MUd C 5
log10 PFU/ml Formation of Filamentous Virions Depends on the M2 LIR The strong conservation of the M2 LIR motif and its requirement
for the accumulation of LC3 at the plasma membrane suggest
that LC3 relocalization benefits the viral life cycle. IAV can bud 244
Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. 244
Cell Host & Microbe 15, 239–247, February 12, 2014 ª2014 Elsevier Inc. Cell Host & Microbe
M2 LIR Motif Required for Influenza A Stability Stability of Influenza A Virions Depends on the M2 LIR
Motif Stability of Influenza A Virions Depends on the M2 LIR
Motif Stability of Influenza A Virions Depends on the M2 LIR
Motif the plasma membrane. The only other known physiological
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plasma membrane results from fusion of vesicles. If true, this
mechanism suggests that the primary biological purpose of
the M2 LIR motif is to deliver appropriate membrane resources
for the generation of virus particles. On this account, the M2 pro-
tein would ensure both the availability of new transport vesicles
by manipulating autophagy downstream of FIP200, and the cor-
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Genetic diversity and population structure analysis based on the high density SNP markers in Ethiopian durum wheat (Triticum turgidum ssp. durum)
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Alemu et al. BMC Genetics (2020) 21:18
https://doi.org/10.1186/s12863-020-0825-x Alemu et al. BMC Genetics (2020) 21:18
https://doi.org/10.1186/s12863-020-0825-x Open Access © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Genetic diversity and population structure
analysis based on the high density SNP
markers in Ethiopian durum wheat (Triticum
turgidum ssp. durum) Admas Alemu1,2*
, Tileye Feyissa1, Tesfaye Letta3 and Bekele Abeyo4 Abstract Background: Ethiopia has been considered as a center of diversity and the second possible center of
domestication of durum wheat. Genetic diversity and population structure analysis in the existing Ethiopian durum
wheat germplasm have enormous importance in enhancing breeding effort and for sustainable conservation. Hence, 192 Ethiopian durum wheat accessions comprising 167 landraces collected from major wheat-growing areas
of the country and 25 improved varieties released from Debre Zeit and Sinana Agricultural Research Centers,
Ethiopia in different years (1994–2010) were assembled for the current study. Results: The panel was genotyped with a High-density 90 K wheat SNP array by Illumina and generated 15,338
polymorphic SNPs that were used to analyze the genetic diversity and to estimate the population structure. Varied
values of genetic diversity indices were scored across chromosomes and genomes. Genome-wide mean values of
Nei’s gene diversity (0.246) and polymorphism information content (0.203) were recorded signifying the presence of
high genetic diversity within this collection. Minor allele frequency of the genome varied with a range of 0.005 to
0.5 scoring a mean value of 0.175. Improved varieties clustered separately to landraces in population structure
analysis resulted from STRUCTURE, PCA and neighbor joining tree. Landraces clustering was irrespective of their
geographical origin signifying the presence of higher admixture that could arise due to the existence of historical
exchanges of seeds through informal seed system involving regional and countrywide farming communities in
Ethiopia. Conclusions: Sustainable utilization and conservation of this rich Ethiopian durum wheat genetic resource is an
irreplaceable means to cope up from the recurrent climate changes and biotic stresses happening widely and
thereby able to keep meeting the demand of durum productivity for the ever-growing human population. Keywords: Ethiopian durum wheat, Genetic diversity, Population structure, SNPs, Landraces, Improved varieties Background for about 95% of world wheat production and durum
covers the other 5% [1]. Modern wheat cultivars generally refer to two species:
hexaploid bread wheat, Triticum aestivum (2n = 6x = 42,
AABBDD), and tetraploid/hard or durum wheat, T. durum (2n = 4x = 28, AABB) used for pasta, macaroni,
couscous and low-rising bread, and the former accounts In Ethiopia, wheat is the second most widely produced
cereal crop after corn and the third most important
staple food behind corn and sorghum [2]. Hard red
wheat accounts for about 75–80% of the national pro-
duction, while durum makes up roughly 10–15% [2]. Wheat has versatile uses in making various foods and
drinks, such as bread, ‘Kolo’ (traditional Ethiopian snack
made from wheat mixed with barley, chickpea and other
legumes and roasted in a clay griddle), ‘Tella’ (traditional * Correspondence: adth14@gmail.com
1Department of Microbial, Cellular and Molecular Biology, Addis Ababa
University, P.O.Box 1176, Addis Ababa, Ethiopia
2Department of Biology, Debre Tabor University, Debre Tabor, Ethiopia
Full list of author information is available at the end of the article * Correspondence: adth14@gmail.com
1Department of Microbial, Cellular and Molecular Biology, Addis Ababa
University, P.O.Box 1176, Addis Ababa, Ethiopia
2Department of Biology, Debre Tabor University, Debre Tabor, Ethiopia
Full list of author information is available at the end of the article Page 2 of 12 Page 2 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 maintained more than 60,000 accessions of different
crops in its gene bank and of these, 7000 are durum
wheat accessions accounting 12% from the total [7, 8]. Besides, up to recent time, agricultural research centers
and institutions have been involved in collecting and
conserving Ethiopian durum wheat landrace accessions
in the country. Due to its uniqueness, Ethiopian durum
wheat has been served as a center of focus for genetic
studies and the source of novel alleles [9–14]. Vavilov
[4] and Zohary [15] reported the presence of high genetic
diversity in Ethiopian durum wheat and recent studies
specified uniqueness of Ethiopian durum landraces form
the Fertile Crescent collections (primary center of domes-
tication) and considered as the possible second center of
domestication for the crop [3]. Durum wheat is long
established in the country and it was likely introduced into
the northern highlands of Ethiopia around 3000 BC [16]. Previous studies indicated the existence of high genetic
variation of cultivated durum wheat in Ethiopia that arises Ethiopian beer), pasta, macaroni, biscuit, cake, and
others. Additionally, wheat straw is commonly used as a
roof thatching material and as animal feed in most
wheat-growing rural areas of Ethiopia. Hence, increasing
wheat production has been a national goal to decrease
the gap between production and human consumption
especially in view of the fastest-growing population as
compared to production. Durum wheat is the result of two successful domesti-
cation events by ancient farmers: first, from wild emmer
(Triticum turgidum ssp. dicoccoides) to domesticated
emmer (T. turgidum ssp. dicoccum) with the loss of fra-
gility of spikes (disarticulation into spikelets), and sec-
ond,
from
cultivated
emmer
to
durum
with
the
appearance of naked kernels or free threshing kernel [3]. Ethiopia is one of the few countries that has been served
as the center of primary gene pool for various crops [4–
6]. Ethiopian biodiversity institute (EBI), formerly known
as Institute
of biodiversity
conservation
(IBC), has Fig. 1 Distribution of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes (B) in 192 Ethiopian
durum wheat accessions Fig. 1 Distribution of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes (B) in 192 Ethiopian of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes Fig. 1 Distribution of SNPs generated from the 90 K Illumina iSelect SNPs array across chromosomes (A) and genomes (B) in 192 Ethiopian
durum wheat accessions Page 3 of 12 Page 3 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Single
nucleotide polymorphisms
(SNPs) are the
most abundant class of DNA markers. Lower rates of
recurrent mutation make them evolutionarily stable. They are excellent markers for studying complex gen-
etic traits and for understanding the genomic evolu-
tion. They have been widely used in genome-wide
association studies, genetic resource characterization,
marker-assisted breeding and genomic selection [26]. Hybridization arrays/microarrays have been used as a
preeminent solution
to
develop SNPs
in
complex
polyploid genomes such as wheat [27]. Once a com-
prehensive SNP data set is available for a species, a
well-designed microarray can be produced; and gener-
ally, the technology is then cost-efficient and the
process
is
relatively
convenient. The
technology
avoids the risk of miscalling diversity on homoeolo-
gous genomes and its power recently increased 100-
fold in wheat moving from 9 K [28] to 820 K [29]
genome-wide SNPs. The 90 K wheat SNP array [27] due to the wide range of agro-ecological conditions
coupled with diverse farmers’ culture [11, 12, 17–22]. Ethiopian farmers have grown durum wheat since imme-
morial time, mostly under adverse environmental condi-
tions and they developed a broad gene pool of durum
wheat landraces adapted to various environmental condi-
tions [23]. In Ethiopia, durum wheat is commonly planted
on heavy black clay soils (vertisols) of the highlands be-
tween 1800 and 2800 masl [23]. Mechanisms of detecting and analyzing genetic diver-
sity have gradually progressed from Mendelian survey of
discrete morphological traits to molecular examinations
of DNA variation [24]. Genetic diversity analysis is a
critical component of plant genetics, breeding, conserva-
tion and evolution [25]. Understanding the existing gen-
etic divergence and distribution of crop species has
paramount importance for conservation and selection of
parents with diverse genetic backgrounds, thereby ren-
dering crop improvement more efficient [22]. Fig. 2 Frequency distribution of Nei’s gene diversity (A), polymorphic information content (PIC) (B) and minor allelic frequency (MAF) (C) of
15,338 polymorphic SNPs generated from Ethiopian durum wheat accessions Fig. 2 Frequency distribution of Nei’s gene diversity (A), polymorphic information content (PIC) (B) and minor allelic frequency (MAF) (C) of
15,338 polymorphic SNPs generated from Ethiopian durum wheat accessions Fig. 2 Frequency distribution of Nei’s gene diversity (A), polymorphic information content (PIC) (B) and minor allelic frequency (MAF) (C) of
15,338 polymorphic SNPs generated from Ethiopian durum wheat accessions Page 4 of 12 Page 4 of 12 Page 4 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Results has been successfully used for genetic diversity ana-
lysis, genome-wide association mapping and construc-
tion of high-density consensus maps in both bread
and durum wheat [12, 30–32]. SNP markers distribution Chromo-
some 1A scored the highest PIC (0.229) and gene di-
versity (0.282) (Table 1). In contrast, the lowest PIC
and genetic diversity score was observed on chromo-
some 7A (PIC = 0.181; gene diversity = 0.217). Chro-
mosomes 2A, 2B, 3A, 3B, 7A and 7B showed slightly
lower polymorphic information content than the aver-
age PIC values of the whole genome. On the other
hand, homoeologous chromosome groups 1, 4, and 5
scored higher Nei’s genetic diversity than the average
genome-wide value. The highest gene diversity, PIC
and MAF were on homoeologous chromosome group
five. Comparable mean values of genetic diversity,
PIC and MAF were scored on A and B genomes. SNPs of which 84.43% was polymorphic. Higher number
polymorphic SNP markers were recorded on B genome
(9013 SNPs) than the A genome (6325 SNPs) in Ethiop-
ian durum wheat accessions (Fig. 1-b). genome was 0.175 ranged from 0.005 to 0.5. Chromo-
some 1A scored the highest PIC (0.229) and gene di-
versity (0.282) (Table 1). In contrast, the lowest PIC
and genetic diversity score was observed on chromo-
some 7A (PIC = 0.181; gene diversity = 0.217). Chro-
mosomes 2A, 2B, 3A, 3B, 7A and 7B showed slightly
lower polymorphic information content than the aver-
age PIC values of the whole genome. On the other
hand, homoeologous chromosome groups 1, 4, and 5
scored higher Nei’s genetic diversity than the average
genome-wide value. The highest gene diversity, PIC
and MAF were on homoeologous chromosome group
five. Comparable mean values of genetic diversity,
PIC and MAF were scored on A and B genomes. SNP markers distribution From 81,587 SNP probes available on the chip, 30,510
SNP calls (23,354 polymorphic SNPs) were reproducible
in the current Ethiopian durum wheat panel. From these
markers, 18,788 SNPs had a known position but only 15,
338 (81.63%) were polymorphic and used for the current
study (Additional file 2: Table S2). The smallest number
of SNP markers were recorded on chromosome 1A (263
SNPs) while the highest on chromosome 2B (2253 SNPs)
(Fig. 1-a). Chromosome 2B also contributed the highest
number of polymorphic SNP markers (1755 SNPs) while
the smallest on chromosome 1A (236 SNPs). Consider-
ing the distribution of SNPs across homoeologous chro-
mosomes, group two scored the highest number of SNP
markers (3639 SNPs of which 78.38% is polymorphic)
while the smallest number on group one with 1709 Molecular characterization of Ethiopian durum wheat
accessions has been investigated in DNA markers with a
very limited number, such as microsatellites [11, 20, 21]. However, except in a single attempt that has made to
characterize Ethiopian durum wheat landraces collected
by EBI siding with Mediterranean durum wheat [12], the
germplasm has not been extensively investigated with a
high density SNP markers. Hence, the present study
aimed to assess the genetic diversity and population
structure of 167 landraces and 25 improved varieties col-
lected and maintained at Debre Zeit and Sinana Agricul-
tural Research Centers, Ethiopia with a 90 K wheat SNP
array. Table 1 Mean values of diversity indices and minor allelic frequency distribution of SNP markers across chromosomes in 192
Ethiopian durum wheat accessions
Chromosome
Nei’s Gene diversity
Polymorphic information content (PIC)
Minor allelic frequency (MAF)
A-Genome
1A
0.282
0.229
0.208
2A
0.221
0.185
0.150
3A
0.246
0.202
0.175
4A
0.248
0.204
0.176
5A
0.272
0.221
0.200
6A
0.265
0.215
0.194
7A
0.217
0.181
0.150
B-Genome
1B
0.254
0.208
0.185
2B
0.229
0.190
0.158
3B
0.233
0.194
0.159
4B
0.279
0.226
0.209
5B
0.265
0.217
0.191
6B
0.246
0.203
0.172
7B
0.235
0.193
0.164
Homoeologous
1
0.259
0.211
0.188
2
0.226
0.188
0.155
3
0.238
0.197
0.166
4
0.264
0.215
0.193
5
0.268
0.218
0.194
6
0.255
0.209
0.182
7
0.226
0.187
0.157
A-Genome
0.2448
0.2015
0.1744
B-Genome
0.2471
0.2035
0.1750
Whole Genome
0.2462
0.2027
0.1748 Page 5 of 12 Page 5 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 genome was 0.175 ranged from 0.005 to 0.5. Genetic diversity analysis The SNP markers exhibited a wide range of poly-
morphic information content (PIC) and Nei’s gene di-
versity
across
chromosomes
and
genomes
of
Ethiopian
durum
wheat. Frequency
distribution
of
SNPs
for
gene
diversity,
polymorphic
information
content, and frequency of the minor allele values of
the genome is presented in Fig. 2-a, Fig. 2-b, and Fig. 2-c, respectively. While a detail of the frequency dis-
tribution of SNP markers across chromosomes is pre-
sented for values of gene diversity (Additional file 4:
Figure S2), PIC (Additional file 5: Figure S3) and
minor allelic frequency (Additional file 6: Figure S4). The overall mean value of polymorphic information
content was 0.203 ranged from 0.01 to 0.375. Nei’s
gene diversity score was varied from 0.01 to 0.5 with
a mean value of 0.246 and the mean MAF of the Genetic stratification and principal component analysis Hence, accessions were grouped into
three clusters based on the STRUCTURE-inferred clus-
tering result with 75, 27 and 90 accessions came to-
gether for sub-populations 1, 2 and 3, respectively
(Additional file 1: Table S1). Landraces gathered on
cluster-one and cluster-three while all improved var-
ieties, except one variety (Selam) that was under cluster
one, assembled on sub-population two. The neighbor-
joining based clustering analysis (Fig. 4) also identified
three clear clusters and except one accession all are
grouped based on the STRUCTURE based stratification. 24.29, 6.61 and 3.74% of the total variance, respectively. The smaller numbers of variance explained by the sec-
ond and consecutive PCs indicated that only few PCs
couldn’t encapsulate the existing genetic variance in
Ethiopian durum wheat. The first PC (PC1) distantly
clustered varieties from landraces and the second PC
grouped the two landrace subgroups (Fig. 5-a). The first
two PCs (PC1 and PC2) clearly clustered the three sub-
populations. However, clustering gets distorted when
additional principal components were considered (Fig. 5-b). Genetic stratification and principal component analysis Genetic stratification and principal component analysis
The optimal sub-population of accessions was inferred
through two approaches: The first method was the
STRUCTURE-based clustering approach that was in-
ferred based on the second order rate of change of the
likelihood (ΔK) (Table 3). The result indicated a clear
peak at K = 3 signifying the optimal sub-populations in Fig. 3 Inference of the optimal numbers of sub-populations (clusters) in Ethiopian durum wheat panel with the Bayesian clustering model in
STRUCTURE (A) and by the discriminant analysis of principal components (DAPC) using adegenet package (B) Fig. 3 Inference of the optimal numbers of sub-populations (clusters) in Ethiopian durum wheat panel with the Bayesian clustering model in
STRUCTURE (A) and by the discriminant analysis of principal components (DAPC) using adegenet package (B) Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 Page 6 of 12 the panel (Fig. 3-a). The second approach was based on
the
discriminant
analysis
of
principal
components
(DAPC) and the result couldn’t show a clear lowest
Bayesian information criterion (BIC) on a specific K
value above which BIC values decreased spontaneously
with simultaneous increment making an elbow at the
optimal K value (Fig. 3-b). However, in this case, it pro-
vided a clue in which somehow less than five clusters
could be optimal. Hence, accessions were grouped into
three clusters based on the STRUCTURE-inferred clus-
tering result with 75, 27 and 90 accessions came to-
gether for sub-populations 1, 2 and 3, respectively
(Additional file 1: Table S1). Landraces gathered on
cluster-one and cluster-three while all improved var-
ieties, except one variety (Selam) that was under cluster
one, assembled on sub-population two. The neighbor-
joining based clustering analysis (Fig. 4) also identified
three clear clusters and except one accession all are
grouped based on the STRUCTURE based stratification. the panel (Fig. 3-a). The second approach was based on
the
discriminant
analysis
of
principal
components
(DAPC) and the result couldn’t show a clear lowest
Bayesian information criterion (BIC) on a specific K
value above which BIC values decreased spontaneously
with simultaneous increment making an elbow at the
optimal K value (Fig. 3-b). However, in this case, it pro-
vided a clue in which somehow less than five clusters
could be optimal. Genetic divergence between landraces and improved
varieties The Bayesian model based stratification and PCA
clustering methods grouped landraces and varieties in
distinct places except in a single variety, Selam, which
was clustered alongside landraces. The numbers of
polymorphic SNPs were slightly higher in varieties
than landraces. Varieties scored higher gene diversity
(0.297), PIC (0.240) and the mean frequency of minor Principal component analysis (PCA) was analyzed with
all Polymorphic SNPs generated from the panel. The
first, second and third principal components explained Fig. 4 Neighbor-joining tree generated based on simple matching dissimilarity coefficients using SNP markers from 192 Ethiopian durum wheat
accessions. Colors of accessions are clusters inferred from STRUCTURE-based analysis Fig. 4 Neighbor-joining tree generated based on simple matching dissimilarity coefficients using SNP markers from 192 Ethiopian durum wheat
accessions. Colors of accessions are clusters inferred from STRUCTURE-based analysis Page 7 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics Fig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A)
Varieties cluster (green, K-2) showing a clear separation from the other two landrace clusters (K1 & K2) using PC1 and PC2. (B) Clustering got
disrupted when considering other PCs Fig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A)
Varieties cluster (green, K-2) showing a clear separation from the other two landrace clusters (K1 & K2) using PC1 and PC2. (B) Clustering got
disrupted when considering other PCs ig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A) Fig. 5 Principal component analysis (PCA) using 15,338 polymorphic SNP markers generated from 192 Ethiopian durum wheat accessions. (A)
Varieties cluster (green, K-2) showing a clear separation from the other two landrace clusters (K1 & K2) using PC1 and PC2. (B) Clustering got
disrupted when considering other PCs allele (0.218) than landraces (gene diversity = 0.213;
PIC = 0.173 & MAF = 0.154) (Table 2). Further analysis of molecular variance between the 25
varieties and 167 landrace accessions indicated higher
genetic variation between the two groups (61.02%) than
individuals within the group (38.98%) (Table 5). Genetic divergence between landraces and improved
varieties However, the number of landraces included in the
present study was higher by six folds than the number of
varieties and that could affect the reliability of the PIC,
genetic diversity and the minor allelic frequency scores
reported in the current study. Genetic clustering via geographic origin The current Ethiopian durum wheat germplasm com-
prises landrace accessions collected from major wheat-
producing areas of the country (Additional file 3: Figure
S1) including Bale, Gondar, Gojjam, Shewa, Tigray, and
Wollo, and 12 Ethiopian durum wheat landraces cur-
rently cultivated in the USA. Genetic variation among clusters Landraces collected from Bale (South-
eastern Ethiopia) grouped in both clusters (44 land-
races in cluster 1 and 24 landraces in cluster 3). The
two landraces collected from Tigray region (North
Ethiopia) were clustered in sub-population one. How-
ever, a landrace collected from Gondar, adjacent to
Tigray, was sub-grouped in cluster three. All twelve
Ethiopian landraces that are now cultivated in the
USA were gathered in sub-population three. Table 4 Analysis of molecular variance (AMOVA) for Ethiopian du
STRUCTURE clustering result
Source of variation
DF
Sum of squares
Variance c
Among populations
2
166,172.20
700.94 Va
Among individuals Within populations
189
240,563.25
636.41 Vb
Total
191
406,735.45
1337.35 Discussions into the different sub-groups irrespective to their geo-
graphic origin. For instance, eight landraces collected
from northeastern Ethiopia (Wollo) were grouped in
sub-population one while 25 landraces from the same
origin clustered in sub-population three (Additional
file 1: Table S1). Landraces collected from central
Ethiopia (Akaki and Shewa) clustered in both sub-
groups; 4 landraces in sub-population one while 19
landraces in sup-population three. However, from the
total eight landraces collected in Bichena (a town in
East Gojjam Zone, west-central Ethiopia), seven were
grouped in sub-population three and the other one
landrace altogether with four landraces collected from
other
parts
of
Gojjam
were
grouped
in
sub-
population one. Landraces collected from Bale (South-
eastern Ethiopia) grouped in both clusters (44 land-
races in cluster 1 and 24 landraces in cluster 3). The
two landraces collected from Tigray region (North
Ethiopia) were clustered in sub-population one. How-
ever, a landrace collected from Gondar, adjacent to
Tigray, was sub-grouped in cluster three. All twelve
Ethiopian landraces that are now cultivated in the
USA were gathered in sub-population three. into the different sub-groups irrespective to their geo-
graphic origin. For instance, eight landraces collected
from northeastern Ethiopia (Wollo) were grouped in
sub-population one while 25 landraces from the same
origin clustered in sub-population three (Additional
file 1: Table S1). Landraces collected from central
Ethiopia (Akaki and Shewa) clustered in both sub-
groups; 4 landraces in sub-population one while 19
landraces in sup-population three. However, from the
total eight landraces collected in Bichena (a town in
East Gojjam Zone, west-central Ethiopia), seven were
grouped in sub-population three and the other one
landrace altogether with four landraces collected from
other
parts
of
Gojjam
were
grouped
in
sub-
population one. Landraces collected from Bale (South-
eastern Ethiopia) grouped in both clusters (44 land-
races in cluster 1 and 24 landraces in cluster 3). The
two landraces collected from Tigray region (North
Ethiopia) were clustered in sub-population one. How-
ever, a landrace collected from Gondar, adjacent to
Tigray, was sub-grouped in cluster three. All twelve
Ethiopian landraces that are now cultivated in the
USA were gathered in sub-population three. Genetic variation among clusters The analysis of molecular variance (AMOVA) re-
vealed the presence of higher genetic variance be-
tween STRUCTURE-inferred sub-populations (52.41%)
than
among
individuals
within
clusters
(47.59%)
(Table 4). The clustering analysis indicated that the SNPs data
couldn’t group landraces clearly based on their geo-
graphical background and accessions were admixed Table 2 Mean of diversity indices and minor allelic frequency distribution of SNPs in landraces and varieties of Ethiopian durum wheat
Accession Type
Sample size
No. of polymorphic SNPs
Nei’s Gene diversity
Polymorphic information content (PIC)
Minor allelic frequency (MAF)
Landrace
167
13,466
0.213
0.173
0.154
Cultivar
25
13,725
0.297
0.240
0.218 Alemu et al. BMC Genetics (2020) 21:18 Page 8 of 12 Alemu et al. BMC Genetics Table 3 Inference of the optimal numbers of clusters existed in Ethiopian durum wheat panel using Delta K Statistics
K
L(K)
stdev
L’(K)
L”(K)
|L”(K)|
Delta K
1
−157,376
3.369
–
–
–
–
2
−138,930
1370.013
18,446.93
−3224.75
3224.75
2.35
3
−123,708
543.0884
15,222.19
−5458.76
5458.76
10.05
4
−113,944
1149.967
9763.425
−1419.46
1419.46
1.23
5
−105,600
708.175
8343.965
−3511.28
3511.28
4.96
6
−100,768
677.594
4832.685
−1326.03
1326.025
1.96
7
−97,261
1353.421
3506.66
580.6019
580.6019
0.43
8
−93,173
1398.676
4087.262
−1491.05
1491.054
1.07
9
−90,577
1708.376
2596.208
635.7119
635.7119
0.37
10
−87,345
2170.333
3231.92
Where;
K = Subpopulations;
Ln (PD) = The log likelihood for each K;
L (K) = An average of 20 values of Ln P(D);
stdev = Standard deviation for 20 values of Ln P(D);
L’(K) = L(K) n – L(K) n-1;
L”(K) = L′(K) n+ 1 −L′(K);
ΔK = |L”(K)|/Stdev;
The gray color of the third line designates the optimal sub-populations of the panel based on the highest delta K value into the different sub-groups irrespective to their geo-
graphic origin. For instance, eight landraces collected
from northeastern Ethiopia (Wollo) were grouped in
sub-population one while 25 landraces from the same
origin clustered in sub-population three (Additional
file 1: Table S1). Landraces collected from central
Ethiopia (Akaki and Shewa) clustered in both sub-
groups; 4 landraces in sub-population one while 19
landraces in sup-population three. However, from the
total eight landraces collected in Bichena (a town in
East Gojjam Zone, west-central Ethiopia), seven were
grouped in sub-population three and the other one
landrace altogether with four landraces collected from
other
parts
of
Gojjam
were
grouped
in
sub-
population one. Genetic diversity of Ethiopian durum wheat Genetic diversity of Ethiopian durum wheat y
p
Genetic diversity is imperative to provide a robust food
security system capable of adapting to recurrent biotic
and abiotic stresses. Genetic diversity analysis is a crucial
step in noticing alleles that could be used as the source
of novel traits with high yielding, resilient for biotic and/
or abiotic stresses and yet delivers satisfied productivity
or in meeting the end-user demands in plant breeding. Ethiopian durum wheat landraces have especially proven
to show a relevant variation for various traits derived
from their potential in adapting to changing environ-
mental conditions [33]. Due to this, Ethiopian durum
wheat germplasm has served as a center of focus for
genetic studies and served as the source of novel QTLs,
genes and gene complexes for many traits [9–14, 34]. Slightly higher number of SNPs (30,510) were repro-
duced in the present study from the 90 K wheat SNP
array than previously reported by Mengistu et al. [12] on
Ethiopian durum wheat (30,155 SNPs) and in Mediterra-
nean durum wheat collections (21,069 SNPs). Genomes
of A and B did not show a significant difference in diver-
sity indices, indicating that they have followed similar Table 4 Analysis of molecular variance (AMOVA) for Ethiopian durum wheat accessions with and without grouping according to
STRUCTURE clustering result
Source of variation
DF
Sum of squares
Variance components
Percentage of variation
Fixation indices
P value
Among populations
2
166,172.20
700.94 Va
52.41
FST = 0.52
Va and FST = 0.000
Among individuals Within populations
189
240,563.25
636.41 Vb
47.59
FIS = 1.00
Vb and FIS = 0.000
Total
191
406,735.45
1337.35 Page 9 of 12 Alemu et al. BMC Genetics (2020) 21:18 Table 5 Analysis of molecular variance) between landraces and varieties
Source of variation
DF
Sum of squares
Variance components
Percentage of variation
Fixation indices
P value
Among populations
1
108,435.72
1228.63 Va
61.02
FST = 0.61
Va and FST = 0.000
Among individuals Within populations
190
298,299.73
784.99 Vb
38.98
FIS = 1.00
Vb and FIS = 0.000
Total
191
406,735.45
2013.62 evolutionary histories in Ethiopian durum wheat land-
races and improved varieties [12]. by simultaneous increment creating an elbow shape [41]. However, it provided a clue in which somehow less than
five clusters could be optimal. Varieties showed a single
distinct cluster and landraces distributed into two distinct
clusters. Genetic diversity of Ethiopian durum wheat Both principal component analysis (PCA) with
the first two components and the neighbor joining cluster-
ing based on simple matching dissimilarity coefficient
proved the former clustering result was optimal showing
three clear clusters. In the current study, clustering was
not based on their geographic origin where landrace ac-
cessions were originally collected in Ethiopia. Mengistu
et al. [12] reported a similar result on a study conducted
in 311 Ethiopian durum wheat accessions (287 landraces
and 24 varieties) collected from major wheat producing
areas of the country. This admixture could be due to the
existence of historical and current exchange of seeds
through informal seed system involving regional and
countrywide farming communities [34]. Ren et al. [36] re-
ported neither geographical nor ecological evidence was
detected in grouping 150 durum wheat accessions with
world-wide origin and noted that the possible reason
could be the existence of gene flow via germplasm ex-
changes among different regions occurred frequently or
that human transfer of genes in history made a very big
admixture. Kabbaj et al. [3] found higher admixtures be-
tween 370 durum wheat accessions included landraces,
varieties and elite lines collected from more than 30 coun-
tries including Ethiopia. However, they observed a very
limited admixture between Ethiopian landraces with other
collections originated world-wide and Ethiopian durum
wheat landraces made a separate cluster and proved the
presence of a unique morphology [10, 34] and represent a
separate sub-species under the name Triticum durum
subs. Abyssinicum or T. aethiopicum [22]. This phenom-
ena placed Ethiopia as a secondary center of origin and di-
versity for durum wheat since the germplasm is distinct
from the primary region of origin of durum wheat, the
Fertile Crescent countries [3]. The 90 K wheat SNP array was a platform made to cap-
ture the most reliable gene-associated SNP markers avail-
able in the wheat genome worldwide and could not enable
to mine new loci. The less number of SNPs reproduced
from the array in the current panel indicates the possibil-
ity of existence of novel alleles and further studies would
be benefited from the employment of both hybridization
and sequencing techniques to provide a thorough descrip-
tion of Ethiopian durum wheat genome. Genetic diversity of Ethiopian durum wheat Comparing with previous reports, higher genetic diver-
sity indices were scored in Ethiopian durum wheat panel
(Table 1) that strengthens the unresolved and ongoing ar-
gument of Ethiopia as the center of origin or domestication
of durum wheat [3]. The result unveiled the presence of
higher genetic diversity in Ethiopian durum wheat that
could arise because of various causes including adaptation
to wider agro-ecology [23], natural crossings due to culti-
vating mixed genotypes in a field and diverse farmers’ cul-
ture of agricultural practices [22, 35]. For instance, Ren
et al. [36] reported mean polymorphic information content
(0.18) and Nei’s gene diversity (0.22) from world-wide col-
lected 150 durum wheat accessions genotyped with 1536
SNP markers. Kabbaj et al. [3] obtained a mean PIC value
of 0.119 from 337 durum wheat accessions included land-
races, varieties and elite lines collected from more than 30
countries genotyped with 35 K Affymetrix Axiom wheat
breeders array. Eltaher et al. [37] reported slightly higher
mean gene diversity (0.3) and PIC (0.23) in 250 winter
wheat
accessions
genotyped
with
Genotyping-By-
Sequencing (GBS) platform. However, unlike the present
study, they only included SNP markers having less than
20% missing information and with minor allelic frequency
(MAF) greater than 5%. As expected, higher PIC and gen-
etic diversity scores were reported in studies using multi-
allelic markers such as SSR, unlike SNPs, that could go be-
yond 0.5 values [38, 39]. Supplementary information pp
y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s12863-020-0825-x. Additional file 1 : Table S1. Accession names and types, cultivated
areas, seed sources and population structure of 192 Ethiopian durum
wheat accessions used for the current study. Additional file 1 : Table S1. Accession names and types, cultivated
areas, seed sources and population structure of 192 Ethiopian durum
wheat accessions used for the current study. Additional file 2 : Table S2. List of polymorphic SNPs, their alleles and
chromosomes used for genetic diversity analysis in Ethiopian durum
wheat accessions. Genetic structure In this study, 192 Ethiopian durum wheat accessions
comprising 167 landraces and 25 improved varieties
were assembled and genotyped with a high density 90 K
wheat SNP array to analyze the existing genetic diversity
and population structure within accessions. Clustering
analysis showed a higher genetic admixture between
landraces despite their geographic origin resulted from Genetic stratification analysis based on the Bayesian clus-
tering model of the second order rate of change of the
likelihood [40] revealed the presence of three subpopula-
tions. However, discriminant analysis of principal compo-
nents based on the Bayesian information criterion (BIC)
couldn’t show the smallest BIC on a specific K value above
which the BIC values spontaneously decreased followed Page 10 of 12 Page 10 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 the probability of finding polymorphisms between any
two random samples while Nei’s gene diversity defined
as the probability of two randomly chosen alleles from
the population is different. Principal component analysis
(PCA) [43] for the genetic relationships among individ-
uals was calculated using a package “SNPrelate” [44] in
R studio [45]. Neighbor-Joining tree based on simple
matching dissimilarity coefficient was constructed using
DARwin var. 6.0.14 [46] and the resulting trees were dis-
played using FigTree var. 1.4.3 [47]. A software package
Arlequin v.3.5.2.2 [48] was used to assess the molecular
variance (AMOVA) between clusters based on STRUCT
URE-inferred subpopulations and between landraces and
varieties. the existence of higher rate of historical seed exchange
throughout the country. Diversity indices revealed the
presence of higher genetic diversity in Ethiopian durum
wheat accessions. Landraces adapted to wider agroecol-
ogy and with the genetic capacity to tolerate various
stresses could be used as a source of unique alleles in
the enhancement of durum breeding through marker
assisted
selection
or
marker
assisted
backcrossing. Hence, sustainable conservation and utilization of Ethi-
opian durum wheat genetic resource is key for future
breeding strategies in Ethiopia and worldwide. Genetic structure analysis Two approaches were implemented to infer the optimal
clusters/subpopulations existed in 192 Ethiopian durum
wheat accessions. First, a Bayesian model-based cluster-
ing approach was used to estimate the optimal subpopu-
lations and the membership probability of each genotype
to the subpopulations using STRUCTURE v.2.3 [49]. To
infer the optimal clusters, an ad hoc quantity (ΔK) ap-
proach was applied that was calculated based on the sec-
ond order rate of change of the likelihood [40]. For this
analysis, 10 sub-populations with 20 independent itera-
tions for each sub-population was done under the ad-
mixture model of population structure with correlated
allele frequencies and 50,000 lengths burn-in period and
100,000 Markov Chain Monte Carlo (MCMC) replica-
tions after burn-in was applied for each iteration. Landraces were originally collected from major wheat-
producing areas of Ethiopia (Additional file 3: Figure S1)
including Bale, Gondar, Gojjam, Shewa, Tigray, and
Wollo, as well as twelve lines, which are originally from
Ethiopia but currently cultivated in the USA. A detail of
accessions is summarized in Additional file 1: Table S1. DNA extraction and SNP genotyping The second approach was based on the discriminant
analysis of principal components (DAPC) implemented
using a package “adegenet” [41] in R studio. In this
method, the optimal clustering solution corresponded to
the lowest Bayesian Information Criterion (BIC) and the
number of clusters determined as the value of K above
which BIC values decreased with simultaneous incre-
ment making an elbow at the optimal cluster [41]. A pooled tissue sample of twenty five one-week-old
seedlings was taken for genomic DNA extraction for
each accession. The DNA extraction was done with
DNeasy 96 Plant Kit (Qiagen GmbH, Hilden, Germany). SNP markers were generated using the Illumina iSe-
lect® 90 K wheat SNP assay comprising 81,587 gene-
associated SNPs [27]. Marker genotypes were called with
the GenomeStudio v2011.1 software package (Illumina,
San Diego, CA, USA) and calls showing residual hetero-
zygosity were entered as missing values before exporting
genotype data from the GenomeStudio. A high-density
consensus map of tetraploid wheat generated by Macca-
ferri et al. [32] was used to identify chromosome posi-
tions of SNPs. The SNPs data used for diversity analysis
is available in Additional file 2: Table S2. Plant material One hundred sixty seven Ethiopian durum landrace ac-
cessions collected from major wheat growing areas of
the country and twenty five improved varieties released
in different years and have been cultivated in Ethiopia
were assembled for the present study. Improved varieties
were released by Debre Zeit Agricultural Research Cen-
ter (DZARC) and Sinana Agricultural Research Center
(SARC), Ethiopia in different years (1994–2010). All
landrace accessions and varieties are maintained by these
two agricultural research centers as a single seed descent
(SSD) progenies. Funding Fingerprinting of accessions was made possible with the financial support
from Bill and Melinda Gates Foundation, the Department for International
Development of the United Kingdom, and the AGER Agroalimentare e
Ricerca-Project “From seed to pasta-Multidisciplinary approaches for a more
sustainable and high quality durum wheat production”. The role of the fund-
ing bodies is limited to direct funding of the fingerprinting of genotypes that
result in this manuscript. 13. Liu W, Maccaferri M, Rynearson S, Letta T, Zegeye H, Tuberosa R, et al. Novel
Sources of Stripe Rust Resistance Identified by Genome-Wide Association
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Plant Sci. 2017;8. https://doi.org/10.3389/fpls.2017.00774. 14. Maccaferri M, Harris NS, Twardziok SO, Pasam RK, Gundlach H, Spannagl M,
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future improvement targets. Nat Genet. 2019;51:885–95. https://doi.org/10. 1038/s41588-019-0381-3. Abbreviations
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l
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allele frequency; MCMC: Markov chain Monte Carlo; PCA: Principal AMOVA: Analysis of molecular variance; BIC: Bayesian information content;
DAPC: Discriminant analysis of principal components; DZARC: Debre Zeit
agricultural research center; EBI: Ethiopian biodiversity institute; MAF: Minor
allele frequency; MCMC: Markov chain Monte Carlo; PCA: Principal
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wheat germplasm. Theor Appl Genet. 2003;107:783–97. https://doi.org/10. 1007/s00122-003-1319-8. 39. References 21. Teklu Y, Hammer K, Huang XQ, Röder MS. Analysis of microsatellite diversity
in Ethiopian Tetraploid wheat landraces. Genet Resour Crop Evol. 2006;53:
1115–26. https://doi.org/10.1007/s10722-005-1146-7. 1. Peng JH, Sun D, Nevo E. Domestication evolution, genetics and genomics
in wheat. Mol Breed. 2011;28:281–301. https://doi.org/10.1007/s11032-011-
9608-4. 1. Peng JH, Sun D, Nevo E. Domestication evolution, genetics and genomics
in wheat. Mol Breed. 2011;28:281–301. https://doi.org/10.1007/s11032-011-
9608-4. 22. Mengistu DK, Kiros AY, Pè ME. Phenotypic diversity in Ethiopian durum
wheat (Triticum turgidum var. durum) landraces. Crop J. 2015;3:190–9. https://doi.org/10.1016/j.cj.2015.04.003. 2. Global Agricultural Information Network. Global agricultural information
network (GAIN) report: Ethiopia. Addis Ababa, Ethiopia; 2018. https://www. fas.usda.gov/regions/ethiopia. Page 12 of 12 Page 12 of 12 Page 12 of 12 Alemu et al. BMC Genetics (2020) 21:18 Alemu et al. BMC Genetics (2020) 21:18 23. Tesemma T, Belay G. Aspects of Ethiopian tetraploid wheats with emphasis on
durum wheat genetics and breeding research. In: Gebre-Mariam H, Tanner DG,
Hulluka M, editors. Wheat research in Ethiopia: a historical perspective. Addis
Ababa, Ethiopia: IAR; 1991. p. 47–71. http://www.eiar.gov.et/. 43. Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D. Principal components analysis corrects for stratification in genome-wide
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ng1847. Ababa, Ethiopia: IAR; 1991. p. 47–71. http://www.eiar.gov.et/. 44. Zheng X, Levine D, Shen J, Gogarten SM, Laurie C, Weir BS. A high-
performance computing toolset for relatedness and principal component
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bioinformatics/bts606. 24. Zhang Q, Maroof MA, Kleinhofs A. Comparative diversity analysis of RFLPs
and isozymes within and among populations of Hordeum vulgare ssp. spontaneum. Genetics. 1993;134:909–16 http://www.ncbi.nlm.nih.gov/
pubmed/8102343. 45. R Development Core team. R: a language and environment for statistical
computing. Vienna: R Foundation for statistical Computing; 2013. http://
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computing. Vienna: R Foundation for statistical Computing; 2013. http://
www.r-project.org/. 25. Peterson G, Dong Y, Horbach C, Fu Y-B. Genotyping-by-sequencing for
plant genetic diversity analysis: a lab guide for SNP genotyping. Diversity. 2014;6:665–80. https://doi.org/10.3390/d6040665. 25. Peterson G, Dong Y, Horbach C, Fu Y-B. Genotyping-by-sequencing for
plant genetic diversity analysis: a lab guide for SNP genotyping. Diversity. 2014;6:665–80. https://doi.org/10.3390/d6040665. 46. Perrier X, Jacquemoud-Collet JP. DARwin software. 2006. http://darwin.cirad. fr/. Accessed 20 Mar 2019. 46. Perrier X, Jacquemoud-Collet JP. DARwin software. 2006. http://darwin.cirad. fr/. Accessed 20 Mar 2019. 26. Ganal MW, Altmann T, Röder MS. SNP identification in crop plants. Curr
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A Radar Object-Based Examination of Rain System
Climatology and Including Climate Variability
Hooman Ayat ( h.ayat@student.unsw.edu.au )
University of New South Wales https://orcid.org/0000-0001-9499-5894
Jason P. Evans
University of New South Wales
Steven C. Sherwood
University of New South Wales
Joshua Soderholm
Australian Bureau of Meteorology
Research Article
Keywords: radar object-based examination, rain system, climatology, climate variability, warming, storms,
intensity, rainfall statistics
Posted Date: August 11th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-783979/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License A Radar Object-Based Examination of Rain System
Climatology and Including Climate Variability
Hooman Ayat ( h.ayat@student.unsw.edu.au )
University of New South Wales https://orcid.org/0000-0001-9499-5894
Jason P. Evans
University of New South Wales
Steven C. Sherwood
University of New South Wales
Joshua Soderholm
Australian Bureau of Meteorology
Research Article
Keywords: radar object-based examination, rain system, climatology, climate variability, warming, storms,
intensity, rainfall statistics
Posted Date: August 11th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-783979/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License A radar object-based examination of rain system
climatology and including climate variability Hooman Ayat1, Jason P. Evans1, Steven C. Sherwood1, Joshua Soderholm2 1 Climate Change Research Centre and ARC Centre of Excellence for Climate Extremes,
University of New South Wales, Sydney, New South Wales, Australia
2 Science and Innovation Group, Australian Bureau of Meteorology, Melbourne, Victoria,
Australia 1 Climate Change Research Centre and ARC Centre of Excellence for Climate Extremes,
University of New South Wales, Sydney, New South Wales, Australia
2 Science and Innovation Group, Australian Bureau of Meteorology, Melbourne, Victoria,
Australia 1 Climate Change Research Centre and ARC Centre of Excellence for Climate Extremes,
University of New South Wales, Sydney, New South Wales, Australia
2 Science and Innovation Group, Australian Bureau of Meteorology, Melbourne, Victoria,
Australia Corresponding author: Hooman Ayat (h.ayat@student.unsw.edu.au) Research Article Keywords: radar object-based examination, rain system, climatology, climate variability, warming, storms,
intensity, rainfall statistics
Posted Date: August 11th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-783979/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1.
Abstract We know the climate is warming and this is changing some aspects of storms, but we
have little knowledge of storm characteristics beyond intensity, which limits our understanding
of storms overall. In this study, we apply a cell-tracking algorithm to 20 years of radar data at a
mid-latitude coastal-site (Sydney, Australia), to establish a regional storm climatology. The
results show that extreme storms in terms of translation-speed, size and rainfall intensity usually
occur in the warm season, and are slower and more intense over land between ~10am and ~8pm
(AEST), peaking in the afternoon. Storms are more frequent in the cold season and often initiate
over the ocean and move northward, leading to precipitation mostly over the ocean. Using
clustering algorithms, we have found five storm types with distinct properties, occurring
throughout the year but peaking in different seasons. While overall rainfall statistics don't show
any link to climate modes, links do appear for some storm types using a multivariate approach. This climatology for a variety of storm characteristics will allow future study of any changes in change. due due to 1 1. Introduction
1 Heavy rainfall is a significant threat to life and property in many parts of the world,
2
especially when it is accompanied by flash floods (Johnson et al. 2016; Allen and Allen 2016). 3
Many studies have shown the potential for climate change to impact rainfall intensity, but how it
4
will affect other storm characteristics (size, translation speed, orientation, etc.) remains largely
5
unexplored. The first step towards exploring the potential future changes is to establish an
6
observed baseline for a wide variety of storm characteristics. 7 Climatological studies can help us to better understand storm characteristics and their
8
(local and remote) drivers in different seasons. Many studies have used gridded datasets (e.g.,
9
global climate models, reanalysis data) to perform climatological studies globally and over
10
specific regions; however, the coarse-resolution of these datasets are often unable to properly
11
capture small scale storms like thunderstorms. Therefore, in order to capture these small scale
12
storms using these datasets, researchers have tried to establish thunderstorm climatologies based
13
on the concept of favourable conditions for thunderstorms, which usually includes a combination
14
of convective available potential energy (CAPE) and vertical wind shear in a region (Ahmed et
15
al. 2019; Brooks et al. 2003; Taszarek et al. 2020; Allen et al. 2011; Groenemeijer et al. 2017). 16
By employing this environmental approach, Allen and Karoly (2014) presented a severe
17
thunderstorm climatology over Australia during 2003–2010, using ERA-Interim reanalysis and
18
reported observation data. They showed that these types of storms are more frequent in
19
December during the afternoon, consistent with the seasonal and diurnal cycle of surface
20
temperature and the maximum availability of heating. Although this approach provides valuable
21
information, environmentally favorable conditions do not necessarily lead to a thunderstorm,
22
causing a misestimation of the thunderstorm frequency. In addition, this approach only provides
23
us with storm frequency and it is unable to provide information on other storm characteristics
24
(Allen and Karoly 2014). 25 Using coarse-resolution datasets, previous authors have tried to investigate the effect of
26
natural climate variability (e.g., El Niño–Southern Oscillation (ENSO), the Indian Ocean Dipole
27
(IOD)) on the rainfall over Australia. Ashok et al. (2003) showed that IOD has significant
28
negative partial correlations with rainfall over the western and southern regions of Australia
29
using an atmospheric general circulation model. 1. Introduction
1 2013) and middle-east (Shwehdi 2005). A
57
limitation of this approach is that it is unable to capture storms without lightning. Additionally, it
58
is only able to provide information regarding storm frequency, not other information like rainfall
59
intensity (Walsh et al. 2016). 60
Rain gauge data and reports of hail, tornado and wind gusts are other means of studying
61
thunderstorm climatology used in many studies (Bhardwaj and Singh 2018; Enno et al. 2013;
62
Saha and Quadir 2016; Pinto et al. 2013; Groenemeijer et al. 2017; Kelly et al. 1985; Doswell et
63
al. 2005). The databases of reported tornadoes, hailstorms, and gust winds in some countries like
64
Australia have a long-term record (Bureau of Meteorology 2021), which can provide valuable
65
information for climatological studies. Using these datasets over Australia, some researchers
66
have found that severe thunderstorms are most prevalent between October and April (Niall and
67
Walsh 2005; Schuster et al. 2005; Davis and Walsh 2008) with a peak between 3 and 7 pm
68
(Griffiths et al. 1993; Schuster et al. 2005), consistent with the findings of Allen and Karoly
69
(2014) using environmental approach and ERA-Interim reanalysis data. Higher frequency of
70
heavy rain events over Australia in summer was also reported by Dare and Davidson (2015),
71
using a high resolution gridded daily rain gauge data. Some efforts have also been made to
72
employ these types of data to investigate relationships between ENSO and storm events, globally
73
(Cook and Schaefer 2008; Cook et al. 2017; Lee et al. 2016, 2013; Lepore et al. 2017) and over
74
Australia (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008; Nicholls et
75
al. 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et al. 2013;
76
King et al. 2014; Ashcroft et al. 2019). Based on these studies, winter-spring rainfall is usually
77
reduced during El Niño and enhanced during La Niña over the eastern and southeastern parts of
78
Australia. Although gauge and reported data provide us with valuable information, the
79 the need for a multivariate rather than bivariate approach to this problem. 1. Introduction
1 Maher and Sherwood
40
(2014) applied a multivariate approach to Australian precipitation to disentangle the multiple
41
sources of large-scale variability using the ERA-Interim and Australian Water Availability
42
Project datasets, and they showed that ENSO, blocking, and the intensity and position of the
43
ridge are driving wintertime precipitation in Australia, with a minor role played by the jet
44
intensity and the IOD. All of these studies investigated the effects of natural climate variability
45
on rainfall intensity and frequency, and the relationships between other characteristics of storms
46
(i.e., size, shape, translation speed, etc.) and climate modes are not understood. 47 One way of studying the thunderstorm climatology is by measuring the occurrence of
48
lightning using satellite instruments such as the Optical Transient Detector (OTD) and/or the
49
Lightning Imaging Sensor (LIS). Dowdy and Kuleshov (2014) produced a climatological map of
50
lightning ground flash density over Australia, which details the seasonal variability of the
51
lightning ground flash densities over land and ocean. They found that during the cooler months,
52
a maximum in lightning activity occurs over the ocean to the east of the continent. Earlier studies
53
by Kuleshov et al. (2001; 2012) had found that the second-highest thunderstorm frequency
54
occurs in the southeast part of Australia along the coastline, most often from spring to early
55
autumn. Similar research has been conducted globally for regions such as the United States and
56
Europe (Taszarek et al. 2020), Brazil (Pinto et al. 2013) and middle-east (Shwehdi 2005). A
57
limitation of this approach is that it is unable to capture storms without lightning. Additionally, it
58
is only able to provide information regarding storm frequency, not other information like rainfall
59
intensity (Walsh et al. 2016). 60 Rain gauge data and reports of hail, tornado and wind gusts are other means of studying
61
thunderstorm climatology used in many studies (Bhardwaj and Singh 2018; Enno et al. 2013;
62
Saha and Quadir 2016; Pinto et al. 2013; Groenemeijer et al. 2017; Kelly et al. 1985; Doswell et
63
al. 2005). The databases of reported tornadoes, hailstorms, and gust winds in some countries like
64
Australia have a long-term record (Bureau of Meteorology 2021), which can provide valuable
65
information for climatological studies. 1. Introduction
1 Allen and Karoly (2014) employed the ECMWF
30
Interim Re-Analysis (ERA-Interim) data and have found that ENSO has a substantial impact on
31
the spatial distribution of severe thunderstorm environments over the continent. In another
32
reanalysis-based study, Hauser et al. (2020) investigated the winter-spring rainfall variability in
33
southeastern Australia (SEA) during El Niño events by quantifying the contribution of clustered
34
mid-latitude weather systems to monthly precipitation anomalies. The authors found that the
35
cluster with below-average rainfall is more frequent compared to the other clusters during El
36
Niño, which confirmed the general suppression of SEA rainfall during these events. Since
37
precipitation in some regions is correlated with more than one large scale driver, and indices are
38
often correlated with each other, the interconnected nature of precipitation dependence suggests
39 2 the need for a multivariate rather than bivariate approach to this problem. Maher and Sherwood
40
(2014) applied a multivariate approach to Australian precipitation to disentangle the multiple
41
sources of large-scale variability using the ERA-Interim and Australian Water Availability
42
Project datasets, and they showed that ENSO, blocking, and the intensity and position of the
43
ridge are driving wintertime precipitation in Australia, with a minor role played by the jet
44
intensity and the IOD. All of these studies investigated the effects of natural climate variability
45
on rainfall intensity and frequency, and the relationships between other characteristics of storms
46
(i.e., size, shape, translation speed, etc.) and climate modes are not understood. 47
One way of studying the thunderstorm climatology is by measuring the occurrence of
48
lightning using satellite instruments such as the Optical Transient Detector (OTD) and/or the
49
Lightning Imaging Sensor (LIS). Dowdy and Kuleshov (2014) produced a climatological map of
50
lightning ground flash density over Australia, which details the seasonal variability of the
51
lightning ground flash densities over land and ocean. They found that during the cooler months,
52
a maximum in lightning activity occurs over the ocean to the east of the continent. Earlier studies
53
by Kuleshov et al. (2001; 2012) had found that the second-highest thunderstorm frequency
54
occurs in the southeast part of Australia along the coastline, most often from spring to early
55
autumn. Similar research has been conducted globally for regions such as the United States and
56
Europe (Taszarek et al. 2020), Brazil (Pinto et al. 1. Introduction
1 High spatio-temporal resolution radar rainfall estimates offer the potential to study the
92
thunderstorms occurring over their coverage areas, particularly over the regions in which gauge
93
observations are usually sparse or unevenly distributed (Ghaemi et al. 2017; Ayat et al. 2018;
94
Moazami et al. 2014). Radar records in many parts of the globe are temporally limited to just
95
over a decade (Allen 2018). However, this time frame is long enough to conduct climatological
96
studies. Previous researchers have employed these datasets to conduct thunderstorm climatology
97
studies over specific regions such as the United States (Ghebreyesus and Sharif 2020; Kuo and
98
Orville 1973; Croft and Shulman 1989; Falconer 1984), Europe (Kaltenboeck and Steinheimer
99
2015; Kreklow et al. 2020; Weckwerth et al. 2011; Overeem et al. 2009; Bližňák et al. 2018;
100
Fairman Jr et al. 2015) and Asia (Chen et al. 2012). 101 Hail particles are readily detected by conventional weather radars due to strong scatter
102
produced by their comparatively larger surface area, or via their quasi-spherical shape which
103
produces distinct differential reflectivity values for dual-polarization radars compared to oblate
104
rain droplets. Using these properties, a few radar-based hail products have been produced and
105
employed to study the climatology of hailstorms over different regions. For instance, Warren et. 106
al (2020) studied the hail climatology in multiple cities in Australia (including the Sydney
107
region) using a radar-based hail product (Maximum Expected Size of Hail; MESH), and found
108
that, on average, damaging hail storms over Sydney occur 32 days per year with a peak during
109
the warm season (November-March). Similar radar-based studies have been conducted over
110
other parts of the globe like Europe (Junghänel et al. 2016; Nisi et al. 2016; Fluck et al. 2021;
111
Lukach et al. 2017; Saltikoff et al. 2010) and the United States (Cintineo et al. 2012; Murillo et
112
al. 2021). 113 Most of the previous radar-based studies employed a pixel-based statistical approach
which limits the storm properties to rainfall/hail frequency and intensity as a function of position. In order to overcome this limitation, a few studies have employed an object-based approach,
where discrete storm objects are identified and characterized. For instance, Haberlie and Ashley
(2019), Poujol et al. (2020) and Prein et al. (2017) applied object-based techniques to radar
products to study the climatology of convective storms in the United States. 1. Introduction
1 91
High spatio-temporal resolution radar rainfall estimates offer the potential to study the
92
thunderstorms occurring over their coverage areas, particularly over the regions in which gauge
93
observations are usually sparse or unevenly distributed (Ghaemi et al. 2017; Ayat et al. 2018;
94
Moazami et al. 2014). Radar records in many parts of the globe are temporally limited to just
95
over a decade (Allen 2018). However, this time frame is long enough to conduct climatological
96
studies. Previous researchers have employed these datasets to conduct thunderstorm climatology
97
studies over specific regions such as the United States (Ghebreyesus and Sharif 2020; Kuo and
98
Orville 1973; Croft and Shulman 1989; Falconer 1984), Europe (Kaltenboeck and Steinheimer
99
2015; Kreklow et al. 2020; Weckwerth et al. 2011; Overeem et al. 2009; Bližňák et al. 2018;
100
Fairman Jr et al. 2015) and Asia (Chen et al. 2012). 101
Hail particles are readily detected by conventional weather radars due to strong scatter
102
produced by their comparatively larger surface area, or via their quasi-spherical shape which
103
produces distinct differential reflectivity values for dual-polarization radars compared to oblate
104
rain droplets. Using these properties, a few radar-based hail products have been produced and
105 distribution of recorded data is highly influenced by the local population (Allen 2018). In
addition, gauge instruments are point measurements and sparse rain gauge networks often fail to
observe the maximum rainfall (Ayat et al. 2018). The limitations of previous approaches have led to the application of remotely-sensed
datasets, which are a growing area of thunderstorm climatology. Some studies (Cecil and
Blankenship 2012; Ni et al. 2017) have applied a satellite-based approach using passive and
active microwave sensors to estimate the global climatology of hail storms. However, images
from these sensors are available only for a few overpasses per day (Ayat et al. 2021a), which
may result in biasing the results depending on the diurnal cycle of convection in the respective
regions (Punge and Kunz 2016). In addition, passive microwave products are capturing the upper
part of the storms in which there might be more hail particles that melt/evaporate at lower
altitudes, leading to false estimation of hail on the ground surface (Ayat et al. 2021b). 1. Introduction
1 Using these datasets over Australia, some researchers
66
have found that severe thunderstorms are most prevalent between October and April (Niall and
67
Walsh 2005; Schuster et al. 2005; Davis and Walsh 2008) with a peak between 3 and 7 pm
68
(Griffiths et al. 1993; Schuster et al. 2005), consistent with the findings of Allen and Karoly
69
(2014) using environmental approach and ERA-Interim reanalysis data. Higher frequency of
70
heavy rain events over Australia in summer was also reported by Dare and Davidson (2015),
71
using a high resolution gridded daily rain gauge data. Some efforts have also been made to
72
employ these types of data to investigate relationships between ENSO and storm events, globally
73
(Cook and Schaefer 2008; Cook et al. 2017; Lee et al. 2016, 2013; Lepore et al. 2017) and over
74
Australia (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008; Nicholls et
75
al. 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et al. 2013;
76
King et al. 2014; Ashcroft et al. 2019). Based on these studies, winter-spring rainfall is usually
77
reduced during El Niño and enhanced during La Niña over the eastern and southeastern parts of
78
Australia. Although gauge and reported data provide us with valuable information, the
79 3 distribution of recorded data is highly influenced by the local population (Allen 2018). In
80
addition, gauge instruments are point measurements and sparse rain gauge networks often fail to
81
observe the maximum rainfall (Ayat et al. 2018). 82
The limitations of previous approaches have led to the application of remotely-sensed
83
datasets, which are a growing area of thunderstorm climatology. Some studies (Cecil and
84
Blankenship 2012; Ni et al. 2017) have applied a satellite-based approach using passive and
85
active microwave sensors to estimate the global climatology of hail storms. However, images
86
from these sensors are available only for a few overpasses per day (Ayat et al. 2021a), which
87
may result in biasing the results depending on the diurnal cycle of convection in the respective
88
regions (Punge and Kunz 2016). In addition, passive microwave products are capturing the upper
89
part of the storms in which there might be more hail particles that melt/evaporate at lower
90
altitudes, leading to false estimation of hail on the ground surface (Ayat et al. 2021b). 1. Introduction
1 122
Although object-based techniques can provide us with more information on storm
123
characteristics, the investigated storm properties in most of these studies were limited to storm
124
number, area, and rainfall intensity, whereas other storm characteristics like storm translation
125
speed, shape and aspect ratio, orientation, direction and volume could also be of interest. In
126
addition, the object-based techniques employed in these studies are limited by the object
127
split/merge issue, which is a common problem in object tracking methods and can lead to
128
calculating misleading storm properties (Muñoz et al. 2018). 129
In this research, we employ the Method of Object-based Diagnostic Evaluation (MODE)
130
Time Domain (MTD), which is modified by the authors so as to handle splitting and merging of
131
objects. We apply this to the Wollongong (near Sydney) radar, which has around 20 years of
132
records, to establish an object-based climatology of precipitation in different seasons over the
133
radar footprint areas (i.e., Greater Sydney, Illawarra and other land/ocean regions within 150 km
134
of the radar). An effort has been made to group the main contributing storms with similar object-
135
based characteristics over this region using clustering algorithms followed by investigating their
136
relationships with different climate modes. 137
This study is presented in seven sections. Section 2 describes the Wollongong radar data
138
and its characteristics. Section 3 introduces the object-based and clustering methods along with
139
the statistics employed in this study. Section 4 describes the study area and section 5 presents the
140
results of the object-based climatology over the study area. Section 6 discusses the results shown
141
in the previous section, and finally, the summary of findings is presented in section 7. 142 Soderholm et al. (2017) employed a cell-tracking algorithm over radar MESH product to study
120
hail climatology in southeast Queensland, Similar efforts have been made in Germany
121
(Thomassen et al. 2020), Italy (Sangiorgio and Barindelli 2020) and Spain (Rigo et al. 2010). 122 Although object-based techniques can provide us with more information on storm
characteristics, the investigated storm properties in most of these studies were limited to storm
number, area, and rainfall intensity, whereas other storm characteristics like storm translation
speed, shape and aspect ratio, orientation, direction and volume could also be of interest. 1. Introduction
1 In
addition, the object-based techniques employed in these studies are limited by the object
split/merge issue, which is a common problem in object tracking methods and can lead to
calculating misleading storm properties (Muñoz et al. 2018). In this research, we employ the Method of Object-based Diagnostic Evaluation (MODE)
Time Domain (MTD), which is modified by the authors so as to handle splitting and merging of
objects. We apply this to the Wollongong (near Sydney) radar, which has around 20 years of
records, to establish an object-based climatology of precipitation in different seasons over the
radar footprint areas (i.e., Greater Sydney, Illawarra and other land/ocean regions within 150 km
of the radar). An effort has been made to group the main contributing storms with similar object-
based characteristics over this region using clustering algorithms followed by investigating their
relationships with different climate modes. This study is presented in seven sections. Section 2 describes the Wollongong radar data
38
and its characteristics. Section 3 introduces the object-based and clustering methods along with
39
the statistics employed in this study. Section 4 describes the study area and section 5 presents the
40
results of the object-based climatology over the study area. Section 6 discusses the results shown
41
in the previous section, and finally, the summary of findings is presented in section 7. 42 1. Introduction
1 In Australia, 4 Soderholm et al. (2017) employed a cell-tracking algorithm over radar MESH product to study
120
hail climatology in southeast Queensland, Similar efforts have been made in Germany
121
(Thomassen et al. 2020), Italy (Sangiorgio and Barindelli 2020) and Spain (Rigo et al. 2010). 122
Although object-based techniques can provide us with more information on storm
123
characteristics, the investigated storm properties in most of these studies were limited to storm
124
number, area, and rainfall intensity, whereas other storm characteristics like storm translation
125
speed, shape and aspect ratio, orientation, direction and volume could also be of interest. In
126
addition, the object-based techniques employed in these studies are limited by the object
127
split/merge issue, which is a common problem in object tracking methods and can lead to
128
calculating misleading storm properties (Muñoz et al. 2018). 129
In this research, we employ the Method of Object-based Diagnostic Evaluation (MODE)
130
Time Domain (MTD), which is modified by the authors so as to handle splitting and merging of
131
objects. We apply this to the Wollongong (near Sydney) radar, which has around 20 years of
132
records, to establish an object-based climatology of precipitation in different seasons over the
133
radar footprint areas (i.e., Greater Sydney, Illawarra and other land/ocean regions within 150 km
134
of the radar). An effort has been made to group the main contributing storms with similar object-
135
based characteristics over this region using clustering algorithms followed by investigating their
136
relationships with different climate modes. 137
This study is presented in seven sections. Section 2 describes the Wollongong radar data
138
and its characteristics. Section 3 introduces the object-based and clustering methods along with
139
the statistics employed in this study. Section 4 describes the study area and section 5 presents the
140
results of the object-based climatology over the study area. Section 6 discusses the results shown
141
in the previous section, and finally, the summary of findings is presented in section 7. 142 Soderholm et al. (2017) employed a cell-tracking algorithm over radar MESH product to study
120
hail climatology in southeast Queensland, Similar efforts have been made in Germany
121
(Thomassen et al. 2020), Italy (Sangiorgio and Barindelli 2020) and Spain (Rigo et al. 2010). 3.1 Radar Data
144 Volumetric rain rates are transformed into
171
a Cartesian grid at a 0.5 km altitude using the Barnes weighting function and a 2.5km constant
172
radius of influence. The final grid has a horizontal resolution of 1 km and a domain size of 300
173
km by 300 km. 174 Rainfall Measuring Mission (TRMM) and the Global Precipitation Measurement Mission
158
(GPM). Satellite overpasses with precipitation are compared with ground radar measurement
159
using the volume matching technique described by Louf et al. (2019), providing a mean
160
calibration value for every pass. Periods of stable calibration are identified, and the mean
161
absolute calibration value for these periods is applied as an offset to the ground radar data. 162
Removal of non-meteorological echoes from reflectivity datasets is challenging. In addition to
163
the ground clutter filtering performed by the signal processor, the technique described by Gabella
164
et al. (2002) is applied using filters for echo continuity and minimum echo area. Unfortunately,
165
this technique is not suitable for removing anomalous propagation which is commonly observed
166
over the adjacent South Pacific Ocean. 167
Reflectivity data is transformed to rain rates using a fitted Z-R relationship derived from
168
9 years of hourly rain gauge data using the Camden Airport AWS (35.04° S, 150.69° E). The A
169
and B coefficients for this relationship were 81 and 1.8 respectively. The maximum rainrate is
170
limited to 100 mm/hr to limit contamination from hail. Volumetric rain rates are transformed into
171
a Cartesian grid at a 0.5 km altitude using the Barnes weighting function and a 2.5km constant
172
radius of influence. The final grid has a horizontal resolution of 1 km and a domain size of 300
173
km by 300 km. 174 Rainfall Measuring Mission (TRMM) and the Global Precipitation Measurement Mission
158
(GPM). Satellite overpasses with precipitation are compared with ground radar measurement
159
using the volume matching technique described by Louf et al. (2019), providing a mean
160
calibration value for every pass. Periods of stable calibration are identified, and the mean
161
absolute calibration value for these periods is applied as an offset to the ground radar data. 162
Removal of non-meteorological echoes from reflectivity datasets is challenging. In addition to
163
the ground clutter filtering performed by the signal processor, the technique described by Gabella
164
et al. 3.1 Radar Data
144 (2002) is applied using filters for echo continuity and minimum echo area. Unfortunately,
165
this technique is not suitable for removing anomalous propagation which is commonly observed
166
over the adjacent South Pacific Ocean. 167 Reflectivity data is transformed to rain rates using a fitted Z-R relationship derived from
168
9 years of hourly rain gauge data using the Camden Airport AWS (35.04° S, 150.69° E). The A
169
and B coefficients for this relationship were 81 and 1.8 respectively. The maximum rainrate is
170
limited to 100 mm/hr to limit contamination from hail. Volumetric rain rates are transformed into
171
a Cartesian grid at a 0.5 km altitude using the Barnes weighting function and a 2.5km constant
172
radius of influence. The final grid has a horizontal resolution of 1 km and a domain size of 300
173
km by 300 km. 174 3.2 Challenges with anomalous propagation
175 Despite all the efforts made in removing non-meteorological echoes, the Wollongong
176
radar site experiences significant anomalous propagation over the ocean within the eastern
177
portion of its coverage. These echoes primarily occur during heat-wave conditions, where strong
178
low-level vertical gradients of humidity and temperature create regions of super refraction for
179
lower elevation scans. The resultant echoes have similar reflectivity gradients, size and shape as
180
precipitation echoes, while also being non-stationary, limiting the effectiveness of any
181
algorithms to remove non-meteorological echoes from reflectivity data (fig S5a; Online
182
Resource 1). In order to reduce the effect of these clutter sources in precipitation estimates, we
183
have opted for a two-step clutter removal process over the whole dataset: 1) Since these clutters
184
often include pixels with low intensity, applying the 3 mm/hr threshold over the convolved data
185
(to detect the storm objects; see section 5.1), effectively removed this clutter throughout the year
186
(fig S5b; Online Resource 1) except some extremes in summer (fig S5d; Online Resource 1). 2)
187
The days with extreme clutter were removed manually from the dataset using maximum daily
188
reflectivity maps (see fig S4; Online Resource 1) such that the days with high values of
189
maximum reflectivity over the ocean and low values over land were considered as a day with
190
extreme clutter over the ocean. 191 3.1 Radar Data
144 This study uses data from a Bureau of Meteorology operational S-band weather radar
145
located near Wollongong, NSW (34.26° S, 150.87° E, 471 m altitude; (Soderholm et al. 2020)). 146
The site experiences partial blocking up to 3 dB in the lowest scan (0.5° elevation) from the
147
northwest to the southeast due to the significant terrain associated with the Great Dividing
148
Range. The archive for this radar started in November 1996 and continues to operate as of 2021. 149
However, the study period is limited to June 2018 in this study. Several hardware and
150
configuration changes have taken place over the last 24 years. Initially, the radar operated on a
151
10-minute volume cycle with 16-level reflectivity data. In December 1999 the number of
152
reflectivity levels was increased to 64. Between October 2010 and January 2021, a major
153
hardware upgrade delivered 160-level reflectivity data and a 6-minute volume cycle. One
154
significant gap is present in the archive from 1/1/1998 to 15/12/1998. 155 To ensure the accuracy of reflectivity values across the entire dataset, an absolute
156
calibration technique is applied using precipitation radar measurements from the Tropical
157 5 Rainfall Measuring Mission (TRMM) and the Global Precipitation Measurement Mission
158
(GPM). Satellite overpasses with precipitation are compared with ground radar measurement
159
using the volume matching technique described by Louf et al. (2019), providing a mean
160
calibration value for every pass. Periods of stable calibration are identified, and the mean
161
absolute calibration value for these periods is applied as an offset to the ground radar data. 162
Removal of non-meteorological echoes from reflectivity datasets is challenging. In addition to
163
the ground clutter filtering performed by the signal processor, the technique described by Gabella
164
et al. (2002) is applied using filters for echo continuity and minimum echo area. Unfortunately,
165
this technique is not suitable for removing anomalous propagation which is commonly observed
166
over the adjacent South Pacific Ocean. 167
Reflectivity data is transformed to rain rates using a fitted Z-R relationship derived from
168
9 years of hourly rain gauge data using the Camden Airport AWS (35.04° S, 150.69° E). The A
169
and B coefficients for this relationship were 81 and 1.8 respectively. The maximum rainrate is
170
limited to 100 mm/hr to limit contamination from hail. 3. Study area
192 The climate of these regions is categorized as humid subtropical, or Cfa based on
195
the Köppen–Geiger classification (Kottek et al. 2006), and is significantly affected by the coastal
196
position, with small interseasonal variations ranging from cool winters to warm and hot summers
197
(Bureau of Meteorology 2016; Dare and Davidson 2015). The mean annual precipitation
198
recorded at Observatory Hill (in Greater Sydney) and Wollongong University (in Illawarra)
199
locations are 1213.4 mm and 1348.6 mm, respectively (Bureau of Meteorology 2013). 200 3. Study area
192 The study area in this research is the radar coverage regions located up to 150 km from
193
the Wollongong radar, which includes coastal regions of Greater Sydney and Illawarra in New
194 6 South Wales. The climate of these regions is categorized as humid subtropical, or Cfa based on
195
the Köppen–Geiger classification (Kottek et al. 2006), and is significantly affected by the coastal
196
position, with small interseasonal variations ranging from cool winters to warm and hot summers
197
(Bureau of Meteorology 2016; Dare and Davidson 2015). The mean annual precipitation
198
recorded at Observatory Hill (in Greater Sydney) and Wollongong University (in Illawarra)
199
locations are 1213.4 mm and 1348.6 mm, respectively (Bureau of Meteorology 2013). 200
Several factors may impact the precipitation over these regions and its seasonality. 201
Generally, precipitation peaks in the first half of the year and decreases in the second half
202
(Bureau of Meteorology 2013). In the summer, the easterly (or inland) trough is a major
203
contributor to rainfall over these regions with a peak in the evening. Its impact can be enhanced
204
by interacting with any upper-level troughs or cold fronts crossing over these regions. Frontal
205
systems also bring rainfall to these regions throughout the year, but mostly in winter when the
206
subtropical ridge moves northward over inland Australia (Bureau of Meteorology 2010). Another
207
source of precipitation over these regions is cut-off lows, which can occur at any time of year but
208
are most common during autumn and winter. These can be intense and last up to a week when
209
formed as part of a blocking pair or east coast lows, in which case they are accompanied by long-
210
lasting heavy rainfall and gusty winds (Bureau of Meteorology 2007). Northwest cloud bands
211
(stretching from northwest to southeast Australia) can also bring precipitation over these regions. 212
They may interact with cold fronts and cut-off lows over southeastern Australia to produce very
213
heavy rainfall over these regions (Reid et al. 2019). Several modes of variability are known to
214
affect precipitation in Australia, However, precipitation over the coastal zone that includes our
215
study area has shown little relationship (Timbal and Hendon 2011; Fita et al. 2017) Method
216 South Wales. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) In
239
addition, with the help of the new approach in tracking the storms, it’s possible to extract storm
240
characteristics with more details from different parts of the storms and better calculate
241
characteristics like translation speed, direction and track length during the lifetime of the storms
242
with high rate of split/merge events. 243
In this research, we are studying the extracted characteristics related to “storms” and
244
“storm objects” using the modified MTD method. The selected threshold to filter the objects is 3
245
mm/hr in the convolved data smoothed by a “3×3-pixel” moving window across the map. The
246
storm-object characteristics of interest in this study include: 1) area: the number of pixels in the
247
storm object; 2) translation speed: the ratio of the distance between the volumetric centroid of
248
two connected objects in time to the temporal resolution of the dataset; 3) maximum intensity:
249
the maximum precipitation rate within a storm object; 4) object average intensity: the average
250
precipitation rate of all cells within a storm object; 5) object volume discharge: the volumetric
251
rain rate that passes through the storm object area during a specified period; 6) aspect ratio: the
252
ratio of the minor and major axis of the fitted ellipse over the storm object; 7) object direction:
253
the compass direction of the line connecting the centroids of two consecutive objects in a
254
sequence, and finally, 8) orientation that is the compass direction of the major axis of the fitted
255
ellipse. 256 Figure 1a represents an example of running the modified MTD on Wollongong radar data
234
during an event that occurred on 2018-10-4. The lines with different colors are showing the 3D-
235
object tracks. In this event, land-originating storms’ parts had a southeastward direction and later
236
were merged with the storms’ parts that had formed over the ocean. Considering split/merge
237
events in this event has shown how successfully this approach could separate storms’ parts over
238
land and ocean before merging which is not possible using the original version of MTD. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD):
217
218
MTD is an extension for MODE to track the storm objects detected in a precipitation map
219
by the MODE algorithm (Clark et al. 2014). Here we employed a modified version of MTD,
220
proposed by Ayat et al. (2021b), that considers split/merge events during the lifetime of a storm. 221
In this method, the “storm objects” at each time step are the connected pixels higher than a
222
specified threshold in the convolved precipitation map (smoothed by an “n⨯n-pixel” moving
223
window across the map). Every “storm object” at each time-step has a unique label number
224
unless it has overlap with another storm object at the previous time-step (each blob in figure 1b). 225
In this case, it takes the label of the storm object at the previous time-step. If a storm-object has
226
overlap with two (or more) storm objects in the previous time step (merging) or two (or more)
227
objects have overlaps with a storm-object at the previous time step (splitting), the storm-
228
object/storm-objects at the current timestep takes/take a new label. Based on these definitions, A
229
“sequence of objects (or 3D objects)” is the connected storm objects in time that has a unique
230
label number and doesn’t have split/merge events during its lifetime (connected blobs with the
231
same colours and numbers in figure 1b). Finally, a “storm” is a group of the 3D objects that are
232
connected via split/merging events (The whole diagram in figure 1b). 233 7 Figure 1a represents an example of running the modified MTD on Wollongong radar data
234
during an event that occurred on 2018-10-4. The lines with different colors are showing the 3D-
235
object tracks. In this event, land-originating storms’ parts had a southeastward direction and later
236
were merged with the storms’ parts that had formed over the ocean. Considering split/merge
237
events in this event has shown how successfully this approach could separate storms’ parts over
238
land and ocean before merging which is not possible using the original version of MTD. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) In
239
addition, with the help of the new approach in tracking the storms, it’s possible to extract storm
240
characteristics with more details from different parts of the storms and better calculate
241
characteristics like translation speed, direction and track length during the lifetime of the storms
242
with high rate of split/merge events. 243 In this research, we are studying the extracted characteristics related to “storms” and
244
“storm objects” using the modified MTD method. The selected threshold to filter the objects is 3
245
mm/hr in the convolved data smoothed by a “3×3-pixel” moving window across the map. The
246
storm-object characteristics of interest in this study include: 1) area: the number of pixels in the
247
storm object; 2) translation speed: the ratio of the distance between the volumetric centroid of
248
two connected objects in time to the temporal resolution of the dataset; 3) maximum intensity:
249
the maximum precipitation rate within a storm object; 4) object average intensity: the average
250
precipitation rate of all cells within a storm object; 5) object volume discharge: the volumetric
251
rain rate that passes through the storm object area during a specified period; 6) aspect ratio: the
252
ratio of the minor and major axis of the fitted ellipse over the storm object; 7) object direction:
253
the compass direction of the line connecting the centroids of two consecutive objects in a
254
sequence, and finally, 8) orientation that is the compass direction of the major axis of the fitted
255
ellipse. 256 The studied storm characteristics in this research include: 1) storm area: the average
257
storm snapshot areas in the storm lifetime; 2) storm volume discharge: the average storm
258
snapshot volume discharge in the storm lifetime; 3) storm average intensity: the average of
259
precipitation rate in the storm lifetime; 4) storm max average intensity: the maximum of storm
260
averaged intensity (calculated at each snapshot) in the storm lifetime; 5) storm translation speed:
261
the area-weighted average translation speed of the storm snapshots in the storm lifetime; 6) storm
262
direction: the area-weighted average direction of the storm snapshots in the storm lifetime; 7)
263
storm contributing objects: The number of root storm objects in the storm graph diagram (e.g. 4.1. Method of Object-based Diagnostic Evaluation (MODE) Time Domain (MTD) in
264
figure 1 storm object numbers 1, 2 and 3 are the root objects in the storm diagram) and 8) storm
265
split/merge event number: the number of split/merge events in the storm lifetime. Note that no
266
thresholds have been applied over the defined storm/storm-object properties in this study. 267 8 8 269
Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (2018-10-04;
270
UTC). Panel (b) illustrates the diagram of a tracked storm with split/merge events. Note that each
271
blob represents a storm object at each time step and each colour shows a sequence (or 3D)
272
object. 273 Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (201 Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (2018-10-04;
270
UTC). Panel (b) illustrates the diagram of a tracked storm with split/merge events. Note that each
271
blob represents a storm object at each time step and each colour shows a sequence (or 3D)
272
object. 273 Fig. 1 Panel (a) shows the 3D-object tracks for storms that occurred on (2018-10-04;
270
UTC). Panel (b) illustrates the diagram of a tracked storm with split/merge events. Note that each
271
blob represents a storm object at each time step and each colour shows a sequence (or 3D)
272
object. 273 4.2. Clustering Analysis
274 The aim of this section is to find storm clusters (types) with similar quantitative
275
characteristics over the study area using both the Agglomerative clustering algorithm and the t-
276
SNE technique. 277 q
4.2.1 t-Distributed Stochastic Neighbor Embedding (t-SNE)
278
279
t-SNE is a statistical technique to visualize high-dimensional data by projecting it on a
280
two or three-dimensional map. Here is a brief overview of the main stages in this method: 1) It
281
starts with constructing a probability distribution of similarities over pairs of events in high-
282
dimensional data such that a similar pair of events have a higher value compared to the one that
283
is less similar; then, 2) another probability distribution of similarities is defined over the points in
284
the low-dimensional map, and finally, 3) the algorithm minimizes the divergence between two
285
distributions using Kullback–Leibler divergence parameter (KL divergence) between the two
286
distributions with respect to the locations of the points in the map. Note that the KL divergence
287
parameter is a measure of how one probability distribution diverges from another using a
288
gradient descent method. For more details, please refer to the original research paper by Maaten
289
and Hinton (2008). 290
4.2.2 Agglomerative clustering
291
292
The agglomerative technique is one of the common types of hierarchical clustering in
293
grouping data based on their similarity. This technique works in a “bottom-up” manner by
294
treating each object as a separate group in the beginning. Next, at each step, the two clusters with
295 4.2.1 t-Distributed Stochastic Neighbor Embedding (t-SNE)
278
279
t-SNE is a statistical technique to visualize high-dimensional data by projecting it on a
280
two or three-dimensional map. Here is a brief overview of the main stages in this method: 1) It
281
starts with constructing a probability distribution of similarities over pairs of events in high-
282
dimensional data such that a similar pair of events have a higher value compared to the one that
283
is less similar; then, 2) another probability distribution of similarities is defined over the points in
284
the low-dimensional map, and finally, 3) the algorithm minimizes the divergence between two
285
distributions using Kullback–Leibler divergence parameter (KL divergence) between the two
286
distributions with respect to the locations of the points in the map. 4.2. Clustering Analysis
274 307
One of the problems with hierarchical clustering is that it doesn’t give information
308
regarding the number of clusters, or where to stop the merging process in the algorithm. In order
309
to overcome this limitation, we have employed the Calinski Harabasz index (CHI) to define the
310
number of clusters which is the optimum value of CHI by increasing the number of clusters. 311
CHI is the ratio of between-cluster variance (VARB) to within-cluster variance (VARW):
312
313
𝐶𝐻𝐼=
𝑉𝐴𝑅𝐵
𝑉𝐴𝑅𝑊×
𝑁−𝐾
𝐾−1
(1)
314
𝑉𝐴𝑅𝐵= ∑
𝑛𝑘 |𝑧𝑘−𝑧|2
𝑁
𝑘=1
(2)
315
𝑉𝐴𝑅𝑊= ∑
∑
𝑛𝑘 |𝑥𝑖−𝑧𝑘|2
𝑛𝑘
𝑘=1
𝐾
𝑘=1
(3)
316
317
Here, N is the population of the data, K denotes the number of clusters, and Zk and z refer
318
to the centroid of cluster k and the entire data, respectively. In the second and third equations, nk
319
is the population of cluster k and xi denotes each member of that cluster (Li et al. 2018). 320 In order to find independent properties as the input for the clustering algorithm, the
correlations between all pairs of the storm-object properties have been calculated and the pairs
with correlation higher than 0.5 are considered as dependent variables and haven’t been used
together as the input in the clustering algorithm. Note that all input data are normalized (ranging
from 0-1) by dividing each input storm property by its maximum and standard deviation. One of the problems with hierarchical clustering is that it doesn’t give information
regarding the number of clusters, or where to stop the merging process in the algorithm. In order
to overcome this limitation, we have employed the Calinski Harabasz index (CHI) to define the
number of clusters which is the optimum value of CHI by increasing the number of clusters. 4.2. Clustering Analysis
274 CHI is the ratio of between-cluster variance (VARB) to within-cluster variance (VARW): 𝐶𝐻𝐼=
𝑉𝐴𝑅𝐵
𝑉𝐴𝑅𝑊×
𝑁−𝐾
𝐾−1
(1)
𝑉𝐴𝑅𝐵= ∑
𝑛𝑘 |𝑧𝑘−𝑧|2
𝑁
𝑘=1
(2)
𝑉𝐴𝑅𝑊= ∑
∑
𝑛𝑘 |𝑥𝑖−𝑧𝑘|2
𝑛𝑘
𝑘=1
𝐾
𝑘=1
(3) 𝐶𝐻𝐼=
𝑉𝐴𝑅𝐵
𝑉𝐴𝑅𝑊×
𝑁−𝐾
𝐾−1
(1)
𝑉𝐴𝑅𝐵= ∑
𝑛𝑘 |𝑧𝑘−𝑧|2
𝑁
𝑘=1
(2)
𝑉𝐴𝑅𝑊= ∑
∑
𝑛𝑘 |𝑥𝑖−𝑧𝑘|2
𝑛𝑘
𝑘=1
𝐾
𝑘=1
(3) 𝐶𝐻𝐼=
𝑉𝐴𝑅𝐵
𝑉𝐴𝑅𝑊×
𝑁−𝐾
𝐾−1
𝑉𝐴𝑅𝐵= ∑
𝑛𝑘 |𝑧𝑘−𝑧|2
𝑁
𝑘=1
𝑉𝐴𝑅𝑊= ∑
∑
𝑛𝑘 |𝑥𝑖−𝑧𝑘|2
𝑛𝑘
𝑘=1
𝐾
𝑘=1 (2)
(3) (3) Here, N is the population of the data, K denotes the number of clusters, and Zk and z refer
318
to the centroid of cluster k and the entire data, respectively. In the second and third equations, nk
319
is the population of cluster k and xi denotes each member of that cluster (Li et al. 2018). 320 4.2. Clustering Analysis
274 Note that the KL divergence
287
parameter is a measure of how one probability distribution diverges from another using a
288
gradient descent method. For more details, please refer to the original research paper by Maaten
289
and Hinton (2008). 290 The agglomerative technique is one of the common types of hierarchical clustering in
293
grouping data based on their similarity. This technique works in a “bottom-up” manner by
294
treating each object as a separate group in the beginning. Next, at each step, the two clusters with
295 9 the most similarity are merged into a bigger cluster and this process continues until all objects
296
are merged into one single big cluster (Subasi 2020). Here we have used the t-SNE algorithm to
297
project our n-dimensional data on a two-dimension map (see fig 9a) and increase the divergence
298
of potential clusters. Then, the agglomerative technique has been employed over the projected
299
data to find the clusters. Similar process has been repeated by applying KMeans clustering
300
algorithm (see fig S3; Online Resource 1). However, based on the density map, the cluster
301
borders have been better recognized by the agglomerative technique. 302 the most similarity are merged into a bigger cluster and this process continues until all objects
296
are merged into one single big cluster (Subasi 2020). Here we have used the t-SNE algorithm to
297
project our n-dimensional data on a two-dimension map (see fig 9a) and increase the divergence
298
of potential clusters. Then, the agglomerative technique has been employed over the projected
299
data to find the clusters. Similar process has been repeated by applying KMeans clustering
300
algorithm (see fig S3; Online Resource 1). However, based on the density map, the cluster
301
borders have been better recognized by the agglomerative technique. 302
In order to find independent properties as the input for the clustering algorithm, the
303
correlations between all pairs of the storm-object properties have been calculated and the pairs
304
with correlation higher than 0.5 are considered as dependent variables and haven’t been used
305
together as the input in the clustering algorithm. Note that all input data are normalized (ranging
306
from 0-1) by dividing each input storm property by its maximum and standard deviation. 4.3. Statistics
321 F1, n an
341
F2,m refer to empirical distribution functions for both variables, and sup is the supremum
342
function. Note that the supremum function of a subset S of a set K is the least element in K th
343
is greater than or equal to all elements of S. 344
In order to model the relationship between two or more variables, in this research, w
345
have employed the Ordinary least squares (OLS) method:
346
347
𝑦= 𝛽𝑋
348
(8)
349
𝛽= 𝑎𝑟𝑔𝑚𝑖𝑛𝑏𝜖𝑅𝑝 𝑆(𝑏)
350
(9)
351
𝑆(𝑏) = (𝑦−𝑋𝑏)𝑇(𝑦−𝑋𝑏)
(10)
352
353
Where n is the number of observations, p is the number of independent variables, y is th
354
n×1 matrix of the dependent parameter, X is the n×p matrix of independent variables. 355
Suppose b is a "candidate" value for the parameter β which is the n×1 matrix o
356
coefficients for independent variables. 357
An effort has been made in this study to investigate the relationships between climat
358
indices (i.e., El Niño–Southern Oscillation (ENSO), Indian Ocean Dipole (IOD), and Souther
359
Annular Mode (SAM)) and storm properties in each cluster (derived from the previous section
360
Since precipitation can be correlated with more than one index, and the indices are ofte
361
correlated with each other (Maher and Sherwood 2014; Taschetto et al. 2011; Mekanik et a
362
2013; Mekanik and Imteaz 2012), we have utilized a multivariate approach rather than
363
bivariate approach to consider the dependency of the climate modes on each other. Equation 1
364
is the model representing the relationship between the three selected indices and each objec
365
based storm properties (in each cluster) for every month using a multiple linear analysis t
366
consider the dependency of the selected indices and include the annual variations. 367
368
𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀
(11)
369
370 (5) (5) (7) (7) Where Dn,m denotes the KS statistic and n,m are the sizes of the two datasets. F1, n and
F2,m refer to empirical distribution functions for both variables, and sup is the supremum
function. Note that the supremum function of a subset S of a set K is the least element in K that
is greater than or equal to all elements of S. 4.3. Statistics
321 Here we employed the non-parametric Kendall’s Tau rank correlation coefficient (tau) to
322
investigate the strength of relationships between variables. This value is derived from the
323
following equation:
324
325
𝑡𝑎𝑢=
𝐶−𝐷
𝐶+𝐷
(4)
326
327
In this equation, C is the number of matched pairs and D is the number of mismatched
328
pairs of the two variables. 329
To determine the significance of the difference between the two data distributions, the
330
nonparametric statistical Kolmogorov-Smirnov test (KS-test) has been employed. This technique
331
is a non-parametric test which doesn’t need the input data distributions to be normal. The null
332 Here we employed the non-parametric Kendall’s Tau rank correlation coefficient (tau) to
322
investigate the strength of relationships between variables. This value is derived from the
323
following equation:
324 𝑡𝑎𝑢=
𝐶−𝐷
𝐶+𝐷
(4) 𝑡𝑎𝑢=
𝐶−𝐷
𝐶+𝐷 (4) In this equation, C is the number of matched pairs and D is the number of mismatched
328
pairs of the two variables. 329 In this equation, C is the number of matched pairs and D is the number of mismatched
328
pairs of the two variables. 329 To determine the significance of the difference between the two data distributions, the
330
nonparametric statistical Kolmogorov-Smirnov test (KS-test) has been employed. This technique
331
is a non-parametric test which doesn’t need the input data distributions to be normal. The null
332 10 hypothesis in this test is that both samples come from a population with the same distribution. 333
This null hypothesis is rejected at the level of ⍺ if:
334
335 hypothesis in this test is that both samples come from a population with the same distribution. 333
This null hypothesis is rejected at the level of ⍺ if:
334
335 hypothesis in this test is that both samples come from a population with the same distribution. 333
This null hypothesis is rejected at the level of ⍺ if:
334
335 𝐷𝑛,𝑚> 𝐶(𝛼)√
𝑛+𝑚
𝑛×𝑚
𝐷𝑛,𝑚= 𝑠𝑢𝑝𝑥|𝐹1,𝑛(𝑥) −𝐹2.𝑚(𝑥)|
(6)
𝐶𝛼= √−𝑙𝑛(
𝛼
2) ×
1
2 𝐷𝑛,𝑚> 𝐶(𝛼)√
𝑛+𝑚
𝑛×𝑚
(5)
336
𝐷𝑛,𝑚= 𝑠𝑢𝑝𝑥|𝐹1,𝑛(𝑥) −𝐹2.𝑚(𝑥)|
337
(6)
338
𝐶𝛼= √−𝑙𝑛(
𝛼
2) ×
1
2
(7)
339
340 𝐷𝑛,𝑚> 𝐶(𝛼)√
𝑛+𝑚
𝑛×𝑚
(5)
336
𝐷𝑛,𝑚= 𝑠𝑢𝑝𝑥|𝐹1,𝑛(𝑥) −𝐹2.𝑚(𝑥)|
337
(6)
338
𝐶𝛼= √−𝑙𝑛(
𝛼
2) ×
1
2
(7)
339
340
Where Dn,m denotes the KS statistic and n,m are the sizes of the two datasets. 4.3. Statistics
321 In order to model the relationship between two or more variables, in this research, we
have employed the Ordinary least squares (OLS) method: 𝑦= 𝛽𝑋
(8)
𝛽= 𝑎𝑟𝑔𝑚𝑖𝑛𝑏𝜖𝑅𝑝 𝑆(𝑏)
(9)
𝑆(𝑏) = (𝑦−𝑋𝑏)𝑇(𝑦−𝑋𝑏)
(10) 𝑦= 𝛽𝑋
(8)
𝛽= 𝑎𝑟𝑔𝑚𝑖𝑛𝑏𝜖𝑅𝑝 𝑆(𝑏)
(9)
𝑆(𝑏) = (𝑦−𝑋𝑏)𝑇(𝑦−𝑋𝑏) (10) Where n is the number of observations, p is the number of independent variables, y is the
354
n×1 matrix of the dependent parameter, X is the n×p matrix of independent variables. 355 Where n is the number of observations, p is the number of independent variables, y is the
354
n×1 matrix of the dependent parameter, X is the n×p matrix of independent variables. 355 Suppose b is a "candidate" value for the parameter β which is the n×1 matrix of
356
coefficients for independent variables. 357 An effort has been made in this study to investigate the relationships between climate
358
indices (i.e., El Niño–Southern Oscillation (ENSO), Indian Ocean Dipole (IOD), and Southern
359
Annular Mode (SAM)) and storm properties in each cluster (derived from the previous section). 360
Since precipitation can be correlated with more than one index, and the indices are often
361
correlated with each other (Maher and Sherwood 2014; Taschetto et al. 2011; Mekanik et al. 362
2013; Mekanik and Imteaz 2012), we have utilized a multivariate approach rather than a
363
bivariate approach to consider the dependency of the climate modes on each other. Equation 11
364
is the model representing the relationship between the three selected indices and each object-
365
based storm properties (in each cluster) for every month using a multiple linear analysis to
366
consider the dependency of the selected indices and include the annual variations. 367 𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀
(11) (11) 𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀 𝑆𝑡𝑜𝑟𝑚 𝑃𝑟𝑜𝑝. = 𝐶0 + 𝐶1 × 𝑁𝑖ñ𝑜 3.4 + 𝐶2 × 𝐼𝑂𝐷 + 𝐶3 × 𝑆𝐴𝑀 11 Where C0 is the Month constant and C1, C2, C3 are Niño 3.4, IOD and SAM coefficients,
371
respectively. Note that, in this equation, each object-based property at every timestep is matched
372
with its monthly climate indices. 4.3. Statistics
321 373
The monthly time series of Niño 3.4, DMI and AAO from NOAA, which refer to ENSO,
374
IOD and Southern Annular Mode (SAM) are accessible from the NOAA Physical Sciences
375
Laboratory (information available online at https://psl.noaa.gov/data/climateindices/list/)
376
4. Results
377
378
The object-based storm properties are compared in different seasons in section 5.1
379
followed by the detailed analyses of the storms originating on land and ocean. Then, the diurnal
380
cycles of storm/storm-object properties are analyzed in section 5.2. Finally, in section 5.3 the
381
main contributing storms with similar object-based properties are clustered using clustering
382
analysis, and the effect of climate variability (i.e., ENSO, IOD and SAM) on these clusters is
383
investigated. Note that by applying the object-based technique over the study period, 1,218,787
384
storm-objects and 35,445 storms have been identified. From the detected storm-objects, 252,636,
385
277406, 371389 and 317356 objects are detected in spring (SON), summer (DJF), autumn
386
(MAM) and winter (JJA), respectively. The corresponding numbers for storms are 8233, 8091,
387
10080, 9041. 388 Where C0 is the Month constant and C1, C2, C3 are Niño 3.4, IOD and SAM coefficients,
371
respectively. Note that, in this equation, each object-based property at every timestep is matched
372
with its monthly climate indices. 373 The monthly time series of Niño 3.4, DMI and AAO from NOAA, which refer to ENSO,
374
IOD and Southern Annular Mode (SAM) are accessible from the NOAA Physical Sciences
375
Laboratory (information available online at https://psl.noaa.gov/data/climateindices/list/)
376 12 5.1. Seasonal analyses of storm/storm-object properties 5.1. Seasonal analyses of storm/storm-object properties 5.1. Seasonal analyses of storm/storm-object properties 389 5.1. Seasonal analyses of storm/storm-object properties
Fig. 2 Storm/storm-object property distributions for different seasons. Note that the bins in the x-
axis are equally spaced in the logarithmic scale except panels e-l. Panels e, h and l are also
presented in polar coordinates. Green, red, brown and blue lines refer to spring, summer, autumn
and winter, respectively. The y-axis in cartesian plots and radius in polar plots show the
normalized frequency ranging from 0-100. Note that storm direction in panel (l) refers to the
direction of the storm motion, and all the angles shown in panels e, h and l are measured from
the positive direction of the x-axis. Figure 2 represents the PDFs of the storm and storm-object properties in different seasons
extracted from the radar data. Storms in summer and spring tend to move towards the east-
southeast (fig 2i) and are larger in size (fig 2a 2p) and volume (fig 2p 2q) compared to the Fig. 2 Storm/storm-object property distributions for different seasons. Note that the bins in the x-
axis are equally spaced in the logarithmic scale except panels e-l. Panels e, h and l are also
presented in polar coordinates. Green, red, brown and blue lines refer to spring, summer, autumn
and winter, respectively. The y-axis in cartesian plots and radius in polar plots show the
normalized frequency ranging from 0-100. Note that storm direction in panel (l) refers to the
direction of the storm motion, and all the angles shown in panels e, h and l are measured from
the positive direction of the x-axis. Figure 2 represents the PDFs of the storm and storm-object properties in different seasons
98
extracted from the radar data. Storms in summer and spring tend to move towards the east-
99
southeast (fig 2i), and are larger in size (fig 2a, 2p) and volume (fig 2p, 2q) compared to the
00
autumn and winter storms which usually move northward. Comparing the mode of PDFs of
01 13 rainfall intensity (fig 2c, 2d, 2m, 2o) shows that typical storms in autumn are more intense
02
compared to the other seasons. 5.1. Seasonal analyses of storm/storm-object properties However, extreme rain intensity is higher the warmer the season,
03
since storms with maximum intensity above 40 mm/hr (top 10% of storms in maximum
04
intensity) during spring, summer, autumn and winter have occurred 1194, 1662, 1182 and 441
05
times, respectively, during the study timeframe. 06 rainfall intensity (fig 2c, 2d, 2m, 2o) shows that typical storms in autumn are more intense
402
compared to the other seasons. However, extreme rain intensity is higher the warmer the season,
403
since storms with maximum intensity above 40 mm/hr (top 10% of storms in maximum
404
intensity) during spring, summer, autumn and winter have occurred 1194, 1662, 1182 and 441
405
times, respectively, during the study timeframe. 406
In autumn, storms tend to move slower (fig 2f, 2n) and look more symmetric (fig 2i) than
407
in other seasons. In summer, storm objects are mostly oriented near 315∘ from the positive x-axis
408
while in autumn and spring the object orientation angles mostly change to 330∘ and in winter
409
storm objects are mostly oriented west-east (fig 2h). Along with having larger and more severe
410
storms in summer than in winter, summertime storms often include more contributing objects
411
and split/merge events during their lifetimes (fig 2j, 2k). Although all of the mentioned
412
differences are statistically significant based on Kolmogorov–Smirnov test, some storm
413
properties clearly vary with seasons such as rainfall intensity and storm direction. However,
414
there are some properties that look about the same in all four seasons like storm-object areas (fig
415
2a). Note that the differences between the storm size in different seasons are clearer in the storm
416
area and volume PDFs (fig 2p, 2q). 417
418 rainfall intensity (fig 2c, 2d, 2m, 2o) shows that typical storms in autumn are more intense
402
compared to the other seasons. However, extreme rain intensity is higher the warmer the season,
403
since storms with maximum intensity above 40 mm/hr (top 10% of storms in maximum
404
intensity) during spring, summer, autumn and winter have occurred 1194, 1662, 1182 and 441
405
times, respectively, during the study timeframe. 406
In autumn, storms tend to move slower (fig 2f, 2n) and look more symmetric (fig 2i) than
407
in other seasons. 5.1. Seasonal analyses of storm/storm-object properties In summer, storm objects are mostly oriented near 315∘ from the positive x-axis
408
while in autumn and spring the object orientation angles mostly change to 330∘ and in winter
409
storm objects are mostly oriented west-east (fig 2h). Along with having larger and more severe
410
storms in summer than in winter, summertime storms often include more contributing objects
411
and split/merge events during their lifetimes (fig 2j, 2k). Although all of the mentioned
412
differences are statistically significant based on Kolmogorov–Smirnov test, some storm
413
properties clearly vary with seasons such as rainfall intensity and storm direction. However,
414
there are some properties that look about the same in all four seasons like storm-object areas (fig
415
2a). Note that the differences between the storm size in different seasons are clearer in the storm
416
area and volume PDFs (fig 2p, 2q). 417
418 In autumn, storms tend to move slower (fig 2f, 2n) and look more symmetric (fig 2i) than
407
in other seasons. In summer, storm objects are mostly oriented near 315∘ from the positive x-axis
408
while in autumn and spring the object orientation angles mostly change to 330∘ and in winter
409
storm objects are mostly oriented west-east (fig 2h). Along with having larger and more severe
410
storms in summer than in winter, summertime storms often include more contributing objects
411
and split/merge events during their lifetimes (fig 2j, 2k). Although all of the mentioned
412
differences are statistically significant based on Kolmogorov–Smirnov test, some storm
413
properties clearly vary with seasons such as rainfall intensity and storm direction. However,
414
there are some properties that look about the same in all four seasons like storm-object areas (fig
415
2a). Note that the differences between the storm size in different seasons are clearer in the storm
416
area and volume PDFs (fig 2p, 2q). 417
418 14 Fig. 3 PDFs of storm properties for storms originating on land (solid lines) and ocean
(dashed lines). All differences between land and ocean distributions are statistically significant
based on the Kolmogorov–Smirnov test. NL indicates the number of storms originating on land
and NO refers to the storms originating on the ocean. 419
420
421
422
423
424
425
426
427
428
429
430
431 Fig. 3 PDFs of storm properties for storms originating on land (solid lines) and ocean
(dashed lines). All differences between land and ocean distributions are statistically significant
based on the Kolmogorov–Smirnov test. NL indicates the number of storms originating on land
and NO refers to the storms originating on the ocean. Fig. 3 PDFs of storm properties for storms originating on land (solid lines) and ocean
(dashed lines). All differences between land and ocean distributions are statistically significant
based on the Kolmogorov–Smirnov test. NL indicates the number of storms originating on land
and NO refers to the storms originating on the ocean. We have also compared the typical and extreme storms’ properties originating on land vs. 426
ocean in different seasons by comparing their PDFs shown in figure 3. The results show that
427
storms in spring and summer mostly initiate over land. However, in autumn and winter storms
428
often originate over the South Pacific Ocean. Land-originating storms are typically larger (fig 3a-
429
d) and move faster (fig 3n-q) than ocean-originating storms in all seasons. In summer and spring,
430
typical land-originating storms have higher maximum (fig 3j-k) and average precipitation
431 15 intensity (fig 3e-f), which is also true for extreme land-originating storms during autumn
compared to their counterparts originating on the ocean. However, typical ocean storms in
autumn and also winter (in terms of precipitation intensity) have slightly higher rain rates
compared with storms originating on land. Therefore, typical ocean-originating storms are more
spatially concentrated with higher rainrate and smaller areas compared to land-originating
storms. In summer and spring, both types of storms (land and ocean) mostly move towards the
east-southeast. However, in autumn and winter, they have different directions. Land storms still
tend to move towards east-southeast, but ocean-storms usually move northward. 440
441
442
Fig. 4 PDFs of storm snapshot properties originating on land and transitioning to the ocean
443
(panels a-d) and originating on the ocean and hitting the land (panels e-l). Solid and dashed lines
444 Fig. 4 PDFs of storm snapshot properties originating on land and transitioning to the ocean
443
(panels a-d) and originating on the ocean and hitting the land (panels e-l). Solid and dashed lines
444 16 are related to the part of the storms that are over land and over the ocean, respectively. 419
420
421
422
423
424
425
426
427
428
429
430
431 St_no
445
refers to the number of storms that start from land and reach the ocean or vice-versa, and
446
St_snp_L and St_snp_O refer to the number of storm snapshots over the land and the ocean,
447
respectively. All differences between land and ocean distributions are statistically significant
448
based on the Kolmogorov–Smirnov test. 449
450
Next, we examine how storm properties change during the transition of storms between
451
land and ocean. Land-originating storms during summer/spring have higher max intensity over
452
land than later over the ocean (fig 4a, b). However, during the autumn and winter, these
453
differences are smaller (fig 4c, d). Summer and spring storms originating over the ocean and
454
reaching land tend to be smaller (fig 4e, f) without much change in rainfall intensity (fig 4i, j). In
455
addition, autumn and winter ocean-originating storms (that reach the land) are also more
456
spatially concentrated (with higher max intensity (fig 4k, l) and smaller sizes) when they are
457
raining over the ocean compared to when they are over land (fig 4g, h). 458
459 are related to the part of the storms that are over land and over the ocean, respectively. St_no
445
refers to the number of storms that start from land and reach the ocean or vice-versa, and
446
St_snp_L and St_snp_O refer to the number of storm snapshots over the land and the ocean,
447
respectively. All differences between land and ocean distributions are statistically significant
448
based on the Kolmogorov–Smirnov test. 449 445
446
447
448
449
450
451
452
453
454
455
456
457
458
459 are related to the part of the storms that are over land and over the ocean, respectively. St_no
445
refers to the number of storms that start from land and reach the ocean or vice-versa, and
446
St_snp_L and St_snp_O refer to the number of storm snapshots over the land and the ocean,
447
respectively. All differences between land and ocean distributions are statistically significant
448
based on the Kolmogorov–Smirnov test. 449
450
Next, we examine how storm properties change during the transition of storms between
451
land and ocean. Land-originating storms during summer/spring have higher max intensity over
452
land than later over the ocean (fig 4a, b). However, during the autumn and winter, these
453
differences are smaller (fig 4c, d). Summer and spring storms originating over the ocean and
454
reaching land tend to be smaller (fig 4e, f) without much change in rainfall intensity (fig 4i, j). In
455
addition, autumn and winter ocean-originating storms (that reach the land) are also more
456
spatially concentrated (with higher max intensity (fig 4k, l) and smaller sizes) when they are
457
raining over the ocean compared to when they are over land (fig 4g, h). 458
459 Next, we examine how storm properties change during the transition of storms between
451
land and ocean. Land-originating storms during summer/spring have higher max intensity over
452
land than later over the ocean (fig 4a, b). However, during the autumn and winter, these
453
differences are smaller (fig 4c, d). Summer and spring storms originating over the ocean and
454
reaching land tend to be smaller (fig 4e, f) without much change in rainfall intensity (fig 4i, j). In
455
addition, autumn and winter ocean-originating storms (that reach the land) are also more
456
spatially concentrated (with higher max intensity (fig 4k, l) and smaller sizes) when they are
457
raining over the ocean compared to when they are over land (fig 4g, h). 458
459 17 460
461 Fig. 5 Average rainfall intensity variation during the storm lifetimes (represented by
seven bins) for storms originating on land (red) and ocean (blue). Only storms with more than
seven timesteps have been selected in these plots. Note that all the storms with more than 7
timesteps are normalized (averaged) into seven bins (timestep). NL and NO refer to the number
of selected storms over land and ocean, respectively. 445
446
447
448
449
450
451
452
453
454
455
456
457
458
459 The shaded areas show the 5 to 95
percentile range and the dashed/solid lines are the medians. Fig. 5 Average rainfall intensity variation during the storm lifetimes (represented by
seven bins) for storms originating on land (red) and ocean (blue). Only storms with more than
seven timesteps have been selected in these plots. Note that all the storms with more than 7
timesteps are normalized (averaged) into seven bins (timestep). NL and NO refer to the number
of selected storms over land and ocean, respectively. The shaded areas show the 5 to 95
percentile range and the dashed/solid lines are the medians. Fig. 5 Average rainfall intensity variation during the storm lifetimes (represented by
2
seven bins) for storms originating on land (red) and ocean (blue). Only storms with more than
3
seven timesteps have been selected in these plots. Note that all the storms with more than 7
4
timesteps are normalized (averaged) into seven bins (timestep). NL and NO refer to the number
5
of selected storms over land and ocean, respectively. The shaded areas show the 5 to 95
6
percentile range and the dashed/solid lines are the medians. 7 Precipitation intensity varies during the lifetime of the storms, as shown in Fig. 5. Storm
intensity generally peaks early in the storm lifetime during all seasons, though more clearly in
the summer. In all seasons excluding winter, ocean-originating storms, on average, have lower
average intensity during their lifetimes compared to their land counterparts. However, the 18 opposite is true in winter, when ocean-originating storms (on average) are more intense. In
473
addition, land-originating storms have a wider range of variability during their lifetimes
474
compared to ocean-originating storms and this range of variability is highest during summer and
475
lowest during the winter. 476 5.2. Diurnal cycle of storm/storm-object properties
477 5.2. Diurnal cycle of storm/storm-object properties
477
478 5.2. Diurnal cycle of storm/storm-object properties
7
8
9
0
Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for
1
different seasons (panels e-j). The results are also presented over land and ocean for different
2
seasons in panels k-z. NL and NO show the number of storm objects in each season over land
3 5.2. Diurnal cycle of storm/storm object properties
Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for
different seasons (panels e-j). 445
446
447
448
449
450
451
452
453
454
455
456
457
458
459 The results are also presented over land and ocean for different
seasons in panels k-z. NL and NO show the number of storm objects in each season over land 80
Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for
481
different seasons (panels e-j). The results are also presented over land and ocean for different
482
seasons in panels k-z. NL and NO show the number of storm objects in each season over land
483 Fig. 6 Diurnal cycle of storm-object properties overall (black; panels a-d) and for
481
different seasons (panels e-j). The results are also presented over land and ocean for different
482
seasons in panels k-z. NL and NO show the number of storm objects in each season over land
483 19 and ocean, respectively. The shaded area shows the range from 5-95% and the dashed/solid lines
4
show the median. 5 show the median. 485
The diurnal cycle varies with season and with the location of storms (land/ocean). 486
Extreme summertime storm-objects have an afternoon peak around 5:00 UTC (15:00 Australian
487
Eastern Standard Time; AEST) in terms of size (fig 6e) and intensity (fig 6f, g). However, winter
488
storms don’t have such a peak during the day (fig 6e-f). Note that the afternoon peak intensity
489
(which exists for all seasons except winter) is mostly related to intense storm-objects over land. 490
In all seasons except winter, high-intensity storm-objects over the ocean are more intense around
491
10:00-23:00 UTC (20:00-09:00 AEST; fig 6l, 6p, 6t, 6m, 6n and 6q) compared to their land
492
counterparts, but in the afternoon and evening, the opposite is true. During spring, summer and
493
autumn, fast-moving ocean storm-objects move faster than land storm-objects during 0:00-10:00
494
UTC (10:00-20:00 AEST) with a peak around 5:00 UTC (15:00 AEST; fig 6n and 6r). However,
495
during the other times of the day, extreme land storm-objects have faster translation speeds. Note
496
that in winter, these peaks are less clear and land storm-objects are mostly faster than their ocean
497
counterparts (fig 6z). 498 521 5.3. Clustering analysis
499 Using clustering algorithms, we have grouped the storms with similar object-based
500
properties over the study area. Here, we employed the Agglomerative clustering technique over
501
the projected multi-dimensional storm data on a 2D map using t-SNE algorithm (more details in
502
section 4.2). The selected input storm properties in the clustering algorithm should not be highly
503
dependent on each other. Therefore, we have calculated the correlation between all pairs of the
504
studied storm-object properties to identify the dependent properties. Figure 7 shows the
505
correlated properties at the significance level of 0.01. By considering a threshold of 0.5 for
506
correlation coefficient, we have found that area vs. volume discharge (fig 7a) and maximum
507
intensity vs. average intensity (fig 7h) are highly dependent on each other and should not be used
508
together as the input in the clustering algorithm. Based on this analysis, the selected input
509
properties for the clustering algorithm are: 1) storm area, 2) storm translation speed, 3) storm
510
max intensity and 4) storm direction which is decomposed into x and y components and have
511
been considered as two independent input variables in the clustering algorithm. 512 The bi-variate histograms in figure 7 also show that storm-objects with higher intensities
513
are generally larger in size and volume (fig 7b, 7c, 7f and 7g). In addition, when the size of the
514
storm-objects increases, the shape of the storm-objects (on average) tends to be more linear (fig
515
7d, 7e). 516 20 Fig. 7 Bivariate histograms for storm-object properties. Tau (correlation coeffici
in the figures are calculated based on Kendall’s tau method Fig. 7 Bivariate histograms for storm-object properties. Tau (correlation coefficient) and
519
P-value in the figures are calculated based on Kendall’s tau method
520
521 Fig. 7 Bivariate histograms for storm-object properties. Tau (correlation coefficient) and
519
P-value in the figures are calculated based on Kendall’s tau method
520 g
g
j
p
p
(
)
P-value in the figures are calculated based on Kendall’s tau method
520 521 21 522
523
Fig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colour on
524
this map shows the density. Panel (b) shows the variation of CHI with the number of clusters. 525
Panels (c) represents the clustered groups of the 2d map (panel a) using the Agglomerative
526
method. 5.3. Clustering analysis
499 They are frequent during the whole year with a frequency peak in
540
spring
541
Type 2 (T2) storms often move south-eastward and include the fastest and largest storms
539
but with low rainfall intensity. They are frequent during the whole year with a frequency peak in
540
spring
541
Type 3 (T3) storms mostly occur during winter with a frequency peak in June, mostly
542
moving northward over the ocean, and include the very slow storms with the smallest size and
543
low intensity compared to the other types. 544
Type 4 (T4) includes the most extreme storm in terms of rainfall intensity and often
545
appears in large sizes moving eastward with low translation speed. They mostly occur during the
546
summer. 547
Type 5 (T5) storms mostly include very fast storms with small sizes and low rainfal
548
intensities that often occur during the winter, mostly moving northward (over the ocean). 549
To further demonstrate the characteristics of each cluster, a video representing the typica
550
storm for each cluster is provided in the supplemental material (Online Resources 2). 551
552
553
554
Fig. 9 Selected regression coefficients for the relationship between climate mode indices
555
and the object-based storm properties in different months. The detail of selection criteria is
556
explained in the manuscript. The coloured bars show the normalized coefficients that are
557
significantly different from zero. Note that the colours represent the storm groups and are
558
matched with the groups’ colours in figure 8. 559
560
We also investigated whether significant relationships exist between storm properties and
561
climate mode indices. Although no statistically significant relationships were found when
562
investigating all storms, we have found some significant relationships between climate indices
563
5
Type 3 (T3) storms mostly occur during winter with a frequency peak in June, mostly
542
moving northward over the ocean, and include the very slow storms with the smallest size and
543
low intensity compared to the other types. 544
Type 3 (T3) storms mostly occur during winter with a frequency peak in June, mostly
542
moving northward over the ocean, and include the very slow storms with the smallest size and
543
low intensity compared to the other types. 5.3. Clustering analysis
499 544
Type 4 (T4) includes the most extreme storm in terms of rainfall intensity and often
545
appears in large sizes moving eastward with low translation speed. They mostly occur during the
546
summer. 547
Type 5 (T5) storms mostly include very fast storms with small sizes and low rainfall
548
intensities that often occur during the winter, mostly moving northward (over the ocean). 549
Type 5 (T5) storms mostly include very fast storms with small sizes and low rainfall
548
intensities that often occur during the winter, mostly moving northward (over the ocean). 549 To further demonstrate the characteristics of each cluster, a video representing the typical
550
storm for each cluster is provided in the supplemental material (Online Resources 2). 551 553
554
Fig. 9 Selected regression coefficients for the relationship between climate mode indices
555
and the object-based storm properties in different months. The detail of selection criteria is
556
explained in the manuscript. The coloured bars show the normalized coefficients that are
557
significantly different from zero. Note that the colours represent the storm groups and are
558
matched with the groups’ colours in figure 8. 559 553
554
Fi
9 S l
t d
i
ffi i
t f
th
l ti
hi
b t
li
t
d i di
555 Fig. 9 Selected regression coefficients for the relationship between climate mode indices
and the object-based storm properties in different months. The detail of selection criteria is
explained in the manuscript. The coloured bars show the normalized coefficients that are
significantly different from zero. Note that the colours represent the storm groups and are
matched with the groups’ colours in figure 8. Fig. 9 Selected regression coefficients for the relationship between climate mode indices
and the object-based storm properties in different months. The detail of selection criteria is
explained in the manuscript. The coloured bars show the normalized coefficients that are
significantly different from zero. Note that the colours represent the storm groups and are
matched with the groups’ colours in figure 8. Fig. 9 Selected regression coefficients for the relationship between climate mode indices
and the object-based storm properties in different months. The detail of selection criteria is
explained in the manuscript. The coloured bars show the normalized coefficients that are
significantly different from zero. 5.3. Clustering analysis
499 Panels (d-h) show the PDFs of different properties for the clustered storms. The annual
527
cycle of each cluster is shown in panel (i). 528
529 ig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colo Fig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colour on
this map shows the density. Panel (b) shows the variation of CHI with the number of clusters. Panels (c) represents the clustered groups of the 2d map (panel a) using the Agglomerative
method. Panels (d-h) show the PDFs of different properties for the clustered storms. The annual
cycle of each cluster is shown in panel (i). Fig. 8 Panel (a) is the projected 5-dimensional storm data on a 2D map. The colour on
4
this map shows the density. Panel (b) shows the variation of CHI with the number of clusters. 5
Panels (c) represents the clustered groups of the 2d map (panel a) using the Agglomerative
6
method. Panels (d-h) show the PDFs of different properties for the clustered storms. The annual
7
cycle of each cluster is shown in panel (i). 8 The results show that there are five storm clusters (types) with similar object-based
properties occurring over the study area. This number is based on the optimum value of CHI
against the number of clusters (fig 9b; for more details see section 4.2.2). Figure 8d-h shows the
PDFs of storm properties for different storm types with similar quantitative characteristics that
have been identified using the Agglomerative and t-SNE algorithms over the study area. Based
on these results, the detected storm types have the following characteristics:
Type 1 (T1) storms have a peak frequency in autumn and include mostly average size
36
storms with the lowest translation speeds but very high rainfall intensities compared to the other
37
groups. They often move towards the north (over the ocean) to the northwest (hit the land)
38 22
Type 2 (T2) storms often move south-eastward and include the fastest and largest storms
539
but with low rainfall intensity. They are frequent during the whole year with a frequency peak in
540
spring
541
Type 2 (T2) storms often move south-eastward and include the fastest and largest storms
539
but with low rainfall intensity. 5. Discussion
586 587
The results presented in Section 4.1 are broadly consistent with previous studies, but with
588
some notable exceptions. For example, during summer, storms are mostly larger, move faster
589
and are accompanied by higher rainfall intensities compared to the storms in winter (fig 2). This
590
is in agreement with the previous studies over Australia reporting that severe thunderstorms are
591
most prevalent in the warm season (Niall and Walsh 2005; Schuster et al. 2005; Davis and Walsh
592
2008; Warren et al. 2020). The extreme summer and spring storms in terms of rainfall intensity
593
are more intense when they are raining over land between 10:00 to 20:00 (AEST; peaking in the
594
afternoon) compared to when they are raining over the ocean, consistent with the diurnal cycle of
595
surface temperature and the maximum availability of heating over land. The diurnal peak of
596
severe thunderstorm over land during the warm season was also reported by Griffiths et al. 597
(1993) in studying the severe thunderstorms in New South Wales, Australia and Schuster et al
598
(2005) in studying the hail climatology of the Greater Sydney, Australia both using reported
599
data. 600
Although previous studies reported that thunderstorms are more prevalent during the
601
warm seasons, we have found that there are more storms during autumn and winter than spring
602
and summer. A reason behind this contradiction is that most previous studies employed ground
603
observations that provide information for storms only over land (Niall and Walsh 2005; Schuster
604
et al. 2005; Davis and Walsh 2008), and, based on our findings, autumn and winter storms
605
mostly initiate from the ocean and tend to move northward causing more precipitation over the
606 587
The results presented in Section 4.1 are broadly consistent with previous studies, but with
588
some notable exceptions. For example, during summer, storms are mostly larger, move faster
589
and are accompanied by higher rainfall intensities compared to the storms in winter (fig 2). This
590
is in agreement with the previous studies over Australia reporting that severe thunderstorms are
591
most prevalent in the warm season (Niall and Walsh 2005; Schuster et al. 2005; Davis and Walsh
592
2008; Warren et al. 2020). 𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡=
𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
(12)
581 Where σ is the standard deviation in this equation, “index coefficient” refers to any
583
calculated coefficient from equation 11 and “index normalized coefficient” is the partial
584
correlation between every index and storm property. 585 5.3. Clustering analysis
499 Note that the colours represent the storm groups and are
matched with the groups’ colours in figure 8. We also investigated whether significant relationships exist between storm properties and
561
climate mode indices. Although no statistically significant relationships were found when
562
investigating all storms, we have found some significant relationships between climate indices
563
and storm properties in each cluster (identified in the previous section) using a multiple
564
regression model described in section 4.3. Based on this approach, 75 regression models have
565
been produced (3 indices × 5 storm properties × 5 clusters). In order to identify robust
566
associations, we have identified instances when at least five coefficients in a row have the same
567
sign (positive or negative), and among them at least three are significantly different from zero. 568
Since these climate indices often have impacts on weather for a period from 3-9 months, this
569
restriction helps us to better exclude those short periods in which precipitation has a statistically
570
acceptable link with climate indices but probably not in reality. Thus, from all of the results, only
571 23 five regressions passed this criterion and are shown in figure 9. The results show that during El-
572
Niño events in cold seasons, T3 and T5 storms have negative correlation with ENSO in cold
573
season with lower rainfall intensity in El Niño and higher rainfall intensity in La-Niña (fig 9a,
574
9b). ENSO also has an impact on T1 and T3 storms' translation speed during the warm season
575
with a positive correlation (faster in El Niño and slower in La-Niña; fig 9c, 9d). Finally, IOD has
576
also shown to have a positive correlation with T1 storms’ translation speed from mid-summer to
577
early winter (Feb, Apr and Jun; fig 9e). Note that in figure 9, all the coefficients have been
578
normalized to derive the partial correlation between every index and storm property as below:
579
580
𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡=
𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
(12)
581
582
Where σ is the standard deviation in this equation, “index coefficient” refers to any
583
calculated coefficient from equation 11 and “index normalized coefficient” is the partial
584
correlation between every index and storm property. 585 five regressions passed this criterion and are shown in figure 9. 5.3. Clustering analysis
499 The results show that during El-
572
Niño events in cold seasons, T3 and T5 storms have negative correlation with ENSO in cold
573
season with lower rainfall intensity in El Niño and higher rainfall intensity in La-Niña (fig 9a,
574
9b). ENSO also has an impact on T1 and T3 storms' translation speed during the warm season
575
with a positive correlation (faster in El Niño and slower in La-Niña; fig 9c, 9d). Finally, IOD has
576
also shown to have a positive correlation with T1 storms’ translation speed from mid-summer to
577
early winter (Feb, Apr and Jun; fig 9e). Note that in figure 9, all the coefficients have been
578
normalized to derive the partial correlation between every index and storm property as below:
579
580
𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡=
𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
(12)
581
582
Where σ is the standard deviation in this equation, “index coefficient” refers to any
583
calculated coefficient from equation 11 and “index normalized coefficient” is the partial
584
correlation between every index and storm property. 585 five regressions passed this criterion and are shown in figure 9. The results show that during El-
572
Niño events in cold seasons, T3 and T5 storms have negative correlation with ENSO in cold
573
season with lower rainfall intensity in El Niño and higher rainfall intensity in La-Niña (fig 9a,
574
9b). ENSO also has an impact on T1 and T3 storms' translation speed during the warm season
575
with a positive correlation (faster in El Niño and slower in La-Niña; fig 9c, 9d). Finally, IOD has
576
also shown to have a positive correlation with T1 storms’ translation speed from mid-summer to
577
early winter (Feb, Apr and Jun; fig 9e). Note that in figure 9, all the coefficients have been
578
normalized to derive the partial correlation between every index and storm property as below:
579
580
𝑖𝑛𝑑𝑒𝑥 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡=
𝑖𝑛𝑑𝑒𝑥 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 × 𝜎 (𝑖𝑛𝑑𝑒𝑥 𝑣𝑎𝑙𝑢𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
𝜎 (𝑠𝑡𝑜𝑟𝑚 𝑝𝑟𝑜𝑝𝑒𝑟𝑡𝑖𝑒𝑠 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑚𝑜𝑛𝑡ℎ)
(12)
581
582
Wh
i
th
t
d d d
i ti
i
thi
ti
“i d
ffi i
t”
f
t
583 5. Discussion
586 616 We have calculated the average wind direction during the rainy days (based on radar
617
data) at 700 hPa and 850 hPa in ERA5 Reanalysis data (see fig S6 and S7; Online Resource 1) to
618
examine whether the storm directions are (on average) following the wind directions in different
619
seasons. The results show that typical storm direction in summer and spring seems to be in
620
agreement with climatological wind fields at 700 hPa in ERA5. This agreement seems to be even
621
better when compared with 850 hPa wind fields since the north westerly storm directions (fig 2e,
622
2l) during these seasons are better matched with the wind directions at this pressure level. 623
However, in autumn and winter, it doesn’t seem to agree well, especially over the ocean. Storms
624
in these seasons mostly move northward over the ocean (fig 2e, 2l). However, the average wind
625
directions in these seasons are south-westerly. A reason might be the climatological conditions during
626
the cold season are not supportive of isolated offshore showers/storms which are more frequent in these
627
seasons. However, further investigations are required by conducting a detailed comparison of wind
628
and storm directions. 629 In all seasons, land and ocean-originating storms tend to peak early in their lifetimes (fig
630
6) consistent with the findings of Ayat et. al. (2021b) and Prein et. al. (2017) over the United
631
States. Additionally, we have found that this peak is more prominent during the summer for
632
severe storms raining over land. Storm characteristics also change during the transition of storms
633
between land and ocean like the decrease in max intensity for summertime land-originating
634
storms when moving over the ocean and wintertime ocean-originating storms when moving over
635
land. These variations are probably related to a change in boundary layer instability in this
636
process, and shows the immediate effect of change in air mass characteristics (land/ocean) on the
637
storms. These characteristics can include surface temperature and humidity, sea/land breezes and
638
topographical interactions, the effects of elevated mixed layers advected over the coast, low-level
639
wind shear and convergence. 640 Using clustering analysis, we have found that there are five storm types with similar
641
object-based properties over this region and described in detail in section 5.3. 5. Discussion
586 The extreme summer and spring storms in terms of rainfall intensity
593
are more intense when they are raining over land between 10:00 to 20:00 (AEST; peaking in the
594
afternoon) compared to when they are raining over the ocean, consistent with the diurnal cycle of
595
surface temperature and the maximum availability of heating over land. The diurnal peak of
596
severe thunderstorm over land during the warm season was also reported by Griffiths et al. 597
(1993) in studying the severe thunderstorms in New South Wales, Australia and Schuster et al
598
(2005) in studying the hail climatology of the Greater Sydney, Australia both using reported
599
data. 600 Although previous studies reported that thunderstorms are more prevalent during the
601
warm seasons, we have found that there are more storms during autumn and winter than spring
602
and summer. A reason behind this contradiction is that most previous studies employed ground
603
observations that provide information for storms only over land (Niall and Walsh 2005; Schuster
604
et al. 2005; Davis and Walsh 2008), and, based on our findings, autumn and winter storms
605
mostly initiate from the ocean and tend to move northward, causing more precipitation over the
606
ocean than land. Thus, the previous studies have not captured the storms over the ocean in these
607
seasons. In addition, most of the previous studies were focused on the severe thunderstorms or
608
storms with deep convective clouds and high storm tops that are often accompanied by
609 24 electrification, which are less frequent during the cold seasons. Using lightning records, Dowdy
610
and Kuleshov (2014) also showed that a maximum in lightning activity during the cooler months
611
occurs over the ocean to the east of Australia, which is consistent with our results. However, they
612
reported a higher frequency of thunderstorms during the warm season. Since storms in cold
613
season are small-scale with low rainfall intensity, probably many of them are not accompanied
614
by lightning to be captured by the sensors. Therefore, a large number of storms over the ocean
615
during this season are probably missed in the mentioned study. 5. Discussion
586 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et
662
al. 2013; King et al. 2014; Ashcroft et al. 2019). While no relationship was found for all storms,
663
we have found that the rainfall intensity in T3 and T5 storm types (that are more frequent in
664
winter) decreases during these events (fig 9a, b). 665 We have found that there are relationships between some object-based storm properties
666
over the study area that have also been reported in previous studies over other regions. For
667
example, we find that storm objects with large volume and size tend to be more linear and are
668
accompanied by higher rainfall intensities (fig 7); this is consistent with the findings of Ayat et. 669
al. (2021b) in comparing a merged ground radar product and a merged satellite precipitation
670
product over the United States, and Prein et al. (2017) in simulating North American mesoscale
671
convective systems with a convection‑permitting climate model. Large-scale heavy storms over
672
the study area mostly fall in T2 and T4 storm categories, and typical T2 and T4 storms showed
673
that they are mostly frontal systems that elongated/oriented parallel to the front borders. 674 In summary, the results are showing that the storm intensity variations are consistent with
675
diurnal/seasonal cycles and are related to climate mode oscillations. However, other
676
characteristics of the storms like storm size and translation speed do not seem to always follow
677
the same relationship. This suggests that further investigations are required to find a more
678
definitive answer to the effect of atmospheric parameters variations (e.g. temperature, humidity,
679
etc.) on storm properties other than intensity. 680 5. Discussion
586 665
We have found that there are relationships between some object-based storm properties
666
over the study area that have also been reported in previous studies over other regions. For
667
example, we find that storm objects with large volume and size tend to be more linear and are
668
accompanied by higher rainfall intensities (fig 7); this is consistent with the findings of Ayat et. 669
al. (2021b) in comparing a merged ground radar product and a merged satellite precipitation
670
product over the United States, and Prein et al. (2017) in simulating North American mesoscale
671
convective systems with a convection‑permitting climate model. Large-scale heavy storms over
672
the study area mostly fall in T2 and T4 storm categories, and typical T2 and T4 storms showed
673
that they are mostly frontal systems that elongated/oriented parallel to the front borders. 674
In summary, the results are showing that the storm intensity variations are consistent with
675
diurnal/seasonal cycles and are related to climate mode oscillations. However, other
676
characteristics of the storms like storm size and translation speed do not seem to always follow
677
the same relationship. This suggests that further investigations are required to find a more
678
definitive answer to the effect of atmospheric parameters variations (e.g. temperature, humidity,
679
etc.) on storm properties other than intensity. 680 end of spring. This type of storm can be accompanied by severe winds. For instance, they
651
contributed to the storm that occurred on 8 January 2003, and brought a severe wind gust with a
652
maximum of 109 km/h. Finally, T4 storms mostly occur in summer and include the extreme
653
high-intensity storms that have very large sizes with slow translation speed. These characteristics
654
of the storms in this group can create devastating flash and riverine floods in this region (fig 8). 655
Their dominant contribution in the East Coast Low event on 1 and 2 February 2005 caused flash
656
flooding in Sydney with reports of 6 cm size hail. 657 Our results in investigating the relationships between climate mode indices and storm
658
properties are consistent with the previous studies over the eastern and southeastern parts of
659
Australia. Those studies have reported a reduction in rainfall intensity during El-Niño events in
660
the cold season (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008;
661
Nicholls et al. 5. Discussion
586 Among these
642
clusters, three storm types might be accompanied by natural disasters, due to their special
643
characteristics. The first storm type (T1) mostly includes the slowest storms that have high
644
rainfall intensities with small areas and mostly move towards the north-northwest with a peak
645
frequency in autumn. These storms can create flash floods if they hit the land like the high
646
impact event that occurred on 30 May 2011 with a dominant contribution of this type of storm
647
and led to flash flooding in some of Sydney's eastern suburbs (e.g., Zetland, Alexandria and
648
Kingsford). T2 storms mostly move towards the southeast and include the largest and fastest
649
storms but with very low rainfall intensity occurring in every season with a peak frequency at the
650 25 end of spring. This type of storm can be accompanied by severe winds. For instance, they
651
contributed to the storm that occurred on 8 January 2003, and brought a severe wind gust with a
652
maximum of 109 km/h. Finally, T4 storms mostly occur in summer and include the extreme
653
high-intensity storms that have very large sizes with slow translation speed. These characteristics
654
of the storms in this group can create devastating flash and riverine floods in this region (fig 8). 655
Their dominant contribution in the East Coast Low event on 1 and 2 February 2005 caused flash
656
flooding in Sydney with reports of 6 cm size hail. 657
Our results in investigating the relationships between climate mode indices and storm
658
properties are consistent with the previous studies over the eastern and southeastern parts of
659
Australia. Those studies have reported a reduction in rainfall intensity during El-Niño events in
660
the cold season (Chung and Power 2017; Risbey et al. 2009a,b; Murphy and Timbal 2008;
661
Nicholls et al. 1996; McBride and Nicholls 1983; Allan et al. 1996; Schepen et al. 2012; Min et
662
al. 2013; King et al. 2014; Ashcroft et al. 2019). While no relationship was found for all storms,
663
we have found that the rainfall intensity in T3 and T5 storm types (that are more frequent in
664
winter) decreases during these events (fig 9a, b). 6. Conclusion
681 Finally, T5 storms are wintertim
723
small scale storms over the ocean, moving northward with high translation speeds. We als
724
studied the connection between climate modes and storm properties for different clusters using
725
linear multivariate approach and the results show that during El-Niño events in cold seasons, T
726
and T5 storms have negative correlation with ENSO in cold season with lower rainfall intensit
727
in El Niño and higher rainfall intensity in La-Niña. In addition, ENSO has an impact on T1 an
728
T3 storms' translation speed during the warm season with a positive correlation (faster in El Niñ
729 The extreme storms in terms of size, intensity and translation speed are more frequent
690
during summer and spring. Storms in these seasons mostly originate on land, move towards the
691
east-southeast, are larger, faster and more intense compared to the storms originating on the
692
ocean. In these seasons, between ~10am and ~8pm (AEST), the extreme storms raining over land
693
(wherever they originate) are larger and have higher rain rate but slower compared to when they
694
are raining over the ocean (with a peak in the afternoon that is consistent with the diurnal
695
maximum of boundary layer instability). However, the opposite is true for storms later at night
696
into early morning. 697 Although severe storms are more frequent during summer and spring, typical storms in
698
autumn have higher rainfall intensity compared to the other seasons. In addition, the storms
699
(including non-severe ones) are more frequent in autumn and winter compared to summer and
700
spring. During the cold season, storms mostly initiate from the ocean and tend to move
701
northward, which causes more precipitation over the ocean than land. Ocean-originating storms
702
during these seasons like summer and spring are typically smaller and move slower but have
703
higher rainfall intensity than the land-originating storms which still tend to move east-
704
southeastward. 705 The results show that the change in the air mass characteristics (land/ocean) can
706
immediately affect the storm properties. For instance, the land-originating storms that cross to
707
the ocean in summer are more intense over land than ocean. However, in winter ocean
708
originating storms that can reach land are more intense over ocean than land. 6. Conclusion
681 In this study, we establish an object-based storm climatology using an S-band weather
682
radar located near Wollongong, NSW (34.26° S, 150.87° E, 471 m altitude) with more than 20
683
years of records (1996-2018). The study area is the radar coverage region (including land and
684
ocean), within 150 km of the radar. Here, we employed the Method of Object-based Diagnostic
685
Evaluation (MODE) Time Domain (MTD) to detect and track the storms. Using this object-
686
based approach helps us to better understand the climatology of storm properties (other than
687
rainfall intensity and frequency) that haven’t been explored in the previous studies over the study
688
area. 689 26 The extreme storms in terms of size, intensity and translation speed are more freque
690
during summer and spring. Storms in these seasons mostly originate on land, move towards th
691
east-southeast, are larger, faster and more intense compared to the storms originating on th
692
ocean. In these seasons, between ~10am and ~8pm (AEST), the extreme storms raining over lan
693
(wherever they originate) are larger and have higher rain rate but slower compared to when the
694
are raining over the ocean (with a peak in the afternoon that is consistent with the diurn
695
maximum of boundary layer instability). However, the opposite is true for storms later at nig
696
into early morning. 697
Although severe storms are more frequent during summer and spring, typical storms
698
autumn have higher rainfall intensity compared to the other seasons. In addition, the storm
699
(including non-severe ones) are more frequent in autumn and winter compared to summer an
700
spring. During the cold season, storms mostly initiate from the ocean and tend to mov
701
northward, which causes more precipitation over the ocean than land. Ocean-originating storm
702
during these seasons like summer and spring are typically smaller and move slower but hav
703
higher rainfall intensity than the land-originating storms which still tend to move eas
704
southeastward. 705
The results show that the change in the air mass characteristics (land/ocean) ca
706
immediately affect the storm properties. For instance, the land-originating storms that cross
707
the ocean in summer are more intense over land than ocean. However, in winter ocea
708
originating storms that can reach land are more intense over ocean than land. 6. Conclusion
681 These changes
709
storm properties during the storm lifetimes might be related to the differences in surfac
710
temperature and humidity, sea/land breezes and topographical interactions, the effects o
711
elevated mixed layers advected over the coast, low-level wind shear and convergence. Howeve
712
further research is needed to find a definitive answer. 713
We have found five types of storms with distinct object-based properties using clusterin
714
techniques. The first storm type (T1) peaks in autumn and mostly includes small-scale and slow
715
moving storms but with high rainfall intensities, often moving northward over the ocean. Th
716
type of storm has the potential to create flash floods when they move offshore. T2 storms are th
717
largest and fastest storms with low rainfall intensities, often moving southeastward with a pea
718
in spring. This storm type can be accompanied by severe gust winds. T3 storms, include th
719
smallest size storms moving northward with low intensities and translation speed and peak
720
winter. T4 storms include the most extreme storms in terms of rainfall intensity with large area
721
often moving slowly towards the east with a peak in summer, and can be a source of devastatin
722
flash and riverine floods over the study area in this season. Finally, T5 storms are wintertim
723
small scale storms over the ocean, moving northward with high translation speeds. We als
724
studied the connection between climate modes and storm properties for different clusters using
725
linear multivariate approach and the results show that during El-Niño events in cold seasons, T
726
and T5 storms have negative correlation with ENSO in cold season with lower rainfall intensi
727
in El Niño and higher rainfall intensity in La Niña In addition ENSO has an impact on T1 an
728 The extreme storms in terms of size, intensity and translation speed are more frequen
690
during summer and spring. Storms in these seasons mostly originate on land, move towards th
691
east-southeast, are larger, faster and more intense compared to the storms originating on th
692
ocean. In these seasons, between ~10am and ~8pm (AEST), the extreme storms raining over lan
693
(wherever they originate) are larger and have higher rain rate but slower compared to when the
694
are raining over the ocean (with a peak in the afternoon that is consistent with the diurna
695
maximum of boundary layer instability). 6. Conclusion
681 However, the opposite is true for storms later at nigh
696
into early morning. 697
Although severe storms are more frequent during summer and spring, typical storms i
698
autumn have higher rainfall intensity compared to the other seasons. In addition, the storm
699
(including non-severe ones) are more frequent in autumn and winter compared to summer an
700
spring. During the cold season, storms mostly initiate from the ocean and tend to mov
701
northward, which causes more precipitation over the ocean than land. Ocean-originating storm
702
during these seasons like summer and spring are typically smaller and move slower but hav
703
higher rainfall intensity than the land-originating storms which still tend to move east
704
southeastward. 705
The results show that the change in the air mass characteristics (land/ocean) ca
706
immediately affect the storm properties. For instance, the land-originating storms that cross t
707
the ocean in summer are more intense over land than ocean. However, in winter ocea
708
originating storms that can reach land are more intense over ocean than land. These changes i
709
storm properties during the storm lifetimes might be related to the differences in surfac
710
temperature and humidity, sea/land breezes and topographical interactions, the effects o
711
elevated mixed layers advected over the coast, low-level wind shear and convergence. Howeve
712
further research is needed to find a definitive answer. 713
We have found five types of storms with distinct object-based properties using clusterin
714
techniques. The first storm type (T1) peaks in autumn and mostly includes small-scale and slow
715
moving storms but with high rainfall intensities, often moving northward over the ocean. Thi
716
type of storm has the potential to create flash floods when they move offshore. T2 storms are th
717
largest and fastest storms with low rainfall intensities, often moving southeastward with a pea
718
in spring. This storm type can be accompanied by severe gust winds. T3 storms, include th
719
smallest size storms moving northward with low intensities and translation speed and peak i
720
winter. T4 storms include the most extreme storms in terms of rainfall intensity with large area
721
often moving slowly towards the east with a peak in summer, and can be a source of devastatin
722
flash and riverine floods over the study area in this season. 6. Conclusion
681 These changes in
709
storm properties during the storm lifetimes might be related to the differences in surface
710
temperature and humidity, sea/land breezes and topographical interactions, the effects of
711
elevated mixed layers advected over the coast, low-level wind shear and convergence. However,
712
further research is needed to find a definitive answer. 713 We have found five types of storms with distinct object-based properties using clustering
714
techniques. The first storm type (T1) peaks in autumn and mostly includes small-scale and slow-
715
moving storms but with high rainfall intensities, often moving northward over the ocean. This
716
type of storm has the potential to create flash floods when they move offshore. T2 storms are the
717
largest and fastest storms with low rainfall intensities, often moving southeastward with a peak
718
in spring. This storm type can be accompanied by severe gust winds. T3 storms, include the
719
smallest size storms moving northward with low intensities and translation speed and peak in
720
winter. T4 storms include the most extreme storms in terms of rainfall intensity with large areas
721
often moving slowly towards the east with a peak in summer, and can be a source of devastating
722
flash and riverine floods over the study area in this season. Finally, T5 storms are wintertime
723
small scale storms over the ocean, moving northward with high translation speeds. We also
724
studied the connection between climate modes and storm properties for different clusters using a
725
linear multivariate approach and the results show that during El-Niño events in cold seasons, T3
726
and T5 storms have negative correlation with ENSO in cold season with lower rainfall intensity
727
in El Niño and higher rainfall intensity in La-Niña. In addition, ENSO has an impact on T1 and
728
T3 storms' translation speed during the warm season with a positive correlation (faster in El Niño
729 27 and slower in La-Niña). Finally, IOD has also shown a positive correlation with T1 storms’
translation speed from mid-summer to early winter (Feb, Apr and Jun). Although previous studies using pixel-based approaches have provided us with valuable
2
information, the outcomes of this research show that the object-based approach can be a
powerful means to better understand other aspects of the storms like storm size, translation
4
speed, direction, aspect ratio, lifetime, track length, etc. 6. Conclusion
681 which was not possible using pixel-
5
based approach. In this study, we have conducted a comprehensive study in understanding the
6
storm climatology. However, this study is limited to a specific region in Australia, and further
7
research is needed to do the same study over the other regions or over a larger scale using
8
regional/global datasets. In addition, there are lots of questions unanswered in understanding the
9
storm structures and the effects of parameters like temperature, humidity, topography, surface
0
type, etc. on the storm properties other than rainfall intensity, which might be better explored
using an object-based approach. 2 Although previous studies using pixel-based approaches have provided us with valuable
732
information, the outcomes of this research show that the object-based approach can be a
733
powerful means to better understand other aspects of the storms like storm size, translation
734
speed, direction, aspect ratio, lifetime, track length, etc. which was not possible using pixel-
735
based approach. In this study, we have conducted a comprehensive study in understanding the
736
storm climatology. However, this study is limited to a specific region in Australia, and further
737
research is needed to do the same study over the other regions or over a larger scale using
738
regional/global datasets. In addition, there are lots of questions unanswered in understanding the
739
storm structures and the effects of parameters like temperature, humidity, topography, surface
740
type, etc. on the storm properties other than rainfall intensity, which might be better explored
741
using an object-based approach. 742
743
Declarations:
744
745
Funding: This work was supported by the Australian Research Council as part of the Center of
746
Excellence for Climate Extremes (CE170100023) for Jason P. Evans and Steven C. Sherwood. 747
748
Conflicts
of
interest/Competing
interests:
The
authors
declare
no
conflicts
of
749
interest/competing interests. 750
751
Availability of data and material: Radar data used in this study are available at
752
https://my.nci.org.au/mancini/project/rq0. 753
754
Code availability: The codes that support the findings of this study are available upon request
755
from the corresponding author. 756
757
References:
758
759
Ahmed, R., M. Latif, S. Adnan, and M. K. Abuzar, 2019: Thunderstorm frequency distribution
760
and associated convective mechanisms over Pakistan. Theor. Appl. Climatol., 137, 755–
761
773, https://doi.org/10.1007/s00704-018-2619-x. 762
Allan, R., J. Lindesay, and D. Parker, 1996: El Niño southern oscillation & climatic
763
variability. 6. Conclusion
681 CSIRO Publishing,. 764
Allen, J., D. Karoly, and G. Mills, 2011: A severe thunderstorm climatology for Australia and
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associated thunderstorm environments. Aust. Meteorol. Oceanogr. J., 61, 143–158. 766
Allen, J. T., 2018: Climate Change and Severe Thunderstorms. 767
——, and D. J. Karoly, 2014: A climatology of Australian severe thunderstorm environments
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1979-2011: Inter-annual variability and ENSO influence. Int. J. Climatol., 34, 81–97,
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https://doi.org/10.1002/joc.3667. 770
——, and E. R. Allen, 2016: A review of severe thunderstorms in Australia. Atmospheric Res.,
771 Declarations: Conflicts
of
interest/Competing
interests:
The
authors
declare
no
conflicts
of
749
interest/competing interests. 750
751 Code availability: The codes that support the findings of this study are available upon request
755
from the corresponding author. 756
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and associated convective mechanisms over Pakistan. Theor. Appl. Climatol., 137, 755–
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Allan, R., J. Lindesay, and D. Parker, 1996: El Niño southern oscillation & climatic
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variability. CSIRO Publishing,. 764
Allen, J., D. Karoly, and G. Mills, 2011: A severe thunderstorm climatology for Australia and
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associated thunderstorm environments. Aust. Meteorol. Oceanogr. J., 61, 143–158. 766
Allen, J. T., 2018: Climate Change and Severe Thunderstorms. 767
——, and D. J. Karoly, 2014: A climatology of Australian severe thunderstorm environments
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1979-2011: Inter-annual variability and ENSO influence. Int. J. Climatol., 34, 81–97,
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https://doi.org/10.1002/joc.3667. 770
——, and E. R. Allen, 2016: A review of severe thunderstorms in Australia. Atmospheric Res.,
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https://openalex.org/W2769087095
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https://europepmc.org/articles/pmc5705562?pdf=render
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English
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Revealing the Radial Effect on Orientation Discrimination by Manual Reaction Time
|
Frontiers in neuroscience
| 2,017
|
cc-by
| 8,825
|
*Correspondence:
Yujun Pan *Correspondence:
Yujun Pan
yujunpan@ems.hrbmu.edu.cn Revealing the Radial Effect on
Orientation Discrimination by Manual
Reaction Time Lixin Liang 1, Yang Zhou 2, 3, Mingsha Zhang 2 and Yujun Pan 1* 1 Department of Neurology, The First Clinical College of Harbin Medical University, Harbin, China, 2 State Key Laboratory of
Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China, 3 Department of Neurobiology, University of
Chicago, Chicago, IL, United States It has been shown that the sensitivity and accuracy of orientation perception in the
periphery is significantly better when the orientations are radial with respect to the
fixation point than when they are tangential. However, since perception and action may
be dissociated, it is unclear whether the perceptual radial effect has a counterpart in
reaction time (RT) of motor responses. Furthermore, it is unknown whether or how
stimulus-response-compatibility (SRC) effect interacts with the radial effect to determine
RT. To address these questions, we measured subjects’ manual RT to grating stimuli that
appeared across upper visual field (VF). We found that (1) RTs were significantly shorter
when a grating was oriented closer to the radial direction than when it was oriented closer
to the tangential direction even though the perceptual accuracies for the more radial
and more tangential orientations were not significantly different under our experimental
condition; (2) This RT version of the radial effect was larger in the left VF than in the right
VF; (3) The radial effect and SRC effect interacted with each other to determine the overall
RT. These results suggest that the RT radial effect reported here is not a passive reflection
of the radial effect in perceptual accuracy, but instead, represents different processing
time of radial and tangential orientations along the sensorimotor pathway. INTRODUCTION The radial effect refers to the observation that the sensitivity and accuracy of orientation perception
in the periphery is significantly better when a stimulus is radially oriented with respect to the
fixation point than when it is tangentially oriented (Rovamo et al., 1982; Bennett and Banks, 1991;
Westheimer, 2003; Sasaki et al., 2006). The radial effect has also been used to explain the induced
effect of vertical disparity (Matthews et al., 2003). Indeed, the anatomical and physiological basis of
the radial effect has been revealed previously (Levick and Thibos, 1982; Leventhal and Schall, 1983;
Schall et al., 1986; Sasaki et al., 2006). Specialty section:
This article was submitted to
Perception Science,
a section of the journal
Frontiers in Neuroscience Specialty section:
This article was submitted to
Perception Science,
a section of the journal
Frontiers in Neuroscience
Received: 20 April 2017
Accepted: 03 November 2017
Published: 24 November 2017 Received: 20 April 2017
Accepted: 03 November 2017
Published: 24 November 2017 Although the radial effect is well established, some questions remain open. First, while previous
studies focused on comparing the perceptual sensitivity and accuracy between radial and tangential
orientations, no study has investigated possible differences in reaction time (RT) of motor responses
to these orientations. Since there are studies demonstrating dissociations between perceptual and
motor measurements under certain conditions (Goodale and Milner, 1992; Goodale, 2014), a
perceptual radial effect does not necessarily imply a RT radial effect. On the other hand, one of
the main purposes of sensory processing is to guide motor response. It is therefore interesting to Keywords: orientation perception, grating stimuli, visual field, SRC, hemisphere asymmetry ORIGINAL RESEARCH
published: 24 November 2017
doi: 10.3389/fnins.2017.00638 Reviewed by: Keywords: orientation perception, grating stimuli, visual field, SRC, hemisphere asymmetry Edited by:
Sheng He, Edited by:
Sheng He,
Institute of Biophysics (CAS), China Institute of Biophysics (CAS), China Reviewed by:
Dirk Kerzel,
Université de Genève, Switzerland
Pinglei Bao,
California Institute of Technology,
United States Edited by:
Sheng He,
Institute of Biophysics (CAS), China
Reviewed by:
Dirk Kerzel,
Université de Genève, Switzerland
Pinglei Bao,
California Institute of Technology,
United States Behavioral Tasks Orientation Discrimination Task (Figure 1A)
A trial began with a red fixation point (16.0 ± 0.2 cd/cm2)
appearing in the center of the screen (14.4 ± 0.2 cd/cm2). The subjects had to initiate fixation after the fixation onset,
and were required to maintain fixation throughout each trial. A trial was excluded if the eye position left a circular window
(3◦radius) centered at the fixation point. After a random
delay (600–1,000 ms), a sine grating (10% contrast, 14.4 cd/m2
mean luminance, 2◦diameter) of either +45◦or −45◦diagonal
grating appeared at one of nine possible locations, which were
horizontally aligned with equal space (2◦) from 8◦left to 8◦
right of the vertical meridian and were 8◦above the fixation
point (Figure 1B). Thus, the positive and negative diagonal
lines from the fixation point went through the centers of the
leftmost and rightmost locations, respectively. Consequently, the
+45◦or −45◦gratings were, respectively, radial and tangential
at the rightmost location, and tangential and radial at the
leftmost location. When the gratings’ location changed from
leftmost to rightmost, either of the two gratings will cover some
intermediate orientations between radial and tangential. Subjects
were instructed to report the self-judged grating orientation (left
or right) as fast as possible by pressing a key accordingly (left
or right) using the index or middle finger of the right hand,
respectively. The inter-trial interval was 2 s. To address these questions, we designed an orientation
discrimination task in which a grating stimulus (+45◦or −45◦)
appeared randomly at one of nine equally spaced locations
in the upper VF. Thus, when the grating appears at one of
these nine locations, its direction with respect to the fixation
point is radial, tangential or some intermediate orientations. We
measured subjects’ manual RT for orientation discrimination
across the range of radial/tangential conditions. Subjects showed
significantly shorter RTs to gratings closer to radial orientation
than closer to tangential orientation, and these differences
were not attributable to the speed-accuracy tradeoff. Such RT
differences decreased as the grating became less radially-oriented
and tangentially-oriented. In addition, the magnitude of radial
effect on RT was significantly larger in the left VF than in
the right VF, which further supports the argument that the
spatial perception is asymmetrically processed between left and
right hemispheres (Corballis et al., 2002; Boulinguez et al.,
2003; Corballis, 2003; Okubo and Nicholls, 2008). Behavioral Tasks Moreover, the
radial effect and SRC effect strongly interact with each other to
determine the overall RT. Citation: Liang L, Zhou Y, Zhang M and Pan Y
(2017) Revealing the Radial Effect on
Orientation Discrimination by Manual
Reaction Time. Front. Neurosci. 11:638. doi: 10.3389/fnins.2017.00638 November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 1 Radial Effect on Orientation Discrimination Liang et al. investigate whether the perceptual radial effect has a motor
counterpart. Second, previous studies only compared orientation
sensitivity
and
accuracy
between
radial
and
tangential
orientations at a given location in the VF. It is unclear how
the radial effect on orientation perception changes as the
orienting direction gradually changes from radial to tangential
directions. Third, stimulus-response-compatibility (SRC) effect
(also known as Simon effect) also affects RT (Fitts and Seeger,
1953; Simon and Rudell, 1967; Wallace, 1971; Whitaker, 1982;
Hommel, 2011; Styrkowiec and Szczepanowski, 2013) and it
has not been investigated whether and how it interacts with the
radial effect. photometer. The subjects sat in front of the monitor at a viewing
distance of 57.5 cm and with their head restrained on a chin rest. Their eye positions were monitored with an infrared image eye
tracker (EyeLink 2000 Desktop Mount, SR Research). Two key
buttons were placed in front of the subjects. We used MATLAB
(Mathworks) with Psychtoolbox extension (Brainard, 1997; Pelli,
1997) on a PC to present stimuli and collect the manual RT data. Stimulus Parameters To reduce performance variations across sessions and subjects,
we used a staircase procedure to determine stimulus parameters
at threshold performance for each subject at the start of each
session (day). For the orientation discrimination task, we varied
the spatial frequency of gratings. For the color discrimination
task, we varied the R or B index of color patches while
keeping the remaining two indices equal to the RGB value
of the gray background. For parameters determination, we
fixed the duration of stimulus appearance (30 ms for gratings
and 50 ms for color patches) and found thresholds at 78%
correct performance by fitting cumulative Gaussian function
to psychometric curves. Across all subjects and sessions, the Color Discrimination Task (Figure 1D) Eighteen right-handed subjects with normal vision (age 18–28,
17 naïve, 7 male and 11 female) participated in the present
experiments. All subjects took part in the first experiment,
named as orientation discrimination task (Figure 1A). Before
the experiments started, each subject was examined about
the self-judged orientation (left or right) to the two gratings
(+45◦and −45◦), by pressing either a left key or a right
key as response of his/her judgment. Then, the subjects were
induced to remain such self-judged orientation throughout
the orientation discrimination experiment. Twelve of eighteen
subjects took part in the second experiment, named as color
discrimination task (Figure 1D). All experiments followed the
guideline of the Institutional Review Board (IRB) of the State
Key Laboratory of Cognitive Neuroscience and Learning, Beijing
Normal University. The protocol was approved by the same
committee. All subjects gave written informed consent in
accordance with the Declaration of Helsinki. The aim of this experiment was to test whether there were
intrinsic group differences in sensorimotor transformation. The
task sequence was identical to the orientation discrimination
task, except that the grating stimulus was replaced by a gray
patch (0.8◦diameter, 14.3 ± 0.3 cd/m2 mean luminance) tinted
either red or blue. Subjects were instructed to report the red
and blue colors as fast as possible by pressing left and right key,
respectively. Response Accuracy Calculation threshold of spatial frequency ranged from 3.8 cycle/degree to
5.6 cycle/degree, and the threshold of color- index ranged from
1 to 5%. We calculated each subject’s response accuracy (correct ratio)
for each grating orientation (+45◦and −45◦) at each tested
location. Experimental Setup All visual stimuli were presented on an LCD monitor (BENQ
XL2720Z, 1,920 × 1,080 pixels, 100 Hz vertical refresh rate)
calibrated and linearized with a Konica Minolta LS-110 November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 2 Radial Effect on Orientation Discrimination Liang et al. FIGURE 1 | Behavioral tasks and the two groups of subjects. (A) Orientation discrimination task. A trial started with a small red fixation point and subjects fixated on
it. After a random delay, a grating appeared in upper visual field. Subjects had to press a left or right key to report the grating orientation (see Methods and Results for
details). (B) Illustration of the grating orientations respect to the fixation point. The dashed circles indicate the grating possible locations across trials and green/red bar
indicates grating with +45◦/−45◦. (C) Illustration of finger responses to the gratings in two groups. (D) Color discrimination task. A trial started with a small red fixation
point and subjects fixated on it. After a random delay, a color patch (red or blue) appeared in upper visual field. Subjects had to press a left or right key to report the
color of stimulus. FIGURE 1 | Behavioral tasks and the two groups of subjects. (A) Orientation discrimination task. A trial started with a small red fixation point and subjects fixated on
it. After a random delay, a grating appeared in upper visual field. Subjects had to press a left or right key to report the grating orientation (see Methods and Results for
details). (B) Illustration of the grating orientations respect to the fixation point. The dashed circles indicate the grating possible locations across trials and green/red bar
indicates grating with +45◦/−45◦. (C) Illustration of finger responses to the gratings in two groups. (D) Color discrimination task. A trial started with a small red fixation
point and subjects fixated on it. After a random delay, a color patch (red or blue) appeared in upper visual field. Subjects had to press a left or right key to report the
color of stimulus SRC Effect Quantification Although the spatial locations of the grating were task-
irrelevant, trials could be separated into SRC-compatible and
SRC-incompatible conditions, based on the spatial relationship
between the location of the grating and the direction of response
key (finger). The SRC-compatible condition included trials in
which the locations of grating and the response key were spatially
compatible (e.g., gratings that appeared in the left VF were
associated with left-key responses; gratings that appeared in the
right VF were associated with right-key responses). In contrast,
the SRC-incompatible condition included trials in which the
grating and the response were spatially opposite (e.g., the gratings
that appeared in the left VF were associated with right-key
responses; gratings that appeared in the right VF were associated
with left-key responses). The RT was calculated by using the same criteria as reported
previously (Zhou et al., 2012, 2017). Briefly, we collected a total of
41,787 trials and excluded 6.18% of them in which subjects broke
fixation or reported incorrectly, or the RTs differed from the
mean by more than 3 standard deviations. For each subject, we
calculated the mean of raw RT as well as the mean of normalized
(differential) RT due to the fact of the existence of RT variation
among subjects. Such RT variation may influence the results of
statistical analysis. To calculate differential RT, we used both subtraction and
division models. First, we set RTs responding to the grating
(+45◦and −45◦) or patch (red and blue) appeared at the vertical
meridian (straight ahead) as baseline condition, and then for
each subject subtract/divide the mean RT at baseline condition
from the mean RT at the other locations under same stimulus
condition, respectively. Since the orientations of two gratings
(+45◦and −45◦) at the vertical meridian location are in the
middle between radial and tangential directions and there is no
spatial SRC effect, the differential RTs predominately reflect the
influence of radial effect on the temporal process of orientation
discrimination. Frontiers in Neuroscience | www.frontiersin.org Reaction Times (RTs) for Orientation
Discrimination Were Significantly Shorter
When a Grating Was Oriented Closer to the
Radial Direction than When It Was Oriented
Closer to the Tangential Direction radial effect on RT, we need to exclude the influence of SRC. To do so, subjects were separated into two groups based on
their subjective preference to the same grating as titled to
left or right (Figure 1C). Group A contained 7 subjects who
preferred the right-key response for the +45◦grating and the
left-key response for the −45◦grating. Group B contained 11
subjects with the opposite preferences. Therefore, we created
experimental conditions in which the SRC conditions were same
for group A and group B subjects (same stimulus location and
same key-press response), but the orientations of the gratings
were opposite, with one being closer to the radial orientation and
the other being closer to the tangential orientation (Figure 1B). The RT difference between two groups of subjects under
the same SRC conditions predominately reflects the radial
effect. To show more detailed results of radial effect on RTs, the
data of differential RTs of two groups are arranged based on
the key-response (manual response), i.e., pressing the same key
but responding to the opposite gratings (+45◦vs. −45◦) in
Figures 2A,B, respectively. Although the manual response is
the same (Figure 2A, left-key response; Figure 2B, right-key
response) and the spatial location of grating is the same (x-axis
showing the horizontal eccentricities of grating), the distribution
pattern of the differential RTs is very different between the two
groups of subjects. Such difference could not be explained by
the SRC effect, because the SRC conditions are identical between
two groups of subjects in both Figures 2A,B. Thus, a reasonable
explanation is that the differences in differential RT between
two groups of subjects are mainly caused by the orientation of
gratings (+45◦vs. −45◦). The differential RTs were shorter when
the gratings were oriented closer to the radial direction than when
they were oriented closer to the tangential direction in both left
(solid line relative to dashed line) and right (dashed line relative
to solid line) VFs. Magnitude of Radial Effect We defined the value of RT difference between two groups of
subjects at each tested location as the magnitude of radial effect
on orientation discrimination at this location. The magnitude of
radial effect was calculated by comparing the absolute average RT
difference between group A subjects and group B subjects. Frontiers in Neuroscience | www.frontiersin.org RESULTS The information of involvement of each subject in the
experiments and the mean of raw RTs under different visual
stimulation conditions (left VF vs. right VF) in orientation and
color discrimination tasks are shown in Tables 1, 2, respectively. However, considering the fact that the large variation of RTs
among individual subjects might affect the results of some
statistical analyses, we calculated the differential RTs (see
methods for details) for each subject and further used them in
various statistical analyses. Since subtraction and division models
showed consistent results, only data of differential RTs from
subtraction model were shown thereafter. We further performed the post-hoc t-tests at each stimulated
location to test the differential RT difference between two groups
of subjects. For left-key response (Figure 2A, groups A subjects
responding to the −45◦grating; group B subjects responding to
+45◦grating), in the left VF the RTs of the group A subjects
were significantly shorter at 3 of 4 locations [t-test, maximum
p = 0.046, t(16) = −2.3] than that of the group B subjects; in TABLE 1 | The mean RTs under different conditions in orientation discrimination
task. RT (ms)
RT (ms)
Subject
Left-key
Right-key
Subject
Left-key
Right-key
Group A
LVF
RVF
LVF
RVF
Group B
LVF
RVF
LVF
RVF
CY
567.3
594
571.5
563.5
GQ
622.3
585.4
648.3
614.4
DY
493
590.1
631.8
508.4
LLX
600
495.6
521.7
507
HZY
561.4
585
638
562.7
LD
620.8
579.6
636.2
592.9
LYY
469.6
506.6
560.5
500.2
LY
509.3
477.7
485.5
484
PY
555.7
568.3
617.1
495
LSK
487.7
458.7
498.3
491.9
YL
474.6
580.2
666.5
479.9
RSF
571.6
570.8
567.9
547.9
YYH
455.7
518.5
578.6
473.8
SJ
599.2
552.6
556.9
599.2
TRM
581.5
531.8
562.9
562.5
WYQ
460.2
446
455.3
426.5
WZY
424.2
421.1
442.9
407.8
XC
488.2
443.2
479.7
516.9
p
g
g
g g
p
j
p
g
+45◦grating), in the left VF the RTs of the group A subjects
were significantly shorter at 3 of 4 locations [t-test, maximum
p = 0.046, t(16) = −2.3] than that of the group B subjects; in
TABLE 2 | The mean RTs under different conditions in color discrimination task. Radial Effect Quantification Considering the fact that subjects’ responses are highly influenced
by the SRC effect (also known as the Simon effect) (Simon and
Rudell, 1967; Wallace, 1971; Whitaker, 1982; Hommel, 2011;
Styrkowiec and Szczepanowski, 2013), which results in the faster
and more accurate responses in SRC compatible conditions
than in SRC incompatible conditions. Thus, to quantify the November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 3 Liang et al. Radial Effect on Orientation Discrimination Reaction Times (RTs) for Orientation
Discrimination Were Significantly Shorter
When a Grating Was Oriented Closer to the
Radial Direction than When It Was Oriented
Closer to the Tangential Direction
We employed the three-way ANOVA analysis [two key responses
(left, right) × two gratings (+45◦, −45◦) × nine locations (−8◦,
−6◦, −4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)] to test the significance
of radial effect on RTs in the orientation discrimination task. The results of ANOVA analysis of the differential RTs showed
a significant interaction between the grating orientations and
grating locations [F(8, 216) = 31.8, p = 1.42e-35], and a significant
interaction between the key-responses and grating locations
[F(8, 216) = 11.5, p = 3.28e-14]. Reaction Times (RTs) for Orientation
Discrimination Were Significantly Shorter
When a Grating Was Oriented Closer to the
Radial Direction than When It Was Oriented
Closer to the Tangential Direction
We employed the three-way ANOVA analysis [two key responses
(left, right) × two gratings (+45◦, −45◦) × nine locations (−8◦,
−6◦, −4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)] to test the significance
of radial effect on RTs in the orientation discrimination task. The results of ANOVA analysis of the differential RTs showed
a significant interaction between the grating orientations and
grating locations [F(8, 216) = 31.8, p = 1.42e-35], and a significant
interaction between the key-responses and grating locations
[F(8, 216) = 11.5, p = 3.28e-14]. RESULTS RT (ms)
RT (ms)
Subject
Left-key
Right-key
Subject
Left-key
Right-key
Group A
LVF
RVF
LVF
RVF
Group B
LVF
RVF
LVF
RVF
CY
698.8
709
687.5
711.1
LLX
448.9
456.6
486.4
445.8
HZY
557
573.7
584
566.5
LD
637
691.2
695.4
657.2
LYY
495.9
529.2
541.2
533.3
LSK
472.6
476.6
520.4
495.4
YL
696.3
697.3
698.5
662.4
RSF
603.7
610.7
582.4
580.6
YYH
588.5
617.8
580.9
564.2
SJ
609.1
627.1
601.1
593.7
TRM
592.6
604.7
675.4
650.4
WZY
389.3
404.9
405.9
405.2 TABLE 1 | The mean RTs under different conditions in orientation discrimination
task. RT (ms)
RT (ms)
Subject
Left-key
Right-key
Subject
Left-key
Right-key
Group A
LVF
RVF
LVF
RVF
Group B
LVF
RVF
LVF
RVF
CY
567.3
594
571.5
563.5
GQ
622.3
585.4
648.3
614.4
DY
493
590.1
631.8
508.4
LLX
600
495.6
521.7
507
HZY
561.4
585
638
562.7
LD
620.8
579.6
636.2
592.9
LYY
469.6
506.6
560.5
500.2
LY
509.3
477.7
485.5
484
PY
555.7
568.3
617.1
495
LSK
487.7
458.7
498.3
491.9
YL
474.6
580.2
666.5
479.9
RSF
571.6
570.8
567.9
547.9
YYH
455.7
518.5
578.6
473.8
SJ
599.2
552.6
556.9
599.2
TRM
581.5
531.8
562.9
562.5
WYQ
460.2
446
455.3
426.5
WZY
424.2
421.1
442.9
407.8
XC
488.2
443.2
479.7
516.9 TABLE 1 | The mean RTs under different conditions in orientation discrimination
task. TABLE 2 | The mean RTs under different conditions in color discrimination task. RT (ms)
RT (ms)
Subject
Left-key
Right-key
Subject
Left-key
Right-key
Group A
LVF
RVF
LVF
RVF
Group B
LVF
RVF
LVF
RVF
CY
698.8
709
687.5
711.1
LLX
448.9
456.6
486.4
445.8
HZY
557
573.7
584
566.5
LD
637
691.2
695.4
657.2
LYY
495.9
529.2
541.2
533.3
LSK
472.6
476.6
520.4
495.4
YL
696.3
697.3
698.5
662.4
RSF
603.7
610.7
582.4
580.6
YYH
588.5
617.8
580.9
564.2
SJ
609.1
627.1
601.1
593.7
TRM
592.6
604.7
675.4
650.4
WZY
389.3
404.9
405.9
405.2 TABLE 2 | The mean RTs under different conditions in color discrimination task. November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 4 Liang et al. Radial Effect on Orientation Discrimination FIGURE 2 | The differential RTs and accuracies of two groups in orientation discrimination task. (A,B) The differential RTs are plotted against the grating horizontal
eccentricity with left-key (A) and right-key (B) response. (C,D) The accuracies are plotted against the grating horizontal eccentricity with left-key (C) and right-key (D)
response. RESULTS Similar results were observed from the RTs of right-key
response (Figure 2B, groups A subjects responding to the +45◦
grating; group B subjects responding to −45◦grating). In the left
VF, the RTs of the group B subjects were significantly shorter
at 3 of 4 locations [t-test, maximum p = 0.042, t(16) = 2.2]
than that of the group A subjects. In contrast, in the right
VF, the RTs of the group A subjects were significantly shorter
than that of the group B subjects at 1 of 4 locations [t-test,
p = 0.010, t(16) = −2.3]. Overall, the differential RTs were significantly shorter when
the grating orientation was closer to the radial direction than
when it was closer to the tangential direction. RESULTS The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs/accuracies of group A and B, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs/accuracies of individual subject in group A and B, respectively. Asterisk denotes the results of post-hoc t-test between two groups: *P < 0.05, **P < 0.01, ***P < 0.001. FIGURE 2 | The differential RTs and accuracies of two groups in orientation discrimination task. (A,B) The differential RTs are plotted against the grating horizontal
eccentricity with left-key (A) and right-key (B) response. (C,D) The accuracies are plotted against the grating horizontal eccentricity with left-key (C) and right-key (D)
response. The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs/accuracies of group A and B, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs/accuracies of individual subject in group A and B, respectively. Asterisk denotes the results of post-hoc t-test between two groups: *P < 0.05, **P < 0.01, ***P < 0.001. [two key responses (left, right) × two gratings (+45◦, −45◦)
× nine locations (−8◦, −6◦, −4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)]
to analyze the variance of response accuracy between different
experimental conditions. The results showed no significant
interaction either between grating orientations and grating
locations [F(8, 216) = 1.4, p = 0.18] or between key responses
and grating locations [F(8, 216) = 1.4, p = 0.18]. Also, the post-
hoc t-test at each location showed no significant difference in
response accuracy between two groups of subjects in left-key
response [Figure 2C, in left VF, minimum p = 0.17, t(16) = −1.5;
in right VF, minimum p = 0.36, t(16) = −1.0]. Similar results
were observed in right-key response condition [Figure 2D, in
left VF, minimum p = 0.16, t(16) = −1.5; in right VF, minimum
p = 0.25, t(16) = −1.2]. These results indicate that the radial effect
on the temporal process of orientation discrimination is not due
to SATO. contrast, in the right VF the RTs of the group B subjects were
significantly shorter than that of the group A subjects at 2 of 4
locations [t-test, maximum p = 0.010, t(16) = 3.7]. The Radial Effect on RT in the Left VF Was
Stronger than That in the Right VF It is commonly known that the speed of object discrimination
could become faster by reducing the response accuracy, which
is known as the SATO. To examine whether the radial effect
on RT was due to SATO, we employed a three-way ANOVA To test whether the radial effect on RT is equally distributed in
the left and right VFs, we performed a four-way ANOVA [two
key responses (left, right) × two gratings (+45◦, −45◦) × four November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 5 Radial Effect on Orientation Discrimination Liang et al. eccentricities (2◦, 4◦, 6◦, 8◦) × two VFs (left, right)]. The results
showed significant interaction among grating × eccentricity ×
VF for differential RTs [F(3, 192) = 38.1, p = 2.14e-20], which
indicated the radial effect on RT is asymmetrically distributed
between the left and right VFs. To further explore the spatial
characteristics of radial effect on RT across VF, we estimated the
magnitude of radial effect at each tested location by calculating
the absolute RT difference between two groups of subjects under
the same key response conditions. SRC effect on the unbalanced tuning curve, we mixed the radial
effect of left-key (Figure 3A) and right-key (Figure 3B) response
together correlated with the location of grating stimulation,
and then the mean radial effect was calculated for each tested
eccentric location. In average, the mixed radial effect was larger
in the left VF than in the right VF (Figure 3C, the error bars will
be described in next paragraph). The paired t-test of mixed radial
effect between 4 tested locations in left VF and other 4 locations
in right VF showed significant difference [t(3) = 3.5, p = 0.040]. However, considering the fact that the few number of samples
(only four paired eccentric locations were tested between left and
right VFs, respectively) might strongly bias the results of t-test,
we used the following method to confirm the above result. We observed the “unbalanced U-shape” tuning curves of the
magnitude of radial effect for both left-key and right-key response
conditions (Figures 3A,B). The magnitude of radial effect was
largest when the grating was either fully radial-oriented or fully
tangential-oriented (the leftmost and rightmost locations) and
became smaller as the grating became less radial-oriented and less
tangential-oriented (closer to the vertical meridian). The RT Differences in Orientation
Discrimination Task between Two Groups
Were Not Caused by Subjects’ Difference
in the Sensorimotor Transformation In the present study, the radial effect was quantified by calculating
the RT differences between two groups. One intuitive argument
is that the observed RT differences might be caused by intrinsic
group differences in the sensorimotor process. Although this is
very unlikely, we nevertheless performed a control experiment to
test it. The Radial Effect on RT in the Left VF Was
Stronger than That in the Right VF the mean radial effect of each subject (n = 11) at each tested
eccentric location was calculated. Paired t-test of this mixed 9 ×
11 matrix showed significant larger radial effect in the left VF
than in the right VF [t(10) = 5.6, p = 2.25e-4]. Error bars in
Figure 3C represents the mean standard deviation of these 11
subjects. differential RTs of group B show dramatically diminished
SRC effect (Figure 4C). Two sample t-test confirmed that the
differential RT differences in group A subjects were significantly
greater than in group B subjects [t(16) = 5.0, p = 0.0010]. Thus, these results clearly showed that radial effect and SRC
effect interact with each other to determine the overall RTs. Such asymmetric distribution of the radial effect was further
confirmed by comparing the differential RT difference (radial
effect) between mirror locations relative to the vertical meridian
(Figure 3D), in which the mean radial effect with standard
deviation at each tested eccentric location was calculated from
the mixed 9 × 11 matrix. Paired t-test showed that the radial
effect was stronger in the left VF than in the right VF at all tested
off-middle locations [maximum p = 0.0035, t(10) = 3.8]. The RT Differences in Orientation
Discrimination Task between Two Groups
Were Not Caused by Subjects’ Difference
in the Sensorimotor Transformation
In the present study, the radial effect was quantified by calculating
the RT differences between two groups. One intuitive argument
is that the observed RT differences might be caused by intrinsic
group differences in the sensorimotor process. Although this is
very unlikely, we nevertheless performed a control experiment to
test it. The Radial Effect on RT in the Left VF Was
Stronger than That in the Right VF In addition,
the radial effect tuning curve also differed between the left and
right VFs in both left-key and right-key response conditions
(Figures 3A,B). One possible reason is that the unbalanced
tuning curve was, at least partially, due to the influence of SRC
effect, i.e., shorter RTs in SRC compatible conditions and longer
RTs in SRC incompatible conditions. To simply exclude the For each key-press condition, we first calculated the mean
differential RT of group A subject (n = 7) at each tested location,
and then calculated the absolute difference (radial effect) between
this mean differential RT and differential RT of each group B
subject (n = 11) at the same tested location. Thus, we created a
9 (locations) × 11 (subjects) matrix of data set for each key-press
condition, respectively. To compare the radial effect between left
and right VFs, these two 9 × 11 matrices were mixed together
based on the eccentric location of grating stimulation, and then FIGURE 3 | The asymmetry of radial effect. (A,B) Tuning curve of the radial effect. The curve (through circles) represents the average of RT differences between two
groups with left-key (A) and right-key (B) responses. (C) The radial effect between left and right VFs. (D) The radial effect between left and right VFs across each
tested horizontal eccentricity. The gray curve (through gray circles) and black curve (through black circles), respectively represent the average of the radial effect in left
and right VFs across each tested horizontal eccentricity. All the error bars denote ±SD. Asterisk denotes the results of paired t-test: **p < 0.01; ***P < 0.001. FIGURE 3 | The asymmetry of radial effect. (A,B) Tuning curve of the radial effect. The curve (through circles) represents the average of RT differences between two
groups with left-key (A) and right-key (B) responses. (C) The radial effect between left and right VFs. (D) The radial effect between left and right VFs across each
tested horizontal eccentricity. The gray curve (through gray circles) and black curve (through black circles), respectively represent the average of the radial effect in left
and right VFs across each tested horizontal eccentricity. All the error bars denote ±SD. Asterisk denotes the results of paired t-test: **p < 0.01; ***P < 0.001. November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 6 Radial Effect on Orientation Discrimination Liang et al. The Radial Effect and SRC Effect Interact
to Determine RT We employed a color discrimination task to test the similarity
of sensorimotor process between two groups. In this task, the
test stimuli and response strategy were exactly same for two
groups of subjects (see details in the Methods). First, SRC
effect was confirmed by a two-way ANOVA analysis [two key
responses (left, right) × two VFs (left, right)]. The results
showed a significant interaction between key response and VF
for the differential RTs [F(1, 11) = 32.0, p =1.47e-04]. Second,
the differential RTs were very similar between two groups for
both left-key (Figure 5A) and right-key (Figure 5B) responses
[left-key response: one-way ANOVA, 2 groups, F(1, 8) = 0.4,
p = 0.53; right-key response: one-way ANOVA, 2 groups,
F(1, 8) = 0.0, p = 0.89]. Also, the differences of differential
RTs in all tested off-middle locations did not reach the
significant level [t-test, minimum p = 0.25, t(10) = −1.2]. Thus,
the difference in differential RTs between the two groups in
orientation discrimination task was not due to the differences in
sensorimotor process between subjects. A line of studies has found that the behavioral performance is
faster and more accurate in SRC-compatible conditions than in
SRC-incompatible conditions (Fitts and Seeger, 1953; Wallace,
1971; Whitaker, 1982), a finding also known as the Simon
effect (Simon and Rudell, 1967; Hommel, 2011; Styrkowiec and
Szczepanowski, 2013). We have shown earlier that the SRC effect
strongly modulated the radial effect (Figures 2A,B). Here, we
employed a three-way ANOVA analysis [two key responses (left,
right) × two gratings (+45◦, −45◦) × nine locations (−8◦, −6◦,
−4◦, −2◦, 0◦, 2◦, 4◦, 6◦, 8◦)] to statistically test the interaction
between radial effect and SRC effect. The results showed that
the interaction between radial effect and SRC (key × grating ×
location) was significant [F(8, 216) = 2.7, p = 0.0069]. Furthermore, the influence of SRC to radial effect was very
different between the two SRC conditions, as shown in Figure 4A
(the averaged radial effect of left and right 4 off-middle locations
in Figures 3A,B, respectively). The averaged radial effect was
greater in the SRC-incompatible condition than in the SRC-
compatible condition in both left and right key responses. Two
sample t-test resulted the significant difference in radial effect
between two SRC conditions [left key response, t(10) = −2.4,
p = 0.037; right key response, t(10) = 6.6, p = 5.81e-05]. Frontiers in Neuroscience | www.frontiersin.org DISCUSSION consistent with the previously reported perceptual radial effect
(Rovamo et al., 1982; Bennett and Banks, 1991; Westheimer,
2003; Sasaki et al., 2006), the former is not a passive reflection
of the latter because under our experimental condition, there was
no significant difference in perceptual accuracy between the more
radial and more tangential orientations. locations in the upper VF, we produced more radial and more
tangential orientations of various degrees. In this way, we were
able to measure the radial effect by comparing the two equally
oblique orientations and avoid the confound of the oblique effect. locations in the upper VF, we produced more radial and more
tangential orientations of various degrees. In this way, we were
able to measure the radial effect by comparing the two equally
oblique orientations and avoid the confound of the oblique effect. A byproduct of the present study is that the RT radial effect
is asymmetric between the left and right VFs: the effect is
larger in the left VF than in the right VF (Figures 3C,D). Such
results are consistent with findings of many previous studies
(Heilman and Van Den Abell, 1979; Sturm et al., 1989; Corballis
et al., 2002; Boulinguez et al., 2003; Corballis, 2003; Okubo and
Nicholls, 2008; Zhou et al., 2012), which indicates that the right
hemisphere of human brain plays a dominant role in processing
spatial information than the left hemisphere does. It has been
reported that the spatial information is dominantly processed in
the left VF by the right hemisphere (e.g., orientation perception),
whereas the non-spatial information (e.g., temporal perception) A byproduct of the present study is that the RT radial effect
is asymmetric between the left and right VFs: the effect is
larger in the left VF than in the right VF (Figures 3C,D). Such
results are consistent with findings of many previous studies
(Heilman and Van Den Abell, 1979; Sturm et al., 1989; Corballis
et al., 2002; Boulinguez et al., 2003; Corballis, 2003; Okubo and
Nicholls, 2008; Zhou et al., 2012), which indicates that the right
hemisphere of human brain plays a dominant role in processing
spatial information than the left hemisphere does. DISCUSSION The differential RT is plotted against the grating horizontal eccentricity in group A (B) and B (C). The solid black curve (through circles)
and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key responses, respectively. Asterisk denotes the results of
post-hoc paired t-tests between left-key and right-key responses in each group: *P < 0.05, **P < 0.01, ***P < 0.001. FIGURE 4 | Mutual interaction between the radial effect and SRC effect. (A) The influence of SRC effect on the radial effect. The radial effect is shown in the
SRC-compatible (gray) and SRC-incompatible (black) conditions with left-key and right-key response, respectively. The error bars denote SD. (B,C) The influence of
the radial effect on SRC effect. The differential RT is plotted against the grating horizontal eccentricity in group A (B) and B (C). The solid black curve (through circles)
and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses, respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key responses, respectively. Asterisk denotes the results of
post-hoc paired t-tests between left-key and right-key responses in each group: *P < 0.05, **P < 0.01, ***P < 0.001. FIGURE 5 | The differential RTs in color discrimination task. The differential RT is plotted against the color patch horizontal eccentricity with left-key (A) and right-key
(B) response. The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses,
respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key
responses, respectively. FIGURE 5 | The differential RTs in color discrimination task. The differential RT is plotted against the color patch horizontal eccentricity with left-key (A) and right-key
(B) response. The solid black curve (through circles) and dashed black curve (through triangles) represent mean differential RTs with left-key and right-key responses,
respectively. The black vertical bars denote ±SEM. The thin solid and dash color lines represent the differential RTs of individual subject with left-key and right-key
responses, respectively. DISCUSSION Orientation processing is a key element of visual perception. Despite the intuitive view that orientation processing is isotropic,
it has been reported that human subjects exhibit perceptual
differences for different orientations, including the oblique effect
(Appelle, 1972; Berkley et al., 1975), the cardinal effect (Rijsdijk
et al., 1980; Carrasco et al., 2001), and the radial effect (Rovamo
et al., 1982; Bennett and Banks, 1991; Westheimer, 2003; Sasaki
et al., 2006). Different from previous radial effect studies,
which focused on comparing orientation sensitivity and accuracy
between radial and tangential orientations, we systematically
assessed the radial effect in term of the processing time along
the sensorimotor pathway by measuring the manual RT to a
+45◦or −45◦grating. The grating appeared randomly at one
of nine equally spaced locations in the upper VF so that its
orientation with respect to the fixation point is radial, tangential
or somewhere in-between (Figure 1B). We found significantly
shorter RTs to gratings closer to radial orientation than those
closer to tangential orientation. Although this RT radial effect was On the other hand, the radial effect strongly affected the
SRC effect too. Based on the relationship between the radial
effect and SRC effect, trials could be separated into two
types (Figure 1C): radial effect and SRC effect matched trials
(group A: radially-oriented conditions were associated with SRC-
compatible conditions and tangentially-oriented conditions were
associated with SRC-incompatible conditions), and radial effect
and SRC effect non-matched trials (group B: radial-oriented
conditions were associated with SRC-incompatible conditions
and tangential-oriented condition were associated with SRC-
compatible condition). Although the SRC conditions were
same, the distributions of the differential RTs were dramatically
different between group A subjects (radial effect and SRC effect
match, Figure 4B) and group B subjects (radial effect and
SRC non-match, Figure 4C). While differential RTs of group
A subjects showed clear SRC effect (circles are smaller than
triangles in the left VF and vice versa in the right VF, Figure 4B), November 2017 | Volume 11 | Article 638 Frontiers in Neuroscience | www.frontiersin.org 7 Radial Effect on Orientation Discrimination Liang et al. FIGURE 4 | Mutual interaction between the radial effect and SRC effect. (A) The influence of SRC effect on the radial effect. The radial effect is shown in the
SRC-compatible (gray) and SRC-incompatible (black) conditions with left-key and right-key response, respectively. The error bars denote SD. (B,C) The influence of
the radial effect on SRC effect. Frontiers in Neuroscience | www.frontiersin.org DISCUSSION It has been
reported that the spatial information is dominantly processed in
the left VF by the right hemisphere (e.g., orientation perception),
whereas the non-spatial information (e.g., temporal perception) When studying the radial effect, one must control for potential
confound from another well-known phenomenon, the oblique
effect, according to which the perception of cardinal orientations
is superior to that of oblique orientations (Appelle, 1972; Berkley
et al., 1975; Furmanski and Engel, 2000). Because of the oblique
effect, we cannot assess the radial effect by comparing different
orientations that include both more cardinal and more oblique
orientations at a given location in the VF. Instead, we only
used gratings of +45◦and −45◦orientations which are equally
oblique. By presenting them at one of the nine equally spaced November 2017 | Volume 11 | Article 638 8 Radial Effect on Orientation Discrimination Liang et al. is dominantly processed in the right VF by the left hemisphere
(Galati et al., 2010). In contrast, it has been also reported that
the process of orientation perception was faster in the right
VF than in the left VF when there were competing orienting
stimuli appeared in the left and right VFs simultaneously (Carlei
and Kerzel, 2017). Indeed, when we put all differential RTs of
two groups of subjects together and then separate them to two
groups based on the grating’s location either in the left or right
VF, one-way ANOVA resulted that the effect of VF–RT was
significant [F(1, 17) = 5.5, p = 0.020]. The mean differential RTs
were significant faster in the right VF than in the left VF [post-
hoc paired t-test, t(17) = 3.8, p = 0.0013], which was consistent
with the above finding of shorter RT in the right VF than in the
left VF. the RTs at several tested locations and in certain trial conditions
(SRC-compatible condition and closer to radial orientation) are
shorter than the RT in the baseline condition (grating in vertical
meridian, with shortest eccentricity). Thus, it is reasonable to
predict that the radial effect should be more obvious if the tested
locations are distributed with an equal eccentricity. Thus, our
results indeed reflect the influence of the radial effect and SRC
effect on RT. Our results indicate that multiple factors interact to determine
the overall RT. DISCUSSION We summarize our finding with the following
simplified model: RT (stimulus, response, location) = Baseline
+ Radial effect (stimulus, response) + SRC effect (stimulus,
response) + interaction (Radial and SRC effects) + VF (location). It would be interesting to investigate the neural mechanisms
underlying these factors and their interactions. We found that the radial effect and SRC effect strongly
interact with each other to determine RT. The radial effect
was significantly larger in the SRC-incompatible condition than
in the SRC-compatible condition, in both the left and right
VFs (Figure 4A). Additionally, the RT difference between SRC-
incompatible and SRC-compatible conditions was larger when
the radial and SRC effects were matched than when they were
unmatched (Figures 4B,C). Further studies are needed to explore
the neural mechanisms underlying the interaction between the
two effects. ACKNOWLEDGMENTS We thank Dr. Ning Qian of Columbia University, New York,
USA, for his helpful suggestions and comments. FUNDING This study is supported by the following foundations: The
Opening Foundation of State Key Laboratory of Neuroscience,
Shanghai Institutes for Biological Sciences, Chinese Academy
of
Sciences
(SKLN-2010A05
and
SKLN-201203)
awarded
to YP; The Opening Foundation of State Key Laboratory
of Cognitive Neuroscience and Learning, Beijing Normal
University (CNLZD1303) awarded to YP. We measured the radial effect by comparing the RTs between
two groups of subjects. It is thus important to rule out systematic
differences in sensorimotor transformation between the two
groups. To this end, we showed that the two groups of subjects
had very similar RTs in a control task on color discrimination
(Figure 5). One may argue that the RT difference among nine tested
locations in the present study might be due to the different
visual sensitivity, because the nine tested peripheral locations
had different eccentricities (Figure 1B). However, our results
cannot be explained by the variation of visual sensitivity with
eccentricity. As shown in Figures 2A,B, 4B,C, for some subjects, AUTHOR CONTRIBUTIONS YZ designed the experiments. LL and YZ collected and analyzed
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conducted in the absence of any commercial or financial relationships that could
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conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Sasaki, Y., Rajimehr, R., Kim, B. W., Ekstrom, L. B., Vanduffel, W., and
Tootell, R. B. (2006). The radial bias: a different slant on visual orientation
sensitivity in human and nonhuman primates. Neuron 51, 661–670. doi: 10.1016/j.neuron.2006.07.021 Copyright © 2017 Liang, Zhou, Zhang and Pan. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
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https://openalex.org/W3172429834
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https://ejournal.umm.ac.id/index.php/jie/article/download/16250/9821
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Indonesian
| null |
ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN DEVISA DI INDONESIA
|
Jurnal Ilmu Ekonomi
| 2,021
|
cc-by-sa
| 4,019
|
Abstract The purpose of this study is to analyze what factors affect
foreign exchange reserves in Indonesia. The focus in this study
is the variables that affect changes in foreign exchange reserves
in the 1999-2019 period are non-oil and gas exports, the rupiah
exchange rate, foreign debt and inflation as independent
variables. Using an open economy model and Keynes's balance
of payments, the analytical tools used in this study are the
PAM regression model (Partial Adjustment Model) and the
adaptive expectation model to see the long-term and short-term
effects of the independent variable on the dependent variable. The results of this study show that simultaneously non-oil and
gas exports, the rupiah exchange rate, foreign debt and
inflation affect Indonesia's foreign exchange reserves, while
partially non-oil exports and the rupiah exchange rate
significantly and positively affect Indonesia's long-term and
short-term foreign exchange reserves, while debt foreign
exchange and inflation are not significant and negatively affect
long-term and short-term foreign exchange reserves. Non-oil
exports, rupiah exchange rate, foreign debt and inflation can
explain Indonesia's foreign exchange reserves of 97%, of which
the remaining 3% is explained by other variables not included
in this study. ANALISIS
FAKTOR-FAKTOR
YANG
MEM
CADANGAN DEVISA DI INDONESIA
Norita Fitria*, Aris Soelistyo, Dwi Susilowati Program Studi Ekonomi Pembangunan, Fakultas Ekonomi dan Bissnis, Universitas Muhammadiyah
Malang, Indonesia *Corresponding Author: noritafityaaa@gmail.com p
g
y
Artikel Info
Article history:
Received 14 April 2021
Revised 18 August 2021
Accepted 30 August 2021
Available online 31 August
2021
Keywords: Cadangan Devisa,
Ekspor Non Migas, Nilai Tukar
Rupiah, Utang Luar Negeri,
Inflasi Jurnal Ilmu Ekonomi (JIE)
Vol. 5, No. 3, August 2021, pp. 451-460 PENDAHULUAN Indonesia
merupakan
negara
berkembang
dimana
keadaan
perekonomian masih di pengaruhi oleh adanya tekanan eksternal dari negara-
negara maju yang berpengaruh terhadap keadaan perekonomian dunia hal ini
di sebabkan karena Indonesia menganut sistem perekonomian terbuka. Keadaan ekonomi dunia yang determinasinya merupakan keadaan
perekonomian negara-negara maju menyebabkan Indonesia sebagai negara
berkembang yang merasakan dampak secara langsung atas kebijakan yang
telah di tetapkan, salah satu dampak yang di rasakan yakni tidak stabilnya nilai
mata uang, tingkat investasi asing yang menurun yang selanjutnya akan
berdampak terhadap tingkat produksi hal ini juga dapat menghambat
pembangunan di Indonesia yang dimana peranan ekonomi sangat penting
untuk dapat melakukan pembangunan. (Asmanto and Suryandari 2009). Peranan Cadangan devisa sangat penting untuk menompang perekonomian
negara karena Cadangan Devisa sendiri di gunakan untuk menjaga kestabilan
negara saat adanya tekanan eksternal yang tidak terduga maupun tekanan dari
dalam negeri yang berdampak pada ketahanan makroekonomi fungsinya untuk
suatu Negara adalah sebagai tolak ukur kekayaan yang di miliki oleh Negara
itu sendiri, memiliki cadangan devisa yang cukup sangat penting yang
fungsinya untuk mendorong kegiatan perdagangan internasional kemudian
fungsi untuk menjaga ketahanan nilai mata uang ketika adanya tekanan
eksternal yang tidak terduga dan fungsi sebagai jenis kekayaan. Ketika neraca 451 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA pembayaran internasional mengalami defisit maka cadangan devisa akan di
gunakan untuk melakukan pembayaran impor dan utang luar negeri sehingga
neraca pembayaran kembali stabil. Untuk menjaga kestabilan moneter,
cadangan devisa di gunakan untuk melakukan intervensi dalam pasar valuta
asing untuk menjaga ketahanan nilai mata uang. Besaran penambahan
cadangan devisa suatu Negara di tentukan oleh kemampuan Negara tersebut
dalam melakukan kegiatan perdagangan internasional dan sistem nilai tukar
yang di tetapkan. Di Indonesia sistem nilai tukar yang di tetapkan adalah sistem
nilai tukar mengambang, jika nilai tukar berfluktuasi atau mengalami
depresiasi maka cadangan devisa di gunakan untuk mengembalikan kestabilan
nilai tukar dengan cara intervensi terhadap pasar valuta asing yang di lakukan
oleh Bank Indonesia untuk mencapai nilai tukar yang di harapkan dan menjaga
nilai tukar rupiah, tujuannya agar dapat menjaga laju inflasi dalam jangka
menengah. Besaran cadangan devisa yang di miliki suatu Negara juga di
pengaruhi oleh derajat keterbukaan ekonomi suatu Negara, sistem
perekonomian yang terbuka akan mendorong transaksi perdagangan
internasional, sehingga permintaan devisa akan semakin besar hal ini akan
menyebabkan terdapat risiko ketimpangan neraca pembayaran internasional,
untuk mencegah hal tersebut terjadi maka pengelolaan cadangan devisa,
pembayaran impor dan utang luar negeri sangat penting untuk pencegahan
krisis. PENDAHULUAN Di dalam perekonomian adanya utang luar negeri jangka pendek
mempresentasikan tingkat kecukupan cadangan devisa Sehingga dalam hal ini
ketersediaan Cadangan Devisa dapat membantu negara untuk mecapai
kestabilan moneter dan ketahanan perekonomian negara. Sehingga
pengelolaan terhadap cadangan devisa sangat penting untuk terus menjaga
posisi cadangan devisa dengan cara melakukan pendorongan akan faktor-
faktor yang dapat menambah cadangan devisa yang di miliki. Sehingga
Indonesia dapat terus melakukan pembangunan dan menjaga kestabilan
perekonomian, yang secara langsung yang akan berdampak terhadap
ketahanan ekonomi ketika ada tekanan yang tidak terduga yang dapat berasal
dari pasar keuangan luar negri maupun tekanan dari keuangan dalam negeri
(Gandhi 2006; Tambunan 2000). Tabel 1. Cadangan Devisa Tahun 2010-2019 (US$)
Tahun
Cadangan Devisa Indonesia
2010
96,210,980,584.043
2011
110,136,597,662.435
2012
112,797,627,833.066
2013
99,386,826,239.083
2014
111,862,594,561.997
2015
105,928,847,088.719
2016
116,369,601,851.059
2017
130,215,330,382.957
2018
120,660,974,090.950
2019
129,186,464,020.187 Tabel 1. Cadangan Devisa Tahun 2010-2019 (US$) 452 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA Berdasarkan Tabel di atas dapat di lihat selama beberapa tahun Cadangan
Devisa di Indonesia mengalami fluktuasi, hal tersebut dapat membahayakan
kestabilan ekonomi di Indonesia apabila terjadi penurunan secara drastis dan
terus menerus sehingga pengelolaan cadangan devisa sangat penting. Ketersediaan Cadangan Devisa yang di miliki tentunya di pengaruhi oleh
beberapa faktor sehingga dalam hal ini untuk dapat menjaga ketahanan posisi
cadangan devisa maka pendeteksian akan faktor-faktor apa saja yang dapat
menambah cadangan devisa yang di miliki harus di lakukan. Penelitian yang di lakukan oleh (Kuswantoro 2017) Pengaruh Inflasi, Kurs
Rupiah, Utang Luar Negri dan Ekspor Terhadap Cadangan Devisa. Adapun
hasil penelitian Secara Simultan Kurs Rupiah, Utang Luar negri dan Ekspor
berpengaruh Positif terhadap Cadangan Devisa. Sedangkan secara parsial
Inflasi negatif tidak berpengaruh terhadap cadangan devisa dan secara parsial
Kurs Rupiah, Utang Luar Negri dan Ekspor berpengaruh positif terhadap
Cadangan Devisa. Selain itu hasil analisis koefisien determinasi menampilkan
bahwa Cadangan Devisa dapat dipaparkan oleh variabel Inflasi, Kurs Rupiah,
Utang Luar negri dan Ekspor sebesar 91,25%. Penelitian oleh(Rochman 2015b)
Pengaruh Inflasi, Kurs, utang Luar Negri dan ekspor Terhadap Cadangan
Devisa. Adapun perolehan hasil penelitian menunjukkan secara simultan
Inflasi, Kurs, Utang Luar Negri dan Ekspor berpengaruh positif terhadap
Cadangan Devisa. Sedangkan secara Parsial Inflasi negatif tidak berpengaruh
terhadap Cadangan Devisa dan Kurs, Utang Luar Negri dan Ekspor
berpengaruh positif terhadap Cadangan Devisa. Adapun hasil koefisien
determinasi menunjukkan Cadangan Devisa dapat di paparkan oleh variabel
Inflasi, Kurs, Utang Luar Negri dan Ekspor sebesar 91,25%. PENDAHULUAN Penelitian
oleh(Sayoga and Tan 2017) Analisis Cadangan Devisa dan Faktor-faktor Yang
Mempengaruhinya. Adapun hasil penelitian menggunakan teknik deskriptif
menujukkan Cadangan Devisa tertinggi terletak pada tahun 2010. Sedangkan
hasil teknik analisis kuantitafi menunjukkan secara simultan Ekspor, Nilai
Tukar Rupiah, Utang Luar Negeri menunjukkan terdapat hubungan signifikan
terhadap Cadangan Devisa. Secara Parsial Utang Luar Negri dan Ekspor
terdapat hubungan positif terhadap Cadangan Devisa sedangkan untuk Nilai
Tukar Rupiah terdapat hubungan negatif terhadap cadangan devisa. Relevansi
penelitian terdahulu dengan penelitian ini adalah faktor yang mempengaruhi
cadangan devisa sedangkan untuk gap penelitian ini adanya penggunaan model
dan alat analisis yang berbeda serta keterbaruan tahun data. Sehingga dapat di
lihat pengaruh jangka pendek dan jangka panjang ekspor non migas, nilai tukar
rupiah, utang luar negeri dan inflasi terhadap cadangan devisa di Indonesia. METODE PENELITIAN Penelitian ini menggunakan pendekatan deskriptif kuantitatif dengan
penentuan obyek penelitian di Indonesia ekspor non migas, nilai tukar rupiah,
utang luar negri dan inflasi sebagai variabel bebas dan cadangan devisa sebagai
variabel terikat. Sumber data yang di gunakan merupakan data sekunder yang
dimana data tersebut di peroleh dari publikasi data Badan Pusat Statistik (BPS)
Indonesia dan data World Bank (Bank Dunia) dengan teknik pengumpulan 453 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA data yang di gunakan yaitu dengan cara dokumentasi, yang kemudian akan di
olah menjadi data Time Series dengan kurun waktu 21 tahun dari 1999-2019. Untuk mencapai tujuan penelitian di gunakan model ekonometrika dengan
Analisis Regresi Partial Adjustment Model (PAM) untuk memperoleh
persamaan jangka pendek dan Model ekspektasi adaptif untuk memperoleh
persamaan jangka panjang selain itu untuk melihat kelayakan data maka akan
di lakukan Uji Asumsi Klasik. data yang di gunakan yaitu dengan cara dokumentasi, yang kemudian akan di
olah menjadi data Time Series dengan kurun waktu 21 tahun dari 1999-2019. Untuk mencapai tujuan penelitian di gunakan model ekonometrika dengan
Analisis Regresi Partial Adjustment Model (PAM) untuk memperoleh
persamaan jangka pendek dan Model ekspektasi adaptif untuk memperoleh
persamaan jangka panjang selain itu untuk melihat kelayakan data maka akan
di lakukan Uji Asumsi Klasik. Regresi Partial Adjustment Model (PAM) untuk estimasi jangka pendek
dalam penelitian ini akan menggunakan formulasi sebagai berikut (Basuki
2014): 𝐷𝐸𝑉𝑡= 𝛽0 + 𝛽1 + 𝐿𝑂𝐺(𝐸𝐾𝑆)𝑡+ 𝛽2𝐿𝑂𝐺(𝐾𝑈𝑅𝑆)𝑡+ 𝛽3𝐿𝑂𝐺(𝐷𝐸𝐵𝑇)𝑡+
𝛽4𝐿𝑂𝐺(𝐼𝑁𝐹)𝑡+ 𝛽5𝐿𝑂𝐺(𝐷𝐸𝑉)𝑡−1 + 𝜀𝑡 ………………… (1) 𝐷𝐸𝑉𝑡= 𝛽0 + 𝛽1 + 𝐿𝑂𝐺(𝐸𝐾𝑆)𝑡+ 𝛽2𝐿𝑂𝐺(𝐾𝑈𝑅𝑆)𝑡+ 𝛽3𝐿𝑂𝐺(𝐷𝐸𝐵𝑇)𝑡+
𝛽4𝐿𝑂𝐺(𝐼𝑁𝐹)𝑡+ 𝛽5𝐿𝑂𝐺(𝐷𝐸𝑉)𝑡−1 + 𝜀𝑡 ………………… (1) 𝛽4
(
)𝑡
𝛽5
(
)𝑡1
𝑡
( )
Model matematika untuk estimasi jangka panjang dengan model Ekspektasi
adaptif(Damondaar N. Gujarati 2007): … (2) )
𝑋𝑡
∗−𝑋𝑡−1
∗
= 𝛾(𝑋𝑡
∗−𝑋𝑡−1
∗
) ……………… (2) HASIL DAN PEMBAHASAN Uji normalitas sendiri di butuhkan karena untuk melihat apakah data
berdistribusi normal dan tidak bias. Uji normalitas tidak hanya di gunakan
untuk melihat probabilitas selain itu dapat di gunakan untuk melakukan uji t,
uji f dan r-square yang selanjutnya terdapat di regresi. Dalam penelitian ini
Nilai probability menunjukkan 0,272453 dimana lebih besar dari 0,05 maka
artinya error berdistribusi normal. Tabel 2 Hasil Uji Normaalitas
Jarque-bera Probabilitas
0,272453 Tabel 2 Hasil Uji Normaalitas
Jarque-bera Probabilitas
0,272453 Tabel 2 Hasil Uji Normaalitas
Jarque-bera Probabilitas
0,272453 Uji Multikolinearitas pada dasarnya adalah adanya hubungan linear di
dalam variabel penjelas. Di dalam kasus multikolinearitas yang terjadi di dalam
time series biasanya di sebabkan oleh adannya common trends di dalam variabel
bebas yang menyebabkan dapat dapat menurun ataupun meningkat dalam
kurun waktu tertentu. Penelitian ini untuk mengetahui apakah adanya
multikolinearitas maka di gunakan model koefisien determinasi regresi
auxiliary dimana apabila nilai koefisien determinasi menunjukkan lebih dari
0,97 maka terdapat multikolinearitas. Dari hasil estimasi model koefisien
determinasi regresi auxiliary dapat di ketahui bahwa koefisien determinasi
auxiliary variabel bebas ekspor non migas, nilai tukar rupiah, utang luar negeri
dan inflasi tidak ada yang lebih besar dari koefisien determinasi model utaman
cadangan devisa (y) dengan demikian dapat di katakan tidak terjadi
multikolinearitas di dalam data ini. Tabel 3 Hasil Uji Multikolinearitas
Variabel Bebas
Nilai Koefisien Determinasi
Ekspor Non Migas
0,68
Nilai Tukar Rupiah
0,64 454 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA Variabel Bebas
Utang Luar Negeri
Inflasi Nilai Koefisien Determinasi Uji Heteroskedastisitas sendiri adalah masalah yang terdapat dalam
regresi dimana terdapat sifat homo atau sama dalam variabel. Dalam penelitian
ini untuk mendeteksi heteroskedastisitas maka di gunakan model glejser
dimana apabila probanilitas chi-square yang di peroleh dari R-square auxiliary
lebih dari 0,05 maka tidak terdapat heteroskedatisitas atau apabila nilai
probabilitas chi-square kurang dari 0,05 maka terdapat heteroskedastisitas. Berdasarkan uji glejser di peroleh nilai prob chi sebesar 0,8858 dimana nilai
tersebut
lebih
besar
dari
0,05. Dengan
demikian
tidak
terdapat
heteroskedasitas. bel 4 Hasil Uji Heteroskedastisitas
Probabilitas Chi-square
0,8858 Uji Autokorelasi sering terdapat di dalam data time series karena
adanya korelasi antar variabel dalam penelitian sebelumnya dimana variabel
tersebut tergantung dengan data tahun-tahun sebelumnya. Dalam penelitian ini
di gunakan model LM Breusch-godfrey untuk mendeteksi autokorelasi, dalam
penelitian ini menggunakan Lag untuk mendeteksi perbandingan nilai antar
Akaike dengan Schwarz yang paling kecil. Berdasarkan output uji LM Breusch
godfrey untuk berbagai panjang lag, lag optimum yang memiliki AIC dan SIC
minimum adalah lag 1. Dari output di peroleh prob chi (1) = 30,37206. Nilai
tersebut lebih besar dari 0,05 sehingga tidak terjadi autokorelasi pada data. Tabel 5 Hasil Uji Autokorelasi
Lag 1
Lag 2
Lag 3
Lag 4
Lag 5
AIC
30,37206
30,41157
30,49847
30,56373
30,64933
SIC
30,67050
30,75974
30,89639
31,01139
31,14673
Hasil Uji Regresi Partial Adjutment Model (PAM)
Tabel 6 Hasil Uji PAM
Variable
Coefficient
t-statistic
probability
LOG(EKS)
0.559055
1233189
0.0000
LOG(KURS)
0.060897
2126704
0.0494
LOG(ULN)
-0.003674
-0.650335
0.9380
LOG(INF)
-0.142250
-1.831627
0.1119
LOG(DEV(-1))
0.433503
2073461
0.0000
C
1.945.567
0.917698
0.3743
R-Squared
0.966371
Adjusted
0.957964
F-Statistic
114.9446 Tabel 5 Hasil Uji Autokorelasi
Lag 1
Lag 2
Lag 3
Lag 4
Lag 5
AIC
30,37206
30,41157
30,49847
30,56373
30,64933
SIC
30,67050
30,75974
30,89639
31,01139
31,14673 Hasil Uji Regresi Partial Adjutment Model (PAM)
Tabel 6 Hasil Uji PAM
Variable
Coefficient
t-statistic
probability
LOG(EKS)
0.559055
1233189
0.0000
LOG(KURS)
0.060897
2126704
0.0494
LOG(ULN)
-0.003674
-0.650335
0.9380
LOG(INF)
-0.142250
-1.831627
0.1119
LOG(DEV(-1))
0.433503
2073461
0.0000
C
1.945.567
0.917698
0.3743
R-Squared
0.966371
Adjusted
0.957964
F-Statistic
114.9446 Hasil Uji Regresi Partial Adjutment Model (PAM)
Tabel 6 Hasil Uji PAM 455 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA Perolehan persamaan untuk jangka pendek sebagai berikut:
LOG (DEV) = 1.945567 + 0.559055 LOG(EKS) + 0.060897 LOG(KURS) -
0.003674LOG(ULN) – 0.142250 LOG(INF) + 0.433503 LOG(DEV(-1)) + e Selanjutnya yaitu melakukan perhitunga koefisien penyesuaian untuk
menentukan persamaan jangka panjang dimana hal tersebut di lakukan dengan
cara membagi koefisien dalam jangka pendek dengan koefisien penyesuaian. Uji Koefisien Determinasi R-square sendiri menggambarkan seberapa besar kemampuan variabel Y
di jelaskan oleh variabel X dalam hal ini semakin mendekati 1 maka
kemampuan variabel X menjelaskan variabel X semakin besar karena 1
menggambarkan 100%. Dalam penelitian ini perolehan R-square sebesar
0.966371 atau sebesar 97%. Dimana artinya variabel X mampu menjelaskan
variabel Y sebesar 97% sedangkan sisanya 3% di jelaskan oleh variabel lain
yang tidak terdapat dalam penelitian ini. Nilai Koefisien Determinasi Perhitungan koefisen penyesuaian dengan perhitungan 1- 0.433503 =0.566497
artinya terdapat perbedaan antara cadangan devisa yang di harapkan dengan
yang terjadi 57% di sesuaikan dalam periode tahunan. Sehingga dari koefisien penyesuaian dengan menggunakan ekspektasi
adaptif maka perolehan persamaan untuk jangka panjang sebagai berikut: LOG (DEV) =0.566497 + 0.986863125 LOG(EKS) + 0.10749748
LOG(KURS) - 0.006485471 LOG(ULN) - 0.251104595 LOG(INF)+e LOG (DEV) =0.566497 + 0.986863125 LOG(EKS) + 0.10749748
LOG(KURS) - 0.006485471 LOG(ULN) - 0.251104595 LOG(INF)+e y
g
p
p
Uji Simultan (Uji F-Statistik) y
g
p
p
Uji Simultan (Uji F-Statistik) Pengujian F-Satatistik di gunakan untuk menguji secara serentak
variabel bebas terhadap variabel terikat. Dalam penelitian di peroleh nilai F-
statstik sebesar 114.9446 yang menunjukkan signifikan dimana nilai tersebut
lebih besar dari nilai F-tabel yang sebesar 3.24 sehingga dapat di simpulkan
menerima H1 yang artinya secara simultan Ekspor Non Migas, Nilai Tukar
Rupiah, Utang Luar Negeri dan Inflasi mempengaruhi Cadangan Devisa di
Indonesia. Tabel 7 Hasil Uji F
F-hitung
F-tabel (α = 5%)
Kriteria
114.9446
3.24
Signifikan
Uji t Statistik Pengaruh Ekspor Non Migas terhadap Cadangan Devisa g
p
g
p
g
Berdasarkan hasil regresi PAM menunjukkan hasil secara jangka panjang
dan jangka pendek ekspor non migas berpengaruh positif dan signifikan
dimana hasil probabilitas uji t menunjukkan hasil sebesar 12.33189
menunjukkan terdapat pengaruh yang signifikan terhadap cadangan devisa. Hasil regresi PAM menunjukkan setiap kenaikan 1% ekspor non migas dapat
meningkatkan cadangan devisa sebesar 56% dalam jangka pendek dan secara
jangka panjang dapat meningkatkan cadangan devisa sebesar 99%. Hal tersebut
sejalan dengan penelitian yang di lakukan oleh (Sukandar 2020) yang
memperoleh hasil bahwa ekspor non migas berpengaruh signifikan terhadap
cadangan devisa. Pada sistem perekonomian terbuka neraca perdagangan
berperan penting hal ini di sebabkan neraca perdagangan yang surplus akan
menabah ketersediaan devisa, laju pertumbuhan pendapatan yang di sebabkan
oleh ekspor akan meyebabkan neraca pembayaran surplus sehingga peranan
ekspor sangat penting bagi keseimbangan neraca pembayaran. Negara dapat
memperoleh aset pembayaran dari ekspor yang berupa valuta asing yang akan
menambah ketersediaan devisa. Selain itu dengan meningkatnya ekspor di
sektor
non
migas
dapat
menstabilkan
perekonomian
karena
laju
pertumbuhannya yang lebih besar di bandingkan dengan sektor migas sehingga
dapat meminimalisir risiko ekonomi Indonesia terhadap perubahan-perubahan
yang terjadi di nilai tukar internasional. Uji t-Statistik j
Pengujian t-statistik di gunakan untuk menguji satu persatu (individual)
variabel bebas terhadap variabel terikat. Dalam penelitian ini di peroleh hasil
uji t-statistik sebagai berikut: 456
j
g
Tabel 8 Hasil Uji t
Variabel
Koefisien
t-hitung t-tabel (α = 5%)
Keterangan
EKS
0.559055
12.33189
2.120
Signifikan 456 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA Variabel
Koefisien
t-hitung t-tabel (α = 5%)
Keterangan
KURS
0.060897
2.126704
2.120
Signifikan
ULN
-0.003674
0.650335
2.120
Tidak Signifikan
INF
-0.142250
-1.831627
-2.120
Tidak Signifikan Pengaruh Utang Luar Negeri Terhadap Cadangan Devisa Berdasarkan hasil regresi PAM yang telah di lakuakan menunjukkan
bahwa secara jangka pendek dan jangka panjang utang luar negeri terhadap
cadangan devisa berpengaruh negatif dan tidak signifikan hal ini karena nilai
uji t sebesar 0.650335 yang lebih kecil daripada nilai t-tabel sehingga di
simpulkan tidak ada hubungan antara utang luar negeri dengan cadangan
devisa. Hasil regresi PAM menunjukkan setiap kenaikan 1% utang luar negeri
secara jangka pendek akan menurunkan cadangan devisa yang di miliki
sebebsar 0,4% sedangkan secara jangka panjang utang luar negeri akan
menurunkan sebesar 0,6% cadangan devisa yang di miliki. Hal tersebut
menunjukkan hasil penelitian tidak sesuai dengan hipotesis yang telah di
tentukan tetapi menurut Boediono apabila utang luar negeri yang di peroleh di
gunakan untuk mengimpor barang-barang yang di gunakan oleh pemerintah
maka tidak terdapat pengaruh terhadap perekonomian dalam negeri selain itu
penelitian sebelumnya yang di lakukan oleh (Susanti 2019)perolehan dari hasil
penelitian tersebut menunjukkan bahwa tidak terdapat pengaruh antara utang
luar negeri terhadap cadangan devisa. Pengaruh Nilai Tukar Rupiah terhadap Cadangan Devisa. Syarat untuk mempertahankan
kestabilan nikai tukar domestik adalah tersedianya cadangan devisa yang lebih
dari cukup agar tercapainya kestabilan perdagangan internasional sehingga
perolehan devisa dari ekspor terus mengalir(Syarifuddin 2015). Pengaruh Nilai Tukar Rupiah terhadap Cadangan Devisa. Berdasarkan hasil Regresi PAM yang telah di lakukan menunjukkan
bahwa secara jangka pendek dan jangka panjang nilai tukar rupiah terhadap
cadangan devisa berpengaruh positif dan signifikan dimana hasil uji t
menunjukkan nilai probabilitas sebesar 2.126704 yang lebih besar dari nilai t-
tabel terdapat pengaruh yang signifikan. Hasil regresi PAM menunjukkan
setiap kenaikan 1% nilai tukar rupiah secara jangka pendek dapat
meningkatkan cadangan devisa sebesar 6% sedangkan secara jangka panjang
dapat meningkatkan cadangan devisa sebesar 11% sehingga setiap nilai tukar
rupiah mengalami apresiasi akan meningkatkan cadangan devisa. Hasil
penelitian ini sejalan dengan penelitian yang di lakukan oleh (Kuswantoro 2017;
Rochman 2015a)hasil dari kedua penelitian tersebut menyatakan bahwa nilai
tukar rupiah berpengaruh signifikan positif. Uang yang sifatnya tidak netral
akan mempengaruhi kegiatan perekonomian Negara sehingga Nilai tukar
sangat penting peranannya untuk menjaga kestabilan harga komoditas dimana
nilai tukar berperan sebagai kunci dari kegiatan perdagangan internasional,
apabila nilai tukar rupiah mengalami apresiasi menunjukkan ekspor sedang
meningkat yang artinya akan menambah aset negara melalui pembayaran 457 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA ekspor yang akan menyebabkan neraca pembayaran internasional surplus
dimana artinya cadangan devisa yang di miliki cukup. Sebaliknya ketika nilai
tukar rupiah mengalami depresi maka harga komoditas akan mengalami
penurunan hal ini akan menaikkan permintaan terhadap ekspor namun
berdampak buruk terhadap impor bahan untuk melakukan produksi sehingga
akan menghambat kegiatan ekonomi, untuk mestabilkan nilai tukar rupiah
maka di butuhkan cadangan devisa untuk membeli valuta asing di pasar valas,
yang akan menyebabkan defisit neraca pembayaran internasional, adanya
peningkatan ekspor secara terus-menerus akan menyebabkan jangka panjang
terhadap apresisasi nilai tukar domestik hal tersebut di sebabkan oleh terjualnya
komoditas domestik secara terus-menerus. Syarat untuk mempertahankan
kestabilan nikai tukar domestik adalah tersedianya cadangan devisa yang lebih
dari cukup agar tercapainya kestabilan perdagangan internasional sehingga
perolehan devisa dari ekspor terus mengalir(Syarifuddin 2015). ekspor yang akan menyebabkan neraca pembayaran internasional surplus
dimana artinya cadangan devisa yang di miliki cukup. Sebaliknya ketika nilai
tukar rupiah mengalami depresi maka harga komoditas akan mengalami
penurunan hal ini akan menaikkan permintaan terhadap ekspor namun
berdampak buruk terhadap impor bahan untuk melakukan produksi sehingga
akan menghambat kegiatan ekonomi, untuk mestabilkan nilai tukar rupiah
maka di butuhkan cadangan devisa untuk membeli valuta asing di pasar valas,
yang akan menyebabkan defisit neraca pembayaran internasional, adanya
peningkatan ekspor secara terus-menerus akan menyebabkan jangka panjang
terhadap apresisasi nilai tukar domestik hal tersebut di sebabkan oleh terjualnya
komoditas domestik secara terus-menerus. KESIMPULAN Perolehan dari penelitian ini secara simultan ekspor non migas, nilai
tukar rupiah, utang luar negeri dan inflasi mempengaruhi cadangan devisa
sedangkan secara parsial hasil uji regresi PAM jangka pendek dan jangka
panjang menunjukkan ekspor non migas dan nilai tukar rupiah bahwa terdapat
hubungan positif dan berpengaruh signifikan terhadap cadangan devisa di
Indonesia, setiap kenaikan ekspor non migas akan menambah cadangan devisa
yang di miliki hal ini karena terjadinya cash inflow kemudian untuk setiap nilai
tukar rupiah terapresiasi akan mengakibatkan ekspor meningkat sehingga akan
meningkatkan cadangan devisa yang di miliki. Sedangkan hasil untuk jangka
pendek dan jangka panjang menunjukkan utang luar negeri dan inflasi
hubungannya adalah negatif dan tidak ada pengaruh dengan cadangan devisa
di Indonesia, sehingga adanya peningkatan atau penambahan dalam utang luar
negeri dan inflasi hal ini akan menurunkan cadangan devisa di Indonesia. Ekspor non migas, nilai tukar rupiah, utang luar negeri dan inflasi dapat
menjelaskan cadangan devisa sebesar 97% sedangkan untuk 3% di jelaskan oleh
faktor lain yang tidak dimasukkan dalam penelitian ini. Pengaruh Inflasi terhadap Cadangan Devisa Berdasarkan hasil Regresi PAM yang telah di lakuakan menunjukkan
bahwa secara jangka pendek dan jangka panjang inflasi terhadap cadangan
devisa berpengaruh negatif dan tidak signifikan dimana hasil uji t
menununjukkan sebesar -1.831627 yang lebih kecil dari nilai t-tabel sehingga
dapat di simpulkan tidak terdapat pengaruh inflasi terhadap cadangan devisa. Hasil regresi PAM menunjukkan setiap kenaikann 1% inflasi akan
menurunkan cadangan devisa yang di miliki secara jangka pendek sebanyak
14% sedangkan secara jangka panjang inflasi akan menurunkan cadangan
devisa sebanyak 25%. Ketika terjadi inflasi hal ini akan berdampak terhadap
kegiatan perekonomian yang dapat menurunkan tingkat produksi dan
mempengaruhi tingkat harga sehingga menyebabkan menurunnya daya beli
masyarakat yang akan menyebabkan jumlah uang beredar sangat banyak yang 458 Fitria
Soelistyo
Susilowati ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA akan menyebabkan depresiasi nilai mata uang. Ketika terjadi depresiasi
pemerintah akan melakukan interfensi terhadap pasar valuta asing dengan
melakukan pembelian valas maka akan ketersediaan cadangan devisa yang di
miliki akan berkurang. Hasil penelitian ini di perkuat dengan sejalannya
dengan penelitian terdahulu yang telah di lakukan (Kuswantoro 2017;
Rochman 2015b)perolehan dari kedua penelitian tersebut menunjukkan bahwa
pengaruh inflasi terhadap cadangan devisa tidak terdapat pengaruh. akan menyebabkan depresiasi nilai mata uang. Ketika terjadi depresiasi
pemerintah akan melakukan interfensi terhadap pasar valuta asing dengan
melakukan pembelian valas maka akan ketersediaan cadangan devisa yang di
miliki akan berkurang. Hasil penelitian ini di perkuat dengan sejalannya
dengan penelitian terdahulu yang telah di lakukan (Kuswantoro 2017;
Rochman 2015b)perolehan dari kedua penelitian tersebut menunjukkan bahwa
pengaruh inflasi terhadap cadangan devisa tidak terdapat pengaruh. Tambunan, T. and Wijanarko. 2000. Perdagangan Internasional Dan Neraca
Pembayaran: Teori Dan Temuan Empiris. LP3ES. Sukandar. 2020. “Pengaruh Ekspor Migas Dan Non Migas Terhadap
Cadangan Devisa Indonesia 2016-2019 Dalam Prespektif Islam.” : 6. g
p
Susanti, Tiyas. 2019. “Pengaruh Utang Luar Negeri, Ekspor,Kurs Dan Inflasi
Terhadap Cadangan Devisa Indonesia.” 8(2): 2019. DAFTAR PUSTAKA Asmanto, Priadi, and Sekar Suryandari. 2009. “Cadangan Devisa, Financial
Deepening Dan Stabilisasi Nilai Tukar Riil Rupiah Akibat Gejolak Nilai
Tukar Perdagangan.” Buletin Ekonomi Moneter dan Perbankan 11(2). g
g
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Basuki, Agus Tri. 2014. “Regresi Model PAM, ECM Dan Data Panel
Dengan Eviews 7.” : 1–75. Damondaar N. Gujarati. 2007. Dasar-Dasar Ekonometrika. Edisi Keti. Jakarta:
Erlangga. Gandhi, Dyah Virgoana. 2006. Seri Kebanksentralan Pengelolaan Cadangan
Devisa Di Bank Indonesia. Kuswantoro, Muchammad. 2017. “Analisis Pengaruh Inflasi, Kurs, Utang
Luar Negeri Dan Ekspor Terhadap Cadangan Devisa Indonesia.”
Tirtayasa Ekonomika 12(1): 146. Rochman, Asep Maulana. 2015. “Pengaruh Inflasi, Kurs Rupiah, Utang Luar
Negri Dan Ekspor Terhadap Cadangan Devisa Indonesia.” Sayoga, Pundy, and Syamsurijal Tan. 2017. “Analisis Cadangan Devisa
Indonesia Dan Faktor-Faktor Yang Mempengaruhinya.” Jurnal
Paradigma Ekonomika 12(1): 25–30. 459 ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA
Fitria
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Susilowati
Sukandar. 2020. “Pengaruh Ekspor Migas Dan Non Migas Terhadap
Cadangan Devisa Indonesia 2016-2019 Dalam Prespektif Islam.” : 6. Susanti, Tiyas. 2019. “Pengaruh Utang Luar Negeri, Ekspor,Kurs Dan Inflasi
Terhadap Cadangan Devisa Indonesia.” 8(2): 2019. Syarifuddin, Ferry. 2015. “Konsep, Dinamika Dan Respon Kebijakan Nilai
Tukar Di Indonesia.” Bank Indonesia (24): 1–101. Tambunan, T. and Wijanarko. 2000. Perdagangan Internasional Dan Neraca
Pembayaran: Teori Dan Temuan Empiris. LP3ES. Fitria
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Susilowati ANALISIS FAKTOR-FAKTOR YANG MEMPENGARUHI CADANGAN
DEVISA DI INDONESIA Susanti, Tiyas. 2019. “Pengaruh Utang Luar Negeri, Ekspor,Kurs Dan Inflasi
Terhadap Cadangan Devisa Indonesia.” 8(2): 2019. p
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Syarifuddin, Ferry. 2015. “Konsep, Dinamika Dan Respon Kebijakan Nilai
Tukar Di Indonesia.” Bank Indonesia (24): 1–101. p
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Predicting attitudes toward ambiguity using natural language processing on free descriptions for open-ended question measurements
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Scientific reports
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Predicting attitudes
toward ambiguity using natural
language processing on free
descriptions for open‑ended
question measurements
OPEN Jimpei Hitsuwari 1,2, Hirohito Okano 1 & Michio Nomura 1* Individual traits and reactions to ambiguity differ and are conceptualized in terms of an individual’s
attitudes toward ambiguity or ambiguity tolerance. The development of natural language processing
technology has made it possible to measure mental states and reactions through open-ended
questions, rather than predefined numerical rating scales, which have traditionally been the dominant
method in psychological research. This study presented three ambiguity-related situations and
responses collected online from 591 participants in an open-ended format. After the analysis with
bidirectional encoder representations from transformers, correlations were calculated using scores
from the numerical evaluation by conventional questionnaire, and a significant moderate positive
correlation was found. Therefore, this study found that attitudes toward ambiguity can be measured
using an open-ended response method of reporting everyday life states. It is a novel methodology that
can be expanded to other scales in psychology and can potentially be used in educational and clinical
situations where participants can be asked to respond with minimal burden. The ambiguous situations faced in the volatility, uncertainty, complexity, and ambiguity (VUCA) era are diverse,
with individual differences in attitudes toward these ambiguous situations1. To measure individual differences,
Lauriola et al.2 developed the Multidimensional Attitude toward Ambiguity Scale (MAAS) based on the Ambigu-
ity Tolerance Scale, which measures individuals’ tolerance degree toward ambiguous situations. This scale has
been validated for construct validity and internal reliability2. The MAAS is utilized globally, with Japanese3 and
Swedish versions4 also being developed. It has been used in numerous behavioral experiments and psychologi-
cal surveys5,6.i However, responding to a predefined numerical rating scale is not necessarily the optimal method to capture
complex mental states and personality traits (People do not usually answer or express their states and emotions
on a yes or no or 1–7 point scale, and most often use natural language.; for review, see7). Considering the recent
popularity of ChatGPT, the development of large language models has made it possible to measure psychologi-
cal states based on natural language, which was quite challenging in the past. For example, in Kjell et al.’s study,
participants had to answer the question, “Overall, in your life, are you satisfied or not?”8. www.nature.com/scientificreports www.nature.com/scientificreports 1Graduate School of Education, Kyoto University, Kyoto, Japan. 2Japan Society for the Promotion of Science,
Tokyo, Japan. *email: nomura.michio.8u@kyoto-u.ac.jp Methods
Th
d This study was approved by the Ethics Committee of the Graduate School of Education at Kyoto University (CPE-
571) and conducted in accordance with relevant guidelines and regulations. We obtained informed consent from
the study participants before their participation. Participants p
A total of 600 native English speakers of British nationality (the language used in most of the previous studies8
is English, so we targeted British nationals referring to those NLP studies) were recruited using an online survey
platform Prolific (https://www.prolific.com/). Nine were excluded because of duplicate IP addresses, extremely
short response times (less than 255 s), and attention-checking errors, resulting in 591 participants (Mage = 43.35,
SD = 14.44, 325 males, 255 females, 11 others) for the final analyses. A question for the attention check (For this
question, select “5. I mildly agree”) was added to MSTAT II (detailed in Procedure section) to exclude participants
who selected anything other than the required answer. They were paid£0.6 as a reward for their participation. Analysis
Th
d l The model for predicting the questionnaire scores was developed by fine-tuning the pre-trained BERT-base-cased
model (https://huggingface.co/bert-base-cased). Closed models like ChatGPT raise scientific reproducibility and
ethical concerns, as the precise architecture and training data are not disclosed, and updates are made without
revealing the differences7. Therefore, for this study, a more open model, BERT, was used. Regarding hyperparam-
eter selection during fine-tuning and final model evaluation, five-fold nested cross-validation (nested CV) was
used. The nested CV has a low bias in estimation accuracy12 and is particularly effective for machine learning
on small samples13. It allows obtaining an estimate of the model’s predictive accuracy, independent of the data
used to build the model (see Supplementary Material for more information). Procedureh The participants provided open-ended responses to three ambiguous situations. The three situations correspond
to the three factors of the MAAS: “How do you typically react when you are uncertain about the responsibilities
of a job? (Discomfort with Ambiguity; DA),” “How do you typically react when ambiguous words like ‘probably,’
‘approximately,’ or ‘perhaps’ are used? (Absolutism; AB),” and “How do you typically react when you are in situ-
ations which can be interpreted in more than one way? (Need for Complexity and Novelty; NC)” The responses
were required to have at least 100 characters (approximately 20 words), and at least 45 s had to pass before
answering the next question. Subsequently, participants responded to a questionnaire containing the MAAS and
the Multiple Stimulus Types Ambiguity Tolerance Scale-II (MSTAT-II)11. The MSTAT-II is a general measure
of ambiguity tolerance and was employed to determine whether it could predict this scale score from the three
situations created from the MAAS (usually, in MAAS, the average of each subscale score is calculated but not
the overall score). Finally, respondents’ demographic data (sex, age, nationality, and education) were collected. Descriptive statistics from the MAAS and MSTAT-II and examples of open-ended responses obtained from the
three texts are presented in Table 1. Predicting attitudes
toward ambiguity using natural
language processing on free
descriptions for open‑ended
question measurements
OPEN They examined the
correlation between the values calculated by bidirectional encoder representations from transformers (BERT),
a large language model, and the scores of the Satisfaction with Life Scale (SWLS)9, which has been convention-
ally used to measure life satisfaction. The BERT regression model transforms the participant’s free text into a
multidimensional vector and uses that vector representation to predict the individual’s questionnaire score. The
results indicated r = 0.74, implying that life satisfaction can be accurately measured using open-ended responses. In another study10, BERT was used to predict the Big Five personality traits based on user comments and posts
comprising fiction (e.g., short stories) in a novel-writing community on Reddit (a bulletin board social site). The results indicated an average performance of r = 0.33, suggesting that personality can be predicted using free
text. The present study asked participants to respond to open-ended questions in three situations (see below in
the Method section) involving ambiguity (from the MAAS subscale), and the obtained texts were analyzed. The
study aimed to determine the extent to which the survey methods consisting of free-text and natural language | https://doi.org/10.1038/s41598-024-59118-z Scientific Reports | (2024) 14:8276 www.nature.com/scientificreports/ processing (NLP) predicted ambiguity tolerance in comparison to conventional numerical scores. Additionally,
this study examined whether the texts answered from the respective MAAS subscales could discriminate between
the respective subscales answered with numerical values. processing (NLP) predicted ambiguity tolerance in comparison to conventional numerical scores. Additionally,
this study examined whether the texts answered from the respective MAAS subscales could discriminate between
the respective subscales answered with numerical values. Discussionhi The findings of this study are novel as they indicate that even free text can predict psychological states
traits8,10 with regard to ambiguity.h Three questions were asked in this study; however, only one question from NC, “How do you typically react
when you are in situations which can be interpreted in more than one way?” was moderately predictive. This
question is more general than the other two questions and applies to various situations. This suggests that refin-
ing situation settings and how questions are asked may allow attitudes toward ambiguity to be measurable, even
with only one open-ended response. The DA, AB, and NC texts showed weak but significant correlations with
their respective scores. Future studies should consider making it possible to discriminate between subscales, for
example, by devising how the questions are asked.h This survey method consisting of free-text and NLP will allow for the measuring of an individual’s personal-
ity in a more ecologically valid form; that is, an open-ended response method when expressing emotions and
states in everyday life8,10,14,15. In Kjell et al.’s study8, questions aimed to examine overall life satisfaction, such as
“Overall, in your life, are you satisfied or not?”; however, in this study, the question was constructed by specifying
the situation and asking the respondent to imagine the situation, where “it can be interpreted in more than one
way.” This allows the use of open-ended surveys that measure not only abstract concepts, such as life satisfaction,
but also other personality traits and psychological states that are more specific.fi p
y
p y
g
pi
While moderate correlation coefficients were observed, aligning with previous studies10, there is scope for
further improvement in correlation by employing alternative language models (e.g., RoBERTa), a topic of inter-
est for future studies. Consistent with previous studies, the results of this study are limited to English-language
data. However, given the translation of the scale into various languages, efforts will be made to globally predict
its scores in open-ended surveys in the future study. Both the MAAS and MSTAT-II used in this study were self-
reported, and future research can attempt to predict a behavior (e.g., decision-making in ambiguous situations)
based on participants’ open-ended responses and BERT scores. In conclusion, this study successfully predicted attitudes toward ambiguity by NLP of open-ended responses
using BERT. Resultsh DA
AB
NC
MSTAT
Text DA
0.28** [0.10, 0.44]
0.12 [− 0.07, 0.29]
0.10 [− 0.08, 0.28]
0.20* [0.02, 0.37]
Text AB
0.12 [− 0.06, 0.29]
0.23* [0.05, 0.40]
0.14 [− 0.04, 0.32]
0.06 [− 0.12, 0.24]
Text NC
0.26** [0.09, 0.42]
0.16† [− 0.02, 0.33]
0.19* [0.01, 0.36]
0.38*** [0.22, 0.53]
All texts
0.34*** [0.18, 0.49]
0.25** [0.07, 0.41]
0.28** [0.11, 0.44]
0.41*** [0.25, 0.55] DA
AB
NC
MSTAT
Text DA
0.28** [0.10, 0.44]
0.12 [− 0.07, 0.29]
0.10 [− 0.08, 0.28]
0.20* [0.02, 0.37]
Text AB
0.12 [− 0.06, 0.29]
0.23* [0.05, 0.40]
0.14 [− 0.04, 0.32]
0.06 [− 0.12, 0.24]
Text NC
0.26** [0.09, 0.42]
0.16† [− 0.02, 0.33]
0.19* [0.01, 0.36]
0.38*** [0.22, 0.53]
All texts
0.34*** [0.18, 0.49]
0.25** [0.07, 0.41]
0.28** [0.11, 0.44]
0.41*** [0.25, 0.55] Table 2. Median correlation coefficient between each text and each questionnaire score. Text DA (Discomfort
with Ambiguity), text AB (Absolutism), and text NC (Need for Complexity and Novelty) were calculated
as free-text responses obtained from open-ended questions adopted from the DA, AB, and NC questions,
respectively. The correlation coefficients in the table show the median correlation coefficients between the
predicted values and true ones for the five test sets in the outer loop (n = 591/5 = 118 or 119). All texts were
calculated by combining these three questions. *** p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.10. Table 2. Median correlation coefficient between each text and each questionnaire score. Text DA (Discomfort
with Ambiguity), text AB (Absolutism), and text NC (Need for Complexity and Novelty) were calculated
as free-text responses obtained from open-ended questions adopted from the DA, AB, and NC questions,
respectively. The correlation coefficients in the table show the median correlation coefficients between the
predicted values and true ones for the five test sets in the outer loop (n = 591/5 = 118 or 119). All texts were
calculated by combining these three questions. *** p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.10. general ambiguity tolerance. Additionally, texts from the DA (r = 0.28, p = 0.002), AB (r = 0.23, p = 0.01), and NC
(r = 0.19, p = 0.04) were weakly correlated with their respective MAAS subscale scores. Data availability Data availability
All data and script are available online (https://osf.io/jza53/?view_only=dbdc4b4c82f94410aed7e5ccbb22a98d) Received: 14 February 2024; Accepted: 8 April 2024 Received: 14 February 2024; Accepted: 8 April 2024 Discussionhi Through the utilization of these technologies, complex human minds can be measured in a way
that is natural to the participants, with little concern that the content of the questionnaire items will influence
participants’ cognitions. Academically, as the scale is translated into other languages, attempts can be made to
predict its scores in open-ended surveys globally to increase its accuracy and discrimination to apply it to social
surveys, education, clinical situations, among other spheres. Resultsh The correlation coefficients between the BERT-predicted and true values of the questionnaire scores when using
free-text responses to the three open-ended questions were calculated (Table 2 presents the medians; see Supple-
mentary Table 1 for the minimum and maximum values). Results indicated that text NC (r = 0.38, p < 0.001) and
the text combining all three texts (r = 0.41, p < 0.001) moderately predicted the MSTAT-II scores, which measure 2
4567890)
Scientific Reports | (2024) 14:8276 |
https://doi.org/10.1038/s41598-024-59118-z
Table 1. MAAS and MSTAT descriptive statistics and examples of free-text responses from the three
situations. M
SD
Examples
DA
4.48
1.12
I get into a bit of a panic, try to work out how to do it on my own, and if I am still unable to, I go to someone with
my tail between my legs
Wait and see; I do not get stressed, I seek to clarify exactly what the responsibilities will be and, if needed, double-
clarify
MA
3.35
1.18
I do not mind them if it is just occasionally, but it can be annoying when they are used in every other sentence
I would feel slightly distrustful and cautious. I have learned never to take assurances at face value. It depends on
who is using such language
NC
4.03
1.18
I always take my time, thinking through the options and how my actions could be interpreted by the people
around me
I react confused, I find it difficult to have to pick a certain path because I always question whether I have chosen
correctly
MSTAT
4.13
0.89 Scientific Reports | (2024) 14:8276 | www.nature.com/scientificreports/ Table 2. Median correlation coefficient between each text and each questionnaire score. Text DA (Discomfort
with Ambiguity), text AB (Absolutism), and text NC (Need for Complexity and Novelty) were calculated
as free-text responses obtained from open-ended questions adopted from the DA, AB, and NC questions,
respectively. The correlation coefficients in the table show the median correlation coefficients between the
predicted values and true ones for the five test sets in the outer loop (n = 591/5 = 118 or 119). All texts were
calculated by combining these three questions. *** p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.10. References 1. Furnham, A. & Ribchester, T. Tolerance of ambiguity: A review of the concept, its measurement and applications. Curr. Psychol. 14, 179–199. https://doi.org/10.1007/BF02686907 (1995). p
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Optimization of the Geometrical Parameters of a Solar Bubble Pump for Absorption-Diffusion Cooling Systems
|
American journal of engineering and applied sciences
| 2,010
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American J. of Engineering and Applied Sciences 3 (4): 693-698, 2010
ISSN 1941-7020
© 2010 Science Publications American J. of Engineering and Applied Sciences 3 (4): 693-698, 2010
ISSN 1941-7020
© 2010 Science Publications American J. of Engineering and Applied Sciences 3 (4): 693-698, 2010
ISSN 1941-7020
© 2010 Science Publications Corresponding Author: N. Dammak, Department of Chemical Engineering and Processes,
National School of Engineers of Gabes (E.N.I.G), Omar Ibn El Khattab Street-6029 Gabes, Tunisia
693 Optimization of the Geometrical Parameters of a Solar Bubble
Pump for Absorption-Diffusion Cooling Systems
1N. Dammak, 1B. Chaouachi, 1S. Gabsi and 2M. Bourouis
1Department of Chemical Engineering and Processes,
National School of Engineers of Gabes (E.N.I.G),
Omar Ibn El Khattab Street-6029 Gabes, Tunisia
2Department of Mechanical Engineering, University Rovira iVirgili,
Avenida dels Països Catalans, 26, 43007 Tarragona, Spain Abstract: Problem statement: The objective of this study was to optimize the geometrical parameters
of a bubble pump integrated in a solar flat plate collector. Approach: This solar bubble pump was part
of an ammonia/water/helium (NH3/H2O/He) absorption-diffusion cooling system. Results: An
empirical model was developed on the basis of momentum, mass, material equations and energy
balances. The mathematical model was solved using the simulation tool “Engineering Equation Solver
(EES)”. Conclusion/Recommendations: Using metrological data from Gabes (Tunisia) various
parameters were geometrically optimized for maximum bubble pump efficiency which was best for a
bubble pump tube diameter of 6 mm, a tube length of 1.5 m, an inclination to the horizontal between
30 and 50° of the solar flat plate collector and a submergence ratio between 0.2 and 0.3. Key words: Bubble pump, absorption-diffusion cycle, ammonia-water, solar cooling r: N. Dammak, Department of Chemical Engineering and Processes,
National School of Engineers of Gabes (E.N.I.G), Omar Ibn El Khattab Street-6029 Gabes, Tunisia
693 INTRODUCTION A new
Generator With Heat Exchanger (GWHX) was
designed, built and tested. The test results of the DAR
with GWHX were compared with baseline tests. The
new generator design increased the cooling COP by as
much as 50% more than the baseline tests while the
cooling capacity remained constant. Srikhirin and
Aphornratana (2002) reported an experimental study
of a DAR cycle. The system was tested with heat input
values between 1000 and 2500 W at a helium pressure
of 6.1 bars. The system cooling capacities were found
to be between 100 and 180 W with a COP between
0.09 and 0.15. Chaouachi and Gabsi (2007) designed
and simulated an absorption diffusion refrigerator
using solar energy for domestic use. They concluded
that the COP would be best when the system was
operated at a low generator temperature and high
pressure. On the other hand, the numerical values of
COP were still low and depended on the power of the
solar bubble pump. Ammonia vapor is separated from the rich solution
(1) in the separator. The vapor (2) then rises inside the
bubble pump and flows through the rectifier where
water vapor condenses (5) to join the weak solution (3). The pure ammonia (6) flows to the condenser,
condenses (7) and flows to the evaporator. At the
evaporator entrance the partial pressure of the ammonia
liquid (7) drops because it mixes with the helium and
ammonia vapor (13) from the absorber, which causes
the refrigerant to evaporate. The ammonia-helium
mixture (8) flowing in parallel through the shell side of
the evaporator/gas heat exchanger enters the absorber
and flows upward in counter flow to the weak solution
entering the absorber at the top (10). In the absorber,
the ammonia vapor is absorbed by the weak solution
and the solution obtained flows down into the reservoir
(11). Helium and ammonia residuals (12) flow toward
the evaporator. INTRODUCTION numerically. The results showed that for the same heat
input, the second configuration desorbed the highest
amount of refrigerant and the first configuration
desorbed the lowest amount. The third configuration
proved to be less efficient in terms of COP than the
second configuration. The first configuration gave the
lowest performance, although heat was supplied
directly to the rich solution. Corresponding Author: N. Dammak, Department of Chemica
The influence of the cycle configuration on the
performance of an absorption-diffusion cooling
system has been investigated by various researchers. Zohar et al. (2007) compared the performance of two
fundamental configurations of a Diffusion Absorption
Refrigeration
(DAR)
cycle,
with
and
without
condensate sub-cooling prior to the evaporator
entrance. They analyzed the performances of the two
cycles by computer simulations and concluded that the
DAR cycle without condensate sub-cooling, at higher
evaporator temperature of about 15°C, shows higher
COP of about 14-20% compared with the DAR cycle
with condensate sub-cooling. Subsequently, they
developed thermodynamic models for three generator
and bubble pump configurations (Zohar et al., 2008):
That is, (a) heat input into the rich solution without heat
transfer to the poor solution; (b) heat input into the rich
solution with heat transfer to the poor solution flowing
in the annular space and (c) heat input into the rich
solution through the poor solution, thus also desorbing
refrigerant from the poor solution, with heat transfer to
the poor solution in the annular space. The performance
of three DAR systems, which differ in their generator
and
bubble
pump
configuration,
was
studied Shelton and Stewart (2002) developed a model for
a bubble pump using two-phase flow correlations to
establish
the
optimum
design
parameters. The
experimental results showed that its performance was
not sensitive to the length of the lift tube; however it
was highly dependant on the submergence ratio. Koyfman et al. (2003) conducted experimental research
on the bubble pump performance. A solution mixture of
organic
solvent
and
hydrochlorofluorocarbon
refrigerant was used as the working fluid. It was found
that the motive head is one of the parameters that most
influences the bubble pump performance. Changing the
motive head by 10% changed the mass flow rates by
about 40%. The authors concluded that a low motive
head provides higher refrigerant flow rates and,
therefore, a higher cooling capacity. The maximum
average generator temperature was below 90°C. Hence,
they concluded that a complete diffusion absorption (
693 Am. J. INTRODUCTION Engg. & Applied Sci., 3 (4): 693-698, 2010 Solar diffusion-absorption cycle: Figure 1 shows a
schematic configuration of the solar heat driven
ammonia/water/helium diffusion-absorption cooling
system, consisting of a bubble pump, an absorber, a
condenser, an evaporator, a rectifier, a heat exchanger
and a reservoir. In this configuration the bubble pump
tubes are integrated in the solar flat plate collector. Solar diffusion-absorption cycle: Figure 1 shows a
schematic configuration of the solar heat driven
ammonia/water/helium diffusion-absorption cooling
system, consisting of a bubble pump, an absorber, a
condenser, an evaporator, a rectifier, a heat exchanger
and a reservoir. In this configuration the bubble pump
tubes are integrated in the solar flat plate collector. refrigerating system that operates with organic solvents
and hydrochlorofluorocarbon refrigerants may be
driven by low temperature heat sources, such as solar
thermal energy or waste heat. Chen et al. (1996)
attempted to improve the current cycle performance of
the Diffusion Absorption Refrigerator (DAR). They
recommended using waste heat from the rectifier to
preheat the weak solution leaving the absorber. A new
Generator With Heat Exchanger (GWHX) was
designed, built and tested. The test results of the DAR
with GWHX were compared with baseline tests. The
new generator design increased the cooling COP by as
much as 50% more than the baseline tests while the
cooling capacity remained constant. Srikhirin and
Aphornratana (2002) reported an experimental study
of a DAR cycle. The system was tested with heat input
values between 1000 and 2500 W at a helium pressure
of 6.1 bars. The system cooling capacities were found
to be between 100 and 180 W with a COP between
0.09 and 0.15. Chaouachi and Gabsi (2007) designed
and simulated an absorption diffusion refrigerator
using solar energy for domestic use. They concluded
that the COP would be best when the system was
operated at a low generator temperature and high
pressure. On the other hand, the numerical values of
COP were still low and depended on the power of the
solar bubble pump. refrigerating system that operates with organic solvents
and hydrochlorofluorocarbon refrigerants may be
driven by low temperature heat sources, such as solar
thermal energy or waste heat. Chen et al. (1996)
attempted to improve the current cycle performance of
the Diffusion Absorption Refrigerator (DAR). They
recommended using waste heat from the rectifier to
preheat the weak solution leaving the absorber. MATERIALS AND METHODS & Applied Sci., 3 (4): 693-698, 2010 Momentum equation from Psys to “0”, from “0” to
“1” and from state “1 to 2” yield (Shelton and Stewart,
2002):
2
2
t
0
2
sys
l
2
l
2
G
1
d
P
P
gz
2
d
−
= ρ
+
−
ρ
(1)
z
sub
H
=
(9)
and:
H
Lsin( )
=
β
(10) Momentum equation from Psys to “0”, from “0” to
“1” and from state “1 to 2” yield (Shelton and Stewart,
2002):
z
sub
H
=
(9) Momentum equation from Psys to “0”, from “0” to
“1” and from state “1 to 2” yield (Shelton and Stewart,
2002): z
sub
H
=
(9) (9) and: 2
2
t
0
2
sys
l
2
l
2
G
1
d
P
P
gz
2
d
−
= ρ
+
−
ρ
(1)
and:
H
Lsin( )
=
β
(10) 2
2
t
0
2
sys
l
2
l
2
G
1
d
P
P
gz
2
d
−
= ρ
+
−
ρ
(1)
and:
H
Lsin( )
=
β 2
2
t
0
sys
l
2
l
2
G
1
d
P
gz
2
d
−
= ρ
+
−
ρ
(1)
H
Lsin( )
=
β 2
0
2
2
1
d
2
d
−
(1)
H
Lsin( )
=
β
(10) H
Lsin( )
=
β
(10) (10) For a bubble pump using nt tubes, the specific mass
flow rate is given by: Where: t
0
l
G
v
=
ρ
(2) (2) t
0
l
G
v
=
ρ t
0
l
G
v
=
ρ 2
1
tot
2
1
m
ntG
.d
4
•
=
π
(11) (11) Momentum equation from Psys to “2” gives
(vertical bubble pump) (Shelton and Stewart, 2002): Mass energy balance at the generator: Mass energy balance at the generator: 2
m
2
sys
m
tp
2
. L
P
P
.g.L
f
. MATERIALS AND METHODS 2
d
ρ ν
=
+ ρ
+
(3) 1
2
3
m
m
m
•
•
•
=
+
(12)
1
1
2
2
3
3
m x
m y
m x
•
•
•
=
+
(13)
2
3
1
g
2
3
1
Q
m h
m h
m h
•
•
•
=
+
−
(14) (3) (12) For a bubble pump integrated in a solar flat plate
collector, Eq. 3 gives: (13) 2
m
2
sys
m
tp
2
. L
P
P
.g.Lsin( )
f
. 2
d
ρ ν
=
+ ρ
β +
(4)
Where: (14) 2
m
2
sys
m
tp
2
. L
P
P
.g.Lsin( )
f
. 2
d
ρ ν
=
+ ρ
β +
(4) (4) Useful solar heat (Sukhatme, 1997; Kalogirou,
2006): Where: u
c
L
f
a
Q
A (
U (T
T )
=
φ −
−
(15) (15) s
poor
poor
2
tp
poor
d
f
k
ρ
ν
=
µ
(5) (5) Liquid, gas and global efficiency are defined as: K and s are the BLASIUS factor (K = 0.316 and
s = -0.25) (Shelton and Stewart, 2002) K and s are the BLASIUS factor (K = 0.316 and
s = -0.25) (Shelton and Stewart, 2002) 2
G
u
m
The gas efficiency : E
Q
•
=
(16) (16) Specific mass flow rate of the rich solution: tot
m
G
. = ν ρ
(6) tot
m
G
. = ν ρ (6) (6) 3
L
u
m
The liquid efficiency : E
Q
•
=
(17)
gl
G
L
The globalefficiency : E
E .E
=
(18) 3
L
u
m
The liquid efficiency : E
Q
•
=
(17) (17) Because of ρpoor >>> ρvapour, the velocity of the poor
solution is given by: gl
G
L
The globalefficiency : E
E .E
=
(18) (18) tot
poor
poor
G
ν
= ρ
(7) Calculation procedure: The basic input data of the
system shown schematically in Fig. 2 were: condenser
temperature (Tc = 45°C), absorber temperature (Tab =
45°C), evaporator temperature (Tev = 15°C). MATERIALS AND METHODS In the present study, the new design of the bubble
pump proposed by Chaouachi and Gabsi (2007) was
optimized. A model for a system consisting of a bubble
pump integrated in a solar flat plate collector was
developed and used to optimize the geometrical
parameters of the solar bubble pump of an
ammonia/water/helium absorption-diffusion cooling
system. An empirical model was developed on the basis of
momentum, mass, material equations and energy
balances. The mathematical model was solved using the
simulation tool “Engineering Equation Solver (EES)”. Theoretical analyses: The following equations are
based on Fig. 2. Frictional losses at the entrance to the
tubes are considered insignificant and the steady state
flow is established. Fig. 1: Schematic view of the absorption diffusion
machine 694
Fig. 1: Schematic view of the absorption diffusion
machine
Fig. 2: Integrated solar bubble pump 4
Fig. 2: Integrated solar bubble pump Fig. 1: Schematic view of the absorption diffusion
machine Fig. 2: Integrated solar bubble pump 694 Am. J. Engg. MATERIALS AND METHODS The
calculation sequence was as follows: (7) Equation 1 and 4 gives
2
2
t
2
2
l
0
G
1
d
neglecting
2
d
−
ρ
: •
Calculate solar intensity (φ) (Liu and Jordan, 1960;
1961) (
)
1.75
1
5
2
tot
l
m
poor
m
2
d
1
L
G
gz
gLsin( )
K
2
d
−
=
ρ
−ρ
β
µ
ρ
(8) •
Calculate the Pressure of the system (Psys) using the
condenser temperature (8) •
Determine the rich solution concentration (x1)
using the P-T-x relationship for the ammonia-water
solution at equilibrium (Zohar et al., 2005) The submergence ratio is defined as: The submergence ratio is defined as: The submergence ratio is defined as: 695 Am. J. Engg. & Applied Sci., 3 (4): 693-698, 2010 ,
( )
,
Fig. 5: Global efficiency Vs submergence ratio
(φav = 0.6 kW m−2, L = 1.5 m and d = 6 mm
(month-8-))
Fig. 6: Liquid efficiency and gas efficiency Vs
submergence ratio (φav = 0.6 kW m−2, L = 1.5
m and d = 6 mm (month-8- )) •
Adjust the outlet temperature of the generator (Tg)
until it satisfies the equality between the energy
balance of the generator (Qg) and the useful solar
heat (Qu) using Eq. 14 and 15 (since the useful heat
available from the collector is transferred to the
rich solution in the generator) •
Determine the poor solution concentration (x3)
using the P-T-x relationship (Zohar et al., 2005) •
Determine the poor solution concentration (x3)
using the P-T-x relationship (Zohar et al., 2005) •
Calculate the refrigerant concentration (y2). The
NH3H2O procedure called from EES provides the
thermodynamic properties of ammonia water
mixtures in saturated conditions •
Calculate the mass flow rate of rich solution (m1),
poor solution (m3) and the mass flow rate of
refrigerant (m2), using Eq. 12 and 13 Fig. 5: Global efficiency Vs submergence ratio
(φav = 0.6 kW m−2, L = 1.5 m and d = 6 mm
(month-8-)) •
Calculate the gas efficiency, the liquid efficiency
and the global efficiency (Eq. 16-18) RESULTS AND DISCUSSION (L = 1.5 m, subav = 0.25, βav = 40°
(month-8-)) p
p
Under the operating conditions Tab = 45°C,
Tc = 45°C, Tev = 15°C, βav = 40°, subav = 0.25,
φ = 0.83 kW m−2, nt = 6 and Ac = 3.8 m2, the cooling
capacity of the diffusion absorption machine was equal
to 450 W. In order to determine the minimal values of the
submergence ratio, the variation in the mass flow rate
of the generated vapor and pumped liquid vs. submergence ratio has been illustrated in Fig. 7. The
intersection of the curve giving the mass flow rate of
the generated vapor and the curve giving both the
generated and pumped solution gives the minimal
submersion ratio (
v
l
v
l
m
m
m givesm
0
•
•
•
•
+
=
=
). Figure 7
shows that the solar flat plate collector cannot be used
with submersion values under 0.18 (the temperature
required in the generator is too high). Figure 8 illustrates the variation of global
efficiency vs. solar intensity for tube diameters between
4 and 6 mm at L = 1.5 m, subav = 0.25 and βav = 40°. Performance is most efficient when the bubble pump
tube diameters are equal to 6 mm (corresponding to φav
= 0.6 kW m−2). This can be explained as follows: If the
bubble pump tube diameters are over 6 mm the flow
rate of the rich solution increases when the heat input is
constant. Thus, the poor solution flow rate increases
and the refrigerant flow rate decreases, resulting in a
decrease in the global efficiency. Fig. 9: Global efficiency Vs tube length (subav = 0.25,
βav = 40° and d = 6 mm (month-8-)) Figure 3 and 4 illustrate the hourly variation of
solar intensity obtained by the Liu and Jordan (1960;
1961) model for the region of Gabes in Tunisia. Solar
intensity was maximum at 11.30 when the inclination
angle of the solar flat plate collector varied between
30 and 50°. This can be explained by the fact that in
winter, when the trajectory of the sun in the sky is low,
the inclination angle must be high. However, it is
recommended for the inclination angle to be low during
the summer months when the sun in the sky is higher
(Solar radiation is perpendicular to the collector). RESULTS AND DISCUSSION Figure 3-9 showed the most significant results
obtained by simulation with the Equation Engineering
Solver (EES) software. 696
Solver (EES) software. Fig. 3: Solar intensity Vs time (day 01/08)
Fig. 4: Solar intensity Vs time (day 01/01)
Fig. 6: Liquid efficiency and gas efficiency Vs
submergence ratio (φav = 0.6 kW m−2, L = 1.5
m and d = 6 mm (month-8- ))
Fig. 7: Mass flow rate of generated vapour and pumped
liquid Vs submergence ratio (φmax = 0.83 kW m−2
(month-8- )) Fig. 3: Solar intensity Vs time (day 01/08)
Fig. 4: Solar intensity Vs time (day 01/01) Fig. 6: Liquid efficiency and gas efficiency Vs
submergence ratio (φav = 0.6 kW m−2, L = 1.5
m and d = 6 mm (month-8- )) 96
Fig. 7: Mass flow rate of generated vapour and pumped
liquid Vs submergence ratio (φmax = 0.83 kW m−2
(month-8- )) Fig. 3: Solar intensity Vs time (day 01/08) Fig. 7: Mass flow rate of generated vapour and pumped
liquid Vs submergence ratio (φmax = 0.83 kW m−2
(month-8- )) Fig. 4: Solar intensity Vs time (day 01/01) 696 Am. J. Engg. & Applied Sci., 3 (4): 693-698, 2010 Fig. 8: Global efficiency Vs solar intensity at different
tube diameters. (L = 1.5 m, subav = 0.25, βav = 40°
(month-8-))
Fig. 9: Global efficiency Vs tube length (subav = 0.25,
βav = 40° and d = 6 mm (month-8-)) Fig. 8: Global efficiency Vs solar intensity at different
tube diameters. (L = 1.5 m, subav = 0.25, βav = 40°
(month-8-)) ratio ranges can be identified. In the first range global
efficiency increases when the submergence ratio
increases. This can be explained by the fact that any
increase in the submergence ratio increases the mass
flow rate of the rich solution. Hence, the mass flow of
the pumped liquid increases and the mass flow of the
generated vapor decreases (Fig. 6) so the global
efficiency (defined as EG.EL) increases. At high
submergence ratios (after reaching the maximum),
however, the global efficiency decreases because the
mass flow rate of the rich solution increases causing the
mass flow rate of the refrigerant to drop sharply (Fig. 6)
compared to the mass flow rate of the poor solution. Fig. 8: Global efficiency Vs solar intensity at different
tube diameters. RESULTS AND DISCUSSION Figure 9 shows that the system pressure has
considerable influence on the global efficiency. This is
because the decrease in the system pressure (decrease in
the absorber and condenser temperature) improves
absorption and the mass flow rate of the rich solution
increases which increases global efficiency. The variation of the global efficiency, gas
efficiency and liquid efficiency of the solar bubble
pump with submergence ratio at different solar flat
plate inclination angles of the solar collector is shown
in Fig. 5 and 6. For inclination angles of the solar flat
plate collector between 30 and 50°, the optimal
submergence ratio varied in the range 0.2-0.3. 7
Global efficiency values for a system pressure Psys
= 17.82 bar (Tc = 45°C) were highest at a tube length of
1.5 m (Fig. 9). When the tube length increases, the
collector area increases so more heat is supplied to the
rich solution witch in turn increases the global
efficiency. When the tubes are too long (over 2 m), the As can be seen in Fig. 5, global efficiency is
sensitive to the submergence ratio. Two submergence 697 Am. J. Engg. & Applied Sci., 3 (4): 693-698, 2010 Liu,
B.Y.H. and
R.C. Jordan,
1960. The
interrelationship and characteristic distribution of
direct, diffuse and total solar radiation. Sol. Energy,
4:
1-19. DOI:
10.1016/0038-
092X(60)90062-1 submergence
ratio
(defined
as
z
sub
H
=
,
where
H
Lsin
=
β ) decreases. Hence, the mass flow rate of the
rich solution and the global efficiency decrease. submergence
ratio
(defined
as
z
sub
H
=
,
where H
Lsin
=
β ) decreases. Hence, the mass flow rate of the
rich solution and the global efficiency decrease. H
Lsin
=
β ) decreases. Hence, the mass flow rate of the
rich solution and the global efficiency decrease. Liu, B. and R.C. Jordan, 1961. Daily insolation on
surfaces tilted towards the equator. ASHRAE
Trans.,
10:
53-53. http://www.osti.gov/energycitations/product.biblio. jsp?osti_id=5047843 ACKNOWLEDGEMENT Zohar, A., M. Jelinek, A. Levy and I. Borde, 2005. Numerical investigation of a diffusion absorption
refrigeration cycle. Int. J. Refrig., 28: 515-525. DOI: 10.1016/j.ijrefrig.2004.11.003 The
researchers
gratefully
acknowledge
the
Spanish Ministry of Science and Innovation for its
financial support (ENE2006-15250). Zohar, A., M. Jelinek, A. Levy and I. Borde, 2007. The
influence of diffusion absorption refrigeration
cycle configuration on the performance. Applied
Therm. Eng.,
27:
2213-2219. DOI:
10.1016/j.applthermaleng.2005.07.025 CONCLUSION A model of an integrated solar bubble pump was
developed. Various parameters were geometrically
optimized for maximum global efficiency by computer
simulation. It was found that efficiency is best for a
bubble pump tube diameter of 6 mm, a tube length of
1.5 m, an inclination angle of the solar flat plate
collector between 30 and 50° and a submergence ratio
in the range 0.2-0.3. In order to determine the minimal
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La Literatura Infantil y su Incidencia en el Desarrollo del Pensamiento Crítico en los Niños de Edad Preescolar
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Ciencia latina
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cc-by
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LA LITERATURA INFANTIL Y SU INCIDENCIA EN
EL DESARROLLO DEL PENSAMIENTO CRÍTICO
EN LOS NIÑOS DE EDAD PREESCOLAR
CHILDREN'S LITERATURE AND ITS IMPACT ON
THE DEVELOPMENT OF CRITICAL THINKING IN
PRESCHOOL CHILDREN
Gloria Guadalupe Lozano López
Universidad Politécnica Salesiana, Ecuador
Lic.Ramiro Rubio.Msc
Universidad Politécnica Salesiana, Ecuador
DOI: https://doi.org/10.37811/cl_rcm.v8i1.10088
La Literatura Infantil y su Incidencia en el Desarrollo del Pensamiento
Crítico en los Niños de Edad Preescolar
Gloria Guadalupe Lozano López 1
Lozanogloria2000@gmail.com
https://orcid.org/0000-0002-3847-0554
Universidad Politécnica Salesiana
Ecuador
Msc. Lic.Ramiro Rubio
wrubio@ups.edu.ec
https://orcid.org/0000-0002-8900-2664
Universidad Politécnica Salesiana
Ecuador
RESUMEN
El desarrollo del pensamiento crítico en los estudiantes es un reto que asumen los docentes de todas las
áreas de estudio, por eso es fundamental que se empleen estrategias efectivas en función de propiciar
estas capacidades de evaluación y análisis, que en efecto pueden aportar una visión más holística acerca
de su entorno. Teniendo en cuenta esta premisa, se planteó esta investigación que buscó analizar la
influencia de la literatura infantil en el desarrollo del pensamiento crítico en niños preescolares de la
Escuela de Educación Básica "1 de marzo" de la provincia de Morona Santiago en Ecuador. Se
estableció un marco teórico que destaca la importancia de la lectura infantil en el desarrollo del
pensamiento crítico. El estudio constó de dos fases: una revisión de la literatura científica sobre la
relación entre la literatura infantil y el pensamiento crítico a través de la contextualización de las dos
variables y, la segunda se trató de un diagnóstico de la población objetivo a través de encuestas a
profesores, niños en edad preescolar y padres de familia. Los resultados revelaron que la literatura
infantil desempeña un papel importante en el desarrollo del pensamiento crítico de los niños en edad
preescolar. Sin embargo, se destacaron áreas susceptibles de mejora, como la selección de materiales
literarios y estrategias didácticas específicas ya que los docentes en múltiples ocasiones recurren a las
mismas lecturas de forma mecanizada y en otras ocasiones, los padres no indagan sobre textos
pertinentes para el mismo fin. A través de esto se formularon sugerencias a todos los relacionados en
esta problemática para potenciar este impacto positivo. En conclusión, el estudio ofrece un enfoque
global al proponer medidas concretas para mejorar tanto la selección de la literatura como la aplicación
de estrategias didácticas. Entre esto también se destacó que los recursos institucionales no deben ser
una limitante para buscar estrategias de mejora.
Palabras clave: literatura infantil, pensamiento crítico, estrategias.
1 Autor principal
Correspondencia: Lozanogloria2000@gmail.com
pág. 7580
Children's Literature and its Impact on the Development of Critical
Thinking in Preschool Children
ABSTRACT
The development of critical thinking in students is a challenge assumed by teachers in all areas of study,
that is why it is essential to use effective strategies in order to promote these evaluation and analysis
skills, which can indeed provide a more holistic view of their environment. With this premise in mind,
this research sought to analyze the influence of children's literature on the development of critical
thinking in preschool children of the "1 de marzo" Basic Education School in the province of Morona
Santiago in Ecuador. A theoretical framework was established highlighting the importance of children's
reading in the development of critical thinking. The study consisted of two phases: a review of the
scientific literature on the relationship between children's literature and critical thinking through the
contextualization of the two variables, and a diagnosis of the target population through surveys of
teachers, preschool children and parents. The results revealed that children's literature plays an
important role in the development of critical thinking in preschool children. However, areas for
improvement were highlighted, such as the selection of literary materials and specific didactic
strategies, since teachers often resort to the same readings in a mechanized way and, on other occasions,
parents do not inquire about relevant texts for the same purpose. Through this, suggestions were made
to all those involved in this problem in order to enhance this positive impact. In conclusion, the study
offers a global approach by proposing concrete measures to improve both the selection of literature and
the application of didactic strategies. It was also emphasized that institutional resources should not be
a limiting factor in the search for improvement strategies.
Keywords: children's literature, critical thinking, strategies
Artículo recibido 07 enero 2024
Aceptado para publicación: 15 febrero 2024
pág. 7581
INTRODUCCIÓN
El presente documento describe la investigación realizada en el cantón Tiwintza, provincia Morona
Santiago, en la Escuela de Educación Básica “1 de Marzo”, en la que se define una muestra poblacional
de 91 padres de familia, 4 docentes y 91 niños de edad preescolar, a través de un enfoque mixto, según
Hernández et al., (2010) y un método de investigación tipo exploratoria. Para lograrlo, se parte del
diagnóstico que identifique la importancia de la Literatura Infantil y que facilite la formulación de
estrategias tomando como base la literatura infantil para el desarrollo del pensamiento crítico en los
niños de preescolar. Los cimientos serán los referentes teóricos; en la propuesta se procede a formular
las estrategias que permitan que el pensamiento crítico sea fortalecido para los niños en edad preescolar
de la Institución de Educación Básica “1 de Marzo”.
Este trabajo halla su justificación, primero que todo en el deber docente de mejorar la calidad de la
enseñanza para sus dicentes, como también en los principios la legislación ecuatoriana y el Ministerio
de Educación (2016), en la forja de los hombres del mañana que planteen soluciones, sean analíticos,
responsables, participativos en sus realidades. Es por ello también que los autores citados, se enfocan
en la necesidad latente de mejoramiento de la educación frente a los nuevos paradigmas que se plantea
la sociedad actual.
La falta de énfasis y comprensión sobre la relevancia de la literatura infantil se convierte en un desafío
significativo para los niños en edad preescolar de la Institución Educativa Básica "1 de Marzo" en el
cantón Tiwintza, provincia de Morona Santiago. Esta problemática se manifiesta en la dificultad de los
estudiantes para identificar personajes, realizar lecturas comprensivas, carecer de motivación, así como
la incapacidad para hacer evaluaciones y críticas sobre los textos literarios.
Considerando este panorama, la falta de enfoque y apreciación hacia la literatura infantil emerge como
un obstáculo relevante para el desarrollo del pensamiento crítico en los niños de preescolar. Esta
dificultad se refleja en la incapacidad de los estudiantes para comprender los textos, identificar
personajes y expresar valoraciones y críticas sobre los contenidos literarios. Además, se nota una
carencia de motivación en el proceso de aprendizaje.
El razonamiento crítico es una habilidad cognitiva que permite a los estudiantes analizar, evaluar y
reflexionar sobre la información de forma objetiva, teniendo en cuenta su edad y nivel de desarrollo.
pág. 7582
Canchihuaman et al. (2018), sostienen que es importante que los docentes promuevan el desarrollo del
pensamiento crítico desde la primera infancia, para que los estudiantes puedan desarrollar su creatividad
y solucionar problemas de forma efectiva a medida que crecen.
De acuerdo al autor, es primordial que los docentes desarrollen el pensamiento crítico desde los niveles
preescolares, con la finalidad de que, al ir avanzando en sus estudios, éstos sean unos referentes para
que puedan desarrollar y dar solución a problemáticas presentadas de acuerdo a su nivel. En ese sentido
Vargas (2019) afirma que la literatura infantil puede ayudar a promover el pensamiento crítico, ya que
expone a los estudiantes a diferentes perspectivas y puntos de vista. En el contexto de este estudio, la
población objeto de estudio es una comunidad rural en Ecuador. La mayoría de los habitantes se dedican
a la agricultura, la ganadería o el comercio. La lectura no es una práctica común en esta comunidad, por
lo que es importante encontrar estrategias para promover el pensamiento crítico a través de la literatura
infantil.
El contexto sociocultural de la comunidad en estudio explica la baja prevalencia de la lectura en el
hogar y la escuela. La mayoría de los habitantes se dedican a actividades agrícolas, ganaderas o
comerciales, lo que les deja poco tiempo para la lectura. Además, las costumbres y tradiciones de la
comunidad no alientan el hábito de la lectura. Es crucial presentar y detallar las estrategias específicas
para fomentar el pensamiento crítico a partir de la literatura infantil, considerando la diversidad
lingüística y cultural de los estudiantes en la Escuela "1 de Marzo". Además, es importante enfatizar la
relación directa entre el desarrollo del lenguaje y el pensamiento crítico, respaldándolo con estudios y
ejemplos de Jean Piaget, lo que podría enriquecer y sustentar de manera más efectiva el apartado
relacionado con el lenguaje y su papel en el desarrollo del pensamiento crítico.
La importancia del pensamiento crítico radica en su estrecha relación con la comprensión lectora,
permitiendo al lector adentrarse en el texto y obtener una comprensión profunda y reflexiva,
especialmente en un mundo cada vez más globalizado. Según Serrano (2008) la lectura trasciende la
simple repetición de palabras; implica una comprensión profunda del contenido, facilitando la
adquisición de conocimiento y la aplicación práctica de los mensajes en la vida cotidiana. El
pensamiento crítico no solo es crucial en el ámbito educativo, sino que también desempeña un papel
esencial en la esfera profesional. Esta perspectiva busca educar para la vida, promoviendo la
pág. 7583
creatividad, la transformación y el cuestionamiento de lo establecido, tanto en el ámbito personal como
profesional.
Los cuestionamientos críticos incitan a las personas a generar conocimiento, provocar cambios y
explorar nuevas alternativas, abriendo caminos hacia respuestas innovadoras y desafiando la
complacencia. Un lector competente distingue sus habilidades al analizar un texto desde múltiples
perspectivas, desglosando sus elementos significativos, evaluando su estructura narrativa, la solidez de
sus argumentos y la credibilidad de las ideas presentadas. Identificar las ideas principales y secundarias
es parte esencial de esta competencia lectora. Por otro lado, según las reflexiones de Lizarazo (2022),
el pensamiento crítico ha sido analizado desde diversas disciplinas como la filosofía, la psicología y la
ciencia, destacando su influencia en el desarrollo educativo. Desde edades tempranas, se considera
crucial que los estudiantes aprendan a discernir y emitir juicios críticos frente a aspectos positivos y
negativos de su entorno.
Existen varios estudios orientados a abordar esta temática, con el propósito de no solo resolver esta
problemática, sino también de fomentar habilidades que permitan a los estudiantes evaluar la literatura
infantil desde una perspectiva crítica. Destacan los trabajos llevados a cabo en instituciones como la
Universidad UNAE, la Universidad UNIANDES, entre otras, las cuales están promoviendo la escritura
para niños y el desarrollo del pensamiento crítico entre los educandos.
Esto motiva a los estudiantes a gozar la lectura de un texto de literatura de cualquier género o tipo, por
lo que la institución educativa debe proveer estos insumos para que los estudiantes enriquezcan sus
conocimientos, desarrollen habilidades críticas y estén prestos a resolver problemáticas de su contexto.
Acordes con los lineamientos curriculares, el Ministerio de Educación del Ecuador (2021), menciona
que:
Los elementos del Currículo se evidencian en el diseño de destrezas básicas que se tornan
imprescindibles y deseables. Las habilidades con criterios de desarrollo necesarios se analizarán en el
transcurso del subnivel para impedir que el estudiante entre en el peligro de exclusión social y rezago
en sus estudios; mientras que, las destrezas con criterios de desempeño deseables pueden desarrollarse
en este subnivel o en el siguiente. (p. 13)
pág. 7584
Esto conlleva la existencia de una comisión encargada de proporcionar los recursos virtuales y digitales
a las Instituciones educativas, las mismas que son elaboradas tomando en cuenta los años de básica en
la que cursan, como también mediante actividades de trabajo que ayuden en el impulso del raciocinio
crítico mediado por la literatura infantil.
En la literatura infantil, se destacan estudios con autores que promueven la didáctica para que los niños,
comprendan lo que su docente les transmite, a lo cual Escalante et al. (2008) afirman que, desde la
enseñanza inicial y preescolar, los niños señalan interés por indagar diferentes materiales de escritura,
que a su vez les induce a expresar ideas, vivencias, emociones, sentimientos, experiencias reales
externalizando su mundo interior. La literatura infantil comprende entonces varios géneros, destacando:
Cuentos de hadas: Son historias fantásticas que involucran momentos mágicos, con personajes
imaginarios y heroicos, lugares atraen a los niños a formar parte de ellos.
Fábulas: Cuentos cortos, en que se personifican a los animales, humanizándolos, lo fascinante es que
dejan una enseñanza o moraleja.
Cuentos populares y leyendas: Son narrativas que pasan de generación en generación y reflejando la
cultura autóctona de los diferentes pueblos y países.
Poesía infantil: Son colecciones de poemas presentados en un estilo lúdico, propio para los niños,
suelen usar la rima y el ritmo para entretener y educar a los niños.
Cuentos de aventuras: Historias llenas de emoción en que los protagonistas se sumergen en viajes y
desafíos, estimulando la imaginación y la exploración de nuevos mundos.
Libros informativos: Proporcionan una amplia gama de temas, como animales, ciencia, historia y
geografía, para que los niños comprendan fácilmente.
Literatura de aprendizaje: Son libros interactivos que enseñan habilidades básicas, como contar,
reconocer colores, letras, a desarrollar la memoria y la percepción.
Cuentos humorísticos: Hacen reír a los niños con situaciones cómicas, juegos de palabras y personajes
risibles.
Literatura multicultural: Exponen diversidad cultural y étnica, con historias y personajes de
diferentes procedencias
pág. 7585
Las interrogantes planteadas apuntan a solucionar esta problemática:
¿Qué estrategias metodológicas pueden aplicarse para fomentar el pensamiento crítico desde la
literatura infantil en niños de edad preescolar?
¿De qué manera la literatura infantil contribuye al desarrollo del pensamiento crítico en los niños
preescolares de la Escuela "1 de Marzo"?
¿Qué parámetros metodológicos pueden emplearse para impulsar la literatura infantil y promover el
desarrollo del pensamiento crítico en la institución educativa?
¿Es viable implementar estrategias metodológicas para fortalecer el pensamiento crítico desde la
literatura infantil en los niños de preescolar en la Escuela de Educación Básica "1 de Marzo"?
Estas preguntas buscan identificar y desarrollar estrategias efectivas que puedan superar la falta de
énfasis en la literatura infantil, mejorando así la comprensión crítica entre los estudiantes de preescolar
en la institución educativa mencionada.
METODOLOGÍA
El trabajo investigativo partió de un enfoque cuantitativo-cualitativo. Cuantitativo pues la información
numérica se trató con estadística descriptiva, la muestra fue determinada a través de fórmula, la
graficación se realizó a través de Excel y enfoque cualitativo porque estos datos obtenidos fueron
sometidos a revisión analítica basada en el marco teórico, donde se realizan descripciones y de hecho
las premisas o preguntas de los cuestionarios, son de tipo descriptivo, hacen referencia a situaciones
referidas precisamente a la literatura infantil y pensamiento crítico.
En el análisis se utilizó el método mixto, que, según Hernández et al., (2010) expresan un grupo de
eventos “empíricos, sistemáticos y críticos de investigación. Como modalidad del presente estudio, se
acudió a la de campo, cuyo análisis de casos se realizó en el aula de la institución educativa 1 de Marzo.
Esta modalidad permitió a la investigadora contactar directamente con los sujetos investigados,
concordando con los objetivos investigativos. Así también se recurrió a la bibliográfica – documental,
misma permitió conocer y profundizar los enfoques, conceptualizaciones, teorías de autores sobre
literatura infantil y el pensamiento crítico, cuyos documentos han sido citados debidamente para una
mejor comprensión y enfoque del estudio.
pág. 7586
Se utilizó la investigación de tipo exploratoria, factible de utilizar en el método cualitativo y
cuantitativo; aquí el estudio se aplica a hechos no indagados con anterioridad, que se quieren examinar
sus características, (Ramos, 2020); el enfoque cualitativo se basa en los estudios de la literatura infantil
y su incidencia para desencadenar pensamiento crítico en los niños. Se usó además el tipo descriptivo,
por detallar al grupo estudiado, como mencionan Guevara et al., (2020) señalan que el objeto de esta
investigación es caracterizar conjuntos homogéneos de fenómenos, con criterios sistemáticos dando una
estructura o el comportamiento de la situación en estudio, para dar información ordenada, comparable
con otras fuentes.
La investigación se centra en la influencia de los textos infantiles en el desarrollo del pensamiento
crítico en la población preescolar de la Escuela de Educación Básica "1 de Marzo". Se dispuso de una
población total 120 estudiantes de preescolar. Además, se incluyeron 120 padres de familia y 4 docentes
correspondientes al nivel de preescolar.
Se tomarán las 4 docentes del grado de preescolar para establecer la muestra de los niños, siendo 120
niños de preescolar y 120 padres de familia de la escuela “1 de Marzo”, los resultados se obtuvieron
aplicando la siguiente formula:
𝑛=
𝑍 2 𝑃𝑄𝑁
𝑍 2 𝑃𝑄 + 𝑒 2 (𝑁 − 1)
𝑍 2 𝑃𝑄𝑁
𝑛= 2
𝑍 𝑃𝑄 + 𝑒 2 (𝑁 − 1)
𝑛=
1.962 ∗ 0.5 ∗ 0.5 ∗ 120
1.962 ∗ 0.5 ∗ 0.5 + 0.052 ∗ (120 − 1)
𝑛=
3.84 ∗ 0.5 ∗ 0.5 ∗ 120
3.84 ∗ 0.5 ∗ 0.5 + 0.0025 ∗ (120 − 1)
𝑛=
115.2
1.2575
𝑛 = 91
Dando como resultado las siguientes muestras.
Tabla 1. Resultado de la población
Muestra estudiantes
Muestra padres de familia
Muestras docentes
91
91
4
pág. 7587
Así, se reúne un total de encuestados de 186, cada uno de los tres grupos contará con una encuesta
dirigida a su grupo correspondiente (padres, docentes, estudiantes).
El instrumento para utilizar es la encuesta, la cual es un método de recopilación de datos para obtener
información y opiniones de un grupo específico de personas; se elaboraron tres formatos para cada
grupo respectivo de encuestados. Por lo general, se utilizan preguntas estandarizadas para recopilar
datos referentes a comportamientos, preferencias, opiniones, actitudes, o características demográficas
de la muestra poblacional (Monserrat, 2015). El cuestionario es un conjunto de preguntas donde los
encuestados expresan su respuesta al requerimiento.
La técnica para analizar los datos recolectados se basa en el uso de la herramienta ofimática de Excel,
misma que permita el conteo o totalización de las preguntas, de acuerdo a las opciones de respuesta,
permite a la vez su traficación y el porcentaje obtenido, de acuerdo con las respuestas.
RESULTADOS Y DISCUSIÓN
Los resultados obtenidos se relacionan con un conjunto común de temas planteados en las preguntas,
los cuales se organizaron considerando los objetivos establecidos en la investigación. La información
proporcionada por los docentes en las preguntas abiertas se analiza para determinar el porcentaje basado
en la frecuencia de repeticiones, lo cual es significativo. Al consultar sobre su experiencia laboral, los
docentes destacaron los beneficios de los textos infantiles en el mejoramiento del pensamiento crítico.
El 100% de los docentes afirmaron que estos textos fomentan el disfrute, la creatividad y la absorción
de la lectura, permitiendo así potenciar la imaginación. Esta práctica genera interés en los estudiantes
por explorar los aspectos narrativos, especialmente durante la etapa evolutiva, en la que los niños son
más susceptibles a asimilar cualidades y adoptar estados de ánimo, deseos y sentimientos como modelos
a seguir (Romero & Silveira, 2019), Esto da lugar a lo que se conoce como Zona de Desarrollo Próximo,
evidenciando que las estrategias metodológicas basadas en la literatura infantil promueven el
pensamiento crítico desde una edad temprana.
Además, al solicitar a los docentes que identifiquen los tipos de literatura infantil que más les gustan a
los estudiantes en base a su experiencia, se evidencia un especial interés en los cuentos infantiles, con
un 75%, en comparación con los libros ilustrados, que representan un 25%. Esta aportación es valiosa,
ya que los niños muestran mayor atención cuando alguien más les lee, especialmente si la literatura está
pág. 7588
acompañada de ilustraciones, ya que estas captan su interés y motivan la exploración continua (Figura
1).
Se propone un listado de textos de literatura infantil y se solicita que se clasifiquen en orden de
importancia, según el criterio personal y profesional de las maestras, considerando cuatro grupos de
textos: libros con imágenes, poesía infantil, fábulas y cuentos de hadas, con una distribución del 25%
para cada categoría. El objetivo es descubrir y aprender mediante la heurística del descubrimiento,
practicando la resolución de problemas y esforzándose por descubrir; cuanta más práctica, mayor
capacidad de generalización. Aunque el gráfico resalta la relevancia de las ilustraciones en el proceso
de lectura, la trama, los personajes y el contenido de los cuentos en sí mismos prevalecen. Esta idea es
respaldada por Soto (2017) quien señala que los cuentos desarrollan micro destrezas del pensamiento
crítico, como la anticipación, las competencias comunicativas, la inferencia, la realización de juicios,
la interpretación y la predicción. Imaginar los diversos escenarios narrados en el cuento potencia la
creatividad y la imaginación; las ilustraciones funcionan mejor cuando es el niño quien lee.
Al analizar los parámetros metodológicos que ayudan al docente a promover la literatura infantil, se
formuló la pregunta sobre cómo se apoya para estimular el pensamiento crítico en los niños. El 75%
indicó un apoyo significativo, mientras que el 25% lo calificó como poco. Esto indica que fomentar el
trabajo en equipo desde una edad temprana, promoviendo círculos de estudio que fomenten la
competitividad académica, refuerza el interés por la literatura infantil y enriquece el lenguaje visual,
promoviendo la imaginación y la fantasía. Respecto a la percepción sobre la importancia de la literatura
infantil y su influencia en el desarrollo del pensamiento crítico en los niños en edad preescolar, el 75%
considera que es muy importante y el 25% la considera importante. Ambos porcentajes reflejan una
percepción positiva, lo que indica la necesidad de aplicar y seguir fomentando la literatura infantil desde
edades tempranas. En este punto, se alinean tanto la motivación intrínseca como la extrínseca, ya que
el aprendizaje por descubrimiento conduce a una tendencia hacia la independencia y la auto
gratificación por los hallazgos propios.
En relación con el tema de estudio, se planteó la pregunta de si es factible establecer estrategias
metodológicas, y el 100% de los docentes respondieron afirmativamente. Este resultado subraya la
necesidad de impulsar el pensamiento crítico en la educación, ya que, en el contexto actual de las
pág. 7589
competencias según la Constitución Ecuatoriana de 2008, el pensamiento crítico debe ser considerado
como una destreza esencial. Además, se evidencia que, dentro del plantel educativo, en la sección de
educación inicial y preparatoria, no existen estrategias metodológicas para mejorar el pensamiento
crítico de los estudiantes, por lo que sería recomendable desarrollar estrategias con el fin de mejorar
este aspecto educativo.
En esta misma dinámica se indagó, si la literatura infantil ha apoyado en distintos ámbitos al desarrollo
de destrezas de los infantes, el 50% señalan que ha posibilitado a mejorar las relaciones interpersonales
y un 50% indican que los niños son más reflexivos; de hecho, esto se corrobora cuando se consultó si
en los libros han encontrado desafíos cognitivos o conceptos complejos, y señalaron con el 75% que sí
y el 25% no, este apunte es relevante, puesto que, el uso de la literatura infantil impulse un esfuerzo
cognitivo; nace entonces la inquietud si los juegos con expresiones verbales , pueden potenciar el
pensamiento crítico de los niños, los encuestados se ratifican con el 100% que ayuda a la expresión
verbal en el desarrollo del pensamiento crítico, bien lo ratifica González, et al. (2014) que manifiesta
que la actividad lúdica permite al niño eliminar sus fronteras y le posibilita a desarrollar su personalidad.
Los resultados muestran que los docentes consideran la factibilidad de establecer estrategias
metodológicas, como se mencionó con anterioridad al no tener establecidas las metodologías
respectivas, no se está cumpliendo al 100% el desarrollo del pensamiento crítico en los estudiantes de
educación inicial.
Con relación a los padres de familia, pues forman parte del conjunto de educación, con lo cual los
representantes conviven más tiempo con los estudiantes, por tal motivo su obligación es realizar el
seguimiento y cumplimiento de las tareas escolares. En esa sintonía y con el fin de analizar cómo la
literatura infantil promociona el desarrollo del pensamiento crítico en los niños, y consecuentes del rol
de los padres en la enseñanza, se solicitó señale tres actitudes y/o comportamientos que ayuden a
desarrollar habilidades de análisis y pensamiento crítico, de los encuestados el 60% manifiestan que
han desarrollado la creatividad, saber pensar antes de contestar y aprender nuevas palabras, el 20% han
encontrado niños motivados, animados y críticos; el 20%, señaló que han desarrollado la curiosidad, el
pensamiento y las emociones; por tanto, los padres son el eje de educación de sus hijos y el interés por
la enseñanza lo asume desde su concepción, lo que honra un compromiso con la escuela, he ahí la
pág. 7590
importancia de forjar una colaboración mutua entre docentes y representantes; sin ser ambiguo o
arbitrario para que relacionen las ideas pertinentes que se hallen en la capacidad de aprendizaje del
estudiante.
En la figura 2, sobre el impulso de la lectura de la familia en el niño, se observa que el 70% ha
respondido como aspecto relevante a la motivación, el 20% señala por instrucciones, el 7,5%
lúdicamente, y el 2,5% de forma obligada, lo que se pone de manifiesto que el padre de familia colabora
en la educación de sus hijos, apoyándose en las tareas en casa, según Martínez et al. (2020) afirman que
los padres de familia son “modelos del desarrollo” (p. 4) del niño al promover su socialización,
formación y crecimiento, asumiendo características específicas; bien se dice que la familia es la base
de la sociedad pues en ella se desarrollan dimensiones del ambiente familiar, la forma de estudio y de
comportamiento en la comunidad; por tanto, a la pregunta del tipo de textos prefiere que su hijo lea para
promover su pensamiento crítico, manifiestan los encuestados que el 84% tiene preferencia por los
cuentos, el 4,9% por los textos de secuencia lógica, el 4,9% la poesía y otros (3,7), y el 2,5% textos
lúdicos, de ahí la importancia de los roles que tienen los involucrados en la educación, ya se dijo que
uno son los padres, el segundo son los docentes y su clima organizacional, y por último el estudiante.
De acuerdo a los resultados obtenido los cuentos son una parte esencial para promover el desarrollo del
pensamiento crítico de los estudiantes de acuerdo a los padres de familia, siendo que dentro de la
institución en el bloque de educación inicial tiene escaso material lector sobre todo cuentos, debido a
esto no se puede reforzar el pensamiento crítico de los estudiantes dentro del establecimiento educativo.
Al evaluar la importancia de la literatura infantil en el desarrollo del pensamiento crítico de los niños y
en coherencia con los parámetros metodológicos, se planteó la pregunta sobre los tipos de textos que
los padres leen con sus hijos. El 43,8% indicó las fábulas, el 36,3% mencionó las historias, el 15% se
refirió a otro tipo de texto y el 4,9% eligió la poesía.
Estos datos se respaldan con la pregunta sobre la frecuencia de lectura de cuentos, donde el 56,8%
respondió que a veces, el 22,2% casi siempre, el 17,3% siempre y el 3,7% casi nunca. Estos resultados
generan preocupación, ya que al asociar el 56,8% que lee a veces con el 3,7% que casi nunca lee, revela
un alto índice de falta o escaso acompañamiento en la lectura con sus hijos. Tal como se mencionó
anteriormente, la carencia de material adecuado para fomentar la lectura en los estudiantes de preescolar
pág. 7591
es evidente, y los resultados de la Figura 3 sugieren que en los hogares también existe una falta de
fomento de la lectura. Esto posiblemente incide en que el estudiante no desarrolle de manera efectiva
su pensamiento crítico.
En esencia la lectura es la base del crecimiento intelectual del niño, Delgado (2022) sostiene que es un
proceso intelectual forjado desde el nivel preescolar, por ser la fase preoperacional del desarrollo del
niño y las actividades deben ser coherentes con las características del niño así, mientras más temprano
se inicia, generará un mejor rendimiento del potencial que se está formando; desde la perspectiva de las
emociones que se manifiesta con la lectura se aprecia que el 96,3% declara alegría, el 2,5% desconocen
y el 1,2% demuestran confusión, sin embargo, el gusto es un buen indicador de la importancia de ser
consecuentes y continuar con el trabajo gestado en el aula y que debe complementarse en casa, con los
padres de familia.
La tercer meta u objetivo es el diseño de tácticas metodológicas factibles para el desarrollo del
pensamiento crítico de los niños, se ha establecido preguntar si su hijo demuestra interés por lo que lee,
los encuestados señalan que el 48,7% a veces demuestran interés, el 46,3% siempre y el 5% nunca, esta
pregunta tiene concordancia. Al plantear si su hijo puede diferenciar entre realidad y ficción al leer
libros infantiles, se establece en los siguientes resultados (figura 4).
En cuanto a la capacidad de diferenciar la realidad y ficción, el 55% señalan poco, el 37,5% mucho y
el 7,5% nada, por ello hay que recurrir al rol del docente en incentivar en los niños y así como en los
padres de familia a asumir su rol; lo importante está en hacer reflexionar que ser padre no solo es el
hecho de matricular a su hijo y despreocuparse, pues es obligación de los padres “propiciar un ambiente
de aprendizaje adecuado en su hogar, organizando espacios dedicados a las obligaciones y
responsabilidades escolares…” (Ministerio de Educación de Ecuador, 2017, Artículo 13, literal f.), por
ello la corresponsabilidad es importante, para generar un desarrollo holístico de sus hijos. Empero al
preguntar sobre en qué medida cree que los libros infantiles pueden estimular la imaginación y la
creatividad del niño el 78,8% señalaron que mucho, el 17,5% medianamente y el 3,7% poco, lo que
corrobora la importancia de promover la participación activa del estudiante, dada la importancia del
aprendizaje por descubrimiento que requiere una situación del contexto como reto permanente a la
inteligencia del aprendiz y conseguir que resuelva problemas e incluso que comparta su aprendizaje.
pág. 7592
Para dar respuesta a las preguntas del antecedente, en ilación con los objetivos, se hace necesario
examinar el tercer grupo encuestado, los estudiantes para analizar cómo la literatura infantil influye en
la potenciación del desarrollo del pensamiento crítico en los niños de preescolar, por ello fue preciso
conocer el lugar mágico el 72% señalan que estarían en un río, el 15% se verían en el bosque y el 13%
en su casa, lo que se acercó a determinar cómo sería el cuento ideal estableciendo que el 80% sería la
familia y el 20% de animales; en los niños de preescolar se manifiestan la curiosidad y es un indicador
que debe ser aprovechado por el docente, según Correa (2009) la narrativa de cuentos permite satisfacer
profundas necesidades, contrastando al niño con su entorno para que lo reinvente desde su perspectiva,
se trata de sumergir al educando en el contexto del cuento, de la trama de la lectura. Dado los resultados
es importante tener un área de lectura donde el niño pueda simular o sentirse a gusto al momento de
leer, ya que esto ayudara a mejor la concentración, al igual que su pensamiento crítico, al ser una
institución con pocos recursos, no es posible tener este tipo de área o ambiente, por lo cual el estudiante
no va a sentirse cómodo y no podrá concentrarse, debido a esto no podrá desarrollar correctamente su
pensamiento crítico.
En el propósito de explorar los fundamentos teóricos que sustentan la literatura infantil y determinar el
aporte al crecimiento del intelecto crítico en los estudiantes de preescolar, se planteó la pregunta del
gusto por los cuentos, el 98.9% respondieron que sí y el 1.1% que no, lo señalado tiene consecuencia
con la pregunta sobre si lee cuentos en su casa ya que el 92,1% dice que sí, el 6,7% que no y el 1,1%
rara vez; por lo visto se determina que los estudiantes gustan del cuento e inclusive se corrobora con la
pregunta le gustaría escribir cuentos donde el 95.5% dijeron que sí, y apenas el 4.5% se pronunciaron
que no, entonces, corresponde a los docentes a seguir utilizando y trabajando en este tipo de literatura
infantil, pues a decir de Oviedo & Páez (2020) señalan que el pensamiento crítico es como piensa en
situaciones educativas que lo favorecen, por ejemplo si se alientan por la investigación educativa.
Con el objeto de diagnosticar la relevancia de la literatura infantil en el desarrollo del pensamiento
crítico se estableció el cuestionamiento de qué emociones siente al leer cuentos, se evidenció que el
83,1% alegría, el 12,4% emoción, el 3,4% responden tristeza y el 1,1% enfado, lo propio cuando se le
preguntó los sentimientos que tiene cuando dibuja alguna escena de un cuento donde el 92.1% respondió
alegría, el 3,4% entre el miedo y la tristeza, en tanto que el 1.1% siente enojo, lo que motiva a escoger
pág. 7593
con diligencia la literatura infantil para motivar el interés en los niños, bien lo señala Oviedo & Páez,
son las situaciones áulicas donde se generan las motivaciones y que los maestros tendrían que estar
preparados y capacitados, en consideración que son niños que absorbe como esponja todo lo que ve,
escucha y hace.
Ahora, en cuanto a diseñar estrategias metodológicas que sean factibles hacia el desarrollo del
pensamiento crítico de los niños, se estableció la pregunta de qué le motiva para crear un cuento (figura
5); De lo señalado, fue oportuna la consulta sobre si le gustaría que le cuenten cuentos donde puede
observar dibujos, y el 98.9% respondió que sí y el 1.1% dijo no, lo que ratifica la importancia de seguir
incentivando la literatura infantil acompañada de gráficas atractivas que llamen la atención de los niños,
donde los espacios de aprendizaje son sitios relevantes y atractivos para los niños de preescolar porque
desarrollan hábitos, independencia, siguen reglas y estimulan la capacidad de tomar decisiones; el
aprendizaje se construye y produce a partir de las situaciones que cambian los esquemas mentales. Las
comunidades construyen ideas sobre su entorno y éstas se replantean y evolucionan para cambiar.
Sin lugar a dudas y desde diferentes perspectivas, es la literatura infantil el pilar sobre el cual se
construye el pensamiento crítico, con la enorme posibilidad de sus diferentes manifestaciones desde lo
poético, histórico, ficción, hasta las novedosas posibilidades que cada estudiante pueda dar en su
pensamiento mientras desarrolla y proyecta su raciocinio. Así entonces, desde las perspectivas del
docente, del padre de familia y del mismo educando, la tierra está abonada para sembrar la semilla de
la lectura como catapulta al pensamiento crítico, bastión del desarrollo humano.
Figura 1. Tipos de Literatura infantil les agrada más a sus estudiantes
Libros ilustrados
25%
Literatura tradicional
Adaptaciones clásicas
Literatura popular.
75%
Ficción
Cuento.
pág. 7594
Figura 2. Manera de promover la lectura en su hijo
Figura 3. Frecuencia de lectura en casa
4%
17%
Siempre
Casi siempre
22%
57%
A veces
Casi nunca
Figura 4. Capacidad del niño de discernir entre la realidad y la ficción al leer libros infantiles
8%
38%
Mucho
Poco
Nada
54%
Figura 5. Que le motiva para crear un cuento
pág. 7595
CONCLUSIONES
El ejercicio integral permitió obtener los siguientes resultados; primero que todo, con relación al
diagnóstico se conoció que los docentes tienen conocimiento de cómo promover el pensamiento crítico
a través de la literatura infantil, sin embargo, se hace necesario ahondar o capacitar en el tema para que
los que desconocen cómo proceder, tengan un mayor campo de posibilidades en el desarrollo de su
labor.
El diagnóstico facilitó una importante información de los docentes y padres de familia con respecto a
su experiencia y conocimiento acerca de la literatura infantil en el desarrollo del pensamiento crítico,
permitiendo afirmar que hay mucho camino por recorrer, sin embargo la actitud tanto de padres de
familia como de los docentes es activa y alentadora en la materia; hay mucho potencial para trabajar en
el objetivo general; de hecho la aplicación de las encuestas despertó inquietud y ganas de trabajar en el
tema, por parte de los encuestados.
Los padres y docentes tuvieron en común en algunas de sus respuestas que la literatura infantil mejora
en los discentes, el vocabulario, la pronunciación, la imaginación, la resolución de conflictos, la
creatividad, la rapidez de pensamiento, sus acciones en sus hogares, las relaciones interpersonales,
genera motivación, disfrute, actitud positiva, sonrisas, mejora su comprensión de los hechos y de las
palabras.
Si bien es cierto, como se estableció a comienzos de la investigación, en la escuela no se cuenta con
suficientes recursos y materiales para trabajar es posible generar espacios e ideas que contribuyan al
aporte de la construcción de conocimiento y como propende la presente investigación, del
fortalecimiento del raciocinio crítico por medio de la Literatura Infantil, lo que implica la consecución
de un número mayor de textos, ya sea en físico, o electrónico con la posibilidad de hacerlo visible por
televisor o computador a los niños de preescolar, ya que como también fue establecido en el trabajo, no
se cuenta con materiales de este tipo dentro de la escuela.
En lo que refiere a la exploración de fundamentos teóricos, permitieron no solamente ampliar los
conocimientos en la materia de estudio, sino que fortalecen la estructura del trabajo, ampliaron los
horizontes, y dieron una orientación con base en la información plasmada. Haciendo referencia al tercer
objetivo, las posibles estrategias requieren una dinámica primero de planeación y orientadora, para que
pág. 7596
las mismas circunstancias y entorno en que se dan, faciliten el ejercicio que sea el vehículo esperado
para conseguir un fortalecimiento del pensamiento crítico.
REFERENCIAS BIBLIOGRAFICAS
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años de las instituciones educativas del nivel inicial de San Juan Pampa-Yanacancha.
http://repositorio.undac.edu.pe/bitstream/undac/335/1/T026_71990891_T.pdf,Ed.
Universidad Nacional "Daniel Alcídes Carrión".
Correa, M. (2009). El cuento, la lectura y la convivencia como valor fundamental en la educación
inicial. Educere, 44, 89-98.
Delgado, V. (2022). La lectura recreativa y su aporte en el aprendiza<je de los niños y niñas en la etapa
pre-operacional. Polo del conocimiento, 7(7), 462-473.
Escalante, D., & Caldera, R. (2008). Literatura para niños: una forma natural de aprender a leer.
Educere, 12(43), 669-678. Obtenido de https://www.redalyc.org/pdf/356/35614570002.pdf
González, C., Solovieva, Y., & Quintanar, L. (2014). El juego de roles sociales: aportes al desarrollo en
la edad preescolar. Avances en Psicología Latinoamericana, 32(2), 287 - 308.
Guevara, G., Verdesoto, A., & Nelly, C. (2020). Metodologías de investigación educativa (descriptivas,
experimentales, participativas, y de investigación-acción). Recimundo, 4(3), 163-173. Obtenido
de https://recimundo.com/index.php/es/article/view/860
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Lizarazo, N. (2022). Aprender a pensar: Propuesta para la estimulación del pensamiento crítico en
niños de preescolar del colegio Luz de la Esperanza, sede B, del corregimiento de Berlín,
municipio de Tona. Universidad Autónoma de Bucaramanga.
Martínez, G., Torres, M., & Ríos, V. (2020). El contexto familiar y su vinculación con el rendimiento
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Oviedo, P., & Páez, R. (2020). Pensamiento crítico en la educación. En R. Páez, & P. Oviedo, Problemas
y posibilidades del pensamiento crítico en la educación (págs. 11-34). Universidad La Salle.
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propuesta didáctica. Educere, 12(42), 505-514. Obtenido de
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Soto, M. (2017). El cuento como mediación pedagógica para el fortalecimiento de la lectoescritura.
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WaterSwap Analysis, a Computation-based Method for the Discovery of Effective and Stable Binding Compounds for Mutant EGFR Inhibition
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WaterSwap Analysis, a Computation-based Method for the
Discovery of Effective and Stable Binding Compounds for
Mutant EGFR Inhibition
Kshipra S. Karnik
Dr Babasaheb Ambedkar Marathwada University
Aniket P. Sarkate
Dr Babasaheb Ambedkar Marathwada University
Vaishanavi S. Jambhorkar
Dr Babasaheb Ambedkar Marathwada University
Pravin Wakte ( pswkshipra16@gmail.com )
Dr Babasaheb Ambedkar Marathwada University https://orcid.org/0000-0002-8118-2963
Research Article
Keywords: EGFR, Waterswap, Virtual screening, Molecular docking, ADMET, Styrylquinoline
Posted Date: October 29th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-981656/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Page 1/10
Abstract
The epidermal growth factor receptor of the tyrosine kinase family has been largely targeted in mutations associated with nonsmall cell lung cancer. EGFR inhibitors have been produced that bind allosterically to the C797S mutant EGFR enzyme. Here, the
Waterswap tool has been used for the interpretation and visualization of trajectories of mutant EGFR-ligand complexes. Virtual
screening of the generated compounds has been carried out along with its molecular docking and ADMET analysis. Out of the
generated library of compounds, the top 15 have been selected. Waterswap calculated the binding free energies of the
compounds and thermodynamic properties of the enumerated compounds were compared with that of standard EAI045. It was
observed that styrylquinoline stabilized better than EAI045. The results show that Waterswap analysis offers a promising new
path in the hunt for improved tools for analyzing and visualizing molecular driving forces in protein-ligand complex simulations.
Introduction
Cancer is the second biggest cause of death in the world, and it is one of the primary causes of morbidity and mortality. In 2015,
cancer claimed the lives of 8.8 million people. Cancer is responsible for about one-sixth of all fatalities worldwide [1]. Despite
decades of attempts to find and develop small molecule anticancer medications, the demand for novel antitumor medicines with
enhanced tumour efficiency, selectivity, and safety remains critical [2, 3].
One of the most significant enzymes in the tyrosine kinase family is the Epidermal Growth Factor Receptor. EGFR is a
transmembrane receptor that is found on the cell surface. The EGFR enzyme is classified as (ErbB1, HER1), ErbB2 (HER2, neu in
rodents), ErbB3 (HER3), and ErbB4 (HER4) based on structural similarities [4]. EGFR and its members have become a popular
target for anticancer therapy, particularly in the treatment of non-small cell lung cancer [5]. Activating mutations in the EGFR gene
had a significant impact on non-small cell lung cancer therapy approaches (NSCLC). The recent identification of novel EGFR
inhibitors was critical in overcoming such mutations [6].
Drugs' affinity for their targets is described by thermodynamic characteristics. As a result, accurate thermodynamic property
calculations have long been a goal of computational chemistry. Hence, accurate thermodynamic property calculations have long
been a computational chemistry goal. In the industry, study of solvation patterns surrounding pharmaceuticals and binding sites
has become a major goal for molecular modelling [7–9]. According to an estimate, hydrophobic surfaces cover 75% of surface
drug sites [10], implying that the hydrophobic effect is one of the most energetic contributions to ligand binding and recognition.
Individual water molecules that serve a crucial stabilising role, for example, by building bridges between the ligand and the
receptor, have been shown by high-resolution co-crystallized structures of protein-ligand complexes [11, 12]. Improved binding
affinity has also been linked to a network of H-bond-enriched water molecules around the ligand [13–15].
The water-swap reaction coordinate was described by Woods et al. as a new reaction coordinate (WSRC) [16]. The ligand and
water are exchanged from being bound to the protein to being solvated by a periodic box of bulk water in this approach. Because
no molecules are moved into a vacuum, the enormous decoupling free energy associated with double decoupling procedures is
avoided. Furthermore, a new approach for restricting the identity of solvent clusters within bulk has been established, allowing the
swap to be accomplished without the requirement for any restrictions on the ligand or swapped water, and this strategy applies to
both buried and solvent-exposed binding sites [16]. The concept behind the water-swap reaction coordinate is that it permits the
identity of water clusters to be limited during simulation. During a simulation, this identity constraint [17] works by modifying how
water molecules are labeled. We used computation-based methods such as virtual screening, molecular docking, and WaterSwap
analysis to find an effective compound with stable binding and mutant EGFR inhibition effect in this study.
Materials And Methods
Insilico methodology
ChemT tool was used for library generation and enumeration. The molecular docking tool, AutoDock Vina, was used for docking
studies of all compounds on the EGFR enzyme. SwissADME tool was used to determine ADMET studies. Waterswap study was
performed on trial version of Cresset’s Flare software.
Page 2/10
ChemT and Library Enumeration
ChemT, a Biochemcore-based software, was used to enumerate the library. In Marvin Sketch, the R-group position on the template
styrylquinoline. The styrylquinoline was then assigned to the identified R-groups through the enumeration model of the software.
EAI045 input determined the standard template. Additionally, few available pharmacokinetic properties such as molecular weight,
H-bond donors, and Lipinski rule of five was set for designing drug-like compounds [18].
Molecular Docking
To investigate the binding pocket, residue interactions, and predicted binding affinity of the enumerated compounds, molecular
docking protocol was carried out using AutoDock tools. Through Chimera modeler, the protein was fetched from the open-source
PDB libraries. Explicit hydrogen bonds were added followed by the removal of water molecules. Using Gastegier charges, ions
were removed and finally, the protein was minimized using the Amber94 force field. The total number of loops was set to 1000 to
ensure maximum minimization of the protein. The minimization was terminated when the energy converged or the root mean
square deviation (RMSD) reached a maximum cut-off of 0.30 Å. Ramachandran plot was set to check the total number of
disallowed residues. Through AutoDock 4.0 GUI, the minimized PDB was converted to a PDBQT file followed by generation of the
grid. To perform flexible docking, torsions were chosen in the selected residues and saved as flex.PDBQT. The configuration file,
which was used to outline the grid area as well as the protein and ligand input, was defined and saved as a text document. All the
ligands were prepared using Gastegier charges and saved as a PDBQT file. The files saved in the fold were called through the
command line under the vina environment to start the docking process. The results for each input ligand were saved as a text file
consisting of docking scores and RMSD values of each ligand conformation which was used for the analysis of data. The output
file was then converted into Maestro readable extension for image saving [19–22].
WaterSwap methodology
The PDB: 5D41 was downloaded initially and full preparation on proteins and ligands was carried out. Under flare protein
preparation wizard, at pH 7.0 small side chains movement were allowed, atoms were removed from residues with incomplete
backbone, along with filling of 1 and 2 residue gaps at 6.00 A° active site size. Then the protein was minimized under the normal
calculation method with a gradient cutoff of 0.200 kcal/mol/A with 1300 maximum iterations.
The ligands were loaded in the sdf format for docking. Docking was performed under the normal calculation method under
Autodock flexible docking protocol. The ligands to dock were already selected, the template ligand was defined, the co-crystallized
ligand was selected for docking grid preparation, the minimized protein was selected and docking calculation was started [16, 23].
For WaterSwap calculation, the protein along with docked ligand was prepared again and minimized as per the above method.
The calculation method was kept custom and no changes were made. The small molecule force field was kept as AMBER with
GAFF version with AM1-BCC as the charge method. The solvate box buffer was kept at 10.0 A. Open MM platform was GPU,
minimized energy tolerance as 0.25 (kcal/mol), the system was equilibrated for 100 ps. For Waterswap, the iterations were kept as
1000 -1300 (1300 for docked ligand), maximum threads were kept on Auto, with Water monitor distance as 7.00 A, the protein
was selected, the ligand was selected along with the proper chain, and the calculation was started.
Result And Discussion
Computational Methods
Database building and Library Screening
The most potent substituted styrylquinoline with the highest antitumour activity was taken from the literature [24], and by
modification, at R1 position a library of around 2000 compounds was generated virtually. Based on the obtained fitness score
using ChemT, the top 1000 hits were screened for further pre-ADMET analysis. All 1000 hits were screened through Lipinski’s Rule
of 5 and around 100 hits were chosen for further study fitting with the criteria. In the virtual screening workflow protocol, all 100
hits were subjected to a flexible docking approach using AutoDock. A score cutoff of -9.20 kcal/mol for docking discarded lowerPage 3/10
scoring compounds selecting 15 highest scoring compounds. Top 15 highest scoring, best pocket fitting, and possessing similar
binding interactions with that of standard EAI045, novel and potent compounds were obtained through virtual screening and were
selected for further studies.
Molecular Docking
Molecular docking was carried out on mutant EGFR enzyme (PDB ID: 5D41) to check the binding pattern and interactions. It was
seen that all 15 molecules docked correctly into the allosteric binding pocket of the protein with common interacting amino acid
residues LYS745, LEU788, ASP855 and PHE856. From Figures 1a and 1b, it was observed that all compounds fit into the
hydrophobic pocket of the protein interacting with the common amino acid residues. The combined docking scores along with
structures are shown in Table 1.
Figure 1b Docked images of top 15 compounds over T790M/C797S mutant EGFR (PDB: 5D41). Blue lines in the figures indicate
π-π stacking interaction and pink lines indicate hydrogen bonding interaction.
ADMET studies
SwissADME website (http://www.swissadme.ch/index.php) was used to quantify the physicochemical and pharmacokinetic
properties of the synthesized derivatives with respect to EAI045. Table 2 shows the analysis of the pharmacokinetic parameters
needed for ADMET study of compounds KSK (1-15).
Table 2
ADMET study of top 15 compounds with EAI045.
Compound
#HB
acceptor
#HB
donors
TPSAa
MLOGPb
GI
absorption
BBB
permeant
Violation
of
Lipinski’s
Rule
Bio availability
Score
Synthetic
Accessibility
EAI045
5
2
110.77
1.93
High
No
0
0.55
3.28
KSK-1
2
2
70.83
3.65
High
No
0
0.55
3.37
KSK-2
5
2
88.68
0.81
High
No
0
0.55
3.99
KSK-3
1
1
42.15
3.75
High
No
0
0.55
3.33
KSK-4
2
1
56.4
3.15
High
No
0
0.55
4.16
KSK-5
2
1
36.36
3.85
High
Yes
0
0.55
3.05
KSK-6
2
0
58.5
3.78
High
No
0
0.55
3.94
KSK-7
3
2
75.27
3.38
High
No
0
0.55
3.34
KSK-8
3
1
100.88
3.91
Low
No
0
0.56
4.39
KSK-9
3
1
70.89
3.41
High
No
0
0.55
4.47
KSK-10
2
1
41.05
3.54
High
Yes
0
0.55
3.54
KSK-11
2
0
29.02
3.18
High
No
0
0.55
3.25
KSK-12
3
1
45.59
3.41
High
Yes
0
0.55
4.19
KSK-13
2
0
67.82
3.98
High
No
0
0.55
3.28
KSK-14
3
1
36.36
3.75
Low
No
0
0.55
3.24
KSK-15
2
1
55.04
3.33
High
Yes
0
0.55
3.41
a TPSA 40Å - 160Å
bMLOGP
must be less than 4.15
Page 4/10
The drug must pass Lipinski’s rule to show good oral absorption in humans [25, 26]. All 15 compounds do not show Lipinski’s rule
violation, passing the criteria. The MLOGP values were also within the acceptable range, showing the compounds' correct drug
absorption and distribution within the body. Almost all the compounds show good GI absorption and some of them were bloodbrain barrier permeant. Through bioavailability scores, it was indicated that a potent compound is available at the site of action.
Overall, the compounds showed satisfactory pharmacokinetic parameters range defined for human use along with good docking
scores [27–29].
Waterswap
When dealing with the cavitation and large-value problem of double decoupling, we used Waterswap, an absolute binding free
energy method. Continuum solvent methods do not include the molecular detail of protein–water, ligand–water, and protein–
water–ligand interaction interactions because they use an explicit model of water [23].
It uses a single simulation to replace the ligand-bound protein with bulk water molecules of the same shape and volume as
shown in Figure 2. An increase in protein-ligand interactions occurs when the protein's resulting cavity is filled with a cluster of
water molecules. With only one reaction coordinate, it's possible to get a more accurate free energy value because it's not the
product of two huge numbers [30].
Moreover, the beneficial waters around the protein-ligand complex are also identified to obtain a stable pocket. These beneficial
waters also become a potential bridge in creating interactions between likely residues and ligand. Both proteins favourable and
ligand favourable waters are important to identify to assess the pocket interactions and their stability [31]. Two different types of
colours are given for the protein depending on its preference i.e., the protein present in green colour states that it prefers ligand,
and protein present in red colour states that it prefers water as indicated in Figure 3. The water box identified by the system further
provides the required binding free energy for the protein and ligand [16].
To analyze Waterswap studies, binding free energy (∆G), shown in Figure 4 comes into play describing the stability of the waters
around the pocket. If ∆Gprotein in the complete simulation decreases, it does not stabilize with the set number of iterations, the
possibility of the existence of unfavourable water distorting the pocket intensity is high. In such cases, the ligand would exit the
simulation box resulting in an inactive compound [32]. The similar identity of ∆Gwater is identified through drastic increase or
instability of the waters resulting in distortions in the protein. This could also result in terminating the protein-ligand interactions.
EGFR protein becomes the perfect example of identifying and comparing the potential waters for the binding affinity and checking
the potency of the complexed ligand. A mutant EGFR protein with known standard EAI045 is initially run for 1300 iterations using
Waterswap to identify all the favourable waters and their binding free energy. Then the ligand is swapped to check the potential
binding affinity and stability of the designed compounds concerning the identified favourable waters. If the binding free energy
results obtained during the complete iterative simulations are similar or better than the results for standard drug EAI045, the
compounds designed are the potential to withstand the waters and show potency.
Figure 5 describes the relative binding free energy graph during the 1300 iterations performed for EAI045 compared to the
styrylquinoline compound identified from the literature. The blue line in the graph shows the average binding free energy change
for the number of iterations. The thermodynamic properties compared in the Figure showed that the styrylquinoline stabilized
better than the standard compound EAI045. Towards the end of the iterations, the graph showed stability after a drop in relative
binding energy in styrylquinoline. Overall, all the attributes taken into account concludes the potential of styrylquinoline
compound as an EGFR inhibitor.
The results show that Waterswap analysis offers a promising new path in the hunt for improved tools for analyzing and
visualizing molecular driving forces in protein-ligand complex simulations.
Conclusion
The binding free energy calculations using the Waterswap analysis for interpreting and visualizing trajectories of EGFR-ligand
complexes have been demonstrated. These visualizations provide precise molecular-level information that should help medicinal
chemists construct better-binding ligands. Although we tested these decompositions on mutant EGFR, we believe they should
Page 5/10
apply to other protein-ligand systems as well. The thermodynamic properties of the enumerated compounds were compared with
that of standard EAI045, and it was observed that styrylquinoline stabilized better than EAI045. The results show that Waterswap
analysis offers a promising new path in the hunt for improved tools for analyzing and visualizing molecular driving forces in
protein-ligand complex simulations.
Declarations
Acknowledgement
The authors K. S. K and P. S. W. are thankful to Indian Council of Medical Research (ICMR) for providing funding in support of
Senior Research Fellowship with Ref No: 3/2/2/33/2018/Online OncoFship/NCD-III. The authors are also thankful to The Head,
Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004 (MS), India, for
providing the laboratory facility and Cresset for providing us with the free trial version of the FlareTM WaterSwap software.
Declaration of Interest
Funding: The project was funded by Indian Council of Medical Research (ICMR) in support of Senior Research Fellowship with Ref
No: 3/2/2/33/2018/Online OncoFship/NCD-III.
Conflicts of interest/Competing interests: The authors declare no competing financial interest.
Availability of data and material: The data that support the findings of this study are available from the corresponding author,
P.S.W, upon reasonable request.
Code availability: Not applicable.
Authors' contributions: All the mentioned authors contributed equally in the preparation of manuscript.
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Tables
Due to technical limitations, table 1 is only available as a download in the Supplemental Files section.
Figures
Figure 1
Page 8/10
a Docked images of top 15 compounds over T790M/C797S mutant EGFR (PDB: 5D41). Blue lines in the figures indicate π-π
stacking interaction and pink lines indicate hydrogen bonding interaction. b Docked images of top 15 compounds over
T790M/C797S mutant EGFR (PDB: 5D41). Blue lines in the figures indicate π-π stacking interaction and pink lines indicate
hydrogen bonding interaction.
Figure 2
(a) Protein pocket filled with ligand. (b) Protein pocket filled with waters.
Figure 3
Interpretation of which protein residues prefer binding to water or ligand. Green represents ligand favourable and red represents
water favourable.
Figure 4
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Swapping of Protein:Water to Protein:Ligand.
Figure 5
The relative binding free energy graph of standard EAI045 and Styrylquinoline.
Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.
Table1Structures.docx
Graphicalabstract.docx
Page 10/10
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Use of apple genetic resources in apple breeding programmes at INRA: Present status and perspectives
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Use of apple genetic resources in apple breeding
programmes at Inra: present status and perspectives Francois Laurens Francois Laurens To cite this version: Francois Laurens. Use of apple genetic resources in apple breeding programmes at Inra: present
status and perspectives. European Malus Germplasm, European cooperative programme for crop
genetic resources networks., Jun 1995, London, United Kingdom. hal-01603372 Use of apple genetic resources in apple breeding
programmes at Inra: present status and perspectives HAL Id: hal-01603372
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Whole-Genome Sequence of the Ancestral Animal-Borne ST398 Staphylococcus aureus Strain S123
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Genome announcements
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To cite this version: Nathalie van Der Mee-Marquet, David Hernandez, Xavier Bertrand, Roland Quentin, Anna-Rita
Corvaglia, et al.. Whole-Genome Sequence of the Ancestral Animal-Borne ST398 Staphylococcus
aureus Strain S123.. Genome Announcements, 2013, 1 (5), pp.e00692-13. 10.1128/genomeA.00692-
13. hal-00926042 Whole-Genome Sequence of the Ancestral Animal-Borne ST398
Staphylococcus aureus Strain S123 Nathalie van der Mee-Marquet,a,b David Hernandez,c Xavier Bertrand,d Roland Quentin,a Anna-Rita Corvaglia,c Patrice Françoisc
Service de Bactériologie et Hygiène, Centre Hospitalier Régional Universitaire, Tours, Francea; Réseau des Hygiénistes du Centre, Centre Hospitalier Universitaire, Tours,
Franceb; UMR 6249 Chrono-environnement Université de Franche-Comté Besançon Franced; Genomic Research Laboratory University of Geneva Hospitals Geneva Marquet,a,b David Hernandez,c Xavier Bertrand,d Roland Quentin,a Anna-Rita Corvaglia,c Patrice Françoisc Nathalie van der Mee-Marquet,a,b David Hernandez,c Xavier Bertrand,d Roland Quentin,a Anna-Rita Corvaglia,c Patrice Françoisc
Service de Bactériologie et Hygiène, Centre Hospitalier Régional Universitaire, Tours, Francea; Réseau des Hygiénistes du Centre, Centre Hospitalier Universitaire, Tours,
Franceb; UMR 6249 Chrono-environnement, Université de Franche-Comté, Besançon, Franced; Genomic Research Laboratory, University of Geneva Hospitals, Geneva,
Switzerlandc Sequence type 398 (ST398) Staphylococcus aureus was originally reported in livestock. We announce the complete genome se-
quence of an ST398 methicillin-susceptible S. aureus strain of animal origin, S123. The genome sequence reveals a wild-type ge-
nome that probably corresponds to an ancestral clone. Received 5 August 2013 Accepted 5 August 2013 Published 29 August 2013
Citation van der Mee-Marquet N, Hernandez D, Bertrand X, Quentin R, Corvaglia A-R, François P. 2013. Whole-genome sequence of the ancestral animal-borne ST398
Staphylococcus aureus strain S123. Genome Announc. 1(5):e00692-13. doi:10.1128/genomeA.00692-13. Received 5 August 2013 Accepted 5 August 2013 Published 29 August 2013 Copyright © 2013 van der Mee-Marquet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Patrice François, patrice.francois@genomic.ch. S tem. Similar to many other sequenced ST398 strains, S123 is de-
void of DNA from phagic elements despite the presence of a
functional type I restriction system. S
equence type 398 (ST398) Staphylococcus aureus was originally
described in livestock (1) but was recently reported as the
causative agent of severe infections in humans (2). An evolution-
ary hypothesis suggests that an ancestral wild-type S. aureus strain
acquired mobile genetic elements triggering host adaptation and
evolution to the methicillin-resistant genotype after insertion of a
staphylococcal cassette chromosome mec (SCCmec) element (3). We report here the genome sequence of a carriage methicillin-
susceptible S. aureus isolate showing sequence type 398 and de-
void of any phage element. We conclude that S123 is potentially an ancestral clone still
missing a phagic element that contributes to host adaptation. Nucleotide sequence accession number. The whole-genome
sequence of S. Whole-Genome Sequence of the Ancestral Animal-Borne ST398
Staphylococcus aureus Strain S123 aureus S123 was deposited in the DDBJ/EMBL/
GenBank databases under the accession number AUPU00000000. ACKNOWLEDGMENTS This study was supported in part by the University Hos
We thank the technicians for their dedicated help. This study was supported in part by the University Hospi
h
k h
h
f
h
d d
d h l This study was supported in part by the University Ho
We thank the technicians for their dedicated help. S. aureus strain S123 was recovered from a pig during a screen-
ing campaign in a Dutch farming area in 2010. Purified genomic
DNA was subjected to whole-genome shotgun sequencing by us-
ing an HiSeq2000 system (Illumina, Inc.). Following fragmenta-
tion, end reparation, and sample tagging, the sequencer produced
21.6 million 100-bp paired-end reads, yielding appreciable cover-
age of around 800. Assembly was performed using Edena 3.0 (4)
and resulted in 49 contigs. The larger contigs showed sizes of
498,000 bp. Overall assembly values were satisfactory, showing a
total length of 2.70 Mbp and an N50 of 152,000 bp. A total of 2,475
predicted coding sequences (CDS) were detected by rapid anno-
tations using subsystems technology (RAST) (5). More than 55%
of the genes were assigned to specific subsystem categories by
RAST (5). In addition to CDS, RAST revealed 77 structural genes,
including 59 tRNA and 18 rRNA genes. Note that S123 contains
two circular plasmids of 15 kb and 4.5 kb at apparent copy num-
bers of 2 and 10, respectively. y
pp
p
y
y
We thank the technicians for their dedicated help. September/October 2013
Volume 1
Issue 5
e00692-13 HAL Id: hal-00926042
https://hal.science/hal-00926042v1
Submitted on 29 May 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. REFERENCES 1. Witte W, Strommenger B, Stanek C, Cuny C. 2007. Methicillin-resistant
Staphylococcus aureus ST398 in humans and animals, Central Europe. Emerg. Infect. Dis. 13:255–258. doi:10.3201/eid1302.060924. 2. van der Mee-Marquet N, François P, Domelier-Valentin AS, Coulomb F,
Decreux C, Hombrock-Allet C, Lehiani O, Neveu C, Ratovohery D,
Schrenzel J, Quentin R, Bloodstream Infection Study Group of the
Réseau des Hygiénistes du Centre (RHC). 2011. Emergence of unusual
bloodstream infections associated with pig-borne–like Staphylococcus au-
reus ST398 in France. Clin. Infect. Dis. 52:152–153. doi:10.1093/cid/ciq053. 3. van der Mee-Marquet N, Corvaglia AR, Valentin AS, Hernandez D,
Bertrand X, Girard M, Kluytmans J, Donnio PY, Quentin R, François P. 2013. Analysis of prophages harbored by the human-adapted subpopula-
tion of Staphylococcus aureus CC398. Infect. Genet. Evol. 18:299–308. 4. Hernandez D, François P, Farinelli L, Osterås M, Schrenzel J. 2008. De
novo bacterial genome sequencing: millions of very short reads assembled
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1 Genome Announcements genomea.asm.org
1 genomea.asm.org
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English
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Social Support for People with Morbid Obesity in a Bariatric Surgery Programme: A Qualitative Descriptive Study
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International journal of environmental research and public health/International journal of environmental research and public health
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Keywords: social support; morbid obesity; bariatric surgery; qualitative study Academic Editor: Paul B. Tchounwou Received: 31 May 2021
Accepted: 15 June 2021
Published: 17 June 2021 International Journal of
Environmental Research
and Public Health Int. J. Environ. Res. Public Health 2021, 18, 6530. https://doi.org/10.3390/ijerph18126530 Article
Social Support for People with Morbid Obesity in a Bariatric
Surgery Programme: A Qualitative Descriptive Study
í
é
á
1
á
1 2
é
2 Public Health 2021, 18,
6530. https://doi.org/10.3390/
ijerph18126530
Academic Editor: Paul B. Tchounwou
Received: 31 May 2021
Accepted: 15 June 2021
Published: 17 June 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
ti
/li
/b /
Citation: Torrente-Sánchez, M.J.;
Ferrer-Márquez, M.;
Estébanez-Ferrero, B.;
Jiménez-Lasserrotte, M.d.M.;
Ruiz-Muelle, A.; Ventura-Miranda,
M.I.; Dobarrio-Sanz, I.;
Granero-Molina, J. Social Support for
People with Morbid Obesity in a
Bariatric Surgery Programme: A
Qualitative Descriptive Study. Int. J. Environ. Res. Public Health 2021, 18,
6530. https://doi.org/10.3390/
ijerph18126530 Article
Social Support for People with Morbid Obesity in a Bariatric
Surgery Programme: A Qualitative Descriptive Study
í
é
á
1
á
1 2
é
2 María José Torrente-Sánchez 1, Manuel Ferrer-Márquez 1,2, Beatriz Estébanez-Ferrero 2,
María del Mar Jiménez-Lasserrotte 3, Alicia Ruiz-Muelle 3, María Isabel Ventura-Miranda
Iria Dobarrio-Sanz 3
and José Granero-Molina 3,4,* María José Torrente-Sánchez 1, Manuel Ferrer-Márquez 1,2, Beatriz Estébanez-Ferrero 2,
María del Mar Jiménez-Lasserrotte 3, Alicia Ruiz-Muelle 3, María Isabel Ventura-Miranda 3
,
Iria Dobarrio-Sanz 3
and José Granero-Molina 3,4,* 1
Hospital HLA Mediterráneo, 04007 Almería, Spain; mjtorrentes@gmail.com (M.J.T.-S.);
Manuferrer78@hotmail.com (M.F.-M.) 1
Hospital HLA Mediterráneo, 04007 Almería, Spain; mjtorrentes@gmail.com (M.J.T.-S.);
Manuferrer78@hotmail.com (M.F.-M.) 2
Hospital Universitario Torrecárdenas, 04009 Almería, Spain; beatriz.estebanez.sspa@juntadeandalucia.es
3
Department of Nursing, Physiotherapy and Medicine, University of Almeria, 04120 Almería, Spain;
mjl095@ual.es (M.d.M.J.-L.); arm297@ual.es (A.R.-M.); mvm737@ual.es (M.I.V.-M.); ids135@ual.es (I.D.-S.)
4
Faculty of Health Sciences, Universidad Autónoma de Chile, Santiago 7500000, Chile
*
Correspondence: jgranero@ual.es 2
Hospital Universitario Torrecárdenas, 04009 Almería, Spain; beatriz.estebanez.sspa@juntadeandalucia.es
3
Department of Nursing, Physiotherapy and Medicine, University of Almeria, 04120 Almería, Spain;
mjl095@ual.es (M.d.M.J.-L.); arm297@ual.es (A.R.-M.); mvm737@ual.es (M.I.V.-M.); ids135@ual.es (I.D.-S.)
4
Faculty of Health Sciences, Universidad Autónoma de Chile, Santiago 7500000, Chile
*
Correspondence: jgranero@ual.es *
Correspondence: jgranero@ual.es Abstract: Background—Morbid obesity (MO) is a chronic metabolic disease affecting physical,
psychological and social wellbeing. Bariatric surgery is a reliable method for losing weight in the long
term, improving the quality of life, body image and social life of people with MO. Current literature
recognises the importance of social support in controlling weight and coping with MO. The objective
of this study was to describe and understand experiences related to social support for patients with
MO included in a bariatric surgery programme. Methods—A qualitative descriptive study, where
data collection included thirty-one interviews with people diagnosed with MO involved in a bariatric
surgery programme. Results—Three main themes emerged from the analysis: (1) accepting the
problem in order to ask for help, (2) the need for close support and (3) professional support: opposing
feelings. Conclusions—A partner, family and friends are the key pillars of social support for those
with MO included in a bariatric surgery programme. Healthcare professionals gave formal support;
the bariatric surgery team provided information, trust and assurance. Nurses provided healthcare
24 h a day, making them the main formal support for people in the bariatric surgery programme.
Citation: Torrente-Sánchez, M.J.;
Ferrer-Márquez, M.;
Estébanez-Ferrero, B.;
Jiménez-Lasserrotte, M.d.M.;
Ruiz-Muelle, A.; Ventura-Miranda,
M.I.; Dobarrio-Sanz, I.;
Granero-Molina, J. Social Support for
People with Morbid Obesity in a
Bariatric Surgery Programme: A
Qualitative Descriptive Study. Int. J. Environ. Res. 1. Introduction Obesity is a chronic disease that is characterised by an accumulation of fat or hyper-
trophy of fatty tissue. Obesity is a public health issue, although the prevalence varies
depending on the country [1]. It affects approximately 13% of the global adult popula-
tion [2]. The prevalence of obesity in Spain is 22.8% of men and 20.5% of women [3]. Obesity is defined as having a body mass index (BMI) > 30 kg/m2, and morbid obesity
(MO) as having a BMI between 40–49.9 kg/m2 [4,5]. Obesity is a multifactorial problem
that is associated with metabolic, endocrine [6] and vascular problems, as well as diabetes
and hypertension [7]. Added to this are external factors, such as poverty [8], unhealthy diet,
lifestyle and physical exercise, as well as socio-cultural factors [9]. MO is a health risk factor,
as it is related to anxiety, depression, body image disorders and sexual dysfunction [10,11]. MO can be prevented with a multidisciplinary approach, including health education, diet,
physical exercise and pharmacological treatment [12–14]. However, therapies or treatments
are not always effective and therefore surgical treatment is required [15]. Bariatric surgery
is a reliable method for losing weight in the long term, improving the quality of life, body
image and social life of people with MO. The criteria for inclusion for a person with MO
in a bariatric surgery programme are: to be an adult with a BMI ≥40 kg/m2 or an adult
with a BMI between 35–39.9 kg/m2 and a serious associated comorbidity, such as type Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Int. J. Environ. Res. Public Health 2021, 18, 6530. https://doi.org/10.3390/ijerph18126530 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 6530 2 of 14 2 diabetes, high blood pressure or hyperlipidemia [16]. Bariatric surgery involves a set
of surgical procedures to treat obesity, prevent the associated morbidity and mortality
and improve the quality of life of obese patients following weight loss. Bariatric surgery
includes restriction techniques that reduce gastric volume [17], malabsorptive techniques
and mixed techniques [18]. q
Physical, psychological, social and cultural factors make living with MO difficult. 2.1. Design A qualitative descriptive study was used. This methodology allows for little-known
experiences to be described from a naturalist perspective without losing their essence
in the analysis process. The study was developed and reported in accordance with the
Consolidated Criteria for Reporting Qualitative Research (COREQ) [42]. 1. Introduction People with MO face physical impediments, difficulty in moving [19], low self-esteem or
self-control [20], disorders associated with negative body image [21], shame and psychoso-
cial stress, all of which can lead to limited social relations, stigma and the loss of quality of
life [22,23]. Facing MO not only requires weight loss but also dealing with psychological
and social problems [24]. Social support refers to the social resources that are available
for people facing a health issue, which are provided by formal and/or informal support
groups. Social support is key in preventing and overcoming chronic pathologies, such as
diabetes, cardiovascular disease, osteoarticular disease or MO [25,26]. Various studies have demonstrated the importance of social support in different
phases of obesity [27]. Social support is associated with making the decision to undergo
bariatric surgery [28], post-surgery results [29–32], stress levels [33], information prior to
treatment [34] and coping strategies [35]. Other studies have explored the experiences
of patients in MO support groups after undergoing bariatric surgery [36,37] or patients
who are expecting bariatric surgery alongside their partners [38,39]. Although the liter-
ature recognises the importance of social support in controlling weight and overcoming
MO, the experiences of people with MO in a bariatric surgery programme have scarcely
been studied. In line with Leahy-Warren’s social support theory [40], social support has a structural
and functional dimension. The structural dimension refers to the social networks of people
with MO, which are divided into informal (family, friends and acquaintances) and formal
(essentially healthcare professionals). The functional dimension is related to the exchange
of activities (informative, instrumental and emotional support), types of social support
received and the perception/satisfaction of them [41]. The objective of this study was to
describe and understand the experiences regarding the social support of people with MO
that were included in a bariatric surgery programme. 2.2. Participants and Setting The researchers used purposive sampling to select people with MO that were included
in a bariatric surgery programme. The participants were recruited by surgeons and nurses
associated with the bariatric surgery programme for people with MO. The criteria for
inclusion were: be a person diagnosed with MO at least 10 years prior to the study, in
the phase prior to surgical intervention, a Spanish speaker, over 18 and having signed an
informed consent form. The exclusion criteria were: rejected participating in the study, not
be included in a bariatric surgery programme or be in a different phase of treatment. Out
of 44 people with MO selected for telephone contact and recruitment, two did not answer
the phone, nine refused to discuss the topic and two did not have time to be interviewed. The interviews took place with a total of 31 participants, of which, 21 were women and
10 were men (Table 1). The study was carried out in two hospitals in the south of Spain
between April 2019 and January 2020. Int. J. Environ. Res. Public Health 2021, 18, 6530 3 of 14 Table 1. Sociodemographic data. Table 1. Sociodemographic data. Participant
Sex
Onset of MO
Marital Status
Job
IDI1
F
Adulthood
Married
Housewife
IDI2
F
Adolescence
Married
Farmer
IDI3
F
Adulthood
Relationship
Housewife
IDI4
F
Adolescence
Single
Unemployed
IDI5
F
Adulthood
Separated
Seamstress
IDI6
F
Pregnancy
Separated
Chef
IDI7
F
Adolescence
Relationship
Administrative assistant
IDI8
F
Adulthood
Married
Healthcare assistant
IDI9
F
Pregnancy
Married
Administrative assistant
IDI10
F
Pregnancy
Married
Farmer
IDI11
F
Pregnancy
Married
Teacher
IDI12
F
Childhood
Married
Administrative assistant
IDI13
F
Adolescence
Married
Housewife
IDI14
F
Adolescence
Single
Unemployed
IDI15
F
Adolescence
Single
Shop assistant
IDI16
F
Pregnancy
Married
Teacher
IDI17
F
Adolescence
Single
Civil servant
IDI18
F
Childhood
Single
Student
IDI19
F
Adolescence
Married
Teacher
IDI20
F
Childhood
Single
Beautician
IDI21
F
Childhood
Relationship
Waitress
IDI22
F
Adolescence
Single
Unemployed
IDI23
M
Adolescence
Married
Engineer
IDI24
M
Adulthood
Married
Salesperson
IDI25
M
Childhood
Single
Farmer
IDI26
M
Adulthood
Married
Salesperson
IDI27
M
Adulthood
Married
Petrol station worker
IDI28
M
Adulthood
Married
Hospitality worker
IDI29
M
Adulthood
Married
Farmer
IDI30
M
Adulthood
Married
Farmer
IDI31
M
Childhood
Married
Unemployed
MO: morbid obesity. IDI: in-depth interview. 2.3. Data Collection Data Analysis
Thematic analysis was carried out using Braun and Clarke’s [43] method with the
following steps: (1) familiarisation with the data—data transcription, reading and re-
reading and annotation of initial ideas; (2) generate initial codes—systematic codification
of data groups; (3) find themes—convert codes into themes; (4) revise themes—verify
codes with themes; (5) define themes—analyse the details of each theme; (6) elaborate the
report—select examples of themes and subthemes, associate the analysis with the research
question and generate the final report. 2.5. Rigor
Credibility: participants and researchers were familiarised with the context of the
study to ensure trust and obtain rich data. Various researchers participated in the codifica-
tion, analysis and interpretation of the data. Transferability: the researchers described the
experiences and context of the study in detail. Reliability and confirmability: the nurses
did the transcriptions and then other members of the research group revised them, verified
them and accepted the participants. Reflexivity: the researchers examined their own values
and preconceptions about the issue at hand before describing the results. 2.6. Ethical Issues
The study was carried out in accordance with the ethical standards established by
the Helsinki Declaration. It was approved by the Ethical Committee of the University of
Almería’s Nursing, Physiotherapy and Medicine Department (protocol number: 45/2018). Prior to the study, informed written consent and permission to record the interview were
obtained. The participants were informed that their participation in the study was inde-
pendent of the bariatric surgery programme. 3. Results
A total of 31 patients, men (32.26%) and women (67.74%) with a mean age = 44.35 years
(SD = 11.57 years, range = 20–66 years) were recruited. The BMI of the participants was
44.5 ± 6.57 kg/m2, MO started during adolescence (32.26%) or in adulthood (32.6%) Table 2. Interview protocol. Stage
Subject
Content/Example Questions
Introduction
Motives, reasons
Learn about your experiences of social support since your inclusion
in a bariatric surgery programme. Ethical issues
Inform about voluntary participation, recording, consent, possibility
of withdrawing and confidentiality. Beginning
Introductory question
Tell me about your experience with MO. Development
Conversation guide
How has it affected your relationship with your friends and other
social relations? How has MO affected your perception of your body and your
hobbies/free time? How has MO affected your life with your partner? How has MO affected your relationship with healthcare providers? 2.5. Rigor Credibility: participants and researchers were familiarised with the context of the
study to ensure trust and obtain rich data. Various researchers participated in the codifica-
tion, analysis and interpretation of the data. Transferability: the researchers described the
experiences and context of the study in detail. Reliability and confirmability: the nurses
did the transcriptions and then other members of the research group revised them, verified
them and accepted the participants. Reflexivity: the researchers examined their own values
and preconceptions about the issue at hand before describing the results. 2.6. Ethical Issues The study was carried out in accordance with the ethical standards established by
the Helsinki Declaration. It was approved by the Ethical Committee of the University of
Almería’s Nursing, Physiotherapy and Medicine Department (protocol number: 45/2018). Prior to the study, informed written consent and permission to record the interview were
obtained. The participants were informed that their participation in the study was inde-
pendent of the bariatric surgery programme. 2.4. Data Analysis Thematic analysis was carried out using Braun and Clarke’s [43] method with the
following steps: (1) familiarisation with the data—data transcription, reading and re-
reading and annotation of initial ideas; (2) generate initial codes—systematic codification
of data groups; (3) find themes—convert codes into themes; (4) revise themes—verify
codes with themes; (5) define themes—analyse the details of each theme; (6) elaborate the
report—select examples of themes and subthemes, associate the analysis with the research
question and generate the final report. 2.3. Data Collection What kind of formal and informal social support was missing for you
after being included in a bariatric surgery programme? What role did technology play in terms of social support in a bariatric
surgical programme? Closing
Final question
Is there anything else you would like to tell me? Appreciation
Thank them for their participation, remind them that their testimony
will be useful and place ourselves at their disposition. MO: morbid obesity. Table 2. Interview protocol. Content/Example Questions 2.3. Data Collection Nurses called participants who fulfilled the inclusion criteria to explain the objectives,
respond to queries and request voluntary participation in the study. Nurses and partici-
pants had established contact prior to the clinical care context. The nurses had a Master’s
degree in qualitative research and they did not need further training. The participants
agreed to be interviewed in a hospital meeting room outside of the surgical ward. Two
nurses with five years of experience working in the bariatric surgery programme carried
out in-depth interviews following the interview protocol (Table 2). Each participant did one
individual, private, in-depth interview that lasted 56 min on average. The nurses collected
field notes to describe the data. Before the interview, participants were informed about
the study, signed consent was obtained and sociodemographic data of the participants
were collected. The participants’ answers were recorded, transcribed and analysed using
ATLAS.ti.9 (ATLAS.ti Scientific Software Development GmbH, Berlin, Germany). Data
collection ceased when the researchers considered that they had reached data saturation. 4 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 nt. J. Environ. Res. Public Health 2021, 18, 6530
4 of 14
Table 2. Interview protocol. Stage
Subject
Content/Example Questions
Introduction
Motives, reasons
Learn about your experiences of social support since your inclusion
in a bariatric surgery programme. Ethical issues
Inform about voluntary participation, recording, consent, possibility
of withdrawing and confidentiality. Beginning
Introductory question
Tell me about your experience with MO. Development
Conversation guide
How has it affected your relationship with your friends and other
social relations? How has MO affected your perception of your body and your
hobbies/free time? How has MO affected your life with your partner? How has MO affected your relationship with healthcare providers? What kind of formal and informal social support was missing for you
after being included in a bariatric surgery programme? What role did technology play in terms of social support in a bariatric
surgical programme? Closing
Final question
Is there anything else you would like to tell me? Appreciation
Thank them for their participation, remind them that their testimony
will be useful and place ourselves at their disposition. MO: morbid obesity. 2.4. 3. Results A total of 31 patients, men (32.26%) and women (67.74%) with a mean age = 44.35 years
(SD = 11.57 years, range = 20–66 years) were recruited. The BMI of the participants was
44.5 ± 6.57 kg/m2, MO started during adolescence (32.26%) or in adulthood (32.6%) Int. J. Environ. Res. Public Health 2021, 18, 6530 5 of 14 5 of 14 (Table 1). Three themes allowed for the description and understanding of the social support
experienced by patients with MO in a bariatric surgery programme (Table 3). Table 3. Themes, subthemes and units of meaning. Themes
Subthemes
Units of Meaning
Accepting the problem in order
to ask for help
Social stigma, the other weight
of obesity
Bullying during childhood, limitations imposed by
others, everything is in my head, a look is worth more
than a thousand words, understanding is better
amongst equals. Acknowledging MO
I do not identify with my body, my health comes first,
physical limitations, acknowledging, self-punishment,
food as comfort and social withdrawal. Bariatric surgery, a shared journey
Need to speak, fear, uncertainty, my only hope, sharing
experiences, sharing decisions and high expectations. The need for close support
The partner, the key support figure
for MO
Emotional support, fear of being left, lack of
communication, suspicion, fear of bariatric surgery and
fear of complications. Necessary company,
unpleasant scrutiny
Feeling observed, discomfort, sharing experiences,
feeling scrutinised, feeling heard, a link to reality,
selfless help and concern for others. Appearing and sharing on
social networks
Looking for information, inspecting results and
Whatsapp groups. Professional support: opposing
feelings
Lack of professional support
Professional indifference, lack of information, lack of
empathy and bariatric surgery is not offered
The bariatric nurse “is
always there”
Trust, assurance, active listening, bariatric surgery team,
nurse as a point of reference, 24/7 telephone contact and
someone in whom to seek comfort. MO: morbid obesity. (Table 1). Three themes allowed for the description and understanding of the social support
experienced by patients with MO in a bariatric surgery programme (Table 3). Table 3. Themes, subthemes and units of meaning. 3.1. Accepting the Problem in Order to Ask for Help Those with MO faced physical, psychological and social problems. Alongside the
associated risks and comorbidites, MO was problematic in relation to the patient’s physical
appearance, image, emotional state and the demands of daily life. Acknowledging the
situation was key in being able to face it, ask for help and find social support. My colleagues are always looking out for me “leave that, I’ll do it,” “careful, you might
fall, be careful”. I feel like they are pitying me . . . I don’t want that! (IDI24) 3.1.1. Social Stigma, the Other Weight of Obesity Those with MO found themselves on the receiving end of insulting comments, which
could lead to feelings of frustration, shame and blame. This situation could happen as early
as childhood, resulting in the victims becoming more and more sensitive and insecure. As
one participant stated, it was possible to interpret any gesture or look as contemptuous: I think they look at me, . . . I don’t go unnoticed and my mind doesn’t rest. If I eat an
ice-cream I think that everyone is thinking “well, that’s why she’s so fat”. I can’t go on 3.1.2. Acknowledging MO Obesity is a chronic disease and the majority of those affected took their time to
acknowledge the problem. They did not feel capable of overcoming it or asking for help
from professionals or their close social circles. Feelings of rejection and denial were very
common in the early phases of acknowledgement, at which point, social support played an
important role: The doctor said that I had to look for solutions but I didn’t want to acknowledge it or look f
g
for help . . . first you have to become aware of the situation yourself. (IDI23) for help . . . first you have to become aware of the situation yourself. (IDI23) Participants suffered from psychological and emotional disorders and despite being
able to share the problem or ask for informal help, they did not find it easy. They recognised
that they did not like themselves or feel liked by others, which limited their social life and
led to them cutting themselves off. As one participant stated, at those times, it was easier
to turn to food than to social support: I sometimes argued with my husband and said “right, now I’m going to have some I sometimes argued with my husband and said “right, now I’m going to have some I sometimes argued with my husband and said “right, now I’m going to have some
chocolate”. Then I regretted it and even made myself sick so that I wouldn’t put on
weight. (IDI1) chocolate”. Then I regretted it and even made myself sick so that I wouldn’t put on
weight. (IDI1) They slowly started to lose contact with those in their social circle, they stopped taking
part in leisure activities and it was difficult to find a partner. They found excuses not to
leave the house, they withdrew into themselves and looked for help within their family: I used to go out more but now I don’t feel like it; I avoid going out with my friends and I I used to go out more but now I don’t feel like it; I avoid going out with my friends and I
go to the beach with my family because I feel more comfortable with them. (IDI8) g
f
g
g
y f
go to the beach with my family because I feel more comfortable with them. 3.1.2. Acknowledging MO (IDI8) MO presented a constant struggle to stay healthy and in shape, which varied de-
pending on gender, according to the participants. Women associated MO with aesthetic
problems; they rejected a body with which they did not identify and avoided looking in
the mirror. Whilst they may recognise their physical limitations and the risk posed to
their health, they placed more importance on the discomfort and shame related to their
body image: I see myself as fat, deformed, I don’t recognise myself. When I get out of the shower, I look
at myself in the mirror and I don’t want to see myself, I’m ashamed of my body. (IDI10) The men did not identify with their bodies either, but MO was more of a health issue
for them. As well as the physical limitations and comorbidities, the exacerbation of prior
conditions and the inability to carry out daily tasks were what worried them the most. As
one participant said, acknowledging the gravity of the situation was the first step in asking
for help: If I were fat but healthy I wouldn’t mind, the problem is that I’m not healthy. I don’t have
a complex, I don’t hide myself, but it’s difficult to deal with alone . . . I need help! (IDI26) like this. (IDI1) Social networks, the media and society’s notion of beauty as being thin led to prejudice
against people with MO. The interviewees found it difficult to buy clothes, find jobs and
establish social relations, both in terms of leisure and finding a partner. The social stigma
around MO had more of an impact than physical problems, which was exemplified by the
pity people with MO face in relation to their limitations: Int. J. Environ. Res. Public Health 2021, 18, 6530 6 of 14 Those with MO found that understanding and empathy came from people who were
in the same situation. Putting yourself in someone else’s shoes is difficult if you have never
experienced the sorts of comments and blaming looks from society when faced with MO. p
g
y
Those with MO turned to patient associations to talk about their problems and experi-
ences and to find social support. 3.1.3. Bariatric Surgery, a Shared Journey After failed attempts to lose weight using other methods, opting for surgery and
joining a bariatric surgery programme was a time in which those with MO preferred not to
be alone. Participants said that bariatric surgery was not part of their original plan due to
fear of the intervention, being judged and the cost of the process. In this phase, rather than
family or friends, their partner was the key pillar of support when making these decisions: My wife would talk to me about obesity and I would change the conversation. She tried
to help me understand that we had a serious problem, that we had to go to the doctor and
even consider bariatric surgery. (IDI27) 7 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 Those in the bariatric surgery programme were scared and unsure of the process. If patients had a partner, they shared those feelings with the partner; they needed their
partner’s positivity to improve their self-acceptance, for the partner to not feel embarrassed
by them and for the partner to encourage them to continue with their daily activities. Those
who did not have a partner shared their experiences with family and friends. As one
participant said, while they were waiting for the intervention, their expectations of the
surgery being a success were high. In fact, they expected a radical change in all areas of
their life, which could lead to disappointment: I want to be thin, positive, focus on the good things. We (partner) talk about it and I tell
him that everything will change, we are going to start from scratch, a new body, different
sexuality, another life. (IDI21) 3.2. The Need for Close Support Those with MO needed support from those with whom they live. Diets, commutes,
work and leisure all required effort for which social support was needed. However, they
were often faced with a lack of understanding; their perceived notion of social stigma
surrounding MO caused an internal conflict that led them to ask for help. 3.2.1. The Partner, the Key Support Figure for MO 3.2.1. The Partner, the Key Support Figure for MO 3.2.1. The Partner, the Key Support Figure for MO Those with MO suffered from stress, insecurity and low self-esteem; it was vital for
their partners to accept them. Although the participants did not express it, they thought
that their partners were not satisfied with them or their physical appearance, which affected
their self-esteem and social or sexual relations. As one participant stated, having their
partner’s support was the driving force they needed to opt for and face the surgical
process together: He tells me that he met me like this (obese), that he loves me like this, that he’s not with
me for my physical appearance but I know it’s impossible . . . I’m embarrassed to have
sexual relations. (IDI6) The participants’ partners worried a great deal about the associated comorbidities. They feared an exacerbation of cardiovascular or respiratory problems (e.g., a heart attack). This led to the partner being in a permanent state of alert and to adapting their lifestyles to
the person with MO. Likewise, the participants had a profound sense of guilt; they knew
that their partner was looking out for them and they did not want them to suffer: She (wife) is scared of something happening to me, of me dying and leaving her alone. I
try to not let my obesity affect my loved ones . . . but it’s difficult. (IDI29) However, this type of support was not universal, and some patients’ partners had
made derogatory comments. As one man said, this led to a fear of being left, which, in
turn, hastened their decision to enter a bariatric surgery programme. In summer, I wear more revealing clothes so my body is on show. If your partner looks at
you and says “look at their bodies, not like yours” you think that they are going to leave
you and you want to have the operation immediately! (IDI 31) My family says “don’t eat this, go for a walk, you need to endure it . . . ” Since I was put
in the bariatric surgery programme, they monitor me and control me all day, it’s stifling.
(IDI14) The patient association brought us together, it was like a big family. We were social and
able to be ourselves, without feeling observed or rejected. (IDI19) The patient association brought us together, it was like a big family. We were social and
able to be ourselves, without feeling observed or rejected. (IDI19) Losing this meeting point (MO patient associations shut down at a local level) had a
negative impact on the participants’ self-esteem. They tried to replace it by using social
networks, such as Facebook, online forums and, above all, Whatsapp, to inform themselves
about advances, pre-operative experiences, post-operative complications and expectations
of the post-surgery period. It was especially mothers with children with MO who turned
to these means of communication: Now we use Whatsapp, that’s how we talk, laugh and cry. For a mother, sharing fears
with someone who is going through the same as you, who is going to understand you, is
priceless. (IDI11) 3.2.2. Necessary Company, Unpleasant Scrutiny Family and friends were social support for those with MO. Nonetheless, some partici-
pants felt self-conscious, scrutinised and uncomfortable in their own homes. The family
was aware of the severe risks involved with MO for the person diagnosed. They monitored
them and hid food, making themselves responsible for the situation not worsening, which
became even more evident when the person with MO was awaiting bariatric surgery: My family says “don’t eat this, go for a walk, you need to endure it . . . ” Since I was put
in the bariatric surgery programme, they monitor me and control me all day, it’s stifling. (IDI14) Int. J. Environ. Res. Public Health 2021, 18, 6530 8 of 14 This situation provoked more anxiety, despair and avoidance behaviours. On occasion,
they resorted to lies, hiding themselves, putting on alarms and waking up in the middle of
the night to eat so that they were not judged in a negative way. They spent years feeling
worried and monitored by their families. As one participant stated, although they knew it
was for their health, over time, they avoided receiving advice from family members: When everyone at home talks about my MO, I get sick of it. I get up from the table and
leave. Then I think and say . . . they’re right. (IDI28) Participants indicated that the support they received from their inner circle was
fundamental in making decisions. The people close to them made them want to fight
and persevere whilst waiting for their bariatric surgery. Their friends could be a source
of support that was even greater than that of their families because they listened to them
without judging them: When I’m feeling down, I turn to a friend and tell her my problems and let off steam. My
family just repeat the doctor’s advice over and over, they don’t listen to me or understand
me. (IDI6) 3.3. Professional Support: Opposing Feelings Healthcare professionals were the key source of formal support for educating patients
and finding a solution to their problem. However, bariatric surgery was not the first choice
of treatment, nor was it explained in doctor or nurse consultations. 3.2.3. Appearing and Sharing on Social Networks Those with MO participated in associations, which allowed for mutual support,
understanding and health education. They shared their experiences, concerns and hopes,
enabling them to feel less lonely and increase social contact. As one of the participants said,
these associations were an extension of the safe space provided by close family, in which
they could share their problems in a social context. The patient association brought us together, it was like a big family. We were social and
able to be ourselves, without feeling observed or rejected. (IDI19) 3.3.1. Lack of Professional Support When patients acknowledged that they could not face MO alone, they looked for
healthcare professionals to guide them in their treatment. However, as some participants
said, they did not find the support they had expected in visits to the doctor, nurse or
dietician. Many healthcare professionals did not show interest or did not have the neces-
sary training to address MO. The patients note that the professionals did not empathise,
listen actively or take their problem seriously, leading them to feel disheartened and
less motivated: During a medical visit, the nurse weighed me on a kitchen scale. I felt awful. Is that why During a medical visit, the nurse weighed me on a kitchen scale. I felt awful. Is that why
I go to the hospital? To be embarrassed? I stopped going (IDI23) 3.3.2. The Bariatric Nurse Is “Always There” From the first contact with the bariatric surgery team, their vision of professional
support changed radically. As one participant said, they slowly gained the confidence and
assurance that they needed, they felt accompanied, listened to, informed and knew that
they could count on their professional support: Speaking to an expert outside of my family is liberating. I can be honest, express my
feelings without anyone judging me . . . this team is just amazing! (IDI1) Along with the social support from family members and close friends, people with
MO needed specialised professional support during the surgical process. The participants
appreciated the specialist nurse who guided and accompanied them from entering the
bariatric surgery programme up until the post-operative phase. They had access to a
24/7 phone service with the bariatric surgery nurse. It allowed them to ask questions,
make appointments, book the surgery, inform themselves and share their fears. Family
members also contacted the service to ask for information, calm their nerves or talk about
their expectations: The specialist nurse manages the situation, always picks up the phone at any hour of the
day. It makes you feel assured and confident, you know that you always have someone
there to inform, help and advise you . . . someone who listens to your fears and worries. (IDI7) of patients sought consultation after advice from other patients or finding information on
the internet: of patients sought consultation after advice from other patients or finding information on
the internet: A neighbour told me that she knew people who had been operated, that it was a good team. I only want them to explain it to me properly . . . to lift that burden. (IDI19) I go to the hospital? To be embarrassed? I stopped going. (IDI23) Dieticians insisted that diet alone allows one to achieve the desired weight loss
objective, and they did not support the idea of bariatric surgery. General practitioners or
nurses did not inform about bariatric surgery in primary care services either. The majority Int. J. Environ. Res. Public Health 2021, 18, 6530 9 of 14 9 of 14 4. Discussion According to Leahy-Warren [40], informal support is key in making the decision to
undergo surgery. In this phase, the partner is a fundamental support figure with whom to
share worries, experiences and conflicts, thus offering assurance [48]. Support from one’s
partner can improve the phase of awaiting surgery [38], especially if both partners are in the
bariatric surgery programme [39]. In accordance with our results, some participants were
on the receiving end of negative gestures or comments, leading to reduced communication
and affection, as well as increased fear of being left [22]. Support from family is key for
people with MO [49] and the more support they have, the more likely they are to consider
bariatric surgery [27]. Our results reflect the overbearing attitudes of family members,
which the people with MO reject, thus leading them to seek support amongst friends [50]. The quality of social support amongst close members of an inner circle can influence weight
gain. In fact, strong social support can be a factor in avoiding weight gain, especially in
men [51]. In terms of professional support, the participants criticised the information they
received on visits to the doctor or nurse, citing a lack of communication, empathy and
possibility to be referred to specialists in bariatric surgery. The patients turned to others
who had undergone surgery, associations or the internet (web pages, forums, etc.) to inform
themselves about the bariatric surgical programme [46]. Their negative opinion changed
radically upon contacting the bariatric surgery team [37], as there was an improvement
in information, communication, support and quality of life while waiting for bariatric
surgery [38]. g
y [
]
Social stigma was an added problem for people with MO [21], as social networks and
the media promoted an idealised body image. The participants reported a lack of access to
quality support for weight control [52], even though they received support from family
and professionals every time they used social networks [53]. Social support could be more
effective in long-term weight loss but there was a lack of evidence on the subject [24]. It
could be important to encourage people with MO to participate in associations to create
a shared social identity [36]. Online support groups can be useful for people with MO
in a bariatric surgery programme, as they can discuss their problems and share their
fears, expectations, challenges and experiences. 4. Discussion The objective of our study was to describe and understand the experiences of social
support amongst people with MO included in a bariatric surgery programme. Leahy-
Warren’s [40] theoretical framework allowed us to study how formal, informal and struc-
tural support influenced their experiences. MO caused physical, psychological and social
problems, as well as emotional and body image issues. According to our results, men
regarded MO as a health issue that also affected social and amorous relationships [22]. Women, on the other hand, were more worried about the aesthetic facet, feeling stuck in
a body that they rejected and did not want to reveal, which, in turn, deteriorated their
social relations [21]. MO limited the daily activities of the patients and although they tried
to gradually adapt [12], their frustration led them to look for solutions. The participants
experienced an initial phase of denial in which they avoided conversations about MO. After failed attempts at losing weight using other methods, their isolation and frustration
led them to join a bariatric surgery programme [44]. However, bariatric surgery is not
always an option, some patients prefer other treatments to avoid risk [37] or to hide their
intentions due to social or economic obstacles [45]. Various studies confirm the positive effects of social support on health and wellbeing
during MO [26], highlighting the importance of the partner, family, extended family
and professionals. Concurring with Willmer and Salzmann-Erikson [46], people with
MO fear bariatric surgery, but they have high expectations for improving their body
image, associated conditions and quality of life. The participants believed that losing
weight would strengthen their personal identity, social relations and participation in public
and professional life [33]. However, their expectations for positive results could lead to
disappointment [44]. The role of weight bias in concerns over body image in bariatric surgery patients
could jeopardise the results of their treatment. Obese people tend to percieve their weight
incorrectly, even after bariatric surgery, patients seem unable to identify changes in their
body image after massive weight loss [47]. Social support could positively influence body Int. J. Environ. Res. Public Health 2021, 18, 6530 10 of 14 10 of 14 image perception and interpersonal body comparisons after bariatric surgery, although
additional studies should be carried out. image perception and interpersonal body comparisons after bariatric surgery, although
additional studies should be carried out. 4. Discussion Healthcare providers must familiarise
themselves with the content of these groups and be cautious when recommending them [35]. Participating in online forums and support groups does not predict stress or weight loss [28],
but they could increase awareness for people with MO regarding their responsibility
in achieving positive outcomes [29]. This social support can be useful for people with
MO in a bariatric surgery programme, although it depends on their level of activity. Professionals must improve their understanding and develop and recommend online
self-help groups [25]. The use of social networks, private groups or public pages requires
careful examination because their effectiveness has not been demonstrated in experimental
studies [54]. Together with early support for patients who take part in a bariatric surgery
programme, life-long, individualised support may be required to optimise the effects
of bariatric surgery. Receiving follow-up visits from a multidisciplinary medical team
or joining support groups may be beneficial in counteracting weight gain after bariatric
surgery [55]. 5. Conclusions Society’s high beauty standards, the media and social networks all contribute to
prejudice towards people with MO. Social stigma, together with physical and psychological
problems, demonstrate the need for social support. The majority of participants with MO
were slow to acknowledge the problem and joining a bariatric surgery programme was
neither an easy decision nor an option they wanted to face alone. They needed information,
company and shared experiences. Those with MO had high expectations for bariatric
surgery, imagining a radical change in their lives, which could lead to disappointment. 11 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 11 of 14 The partners of people with MO were the key social support figures for them as they
needed support to face the surgical process. However, those with MO were sometimes
on the receiving end of disrespectful comments, leading to fear of being left by their
partners. The partners were in a constant state of alert, worrying about the potential
exacerbation of comorbidities, which provoked feelings of blame amongst the people with
MO. Family and friends were also members of the participants’ social support groups. Families were aware of the risks of living with MO and therefore monitored and scrutinised
the person’s lifestyle. Those with MO felt self-conscious and uncomfortable, thus their
friends substituted family as social support. Healthcare professionals were the main source
of formal support, but people with MO did not find the support they expected in general
practitioners, nurses or dieticians. Some healthcare professionals showed little interest in
the issue and lacked specific training to be able to guide the person with MO in making
decisions. The majority of people with MO reached the bariatric surgery consultant either
through advice from people who had already undergone the surgery or information found
on the internet. This situation changed dramatically upon the first contact with the bariatric
surgery team as the people with MO felt confident, assured, accompanied, listened to and
informed. Those with MO needed a specialised healthcare professional who centralised
the information and accompanied them throughout the whole surgical process. The nurse
was the main source of support for those with MO in the bariatric surgery programme;
they had access to a 24/7 phone service in which the bariatric surgery nurse attended to
their needs. Our study pointed to implications for clinical practice, as well as new research
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in their decision to undergo bariatric surgery. Organising support groups among patients
themselves, or with family or friends, managed by bariatric surgery nurses, could help
to improve shortcomings and support decision making. Social support can also help to
generate more realistic perspectives regarding outcomes after bariatric surgery in patients. The role of healthcare professionals, friends, partners and/or social networks as a means
of support for weight control after bariatric surgery may be fundamental and should be
investigated further. Providing images of before and after surgery, as well as explaining
that the results are not the same in all patients, could be helpful to patients’ perceptions of
their improvements. Author Contributions: Conceptualisation, J.G.-M., M.J.T.-S. and M.F.-M.; methodology, B.E.-F. and
M.d.M.J.-L.; formal analysis, A.R.-M. and I.D.-S.; investigation, M.I.V.-M.; data curation, A.R.-M. and B.E.-F.; writing—original draft preparation, M.J.T.-S. and M.F.-M.; writing—review and editing,
J.G.-M. and M.F.-M.; supervision, M.J.T.-S. and J.G.-M. All authors have read and agreed to the
published version of the manuscript. Funding: Thanks to the Research Group Health Sciences CTS-451 and the Health Research Center
from the University of Almería for their support. Institutional Review Board Statement: The study was conducted according to the guidelines of the
2013 Helsinki Declaration. The protocol was approved by the Ethics and Research Committee of the
Department of Nursing, Physiotherapy and Medicine (Nº. 45/2018). Informed Consent Statement: Informed consent was obtained from all participants involved in
the study. Informed Consent Statement: Informed consent was obtained from all participants involved in
the study. Data Availability Statement: Data are available from the authors (J.G.-M., M.J.T.-S. and M.F.-M.)
upon reasonable request. Acknowledgments: Our sincere gratitude is due to all participants. Acknowledgments: Our sincere gratitude is due to all participants. Conflicts of Interest: The authors declare no conflict of interest. 12 of 14 12 of 14 Int. J. Environ. Res. Public Health 2021, 18, 6530 1.
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$$\ell _2$$-penalized approximate likelihood inference in logit mixed models for regional prevalence estimation under covariate rank-deficiency
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Metrika (2022) 85:459–489
https://doi.org/10.1007/s00184-021-00837-y Metrika (2022) 85:459–489
https://doi.org/10.1007/s00184-021-00837-y B Joscha Krause
krause@uni-trier.de ℓ2-penalized approximate likelihood inference in logit
mixed models for regional prevalence estimation under
covariate rank-deficiency Joscha Krause1
· Jan Pablo Burgard1 · Domingo Morales2 Received: 1 October 2020 / Accepted: 28 July 2021 / Published online: 17 August 2021
© The Author(s) 2021 1
Department of Economic and Social Statistics, Trier University, Universitätsring 15, 54296 Trier,
Germany 2
Operations Research Center, University Miguel Hernández de Elche, Elche, Spain 1 Introduction Regional prevalence estimation is an essential element of modern epidemiologic
research (Branscum et al. 2008; Stern 2014; Burgard et al. 2019). Policymakers and
health care providers need reliable information on regional disease distributions to plan
comprehensive health programs. Depending on the disease of interest, corresponding
figures may not be recorded in registries and must be estimated from survey data
instead. However, national health surveys often lack in sufficient local observations
due to limited resources. As a result, regional prevalence estimates based on survey
data can be subject to unacceptable uncertainty due to large sampling variances. Small
area estimation (SAE) solves this problem by linking a response variable of interest
to statistically related covariates by means of a suitable statistical model. The obser-
vations from multiple regions are combined and jointly used for model parameter
estimation. Regional prevalence estimates are obtained via model prediction, which
allows for an increase in the effective sample size relative to classical direct estimation. See Rao and Molina (2015) for an overview on SAE. In practice, the efficiency advantage of SAE methods over direct estimators is
mainly determined by two aspects: (i) finding a suitable model type to describe the
response variable, and (ii) having covariate data with explanatory power. Regarding
the first aspect, since regional prevalence is usually stated as proportion (number of
sick persons divided by the total number of persons), binomial, Poisson or negative
binomial mixed models are canonical choices. The binomial-logit approach has been
used for regional proportion estimation in the past, for instance by Molina et al. (2007), Ghosh et al. (2009), Chen and Lahiri (2012), Erciulescu and Fuller (2013),
López-Vizcaíno et al. (2013), López-Vizcaíno et al. (2015), Burgard (2015), Militino
et al. (2015), Chambers et al. (2016), Hobza and Morales (2016), Liu and Lahiri
(2017) and Hobza et al. (2018). The Poisson or negative binomial mixed models were
applied to estimate small area counts or proportions by Berg (2010), Chambers et al. (2014), Dreassi et al. (2014), Tzavidis et al. (2015) and Boubeta et al. (2016, 2017),
among others. Marino et al. (2019) propose a semiparametric approach allowing for a
flexible random effects structure in unit-level models. Ranalli et al. (2018) introduced
benchmarking for logistic unit-level. Concerning the second aspect, medical routine
data provided by official statistics or health insurance companies have been found to
be promising data bases for regional prevalence estimation. Abstract Regional prevalence estimation requires the use of suitable statistical methods on
epidemiologic data with substantial local detail. Small area estimation with medical
treatment records as covariates marks a promising combination for this purpose. How-
ever, medical routine data often has strong internal correlation due to diagnosis-related
groupingintherecords.Dependingonthestrengthofthecorrelation,thespacespanned
by the covariates can become rank-deficient. In this case, prevalence estimates suffer
from unacceptable uncertainty as the individual contributions of the covariates to the
model cannot be identified properly. We propose an area-level logit mixed model for
regional prevalence estimation with a new fitting algorithm to solve this problem. We
extend the Laplace approximation to the log-likelihood by an ℓ2-penalty in order to
stabilize the estimation process in the presence of covariate rank-deficiency. Empiri-
cal best predictors under the model and a parametric bootstrap for mean squared error
estimation are presented. A Monte Carlo simulation study is conducted to evaluate
the properties of our methodology in a controlled environment. We further provide
an empirical application where the district-level prevalence of multiple sclerosis in
Germany is estimated using health insurance records. Keywords Generalized linear mixed models · Laplace approximation · Multiple
sclerosis · Prevalence mapping · Small area estimation 2
Operations Research Center, University Miguel Hernández de Elche, Elche, Spain 123 123 460 J. Krause et al. 1 Introduction Exemplary applications
were provided by Tamayo et al. (2016), Burgard et al. (2019), and Breitkreutz et al. (2019). However, using medical routine data as covariates can be problematic, especially
within logit mixed models. Medical treatment frequencies are typically recorded and
coded into diagnosis groups, for instance on ICD-3 level (World Health Organization
2018). This context-related segmentation can induce strong correlation between med-
ical treatment frequencies for diseases that are closely related in terms of comorbidity,
such as diabetes and hypertension (Long and Dagogo-Jack 2011). If two or more
diagnoses from the auxiliary data set are strongly correlated, the space spanned by the
covariates can become rank-deficient. In that case, it is not possible to accurately sep-
arate the individual contributions of the covariates to the description of the response
variable. Model parameter estimates suffer from high variance and model predic- 123 ℓ2-penalized approximate... 461 tions for regional prevalence are not reliable. This is particularly problematic for logit
mixed models, as model parameter estimation already relies on approximate inference
in the absence of rank-deficiency (Breslow and Clayton 1993). The respective like-
lihood integral does not have a closed-form solution, which requires techniques like
the Laplace approximation to find a proper substitute as objective function. Therefore,
when approximate inference is to be performed on a rank-deficient covariate space,
methodological adjustments are required to allow for reliable results. In this paper, we propose a modification to the maximum likelihood Laplace (ML-
Laplace) algorithm for model parameter estimation (e.g. Demidenko 2013; Hobza
et al. 2018) in a logit mixed model under covariate rank-deficiency. We draw from
theoretical insights on ridge regression (Hoerl and Kennard 1970) and extend the
Laplace approximation to the log-likelihood function by the squared ℓ2-norm of the
regression parameters (ℓ2-penalty). This adjustment reduces the variance of model
parameter estimates considerably and improves approximate inference in the presence
of strong covariate correlation. To the best of our knowledge, ℓ2-penalization has only
been studied for standard ML estimation in fixed effect logit models, for instance by
Schaefer et al. (1984), Cessie and Houwelingen (1992), and Pereira et al. (2016). We
are not aware of corresponding studies for logit mixed models based on ML-Laplace
estimation, especially not in the context of SAE. An area-level binomial logit mixed model for regional prevalence estimation is
presented. 1 Introduction Following Jiang and Lahiri (2001) and Jiang (2003), we derive empirical
best predictors (EBPs) under the model and present a parametric bootstrap estimator
for their mean squared error (MSE). Thereafter, we state the Laplace approximation
to the log-likelihood function and demonstrate ℓ2-penalized approximate likelihood
(ℓ2-PAML) estimation of the model parameters. We further show how the tuning
parameter for the ℓ2-penalty can be chosen in practice. A Monte Carlo simulation study
is conducted to study the behavior of ℓ2-PAML estimation under different degrees of
covariate correlation. And finally, the proposed methodology is applied to regional
prevalence estimation in Germany. We use health insurance records of the German
Public Health Insurance Company (AOK) and inpatient diagnosis frequencies of the
Diagnosis-Related Group Statistics (DRG-Statistics) to estimate district-level multiple
sclerosis prevalence. The remainder of the paper is organized as follows. In Sect. 2, we present the
model and its EBP. We further address MSE estimation. In Sect. 3, we present the
Laplace approximation and the technical details for ℓ2-PAML. Section 4 contains a
Monte Carlo simulation study. Section 5 covers the application to regional prevalence
estimation. Section 6 closes with some conclusive remarks. 2.1 Formulation For the subsequent derivation, we rely on model-based inference in a finite population
setting. Let U be a finite population of size |U| = N. Suppose that U is partitioned
into D domains Ud of size |Ud| = Nd. That is to say, U = ∪D
d=1Ud, Ud1 ∩Ud2 = ∅, For the subsequent derivation, we rely on model-based inference in a finite population
setting. Let U be a finite population of size |U| = N. Suppose that U is partitioned
into D domains Ud of size |Ud| = Nd. That is to say, U = ∪D
d=1Ud, Ud1 ∩Ud2 = ∅, 123 462 J. Krause et al. d1 ̸= d2, and D
d=1 Nd = N. Let S be a sample of size |S| = n that is drawn from
U. For simplicity, assume the sampling scheme is such that there are domain-specific
subsamples Sd of size |Sd| = nd with fixed nd > 0 for all d = 1, ..., D. Thus, we
have S = ∪D
d=1Sd and D
d=1 nd = n. Let y be a dichotomous response variable with
potential outcomes {0, 1}. Denote the realization of y for some individual i ∈Ud by
yid. Note that we use the same symbol for a random variable and its realizations in
order to avoid overloading the notation. Define yd =
i∈sd yid as the sample total
(count) of y in domain Ud. Let x = {x1, ..., x p} be a set of covariates statistically
related to y. Denote xd as a 1 × p vector of aggregated (domain-level) realizations
of x. Suppose that corresponding information is retrieved from administrative records
and not calculated from the sample S. In what follows, we present an area-level
logit mixed model for estimating the domain totals Yd =
i∈Ud yid or proportions
pd = Yd/Nd of the response variable. Let us consider a set of random effects such
that {vd : d = 1, . . . , D} are independent and identically distributed according to
vd ∼N(0, 1). In matrix notation, we have normally distributed random effects v =
col
1≤d≤D(vd) ∼ND(0, I D)
(1) (1) and, hence, their probability density function (PDF) is stated as and, hence, their probability density function (PDF) is stated as fv(v) = (2π)−D/2 exp
−1
2 v′v
. 2.1 Formulation (2) (2) The model assumes that the distribution of the response variable yd, conditioned to
the random effect vd, is yd|vd ∼Bin(nd, pd),
d = 1, . . . , D,
(3) (3) and that the natural parameter fulfills ηd = log
pd
1 −pd
= xdβ + φvd,
d = 1, . . . , D,
(4) (4) where φ > 0 is an standard deviation parameter, β = col1≤r≤p(βr) is the vector of
regression parameters and xd = col′
1≤r≤p(xdr). We complete the model definition by
assuming that the domain-specific sample counts yd are independent when conditioned
on the random effects v. Therefore, the conditional PDF of y = col1≤d≤D(yd) given
v is stated as P(y|v) =
D
d=1
P(yd|vd),
P(yd|v) = P(yd|vd) =
nd
yd
pyd
d (1 −pd)nd−yd, (5) (5) where where pd =
exp {xdβ + φvd}
1 + exp {xdβ + φvd} =
exp{ηd}
1 + exp{ηd},
1 −pd =
1
1 + exp{ηd}. (6) (6) 123 123 ℓ2-penalized approximate... 463 Further, the unconditional PDF of y is Further, the unconditional PDF of y is P(y) =
RD P(y|v) fv(v) dv =
RD ψ(y, v) dv,
(7) P(y) =
RD P(y|v) fv(v) dv =
RD ψ(y, v) dv,
(7) P(y) =
RD P(y|v) fv(v) dv =
RD ψ(y, v) dv,
(7)
with (7) with with with with ψ(y, v) = (2π)−D
2 exp
−v′v
2
D
d=1
nd
yd
exp {yd(xdβ + φvd)}
1 + exp {xdβ + φvd}nd
= (2π)−D
2
D
d=1
nd
yd
exp
−v′v
2
exp
p
k=1
D
d=1
ydxdk
βk + φ
D
d=1
ydvd
−
D
d=1
nd log (1 + exp {xdβ + φvd})
. (8) ψ(y, v) = (2π)−D
2 exp
−v′v
2
D
d=1
nd
yd
exp {yd(xdβ + φvd)}
1 + exp {xdβ + φvd}nd = (2π)−D
2
D
d=1
nd
yd
exp
−v′v
2
exp
p
k=1
D
d=1
ydxdk
βk + φ
D
d=1
ydv
−
D
d=1
nd log (1 + exp {xdβ + φvd})
. (8) 2.2 Prediction Hereafter, we obtain EBPs under the area-level logit mixed model introduced in
Sect. 2.1. For this, wefirst derivebest predictors (BPs) inapreliminarysettingwhereall
model parameters θ := (β′, φ) are assumed to be known. Then, the EBPs are obtained
by replacing the full parameter vector θ by consistent estimators ˆθ := ( ˆβ
′, ˆφ). Note
that calculating the EBP requires Monte Carlo integration over the random effect
PDF, which can be computationally infeasible for some applications. Therefore, we
also state two alternative predictors that do not rely on Monte Carlo integration and
are easier to apply in practice. We start with the EBPs. Recall the definition of the
conditional PDF P(y|v) from the last section. For the domain-specific component
P(yd|vd), we can write P(yd|vd) =
nd
yd
pyd
d (1 −pd)nd−yd =
nd
yd
exp {yd(xdβ + φvd)}
1 + exp {xdβ + φvd}nd
= exp
log
nd
yd
+ yd(xdβ + φvd) −nd log
1 + exp{xdβ + φvd}
.(9) The probability density function of v is The probability density function of v is The probability density function of v is f (v) =
D
d=1
f (vd),
f (vd) = (2π)−1/2 exp
−1
2 v2
d
. (10) (10) The BP of pd = pd(θ, vd) is given by the conditional expectation ˆpd(θ) = Eθ[pd|y]. Due to the conditional independence of the response realizations given the random
effects, we have Eθ[pd|y] = Eθ[pd|yd] and The BP of pd = pd(θ, vd) is given by the conditional expectation ˆpd(θ) = Eθ[pd|y]. Due to the conditional independence of the response realizations given the random
effects, we have Eθ[pd|y] = Eθ[pd|yd] and 123 123 464 J. Krause et al. Eθ[pd|yd] =
R
exp{xdβ+φvd}
1+exp{xdβ+φvd} P(yd|vd) f (vd) dvd
R P(yd|vd) f (vd) dvd
= Nd(yd, θ)
Dd(yd, θ) = Nd(yd, θ)
Dd(yd, θ),
(11) (11) where Nd = Nd(yd, θ), Dd = Dd(yd, θ), Nd = Nd(yd, θ) and Dd = Dd(yd, θ) are
functions of the model parameters and the domain-specific sample counts. 2.2 Prediction They are
stated as follows: Nd =
R
exp{xdβ + φvd}
1 + exp{xdβ + φvd} exp
log
nd
yd
+ yd xdβ + φydvd
−nd log
1 + exp{xdβ + φvd}
f (vd) dvd,
Dd =
R
exp
log
nd
yd
+ yd xdβ + φydvd −nd log
1 + exp{xdβ + φvd}
f (vd) dvd,
Nd =
R
exp{xdβ + φvd}
1 + exp{xdβ + φvd} exp
φydvd −nd log
1 + exp{xdβ + φvd}
f (vd) dvd,
Dd =
R
exp
φydvd −nd log
1 + exp{xdβ + φvd}
f (vd) dvd. We can conclude that the EBP of pd is ˆpd(ˆθ). However, its quantification requires
integration over the random effect PDF f (vd). As the logit mixed model belongs to
the family of generalized linear mixed models, this cannot be performed analytically. Instead, we apply Monte Carlo integration and approximate the EBP as follows: 1. Estimate ˆθ = ( ˆβ, ˆφ). (β, φ)
2. For k = 1, . . . , K, generate v(k)
d
i.i.d. N(0, 1) and v(K+k)
d
= −v(k)
d . 3 Calculate ˆpd(ˆθ) = ˆNd/ ˆDd where (β, φ)
2. For k = 1, . . . , K, generate v(k)
d
i.i.d. N(0, 1) and v(K+k)
d
= −v(k)
d . ˆ
ˆ
ˆ d
3. Calculate ˆpd(ˆθ) = ˆNd/ ˆDd, where 3. Calculate ˆpd(ˆθ) = ˆNd/ ˆDd, where ˆNd =
1
2K
2K
k=1
exp{xd ˆβ + ˆφv(k)
d }
1 + exp{xd ˆβ + ˆφv(k)
d }
exp
ˆφydv(k)
d
−nd log
1 + exp{xd ˆβ + ˆφv(k)
d }
,
ˆDd =
1
2K
2K
k=1
exp
ˆφydv(k)
d
−nd log
1 + exp{xd ˆβ + ˆφv(k)
d }
. ˆDd =
1
2K
2K
k=1
exp
ˆφydv(k)
d
−nd log
1 + exp{xd ˆβ + ˆφv(k)
d }
. The EBP of pd can be used to obtain the predictor ˆYd = Nd ˆp(ˆθ) of the domain total
Yd. The EBP of pd can be used to obtain the predictor ˆYd = Nd ˆp(ˆθ) of the domain total
Yd. We now state two alternative predictors that do not rely on Monte Carlo integration. The first is a synthetic predictor. 2.3 Mean squared error estimation In order to assess the reliability of the obtained predictions for pd, we use the mean
squared error. It is generally characterized by MSE( ˆpd) = E[( ˆpd −pd)2]. However,
MSE( ˆpd) cannot be quantified directly and must be estimated instead. For this, we
apply a parametric bootstrap as demonstrated by González-Manteiga et al. (2007) and
Boubeta et al. (2016). It is performed as follows. 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ
′, ˆφ). 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ
′, ˆφ). 2. Repeat B times with b = 1, ..., B: 2. Repeat B times with b = 1, ..., B: (a) Generate v(b)
d
∼N(0, 1), y(b)
d
∼Bin(nd, p(b)
d ), d = 1, . . . , D,
where p(b)
d
=
exp
xd ˆβ+ ˆφv(b)
d
1+exp
xd ˆβ+ ˆφv(b)
d
. (b)
(b) For each bootstrap sample, calculate the estimator ˆθ
(b) and the EBP ˆp(b)
d
=
ˆp(b)
d (ˆθ
(b)) as stated above. (b) For each bootstrap sample, calculate the estimator ˆθ
(b) and the EBP ˆp(b)
d
=
ˆp(b)
d (ˆθ
(b)) as stated above. 3. Output: mse( ˆpd) = 1
B
B
b=1
ˆp(b)
d
−p(b)
d
2. 2.2 Prediction It is characterized by a regression-synthetic prediction
from the area-level logit mixed model without considering the random effect. On that
note, the synthetic predictor of pd is obtained according to ˜psyn
d
=
exp{xd ˆβ}
1 + exp{xd ˆβ}
,
(12) (12) which constitutes the synthetic predictor ˜Y syn
d
= Nd ˜psyn
d
for Yd. The plug-in predictor
is obtained along the same lines, but includes the random effects vd as well as the which constitutes the synthetic predictor ˜Y syn
d
= Nd ˜psyn
d
for Yd. The plug-in predictor
is obtained along the same lines, but includes the random effects vd as well as the ℓ2-penalized approximate... 465 variance parameter φ. For the prediction of pd, we have variance parameter φ. For the prediction of pd, we have ˜p plug
d
=
exp{xd ˆβ + ˆφ ˆvd}
1 + exp{xd ˆβ + ˆφ ˆvd}
,
(13) (13) where ˆvd is a predictor for the random effect vd. We describe how to calculate the
corresponding predictors in Sect. 3. Finally, the plug-in predictor of Yd is ˜Y plug
d
=
Nd ˜p plug
d
. where ˆvd is a predictor for the random effect vd. We describe how to calculate the
corresponding predictors in Sect. 3. Finally, the plug-in predictor of Yd is ˜Y plug
d
=
Nd ˜p plug
d
. 3 Penalized model parameter estimation In this section, it is demonstrated how model parameter estimation in the area-level
logit mixed model under covariate rank-deficiency is performed. The foundation of
our estimation strategy is the ML-Laplace algorithm (e.g. Demidenko 2013; Hobza
et al. 2018). That is to say, the integrals in the likelihood function are approximated via
the Laplace method and the result is maximized with a Newton-Raphson algorithm. However, in light of the comments in Sect. 1 and prior to maximization, we extend
the approximated likelihood function by the squared ℓ2-norm of β to account for the
negative effects of covariate rank-deficiency. With this, we obtain a penalized version
approximated likelihood, which is then maximized to obtain reliable model parameter
estimates.Werefertothisprocedureasℓ2-penalizedapproximatemaximumlikelihood
(ℓ2-PAML) estimation. 123 J. Krause et al. 466 3.1 Laplace approximation We first perform the Laplace approximation of the likelihood function. Let h : R
→R
be a continuously twice differentiable function with a global maximum at x0. This is
to say, assume that h′(x0) = 0 and h′′(x0) < 0. A Taylor series expansion of h(x)
around x0 yields h(x) = h(x0) + h′(x0)(x −x0) + 1
2h′′(x0)(x −x0)2 + o
|x −x0|2
≈h(x0) + 1
2h′′(x0)(x −x0)2. (14) The univariate Laplace approximation is The univariate Laplace approximation is The univariate Laplace approximation is ∞
−∞
eh(x) dx ≈
∞
−∞
eh(x0) exp
−1
2
−h′′(x0)
(x −x0)2
dx
= (2π)1/2
−h′′(x0)
−1/2eh(x0)
∞
−∞
exp
−1
2
x−x0
(−h′′(x0))−1/2
2
(2π)1/2
−h′′(x0)
−1/2
d
= (2π)1/2
−h′′(x0)
−1/2eh(x0). (15 ∞
−∞
eh(x) dx ≈
∞
−∞
eh(x0) exp
−1
2
−h′′(x0)
(x −x0)2
dx = (2π)1/2
−h′′(x0)
−1/2eh(x0)
∞
−∞
exp
−1
2
x−x0
(−h′′(x0))−1/2
2
(2π)1/2
−h′′(x0)
−1/2
dx
(2 )1/2
h′′(
)
−1/2 h(x0)
(15) = (2π)1/2
−h′′(x0)
−1/2eh(x0)
∞
−∞
exp
2
(−h′′(x0))−1/2
(2π)1/2
−h′′(x0)
−1/2
dx
= (2π)1/2
−h′′(x0)
−1/2eh(x0). (15) = (2π)1/2
−h′′(x0)
−1/2eh(x0). (15) (15) Let us now approximate the log-likelihood of the area-level logit mixed model. Recall
that v1, . . . , vD are independent and identically distributed according to vd ∼N(0, 1),
and that Let us now approximate the log-likelihood of the area-level logit mixed model. Recall
that v1, . . . , vD are independent and identically distributed according to vd ∼N(0, 1),
and that yd|vd
ind
∼Bin(nd, pd),
pd = pd(vd) =
exp {xdβ + φvd}
1 + exp {xdβ + φvd},
d = 1, . . . , D. Thus, y1, . . . , yD are unconditionally independent with marginal probability density Thus, y1, . . . 3.1 Laplace approximation (18) From there, we can state the log-likelihood function under the model, which is given
by From there, we can state the log-likelihood function under the model, which is given
by l =
D
d=1
ld = log P(yd). l =
D
d=1
ld = log P(yd). Using the results of the Laplace approximation, we obtain Using the results of the Laplace approximation, we obtain ld ≈l0d(θ) = log
nd
yd
−1
2 log ξ0d −v2
0d
2
+yd(xdβ + φv0d)nd log
1 + exp{xdβ + φv0d}
,
(19) ld ≈l0d(θ) = log
nd
yd
−1
2 log ξ0d −v2
0d
2 +yd(xdβ + φv0d)nd log
1 + exp{xdβ + φv0d}
,
(19) (19) where p0d = pd(v0d) and ξ0d = 1 + φ2nd p0d(1 −p0d). where p0d = pd(v0d) and ξ0d = 1 + φ2nd p0d(1 −p0d). where p0d = pd(v0d) and ξ0d = 1 + φ2nd p0d(1 −p0d). 3.1 Laplace approximation , yD are unconditionally independent with marginal probability density
∞ P(yd) =
∞
−∞
P(yd|vd) f (vd) dvd
=
∞
−∞
nd
yd
exp
yd(xdβ + φvd} −nd log
1 + exp{xdβ + φvd}
·(2π)−1/2 exp{−1
2v2
d} dvd = (2π)−1/2
nd
yd
·
∞
−∞
exp
−v2
d
2 + yd(xdβ + φvd) −nd log
1 + exp{xdβ + φvd}
dvd
(2 ) 1/2
nd
∞
h(
)
d
(16 P(yd) =
∞
−∞
P(yd|vd) f (vd) dvd
=
∞ nd
exp
yd(xdβ + φvd} −nd log
1 + exp{xdβ + φvd}
P(yd) =
∞
−∞
P(yd|vd) f (vd) dvd
=
∞
−∞
nd
yd
exp
yd(xdβ + φvd} −nd log
1 + exp{xdβ + φvd}
·(2π)−1/2 exp{−1
2v2
d} dvd = (2π)−1/2
nd
yd
·
∞
−∞
exp
−v2
d
2 + yd(xdβ + φvd) −nd log
1 + exp{xdβ + φvd}
dvd
= (2π)−1/2
nd
yd
∞
−∞
exp
h(vd)
dvd,
(16)
where
h(vd) = −v2
d + yd(xdβ + φvd) −nd log
1 + exp{xdβ + φvd}
(17) P(yd) =
∞
−∞
P(yd|vd) f (vd) dvd =
∞
∞
P(yd|vd) f (vd) dvd =
∞
−∞
nd
yd
exp
yd(xdβ + φvd} −nd log
1 + exp{xdβ + φvd}
·(2π)−1/2 exp{−1
2v2
d} dvd = (2π)−1/2
nd
yd
∞
2 ·
∞
−∞
exp
−v2
d
2 + yd(xdβ + φvd) −nd log
1 + exp{xdβ + φvd}
dvd = (2π)−1/2
nd
yd
∞
−∞
exp
h(vd)
dvd,
(16) = (2π)−1/2
nd
yd
∞
−∞
exp
h(vd)
dvd,
(16) (16) (16) here where h(vd) = −v2
d
2 + yd(xdβ + φvd) −nd log
1 + exp{xdβ + φvd}
. (17) (17) 123 123 467 ℓ2-penalized approximate... Note that for the maximizer of h(·), denoted by v0d, the first derivative is h′(v0d) = 0,
and the second derivative is characterized by h′′(v0d) < 0. By applying (15) in vd =
v0d, we get P(yd) ≈
nd
yd
·
1 + φ2nd pd(v0d)(1 −pd(v0d))
−1/2
· exp
−v2
0d
2 + yd(xdβ + φv0d) −nd log
1 + exp{xdβ + φv0d}
. Step 1: Log-likelihood approximation In order to maximize h(vd), we need to quantify its first and second derivatives. These
are h′(vd) = −vd + φ
yd −nd pd(vd)
(21)
h′′(vd) = −
1 + φ2nd pd(vd)(1 −pd(vd))
(22) (21) (22) for all d = 1, ..., D. The Newton-Raphson algorithm maximizes h(vd) = h(vd, θ),
defined in (17), for fixed θ = (β′, φ) = θ0. The updating equation is v(k+1)
d
= v(k)
d
−h′(v(k)
d , θ0)
h′′(v(k)
d , θ0)
,
(23) (23) where k denotes an iteration of the procedure. where k denotes an iteration of the procedure. where k denotes an iteration of the procedure. 3.2 ℓ2-penalized approximate maximum likelihood The approximated log-likelihood function is expanded by the squared ℓ2-norm of
the regression coefficients β to account for strong correlation between covariates in
x1, . . . , xD. We obtain the penalized maximum likelihood problem ˆθ = argmax
θ∈Rp+1 l pen(θ),
l pen(θ) =
D
d=1
l0d(θ) −λ∥β∥2
2,
(20) (20) where l0d(θ) is defined in (19) and λ > 0 is a predefined tuning parameter. Maximiza-
tion is performed via a Newton-Raphson algorithm. However, note that the Laplace
approximations of l1, ...,lD depends on the maximizers of h(v1), ..., h(vD), which
in turn depend on l1, ...,lD. Therefore, the maximization of (20) must contain two
steps that are performed iteratively and conditional on each other. The first step is the
approximation of the log-likelihood by maximizing h(v1), ..., h(vD). The second step
is the maximization of l pen(θ) given the results of the first step. This is demonstrated
hereafter. 123 J. Krause et al. 468 ep 1: Log-likelihood approximation Step 2: Penalized maximization ∂η0dr
∂φ
= 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2
0d)∂p0d
∂φ
= φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2
0d)],
∂η0d
∂βr
= φ2v0dndxdr p0d(1 −p0d)[1 −6p0d + 6p2
0d],
∂η0d
∂φ
= 2nd p0d(1 −p0d) + 2φnd1 −2p0d)∂p0d
∂φ
∂ ∂η0dr
∂φ
= 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2
0d)∂p0d
∂φ
= φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2
0d)],
∂ ∂η0dr
∂φ
= 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2
0d)∂p0d
∂φ
= φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2
0d)],
∂η0d = φ2v0dndxdr p0d(1 −p0d)[1 −6p0d + 6p2
0d], +2φnd(1 −2p0d)p0d(1 −p0d)v0d + φ2ndv0d(1 −6p0d + 6p2
0d)∂p0d
∂φ
= nd p0d(1 −p0d)[2 + 2φ(1 −2p0d)v0d + 2φ(1 −2p0d)v0d
+φ2v2
0d(1 −6p0d + 6p2
0d)]. For the domain-specific likelihood component l0d, this yields to ∂2l0d
∂β2r
= −1
2
∂η0dr
∂βr ξ0d −η2
0dr
ξ2
d
−ndx2
dr p0d(1 −p0d), ∂2l0d
∂β2r
= −1
2
∂η0dr
∂βr ξ0d −η2
0dr
ξ2
d
−ndx2
dr p0d(1 −p0d),
∂2l0d
∂βs∂βr
= −1
2
∂η0dr
∂βs ξ0d −η0drη0ds
ξ2
d
−ndxdr xdjs p0d(1 −p0d),
∂2l0d
∂φ∂βr
= −1
2
∂η0dr
∂φ ξ0d −η0drη0d
ξ2
d
−v0dndxdr p0d(1 −p0d),
∂2l0d
∂φ2 = −1
2
∂η0d
∂φ ξ0d −η2
0d
ξ2
d
−v2
0dnd p0d(1 −p0d). ∂2l0d
∂βs∂βr
= −1
2
∂η0dr
∂βs ξ0d −η0drη0ds
ξ2
d
−ndxdr xdjs p0d(1 −p0d), ∂2l0d
∂φ∂βr
= −1
2
∂η0dr
∂φ ξ0d −η0drη0d
ξ2
d
−v0dndxdr p0d(1 −p0d), ∂2l0d
∂φ2 = −1
2
∂η0d
∂φ ξ0d −η2
0d
ξ2
d
−v2
0dnd p0d(1 −p0d). As for the first partial derivatives applying these equations to all domain-specific
likelihood components l01, ...,l0D and considering the ℓ2-penalty, we end up with As for the first partial derivatives applying these equations to all domain-specific
likelihood components l01, ...,l0D and considering the ℓ2-penalty, we end up with ∂2l pen
∂β2r
=
D
d=1
∂2l0d
∂β2r
−2λ,
∂2l pen
∂βs∂βr
=
D
d=1
∂2l0d
∂βs∂βr
,
∂2l pen
∂φ∂βr
=
D
d=1
∂2l0d
∂φ∂βr
,
∂2l pen
∂φ2
=
D
d=1
∂2l0d
∂φ2 . (25) (25) For r, s = 1, . . . , p + 1, define the components of the score vector For r, s = 1, . . . Step 2: Penalized maximization We continue with maximizing the penalized approximate log-likelihood function. Regarding the first partial derivatives of l pen with respect to β1, ..., βp and φ, it holds
that ∂p0d
∂βr
= xdr p0d(1 −p0d) = xdr(p0d −p2
0d), ∂p0d
∂φ
= v0d p0d(1 −p0d) = v0d(p0d −p2
0d),
η0dr = ∂ξ0d
∂βr
= φ2ndxdr[p0d −3p2
0d + 2p3
0d],
η0d = ∂ξ0d
∂φ = 2φnd p0d(1 −p0d) + φ2nd(1 −2p0d)∂p0d
δφ
= φnd p0d(1 −p0d)[2 + φ(1 −2p0d)v0d]. For the domain-specific likelihood component l0d, this yields to ∂l0d
∂βr
= −1
2
η0dr
ξ0d
+ (yd −nd p0d)xdr,
∂l0d
∂φ = −1
2
η0d
ξ0d
+ (yd −nd p0d)v0d. With the application of these equations to all domain-specific likelihood components
l01, ...,l0D and the consideration of the ℓ2-penalty, we finally obtain With the application of these equations to all domain-specific likelihood components
l01, ...,l0D and the consideration of the ℓ2-penalty, we finally obtain ∂l pen
∂βr
=
D
d=1
∂l0d
∂βr
−2λβr,
∂l pen
∂φ
=
D
d=1
∂l0d
∂φ . (24) (24) 123 ℓ2-penalized approximate... 469 For the second partial derivatives, it holds that For the second partial derivatives, it holds that For the second partial derivatives, it holds that ∂η0dr
∂βs
= φ2ndxdr xdjs[p0d(1 −p0d) −6p2
0d(1 −p0d) + 6p3
0d(1 −p0d)]
= φ2ndxdr xdjs p0d(1 −p0d)[1 −6p0d + 6p2
0d], βs
= φ2ndxdr xdjs p0d(1 −p0d)[1 −6p0d + 6p2
0d],
∂η0dr
∂φ
= 2φndxdr p0d(1 −p0d)(1 −2p0d) + φ2ndxdr(1 −6p0d + 6p2
0d)∂p0d
∂φ
= φndxdr p0d(1 −p0d)[2(1 −2p0d) + φv0d(1 −6p0d + 6p2
0d)],
∂η0d
∂βr
= φ2v0dndxdr p0d(1 −p0d)[1 −6p0d + 6p2
0d],
∂η0d
∂φ
= 2nd p0d(1 −p0d) + 2φnd1 −2p0d)∂p0d
∂φ
+2φnd(1 −2p0d)p0d(1 −p0d)v0d + φ2ndv0d(1 −6p0d + 6p2
0d)∂p0d
∂φ
= nd p0d(1 −p0d)[2 + 2φ(1 −2p0d)v0d + 2φ(1 −2p0d)v0d
+φ2v2
0d(1 −6p0d + 6p2
0d)]. Step 2: Penalized maximization , p + 1, define the components of the score vector U0r = ∂l pen
∂βr
,
U0p+1 = ∂l pen
∂φ ,
(26) (26) 123 123 123 J. Krause et al. 470 as well as the Hessian matrix as well as the Hessian matrix as well as the Hessian matrix H0rs = H0sr = ∂2l pen
∂βs∂βr
,
Hrp+1 = Hp+1r = ∂2l pen
∂φ∂βr
,
H0p+1p+1 = ∂2l pen
∂φ2 . (27) (27) In matrix form, we have U0 = U0(θ) =
col
1≤r≤p+1(U0r) and H0 = H0(θ) = In matrix form, we have U0 = U0(θ) =
col
1≤r≤p+1(U0r) and H0 = H0(θ) = (H0rs)r,s=1,...,p+1. The Newton-Raphson algorithm maximizes l pen(θ), with fixed
vd = v0d, d = 1, . . . , D. Let k denote the index of iterations. The corresponding
updating equation is θ(k+1) = θ(k) −H−1
0 (θ(k))U0(θ(k)). (28) (28) Complete ℓ2-PAML algorithm (c) k ←k + 1. 3. Output: ˆθ = θ(k), ˆvd = v(k)
d , d = 1, . . . , D. We remark that the output of the ℓ2-PAML algorithm gives estimates ˆθ of the model
parameters θ and mode predictions ˆv of the random effects vd, d = 1, ..., D. We remark that the output of the ℓ2-PAML algorithm gives estimates ˆθ of the model
parameters θ and mode predictions ˆv of the random effects vd, d = 1, ..., D. Complete ℓ2-PAML algorithm The final algorithm containing both steps is performed as follows. 1. Set the initial values k = 0, θ(0), θ(−1) = θ(0) + 1, v(0)
d
= 0, v(−1)
d
= 1, d =
1, . . . , D. 2. Until ∥θ(k) −θ(k−1)∥2 < ε1, |v(k)
d
−v(k−1)
d
| < ε2, d = 1, . . . , D, do 1. Set the initial values k = 0, θ(0), θ(−1) = θ(0) + 1, v(0)
d
= 0, v(−1)
d
= 1, d =
1, . . . , D. (k)
(k
1)
(k)
(k
1) 1. Set the initial values k = 0, θ(0), θ(−1) = θ(0) + 1, v(0)
d
= 0, v(−1)
d
= 1, d =
1, . . . , D. (k)
(k
1) ,
,
+ ,
d
,
d
1, . . . , D. 2. Until ∥θ(k) −θ(k−1)∥2 < ε1, |v(k)
d
−v(k−1)
d
| < ε2, d = 1, . . . , D, do ,
,
2. Until ∥θ(k) −θ(k−1)∥2 < ε1, |v(k)
d
−v(k−1)
d
| < ε2, d = 1, . . . , D, do (a) Apply algorithm (23) with seeds v(k)
d , d = 1, . . . , D, convergence tolerance (a) Apply algorithm (23) with seeds v(k)
d , d = 1, . . . , D, convergence tolerance
ε2 and θ = θ(k) fixed. Output: v(k+1)
d
, d = 1, . . . , D. (a) Apply algorithm (23) with seeds v(k)
d , d = 1, . . . , D, convergence tolerance
ε2 and θ = θ(k) fixed. Output: v(k+1)
d
, d = 1, . . . , D. (a) Apply algorithm (23) with seeds v(k)
d , d = 1, . . . , D, convergence tolerance
ε2 and θ = θ(k) fixed. Output: v(k+1)
d
, d = 1, . . . , D. d
ε2 and θ = θ(k) fixed. Output: v(k+1)
d
, d = 1, . . . , D. k (b) Applyalgorithm(28)withseedθ(k),convergencetoleranceε1 andv0d = v(k+1)
d
fixed, d = 1, . . . , D. Output: θ(k+1). (b) Applyalgorithm(28)withseedθ(k),convergencetoleranceε1 andv0d = v(k+1)
d
fixed, d = 1, . . . , D. Output: θ(k+1). (c) k ←k + 1. (b) Applyalgorithm(28)withseedθ(k),convergencetoleranceε1 andv0d = v(k+1)
d
fixed, d = 1, . . . , D. Output: θ(k+1). 3.3 Tuning parameter choice and information criterion In the technical descriptions of Sect. 3.2, we assumed that the tuning parameter λ
had been defined prior to model parameter estimation. In practice, it has to be found
empirically from the sample data. Note that this aspect is crucial for the effectiveness of
the proposed method. On the one hand, if λ is chosen too small, the ℓ2-PAML approach
cannot sufficiently stabilize model parameter estimates in the presence of covariate
rank-deficiency. On the other hand, if λ is chosen too large, the shrinkage induced
by penalization dominates the optimization problem and resulting model parameter
estimates are heavily biased. Finding an appropriate value for the tuning parameter is
oftendoneviagridsearch,ascanbeseenforinstanceinBergstraandBengio(2012)and
Chicco (2017). We define a sequence of candidate values {λq}Q
q=1, where λq > λq+1. For each candidate value λq model parameter estimation as demonstrated in Sect. 3.2
is performed. The results of model parameter estimation have to be evaluated by a 471 ℓ2-penalized approximate... suitable goodness-of-fit measure. For our application, we choose the non-corrected
Bayesian information criterion (BIC; Schwarz 1978). Alternative measures would be
the generalized cross-validation criterion (Craven and Wahba 1979) or the Akaike
information criterion (Akaike 1974). For given candidate value λq, let ˆβ(λq) and
ˆφ(λq) be the estimators of β and φ, respectively. Further, let ˆvd(λq) be the mode
predictor of vd. The Laplace non-corrected BIC is given by BIC(λq) = p log(D) −2lapp(λq),
(29) (29) where the second term is the Laplace approximation (19) to the log-likelihood, that is lapp(λq) = lapp
ˆβ(λq), ˆφ(λq), ˆv1(λq), . . . , ˆvD(λq)
=
D
d=1
log
nd
yd
+
D
d=1
−1
2 log ˆξd(λq) −ˆvd(λq)2
2
+
yd(xd ˆβ(λq) + ˆφ(λq)ˆvd(λq))
−nd log
1 + exp{xd ˆβ(λq) + ˆφ(λq)ˆvd(λq)}
, where ˆξd(λq) = 1 + ˆφ(λq)2nd ˆpd(λq)(1 −ˆpd(λq)),
ˆpd(λq) =
exp
xd ˆβ(λq) + ˆφ(λq)ˆvd(λq)
1 + exp
xd ˆβ(λq) + ˆφ(λq)ˆvd(λq)
. For all λ1, ..., λQ, the following algorithm is performed: For all λ1, ..., λQ, the following algorithm is performed: 1. Apply the ℓ2-PAML algorithm to obtain ˆθ(λq) and ˆv1(λq), ..., ˆvD(λq). 2. Calculate ˆpd(λq) and ˆξd(λq), d = 1, ..., D. 1. Apply the ℓ2-PAML algorithm to obtain ˆθ(λq) and ˆv1(λq), ..., ˆvD(λq). 1. Apply the ℓ2-PAML algorithm to obtain ˆθ(λq) and ˆv1(λq), ..., ˆvD(λq). 2. Calculate ˆpd(λq) and ˆξd(λq), d = 1, ..., D. 3. Calculate BIC(λq) according to (29). 3.3 Tuning parameter choice and information criterion q
q
3. Calculate BIC(λq) according to (29). After the algorithm is finalized, the optimal tuning parameter λopt can be defined as
the candidate value that minimizes the BIC. After the algorithm is finalized, the optimal tuning parameter λopt can be defined as
the candidate value that minimizes the BIC. However, due to the non-convexity of the underlying optimization problem for ℓ2-
PAML estimation, the behavior of the BIC along the tuning parameter sequence can be
volatile to the extent that it may be characterized by multiple local minima. Therefore,
we further apply cubic spline smoothing by defining BIC(λ) = f (λ) + ϵq, where
f (λ) is a twice differentiable function and ϵq ∼N(0, ψ). The cubic spline estimate
ˆf of the function f is obtained from solving the optimization problem min
f ∈F
Q
q=1
BIC(λq) −f (λq)
2 + δ
f ′′(λ)2 dλ,
(30) (30) 123 472 J. Krause et al. J. Krause et al. where F = { f : f is twice differentiable} denotes the class of twice differentiable
functions and δ > 0 is a smoothing parameter. After ˆf has been obtained, the optimal
tuning parameter value λopt is defined as the minimizer of the smoothed function, that
is is λopt = argmin
λ∈{λq}Q
q=1
ˆf (λ). (31) (31) 4.1 Setup Hereafter, the performance of the ℓ2-PAML approach is evaluated under controlled
conditions. For this, we conduct a Monte Carlo simulation study with K = 500
iterations that are indexed by k = 1, ..., K. We generate synthetic data according to yd ∼Bin(nd, pd), pd =
exp
β0 + xdβ1 + φvd
1 + exp
β0 + xdβ1 + φvd
,
β0 = −0.2, β1 = 0.315,
d = 1, ..., D, where nd = 100, 15 as column vector of five ones, and φ = 0.4. The random effect vd
is drawn from a standard normal, as defined in Sect. 2.1. For the covariate vector xd, we
consider four different settings {A, B, C, D} with respect to the dependency between
the auxiliary variables. This is done in order to test the methodology under different
covariate correlation situations. In the A-setting, we have orthogonal covariates that
are generated according to xrd ∼U(0.7, 1.2), r = 1, ..., 5. For the remaining three
settings, we choose x1d ∼U(0.7, 1.2),
xrd = α(zd + ρx1d),
zd ∼U(0, 0.2),
r = 2, ..., 5, where ρ is a parameter controlling the dependency between x1d and xrd, and α is
a parameter harmonizing the variance of the random variables over settings. In the
B-setting, there is medium correlation with 20-50% on a percentage scale for the
product-moment correlation coefficient. For the C-setting, we have correlation with
about 50-75%. And in the D-setting, we have a strong correlation with 80-90%. Note
that the latter mimics situations of quasi rank-deficiency, which are of special interest. In addition to covariate correlation, we let the total number of areas D vary over
scenarios in order to evaluate the method under different degrees of freedom. Overall,
we consider 8 simulation scenarios: A.1 : D = 50,
A.2 : D = 100,
B.1 : D = 50, ρ = 0.3, α = 2.0, B.2 : D = 100, ρ = 0.3, α = 2.0,
C.1 : D = 50, ρ = 0.9, α = 1.5, C.2 : D = 100, ρ = 0.9, α = 1.5,
D.1 : D = 50, ρ = 1.5, α = 0.7, D.2 : D = 100, ρ = 1.5, α = 0.7. 4.2 Model parameter estimation results The target of this subsection is to study the fitting behavior of the ℓ2-PAML algorithm. Define θ := (β0, β′
1, φ). For a given estimator ˆθr ∈ˆθ of the model parameter θr,
r = 1, ..., p + 1, we consider the following performance measures: Bias( ˆθr) = 1
K
K
k=1
ˆθ(k)
r
−θr
, MSE( ˆθr) = 1
K
K
k=1
ˆθ(k)
r
−θr
2
,
(32) (32) where θ(k)
r
is the value that ˆθr takes in the k-th iteration of the simulation and θr
denotes the true value. As θr = 0.3 for all components of β1, we average the perfor-
mance measures for the regression parameters. Table 1 contains the results for model
parameter estimation. We start with the regression parameters β = (β0, β′
1)′. It can be seen that the
ℓ2-PAML algorithm obtains more efficient estimates than the ML-Laplace approach. Its MSE is significantly smaller in all considered scenarios. The largest efficiency
gains are obtained in the D-scenarios, which include strong covariate correlation. This could be expected from theory, as the ℓ2-penalty was introduced by Hoerl and
Kennard (1970) in order to improve the fitting behavior in these settings. However,
we also see that under orthogonal covariates (A-scenarios), the ℓ2-PAML algorithm
still outperforms the ML-Laplace approach. This is because approximate likelihood
inference introduces additional uncertainty to model parameter estimation. Here, the
ℓ2-penalty stabilizes the shape of the objective function, which allows for efficiency
gains even without covariate correlation. Yet, the increased efficiency comes at the cost
of an increased bias. The slope parameters β1, which are penalized while applying the
ℓ2-PAML algorithm, are estimated with larger bias relative to the ML-Laplace method. Please note that this is in line with theory. Hoerl and Kennard (1970) showed that the
ℓ2-penalty affects the bias-variance trade-off the researcher typically encounters in
ML estimation. It increases the bias in order to reduce the variance, which ultimately
allows for a smaller MSE when the regularization parameter λ is chosen appropriately. This is also becomes evident when looking at the distribution of regression param-
eter estimates. Figure 1 shows boxplots of the absolute deviation | ˆβr −βr|, βr ∈β1,
over all Monte Carlo iterations and for different simulation scenarios. 4.1 Setup A.1 : D = 50,
A.2 : D = 100,
B.1 : D = 50, ρ = 0.3, α = 2.0, B.2 : D = 100, ρ = 0.3, α = 2.0,
C.1 : D = 50, ρ = 0.9, α = 1.5, C.2 : D = 100, ρ = 0.9, α = 1.5,
D.1 : D = 50, ρ = 1.5, α = 0.7, D.2 : D = 100, ρ = 1.5, α = 0.7. 12 3 473 ℓ2-penalized approximate... The objective is to estimate the domain proportion pd, d = 1, ..., D. We compare
two model parameter estimation approaches for the logit mixed model described
in Sect. 2.1: a non-penalized approach that is obtained from maximizing lapp
(Laplace-ML), and the ℓ2-penalized approach through maximizing l pen (ℓ2-PAML),
as described in Sect. 3. We evaluate the simulation outcomes with respect to three
aspects: (i) model parameter estimation, (ii) domain proportion prediction, and (iii)
MSE estimation based on the parametric bootstrap in Sect. 2.3. The results are sum-
marized in the following subsections. 4.2 Model parameter estimation results In each quarter,
the distribution yielded by the ℓ2-PAML algorithm is displayed on the left, while the
the one obtained by the ML-Laplace algorithm is located on the right. We see that the 123 123 474 J. Krause et al. Table 1 Model Parameter Estimation Results
Scen
Method
Bias(β0)
MSE(β0)
Bias(β1)
MSE(β1)
Bias(φ)
MSE(φ)
A.1
ML-Laplace
−0.2760
2.3958
0.0059
0.4972
0.1691
0.0344
A.1
ℓ2-PAML
0.6752
1.4878
−0.1889
0.1908
0.1832
0.0416
A.2
ML-Laplace
−0.3861
1.1191
0.0317
0.2364
0.1870
0.0381
A.2
ℓ2-PAML
0.5021
0.9434
−0.1522
0.1215
0.1985
0.0430
B.1
ML-Laplace
−0.3128
0.6994
0.0132
0.9795
0.1699
0.0345
B.1
ℓ2-PAML
0.2271
0.5077
−0.1197
0.3004
0.1819
0.0406
B.2
ML-Laplace
−0.3086
0.3347
0.0136
0.4284
0.1898
0.0390
B.2
ℓ2-PAML
0.1501
0.2963
−0.1007
0.1534
0.2014
0.0441
C.1
ML-Laplace
−0.3380
0.8412
0.0060
3.1709
0.1621
0.0325
C.1
ℓ2-PAML
0.4065
0.8252
−0.0979
0.7336
0.1806
0.0409
C.2
ML-Laplace
−0.3096
0.3974
0.0087
1.7727
0.1813
0.0359
C.2
ℓ2-PAML
0.2329
0.3875
−0.0674
0.3062
0.1931
0.0413
D.1
ML-Laplace
−0.3345
0.7745
0.0186
11.7489
0.1641
0.0328
D.1
ℓ2-PAML
0.2697
0.6943
−0.0939
3.1089
0.1852
0.0416
D.2
ML-Laplace
−0.2907
0.3461
0.0128
5.6289
0.1868
0.0380
D.2
ℓ2-PAML
0.1016
0.3210
−0.0615
1.7118
0.2021
0.0448 12 ℓ2-penalized approximate... 475 l2-PAML
ML-Laplace
0.0
1.0
2.0
Scenario A.1
Absolute deviation
0
1
2
3
Scenario B.1
Absolute deviation
0
2
4
6
8
Scenario C.1
Absolute deviation
0
5
10
15
Scenario D.1
Absolute deviation
l2-PAML
ML-Laplace
l2-PAML
ML-Laplace
l2-PAML
ML-Laplace
Fig. 1 Absolute deviation of regression parameter estimates l2-PAML
ML-Laplace
0.0
1.0
2.0
Scenario A.1
Absolute deviation 0
1
2
3
Scenario B.1
Absolute deviation
l2-PAML
ML-Laplace Absolute deviation 0
5
10
15
Scenario D.1
Absolute deviation
l2-PAML
ML-Laplace
stimates 0
2
4
6
8
Scenario C.1
Absolute deviation
l2-PAML
ML-Laplace Scenario D.1 Fig. 1 Absolute deviation of regression parameter estimates boxes and whiskers of the ℓ2-PAML algorithm are much shorter than those of the ML-
Laplace method. This implies that the deviations from the true value are much smaller
under penalization for the vast majority of cases. Accordingly, the fitting behavior is
overall stabilized. Concerning φ, the results are different. The standard deviation parameter estima-
tion is not influenced by the covariate correlation. An intuitive explanation for this
phenomenon is that p0d is not affected by the collinearity of xd, and that the diagonal
element H0p+1p+1 of the Hessian matrix depends on xd only through p0d. 4.3 Domain proportion prediction results The target of this subsection is to investigate the behavior of the EBP of pd, d =
1, ..., D. We consider absolute bias, MSE, relative absolute bias, and relative root
mean squared error as performance measures. For a domain proportion prediction in
the k-th iteration of the simulation study, define ¯pd = 1
K
K
k=1
p(k)
d , RBd =
K
k=1 | ˆp(k)
d
−p(k)
d |
K| ¯pd|
, ¯pd = 1
K
K
k=1
p(k)
d , RBd =
K
k=1 | ˆp(k)
d
−p(k)
d |
K| ¯pd|
,
REd =
1
K
K
k=1( ˆp(k)
d
−p(k)
d )2
| ¯pd|
, d = 1, . . . , D. REd =
1
K
K
k=1( ˆp(k)
d
−p(k)
d )2
| ¯pd|
, d = 1, . . . , D. Further, let Further, let Bd = 1
K
K
k=1
| ˆp(k)
d
−p(k)
d |, Ed = 1
K
K
k=1
( ˆp(k)
d
−p(k)
d )2, d = 1, . . . , D. The performance measures are then given by The performance measures are then given by ABias = 1
D
D
d=1
Bd, RABias = 1
D
D
d=1
RBd,
MSE = 1
D
D
d=1
Ed, RRMSE = 1
D
D
d=1
REd. The results obtained from the simulation study are summarized in Table 2. We observe
that ℓ2-PAML improves domain total prediction performance in terms of all consid-
ered performance measures and for all implemented simulation scenarios, including
those without covariate correlation. This is in line with the simulation results for
model parameter estimation from the last subsection. The ℓ2-penalty stabilizes the
estimation performance even for orthogonal covariates due to the necessary Laplace
approximation. However, the strongest efficiency gains in terms of the MSE relative
to the ML-Laplace algorithm are obtained in the C- and D-scenarios, where we have
strong covariate correlation. Against the backhground of Hoerl and Kennard (1970),
this could be expected from theory, as ℓ2-penalization is known to be particularly
useful in the presence of (quasi-)multicollinearity. Overall, we can conclude that the
ℓ2-PAML algorithm not only improves model parameter estimation, but also domain
total prediction in any setting. 4.2 Model parameter estimation results This is why,
we expect that the asymptotic behavior of the ML-Laplace and ℓ2-PAML estimators
of φ will be not (or almost not) affected by the covariate correlation. Concerning the comparison of the two fitting algorithms, the ℓ2-PAML approach
increases the efficiency of regression parameter estimation. On the other hand, the
efficiency of standard deviation parameter estimation is impaired relative to the ML-
Laplace approach. In general, both methods overestimate the true value. This is likely
due the involved Laplace approximation in both algorithms. It is known to induce
bias to model parameter estimation, as for instance addressed by Jiang (2007), p. 131. However, the bias for the ℓ2-PAML algorithm is larger, as it implements additional
shrinkage of the regression parameters through the ℓ2-penalty. The regression parame-
ter estimates are drawn to zero (to some extent), which causes a larger proportion of the
target variable’s variance to be attributed to the random effect. This leads to a stronger
overestimation of the random effect standard deviation. Nevertheless, we will see in
the next subsection that the efficiency advantage in regression parameter estimation
overcompensates the loss in standard devation parameter estimation accuracy. 123 476 J. Krause et al. 4.4 Mean squared error estimation results Krause et al. Table 3 Mean Squared Error
Estimation Results
Scen
MSE
mse
RBias
RRMSE
A.1
0.001735
0.001687
−0.027801
0.097148
A.2
0.001729
0.001676
−0.030432
0.089698
B.1
0.001879
0.001851
−0.014858
0.090956
B.2
0.001851
0.001842
−0.004771
0.085181
C.1
0.001300
0.001275
−0.019039
0.171667
C.2
0.001333
0.001276
−0.042769
0.168694
D.1
0.001605
0.001613
0.004951
0.124678
D.2
0.001640
0.001610
−0.018371
0.119636 with respect to practice, a RRMSE ranging from 8.5% to 17.2% is a solid result for
uncertainty estimation. with respect to practice, a RRMSE ranging from 8.5% to 17.2% is a solid result for
uncertainty estimation. with respect to practice, a RRMSE ranging from 8.5% to 17.2% is a solid result for
uncertainty estimation. 4.4 Mean squared error estimation results The target of this subsection is to study the performance of the parametric bootstrap
for MSE estimation. We employ B = 500 bootstrap replicates in order to approximate 123 477 ℓ2-penalized approximate... Table 2 Domain Proportion Prediction Results
Scen
Method
ABias
MSE
RABias
RRMSE
A.1
ML-Laplace
0.033342
0.001781
0.047429
0.059960
A.1
ℓ2-PAML
0.032920
0.001735
0.046830
0.059188
A.2
ML-Laplace
0.033094
0.001749
0.047128
0.059465
A.2
ℓ2-PAML
0.032911
0.001729
0.046870
0.059132
B.1
ML-Laplace
0.034811
0.001922
0.053363
0.066946
B.1
ℓ2-PAML
0.034416
0.001879
0.052758
0.066201
B.2
ML-Laplace
0.034372
0.001872
0.052849
0.066259
B.2
ℓ2-PAML
0.034180
0.001851
0.052557
0.065877
C.1
ML-Laplace
0.028710
0.001344
0.036400
0.046199
C.1
ℓ2-PAML
0.028228
0.001300
0.035794
0.045434
C.2
ML-Laplace
0.028748
0.001355
0.036663
0.046597
C.2
ℓ2-PAML
0.028539
0.001333
0.036399
0.046227
D.1
ML-Laplace
0.032102
0.001652
0.044746
0.056371
D.1
ℓ2-PAML
0.031651
0.001605
0.044116
0.055569
D.2
ML-Laplace
0.032197
0.001663
0.045118
0.056794
D.2
ℓ2-PAML
0.031972
0.001640
0.044802
0.056401 the prediction uncertainty under the model. For a MSE estimate in the k-th iteration
of the simulation study, define MSEd = 1
K
K
k=1
( ˆY (k)
d
−Y (k)
d )2,
msed = 1
K
K
k=1
mse( ˆY (k)
d ),
mse = 1
D
D
d=1
msed, where ˆY (k)
d
and mse( ˆY (k)
d ) are the EBP of Yd and its bootstrap MSE estimator (see
Sect. 2.3), respectively. We consider the following performance measures where ˆY (k)
d
and mse( ˆY (k)
d ) are the EBP of Yd and its bootstrap MSE estimator (see
Sect. 2.3), respectively. We consider the following performance measures RBias = 1
D
D
d=1
msed −MSEd
MSEd
,
RRMSE = 1
D
d=1
1
D
D
d=1(msed −MSEd)2
MSEd Table 3 summarizes the simulation results. We see that the parametric bootstrap estima-
tor shows a decent performance overall. There is a slight tendency for underestimation. However, with a relative bias of less then 4.3% for approximate likelihood inference
with a generalized linear mixed model, this is negligible. With regards to the RRMSE,
we see that the parametric bootstrap is more efficient under orthogonal covariates (A-
scenarios) and medium correlation (B-scenarios). In the C- and D-scenarios, which
employ stronger covariate correlation, the RRMSE becomes larger. This is in line with
the results of Sect. 4.2. In these scenarios, the model parameter estimates are subject
to larger variation, which affects the bootstrap due to its parametric construction. Yet, 3 478 J. 5.1 Data description and model specification In what follows, we apply the ℓ2-PAML approach to estimate the regional prevalence
of multiple sclerosis in Germany. For this, we consider the German population of
the year 2017. It is segmented into 401 administrative districts and contains about
82 million individuals. The districts correspond to the domains in accordance with
Sect. 2.1. The required demographic information is retrieved from the German Federal
Statistical Office and based on the methodological standards described in Statistisches
Bundesamt (2016). As model response y, we define a binary variable with realizations yid =
1 person has multiple sclerosis
0 else yid =
1 person has multiple sclerosis
0 else for some i ∈Ud. The objective is to estimate pd = Yd/Nd with Yd =
i∈Ud yid
for all German districts. In order to define whether a person has multiple sclerosis,
we rely on an intersectoral disease profile provided by the Scientific Institute Institute
of the AOK (WIdO). It is based on multiple aspects, including medical descriptions,
inpatient diagnoses, and ambulatory diagnoses. The necessary sample counts for y are
based on health insurance records provided by the AOK. In particular, we use district-
level prevalence figures of the AOK insurance population in 2017 that are based on the
intersectoral disease profiles. The AOK insurance population is the biggest statutory
health insurance population of the country with roughly 26 million individuals in
2017 (AOK Bundesverband 2018). Note that the German health insurance system has
a rather unique separation between statutory and private health insurance. Usually, this
has to be accounted for in order to produce reliable prevalence estimates. However,
Burgard et al. (2019) showed that model-based inference using covariate data with
sufficient explanatory power can overcome this issue. 479 ℓ2-penalized approximate... As auxiliary data source, we use district-level inpatient diagnosis frequencies of
the German DRG-Statistics that are provided by the German Federal Statistical Office
(Statistisches Bundesamt 2017). The data set contains figures on how often a given
disease has been recorded in hospitals within a year. Both main and secondary diag-
noses are considered. With respect to diagnosis grouping, the records are provided on
the ICD-3 level (World Health Organization 2018). Note that the DRG-Statistics are a
full census of all German hospitals. Thus, the corresponding records cover the entire
population, as required for the model derivation in Sect. 2.1. 5.1 Data description and model specification However, a drawback
of the data set’s richness is that we have to choose a suitable set of predictors x out
of approximately 3000 potential covariates. Naturally, it is not feasible to apply an
exhaustive stepwise strategy that is often used in the context of variable selection, as
for instance demonstrated by Yamashita et al. (2007). Instead, we apply a heuristic strategy based on the premise that the objective is
to find a covariate subset with sufficient explanatory power for our purpose. First,
we isolate the 20 variables of the DRG-Statistics that have the strongest correlation
with the AOK records on G35, which is multiple sclerosis on the ICD-3 level. The
variables are arranged in decreasing order with respect to their correlation. Next, we
use the ℓ2-PAML algorithm from Sect. 3.2 to perform model parameter estimation for
p covariates, where p ∈{2, 3, ..., 20}. That is to say, we start with the 2 covariates
that have the strongest correlation to G35, and then sequentially increase the number
of predictors up to 20. For every result of model parameter estimation, we calculate
the Laplace non-corrected BIC in (29). Then, we select the covariate subset that corre-
sponds to the model fit which minimizes the BIC. The BIC curve over all considered
covariate set cardinalities is displayed in Fig. 2. We see that the curve has an odd
evolution over the covariate sets. This can be attributed to three reasons. Firstly, due
to the non-linearity of the link function, the covariate sorting is guaranteed to orga-
nize the covariates in descending order with regards to their relevance for the target
variable. Secondly, due to the strong correlation between them, the covariate contri-
butions interfer with each other. That is to say, when including an additional covariate
into the active set, the contributions of the previously contained covariates can change
considerably. And finally, as already addressed in Sect. 3.3, the non-convexity of the
optimization problem further leads to irregularities in the BIC curve. Despite these issues, the BIC curve has a clear minimum that is located at p = 9. Therefore, we isolate the 9 DRG-Statistics variables that have the strongest correlation
with the AOK records on G35. Thereafter, we perform a parametric bootstrap to esti-
mate the standard deviation of each model parameter estimate ˆθ j ∈ˆθ, j = 1, ..., p+1,
to evaluate its significance in terms of the p-value. 5.1 Data description and model specification The parametric bootstrap is
described as follows: 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ
′, ˆφ). 1. Fit the model to the sample and calculate the estimator ˆθ = ( ˆβ
′, ˆφ). 2. Repeat B times with b = 1, ..., B: 2. Repeat B times with b = 1, ..., B: 2. Repeat B times with b = 1, ..., B: (a) Generate v(b)
d
∼N(0, 1), y(b)
d
∼Bin(nd, p(b)
d ), d = 1, . . . , D, where p(b)
d
=
exp
xd ˆβ+ ˆφv(b)
d
1+exp
xd ˆβ+ ˆφv(b)
d
. (b) For each bootstrap sample, calculate the estimator ˆθ
(b). 123 123 J. Krause et al. 480 5
10
15
20
979780
979790
979800
979810
Number of Predictors
Bayesian Information Criterion
Fig. 2 BIC over covariate set cardinalities
3. Output: sd( ˆθ j) =
1
B
B
b=1
ˆθ(b)
j
−1
B
B
k=1 ˆθ(k)
j
2, j = 1, ..., p + 1. Based on the estimated standard deviations, we calculate test statistics for a sequence
of t-tests under the null hypothesis H0 : θ j = 0, j = 1, ..., p + 1. For a given
θ j ∈θ, the test statistic is given by t j = ˆθ j/sd( ˆθ j) and follows a standard normal
distribution. The test statistic values are located in the pdf of the standard normal to
obtain their respective p-values. We delete every predictor that corresponds to a model
parameter that is not relevant on at least a 10% significance level. The entire procedure
is summarized hereafter: 5
10
15
20
979780
979790
979800
979810
N
b
f P
di t
Bayesian Information Criterion Number of Predictors Fig. 2 BIC over covariate set cardinalities 3. Output: sd( ˆθ j) =
1
B
B
b=1
ˆθ(b)
j
−1
B
B
k=1 ˆθ(k)
j
2, j = 1, ..., p + 1. Based on the estimated standard deviations, we calculate test statistics for a sequence
of t-tests under the null hypothesis H0 : θ j = 0, j = 1, ..., p + 1. For a given
θ j ∈θ, the test statistic is given by t j = ˆθ j/sd( ˆθ j) and follows a standard normal
distribution. 123 5.1 Data description and model specification The test statistic values are located in the pdf of the standard normal to
obtain their respective p-values. We delete every predictor that corresponds to a model
parameter that is not relevant on at least a 10% significance level. The entire procedure
is summarized hereafter: Based on the estimated standard deviations, we calculate test statistics for a sequence
of t-tests under the null hypothesis H0 : θ j = 0, j = 1, ..., p + 1. For a given
θ j ∈θ, the test statistic is given by t j = ˆθ j/sd( ˆθ j) and follows a standard normal
distribution. The test statistic values are located in the pdf of the standard normal to
obtain their respective p-values. We delete every predictor that corresponds to a model
parameter that is not relevant on at least a 10% significance level. The entire procedure
is summarized hereafter: 1. Find the 20 covariates with the strongest correlation to y g
2. Perform model parameter estimation for p ∈{2, 3, ..., 20} predictors 3. Find the number of predictors that minimizes the BIC 4. For the BIC-minimal predictor set, perform a parametric bootstrap to estimat
standard deviations for the model parameter estimates 5. Perform t-tests to evaluate their significance and delete insignificant predictors The proposed strategy yields us the final covariate set x which consists of p = 5
predictors. The selected covariates are briefly characterized as follows: • X1: G43 (Migraine, secondary diagnosis) • X2: M20 (Acquired deformities of fingers and toes, main diagnosis) • X3: E66 (Overweight and obesity, main diagnosis) • X4: E04 (Other nontoxic goiter, main diagnosis) • X5: G35 (Multiple sclerosis, secondary diagnosis) Please note that the association of these variables with multiple sclerosis is the result
of district-level correlation. It does not directly imply person-level comorbidities in 123 481 ℓ2-penalized approximate... Table 4 Estimation results for final model specification
Parameter
Estimate
Std.Dev. p-value
95%-Conf.Int. β0
−5.79912
0.00640
0.00000
[−5.81167; −5.78657]
β1
−0.00092
0.00009
0.00000
[−0.00110; −0.00073]
β2
0.00026
0.00009
0.00380
[0.00009; 0.00043]
β3
−0.00030
0.00007
0.00001
[−0.00043; −0.00017]
β4
−0.00081
0.00006
0.00000
[−0.00093; −0.00068]
β5
0.00154
0.00009
0.00000
[0.00136; 0.00173]
φ
0.10939
0.00551
0.00000
[0.09860; 0.12019] a medical sense. Applying the ℓ2-PAML algorithm on the final covariate set yields
us the final model specification that we use for regional prevalence estimation. It is
summarized in Table 4. 5.2 Results Let us now investigate the results of prevalence estimation. The national prevalence
D
d=1 Yd/ D
d=1 Nd · 100% is estimated at 0.296%. Based on the parametric boot-
strap, we calculate a 95% confidence interval of [0.293%; 0.300%]. This implies that
the estimated total number of persons with multiple sclerosis ranges approximately
from 239000 to 246000, which is in line with reference figures on this topic. The Cen-
tral Research Institute of Ambulatory Health Care in Germany estimated that in 2017
about 240000 individuals had multiple sclerosis (Müller 2018). The regional distribu-
tion of prevalence estimates on the district-level is displayed in Fig. 3. We observe a
prevalence discrepancy between the western and eastern parts of Germany. The esti-
mated prevalence in western Germany are overall higher than in eastern Germany. Further, we observe regional clustering with higher prevalence in the central-northern
and central-southern parts of Germany. This is also consistent with reference studies. Similar patterns have been found by Central Research Institute of Ambulatory Health
Care in Germany (Müller 2018) and Petersen et al. (2014). Overall, the estimates are
plausible in both level and geographic distribution. Figure 3 shows the distributions of district-level prevalence estimates for the EBPs
under both ℓ2-PAML and the classical ML-Laplace method. The ML-Laplace results
are displayed in black, the ℓ2-PAML results are plotted in red. We see that the means
of the distributions are almost identical. However, the ℓ2-PAML distribution shows
considerably less variance than the ML-Laplace distribution. This is in line with both
theory and the simulation study, which both suggest stabilizing effects through ℓ2-
penalization. This is further evident when looking at the summarizing quantiles of both predictive
distributions. They are displayed in Table 5 . We see that the ℓ2-PAML estimates are
more more focussed around the mean and do not show as strong of outliers at the tails
of the distribution compared to ML-Laplace. Figure 5 displays the root mean squared error estimates rmse( ˆpd) =
mse( ˆpd)
for the prevalence estimates in Fig. 3, where mse( ˆpd) is obtained from the paramet-
ric bootstrap procedure described in Sect. 2.3. It becomes evident that there are no
obvious spatial patterns in the RMSE estimates. We neither observe a particular depen-
dency on the domain sizes nor on the prevalence estimates themselves. 5.1 Data description and model specification The confidence intervals for the parameters are calculated
according to ˆθ j ± t(D,1−α/2)sd( ˆθ j), j = 1, ..., p + 1, where t(·) is the corresponding
quantile of t-distribution with D degrees of freedom and significance level α. The
BIC value of the upper model specification is 979754 and therefore even better than
the optimal fit with p = 9 in Fig. 2. This suggests that the used model specification
was a reasonable choice given the considered data basis. Further, observe that the
estimated value for the standard deviation parameter φ is considerably larger than all
slope parameters β1, ..., β5. This implies that the random effects v1, ..., vD are clearly
evident in the empirical distribution of p1, ..., pD. Therefore, we can conclude that
using a mixed effect model in this context was a necessary choice. We further look at the internal correlation structure of the considered predictors in
order to assess the demand for ℓ2-penalization in the application. For this, we look
at the empirical correlation matrix for the five selected DRG-Statistics variables. It is
given as follows: ϱxx =
⎛
⎜⎜⎜⎜⎝
1.00 0.95 0.93 0.85 0.94
0.95 1.00 0.94 0.87 0.95
0.93 0.94 1.00 0.88 0.95
0.85 0.87 0.88 1.00 0.88
0.94 0.95 0.95 0.88 1.00
⎞
⎟⎟⎟⎟⎠ ϱxx =
⎛
⎜⎜⎜⎜⎝
1.00 0.95 0.93 0.85 0.94
0.95 1.00 0.94 0.87 0.95
0.93 0.94 1.00 0.88 0.95
0.85 0.87 0.88 1.00 0.88
0.94 0.95 0.95 0.88 1.00
⎞
⎟⎟⎟⎟⎠ We observe that (beside the main diagonal elements), the correlation values range
from 0.85 to 0.95, or 85% to 95% on a percentage scale. This suggests that the internal
correlation structure is very strong and comparable to the D-scenarios of our simulation
study. Therefore, we conclude that using ℓ2-penalization is reasonable in this context. However, note that some of this correlation is due to the size as a result of district-
level aggregation. Again, this does not directly resemble medical comorbidity on an
individual level. 123 482 J. Krause et al. Table 5 Quantiles of the EBP distributions
Method
Min
0.25
0.50
Mean
0.75
Max
ML-Laplace
0.171%
0.259%
0.297%
0.301%
0.336%
0.513%
ℓ2-PAML
0.209%
0.269%
0.295%
0.300%
0.324%
0.471% 5.2 Results However, with
respect to the overall level of RMSE estimates, we can conclude that our estimates
are more efficient than direct estimates ˆpdir
d
= yd/nd, d = 1, ..., D, that are exclu-
sively obtained from the health insurance records. Their standard deviation is given
by sd( ˆpdir
d ) =
ˆpdir
d (1 −ˆpdir
d ). 123 ℓ2-penalized approximate... 483 0.200% to 0.245%
0.245% to 0.290%
0.290% to 0.335%
0.335% to 0.370%
0.370% and higher 0.200% to 0.245%
0.245% to 0.290%
0.290% to 0.335%
0.335% to 0.370%
0.370% and higher Fig. 3 Results of prevalence estimation A one-to-one comparison of rmse( ˆpd) and sd( ˆpdir
d ) per domain is visualized in
Fig. 6. The ordinate measures sd( ˆpdir
d ) and the abscissa measures rmse( ˆpd). The red
line marks the bisector, which indicates equality between the two. We observe that
rmse( ˆpd) is always smaller than sd( ˆpdir
d ) by quite a margin. Thus, given the reason-
able performance of the parametric bootstrap for MSE estimation in the simulation, we
can conclude that our estimates mark an improvement over the direct estimates. There
is a slight positive relation between the two measures. That is to say, a relatively large
sd( ˆpdir
d ) is accompanied by a relatively large rmse( ˆpd) on expectation. However, the
trend is only vaguely visible. Finally, let us look at the distribution of random effect predictions over domains. They are visualized in Fig.7. The bars of the histogram correspond to the probability
density of the mode predictors in the respective interval of the support. The red line is 123 484 J. Krause et al. 0.001
0.002
0.003
0.004
0.005
0
200
400
600
800
1000
Distribution of predictions
N = 401 Bandwidth = 0.0001798
Density
ML−Laplace
l2−PAML
Fig. 4 Comparision of the EBPs Distribution of predictions 0.001
0.002
0.003
0.004
0.005
0
200
400
600
800
1000
Distribution of predictions
N = 401 Bandwidth = 0.0001798
Density
ML−Laplace
l2−PAML
Fig. 4 Comparision of the EBPs Fig. 4 Comparision of the EBPs the result of a kernel density estimation over their realized values. We observe that the
distribution is very close to normal. This is in line with the theoretical developments
from Sect. 2.1. Overall, it can be concluded that the ℓ2-PAML approach in the area-
level logit mixed model was a sensible choice for the considered application. the result of a kernel density estimation over their realized values. We observe that the
distribution is very close to normal. This is in line with the theoretical developments
from Sect. 2.1. Overall, it can be concluded that the ℓ2-PAML approach in the area-
level logit mixed model was a sensible choice for the considered application. 6 Conclusion Regional prevalence estimation is an important issue to monitor the health of the
population and for planning capacities of a health care system. A good covariate on
the prevalence of a disease can be typically obtained from medical treatment records
such as the DRG-Statistics in Germany. We proposed a new small area estimator for
regional prevalence that copes with two major issues in this context. First, typically
health surveys do not have a large sample and the sample size is mainly dedicated
to allow for the estimation of national figures. Within regional entities, therefore,
the sample size is very small. Applying classical design based or model assisted
estimators on these small sample sizes leads to very high standard errors for many
regions. Our small area estimator is capable of overcoming this issue by using a model
based approach. The second problem we tackle is, that the best covariates at hand,
typically have high correlations between each other. This leads to numerical problems
inhibiting the exploitation of these covariates. To overcome this problem we propose
to use a ℓ2-penalization approach. This leads to the need for revising the parameter
estimation procedure and to adapt it to the new requirements. We provide therefore ℓ2-penalized approximate... 485 0.005 to 0.010
0.010 to 0.015
0.015 to 0.020
0.020 to 0.025
0.025 and higher
Fig. 5 Results of RRMSE estimation Fig. 5 Results of RRMSE estimation a novel Laplace approximation to a logit mixed model with ℓ2 regularization. This
estimation procedure is applicable for other purposes such as classical logit mixed
model estimation with ℓ2-penalization. The prevalence estimation maps of Sect. 5 show some clusters of small areas with
high or low prevalence. This fact indicates that modeling spatial correlation by intro-
ducing, for example, simultaneous autoregressive random effects, might benefit the
final predictions. Combining this additional generalization with the robust penalized
approach is thus desirable. However, it is not an easy theoretical task and deserves
future independent research. In a Monte Carlo simulation study we show that the
proposed estimation approach ℓ2-PAML yields stable parameter estimates even under
strong correlations of the covariates. This simulation results underpin the theoreti-
cal arguments. Finally, we applied this newly derived estimator to the prediction of 123 486 J. Krause et al. 486
J. Krause et al. Declarations Conflict of interests The authors declare they have no competing interests. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence,
and indicate if changes were made. The images or other third party material in this article are included
in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons licence and your intended use is not permitted
by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 6 Conclusion 0.01
0.02
0.06
0.07
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.03
0.04
0.05
Standard Deviation, Direct Estimator
Root Mean Squared Error, l2-PAML
Fig. 6 Comparision of estimation uncertainty
Domain-Level Random Effect Realizations
Value
Density
-3
-2
-1
0
1
2
3
0.0
0.1
0.2
0.3
0.4
Fig. 7 Distribution of random effect mode predictions 0.01
0.02
0.06
0.07
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.03
0.04
0.05
Standard Deviation, Direct Estimator
Root Mean Squared Error, l2-PAML Fig. 6 Comparision of estimation uncertainty Domain-Level Random Effect Realizations
Value
Density
-3
-2
-1
0
1
2
3
0.0
0.1
0.2
0.3
0.4
Fig 7 Distribution of random effect mode predictions Domain-Level Random Effect Realizations
Value
Density
-3
-2
-1
0
1
2
3
0.0
0.1
0.2
0.3
0.4
Fig. 7 Distribution of random effect mode predictions Domain-Level Random Effect Realizations Domain-Level Random Effect Realizations Value
-3
-2
-1
0
1
2
3
0.0
Fig. 7 Distribution of random effect mode predictions
123 Fig. 7 Distribution of random effect mode predictions 123 123 123 ℓ2-penalized approximate... 487 district-level multiple sclerosis prevalence and obtained estimates with a considerably
low root mean squared error. Hence, we recommend using our new approach for the
regional prevalence estimation. district-level multiple sclerosis prevalence and obtained estimates with a considerably
low root mean squared error. Hence, we recommend using our new approach for the
regional prevalence estimation. Funding Open Access funding enabled and organized by Projekt DEAL. This research is supported by the
Spanish grant PGC2018-096840-B-I00, by the grant “Algorithmic Optimization (ALOP) - Graduate School
2126”fundedbytheGermanResearchFoundation,aswellastheresearchproject“Gesundheitsatlas”funded
by the Scientific Institute of the German Public Health Insurance Company. Data availability The demographic data as well as the DRG-Statistic data used in this study are available
on request from the German Federal Statistical Office. The health insurance records are property of the
German Public Health Insurance Company and subject to special privacy restrictions under the national
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l J International Journal for Crime, Justice and Social Democracy Document Version:
Publisher's PDF, also known as Version of record Abstract Emerging from critical conferences in the early 1970s involving academic researchers, community-based
workers and activists, critical social research challenged the role and legitimacy of mainstream social
sciences in their support of social orders fractured by class, ‘race’, sectarianism, gender, sexuality and age. This article opens with a brief reflection on the emergence and consolidation of critical social theory as the
foundation and context for research that challenges state-institutionalised power and authority. It draws
on long-term, in-depth primary research into the operational policies and practices of policing and
incarceration, exploring the profound challenges involved in bearing witness to the ‘pain of others’. Recounting personal testimonies ‘from below’, revealing institutionalised deceit and pursuing ‘truth
recovery’, it argues that dissenting voices are the foundation of hope, resistance and transformation. Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of
Research and Resistance Phil Scraton1
Queen’s University Belfast, United Kingdom Phil Scraton1 Phil Scraton1
Queen’s University Belfast, United Kingdom Queen’s University Belfast, United Kingdom Open Access
Thi
h Open Access
This research has been made openly available by Queen's academics and its Open Research team. We would love t
this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 IJCJ&SD 9(4) 2020 ISSN 2202-8005 Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of
Research and Resistance Introduction In his classic text Ways of Seeing, the artist John Berger encouraged wide public debate about how
selectivity afforded to images ‘establish our place in the surrounding world’. The photographer’s ‘choice
of subject’, the painter’s ‘marks … on the canvas or paper’ and the consequent ‘perception or appreciation
of an image’ are filtered by ‘our way of seeing’ (Berger 1972: 10). Apparently impulsive, the immediacy
and spontaneity of observation are informed by prior knowledge and understanding. Reflecting on her
experiences as a war journalist witnessing brutality and brutalisation, Susan Sontag (2003: 125–126)
observes that outsiders can neither ‘understand’ nor ‘imagine how dreadful, how terrifying and how
normal’ are the ‘ordeals of others’. She was driven by a determined resolve to reveal the reality of ‘faraway
suffering’, informing and encouraging interventions that might ‘do something to alleviate it’. Situating
herself in the presence of those ordeals, photographing and reporting the deprivations endured by
civilians caught in the crossfire, Sontag introduced an international audience to a ‘way of seeing’ that
otherwise would have remained invisible. While distinct in focus and content, Berger and Sontag illustrate
the significance of proximity, interpretation and representation in informing knowledge and
understanding. Seemingly unique, the ‘caught in the moment’ gaze of the individual, however, cannot be understood in
isolation from its broader social, political and economic contexts. An instant in time and place is framed by
the prevailing ideologies and their contestation. As Foucault (1980) demonstrates, through mechanisms,
techniques and procedures central to the maintenance and legitimacy of the state’s established order,
‘knowledge’ embedded in official discourse reflects and reproduces ‘regimes of truth’. In this context,
‘truth’ is manufactured, transmitted and reproduced. Acts and events are ascribed meaning, understanding
is inhibited, and agency is diminished. A key component of critical social research is its interrogation of
the processes through which legitimacy is ascribed to specific events thus elevating interpretation to fact. It enquires ‘beneath the surface of appearances’ to reveal ‘how social systems really work’ and ‘how
ideology or history conceal the processes which oppress and control people’ (Harvey 1990: 6). Revealing the mechanisms through which knowledge is ascribed legitimacy, critical social research
questions the premises on which public information is considered reliable, challenging the claims made
for ‘value-free’ ‘scientific’ analysis within social sciences. Further, it explores the processes through which
dissenting accounts emerge and their significance in the advancement of alternative discourses. Introduction Thus, it
‘rejects the premise that knowledge … is value-free or value-neutral’ but ‘derived and reproduced,
historically and contemporaneously, in the structural relations of inequality and oppression that underpin
established social orders’ (Scraton and Chadwick 2013: 107). This article focuses on distinct but related research conducted over four decades in the United Kingdom,
reflecting the significance of personal and collective testimonies in recovering truth otherwise lost in the
interminable processes, obstructive investigations and deceitful obfuscations of official procedures and
discourse. Gathering testimonies, derived in ‘being there’ and ‘bearing witness’, reflects Sontag’s mission
as an investigative journalist. It also establishes critical social research as fundamental to knowledge
acquisition and truth recovery. Taking Berger literally, what follows identifies and engages the ‘view from
below’ or, in the case of incarceration, the ‘view from inside’. Researching within and alongside complex
communities enduring social, political and economic marginalisation challenges the power dynamic and
academic conceit inherent in mainstream social sciences. Keywords State power; legitimacy; regimes of truth; structural inequality; critical social research; incarceration;
Ireland; Hillsborough. Please cite this article as:
Scraton P (2020) Fractured lives, dissenting voices, recovering ‘truth’: Frontiers of research and
resistance. International
Journal
for
Crime,
Justice
and
Social
Democracy
9(4):
1-15. https://doi.org/10.5204/ijcjsd.1686 Except where otherwise noted, content in this journal is licensed under a Creative Commons Attribution 4.0
International Licence. As an open access journal, articles are free to use with proper attribution. ISSN: 2202-
8005 © The Author(s) 2020 Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance www.crimejusticejournal.com Power, Legitimacy and State Authority For, the ‘sociological imagination’ was ‘not
a fashion or an aesthetic choice’ but a means through which ‘the struggles of oppressed people for equality,
self-determination and social justice’ could be advanced (Krisberg 1975: 19). Having previously focused on ‘how social actors define each other and their environments’, Becker (1974:
60) recognised the significance of structural ‘differentials in the power to define’ invested in ‘elites, ruling
classes, bosses, adults, men, Caucasians, superordinate groups generally’. They were discernible in
‘primitive forms of control’ and, more significantly, ‘by controlling how people define the world’. Davis
(1975: 205) identified inequality as essential to the success of advanced capitalism, evident and
reproduced in the complex intersections of ‘class, ethnicity, sex and political and economic’ relations. Those holding political and economic power set an agenda ‘detrimental to the interests and needs’ of the
powerless. This emerging body of work was neither homogenous nor uncontested, identifying as ‘new’,
‘radical’ or ‘critical’ criminology (Taylor, Walton and Young 1973, 1975). It affirmed the ‘diverse and
unique world of everyday life, the claimed location of interactionists, yet it adapted and contextualized
new deviancy theory’ prioritising ‘the dynamics of power and the processes which underpinned its
legitimacy’ (Scraton and Chadwick 1991: 165). Built on these foundations, critical analysis of the State’s definition and operational dynamics of
‘reasonable’ force (policing) and ‘humane’ containment (prisons) prioritises the processes through which
public order, social discipline and civil obedience are sustained. State authority is the concrete
manifestation of the relationship between power and legitimacy. All states, whatever their form, claim
authority for institutional powers to govern and regulate their subjects. In exercising institutional power,
legitimacy is secured and maintained through regulatory processes embodied in the administration of the
‘rule of law’—policing, courts, incarceration. State authority is absolute and determining, albeit mediated
by the extent to which distinct regimes accommodate participatory democracy. It holds the ‘monopoly of
the legitimate use of physical force within a given territory’ (Weber 2009: 77–78, emphasis in original),
institutionalised in the relationships between geographical boundaries, political autonomy and legal
jurisdiction. Poulantzas (1978: 81) argued that the State’s monopoly on the legitimate use of violence ‘underlies the
techniques of power and mechanisms of consent: it is inscribed in the web of disciplinary and ideological
devices; and even when not directly exercised, it shapes the materiality of the social body upon which
domination is brought to bear’. Power, Legitimacy and State Authority Identifying key moments in the emergence and consolidation of critical social theory and its application to
the ‘ordeals of others’ formed the underlying premise of the text Power, Conflict and Criminalisation
(Scraton 2007). It acknowledged the radical shift in social sciences that had challenged academic
orthodoxies, not least the contributions of critical interventionists in the USA compelled to face down the
fierce threat of McCarthyism. In this hostile climate within sociology, Wright Mills (1959: 20) was
determined to establish an alternative discourse to the ‘inhibitions, obscurities and trivialities’
underpinning the ‘bureaucratic techniques’ and ‘methodological pretensions’ useful to ‘the corporation, IJCJ&SD 2
2020 9(4) IJCJ&SD 2
2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance army and the state’. Functionalist orthodoxy, bereft of ‘sociological imagination’ and leaving political-
economic interests undeclared, served and serviced post-war regeneration reinforcing mainstream,
traditional hierarchical values as norms. These were values that sustained social stratification, gendered
families, racial superiority, homogenous sexuality, compliant youth and law-abiding citizens. army and the state’. Functionalist orthodoxy, bereft of ‘sociological imagination’ and leaving political-
economic interests undeclared, served and serviced post-war regeneration reinforcing mainstream,
traditional hierarchical values as norms. These were values that sustained social stratification, gendered
families, racial superiority, homogenous sexuality, compliant youth and law-abiding citizens. Within a decade, in a highly charged public address, Howard Becker (1967: 240) considered critical social
analysis was ‘caught in a crossfire’ of political values. State institutions and mega-corporations had
financed and incorporated sociological research to help deliver their exploitative agendas. Calling for
observational research into polarised social divisions, sufficiently immersed ‘to have a perspective’, Becker
(1967: 240) challenged the mantra of ‘value-freedom’, demanding that social scientists identify ‘whose
side we are on’. Alvin Gouldner, also rejected the proposition that social science research could claim
value-neutrality, reiterating the significance of structural relations evident in manifestations of power,
legitimacy and authority. As ‘new’ capitalism advanced, functionalist academic research had spawned a
generation of economic expansionists, social engineers and welfare technicians. Railing against social
research that patronised the ‘concrete and smaller worlds’ of everyday life with researchers cast as ‘zoo-
keepers of deviance’, Gouldner (1973: x) argued for ‘reflexive’ analysis locating social and cultural
relations in material history and the political-economic present. Power, Legitimacy and State Authority The threat of physical punishment, from incarceration through to solitary
confinement and state execution, is internalised through ‘mechanisms of fear’. For Hall (1985), the
conjuncture identified by Poulantzas as authoritarian statism more appropriately should be understood
as authoritarian populism, generated by the purposeful construction and manipulation of popular consent. In their highly influential work, Hall et al. (1978: 319) proposed that the underlying crisis in the UK www.crimejusticejournal.com IJCJ&SD 3
2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance economy had generated crises ‘in political legitimacy, in social authority, in hegemony and in class struggle
and resistance’. economy had generated crises ‘in political legitimacy, in social authority, in hegemony and in class struggle
and resistance’. What followed was a relentless, dogmatic commitment—in government policy supported by compliant
mainstream media—to regulate popular dissent, community-based movements and increasingly strong
trade union resistance. It represented a ‘qualitative shift in the balance and relations of force: the law, the
police, administrative regulation, public censure’ (Hall et al. 1978: 278). What had been progressive, in
retrospect modest, advances in welfare, education, social housing and workers’ rights were redefined
within a right-wing media as responsible for social upheaval, economic decline and the subversion of
democracy. Within a year of its 1979 election, the British Conservative Government introduced extensive
surveillance and regulatory mechanisms across social policy, welfare support and criminal justice
alongside trade union regulation. In 1969, the British Army had been deployed on the streets of Northern Ireland, ostensibly to quell civil
unrest and to work alongside the secret services and the Royal Ulster Constabulary. Direct rule by the
British Government was imposed in 1972 with the virtual suspension of civil rights. This included
internment without trial, implementation of emergency powers, progressing non-jury trials and
withdrawing ‘special category status’ from politically affiliated prisoners. Within the UK, ‘laws became
increasingly prohibitive and repressive, the police operated outside the checks and balances of political
accountability, sentencing hardened and prison conditions worsened’ (Scraton 2007: 228). This revealed
the authoritarian state’s reaction to criminalise ‘an unlimited reservoir of acts’ while characterising its
interventionism as ‘warfare’ (Christie 1994: 24)—a war on drugs, terrorism, unions and welfare claimants. This relentless shift to authoritarianism consolidated the New Right’s political agenda. Power, Legitimacy and State Authority Having presided over the deaths of ten prisoners on hunger strike in the North of Ireland and inner-city
uprisings in England’s Black communities, Prime Minister Margaret Thatcher projected her UK
Government’s ideological purpose: At one end of the spectrum are the terrorist gangs within our borders, and the terrorist
states which finance and arm them. At the other are the hard Left, operating inside our
system, conspiring to use union power and the apparatus of local government to break, defy
and subvert the law … now the mantle has fallen on us to conserve the very principle of
Parliamentary democracy and the rule of law itself. (Thatcher 1984) Legitimate trade union action and local authority opposition were placed on a spectrum alongside
terrorism. The die was cast. Structural constraints on individuals, families and communities marginalised by class, gender, sexuality,
‘race’ and age hardened under Thatcherism and, in the USA—its closest political-ideological ally—
Reaganism. These overarching determining contexts shackled potential, diminished hope and hobbled
agency. Having introduced draconian legislation, the UK Government used criminal justice agencies to
stifle dissent from within Black communities, trades unions, young people and politically affiliated
prisoners in the North of Ireland. It declared war on Argentina claiming ‘ownership’ of the Malvinas and
legitimacy for its intervention. As state authoritarianism consolidated its political and ideological mission,
critical social research confirmed its guiding principles: ‘bearing witness, gathering testimonies, sharing
experiences, garnering the view from below and exposing the politics and discourses of authoritarianism’
(Scraton 2007: 240). IJCJ&SD 4
2020 9(4) IJCJ&SD 4
2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance The ‘Healthy Prison’ Oxymoron In the early 1970s, I taught a Criminology class in the Victorian-built Walton Prison, now HMP Liverpool. Guards were consciously intimidating, some openly wearing National Front pins. The jail was rat-infested and
stank of urine. Without night access to toilets, prisoners, three to a cell, wrapped excreta in newspaper and
threw it from cell windows. Each morning, it was collected by prisoners on the ‘shit detail’. These were the
conditions endured by prisoners often locked down 23 in every 24 hours. Strangeways, now HMP Manchester,
was Walton’s Victorian sibling. MOBS KILL SEX INMATES AND BURN PRISON
11 DIE IN JAIL RIOT
(Daily Mirror) Unattributed allegations had been encouraged by inducements,
remaining uncontested by official negotiators inside the prison throughout. The failure to deny became
the substance of verification. Lord Justice Woolf was appointed to lead a comprehensive inquiry and make recommendations for
systemic change in prison regimes, management and staffing prioritising prisoner rehabilitation. Intended
to underpin root-and-branch reform throughout the prison estate, his report made 12 recommendations
and 204 proposals. They were not realised. Twenty-five years later, reflecting on the failure to liberalise
prison regimes, he condemned ‘competition between politicians’ to be ‘tough on crime … for that read
prisoners’ (The Guardian 1 April 2015). Throughout the post-Strangeways decades, reflecting the
profound authoritarian shift discussed earlier, successive Conservative and Labour Governments cast the
net of criminalisation ever-widely, imposing increasingly longer jail sentences. Most prisons functioned
not as places of reform and progression but as human warehouses—sites of regression. The inhumane
conditions that prevailed in Strangeways persisted: overcrowded and unhygienic cells, weak management,
aggressive regulation and minimal opportunity for work/education. What were the institutionalised consequences of this failure? In 2017, the Strangeways Independent
Monitoring Board supplied the answer: ‘decent, humane, safe accommodation in which prisoners might
find a degree of self-respect’ were unfeasible, given ‘the squalid, vermin-infested, damp environment more
reminiscent of Dickensian England’ (IMB 2017: 7).2 Prisoners were subjected to ‘vermin in the residential
accommodation’, a ‘plague of rats’ in the shower block, with bedding, clothing and belongings ‘soiled by
vermin’. Residential areas were ‘unhygienic, unhealthy, environmentally dangerous’ (IMB 2017: 18). The
inhumanity and neglect, against which the 1990 protesters had railed, prevailed. An inspection at another Victorian prison, HMP Liverpool, condemned its ‘abject failure … to offer a safe,
decent and purposeful environment’.3 Its regime was ‘unacceptably poor’, the prison ‘so dirty, infested and
hazardous to health’ it ‘could not be cleaned’. Inspectors ‘could not recall having seen worse living
conditions’ (HMCIP 2018: 5–6). Three decades beyond the Strangeways uprising and Woolf’s mild
reformism, conditions in Manchester and Liverpool prisons, among others, remain uninhabitable. Meaningful work and recreation are unavailable to most prisoners languishing for excessive periods in
locked cells, their physical and mental health compromised. The severe criticisms levelled at these regimes by monitors and inspectors are consistent with devastating
inspection assessments at Northern Ireland’s main male prison, Maghaberry. In 2015, persistent long-term
overcrowding in ‘unfit for purpose’ accommodation was condemned by the prison’s independent
monitors. MOBS KILL SEX INMATES AND BURN PRISON
11 DIE IN JAIL RIOT
(Daily Mirror) 20 DEAD
SEX OFFENDERS HANGED AND CASTRATED
(Manchester Evening News) On the morning of 2 April 1990, gruesome headlines dominated the front pages of UK tabloids. A hastily
constructed perimeter fence encircled the Strangeways prison as live radio and television confidently
reported the extremity of prisoners’ violence. They claimed a rampaging mob had destroyed a wing of the
insanitary and overcrowded jail, set a fire, raided prison hospital drugs cabinets and slaughtered fellow
prisoners. A small group occupied the prison’s roof, hurling tiles and masonry at officers below. As days
passed, the headlines continued: ‘Carnage in the Cages’; ‘Quake Britain’; ‘Tortured’; ‘Lynched in Jail
Horror’; ‘Prison Mob Hang Cop’; ‘Bodies cut up and dumped in sewer’. The reported execution of 11
became 20, then 30. As non-participant prisoners were dispersed to other prisons, further gruesome
details emerged. Sex offenders had been targeted, put before kangaroo courts, beaten, castrated and
executed, their bodies butchered. By day, negotiations faltered; by night, hovering helicopters blasted music to exhaust the protesters. As
the unprecedented occupation continued for 25 days, calls for military intervention mounted. Prisoners
shouted grievances from the rooftop, holding aloft their unequivocal message scrawled on a white sheet:
NO DEAD. Whatever the Home Office negotiators knew, they remained silent regarding casualties. It
transpired that while some prisoners jailed for sex offences had been assaulted, allegations of torture and
death were false. Gruesome events, graphically transmitted in media quotes from unidentified witnesses,
had not happened. There had been no executions, no prisoners hung from balconies, none butchered nor
castrated. In an instant, a myth had been created, and it remained uncorrected for several weeks. As the small dishevelled group retreated from their occupation, well aware they faced severe punishment,
the media fell silent. The ‘Strangeways Massacre’ had been the most remarkable story in contemporary
English penal history. Condemned by Prison Governor Brendan Friel as an ‘explosion of evil’, its promotion
had been engineered, a direct consequence of unofficial reports and off-the-record briefings by prison IJCJ&SD 5
2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance officials, guards and their association. Unattributed allegations had been encouraged by inducements,
remaining uncontested by official negotiators inside the prison throughout. The failure to deny became
the substance of verification. officials, guards and their association. MOBS KILL SEX INMATES AND BURN PRISON
11 DIE IN JAIL RIOT
(Daily Mirror) Most prisoners, particularly the vulnerable, were offered no purposeful activity. A landing
culture dominated by boredom, drug-taking, bullying and self-harm was the norm. Within a debilitating
atmosphere of depression, mental ill-health pervaded, treatment programs were non-existent, and ‘at-risk’
prisoners were poorly monitored. Maghaberry’s deficiencies were endemic. A full independent inspection followed (HMCIP/CJINI 2015). In three of four assessment categories—
safety, respect and dignity, purposeful activity (World Health Organisation 1999)—the prison was ranked
‘poor’. Its management, conditions and regime were trapped in a downward spiral endangering prisoners’
lives. The Chief Inspector condemned the prison as ‘one of the worst prisons [we] have ever seen and the
most dangerous … Dickens could write about Maghaberry without batting an eyelid’ (BBC News 5
November 2015). Unsafe and unstable, Northern Ireland’s main prison had descended into crisis. The severe condemnation of Manchester, Liverpool and Maghaberry prisons reflects institutional failures
in the operational policies and practices prevalent throughout most prisons: overcrowding, long periods
of lockdown, minimal work opportunities, drug use, bullying and hopelessness. These are the ingredients
of revolt. In the aftermath of Strangeways, rather than positive regimes emerging to stimulate hope and
recovery, punitive regimes of fear and degradation prevailed. It is a direction of travel consistent with the
USA. In analysing California’s ‘distinctive new form and function of the prison’, Simon (2007: 142–143) www.crimejusticejournal.com IJCJ&SD 6
2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance considers it ‘a space of pure custody, a human warehouse or even a kind of waste management facility’. Populist politicians relentlessly promote all prisoners as inherently ‘dangerous’, recasting harsh rhetoric
as a commitment to protecting ‘the wider community’. In this regressive climate, the ‘transformation of
the prisoner through penitence, discipline, intimidation, or therapy’ has been abandoned in favour of
incarcerating ‘people whose propensity for crime makes them appear an intolerable risk for society’. As
Simon (2007: 142–143) concludes, maintaining ‘order’ within the ‘toxic-waste-dump prison increasingly
relies on total segregation of the prisoners considered to be the most threatening’. It offers no correctional
pretence and no commitment to reform—only an inhumane reality of unconditional containment, social
isolation and persistent lockdown. What are the consequences of such institutionalised inhumanity? MOBS KILL SEX INMATES AND BURN PRISON
11 DIE IN JAIL RIOT
(Daily Mirror) As Sim (2009: 130–131) states, prisons
have an ‘extraordinary capacity for inflicting pain and inculcating fear, and if need be terror, into the lives
of the confined’. Through its inherent culture, incarceration generates ‘social harm’ derived in ‘systemic
discourses of punitive degradation’ further damaging the ‘lives of those whose sense of psychological and
material well-being’ has been ‘fractured by their pre- and post-prison experiences’ (Sim 2009: 130–131). Regarding penal expansionism’s populist manifesto, Law’s research with women prisoners in the USA
demonstrates that mass incarceration plays to a banal ‘tough on crime’ agenda. It has resulted in ‘the
criminalization of more activities, leading to higher rates of arrest, prosecution and incarceration while
shifting money and resources away from other public entities—education, housing, health care, drug
treatment and other societal supports’ (Law 2009: 169). Gilmore (2007: 13) questions how incarceration is ‘supposed to produce stability through controlling what
counts as crime’. She reflects on centuries ‘of experience into conflicting and generally overlapping
explanations for why societies decide they should lock people out by locking them in’ (2007: 13). Put
directly, ‘those locked up cannot make trouble outside’. Penal reality is a ‘mix of care, indifference,
compulsory training, and cruelty to people in cages’ (Gilmore 2007: 13). For most prisoners subjected to
this reality, ‘the acquisition of sobriety and skills’ and the adoption of ‘alternative lifestyles’ are
unrealisable. Liberal reformist claims for prisoner rehabilitation have little traction in retributive regimes
prioritising incapacitation. Rehabilitation is reduced to aspirational rhetoric, overwhelmed by a persistent
and growing wave of ‘penal populism … dehumanising criminals as dangerous and irredeemable outsiders’
giving ‘legitimacy to a whole array of symbolic and draconian practices’ (Di Giorgi 2013: 34). Additional to
dissuading the offender from committing further crimes, the severity of the prison sentence and the
inhumane conditions in which it is served are proposed as deterrents to others. Neither objective is
achieved. Thus, penal reformism is exposed as deceit; that via some magical process, conducted within a punitive
and destructive environment of institutionalised deprivation, prisoners emerge not only unscathed by
captivity but equipped to face a hostile world. Such groundless assumptions neglect the harm and
consequences inflicted on prisoners ‘in our name’. Prisons create and reproduce ‘populations of
incomplete and wounded lives … inside the prison or outside the prison’ (Quinney 2006: 270). MOBS KILL SEX INMATES AND BURN PRISON
11 DIE IN JAIL RIOT
(Daily Mirror) ‘Prisoner’
or ‘non-prisoner’, within the walls or in the community, ‘no-one escapes the damage caused by the fact
that prison exists’ (Quinney 2006: 270). Its deprivations and consequences are ‘pervasive’. Whether
‘economic, social, psychological’ or ‘spiritual’, the ‘injuries caused by the prison are shared by all’ (Quinney
2006: 270). Yet the pains of confinement are generated and sustained in ‘our’ name, financed through ‘our’
financial contributions. Given the emotional, social and political dislocation inflicted selectively on
individuals and communities, the full societal cost cannot be reduced to economics. It is ironic that in times of severe political-economic crisis, an increasingly popular liberal reformist
defence for incarceration is that it provides a safe harbour from destitution—poverty, homelessness,
alcohol and drugs. While prisons might provide marginal respite from harsh realities in communities, they
are places that reinforce impoverishment while encouraging prescribed and illicit drug dependency. Half
a century has passed since Goffman (1968: 24–25) observed that those entering prison from relatively
stable circumstances are ‘immediately stripped of support’ while subjected to ‘a series of abasements, IJCJ&SD 7
2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance degradations, humiliations and profanations of self’ inflicted ‘around the clock’. Relentless incarceration
generates a dislocated state of ‘permanent civil dispossession’, abrogating prisoners’ political ‘rights’. Through objectifying procedures imposed on arrival—‘photographing, weighing, fingerprinting, assigning
numbers, searching, listing personal possessions for storage, undressing, bathing, disinfecting, haircutting,
issuing institutional clothing, rules’—the prisoner is ‘shaped and coded into an object that can be fed into
the administrative machinery of the establishment’ (Goffman 1968: 25–26). Institutionalised objectification underpinned the Strangeways uprising, generating persistent volatility
against penal authoritarianism. Our research into the uprising and hostage-taking in Scotland’s Peterhead
Prison recorded prisoners’ testimonies revealing the parlous conditions they endured (Scraton, Sim and
Skidmore 1991). Multiple deprivations were exposed in a deficient regime within a remote jail built in the
1880s. The ‘totality of the institution’ was ‘underwritten by a degree of totality or absolutism in power
relations which virtually strip the prisoner of civil rights, personal consultation and democratic process’
while imposing an ‘unrelenting imposition of authority’ (Scraton, Sim and Skidmore 1991: 61–62). As
Mandela (1994: 340–341) concluded, prisons are ‘designed to break one’s spirit and destroy one’s resolve’. MOBS KILL SEX INMATES AND BURN PRISON
11 DIE IN JAIL RIOT
(Daily Mirror) Their regimes purposefully ‘exploit every weakness, demolish every initiative, negate all signs of
individuality—all with the idea of stamping out that spark that makes each of us human and each of us
who we are’. The ‘challenge for every prisoner’ is ‘how to survive prison intact, how to emerge from a
prisoner undiminished’. Distinctive in time and specific in circumstances, what unifies these accounts is the ‘view from inside’. In
applying the principles of critical analysis, bearing witness to the events and gathering testimonies from
those suffering the institutional deprivations and arbitrary punishments administered behind closed
doors, ‘truth’ is recovered. These testimonies, collected independently and over decades, provide a
consistent and informed counter to official versions propagandised relentlessly at stage-managed press
conferences and in evidence to official inquiries. Once headlined in public discourse, however, the
propaganda becomes virtually impossible to unravel and reverse. From War to Peace: Women’s Incarceration in Northern Ireland The 1998 Northern Ireland (Sentences) Act was central to the settlement and established the legislative
framework to release on licence prisoners affiliated to a paramilitary organisation. In 2003, the Human
Rights Commission initiated in-depth research into the conditions experienced by non-politically affiliated
women prisoners held in a high-security unit within Northern Ireland’s largest male jail (Scraton and
Moore 2005). As the initial report was published, women prisoners were transferred to a medium-security
unit within a male young offenders’ institution, and the research was extended (Scraton and Moore 2007). The research revealed a prison estate unfit for purpose. Incarcerating women in a male jail imposed severe
restrictions on their movement, association, work and educational opportunities. With no prison staff
recruited since the early 1990s and the early release of politically affiliated prisoners, there remained a
surplus of mid-career, predominantly male, Protestant prison managers and officers. Many had limited
experience of prisoner contact, few with women prisoners. A culture of non-engagement persisted; contact
was reactive rather than proactive in conditions where often distressed and vulnerable women—and
young men—were locked down for long periods without opportunities to participate in constructive
activities. Further, serious failures in oversight and monitoring were institutionalised. In-depth interviews revealed that the women’s lives in their communities often amounted to ‘virtual’
imprisonment. Many were locked into poverty, child-rearing, primary parenting and caring
responsibilities in neighbourhoods ravaged by illicit and prescription drugs. Over one-third of Northern
Ireland’s children live on or below the poverty line, affecting schooling, health and welfare. Held in a prison
regime designed, administered and prioritised to accommodate men reflected women’s experiences of
marginalisation within their communities. Negotiating the regime exacerbated the profound impact of
maintaining family contact, particularly with children, and the often-traumatising experiences of ‘family’
visits. On their release, women—particularly mothers—labelled ‘former prisoner’ were subjected to
stigma, humiliation and shame associated with their incarceration. Ciara was 16, a child in an adult jail. She self-harmed, and no part of her visible body was free from tears
or cuts. Her only clothing was a canvas pinafore dress—its Velcro fastening removed to prevent her
scouring her skin. Held in the prison hospital for nine days, she had been returned to a punishment and
segregation cell. It was bare except for a solid concrete plinth, a canvas blanket and a cardboard potty as a
toilet. From War to Peace: Women’s Incarceration in Northern Ireland In 2003, under its unique powers of entry, the Northern Ireland Human Rights Commission visited HMP
Maghaberry, widely considered one of the ‘most complex’ prisons in Europe due to its diverse population: men;
women; remand; short-term/medium-term/life sentence prisoners; asylum seekers; sex offenders; ‘political’
prisoners. Commissioners, profoundly concerned by the conditions and regime they experienced, initiated
research specifically focused on women prisoners. From 1969, for almost three decades, the six counties of Northern Ireland were at war, occupied by the
British military and secret intelligence services. During the Conflict, in a population of approximately 1.6
million, 3636 people died, including over 2000 civilians (McKittrick et al. 2004: 1477). Hillyard et al. (2003) note that 88,000 households lost a close relative; a further 50,000 households had a resident
injured. As a direct consequence of sectarian intimidation and threats, approximately 28,000 people lost
employment, and 54,000 families were relocated. Throughout the Conflict, the North was policed and
regulated by special powers and non-jury trials. In August 1971, the internment of ‘political’ activists
without trial was introduced and institutionalised; 2357 men and women were arrested and subjected to
harsh interrogation (McEvoy 2001). Boyle, Hadden and Hillyard (1975: 32) note that in imposing Direct
Rule powers, the UK Government established a ‘new system of arrest and detention’. Initially, those prisoners claiming ‘political’ affiliation were granted ‘special category status’. In 1976,
however, the British State reclassified politically affiliated prisoners as ‘ordinary’ criminals. In response,
Republican prisoners refused to wear prison clothes or conform to prison rules. Draped only in coarse
blankets, they were confined to strip cells. Following a series of beatings by guards, they embarked on a
‘dirty protest’, smearing excrement on cell walls. Hunger strikes followed, and 10 men died (McKeown
2001; Scraton 2020). IJCJ&SD 8
2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance The politics of incarceration throughout the Conflict were driven by the State’s steadfast commitment to
criminalising and containing politically affiliated prisoners. In April 1998, following four years of uneasy
peace negotiations, the UK and Irish Governments signed the Good Friday Agreement, setting the
constitutional foundation for devolution powers to a democratically elected Northern Ireland Assembly. From War to Peace: Women’s Incarceration in Northern Ireland Unsurprisingly, she had attempted to hang herself: I hear voices, [they] tell me to cut myself. And I release the pain as well. It’s terrible, so it is. You sleep and you keep changing positions ... Just look what they make me go to the toilet
in. That’s for night time … It’s a disgrace … They don’t give me underwear or nothing … just
a wee sanitary towel and that’s it … stuck in the cell with a camera ... no privacy or nothing. Your dignity’s taken away. They just said, ‘It’s your own fault you’re behind the door’. (Moore and Scraton 2014: 134–136) Such were the conditions that led to prisoners self-harming and taking their own lives. The research interviews and observation further exposed the fiction of penal reformism and the lip-service
paid to the ‘healthy prison’ agenda. Women prisoners were judged and treated according to their
perceived femininity and the tropes associated with female conformity. They were consistently
disrespected, not least when escorted within the prison, walking a gauntlet of shouted sexist abuse from
young men in the male blocks. Invariably, abuse focused on their bodies and appearance. With workshops
and education prioritised for young male prisoners, purposeful or creative activity for women prisoners
was minimal. Most suffered mental ill-health, often as a consequence of imprisonment within a deficient
regime. Ciara’s comments demonstrate how women’s vulnerability was heightened through punitive IJCJ&SD 9
2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance isolation. Further, the claims made about rehabilitation, resettlement and reintegration on release
amounted to cruel deceit. isolation. Further, the claims made about rehabilitation, resettlement and reintegration on release
amounted to cruel deceit. As McCulloch and Scraton (2009: 11) propose, ‘the violence of incarceration is historically, socially and
culturally imprinted on the foundations of the prison’. For Ciara and the women interviewed, the pain of
incarceration was a combination of mounting depression, mental ill-health and physical debilitation. The
research developed to include the incarceration of men and young people within the context and legacy of
the Conflict (Scraton 2015). Focusing on institutional and interpersonal power relations, it questioned the
political legitimacy afforded to carceral regimes, their management and operation. From War to Peace: Women’s Incarceration in Northern Ireland Limited educational
provisions, inadequate or insufficient opportunities for skills development, long periods of lockdown and
mental ill-health extended throughout the penal estate. The research findings were supported by reports
from the Independent Monitoring Boards and the Inspectorates. The cumulative evidence presented above challenges the social, political and ideological justifications
advanced for prisons as places of opportunity where individuals—many suffering from mental ill-health,
deep-seated addictions and inescapable poverty—can exit prison gates with hope and purpose. It exposes
the operational reality of life in jail as oppressive, contextualised by policies of containment, security and
institutional control rather than opportunity, safety and personal development. The abject failure of penal
reform contradicts claims for the legitimacy of incarceration, thus returning the focus to decarceration and
abolition. In seeking the eventual abolition of mass imprisonment, however, Davis (2003: 103–104) argues, the
challenge of campaigning for ‘more humane, habitable environments’ while not ‘bolstering the
permanence of the prison system’. Its resolution requires sociological imagination and political will to
create a ‘constellation of alternative strategies and institutions … revitalization of education at all levels’
alongside a ‘health system providing free physical and mental health care to all’ (Davis 2003: 103–104). On the road to abolition, reducing the prison population—decarceration—requires a societal commitment
to developing and funding a ‘justice system based on reparation and reconciliation rather than retribution
and vengeance’. Hillsborough: Justice Delayed is Justice Denied In the
Liverpool Daily Post (18 April 1989), John Williams was unequivocal: ‘gatecrashers’ had ‘wreaked their
fatal havoc’; ‘uncontrolled fanaticism and mass hysteria’ had ‘literally squeezed the life out of men, women
and children’. It was ‘yobbism at its most base’ as ‘Scouse killed Scouse’. The allegations that followed became increasingly sinister. Unnamed police officers claimed that while
rescuing the dying, they were assaulted by Liverpool fans who stole from the dead. The Star (18 April
1989) claimed that ‘Ticketless thugs staged crush to gain entry’. They had ‘attacked an ambulance man,
threatened firemen and punched and urinated on policemen as they gave the kiss of life to stricken victims’. The following day, the allegations were echoed by five national newspapers: ‘Dead Fans Robbed by Drunk
Fans’ (Daily Star); ‘They were drunk and violent and their actions were vile’ (Mail); ‘Police Accuse Drunken
Fans: Police saw “sick spectacle of pilfering from the dying”’ (Express); ‘Fury as police claim fans robbed
victims’ (Mirror); ‘Police tell MP of attacks on them as they helped injured’ (Telegraph). The Sun cleared its
front page: ‘THE TRUTH’: ‘Some fans picked pockets of victims; Some fans urinated on the brave cops;
Some fans beat up PC giving life kiss’. A ‘high-ranking police officer’ claimed that a dying young woman
had been sexually abused: ‘fans were just acting like animals’. In the public’s mind, the die was cast. Four months later, the Home Office Inquiry published an interim report concluding that overcrowding was
the ‘main cause’ of the disaster, the ‘main reason’ being a serious failure in police control (Taylor 1989). Contrary to earlier press coverage, the report berated the Match Commander and senior police officers for
their collective failure on the day, exacerbated by their evasiveness before the inquiry. Preliminary inquest
proceedings heard uncontested pathology findings on each of the deceased. The Coroner ordered that
blood alcohol levels of all who died, including children, should be recorded. His unprecedented decision
further fuelled the allegation that alcohol consumption had contributed to the deaths. In November 1991, following the Director of Public Prosecutions’ decision not to proceed with criminal
prosecutions, the full inquests were resumed. Remarkably, the Coroner imposed a 3.15 pm cut-off on
evidence. Without taking medical opinion or providing clinical evidence, he claimed that by that time all
who died would have suffered irreversible fatal injuries. It was a claim without substance. Hillsborough: Justice Delayed is Justice Denied On 15 April 1989, as an FA Cup semi-final kicked off at Sheffield’s Hillsborough Stadium, compression in a
tightly-packed crowd on standing terraces led to the death of 96, mostly young Liverpool soccer fans. Four
hundred were seriously injured, thousands traumatised. To relieve congestion outside, police officers had
opened an exit gate allowing over 2000 fans to enter an unfamiliar stadium. Descending a steep tunnel, they
emerged at the rear of already overcrowded pens—fences to the sides, to the front and no means of retreat. As people were dying, the Police Commander told officials that fans had forced entry. Broadcast
internationally, his lie blamed fans for the disaster. Further elaborated, the unsubstantiated police version
dominated the headlines: fans had arrived ‘late’, without tickets, many drunk, violent and abusive. These
unproven allegations deflected responsibility, then infected the investigations and inquests, the latter
concluding that the deaths were ‘accidental’. In this hostile climate, the Hillsborough Project was founded. Its
research provides a critical analysis of the causes, circumstances and aftermath. The day after the disaster, Prime Minister Margaret Thatcher, her Press Secretary Bernard Ingham and
Home Secretary Douglas Hurd arrived in Sheffield. Subsequently, Ingham wrote that the Chief Constable
told Thatcher, ‘there would have been no Hillsborough if a mob, who were clearly tanked up, had not tried
to force their way into the ground’ (Personal Correspondence, dated 13 July 1994). Within days, allegations
from senior police officers and local Conservative politicians dominated media coverage, and a public
inquiry was appointed, led by Lord Justice Taylor (Coleman et al. 1990; Scraton, Jemphrey and Coleman
1995). Accepting the veracity of police briefings, the press minimised accounts from survivors and
witnesses that painted a markedly different picture. IJCJ&SD 10
2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance The relentless condemnation of fans’ behaviour, vilified for causing death and physical injury, devastated
already traumatised survivors. On 17 April, the Star claimed the tragedy was the consequence of a ‘crazed
surge’. The Yorkshire Post reported ‘thousands of latecomers’ without tickets had caused a ‘fatal charge’. Writing in the Evening Standard (17 April 1989) Peter McKay condemned ‘the tribal passions of Liverpool
supporters’ who had ‘literally killed themselves and others to be at the game’. Jacques Georges, the UEFA
President, condemned ‘beasts waiting to charge into the arena’ (Liverpool Echo, 17 April 1989). Hillsborough: Justice Delayed is Justice Denied Further, in
consultation with police advisers, the Coroner selected witnesses, including local residents, publicans and
police officers, whose collective testimony affirmed that ticketless, drunk and aggressive fans had arrived
at the stadium determined to force entry. Five months later, following his selective summary of evidence
from 230 witnesses, he directed the jury to a majority ‘accidental death’ verdict. It devastated the families and survivors. A bereaved mother stated, ‘The inquests were a farce. The Coroner
clearly directed the jury … he got what he wanted’. Another mother stated: ‘… the jury could only arrive at
one verdict after the Coroner’s performance’ (Scraton 2016: 219). The reversal of the public inquiry
findings was welcomed by South Yorkshire senior police officers and the Chairman of the Police Federation
who addressed bereaved families, live on television, ‘What more do they want?’ Using an unfortunate
metaphor, another senior police officer involved proclaimed that it was ‘time to bury the past’ (Scraton,
Jemphrey and Coleman 1995: 261). Two years later, a High Court judicial review confirmed the Coroner’s direction as ‘impeccable’. In 1997,
an unprecedented judicial ‘scrutiny’ of ‘new’ evidence was initiated by the Labour Government. Despite
compelling evidence that police statements had been altered by a team appointed within the South
Yorkshire Police, it concluded that there was no justification for a further public inquiry (Stuart-Smith
1998). By this time, two detailed alternative reports had been published by the Hillsborough Project
(Coleman et al. 1990; Scraton, Jemphrey and Coleman 1995). Their findings were consolidated by Jimmy IJCJ&SD 11
2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Res Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance McGovern’s award-winning 1996 drama–documentary Hillsborough and a comprehensive analysis,
Hillsborough: The Truth (Scraton 1999, 2016). The analysis revealed the full extent and impact of altered
police statements, which had been ordered by the South Yorkshire Police and its solicitor. Despite
widespread publicity, this extensive body of research and analysis was ignored for a further decade. In
2009, the families’ persistent campaign led to the appointment of a unique independent panel of inquiry. Scraton drafted the application, headed its research and was the primary author of its comprehensive
report. The panel’s researchers analysed two million documents from 80 organisations and individuals. Hillsborough: Justice Delayed is Justice Denied Two years
later, it delivered a comprehensive report: its 12 chapters and 153 findings established a catalogue of
official failures (Hillsborough Independent Panel 2012). The research exonerated the fans. New criminal
investigations followed, alongside a review of policing by the Independent Police Complaints Commission
(now the Independent Office for Police Conduct). The Attorney-General quashed the inquest verdicts,
ordering new inquests. Following five preliminary hearings, supported by legal aid, full inquests ran for
two years, presided over by a High Court Judge. In 2016, the jury returned a verdict of unlawful killing. In
addition, it recorded egregious failings: 10 by the stadium owners, architects, safety engineers and
ambulance service; 15 by the South Yorkshire Police. The verdict exonerated the fans, overturning the
accidental death verdict so heavily influenced by the Coroner’s direction a quarter of a century earlier.4 Throughout two decades, senior South Yorkshire police officers, the Chief Constable and their lawyers, the
Police Federation, Members of Parliament and civilian witnesses closely associated with the police
purposefully and collectively attributed full responsibility for the disaster to fans, including survivors and
those who died. Their statements generated and sustained a persistent myth regarding fans’ culpability,
influencing the media, the Coroner, appeal court judges, the judicial scrutiny of ‘new’ evidence, government
ministers and senior politicians. The detailed analysis and substantial recommendations of the
Hillsborough Project’s 1990 and 1995 reports, precursors to the Independent Panel’s comprehensive
findings two decades later, were ignored. Finally, the 2016 verdict silenced the highly vocal and much-
publicised vilification directed against those who survived Hillsborough. However, those involved in the
construction and promulgation of the myth failed to accept responsibility or apologise for the additional
pain and suffering inflicted on bereaved families and survivors. Critical Research: The ‘Echo’ and the ‘Answer’ This brief excursion into my critical work on prisons, the legacy of civil war in the North of Ireland and
three decades of investigative research into the Hillsborough aftermath demonstrates a driving imperative
to recover truth and pursue justice through ‘bearing witness’, accessing alternative accounts, analysing
‘hidden’ data and recording abuses of state power. In challenging the obfuscations of ‘official discourse’
and ‘scientific’ knowledge, investigative research positions the immediacy of ‘personal troubles’ within
their broader context as ‘public issues’ (Wright Mills 1959). Despite its claims for value-neutrality, official
discourse is no guarantor of ‘truth’. Its legitimacy is derived in the status quo and the maintenance of
dominant vested interests. Rather, the truth must be sought in alternative oppositional accounts: the ‘view
from below’. In its recovery, truth holds the powerful—whether private or state agencies, corporations or
individuals—to account. It does so on ‘their’ terms, using the rule of law and, where appropriate, engaging
international standards and building oppositional alliances. Its fulfilment, however, necessitates a
‘fundamental shift in the structural relations and determining contexts of power which marginalize and
exclude [individuals] from effective participation in their destinies’ (Scraton and Haydon 2002: 326). While diverse in focus, the research discussed here exposes the myth of value-freedom, demonstrating
that regulatory institutions do not achieve the value-neutrality they claim. Rather, these institutions are
woven into the fabric of inequality, marginalisation and criminalisation, which is evident in the power
imbalance derived in and sustained by the determining contexts of class, ‘race’, gender and sexuality. Multiple deprivations are rooted in endemic poverty and structural inequality. There is consistent
evidence in cross-jurisdictional prison research that deprivation and punishment are bedfellows. Working-class communities disproportionately endure cuts in employment, essential services, health care, IJCJ&SD 12
2020 9(4) www.crimejusticejournal.com www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance welfare and education while living in poor, high-density housing, reliant on food banks. In June 2017, 72
people were killed and many more bereaved and displaced as a consequence of a foreseeable fire in a
neglected social housing scheme administered by one of London’s most affluent boroughs. The Grenfell
Tower fire is testimony to institutional failure in state–community relations and public consultation
(Bulley, Edkins and El-Enany 2019). It is a failure derived, legitimated and reproduced in the structural
relations of inequality and oppression that constitute, maintain and reproduce the established socio-
economic order and its state institutions. Critical Research: The ‘Echo’ and the ‘Answer’ While this article was under review, it became dramatically apparent that a highly contagious and deadly
disease, COVID-19, was escalating rapidly into a pandemic. With no known vaccine, it claimed many lives,
closed down entire cities and towns, factories and businesses, shops and social venues. As people isolated
in their homes, instructed by the State to end all direct personal contact, it appeared no one was immune
from contracting the virus. Inevitably and unsurprisingly, however, it continues to impact
disproportionately on the most marginalised communities where physical, spatial and social distancing
are not possible, and medical capacity is minimal (Davis 2020; Giroux 2020). Unrelated, but at the height
of the pandemic, in Minneapolis a White police officer was filmed kneeling on the neck of a prostrate Black
man while other officers watched on passively and approvingly. Under the knee, George Floyd died. Under
the banner ‘Black Lives Matter’, his death triggered unprecedented international protests directed against
racist policing and colonial heritage—the visible manifestation of racist oppression. Clearly distinct in context and circumstances, the impact of the Grenfell fire, the marginalisation laid bare
by a pandemic and the use of state violence against the Black community have shared roots in societies
whose administration of state power and political and economic reproduction are dependent on
maintaining and sustaining structural inequalities. Critical social research does not ‘hover in the air’ above
seemingly random events unfolding ‘on the ground’ and managed by state institutions. As this article
demonstrates, working with families and communities campaigning for ‘justice’ for loved ones in prison,
in the aftermath of disasters or conflict reflects Sontag’s commitment to deploying her photographic skills
as a chronicler of oppression, exposing the pain of ‘others’. Within communities riven by social, political and economic exclusion and ravaged by prescription and
street drugs, the persistent challenge is to establish programs that share knowledge, consult inclusively
and support active participation across the lifespan. These programs should celebrate diversity while
eliminating fear and defeating discrimination in all its complex, interrelated forms. They should respond
with alacrity, compassion and resources to mental ill-health crises. As Giroux (2002) states, defeating
exclusion requires generating and sustaining a politics of hope derived in the advancement of learning and
the creation of opportunity. Correspondence: Phil Scraton, Professor Emeritus, School of Law, Queen’s University, Main Site Building,
Belfast, BT7 1NN, Northern Ireland. Email: p.scraton@qub.ac.uk Critical Research: The ‘Echo’ and the ‘Answer’ g
llsborough Independent Panel web-site includes its report and all supporting documents: 4 The Hillsborough Independent Panel web-site includes its report and all supporting documents:
https://discovery.nationalarchives.gov.uk/details/r/C14176659 See also: http://hillsboroughinquests.independent.gov.uk/
(temporarily unavailable at the time of publishing due to ongoing legal proceedings). g
p
p
pp
g
https://discovery.nationalarchives.gov.uk/details/r/C14176659 See also: http://hillsboroughinquests.independent.gov.uk/
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Press. Christie N (1994) Crime Control as Industry. 2nd ed. London: Routledge. Coleman S, Jemphrey A, Scraton P and Skidmore P (1990) Hillsborough and After: The Liverpool Experience. Liverpool: Liverpool City Council. Coleman S, Jemphrey A, Scraton P and Skidmore P (1990) Hillsborough and After: The Liverpool Experience. Liverpool: Liverpool City Council. Davis A (2003) Are Prisons Obsolete? New York: Seven Stories Press. Davis A (2003) Are Prisons Obsolete? New York: Seven Stories Press. Davis M (2020) The Monster Enters: COVID-19, Avian Flu and the Plagues of Capitalism. New York: OR (
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Dorfman A (2004) Manifesto for Another World: Voices from Beyond the Dark. New York: Seven Stori Dorfman A (2004) Manifesto for Another World: Voices from Beyond the Dark. New York: Seven Stories Press. Foucault M (1980) Power/Knowledge: Selected Interviews and Other Writings 1972–1977. Critical Research: The ‘Echo’ and the ‘Answer’ Alongside a commitment to social and economic justice, as the research in prisons and with young people
in the North of Ireland demonstrates, is the creation of inclusive dialogue prioritising critical thought and
informing political action. In a well-quoted passage, Dorfman (2004: 10) states that those who bear
witness to torture, degradation or the suffering of others could not ‘live with themselves if they did nothing
… could not stain their lives by remaining silent’. Witnessing suffering yet ‘doing nothing’ redefines
observers as ‘accomplices’—so it is with critical social research. In challenging the ‘darkness of apathy’
and ensuring that the ‘voices’ of the marginalised are eminent, the excluded ‘receive the echo and answer’
that is their right. Correspondence: Phil Scraton, Professor Emeritus, School of Law, Queen’s University, Main Site Building,
Belfast, BT7 1NN, Northern Ireland. Email: p.scraton@qub.ac.uk IJCJ&SD 13
2020 9(4) www.crimejusticejournal.com Phil Scraton: Fractured Lives, Dissenting Voices, Recovering ‘Truth’: Frontiers of Research and Resistance 1 School of Law, Queens University Belfast, p.scraton@qub.ac.uk. This article was first presented as a plenary presentation to the
Interdisciplinary Workshop on State Violence, Macquarie University, Australia in December 2018. It is derived from three
substantial research projects spanning three decades: The Violence of Incarceration; The Context, Circumstances and
Consequences of the Hillsborough Disaster; Childhood, Transition and Marginalisation. While distinct in focus, the projects
share an imperative of critical social research—foregrounding and contextualising the ‘view from below’ or, in the case of the
prison research, the ‘view from inside’. Many thanks to the organisers of and participants in the Workshop, co-researchers on
the projects, to the reviewers whose comments were very helpful and, as ever, to Deena Haydon. 2 Independent Monitoring Boards (IMBs) are established at all prisons, independent of prison management, with powers of
entry to monitor operational policies and practices and hear prisoners’ complaints or concerns. 3 Prison inspections are conducted usually at five-year intervals and over two weeks at all prisons by Her Majesty’s Inspectorate
of Prisons (HMIP), an independent body headed by a Chief Inspector (HMCIP). Their reports provide an assessment based on
the World Health Organisation’s ‘Healthy Prison’ criteria. 3 Prison inspections are conducted usually at five-year intervals and over two weeks at all prisons by Her Majesty’s Inspectorate
of Prisons (HMIP), an independent body headed by a Chief Inspector (HMCIP). Their reports provide an assessment based on
the World Health Organisation’s ‘Healthy Prison’ criteria. HMCIP (2018) Report on an Unannounced Inspection of HMP Liverpool 4–15 September 2017.
www.justiceinspectorates.gov.uk/hmiprisons/wp-content/uploads/sites/4/2018/01/HMP-Liverpool-Web-
2017.pdf Moore L and Scraton P (2014) The Incarceration of Women: Punishing Bodies, Breaking Spirits. London: Palgrave
Macmillan. McKittrick D, Kelters S, Feeney B, Thornton C and McVea D (2004) Lost Lives: The Stories of the Men, Women and
Children Who Died as a Result of the Northern Ireland Troubles. Edinburgh and London: Mainstream Publishing. Moore L and Scraton P (2014) The Incarceration of Women: Punishing Bodies, Breaking Spirits. London: Palgrave
Macmillan. McKittrick D, Kelters S, Feeney B, Thornton C and McVea D (2004) Lost Lives: The Stories of the Men, Women and
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RESEARCH AND EDUCATION IN THE MEDICAL AREA
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International journal of research - granthaalayah
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ABSTRACT The training of health professionals poses a great challenge in
developing countries around the world. Medicine of the XXI century,
represented in health careers, has a direct relationship with scientific and
technical development, involves, observation, the search for knowledge
about the problems that affect the health and biological well-being of the
human being. This work implies a great ethical commitment to society,
hence the importance of strengthening research capacity in both
undergraduate and graduate studies. Developing human talent is a task
that must be carried out continuously from the beginning of the medical
career. Preserving life and preventing serious complications in the
vulnerable population due to known or unknown pathologies is an
unavoidable function of all health personnel, this is only achieved by
training future professionals in the best way to guarantee the survival of
future generations against the pathogenic microorganisms that can cause
death. Teachers must implement the necessary strategies to motivate new
professionals. Curiosity and interest must be strengthened throughout all
medical careers. Article Citation: MSC. Alida Bella
Vallejo López, DRA. Josefina
Ramirez Amaya, MSC. Juana Kou
Guzman, ING. Cesar Noboa, MSC. Oswaldo Francisco Zavala Palacios,
and MSC. Narcisa Liliana Durán
Ávila. (2020). RESEARCH AND
EDUCATION IN THE MEDICAL
AREA. International Journal of
Research -GRANTHAALAYAH, 8(9),
43-48. ,
,
Oswaldo Francisco Zavala Palacios,
and MSC. Narcisa Liliana Durán
Ávila. (2020). RESEARCH AND
EDUCATION IN THE MEDICAL
AREA. International Journal of
Research -GRANTHAALAYAH, 8(9),
43-48. Received Date: 31 August 2020
Accepted Date: 24 August 2020 Keywords:
Research
Education
Health
Medicine © 2020 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original author and source are credited. 43 RESEARCH AND EDUCATION IN THE MEDICAL AREA MSC. Alida Bella Vallejo López *1
, DRA. Josefina Ramirez Amaya 2
, MSC. Juana Kou Guzman 3
, ING. Cesar Noboa-Teran 4
, MSC. Oswaldo Francisco
Zavala Palacios 5
, MSC. Narcisa Liliana Durán Ávila 6 *1, 2, 3, 4, 5 Guayaquil University, Faculty of Medical Sciences, Ecuador
6 Laboratory Jesus De Nazareth, Faculty of Medical Sciences, Ecuador DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 ISSN (Online): 2350-0530 International Journal of Research -GRANTHAALAYAH
ISSN (Print): 2394-3629 September 2020, Vol 8(09), 43 – 48
DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353
RESEARCH AND EDUCATION IN THE MEDICAL AREA
MSC. Alida Bella Vallejo López *1
, DRA. Josefina Ramirez Amaya 2
, MSC.
Juana Kou Guzman 3
, ING. Cesar Noboa-Teran 4
, MSC. Oswaldo Francisco
Zavala Palacios 5
, MSC. Narcisa Liliana Durán Ávila 6
*1, 2, 3, 4, 5 Guayaquil University, Faculty of Medical Sciences, Ecuador
6 Laboratory Jesus De Nazareth, Faculty of Medical Sciences, Ecuador
DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 ISSN (Online): 2350-0530 International Journal of Research -GRANTHAALAYAH
ISSN (Print): 2394-3629 September 2020, Vol 8(09), 43 – 48
DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353
RESEARCH AND EDUCATION IN THE MEDICAL AREA
MSC. Alida Bella Vallejo López *1
, DRA. Josefina Ramirez Amaya 2
, MSC. Juana Kou Guzman 3
, ING. Cesar Noboa-Teran 4
, MSC. Oswaldo Francisco
Zavala Palacios 5
, MSC. Narcisa Liliana Durán Ávila 6
*1, 2, 3, 4, 5 Guayaquil University, Faculty of Medical Sciences, Ecuador
6 Laboratory Jesus De Nazareth, Faculty of Medical Sciences, Ecuador
DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 ISSN (Online): 2350-0530
ISSN (Print): 2394-3629 International Journal of Research -GRANTHAALAYAH
September 2020, Vol 8(09), 43 – 48
DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 DOI: https://doi.org/10.29121/granthaalayah.v8.i9.2020.1353 Article Type: Research Article 2. BACKGROUND Research is as old as humanity and has grown over time, like science to the present day. The emergence of the
great philosophical, scientific and mathematical movements to understand the great phenomena of the world, from
a deeper and more rational vision, had a great impulse after Christ. Among the most prominent figures of scientists in the history of mankind who made contributions to scientific
research, we can mention Galileo Galilei, considered the father of the scientific method, Hippocrates known as the
father of medicine, among others. The word investigate comes from the Latin investigare derived from vestigiun which means to look for a clue. (Universia México 2016) affirms that: Research is a quality of the human being, for the search for knowledge and
truth. Investigating means searching and discovering, in order to develop new theories in different disciplines or
modify existing ones. But this capacity, if it already exists in each person, must be developed, and the best way to do
it is by promoting it from youth, through the development of academic papers. For Carlos Borsotti (1989) “… research is a process through which an attempt is made to respond to scientific
problems, through systematic procedures, which include the production of valid and reliable information”. Therefore, research should be a fundamental educational practice for all students, especially in the medical
education curriculum, both undergraduate and graduate. g
g
Since Wilhelm Von Humboldt (1810) added research units to the university process, the value of the
institutional mission of the University has increased, by generating new knowledge, to transcend and not remain
stuck in theories considered as absolute truths. (Deiters, H. 1960). (
)
This is another way to enrich future professionals educationally, to prepare them with the capacity for inquiry,
to face the problems and challenges of science and society. (Naranjo R. 2013) "The research allows to expand knowledge, based on data on any topic of social or scientific interest for
academic purposes" (Coelho, 2019). Research requires time and dedication, the researcher must be interested in a
topic and have a curiosity to investigate, in order to solve a puzzle. So, although all human beings have the ability to
investigate, not everyone is interested in doing it, so it is clear that to investigate there must be motivation. Research and Education in The Medical Area The benefits obtained range from economic growth to the discovery of new technologies that provide greater
comfort to people and new treatments for diseases. Thousands of investigations could be mentioned that have
changed the world, generating comforts. Going through the creation of the microscope, vaccines, X-rays, the
decomposition of the atom, the study of DNA and the creation of new academic areas to learn about various topics,
nothing would be the same, if it had not been investigated. (Vallejo A. 2020). In a globalized context, research plays an important role in the training process of future professionals in the
health area, considering that the area of medicine is constantly updating knowledge, methodologies and procedures,
to improve the quality of healthcare processes. In addition, they must be informed of new health problems that arise
in the world, considering that viruses, bacteria and microbial agents evolve and appear with increasing
aggressiveness. gg
The current trend in the world is to conduct research of public utility, especially when it comes to biomedical
research. Institutions must support not only theoretical academic training, but also training in the pillars of research
such as the concepts of basic science and statistics, the mechanism by which the relevance of a medical or scientific
study is presented or established, regardless of the area in which it has been developed. The impulse for the development of research, on the part of doctors, must begin with profound modifications in
the study plans of medical careers. The way we teach medicine and its related careers must be changed. Teaching
should be presented as a guide to understanding, developing critical thinking, achieving independence of judgment,
and preparing for change. Every medical student must be prepared to prevent and attend to the priority health
problems of the population in an ethical framework, but also to develop their eventual capacity to carry out research,
regardless of their final destination: generalist, specialist, Ph DV (Norero, Colomba. 2000). International Journal of Research -GRANTHAALAYAH 1. INTRODUCTION Research plays a decisive role in the learning process, to increase student knowledge, allowing comprehensive
academic development. Research is considered an activity aimed at obtaining significant findings that increase
human knowledge and science, promotes the development of skills and the discovery of new facts, technological
advances. Bijarro, Francisco (2007): It allows to develop improvements in all areas of knowledge; Thanks to research, it was possible to discover
various inventions that are indispensable to society today. In this way, the researcher can understand the needs and
problems that influence man's life. Research expands knowledge. Scientific development brings with it a cultural
evolution for a better adaptation to the modern world. 43 Research and Education in The Medical Area Research and Education in The Medical Area RESEARCH IN THE MEDICAL AREA Medicine, like all science, needs research to ensure advances in knowledge. Research in medicine contributes
to: understanding the biological processes of the human being, carrying out prevention, detection, diagnosis,
treatment and looking for the causes of diseases. Medical practice also includes the control of the health problems of
the population. To contribute to scientific advancement, there are various types of health research, such as biomedical,
epidemiological, clinical, and health education research at the individual and population levels. It is important to
recognize that there are health problems of various kinds, and to face them, learning in research during professional
training must be valued. All research must be carried out in accordance with scientific and ethical principles, both in the theoretical and
in the experimental part, always having as a priority to avoid risks of all kinds to the life and integrity of people. If there were no researchers in the medical area, we would not understand the complexity of the human body,
nor would the function of each organ, the cure or treatment of diseases not have been found, there would be no
vaccines, there would be no known DNA. They would not perform surgeries. Man is always exposed to diseases to
investigate. Research takes time, dedication and sacrifice and, in most cases, it is not rewarded. This leads to the need to
train educators and other related professionals to learn theories, methods and research techniques, so that they can
analyze and interpret in their professional contexts. (Martinez, G., 2007) Society must understand and recognize that research is important in the life of man, it is the impulse that those
who are inclined to investigate need. Medical personnel have the fundamental responsibility to protect, ensure,
restore and guarantee health, this mission can only be effectively fulfilled by well-trained professionals both
academically and ethically, with a vocation for service and with the desire to investigate to expand their services
every day, services, knowledge. That the doctor is up to date will always be a professional, moral and social
obligation. 2. BACKGROUND With the evolution of technologies, man adapts to new ways of communicating, studying and researching,
everyone gets involved in one way or another in this new scheme of society, the digital age has come to stay, and
while it has great advantages, it also has some disadvantages. This is evident in those people who have not yet
mastered technology and computing, however it is important to join the new trend to constantly update. 44 MSC. Alida Bella Vallejo López, DRA. Josefina Ramirez Amaya, MSC. Juana Kou Guzman, ING. Cesar Noboa, MSC. Oswaldo
Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila Currently the world trend is network connectivity. Internet allows to know more quickly all the studies,
discoveries and changes that occur every minute, knowledge evolves and science advances, everyone watches this
change carefully. Despite this, few are interested in investigating. One of the highlights of information technology is
that it turns out to be a very useful medium for researchers. The new era brings the creation of the "virtual
university", before which some projections indicate the disappearance of the structure of the traditional university
in its current configuration. The reason for this is that the "virtual university" has several advantages over the
traditional one, as it encourages collaboration between universities and professors who are in remote locations,
allowing students to easily access top-notch libraries from different institutions. (Akyeampong, 1998) The number of medical researchers has decreased significantly in recent years, with a significant decrease in
the interest of students to continue researching in their professional lives. "There is constant global concern about
the decline of so-called 'medical researchers.' Students no longer perceive a career with solid research bases as viable
for their professional future” (Herrera Miranda GL, 23 (2)). International Journal of Research -GRANTHAALAYAH 3. WHY DO UNIVERSITIES NEED TO INVESTIGATE? Research and education complement each other, especially in universities; since research is one of the
fundamental pillars in the training of students, even more so in the area of health, because it allows increasing
scientific production. The entities that regulate the quality of education in various countries of the world demand
that education should be promoted beyond the classroom, the evaluation of a training center also depends to a large
extent on how much it contributes to universal knowledge. These contributions help to improve the world by
discovering and exploring new ways of solving everyday problems in the social field or of a biological nature in the
area of medicine. By definition, the university is the institution dedicated to higher education, (one that provides specialized
knowledge of each branch of science) that is made up of several faculties and that grants the corresponding academic 45 Research and Education in The Medical Area Research and Education in The Medical Area degrees. For his part (Albert, 2007) considers that “Research in general is an activity aimed at acquiring new
knowledge; regardless of what that knowledge is, it can refer to the arts, letters or knowledge”. degrees. For his part (Albert, 2007) considers that “Research in general is an activity aimed at acquiring new
knowledge; regardless of what that knowledge is, it can refer to the arts, letters or knowledge”. The university is the best place to promote scientific activity. In this way, it is possible to enrich production
systems, obtain updated information and attract economic resources, as well as establish scientific projects with
greater risk and potential return (Nuñez & Montalvo, 2014). Universities must impact, leave a mark in a region or
country, beyond graduates, and one way to do this is through research. (Mazuera Arias, R. 2016). In the health area, the Medicine career and its related careers have the obligation to know health issues, so
teaching, the most relevant content and research are important for better learning. The student should not be a data bank, but a restless being, capable of questioning and solving problems (Norero
V., Colomba. 2000). The development of investigative competences allows expanding the field of knowledge. In the
21st century, transformations are required to improve the educational-institutional model. International Journal of Research -GRANTHAALAYAH 3. WHY DO UNIVERSITIES NEED TO INVESTIGATE? (Gómez, J.; Diéguez, P.;
Gómez 2014) Universities are committed to promoting research, governments are favored by developing technological and
scientific inventions for tomorrow, (Becker & Pera Espinoza, 2019) express that “the economic success or failure of
a nation no longer depends on technology or machinery that he owns, but what he does with his people”. This
suggests that the population generates economic and technological income for the benefit of a nation thanks to its
contribution to the people through its research and that by working on this research topic, jobs are generated,
increasing the value in the productive and economic system to improve, quality of life in all areas. 4. VISION OF THE WORLD The countries' commitment must be aimed at promoting all fields of science, generating new knowledge and
deepening existing ones. Any complete investigation will always be the starting point of a new investigation,
expanding more and more the imagination, ideas, theories, of the phenomena that surround us. The United Nations Educational, Scientific and Cultural Organization (UNESCO, 2013), in addition to China, the
European Union, Japan, the Russian Federation and the United States still account for 72% of the world's research
population. While in Latin America and South Asia they concentrate 8% and 23.3% of the world population of
researchers, respectively, the more or less comparable mode is: 3.6% in the first and 3.1% in the second, which
represents a low rate of researchers. The National Science Foundation (National Science Board, 2018) published that the United States leads the
world in research and development (R&D) spending with 496,000 million dollars (26% of the world total), followed
by China with 21% ($ 408 billion). In all areas of the university educational community, research work will always be productive for those
involved, and it would be much more effective if it were implemented in the curricula, throughout all university
careers. It is urgent, then, to promote research as a resource for the autonomous production of knowledge, which
depends not only on the creation of individual and group expertise and learning, but also on the efficient
management and organization of investigative processes In developed countries there is evidence of the support given to researchers, however in developing countries
much remains to be done. A change of mentality to facilitate administrative procedures such as the provision of
infrastructure, in addition to financial support, this process should be seen as an investment, which will benefit the
country. A change in the focus of the subjects or courses taught in undergraduate becomes necessary to achieve a
consolidated course of research fundamentals, as well as the dedication of a fixed time during the undergraduate
career to achieve the necessary motivation in the student and that you undertake a research project on your own (or
with a suitable mentor) and ultimately stand out. (Ávila, M.J. 2014) SOURCES OF FUNDING This research received no specific grant from any funding agency in the public, commercial, or not-for-profit
sectors. 5. CONCLUSIONS The scientific advances achieved in the new century are the result of the work carried out by researchers, in all
areas of knowledge. Research in the new educational context is an indispensable factor for Higher Education Institutions, teachers
must involve students in scientific activities, to strengthen the learning of future professionals. 46 MSC. Alida Bella Vallejo López, DRA. Josefina Ramirez Amaya, MSC. Juana Kou Guzman, ING. Cesar Noboa, MSC. Oswaldo
Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila MSC. Alida Bella Vallejo López, DRA. Josefina Ramirez Amaya, MSC. Juana Kou Guzman, ING. Cesar Noboa, MSC. Oswaldo
Francisco Zavala Palacios, and MSC. Narcisa Liliana Durán Ávila In the medical area, there is a great responsibility to protect the life and health of the population, hence the
importance of promoting a culture of researchers with ethical values, who provide new knowledge. The countries that investigate generate knowledge that benefits society with technological and economic
development and progress. All countries must adopt government and financial policies to support training programs
and research skills development, providing infrastructure, legal resources and incentives to researchers, which will
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(CIDE). Colección investigamos No 5. Edita: © Secretaría General Técnica. Madrid. [10] Martínez, G. R. A. 2007. La investigación en la práctica educativa: guía metodológica de la investigación para
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http://www.ems.sld.cu/index.php/ems/article/view/1579 [15] Vallejo López, A. (2020). El papel del docente universitario en la formación de estudiantes investigadores
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http://www.ems.sld.cu/index.php/ems/article/view/1579 [16] Vallejo López, A., Daher Nader, J., & Rincón Rios, T. (2020). Investigación y creatividad para el desarrollo de
competencias científicas en estudiantes universitarios de la salud. EducacióN MéDica Superior, 34(3). Recuperado de http://www.ems.sld.cu/index.php/ems/article/view/1606/1033 [16] Vallejo López, A., Daher Nader, J., & Rincón Rios, T. (2020). Investigación y creatividad para el desarrollo de
competencias científicas en estudiantes universitarios de la salud. EducacióN MéDica Superior, 34(3). Recuperado de http://www.ems.sld.cu/index.php/ems/article/view/1606/1033 48 International Journal of Research -GRANTHAALAYAH
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Community Radio for cohesion and integration during Kenya’s 2017 post-election violence: A study of Migori County
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African social science and humanities journal
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cc-by-sa
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African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj *Corresponding author *Corresponding author Abstract: Kenya’s elections are mostly marred with violence, especially
after announcements of presidential results. Peace and media experts on the
other hand have blamed Kenyan media for fueling cases of post-poll
conflicts. It is in this understanding that this study assessed the contents of
community radios in Migori County to investigate the role they played in
promotingcohesionandintegrationduringthe2017Kenya’spost-election
violence. The research, which was conducted in Migori town, studied five
radio stations namely; Milambo, Onagi, Anyuola, Rameny and Tarumbeta
which are located within Migori County. The specific objectives were: to
assess the extent to which the 2017 post-election violence affected residents
of Migori town; to establish if the five stations developed contents that
promoted cohesion and integration during the violence and to assess the
extent to which the contents aired on the radio stations promoted cohesion
and integration among residents of Migori town. Study was a survey that utlilised mixed research approach where
qualitative and quantitative methods of data collection were used. Systematic random sampling was employed to
administer 399 questionnaires. For qualitative data, 21 key informants were purposeively selected and interviewed. The quantitative data collected was analysed using SPSS while qualitative data was analyzed using content
analysis. From the study it was found and concluded that Community Radio, if professionally used, promotes
cohesion and integration during post-poll conflicts. The study has recommended that in order to reduce incessant
post-poll violence in Kenya, the relevant stakeholders should work together to air contents that enhance cohesion
and integration in order to curb violence recurrence. Policies should be developed and funding availed to help
facilitate matters media and peace. )
Article detail
Received: 27 September 2021
Accepted: 29 December 2021
Published: 12 January 2022
Conflict of Interest: The author/s
declared no conflict of interest. Community Radio for cohesion and
integration during Kenya’s 2017 post-election
violence: A study of Migori County This article is published by JFP
Publishers in the African Social
Science and Humanities Journal
(ASSHJ). Volume 3, Issue 1, 2022. ISSN: 2709-1309 (Print) Nicholas Anyuor1*, Rosechieng’2
1&2Communication, Journalism and Media Studies, Rongo University, Kenya,
nanyuor@gmail.com1, achijebet@gmail.com2 This article is distributed under a
Creative Common Attribution (CC
BY-SA 4.0) International License. Article detail Received: 27 September 2021
Accepted: 29 December 2021
Published: 12 January 2022 Conflict of Interest: The author/s
declared no conflict of interest. Conflict of Interest: The author/s
declared no conflict of interest. Keywords – Community Radio, Integration and cohesion, Post-election violence, Vernacular radio African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj 1. INTRODUCTION Media are powerful influencers that play crucial role in shaping opinions of members of the public and their world
views (Mcombs, 1994); ‘the media therefore through various forms of programmes provide a space where
societies can dialogue and articulate solutions to problems that easily trigger conflicts among them and promote
peaceful co-existence’(Mwangi,2017: 1). African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |43 African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj https://journals.jfppublishers.com/asshj
Role of media, particularly community radio in promoting peace, has been witnessed globally. In Columbia, for
example, community radio was included in peace-building initiatives after the signing of peace agreement between
the Colombian State and the FARC EP guerrillas (Vega et al 2019) Role of media, particularly community radio in promoting peace, has been witnessed globally. In Columbia, for
example, community radio was included in peace-building initiatives after the signing of peace agreement between the Colombian State and the FARC-EP guerrillas (Vega et al., 2019). Since 1997, with Decree 1447, the Colombian State had said that community radio should be a space for community
reflection, consultation, and conflict resolution in the country. As a result, during the last 20 years, community radio
stations emerged and generated popular and citizen reflections on peace and conflict, which translated into
communicative processes with the capacity to foster diversity, intercultural dialogue, and tolerance. As a
consequence, community media became privileged partners in conflict resolution and reconciliation (Pereira &
Cadavid, 2011 in Vega et al., 2019: 1392). In Africa, Mwangi (2017) argues, the influence of radio has continued to grow and has been utilised in peace
building process and in conflict resolution. Community radios have been used in peace building in some war-torn
countries. This has led to attainment of certain level of cohesion and integration among the people in the regions. In countries like Burundi, Rwanda, Kenya, among others, community radios have been accused of being
destructive,butatcertainpointtreasuredfortheirconstructivecontents.In,urundi,forinstance,Ijambostudio’s
role in promoting cohesion among the people cannot be gainsaid. It brought together journalists from different
tribes who developed peace-building programmes to restore order and integration in this country (Gilboa, 2002 in
Mwangi, 2017). 1. INTRODUCTION The setting up of studio Ijambo in 1995 saw a team of journalists drawn from Hutu and Tutsi ethnic groups initiate
programs aimed at promoting dialogue, reconciliation and peaceful co-existence…this
enabled
the
two
tribes
reach
a
common ground in the conflict that afflicted the region (Gilboa, 2002 in Mwangi, 2017: 1). In Kenya, the need to have community and vernacular radios made the government to liberate airwaves, offering
invaluable opportunities for the opening of many radio stations-the commercial radios, faith-based radios, among
others. Some of these stations have also been accused of fueling violence while others have been promoting peace
among conflicting parties. For example, KASS FM, a vernacular radio station in the Rift Valley region, was accused
of fueling 2007/08 post-poll violence after by introducing partisan programming (Waki et al., 2008). On the other
hand, some radios developed peace-builiding programmes in efforts to restore cohesion and integration in Kenya
after the 2007/08 post-election violence. Community-cum-faith-based Radio Amani, based in Nakuru County,
developed peace-building programme that ‘provided a platform for peace building through community
participation’(Wakietal.,2008:2). Therefore, in asmuchas community radios can be destructive, they can also be used to air contents that promote
peace as has been witnessed in different parts of the world. It is in this background that this study assessed the role
community radio played in promoting cohesion and integration during the 2017 post-election violence in Kenya. 2. LITERATURE SURVEY 2. LITERATURE SURVEY 2.1. Kenya’s 2017 post-election violence 2.1. Kenya’s 2017 post-election violence 2.2. The impact of Community Radio in the society Communication has been critical in the society as it is an ingredient that fuels development in all aspects. To
communicate to wider audience, mass media such as radio, are invaluable. Radio communication ensures that
information reaches the right and the relevant audience and offers opportunity for the people to exchange ideas. Community radio is important in the society as it offers communication services beyond commercial; not-for profit
and acts as a watchdog (Seidu, 2011). In some countries such as Australia, community media have changed the
societies into positive thinking about themselves and community at large. This was evident in the response of
ustralia’sindigenouscommunities to the contents of their community media-as viewed by the Aboriginal and
Torres Strait Islander people who trusted community media as a player of central role in organizing lives of the
local people (Romano, 2010). These indigenous audiences, accordingtoRomano(2010),describetheircommunitymediaas‘aprimarysource
of news and information that helps to sustain social networks, promote social self-esteem, nourish community
identities and kinship ties, maintain language and culture, educate people and support cultural dialogue’
(Meadows, et al., 2007: 51-68 in Romano, 2010: 23). It is through community radio stations that in some countries
such as South Africa, public journalism has been promoted, even though licenses here are given with stringent
rules (Broadcasting Act No. 4 of 1999 in Davidson, 2020). This kind of framework makes community radios have
their missions and identities that are different from public and commercial broadcastings. In Nigeria, community
radios that have been established have spurred development and understanding among the people towards the
good of the communities. It is argued that even though the Nigeria’s population is over 150 million, newspaper
circulation is low and the papers majorly use English, a language which is not understood by many in this West
Africa State; leading to increasing influence of radio stations where, for example, Radio Nigeria claims more than
100 million listeners while Raypower FM claims to reach 23 million listeners (UNESCO, 2008 in Awoshakin, 2020). This kind of proliferation of community radio outlets allowed journalists to play closer attention to community
issues where they communicate these in their local languages and therefore, effectively engaging local communities
(Awoshakin, 2020). 2.2. The impact of Community Radio in the society Awoshakin (2020) argues that this scenario has been possible for the reporting of health
concerns and political engagements; that through community radios, journalists are in positiontoreportthe‘issues
that the local communities were wrestling with, brought local people into the studios to have conversations with
politicians, and broadcast these conversations in local languages’ (woshakin, 2010: 57). By adhering to this
globally, the community radios have improved development initiatives at grassroot levels where they serve (Seidu,
2011). In East Africa, even though community radios have been sources of violence in some countries such as
Rwanda, according to Ogenga (2020), they can also be used to mobilise and educate people on how to share
resources in efforts to avoid conflicts. For example, a successful peace journalism strategy thatendedorratherreducedtheLord’sResistancermy
conflict has been witnessed in Uganda sometime. Ugandan military resorted to use of radio after failed military
interventions. The military established Radio Freedom for peace. This initiative drove away fighters, including
child soldiers, from LRA back to their homes and handing over the firearms to the government (Laker, 2020). This
means community radios are powerful and can be used to influence and shape communities. Therefore, it is on this
note that this study was conducted to assess the impact of community radio in cohesion and integration during the
2017 post-election in Migori, Kenya. 2.1. Kenya’s 2017 post-election violence ThevotingprocessofKenya’s2017electionswaspeacefuluntilUhuruKenyattawasdeclaredwinnerby3lectoral
body (Mutahi, 2019). Opposition supporters from different regions including Migori prtested Uhuru’s victory.
Police then acted to quell the violence (Humann Rights Watch, 2017); however, they were using force and live
ammunitions and many people in these regions became victims (Mutahi, 2019). A lot of crimes such as sexual
violence were committed during this poll violence not only by locals but also by State agents such as police officers
(Reliefweb, 2021). According to Muhumuza (2017), 92 people were killed during the months of election violence in
2017 while 86 cases of sexual or gender-based violence were reported. African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |44 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj 2.2. The impact of Community Radio in the society 2.4. Impact of Community Radio in cohesion and integration TheconceptofmediaandpeaceorratherpeacejournalismwasdevelopedbyascholarJohan7altung’in1960s
(Youngblood, 2020). Youngblood (2020) argues that journalism that promotes cohesion should focus on the
importance of language that does not victimize other parties. This focus of using media in promoting peace would
address conflicts that have been witnessed across the globe. Youngblood (2020) accuses Kenyan media of playing
negative role when it comes to political conflict; giving an example with 2008/2007 election violence, arguing that
media was ill prepared to cover it. Ogenga (2020), however, argues that community radio can be good media
outlets or platforms for peace building and cohesion. He asserts that community radios can perform this act of
enhancing cohesion in the society since they ‘are owned by the community and serve the interest of the
community’(Ogenga,2020: 23). It is in this backdrop that community radio can be used to promote cohesion and
integration among the people. But Ogenga (2020) avers that for community radio to succeed in matters cohesion and integration, members of
the public must be involved in discussing matters peace using language that is understood well by the community. It is through these radios that locals can easily be mobilized and educated on the importance of peace and on
sharing of natural resources to avoid conflicts. 2.3. Community Radio in Kenya In Kenya, community radio can also be referred to as ‘rural radio’, ‘people’s radio’, (Ochichi, 2013). Kenya
produced the first Community Radio (CR), the Homa Bay CR in May 1982 in the current Homa Bay County, then
Nyanza Province (Quarmyne, 2006). Currently, community based radio stations in Kenya have increased in African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |45 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj
number from 10 in 1999 to over 120 in 2015 (Oluoch, 2020). Oluoch (2020) argues that such stations give invaluable
local broadcasting where matters development are addressed. African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj number from 10 in 1999 to over 120 in 2015 (Oluoch, 2020). Oluoch (2020) argues that such stations give invaluable
local broadcasting where matters development are addressed. 3. PROBLEM STATEMENT Political violence has not only been witnessed in Kenya, but it has been a global concern. Media on the other hand,
regardless of the outlet, have been accused of fueling such violence. For example, in Rwanda, media outlets-radio
and newspapers-were blamed for fueling the 1994 Genocide that left almost one million people dead in a span of 90
days.Kenya’s2007/2008post-election violence never went without a blame on the media especially community
radios. On the other hand, vernacular and community radios’ contents have been utilised in promoting peace
among warring communities worldover. This was witnessed in Uganda to curb the Lord Resistance rmy’s
incessant conflict. In 2017, Kenyans witnessed post-election violence where some people were killed and others
displaced, and counties such as Migori were classified as hotspots. But eventually peace and harmony were
restored. Was the community radio one of the actors used to promote peace, especially in Migori County? There is
no single study that has been conducted to find out the role community radios played in peace-building process
during 2017 post-electionviolenceinKenya’sMigoriounty.Thisisthegapthestudyhasfilledafterinvestigating
the role five community radios based in Migori County played in promoting cohesion and integration in Migori
during the 2017 post-poll violence. Key findings y
g
The first research question was to find out the extent to which the 2017 post-election violence affected residents of
Migori town. To answer this, we compared percentage of responses from 399 questionnaires and from 21 key
informants. The tables below show various results. y
g
The first research question was to find out the extent to which the 2017 post-election violence affected residents of
Migori town. To answer this, we compared percentage of responses from 399 questionnaires and from 21 key
informants. The tables below show various results. Table 1: Are you a resident of Migori town? Responses
Frequencies
Valid Percent (%)
Yes
310
80.9
No
73
19.1
Total
383
100.0
Table 2: Did you experience the effects of 2017 post-election violence in Migori town? Responses
Frequancies
Valid Percent (%)
Yes
266
69.5
No
117
30.5
Total
383
100 Table 1: Are you a resident of Migori town? Responses
Frequencies
Valid Percent (%)
Yes
310
80.9
No
73
19.1
Total
383
100.0 Table 2: Did you experience the effects of 2017 post-election violence in Migori town? Responses
Frequancies
Valid Percent (%)
Yes
266
69.5
No
117
30.5
Total
383
100 5. DATA ANALYSIS AND DISCUSSIONS
5.1. The key findings and analysis This study used interview schedules and questionnaires as tools of data collection. A total of 399 questionnaires
were administered to residents of Migori town. Out of the 399 questionnaires, 383 were filled and returned. This is
96% of the respondents, which is enough population for a study. 21 interviews were conducted where media,
cohesion and integration, and human rights stakeholders were selected as the key informants. Out of this, 19 key
informants responded; giving 95%, which is good response for research. This study looked at the contents of five
community radio stations in Migori County, namely; Milambo, Onagi, Anyuola, Rameny and Tarumbeta. From the
study, the quantitative data results were as follow: 4. RESEARCH METHODOLOGY 4. RESEARCH METHODOLOGY The study used mixed research approach where both quantitative and qualitative data collection methods were
used. It employed systematic random sampling to administer 399 questionnaires from a population of 71,660
residentsofMigoritown.SamplesizewascalculatedusingYamane’sformulatoget399respondentsfrom71,660
residents of Migori town, according to Census Report of 2019. For qualitative data, 21 key informants, purposively
selected from relevant stakeholders, were interviewed. They were drawn from the newsrooms of the community
radio stations and peace stakeholders in Migori County. Migori County was selected for the study since it had been
identifiedasoneofthehotspotsduringthe2017pollclashes,accordingtoreportpublishedbyhina’sXinhuain
2018; and is also a cosmopolitan county (Star, April 14, 2017) that is comprising different tribes and races such as
Asia, Somali, Luhyia, Kuria, Kikuyu, Kisii, among others and therefore, call for peace, cohesion and integration
during this period was crucial. The investigated the contents of five radio stations namely; Milambo, Onagi, Anyuola, African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |46 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj Rameny and Tarumbeta, because they are the main community radios in Migori County with wider coverage of the
county and Migori town. Rameny and Tarumbeta, because they are the main community radios in Migori County with wider coverage of the
county and Migori town. First analysis y
Based on the responses from the participants, 69.5% (266 respondents) were affected by the post-election violence
in Migori town while 117 respondents (30.5%) disagreed. From qualitative data, 19 key informants responded from
a total of 20. This is a good response of 95% for research. From the 19 key informants, 17 (89.5%) agreed that indeed
they were extremely affected by the 2017 post-election violence in Migori town. However, 10.5 % (Two key
informants) said people were affected but mildly. During the interviews, participant A, a journalist in Migori with
one of the community radio stations under study, averred that the violence indeed affected residents of Migori
town. He gave an example with the People Living with Disabilities (PLWDs), women and children who were
‘badlyaffectedbythesituation.’ I did news stories about PLWDs to highlight their plight. The blind and the crippled were extremely affected. The
blind could not see, the crippled could not run away from the danger. We also ran stories about children and women
who were most vulnerable during the violence. It was sad that even some children were shot by police, and women and
girls gang raped by State agents. The situation was bad here (Participant A, March 14, 2021). I did news stories about PLWDs to highlight their plight. The blind and the crippled were extremely affected. The
blind could not see, the crippled could not run away from the danger. We also ran stories about children and women
who were most vulnerable during the violence. It was sad that even some children were shot by police, and women and
girls gang raped by State agents. The situation was bad here (Participant A, March 14, 2021). The comment concurs with literature by many scholars and reports by several agencies about the 2017 post-election
violence in Kenya. Mutahi (2019) argues that people from Kisumu, Migori, Homa Bay, Mathare, Kibra, Kisii were
heavilyaffectedbytheprotestsafterUhuruKenyattawasdeclaredwinneroftheugust8,2017election,‘with24 The comment concurs with literature by many scholars and reports by several agencies about the 2017 post-election
violence in Kenya. Mutahi (2019) argues that people from Kisumu, Migori, Homa Bay, Mathare, Kibra, Kisii were
heavilyaffectedbytheprotestsafterUhuruKenyattawasdeclaredwinneroftheugust8,2017election,‘with24 ican Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online |47 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj fatal police shootings confirmed by Human Rights organisations during thosefatefulday’(Mutahi,2019: 5). First analysis In a report by International Federation for Human Rights (FIDH) and the Kenya Human Rights Commission
(KHRC) in Reliefweb (2021), sexual violence was witnessed in Migori during the 2017 violence. According to a rape
victims interviewed by FIDH in Migori town, police officers were also rape agents. Therefore, the 2017 post-
election violence in Kenya affected people of Migori town differently. y
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The second research question sought to establish if the five radio stations in the study developed contents that
promoted cohesion and integration among residents of Migori town during the 2017 Post-Election Violence. Table 3: Did you listen to any of the following radio stations based in Migori County, during the 2017 post-election vio
Anyuola, Rameny and Tarumbeta? Table 3: Did you listen to any of the following radio stations based in Migori County, during the 2017 post-election violence? Milambo, Onagi,
Anyuola, Rameny and Tarumbeta? Responses
Frequencies
Percentage (%)
Yes
300
78.3
No
83
21.7
Total
383
100 From the results, 78% (300 respondents) listened to at least one of the named radio stations during the 2017 post-
election violence. The listenership was as indicated below:
Name of the station
Percentage (%)/100%
Milambo FM
50
Onagi Radio
31
Anyuola FM
8
Tarumbeta
6
Rameny
5 The third research question was on how the contents aired on the five community radio stations promoted
cohesion and integration among residents of Migori town during 2017 Post-Election violence. The results from the
questionnaires and interviews conducted during the study were as follow: _____________________________________________________________________________________________
Table 4 (i): Did this station (identified in b above) have programmes and news items on matters peace? Responses
Frequencies
Percentage (%)
Yes
280
73
No
103
27
Total
383
100
Table 4 (ii): If yes, in 4 (i), were the contents geared towards cohesion and integration among residents of Migori town? Responses
Frequencies
Percentage (%)
Yes
250
65
No
133
35
Total
383
100
Table 4 (iii): If yes in 4 (ii) above, to what extent if you were to rate between 1 (Unsatisfied) and 10 (Very Satisfied),
did the contents aired impact peace, cohesion and integration on residents of Migori? Responses
Frequancies
Percentage (%)
Unsatisfied
23
6
@on’tknow
32
8.4
Satisfied
256
66.8
Very Satisfied
72
18.8
Total
383
100 Table 4 (i): Did this station (identified in b above) have programmes and news items on matters peace? Second analysis These were busy days for us and we also gave bonus/extra
airtime for the paid shows as way of partnering with communities and peace agencies (Participant C, April 18, 2021). Some communities such as Luhya, Kuria, Luo, which are the majority in Migori County, also organized peace
programmes that were aired on our radio almost on a daily basis. Morning hours we invited religious leaders to pray
and offer spiritual guidance to people on matters peace. These were busy days for us and we also gave bonus/extra
airtime for the paid shows as way of partnering with communities and peace agencies (Participant C, April 18, 2021). These versions agree with the opinions of Media and Peace scholars who argue that it is the role of the community
radios to promote peace in the society and to enhance cohesion and integration during times of violence. Ogenga
(2020) observes that community radio can play imperative role in conflict resolution and peace-building in the
society. He avers that for community radio to effectively perform its role, members of the said community should
be engaged in discussing matters peace. Through this, these members find forum to engage and educate
themselves on the importance of peace within the community and society at large. In table 4 (ii), the result shows that the contents aired were majorly aimed at promoting cohesion and
integration. From the data collected by use of questionnaires, 250 respondents (65%) agree that the stations aired
contents that were geared towards cohesion and integration, while 133 respondents (35%) disagreed. This again
concurs with the results of qualitative data from key informants where all the 19 respondents (100%) agreed that
the stations aired peace contents during the 2017 post-poll violence in Kenya. Participants D & E who work with peace organisations within Migori County argued that as organisations, they
were invited by the community radio stations during talk shows to discuss matters living together; co-exitence
among residents of Migori. Even though they were invited by the stations, at times they would pay for airtime to
address members of the public on issues cohesion and integration. Yes we partnered with these radio stations. They invited us to specifically engage people on matters cohesions and
integration. First analysis Responses
Frequencies
Percentage (%)
Yes
280
73
No
103
27
Total
383
100 Table 4 (iii): If yes in 4 (ii) above, to what extent if you were to rate between 1 (Unsatisfied) and 10 (Very Satisfied),
did the contents aired impact peace, cohesion and integration on residents of Migori? Responses
Frequancies
Percentage (%)
Unsatisfied
23
6
@on’tknow
32
8.4
Satisfied
256
66.8
Very Satisfied
72
18.8
Total
383
100 African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |48 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj Second analysis Table 4 (i) shows that 73% of the respondents (280 respondents) agreed that the five radio stations aired contents
that were promoting peace in Migori town during the post-poll violence of 2017 while 103 respondents (27%)
denied. This corroborates what was found during the key informants interviews where 15 key informants (78.9%)
agreed that the contents aired promoted peace. 2 key informants (10.5%) disagreed. Participants B&C, whoareprogrammes’editorsinoneofthefiveradiostations studied agree that during the
post-election violence of 2017, the radio station that they work for aired many programmes and ran several news
stories about the violence and peace. They remarked that they developed initiative where political, community and
religious leaders were invited to free talk-shows to address local communities on the need for peace. g
p
We had many programmes on cohesion and integration for the people of Migori. We invited political, religious and
community leaders during talk-shows to talk to people about the need for peace, cohesion and integration among
themselves. It actually worked well for us. I hosted a number of talk-shows; both free and paid ones (Participant B,
April 18, 2021). We had many programmes on cohesion and integration for the people of Migori. We invited political, religious and
community leaders during talk-shows to talk to people about the need for peace, cohesion and integration among
themselves. It actually worked well for us. I hosted a number of talk-shows; both free and paid ones (Participant B,
April 18, 2021). Even though these stations hosted free talk-shows and ensured those invited were from different communities and
backgrounds, some peace organisations paid for the airtime during the talk-shows to have discussions with people
about peace. However, these radio stations still offered extra airtime for free for such programmes. Even though these stations hosted free talk-shows and ensured those invited were from different communities and
backgrounds, some peace organisations paid for the airtime during the talk-shows to have discussions with people
b
t
H
th
di
t ti
till
ff
d
t
i ti
f
f
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h about peace. However, these radio stations still offered extra airtime for free for such programmes. Some communities such as Luhya, Kuria, Luo, which are the majority in Migori County, also organized peace
programmes that were aired on our radio almost on a daily basis. Morning hours we invited religious leaders to pray
and offer spiritual guidance to people on matters peace. African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) Second analysis However, sometimes we as the organisations would buy airtime to have our own related programmes run
on air; though this also sometimes proved difficult when there were had no enough funding (Participant D, April 17,
2021). Participant E argued that the conflict situation was so extreme that at times they would set an agenda of consoling
the victims. This means they would send messages to particular families they knew had been affected by the
violence and educate them, and other vulnerable ones on what to do in such scenarios. We went to the extent of consoling the victims we knew. During our talk-shows and while on air we would also
educate them on how to handle their specific situations and caution them. We also reached out to the relatives of the
victims to inform them about the loss because some were not aware (Participant E, April 17, 2021). African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |49 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj This kind of spreading messages of cohesion and integration during violence is one of the key roles played by the
media, moreso community radios. For example, such strategy was witnessed in Uganda sometime back during the
Lord’sResistancermyconflicts. Military intervention failed and so a community radio, Radio Freedom, was started
in7ulutownin1998,andlater,a‘numberoffightersslowlybeganescapingfromtheLRcamps-a positive step in
the search forpeace’(Laker,2020:16-17). In table 4 (iii), 328 respondents (85.6%) were either satisfied or were very satisfied with the kind of cohesion
and integration messages that were passed through the five radio stations, while 55 respondents (14.4%) were not
satisfied (6%) or didn’tknow(8.4%). From the qualitative data, 17 key informants (89.5%) were satisfied with the
contents while 2 (10.5%) said they felt not satisfied; that more would have been done to enhance cohesion and
integration. Participant F, for example, a programme host in one of the stations under study, said that during their
evaluations through in-calls, they realized many locals were satisfied with the kind of cohesion and integration
messages the particular radio station was passing across. Some confessed that locals were changing their minds
into embracing each other in the villages after listening to the programmes on the radios: This violence went far. People resorted to settling their political scores around. 7. CONTRIBUTIONS TO SCIENTIFIC, COMMUNITY AND FUTURE RESEARCH This is a social science paper whose findings are key in finding peaceful way of life of a community as a result of
effective and proper use of community radio in promoting cohesion and integration. It reveals the destructiveness
and constructiveness of community radios and gives scientific recommendations that when properly and effciently
implemented will be of great significance to communities and the society at large. By doing this, this research
spurs majorly Sustsianable Development Goal 16 that talks about need for Peace, Justice and Strong Instituions. More related study can be conducted to find out if social media can be utilised in a communal way by members of
communities on matters peace, cohesions and integration. Second analysis But through our programmes and talks
about the need to live together peacefully, many confessed to us that they were engaging in political strategies for
peace. They were changing their mindsets (Participant F, April 17, 2021). This is similar to what was evident with the military radio, the Radio Freedom in Gulu, Uganda whose impact was
felt when the LRA conflict ended (Laker, 2020). Therefore, community radio stations in Migori County in one way
or the other promoted peace, cohesion and integration among residents of Migori town during the 2017 post-
election violence in Kenya. 6. RESEARCH IMPLICATIONS 6. RESEARCH IMPLICATIONS States and global peace agencies should fund establishment of peace community radios in conflict-prone areas to
air contents that aim at promoting peace, cohesion and integration. Policies and guidelines should be developed on
how community radios worldover can engage communities they are serving on issues of cohesion and integration. Journalists and other media practitioners should be trained on peace journalism at all costs. Political, religious and
community leaders should be actively engaged in peace initiatives and trained on how to handle media on matters
cohesion and integration. Community radios can also have contents on resource allocation and good governance in
efforts to enhance harmony and integration among the community members they are serving. 9. FUNDING 9. FUNDING This research paper didn’treceiveanyinternal or external funding. References Awoshakin, T. (2010). Civic Journalism Initiatives in Nigeria. In Romano, A. (Ed). International Journalism and
Democracy: Civic Engagement Models from Around the World. New York: Routledge Tylor & Francis
Group. Colin, F. & Sonia, R. (2002). Community Radio for Change and Development. New Delhi: SAGE Publications. Davidson, B. (2010). Public Journalism in South Africa: Experiences and Experiments with Local and Community
Media. In Romano, A. (Ed). International Journalism and Democracy: Civic Engagement Models from
Around the World. New York: Routledge Tylor & Francis Group. Forges, A. D. (2007). Call to Genocide: Radio in Rwanda, 1994. In Thompson, A. (Ed). The Media and Rwanda
Genocide. Kampala: Fountain Publishers. Forges, A. D. (2007). Call to Genocide: Radio in Rwanda, 1994. In Thompson, A. (Ed). The Media and Rwanda
Genocide. Kampala: Fountain Publishers. Human Rights Watch. (2017). Kenya: Post-Election Killings, Abuse: Investigate Police Use of Excessive Force:
Uphold Right to Peaceful Protest. Available at: https://www.hrw.org/news/201708/27/kenya-post-election-
killings-abuse. Laker, G. (2020). Peace Journalism in the LRA Conflict. In Ogenga, F. (Ed). Peace Journalism in East Africa: a
Manual for Media Practitioners. London & New York: Routledge Tylor & Francis Group. Maweu, J. (2020). The Conversation. Nairobi. Available at: https://the conversation.com/how-community-radio-
has-contributed-to-building-peace-a-kenyan-case-study-416222. Muhumuza, R. (2017). Kenya Watchdog says 92 people killed in Election Violence. Available at:
https://apnews.com/article/6c6862192c48c16992653a4972a3c3 Muhumuza, R. (2017). Kenya Watchdog says 92 people killed in Election Violence. Available at:
https://apnews.com/article/6c6862192c48c16992653a4972a3c3 Mutahi, P. (2019). Violence, SecurityandthePolicingofKenya’s20173lections.Journalof3asternfricanStudies
13(2). 1-19. Available at: https://www.researchgate.net/publication/331793528-violence-security-and-the-
policing-of-kenya’s-2017-elections p
g
y
Ochichi, J. P. (2013). Assessment of Community Radio in the Context of Rural Development in Kenya. Nairobi:
University of Nairobi. Ogenga, F. (2020). Thinking About Community Radio and Beyond for Conflict Management in the North Rift. In
Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual for Media Practitioners. London & New York:
Routledge Tylor & Francis Group. Oluoch, J. (2020). Re-Situating Local Mass media: A Tool for Peacebuilding among the Abakuria in Kenya. In
Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual for Media Practitioners. London & New York:
Routledge Tylor & Francis Group. g
y
p
Quarmyne, A.T. (2006). From Homa Bay to Ada: Lessons on Community Radio, Access and Participation. A paper
presentedattheWorldPressCreedom@ayonference,“Media,@evelopmentandPoverty3radication.”Sri
Lanka. Reliefweb. (2021). Kenya: Sexual Violence Committed during 2017 Elections must be Accounted for in the Run-up
to 2022 Elections. A Report by International Federation for Human Rights & Kenya Human Rights
Commission. Available at: https://reliefweb.int/report/kenya/kenya-sexual-violence-committed-during-2017- Romano, A. (2010). 8. CONCLUSION The research findings indicate how important community radios are in promoting peace, cohesion and integration. Through these radios, programmes and talk-shows can be organized to discuss need to peaceful co-existence with
each other. Again, it is through community radios that local people can be engaged to address each other, and
engage one another in enhancing peace, cohesion and integration. Apart from such messages and engagements,
community radios can inform and educate people on how to handle conflicts, resource allocations and sharing, and African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |50 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj
how to channel information to the relevant authorities. Therefore, community radios are key in promoting cohesion
and integration for a better governance. Milambo, Anyuola, Tarumbeta, Onagi and Rameny community radios played
role in promoting cohesion and integration in Migori County during 2017 post-election violence. African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj how to channel information to the relevant authorities. Therefore, community radios are key in promoting cohesion
and integration for a better governance. Milambo, Anyuola, Tarumbeta, Onagi and Rameny community radios played
role in promoting cohesion and integration in Migori County during 2017 post-election violence. how to channel information to the relevant authorities. Therefore, community radios are key in promoting cohesion
and integration for a better governance. Milambo, Anyuola, Tarumbeta, Onagi and Rameny community radios played
role in promoting cohesion and integration in Migori County during 2017 post-election violence. African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) References Deliberation and Journalism. In Romano, A. (Ed). International Journalism and Democracy: African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |51 African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj
Civic Engagement Models from Around the World. New York: Routledge Tylor & Francis Group. Seidu, A., Alhassan, A., & Abdulai A. (2011). The Role of Community Radio in Livelihood Improvement: The Case
of Simli Radio. The Journals of field actions. 5, 2011. Available at:
https://journals.openedition.org/factsreporting/869
The Star. (April 14, 2017). Migori is Cosmopolitan County. The Xinhua. (2017). Over 150 Kenyans injured in Post-Election Violence Chaos: Agencies. Available at:
https://www.xinhuanet.com/english/2017-08/13/c-136522704.htmhinhuaNet
Waki, P., & McFadyen, G. (2008). Report from the Commission of Inquiry into Post-Election Violence. Nairobi. Youngblood, S. (2020). Peace Journalism Approach. In Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual
for Media Practitioners. London & New York: Routledge Tylor & Francis Group. African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj
Civic Engagement Models from Around the World. New York: Routledge Tylor & Francis Group.
Seidu, A., Alhassan, A., & Abdulai A. (2011). The Role of Community Radio in Livelihood Improvement: The Case
of Simli Radio. The Journals of field actions. 5, 2011. Available at:
https://journals.openedition.org/factsreporting/869
The Star. (April 14, 2017). Migori is Cosmopolitan County.
The Xinhua. (2017). Over 150 Kenyans injured in Post-Election Violence Chaos: Agencies. Available at:
https://www.xinhuanet.com/english/2017-08/13/c-136522704.htmhinhuaNet
Waki, P., & McFadyen, G. (2008). Report from the Commission of Inquiry into Post-Election Violence. Nairobi.
Youngblood, S. (2020). Peace Journalism Approach. In Ogenga, F. (Ed). Peace Journalism in East Africa: a Manual
for Media Practitioners. London & New York: Routledge Tylor & Francis Group. African Social Science and Humanities Journal (ASSHJ)
https://journals.jfppublishers.com/asshj African Social Science and Humanities Journal (ASSHJ) - ISSN: 2709-1309 (Print), 2709-1317 (Online) |52
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Cross-sectional associations between multisensory impairment and brain volumes in older adults: Baltimore Longitudinal Study of Aging
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Scientific reports
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www.nature.com/scientificreports www.nature.com/scientificreports Cross‑sectional associations
between multisensory impairment
and brain volumes in older adults:
Baltimore Longitudinal Study
of Aging
OPEN Sensory impairment and brain atrophy is common among older adults, increasing the risk of
dementia. Yet, the degree to which multiple co-occurring sensory impairments (MSI across vision,
proprioception, vestibular function, olfactory, and hearing) are associated with brain morphometry
remain unexplored. Data were from 208 cognitively unimpaired participants (mean age 72 ± 10 years;
59% women) enrolled in the Baltimore Longitudinal Study of Aging. Multiple linear regression
models were used to estimate cross-sectional associations between MSI and regional brain imaging
volumes. For each additional sensory impairment, there were associated lower orbitofrontal gyrus
and entorhinal cortex volumes but higher caudate and putamen volumes. Participants with MSI had
lower mean volumes in the superior frontal gyrus, orbitofrontal gyrus, superior parietal lobe, and
precuneus compared to participants with < 2 impairments. While MSI was largely associated with
lower brain volumes, our results suggest the possibility that MSI was associated with higher basal
ganglia volumes. Longitudinal analyses are needed to evaluate the temporality and directionality of
these associations. Keywords Sensory, Neuroimaging, Brain aging, Olfactory impairment Keywords Sensory, Neuroimaging, Brain aging, Olfactory impairment Sensory impairment in the form of hearing and/or vision loss, proprioceptive impairment, lower vestibular
function, and impaired olfaction is prevalent among older adults1,2. Among older Americans, an estimated 33%
have hearing impairment3, 24% have olfactory impairment4, and 18% have vision impairment3,5. Importantly,
prior research suggests that sensory impairments might be an early sign of cognitive decline6,7 and dementia8. p
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Studies have linked multiple sensory impairments (multisensory impairment [MSI]) to cognitive and physi-
cal decline9,10 and increased risk of mortality among older adults11. Emerging evidence show an association
between single sensory impairments and altered brain structure12,13. Yet, whether the co-occurrence of these
sensory impairments leads to lower volumes in each brain region or additional surrounding brain regions
remains unclear. With neuroimaging evidence linking brain atrophy and cognitive change14, identifying sen-
sory impairments or combinations of MSI associated with brain structure could provide novel mechanistic
insights and modifiable or treatable targets involved between multiple sensory loss and cognitive impairment
and dementia risk.h This study examined whether single and multiple sensory impairments are associated with relative brain
volumes across several regions among cognitively unimpaired older adults. Though this study is exploratory,
we hypothesized that a greater number of sensory impairments would be associated with smaller brain volumes
and as a corollary larger ventricular space. 1Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. 2Center
on Aging and Health, Johns Hopkins University, 2024 E. Monument Street, Suite 2‑700, Rm 2‑726, Baltimore,
MD 21205, USA. 3Intramural Research Program, National Institute on Aging, Baltimore, MD, USA. 4Department of
Health and Community Systems, University of Pittsburgh School of Nursing, Pittsburgh, PA, USA. 5Department of
Otolaryngology, Johns Hopkins School of Medicine, Baltimore, MD, USA. *email: awaniga1@jhu.edu | https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 www.nature.com/scientificreports/ Methods
Participants Participants were enrolled in the Baltimore Longitudinal Study of Aging (BLSA), an ongoing longitudinal cohort
study conducted by the National Institute on Aging Intramural Research Program15. The BLSA recruits par-
ticipants aged 20 years and older with no major physical or cognitive impairments or chronic diseases, except
controlled hypertension. At each visit, participants undergo comprehensive physical, cognitive, sensory assess-
ments, along with neuroimaging exams. Visits are scheduled every 1–4 years, depending on age (participants
under age 60 visited every 4 years, those aged 60–79 years old visited every 2 years, and those aged 80 and above
visited annually). y
Eligibility criteria for this cross-sectional study include: (1) aged 50 years or older and (2) free of mild cogni-
tive impairment or dementia based on a two-step assessment of neurocognitive testing. First, neurocognitive data
of participants were analyzed, and cognitive impairment was defined if their Clinical Dementia Rating score were
≥ 0.516 or if they had > 3 errors on the Blessed Information-Memory-Concentration test17. Second, the diagnosis
of dementia and Alzheimer’s Disease were based on the Diagnostic and Statistical Manual of Mental Disorders, 3rd
ed., Revised18 and the National Institute of Neurological and Communication Disorder and Stroke-Alzheimer’s
Disease and Related Disorders19. Mild cognitive impairment was defined according to the Petersen criteria20.i A total of 775 participants had at least one of five sensory measures collected from December 2015 to Decem-
ber 2018 (Fig. 1). Approximately 54% had all five sensory measures collected at their most recent visit (n = 420). Along the 420 participants, 241 participants also had a brain MRI scan during the same visit. Thirteen partici-
pants were excluded due to cognitive impairments and twenty participants were excluded because they were
younger than 50 years. The final analytic sample was 208 participants ≥ 50 years old who were cognitively unim-
paired with complete data collected for sensory measures and brain MRI. All participants provide written informed consent at each study visit, and the study protocol was approved by
the Institutional Review Board of the National Institutes of Health Intramural Research Program15. All experi-
ments were performed in accordance with U.S. Common Rule, 45 CFR 46. Sensory impairment Vision Vision
Vision was assessed in four ways: visual acuity, visual fields, contrast sensitivity, and stereo acuity. A particip
was considered visually impaired if they had impairment in any of these features. Visual acuity. Presenting visual acuity was based on better eye acuity from an ETDRS (Early Treatment of
Diabetic Retinopathy Study) eye chart. Participants were given the ETDRS eye chart and asked to correctly
read at least 3 letters out of 5 letters. Scores were calculated using the average of the logarithm 10 transfer of the
minimum angle of resolution of the better eye. A score larger than 0.30 was defined as impaired visual acuity24,25. Visual fields. Visual fields were measured with the area of vision that the participant can see using a single
intensity visual field test. The test was given to each eye separately but combined to generate binocular fields
using an algorithm. The visual fields were separated into the central field (56 points), the upper peripheral field
(18 points), and the lower peripheral field (22 points). Scores were calculated by the total number of points
missed (out of a total of 96 points) on the binocular visual field test, and visual field impairment was defined as
a score greater than one standard deviation from the population mean26. Contrast sensitivity. Contrast sensitivity was measured by the ability to discern between shades using a Pelli–
Roboson chart positioned 1 m away, which is a 90 × 60 cm (36 × 24 inches) wall chart with 8 lines of letters with
different contrasts. Each letter was 4.9 × 4.9 cm (2 × 2 inches) large, and there were 6 letters per line. The left 3
letters had more contrast than the right triplet. The contrast of the letters declined from the top to the bottom
and from the left to the right of the chart. The top left had the highest contrast, 100%, and the bottom right had
the lowest contrast, 0.6%. Scores were recorded as the logarithmic contrast sensitivity (1/contrast) of the line that
at least 2 letters of the right triplet were correctly seen. Impairment was defined as the log of contrast sensitivity
< 1.55, based on published population-based studies of older adults aged 60 years and older24,25. Stereo acuity. Stereo acuity measures depth perception which was categorized using the Randot Stereo Vision
Test27,28. Sensory impairment The test booklet was presented upright in front of the participant who was asked to identify stereo
images of decreasing depth differentials. The minimum depth differential in seconds of arcs was recorded (pos-
sible values: 800, 400, 200, 140, 100, 80, 60, 50, or 40), and if the value was less than or equal to 80, the participant
was defined as impaired for stereo acuity24,25. Vestibular function
b l
f f
Vestibular function was assessed by saccular function and semicircular canal function. A participant was con-
sidered impaired in vestibular function if they had impairment in any of these features. Saccular function. Saccular function was measured using the cervical vestibular-evoked myogenic potential
(cVEMP). During the test, participants sat on a chair and had electromyographic (EMG) electrodes placed on
their sternocleidomastoid muscle and sternoclavicular junction bilaterally. The ground electrode was placed
on the manubrium. Sound stimuli (500 Hz and 125 dB tone bursts) were delivered to evoke cervical myo-
genic potentials, which were recorded and normalized for background EMG. Saccular function impairment was
defined if cVEMP was absent bilaterally12. Semicircular canal function. Semicircular canal function was measured by vestibulo-ocular reflex (VOR) gain. VOR was determined using the Video Head Impulse Testing (VHIT), and VOR gain, the ratio of eye velocity to
head velocity was determined using the EyeSeeCam system (Interacoustics, Eden Prarie, MN) by placing it at
the same plane of the horizontal semicircular canals. During VHIT, the participant’s head was placed 30-degree
down from the horizontal axis initially. Then, the participant was asked to fix their gaze on a 1.5-m away wall
while the head was moved randomly 150–250 degrees per second in the horizontal plant toward the right or left. Each side was completed at least 10 times. The EyeSeeCam captured the eye and head velocity to calculate VOR
gain. Semicircular canal function impairment was defined if VOR gain was smaller than 0.712. Proprioception Proprioception was defined by a threshold for perception of passive ankle movement. Participants’ bare feet were
placed on two pedals at a neutral position. The right pedal has a motor that directs its movement, while the left
pedal is moved by the participant. While blindfolded, the participant was asked to move their left foot to match
the rotation angle of the right foot driven by the motor pedal29. There were four trials with a sequential pedal
direction: plantarflexion, dorsiflexion, dorsiflexion, and plantarflexion. Proprioception impairment was defined
as the average of best plantarflexion and best dorsiflexion was > 2.2 degrees, based on established thresholds for
older adults30. MRI‑measured brain volumesh The primary study outcome is regional brain volumes measured by MRI. Brain MRI data were acquired using a
3T Philips Achieva scanner, with a T-1 volumetric scan magnetization prepared rapid acquisition with gradient
echo scan (MPRAGE: repetition time = 6.8 ms, echo time = 3.2 ms, flip angle = 8°, image matrix = 256 × 256, 170
slices, pixel size = 1 × 1 mm, slice thickness = 1.2 mm; sagittal acquisition). Multi-atlas region Segmentation using
Ensembles (MUSE) software was used to obtain anatomical labels and regional brain volumes21,22. There are
48 regional volumes of interest that broadly include total brain; gray and white matter; cerebellum; ventricular
space; frontal, temporal, parietal, and occipital lobes; and specific regions within. Intracranial volume (ICV)
was approximated using a template warping algorithm by estimating ICV residuals based on centering at age
70 years in the larger BLSA sample23. Figure 1. Baltimore longitudinal study of aging (BLSA) participant flowchart (n = 208). Figure 1. Baltimore longitudinal study of aging (BLSA) participant flowchart (n = 208). https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Olfaction Olfaction was measured with the number of correctly identified odors on the 16-item Sniffin’ Sticks Odor Iden-
tification test. Participants were randomly assigned to one of the two odor identification tests, A or B. Olfactory
impairment was defined if the score was below the 10th percentile (8 for test A and 7 for test B) in the current
sample29,31. https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Covariates Covariates in this study include age (years), sex, race, body mass index (BMI; kg/m2), usual gait speed (m/s),
intracranial volume, and years of education. Age, sex, race, and years of educations collected via staff adminis-
tered surveys. Usual gait speed (m/s) was measured during an 6 m overground walking test. BMI was calculated
as weight (measured via digital scale) divided by height squared (measured via stadiometer). Intracranial volume
(cm3) was also added as a covariate and estimated at age 70 years using linear mixed effects models. Each covari-
ate has been separately associated with sensory impairments and brain volumes15,23,34–37. Statistical analysis Independent t-tests or chi-square tests were performed to compare the demographic characteristics of the 208
study participants. Sensory impairments were analyzed dichotomously and included five types: vision, proprio-
ceptive, vestibular, olfactory, and hearing impairment. Multisensory impairment (MSI) was defined when the
individual had two or more co-occurring sensory impairments. Each regional brain volume outcome was treated as a continuous variable using multivariable linear regres-
sion models. We analyzed cross-sectional associations of sensory impairments with brain volumes in four ways,
differentiated based on how sensory impairments (SI) were operationalized. First, each sensory impairment
(yes/no) was included as a predictor. Second, MSI was analyzed as a count (number of sensory impairments,
ranging from 0 to 5). Third, MSI was analyzed as a categorical variable (MSI ≥ 2 sensory impairments vs. MSI
< 2 sensory impairments) to capture co-occurring sensory impairments.f For each model comparing differences in brain volumes by categorical impairment variables, sensitivity
analyses were conducted by redefining the reference of “no impairment” to those with no sensory impairment
across any of the five sensory functions (n = 68). Though this study is exploratory and multiple comparisons were
conducted, we added a correction for multiple comparison testing (n = 336 comparisons) using Benjamin–Hoch-
berg false discovery rate (FDR)38. g
y
All models were adjusted for age (years), sex, race, body mass index (BMI; kg/m2), years of education, usual
gait speed (m/s), and intracranial volume. Two-tailed hypothesis testing with an alpha level = 0.05 was used to
determine statistical significance. All statistical analysis was performed using Stata version 16.0 (Stata Corpora-
tion, College Station, TX; https://www.stata.com/stata16/). Hearing g
Hearing was measured with a speech frequency pure-tone average (PTA) at thresholds of 0.5, 1, 2, and 4 kHz. Participants were seated in a soundproof chamber under unaided conditions with an automated testing device
(Virtual Equipment Co., Audiometer Model 320). The hearing score was both a categorical and continuous vari-
able, with the former defined by the common-used WHO defined categories: normal (PTA < 25 dB), mild loss
(25–40 dB), moderate loss (41–70 dB), and severe loss (> 70 dB). Participants were defined as hearing impaired
if their PTA was ≥ 25 dBHL32,33. Results Demographic characteristics of the final analytic sample (n = 208) by sensory impairment are summarized in
Table 1. The overall mean age was 72.1 years with a standard deviation of 10.1 years (range 50–95 years) and
59% were women. Participants were well-educated (mean years of education 17.7 years, SD = 2.6 years) and
functioning with a mean usual gait speed of 1.17 m/s (SD = 0.22 m/s). Participants with vestibular impairment tended to be older on average than other groups (mean age 80.1 years,
SD = 7.3 years), while participants with proprioceptive impairment tended to be younger than other groups on
average (mean age 74.3 years, SD = 12.5 years). For all sensory impairments except proprioception, the mean
age was higher for those who were impaired than the unimpaired. Those with hearing impairments tended to be
men (p = 0.006) and White (p = 0.007). Participants with impaired vision and vestibular function had significantly
slower usual gait speed than participants without these impairments.h The distribution of sensory impairments by number and type are shown in Table 2. Among 208 participants,
138 participants (66.5%) had no or only one sensory impairment. The most prevalent sensory impairment was
hearing (44.7%) whereas 9.6% had proprioceptive impairment. Forty-four participants (21.1%) had two sensory
impairments with vision and hearing the most common pair (n = 20, 9.6%), followed by vestibular and hearing
(n = 8, 3.8%). For multiple co-occurring impairments, nineteen participants (9.1%) had three, five participants
(2.4%) had four and two participants had five. Single sensory impairment and brain volumes Participants with vision impairment had lower frontal gray matter volume ( β = − 3.00 cm3, SE = 1.45, p = 0.041)
and inferior temporal gyrus volume ( β = − 0.89 cm3, SE = 0.24, p < 0.001) compared to participants without vision
impairment in fully adjusted models, the latter association remained statistically significant after the FDR analysis
(Table 3). Participants with proprioceptive impairment had higher ventricular space ( β = 10.10 cm3, SE = 3.84,
p = 0.009) and frontal lobe white matter ( β = 5.10 cm3, SE = 2.53, p = 0.046), and caudate volumes ( β = 0.38 cm3,
SE = 0.16, p = 0.019), and lower entorhinal cortex volume ( β = − 0.26 cm3, SE = 0.10, p = 0.008) than partici-
pants without proprioceptive impairment. Participants with vestibular impairment had lower superior frontal
gyrus volume ( β = − 0.94 cm3, SE = 0.38, p = 0.015), lower orbitofrontal gyrus volume ( β = − 0.91 cm3, SE = 0.43,
p = 0.033), lower superior parietal lobe volume ( β = − 1.00 cm3, SE = 0.29, p = 0.001), lower superior occipital
gyrus volume ( β = − 0.35 cm3, SE = 0.17, p = 0.047), and lower inferior occipital gyrus volume ( β = − 0.57 cm3,
SE = 0.23, p = 0.016) than those without vestibular impairment. The lower superior parietal lobe volume associa-
tion remained statistically significant after the FDR analysis. Participants with olfactory impairment had lower https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Table 1. Characteristics of study population by composite sensory impairments (n = 208). Independent
t-test was performed for age, body mass index, years of education, and usual gait speed. Chi-square test was
performed for sex, race, and APOE e4 allele. Total
Vision impairment
Proprioception impairment
(n = 208)
Yes
No
p-value
Yes
No
p-value
Participant, no. (%)
208
66 (31.7)
142 (68.3)
20 (9.6)
188 (90.4)
Age (years), mean (sd)
72.1 (10.1)
76.1 (8.7)
70.2 (10.1)
< 0.001
74.3 (12.5)
71.9 (9.8)
0.312
Women, no. (%)
122 (58.7)
38 (57.6)
84 (59.2)
0.830
10 (50.0)
112 (59.6)
0.408
Body Mass Index (kg/m2), mean (sd)
27.3 (4.4)
26.3 (3.8)
27.8 (4.5)
0.028
27.4 (3.6)
27.3 (4.5)
0.935
White, no. Single sensory impairment and brain volumes (%)
128 (61.5)
43 (65.2)
85 (59.9)
0.718
12 (60.0)
116 (61.7)
0.325
Years of education, mean (sd)
17.7 (2.6)
18.2 (2.6)
17.5 (2.6)
0.106
17.8 (2.1)
17.7 (2.6)
0.914
Usual pace gait speed (m/s), mean
(sd)
1.17 (0.22)
1.11 (0.23)
1.20 (0.21)
0.005
1.09 (0.21)
1.18 (0.22)
0.069
APOE e4 allele, no. (%)
50 (24.0)
13 (19.7)
37 (26.1)
0.318
3 (15.0)
47 (25.0)
0.320
Vestibular impairment
Olfaction impairment
Hearing impairment
Yes
No
p-value
Yes
No
p-value
Yes
No
p-value
Participant, no. (%)
38 (18.3)
170 (81.7)
28 (13.5)
180 (86.5)
93 (44.7)
115 (55.3)
Age (years), mean (sd)
80.1 (7.3)
70.3 (9.7)
< 0.001
78.4 (6.9)
71.1 (10.1)
< 0.001
77.5 (8.3)
67.7 (9.1)
< 0.001
Women, no. (%)
20 (52.6)
102 (60.0)
0.404
13 (46.4)
109 (60.6)
0.158
45 (48.4)
77 (67.0)
0.007
Body Mass Index (kg/m2), mean (sd)
26.4 (4.4)
27.5 (4.4)
0.157
26.2 (4.0)
27.5 (4.4)
0.164
26.9 (4.1)
27.7 (4.6)
0.185
White, no. (%)
24 (63.2)
104 (61.2)
0.376
13 (46.4)
115 (63.9)
0.051
69 (74.2)
59 (51.3)
0.006
Years of education, mean (sd)
18.2 (2.4)
17.6 (2.7)
0.274
18.5 (2.0)
17.6 (2.7)
0.081
18.1 (2.7)
17.5 (2.5)
0.094
Usual pace gait speed (m/s), mean
(sd)
1.08 (0.23)
1.20 (0.21)
0.003
1.18 (0.26)
1.17 (0.21)
0.838
1.14 (0.21)
1.20 (0.22)
0.054
APOE e4 allele, no. (%)
6 (15.8)
44 (25.9)
0.188
8 (28.6)
42 (23.3)
0.546
21 (22.6)
29 (25.2)
0.658 Table 1. Characteristics of study population by composite sensory impairments (n = 208). Independent
t-test was performed for age, body mass index, years of education, and usual gait speed. Chi-square test was
performed for sex, race, and APOE e4 allele. orbitofrontal gyrus volume ( β = − 0.94 cm3, SE = 0.50, p = 0.047) and lower posterior cingulate gyrus volume
( β = − 0.34 cm3, = 0.16, p = 0.035).h
3 The associations between vision impairment and inferior temporal gyrus ( β = − 1.33 cm3, SE = 0.41, p = 0.002)
and proprioception impairment and entorhinal cortex ( β = − 0.40 cm3, SE = 0.18, p = 0.035) were robust to sensi-
tivity analyses comparing each sensory impairment with participants who had no sensory impairments (n = 68). The associations between proprioception impairment with ventricular space (p > 0.05) and olfactory impairment
with posterior cingulate gyrus (p > 0.05) were not robust to sensitivity analyses. MSI and brain volumes In fully adjusted models, participants with two or more co-occurring sensory impairments (n = 70, 34%) had
lower mean volume in the frontal gray matter ( β = − 3.20 cm3, SE = 1.62, p = 0.05), parietal lobe gray matter
( β = − 2.51 cm3, SE = 0.97, p = 0.01), superior frontal gyrus ( β = − 1.01 cm3, SE = 0.34, p = 0.003), orbitofrontal gyrus
( β = − 0.91 cm3, SE = 0.38, p = 0.018), superior parietal lobe ( β = − 0.68 cm3, SE = 0.27, p = 0.011), and precuneus
( β = − 0.74 cm3, SE = 0.35, p = 0.038) compared to those with less than two sensory impairments (Table 4, second
column). None of the results remained statistically significant after FDR correction.h y
git
These results were not robust when changing the reference to participants with no sensory impairments. However, the sensitivity analyses showed that those with two or more sensory impairments had lower amygdala
volume ( β = − 0.04 cm3, SE = 0.02, p = 0.04) compared to those with no sensory impairments. Count of sensory impairments and brain volumes
h
dd
l
d Each additional sensory impairment was associated with lower mean volume of the orbitofrontal gyrus
( β = − 0.35 cm3, SE = 0.17, p = 0.04) and in the entorhinal cortex ( β = − 0.09 cm3, SE = 0.03, p = 0.006) in fully
adjusted models (Table 4, first column). Only the association with entorhinal cortex remained statistically sig-
nificant after FDR correction. There was also a higher mean volume in the caudate ( β = 0.14 cm3, SE = 0.05,
p = 0.006) and in the putamen ( β = 0.13 cm3, SE = 0.06, p = 0.043) of the basal ganglia though none of the results
remained statistically significant after FDR correction. Single sensory impairment and brain volumes However, the other associations
had similar magnitude and directionality as reported above but did not reach statistical significance (p > 0.05). Sensory impairment patterns with brain volumes For participants with two co-occurring sensory impairments (MSI = 2), there were 8 observed combinations;
for participants with three sensory impairments (MSI = 3), there were 8 observed combinations; for participants
with four types of sensory impairments (MSI = 4), there were 2 observed combinations (Table 2). Scientific Reports | (2024) 14:9339 | https://doi.org/10.1038/s41598-024-59965-w www.nature.com/scientificreports/ Table 2. Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VES
vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Sensory impairment patterns with brain volumes Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VES
vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Table 2. Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VES
vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Scientific Reports | (2024) 14:9339 | Sensory impairment patterns with brain volumes Numbers of participants
Numbers of sensory impairment
0
68 (33.0%)
1
70 (33.5%)
2
44 (21.1%)
3
19 (9.1%)
4
5 (2.4%)
5
2 (1.0%)
Combinations of two impairments
VS + H
20 (9.6%)
VES + H
8 (3.8%)
SML + H
4 (1.9%)
VS + SML
3 (1.4%)
VS + VES
3 (1.4%)
VS + PROP
2 (1.0%)
VES + SML
2 (1.0%)
PROP + H
2 (1.0%)
VES + PROP
0 (0.0%)
PROP + SML
0 (0.0%)
Combinations of three impairments
VS + SML + H
6 (2.9%)
VS + VES + H
5 (2.4%)
VS + PROP + H
2 (1.0%)
VES + SML + H
2 (1.0%)
VS + VES + PROP
1 (0.5%)
VS + VES + SML
1 (0.5%)
VES + PROP + H
1 (0.5%)
PROP + SML + H
1 (0.5%)
VES + PROP + SML
0 (0.0%)
VS + PROP + SML
0 (0.0%)
Combinations of four impairments
VS + VES + SML + H
3 (1.4%)
VS + VES + PROP + H
2 (1.0%)
VS + VES + PROP + SML
0 (0.0%)
VS + PROP + SML + H
0 (0.0%)
VES + PROP + SML + H
0 (0.0%) Numbers of participants
Numbers of sensory impairment
0
68 (33.0%)
1
70 (33.5%)
2
44 (21.1%)
3
19 (9.1%)
4
5 (2.4%)
5
2 (1.0%)
Combinations of two impairments
VS + H
20 (9.6%)
VES + H
8 (3.8%)
SML + H
4 (1.9%)
VS + SML
3 (1.4%)
VS + VES
3 (1.4%)
VS + PROP
2 (1.0%)
VES + SML
2 (1.0%)
PROP + H
2 (1.0%)
VES + PROP
0 (0.0%)
PROP + SML
0 (0.0%)
Combinations of three impairments
VS + SML + H
6 (2.9%)
VS + VES + H
5 (2.4%)
VS + PROP + H
2 (1.0%)
VES + SML + H
2 (1.0%)
VS + VES + PROP
1 (0.5%)
VS + VES + SML
1 (0.5%)
VES + PROP + H
1 (0.5%)
PROP + SML + H
1 (0.5%)
VES + PROP + SML
0 (0.0%)
VS + PROP + SML
0 (0.0%)
Combinations of four impairments
VS + VES + SML + H
3 (1.4%)
VS + VES + PROP + H
2 (1.0%)
VS + VES + PROP + SML
0 (0.0%)
VS + PROP + SML + H
0 (0.0%)
VES + PROP + SML + H
0 (0.0%) Table 2. Table 2. Distribution of multisensory impairments (n = 208). VS visually impaired, H hearing impaired, VE
vestibular impaired, SML olfactory impaired, PROP proprioceptive impaired. Discussion
Th
d
h This study characterized cross-sectional associations between co-occurring sensory impairments and brain
volumes in cognitively unimpaired adults aged 50 years and older. Combinations of MSI that include vision,
proprioceptive, and/or vestibular impairments were associated with lower brain volumes in the frontal gray
matter, superior frontal gyrus, orbitofrontal gyrus, superior parietal lobe, and entorhinal cortex regions. Further,
there is a possibly that MSI is uniquely associated with the lower amygdala volume. Oppositely, MSI was associ-
ated with higher volumes in the basal ganglia region, suggesting a possible compensatory mechanism within
this region as the brain attempts to adapt to brain atrophy in other regions. Collectively, these results highlight
potential associations between MSI and brain structure so that future longitudinal research focusing on these
regions may be performed to understand the temporality of these associations.i g
y
p
p
y
It is unclear the specific sensory impairments that drive the detected association between MSI and lower
volume in the orbitofrontal gyrus. However, in the single sensory impairment analyses, vestibular and olfactory
impairment separately were significantly associated with lower orbitofrontal gyrus volume. This suggests that
a combination of vestibular and/or olfactory impairments may reflect or result from lower orbitofrontal gyrus
volume, but the sample with both vestibular and olfactory impairment is too small to produce stable estimates. This is supported by animal studies showing that neural pathways between sensory cortices and the orbitofrontal
gyrus exhibit diminished numbers of neural connections in animals with sensory impairments39,40. It is important
to acknowledge that this finding was not robust when accounting for multiple testing.h gi
g
g
p
g
The observed association between MSI and lower volume in the entorhinal cortex is consistent with kno
inputs from the vestibular system into the entorhinal cortex, which contains a neuronal population called Scientific Reports | (2024) 14:9339 | https://doi.org/10.1038/s41598-024-59965-w www.nature.com/scientificreports/ Table 3. Differences in mean regional brain volumes (cm3) for each separate sensory impairment^. Table 3. Differences in mean regional brain volumes (cm3) for each separate sensory impairment^. All are
multivariable linear regression models adjusted for age, sex, race, BMI (body mass index), icv70 (intracranial
volume at age 70 years old), and years of education. ^The reference group for each column is no impairment
in that specific sensory function. *p < 0.05; **p < 0.01; ***p < 0.001. # The association remained statistically
significant after the FDR analysis. Discussion
Th
d
h All are
multivariable linear regression models adjusted for age, sex, race, BMI (body mass index), icv70 (intracranial h
Vision impairment
Proprioceptive impairment
Vestibular impairment
Olfactory impairment
Hearing
impairment
Beta coefficient (SE)
Cerebellum
1.867 (1.593)
− 4.054 (2.290)
− 0.475 (1.960)
1.579 (2.273)
− 0.162 (1.677)
Total brain
0.940 (6.412)
16.157 (9.613)
− 10.873 (7.914)
6.196 (8.725)
2.909 (6.763)
Ventricular space
0.473 (2.584)
10.095 (3.836)**
− 1.685 (3.203)
3.900 (3.510)
0.634 (2.726)
Gray matter
− 4.846 (3.846)
− 1.238 (5.829)
− 6.684 (4.765)
0.235 (5.260)
0.628 (4.074)
White matter
5.145 (3.676)
7.734 (5.549)
− 2.478 (4.577)
1.873 (5.030)
1.500 (3.896)
Frontal lobe
− 0.604 (2.544)
7.129 (3.808)
− 2.322 (3.151)
− 2.155 (3.463)
1.809 (2.681)
Temporal lobe
− 0.932 (1.378)
− 0.377 (2.083)
− 1.798 (1.706)
0.872 (1.879)
1.173 (1.454)
Parietal lobe
− 0.033 (1.463)
0.720 (2.208)
− 2.657 (1.805)
0.910 (1.992)
− 0.107 (1.544)
Occipital lobe
− 0.136 (1.168)
1.977 (1.758)
− 1.520 (1.445)
0.691 (1.591)
− 1.024 (1.230)
Frontal gray matter
− 2.997 (1.454)*
2.030 (2.213)
− 2.194 (1.815)
− 1.839 (1.997)
0.723 (1.549)
Temporal gray matter
− 1.710 (0.902)
− 1.390 (1.370)
− 0.886 (1.126)
− 0.177 (1.239)
1.042 (0.957)
Parietal gray matter
− 1.018 (0.881)
− 0.333 (1.334)
− 2.057 (1.087)
0.361 (1.204)
− 0.400 (0.932)
Occipital gray matter
− 0.571 (0.801)
0.574 (1.210)
− 0.946 (0.992)
0.250 (1.093)
− 0.657 (0.845)
Frontal white matter
2.393 (1.687)
5.098 (2.534)*
− 0.128 (2.103)
− 0.316 (2.310)
1.086 (1.787)
Temporal white matter
0.778 (0.877)
1.014 (1.325)
− 0.912 (1.088)
1.049 (1.195)
0.131 (0.927)
Parietal white matter
0.985 (0.874)
1.054 (1.322)
− 0.600 (1.087)
0.549 (1.194)
0.294 (0.925)
Occipital white matter
0.435 (0.535)
1.403 (0.803)
− 0.574 (0.663)
0.441 (0.729)
− 0.366 (0.565)
Superior frontal gyrus
− 0.578 (0.309)
0.381 (0.470)
− 0.938 (0.381)**
− 0.629 (0.422)
0.408 (0.327)
Middle frontal gyrus
− 0.397 (0.393)
0.454 (0.594)
0.044 (0.488)
0.152 (0.536)
0.350 (0.415)
Inferior frontal gyrus
− 0.199 (0.218)
0.307 (0.329)
− 0.091 (0.271)
0.070 (0.298)
0.018 (0.231)
Medial frontal cortex
− 0.017 (0.069)
0.023 (0.105)
− 0.113 (0.086)
− 0.003 (0.095)
− 0.010 (0.073)
Orbitofrontal gyrus
− 0.618 (0.345)
0.338 (0.524)
− 0.914 (0.426)*
− 0.936 (0.469)*
0.139 (0.367)
Precentral gyrus
− 0.298 (0.258)
0.187 (0.391)
0.338 (0.320)
− 0.228 (0.353)
0.021 (0.273)
Postcentral gyrus
− 0.030 (0.266)
− 0.355 (0.401)
− 0.412 (0.329)
0.127 (0.363)
0.069 (0.281)
Superior parietal lobe
− 0.239 (0.241)
− 0.046 (0.365)
− 0.999 (0.291)**#
− 0.205 (0.329)
0.160 (0.255)
Supramarginal gyrus
− 0.169 (0.224)
0.280 (0.337)
− 0.394 (0.276)
0.111 (0.305)
0.038 (0.236)
Angular gyrus
− 0.255 (0.276)
− 0.526 (0.416)
0.316 (0.342)
0.196 (0.376)
− 0.399 (0.290)
Precuneus
− 0.346 (0.320)
0.233 (0.485)
− 0.529 (0.397)
0.202 (0.438)
− 0.300 (0.338)
Superior temporal gyrus
0.125 (0.194)
0.159 (0.293)
− 0.233 (0.240)
− 0.190 (0.264)
0.289 (0.204)
Middle temporal gyrus
− 0.328 (0.340)
− 0.923 (0.511)
− 0.242 (0.423)
− 0.122 (0.465)
− 0.250 (0.359)
Inferior temporal gyrus
− 0.889 (0.240)***#
− 0.199 (0.374)
− 0.040 (0.308)
− 0.109 (0.338)
0.410 (0.260)
Hippocampus
− 0.086 (0.092)
0.059 (0.139)
− 0.118 (0.114)
-0.046 (0.125)
− 0.162 (0.096)
Parahippocampus
− 0.069 (0.091)
− 0.060 (0.137)
− 0.132 (0.112)
0.010 (0.124)
− 0.044 (0.096)
Entorhinal cortex
− 0.089 (0.066)
− 0.264 (0.098)**
− 0.104 (0.082)
− 0.115 (0.090)
− 0.034 (0.070)
Amygdala
− 0.018 (0.030)
− 0.034 (0.045)
− 0.020 (0.037)
− 0.014 (0.041)
− 0.050 (0.031)
Fusiform gyrus
− 0.151 (0.218)
− 0.177 (0.329)
0.029 (0.271)
0.145 (0.297)
0.294 (0.229)
Superior occipital gyrus
0.089 (0.142)
0.030 (0.214)
− 0.348 (0.174)*
0.051 (0.193)
0.141 (0.149)
Middle occipital gyrus
− 0.306 (0.179)
0.156 (0.272)
0.052 (0.224)
− 0.083 (0.246)
− 0.156 (0.190)
Inferior occipital gyrus
− 0.108 (0.192)
− 0.116 (0.289)
− 0.567 (0.234)*
− 0.014 (0.261)
− 0.234 (0.202)
Occipital pole
− 0.108 (0.142)
0.282 (0.214)
0.190 (0.176)
− 0.071 (0.194)
0.002 (0.150)
Cuneus
0.146 (0.178)
0.018 (0.269)
0.147 (0.221)
0.120 (0.243)
-0.058 (0.188)
Anterior cingulate gyrus
− 0.064 (0.160)
0.470 (0.240)
0.024 (0.199)
− 0.176 (0.218)
0.222 (0.168)
Posterior cingulate gyrus
− 0.143 (0.117)
0.150 (0.177)
0.024 (0.146)
− 0.336 (0.158)*
-0.089 (0.124)
Middle cingulate gyrus
− 0.090 (0.137)
0.205 (0.206)
− 0.093 (0.169)
0.055 (0.186)
0.163 (0.144)
Caudate
0.170 (0.107)
0.378 (0.160)*
0.063 (0.133)
0.254 (0.145)
0.084 (0.113)
Globus pallidus
0.024 (0.040)
0.0130 (0.060)
− 0.022 (0.050)
− 0.020 (0.055)
0.065 (0.042)
Putamen
0.119 (0.128)
0.189 (0.193)
0.159 (0.159)
0.267 (0.174)
0.085 (0.135)
Thalamus
− 0.108 (0.144)
0.321 (0.217)
0.091 (0.179)
0.186 (0.196)
− 0.026 (0.152) https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Table 4. Discussion
Th
d
h Associations between MSI (as a count and in categories) and mean regional b
(n = 208). SI sensory impairment, MSI multisensory impairment. All are multivariable l
dj
t d f
BMI (b d
i d
) i
70 (i t
i l
l
t
70
Continuous SI ranging from 0 to 5 impairments
Categorical MSI
(MSI ≥ 2 vs. Table 4. Associations between MSI (as a count and in categories) and mean regional brain volumes (cm3)
(n = 208). SI sensory impairment, MSI multisensory impairment. All are multivariable linear regression models
adjusted for age, sex, race, BMI (body mass index), icv70 (intracranial volume at age 70 years old), and years
of education. *p < 0.05; **p < 0.01; ***p < 0.001. # The association remained statistically significant after the FDR
analysis. www.nature.com/scientificreports/ y
gg
g
g
g
y
g
g
p y
g
Two or more co-occurring sensory impairments were associated with lower volumes in the gray matter of
the frontal and parietal lobes, in the superior frontal gyrus and superior parietal lobe, and the precuneus. Our
findings suggest that these gray matter associations may be driven by vision impairment and the frontal and
parietal lobe associations driven by vestibular impairment. However, caution in interpreting these exploratory
findings should be noted since the prevalence of various patterns was low and subject to type 1 error due to
multiple testing and did not remain statistically significant after accounting for multiple testing.hhii g
y
git
g
g
There are limitations to acknowledge. The first limitation is the small sample size, that was defined with par-
ticipants who had all five sensory measures plus complete brain MRI scans. Second, the generalizability of the
findings is narrow because BLSA participants tend to have higher education and better health than the general
older adult population. Third, the prevalence of MSI with three or more sensory impairments observed was low,
yielding low statistical power. Fourth, the cross-sectional design does not account for temporality and thus the
direction of association is unclear. Fifth, multiple statistical tests were performed, increasing the likelihood of
type 1 error. Though this was an exploratory study, we performed FDR analyses in the sensitivity analyses to
address issues with multiple comparison testing. Yet, when doing so might increase type II error that reduces
the detection of true associations.h This study has multiple strengths. First, sensory impairments were comprehensively measured across a large
sample of older adults. Second, brain volumes were quantified via brain MRI scans. Third, this study was able to
describe specific patterns of MSI and explore their relationships to brain volumetric measures. pi
p
p
p
In conclusion, this study found that higher numbers of sensory impairments were linked to higher ventricular
volumes and lower brain volumes, primarily in the superior frontal gyrus, orbitofrontal gyrus, and precuneus. In contrast, those living with MSI had higher volumes in the basal ganglia regions. Future research is needed to
explore longitudinal associations to evaluate whether multiple sensory impairments lead to accelerated brain
atrophy and whether there are some brain regions may be preserved or activated (i.e., the basal ganglia) in
response, and assess whether these relationships are linked to cognitive outcomes. www.nature.com/scientificreports/ cells which are involved in spatial navigation41. Indeed, vestibular impairment has been associated with entorhinal
cortex atrophy in prior work42. These findings are also consistent with a previous study in rats, which performed
deafferentation injury surgeries on the entorhinal cortex of rats’ brains and assessed the rats’ performance using
sensory integration testing. By analyzing histological brain slides and performance scores, researchers concluded
that rats with lesions in the entorhinal cortex showed sensory integration deficits and behavioral change43. This
suggests that the entorhinal cortex plays a role in multisensory function. Future research into the biological
mechanisms behind the link between MSI and both the orbitofrontal gyrus and entorhinal cortex in humans
is warranted. In sensitivity analyses comparing MSI versus no sensory impairment, a possible unique association was
revealed between two or more sensory impairments and lower amygdala volumes. If this association is true, this
is consistent with the amygdala’s role in the processing of sensory information44. However, this finding did not
remain statistically significant when accounting for multiple testing. Without replication of this finding in larger
studies, this relationship is likely spurious since it was not detected in the main analysis. MSI and proprioceptive impairment alone were associated with a higher mean volume in the basal ganglia
region in the main analysis but did not remain statistically significant after multiple testing correction. Still, a
possible explanation for this potential finding is that the basal ganglia region may play a compensatory role in
how the brain adapts to MSI. The basal ganglia are involved in both motor control and cognitive function45 and
are thought to work with cortical regions in executing stereotyped motor and cognitive actions under cortical
volitional control. Conceivably, with the loss of sensory-driven cortical control, “implicit” behaviors and actions
stored in the basal ganglia are increasingly activated or are lost more slowly than other brain regions. A previous
study using a smaller sample of BLSA participants also demonstrated that worse vestibular function was related
to higher volumes of the basal ganglia region, the caudate and putamen, specifically42. Also, this finding might
coincide with the basal ganglia tending to be last to atrophy and lose function according to the “first in, last out”
principle46. Collectively, these findings suggest the possibility that proprioceptive and/or vestibular dysfunction
may trigger a higher utilization of the basal ganglia, which indirectly mitigates this region’s atrophy with age. www.nature.com/scientificreports/ Also, leveraging machine
learning approaches (e.g., random forest) using MSI information to detect or predict changes in brain volumes
are warranted. Such work may help provide mechanistic insights linking sensory impairments with aging brain
and detection of cognitive decline. Replication is needed given the exploratory nature of the findings and the
possibility of false discovery. Data availability y
Because of the sensitive nature of the data collected for this study, requests to access the data set from qualified
researchers trained in human subject confidentiality protocols may be sent to the Intramural Research Program
of the National Institute on Aging at https://blsa.nih.gov. Received: 29 November 2023; Accepted: 17 April 2024 Received: 29 November 2023; Accepted: 17 April 2024 Discussion
Th
d
h MSI < 2)
Beta coefficient (SE)
Cerebellum
0.055 (0.762)
− 1.382 (1.762)
Total brain
1.679 (3.158)
− 5.286 (7.140)
Ventricular space
1.574 (1.269)
1.747 (2.879)
Gray matter
− 2.197 (1.897)
− 8.332 (4.265)
White matter
2.255 (1.813)
1.605 (4.117)
Frontal lobe
0.359 (1.254)
− 1.429 (2.835)
Temporal lobe
− 0.180 (0.680)
− 1.053 (1.537)
Parietal lobe
− 0.255 (0.721)
− 2.722 (1.620)
Occipital lobe
− 0.195 (0.576)
− 0.216 (1.302)
Frontal gray matter
− 0.941 (0.721)
− 3.197 (1.622)*
Temporal gray matter
− 0.499 (0.447)
− 1.334 (1.010)
Parietal gray matter
− 0.648 (0.433)
− 2.510 (0.970)*
Occipital gray matter
− 0.338 (0.395)
− 0.579 (0.893)
Frontal white matter
1.300 (0.831)
1.768 (1.886)
Temporal white matter
0.319 (0.433)
0.282 (0.980)
Parietal white matter
0.393 (0.431)
− 0.211 (0.978)
Occipital white matter
0.142 (0.264)
0.363 (0.597)
Superior frontal gyrus
− 0.241 (0.153)
− 1.014 (0.340)**
Middle frontal gyrus
0.055 (0.194)
− 0.15 (0.439)
Inferior frontal gyrus
− 0.017 (0.108)
− 0.118 (0.243)
Medial frontal cortex
− 0.022 (0.034)
0.015 (0.077)
Orbitofrontal gyrus
− 0.351 (0.170)*
− 0.914 (0.382)**
Precentral gyrus
− 0.024 (0.128)
− 0.129 (0.289)
Postcentral gyrus
− 0.078 (0.131)
− 0.356 (0.296)
Superior parietal lobe
− 0.213 (0.118)
− 0.679 (0.265)**
Supramarginal gyrus
− 0.051 (0.110)
− 0.456 (0.248)
Angular gyrus
− 0.130 (0.136)
− 0.302 (0.307)
Precuneus
− 0.182 (0.158)
− 0.740 (0.354)*
Superior temporal gyrus
0.049 (0.096)
− 0.0001 (0.216)
Middle temporal gyrus
− 0.287 (0.167)
− 0.548 (0.378)
Inferior temporal gyrus
− 0.168 (0.122)
− 0.367 (0.276)
Hippocampus
− 0.075 (0.045)
− 0.114 (0.102)
Parahippocampus
− 0.052 (0.045)
− 0.119 (0.101)
Entorhinal cortex
− 0.089 (0.032)**#
− 0.081 (0.074)
Amygdala
− 0.024 (0.015)
− 0.043 (0.033)
Fusiform gyrus
0.032 (0.108)
− 0.175 (0.243)
Superior occipital gyrus
0.007 (0.070)
− 0.038 (0.158)
Middle occipital gyrus
− 0.094 (0.089)
− 0.183 (0.201)
Inferior occipital gyrus
− 0.181 (0.094)
− 0.211 (0.213)
Occipital pole
0.025 (0.070)
− 0.065 (0.159)
Cuneus
0.064 (0.088)
0.197 (0.198)
Anterior cingulate gyrus
0.064 (0.079)
0.106 (0.179)
Posterior cingulate gyrus
− 0.078 (0.058)
− 0.173 (0.131)
Middle cingulate gyrus
0.028 (0.067)
0.006 (0.152)
Caudate
0.143 (0.052)**
0.213 (0.119)
Globus pallidus
0.015 (0.020)
0.017 (0.045)
Putamen
0.128 (0.063)*
0.173 (0.143)
Thalamus
0.041 (0.071)
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22, 295–301 (2012). 2. Correia, C. et al. Global sensory impairment in older adults in the United States. J. Am. Geriatr. Soc. 64, 306–313 (2016). 3. Crews, J. E. & Campbell, V. A. Vision impairment and hearing loss among community-dwelling older Americans: Implications
for health and functioning. Am. J. Public Health 94, 823–829 (2004). g
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4. Murphy, C. Prevalence of olfactory impairment in older adults. JAMA 288, 2307 (2002). https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | Acknowledgementsh g
The authors of this manuscript thank the participants and staff in the Baltimore Longitudinal Study of Aging
for their contributions. https://doi.org/10.1038/s41598-024-59965-w Scientific Reports | (2024) 14:9339 | www.nature.com/scientificreports/ Competing interests
Th
h
d
l The authors declare no competing interests. Author contributions JAS, YA, AAW, and CT conceptualized the project. All authors participated in the analysis, interpretation and
in drafting and critically reviewing the manuscript for intellectual content and developed the final version for
submission. All authors read and approved this manuscript. Funding YA, ALG, JAS and AAW were supported by Grant Number R01AG061786 from the National Institute on Aging,
National Institutes of Health. AAW was supported by Grant Number K01AG076967 from the National Institute
on Aging, National Institutes of Health. The Baltimore Longitudinal Study of Aging was supported by the Intra-
mural Research Program of the National Institute on Aging (ZIAAG000015 and ZIAAG000191). © The Author(s) 2024 Additional information Correspondence and requests for materials should be addressed to A.A.W. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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McNally, A, Kallonen, T, Connor, C, Abudahab, K, Aanensen, DM, Horner, C, Peacock, SJ, Parkhill, J,
Croucher, NJ & Corander, J 2019, 'Diversification of colonization factors in a multidrug-resistant Escherichia coli
lineage evolving under negative frequency-dependent selection', mBio, vol. 10, no. 2, e00644-19. https://doi.org/10.1128/mBio.00644-19 Link to publication on Research at Birmingham portal Link to publication on Research at Birmingham portal Publisher Rights Statement:
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McNally, A, Kallonen, T, Connor, C, Abudahab, K, Aanensen, DM, Horner, C, Peacock, SJ, Parkhill, J,
Croucher, NJ & Corander, J 2019, 'Diversification of colonization factors in a multidrug-resistant Escherichia coli
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the work immediately and investigate. Download date: 24. Oct. 2024 RESEARCH ARTICLE
Ecological and Evolutionary Science RESEARCH ARTICLE
Ecological and Evolutionary Science
crossm RESEARCH ARTICLE
Ecological and Evolutionary Science
crossm Citation McNally A, Kallonen T, Connor C,
Abudahab K, Aanensen DM, Horner C, Peacock
SJ, Parkhill J, Croucher NJ, Corander J. 2019.
Diversification of colonization factors in a
multidrug-resistant Escherichia coli lineage
evolving under negative frequency-dependent
selection. mBio 10:e00644-19. https://doi.org/
10.1128/mBio.00644-19. Citation McNally A, Kallonen T, Connor C,
Abudahab K, Aanensen DM, Horner C, Peacock
SJ, Parkhill J, Croucher NJ, Corander J. 2019.
Diversification of colonization factors in a
multidrug-resistant Escherichia coli lineage
evolving under negative frequency-dependent
selection. mBio 10:e00644-19. https://doi.org/
10.1128/mBio.00644-19. Diversification of Colonization Factors in a Multidrug-Resistant
Escherichia coli Lineage Evolving under Negative Frequency-
Dependent Selection Alan McNally,a Teemu Kallonen,b,c Christopher Connor,a Khalil Abudahab,b David M. Aanensen,b Carolyne Horner,d
Sharon J. Peacock,b,e,f
Julian Parkhill,b Nicholas J. Croucher,g Jukka Coranderb,c,h aInstitute of Microbiology and Infection, University of Birmingham, Birmingham, United Kingdom
bInfection Genomics, Wellcome Sanger Institute, Cambridge, United Kingdom
cDepartment of Biostatistics, University of Oslo, Oslo, Norway
dBritish Society of Antimicrobial Chemotherapy, Birmingham, United Kingdom
eDepartment of Medicine, University of Cambridge, Cambridge, United Kingdom
fLondon School of Hygiene and Tropical Medicine, London, United Kingdom
gFaculty of Medicine, School of Public Health, Imperial College, London, United Kingdom
hDepartment of Mathematics and Statistics, University of Helsinki, Helsinki, Finland on May 8, 2019 by guest
http://mbio.asm.org/
aded from ABSTRACT
Escherichia coli is a major cause of bloodstream and urinary tract infec-
tions globally. The wide dissemination of multidrug-resistant (MDR) strains of ex-
traintestinal pathogenic E. coli (ExPEC) poses a rapidly increasing public health
burden due to narrowed treatment options and increased risk of failure to clear an
infection. Here, we present a detailed population genomic analysis of the ExPEC
ST131 clone, in which we seek explanations for its success as an emerging patho-
genic strain beyond the acquisition of antimicrobial resistance (AMR) genes. We
show evidence for evolution toward separate ecological niches for the main clades
of ST131 and differential evolution of anaerobic metabolism, key colonization, and
virulence factors. We further demonstrate that negative frequency-dependent selec-
tion acting across accessory loci is a major mechanism that has shaped the popula-
tion evolution of this pathogen. IMPORTANCE Infections with multidrug-resistant (MDR) strains of Escherichia coli are
a significant global public health concern. To combat these pathogens, we need a
deeper understanding of how they evolved from their background populations. By
understanding the processes that underpin their emergence, we can design new
strategies to limit evolution of new clones and combat existing clones. By combin-
ing population genomics with modelling approaches, we show that dominant MDR
clones of E. coli are under the influence of negative frequency-dependent selection,
preventing them from rising to fixation in a population. Furthermore, we show that
this selection acts on genes involved in anaerobic metabolism, suggesting that this
key trait, and the ability to colonize human intestinal tracts, is a key step in the evo-
lution of MDR clones of E. coli. Editor Julian E. Davies, University of British
Columbia Copyright © 2019 McNally et al. Diversification of Colonization Factors in a Multidrug-Resistant
Escherichia coli Lineage Evolving under Negative Frequency-
Dependent Selection The lack of phylogeographic signal and phylogenetic
structure based on host source suggests rapid global dispersal and frequent host
transitions within clade C (14). Research has suggested that the acquisition of fluoro-
quinolone resistance via point mutations in DNA gyrase and DNA topoisomerase genes
was the primary driver in the rapid emergence of clade C, alongside the predated
acquisition of well-defined ExPEC virulence factors (11, 12). Later work also suggested
that clade C E. coli ST131 may dominate as a successful MDR clade due to the ability to
offset the fitness cost of MDR plasmid acquisition and maintenance via compensatory
mutations in gene regulatory regions (14). Genome-wide association studies (GWAS)
have been used to identify loci and lineage-specific alleles significantly associated with
clade C E. coli ST131, which suggested a secondary flagellum locus encoding lateral
flagella (Flag-2 [15]), and a number of hypothetical proteins and promoter regions as
being clade C E. coli ST131-associated loci (14). Recent work on E. coli causing bacteremia provided compelling evidence that
resistance to antimicrobials has not been the major driver of the success of ST131 (16). An analysis of a large 11-year population survey across the United Kingdom showed
that ST131 rapidly stabilized at a level of approximately 20% after its emergence around
2002 in the United Kingdom. This was far in excess of already-resident MDR clones, such
as ST88 or ST405. Nevertheless, the overall prevalence of resistance phenotypes re-
mained approximately constant in the population. Furthermore, most currently known
major ExPEC clones (primarily ST12, ST73, ST95, and ST69, the last of which also rapidly
emerged in 2002) show a similar stable population frequency across the 10 years
following the introduction of ST131, despite exhibiting far less extensive resistance
profiles. These observations suggested the distribution of ExPEC strains was shaped by
negative frequency-dependent selection (NFDS) (16). NFDS describes the situation in
which a given phenotype is most beneficial to a population when it is rare, such as the
emergence of a new antigen or resource-use strategy. This is because as the phenotype
becomes common it either becomes costly, such as when antigens are recognized by
an increased proportion of hosts, or less beneficial, such as when strains compete more
fiercely for the same resources. Recently, a multilocus NFDS model of postvaccination Streptococcus pneumoniae
population dynamics was described (17). Diversification of Colonization Factors in a Multidrug-Resistant
Escherichia coli Lineage Evolving under Negative Frequency-
Dependent Selection This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license. KEYWORDS AMR, Escherichia coli, evolutionary genomics, negative frequency-
dependent selection E
scherichia coli is now the most common cause of bloodstream infections in the
developed world, outnumbering cases of Staphylococcus aureus bacteremia by 2:1
(1). E. coli is also the most common cause of urinary tract infections (UTI), which in turn
are among the most common bacterial infections in the world (2). Bacteremia and UTI
are caused by a subset of E. coli termed extraintestinal pathogenic E. coli (ExPEC). ExPEC E
s
d March/April 2019
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e00644-19 ® ®
mbio.asm.org
1 mbio.asm.org
1 ® McNally et al. are not a phylogenetically distinct group of E. coli but rather represent strains which
have acquired virulence-associated genes that confer the ability to invade and cause
disease in extraintestinal sites (3). Genes associated with virulence that confer the
ability to adhere to extraintestinal tissues, to sequester extracellular iron, and to evade
the nonspecific immune response and toxins resulting in localized tissue destruction
have all been described as essential in the process of ExPEC pathogenesis (4). The problem presented by the scale of ExPEC infections is exacerbated by the
number of cases involving multidrug-resistant (MDR) strains (1, 5, 6). Epidemiological
surveys report as many as 60% of UTI ExPEC isolates as being resistant to three or more
classes of antibiotics and as many as 50% of bacteremia isolates (5, 6). The increase in
MDR ExPEC prevalence has been rapid and primarily attributable to a small number of
ExPEC lineages (5). The most common of these is the E. coli ST131 lineage, which has
rapidly become a dominant cause of ExPEC UTI and bacteremia globally (5–7). E. coli
ST131 is particularly associated with carriage of the CTX-M class of extended-spectrum
-lactamase (ESBL) which confers resistance to 3rd-generation cephalosporins (7), and
there have been a small number of reports of E. coli ST131 isolates carrying metallo-
-lactamases conferring resistance to carbapenems (8). The carriage of these resistance
genes is driven by the acquisition and stable maintenance of large MDR plasmids (9). The phylogenetic structure of E. coli ST131 is well characterized (10–14) and shows
the emergence of a globally disseminated MDR-associated clade C from primarily
drug-susceptible clades A and B. March/April 2019
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e00644-19 RESULTS Species-wide distribution of the E. coli accessory genome. The multilocus NFDS
model devised to explain the impact of vaccination on the S. pneumoniae population
was applied to the 1,094 systematically sampled E. coli isolates from the BSAC collection
(16). Collected between 2001 and 2011, this collection revealed that the sequence
clusters, or strains, corresponding primarily to sequence types ST69 and ST131 rose
rapidly at the start of the sampling period, prior to stabilizing at frequencies of 8% and
18%, respectively. The rise of ST131 was primarily driven by increases in MDR clade C
isolates, although isolates from ST69 and ST131 clade B rose in frequency despite them
lacking such a resistance profile. It is possible this represents drift in a neutral popu-
lation. However, this seemed unlikely, given that the invading genotypes rose quickly
and displaced some strains, such as ST10, ST14, and ST144, while leaving the common
ST73 and ST95 at quite stable frequencies. Alternatively, these strains could each be
filling one distinct separate niche. However, these two strains displace multiple other
genotypes, suggesting no strict demarcation between niches. Additionally, there was
little evidence of any strain being associated with a range of unique traits. Using the
previous analysis of gene content with Roary, the 18 strains with at least ten repre-
sentatives in the population had a mean of 16.7 private genes (range, 1 to 49), defined
as those loci present at 95% in one strain and 5% in all others. However, this does not mean the association between sequence clusters and
accessory loci was random. The same Roary analysis identified 8,311 intermediate-
frequency genes, present in between 5% and 95% of the overall population analyzed
by Kallonen et al. (16) (see Fig. S2 in the supplemental material). Their distribution
exhibited a strong association with individual sequence clusters, as defined by an
analysis of the core genome (see Materials and Methods), in agreement with recent
k-mer-based analyses of this population that showed divergence in the core and
accessory genome was strongly correlated (19). Hence, as in S. pneumoniae, strains
appear to be defined by a characteristic combination of common accessory loci, rather
than distinctive private sequences (14, 20). This suggests sequence clusters may be
ecologically distinct but with overlapping niches. We therefore tested whether the population-level frequencies of these intermediate
frequency genes were conserved, as following perturbation by vaccination in S. pneu-
moniae. Diversification of Colonization Factors in a Multidrug-Resistant
Escherichia coli Lineage Evolving under Negative Frequency-
Dependent Selection Frequencies of accessory genes were found to
be highly conserved across multiple populations on different continents, despite these
populations themselves being composed of different strains, as defined by core ge-
nome sequences. Detailed modeling and functional analysis indicated changes in strain
prevalence could be explained by NFDS driving accessory loci toward equilibrium mbio.asm.org
2 ® Colonization Factors in MDR E. coli frequencies, through mechanisms involving interactions with other bacteria, hosts, or
mobile elements (17). The levels of the selective force were estimated to be similar
across the populations and manifested in the maintenance of stable population
frequencies of accessory loci, despite a substantial perturbation of the population by
the introduction of the pneumococcal vaccine (17). Here, we synthesized different genomic analyses to provide a detailed view of the
ecology and evolution of ST131. We provide evidence of NFDS shaping the species-
wide carried E. coli population using 1,094 systematically sampled bacteremia isolates
from the British Society for Antimicrobial Chemotherapy (BSAC) collection. These
genomes, and those collated from previous large-scale phylogenomic studies (11–14,
16, 18), allowed a high-resolution analysis of E. coli ST131 using a total of 862 genomes,
revealing the steps in the clades’ diversification. This found clade C to have accumu-
lated significantly elevated allelic diversity, particularly enriched for genes involved in
anaerobic metabolism and other loci important for colonization of the human host by
ExPEC. Our data suggest the evolution of the MDR phenotype is part of a wider
ongoing adaptation toward prolonged human colonization that permits expansion
despite NFDS due to an adaptive radiation through diversification of metabolic and
antigenic loci. March/April 2019
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e00644-19 RESULTS This shows the changing correlation of gene
and Spearman methods, in each year relative to those observed in 2001. Both measures indicate a divergence in
until 2010, at which point there is a reversion to the frequencies seen in the original population. (C) Emergence
d). The frequencies of the subclades of ST131 are shown by the red dashed lines. 0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
Gene frequencies in 2001
Gene frequency ranges post−2001
A 0.80
0.85
0.90
0.95
2002.5
2005.0
2007.5
2010.0
Year
Correlation with 2001 frequencies
Method
Pearson
Spearman
B B A Method
Pearson
Spearman on May 8, 2019 by guest
http://mbio.asm.org/
Downloaded from C 0.00
0.05
0.10
0.15
0.20
2002.5
2005.0
2007.5
2010.0
Year
Frequency in population
Strain
SC9
CladeA
CladeB
CladeC
SC13
C 0
0
0
Frequency in population Strain
SC9
CladeA
CladeB
CladeC
SC13 ay 8, 2019 by guest FIG 1 Summarizing the population dynamics of the British Society for Antimicrobial Chemotherapy extraintestinal pathogenic E. coli collection. These isolates
were collected from bacteremia cases around the United Kingdom between 2001 and 2011. (A) Conservation of gene frequencies. Each point corresponds to
one of the 6,824 genes identified by Roary in the BSAC collection with mean frequencies between 0.05 and 0.95 across all years. Error bars indicate the full
range observed across annual samples. (B) Correlation of gene frequencies with those observed in 2001. This shows the changing correlation of gene
frequencies, calculated by both the Pearson and Spearman methods, in each year relative to those observed in 2001. Both measures indicate a divergence in
gene frequencies as ST69 and ST131 emerge, until 2010, at which point there is a reversion to the frequencies seen in the original population. (C) Emergence
of ST69 (SC9, in orange) and ST131 (SC13, red). The frequencies of the subclades of ST131 are shown by the red dashed lines. samples from until 2008, as might be expected with neutral drift (Fig. 1B), as ST131 and
ST69 became more prevalent (Fig. 1C). However, the gene frequencies “bounced back”
toward their original frequencies in 2008 (21), as indicated by the increased correlation
with the 2001 frequencies (Fig. 1C). This elevated correlation was sustained in subse-
quent years, despite the persistently high frequencies of ST131 or ST69, suggesting a
reconfiguration of other lineages in the population. Multilocus NFDS modeling of the E. coli population. RESULTS Comparisons between the pre-ST131 2001 samples and subsequent data from
up to 2011 found strong linear correlations between the prevalences of their
intermediate-frequency genes (Fig. 1A, see also Fig. S3A). This is consistent with these
loci existing at “equilibrium” frequencies, determined by their costs and frequency-
dependent benefits. These correlations with the first sample, in 2001, weakened with mbio.asm.org
3 March/April 2019
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e00644-19 ® McNally et al. 0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
Gene frequencies in 2001
Gene frequency ranges post−2001
A
0.80
0.85
0.90
0.95
2002.5
2005.0
2007.5
2010.0
Year
Correlation with 2001 frequencies
Method
Pearson
Spearman
B
0.00
0.05
0.10
0.15
0.20
2002.5
2005.0
2007.5
2010.0
Year
Frequency in population
Strain
SC9
CladeA
CladeB
CladeC
SC13
C
FIG 1 Summarizing the population dynamics of the British Society for Antimicrobial Chemotherapy extraintestinal pathogenic E. coli collection. These isolates
were collected from bacteremia cases around the United Kingdom between 2001 and 2011. (A) Conservation of gene frequencies. Each point corresponds to
one of the 6,824 genes identified by Roary in the BSAC collection with mean frequencies between 0.05 and 0.95 across all years. Error bars indicate the full
range observed across annual samples. (B) Correlation of gene frequencies with those observed in 2001. This shows the changing correlation of gene
frequencies, calculated by both the Pearson and Spearman methods, in each year relative to those observed in 2001. Both measures indicate a divergence in
gene frequencies as ST69 and ST131 emerge, until 2010, at which point there is a reversion to the frequencies seen in the original population. (C) Emergence
of ST69 (SC9, in orange) and ST131 (SC13, red). The frequencies of the subclades of ST131 are shown by the red dashed lines. 0.75
1.00
001
0.80
0.85
0.90
0.95
2002.5
2005.0
2007.5
2010.0
Year
Correlation with 2001 frequencies
Method
Pearson
Spearman
B
0.00
0.05
0.10
0.15
0.20
2002.5
2005.0
2007.5
2010.0
Year
Frequency in population
Strain
SC9
CladeA
CladeB
CladeC
SC13
C
s of the British Society for Antimicrobial Chemotherapy extraintestinal pathogenic E. coli collection. These isolates
d the United Kingdom between 2001 and 2011. (A) Conservation of gene frequencies. Each point corresponds to
n the BSAC collection with mean frequencies between 0.05 and 0.95 across all years. Error bars indicate the full
Correlation of gene frequencies with those observed in 2001. March/April 2019
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e00644-19 mbio.asm.org
4 RESULTS To obtain a population-wide
view of these dynamics, the previously described multilocus NFDS model was applied
to this data set to test whether these strain dynamics were consistent with selection of
the phenotypes encoded by a set of accessory loci, L. L was primarily composed of the
7,204 genes, identified by Roary, present at intermediate frequencies (between 5% and
95%) in the 2001 sample. These prevalences were assumed to represent the equilibrium
frequencies, el, determined by NFDS in an unperturbed population (17). As only a single
sample was available that predated the emergence of ST69 and ST131, these el
estimates are likely to be associated with considerable noise; however, the bounce back mbio.asm.org
4 March/April 2019
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e00644-19 ® Colonization Factors in MDR E. coli of accessory locus frequencies toward these el suggests they are likely to be accurate
for a high proportion of the modeled loci. Additionally, L included seven antibiotic
resistance phenotypes present in the same intermediate frequency range. Although
mechanistic models disagree as to whether antibiotic resistance is under NFDS directly
(22) or linked to other loci under balancing selection (23), the stability of individual
phenotype frequencies in this population (16) suggests this simplifying assumption is
reasonable. These were then simulated as evolving under NFDS; a fraction pf evolved
under strong NFDS, determined by the parameter f, while the rest evolved under weak
NFDS, according to parameter w (see Materials and Methods). g
p
w
The model was initialized with the 2001 population, which was seeded with
genotypes observed in later years at a low level, representing the possibility they were
present in the population but unsampled. Subsequent simulation with a Wright-Fisher
framework included these post-2001 genotypes migrating into the population at a rate
m. Each isolate was assigned to a sequence cluster, or strain, through identifying
clusters in the previously published hierBAPS analysis that approximately corresponded
to a multilocus sequence typing clonal complex (see Materials and Methods). The
invasion of the resident population by the sequence clusters corresponding to ST131
and ST69 was driven by an increased reproductive fitness relative to the rest of the
population, parameterized in the model as r. Fitting this model through approximate
Bayesian computation (ABC) using BOLFI estimated the parameters listed in Table S2a,
which identified significant evidence for NFDS (f and pf greater than their respective
lower bounds used in fitting). RESULTS The 95% credibility interval w was not higher than the
lower bound used in fitting, which might represent a genuine absence of NFDS acting
on these loci or a lack of power to detect NFDS in this population over this interval. The
conclusion that NFDS acting on at least a subset of accessory loci accounts for the
observed population dynamics better than equivalent simulations in which NFDS is
negligible provides a quantitative gene-level model that explains the previous strain-
level observations of Kallonen et al. (16). These simulations successfully reproduced several aspects of the observed data
(Fig. 2 and S3B). Both ST131 and ST69 rapidly spread through the population, before
stabilizing at an equilibrium frequency of 20%. This does not occur at the expense of
the established common clones, such as ST73 and ST95. Instead, in accordance with the
genomic data, the displaced sequence clusters include ST10, ST14, ST144, and ST405. These patterns are qualitatively distinct from an equivalent neutral model fit (Fig. 2,
bottom). In these simulations lacking NFDS, both ST131 and ST69 are predicted to
exponentially increase in frequency rather than rapidly stabilize at a frequency that
permits coexistence with the already-resident strains. All other strains commensurately
decrease at accelerating rates, proportionate to their original prevalence, meaning the
biggest falls are observed for ST73 and ST95, which were actually unperturbed by the
invading strains. The greater invasion rate of ST131 relative to ST69 is an artifact of its
higher prevalence in the overall data set, meaning it is seeded at a higher level, rather
than a true ecological difference. Factors affecting the emergence of ST131. The estimate of the fitness advantage
driving the invasion of ST69 and ST131, r, is contingent on aspects of the model
structure. The absence of population genomic data predating 2001, or from other
locations, makes it difficult to model the initial invasion of ST131. Therefore, isolates
from 2002 onwards were seeded in the initial population at a frequency of 103 to
replicate the likely scenario in which these genotypes were already present in the
United Kingdom or Ireland in 2001 but not captured by the first sample. Increasing the
level at which these strains were seeded by 10-fold resulted in a slightly increased final
frequency (see Fig. S4). This frequency should represent an approximate upper bound,
beyond which it would be unlikely that the strains would have remained unsampled in
2001. mbio.asm.org
6 RESULTS Decreasing the seeding level had an almost negligible effect on the simulated
dynamics. Hence, the inferred parameters are not markedly sensitive to the seeding
level used in the first time step of the simulations. mbio.asm.org
5 March/April 2019
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e00644-19 ® McNally et al. FIG 2 Simulations of changes in the BSAC extraintestinal pathogenic E. coli population evolving under multilocus NFDS. Genomic data (top) and median
frequencies (middle) observed from 100 simulations run with the best-matching parameter set identified by fitting the model with BOLFI. This corresponded
to f 0.029, r 0.179, m 0.001, pf 0.425, and w 0.0048. Each column corresponds to a sequence cluster identified by hierBAPS (see Materials and
Methods) and is annotated with the predominant sequence type with which it is associated. Each bar indicates the frequency of the sequence cluster in
(Continued on next page)
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d from FIG 2 Simulations of changes in the BSAC extraintestinal pathogenic E. coli population evolving under multilocus NFDS. Genomic data (top) and median
frequencies (middle) observed from 100 simulations run with the best-matching parameter set identified by fitting the model with BOLFI. This corresponded
to f 0.029, r 0.179, m 0.001, pf 0.425, and w 0.0048. Each column corresponds to a sequence cluster identified by hierBAPS (see Materials and
Methods) and is annotated with the predominant sequence type with which it is associated. Each bar indicates the frequency of the sequence cluster in
(Continued on next page) March/April 2019
Volume 10
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e00644-19 ® Colonization Factors in MDR E. coli The estimate of r was also affected by the simulated population size. To overcome
the greater stochasticity inherent to smaller populations, r must be higher for ST69 and
ST131 for them to consistently rise from their low initial frequencies in the early phases
of the simulations. Hence, the same r with a 10-fold smaller population than was
computationally feasible for fitting (5 104) results in lower median frequencies for
ST69 and ST131, whereas maintaining r with a 20-fold increased population size results
in the emerging strains reaching a higher prevalence by the end of the simulations (see
Fig. S5A). RESULTS Hence, as the effective population size of E. coli across the United Kingdom
and Ireland is likely to be greater than 5 104, the fitness advantage driving these
emerging strains to invade is probably lower than the r estimated here. The emergence driven by r is limited by NFDS, which generally constrains the
invasion of new clusters of genetically coherent isolates, as the traits with which they
are associated have a smaller net benefit as they increase in frequency. Simulations in
which ST69 and ST131 have the same fitness benefit, r, in the absence of NFDS confirm
this advantage would be sufficient to dominate the population in a neutrally evolving
population (Fig. S5B). This inhibition would be expected to most strongly affect lineages with very similar
sets of accessory loci. As the MDR isolates of clade C share the most recent common
ancestor of the three clades, around 25 years ago (11), their expansion would be
expected to be subject to the strongest inhibition by NFDS. Their rapid rise suggests
the effects of NFDS may have been ameliorated by the diversification of accessory loci,
such that the per locus constraint of the equilibrium frequencies does not limit the
strain’s overall prevalence. Extensive variation suggestive of this diversity has previously
been noted with the capsules expressed within clade C (23). Therefore, the distributions
of intermediate frequency loci within each sequence cluster and the clades of ST131
were analyzed by estimating the overall gene content through a Heap’s law analysis of
pangenome curves (24) and through pairwise analysis of gene content similarity by
estimating genomic fluidity (25). Comparing the metrics across the sets of isolates
revealed that ST131 was one of a small number of clades that had an open pangenome
but low genomic fluidity (Fig. S4B). This implies each pair of isolates is similar, reflecting
their recent shared ancestry; but, many isolates have undergone different diversifying
recombinations at one or more loci present at an intermediate frequency in the wider
population. This can account for the overall clade containing many loci in total, despite
its recent origin. This is consistent with the adaptive radiation of a successful strain
expanding in its population size. g
consecutive time periods, from left to right. The bars are colored according to the number of antibiotic resistance phenotypes associated with the isolates within
the sequence cluster at different time points. (Bottom) The equivalent best fit in the absence of NFDS. Only sequence clusters reaching a frequency of at least
2.5% at one time point in the genomic sample are shown; the full results of the simulation, including measures of between-simulation variation, are shown
in Fig. S3. March/April 2019
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e00644-19 mbio.asm.org
7 RESULTS The taxa are
color coded by BAPS grouping (Table S1) and show clade A (green, BAPS-3), clade B (red, yellow, and
purple, BAPS-2, -4, and -5, respectively), and clade C (blue, BAPS-1). FIG 3 (A) Maximum likelihood phylogeny of 862 E. coli ST131 strains. The phylogeny was inferred using
RAxML with a GTR Gamma model of substitution, on an alignment of concatenated core CDS as
determined by Roary. (B) PANINI plot of the accessory genome content of all 862 strains based on a tSNE
plot. The plot is a diagrammatical representation of the relatedness of each strain based on the
presence/absence of accessory genes and is presented as a two dimensional representation. The taxa are
color coded by BAPS grouping (Table S1) and show clade A (green, BAPS-3), clade B (red, yellow, and
purple, BAPS-2, -4, and -5, respectively), and clade C (blue, BAPS-1). discernible substructuring into distinct groups. This concurs with the previous analysis
of the gene content of clade C and the previous finding of multiple accessory genome
subclusters within this set of isolates (14). Low-frequency accessory genes suggest clade A and clade B/C E. coli ST131
rarely cohabit the same ecological spaces. To identify which aspects of the accessory
genome differed between the clades of ST131, the distributions of the 32,631 sets of
orthologous genes identified by Roary were analyzed. Characterizing the full set of
loci present at intermediate frequencies was not feasible, as even focusing on the 3,354
present at between 5% and 95% frequency revealed that the majority of these were
present at a frequency 20% (see Fig. S1). Therefore, the search was refined to
clade-specific genes occurring at a frequency of 95% in one clade but at 5% in the
other two clades. In both clade A and clade B/C, the overwhelming majority of low-frequency acces-
sory genes encode hypothetical proteins (64.4% clade A, 58% clade B/C). Excluding the
hypothetical proteins from the analysis showed an unexpected bias in functional gene
categories differentially observed in the lineages (Fig. 4). The most common gene types
were functional phage, plasmid, and other mobile genetic element (MGE) genes, with
more private phage genes present in clade B/C than in clade A. Conversely, there were
more private plasmid genes in clade A than in clade B/C, despite the presence of a
diverse number of MDR plasmids within clade C (14). RESULTS To better understand this diversification and the basis for the high fitness of ST131
represented by r, a comprehensive genomic data set encompassing all known ST131
genome sequences was created to understand the unique characteristics of the
ST131 lineage, with particular focus on the successful clades B and C. Core and accessory genomic structure of the ST131 population. A maximum
likelihood phylogeny generated from an alignment of concatenated core coding DNA
sequences (CDS) from all 862 genomes confirmed the earlier consensus three-clade
structure of the lineage (Fig. 3A), and in agreement with previous studies, there was no
strong phylogeographic signal or host source clustering evident in the phylogeny
(https://microreact.org/project/BJKoeBt2b). To confirm that the collation of the 862
genomes was consistent with previous descriptions of the accessory genome distribu-
tion in ST131, isolate relatedness based on shared accessory gene content was visual-
ized as a two-dimensional projection using PANINI (Fig. 3B) (26). Clades A and B largely
resided in dense clusters at the periphery of the projection. In contrast, clade C isolates
were more diffuse, overlapping with some clade B isolates, forming a cloud with March/April 2019
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e00644-19 mbio.asm.org
7 ® McNally et al. FIG 3 (A) Maximum likelihood phylogeny of 862 E. coli ST131 strains. The phylogeny was inferred using
RAxML with a GTR Gamma model of substitution, on an alignment of concatenated core CDS as
determined by Roary. (B) PANINI plot of the accessory genome content of all 862 strains based on a tSNE
plot. The plot is a diagrammatical representation of the relatedness of each strain based on the
presence/absence of accessory genes and is presented as a two dimensional representation. The taxa are
color coded by BAPS grouping (Table S1) and show clade A (green, BAPS-3), clade B (red, yellow, and
purple, BAPS-2, -4, and -5, respectively), and clade C (blue, BAPS-1). McNally et al. FIG 3 (A) Maximum likelihood phylogeny of 862 E. coli ST131 strains. The phylogeny was inferred using
RAxML with a GTR Gamma model of substitution, on an alignment of concatenated core CDS as
determined by Roary. (B) PANINI plot of the accessory genome content of all 862 strains based on a tSNE
plot. The plot is a diagrammatical representation of the relatedness of each strain based on the
presence/absence of accessory genes and is presented as a two dimensional representation. March/April 2019
Volume 10
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e00644-19 RESULTS Together, this suggests that clade
A strains of E. coli ST131 and clade B/C strains of E. coli ST131 are exposed to different
plasmid and phage pools, an observation which is most parsimoniously explained by
them rarely sharing the same ecological habitats at the same time. Clade A contained the highest number of loci exclusive to a lineage (27) despite
constituting the least-sampled clade. This is likely explained by the fact that clade A is
on the longest branch of the ST131 phylogeny, and as such, has had most evolutionary
time to accumulate differences in the gene repertoire. Clade B had only 2 exclusive loci
and clade C had 18. When clades B and C were combined against clade A, there were
60 loci exclusively present in the B/C combination. The majority of clade A private
genes encode hypothetical proteins, while those private to clade C encode DNA mbio.asm.org
8 FIG 4 Bar chart depicting functional classes of accessory genes differentially present in clade A and clade B/C E. coli ST131. Functional classes
are based on GO classes as described in Materials and Methods. *, significant difference exists between clade A and clade C as determined by
t test. Colonization Factors in MDR E. coli ® Colonization Factors in MDR E. coli on May 8, 2019 by
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aded from FIG 4 Bar chart depicting functional classes of accessory genes differentially present in clade A and clade B/C E. coli ST131. Functional classes
are based on GO classes as described in Materials and Methods. *, significant difference exists between clade A and clade C as determined by
t test. modification proteins and metabolic functions. The genes private to clade B/C com-
bined also encode hypothetical proteins and metabolic functions, notably, five dehy-
drogenase enzymes involved in anaerobic metabolism labeled yihV, garR_3, fadJ, fdhD,
and gnd in our data set. A BLAST analysis against the NCBI nonredundant database
suggested that the dehydrogenase enzyme gene annotated as pdxA in our Roary data
set was confined to clade C ST131 strains. These dehydrogenase enzyme genes were
found to be present across phylogroup B2 E. coli strains (of which ST131 is a member)
through blastn searches of the NCBI nonredundant database. March/April 2019
Volume 10
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e00644-19 RESULTS coli ST131, based on concatenated core CDS, with the presence of alternative alleles of 64 loci
involved in anaerobic metabolism. Each blue box along the top of the tree annotation represents an individual anaerobic metabolism gene, and its presence
in the ST131 population is indicated by a blue line. The inset is a bar chart displaying the proportions of the accessory pangenome that are occupied by genes
involved in anaerobic metabolism for ST131 clade A, clade B, subsampled clade C versus clade A, and subsampled clade C versus clade B. P 0.042 for clade
C versus clade A and P 0.086 for clade C versus clade B. Error bars represent standard errors of the means. Significance was determined using the median
value P value from chi-square tests performed on random subsamples of the C clade. sensor kinase for activation of the cobalamin biosynthesis operon. These anaerobic
metabolism genes would normally be considered essential to E. coli. Interrogation of
the pangenome matrix for the data set showed that versions of these genes existed as
core genome loci. Comparison of the nucleotide sequences of the clade-specific loci
with their core genome counterparts showed that these were not genes private to
clade B/C per se but rather represented multiple unique alleles of genes that are core
to the ST131 population, which differ at the nucleotide sequence level by more than
5%. This suggests a unique selection pressure is acting on these core genes in clade B/C
compared to those in clade A. The discovery of these core genes that display allelic
variation is explained by the fact that the percent nucleotide identity cutoff applied in
the formation of our pangenome matrix is 95%, meaning that any alternative alleles of
core genes differing by 5% or more will appear in our matrix as accessory genes. Further scrutiny of low-frequency loci in clade B/C also identified alternative alleles
of a large number of well-characterized extraintestinal pathogenic E. March/April 2019
Volume 10
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e00644-19 RESULTS Therefore, these loci are
not unique to clade C ST131 and were either acquired by an ancestral clade B/C strain
or have been lost by clade A. High diversity in core anaerobic metabolism genes unique to clade B/C. An
analysis of accessory loci private to clade B/C (present in 95% of that population)
identified two separate loci encoding 3-hydroxyisobutyrate dehydrogenase enzymes
and loci encoding 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-CoA) dehydrogenase,
6-phosphogluconate dehydrogenase, and formate dehydrogenase. An analysis of clade
B/C loci circulating at a low frequency of 20% also identified a significant overrep-
resentation of genes encoding dehydrogenase enzymes involved in anaerobic metab-
olism (a total of 64 loci), including seven variants of formate dehydrogenase. There
were also seven variants of the eutA gene found in the ethanolamine utilization
pathway (the eut operon) and a distinct version the cobW gene, which encodes the mbio.asm.org
9 ® McNally et al. Clade A
Clade C vs. Clade A
Clade B
Clade C vs. Clade B
0.000
0.005
0.010
0.015
0.020
0.025
Proportion of Anaerobic Metabolic
Genes in Acessory Pangenome
*
FIG 5 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of 64 loci
involved in anaerobic metabolism. Each blue box along the top of the tree annotation represents an individual anaerobic metabolism gene, and its presence
in the ST131 population is indicated by a blue line. The inset is a bar chart displaying the proportions of the accessory pangenome that are occupied by genes
involved in anaerobic metabolism for ST131 clade A, clade B, subsampled clade C versus clade A, and subsampled clade C versus clade B. P 0.042 for clade
C versus clade A and P 0.086 for clade C versus clade B. Error bars represent standard errors of the means. Significance was determined using the median
value P value from chi-square tests performed on random subsamples of the C clade. Clade A
Clade C vs. Clade A
Clade B
Clade C vs. Clade B
0.000
0.005
0.010
0.015
0.020
0.025
Proportion of Anaerobic Metabolic
Genes in Acessory Pangenome
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aded from FIG 5 Annotation of a maximum likelihood phylogeny of E. mbio.asm.org
10 RESULTS Cell
division Capsule on May 8, 2019 by
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aded from FIG 6 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of loci involved
in capsule production, cell division, iron acquisition, pili/fimbriae production, flagella, and MDR efflux pumps. Each box represents an individual gene, and its
presence in the ST131 population is indicated by an appropriately colored line. from other sources. Given that these alleles differ from the normal conserved versions
of genes by 5% at the nucleotide level, it is implausible that these alleles would be
arising repeatedly and independently via mutation. Instead, the most parsimonious
explanation is that the minor frequency alternative alleles are being distributed
through the population via recombination. This conclusion is supported by the fact that
every one of the allele variants identified in our analysis has 100% nucleotide identity
matches with genes present in other E. coli in the NCBI nonredundant database. g
Given that our data set is biased toward clade C genomes, we performed compar-
ative analyses of the frequency with which allelic diversity occurs in anaerobic metab-
olism genes. We randomly subsampled clade C 100 times and compared equal num-
bers of clade A, B, and C genomes for allelic diversity. Our data show that even when
randomly subsampling clade C, the levels of diversity observed in anaerobic metabo-
lism genes is significantly higher than in clade A, providing evidence that the accumu-
lation of sequence diversity is specific to the MDR clade C, despite this clade having the
most recent common ancestor of the three (Fig. 5). Finally, we sought to exclude the possibility that the presence of these allelic
variants was skewed by some form of geographically localized expansion of variants. To do this, we compared the relative frequencies of all accessory genes, highlight-
ing the allele variants in anaerobic metabolism, capsule, cell division, MDR efflux,
iron acquisition, fimbriae, and flagella present in U.K. versus non-U.K. isolate
genomes (see Fig. S6). March/April 2019
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e00644-19 RESULTS coli virulence-
associated genes: antigen 43 (7 alternative alleles); heavy metal resistance, such as
arsenic (5 loci), copper (4 loci), and mercury (5 loci); capsule biosynthesis (20 loci); cell
division and septation (14 loci); antibiotic resistance to chloramphenicol (3 loci), mac-
rolides (2 loci), rifampin (1 locus), and MDR efflux pumps (21 loci); iron acquisition (39
loci); curli and type I fimbriae and P pili (42 loci); lateral and classical flagella (26 loci);
and LPS synthesis (9 loci). These variant loci represent alternative alleles of genes found
widely across the E. coli phylogeny, indicating there are multiple allelic variants of
important genes that are confined to clade B/C of the E. coli ST131 lineage. We sought to determine the distribution of this allelic diversity across the E. coli
ST131 phylogeny by annotating the tips of the phylogenetic tree with the presence/
absence of each of the anaerobic metabolism (Fig. 5) and capsule, cell division, MDR
efflux, iron acquisition, pili, and flagellum divergent loci (Fig. 6). Our analysis shows that
each alternative allele occurs at very low frequency, but that alleles are randomly
distributed throughout the phylogeny of the C clade and are exclusive to clade C. An
analysis of the source attribution of isolates displaying this allelic diversity showed that
80% of isolates displaying allelic diversity were human isolates (32% of those blood-
stream isolates, 20% UTI isolates, 2% commensal, and the remainder of an unknown
source), 5% were avian isolates, 10% companion animal isolates, and the remainder mbio.asm.org
10 ® Colonization Factors in MDR E. coli Capsule
Cell
division
Iron acquisition
Pili/fimbriae
Flagella
MDR efflux
FIG 6 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of loci involved
in capsule production, cell division, iron acquisition, pili/fimbriae production, flagella, and MDR efflux pumps. Each box represents an individual gene, and its
presence in the ST131 population is indicated by an appropriately colored line. Capsule
Cell
division
Iron acquisition
Pili/fimbriae
Flagella
MDR efflux
FIG 6 Annotation of a maximum likelihood phylogeny of E. coli ST131, based on concatenated core CDS, with the presence of alternative alleles of loci involved
in capsule production, cell division, iron acquisition, pili/fimbriae production, flagella, and MDR efflux pumps. Each box represents an individual gene, and its
presence in the ST131 population is indicated by an appropriately colored line. RESULTS Iterative chi-square tests were performed to assess
significance, as described in Materials and Methods. *, P 0.05; **, P 0.01; ***, P 0.001. 0.00
0.05
0.10
0.15
0.20
0.25
Phage
Genetic Processing
Mobile Genetic Elements
Metabolism
Protein of Unknown Function
Plasmid
Molecular Transport
Virulence
Environmental Interaction
Conjugal Transfer
Miscellaneous
Adhesion
Anaerobic Metabolism
Antimicrobial Resistance
Cell Division
Outer Membrane Proteins
Iron Acquisition
Inner Membrane Proteins
Cell Wall
Flagella
Ribosome
Proportion of accessory pangenome
Subsampled ST131
ST73
*
* * *
* *
*
*
*
*
* * *
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
* * *
* * *
* * *
A 0.00
0.05
0.10
0.15
0.20
0.25
Phage
Genetic Processing
Mobile Genetic Elements
Metabolism
Protein of Unknown Function
Plasmid
Molecular Transport
Virulence
Environmental Interaction
Conjugal Transfer
Miscellaneous
Adhesion
Anaerobic Metabolism
Antimicrobial Resistance
Cell Division
Outer Membrane Proteins
Iron Acquisition
Inner Membrane Proteins
Cell Wall
Flagella
Ribosome
Proportion of accessory pangenome
Subsampled ST131
ST95
*
* * *
* *
* *
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
B B A on May 8, 2019 by
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aded from FIG 7 Bar charts depicting the compositions of the accessory genomes of ST73 (A) and ST95 (B) compared to a repetitively sampled clade C ST131. The
proportions of the accessory genome are plotted against manually assigned functional categories. Hypothetical proteins are responsible for the majority of the
accessory pangenome and are omitted from the graphs. Error bars are standard errors of the means. Iterative chi-square tests were performed to assess
significance, as described in Materials and Methods. *, P 0.05; **, P 0.01; ***, P 0.001. of the most dominant lineages associated with clinical extraintestinal disease alongside
ST131 (5, 16) but are predominantly non-MDR lineages and rarely associated with MDR
plasmids (16). As with our interclade comparisons, we randomly subsampled clade C
ST131 100 times to allow equal numbers of genomes per lineage to be compared. RESULTS Our data showed a strong linear relationship between the
frequency of genes in the two populations, indicating that the data were not biased
by expansion of alleles in a given geographical location, and that this accumulated
diversity was equally as likely to happen in any given strain independent of its
geographical origin. Allelic diversity of anaerobic metabolism genes in clade C ST131 is not ob-
served in other dominant ExPEC lineages. The possibility exists that the above
observations made for clade C of E. coli ST131 simply reflect the general evolutionary
path of a successful commensal and extraintestinal pathogen. To test this, we per-
formed an identical analysis on the pangenome of 261 ST73 isolates and of 160 ST95
isolates from the U.K. BSAC population survey (16). E. coli ST73 and ST95 represent two mbio.asm.org
11 March/April 2019
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e00644-19 ® McNally et al. 0.00
0.05
0.10
0.15
0.20
0.25
Phage
Genetic Processing
Mobile Genetic Elements
Metabolism
Protein of Unknown Function
Plasmid
Molecular Transport
Virulence
Environmental Interaction
Conjugal Transfer
Miscellaneous
Adhesion
Anaerobic Metabolism
Antimicrobial Resistance
Cell Division
Outer Membrane Proteins
Iron Acquisition
Inner Membrane Proteins
Cell Wall
Flagella
Ribosome
Proportion of accessory pangenome
Subsampled ST131
ST73
*
* * *
* *
*
*
*
*
* * *
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
* * *
* * *
* * *
0.00
0.05
0.10
0.15
0.20
0.25
Phage
Genetic Processing
Mobile Genetic Elements
Metabolism
Protein of Unknown Function
Plasmid
Molecular Transport
Virulence
Environmental Interaction
Conjugal Transfer
Miscellaneous
Adhesion
Anaerobic Metabolism
Antimicrobial Resistance
Cell Division
Outer Membrane Proteins
Iron Acquisition
Inner Membrane Proteins
Cell Wall
Flagella
Ribosome
Proportion of accessory pangenome
Subsampled ST131
ST95
*
* * *
* *
* *
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
A
B
FIG 7 Bar charts depicting the compositions of the accessory genomes of ST73 (A) and ST95 (B) compared to a repetitively sampled clade C ST131. The
proportions of the accessory genome are plotted against manually assigned functional categories. Hypothetical proteins are responsible for the majority of the
accessory pangenome and are omitted from the graphs. Error bars are standard errors of the means. March/April 2019
Volume 10
Issue 2
e00644-19 DISCUSSION The evolutionary events that led to the emergence of E. coli ST131 have been an
intense focus of research, with consensus opinion suggesting that following the
acquisition of key ExPEC virulence factors, the acquisition of fluoroquinolone resistance
in the 1980s by the clade C sublineage of ST131 was a key event in that emergence (11,
12). However, a recent nationwide U.K. population survey rejected this hypothesis and
suggested that success of the major ExPEC clones is not dictated by resistance traits
(16). Here, we combined an analysis of population frequency dynamics with an analysis
of the pangenome of the ST131 lineage, strongly suggesting that this species’ popu-
lation structure and dynamics are shaped by NFDS acting on genomic islands. Such
multilocus NFDS is able to account for how an otherwise stable population was
disrupted by the invasion of ST131 and ST69, displacing some lineages while leaving
other, largely antibiotic-susceptible, genotypes at almost untouched prevalence. Previous work has suggested that clade C strains of E. coli ST131 undergo reduced
levels of detectable core genome recombination compared to those of other phylo-
group B2 E. coli (30) or ST131 clade A strains (14). We have previously postulated that
this may be a result of ecological separation between clade C strains and other
common ExPEC (14, 30). Our analysis of nearly 900 genomes has allowed us to
interrogate accessory gene movement to a far greater resolution than previously
possible. From the analysis of the accessory genome, we identified thousands of
plasmid, phage, and other mobile genetic element genes which are private to clade A
and the combined clade B/C. Such an observation is a classic signature of ecological
separation of the two populations (31, 32), particularly given that the genetic distance
between clade A and clade B/C is much smaller than between other lineages and
species from which the circulating genes are also found in the NCBI nonredundant
database. Our analysis also identified a significantly increased level of sequence diversity in
genes involved in key host colonization processes in clade C. This diversity was
uncovered through our pangenome analysis as allelic variants of core genes. Primary
among these is a large number of genes involved in anaerobic metabolism, including
seven allelic variants of the formate dehydrogenase gene, as well as allelic variants of
genes involved in ethanolamine utilization and cobalamin biosynthesis. RESULTS Our
analysis showed higher levels of plasmid, phage, and hypothetical proteins in the
accessory genomes of both ST73 and ST95 than in ST131 (Fig. 7). ST73 and ST95
displayed similar ratios of alternative alleles in adhesins, cell division, and septation
genes and multiple iron acquisition genes as observed in ST131 but a higher diversity
in virulence-associated genes. However, enrichment in allelic variation in anaerobic
metabolism genes was significantly higher in any given subsampled set of clade C
ST131 genomes than in both lineages. This supports the hypothesis that the observa-
tion of increased diversity accumulating in anaerobic metabolism genes is not a more
general extraintestinal pathogenic E. coli trait but is particularly enriched in the ST131
lineage. It also suggests that this is independent of diversification of adhesins or
virulence factors, which was enhanced in the ST73 and ST95 lineages. The accumulation of nucleotide diversity in a given set of loci can often be
interpreted as a signature of some form of selection occurring on those genes. However, the low levels of frequency of any given allele across clade C strains
contradict a hypothesis for positive selection, where one would expect successful or
beneficial alleles to sweep to a high frequency or fixation. Indeed, a comparison of the
sequences of each of anaerobic metabolism loci in which three or more alleles were
observed identified just three loci which showed a mixture of weakly positive and
highly negative Tajima’s D scores, indicating no common selective mechanism in these
loci (Table S2b). However, these results can be reconciled with a lineage evolving under NFDS. Different resource use strategies can facilitate coexistence between competing strains,
such those cocolonizing a host, resulting in frequency-dependent selection (28, 29). This would explain the sustained intermediate frequencies of genes encoding dehy-
drogenases over multiple years (see Fig. S7). Hence, this diversification of metabolic loci
could represent the adaptive radiation of particular traits within a successful genetic
background, able to efficiently compete with the resident E. coli population through a
diverse panel of metabolic capacities suited to exploiting resources under anaerobic
conditions. mbio.asm.org
12 ® Colonization Factors in MDR E. coli March/April 2019
Volume 10
Issue 2
e00644-19 DISCUSSION The pivotal role
of ethanolamine production and cobalamin biosynthesis in the ability of Gram-negative
pathogens to outcompete bacteria in the human intestine is well documented (33, 34),
and this phenomenon only occurs when supported by an increased ability to perform
anaerobic respiration in the presence of inflammation (33). It has been shown that MDR
E. coli ST131 is able to colonize the gastrointestinal tract of humans for months or years
in the absence of antibiotic selection (35, 36) and that this colonization results in a
displacement of the E. coli colonizing the host prior to exposure to the MDR strain (35). While this diversity in anaerobic metabolism genes was unique to clade C ST131, the
allelic variation observed in other human colonization and virulence factors, such as
iron acquisition, fimbriae, and cell division, was also observed in two of the other most
commonly isolated lineages of E. coli from extraintestinal infections, ST73 and ST95. This diversity likely reflects selection occurring on genes important for ExPEC patho-
genesis. Iron acquisition is well characterized as a key virulence determinant in ExPEC,
with the ability to initiate successful UTI completely abrogated in the absence of
functional iron acquisition systems (37). Recent experimental vaccine work exploiting
siderophore production by ExPEC has shown it to be highly effective in rodent models
on ExPEC UTI (38). The importance of iron acquisition can also explain many of the MDR
efflux allele variants seen in this data set, with half occurring in the acrD gene, which
has been experimentally shown to play a role in iron acquisition in E. coli (39). We
identified multiple alleles of genes in the type 1 fimbria operon and in genes in the P
pilus operon, which are classical virulence determinants in UTI (40), and multiple genes
involved in capsule biosynthesis, which we have previously reported as being a hot
spot for recombination in E. coli ST131 (13, 41). We also identified multiple alleles of mbio.asm.org
13 March/April 2019
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e00644-19 ® McNally et al. genes involved in controlling incomplete septation and filamentous growth, which is a
crucial process in the formation of the filamentous intracellular bacterial communities
(IBCs) which are thought to be fundamental in the ability of ExPEC to survive inside
bladder epithelial cells and cause UTI (42). DISCUSSION There are a small number of allelic variants
in anaerobic metabolism genes also present in ST73 and ST95, possibly reflecting
recent experimental studies suggesting a crucial role for the cytochrome bd oxidase
system in the ability to cause urinary tract infection (43). Also, previous studies using
saturated mutagenesis techniques and studying global transcriptional patterns during
urinary tract infection of ExPEC strains have suggested a key role for dehydrogenase
enzymes involved in anaerobic metabolism in the ability to cause pathology in the
mammalian urinary tract (27, 44, 45). y
Recent modeling data on why drug-resistant and drug-susceptible populations of
bacteria coexist highlighted that any factors which increase the duration of colonization
in a human host will also increase the selective pressure for them to evolve antibiotic
resistance (23). Hence, both the success of ST131 in invading the population and the
association of many isolates in this lineage with an MDR phenotype would be consis-
tent with the accumulating diversity in anaerobic metabolism loci facilitating enhanced
persistence within its host, perhaps through an improved ability to outcompete
resident commensal E. coli strains. The fact that this selection is only seen in clade C of
ST131 suggests that this occurred around the time of the emergence of the lineage as
a human clinical threat (13) alongside the development of fluoroquinolone resistance. Subsequent acquisition of MDR plasmids, and the consequent selection for an ability to
offset the fitness costs of long-term MDR plasmid maintenance (14), is likely to have
occurred as a result of prolonged exposure to selective antibiotic environments during
colonization of humans. Nevertheless, neither anaerobic metabolism genes nor antibi-
otic resistance loci have swept to fixation in ST131, reflecting their fluctuating but
stable prevalence in the broader E. coli population. This diversification can instead be explained by NFDS, under which these genes are
beneficial when rare, because they provide an advantage over cocolonizing strains which
will typically lack the same metabolic capacities. However, as these traits become more
common as ST131 expands, representatives of this lineage will more commonly encounter
one another, therefore necessitating further diversification for different clade C represen-
tatives to sustain their advantage over competitors. DISCUSSION Similarly, the capsule locus diversifica-
tion previously observed within clade C, resulting in the capsule synthesis locus corre-
sponding to a “hot spot” of recombination (23), could result from NFDS on variable antigens
(46), with the host immune system selecting for a diversity of capsule structures as the
dominant type becomes more common following ST131’s emergence (16). yp
g
g
This study presents evidence for two complementary aspects of diversification
within the E. coli ST131 lineage. The first is ecological niche separation across a lineage,
whereby the different clades of ST131 find themselves competing for similar niches,
resulting in them very rarely cohabiting the same habitat. This then results in the
formation of distinct subclades within ST131. The second is NFDS across a species
within the specific environment of the human gut and the diversification of key genes
involved in the colonization of the human gut niche to escape the constraints imposed
by NFDS. Further studies are required to fully determine the extent to which niche
separation and NFDS are either separate or linked processes. Determining whether loci
subject to NFDS are also those that determine niche adaptation will be integral to this
process. Understanding the processes that govern the epidemiological dynamics of
dominant E. coli lineages, and those of similar pathogens causing bloodstream infec-
tions, is critical for addressing the public health threat of antibiotic resistance. March/April 2019
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e00644-19 MATERIALS AND METHODS Genome data. For the NFDS modeling study, the 1,094 isolates from the BSAC resistance surveillance
bacteremia collection from the United Kingdom and Ireland between 2001 and 2011 were used. These
were described by Kallonen et al. (16). For the ST131-specific study, a collection of 862 E. coli ST131 genomes (see Table S1 in the
supplemental material), of which 684 were previously sequenced as part of phylogenomic investigations mbio.asm.org
14 March/April 2019
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e00644-19 ® Colonization Factors in MDR E. coli of the ST131 lineage (10, 11, 13, 14, 16, 47), was analyzed. An additional 184 ST131 isolates from the BSAC
bacteremia resistance surveillance project conducted in the United Kingdom and Ireland between 2001
and 2011 (indicated in Table S1) were included. Together, this collection represents bacteria isolated
from invasive disease (bloodstream infections, 504 isolates; 58.5%), human asymptomatic carriage (21
isolates, 2.4%), disease resulting from intestinal carriage (UTI, 163 isolates; 18.9%), and bacteria isolated
from a range of veterinary livestock, pets, wild birds, and the wider environment (67 isolates, 7.8%). This
provides a data set isolated between 1967 and 2013, providing as broad a view of isolate origin as
possible within existing data sets. In an attempt to avoid any issues arising from different assembly or annotation metrics employed in
the previous projects, only raw sequence data were downloaded in fastq format using the previously
published accession data. De novo assembly was then performed on all the genomes using Velvet (48)
and annotation using Prokka (49) as previously described (16). A pangenome of the entire data set was
constructed using Roary with 95% identity cutoff (50). A concatenated core CDS alignment was made
from the Roary output, and a maximum likelihood phylogenetic tree was constructed from the alignment
using RaxML version 8.2.8 (51) and the GTR model with Gamma rate heterogeneity. For comparative lineage analysis, 264 ST73 genomes and 162 ST95 genomes that were sequenced
and fully characterized as part of the U.K. BSAC genome study (16) were utilized. Multilocus NFDS modeling. Multilocus NFDS modeling used genomic data from the previous
publication analyzing the population dynamic of bloodstream infection E. coli isolates in the United
Kingdom (16). This first required splitting the population into genetically coherent clusters of isolates. MATERIALS AND METHODS This is necessary, as the sequence cluster (or “strain”) frequencies are used for model fitting; therefore,
the clusters must be coherent enough to behave reproducibly but large enough to overcome the
stochasticity of the model. The previous hierBAPS analysis of the core genome provided three levels of
hierarchical clustering (52). The highest-level clusters corresponded to individual common strains in
some cases but polyphyletic collections of diverse genotypes in others. Therefore, to ensure the
sequence clusters used in the model were of similar levels of diversity, available sequence typing data
were used to identify the level of clustering that corresponded to clonal complexes. Each of the
highest-level hierBAPS clusters was tested by constructing links between single- and double-locus
variants. If the isolates formed a single clonal complex, the cluster was defined as a sequence cluster. Otherwise, the second level of clustering was tested in the same way; if these groups represented a
clonal complex, they were used as a sequence cluster. Otherwise, the third most fine-grained level of
clustering was used to define a sequence cluster. This identified 56 sequence clusters across the
population. The multilocus NFDS model also requires the definition of a set of L loci, each of which is associated
with an equilibrium population-wide frequency, el. The majority of these correspond to accessory genetic
loci, identified as sets of orthologous sequences by a previous Roary analysis (16). Only the 7,204 present
at between 5% and 95% frequency in the first sample, from 2001, were included in L and therefore
modelled as evolving toward el under NFDS. The set of L loci was supplemented with seven loci associated with resistance phenotypes if they
were present within this frequency range in 2001: amoxicillin, clavulanic acid, ciprofloxacin, cefuroxime,
gentamicin, piperacillin-tazobactam, and trimethoprim. The first six of these were directly inferred from
the previously published analysis. Trimethoprim was instead inferred from the sul and dfrA alleles
identified by Roary; data from the Cambridge University Hospitals collection (16) was used to train
a model constructed with the randomForest R library (https://cran.r-project.org/web/packages/
randomForest/), which had 93% accuracy when applied back to the training data set. This was used to
infer resistance phenotypes for the BSAC collection. Analysis used the heterogeneous multilocus NFDS model described previously (17), modified to treat
a vaccine cost, v, as a fitness advantage, r. March/April 2019
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e00644-19 MATERIALS AND METHODS This represents
the likely scenario in which they were present in the sampled region in 2001 but were not observed in
the necessarily finite set of sequenced isolates. The parameter m represented the rate at which all isolates
entered the population through migration; this was biased to import all sequence clusters at the same
rate, to avoid any fits in which high rates of migration would artifactually replicate the population
observed in the later years of the collection (17). Model fitting to genomic data. As in Corander et al. (17), the simulation model was fitted through
approximate Bayesian computation (ABC) using the BOLFI algorithm, which has been shown to accel-
erate ABC inference 1,000 to 10,000 times without loss of accuracy (53). The prior constraints placed on
the parameter values were as follows: the lower bound on all parameters was set to 0.0009; the upper
bounds were 0.99 for ri, 0.2 for m, 0.99 for pf, 0.03 for f, and 0.005 for w. We used 500 iterations of the
BOLFI algorithm to minimize the Jensen-Shannon divergence of the sequence cluster frequencies in the
genomic data and in the simulations, as ascertained through randomly sampling discrete sets of isolates
in accordance with the size and timings of the genomes selected for sequencing from the original
collection. Convergence of BOLFI was monitored after every 100 iterations and the approximate
likelihood estimate was assessed to have been stabilized after 500 iterations (53). The 95% posterior
credible intervals for the parameters were obtained using three generations of sequential Monte Carlo
sampling with the same default settings as used in Corander et al. (17). The neutral model was fitted by
fixing pf, f, and w at zero and estimating r and m through 500 iterations of the BOLFI algorithm,
followed by sequential Monte Carlo sampling, as with the full model. Accessory genome analysis. The pangenome matrix from Roary was utilized to investigate the
presence of clade-specific loci. The PANINI tool was used with the default setting to visualize the
accessory gene-sharing patterns in the population available at https://microreact.org/project/BJKoeBt2b
(26). PANINI has been demonstrated to provide efficient complementary visual means to phylogenetic
trees to accurately extract both distinct lineages present in a population-wide genomic data set and to
highlight clusters within lineages, which are explained by rapidly occurring, homoplasic alterations, such
as phage infection. MATERIALS AND METHODS To simulate the invasion of the resident population by ST131
and ST69, all individuals, i, of the two sequence clusters containing ST131 (and related sequence types
ST5640, ST5387, ST5494, ST5432, and ST5630) and ST69 (and related sequence type ST106) were
assigned the same fitness advantage, ri r. For all other individuals in the population, ri 0. Hence, the
function defining the number of progeny, Xi,t, produced by i at time t was Xi,t Pois
Nt1 ri1 m1 f
i,t 1 w
i,t In this formula, density-dependent competition is parameterized by the carrying capacity , set at
50,000 to represent a large population that is still computationally feasible, and the total number of cells
in the simulated population at t, Nt. The strength of NFDS was determined by the parameters pf, f, and
w. As previously, the set of L accessory loci and resistance phenotypes were ordered according to the
statistic Δl: l
fl,t0 el
2
1 el1 el where fl,t0 is the mean post-2001 locus frequency. If the L loci and phenotypes considered to be under
NFDS were ordered by ascending values of Δl, then lf was the highest ranking locus meeting the criterion
l lf
L pf. This determined the strength of NFDS acting on each locus. Therefore, the effect of NFDS on the
reproductive fitness of individual i depended on which loci were encoded in its genome, as represented
by the binary variable gi,l, and on the deviation of their simulated frequencies at time t, fl,t, from their
corresponding equilibrium frequencies: mbio.asm.org
15 March/April 2019
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e00644-19 ® McNally et al. McNally et al. and i,t
l
lf 1
L
gi,l el fl,t These summed deviations served as the exponents for the NFDS terms of the reproductive fitness,
with i,t and f corresponding to those loci under stronger NFDS, and i,t and w corresponding to those
loci under weaker NFDS. The simulations were initialized with a random selection of genotypes from the genomic data,
which were biased such that those isolates observed in 2001 were represented at 1,000-fold greater
frequency than genotypes collected in later years. This was necessary to “seed” the initial population with
ST131 and ST69, to facilitate their expansion in a realistic manner in subsequent years. March/April 2019
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e00644-19 MATERIALS AND METHODS Roary was run on the entire data set using the default 95% sequence identity
threshold to cluster genes, allowing the separation of genes based on allelic as well functional
differences. Based on a frequency distribution histogram (Fig. S1), a locus as assigned as being clade
specific if it occurred at a frequency 95% in one clade and at 5% in the other two clades. Loci
identified as clade specific were functionally annotated by performing a tblastn analysis of the nucleotide
sequence of the loci against the NCBI nonredundant database. Functional categorization of pangenomes. To assess the functional composition of the accessory
pangenome, Gene Ontology (GO) terms were assigned to gene sequences from the pangenome. Briefly,
representative sequences from the pan genome of ST131 were mapped to orthologous groups in the
bactNOG database using the eggNOG emapper utility (54) Mapping was performed using the diamond
search algorithm. The output from eggNOG was filtered to remove orthologous groups with no GO
terms. The number of genes mapping to an orthologous group was used as a score for GO term
enrichment analysis. y
Comparisons of lineage and clade-specific loci. To compare lineage pangenomes while account-
ing for differences in the number of genomes, a sampling approach was utilized. Specifically, a subset
with a size equal either to the number of ST73 or ST95 genomes was selected at random from the ST131
clade C, while ensuring a cross representation of the phylogenetic diversity of the clade. The functional
enrichment of genes in the subset was quantified and statistically compared to the ST73 or ST95
pangenome using a chi-square test. This process was repeated 100 times to produce 100 P values, from
which the median P value was calculated. Utilizing the same subsampling approach, the pangenome
composition of clade C ST131 genomes was compared to both the clade A and clade B pangenomes. Chi-square statistical tests were performed to assess the significance of the observed differences in
functional enrichment. Briefly, with each iteration of the sampling procedure, a chi-square test was
performed using the functional proportion of the subsampled pangenome as the observed value and the
proportion for ST73 or ST95 as the expected value. This generated 100 P values from which one can use
the average, maximum, or median to assess significance of the observed differences. In addition,
proportional Z statistic tests were also performed to assess the significance of the observed difference. REFERENCES 1-producing Escherichia coli isolates that belong to the successful and
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.org/10.3201/eid1910.130309. 11. Stoesser N, Sheppard AE, Pankhurst L, de Maio N, Moore CE, Sebra R,
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vanh R, Wyllie D, Sokurenko E, Manges AR, Johnson TJ, Price LB, Peto
TEA, Johnson JR, Didelot X, Walker AS, Crook DW, Modernizing Medical
Microbiology Informatics Group (MMMIG). 2016. MATERIALS AND METHODS The measurements from the 100 replicates of the subsampling procedure were used to generate an
average for the proportions as well as to estimate the variance. The tests were conducted using the
proportional measurements from ST73 and ST95 as the “true” means and quantifying how distinct the
ST131 subsamples were from these reference values. Comparisons of lineage and clade-specific loci. To compare lineage pangenomes while account-
ing for differences in the number of genomes, a sampling approach was utilized. Specifically, a subset
with a size equal either to the number of ST73 or ST95 genomes was selected at random from the ST131
clade C, while ensuring a cross representation of the phylogenetic diversity of the clade. The functional
enrichment of genes in the subset was quantified and statistically compared to the ST73 or ST95
pangenome using a chi-square test. This process was repeated 100 times to produce 100 P values, from
which the median P value was calculated. Utilizing the same subsampling approach, the pangenome
composition of clade C ST131 genomes was compared to both the clade A and clade B pangenomes. p
g
p
p
g
Chi-square statistical tests were performed to assess the significance of the observed differences in
functional enrichment. Briefly, with each iteration of the sampling procedure, a chi-square test was
performed using the functional proportion of the subsampled pangenome as the observed value and the
proportion for ST73 or ST95 as the expected value. This generated 100 P values from which one can use
the average, maximum, or median to assess significance of the observed differences. In addition,
proportional Z statistic tests were also performed to assess the significance of the observed difference. The measurements from the 100 replicates of the subsampling procedure were used to generate an
average for the proportions as well as to estimate the variance. The tests were conducted using the
proportional measurements from ST73 and ST95 as the “true” means and quantifying how distinct the
ST131 subsamples were from these reference values. mbio.asm.org
16 ® Colonization Factors in MDR E. coli Analysis of diversity observed in genes involved in anaerobic metabolism. The sequences of 64
anaerobic metabolic genes in which allelic diversity was observed were extracted from individual
genomes. The nucleotide sequences were then clustered at 80% identity and 80% length using CD-HIT,
which was run using the accurate flag and “word size” of 5 (55). MATERIALS AND METHODS An additional CD-HIT script was used to
extract gene sequences for clusters with more than 3 genes, the minimum required by MEGA-CC for
analysis. The sequences were then aligned using Muscle with default settings (56). The resulting
alignment files were analyzed in MEGA-CC to produce measurements of Tajima’s D (57). ST131 clade-specific SNPs. To visualize the ST131 clades A, B, C, C1, and C2 within the ML tree and
the PANINI clustering we identified clade-specific single-nucleotide polymorphisms (SNPs) (Table S1) as
previously described (16). Data availability. Accession numbers for the reads used in this study are listed in Table S1 with
information of year and place of isolation and the results of the in silico PCR for clade-specific SNPs. ACKNOWLEDGMENTS This work was supported by the Health Innovation Challenge Fund (HICF-T5-342 and
WT098600), a parallel funding partnership between the U.K. Department of Health and
Wellcome. The views expressed in this publication are those of the authors and not
necessarily those of the Department of Health, Public Health England, or Wellcome. T.K. was funded by the Norwegian Research Council JPIAMR (grant number 144501). J.C. was funded by the ERC (grant number 742158). N.J.C. is funded by a Sir Henry Dale
Fellowship, jointly funded by the Wellcome Trust and Royal Society (grant number
104169/Z/14/Z). C.C. is funded by the Wellcome MIDAS doctoral training program
at UoB. May 8, 2019 by guest Supplemental material for this article may be found at https://doi.org/10.1128/mBio
.00644-19. Supplemental material for this article may be found at https://doi.org/10.1128/mBio
00644-19. FIG S1, PDF file, 0.3 MB. FIG S2, PDF file, 14.5 MB. FIG S3, PDF file, 0.3 MB. on May 8, 2019 by guest
http://mbio.asm.org/
aded from FIG S4, PDF file, 0.1 MB. FIG S5, PDF file, 0.1 MB. FIG S6, JPG file, 0.1 MB. FIG S7, PDF file, 0.1 MB. TABLE S1, CSV file, 0.1 MB. TABLE S2, PDF file, 0.3 MB. on May 8, 2019 by guest
p://mbio.asm.org/ March/April 2019
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e00644-19 mbio.asm.org
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A multilayer perceptron-based model applied to histopathology image classification of lung adenocarcinoma subtypes
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OPEN ACCESS 1Key Laboratory of Geophysical Exploration Equipment, Ministry of Education, College of
Instrumentation and Electrical Engineering, Jilin University, Changchun, China, 2Department of
Thoracic Surgery, The First Hospital of Jilin University, Changchun, China, 3Department of Pathology,
Central Hospital Affiliated to Shenyang Medical College, Shenyang, China, 4Center for Reproductive
Medicine and Center for Prenatal Diagnosis, The First Hospital of Jilin University, Changchun, China CITATION
Liu M, Li L, Wang H, Guo X, Liu Y, Li Y,
Song K, Shao Y, Wu F, Zhang J, Sun N,
Zhang T and Luan L (2023) A multilayer
perceptron-based model applied to
histopathology image classification of lung
adenocarcinoma subtypes. Front. Oncol. 13:1172234. doi: 10.3389/fonc.2023.1172234 Objective: Lung cancer is one of the most common malignant tumors in humans. Adenocarcinoma of the lung is another of the most common types of lung cancer. In clinical medicine, physicians rely on the information provided by pathology tests
as an important reference for the fifinal diagnosis of many diseases. Thus,
pathological diagnosis is known as the gold standard for disease diagnosis. However, the complexity of the information contained in pathology images and
the increase in the number of patients far exceeds the number of pathologists,
especially in the treatment of lung cancer in less-developed countries. COPYRIGHT
© 2023 Liu, Li, Wang, Guo, Liu, Li, Song,
Shao, Wu, Zhang, Sun, Zhang and Luan. This
is an open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms. Methods: This paper proposes a multilayer perceptron model for lung cancer
histopathology image detection, which enables the automatic detection of the
degree of lung adenocarcinoma infifiltration. For the large amount of local
information present in lung cancer histopathology images, MLP IN MLP (MIM)
uses a dual data stream input method to achieve a modeling approach that
combines global and local information to improve the classifification
performance of the model. In our experiments, we collected 780 lung cancer
histopathological images and prepared a lung histopathology image dataset to
verify the effectiveness of MIM. TYPE Original Research
PUBLISHED 18 May 2023
DOI 10.3389/fonc.2023.1172234 Frontiers in Oncology KEYWORDS A multilayer perceptron-based
model applied to histopathology
image classification of lung
adenocarcinoma subtypes OPEN ACCESS
EDITED BY
Marco Rengo,
Sapienza University of Rome, Italy
REVIEWED BY
James C. L. Chow,
University of Toronto, Canada
Yee Kai Tee,
Tunku Abdul Rahman University, Malaysia
*CORRESPONDENCE
Tianyu Zhang
zty@jlu.edu.cn
Lan Luan
luanlan33@symc.edu.cn
RECEIVED 02 March 2023
ACCEPTED 05 May 2023
PUBLISHED 18 May 2023
CITATION
Liu M, Li L, Wang H, Guo X, Liu Y, Li Y,
Song K, Shao Y, Wu F, Zhang J, Sun N,
Zhang T and Luan L (2023) A multilayer
perceptron-based model applied to
histopathology image classification of lung
adenocarcinoma subtypes. Front. Oncol. 13:1172234. doi: 10.3389/fonc.2023.1172234 1 Introduction Morphological observation of tumor
cells under a microscope revealed that different types of tumor cells
have different nuclei, cell size, and morphology (5). However,
sometimes the microscope cannot clearly identify tumor type;
hence, tumor type has to be further determined by the indexes
obtained by immunohistochemistry. If the tumor is small, then
immunohistochemistry cannot also give a good judgment. Current
treatment methods for lung cancer are limited, irreversible, and
harmful to the human body. Therefore, accurate diagnosis prior to
lung cancer treatment is necessary to avoid unnecessary harm
to patients. Deep learning has become widely used in different fields with the
rapid development of artificial intelligence (6). and increasing studies
are showing the reliability of deep learning algorithms in medical
image analysis (7). Some examples are as follows: Hua et al. (8)
applied the DBN to classify lung nodules as malignant or benign with
a sensitivity rate of 73.40% and a specificity rate of 82.20%.Roy et al. (9) studied a system based on fuzzy inference system for the image
classification of cancerous and non-cancerous lung tumors. In a
paper published in 2018 at New York University School of Medicine
(10), a deep learning-based triple classification task for non-small cell
lung cancer histopathology slices was explored, and verified the
reliability of CNNs applied to pathology tasks. In 2019, X. Wang
et al. (11) proposed a new method based on weakly supervised
learning to solve the classification problem of pathological sections of
lung cancer. W. Shen et al. (12) designed an end-to-end deep learning
architecture, the multi-crop CNN, for low- and high-malignancy
lung nodule classification. Kriegsmann et al. (13) used a CNN-based
model for the classification of common lung cancer subtypes. The
study highlights the potential of CNN image classification models for The MLP IN MLP (MIM) proposed in this study is also
architected as a MLP, but it notices the information features
between patches and the local information within each patch. In
particular, we regarded the local patches (e.g., 16×16) as “visual
sentences” and further divided them into smaller patches (e.g., 4×4)
as “visual words.” A combination of the global feature information
of “visual sentences” and the local feature information of “visual
words” was used for modeling. In comparison to CNN and VT,
MIM’s network is considered lightweight and less computationally
intensive, while still being able to effectively extract both global and
local information. 1 Introduction tumor differentiation. Taken together, this demonstrates that deep
learning tools are feasible for some aspects of renal pathology
classification. Khademi et al. (14) proposed a novel spatio-temporal
fusion model using convolutional autoencoder to extract features
from CT scans and Swin Transformer to process the time series
features of clinical data. The model shows high accuracy and stability
in the malignancy prediction of lung cancer. Lung cancer has become one of the major cancer diseases in the
world (1). Breast cancer and prostate cancer are more common
among women and men, respectively, but the incidence and
mortality rates of both cancers are much lower than those of lung
cancer. Lung adenocarcinoma is the most common type of lung
cancer, accounting for about 50% of lung cancers (2). Microinvasive
lung adenocarcinoma is an early type of lung cancer that is detected
in a timely manner whose tumor cells can be completely removed
through radical surgery (3). Invasive lung adenocarcinoma is
relatively more malignant and more complicated to treat than
noninvasive ones. Timely and accurate determination of the type
of lung cancer can greatly improve the cure rate of patients. Convolutional neural networks (CNNs) (15) and vision
transformers (VTs) (16) have emerged as popular deep learning
models for various computer vision tasks, including image
segmentation and classification (17). While CNNs and VTs have
shown promising results, they often require complex network
architectures and high computational costs. To address these
challenges, a new model called MLP-Mixer (18) has been proposed
by the Google team. Unlike CNNs and VTs, MLP-Mixer relies
entirely on multilayer perceptron (MLP) and does not use any
convolutional operations or self-attentive mechanisms, resulting in
a simpler network architecture and lower computational
requirements. Therefore, MLP-Mixer offers a promising alternative
for efficient and effective image processing tasks. MLP-Mixer only
applies MLP to process the global information between patch
sequences to achieve classification tasks, which can obtain a
performance comparable to those of CNN and VT. The
morphology and volume of cell nuclei in histopathological images
are different; therefore, these images have a large amount of local
information. Moreover, MLP-Mixer destroys the local structure of
patches and cannot capture local information; hence, the application
of MLP-Mixer model to histopathological images has a shortage. The most reliable scientific basis for physicians to diagnose lung
cancer is through pathology (4). OPEN ACCESS Results: The MIM achieves a diagnostic accuracy of 95.31% and has a precision,
sensitivity, specificity and F1-score of 95.31%, 93.09%, 93.10%, 96.43% and
93.10% respectively, outperforming the diagnostic results of the common
network model. In addition, a number of series of extension experiments
demonstrated the scalability and stability of the MIM. Conclusions: In summary, MIM has high classifification performance and
substantial potential in lung cancer detection tasks. histopathology image, lung cancer, deep learning, multilayer perceptron,
image classification 01 Frontiers in Oncology frontiersin.org 10.3389/fonc.2023.1172234 Liu et al. 2.2 Data preprocessing Dataset pre-processing was performed, and images were
subjected to resizing, data enhancement, and data normalization
operations. All images were resized to 256×256 pixels. Five types of
enhancement techniques (horizontal flip, rotation, scaling, height
shift, and width shift) were then applied on the dataset, where the
rotation was set to 15° and the height shift and width shift were set
to 5% of the image size. These enhancement methods alter the
relative position of cancer in histopathological images; therefore,
the training set was expanded for training the MIM and no data
enhancement operation was taken for the test set. The tumor tissues
were extracted and sent to the pathology department; thus, each
doctor may use different stains in the process of making tumor
sections because of different personal habits. An image 1 Introduction (c) In this stage, the pre-processed data are input into MIM
Layer, and the global information features and local
information features in it are extracted using a multilayer
perceptron. (c) In this stage, the pre-processed data are input into MIM
Layer, and the global information features and local
information features in it are extracted using a multilayer
perceptron. (c) In this stage, the pre-processed data are input into MIM
Layer, and the global information features and local
information features in it are extracted using a multilayer
perceptron. (d) In this stage of classification, the output feature maps are
classified using the standard classification head. (e) Finally, our proposed MLP IN MLP is evaluated and the
model is evaluated using four metrics: accuracy, precision,
specificity, sensitivity and F1-score. (e) Finally, our proposed MLP IN MLP is evaluated and the
model is evaluated using four metrics: accuracy, precision,
specificity, sensitivity and F1-score. The main contributions of this paper are as follows. (1) A new
deep learning based model (i.e., MIM) is proposed, which only uses
multi-layer perceptron to do feature extraction for visual sentences
and visual words respectively, which can extract local features and
global features sufficiently to improve the overall classification
performance. (2) A triple classification task was performed to
identify the degree of lung adenocarcinoma infiltration, and a
digital pathology image dataset for lung adenocarcinoma
infiltration detection was prepared in collaboration with
pathologists. The experimental results proved that the MIM model
can effectively determine the degree of lung cancer infiltration and
achieved a good performance of 95.31%. (3) The MIM model has
good performance in detecting the degree of lung adenocarcinoma
infiltration and shows good generalization performance in other
cancer histopathology image classification tasks. 1 Introduction The proposed model was applied to a private
lung cancer histopathology dataset. The workflow of MIM is shown
in Figure 1: in (a) We use the lung cancer histopathology dataset provided by
the First Hospital of Jilin University as the training set, and
the details of this dataset are described in Section2. (a) We use the lung cancer histopathology dataset provided by
the First Hospital of Jilin University as the training set, and
the details of this dataset are described in Section2. D
A
B
E
C
FIGURE 1
Workflow of the proposed MLP IN MLP model: (A) train date, (B) date process, (C) train process, (D) output classification results, (E) Assessing performance. 1
ow of the proposed MLP IN MLP model: (A) train date, (B) date process, (C) train process, (D) output classification results, (E) Assessing p 02 Frontiers in Oncology frontiersin.org Liu et al. 10.3389/fonc.2023.1172234 different departments, and the diagnosis was collated and labeled by
four experienced doctors over a period of 3 months to ensure that
each sample had considerable clinical features. Based on this
approach, we cropped and cut the histopathological images and
gave specific labels for each cut. The dataset includes two parts:
training set and test set. Each part contains three categories
(Normal, Infiltration, and Micro_infiltration). In the training set
there are 7842 data, Infiltration and Micro_Infiltration include
2,614 and 2,614 digital pathology images respectively, and the
remaining 2,614 pathology data are Normal. A total of 870 digital
pathological images are included the test set. Among which, each
category contains 290 lung pathological images. This dataset
contains 8712 lung pathology images of size 2048×1500. Among
the invasive and microinvasive lung adenocarcinomas, the tumors
were predominantly of the adnexal type of growth, whereas
microinvasive adenocarcinomas had infiltration foci of ≤0.5 cm in
maximum diameter and were of the follicular, papillary,
micropapillary, or solid type. Infiltrating adenocarcinomas had
infiltration foci with a minimum diameter of >0.5 cm. Normal
represented a benign area of lung tissue. Some samples of the
specific data are shown in Figure 2. (b) During the data processing, we expanded the dataset by
performing data enhancement operation, and then finally
did image normalization. (b) During the data processing, we expanded the dataset by
performing data enhancement operation, and then finally
did image normalization. 2.4 Methods The overall structure of MLP-Mixer consists of three main parts
as follows: What makes the MLP-Mixer model effective is that it can fully
handle the relationship between several input patches to achieve
global information extraction. However, this method also has an
obvious drawback, that is, after the input image is split into several
patches, it spreads each patch into a 1D vector and thus destroys the
local information inside each patch. Therefore, we believe that
MLP-Mixer modeling does not take into account the information
inside each input patch. 1. Per-patch Fully-connected: Linearly map the input image
into a number of p×p×3-sized image patches, flatten each
patch and linearly map it into a two-dimensional vector. The input image is thus converted into a two-dimensional
(S,D) tensor, where S represents the number of patches and
D represents the flattened dimensions of each patch. Specifically, S = HW
P2 (where H and W represent the height
and width of the input image, respectively, and p represents
the size of each patch), and D=3×p2 (where 3 represents the
number of channels in the input image). 1. Per-patch Fully-connected: Linearly map the input image
into a number of p×p×3-sized image patches, flatten each
patch and linearly map it into a two-dimensional vector. The input image is thus converted into a two-dimensional
(S,D) tensor, where S represents the number of patches and
D represents the flattened dimensions of each patch. Specifically, S = HW
P2 (where H and W represent the height
and width of the input image, respectively, and p represents
the size of each patch), and D=3×p2 (where 3 represents the
number of channels in the input image). By analyzing the lung histopathology dataset, we found that the
histological features of lung cancer can be broadly classified as
adnexal, alveolar, papillary, micropapillary, and solid (21). The cells
that are likely to be presented in high magnification view are mostly
tubular, columnar oval, and peg-shaped. Pathologists rely on
information about these complex shape features to diagnose the
type of cancer and the degree of infiltration. 2. Mixer Layer: This layer consists of N identical layers. Figure 3
shows the internal structure of the layers. Each layer consists
of two types of MLPs: channel-mixing MLP and token-
mixing MLP. Token-mixing MLP acts on the columns of a
2D table and learns the feature information between different
spatial locations; it operates independently on each channel. 2.1 Dataset A unique dataset of lung cancer histopathological images
provided by the First Hospital of Jilin University was used. The
histopathological images were derived from 780 cases. Each case
was given a clear diagnosis by a joint consultation of doctors from A
B
C
FIGURE 2
Some representative examples of lung adenocarcinoma data: (A) microinvasive lung adenocarcinoma, (B) invasive lung adenocarcinoma, and (C)
images of normal lung tissue. C B FIGURE 2
Some representative examples of lung adenocarcinoma data: (A) microinvasive lung adenocarcinoma, (B) invasive lung adenocarcinoma, and (C)
images of normal lung tissue. 03 03 Frontiers in Oncology frontiersin.org Liu et al. 10.3389/fonc.2023.1172234 10.3389/fonc.2023.1172234 degradation over deep gradients and the accuracy
degradation of the training set. Layer normalization (20) is
a key part of the model for stable training and faster
convergence. normalization operation was performed to turn the pathological
images into black and white to avoid the color of the stain as a
feature during model learning. 3. Finally, MLP-Mixer uses a standard classification head with
the global average pooling layer followed by a linear
classifier. 3. Finally, MLP-Mixer uses a standard classification head with
the global average pooling layer followed by a linear
classifier. 2.3 MLP-mixer The MLP-Mixer does not use convolution operations or self-
attentive mechanisms. Its architecture is based entirely on repeated
application of multilayer perceptrons (MLPs) on spatial locations or
feature channels, relying only on basic matrix multiplication
routines, changes in data layout (reshaping and transposition),
and scalar nonlinearities. Therefore, the model has a simple
network and low computational effort. A simple MLP-based
model that can compete with the best CNN and VT models
available today was constructed. MLP-Mixer implements the convolutional and pooling layers of
CNNs and the self-attentive layer of Transformer architecture using
the two MLP types and demonstrates a simple MLP-based model
that achieves competitiveness with current CNN or VT models,
because MLPs require only simple mathematical operations. 2.4 Methods Channel-mixing MLP acts on the rows of a 2D table and
learns the feature information between different channels; it
runs independently on the same spatial location. The output
dimensions of both MLPs always remain the same as the
input dimensions. The idea is to explicitly distinguish
between per-position (channel-mixing) operations and
cross-position (token-mixing) operations. In addition,
other standard components are introduced, such as the
residual structure (skip-connections) (19), which improves
the ability of information interaction and avoids network 2. Mixer Layer: This layer consists of N identical layers. Figure 3
shows the internal structure of the layers. Each layer consists
of two types of MLPs: channel-mixing MLP and token-
mixing MLP. Token-mixing MLP acts on the columns of a
2D table and learns the feature information between different
spatial locations; it operates independently on each channel. Channel-mixing MLP acts on the rows of a 2D table and
learns the feature information between different channels; it
runs independently on the same spatial location. The output
dimensions of both MLPs always remain the same as the
input dimensions. The idea is to explicitly distinguish
between per-position (channel-mixing) operations and
cross-position (token-mixing) operations. In addition,
other standard components are introduced, such as the
residual structure (skip-connections) (19), which improves
the ability of information interaction and avoids network Digital pathological images are more highly complex than
natural images with rich details and local information; therefore,
the application of MLP-Mixer to medical pathology will be
somewhat limited. It will fail to tap the cell and tissue features in
different locations if the granularity of patch segmentation is not
good enough. This paper proposes an MIM architecture to address
the shortcomings of MLP-Mixer. The proposed model considers the
global and local information between patches and the internal
information of each patch, improving the performance of the
model. The specific network structure is shown in Figure 4. We partitioned the input 2D image into n patches uniformly and
without overlapping. Let (p, p) be the resolution of each patch and
X = [X1, X2, …, Xn] ∈Rn×p×p×3. Each patch was spread into a 1D FIGURE 3
Detailed structure of Mixer Layer. 04 Frontiers in Oncology frontiersin.org Liu et al. Liu et al. 10.3389/fonc.2023.1172234 FIGURE 4
Internal structure of MLP IN MLP. vector with a length of 3 × p². Finally, linear mapping was performed
to obtain a 2D tensor table about patches (n, c) i.e. X∈Rn×c. 2.4 Methods MLP-
Mixer just designs a mixer layer to handle the relationship between n
image patch sequences, unlike MIM, where two standard mixer layers
extract the global and local information in the image. In MIM, we
consider each patch as a visual sentence representing an image. Each
patch will be further divided into m sub-patch visual words, that is, a
visual sentence consists of a series of visual words: Xi ! [Xi1, Xi2, …,
Xim] ∈Rn×P×P×3, where Xij ∈Rs×s×3 is the j-th visual word of the i-th
visual sentence, and (s, s) is the resolution size of the subpatch, j=1, 2,
…, m. Each subpatch was also spanned into a 1D vector during the
segmentation of the subpatch, and the 2D tensor table (n’, c’) about
the subpatch was obtained after linear mapping. For example, the size
of the visual sentence is 16×16×3. Here, p=16; therefore, the size of
the visual word is 8×8×3. Each visual sentence was divided into four
visual words, that is Xij ∈R8×8×12. words. For example, in a patch of human face, a word corresponding
to the eye is more related to other words of eyes and interacts less
with forehead part. For the sentence level, the sequence of visual
words are transformed into the domain of visual sentence by linear
projection and added into the visual sentence as follows: Ul
i = Xl
i + FC(LN(Yl
ij))
(3) (3) With the above addition operation, the representation of
sentence sequences is augmented by word-level features. We used
the standard mixer layer for transforming the visual sentences as
follows: Sl
i = Ul
i + (W2s(W1(LN( Ul
i))T))T
(4)
Ql
i = Sl
i + (W4s(W3(LN( Sl
i))T))T
(5) Sl
i = Ul
i + (W2s(W1(LN( Ul
i))T))T
(4) (4) Ql
i = Sl
i + (W4s(W3(LN( Sl
i))T))T
(5) (5) In MIM, we have two data flows in which one flow operates
across the visual sentences, and the other processes the visual words
inside each sentence. For the visual words, we utilize a small mixer
layer as follows to explore the relation between visual words: This big mixer layer is used for modeling the relationships
among sentence sequences. Frontiers in Oncology 2.5 Hyperparameter settings proposed MIM and other networks. The results are shown in
Table 2. The accuracy, precision, sensitivity, specificity and F1-
score of MIM on the test set were 95.31%, 93.09%, 93.10%, 96.43%
and 93.10%, respectively. The accuracy, precision, sensitivity,
specificity and F1-score of MLP-Mixer on the test set were
92.43%, 88.97%, 88.96%, 94.24% and 88.97%, respectively. The
five evaluation metrics of the proposed MIM were on average
nearly 3% higher than those of MLP-Mixer. In addition, MIM
had higher evaluation metrics than ResNet50 and Swin
Transformer (25). The effectiveness of modeling using a
combination of global and local information in the MIM model
was demonstrated for the large amount of local information present
in lung adenocarcinoma. proposed MIM and other networks. The results are shown in
Table 2. The accuracy, precision, sensitivity, specificity and F1-
score of MIM on the test set were 95.31%, 93.09%, 93.10%, 96.43%
and 93.10%, respectively. The accuracy, precision, sensitivity,
specificity and F1-score of MLP-Mixer on the test set were
92.43%, 88.97%, 88.96%, 94.24% and 88.97%, respectively. The
five evaluation metrics of the proposed MIM were on average
nearly 3% higher than those of MLP-Mixer. In addition, MIM
had higher evaluation metrics than ResNet50 and Swin
Transformer (25). The effectiveness of modeling using a
combination of global and local information in the MIM model
was demonstrated for the large amount of local information present
in lung adenocarcinoma. Finding the optimal values of hyperparameters is one of the
crucial tasks for building a robust model. Besides fully extracting
image information, the hyperparameters values have a great
influence on the fast convergence of the model. MIM was used to
train the image of the lung cancer pathological tissue dataset for 200
epochs. In each epoch, batch size is set to 256. It uses a method to
train the lung adenocarcinoma infiltration level recognition task
from scratch. AdamW (23) was used as the optimizer, and its
parameters were set as 3×10−5 learning rate. In addition, the
dropout ratio (24) was set to 0.1 in the training process. The superior performance of MIM can be further demonstrated
by the correlation model confusion matrix shown in Figure 5. In the
test confusion matrix, the classifications of normal lung
pathological data are very accurate. The classification errors of
ResNet and Swin Transformer are shown in Figures 5B and C,
respectively. Both methods misclassified Infiltration as
Micro_infiltration. 2.7 Performance metrics Accuracy, precision, sensitivity, specificity and F1-score were
calculated to evaluate the performance of MIM. Here, accuracy
indicates the proportion of the number of samples correctly
predicted by the model to the total number of samples. Precision
indicates the proportion of samples correctly predicted by the
model as a percentage of all samples predicted by the model to be
positive. Sensitivity indicates the proportion of the number of
samples correctly predicted by the model as a percentage of the
total number of positive samples; the specific value indicates the
proportion of the number of samples correctly predicted by
the model as a percentage of the total number of negative
samples. F1-score combines the results of precision and
sensitivity. True positive, true negative, false positive, and false
negative were used in the definition of these five criteria in Table 1. 3 Result A hybrid dataset consisting of the publicly available
pathological dataset of gastric cancer from Northeastern
University (26) and our private dataset was used to come up with
extended experiments to explore the performance of the model on
lung tissue and other histopathological data. The experimental
setup was the same as the main experimental setup. The gastric 2.4 Methods In summary, the inputs and outputs of
the MIM layer include the sequence of visual word and sequence of
visual sentences as shown in Figure 4; hence, MIM can be
formulated as: Zl
ij = Xl
ij + (W2s(W1(LN( Xl
ij))T))T
(1)
Yl
ij = Zl
ij + (W4s(W3(LN( Zl
ij))T))T
(2) (1) Y , Q = MIM(X , U)
(6) (6) (2) In our MIM layer, the small mixer layer is used to model the
relationship between visual words for local feature extraction, and
the big mixer layer captures the intrinsic information from the
sequence of visual sentences. We built the MIM network by stacking
the MIM layer for N times. Finally, MIM uses a standard
classification head with the global average pooling layer, followed
by a linear classifier. where l = 1, 2, …, L is the index of the l-th block, L is the total number
of stacked blocks, T stands for transpose, s is an element-wise
nonlinearity (GELU) (22), W is a fully connected layer process, Z
is the output after token-mixing MLP, and Y is the output after
channel-mixing MLP. This process builds the relationship among
visual words by computing the interactions between any two visual Frontiers in Oncology
05 05 frontiersin.org frontiersin.org 10.3389/fonc.2023.1172234 Liu et al. 2.6 Experimental environment The experiments were carried out on a local workstation with 32
GB RAM and Windows 10 operating system. The GPU of the
workstation consists of NVIDIA RTX 3090 and CPU Intel Core i9-
10875H 2.30 GHz. We used the Python 3.7 programming language
and PyTorch 1.8.0. 2.5 Hyperparameter settings In Figure 5D, the misclassification of MLP-
Mixer is due to the confusion caused between Infiltration and
Micro_infiltration. The main reason for these phenomena are that
the Infiltration and Micro_infiltration samples contain tumors, but
the only difference is whether the infiltrated area is more than
5 mm. Moreover, the model gave wrong judgments for some
samples near the threshold. As shown in Figure 5A, MIM
effectively alleviated these problems. Using MIM, only 21
Infitration data were incorrectly predicted as Micro_infiltration
data, and 13 Micro_Infitration data were incorrectly predicted as
Infitration data. The effectiveness of the MIM modeling approach
using local and global information was further demonstrated in
terms of the degree of infiltration of lung adenocarcinoma, allowing
the effective identification of differences in infiltrative and
microinfiltrative pathological features. 3.1 Experimental results and analysis In the test phases, accuracy, precision, sensitivity, specificity and
F1-score were calculated to evaluate the performance of the TABLE 1 Criteria and corresponding definitions for image global detection evaluation. TABLE 1 Criteria and corresponding definitions for image global detection evaluation. Criterion
Definition
Criterion
Definition
Accuracy
TP + TN
TP + TN + FP + FN
Precision
TP
TP + FP
Sensitivity
TP
TP + FN
Specificity
TN
TN + FP
F1-score
2 Precision Sensitivity
Precision + Sensitivity TABLE 1 Criteria and corresponding definitions for image global detection evaluation. Criterion
Definition
Criterion
Definition
Accuracy
TP + TN
TP + TN + FP + FN
Precision
TP
TP + FP
Sensitivity
TP
TP + FN
Specificity
TN
TN + FP
F1-score
2 Precision Sensitivity
Precision + Sensitivity 06 10.3389/fonc.2023.1172234 Liu et al. TABLE 2 Results of MLP IN MLP and other networks on private datasets (In %). TABLE 2 Results of MLP IN MLP and other networks on private datasets (In %). Model
Accuracy
Precision
Sensitivity
Specificity
F1-score
MLP IN MLP
95.31
93.09
93.10
96.43
93.10
MLP-Mixer (18)
92.43
88.97
88.96
94.24
88.97
ResNet50 (19)
93.95
91.46
91.14
95.36
91.30
Swin (25)
94.60
92.48
92.06
95.85
92.27 histopathology database from Northeastern University has three
sizes (160×160, 120×120, and 80×80 pixels), and two data types
(normal and abnormal). Some examples are shown in Figure 6. In
this experiment, 160×160 size data were used and resized to the
same size (256×256) as our private dataset during data processing. We use 10080 normal data images and 6670 abnormal data images. Considering the number of training and test sets in our private
dataset, we randomly partitioned the training and test sets in a 9:1
ratio for the histopathological data of gastric cancer. The mixed data set is a five-category task. The combined dataset’s data settings are
shown in Table 3. Six representative models were selected for the comparative
experiments with MIM, including two CNN models [VGG16 (27)
and ResNet50] two VT models [ViT and XCiT (28)], and two MLP
models [ResMLP (29) and MLP-Mixer]. The experimental settings
were the same as the previoussetting. Table 4 shows the results of
the models on the test set of the combined dataset. 3.1 Experimental results and analysis ViT obtained the
highest accuracy rate of 95.50% among the six models, and MLP- D
A
B
C
FIGURE 5
The confusion matrix obtained by the model on a private dataset: (A) MLP IN MLP, (B) Swin Transformer, (C) ResNet50, (D) MLP-Mixer. A B B A C D D C 07 Frontiers in Oncology frontiersin.org Liu et al. Liu et al. 10.3389/fonc.2023.1172234 A
B
FIGURE 6
Some examples in the gastric cancer dataset: (A) normal stomach tissue data, (B) abnormal stomach tissue data. B FIGURE 6
Some examples in the gastric cancer dataset: (A) normal stomach tissue data, (B) abnormal stomach tissue data. dataset, LC25000 (30), was incorporated to the private dataset, and
extended experiments were performed on the hybrid dataset to
validate the generalization ability of MIM on other lung cancer
subtype pathology datasets. The experimental setup was the same as
the main experimental setup. The LC25000 dataset has five different
data types about lung and colon histopathology: normal lung
histopathology image (Lung_n), lung squamous carcinoma
histopathology image (Lung_scc), lung adenocarcinoma
histopathology image (Lung_aca), normal colon histopathology
image (Colon_n), and colon adenocarcinoma histopathology
image (Colon_aca). Some examples are shown in Figure 7. The
dataset has 4500 images in the training set and 500 images in the
test set for each type, for a total of 25000 images for the five data Mixer had the lowest accuracy rate of 91.45%. Among the models,
the proposed MIM model achieved the highest test accuracy of
96.50%, which was nearly 1% higher than the accuracy of ViT and
nearly 5% more accurate than MLP-Mixer. In addition, the
precision, sensitivity, specificity and F1-score of MIM were also
the highest at 93.95%, 93.89%, 98.91% and 93.92%, respectively. The
results show the effectiveness of the proposed MIM model. Mixer had the lowest accuracy rate of 91.45%. Among the models,
the proposed MIM model achieved the highest test accuracy of
96.50%, which was nearly 1% higher than the accuracy of ViT and
nearly 5% more accurate than MLP-Mixer. In addition, the
precision, sensitivity, specificity and F1-score of MIM were also
the highest at 93.95%, 93.89%, 98.91% and 93.92%, respectively. The
results show the effectiveness of the proposed MIM model. 3.2.2 Extended experiment on combined dataset:
private data and public data Model
Accuracy
Precision
Sensitivity
Specificity
F1-score
MLP IN MLP
96.50
93.95
93.89
98.91
93.92
ResNet50 (19)
Vgg16 (27)
XCiT (28)
ViT (16)
ResMLP (29)
MLP-Mixer (18)
94.90
93.68
95.05
95.50
94.87
91.45
92.44
93.70
87.40
92.49
92.43
87.12
92.34
93.53
89.66
92.37
92.10
87.19
98.46
98.12
97.85
98.53
98.39
97.62
92.39
93.62
88.53
92.43
92.27
87.16 A
B
C
D
E
FIGURE 7
Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung
adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. B D A C D C B E A FIGURE 7
Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung
adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. not detected; therefore, the accuracy of MIM in this set was 97.42%. For colon adenocarcinoma histopathological images, 996 images
were detected by the MIM model, and only four images were not
detected; therefore, the accuracy of the model for colorectal images
was 99.60%. The LC2500 dataset contains lung cancer subtypes
different from invasive lung cancer in addition to the colon
histopathological data that are also present. The findings indicate
that MIM has good classification performance between different lung
cancer subtypes and performs well for colon histopathological data. types. Both datasets contain normal lung histopathology data;
hence, we set all normal lung tissue data as one category, forming
a seven-category hybrid dataset. Table 5 shows the data settings. The results of the comparison tests between MIM and six classical
deep learning models in the lung cancer and colon cancer detection
task are shown in Table 6. Obviously, among the six classical models,
ConViT (33) performed the best in the test set with accuracy,
precision, sensitivity, specificity and F1-score values of 97.83%,
96.70%, 96.43%, 99.59% and 96.52%, respectively. The accuracy,
precision, sensitivity, specificity and F1-score values of MIM on the
test set were 98.09%, 97.17%, 97.04%, 99.67% and 97.11%,
respectively. The specific detection results of MIM are shown in
Table 7. For the five types of lung pathological images, 2309 images
were correctly detected by the MIM model, and only 61 images were 3.2.2 Extended experiment on combined dataset:
private data and public data In addition to exploring the sensitivity between different
histopathologies, we also incorporated other lung cancer subtypes
to further increase the complexity of the experiment. The public TABLE 3 Data setting for training and test sets. TABLE 3 Data setting for training and test sets. Class/Dataset
Train
Test
Sum
Micro_infiltration
2614
290
2904
Infiltration
2614
290
2904
Normal
2614
290
2904
Stomach_Abnormal
6013
657
6670
Stomach_Normal
9072
1008
10080
Sum
22927
2535
25462 08 Liu et al. 10.3389/fonc.2023.1172234 Liu et al. TABLE 4 Comparison results of models on the test set of the combined dataset (In %). TABLE 4 Comparison results of models on the test set of the combined dataset (In %). TABLE 4 Comparison results of models on the test set of the combined dataset (In %). Model
Accuracy
Precision
Sensitivity
Specificity
F1-score
MLP IN MLP
96.50
93.95
93.89
98.91
93.92
ResNet50 (19)
Vgg16 (27)
XCiT (28)
ViT (16)
ResMLP (29)
MLP-Mixer (18)
94.90
93.68
95.05
95.50
94.87
91.45
92.44
93.70
87.40
92.49
92.43
87.12
92.34
93.53
89.66
92.37
92.10
87.19
98.46
98.12
97.85
98.53
98.39
97.62
92.39
93.62
88.53
92.43
92.27
87.16
A
B
C
D
E
FIGURE 7
Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung
adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. Model
Accuracy
Precision
Sensitivity
Specificity
F1-score
MLP IN MLP
96.50
93.95
93.89
98.91
93.92
ResNet50 (19)
Vgg16 (27)
XCiT (28)
ViT (16)
ResMLP (29)
MLP-Mixer (18)
94.90
93.68
95.05
95.50
94.87
91.45
92.44
93.70
87.40
92.49
92.43
87.12
92.34
93.53
89.66
92.37
92.10
87.19
98.46
98.12
97.85
98.53
98.39
97.62
92.39
93.62
88.53
92.43
92.27
87.16
A
B
C
D
E
FIGURE 7
Some examples in the LC25000 dataset: (A) lung squamous carcinoma histopathology picture, (B) normal lung histopathology image, (C) lung
adenocarcinoma histopathology image, (D) normal colon histopathology image, (E) colon adenocarcinoma histopathology image. Frontiers in Oncology
frontiersin.org
09 4 Discussion First, in our experiments, we found that the performance is better
when the number of MIM blocks is set to 8, when the parameter size TABLE 5 Data setting for training and test sets. TABLE 5 Data setting for training and test sets. g
g
Class/Dataset
Train
Test
Sum
Micro_infiltration
2614
290
2904
Infiltration
2614
290
2904
Lung_aca
4500
500
5000
Lung_scc
4500
500
5000
Lung_n
7114
790
7904
Colon_aca
4500
500
5000
Colon_n
4500
500
5000
Sum
30342
3370
33712 09 Frontiers in Oncology Liu et al. 10.3389/fonc.2023.1172234 TABLE 6
A comparison of image classification results on the mixed test set. TABLE 6
A comparison of image classification results on the mixed test set. TABLE 6
A comparison of image classification results on the mixed test set. Model
Accuracy
Precision
Sensitivity
Specificity
F1-score
MLP IN MLP
98.09
97.17
97.04
99.67
97.11
EfficientNet (31)
96.96
95.22
95.45
99.32
95.34
InceptionV2 (32)
96.76
95.29
95.31
99.43
95.30
Swin (24)
97.51
96.09
95.97
99.53
96.03
ConViT (33)
97.83
96.70
96.43
99.59
96.52
gMLP (34)
96.41
96.09
95.16
99.35
95.63
MLP Mixer (17)
95.54
92.71
92.07
99.00
92.39
(In %). TABLE 7 The result of MLP IN MLP in classification task. Data Type
Correct
Incorrect
Accuracy
Lung histopathology image
2039
61
97.42
Colon histopathology image
996
4
99.60
Sum
3305
65
98.09
(In %). positively correlated with the number of patches. The fullest
possible exploitation of the internal local information of patches
should also be considered. Therefore, the resolutions of vision
sentences and vision words were set to 16×16 and 4×4, respectively,
considering the computational power and training time of the model. of MIM is about 165 MB. We had 7842 training data and used 200
epochs to train MIM, and the total time was about 230 minutes. We
lightened the model according to the actual needs of clinical work
(35). First, we used a quantization technique to reduce the number of
bits required to store each weight from 32 bits to 16 bits. Then, the
size of the model parameters was reduced to 83 MB. Consequently,
the classification performance was almost unchanged, and the total
training time was reduced by about 55 minutes. Third, the optimizer and activation function should be chosen
carefully. Publisher’s note All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. 5 Conclusion This work was supported in part by the Science and Technology
Department of Jilin Province of China under Grants 20210204193YY,
20220203133SF, and YDZj202201ZYTS137, by the National Key
Research and Development Project of China under Grant
2019YFC0409105, and by the Shenyang Science and Technology
Plan Public Health R&D Special Project under Grant 21-172-9-18. Support by Graduate Innovation Fund of Jilin University. In the experiments, MIM was tested on a lung cancer pathology
dataset with 95.31% accuracy, showing the model’s potential in lung
cancer determination tasks. The lung pathological images have a
large amount of information on histological shapes, such as pegs
and papillae. The local structure of patches was destroyed in the
MLP-Mixer model. Compared with the conventional MLP-Mixer,
MIM can better preserve and model local information for lung
histopathology identification tasks. In the extended experiments,
MIM is more efficient and performs better than CNN, MLP and VT
models proving its powerful generalization ability. Ethics statement Written informed consent was obtained from the individual(s)
for the publication of any potentially identifiable images or data
included in this article. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. Data availability statement The original contributions presented in the study are included
in the article/supplementary material. Further inquiries can be
directed to the corresponding authors. 4 Discussion Although various optimizers (e.g., stochastic gradient
descent (36) and Lagrangian optimizer) and activation functions
(e.g., CroReLU) (37, 38) are implemented with deep networks to
solve different classification problems, we applied AdamW and GELU
because of their efficiency in the proposed architecture with our
datasets. AdamW solves the problem of parameter over-fitting with
Adam optimizer (39) by introducing the L2 regularization terms of
parameters in the loss function. It is the fastest optimizer for gradient
descent speed and training neural networks that is used in all models. GELU has been widely used in the recent Transformer model. Randomness is introduced to make the model training process
more robust and avoid the problem of gradient disappearance well. Loss functions have an important role in a deep network architecture. Hybrid loss functions have been proposed in several works to
improve the performance of model. We used the default loss
function [i.e., cross-entropy (40)] because of its low computational
cost and efficiency with our pathological images. Second, the segmentation granularity of patches varies in terms of
the feature richness that can be extracted from the image. By contrast,
the feature extraction layer of MIM has two different data streams,
namely, visual sentences and visual words, and MIM was designed to
capture the global feature information between patches and mine the
local feature information within patches. In the pre-experiments, we
set different resolutions for the visual sentences and visual words. For
the fairness of the experiments, the number of feature extraction
layers in the experiments was set to 8. The rest of the experimental
settings and the datasets used were the same as those in the main
experiments. The specific experimental details and results are shown
in Table 8. The highest accuracy rate of 95.31% was achieved with a
visual sentence resolution of 16 and a visual word resolution of 4. The
finer the granularity of patch segmentation, the more the number of
patches. Additionally, the amount of model computation was TABLE 8 Comparison test of different patch sizes. Model
Visual Sentences
Visual word
Accuracy
MIM
32
8
93.24
32
4
94.12
16
8
94.78
16
4
95.31
(In %). Model
Visual Sentences
Visual word
Accuracy
MIM
32
8
93.24
32
4
94.12
16
8
94.78
16
4
95.31
(In %). 10 Frontiers in Oncology frontiersin.org Liu et al. Liu et al. 10.3389/fonc.2023.1172234 9. Roy TS, Sirohi N, Patle A. Classification of lung image and nodule detection using
fuzzy inference system, in: International conference on computing, communication &
automation IEEE (2015), 1204–7. Author contributions Finally, although the proposed model showed a prominent
performance in the classifying degree of lung cancer infiltration,
this study encountered some challenges. Our work was limited to two
types of lung cancer data, namely, infiltration and microinfiltration,
because of data inadequacy. Moreover, we only considered digital
pathological images. Other medical imaging techniques, such as
computed tomography and magnetic resonance imaging, can also
be taken under consideration in future work. In addition, this work
can be extended to patients’ medical history (age, gender, physical
condition, etc.) with image datasets for more accurate predictions. The conception or design of the work: ML, LYL, and TZ; the
acquisition, analysis, or interpretation of data: YGL, FW, TZ, and
YPL; drafting the work or revising: YS, XG, and HW; provide
approval for publication of the content: NS, JZ, and KS; agree to be
accountable for all aspects of the work: ML and LL. All authors
contributed to the article and approved the submitted version. 11. Wang X, Chen H, Gan C. Weakly supervised deep learning for whole slide lung
cancer image analysis. IEEE Trans cybernetics (2019) 50(9):3950–62. doi: 10.1109/
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Bioactive Contents of Different Parts of Strawberry Tree (Arbutus unedo L.) Plant
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DergiPark (Istanbul University)
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ÖZ Çalışma, Piraziz (Giresun) ilçesinde yetiştirilen kocayemiş bitkisinin farklı kısımlarının (yaprak,
çiçek ve meyve) toplam fenolik, toplam flavonoid ve antioksidan aktivitesini (DPPH ve FRAP
testlerine göre) belirlemek amacıyla yürütülmüştür. En yüksek toplam fenolik içeriği yaprak (1.10
g 100 g-1) ve çiçekte (1.08 g 100 g-1) belirlenirken, en düşük meyvede (0.81 g 100 g-1) tespit
edilmiştir. Toplam flavonoid içeriği en yüksek yaprakta (3.38 g 100 g-1), en düşük ise meyvede (0.04
g 100 g-1) bulunmuştur. En yüksek antioksidan aktivite her iki yönteme göre de yaprak (sırasıyla
7.72 mmol 100 g-1 ve 2.58 mmol 100 g-1) ve çiçekte (sırasıyla 7.84 mmol 100 g-1 ve 2.58 mmol 100
g-1) tespit edilirken, en düşük meyvede (sırasıyla 1.21 mmol 100 g-1 ve 1.50 mmol 100 g-1)
belirlenmiştir. Sonuç olarak, incelenen özellikler bakımından en iyi sonuçlar bitkinin yaprak ve
çiçek kısımlarından elde edilmiştir. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının
Biyoaktif İçerikleri ehmet Fikret BALTA1
, İzzet YAMAN1
, Orhan KARAKAYA2*
, Umut ATEŞ2
1Bahçe Bitkileri, Ziraat Fakültesi, Ordu Üniversitesi, Türkiye
2Bahçe Bitkileri, Ziraat Fakültesi, Sakarya Uygulamalı Bilimler Üniversitesi, Türkiye Anahtar Kelimeler: Yaprak, çiçek, meyve, fenolik, antioksidan. * Sorumlu yazar : orhankarakaya7@gmail.com Keywords: Leaves, flower, fruit, phenolics, antioxidant. Research Article Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023
Received: 21-Nov-2023 Accepted: 26-Dec-2023
homepage: https://dergipark.org.tr/tr/pub/joinabt
https://doi.org/10.58728/joinabt.1393739 Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023
Received: 21-Nov-2023 Accepted: 26-Dec-2023
homepage: https://dergipark.org.tr/tr/pub/joinabt
https://doi.org/10.58728/joinabt.1393739 ABSTRACT unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki
yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyv
ile ağaç çileği olarak da bilinmektedir [5]. Ülkemizde bölgelere göre d
yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, d
yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. un
gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her
süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7
olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli m
değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A
kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta o
dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda ku
A. unedo‘nın meyve verme süresi diğer pek çok türe göre çok daha
meyvelerinin kış aylarında veya kış aylarına yakın bir zamanda
alternatif bir besin kaynağı sağlamaktadır [7]. Yüksek biyokimy
bozukluklar, kalp damar hastalıkları, kanser, damar sertleşmesi, b
hipertansiyon gibi pek çok hastalığa karşı da koruyucu etkisinin
söktürücü, enfeksiyon önleyici ve yaşlanmayı geciktirici etkilere s
çiçeklerinin astım hastalığı ve yapraklarından hazırlanan çayların ise
hastalıklarına karşı koruyucu etkisi bulunmaktadır [16, 17]. Bu çalış
kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flav
belirlemek amacıyla yürütülmüştür. Materyal ve Yöntem
Materyal
Çalışma, Giresun iline bağlı Piraziz ilçesinde yürütülmüştür. Çalışm
yetişen kocayemiş genotipine ait yaprak, çiçek ve meyveler oluşturmu
Yöntem
Kocayemiş bitkisinden alınan yaprak ve çiçek örnekleri gölge bir
örnekleri, meyvenin kendine özgü rengini aldığı zamanda hasat edilm
biyokimyasal analizler yapılıncaya kadar -20°C’de muhafaza edilmişt
toplam fenolik, toplam flavonoid ve antioksidant aktivite (DP Giriş Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alanına sahip olup, Asya, Avrupa ve
Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkânı bulmuştur [1]. Ülkemizde ise
Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayılış göstermektedir. Ülkemiz doğal
florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne L. en önemli kocayemiş türleridir
[2]. Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alanına sahip olup, Asya, Avrupa ve
Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkânı bulmuştur [1]. Ülkemizde ise
Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayılış göstermektedir. Ülkemiz doğal
florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne L. en önemli kocayemiş türleridir
[2]. A. unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki habitusuna [3] ve yuvarlak veya
yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyve görünüşlerinin çileğe benzemesi
ile ağaç çileği olarak da bilinmektedir [5]. ABSTRACT Ülkemizde bölgelere göre değişmekle birlikte; dağ çileği, dağ
yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, davulga, davulgı üzümü, piridim,
yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. unedo bitkisi gerek görsel özellikleri
gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her dem yeşil olan bitki, formu, uzun
süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7] ve meyve tutumundan meyve
olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli meyveleri [8] ile süs bitkisi olarak
değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A. unedo bitkileri çit bitkisi olarak
kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta olmak üzere, jöle, marmelat, reçel,
dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda kullanılmaktadır [11, 2, 7, 12, 13]. A. unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki habitusuna [3] ve yuvarlak veya
yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyve görünüşlerinin çileğe benzemesi
ile ağaç çileği olarak da bilinmektedir [5]. Ülkemizde bölgelere göre değişmekle birlikte; dağ çileği, dağ
yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, davulga, davulgı üzümü, piridim,
yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. unedo bitkisi gerek görsel özellikleri
gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her dem yeşil olan bitki, formu, uzun
süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7] ve meyve tutumundan meyve
olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli meyveleri [8] ile süs bitkisi olarak
değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A. unedo bitkileri çit bitkisi olarak
kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta olmak üzere, jöle, marmelat, reçel,
dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda kullanılmaktadır [11, 2, 7, 12, 13]. A. unedo‘nın meyve verme süresi diğer pek çok türe göre çok daha uzundur [5, 2]. Özellikle olgun
meyvelerinin kış aylarında veya kış aylarına yakın bir zamanda hasadının yapılmasıyla insanlara
alternatif bir besin kaynağı sağlamaktadır [7]. Yüksek biyokimyasal içeriği sayesinde nörolojik
bozukluklar, kalp damar hastalıkları, kanser, damar sertleşmesi, böbrek rahatsızlıkları, romatizma,
hipertansiyon gibi pek çok hastalığa karşı da koruyucu etkisinin yanında, kabızlığı giderici, idrar
söktürücü, enfeksiyon önleyici ve yaşlanmayı geciktirici etkilere sahiptir [14, 15]. Bunun yanında
çiçeklerinin astım hastalığı ve yapraklarından hazırlanan çayların ise romatizma, idrar yolu ve böbrek
hastalıklarına karşı koruyucu etkisi bulunmaktadır [16, 17]. Bu çalışmada kocayemiş bitkisinin farklı
kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flavonoid ve antioksidan aktivitesini
belirlemek amacıyla yürütülmüştür. Materyal Çalışma, Giresun iline bağlı Piraziz ilçesinde yürütülmüştür. Çalışmanın materyalini Piraziz ilçesinde
yetişen kocayemiş genotipine ait yaprak, çiçek ve meyveler oluşturmuştur. Yöntem Kocayemiş bitkisinden alınan yaprak ve çiçek örnekleri gölge bir ortamda kurutulmuştur. Meyve
örnekleri, meyvenin kendine özgü rengini aldığı zamanda hasat edilmiştir. Alınan tüm bitki örnekleri
biyokimyasal analizler yapılıncaya kadar -20°C’de muhafaza edilmiştir. Biyokimyasal özellikler olarak
toplam fenolik, toplam flavonoid ve antioksidant aktivite (DPPH ve FRAP testlerine göre)
belirlenmiştir. ABSTRACT ABSTRACT The study was carried out to determine the total phenolics, total flavonoids and antioxidant activity
(according to DPPH and FRAP tests) of different parts (leaves, flower and fruit) of the strawberry
tree plant grown in Piraziz (Giresun) district. The highest total phenolic content was determined in
leaves (1.10 g 100 g-1) and flower (1.08 g 100 g-1), while the lowest was detected in fruit (0.81 g
100 g-1). The highest total flavonoid content was found in the leaves (3.38 g 100 g-1) and the lowest
in the fruit (0.04 g 100 g-1). The highest antioxidant activity was detected in leaves (7.72 mmol 100
g-1 and 2.58 mmol 100 g-1, respectively) and flower (7.84 mmol 100 g-1 and 2.58 mmol 100 g-1,
respectively) according to both methods, while the lowest was determined in fruit (1.21 mmol 100
g-1 and 1.50 mmol 100 g-1, respectively). As a conclusion, the best results in terms of the investigated
properties were obtained from the leaves and flower parts of the plant. Keywords: Leaves, flower, fruit, phenolics, antioxidant. * Sorumlu yazar : orhankarakaya7@gmail.com Publisher: Sakarya University of Applied Sciences Publisher: Sakarya University of Applied Sciences https://www.subu.edu.tr/tr Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/ BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Giriş
Kocayemiş (Arbutus unedo L.) dünya üzeri
Afrika’nın ılıman iklim koşullarında doğ
Karadeniz, Marmara, Akdeniz ve Ege bölge
florası içerinde de yer alan Arbutus unedo L
[2]. A. unedo her dem yeşil olup 1.5-9 m ara
yuvarlağa yakın turuncu-kırmızı meyvelere
ile ağaç çileği olarak da bilinmektedir [5]. Ü
yemişi, ayı yemişi, kocakarıyemişi, kara y
yağma, enderek ve zefre yemişi gibi farklı is
gerekse besleyici özellikleri ile pek çok kull
süre bitki üzerinde görülebilen gösterişli
olumuna kadar geçen sürede sarıdan kırmız
değerlendirilmektedir [9, 10]. Bunun yanın
kullanılmaktadır [3]. Çileğe benzeyen meyv
dondurma, pastacılıkta, alkollü içecek yapım
A. unedo‘nın meyve verme süresi diğer pe
meyvelerinin kış aylarında veya kış aylar
alternatif bir besin kaynağı sağlamaktadı
bozukluklar, kalp damar hastalıkları, kans
hipertansiyon gibi pek çok hastalığa karş
söktürücü, enfeksiyon önleyici ve yaşlanm
çiçeklerinin astım hastalığı ve yapraklarınd
hastalıklarına karşı koruyucu etkisi bulunm
kısımlarının (yaprak, çiçek ve meyve) top
belirlemek amacıyla yürütülmüştür. ABSTRACT Materyal ve Yöntem
Materyal
Çalışma, Giresun iline bağlı Piraziz ilçesin
yetişen kocayemiş genotipine ait yaprak, çiç
Yöntem
Kocayemiş bitkisinden alınan yaprak ve Giriş
Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alanına sahip olup, Asya, A
Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkânı bulmuştur [1]. Ülke
Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayılış göstermektedir. Ülke
florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne L. en önemli kocayemiş
[2]. A. unedo her dem yeşil olup 1.5-9 m arasında boylanabilen bitki habitusuna [3] ve yuva
yuvarlağa yakın turuncu-kırmızı meyvelere sahip bir türdür [4]. Meyve görünüşlerinin çileğe b
ile ağaç çileği olarak da bilinmektedir [5]. Ülkemizde bölgelere göre değişmekle birlikte; dağ
yemişi, ayı yemişi, kocakarıyemişi, kara yaprak, andrana, andıra, davulga, davulgı üzümü
yağma, enderek ve zefre yemişi gibi farklı isimler verilmiştir [6]. A. unedo bitkisi gerek görsel
gerekse besleyici özellikleri ile pek çok kullanım alanına sahiptir. Her dem yeşil olan bitki, fo
süre bitki üzerinde görülebilen gösterişli çan şeklinde çiçekleri [7] ve meyve tutumund
olumuna kadar geçen sürede sarıdan kırmızıya kadar değişen renkli meyveleri [8] ile süs bitk
değerlendirilmektedir [9, 10]. Bunun yanında, çalımsı formda olan A. unedo bitkileri çit bitk
kullanılmaktadır [3]. Çileğe benzeyen meyveleri taze tüketim başta olmak üzere, jöle, marme
dondurma, pastacılıkta, alkollü içecek yapımında ve tıbbi alanlarda kullanılmaktadır [11, 2, 7
A. unedo‘nın meyve verme süresi diğer pek çok türe göre çok daha uzundur [5, 2]. Özell
meyvelerinin kış aylarında veya kış aylarına yakın bir zamanda hasadının yapılmasıyla
alternatif bir besin kaynağı sağlamaktadır [7]. Yüksek biyokimyasal içeriği sayesinde
bozukluklar, kalp damar hastalıkları, kanser, damar sertleşmesi, böbrek rahatsızlıkları, ro
hipertansiyon gibi pek çok hastalığa karşı da koruyucu etkisinin yanında, kabızlığı gide
söktürücü, enfeksiyon önleyici ve yaşlanmayı geciktirici etkilere sahiptir [14, 15]. Bunu
çiçeklerinin astım hastalığı ve yapraklarından hazırlanan çayların ise romatizma, idrar yolu
hastalıklarına karşı koruyucu etkisi bulunmaktadır [16, 17]. Bu çalışmada kocayemiş bitkis
kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flavonoid ve antioksidan a
belirlemek amacıyla yürütülmüştür. Materyal ve Yöntem
Materyal
Çalışma, Giresun iline bağlı Piraziz ilçesinde yürütülmüştür. Çalışmanın materyalini Piraziz
i
k
i
i i
i
k
i
k
l
l Giriş
Kocayemiş (Arbutus unedo L.) dünya üzerinde geniş bir yayılım alan
Afrika’nın ılıman iklim koşullarında doğal olarak yetişme imkân
Karadeniz, Marmara, Akdeniz ve Ege bölgelerinde doğal olarak yayıl
florası içerinde de yer alan Arbutus unedo L. ve Arbutus andrachne
[2]. A. Antioksidan aktivite (mmol 100 g-1) Antioksidan aktivite (mmol 100 g-1) Bitki örneklerin antioksidan aktivitesi DPPH ve FRAP yöntemlerine göre belirlenmiştir. DPPH
yöntemine göre antioksidan aktivitesi Blois (1958)’in rapor ettiği yöntem kullanılarak tespit edilmiştir. Hazırlanan stok çözeltiden yaprak ve çiçek örnekleri için 0.02 mL ve meyve örneği için 0.1 mL alınmış
ve son hacim metanol ile 3 mL’ye tamamlanmıştır. Daha sonra örneklerin üzerine 1 mL DPPH çözeltisi
ilave edilmiştir. Hazırlanan örnekler spektrofotometrede 517 nm dalga boyunda okunmuştur. Elde
edilen absorbans değerleri mmol 100 g-1 olarak ifade edilmiştir. FRAP yöntemine göre antioksidan aktivitesi Benzie ve Strain (1996) tarafından bildirilen yönteme göre
belirlenmiştir. Yaprak, çiçek ve meyve örnekleri için hazırlanan stok çözeltiden 0.02 mL alınarak,
üzerine 1.23 mL fosfat tamponu ve 1.25 mL potasyum ferrik siyanit ilave edilmiştir. Hazırlanan örnekler
50°C’de 30 dk su etüvde inkübe edildikten sonra üzerine 1.25 mL TCA ve 0.25 mL demir klorür
eklenmiştir. Örneklerin absorbans değerleri spektrofotometrede 415 nm’de okunmuştur. Elde edilen
sonuçlar mmol 100 g-1 olarak ifade edilmiştir. İstatistiksel Analizler Verilerin değerlendirilmesinde JMP 16 (deneme sürümü) istatistik paket programı kullanılmıştır. İncelenen özellikler bakımından bitki kısımları arasındaki farklılık Tukey çoklu karşılaştırma yöntemine
göre %5 önem seviyesinde belirlenmiştir. Toplam flavonoid (g 100 g-1) Toplam flavonoid (g 100 g-1) Toplam flavonoid içeriği Chang ve ark. (2002)’nın bildirdiği yönteme göre belirlenmiştir. Yaprak, çiçek
ve meyve örnekleri için hazırlanan stok çözeltiden 0.1 mL alınarak, üzerine 4.2 mL metanol, 0.1 mL
alüminyum nitrat ve 0.1 mL amonyum asetat ilave edilmiştir. Hazırlanan örneklerin absorbans değerleri
spektrofotometrede 415 nm dalga boyunda okunmuştur. Elde edilen sonuçlar g 100 g-1 olarak ifade
edilmiştir. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri 4.7 mL’ye tamamlanmış ve üzerine 0.1 mL folin reaktifi ve 0.3 mL sodyum karbonat ilave edilmiştir. Hazırlanan örnekler spektrofotometrede 760 nm’de okunmuştur. Elde edilen absorbans değerleri g 100
g-1 olarak ifade edilmiştir [18]. 4.7 mL’ye tamamlanmış ve üzerine 0.1 mL folin reaktifi ve 0.3 mL sodyum karbonat ilave edilmiştir. Hazırlanan örnekler spektrofotometrede 760 nm’de okunmuştur. Elde edilen absorbans değerleri g 100
g-1 olarak ifade edilmiştir [18]. Toplam fenolik (g 100 g-1) Toplam fenolik içeriği Folin-Ciocalteu yöntemine göre tespit edilmiştir. Hazırlanan stok çözeltiden
yaprak ve çiçek örnekleri için 0.1 mL ve meyve örnekleri için ise 0.5 mL alınarak, son hacim saf su ile Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 106 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Bulgular ve Tartışma Meyve ve sebzelerde önemli miktarda bulunan biyoaktif bileşikler insan sağlığını teşvik eder ve kronik
hastalıklar başta olmak üzere kanser ve kardiyovasküler hastalıklar gibi pek çok hastalığın riskini
azaltmada önemli rol oynar [22]. Antioksidanlar, hastalıklara neden olduğu bilinen düzensiz reaktif
oksijen türleri ve serbest radikallerin zararlı etkisini önleyerek vücudun savunma sistemine önemli
derecede katkı sağlarlar [23]. Gıdalardan alınan antioksidanların vücutta üretilenlere göre daha yüksek
koruma sağladığı bildirilmiştir [24]. Bu durum, fenolik ve antioksidan içeriği yüksek yenilebilir yabani
meyve türlerinin tüketiminin önemini arttırmaktadır. Öyle ki kocayemiş de zengin besin içeriğiyle bu
anlamda önemli bir yere sahiptir [4, 11, 12]. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği arasındaki farklılık önemli bulunmuştur
(p<0.05). Toplam fenolik içeriği bakımından en yüksek değerler yaprak (1.10 g 100 g-1) ve çiçekte (1.08
g 100 g-1) belirlenirken, en düşük değerler meyvede (0.81 g 100 g-1) tespit edilmiştir (Şekil 1). Toplam
fenolik içeriği, Lapseki (Çanakkale) yöresinde yetişen Arbutus unedo türüne ait çiçeklerde 20.7 g 100
g-1 ve meyvelerde 1.40 g 100 g-1 [25], Artvin ilinde yetişen Arbutus andrachne türünün çiçeklerinde Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 107 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri 4.40 g 100 g-1 ve meyvelerinde 0.70 g 100 g-1 [26], Cezayir’de yetişen Arbutus unedo türüne ait
yapraklarda 17.5 g 100 g-1 ve meyvelerde 1.30 g 100 g-1 [27] olarak bildirilmiştir. Araştırıcıların
bulguları değerlendirildiğinde kocayemiş bitkisinde toplam fenolik içeriği bakımından en yüksek
değerlerin yaprak ve çiçekte, en düşük ise meyvelerde olduğu görülmektedir. Benzer sonuçlar mevcut
çalışmada da kaydedilmiştir. Buna karşılık toplam fenolik içeriği bakımından yaprak ve çiçeklerden elde
edilen değerler araştırıcıların bulgularından farklılık gösterirken, meyveden elde edilen değerler benzer
bulunmuştur. Görülen farklılıkların genotipten, ekolojik koşullardan ve meyvenin olgunluk
durumundan kaynaklı olduğu düşünülmektedir. Şekil 1. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Şekil 1. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Kocayemiş bitkisinin farklı kısımlarının toplam flavonoid içeriği arasındaki farklılık önemli
bulunmuştur (p<0.05). En yüksek toplam flavonoid içeriği yaprakta (3.38 g 100 g-1), en düşük ise
meyvede (0.04 g 100 g-1) bulunmuştur (Şekil 2). Toplam flavonoid içeriğini Saral ve ark. (2017) Arbutus
andrachne türüne ait çiçeklerde 11.42 g 100 g-1 ve meyvelerde 0.62 g 100 g-1 olarak belirlerken, Asmaa
ve ark. (2019) Arbutus unedo türünün yapraklarında 6.52 g 100 g-1 ve meyvelerinde 0.04 g 100 g-1 olarak
tespit etmişlerdir. Genel olarak değerlendirildiğinde yaprak ve çiçeklerin, meyvelere göre daha yüksek
toplam flavonoid içeriğine sahip olduğu görülmektedir. Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 1.1
Rakip Çıkarlar Yazarlar arasında herhangi bir çıkar çatışması yoktur. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Şekil 4. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (FRAP testine göre) Şekil 4. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (FRAP testine göre) Sonuçlar Kocayemiş bitkisinin farklı kısımlarının (yaprak, çiçek ve meyve) toplam fenolik, toplam flavonoid ve
antioksidan aktivitesinin (DPPH ve FRAP testlerine göre) incelendiği çalışmada, bitki kısımları arasında
incelenen özellikler bakımından önemli farklılıklar belirlenmiştir. Toplam flavonoid içeriği hariç, diğer
incelenen özellikler bakımından en yüksek değerler yaprak ve çiçekte belirlenirken, en düşük ise
meyvede tespit edilmiştir. Toplam flavonoid içeriği ise en yüksek yaprakta, en düşük ise meyvede
bulunmuştur. Sonuç olarak, insan sağlığını teşvik eden maddeler bakımından yaprak ve çiçek bitki
kısımlarının kayda değer sonuçlar verdiği tespit edilmiştir. Bitkinin bu kısımlarının farklı ürünlere
işlenerek değerlendirilme potansiyelinin yüksek olduğu düşünülmektedir. 1.2
Yazarların Katkıları Tüm yazarlar makaleye eşit katkı sunmuştur. Tüm yazarlar makaleye eşit katkı sunmuştur. Bulgular ve Tartışma Mevcut çalışmada da benzer sonuçlar
kaydedilmiştir. Ancak yaprak ve çiçeklere ait toplam flavonoid değerleri araştırıcıların bulgularından
düşük bulunmuştur. Toplam flavonoid içeriği bakımından görülen farklılıkların başta genetik yapıdan
ve ekolojik koşullardan olmak üzere meyvenin olgunluk durumu ile olgunlaşma dönemindeki
güneşlenme süresi ve gece ile gündüz arasındaki sıcaklık farklılığından kaynaklı olabilir. Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 108 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Şekil 2. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Şekil 2. Kocayemiş bitkisinin farklı kısımlarının toplam fenolik içeriği Antioksidan aktivitesi bakımından kocayemiş bitkisinin farklı kısımları arasında önemli bir farklılık
belirlenmiştir (p<0.05). En yüksek antioksidan aktivite her iki yönteme göre de (DPPH ve FRAP) yaprak
(sırasıyla, 7.72 mmol 100 g-1 ve 2.58 mmol 100 g-1) ve çiçekte (sırasıyla, 7.84 mmol 100 g-1 ve 2.58
mmol 100 g-1) tespit edilirken, en düşük meyvede (sırasıyla, 1.21 mmol 100 g-1 ve 1.50 mmol 100 g-1)
belirlenmiştir (Şekil 3 ve Şekil 4). İlgili çalışmalarda DPPH testine göre antioksidan aktivitesi, Arbutus
unedo türüne ait meyvelerde çiçeklere göre daha yüksek bildirilirken, FRAP testine göre ise çiçeklerde
meyvelere göre daha yüksek bulunmuştur [27]. Farklı bir çalışmada FRAP testine göre Arbutus
andrachne türünün çiçeklerinde meyvelerine göre daha yüksek antioksidan aktivite rapor edilmiştir
[26]. Benzer sonuçlar Arbutus unedo türünde de bildirilmiştir [25]. Mevcut çalışmada da en yüksek
antioksidan aktivitesi sırasıyla yaprak, çiçek ve meyvede belirlenmiştir. Şekil 3. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (DPPH testine göre) Şekil 3. Kocayemiş bitkisinin farklı kısımlarının antioksidan aktivitesi (DPPH testine göre) Journal of Agricultural Biotechnology (JOINABT) 4(2), 105-112, 2023 109 BALTA et al. Kocayemiş (Arbutus unedo L.) Bitkisinin Farklı Kısımlarının Biyoaktif İçerikleri Kaynakça [1] Karadeniz, T., Kalkışım, Ö., & Şişman, T. (2003). Trabzon çevresinde yetişen kocayemiş (Arbutus
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https://www.nature.com/articles/s41598-021-91279-z.pdf
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English
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Anti-demineralizing protective effects on enamel identified in experimental and commercial restorative materials with functional fillers
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Scientific reports
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cc-by
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Anti‑demineralizing protective
effects on enamel identified
in experimental and commercial
restorative materials
with functional fillers
OPEN Matej Par1,2*, Andrea Gubler2, Thomas Attin2, Zrinka Tarle1 & Tobias T. Tauböck2 The aim of this study was to investigate whether experimental and commercial dental restorative
materials with functional fillers can exert a protective anti-demineralizing effect on enamel that is
not immediately adjacent to the restoration. Four experimental resin composites with bioactive glass
and three commercial restorative materials were investigated. Enamel blocks were incubated in a
lactic acid solution (pH = 4.0) at a standardized distance (5 mm) from cured specimens of restorative
materials. The lactic acid solution was replenished every 4 days up to a total of 32 days. Surfaces of
enamel blocks were periodically evaluated by Knoop microhardness measurements and scanning
electron microscopy. The protective effect of restorative materials against acid was identified as
enamel microhardness remaining unchanged for a certain number of 4-day acid addition cycles. Additionally, the pH of the immersion medium was measured. While enamel microhardness in the
control group was maintained for 1 acid addition cycle (4 days), restorative materials postponed
enamel softening for 2–5 cycles (8–20 days). The materials capable of exerting a stronger alkalizing
effect provided longer-lasting enamel protection. The protective and alkalizing effects of experimental
composites improved with higher amounts of bioactive glass and were better for conventional
bioactive glass 45S5 compared to a fluoride-containing bioactive glass. Scanning electron micrographs
evidenced the protective effect of restorative materials by showing a delayed appearance of an
etching pattern on the enamel surface. A remotely-acting anti-demineralizing protective effect on
enamel was identified in experimental composites functionalized with two types of bioactive glass, as
well as in three commercial ion-releasing restorative materials. As secondary caries represents one of the main reasons for the failure of composite restorations1, bioactive glasses
(BGs) have been investigated as interesting dopants for restorative resin composites due to their potential for
releasing remineralizing ions2,3, neutralizing acid4,5, and precipitating hydroxyapatite6,7. BGs encompass vari-
ous formulations with adjustable relative ratios of constituent elements. The composition of a particular BG
determines its network structure, which in turn reflects on its properties8. In this way, subtle compositional
adjustments can be made to tailor reactivity, solubility, and ion release for a desired therapeutic effect9. Among
various BG compositions that have been investigated as functional additives for methacrylate-based restorative
composites, fluoride-modified BGs appear especially promising due to their ability for releasing fluoride ions
when exposed to an aqueous medium2. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Endodontics and Restorative Dentistry, School of Dental Medicine, University of Zagreb,
Gunduliceva 5, Zagreb, Croatia. 2Department of Conservative and Preventive Dentistry, Center for Dental
Medicine, University of Zurich, Plattenstrasse 11, Zurich, Switzerland. *email: mpar@sfzg.hr Scientific Reports | (2021) 11:11806 Anti‑demineralizing protective
effects on enamel identified
in experimental and commercial
restorative materials
with functional fillers
OPEN These ions can be incorporated into the enamel to render it more resist-
ant to demineralization10. An additional potential benefit of fluoride-modified BGs is their capability to form a
fluorapatite layer on the composite surface11, which can be envisioned as a possible sealing agent for marginal
gaps12. Besides experimental BG formulations containing various therapeutic elements, the conventional Hench’s
BG 45S5 formulation has also been extensively investigated as a possible filler for resin composites3–6,13. Notwith-
standing the unavoidable trade-offs between its high reactivity and stability of mechanical properties14 and the 1Department of Endodontics and Restorative Dentistry, School of Dental Medicine, University of Zagreb,
Gunduliceva 5, Zagreb, Croatia. 2Department of Conservative and Preventive Dentistry, Center for Dental
Medicine, University of Zurich, Plattenstrasse 11, Zurich, Switzerland. *email: mpar@sfzg.hr | https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 www.nature.com/scientificreports/ Table 1. Bioactive glass and reinforcing fillers used in experimental composites. Bioactive glass 45S5
Experimental fluoride-
containing bioactive glass
Inert barium glass
Silica
Particle size (d50)
3 µm
3 µm
1 µm
5–50 nm
Composition (wt%)
45.0% SiO2
24.5% CaO
24.5% Na2O
6.0% P2O5
33.5% SiO2
33.0% CaO
10.5% Na2O
11.0% P2O5
12.0% CaF2
55.0% SiO2
25.0% BaO
10.0% Al2O3
10.0% B2O3
> 99.8%
SiO2
Silanization (wt%)
None
None
3.2
4–6
Manufacturer
Schott, Mainz, Germany
Schott, Mainz, Germany
Schott, Mainz, Germany
Evonik, Hanau, Germany
Product name / LOT
G018-144 / M111473
Experimental batch
GM27884 / Sil13696
Aerosil R 7200 / 157,020,635 Table 1. Bioactive glass and reinforcing fillers used in experimental composites. Table 1. Bioactive glass and reinforcing fillers used in experimental composites. Table 2. Composition of experimental composites. Material designation
Filler composition (wt%)
Total filler ratio (wt%)
Bioactive glass 45S5
Experimental
fluoride-containing bioactive
glass
Reinforcing fillers (inert
barium glass : silica = 2:1)
Control
0
0
70
70
C-10
10
0
60
70
C-20
20
0
50
70
E-10
0
10
60
70
E-20
0
20
50
70 Table 2. Composition of experimental composites. lack of fluoride release, the traditional BG 45S5 composition represents a promising candidate for functional-
izing resin composites4,6,15,16. BG embedded within resin composites dissolves when exposed to an aqueous medium, releasing its con-
stituent elements into the solution2. This ion release is accompanied by an alkalizing effect11. The release of
remineralizing ions and the increase of the solution pH can be employed to protect dental hard tissues against
demineralization caused by bacterial acids. Anti‑demineralizing protective
effects on enamel identified
in experimental and commercial
restorative materials
with functional fillers
OPEN Such a protective effect has been demonstrated in experimental BG-
functionalized composites intended for use as orthodontic adhesives; these materials showed the potential for
protecting enamel adjacent to bonded brackets against the formation of white spots17–21. To investigate whether
the protective effect of BG-functionalized composites can extend beyond the dental hard tissues immediately
adjacent to the material22, the present in vitro study used enamel blocks incubated in a lactic acid solution at a
standardized distance from cured specimens of restorative materials. Experimental composites functionalized
with 10 or 20 wt% of conventional BG 45S5 and an experimental fluoride-modified BG were compared to three
commercial restorative materials capable of exerting an anti-demineralizing protective effect. Microhardness
(MH) was chosen as a convenient indicator of structural integrity of the enamel surface23 exposed to simulated
cycles of acid attack. Additionally, enamel surface morphology and pH changes in the immersion solution were
evaluated. The null hypotheses were that the evaluated parameters (enamel MH, enamel surface morphology,
and pH of the immersion solution) would: (1) remain unchanged during the simulated acid attack; and (2) not
differ among the investigated restorative materials. Figure 1. Experimental design. Figure 1. Experimental design. Konstanz, Germany; shade: A2, LOT: 1903000819), a giomer (Beautifil II, Shofu, Kyoto, Japan; shade: A2, LOT:
041923), and a resin-based “alkasite” material (Cention, Ivoclar Vivadent, Schaan, Liechtenstein; shade: universal,
LOT: XL7102). The alkasite material contains two types of reactive filers: an ionomer glass based on a calcium
barium alumino-fluoro-silicate, and a calcium fluoro-silicate glass25,26. Enamel blocks. One hundred twenty-eight intact human third molars were collected as by-products of
regular dental treatment. Patients had given written informed consent to use the teeth for research purposes, and
all teeth were irreversibly anonymized immediately after extraction. Under these terms, the research complied
with the use of anonymized biological material and, consequently, authorization from the local ethics committee
was not required (Federal Act on Research involving Human Beings (Human Research Act; article 2, paragraph
2)). The teeth were stored at 8 °C in 0.1% thymol solution and used within 6 months of extraction. h
y
Enamel blocks (1 block per tooth; 3 × 3 × 1 mm) were prepared from buccal surfaces using a low-speed preci-
sion cutting machine (IsoMet, Buehler, Lake Bluff, IL, USA). The buccal sides of enamel blocks were ground using
P4000 silicon carbide paper27 (Buehler; 2 min at 30 rpm, median particle size = 2.5 µm). After preparation, the
enamel blocks were stored in a phosphate-buffered saline solution and used within five days of preparation. From
the total of 128 enamel blocks, 64 were used for the MH and pH measurements, while 64 were used for the scan-
ning electron microscopy (SEM) study, as described in the schematic representation of the study design in Fig. 1. Restorative material specimens. Discoid specimens (diameter = 7 mm, thickness = 2 mm) were pre-
pared by casting the restorative materials into custom-made polyoxymethylene molds, covering their surfaces
with Mylar foils, and flattening using glass plates. The glass ionomer material was left to set in the mold for
15 min, while the light-curable materials (giomer and experimental composites) and the dual-curable alkasite
material were illuminated using a violet-blue LED curing unit (Bluephase PowerCure, Ivoclar Vivadent, Schaan,
Liechtenstein; emission wavelength range: 390–500 nm, radiant exitance: 1,340 mW/cm2) for 20 s, resulting in
a total radiant exposure of 26.8 J/cm2). To simulate a clinically realistic scenario, the specimens were exposed
to the immersion medium within 15 min after preparation. Materials and methods
i
l
i Experimental resin composites. Experimental resin composites were prepared as described in previ-
ous studies11,24. The resin system contained bisphenol-A-glycidyldimethacrylate (Bis-GMA, Merck, Darmstadt,
Germany) and triethylene glycol dimethacrylate (TEGDMA, Merck) in a ratio of 60:40 wt%. Camphorquinone
(0.2 wt%; Merck) and ethyl-4-(dimethylamino) benzoate (0.8 wt%; Merck) were added to photoactivate the resin
system. The components of the resin system were mixed using a magnetic stirrer for 48 h.hi The composition of fillers used to prepare experimental composites is given in Table 1. BG 45S5, inert barium
glass, and silica were obtained from commercial vendors. The experimental fluoride-modified experimental
BG with theoretical network connectivity similar to that of BG 45S5 (2.1)8 was prepared on-demand by Schott
(Mainz, Germany) via melt-quench route. By using similar preparation and grinding procedures, comparable
particle sizes were obtained for both BG types.i Experimental composites with a total filler ratio of 70 wt% were prepared by replacing 0, 10, or 20 wt% of
silanized reinforcing fillers (barium glass and silica) with two types of unsilanized BG (Table 2). The experimen-
tal composite containing only reinforcing fillers was used as an inert control. The composites were prepared by
mixing the resin system and the fillers for 5 min in a dual asymmetric centrifugal mixing system (Speed Mixer
TM DAC 150 FVZ, Hauschild & Co. KG, Hamm, Germany) at 2,000 rpm, and deaerating in vacuum for 48 h. mixing the resin system and the fillers for 5 min in a dual asymmetric centrifugal mixing system (Speed Mixer
TM DAC 150 FVZ, Hauschild & Co. KG, Hamm, Germany) at 2,000 rpm, and deaerating in vacuum for 48 h. Materials and methods
i
l
i In addition to the experimental composites, three commercial restorative materials with an acid-protective
capability were used as references: a reinforced glass ionomer restorative (ChemFil Rock, Dentsply Sirona, In addition to the experimental composites, three commercial restorative materials with an acid-protective
capability were used as references: a reinforced glass ionomer restorative (ChemFil Rock, Dentsply Sirona, Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z www.nature.com/scientificreports/ Enamel blocks incubated with specimens of restorative materials (n = 8 per material)
Control
C-10
C-20
E-10
E-20
Beautifil II
ChemFil
Rock
Cention
Added 5 mL of lactic acid (pH = 4.0)
and replenished after 4, 8, 12, 16, 20, 24, 28, 32 days
After 4, 8, 16, 32 days:
•
withdrawn two enamel blocks per
material group for scanning electron
microscopy
After 0, 4, 8, 12, 16, 20, 24, 28, 32 days:
•
measured microhardness of enamel
•
measured pH of immersion medium
128 enamel blocks (3 x 3 x 1 mm)
64 enamel blocks
for microhardness and pH study
64 enamel blocks
for scanning electron microscopy study •
withdrawn two enamel blocks per
material group for scanning electron
microscopy www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 3. Knoop microhardness of enamel blocks (mean values with standard deviations in parentheses). Same
letters denote statistically similar microhardness values within a material. Time
No. of acid addition
cycles
Material
Control
C-10
C-20
E-10
E-20
Beautifil II
ChemFil
Cention
Initial
0
243.8 (38.8) a
243.7 (39.2) ab
255.2 (42.9) a
262.4 (20.5) a
233.8 (45.0) a
252.7 (33.9) a
267.6 (22.2) a
261.9 (28.6) ab
4 days
1
228.3 (29.8) a
254.2 (32.6) a
267.6 (38.1) a
259.3 (25.1) a
263.8 (21.5) a
245.9 (27.2) a
260.5 (22.2) ab
276.4 (29.8) a
8 days
2
174.9 (32.3) b
207.2 (27.3) bc
256.6 (33.9) a
231.1 (34.1) a
245.4 (40.3) a
203.1 (33.1) ab
244.8 (23.8) abc
269.9 (20.5) a
12 days
3
145.8 (26.8) bc
178.5 (29.8) bc
239.7 (40.4) a
220.6 (22.1) b
221.0 (35.1) ab
201.6 (42.7) ab
221.4 (32.7) bc
239.3 (27.8) ab
16 days
4
107.1 (33.0) cd
136.1 (23.4) d
230.2 (51.8) a
167.2 (32.5) c
174.9 (37.5) bc
175.3 (33.1) b
203.5 (39.5) bc
212.8 (28.2) bc
20 days
5
81.8 (13.7) de
113.9 (32.6) d
207.7 (49.7) ab
123.5 (32.4) d
136.1 (40.0) c
156.9 (36.8) bc
198.5 (47.6) c
175.0 (50.1) c
24 days
6
40.7 (20.3) ef
47.1 (11.6) e
147.4 (51.6) bc
55.5 (30.7) e
69.4 (27.9) d
99.3 (36.5) d
129.4 (44.0) d
98.2 (50.3) d
28 days
7
29.4 (10.6) f
38.6 (14.8) e
145.1 (48.1) bc
43.0 (23.2) e
59.2 (19.1) d
106.0 (29.1) cd
112.4 (49.6) d
70.5 (30.6) d
32 days
8
25.3 (5.3) f
30.8 (9.2) e
122.3 (41.5) c
36.0 (11.4) e
50.0 (16.0) d
88.9 (35.3) d
113.4 (48.6) d
63.8 (19.5) d Table 3. Knoop microhardness of enamel blocks (mean values with standard deviations in parentheses). Same
letters denote statistically similar microhardness values within a material. Immersion in lactic acid. The enamel blocks and restorative material specimens were immersed in closed
vials (Eppendorf; Hamburg, Germany) containing 5 mL of lactic acid solution (pH = 4.0). This experimental
setup was adopted and modified according to previous studies28–30. Each vial contained one enamel block and
one restorative material specimen, which were held at a standardized distance of 5 mm. The immersion solution
was agitated using a horizontal laboratory shaker at a speed of 30 revolutions per minute. The environmental
temperature ranged between 23–24 °C. www.nature.com/scientificreports/ Surface MH of enamel blocks and pH of the immersion solution were
measured at the following time points: 4, 8, 12, 16, 20, 24, 28, and 32 days. At each time point, the immersion
medium was replenished with 5 mL of fresh lactic acid solution (pH = 4.0). Microhardness measurements. Knoop MH measurements were performed on the buccal sides of
enamel blocks using a digital hardness tester (model no. 1600–6106; Buehler). Indentations were made under a
load of 100 g and a dwell time of 20 s at random positions. The indentations were evaluated within 2 min after
preparation, with a resolution of 0.015 µm31. Per each specimen and time point, five replicate indentations were
made and their mean values were considered as a statistical unit. Eight specimens were used per material (n = 8). pH measurements. A calibrated pH electrode (780 pH Meter, Metrohm, Herisau, Switzerland) was
immersed in the solution, and pH values were recorded with a resolution of 0.01 pH units. Per each specimen
and time point, three replicate measurements were made and their mean values were considered as a statistical
unit. Eight specimens were used per material (n = 8). Scanning electron microscopy. Enamel blocks for the SEM study (n = 8 per material) were subjected to
the same acid exposure protocol as the specimens for the evaluation of MH and pH. Upon reaching the time
points of 4, 8, 16, and 32 days, two enamel blocks per material were withdrawn, rinsed with distilled water, dried,
and sputtered with 5 nm of gold. A scanning electron microscope (SEM; GeminiSEM 450, Zeiss, Oberkochen,
Germany) was used at 10 kV and 10,000 × magnification to evaluate surface morphology of enamel blocks. Statistical analysis. Normality of distribution and homogeneity of variances were checked using Levene’s
and Shapiro–Wilk’s tests, respectively. Within each restorative material, MH and pH values were compared
among time points using repeated-measures ANOVA with Bonferroni correction for multiple comparisons. The statistical analysis was performed using SPSS (version 20, IBM, Armonk, NY, USA) at an overall level of
significance of α = 0.05. 1. A significant initial pH increase, followed by a plateau at pH = 6–7. Materials: Control, E-10, Beautifil II, and
ChemFil. Figure 1. Experimental design. Per material, 16 specimens were prepared (8 for the
MH and pH study; and 8 for the SEM study). https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | Discussionh This study showed that restorative materials with functional fillers exert an anti-demineralizing effect when
incubated with enamel blocks in an acid solution. This effect was evidenced by MH of enamel blocks remaining
unimpaired over a material-dependent number of acid attack cycles. To demonstrate that the protective effect
does not depend on the close proximity of restorative materials to dental hard tissues, enamel blocks and restora-
tive material specimens were separated by a distance of 5 mm. The protective effect was material-dependent and
identified for the experimental BG-containing composites, as well as for three commercial restorative materials,
leading us to reject both null hypotheses.h g
j
yp
The composition of experimental composites with 10 and 20 wt% of BG followed a previous study in which
these materials were shown to neutralize acid similarly to the alkasite material and better than a giomer and
a glass ionomer11. These results motivated the present study, which aimed to investigate whether the acid-
neutralizing and ion-releasing activities of experimental BG-composites can protect remote enamel surfaces
against demineralization caused by lactic acid. g
y
Notwithstanding the complex pathophysiology of tooth caries, the initial damage to the enamel surface stems
from an imbalance in the dissolution and precipitation processes32. While various sophisticated approaches are
beneficial for in-depth evaluations of caries progression23, simple measurements of enamel surface MH indicat-
ing initial structural changes due to acid exposure have proven useful for evaluations of the anti-demineralizing
effect of restorative materials33–35. Therefore, our study employed periodic replenishing of the lactic acid solution
and evaluated the number of acid addition cycles that enamel blocks withstood before showing signs of surface
softening. The initial enamel MH in all experimental groups was within the usual MH range for sound enamel36. Statistical comparisons of those initial MH values and MH measured after successive acid additions enabled
identifying the point of significant MH decrease, which was considered to signify initial enamel softening. Th
b l
f h
d
l f
l
d
l
b
d y
g
p
gi
g
yt
g
The capability of the investigated materials for protecting enamel against demineralization is based on two
main effects, namely the neutralization of acid and the release of remineralizing ions2,11. The latter mechanism
shifts the dissolution/precipitation dynamics between the enamel hydroxyapatite and the immersion solution
towards precipitation. Results MH values of enamel blocks measured after acid addition cycles are shown in Table 3. Repeated additions of acid
solution significantly decreased enamel MH in all groups. The protective effect of restorative materials against
acid was identified as enamel MH remaining statistically similar to initial MH values for a certain number of
acid additions. The enamel MH in the control group was maintained for up to 1 acid addition and showed a
statistically significant decrease thereafter. In all other groups, enamel MH was maintained for more acid addi-
tion cycles, remaining statistically similar to initial MH values for up to 2 cycles (E-10 and ChemFil), 3 cycles
(C-10, E-20, and Beautifil II), 4 cycles (Cention) and 5 cycles (C-20). The number of acid additions over which
enamel MH remained statistically similar to the initial values is summarized in Fig. 2.hh y
g
The pH changes in the immersion solution are shown in Fig. 3. The initial pH (measured before incubating
enamel blocks and restorative materials) was 4.0 in all groups. Statistically significant pH changes were observed
after periodic acid additions, leading to the following three patterns in the pH curves: https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | ntificreports/
0
1
2
3
4
5
Control
E-10
ChemFil
C-10
E-20
Beaufill II Cenon
C-20
s
n
oitid
d
a dic
a f
o r
e
b
m
u
N
Figure 2. Number of acid addition cycles for which enamel surface MH remained statistically similar to initial
values. www.nature.com/scientificreports/ Figure 2. Number of acid addition cycles for which enamel surface MH remained statistically similar to initial
values. Figure 2. Number of acid addition cycles for which enamel surface MH remained statistically similar to initial
values. 2. A transient peak at pH = 8–10 after 4 days, followed by a pH drop and stabilization at pH = 6–7. Materials:
C-10, E-20, and Cention.i 2. A transient peak at pH = 8–10 after 4 days, followed by a pH drop and stabilization at pH = 6–7. Materials:
C-10, E-20, and Cention. 3
A significant initial pH increase followed by a plateau at pH
9 10 Material C 20 C
0,
0, a d Ce t o . 3. A significant initial pH increase, followed by a plateau at pH = 9–10. Material: C-20. Results The pH changes in patterns (1) and (2) converged to a plateau at pH = 6–7; this was the most common
behavior identified in 7 out of 8 tested materials. Pattern (3) was observed for only one material (C-20) capable
of maintaining alkaline pH over the whole observation period.t g
p
p
SEM images of enamel surfaces after 4, 8, 16, and 32 days of immersion in the acid solution are shown in
Fig. 4 for the experimental materials and Fig. 5 for the commercial materials. After 4 days, enamel surfaces in
all groups showed a scratch pattern from grinding, while enamel blocks immersed with Cention additionally
showed a precipitate on their surface. Over the subsequent time points (8, 16, and 32 days), the scratch patterns
gradually became less visible and were replaced with etching patterns. For C-20, Beautifil II, and Cention, the
scratch pattern remained visible up to 16 days, whereas for other materials they were replaced by etching patterns
at earlier times (4–8 days). After 32 days, etching patterns were clearly observable for all materials. Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z Discussionh Sufficiently saturating the immersion solution with calcium and phosphate ions can pre-
vent enamel demineralization even in moderately acidic environments32. A strong alkalizing effect is therefore
not necessary for a protective effect if sufficient concentrations of remineralizing ions are released. This consid-
eration can explain the finding that E-10, Beautifil II, and ChemFil demonstrated a better protective effect than
the control composite by maintaining enamel MH unchanged for a longer time (8–12 days vs. 4 days for the
control composite) despite showing pH curves similar to that of the control composite. Discussionh a
b
b
b
b
b
b
b
b
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
Control
microhardness
unchanged
a
b
bc
bc
bc
bc
bc
bc
c
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
C-20
microhardness
unchanged a
b
b
b
b
b
b
b
b
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
Control
microhardness
unchanged a
b
c
c
c
c
c
c
c
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
C-10
microhardness
unchanged a
b
bc
c
b
b
b
b
b
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
E-10
microhardness
unchanged a
b
b
b
b
b
b
b
b
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
Beaufil II
microhardness
unchanged a
b
c
c
c
c
c
c
c
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
E-20
microhardness
unchanged Beaufil II Time (days) a
b
c
d
e
e
e
e
e
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
Cenon
microhardness
unchanged Time (days) Figure 3. pH changes (mean values ± standard deviations) of the immersion medium and time periods for
which enamel microhardness remained unchanged compared to initial values. Same letters denote statistically
similar pH values within a material. capability, namely C-10, C-20, E-20, and Cention. For these materials, the synergistic effect of the alkalization
and ion release led to a longer-lasting protective capability. Discussionh A comparatively better
protective effect (MH unchanged for 12–20 days) was identified for the materials with a pronounced alkalizing Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z www.nature.com/scientificreports/ a
b
b
b
b
b
b
b
b
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
Control
microhardness
unchanged
a
b
c
c
c
c
c
c
c
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
C-10
microhardness
unchanged
a
b
bc
bc
bc
bc
bc
bc
c
0
1
2
3
4
5
6
7
8
9
10
0
4
8
12
16
20
24
28
32
pH
Time (days)
C-20
microhardness
unchanged
a
b
bc
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Time (days)
E-10
microhardness
unchanged
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pH
Time (days)
E-20
microhardness
unchanged
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b
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0
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4
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6
7
8
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pH
Time (days)
Beaufil II
microhardness
unchanged
a
b
b
b
b
b
b
b
b
0
1
2
3
4
5
6
7
8
9
10
0
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8
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16
20
24
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32
pH
Time (days)
ChemFil
microhardness
unchanged
a
b
c
d
e
e
e
e
e
0
1
2
3
4
5
6
7
8
9
10
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16
20
24
28
32
pH
Time (days)
Cenon
microhardness
unchanged
Figure 3. pH changes (mean values ± standard deviations) of the immersion medium and time periods for
which enamel microhardness remained unchanged compared to initial values. Same letters denote statistically
similar pH values within a material. Discussionh Th
H i
b
d f
th
t
l
b
tt ib t d t th di
l ti
f
l37
hi h l d t The pH increase observed for the control group can be attributed to the dissolution of enamel37 which led to
acid neutralization, as previously reported in studies that incubated enamel blocks in lactic acid solution28,30. As
the control group showed a plateau at the pH = 6–7 throughout the whole observation period, the alkalizing effect
in other groups had to be sufficiently pronounced in order to become distinguishable from the acid neutralization
by enamel blocks alone. Such an effect was observed for C-10, E-20, and Cention showing a transient alkalization,
and for C-20 which showed a more notable alkalization by maintaining a plateau at pH = 9–10. The observation
of the alkalizing effect of E-10, Beautifil II, and ChemFil being similar to that of the control composite does not The pH increase observed for the control group can be attributed to the dissolution of enamel37 which led to
acid neutralization, as previously reported in studies that incubated enamel blocks in lactic acid solution28,30. As
the control group showed a plateau at the pH = 6–7 throughout the whole observation period, the alkalizing effect
in other groups had to be sufficiently pronounced in order to become distinguishable from the acid neutralization
by enamel blocks alone. Such an effect was observed for C-10, E-20, and Cention showing a transient alkalization,
and for C-20 which showed a more notable alkalization by maintaining a plateau at pH = 9–10. The observation
of the alkalizing effect of E-10, Beautifil II, and ChemFil being similar to that of the control composite does not https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | www.nature.com/scientificreports/ Figure 4. Scanning electron microscopy images of the surfaces of enamel blocks immersed with the control
composite and the composites containing conventional (C-10 and C-20) and experimental (E-10 and E-20)
bioactive glass. Figure 4. Scanning electron microscopy images of the surfaces of enamel blocks immersed with the control
composite and the composites containing conventional (C-10 and C-20) and experimental (E-10 and E-20)
bioactive glass. necessarily imply that these materials lack an acid-neutralizing effect. Discussionh These materials indeed demonstrated a
moderate acid-neutralizing effect in a previous study11; however, by measuring pH at the end of four-day acid
addition cycles in the present study, their contributions to the pH increase were indiscernible from the acid-
neutralizing effect of the dissolution of enamel blocks.f gf
As the acid-neutralizing effect was eventually exhausted for all materials except C-20, their pH curves con-
verged to the plateau values of the control composite at pH = 6–7. Although C-20 maintained a high pH of 9–10
throughout the whole observation period, MH measurements showed that its protective effect on enamel was
exhausted after 20 days. This finding suggests that an apparently long-lasting alkalizing capability of C-20 was not
sufficient to protect enamel against demineralization. Although the pH = 9–10 was consistently measured at the
end of each four-day acid addition cycle, repeatedly exposing the enamel to the low-pH solution undersaturated
with calcium and phosphate ions at the beginning of each cycle produced cumulative enamel damage, which was
not remineralized over the rest of the cycle despite the alkaline pH being attained towards the end of each cycle. This reasoning also implies that the commonly used approach of measuring the alkalizing effect of restorative
materials after particular time intervals2,4,6,38 does not necessarily indicate protection against demineralization
because it does not take into account the demineralization that occurs before reaching a particular time point.f g
p
p
Whereas all investigated commercial restorative materials showed a better protective effect than the control
composite, the best performance among them was identified in a recently launched “alkasite” restorative material
(Cention). In addition to showing a transient alkalizing capability, this material also showed the longest protective
effect among the commercial materials. It is interesting to note that the other two commercial materials protected
enamel better than the control composite, even though their pH curves indicated an acid neutralization capabil-
ity similar to that of the control. Their protective effect was apparently more reliant on ion release, particularly
of fluoride ions which can be incorporated into demineralized enamel over a 4-day acid exposure cycle, thereby
rendering enamel more acid-resistant to the acid attack in the next cycle39.hf g
y
The results for the BG-functionalized composites show that (I) the protective and alkalizing effects were
dose-dependent, and (II) the protective and alkalizing effects were better for composites functionalized with
the conventional BG 45S5 compared to the experimental fluoride-containing BG. Scientific Reports | (2021) 11:11806 | Discussionh Since these differences imply
various extents of material degradation due to the dissolution of functional fillers11, the ion release profiles vs. the stability of mechanical properties of experimental composites with these two BG types are being addressed
in ongoing studies. These studies will also include the commercial material Cention since the evidence of its
high reactivity (protective and alkalizing effects better than all other tested materials except C-20) makes it an
interesting candidate for studying long-term degradation of mechanical properties. Although Cention lacks true self-adhesive properties, it was initially marketed as capable of being placed
without an adhesive system due to the assumed capability for sealing marginal gaps via hydroxyapatite precipi-
tation. These recommendations have recently been revised by the manufacturer, indicating that an adhesive
system (preferably the proprietary product Cention Primer) should be applied before restoring the cavity using Scientific Reports | (2021) 11:11806 | https://doi.org/10.1038/s41598-021-91279-z www.nature.com/scientificreports/ Figure 5. Scanning electron microscopy images of the surfaces of enamel blocks immersed with commercial
reference materials. Figure 5. Scanning electron microscopy images of the surfaces of enamel blocks immersed with commercia
reference materials. Cention. Therefore, clinical application of this new “alkasite” material type can be regarded as being similar to
other dual-curing bulk-fill composites with the additional benefit of protective effect on surrounding dental hard
tissues through ion release and alkalization.hifh The SEM micrographs support the finding of the protective effect of restorative materials on enamel. The
anti-demineralizing effect was identified as the grinding scratch pattern on the enamel surface remaining visible
over a material-dependent period of acid immersion30. As the protective effect was exhausted by the periodical
replenishing of the acid solution, the scratch pattern progressively became shallower and was eventually replaced
by etching patterns. An illustrative example of such a gradual transition was observed for C-20, which showed
a gradual thinning of the scratch pattern over 4–16 days, followed by an emergence of the etching pattern after
32 days. y
Distinctive etching patterns of Type 1 and 2 according to Silverstone et al.40 were observed on SEM micro-
graphs. For example, Type 1 etching pattern characterized by enamel dissolution being more pronounced in
prism cores was observed after 32 days for the control group, while Type 2 etching pattern in which dissolution
occurs preferentially on prism peripheries was observed after 32 days for C-20 and ChemFil. Conclusions Restorative materials functionalized with reactive fillers showed an anti-demineralizing effect on enamel blocks
that were 5 mm away from material specimens. The remotely-acting protective effect was identified in experi-
mental composites functionalized with two types of bioactive glass, as well as in three commercial restorative
materials. Although the materials which showed pronounced alkalizing capability also showed a longer-lasting
protective effect, the protection of enamel against demineralization was also attainable without a pronounced
alkalization. www.nature.com/scientificreports/ under the conditions of alkaline pH at the time point of 4 days25. As the pH dropped over subsequent time points,
the precipitate was dissolved, revealing the underlying scratch pattern on the enamel surface. under the conditions of alkaline pH at the time point of 4 days25. As the pH dropped over subsequent time points,
the precipitate was dissolved, revealing the underlying scratch pattern on the enamel surface. A simple demineralization model using periodically replenished acid solution was employed in this study
because it was a convenient and cost-effective means for demonstrating the protective effects of experimental
composites30. As such a model does not simulate clinically realistic slowly-advancing subsurface lesions, a mul-
tispecies biofilm model41 is planned to be used in a future study that will evaluate the capability of experimental
bioactive glass-containing composites for preventing the progression of carious lesions along restoration margins. Received: 9 March 2021; Accepted: 25 May 2021 Received: 9 March 2021; Accepted: 25 May 2021 Received: 9 March 2021; Accepted: 25 May 2021 References Remineralization of demineralized enamel via calcium phosphate nanocomposite. J. Dent. Res. 91, 979–984 (2012).i 11. Par, M., Attin, T., Tarle, Z. & Tauböck, T. T. A new customized bioactive glass filler to functionalize resin composites: Acid-
neutralizing capability, degree of conversion, and apatite precipitation. J. Clin. Med. 9, 1173. https://doi.org/10.3390/jcm9041173
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2. Khvostenko, D., Hilton, T. J., Ferracane, J. L., Mitchell, J. C. & Kruzic, J. J. Bioactive glass fillers reduce bacterial penetration into
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The authors declare no competing interests. Author contributions M.P. study conception, experimental design, funding acquisition, formal analysis and data interpretation, statisti-
cal analysis, visualization, wrote manuscript. A.G. performed experiments, data acquisition and interpretation,
resources, revised and approved final manuscript. T.A. study conception, experimental design, funding acquisi-
tion, supervision, data interpretation, project administration, resources, revised and approved final manuscript. Z.T. study conception, supervision, data interpretation, project administration, revised and approved final manu-
script. T.T.T. study conception, experimental design, funding acquisition, supervision, data interpretation, project
administration, resources, revised and approved final manuscript. Fundingh g
This work was financially supported by the Research Fund of the Swiss Dental Association SSO (grant number
321-20). Matej Par’s research stay at University of Zurich was funded by a grant from the Swiss Federal Depart-
ment of Economic Affairs, Education and Research (2019.0221). References et al. Deposition of fluoride on enamel surfaces released from varnishes is limited to vicinity of fluoridation site. Clin
Oral Investig. 11, 83–88 (2007). g
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with unsilanized bioactive glass fillers. Sci. Rep. 10, 15237 (2020). gi
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Mater. 35, 1757–1768 (2019).hf 6. Kelić, K., Par, M., Peroš, K., Šutej, I. & Tarle, Z. Fluoride-releasing restorative materials: The effect of a resinous coat on ion release
Acta Stomatol. Croat. 54, 371–381 (2020). (
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conditions. Clin. Oral Investig. 21, 2689–2693 (2017). https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 | www.nature.com/scientificreports/ 28. Kaga, M. et al. Inhibition of enamel demineralization by buffering effect of S-PRG filler-containing dental sealant. Eur. J. Oral Sci. 122, 78–83 (2014).fi 29. Kaga, N. et al. Protective effects of GIC and S-PRG filler restoratives on demineralization of bovine enamel in lactic acid solu
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0. Kakuda, S., Sidhu, S. K. & Sano, H. Buffering or non-buffering; an action of pit-and-fissure sealants. J. Dent. 43, 1285–1289 (2015) 30. Kakuda, S., Sidhu, S. K. & Sano, H. Buffering or non-buffering; an action of pit-and-fissure sealants. J. Dent. 43, 1285–1289 (2015). 31 P
M M
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T T l
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ties of bulk-fill and conventional resin composites. Sci. Rep. 10, 10560 (2020). hf
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-fill and conventional resin composites. Sci. Rep. Additional information Correspondence and requests for materials should be addressed to M.P. Correspondence and requests for materials should be addressed to M.P. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-91279-z Scientific Reports | (2021) 11:11806 |
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An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
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Zenodo (CERN European Organization for Nuclear Research)
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An Appraisal of Insecurity and the Consequences on Residents in
Calabar Metropolis, Cross River State, Nigeria Agbor, Emmanuel A.1, Obongha, Ukpali E.2, Ita, Emmanuel. E.3
1,2,3Department of Urban and Regional Planning Cross River University of Technology, Calabar ABSTRACT: The Ancient city of ‘CALABAR’ often interpreted as “Come And Live And Be At Rest” has experienced insecurity
and safety for over a decade due to rapid population growth and urbanization. The urban settings have also influenced criminal
activities and disorder especially the physical form of the metropolis and its diverse economic activities. The increase crime rate is
also attributed to the city’s social activity pattern and the structure of its transportation network which distributes traffic to all the
neighborhoods within and around Calabar Metropolis. The aim of this study is to appraise the insecurity situation in the ancient city
of Calabar and the consequences on the residents in the metropolis. The specific objectives are; to identify the major types of
criminal activities in Calabar metropolis; to determine the cause of such criminal activities in the study area; and to examine the
physical form and structure of the area under study. The Pearson’s Product Moment statistical techniques was employed to test the
relationship between population and crime. At 4.345 degree of freedom and 0.05 level of significance, the P-value was less than the
chosen alpha of .05, the null Hypothesis was therefore rejected confirming a significant relationship between population and crime
in the study area. The study further revealed increase criminal activities in the inner-city neighborhoods and the Gated and rich
neighborhoods in the study area due to the deteriorating quality of urban life and poor urban planning and design. KEYWORDS: Crime, Felonies, Misdemeanors, Population, Safety, Security. International Journal of Social Science And Human Research
ISSN(print): 2644-0679, ISSN(online): 2644-0695
Volume 04 Issue 07 July 2021
DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586
Page No: 1901-1915 International Journal of Social Science And Human Research
ISSN(print): 2644-0679, ISSN(online): 2644-0695
Volume 04 Issue 07 July 2021
DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586
Page No: 1901-1915 International Journal of Social Science And Human Research
ISSN(print): 2644-0679, ISSN(online): 2644-0695
Volume 04 Issue 07 July 2021
DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586
Page No: 1901-1915 Volume 04 Issue 07 July 2021
DOI: 10.47191/ijsshr/v4-i7-46, Impact factor-5.586
Page No: 1901-1915 INTRODUCTION The world is experiencing the second phase of urbanization phenomenon since the industrial revolution in the mid-19th century. The
current rate of urbanization and urban growth is unprecedented. The United Nations survey in 1800, revealed that only 2.00 percent
of the world population was urbanized. By 1850, not only more than 5.0 percent of the global population was urbanized and no
society had more than half of its population living in cities except London which had more than one million people. The percentage
of world’s population living in urban centres increased between 1800 and 1985 from 14.00 percent to 43.00 percent as indicated
below on table 1. Currently in year 2020 the world urban population has increased to 2.9 billion as reported by the United Nations
and Population Reference Bureau (1982). Currently, most African nations are experiencing the fastest rate of urbanization in the world without a corresponding
increase in the essential basic infrastructure which is creating serious economic, social, political and environmental related problems. Between 1970-1995 the average African country’s urban population grew by 5.2 percent per annum while its Gross Domestic
Product (GDP) declined by 0.66 percent per annum Hicks (1998). Urban population growth has been on the increase in Nigeria
since the pre-colonial era. Okeke (1998) asserts that Nigeria urban centres are amongst the fastest growing cities in the world. For
instance, in 1921 Nigeria had a total of 29 urban centres. By 1931, the number drop to 27 as a result of the political boundary
delineations between the Federal Republic of Nigeria and the British Cameroun protectorate. The number of urban centres reached
56 in 1953 and between 1963 and 1991 the number of urban centres rose to 182 and 359 respectively. There was also an increase in
the percentage urban population. A total of 7.18% of Nigeria’s population lived in urban centres while 92.82% were rural inhabitants. The percentage rose
to 19.30% and 36.30% respectively between 1963 and 1991 as indicated on table 1 below. The United Nation (2000) projected that
by the end of 2050 half of the world’s population will live in urban centres. INTRODUCTION IJSSHR, Volume 04 Issue 07 July 2021 Page 1901 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
Table 1: Number and growth of urban cities in Nigeria (Population 20,000 person)
Year
Population
(in thousand)
Total population
(in thousand)
No
of
Urban
Cities
%
of
Urban
Pop
% from 1921
Rural
Urban
Rural
Urban
1921
17.375
1,345
18,720
29
7.18
1931
18,625
1,431
20,056
27
7.14
0.42
2.79
1953
27.166
3,237
30,403
56
10.65
1.42
2.76
1963
44.925
10,745
55,670
182
19.30
2.29
5.01
1991
57,185
31,807
88.992
359
36.3
1.70
4.52
Source: NPC, 1991 Population census An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross R rity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The increasing Nigeria urban centres and population is occurring without generating the needed resources and employment
opportunities to accommodate the surge in population. The concentration of economic activities in urban centres creates disparity
in the economic growth between urban and rural areas. The less privileged and poor migrants who move from rural areas to urban
centres in search of employment, security and safety, find themselves living in slum settlements within and around urban centres. As more people moved into an urban center voluntarily or forced by administrative compulsion, many social maladies become
noticeable in the city. This group of people who suffer extreme deprivation and exclusion later indulge into a wide range of criminal
activities. Crime is an action or omission which constitute an offence and is punishable by law. In modern criminal law crime does
not have any simple universally accepted definition. A crime or offence is an act lawfully not only to individuals but also to a
community, society or the states (Wikipedia Encyclopedia, 2008). Criminologists have grouped crime into two major categories –
felonies and misdemeanors. According to Paul Tappen, crime is an international act or omission in violation of criminal law
committed without defence or justification and sanctional by state as a felony or misdemeanor. This study seeks to appraise the internal security and safety situation in the ancient city of Calabar arising from rapid
population and urbanization, the physical structure and planning of the metropolis. II. There has been immense increase in population and urban expansion over the years in Calabar Metropolis. The official national
population census conducted during the colonial administration revealed that Calabar had a total of 46,705 people in 1953 and
100,628 people in 1963 with percentage growth rate of 1.09. The next and most resent official census reported a total of 329,000 in
1991 and 375,196 in 2006 respectively. Urban population growth becomes a significant problem when its consequences impact negatively on the environmental
resources and threats to human lives. Among the most serious problems confronting Calabar Metropolis and its inhabitants include
lack of employment opportunities, increasing poverty, housing shortage and expansion of squatter settlements, growing insecurity
and rising crime rates, uncoordinated urban development and increasing vulnerability to disaster which are the basic issues of
mankind in the societies. The ancient city of Calabar is experiencing high rate of insecurity resulting from the presence of less privilege (poor) who
suffer extreme deprivation and exclusion. These poor migrants who migrated from rural areas to Calabar in search of security, safety
and gainful employment are found living in slum settlements without basic facilities and services. Calabar Metropolis has witnessed
a wide range of criminal activities over a decade such as kidnapping, armed robbery, rape, murder, burglary, auto theft, arson etc. These criminal activities occur in different geographical locations and settings (neighbourhoods) such as Bay side,
Anantigha, Watt Market, Ekpo Abasi, Akim Qua, Big Qua, Ikot Ansa etc. Other locations that cluster criminal activities are
entertainment clubs, super markets, stores, major motor parks and residential neighbourhoods, example the State, Federal and
Private Housing Estates inhabited by relatively well-to-do, young and adults, abandoned and uncompleted public and private
property etc. III.
AIM OF THE STUDY IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in
Page 1902
III. AIM OF THE STUDY
The aim of this study is to appraise the security and safety situation in Calabar Metropolis with a view to finding solution to the
problem. Objectives
1. To identify the major types of criminal activities in Calabar Metropolis
2. To determine the causes of such criminal activities in the study area
3. To examine the physical form and structure of the study area The aim of this study is to appraise the security and safety situation in Calabar Metropolis with a view to finding solution to the
problem. Objectives 1. To identify the major types of criminal activities in Calabar Metropolis 2. To determine the causes of such criminal activities in the study area 3. To examine the physical form and structure of the study area IJSSHR, Volume 04 Issue 07 July 2021 Page 1902 Page 1902 IV.
STUDY AREA The ancient city of Calabar has since 1967 remain the centre of activities and the administrative headquarters of the Cross River
State Government of Nigeria. Calabar is located between latitude 4056’ and 5004’ North of the Equator and longitude 8018’ and
8024’ East of the Greenwich Meridian. The City is on the peninsular between the Calabar River on the nearest buildable land to the
sea. Calabar remains one of the oldest city and port in the West African Sub-region and a slave depot along the Bight of Biafra
(Bonny). It was the first national Administrative capital of Nigeria from 1882-1906 during the early British rule in Nigeria. Calabar
is a dynamic urban centre with a rural-urban migrant population. The population of Calabar urban had been estimated to be 82,100
in 1975;159,599 in 1978; 140,200 in 1985, 217,800 in 1995; 320,862 in 1991 and 375,196 in 2006. The indigenes are the Efiks, the
Quas and Efuts who are engaged in commercial, industrial, educational and small scale farming. Climatically, Calabar falls within
the rain forest belt and the vegetation is mainly tropical. Fig 1: Map of Nigeria Showing Cross River State
Source: Cross River GIA Fig 1: Map of Nigeria Showing Cross River State
Source: Cross River GIA FIG. 2: Map of Cross River State Showing Calabar Metropolis FIG. 2: Map of Cross River State Showing Calabar Metropolis FIG. 2: Map of Cross River State Showing Calabar Metropolis FIG. 2: Map of Cross River State Showing Calabar Metropolis IJSSHR, Volume 04 Issue 07 July 2021 Page 1903 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
Figure 3: Map of Calabar showing the Study Area
Source: Cross River GIA he Consequences on Residents in Calabar Metropol
Figure 3: Map of Calabar showing the Study Area
Source: Cross River GIA he Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
Figure 3: Map of Calabar showing the Study Area
Source: Cross River GIA An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria urity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Figure 3: Map of Calabar showing the Study Area
Source: Cross River GIA An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian
Police recorded about 26 criminal offences between 1994 and 2003 as shown on table 3 below An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian
Police recorded about 26 criminal offences between 1994 and 2003 as shown on table 3 below. A summary of crime statistics compiled by the research department of Clean Foundation in collaboration with the Nigerian
Police recorded about 26 criminal offences between 1994 and 2003 as shown on table 3 below. ble 3: Crime Statistics in Nigeria (1994-2003)
OFFENCES
RECORDED INCIDENCE
Armed Robbery
25,352
Assault
377.568
Child Stealing
1,320
False Pretence
110,913
Forgery of Currency Note
6,759
Murder
17,448
Attempted Murder
2,434
Theft, Larceny and others
505,075
Burglary
64,863
Demand/Menace
1,824
Man Slaughter
259
Suicide
2,305
Attempted Suicide
754
General Hamm and Wounding
156,328
Coining Offence
211
Gambling
3,330
Rape
22,151
Slave Dealing
196
Breach of Public Peace
72.009
Perjury
903
House Breaking
72,574
Store Breaking
39,724
Bribery and Corruption
1,690
Escape from Lawful Custody
4,699
Kidnapping
3,589
Unnatural Offences
4,366
Total
1,488,644
Source: Clean Foundation, 2003 Global trends have shown a tremendous increase in crime rates over the years. Reports have shown an increase in crime
from 2,000 to 2,300 for every 1000 people between 1980-2000. This showed that 60 percent of urban residents in developing
countries have been victims of crime, over a five-year period with victimization rate of 70 percent in parts of Latin America and the
Caribbean and Africa. According to the UN-Habitat, (2007) cities in Africa have the highest reported cases of burglary, with
victimization rates of over 8 percent of the population. In the year 2000, the police in South Africa recorded 460 robberies for every
100,000 people. V.
LITERATURE REVIEW The whole world including Sub-Saharan Africa (SSA) are experiencing high rare of population growth and urban expansion. The
United Nation (2008) reported that only 20 million people were living in urban areas in Sub-Saharan Africa. Todaro (2000) observed
that the SSA region recorded the highest urban growth rate in the world with an average of 5% per annum. Hove, Ngwerume and
Muchemwa (2013) reported a total urban population of 250 million. Accordingly, the United Nation Population Fund*UNPF 2000)
concluded that at this rate of growth, urban population will double between 2000 and 2030 resulting to serve social, economic,
physical and environmental consequences. The poor migrant population in urban centres will create informal settlements with extreme human insecurity resulting to
high rate of criminal offences ranging from major crimes referred to as felonies or predatory crimes and minor crimes otherwise
known as misdemeanors. According to Obeng-Odoom (2011) these poor migrants constitute part of the urban poor who suffer
extreme deprivation and exclusion. Rakisits (2008) also observed the growing number of street children like the Amajeris or
Skolombos in the Northern and Southern States in Nigeria and destitute in our urban areas who survive through begging, stealing,
casual work or crime as major consequences of deprivation. ‘Crime’ does not have a universally accepted definition. It has different
meaning by different schools of thought. Paul Tappen, a criminologist, defined crime as an international act or omission in violation
of criminal law submitted without defence or justification and sanctioned by state as a felony or misdemeanor. Felonies are major
or serious crimes that attract death sentence or incarceration in a prison for several years by government. These include kidnapping,
murder, rape, arm robbery, burglary etc. Misdemeanor or minor crimes are unserious crimes such as shoplifting, petty theft, pick
pocking, simple assault etc which attract conferment in a local jail for at least a year. Table 2: Major Crime Incidence in the Urban Areas and Rates per 100,000 Populations. S/NO
CRIME
INCIDENCE
CRIME INDEX
I
Murder
19,645
7.4
Ii
Forcible Rape
957,679
36.1
Iii
Robbery
537,050
202.1
Iv
Aggravated Assault
1,029,814
388
V
Burglary
2,501,524
943
Vi
Larceny
7,894,620
2,925.9
Vii
Auto Theft
1,395,192
525.9
Viii
Arson
88,887
44.3
Total
13,562,501
5,078.9
Source: FBI (UCR), 1996 : Major Crime Incidence in the Urban Areas and Rates per 100,000 Populations. IJSSHR, Volume 04 Issue 07 July 2021 Page 1904 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria the most important environmental variables. Security has been defined by the Oxford Learner’s Dictionary as “the state of being
free from danger or threat” and is one of the basic human needs. Human security protects communities and their inhabitants from
insecurity and violence. Safety on the other hand refers to the environmental factors whose absence may lead to occurrence of crime
and gives rise to threat and risk (Kelly and Dian 2009). It is interesting to note that the UN-Habitant (2007) have placed urban safety
and security within the wider perspective of human security in our societies. According to Ogboi (2013) the level of safety differ
among urban neigbourhoods especially the inner city and the suburban context. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria In Johannesburg for instance, 30 percent of residents were reported to have been victims of robbery. Studies in urban and regional planning have continuously placed safety and security concerns within spatial context by
examining design and policy intervention that can create defensible space (Newman, 1972; Frank and Engelke, 2001; Handy,
Boarnet, Ewing and Killingswarth 2002). Environmental criminologists have placed much emphasize on the importance of
geographical locations and architectural designs as factors that influence crime and its prevalence. It has been urged that certain
urban design patterns such as neighbourhood design, street layout, avenues, alleys, boulevards, private homes and business
establishments attract criminals and remain “Hotspots” and sites of high crime and deviate rates. Stark (1987) addresses such
neighbourhood or communities as deviant neighbourhood which are characterized by dense population, with poor and overcrowded
conditions that increase the temptation and opportunity to involve in crime and deviant behavior. The built environment (neighbourhood) also present environmental constraints and opportunities which may be
characterized by physical disorder (litter) and social disorder (crime). According to Ross et al (2001); Klinenberg (2002) living in a
neighbourhood with such high physical and social disorder generates stress and fear. On the contrary, clean and safe neighbourhood
attract outdoor activities. Recent studies have tried to acertain the promotion of a safety and healthy community through the
manipulation of urban design, land use and transportation. (Greenwald and Boarnet, 2002); Safety and security have been seen as IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1905 VII.
RESEARCH FINDINGS AND DISCUSSIONS VII. RESEARCH FINDINGS AND DISCUSSIONS PRESENTATION OF RESULT Table 4: Trend of population for Calabar metropolis based on official population census
S/n
Year
Population
Percentage (%)
1
1953
46,705
5.45
2
1963
100,628
11.82
3
1991
329,000
38.64
4
2006
375,196
44.06
TOTAL
851,529
100.00 Table 4: Trend of population for Calabar metropolis based on official population census d of population for Calabar metropolis based on official population census The results in Table 3 shows the trends in population for Calabar metropolis based on official population census revealed
a total population of 851,529. While year 2006 had the highest population of 375,196 (44.06%), followed by 1991 with a population
of 329,000 (38.64%), while the least was 1953 which has a population of46,705 (5.45%).. This indicates that there is a geometric
increase in the population of the respondents in Calabar metropolis. The result is also presented in a bar chart as shown in Figure 4. The results in Table 3 shows the trends in population for Calabar metropolis based on official population census revealed
a total population of 851,529. While year 2006 had the highest population of 375,196 (44.06%), followed by 1991 with a population
of 329,000 (38.64%), while the least was 1953 which has a population of46,705 (5.45%).. This indicates that there is a geometric
increase in the population of the respondents in Calabar metropolis. The result is also presented in a bar chart as shown in Figure 4. al population of 851,529. While year 2006 had the highest population of 375,196 (44.06%), followed by 1991 with a popula
29,000 (38.64%), while the least was 1953 which has a population of46,705 (5.45%).. This indicates that there is a geom
ease in the population of the respondents in Calabar metropolis. VI. The research study made use of the primary and secondary data. The official national population census data was obtained from the
National Population Commission while the last official national census of 2006 was projected to 2020 at 3.0% growth rate. Data on
crime was obtained from the Cross River State Criminal Intelligence Bureau (SCIB) Headquarters Calabar. Oral personal interview
and direct observation were adopted to indentify crime hotspots and most especially the physical form of the city and its social and
economic activity pattern, the structure of the city’s street network and neighborhood design. The Pearson’s Product Moment (PPM) statistical instrument was employed to explore the relationship between population
and crime variables as shown on tables 4 and 5 below. VII.
RESEARCH FINDINGS AND DISCUSSIONS The result is also presented in a bar chart as shown in Figu
Figure 4: Bar chart of trends in population for Calabar metropolis based on official population census
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1953
1963
1991
2006
TOTAL
1
2
3
4
POPULATION
Percentage (%) Figure 4: Bar chart of trends in population for Calabar metropolis based on official population census
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1953
1963
1991
2006
TOTAL
1
2
3
4
POPULATION
Percentage (%) Figure 4: Bar chart of trends in population for Calabar metropolis based on official population cen IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1906 Page 1906 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
Table 5: Estimated population figure for Calabar Metropolis
S/n
Year
Estimated population
Number of crime committed
1
2006
375,196
326
2
2007
367, 458
211
3
2008
374,363
291
4
2009
386,989
172
5
2010
375,882
152
6
2011
365,605
156
7
2012
379, 553
204
8
2013
386,277
319
9
2014
398,045
325
10
2015
422,286
364
11
2016
448,003
305
12
2017
475,286
335
13
2018
505,231
337
14
2019
534,938
250
15
2020
567,515
500
Total
3, 737, 581
4247 ecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
population figure for Calabar Metropolis An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cros It is evident from the result in table 5 of the estimated population figure for Calabar Metropolis that the total estimated population
for the fifteen years (2006-2020) is 3, 737, 581 and the corresponding number of crime committed within these years amounted to
4,247. Year 2020 had the highest crime rate of 500; this was followed by year 2015 that has a crime rate of 364 cases. While the
least was year 2010 with an estimated crime rate of 152 accordingly. This implies that there is an arithmetic increase in the rate of
crime committed between 2006-2020. The result is further presented in the bar chart in figure 5 for the fifteen years (2006-2020) is 3, 737, 581 and the corresponding number of crime committed within these years amounted to
4,247. It is evident from the result in table 5 of the estimated population figure for Calabar Metropolis that the total estimated population
for the fifteen years (2006-2020) is 3, 737, 581 and the corresponding number of crime committed within these years amounted to
4,247. Year 2020 had the highest crime rate of 500; this was followed by year 2015 that has a crime rate of 364 cases. While the
least was year 2010 with an estimated crime rate of 152 accordingly. This implies that there is an arithmetic increase in the rate of
crime committed between 2006-2020. The result is further presented in the bar chart in figure 5 VII.
RESEARCH FINDINGS AND DISCUSSIONS Year 2020 had the highest crime rate of 500; this was followed by year 2015 that has a crime rate of 364 cases. While the
least was year 2010 with an estimated crime rate of 152 accordingly. This implies that there is an arithmetic increase in the rate of
crime committed between 2006-2020. VII.
RESEARCH FINDINGS AND DISCUSSIONS The result is further presented in the bar chart in figure 5
Figure 5: Estimated population figure for Calabar Metropolis
0
100000
200000
300000
400000
500000
600000
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
S/n
Year
Estimated population
Number of crime committed Figure 5: Estimated population figure for Calabar Metropolis
0
100000
200000
300000
400000
500000
600000
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
S/n
Year
Estimated population
Number of crime committed Figure 5: Estimated population figure for Calabar Metropolis IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in
Page 1907
Table 6: Major crime statistics in Calabar metropolis (2006-2020)
S/n
Year
Kidnapping
Cultism
Rape
Burglar
y
Murder
Arson
Armed
robbery
Theft
Auto
theft
Vandalism
1
2006
7
-
9
10
18
5
37
240
-
-
2
2007
5
-
7
6
7
3
15
168
-
-
3
2008
4
-
16
9
15
2
21
224
-
-
4
2009
8
-
11
4
15
5
25
103
1
-
5
2010
4
-
8
7
15
-
18
99
1
- Table 6: Major crime statistics in Calabar metropolis (2006-2020) Page 1907 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
6
2011
6
-
5
4
22
2
31
86
-
-
7
2012
7
-
16
5
31
3
36
106
-
-
8
2013
9
-
12
10
24
-
63
201
-
-
9
2014
7
-
15
8
26
2
24
243
-
-
10
2015
4
-
11
6
12
2
14
315
-
-
11
2016
9
-
15
16
5
3
45
199
-
-
12
2017
15
-
12
9
19
5
52
220
2
1
13
2018
6
6
11
5
22
1
38
238
5
1
14
2019
7
-
4
6
24
4
30
172
3
-
15
2020
8
4
35
10
26
10
41
342
4
16
Tot
al
106
10
187
115
281
47
490
2956
16
18
It can be discerned from the statistical analysis in Table 6 of the major crime statistics in Calabar metropolis (2006-2020), theft has
the highest with a total of 2956, this was followed by armed robbery 490 while those with murder cases came third with a total of
291 while the least are those who constitute the categories of cultism with a total of 10. VII.
RESEARCH FINDINGS AND DISCUSSIONS The results are presented in the bar chart in
Figure 6 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cros rity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria It can be discerned from the statistical analysis in Table 6 of the major crime statistics in Calabar metropolis (2006-2020), theft has
the highest with a total of 2956, this was followed by armed robbery 490 while those with murder cases came third with a total of
291 while the least are those who constitute the categories of cultism with a total of 10. The results are presented in the bar chart in
Figure 6 Figure 6: Bar chart of major crime statistics in Calabar metropolis (2006-2020)
0
500
1000
1500
2000
2500
3000
Major Crime Statistics in Calabar Metropolis
(2006-2020)
2006-2020 Figure 6: Bar chart of major crime statistics in Calabar metropolis (2006-2020)
0
500
1000
1500
2000
2500
3000
Major Crime Statistics in Calabar Metropolis
(2006-2020)
2006-2020 Figure 6: Bar chart of major crime statistics in Calabar metropolis (2006-2020) VIII.
TEST OF HYPOTHESIS Ho:
There is no significant relationship between population and crime in Calabar metropolis. The variables involved in this hypothesis are population and crime. To test this hypothesis, Pearson’s Product Moment
Correlation Statistics was employed. The choice of this statistical tool was because the scored were measured and the researcher
intends to explore relationship between the variables under study. The result is presented in Table 7. IJSSHR Volume 04 Issue 07 July 2021
www ijsshr in
Page 1908
Table 7: Relationship between population and crime rate in Calabar metropolis (N=4247)
Variables
∑x
∑y
∑x2
∑y2
∑xy
df
LS
r-cal
p-value
Population
(x)
63356
1017090
513905720
4245
0.05
.78
.000
Crime
rate
(y)
66664
2554366 Table 7: Relationship between population and crime rate in Calabar metropolis (N=4247)
Variables
∑x
∑y
∑x2
∑y2
∑xy
df
LS
r-cal
p-value
Population
(x)
63356
1017090
513905720
4245
0.05
.78
.000
Crime
rate
(y)
66664
2554366 IJSSHR, Volume 04 Issue 07 July 2021 IJSSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in Page 1908 Page 1908 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The result obtained in Table 7 revealed that with 4247 respondents as the estimated population figure for Calabar Metropolis. While X scores has a sum of 63356 sum of all scores squared of 1017090 while crime rate (y) scores has a sum of 66664 and sum
of all scores squared of 2554366. The sum of product of x and y scores is 513905720 accordingly. At 4,345 degrees of freedom and
0.05 level of significance, the p-value was found to be .000 which is less than the chosen alpha of .05. Thus, the null hypothesis is
rejected. This implies that there is a significant relationship between population rate and crime rate in Calabar metropolis. Aptly
put, the greater the population the higher the rate of crime in Calabar metropolis. Calabar Metropolis comprises of Calabar Municipality and Calabar South. The study revealed that the inner city neighborhoods are
the crime Hotspots and further identified other crime endemic areas in the metropolis as shown on fig. 7. SSHR, Volume 04 Issue 07 July 2021 www.ijsshr.in
Page
Parliamentary Extension
Ikot Effanga
Jonathan By Pass
Akai Effa
CALABAR MUNICIPALITY
Satellite Town
Obutong
AsariIso
IkotIshie
Ikot Ansa
Ekorinim
Lemna
INNER CITY NEIGHBORHOODS:
Henshaw Town
Duke Town
Watt Market
Etim Edem Park
CALABAR SOUTH
Efut Ekondo
Efut Abua
Edibe-Edibe
Mbukpa
Ekpo Abasi
Anatigha
Esuk Nsidung
Afokang
Itiat Ekpe
(Bayside)
FIG 7. CRIME HOTSPOTS IN THE CALABAR METROPOLIS
INNER CITY NEIGHBORHOODS:
Akim Qua Town
Edeba Town
Big Qua Town
Diamond Town
Essien Town
Source: Field Survey (2020)
Inner City Neighborhoods
- - - Local Govt. Boundary
Surrounding Settlements FIG 7. CRIME HOTSPOTS IN THE CALABAR METROPOLIS Lemna Itiat Ekpe
(Bayside) Ekpo Abasi Inner City Neighborhoods
- - - Local Govt. Boundary
Surrounding Settlements Source: Field Survey (2020) IJSSHR, Volume 04 Issue 07 July 2021 Page 1909 Page 1909 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The geographic locations, architectural and urban design patterns, such as neighborhood design, street layouts, avenue,
alley ways, boulevards, private homes and business establishments like the popular Watt market, Eka Ika Qua and Mbukpa market
among others in the Calabar metropolis are the primary factors that influence crime and its prevalence. Other locations are Jonathan
Bye-pass, Parliamentary extension, Akai Effa, Ikot Ishie, Ikot Ansa, etc in Calabar Municipality and Anatigha, Ekpo Abasi, Mbukpa,
Edibe-Edibe etc in Calabar South. Table 6 above shows constant increase in crime except for auto theft, vandalism and cultism from 2012-2014 which is the
last quarter of the previous administration and from 2015-2020 towards the end of the present administration. Criminal activities
dropped significantly during CONVID-19 pandemic which held criminal elements hostage (indoors) for fear of the deathly virus
which is on-going in both developed and developing nations of the world. The year 2020 also witnessed serious civilian protest
against the Nigerian Police Force tagged ENDSARS along the streets of Nigerian urban centres and Calabar in particular. This
protest led to insecurity and safety of life and property in the Calabar metropolis. The period recorded an increase in theft, arm
robbery, rape, murder, vandalism, burglary, arson, kidnapping, auto-theft and cultism respectively as shown on table 6 above. Within
the period under study several public and private offices, business and commercial buildings were set ablaze, theft, arm robbery,
rape, murder and vandalism were at their peak. Below are places destroyed and looted within Calabar Metropolis during the ENDSARS protest - the Calabar International
Conference Centre, the Newspaper Cooperation (Chronicle), the Cross River State Ministry of Works at Ekorinim which comprises
the Vehicle Inspection Office (VIO), Government Warehouse, Training School, Mechanical Workshops, Cross River Independent
Electoral Commission (INEC), West African Examination Council Office, Governor’s Petrol Station, Garment Factory, Roll Back
Malaria Office, Senators Houses, Access Bank. First Bank, National Television Authority (NTA) Value Mart Supermarket,
University of Calabar Microfinance Bank etc. IJSSHR, Volume 04 Issue 07 July 2021 Page 1910 Page 1910 www.ijsshr.in Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria
The study revealed that residents in the crime endemic neighbourhoods (Inner city) of the metropolis are imporrished
ms of resources availability with fewer opportunities to bridge the gap between the rich and the poor hence the tendency
ulge in criminal activities. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Among these crime endemic neighborhoods are Duke Town, Henshaw Town, Esuk, Watt Market, Etim Edem in Calab
th and Akim Qua, Big Qua Town, Diamond Town, Essien Town, Edeba, Ika-Ika Qua Market in Calabar Municipality. T
eriorating conditions and quality of life in these neighborhoods makes them criminogenic. The study further revealed that vio An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria The study revealed that residents in the crime endemic neighbourhoods (Inner city) of the metropolis are imporrished in
terms of resources availability with fewer opportunities to bridge the gap between the rich and the poor hence the tendency to
indulge in criminal activities. The study revealed that residents in the crime endemic neighbourhoods (Inner city) of the metropolis are imporrished in
terms of resources availability with fewer opportunities to bridge the gap between the rich and the poor hence the tendency to
indulge in criminal activities. Among these crime endemic neighborhoods are Duke Town, Henshaw Town, Esuk, Watt Market, Etim Edem in Calabar
South and Akim Qua, Big Qua Town, Diamond Town, Essien Town, Edeba, Ika-Ika Qua Market in Calabar Municipality. The
deteriorating conditions and quality of life in these neighborhoods makes them criminogenic. The study further revealed that violet
crime and delinquency rates are more prevelant among the lower socio-economic groups living in these neighborhoods and other
crime locations. IJSSHR, Volume 04 Issue 07 July 2021 Page 1911 www.ijsshr.in XI.
RECOMMENDATIONS Crime can be reduced through the encouragement of neighborhood watch and frequent policing of target areas of crime hotspots in Crime can be reduced through the encouragement of neighborhood watch and frequent policing of target areas of crime hotspots in
Calabar Metropolis. Government should have democratized urban design and planning to accommodate all social groups) that is
Integration between urban and suburban areas to reduce high crime rate. There should be stiffer penalties, faster court trails and
sentences for crime victims. The public should be informed through public media to always take precautionary measures to safeguard
their cars and home doors. The use of hard drugs by the youths should be closely monitored by the Nigerian Police Force. Brujak
(1999) observed that the higher the proportion of young people especially the male counterpart in a city, the greater the incidence
of violent crime. He further contended that the youth populations are more predisposed to crime than the elderly people. This shows
that there is a relationship between age structure of population of a city and incidence of crime. In its study, the FBI (1996) crime index shows that about 55.6 percent of offenders are between 18-25 years of age. Orphanage homes in the Calabar Metropolis should be a transformed as foundation for the welfare of youths and should be equipped
to deal with the problem of child abandonment and street children popularly known as Skolombo in Calabar Metropolis, Amajiris
and Damiskas in Northern Nigerian cities, Agberos and Alayes in Western parts of Nigeria and touts, street urchins and juveniles. The higher income class should be informed through public media to avoid extravagant and flamboyant lifestyles. Government and
the organize private sector should introduce youth empowerment programme or employment opportunities for the poor youths in
our societies to reduce inequality and denial justice which fosters and engenders criminal activities. There should be synergy among
ethnic, religious and political groups to co-exist in harmony with one and another. Government curfew laws should be enforced to
reduce crime in hot spots of the Calabar metropolis as shown on fig 7. Urban and regional planners should develop safety base designs for urban spaces such as residential and commercial
neighborhoods, parks and recreational grounds, street networks and urban renewed projects. CAUSES OF CRIME IN CALABAR METROPOLIS Oral interview of selected groups of residents in the inner city neighborhoods and other crime locations and hotspots in th
metropolis revealed the following factors as being responsible for crime in the study area. Extravagant and flamboyant lifestyles; Driving and walking alone on dark streets; Building of high wall fences in rich residential
neighborhoods; Poverty and deprivation; Leaving with house doors open; Leaving car keys in the ignition; Exposing valuable
properties in the car; Indecent dressing code by women; Disintegration of urban and suburban areas through urban planning and
architectural design; Poor policing of crime hotspots and lack of patrol vehicles; Unemployment; Rapid population and urbanization;
Drug usage; The structure of political power and corruption; Religion and intergenerational transmission of violence; Family
conditions; Lack of access to legitimate opportunities for minority groups; Deteriorating condition of urban neighborhoods and
quality of life; Neighborhood streets with poor linear visibility and dead-ends; Incessant use of artifacts which include grafitis on
city buildings and walls such as “Beware of dogs”, cars parked at owner’s risk etc are criminogenic; Poor funding of security
personnel; Lack of security gargets such as CCTV cameras; Poor urban planning, design and management. X.
CONSEQUENCES OF CRIME ON RESIDENTS OF CALABAR METROPOLIS
There has been serious problem of insecurity and safety due to high rate of criminal activities in the Calabar Metropolis.
Many business have folded up and businessmen have loss their savings to armed robbers and fraudsters in recent times.
Rape victims for instance have suffered humiliation, embarrassment shame and serious depression. The psychological and
physical trauma will live with the residents for a long time.
Many business have folded up and businessmen have loss their savings to armed robbers and fraudsters in recent times.
Rape victims for instance have suffered humiliation, embarrassment shame and serious depression. The psychological and
physical trauma will live with the residents for a long time.
Millions of Naira, personal properties and many lives have been lost especially during the ENSARS protest in Calabar. Insurance claims and premium are now high and many victims of vandalization may not be able to cope. Most victims
and property owners suffered physical assault and majority are permently disabled due to high medical cost.
Millions of Naira, personal properties and many lives have been lost especially during the ENSARS protest in Calabar. Insurance claims and premium are now high and many victims of vandalization may not be able to cope. Most victims
and property owners suffered physical assault and majority are permently disabled due to high medical cost.
Under the present economic down turn government may find it difficult to maintain the cost of security systems and many
other crime prevention measures and apparatus. There is general fear in the system especially the frequent cases of
kidnapping and assassination of high profile politicians and businessmen within the metropolis and its environ.
Under the present economic down turn government may find it difficult to maintain the cost of security systems and many
other crime prevention measures and apparatus. There is general fear in the system especially the frequent cases of
kidnapping and assassination of high profile politicians and businessmen within the metropolis and its environ. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Oral interview with the Nigerian Police Force in Calabar revealed Youth involvement in crime from the age of 12-21 and
above which according to them is a reflection of the degree of social disorganization and deteriorating quality of urban life. According to the police force these crime endemic locations have the highest population of youth gangs who have masterminded
the kidnapping of high profile government officials and business mogul for ransom to get rich quick. The study further revealed high incidence of crime in the Gated and rich neighborhoods such as the Satellite Town,
Ekorinim, State Housing Estate, Federal Housing Estate, House of Assembly village and other private housing estates within the
metropolis. These rich neighborhoods are characterized with high wall fences which negates the Eye-on-the-street theory by Ross
(2000) and Eyler and Vest (2002), Narrow and winding lonely streets with Dead-ends, and grafitis on fences and buildings such as
“Beware of dogs”, car parked at owners risk” etc which attract criminality as shown on the plates below. HR, Volume 04 Issue 07 July 2021 www.ijsshr.in
Pa IJSSHR, Volume 04 Issue 07 July 2021 Page 1912 Page 1912 An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria XII.
CONCLUSION Urbanization is a product of rapid population growth of an area. It is an important component for the economic and social
development of an area with its attendant consequences such as unemployment, poverty, crime, insecurity and safety. Calabar
Metropolis has witnessed a wide range of criminal activities, at different geographical locations by both physical and social disorder. Other factors that influenced crime and its prevalence in Calabar Metropolis include architectural and urban design pattern such as
neighborhood design, the structure of the city’s street layouts, private homes, offices business establishments etc. It must be noted that security and safety are the major objectives of physical planning to reduce crime rate in our urban
centres. Urban planners should develop appropriate designs and opportunities to minimize the rate of criminal activities in the urban
centres. Government in partnership with the private sector should employ appropriate security apparatus, skill security personnel,
modern technology and other measures to promote the security and safety of residents in the study area. An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria activities in the cities. Other crime prevention technology such as electric fencing, security breach alarm systems, auto-breach sensor
and so on, should be utilized to reduce auto-theft, license plate recognition technology, and the use of Geographic positioning
systems (GPS) should also be used to monitor criminal activities in our urban areas. ACKNOWLEDGEMENTS We acknowledge the Authors whose materials and publications guided us in this research as indicated in the Bibliography section. We also want to thank our wives and children who allowed us to be away from them at the course of this research and the cab man
who took us round the city of Calabar in order to get important information and photographs needed for this study. Above all, we
give God the Glory for wisdom, understanding and knowledge bestowed on us to complete this work. XI.
RECOMMENDATIONS The “Eye on the street” defined by Jacobs (1961) as a natural surveillance that is a deterrent of criminal activities should
be adopted in street network design. It is seen as the resident ability to view public streets. The design orientation of buildings with
windows directly facing the street increases natural surveillance for residents of rich neighborhoods such as Ekorinim, Satellite
Town, Public and private residential estates in Calabar Metropolis. High wall fences and heavy landscaping should be discouraged
in rich neighborhoods as they block view and create hideouts for criminal activities like rape and handbag snatching. Street lighting
along major and minor streets should be encouraged as well as public open spaces and parking lots to scare criminals and hoodlums. Undeveloped plots of land should be cleared and maintained while uncompleted buildings and open parks should be policed more
frequently by law enforcement officers. The Cross River State Government should key into the crime prevention technology in
major urban areas in the state through the use of video surveillance (Close Circuit Television Camera (CCTV) to monitor criminal IJSSHR, Volume 04 Issue 07 July 2021 Page 1913 www.ijsshr.in REFERENCES 1) Brantingham, P. (2005) “Daily Urban Crime and Disorder: Is there a simple solution? Canada. Institute for Urban Research
Studies. Simon Frazer University. 2) Bryjak, G. J. (1999) “Multiple Reasons for Lower Crime Rates” San Diego Union Tribune. March 28. 2) Bryjak, G. J. (1999) “Multiple Reasons for Lower Crime Rates” San Diego Union Tribune. March 28. 3) Federal Bureau of Investigation (1996) “Uniform Crime Report” Washington D.C. Government Offi 4) Federal Bureau of Investigation (1996) Crime in the United States” Washington D.C. Government office. 4) Federal Bureau of Investigation (1996) Crime in the United States” Washington D.C. Government office. 5) Frank L. D. and Engelke P. O. (2001) “The Built Environment and Human Activity Patterns: Exploring the Impacts of
Urban Form and Public Health”. Journal of Planning Literature. Vol.16 No.2 Pp. 202-218. 5) Frank L. D. and Engelke P. O. (2001) “The Built Environment and Human Activity Patterns: Exploring the Impacts of
Urban Form and Public Health”. Journal of Planning Literature. Vol.16 No.2 Pp. 202-218. 6) Greenwald M. and Boarnet M. G. (2002). The Built Environment as a Determination of Walking Behaviour: Analyzing
Non-work Pedestrian Travel in Portland, Oregon”, Transportation research record. Journal of Transportation Research
Board, No. 1780, TRB, National Research Council, Pp. 32-42. 6) Greenwald M. and Boarnet M. G. (2002). The Built Environment as a Determination of Walking Behaviour: Analyzing
Non-work Pedestrian Travel in Portland, Oregon”, Transportation research record. Journal of Transportation Research
Board, No. 1780, TRB, National Research Council, Pp. 32-42. 7) Handy S. (1996) “Urban Form and Pedestrian Choices Study of Austin Neighborhood”. Transportation Research Record
1522, TRB. National Research Council. 7) Handy S. (1996) “Urban Form and Pedestrian Choices Study of Austin Neighborhood”. Transportation Research Record
1522, TRB. National Research Council. 8) Handy S. L., Boarnet M. Ewing R. and Killingworth R. E. (2002) “How the Built Environment affects Physical Activity;
views from Urban Planning”. American Journal of Preventive Medicine, Vol. 23. Nov. 2 (Supplement), Pp. 64-73. 8) Handy S. L., Boarnet M. Ewing R. and Killingworth R. E. (2002) “How the Built Environment affects Physical Activity;
views from Urban Planning”. American Journal of Preventive Medicine, Vol. 23. Nov. 2 (Supplement), Pp. 64-73. 9) Hicks, J. REFERENCES (1998) “Enhancing the Productivity of Urban Africa” Being paper Published in the Proceedings of an International
Conference Research Community for the Habitat Agenda: Linking Research and Policy for the Sustainability of Human
Settlement held in Geneva, July 6-8. 1998. 9) Hicks, J. (1998) “Enhancing the Productivity of Urban Africa” Being paper Published in the Proceedings of an International
Conference Research Community for the Habitat Agenda: Linking Research and Policy for the Sustainability of Human
Settlement held in Geneva, July 6-8. 1998. y
10) Hove, M., Ngwerume E. T. and Muchemwa C. (2013) “The Urban crisis in Sub-Saharan Africa: A threat to Human Security
and Sustainable Development”. Stability: International Journal of Security and Development 2(1). 11) Jacobs, J. (1961) “The Death and Life of Great American cities, Vintage Books, New York. 12) Kelly E. & Dian D. C. (2009) Securing the Built Environment: An Analysis of Crime Prevention through Environmental
Design Ball University, Munie Indiana. ) Kelingberg, E. (2002) Heat Wave: A social autopsy of disorder in Chicago. The University of Chicago P 14) Newman, O. (1972) Dejensible space: Crime Prevention through Urban Design. New York. Macmillan. 15) Nnodu V. C, Okoye, Onwuka (2008) Urban Environmental problems in Nigeria. Rex Charles and Patrick Limited,
Booksmith House, Harmony Place, Nimo, Anambra State, Nigeria. ) NPC (1998), 1991 Population Census of the Federal Republic of Nigeria. Nigeria Population Commissio 17) Obeng-odoom, F. (1981) :Informal sector in Ghana under Siege” Journal of Developing Societies, Vol. 27 no. 3-4 DOI:
http:/dxi.doi/org/10.1177/0169796x1102700406. 18) Ogboi, K. C. (2013) “Enhancing Urban Security and safety in Nigeria through Parks, S. E. Honsemann R. A. and Brownson
R. C. (2002) Differential Correlates of Physical Activity in Urban and Rural Adults of various socioeconomic Background
in the U.S. Journal of Epidemolog and Community Health. Vol. 57, Pp. 29-35. IJSSHR, Volume 04 Issue 07 July 2021 Page 1914 Page 1914 www.ijsshr.in An Appraisal of Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nigeria Insecurity and the Consequences on Residents in Calabar Metropolis, Cross River State, Nige 19) Okeke, D. C. (2002) Environmental and Urban Renewal Strategies: Theoretical and Analytical Frameworks. Institute for
Development Studies, University of Nigeria, Enugu Campus, Nigeria. 20) Rakisits, C. (2008) “Child soldiers in East of Democratic of Congo”. Refugee Survey Quarterly, 27(4): 108-122. DOI:htt:dxi.doi.org/10.1093 req/hdno54. 21) Rose, C. E. (2000)”Walking Exercising, and smoking: Does neighborhood matter? Social Science and Medicine. Vol. 51,
No. 2, Pp.265-274. 22) Stark, R. (1987) “Deviant Places: A theory of the Ecology of Crime” Criminology V 23) Todaro, M. (2000) Economic Development, Essex: Pearson Education Limited. 23) Todaro, M. (2000) Economic Development, Essex: Pearson Education Limited. 24) United Nations, (1996) An Urbanizing World: Global Report on Human Settlements 1996. A United Nation Publication. New York. 25) UN-Habitat (2008) State of the World’s Cities Report, Nairobi, Kenya: UN-Habitat United Nations Population Fund
(2007), State of World Population 2007: Unleashing the potential of urban growth, accessed online at www.un7pa, on 3
Jan. 2013. United Nations Department of Economic and Social Affairs (DESA) 2011) Population Distribution,
Urbanization, Internal Migration and Development: An International Perspective. New York City, NY. IJSSHR, Volume 04 Issue 07 July 2021 Page 1915 www.ijsshr.in
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Mössbauer Spectroscopy in Heterogeneous Catalysis
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Advances in catalysis
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Mo¨ ssbauer Spectroscopy in
Heterogeneous Catalysis JEAN-MARC M. MILLET
Institut de Recherches sur la Catalyse et l’Environnement de Lyon, IRCELYON, UMR 5256 CNRS-
Universite´ Claude-Bernard, Lyon 1, 2 avenue A. Einstein, F-69626 Villeurbanne Cedex, France; E-mail:
jean-marc.millet@ircelyon.univ-lyon1.fr Mo¨ ssbauer spectroscopy dates back to 1958 and for characterization of a solid catalyst was
first used in 1971; the technique achieved a high point in the 1980s and has been applied only
modestly for catalyst characterization since then. The declining use of the technique may be
related to the experimental difficulties and the complexity of the spectral analysis. However, the
adaptability of Mo¨ ssbauer spectroscopy to the investigation of catalysts under working conditions
and the progress made in the field are evidently leading to a reawakening of interest in its
application in catalysis. The value of Mo¨ ssbauer spectroscopy in catalyst characterization is
illustrated by results of investigation of Fischer–Tropsch catalysts by 57Co Mo¨ ssbauer emission
spectroscopy performed at temperatures up to 723 K and pressures up to 40 bar corresponding to
industrial working conditions. Furthermore, synchrotron Mo¨ ssbauer techniques have reached a
level of maturity that today allow their use for applications from which catalysts characterization
would benefit. This review includes a brief summary of the technique and an overview of ap-
plications in catalysis. The majority of investigations have dealt with the isotopes 57Fe, 119Sn, and
57Co (in emission mode). In contrast, investigations focusing on other isotopes, such as 197Au,
121Sb, 125Te, 193Ir, or 99Ru, have been limited in number but very fruitful. Opportunities for future
applications to working catalysts are presented and discussed. New techniques related to the use
of synchrotron radiation as energy source are emphasized. Such techniques have been applied to
isotopes other than 57Fe, such as 169Tm, 119Sn, 83Kr, 181Ta, 151Eu, 161Dy, and 149Sm. The in-
vestigation of isotopes such as 197Au, 197Pt, 61Ni, 186W, 99Ru, and 99Rh, is also possible and
should strongly benefit catalyst characterization. An effort has been made here to present the
techniques, results, and developments in a didactic manner. I. Introduction The nature and breadth of the physical techniques used to investigate solid catalysts
continue to increase rapidly in complexity. This statement pertains specifically to
Mo¨ ssbauer spectroscopy, which was applied to the characterization of solid catalysts
as early as 1971 (1). A retrospective analysis of the use of Mo¨ ssbauer spectroscopy in
catalysis showed that it has consistently accounted for 3–10% of the communica-
tions presented at the International Congresses on Catalysis (ICC) (7%t at the ICC
in Paris in 2004). Such continuity over the years reflects the high value of this
technique in catalyst characterization. The theoretical basis of Mo¨ ssbauer spectroscopy as well as its applications to
catalyst characterization was reviewed in Advances in Catalysis in 1989 (2). This
thorough article summarizes the physical basis of the technique and significant
contributions to the characterization of solid catalysts. Since 1989, Mo¨ ssbauer
spectroscopy has not underwent major developments, and its applications to catal-
ysis have been largely limited to catalysts that were not in reactive atmospheres,
notwithstanding the impressive advances that have been made with other techniques
in characterizing catalysts under working conditions. Because this volume is focused on characterization of working catalysts, this
chapter is short, including a brief summary of the technique and the underlying
physical principles, with a focus on its potential and limitations for investigation of
catalysts as they function. Only the essential aspects necessary to understand the
technique are presented here. The summary of recent applications emphasizes in-
vestigations with less commonly used isotopes and on investigations of catalysts at
temperatures and pressures relevant to catalytic reactions. The practical aspects of
the use of Mo¨ ssbauer spectroscopy are not treated in detail, and only a few examples
are discussed. Readers searching for more complete reviews of research done
using Mo¨ ssbauer spectroscopy should consult the reviews published in recent years
(3–6). This article concludes with an evaluation of potential innovations that may
arise in the near future and that have not yet been applied for investigations of
heterogeneous catalysis. Mo¨ ssbauer Spectroscopy in
Heterogeneous Catalysis Abbreviations: ADC, analog-to-digital converter; CEMS, conversion electron Mo¨ ssbauer spectroscopy;
DCEMS, depth selective conversion electron Mo¨ ssbauer spectroscopy; DFT, density functional theory;
EMS, emission Mo¨ ssbauer spectroscopy; ESRF, European synchrotron radiation facility; FeZSM-5,
iron-containing Zeolite Society Mobil constructed from five-membered-ring building units; GC, gas
chromatography; HTP, high throughput; IMS, imaging Mo¨ ssbauer spectroscopy; MCM-41, mobil
composite material/mesoporous composite materials of type 41; MERDJ, Mo¨ssbauer Effect Reference
and Data Journal; NBD, nuclear Bragg diffraction of synchrotron radiation; NFS, nuclear forward
scattering of synchrotron radiation; NIS, nuclear inelastic scattering of synchrotron radiation; NQES,
nuclear quasi-elastic scattering of synchrotron radiation; NR, nuclear reflectometry of synchrotron
radiation; NRS, nuclear resonance scattering; NSAS, small-angle scattering of synchrotron radiation;
SCA, single channel analyzer; SOD, second-order Doppler shift; XANES, X-ray absorption near edge
spectroscopy; ZSM-5, Zeolite Society Mobil constructed from five-membered-ring building units. 1 A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES Mo¨ ssbauer spectroscopy provides measurements of the resonant absorption of
g-rays by nuclear transitions from a ground state to an excited state. Like other
nuclear techniques, it is based on a phenomenon that is specific to a given isotope
and for which no interference from other isotopes is possible. In conventional Mo¨ ssbauer spectroscopy, X-rays with energies corresponding to
nuclear transitions (5–150keV) can be produced only by use of radioactive sources
containing a parent isotope of the absorbing nucleus in an appropriate excited state
from which it decays into the ground state with emission of a g-quantum. For spec-
troscopic applications, the g-radiation must be variable. The chemical perturbations 2 at the nuclear levels are very small relative to the transition energy (i.e., 108–1011). Variations of the energy of the parent radiation of this order of magnitude can be
achieved by the Doppler effect. For this purpose the radiation source has to be
moved linearly at a low velocity v (generally few mm/s) that will allow variation of the
incident energy E0: E ¼ E0 1 v
c
. ð1Þ ð1Þ Experimentally, the most convenient way to achieve such movement is to fix
the source on a constant-acceleration oscillator allowing imposition of velocities in
positive and negative direction depending on whether the source moves toward or
away from the absorber: E ¼ E0 (17v/c) (Fig. 1). For convenience, Mo¨ ssbauer
spectrum is taken to correspond to the plot of the intensity of the transmitted or
diffused X-rays as a function of the Doppler velocity v instead of the corresponding
energy. The observation of resonance is governed by the probability of emission (in the
source) and absorption (in the absorber) of the gamma photons. This probability
(Eq. (2)) is named the Lamb–Mo¨ ssbauer or recoil-free factor. The probability of the
interaction of a photon with a nucleus that is at the basis of the absorption is
proportional to the square of the matrix element of the interaction Hamiltonian
which excites the initial state i to the final state f: f ¼ exp
4p2E2
l x2
ðhcÞ2
! ,
ð2Þ ð2Þ where x is the vibrational amplitude of the absorbing or emitting atoms in the solid
lattice, Eg the energy of the photon, and h is Planck’s constant. A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES The factor f can be
expressed in the Debye model as follows: f ¼ exp
3ER
2kTD
1 þ 4
T
TD
2 Z T=TD
0
y dy
ey 1
"
#
(
)
,
ð3Þ ð3Þ sample
detector
radioactive
source
FIG. 1. Basic apparatus for measuring a Mo¨ ssbauer spectrum in transmission mode. FIG. 1. Basic apparatus for measuring a Mo¨ ssbauer spectrum in transmission mode. 3 where ER is the recoil energy ER ¼ E2
Y=2Mc2
; T the temperature, TD the Debye
temperature of the solid, and y ¼ hv=kT: When T5TD and T5TD, the expression
may be approximated by where ER is the recoil energy ER ¼ E2
Y=2Mc2
; T the temperature, TD the Debye
temperature of the solid, and y ¼ hv=kT: When T5TD and T5TD, the expression
may be approximated by f TTD ¼ exp
3ER
2kTD
1 þ 2p2
3
T
TD
2
"
#
(
)
and
f TTD ¼ exp 6ER
kTD
T
TD
. ð4Þ ð4Þ ð4Þ These equations are useful because they allow visualization of practical limits of the
technique in terms of suitable isotopes, characteristics of the solids, and exper-
imental conditions. Indeed, to have sufficiently large values of f, M should not be
too small (Z40), Eg not too large (r150 keV), TD not too small (which corresponds
to relatively strong chemical bonds), and T not too high. y
g
)
g
As a result of these constraints, 79 isotopes have been found for which the
Mo¨ ssbauer effect can be observed. For 38 of them, chemical perturbations of
the nuclear levels result in resonance shifts or splittings of the order or greater than
the observed width Gexp of the resonance line (this parameter is defined in the next
paragraph (Eq. (9)) and can thus be used for Mo¨ ssbauer spectroscopy. This list of
38 isotopes may be further restricted because of experimental difficulties encoun-
tered in their use. These difficulties are related either to their large recoil energies,
which require a very low recording temperature for the spectra (4.2 K), or the short
half-lives of the parent nucleides used as sources. A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES For higher values of t, the proportionality no longer holds, and corrections
have to be made to use the intensity values, for example, to establish the relative
amounts of different phases. Equation (5) gives the maximum value of the resonance absorption cross-section,
which varies as follows with the energy: s ¼ s0 1 þ 4ðE E0Þ2
G2
0
"
#1
,
ð7Þ ð7Þ where E is the incident energy and G0 the width of the excited nuclear state. This
width is given by Heisenberg’s uncertainty relationship: G0 ¼ h
2pt ,
ð8Þ ð8Þ where t is the mean (or natural) exponential lifetime of the excited state (1.44 the
half-lifetime). Because the source radiation and the absorber cross-section have line
width values of G0, the minimum width actually observable in a transmission
experiment is 2G0. This corresponds approximately to 0.2 mm/s of velocity Doppler
shift for 57Fe (and 0.65 mm/s for 119Sn). The broadening of the line width beyond
this theoretical value is related to inhomogeneities arising from imperfections in the
source or the absorber or from a saturation effect. The observed experimental line
width is evaluated, and an approximate value can be calculated from the following
equation: ð9Þ Gexp ¼ G0 2 þ 0:27tA
ð
Þ,
ð9Þ where tA, called sample or absorber thickness, equals fAnAas0t, with fA being the
Lamb–Mo¨ ssbauer factor of the absorber, nA the number of resonant isotope species
per cubic centimeter, and a the fractional abundance of the Mo¨ ssbauer isotope. Generally, tA has to be optimized experimentally, and values close to or smaller
than 1 are used. Because the cross-section has a Lorentzian shape 1/(1+x2) form
around E0 (Eq. (7)), the resonance peak has the same shape. A. PHYSICAL PRINCIPLES AND CURRENT CAPABILITIES In the former case, cryogenic
cooling of both the source and the sample during the experiment may be needed; in
the latter case, the manufacturing of the source requires rapid access to a nuclear
reactor or accelerator. In practice, by far the most commonly used Mo¨ ssbauer isotopes in heterogeneous
catalysis (>90% of all publications) are 57Fe and 57Co (in the emission mode). 119Sn represents more than half of the remaining investigations, and most of the
others have been done with a group of nuclei including 197Au,193Ir, 121Sb, 125Te,
and 151Eu. In view of the importance of Ni, Ta, and Ru in catalysis, Mo¨ ssbauer investi-
gations of 61Ni, 181Ta, or 99Ru would have been of great interest. Unfortunately, the
aforementioned difficulties encountered with these isotopes lead to the realization
that although Mo¨ ssbauer spectroscopy is in principle possible, almost no investi-
gations have been reported using these isotopes. The magnitude of the observed absorption depends on the absorption cross-
section for g-rays to excite a transition between the nuclear ground and excited
states at resonance. This cross-section per nucleus s0 is given by the following
equation: s0 ¼ 1
2p
h2c2
E2
0
2Ie þ 1
2Ig þ 1
1
ð1 þ aÞ ,
ð5Þ ð5Þ where c is the velocity of light, E0 the transition energy, Ie and Ig the excited and
ground state spins, and a the internal conversion coefficient (a describes the relative
probability of radiative (g-ray) and non-radiative (electron conversion) processes of
the transition (2)). 4 The magnitude of the observed resonance depends not only on s0 but also on the
effective thickness of the absorber, which may be expressed as follows: The magnitude of the observed resonance depends not only on s0 but also on the
effective thickness of the absorber, which may be expressed as follows: t ¼ nf s0,
ð6Þ ð6Þ t ¼ nf s0, where n is the number of resonant isotope species per square centimeter. For small
values of t (r0.5) there is proportionality between the observed resonance intensity
and t. For higher values of t, the proportionality no longer holds, and corrections
have to be made to use the intensity values, for example, to establish the relative
amounts of different phases. where n is the number of resonant isotope species per square centimeter. For small
values of t (r0.5) there is proportionality between the observed resonance intensity
and t. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS The nuclear ground and excited levels involved in the Mo¨ ssbauer transition are
shifted or split because of the electrostatic interactions between the nuclear charge
and the surrounding electric charge (Fig. 2). The first interaction, sometimes called
the electric monopole interaction, shifts only the nuclear levels and is related to the
perturbation resulting from the electrons inside the nuclear volume. This shift is 5 5 absorber
energy
-3/2
+3/2
-1/2
+1/2
±1/2
±3/2
+1/2
-1/2
source
I=3/2
I=1/2
m
δ
δ
∆
δ
Transmission
Transmission
Transmission
velocity
velocity
velocity
FIG. 2. Schematic representation of the hyperfine interactions in the case of a D transition 1/2-3/2
like that of 57Fe. Transmission Transmission FIG. 2. Schematic representation of the hyperfine interactions in the case of a D transition 1/2-3/2
like that of 57Fe. different for the ground and excited states because the nuclear volumes are different
in the two states. Furthermore, these shifts are different for the source and the
absorber because emitting and absorbing nuclei have different electron densities
inside the nuclear volumes because of different chemical states and electronic en-
vironments, and the absorption will be observed at a velocity characteristic of the
absorber relative to the source. This measured shift, called isomer shift (IS), is
expressed as follows: IS ¼ 2
3 e2pZSðZÞD r2
rAð0Þ rSð0Þ
,
ð10Þ ð10Þ where D r2
is the variation of the mean-square radius of the nucleus between the
excited and ground states of the Mo¨ ssbauer transition, S0ðZÞ is a correction for the
relativistic effects, and the term in brackets, rAð0Þ rSð0Þ; the difference between
the electron density at the nucleus in the absorber A and the source S. Isomer shifts measured and defined as in Eq. (10) depend on the source. For a
comparison of results reported by different authors, these must be stated with
respect to that of a standard compound. In the case of 119Sn, the usual standards 6 are BaSnO3 and SnO2, and in the case of 57Fe, they are a-Fe metal or sodium
nitroprusside. The latter compound is, however, used less and less. For Mo¨ ssbauer
nucleides other than these two, there are generally no specific prescriptions regard-
ing standards. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS In some cases, the natural line width is very broad relative to those
of iron or tin, or the change in nuclear radius between the ground and the excited
state is very small so that the measurement of isomer shifts is difficult or virtually
impossible. The use of an isomer shift standard thus became insignificant. Although IS is independent of T, variations with temperature are observed
experimentally because ISOBS is in fact a linear combination of IS and a second shift
which is sample-dependent, being called a second-order Doppler shift (SOD): ISOBS ¼ IS þ ISSOD. ð11Þ ð11Þ This shift arises from the relative motions associated with atomic vibrations, via a
second-order relativistic effect; it is equal to the following: This shift arises from the relative motions associated with atomic vibrations, via a
second-order relativistic effect; it is equal to the following: ISSOD ¼ V 2
2c2 Ey,
ð12Þ ð12Þ where V 2
is the mean-square velocity of the probe nucleus in the sample, which
can be derived from the Debye model of the vibrations: ISSOD ¼ 1
2c
9kTD
8M
1 þ 8
T
TD
4 Z
T
TD
0
y3dy
ey 1
"
#
. ð13Þ ð13Þ It is evident that this term depends on the temperature and also on the source and
the absorber (through M and TD). A second perturbation of the nuclear levels is caused by the magnetic interaction
between the nuclear magnetic moment and the surrounding electron spin density,
creating an electric field gradient at the nucleus. This electric field gradient is a
tensor that is described by the spatial orientations of its three principal axes UXX,
UYY, and UZZ. This interaction partially removes the degeneracies of nuclear states in which the
nuclei have non-zero quadrupole moments Q (spin I>1) into 2I+1 nuclear states
characterized by the value of the magnetic quantum number mI, and it shifts them. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS In the general case when the electrical field gradient has no axial symmetry, the
energy shift of the levels can be expressed as follows: EQ ¼ eUZZQ 3m2
I IðI þ 1Þ
4Ið2I 1Þ
1 þ Z2
3
1=2
,
ð14Þ ð14Þ where Q is the quadrupole moment of the nucleus in the excited state of the
Mo¨ ssbauer transition, mI the magnetic quantum number, UZZ the main component
of the electric field gradient tensor
UZZ
j
j4 UYY
j
j UXX
j
j
ð
Þ; and Z the asymmetry
parameter ðZ ¼ ðUXX UYYÞ=UZZÞ; which is zero in the case of axial symmetry. In the case of 57Fe, the ground state is an I ¼ 1/2 state without a quadrupole
moment and is thus not affected by the electric field gradient. The first excited state
is an I ¼ 3/2 state with a quadrupole moment and has a degeneracy of 4 that is 7 partially lifted. It is symmetrically split into two substrates. This splitting, called
quadrupolar splitting (QS or D), is given by the following: partially lifted. It is symmetrically split into two substrates. This splitting, called
quadrupolar splitting (QS or D), is given by the following: D ¼ 1
2 eUZZQ 1 þ Z2
3
1=2
. ð15Þ ð15Þ As a result, the Mo¨ ssbauer spectrum will consist of two lines, called a doublet,
with a 2G0-line width and separated by D, the isomer shift being measured at the
center of the doublet (Fig. 2). The relative intensities of the two absorption lines of
the doublet depend on the orientation of Uzz relative to the incident X-ray direction
(Table I). For a fine powder absorber, in which all directions are uniformly
distributed, the two lines of the corresponding doublet have equal intensities. A third perturbation of the nuclear energy levels occurs in the presence of a
magnetic field and yields different energies for the different orientations of the
nuclear magnetic moment mI relative to the field direction. Each nuclear level of
non-zero spin is shifted into 2I+1 sub-levels reached by shifting it by – gmNmIH,
where H is the magnitude of the magnetic field, g the nuclear factor of the con-
sidered sub-level, and mN the nuclear Bohr magneton. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS For example, in the case of iron, the ground level will undergo a symmetric
splitting (I ¼ 1/2, mI ¼ +1/2, mI ¼ 1/2) and the first excited state is split into four
sub-levels (I ¼ 3/2, mI ¼ +3/2, mI ¼ +1/2, mI ¼ 1/2, mI ¼ 3/2). Among the
eight possible transitions between the two sub-levels of the ground state and the
four sub-levels of the excited state only six are allowed by the quantum mechanical
selection rule, and the resonance spectra consist of six lines with a 2G0-line width
called a sextet. The isomer shift is measured at the center of the sextet (Fig. 2). H is
proportional to the difference between the positions of two lines of the spectrum;
the outermost ones are generally considered in the data analysis for better precision. TABLE I
Line Position and Relative Intensities in 57Fe Spectra Obtained in the Presence of an Electric Field Gradient
(Doublet) and both an Electric Field Gradient and a Magnetic Field (Sextet)
Line
Spin state
Line position (mm/s)
Relative intensities
Ig
Ie
Doublet
1
71/2
71/2
dD/2
2+3sin2ygVZZ
2
71/2
73/2
d+D/2
3(1+cos2ygVZZ)
Sextet
1
+1/2
3/2
d(Z+3)(z/2)+e
3(1+cos2ygH)
2
+1/2
1/2
d(Z+1)(z/2)e
2(2sin2ygH)
3
+1/2
+1/2
d(Z1)(z/2)e
1(1+cos2ygH)
4
1/2
1/2
d(Z1)(z/2)e
1(1+cos2ygH)
5
1/2
+1/2
d(Z+1)(z/2)e
2(2sin2ygH)
6
1/2
+3/2
d+(Z+3)(z/2)+e
3(1+cos2ygH)
ygVZZ and ygH are respectively the angles between the gamma ray direction, Vzz and H. Ig and Ie are the
spin states of the ground and excited states; D the quadrupolar splitting (Eq. (15)), and e the quadrupolar
shift parameter (Eq. (16)). Z the ratio between the g factor of the ground (gi) and excited state (ge), and
z ¼ gemNH. TABLE I
Line Position and Relative Intensities in 57Fe Spectra Obtained in the Presence of an Electric Field
(Doublet) and both an Electric Field Gradient and a Magnetic Field (Sextet) TABLE I ygVZZ and ygH are respectively the angles between the gamma ray direction, Vzz and H. Ig and Ie are the
spin states of the ground and excited states; D the quadrupolar splitting (Eq. (15)), and e the quadrupolar
shift parameter (Eq. (16)). Z the ratio between the g factor of the ground (gi) and excited state (ge), and
z ¼ gemNH. B. HYPERFINE INTERACTIONS AND MO¨ SSBAUER PARAMETERS 8 In the presence of a small simultaneous electric quadrupolar interaction, the split
levels are shifted by an amount e, which is called the quadrupole shift parameter
and is given by the following: ¼
e2UzzQ
8
½2 ð3 Z cos 2fÞsin2y,
ð16Þ ð16Þ where f and y are the azimuthal and polar angles of the magnetic field direction in
the principal axes coordinate system of the electric field gradient. The relative in-
tensities of the six lines of the sextet depend on the orientation of the magnetic field
relative to the incident g-ray beam (Table I). The magnetic field can have several origins and may be written as the sum of up
to five contributions that can be added vectorially to give the following expression: ð17Þ H ¼ HS þ HL þ HD þ HM þ HE,
ð17Þ where HS, HL, and HD are, respectively, the fields attributed to the s-electron spin
polarization term, to the on-site orbital electron current, and to the dipole inter-
action related to the local electron spin moments on the probe site. HM is the field
resulting from the magnetization of the particle within which the nucleus is located. It depends on the shape and magnetic domain structure of the particles. HE is an
external magnetic field that can be applied to the sample. C. SPECTRAL ANALYSIS AND INTERPRETATIONS In the preceding section the hyperfine parameters (d, D, H, and f ) that can be
extracted by spectral analysis were presented. These parameters have complex re-
lationships with physico-chemical properties that are presented in a rather simpli-
fied manner in the following section, showing how local electronic, magnetic,
structural, and chemical environments can be characterized. Equation (10) shows that the isomer shift IS is a direct measure of the total
electronic density at the probe nucleus. This density derives almost exclusively from
s-type orbitals, which have non-zero electron densities at the nucleus. Band
electrons, which have non-zero occurrence probabilities at the nucleus and s-type
conduction electrons in metals may also contribute, but to a lesser extent. Figure 3
shows the linear correlation that is observed between the experimental values of
121Sb Mo¨ ssbauer isomer shift and the calculated values of the valence electron
density at the nucleus rvð0Þ. The total electron density at the nucleus rAð0Þ (Eq. 10)
is the sum of the valence electron density rvð0Þ and the core electron density rcð0Þ,
which is assumed to be constant. This density is not only determined by the
s-electrons themselves but also by the screening by other outer electrons (p-, d-, or
f-electrons) and consequently by the ionicity or covalency and length of the chem-
ical bonds. IS is thus a probe of the formal oxidation state of the isotope under
investigation and of the crystal field around it (high- and low-spin Fe2+ may be
differentiated). The variation of IS with temperature can be used to determine the
Debye temperature of a compound (see Eq. (13)). 9 9 9 FIG. 3. Linear correlation between the experimental values of the 121Sb Mo¨ ssbauer isomer shift d
(relative to BaSnO3) and the calculated values of the valence electron density rvð0Þ at the nucleus for a
series of compounds. rsSð0Þ ¼ rvð0Þ þ rcð0Þ; where rcð0Þ is the core electron density, which is assumed
constant. Figure according to Lippens (7). FIG. 3. Linear correlation between the experimental values of the 121Sb Mo¨ ssbauer isomer shift d
(relative to BaSnO3) and the calculated values of the valence electron density rvð0Þ at the nucleus for a
series of compounds. rsSð0Þ ¼ rvð0Þ þ rcð0Þ; where rcð0Þ is the core electron density, which is assumed
constant. Figure according to Lippens (7). C. SPECTRAL ANALYSIS AND INTERPRETATIONS The quadrupolar splittings depend mainly on the electric field gradient at the
nucleus, which is caused by the spatial distribution of the electrons around it, via
electric Coulomb forces. Both electrons (valence contribution Uval) and neighboring
anions and cations (lattice contribution Ulat) in the vicinity of the nucleus contrib-
ute to this electric field gradient U, which can be expressed as follows: U ¼ Uvalð1 RÞ þ Ulatð1 gÞ,
ð18Þ ð18Þ where R and g are screening and anti-screening Sternheimer parameters. Values of
R range between 0 and 1, and values of g between 10 and 100. Because of these
two contributions, the quadrupolar splitting is strongly related to site symmetry and
local structure. For example, in the case of iron, if the site being characterized is a
Fe3+(4sx3d 5) ion, inner atomic shells are spherically symmetric, and qval is the 10 major component of the electric field gradient). In contrast, Fe2+(4sx3d 6) ions have
asymmetric distributions of their valence electrons, and qval has a dominant con-
tribution to the electric field gradient. The quadrupolar splitting characterizes the
degree of local asymmetry at the probe site. Such local asymmetry is caused by
various forms of structural, chemical, and magnetic disorder, such as lattice defects,
chemical substitutions of neighboring anions, local moment disorder in magneto-
volume and magneto-elastic materials that can be characterized and quantified in
solid catalysts. For example, the Mo¨ ssbauer spectrum of a BaFe3Al9O19 catalyst for combustion
of methane is given in Fig. 4 (8). This spectrum has been fitted with four sextets that
correspond to the four octahedral crystallographic sites of the structure that may be
occupied by the ferric ions, thus leading to a different atomic environment for each
of them. From the relative intensities of the four sextets used for the fit and con-
sidering equal f factors for the corresponding species, it is possible to determine the
respective occupation of these sites by iron. Alternatively, only a complete Rietveld
analysis of the powder X-ray diffraction pattern of the catalyst would have enabled
such a determination. The internal magnetic field H is a reliable signature of a given species and allows a
good identification of magnetic phases containing these species. C. SPECTRAL ANALYSIS AND INTERPRETATIONS Assuming that
particles for which trotL and tr>tL contribute exclusively to one of the two com-
ponents (paramagnetic and magnetic), the temperature at which tr ¼ tL and at
which the hyperfine split takes place can be determined; V is calculated from Eq. (19) at the temperature T at which the spectral areas of the two components are
equal). The dependence of the two spectral areas on temperature in the range in
which both components are observed yields the particle size distribution. One can often describe a solid catalyst as a dispersed set of active species on the
surface of a support, present as more or less isolated atoms or small particles. These
species, because of their small size and their bonding to the support, present specific
features different from those of bulk material. Many studies of supported catalysts
have shown that the f factor for small particles is influenced by the vibration of the
particles relative to the atomic environment. It may therefore be difficult to unravel
contributions of surface vibrations to the f factors of small particles. On the other
hand, when active species are weakly adsorbed at the surface of a support, they can
also vibrate or even diffuse on the surface, leading to a decrease of their f factors
and to a broadening of the lines in the Mo¨ ssbauer spectra that can result in the
disappearance of the signal. Furthermore, if they only vibrate, the mean-square
vibration amplitude perpendicular to the surface is not the same as that parallel to
the surface, leading to an anisotropy of the f factor. This effect (referred to as
Karyagin–Goldanskii effect) implies a dependence of f upon y (the angle between
Uzz and the direction of the incident g-radiation). In the case of a doublet, an
asymmetry of the intensity of the two lines is observed; this may complicate spectral
analysis. Another difficulty may arise from the deviation from a Lorenzian line shape of
the absorption signal. Such deviation corresponds to either homogeneous or in-
homogeneous broadening. Inhomogeneous broadening relative to the natural line
width is caused by variable environments of the probe nuclei that create variations
in the electronic or structural characteristics. C. SPECTRAL ANALYSIS AND INTERPRETATIONS In catalyst char-
acterization, one of the major interests in this last parameter relates to its variation
with temperature, which can be used to evaluate particles sizes and particle size
distributions when the particles are small enough. Indeed, when particles of a
magnetic phase are small, a collective reorientation of the magnetic moment is
observed in these particles. In a large particle, once a particular orientation has been
reached below the magnetic ordering temperature, it does not change, and an in-
ternal magnetic field is observed leading to a sextet in the Mo¨ ssbauer spectrum. When particles are small, the thermal excitation energy can be sufficiently high to
invert all of the spins simultaneously, thereby reversing the magnetization of the
lattice. If the relaxation time (tr) of these inversions is smaller than the nuclear
Larmor precession time (tL), the magnitude of the magnetic field at the nucleus
averages to zero, and no magnetic splitting is observed. The solid behaves as a
paramagnetic solid (superparamagnetic behavior), which results in the observation
of a doublet instead of a sextet in the Mo¨ ssbauer spectrum. x107
Velocity (mm/s)
Counts
1.36
1.34
1.32
1.30
-10
-5
0
5
10
FIG. 4. Mo¨ ssbauer spectrum of a BaFe3Al9O19 catalyst recorded at 298 K in air. Solid lines are derived
from least-square fits. Figure according to Naoufal et al. (8). FIG. 4. Mo¨ ssbauer spectrum of a BaFe3Al9O19 catalyst recorded at 298 K in air. Solid lines are derived
from least-square fits. Figure according to Naoufal et al. (8). 11 11 The relaxation time can be expressed in a simplified manner as follows: tr ¼ t0eKV=kT,
ð19Þ ð19Þ where K is the anisotropy constant and V the volume of the particle. It is clear that
tr increases with decreasing temperature and can become larger than tL. In such
cases, the spectrum changes, and a sextet is again observed. A simplified method was proposed by Ku¨ ndig et al. (9) allowing evaluation of the
particle size and the size distribution of a solid by analysis of its spectrum as a
function of temperature. By variation of the temperature it is possible to follow the
variation of the relative spectral areas of the sextet and doublet. C. SPECTRAL ANALYSIS AND INTERPRETATIONS This broadening can be considered to
be a consequence of the superposition of many sub-spectra and can relatively easily
be taken into account by fitting the experimental spectra considering a distribution
of sub-spectra with Lorenzian line shapes. Homogeneous broadening originates
from time variation in the electronic environment. Such effects, referred to as re-
laxation effects, imply the modification of the line width of the elemental spectrum. These broadenings may coexist and significantly complicate the analysis and in-
terpretation of Mo¨ ssbauer spectra. 12 D.1. Emission Mo¨ssbauer Spectroscopy (EMS) In Mo¨ ssbauer absorption spectroscopy, as described in the preceding section, an
emitting moving source with a single-line resonance is used to scan the energy levels
of an absorber that is the material under investigation. In contrast, in Mo¨ ssbauer
emission spectroscopy, the source corresponds to the material under investigation
and a reference single-line absorber has to be used. The studied emitting material
can be moved but most of the times it is the reference absorber that is moved
(Fig. 5). This technique has been used successfully to investigate CoMo/Al2O3
catalysts, which are well known for their use in hydrotreating processes. The tech-
nique has contributed to identify the active species of these catalysts (10,11). The catalysts to be investigated with this method should be synthesized with
radioactive 57Co; this constitutes the major experimental challenge of the technique. Another challenge may be encountered in the interpretation of the MES spectra. This interpretation may be hampered by the so-called chemical aftereffects. The
transition from 57Co to 57Fe occurs in the catalyst via electron capture, whereby the
electronic structure of the atom is changed. This change is followed by a reshuffling
of the electrons in the material that may take time to occur, especially if the material
is an insulator. If this required time is longer than the lifetime of the excited state,
the probed electronic structure and, thus, the energy levels may have changed. Therefore, EMS is generally applied as a fingerprint technique, with spectra being
compared to each other and to those of reference compounds. radioactive
sample
reference absorber
radioactive
sample
detector
detector
FIG. 5. Basic equipment for measuring an emission Mo¨ ssbauer spectrum. The sample is prepared with
a radioactive isotope and used as the source. FIG. 5. Basic equipment for measuring an emission Mo¨ ssbauer spectrum. The sample is prepared with
a radioactive isotope and used as the source. 13 13 The single-line absorber is generally potassium (or sodium) nitroprusside,
K4Fe(CN)6,3H2O, enriched with 57Fe. The single-line absorber is generally potassium (or sodium) nitroprusside,
K4Fe(CN)6,3H2O, enriched with 57Fe. Besides hydrotreating catalysts (12–20), supported cobalt Fischer–Tropsch
catalysts (21–25) and multi-phasic molybdate-containing catalysts for propene
oxidation (26) have also been characterized by this technique. The technique can
also be applied for investigations involving 107Cd, 133Cs, 195Pt, 99Rh, and 83Rb. D.2. Conversion Electron Mo¨ssbauer Spectroscopy (CEMS) A. IDENTIFICATION OF CATALYST COMPONENTS Mo¨ ssbauer spectroscopy has been quite successful in identifying catalyst com-
ponents. Mo¨ ssbauer spectroscopy provides quantitative site populations, easily
discriminating between various metal oxidation states and anion coordinations, and
it can lead to phase compositions or distributions between phases of the isotope
under investigation. It also gives quantitative population distributions of local dis-
tortion environments and local chemical environments, via extracted quadrupolar
splitting distributions. 57 The majority of investigations in this category has dealt with the isotopes 57Fe,
119Sn, and 57Co (in emission mode). In contrast, investigations focusing on other
isotopes, such as 197Au, 121Sb, 125Te, 193Ir, or 99Ru, have been limited in number but
very fruitful. D.2. Conversion Electron Mo¨ssbauer Spectroscopy (CEMS) It is possible to analyze the fluorescence radiation emitted from the decay of the
excited state in the solid instead of the absorption of the gamma X-rays. This can be
done by detecting and analyzing the conversion electrons arising from the internal
conversion process in which the de-excitation leads to the ejection of an inner-shell
electron instead of being used in the production of a g-radiation (Fig. 6). This
internal conversion process is much more frequent than X-ray emission. The initial
kinetic energy of an ejected conversion electron is equal to the energy of the tran-
sition minus its original atomic binding energy. Because emitted electrons lose
energy as they interact with the sample, their path length is limited to 0.1–0.2 mm,
and therefore CEMS is a surface-sensitive technique. It is possible to analyze the
energy distribution of the ejected electrons and to obtain spectra with various depth
resolutions (depth-selective CEMS is called DCEMS). Ultrahigh-vacuum and high-
resolution spectrometers allow analysis of surface layers with thicknesses as low as
1 nm with almost atomic-scale depth resolution. This technique is used only infrequently in catalysis research. For finely dispersed
catalysts, the size of the particles is smaller than the depth analyzed, and spectra are radioactive
source
e-
channeltron
high vaccuum
chamber
FIG. 6. Basic equipment for measuring a conversion electron Mo¨ ssbauer spectrum. FIG. 6. Basic equipment for measuring a conversion electron Mo¨ ssbauer spectrum. 14 consequently similar to those recorded in the conventional transmission mode. Furthermore, the high-resolution analysis of a fine-powder sample is sometimes
difficult to achieve, and the use of ultrahigh vacuum may lead to surface modi-
fication of the sample. Therefore, CEMS has found limited applications in the field
of catalysis. y
This technique has been reviewed recently, and applications to surface charac-
terization were described in Reference (27). It is noteworthy that a low-temperature apparatus has been described recently
that allows g-ray and conversion electron Mo¨ ssbauer spectroscopy measurements at
the same time, in the temperature range from 10 K to room temperature (28). III. Applications of Mo¨ ssbauer Spectroscopy in Heterogeneous
Catalysis In this section, we summarize results characterizing catalytic materials by
Mo¨ ssbauer spectroscopy. An analysis of the literature leads to the following clas-
sifications of the objectives of this work: 1. Identification of catalyst components in terms of active phase or active sites and
search for correlations between these components and one or more of the cat-
alytic properties. 1. Identification of catalyst components in terms of active phase or active sites and
search for correlations between these components and one or more of the cat-
alytic properties. 2. Investigations of catalysts under working conditions and analysis of activation
or deactivation processes that may take place with time on stream. 2. Investigations of catalysts under working conditions and analysis of activation
or deactivation processes that may take place with time on stream. 3. Study of chemisorption of reactants or molecular probes. Progresses and recent results obtained in each category are presented below. Our
intent is to give a broad view of the results generated recently, and this review is not
exhaustive. A.1. Iron-Containing Catalysts 57Fe Mo¨ ssbauer spectroscopy has been used extensively to characterize catalysts,
constituting the large majority of the Mo¨ ssbauer investigations of catalysts. The 15 publications dealing with the Mo¨ ssbauer spectroscopy of iron-containing catalysts
cover a broad range, and only a partial coverage of the work is included here. These
investigations focus mainly on well-known reactions for which iron-containing
catalysts are among the best. These reactions are the Fischer–Tropsch synthesis
(29–50), ethylbenzene dehydrogenation (51–58), ammonia synthesis (59–71), the
water gas shift (72–75), and oxidation reactions (76–90). In addition to these cat-
alysts for these reactions, several others have been the focus of Mo¨ ssbauer inves-
tigations, including those concerned with catalytic materials that have attracted
recent attention, such as iron supported on ZrO2 (91–104) or MgO, either directly
prepared from MgO (105–107) or from substituted hydrotalcite (108–111). Zeolites
and mesoporous materials (112–125), especially ZSM-5 (120–125) and MCM-41
(112–128) were also investigated. Furthermore, bimetallic alloys have also been
characterized by Mo¨ ssbauer spectroscopy (129–149) to determine the active phases
and active sites. A.2. Tin-Containing Catalysts The characterization of tin-containing catalysts by Mo¨ ssbauer spectroscopy has
been focused mainly on supported bimetallic alloys (150–162). These catalysts,
which consist of Pt, Pd, Rh, or Ru as primary metals with Sn used as a promoter,
are generally supported on SiO2 or Al2O3. These catalysts are used in reforming and
hydrogenation processes in the petroleum industry (151,152) and have recently
drawn attention for other applications, such as low-temperature CO oxidation,
alkane and alcohol dehydrogenation, metathesis reactions (153–160), and even ni-
trate removal for drinking water processing (161). The Mo¨ ssbauer transition in the isotope 119Sn is of the same type as that oc-
curring in 57Fe (1/2-3/2). Depending upon the presence or absence of an electric
field gradient at the nucleus, single lines or doublets are observed. Mo¨ ssbauer
spectroscopy appears to be efficient for identification and quantifications of various
compounds that are formed, depending upon the nature of the support, the metal
relative contents and the preparation conditions; these include Pt3Sn, PtSn, PtSn2,
or SnO and SnO2. Preparation of these catalysts by the contacting of silica-
supported rhodium with tetra-n-butyl-tin, led to relatively stable solids with
organometallic fragments bonded to the rhodium surface. These materials may
exhibit very high selectivities and activities for hydrogenation of a,b unsaturated
aldehydes or nitrobenzene (159). Mo¨ ssbauer spectroscopy has allowed identifica-
tion of all the species that were encountered when tetra-n-butyl-tin interacted with
the surfaces of silica or silica-supported rhodium (Fig. 7). At room temperature,
tetra-n-butyl-tin was physically adsorbed on the surfaces (characterized by a single
line d ¼ 1.42 mm/s). After reaction under H2 at 373 K, the formation of grafted
organometallic fragments on the rhodium surface (characterized by a doublet with
d ¼ 1.39 and D ¼ 2.26 mm/s) as well as a well-defined bimetallic RhSn compound
(characterized by a doublet d ¼ 1.42 and D ¼ 0.85 mm/s) were identified, whereas
with pure silica, surface-bound Si–O–Sn(n-C4H9)3 moieties were observed
(characterized by a doublet d ¼ 1.39 and D ¼ 2.69 mm/s). After treatment at
523 K, the rhodium-grafted organometallic species were completely decomposed, 16 x106
x106
6.165
1.70
(a)
(b)
(c)
(d)
1.68
1.66
1.64
6.160
6.155
6.150
6.145
velocity (mm/s)
velocity (mm/s)
velocity (mm/s)
velocity (mm/s)
x106
x106
5.72
5.73
9.94
9.95
9.93
9.92
5.71
5.70
5.69
5.68
FIG. 7. A.2. Tin-Containing Catalysts Experimental Mo¨ ssbauer spectra of samples recorded at 78 K: (a) Sn(n-C4H9)4 physisorbed on
SiO2, (b) SiOSn-(n-C4H9)3, (c) Rhs[Sn(n-C4H9)2]/SiO2, and (d) Rhs[Sn(n-C4H9)2]/SiO2 treated at 523 K. Solid lines are derived from least-square fits. Figure according to Millet et al. (158). x106
1.70
(b)
(d)
1.68
1.66
1.64
velocity (mm/s) x106
6.165
(a)
6.160
6.155
6.150
6.145
velocity (mm/s) (b) (a) (d) d)
y (
)
velocity (mm/s)
x106
9.94
9.95
9.93
9.92 (c)
y (
)
velocity (mm/s)
x106
5.72
5.73
5.71
5.70
5.69
5.68 velocity (mm/s) velocity (mm/s) FIG. 7. Experimental Mo¨ ssbauer spectra of samples recorded at 78 K: (a) Sn(n-C4H9)4 physisorbed on
SiO2, (b) SiOSn-(n-C4H9)3, (c) Rhs[Sn(n-C4H9)2]/SiO2, and (d) Rhs[Sn(n-C4H9)2]/SiO2 treated at 523 K. Solid lines are derived from least-square fits. Figure according to Millet et al. (158). and formation of a well-defined bimetallic RhSn compound was observed. Because
the isomer shift of Pt–Sn alloys of varying composition varies linearly with the Sn
mole fraction (160), it was possible to determine the composition of the alloy-like
structures and consequently the relative amount of the grafted species. This quan-
tification was confirmed by use of X-ray photoelectron spectroscopy. Mesoporous tin-containing analogues of MCM-41 and tin oxide-modified me-
soporous SBA-15 have also been characterized by Mo¨ ssbauer spectroscopy
(162,163). In the first case, the results suggest that tin was incorporated in the
structure of the silicate and, in the second, they indicated that two types of sup-
ported tin species were formed, depending on the tin content. One would corre-
spond to atomically isolated species stabilized in the wall of the pore and susceptible
to reduction to Sn2+ under reductive treatment conditions and the other to large
oxide clusters distributed in the external pore structure. A.3. Gold-Containing Catalysts Highly dispersed supported gold has been shown to have high catalytic activity
for various oxidation reactions 197Au spectroscopy has emerged as one of the 17 methods of choice for investigation of catalysts consisting of gold particles on oxide
supports, such as TiO2, Fe2O3, SiO2, Al2O3, MgO, and MnOx (164–170). The 197Au
resonance is characterized by Ig and Ie, respectively, equal to 3/2 and 1/2. Con-
sequently, the presence of an electric field gradient at the nucleus does not affect the
excited state, although the ground state is symmetrically split into two sub-states. This splitting leads to the presence of doublets in the spectra. Early investigators proposed the presence of both metallic and ionic gold species
(AuIII) in the active catalysts (165), but more recent investigations suggest the pres-
ence of only metallic species (166,167). Mo¨ ssbauer spectra obtained for gold cat-
alysts supported on TiO2 and SiO2 are shown in Fig. 8. The solid lines in the figure
are derived from least-squares fits and show both the total fits and the sub-spectra. The spectra are fitted using a single line (IS ¼ 1.22 mm/s), which is attributed to
bulk gold, and a doublet that has been attributed to surface metallic species
(1.5 mm/s oIS o0.75 mm/s and 1.25 mm/soQSo2.5 mm/s). Indeed, since the
earlier Mo¨ ssbauer investigations of gold compounds (168,169), it was observed that
isomer shifts (IS) and quadrupolar splittings (QS) vary over a wide range. This variation could have rendered difficult the attribution of oxidation states to
the detected species, but it was shown that a linear correlation between IS and
QS values exists for AuI and AuIII compounds (170). This relationship has been 100.0
Au/TiO2
5.2 nm
Au/TiO2 spent
propene epox
Au/TiO2/SiO2
3.2 nm
Au/SiO2
6 - 30 nm
99.5
99.0
100.0
100.0
99.5
99.0
98.5
99.5
99.0
100.0
99.5
-10 -8
-6
-4
-2
0
Velocity (mm/s)
Transmission (%)
2
4
6
8
10
FIG. 8. Mo¨ ssbauer spectra recorded at T ¼ 4.2 K of gold catalysts on various supports used for
propene epoxidation. The average size of the particles is given in the legend. Figure according to
Goossens et al. (165). FIG. 8. Mo¨ ssbauer spectra recorded at T ¼ 4.2 K of gold catalysts on various supports used for
propene epoxidation. The average size of the particles is given in the legend. Figure according to
Goossens et al. (165). A.3. Gold-Containing Catalysts 18 explained by the fact that for a given oxidation state the effective charge on the gold
atom does not vary as a result of p-back donation, and only transfers between the
d- and s-shells are observed, which affect both IS and QS values. This is possible
because the lattice contribution to QS is at least one order of magnitude smaller
than the contribution of the valence electrons (Eq. (18)). To determine whether the
observed doublet could be attributed to ionic species, the IS/QS combinations have
thus been considered, and it has been observed that they did not fit within ranges
corresponding to AuI and AuIII. Moreover, the isomer shifts always remained close
to that of the bulk species. It was thus proposed that they correspond to metallic
surface species, the presence of a QS being only related to the large electric field
gradient generated at the surface of the gold particles (166). The Au/TiO2 spectrum
(upper plot of Fig. 8) still showed a small contribution that might be attributed to
AuV that has not been explained but which disappeared after catalysis and was
never observed on any other support. A recent study on Au/Fe2O3 confirmed the
exclusive presence of metallic gold Au0 on the catalysts but only after heat treat-
ment at high temperature (4001 C) (170). At lower temperature, at which the
catalysts were more active, both AuIII (probably related to the presence of
AuOOH,H2O) and Au0 were observed. Although discrepancies remain in the lit-
erature on the existence and role of the gold species, Au0 and/or AuIII were most of
the time identified by Mo¨ ssbauer spectroscopy in the catalysts and may both play
a role; conversely AuI was never observed in the catalysts suggesting that its
involvement is very unlikely. A.4. Antimony-Containing Catalysts 121Sb Mo¨ ssbauer spectroscopy has been used to investigate the tin-antimony
oxide catalysts used for selective oxidation of hydrocarbons (171). Recent inves-
tigations have been conducted to characterize FeSbO catalysts for ammoxidation of
propene (172) and VSbO (173) and MoVSbNbO (174) catalysts for ammoxidation
of propane. p
p
The Mo¨ ssbauer transition for the 121Sb isotope is of the type 5/2-7/2. Broad,
asymmetric lines indicating unresolved quadrupolar split spectra are observed for the
oxide compounds (Fig. 9). However, the relative variation of the nuclear radius
D r2
is rather large, and therefore the isomer shift for this system becomes a
sensitive parameter not only for changes in oxidation state but also for changes in the
local environment. Isomer shifts allow the identification and characterization of
various ionic species. In the cases of VSbO and FeSbO, the investigations evidenced
the presence of b-Sb2O4 formed during the synthesis or by segregation under catalytic
condition. In the case of MoVSbNbO, it allowed a determination of the relative SbIII/
SbV contents of the two main phases of the complex catalysts. The results obtained in
the latter case were consistent with X-ray absorption near-edge spectra (174). A.5. Catalysts Containing other Elements Other characterization of catalysts have dealt with isotopes such as 125Te, 193Ir,
99Ru and 51Eu. The tellurium-containing catalysts were MoVTeNbO ammoxidation
catalysts (175) and Te-doped Cr2O3 hydration–dehydration catalysts (176) and the 19 19 Iz = 7/2
Iz = 5/2
±7/2
m
Electrical quadrupole interaction
±5/2
±3/2
±1/2
±5/2
±3/2
±1/2
FIG. 9. Schematic representation of the electrical quadrupolar splitting in the case of a transition 5/
2-7/2 like that of 121Sb and corresponding the experimental spectra of FeSbO catalysts (173). Electrical quadrupole interaction FIG. 9. Schematic representation of the electrical quadrupolar splitting in the case of a transition 5/
2-7/2 like that of 121Sb and corresponding the experimental spectra of FeSbO catalysts (173). iridium-containing catalysts, iridium/iron bimetallic methanol synthesis catalysts
(177); 99Ru and 51Eu spectroscopies were applied for the characterization of carbon-
supported ruthenium–tin catalysts (used for the reduction of a,b-unsaturated
aldehydes) (178) and the study of the hydrogen reduction of supported europium
catalysts (179). These investigations are scarce, and all the published data have been
cited here to illustrate the breadth of the applications of Mo¨ ssbauer spectroscopy. B. INVESTIGATIONS OF CATALYSTS IN REACTIVE ATMOSPHERES AND UNDER WORKING
CONDITIONS Characterization of catalysts in reactive atmospheres and during catalysis has led
to insights that have not emerged from other characterizations. The introduction of
EMS (57Co) to investigate Co–Mo/Al2O3 hydrodesulfurization catalysts in reactive
atmospheres was a key technological advance in the 1980s; it provided the first
detailed insight into the nature of the promoter atoms in the active sulfided state of
the catalysts and showed that the catalytic activity was related to the presence of the
Co–Mo–S edge structures (10,11). Since that time, Mo¨ ssbauer spectroscopy has been used widely to characterize
catalysts in reactive atmospheres, leading to continuous progress in the under-
standing of structure/catalytic property relationships (180–194). 20 20 Methodologies for investigation of solid catalysts under real working condi-
tions with simultaneous measurements of catalytic activity data have advanced
markedly in recent years, and Mo¨ ssbauer spectroscopy has played an important
role. Only isotopes that can be investigated at room temperature or higher
temperatures (57Fe,57Co, 119Sn, and 151Eu) have been used in this context; they
are powerful because they allow the characterization of the local atomic environ-
ment of the working catalytic species, giving rise to unique fingerprints of the
catalytic sites. It is important in this section to differentiate between the catalyst characteriza-
tions with Mo¨ ssbauer spectroscopy under real working conditions and those per-
formed under controlled environments after quenching of the reaction. Only
recently (in the preceding 12 years) has this distinction been made with some
clarity; the unclear term ‘‘in situ’’ has been used often in the literature for both types
of investigations; the reader should be aware of this issue and carefully review the
experimental conditions when the term ‘‘in-situ’’ is used. B.1. Investigations of Functioning Catalysts Investigations of functioning catalysts with Mo¨ ssbauer spectroscopy have been
performed for a wide range of samples and applications. The reactions include
hydrodesulfuration (15), the Fischer–Tropsch reaction (20,180), selective oxidation
or oxidative dehydrogenation (181–186), and acetonitrile synthesis (187). We have seen in the first part of this article that the time required for obtaining a
Mo¨ ssbauer spectrum is rather long. The application of the technique to follow time-
dependent changes has been limited, and in most cases it is catalysts operating
under steady-state conditions that have been characterized. A great advantage of
Mo¨ ssbauer spectroscopy is that the measurements can be done under extreme
working conditions, at both, high pressures and high temperatures. This essential
point, which sets Mo¨ ssbauer spectroscopy apart from numerous other methods for
characterization of catalysts, is illustrated by the example of an industrial cobalt-
containing Fischer–Tropsch catalyst investigated by
57Co Mo¨ ssbauer emission
spectroscopy (24). A cell has been constructed that allows investigation of catalysts
at temperatures up to 723 K and pressures up to 20 bar with injection of steam at
10 bar, permitting the production of wax and continuous catalyst test runs lasting
up to 2 months (Fig. 10). The stainless steel cell is composed of two compartments. The inner one which corresponds to the high temperature and high pressure reactor
and the outer one which is there to safely confine the radioactive 57Co in case the
inner one fails. The thickness of the beryllium windows of the two compartments
have been optimized to support high pressure without absorbing too much g-rays. The catalyst is placed onto a sintered metal filter with pore size diameter of 5 mm
that allows the reactive gas to flow from top to bottom through the catalyst powder. This filter also allows draining of the produced wax downwards in a specially
designed reservoir. It is stressed that only few physical chemical techniques can be
used under such drastic conditions. To investigate the deactivation of these catalysts, a 57Co-containing catalyst was
prepared by adding ca. 1000 ppm of 57Co to a cobalt and platinum supported on 21 FIG. 10. Photograph and schematic representation of high-pressure Mo¨ ssbauer emission spectroscopy
cell (24). FIG. 10. Photograph and schematic representation of high-pressure Mo¨ ssbauer emission spectroscopy
cell (24). alumina catalyst. B.1. Investigations of Functioning Catalysts This catalyst, the initial composition of which was 15Co/0.025Pt/
100Al2O3, was then reduced in hydrogen at 653 K for 16 h (20). The Mo¨ ssbauer
emission spectrum showed about 70% reduction of cobalt. The catalyst was sub-
sequently treated at 423 K in the cell in a hydrogen–water gas mixture with H2/
H2O ¼ 1 (molar) at 1, 5, and 10 bar, corresponding to a water partial pressures of
0.5, 2.5, and 5 bar, respectively. The Mo¨ ssbauer emission spectra showed that the
amount of metallic cobalt increased from 70% at 1 bar to 86% at 10 bar total
pressure. These results show that the catalysts resisted oxidation under the con-
ditions used and became more and more reduced when the total pressure (and thus
the water partial pressure) increased. The investigation also showed that water was
not responsible for the deactivation of the cobalt-containing Fischer–Tropsch cat-
alysts. It was inferred that the deactivation should better be described as a surface
poisoning or a carbon deposition, as had been proposed (188,189). alumina catalyst. This catalyst, the initial composition of which was 15Co/0.025Pt/
100Al2O3, was then reduced in hydrogen at 653 K for 16 h (20). The Mo¨ ssbauer
emission spectrum showed about 70% reduction of cobalt. The catalyst was sub-
sequently treated at 423 K in the cell in a hydrogen–water gas mixture with H2/
H2O ¼ 1 (molar) at 1, 5, and 10 bar, corresponding to a water partial pressures of
0.5, 2.5, and 5 bar, respectively. The Mo¨ ssbauer emission spectra showed that the
amount of metallic cobalt increased from 70% at 1 bar to 86% at 10 bar total
pressure. These results show that the catalysts resisted oxidation under the con-
ditions used and became more and more reduced when the total pressure (and thus
the water partial pressure) increased. The investigation also showed that water was
not responsible for the deactivation of the cobalt-containing Fischer–Tropsch cat-
alysts. It was inferred that the deactivation should better be described as a surface
poisoning or a carbon deposition, as had been proposed (188,189). With the same type of cell, it has recently been possible to investigate
the sulfidation of calcined Co–Mo/Al2O3 hydrodesulfurization catalysts under 22 realistic hydrotreatment conditions, with an activation of the catalysts at 673 K and
40 bar (20). B.1. Investigations of Functioning Catalysts In another example, a complex multi-component BiMoCoFeO catalyst used in the
partial oxidation of propene to acrolein was characterized by Mo¨ ssbauer spectros-
copy. This example has been chosen because it provides a good demonstration of the
high efficiency of Mo¨ ssbauer spectroscopy for the characterization of working
catalysts (181,182). y
(
)
In this investigation, Co1xFexMoO4 as pure solid solutions and in mixtures with
Bi2(MoO4)3 were characterized at 688 K under the conditions of propene oxidation
to acrolein (C3H6:O2:N2 ¼ 1:1:5.6 (molar ratios) at atmospheric pressure). The
mechanical mixture of the mixed iron and cobalt molybdate with the bismuth
molybdate has been shown to be a good model for the industrial catalysts, since
both selectivity to acrolein and propene conversion larger than 95% could be
simultaneously achieved on such catalyst (190). The data showed that under
catalytic reaction conditions, no reaction was taking place between the two
solid phases, which remained stable except when iron loadings were high and
Fe2(MoO4)3 was formed. A comparison of the data obtained with the pure solid
solution and the mixtures of phases showed that cobalt stabilized Fe2+ sites in
the solid solution while Bi2(MoO4)3 stabilized Fe3+ sites in the solid solution. The
presence of both species was proposed to be a key to the mild oxidation of propene
by creating a high electrical conductivity, which favors the electron exchange in
the Mars–van Krevelen mechanism. Furthermore, because iron sites have different
environments in a and b polymorphic forms of Co1xFexMoO4, it was possible
to determine the relative ratio of the two forms under the conditions of the catalytic
test. It was confirmed that the b-type solid solution was more efficient than the
a-type. Because the former was preferentially formed at high iron contents, it has
been suggested that an optimal content of iron was present, which was sufficiently
high to stabilize the b-phase but not too high, preventing the formation of ferric
molybdate. C. INVESTIGATION OF CHEMISORPTION OF REACTANTS AND MOLECULAR PROBES Heterogeneous catalysis results from interactions between molecules in a gas or
liquid phase and active sites at the surface of a solid. Mo¨ ssbauer spectroscopy,
which is an elemental probe technique, may be used to investigate adsorption sites
containing appropriate isotopes and thereby gain information about adsorption
and reaction processes. Several examples of chemisorption investigations with Mo¨ ssbauer spectroscopy
appeared in the literature of the 1980s (2). In contrast, only very few investigations
were reported in recent years. This lack may be explained on the one hand by the
development of other techniques such as infrared spectroscopy, which are better
adapted, more reliable, and easier to perform than Mo¨ ssbauer spectroscopy, and on
the other by the difficulties of interpretation of Mo¨ ssbauer spectra. These difficulties have several origins. For example, f factors of surface species,
especially in their component perpendicular to the surface, will change extensively
as a result of the adsorption or desorption of molecules. In the case of carbon-
supported Pt–Fe catalysts, the doublet corresponding to iron atoms at the surface
of the alloy particles was detected only when hydrogen was adsorbed (195). In the
case of small particles dispersed on a support, the discrimination of resonant species
at the surface of the particles from bulk species is possible only if the former are
sufficiently numerous compared to the latter. This requirement implies a particle
size that does not exceed about 20 nm. Furthermore, these surface species are not
always easily differentiated from bulk species. The major parameter that allows
such differentiation is the quadrupolar splitting which may strongly vary because of
the larger electric field gradient related to the structural discontinuity at the surface. In the case of magnetic compounds, the hyperfine field at the surface has to be
different from that in the bulk. Though agreement seems to have been reached on
this point, it remains questionable in most cases whether the change may be de-
tectable. Furthermore, it is often difficult to separate surface effects from size effects
(such as superparamagnetism or collective magnetic excitations). Chemisorbed
molecules have been shown to affect the total magnetic anisotropy constant of small
particles and thus change their superparamagnetic relaxation times. Partial recon-
struction of the surface may also occur and complicate the data analysis even more. Among the few investigations reported on chemisorption of molecules, two are
cited. B.2. Investigations of Quenched Catalysts It is not always possible to record Mo¨ ssbauer spectra under catalytic reaction
conditions, and characterization with this technique sometimes requires that spectra
be measured at room temperature or at cryogenic temperatures. The latter limi-
tation pertains to investigations involving isotopes that, by their very nature, re-
quire low temperature and to iron when phases are superparamagnetic at room
temperature or higher temperatures. Most of the work reported in this category
concerns 57Fe; however, some investigations have been reported that deal with the
characterization of PtSn particles (with the isotope 119Sn) used in the selective
hydrogenation of crotonaldehyde (which occurs at 353 K), the oxidation of CO
(which occurs at room temperature) or the dechlorination of 1,2-dichloroethane
(which occurs at 473 K) (191–193). In these investigations the spectra were recorded
at 77 K under the reactive atmosphere after rapid quenching. It is noteworthy that Mo¨ ssbauer spectroscopy applies not only to solid–gas
reactions but also to solid–liquid reactions. It has been used, for example, to 23 23 characterize (at 77 K) active site heterogeneity in pyrolyzed carbon-supported iron
porphyrin catalysts for the electrochemical reduction of oxygen (194). characterize (at 77 K) active site heterogeneity in pyrolyzed carbon-supported iron
porphyrin catalysts for the electrochemical reduction of oxygen (194). C. INVESTIGATION OF CHEMISORPTION OF REACTANTS AND MOLECULAR PROBES Both concern N2 chemisorption on FeZSM-5 (196) and on small aFe particles
supported on carbon (197). In the first case, the affinity of nitrogen for certain
ferrous centers after adsorption at 623 K was clearly evidenced. In the second case
nitrogen was shown to react with surface iron atoms only at temperatures up to
700 K, resulting in a number of different types of environments for the latter
depending upon the number of nitrogen atoms in their proximity. The other such
investigations that have been reported deal with the adsorption of gases such as H2,
CO, and SO2 (198,199). 24 There are reports of the use of iron species as probes (chemisorbed species) to
characterize supports and their adsorption properties. Burger et al. (200) used
Mo¨ ssbauer spectroscopy to characterize submicroscopic droplets of Sn(IV) and
Fe(III) complexes carried in an alkane/naphthalene mixture. The analysis of the
Mo¨ ssbauer parameters gave a qualitative picture regarding the solution structure
inside the pores and the adsorption and wetting properties of the solid. Pol’shina et al. (201) investigated the introduction of heteroatoms such as O or N
into active carbons by measuring the adsorption of ferric ions. Analysis by
Mo¨ ssbauer spectroscopy allowed them to distinguish and quantify ferric ions that
form surface complexes with O-containing groups (carboxylate complexes). A.1. Mo¨ssbauer Spectroscopy of Catalysts under Working Conditions The preceding several years have witnessed an evolution in Mo¨ ssbauer investi-
gations in catalysis toward investigations of catalysts under working conditions,
consistent with a general trend in spectroscopic characterization of catalysts. The
trend in spectroscopy of catalysts is toward extreme conditions of temperature and
pressure. Mo¨ ssbauer spectroscopy is well suited to such conditions. The development is still at an early stage, and so far there has been no report
illustrating the coupling of Mo¨ ssbauer spectroscopy of a working catalyst with
another technique, except for on-line gas chromatography for product analysis. There is a great interest in simultaneous catalyst characterization with comple-
mentary spectroscopic methods applied in ways that ensure that the data are all
internally consistent. Mo¨ ssbauer spectroscopy is expected to play a role in such
combined investigations of catalysts under working conditions (202). A.2. Mo¨ssbauer Spectroscopy of Catalysts below 4.2K With the development of nanoscaled catalysts, several recent studies have pointed
out the great interest of recording Mo¨ ssbauer spectra below 4.2 K up to 0.055 K
(100,203,204); it allowed the identification at the surface of various support such as
mesoporous silica or ZrO2, the formation of nanometric iron oxide clusters; this
identification was not possible in classical low temperature studies conducted above
4.2 K, which concluded to the presence of larger particles. The analysis by
Mo¨ ssbauer spectroscopy performed at lower temperature enabled to show that
these larger particles were agglomerates of nanometric iron clusters and allowed to
reach another level of resolution of ferric particles structures (204). Technological Advances and Theoretical Evaluation of Mo¨ssbauer Parameters In the preceding decade there have been no drastic changes in the design of
Mo¨ ssbauer spectrometers. However, two new innovations have recently been 25 25 proposed that both reduce the acquisition time and improve the precision of the
measurements (205,206). One of the problems of the conventional method of Mo¨ ssbauer spectroscopy is
the occurrence of pulse overlapping at high-count rates. The single channel analyzer
(SCA) that is generally used to register pulses of the signal almost always records
interfering noise pulses. Such pulse overlapping disturbs the amplitude spectrum,
reduces the signal-to-noise ratio, and limits the maximum count rate of the data
acquisition system, thereby increasing the duration of an experiment. To solve this problem, new instrumentation has been proposed that includes a fast
analog-to-digital converter (ADC) together with a modern digital signal processor,
which significantly increases the count rate as a consequence of the elimination of
pulse overlapping (205). The proposed instrumentation allows calculation of the
exact amplitude of each pulse, which may be combined with the current velocity
discrimination, therefore leading to the recording of a two-dimensional Mo¨ ssbauer
spectrum (Fig. 11). The application of such two-dimensional data acquisition sys-
tems allows one to choose the optimal energy window in the amplitude spectrum
after the experiment and to measure g-quanta absorption and X-ray emission spectra
simultaneously in the same transmission experiment. The new instruments also re-
duce data acquisition time, because amplitude and Mo¨ ssbauer spectra are collected
simultaneously. Even more interesting is a new spectrometer that has been built with a novel
moving system that synchronizes the movement of the radioactive source with
that of the detector (206). This system allows the accumulation of Mo¨ ssbauer
spectra either in constant acceleration or constant velocity modes, therefore
giving better signal-to-noise ratios and also reducing the time necessary for
accumulation of a spectrum. Narrower line widths were also obtained with this
equipment design. 80000
70000
60000
50000
40000
Counts
Energy, Kev
Velocity, mm/s
30000
20000
10000
0 3
7
11
15
19 -9 -7-5 -3 -11 3 5 7 9
FIG. 11. Two-dimensional Mo¨ ssbauer spectrum of an iron foil, measured with a proportional counter. Figure according to Maltsev et al. (205). 80000
70000
60000
50000
40000
Counts
Energy, Kev
Velocity, mm/s
30000
20000
10000
0 3
7
11
15
19 -9 -7-5 -3 -11 3 5 7 9 FIG. 11. A.4. Mo¨ssbauer Spectroscopy and High-Throughput Catalyst Characterizatio The implementation of combinatorial chemistry and automated methods for
rapid synthesis, testing, and characterization of catalysts, has opened a wide range
of new opportunities in catalysis. However, so far, Mo¨ ssbauer spectroscopy has not
been introduced into this methodology. Two hurdles must be overcome for
Mo¨ ssbauer spectroscopy to become important in high-throughput catalyst char-
acterization: the system for recording spectra must be scaled down, and the data
acquisition and exploitation systems must be adapted. The first condition could easily be fulfilled if progress made in spatial resolution
were exploited. For example, an instrument has been developed that allows the
measurement of Mo¨ ssbauer spectra of metallurgical slags with a spatial resolution
of approximately 500 mm (209,210). By use of a 57Co source with high-specific
activity, the g-ray beam can be collimated from the conventional diameter of ap-
proximately 1 cm to a diameter of 500 mm. The sample is mounted in an epoxy resin,
ground to a thin disk, and then mounted on an x–y stage directly behind a lead
shield with a 500 mm hole. This technology should be easily applied to a sample set
such as those generated with synthesis robots. With regard to the second condition, advances in Mo¨ ssbauer data analysis have
progressed so far that it is currently possible to handle the fitting processes of a
Mo¨ ssbauer spectrum in a completely automated manner by using genetic algo-
rithms and fuzzy logic; both software and hardware have been developed (211,212). Nevertheless, it is not trivial in many cases to relate the extracted hyperfine pa-
rameters to the underlying structure and properties of the material without a sig-
nificant amount of experience, and many pitfalls have been documented (213). The development of such integrated approaches to spectral analysis will benefit
greatly from the large data bases and reference banks developed with the
Mo¨ ssbauer Effect Data Indexes covering 1958–1976 and the Mo¨ssbauer Effect
Reference and Data Journal database (MERDJ: Vols. 1–19) covering 1976 to the
present (http://www.unca.edu/medc/Journal.html). Technological Advances and Theoretical Evaluation of Mo¨ssbauer Parameters Two-dimensional Mo¨ ssbauer spectrum of an iron foil, measured with a proportional counter. Figure according to Maltsev et al. (205). 26 Furthermore, the theoretical evaluation of the Mo¨ ssbauer hyperfine parameters
(isomer shift (IS), quadrupolar splitting (QS), and magnetic hyperfine field (H)) is
now possible with first-principles electronic structure calculations based on density
functional theory (DFT) (207,208). The quality of the data depends on the size of
the atomic cluster chosen to represent the solid and the charge densities around the
nucleus under investigation. A detailed understanding of the relationships between
the electrical field gradient or magnetic field and their various causes can come only
from comparisons with sufficiently complete electronic structure calculations. Such
calculations for realistic local and longer-range environments are rare and are very
much needed, especially for the characterization of surface species. A.5. Mo¨ssbauer Spectroscopy for Characterization of Membranes and Monoliths Another advantage of Mo¨ ssbauer spectroscopy that should benefit future re-
search is that it can be used in a non-destructive way. This advantage should allow
the investigation of membranes and monoliths without them being damaged, even 27 27 sample
detector
radioactive
source
FIG. 12. Basic equipment for measuring a Mo¨ ssbauer spectrum in diffusion mode. radioactive
source FIG. 12. Basic equipment for measuring a Mo¨ ssbauer spectrum in diffusion mode. under catalytic reaction conditions. This application can be achieved by experi-
ments in the scattering or diffusion mode (Fig. 12). Furthermore, local micro
heterogeneities in a monolithic or membrane sample may be characterized by use of
an imaging Mo¨ ssbauer spectroscopic device system (IMS), which can record 256
simultaneous Mo¨ ssbauer spectra along a sample. The experimental system, which
has been applied for the characterization of metallic ribbons, is based on a linear
position-sensitive proportional counter that facilitates measurements with a spatial
resolution on a length of about 50 mm (214). Other improvements in spatial resolution in the future should allow more
accurate investigation of conventional powdered samples such as catalysts. This will
likely be made possible as a result of developments in synchrotron investigations
of nuclear forward scattering (presented in the next section) (215). The development
of a Mo¨ ssbauer electron microscope that would focus conversion electrons using
conventional electron optics has also been mentioned (216). B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation Synchrotrons produce photons with energies in the range of nuclear Mo¨ ssbauer
transitions and can, in principle, be used to excite these transitions. However, syn-
chrotron radiation can be monochromatized to only about 1 meV with new mon-
ochromators. Because the accessible nuclear levels are extremely narrow (between
109 and 108 eV), it is only about 103 of the incident photons that can excite the
nuclear levels (excitation cross-section could be as much as 103 G0). This is far
weaker than radiation that is non-resonantly scattered by the electronic processes in
the solid arising from the scattering of the entire 1 meV width of the incident
radiation. This limitation has been overcome by use of the property of synchrotron radi-
ation that can be emitted from electron clouds (bunches) circulating in the storage
rings. Bunches are a few centimeters in length, corresponding to
100 ps, and the
time between two successive pulses is 2.8 ns to 2.8 ms (at the European synchrotron
radiation facility). When a material absorbs X-rays, resonant and non-resonant
scattering occurs. However, because of the lifetime of the nuclear excited state, the
resonant scattering is delayed with respect to the non-resonant scattering (the nat-
ural lifetime of the first nuclear excited state for 57Fe is 141 ns). By suppressing data
acquisition during the short non-resonant pulses, it is possible to measure only the
nuclear forward scattered intensity. A typical NFS experiment may be described as follows (218): the pulsed syn-
chrotron radiation impinges on the sample as bunches, as they are produced in the
storage ring (Fig. 13a). The typical pulse duration is
100 ps, and the period of
pulse repetition is of the order of 200 ns, corresponding to the electron bunch
distance. A fast detector records the transmitted and forward scattered radiation,
and the time evolution of this intensity is used to discriminate the resonant from the
non-resonant intensity (Fig. 13b). The black spikes corresponding to the exciting
pulses, which define the ‘‘time zero’’ of the nuclear excitation are suppressed by a
gating acquisition system in the course of the experiment. The signal between these
spikes represents the nuclear forward scattered intensity that is recorded and which
is delayed with respect to the exciting pulse as a consequence of the lifetime of the
nuclear excited states. B. FUTURE DEVELOPMENTS AND APPLICATIONS The main future developments of Mo¨ ssbauer spectroscopy will undoubtedly be
based on the use of synchrotron radiation as a source for Mo¨ ssbauer experiments,
taking advantage of its high flux, brilliance, pulse structure, and energy tunability. In the field of catalysis, new experimental opportunities include the use of parentless
Mo¨ ssbauer isotopes and time resolution. Catalyst characterizations should be pos-
sible over a wide range of time scales, including investigations of activation and
deactivation under working conditions on time scales of tens to hundreds of sec-
onds, investigations of transport processes such as diffusion on time scales from
micro- to picoseconds, and investigations of processes such as relaxation and elec-
tron transfers on the intrinsic lifetime scale of the Mo¨ ssbauer nuclei. All these techniques are referred to as nuclear resonance scattering (NRS) of
synchrotron radiation; they include a wide spectrum of experimental techniques,
such as nuclear forward scattering (NFS), nuclear inelastic scattering (NIS), nuclear 28 28 Bragg diffraction (NBD), nuclear small-angle scattering (NSAS), nuclear reflect-
ometry (NR), nuclear quasi-elastic scattering (NQES), and quasi-elastic and
inelastic scattering with nuclear resonance energy analysis (217). In this review, we
focus only on NFS and NIS, which are the two main techniques, and we attempt to
describe the advantages of using them for the characterization of solid catalysts. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation Figure 14 shows three 57Fe case studies of the time behavior of the photons re-
emitted in the forward direction and a comparison with the typical spectra obtained
in Mo¨ ssbauer spectroscopy. Figure 14a corresponds to the case for which there is
no hyperfine interaction. The nuclear levels are not split, and only one transition
between ground and excited state is possible. In that case, the Mo¨ ssbauer spectrum
shows a single-absorption line and contains only g-quanta of equal energy. In the
presence of an electric field gradient (Fig. 14b), the splitting of the excited state is 29 29 (a)
(b)
200 ns
sample
detector
200 ns
FIG. 13. Basic equipment for an NFS experiment (a) and time structure of the detected radiation (b). (a) detector sample (b) FIG. 13. Basic equipment for an NFS experiment (a) and time structure of the detected radiation (b). 13. Basic equipment for an NFS experiment (a) and time structure of the detected radiation (b). observed, and the Mo¨ ssbauer spectrum has two absorption lines (a doublet). Accordingly, the NFS spectrum shows characteristic modulations caused by the
interference of the two forward re-emitted radiations containing g-quanta with two
different energies (quantum beats). The equal probability of the two transitions is
directly evidenced by the symmetry of the doublet and is evaluated on the basis of
the contrast between the maximum and the minimum of the beats. This contrast
vanishes with increasing inequality of the two probabilities. When a magnetic hyperfine field is present (Fig. 14c), both ground and excited
states split into sub-levels, which leads to the observation of six absorption lines
corresponding to the six transitions allowed by selection rules (sextet) in the
Mo¨ ssbauer spectrum. In the NFS spectrum, these transitions give rise to interfer-
ences and the superposition of quantum beats of different frequencies. The latter
superposition leads to complex patterns that are difficult to interpret. In that case,
the intrinsic polarization of the incident synchrotron radiation (the radiation is
linearly polarized in the plane of the synchrotron ring) may advantageously be used
to reduce the number of free parameters (i.e., to reduce the number of hyperfine
radiation components and consequently reduce the number of quantum beats). The
polarization of the synchrotron radiation mentioned above is an important pro-
perty of the source, which is different from the source used for conventional
Mo¨ ssbauer experiments, because radioactive sources emit non-polarized radiation. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation The mechanism producing synchrotron radiation guarantees almost complete linear 30 velocity (mm/s)
time (ns)
FIG. 14. Examples of experimental NFS time patterns with corresponding schematic conventional
Mo¨ ssbauer spectra: (a) stainless steel (221), (b) iron porphyrin complex [Fe(CH3COO)(TPpivP] (spec-
trum taken at 30 K in a 4 T field) (222), and (c) a-Fe (221). FIG. 14. Examples of experimental NFS time patterns with corresponding schematic conventional
Mo¨ ssbauer spectra: (a) stainless steel (221), (b) iron porphyrin complex [Fe(CH3COO)(TPpivP] (spec-
trum taken at 30 K in a 4 T field) (222), and (c) a-Fe (221). polarization. Furthermore, optical elements can convert synchrotron radiation into
circularly polarized radiation with high efficiency. When experimental NFS spectra of samples with finite thickness are recorded, the
time decay also shows modulations attributed to coherent multiple scattering oc-
curring in solids (dynamical beats). These superimposed modulations have different
(non-periodic) time modulation and are taken into account when the spectra are
fitted. At this point, if we consider the Mo¨ ssbauer parameters calculated in conventional
Mo¨ ssbauer spectroscopy, the isomer shift (IS) is not accessible from NFS spectra,
because the latter only shows interference between radiation fields corresponding to
different radiations. Isomer shifts will have to be measured relative to a second
reference absorber that must be placed behind or in front of the sample along the
g-ray beam. When the nuclei are subjected to an electrical quadrupole interaction, the NFS
pattern shows quantum beats with a single frequency corresponding to the energy
difference between the sub-levels of the exited state (DEQ), which is equal to the 31 quadrupolar splitting (D). The time dependence of the intensity of the delayed
radiation can be expressed approximately as follows: quadrupolar splitting (D). The time dependence of the intensity of the delayed
radiation can be expressed approximately as follows: IðtÞ exp
t
t
cos2 DEQt
2_
,
ð20Þ ð20Þ where t is the natural lifetime of the nuclear transition (t ¼ 141 ns for 57Fe). This
expression contains two time-dependent terms. The first, exp ðt=tÞ; corresponds to
the nuclear decay and the second, cos2ðDEQt=2_Þ; to the quantum beat pattern;
DEQ, which is equal to h2p/F (F is the period of the quantum beats), can be
estimated using the following approximate relationship: DEQ½mm=sF½ns 87. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation ð21Þ ð21Þ For magnetic compounds, the determination of the internal magnetic field re-
quires the decomposition of the complex spectra of multi-quantum beats. Computer
codes now allow one to attempt to perform such decomposition and to interpret the
spectra. The determination of the complex quantum beat structure requires spectra
recorded over long periods for high accuracy. In spite of that, analysis of complex
spectra remains very often difficult and NFS does not compete against conventional
Mo¨ ssbauer spectroscopy. However, it offers a major advantage over the latter
technique by being a time-resolved technique, with accumulation times in the range
of minutes or less. This advantage is gained without loss of the advantages of the
Mo¨ ssbauer technique, such as site selectivity, quantitative analysis, and applica-
bility to catalysts under extreme working conditions (high pressure and high tem-
perature). The NFS technique benefits from the outstanding properties of
synchrotron radiation, such as the high collimation of the photon beam that pre-
vents Mo¨ ssbauer line asymmetry resulting from the finite dimensions of the source. Furthermore, it can easily be combined with several complementary spectroscopic
techniques. As in conventional Mo¨ ssbauer spectroscopy, 57Fe is the most widely used isotope
in nuclear resonant scattering of synchrotron radiation. Experiments with other
isotopes, such as 169Tm, 119Sn, 83Kr, 181Ta, 151Eu, 161Dy, and 149Sm have been
reviewed recently (219). Other isotopes with very short half-life time sources should
also be usable. The applicability of such isotopes represents another strong advan-
tage that can be exploited in the future for catalyst characterization. The inves-
tigation of isotopes such as 197Au (Eg ¼ 77.34 keV; source, 197Pt, half-life, 18 h);
61Ni (Eg ¼ 67.40 keV; source, 61Co, half-life, 99 min); 186W (Eg ¼ 122.5 keV; source,
186Re, half-life, 90 h);
99Ru (Eg ¼ 89.36 keV; source,
99Rh, half-life, 16 days);
or 125Te (Eg ¼ 35.46 keV; source, 125I, half-life, 60 days) may be expected. g
As was stated in Section II.A, the energy resolution of the radioactive sources
used in conventional Mo¨ ssbauer spectroscopy is typically 109 eV. This resolution
is determined by the natural line width and the maximum energy range obtained by
Doppler-shifting techniques. In the case of synchrotron radiation, the energy res-
olution, which is related to the time period following the excitation of the isotope, is
superior to that in conventional Mo¨ ssbauer spectroscopy. B.1. Nuclear Forward Scattering (NFS) of Synchrotron Radiation This period can be as
short as 2.8 ms, which leads to an energy resolution of about 1010 eV. However, the 32 32 recordable period is determined by the bunch separation, which depends on the
operationing mode of the synchrotron. The energy resolution, which depends on
this period, will be superior to that of the radioactive source only if the bunch
separation is larger than the lifetime of the excited nuclear state. The reported
examples of experimental characterization of solids mainly concern biological
inorganic solids (220) or geological samples (221). In the latter case, the measure-
ments were made at pressures up to 1.5 Mbar. B.2. Nuclear Inelastic Scattering (NIS) of Synchrotron Radiation The one phonon term is given by where f is the Lamb–Mo¨ ssbauer factor, d(E ) a Dirac function describing the elas-
tic part of absorption (zero phonon term) and Snp(E ) a series of terms from
which the npth term represents the inelastic absorption accompanied by creation -
(annihilation) of np phonons. The one phonon term is given by S1ðEÞ ¼
ERgð E
j jÞ
Eð1 ebEÞ
ð23Þ ð23Þ and the subsequent terms by the following when the harmonic approximation is
valid: and the subsequent terms by the following when the harmonic approximation is
valid: S1ðEÞ ¼ 1
np
Z þ1
1
S1ðE0ÞSnp1ðE E0Þ dE0. ð24Þ ð24Þ In these expressions, b ¼ ðkTÞ1; ER ¼ _2k2=2Mis the recoil energy of a free nu-
cleus; k the wave vector of the g-ray quantum and M the mass of the nucleus. The
function g(E) is the normalized density of phonon states: gðEÞ ¼ V 0
1
ð2pÞ3
X
j
Z
d~qd E _ojð~qÞ
;
ð25Þ ð25Þ where V 0 is the volume of the unit cell, and the index j enumerates the branches of
the dispersion relation ½_ojð~qÞ with ~q being the phonon momentum. The integral is
taken within the first Brillouin zone. This technique, besides allowing determination of the Lamb–Mo¨ ssbauer factor,
provides direct access to the density of phonon states for the probe isotope in a
solid. It thus provides information about lattice dynamics that is excluded by the
limitations of Mo¨ ssbauer spectroscopy. This technique could be valuable in inves-
tigations of adsorption with the adsorbing element as the probe and showing
the modifications brought about by the adsorbate on the dynamic properties of
the probe. The frequency distribution of vibrations can be different in various directions,
although Mo¨ ssbauer spectroscopy did not show it (225). Few examples are available
to illustrate the use of the technique. Furthermore, most of these examples have been
chosen to show basic applications of the technique. Temperature and pressure de-
pendences of nuclear inelastic absorption in iron metal (a-Fe) have been investigated
(226), as has the anisotropy dependence in a 57FeBO3 single crystal (225). NIS has
also been used to investigate complexes in solution (57Fe[2,20-(bipyridyl)3]Cl2 in
water and in glycerol) (227). A recent publication reports the characterization of
perovskite-related oxides ((Sr,Ca)(Fe,Co)O3d and (Ba,Ca)(FeCo)O3d) that may
find applications in fuel cells or oxygen-permeable membranes (228). B.2. Nuclear Inelastic Scattering (NIS) of Synchrotron Radiation This technique comprises measurement of the energy distribution of phonons
excited by the inelastic absorption of X-rays at the resonance. The Mo¨ ssbauer effect
comprises the absorption and re-emission of X-rays that occur in an elastic manner. This excitation can also be achieved if the radiation energy is not exactly equal to
the resonance energy E0, but instead is equal to E07DE when a phonon of energy
DE is annihilated providing the lacking energy or a phonon of energy DE is created,
taking up the excess energy. Annihilation or emission of several phonons can be
involved in the process. When resonance absorption takes place with detuned syn-
chrotron radiation, the delayed re-emitted g-quanta or conversion electrons are
emitted into a solid angle of 4p and can be counted by a detector placed at a
position out of the incoming beam (Fig. 15). The normalized probability of nuclear inelastic absorption W(E) can be decom-
posed in terms of a multi-phonon expansion (222,223): WðEÞ ¼ f
dðEÞ þ
X
1
np¼1
SnpðEÞ
! ,
ð22Þ ð22Þ Resonant
sample
Ionization
chamber
Detector #1
Detector #2
High-heat-load
monochromator
High-resolution
monochromator
Storage ring
Undulator
e−
Detector #1
Detector #2
FIG. 15. Basic equipment for measuring a nuclear inelastic scattering spectrum. Detector 1 measures
the intensity of the incoherent nuclear forward scattering, which proceeds both elastically and inelas-
tically; detector 2 measures only the intensity of the coherent nuclear forward scattering, which proceeds
elastically. Figure according to Ru¨ ffer and Chumakov (224). Resonant
sample Detector #2 High-heat-load
monochromator FIG. 15. Basic equipment for measuring a nuclear inelastic scattering spectrum. Detector 1 measures
the intensity of the incoherent nuclear forward scattering, which proceeds both elastically and inelas-
tically; detector 2 measures only the intensity of the coherent nuclear forward scattering, which proceeds
elastically. Figure according to Ru¨ ffer and Chumakov (224). 33 where f is the Lamb–Mo¨ ssbauer factor, d(E ) a Dirac function describing the elas-
tic part of absorption (zero phonon term) and Snp(E ) a series of terms from
which the npth term represents the inelastic absorption accompanied by creation -
(annihilation) of np phonons. B.2. Nuclear Inelastic Scattering (NIS) of Synchrotron Radiation In the latter
case, a soft phonon peak in the NIS spectra was found to be correlated with oxygen
ordering in the structure. Changes were also observed upon absorption of CO2 that
have unfortunately not been interpreted because of the complexity of the spectra. Nuclear resonant forward scattering and inelastic scattering of synchrotron ra-
diation constitute a promising field for development of catalyst characterization in
the future. For the characterization of solid catalysts, NFS has the advantage over 34 Mo¨ ssbauer spectroscopy of being a time-resolved spectroscopic technique. This
characteristic opens new areas of research, particularly because it also combines in
the same time all the advantages of Mo¨ ssbauer spectroscopy in terms of sensitivity
(it is even more sensitive for hyperfine parameter effects than conventional
Mo¨ ssbauer spectroscopy) and in terms of applicability under catalytic reaction
conditions. It still appears, however, for complex systems, such as those often
encountered in catalysis, difficult to analyze the time-domain spectra relative to
energy-domain spectra, and for the time being NFS should be considered to be
complementary to conventional Mo¨ ssbauer spectroscopy and not a replacement of
it. In the future, NIS may also become an extremely efficient tool for characterizing
adsorption, although the energy resolution for recording molecular vibrations,
which is at present time about 5 cm1, has to be improved. V. Conclusions This review includes a short summary of the principles of Mo¨ ssbauer spectroscopy
and of recent applications of the technique in heterogeneous catalysis. The fre-
quency of applications of the technique in this field has remained nearly constant,
but the applications to investigations of catalysts under controlled conditions and
under catalytic reaction conditions, with characterizations of activation, reaction,
and deactivation processes has increased significantly in the preceding few years. Although Mo¨ ssbauer spectroscopy is characterized by complexity in the experi-
mental technique as well as in the interpretation and analysis of spectra, if offers
for samples including those incorporating iron, cobalt, gold, and other elements
of catalytic interest the capability for (i) determination of accurate oxidation states
of the metals, (ii) resolution of microcrystalline or poorly crystalline phases, (iii)
quantification of coordination and local symmetry site populations, (iv) character-
ization of various electronic, magnetic, and (v) structural phase transitions; and
quantification of chemical, structural, and magnetic disorder. A section of this review is devoted to the potential areas of likely future devel-
opments in Mo¨ ssbauer spectroscopy in the field of catalysis. In view of the con-
siderable value of spectroscopic measurements made of catalysts while they are
working, Mo¨ ssbauer spectroscopy, which is relatively well adaptable to such con-
ditions, is expected to gain interest in the catalysis community. Cells have already
been adapted to conditions of high pressure and high temperature. Furthermore,
these cells provide flexibility, allowing the coupling of Mo¨ ssbauer spectroscopy with
other spectroscopic techniques. New experimental setups continue to be developed
and should improve the precision of the measurements and reduce the acquisition
time (which still remains a handicap for the technique). Synchrotron Mo¨ ssbauer
spectroscopy in particular deserves more attention because it can become a useful
for investigating catalytic reactions on many types of catalysts. The use of syn-
chrotron radiation for Mo¨ ssbauer spectroscopy was proposed as early as 1974, and
it is now becoming accessible to non-experts. NFS of synchrotron radiation allows
experiments that are in many aspects very similar to conventional Mo¨ ssbauer
spectroscopy, and they offer the benefit of combining all the advantages of 35 conventional Mo¨ ssbauer spectroscopy, with the time-resolution of NFS at a time
scale interesting for catalyst characterizations. This latter advantage is conspicu-
ously missing in conventional Mo¨ ssbauer spectroscopy when compared with other
spectroscopic techniques for characterizing catalysts under working conditions. V. Conclusions In
the future, NIS experiments may bring new insights in catalyst characterization,
and these techniques should also broaden the field of application of Mo¨ ssbauer
spectroscopy to allow the investigation of important isotopes with very short half-
lifetime sources, such as 61Ni and 99Ru. It is hoped that the synthetic presentation of NFS and NIS techniques given in
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157, 15 (2004). 207. Guenzburger, D., Ellis, D.E., and Zeng, Z., Hyp. Interact. 113, 25 (1998). 41 208. Paulsen, H., and Trautwein, A.X., Top. Curr. Chem. 235, 197 (2004). 209. McCammon, C.A., Chaskar, V., and Richards, G.G., Meas. Sci. Technol. 2, 657 (1991). 210. McCammon, C.A., Hyp. Interact. 92, 1235 (1994). 211. de Souza, P.A. Jr., Hyp. Interact. 113, 383 (1998). 212. de Souza, P.A. Jr., Garg, R.V., and Garg, K., Hyp. Interact. 112, 275 (1998). 213. Rancourt, D.G., Hyp. Interact. 117, 3 (1998). 214. Smith, P.R., Cashoin, J.D., and Brown, L.J., Hyp. Interact. 71, 1503 (1992). 215. Gerdau, E., and DeWaard, H., Hyp. Interact. 123/124 (1999). 216. Rancourt, D., and Klingelho¨ fer, G., Fourth Seeheim Workshop on Mo¨ ssbauer Spectroscopy
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611 (1999). 220. Gru¨ nsteudel, H.F., Haas, M., Leupold, O., Mandon, D., Matzanke, B.F., Meyer-Klaucke, W.,
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Phys. Rev. B 56, 10758 (1997). 226. Chumakov, A.I., Ru¨ ffer, R., Baron, A.Q.R., Gru¨ nsteudel, H., and Gru¨ nsteudel, H.F., Phys. Rev. B
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1. INTRODUCTION In Ethiopia, agriculture remains among the most important economic sectors. It produces about one-third of GDP
and employs 70 percent of the workforce, accounting for 80 percent of the country’s merchandise exports (EATA,
2014). Agriculture is the leading sector of Ethiopian economy as well the overall economic growth of the country
largely depends on the agricultural sector. However, Ethiopian agriculture remains characterized by small-scale
subsistence production systems where crop and livestock yields are very low(Desta and Almaz, 2015).Although
agricultural productivity is very much affected due to variability of rainfall and drought(Sensen, 2013). Even if agriculture status is the backbone of the Ethiopian economy, agricultural practice has been
traditionally dominated for centuries by small-scale farmers and its performance has long been adversely affected
by shortage of rain and water that left many to sustain their lives on famine relief support(Abebe et al., 2011,
Temesgen et al., 2018). From the total production of Ethiopia’s, about 97% food crops are produced by rain-fed
agriculture, whereas only 3% is from irrigated agriculture (FAO, 2015). Because of high dependency on rain-fed
agriculture, other topographic and low adaptive capacity along with other related factors, Ethiopia ranks the ninth
most susceptible country in the world to natural disasters and weather related shocks (Tongul and Hobson, 2013). On the other hand, Ethiopia has abundant water resources, its agricultural system does not yet fully benefit from
the technologies of water management and irrigation(Seleshi et al., 2010). According toNahusenay and Madhu( 2015),one of the aspects given attention by the government is to improve
food and agricultural production through the strengthening small scale irrigation. The net farm income is the most
watched indicator of farm sector well-being, as it captures and reflects the entirety of economic activity across the
range of production processes, input expenses, and marketing conditions that have persisted during a specific time
period. There are different mechanisms by which irrigated agriculture can improve household income and food
security, through: increasing farm production and productivity that helps very poor households meet the basic
needs, protecting against risks of crop loss due to erratic, insufficient rainwater supplies, promoting greater use of
yield enhancing farm inputs and creation of additional employment(Agidew, 2017). Agricultural intensification is presumed to be a necessary pre-condition for the development of the
agricultural sector in Ethiopia. Impact of Small-Scale Irrigation Schemes on Smallholders
Household Income and Food Security in Ethiopia: Review
Abdulsemed Abanega Abdurahman1 Ahmed Mohammed Abachebsa2
1.Department of Agricultural Economics and Agribusiness Management, Jimma University P.O. Box. 307,
Jimma, Ethiopia
2.Department of Natural Resource Management; Jimma University; P.O. Box. 307, Jimma, Ethiopia Abdulsemed Abanega Abdurahman1 Ahmed Mohammed Abachebsa2
Department of Agricultural Economics and Agribusiness Management, Jimma University P.O. Box. 307,
Jimma, Ethiopia
2.Department of Natural Resource Management; Jimma University; P.O. Box. 307, Jimma, Ethiopia Abstract In Ethiopia, agriculture remains among the most important economic sectors and has been traditionally dominated
for centuries by small-scale farmers. Its performance has long been adversely affected by shortage of rain and
water to sustain their lives on famine relief support. From the total production of Ethiopia’s, about 97% food crops
are produced by rain-fed agriculture, whereas only 3% is from irrigated agriculture. On the other hand, Ethiopia
has abundant water resources, but not yet fully benefited from technologies of water management and irrigation. The aim of this paper is to identify the impact of small-scale irrigation on household income and food security
reviewing different literature. Different studies indicated that access to irrigation water can enable farmers to adopt
new technologies and intensify cultivation, leading to increased productivity, production, and greater returns from
farming. Access to irrigation enables farmer to grow crops more than once a year increasing production, income
and improve their food security status. This review concludes small-scale irrigation is one of the possible solutions
to increase smallholder household income and food needs in the country. But there are different challenges
hindering the irrigation development by smallholder households like lack of market at the time of harvest, distance
from water sources, knowledge on the efficient use of irrigation, and linkage between micro finance institutions
and smallholder horticultural crop producers are some constraints. These constraints have to be solved to increase
participation of stallholder household in irrigation technology to improve their living standards through this sector. Keywords: Agriculture, Ethiopia, Small scale irrigation, Water resource
DOI: 10.7176/JBAH/11-1-05 Publication date: January 31st 2021 www.iiste.org www.iiste.org Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.11, No.1, 2021 Impact of Small-Scale Irrigation Schemes on Smallholders
Household Income and Food Security in Ethiopia: Review
Abdulsemed Abanega Abdurahman1 Ahmed Mohammed Abachebsa2
1.Department of Agricultural Economics and Agribusiness Management, Jimma University P.O. Box. 307,
Jimma, Ethiopia
2.Department of Natural Resource Management; Jimma University; P.O. Box. 307, Jimma, Ethiopia 1. INTRODUCTION To this end, various government and non-governmental organizations (NGOs),
among others, initiated small-scale irrigation schemes throughout the country(Kinfe et al., 2012).The importance
of small-scale irrigation for improving farmers’ income and reducing rural poverty has been emphasized in various
literatures and small-scale irrigation schemes play a huge role to increase income of the society(Ayana, 2016). The
current Ethiopian government has undertaken various activities to expand irrigation in the country. The country’s 40 Journal of Biology, Agriculture and Healthcare ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.11, No.1, 2021 Agricultural Development Led Industrialization strategy considers irrigation development as a key strategy for
sustainable agricultural development. Thus, irrigation development, particularly small-scale irrigation is planned
to be accelerated(MoFED, 2010). The local institutions and the rural infrastructure services can improve the
awareness of people regarding irrigation and he expansion of small-scale irrigation can be an important strategy
to increase income, build-up assets, increase total expenditure, and spend more income on improved agricultural
technologies(Woldegebrial et al., 2015). Irrigation in Ethiopia contributes to increase farmers‟ income, household resilience and buffering livelihoods
against shocks and stresses by producing higher value crops for sale at market and to harvest more than once per
year. In turn, this provided them to build up their assets, buy more food and non-food household items, educate
their children, and reinvest in further increasing their production by buying farm inputs or livestock. However, the
benefits are very unevenly distributed among households(Eshetu et al., 2010). Therefore, the aim of this paper is
to contribute literature on impact of small-scale irrigation schemes on smallholder farmers’ incomes and food
security and also challenges and opportunity for smallholder farmers to use irrigation in Ethiopia. 2. Basic Concept and Definitions In general terms, irrigation can be defined as the artificial supply of water to supplement natural precipitation or
substitute for it for the purpose of agricultural production(Mohamed, 2006). According to Indhumathi et al., (2017)
Water is most powerful natural resource as it has great influences in health and wealth of the people and production
of food. Thus, water has great role in survival and comfortable life of living things. We are depending on the
water resources for development of agriculture. Besides various other uses of water, the largest use of water in
the world is for irrigating land. The same report by MoWE( 2013), indicated that water is a basic need for human
beings and animals. It is essential for their metabolic processes. It is used to build healthy workforce, ensuring
food security, provision of clean energy for agriculture and industry. Food security is defined in different ways by different organizations around the world. For example, in 1996,
the definition of food security was agreed and accepted at the world food summit in Rome and it was defined food
security as a physical and economic access by all people at all times to sufficient, safe and nutritious food to meet
their dietary needs and food preference for an active and healthy life(Todaro and Smith, 2011). According to
FAO(2011), food security contains different features, such as food availability, accessibility, utilization and
stability. Food availability refers to the existence of food from own production or on the markets. It is a
combination of domestic foods production, marketable imports and food aid. Food access is guaranteed when
households and all individuals within them have adequate resources, through access options. Food utilization
refers to nutritional and safety aspects of food security to meet adequate diet, clean water, sanitation and health
care to search a state of nutritional wellbeing. Food stability complements the previous factors by stressing that
food must be available, accessible, affordable and properly utilized on a continuous basis. 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia Access to such small scale irrigation can significantly improve income level and food security status of beneficiary
households(Eshetu and Young-Bohk, 2017). According to Lipton et al. (2003), irrigation increases productivity
by reducing crop losses that were due to limited water supply, enabling cultivation during the lean season, and
making crop production possible on lands where rain fed agriculture is infeasible. The other study shows that farm
productivity is positively correlated with the availability of farm power coupled with efficient farm implements
and their sustainable utilization. On the other hand the report of (MoA, 2015) shows that Agricultural
mechanization not only enables efficient utilization of various inputs such as seeds, fertilizers, plant protection
chemicals and water for irrigation but also it helps in poverty alleviation by making farming an attractive enterprise. In Ethiopia, nearly all small farmers are still using traditional hand tools and oxen plow, which greatly is restricting
crop productivity and production. Irrigated agriculture plays a central and dynamic role in the improvement of rural livelihoods, but is often
subject to criticisms of inefficiency in water use, high capital and recurrent costs and lack of sustainability
associated with inequity in the distribution of both land and water(Reinders, 2011). Although irrigation leads to
increased agricultural income and production diversity, and this leads to improved diets and access to irrigation
has the potential to improve both household income and the diversity of crops that farmers produce. Increasing
household income, in turn, leads to higher dietary diversity, while increases in production diversity do not
contribute to increases in dietary diversity over and above the effect of income (Passarelli et al., 2018). y
y
(
)
According to Garbero and Songsermsawas (2018), Small-scale farmers in the developing world face multiple
challenges that limit their opportunities to achieve higher agricultural productivity and improve their living
conditions. One promising channel to help farmers attain more desirable agricultural outcomes is to increase their
access to water, an important input for agricultural activities. Several studies have noted the positive and significant
benefits of irrigation infrastructure on agriculture. Although, small-scale irrigation development has a positive
impact on food security status of rural households and a much higher proportion of those who are poor are non-
irrigating rather than irrigating households. Thus, the food insecurity occurrence in non-irrigating households is
greater than in irrigating households1(Muez, 2014). 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia The main income sources of Ethiopian rural households were
cropping, livestock and off-farm activities and the irrigating households have significantly larger mean annual
income than non-irrigating households. Although the poverty analysis indicated that a much higher proportion of
poor are non-irrigating than irrigating households, different studies revealed that there is positive significant impact
on the income of participant household income as a result of participation in small-scale irrigation(Getaneh,
2011;Temesgen et al., 2018). g
)
According to Awulachew (2010), with high potential, if it is successfully operated, irrigation in Ethiopia
could play a significant role in the agricultural transformation of the country, contributing up to ETB 140 billion
to the economy. Furthermore, another study done by Tsegazeab (2015) micro irrigation is one of the most useful
irrigation systems designed to increase production and productivity; and reduces risk, related with rainfall
variability and increases income of rural farm households and not surprisingly, planners, researchers, development
practitioners, and donors emphasize the importance of micro irrigation in their policy recommendations and actual
measures. According to Aam (2013), small-scale irrigation has immense potential to improve agricultural productivity
and incomes of poor rural households. Irrigation in fact is nothing but is a continuous and reliable water supply to
different crops in accordance with their water requirement. According to Seleshi et al.(2005), agricultural
production in Ethiopia has affected by increasing population, deforestation and land distribution. This reflected in
a decrease in household production and grazing land. Hence, it has become a common phenomenon to ask for
emergency food assistance for acutely food insecure people in Ethiopia. The adoption of new technology (e.g. irrigation) is the major powerful for agricultural growth and poverty reduction(Norton et al., 2010). This indicates
that investment in irrigation can increase farmer’s independence on rainfall, it increases irrigated farmland, it
generates employment, it promotes farmers to produce two or three times in a year and use more of chemical
inputs. Small scale irrigation in developing countries was considered as a means of increasing production, reducing
the dependence on rainfall and provides jobs to the poor(Chazovachii, 2012). Although small-scale irrigation is believed in helping to address irregular rainfall pattern problem there by
reducing rural poverty, food insecurity as well as improving the overall contribution of agriculture to the national
economy(Tizita, 2017), it increases land productivity, crop yields, adoption of mineral fertilizers and enables to
diversify into non-conventional and market-oriented products (Eshetu et al., 2010). 3. History of Irrigation development and participation condition in Ethiopia Ethiopia has a long history of traditional irrigation systems. Simple river diversion still is the dominant irrigation
system in Ethiopia. According to Seleshi et al. (2010), estimates the irrigation potential of Ethiopia may be as
large as 4.3 million hectares. Traditional irrigation schemes cover more than 138,000 hectares whereas modern
small-scale irrigation covers about 48,000 hectares. The total current irrigation covers only about 6% of the
estimated potential land area. According to Gebremedhin and Asfaw( 2015), irrigation was practiced during ancient times in Ethiopia even
if its exact date of emergence is unknown. Ancient use of irrigation water was through use of surface irrigation
methods and spate irrigation types. Modern irrigation was started at the Awash River basin with bilateral
cooperation of Ethiopia and Dutch company. Ethiopia is a rich country in water resource and most of the time it
is termed as a water tower of east Africa because of its abundant water resource availability(Adugna , 2014). Although Ethiopia has a huge potential of water resource which accounts 122 billion meter cube annual surface
runoff and 2.9 billion meter cube groundwater, it is characterized by uneven spatial and temporal
distributions(Tesfa, 2015). But, Ethiopia is using a very little of its abundant water resource potential for irrigated
agriculture(ATA, 2016). The study taken by Gebremedhin (2015), indicated that the policies and strategies of
Ethiopia strongly supports the irrigation developments especially the small scale irrigation through the Water
Sector Development Programs (WSDP) and Ethiopian Irrigation Development Plan (IDP). This irrigation
development is mainly expressed in the development of small-scale irrigation schemes by governments, donors
and NGOs. Irrigation is believed as a key for food security and poverty reduction in Ethiopia. As a result,
developments in the Ethiopian irrigation system have shown great advancements so as to assure Ethiopian
livelihoods especially in the rural areas. However, the contribution of irrigation to the national economy as
compared to its potentials is insignificant(Woldegebrial et al., 2017). 41 Journal of Biology, Agriculture and Healthcare www.iiste.org iste.org 5. Challenges and Opportunities of Irrigation Development in Ethiopia
There are different challenges that hinder the irrigation development by sma 5. Challenges and Opportunities of Irrigation Development in Ethiopia
There are different challenges that hinder the irrigation development by small holder households in Ethiopia. According to Temesgen (2017), out of the challenges pointed out by farmers, lack of market at the time of harvest
holds the first rank, distance of farm land from irrigation water source, lack of knowledge on the efficient use of
irrigation by the farmers, lack of market and linkage, lack of awareness on the sustainable use of water and lack
of linkage between micro finance institutions and smallholder horticultural crop producers are some constraints. Although the major small-scale irrigation problems identified by different studies throughout the country indicated
that, financial constraints especially for the purchase of motor pumps, shortage of agricultural inputs specially
improved seed and pesticides, high cost of irrigation, shortage of water pump technologies, spare parts and gabions,
technical problems such as maintenance of motor pumps, insufficient market information and market networks,
shortage of ponds and diversion, infrastructure specially road and storage, theft of fruits, diseases and pests such
as rust, root ruts, ball worm, blights, powdery mildew, gummosis and water borne diseases are among the problems
(Nahusenay and Madhu, 2015). Another study taken by Gebremedhin and Asfaw (2015), identified the challenges for Ethiopian irrigation
development are: Inadequate awareness of irrigation water management as in irrigation scheduling techniques,
water saving irrigation technologies, water measurement techniques, operation and maintenance of irrigation
facilities, inadequate knowledge on improved and diversified irrigation agronomic practices, shortage of basic
technical knowledge on irrigation pumps, drip irrigation system, sprinkler irrigations, surface and spate irrigation
methods, inadequate baseline data and information on the development of water resources, lack of experience in
design, construction and supervision of quality irrigation projects, low productivity of existing irrigation schemes,
inadequate community involvement and consultation in scheme planning, construction and implementation of
irrigation development and poor economic background of users for irrigation, infrastructure development are some
challenges. Another study taken by Tizita(2017) shows that lack of suitable land that can be used for irrigation
(irrigable farm land) was the major constraints for irrigation non-user households. On the other hand, there are many opportunities that enhance irrigation development in Ethiopia. From these
opportunities the policies and strategies of Ethiopia strongly supports the irrigation developments(Tadesse and
Baihilu, 2017). 5. Challenges and Opportunities of Irrigation Development in Ethiopia
There are different challenges that hinder the irrigation development by sma Another study taken by Amede(2014), identified the following future opportunities: high water
potential, high commitment of the Ethiopia government, donors and NGOs to support irrigation management and
development activity. There is also availability of favorable climate condition and ground water in Ethiopia. It can
be used as an opportunity to produce more than once a year if the farmers could be able to access the ground water
by means of using different types small-scale irrigation (Temesgen, 2017). 4. Impacts of Small-scale Irrigation on income and Food Security in Ethiopia It also improves farm
households‟ diet, incomes, health and food security(Torell and Ward, 2010). Hence irrigated agriculture is
accepted as essential in increasing land productivity, enhancing food security, earning higher and more stable
incomes and increasing for multiple cropping and crop diversification(Smith, 2004). Access to irrigation positively
and significantly affects household food security and irrigation user households were more likely to be food secure
as compared to irrigation non-users, holding other variables constant. This implies that irrigation enables
households to grow food crops more than once a year, hence increased production, income and food availability 42 Journal of Biology, Agriculture and Healthcare Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.11, No.1, 2021 www.iiste.org iste.org of the household(Tizita., 2017). Although irrigation expansion has been regarded as a promising approach to ensure food and livelihood
security in the face of climate change and population growth while only 4% of cultivated area in sub-Saharan
Africa is currently irrigated, the potential for the expansion of irrigation in the region is large and investments are
accelerating(Giordano et al., 2012). Previous investments in irrigation by donors and governments have focused
on developing large scale irrigation schemes, but evidence suggests that the greatest gains in terms of profitability
and sustainability will come from investments in small scale irrigation (Xie et al., 2014). In general irrigation in Ethiopia is considered as a basic strategy to alleviate poverty and reduce food security. It is useful to transform the rain-fed agricultural system which depends on rainfall into the combined rain-fed and
irrigation agricultural system. This is believed to be the most prominent way of sustainable development in the
country. However, the development of irrigation practices in Ethiopia has to be investigated so as to seriously
know the history of irrigation emergence and its subsequent developments(Gebremedhin, 2015). 6. Conclusions Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.11, No.1, 2021 and linkage, lack of awareness on the sustainable use of water and lack of linkage between micro finance
institutions, smallholder horticultural crop producers, shortage of agricultural inputs specially improved seed and
pesticides, high cost of irrigation, shortage of water pump technologies and infrastructure specially road and
storage are some constraints . On the other hand, there are different opportunities that increase irrigation
development in Ethiopia. i.e. High-water potential, effective utilization of small-scale irrigation scheme
infrastructure through diversification of uses to meet various needs for water such as domestic, irrigation, the
interest of development agencies to give education and training, policies and strategies of Ethiopia strongly
supports the irrigation developments. and linkage, lack of awareness on the sustainable use of water and lack of linkage between micro finance
institutions, smallholder horticultural crop producers, shortage of agricultural inputs specially improved seed and
pesticides, high cost of irrigation, shortage of water pump technologies and infrastructure specially road and
storage are some constraints . On the other hand, there are different opportunities that increase irrigation
development in Ethiopia. i.e. High-water potential, effective utilization of small-scale irrigation scheme
infrastructure through diversification of uses to meet various needs for water such as domestic, irrigation, the
interest of development agencies to give education and training, policies and strategies of Ethiopia strongly
supports the irrigation developments. In general irrigation have a great role in economic development and in increments of food security and
Ethiopia has high potential of water resource that can help irrigation development from African countries but, still
she didn’t use this potential efficiently and the various actors of this sectors have to give attention to utilize this
resource efficiently. 6. Conclusions Irrigation plays an important role in the performance of agriculture and improvement of rural livelihoods which
increases income from agriculture sector and reduce the risk of crop failure as a result of erratic rainfall. It increases
farm production and productivity that helps poor households to meet the basic needs, protecting against risks of
crop loss due to erratic, unreliable or insufficient rainwater supplies, promote greater use of yield enhancing farm
inputs and create additional employment. Also, small-scale irrigation development has a positive impact on food
security status of rural households and a much higher proportion of those who are poor are non-irrigating rather
than irrigating households. Ethiopia is a rich country in water resource, but using a very little of its abundant water
resource potential for irrigated agriculture. While investment in irrigation can increase farmer’s independence on
rainfall, it increases irrigated farmland, generates employment, promotes farmers to produce two or three times in
a year and use more of chemical inputs. There are different challenges that hinder the irrigation development by small holder households in Ethiopia. Out of these challenges written in different papers, lack of market at the time of harvest holds, distance of farm
land from irrigation water source, lack of knowledge on the efficient use of irrigation by the farmers, lack of market 43 Journal of Biology, Agriculture and Healthcare www.iiste.org
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.11, No.1, 2021
and linkage, lack of awareness on the sustainable use of water and lack of linkage between micro finance
institutions, smallholder horticultural crop producers, shortage of agricultural inputs specially improved seed and
pesticides, high cost of irrigation, shortage of water pump technologies and infrastructure specially road and
storage are some constraints . On the other hand, there are different opportunities that increase irrigation
development in Ethiopia. i.e. High-water potential, effective utilization of small-scale irrigation scheme
infrastructure through diversification of uses to meet various needs for water such as domestic, irrigation, the
interest of development agencies to give education and training, policies and strategies of Ethiopia strongly
supports the irrigation developments. References
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https://openalex.org/W4387950240
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https://www.nature.com/articles/s41440-023-01450-4.pdf
|
English
| null |
Skin sodium content as a predictor of blood pressure response to renal denervation
|
Hypertension research
| 2,023
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cc-by
| 8,253
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Abstract Patients with treatment resistant hypertension (TRH) are known to have elevated sodium (Na) content in muscle and skin. Renal denervation (RDN) emerged as an adjacent therapeutic option in this group of patients. This analysis aimed at
evaluating whether tissue Na content predicts blood pressure (BP) response after RDN in patients with TRH. Radiofrequency-
device based RDN was performed in 58 patients with uncontrolled TRH. Office and 24-h ambulatory BP were measured at
baseline and after 6 months. To assess tissue Na content Na magnetic resonance imaging (Na-MRI) was performed at baseline
prior to RDN. We splitted the study cohort into responders and non-responders based on the median of systolic 24-h
ambulatory blood pressure (ABP) reduction after 6 months and evaluated the association between BP response to RDN and
tissue Na content in skin and muscle. The study was registered at http://www.clinicaltrials.gov (NCT01687725). Six months
after RDN 24-h ABP decreased by −8.6/−4.7 mmHg. BP-Responders were characterized by the following parameters: low
tissue sodium content in the skin (p = 0.040), female gender (p = 0.027), intake of aldosterone antagonists (p = 0.032), high
baseline 24-h night-time heart rate (p = 0.045) and high LDL cholesterol (p < 0.001). These results remained significant after
adjustment for baseline 24-h systolic BP. Similar results were obtained when the median of day-time and night-time ABP
reduction after 6 months were used as cut-off criteria for defining BP response to RDN. We conclude that in addition to
clinical factors including baseline 24-h ABP Na-MRI may assist to select patients with uncontrolled TRH for RDN treatment. Keywords Renal denervation
●Tissue sodium
●Sodium MRI ARTICLE ARTICLE * Roland E. Schmieder
roland.schmieder@uk-erlangen.de Merve Guenes-Altan1
●Axel Schmid2
●Dennis Kannenkeril1
●Peter Linz2
●Christian Ott1
●Agnes Bosch1
●
Mario Schiffer1
●Michael Uder2
●Roland E. Schmieder1 Received: 17 July 2023 / Revised: 24 August 2023 / Accepted: 26 August 2023 / Published online: 25 October 2023
© The Author(s) 2023. This article is published with open access Hypertension Research (2024) 47:361–371
https://doi.org/10.1038/s41440-023-01450-4 Hypertension Research (2024) 47:361–371
https://doi.org/10.1038/s41440-023-01450-4 1
Department of Nephrology and Hypertension, University Hospital
Erlangen, Friedrich-Alexander University Erlangen-Nuremberg
(FAU), Erlangen, Germany 2
Institute of Radiology, University Hospital Erlangen, Friedrich-
Alexander University Erlangen-Nuremberg (FAU),
Erlangen, Germany Skin sodium content as a predictor of blood pressure response to
renal denervation Merve Guenes-Altan1
●Axel Schmid2
●Dennis Kannenkeril1
●Peter Linz2
●Christian Ott1
●
Mario Schiffer1
●Michael Uder2
●Roland E. Schmieder1 * Roland E. Schmieder
roland.schmieder@uk-erlangen.de
1
Department of Nephrology and Hypertension, University Hospital
Erlangen, Friedrich-Alexander University Erlangen-Nuremberg
(FAU), Erlangen, Germany
2
Institute of Radiology, University Hospital Erlangen, Friedrich-
Alexander University Erlangen-Nuremberg (FAU),
Erlangen, Germany Study cohort All patients who participated in our study were aged 40–77
years, had uncontrolled TRH with 3–10 antihypertensive
drugs and were eligible for Na-MRI examination without
MRI contraindication. True hypertension was confirmed by
24-h
ambulatory
blood
pressure
(ABP)
measurement
(average 24-h ABP ≥130/80 mmHg). All patients fulfilled
the following exclusion criteria: No known secondary cause
of HTN including hyperaldosteronism, no significant renal
artery pathologies, no prior RDN and no known contra-
indication for RDN procedure (e.g. renal artery stenosis >
50%, implanting of renal stents). Study design Our single-center, post-hoc study includes 58 patients with
uncontrolled TRH who underwent RDN and Na-MRI. The patients participated in the “Renal Denervation in
Treatment Resistant Hypertension” trial, an investigator
initiated study program performed only in our Erlanger
center. The study was registered at http://www.clinicaltrials. gov (NCT01687725). All patients were followed up for Several studies have documented an accumulation of Na
without simultaneously commensurate water which leads to
the concept that there must exist non-osmotic storage of Na
in tissue, for example in skin and muscle [13–16]. Na
magnetic resonance imaging (Na-MRI) can be used to
visualize and quantify Na content in tissue. Using this non-
invasive technique elevated Na content in muscle and skin
has been detected in patients with hypertension and in
particular in patients with TRH [17]. Moreover, we pre-
viously analyzed the impact of RDN on tissue Na content in
patients with treatment resistant hypertension and did not
observe any change in muscle or skin Na content 6 months
after RDN [18]. The objective of the present study was to
analyze whether tissue Na content in patients with TRH
serves as a determinant of BP response in RDN. 6 months at the Clinical Research Centre of the Department
of Nephrology and Hypertension, University Hospital
Erlangen-Nuremberg, Germany (www.crc-erlangen.de). The respective study protocol was approved by the local
Ethical Review Committee (ethics committee of the Uni-
versity of Erlangen-Nuremberg) and the study was con-
ducted according to the Declaration of Helsinki and Good
Clinical Practice. Written informed consent was obtained
from all patients and prior to study inclusion. Introduction treatment resistant hypertension (TRH) among patients with
HTN was approximately 10% [3]. Arterial hypertension (HTN) is highly prevalent worldwide
and a major risk factor for cardiovascular disease (CVD)
and stroke. In 2016, it was estimated that 46.0% of adults in
the United States suffered from HTN [1]. A multinational
study in adults revealed that 55.6% of patients were aware
of their diagnosis with only 17.1% having adequately
controlled
HTN
[2]. Furthermore,
the
prevalence
of Increased activity of the sympathetic nervous system
(SNS) activity and increased sodium (Na) retention have
been found to play an important role in the pathogenesis of
TRH while both mechanisms are interconnected to each
other [4–6]. High SNS activity results in increased Na and
water retention [7, 8]. This enhances vasoconstriction due
to vasoactive hormones and induces hypertrophic response
of the myocardium and vessels [9]. Thus, targeting
increased SNS activity appears to be an attractive treatment
strategy [10]. The ESH 2021 and ESC 2023 consensus statements
recommend RDN as an adjacent treatment option to achieve
BP control in patients with uncontrolled TRH in addition to
pharmacotherapy and lifestyle changes [11, 12]. Since blood
pressure (BP) response after RDN has a large variability and
there exist patients with poor BP response after RDN, the
need to identify determinants of BP response to RDN have
been underlined in the consensus statements [11, 12]. M. Guenes-Altan et al. 362 Graphical Abstract Clinical characteristics We included 58 patients in our study with a mean age of 62
years. Most patients were male and overweight. About half
of the patients had type 2 diabetes (T2D). All patients had
uncontrolled TRH with 6.2 (3.0–10.0) antihypertensive
drugs on average. Four patients were not on diuretic therapy
because of contraindications and drug intolerance. The
detailed clinical characteristics are shown in Table 1. Blood pressure The average 24-h ABP in our study cohort was 157/87 mmHg
and the office BP was 158/87 mmHg. Six months after RDN
24-h ABP was reduced by −8.6/−4.7 mmHg and office BP
was reduced by −12.9/−7.7 mmHg (all p < 0.001, see
Table 2). The average 24-h ABP in our study cohort was 157/87 mmHg
and the office BP was 158/87 mmHg. Six months after RDN
24-h ABP was reduced by −8.6/−4.7 mmHg and office BP
was reduced by −12.9/−7.7 mmHg (all p < 0.001, see
Table 2). Medication change (conducted according to the discre-
tion of the primary care physician) did not differ between
the two groups (p = 0.320): At 6 months, decrease in
number of antihypertensive drugs took place in 37.0% of
responders
versus
23.1%
in
non-responders,
whereas
increase in medication number occurred in 25.9% in
responders and 23.1% in non-responders. Assessments Baseline assessments included office and 24-h ABP mea-
surements, Na-MRI examination, collection of demographic Skin sodium content as a predictor of blood pressure response to renal denervation 363 by performing Pearson’s test. Subsequently, since baseline
BP predicted the BP change in many previous RDN studies
related to Wilders principle “law of initial value” [24], we
adjusted our univariate approach only to 24-h, day-time and
night-time systolic ABP. A two-sided p value of <0.05 was
considered statistically significant. data and antihypertensive medication as well as physical
examination, standard blood and urine tests. Office and
ambulatory BP were measured with validated devices fol-
lowing the recommendations of the European Society of
Hypertension/European Society of Cardiology [19, 20]. Office BP was assessed after a rest of at least 5 min and
repeated twice in a sitting position with a validated auto-
matic device. Ambulatory BP was conducted with a vali-
dated device (Mobilograph, IEM, Aachen, Germany) and
mean values for 24-h, day-time and night-time ABP were
calculated according to the published recommendations. Estimated glomerular function (eGFR) was calculated using
the Chronic Kidney Disease Epidemiology Collaboration
(CKD-EPI) formula [21]. Adverse events occurring during
the trial were recorded at each visit. Na-MRI measurements At baseline, skin Na content was 20.9 ± 4.3 AU and muscle
Na content was 20.6 ± 4.4 AU. Six months after RDN we
observed no change in skin (20.9 ± 4.3 AU versus baseline
21.1 ± 4.9 AU; p = 0.915) and muscle (20.6 ± 4.4 AU ver-
sus baseline 20.9 ± 4.0 AU; p = 0.683) Na content. At baseline, skin Na content was 20.9 ± 4.3 AU and muscle
Na content was 20.6 ± 4.4 AU. Six months after RDN we
observed no change in skin (20.9 ± 4.3 AU versus baseline
21.1 ± 4.9 AU; p = 0.915) and muscle (20.6 ± 4.4 AU ver-
sus baseline 20.9 ± 4.0 AU; p = 0.683) Na content. Na-MRI measurements Skin and muscle Na content in the left lower leg were
measured non-invasively with a clinical 3.0 T MR system
(Magnetom Skyra, Siemens Healthineers, Erlangen, Ger-
many) using a transit/receive Na RF birdcage knee coil
(32.6 MHz, Stark Contrast, Erlangen, Germany) at baseline
and 6 months after RDN. A detailed description of the
procedure, as well as reliability and accuracy have been
shown previously [9, 23]. RDN procedure A radiofrequency-based Symplicity-Flex catheter (Sympli-
city by Ardian Inc, Palo Alto, CA, USA) was used for RDN
procedure. A renal angiogram was previously performed to
exclude renal artery abnormalities. As previously described
[22], the femoral artery was accessed with standard endo-
vascular technique and renal arteries of both sides were
treated in one session. Up to 6 radiofrequency ablations
(energy delivery for up to 120 s and 8 watts each) were
applied longitudinally and rotationally within each artery to
achieve a full 4-quadrant ablation. Visceral pain during the
procedure was managed with anxiolytics and narcotics and
patients were given 500 IE heparin. Correlation analysis We also did not observe any correlation between
baseline
24-h
systolic
ABP
and
skin
Na
content
(r = −0.070, p = 0.601). heart rate. We also did not observe any correlation between
baseline
24-h
systolic
ABP
and
skin
Na
content
(r = −0.070, p = 0.601). heart rate. We also did not observe any correlation between
baseline
24-h
systolic
ABP
and
skin
Na
content
(r = −0.070, p = 0.601). Correlation analysis triceps surae (AU)
20.6 ± 4.4
Sodium content in skin (AU)
20.9 ± 4.3
Office BP
Office systolic BP (mmHg)
158 ± 23
Office diastolic BP (mmHg)
87 ± 16
Office heart rate (bpm)
71 ± 15
24-h ABP
24-h systolic ABP (mmHg)
157 ± 16
24-h diastolic ABP (mmHg)
87 ± 13
24-h ambulatory heart rate (bpm)
67 ± 12
Day-time systolic ABP (mmHg)
159 ± 16
Day-time diastolic ABP (mmHg)
89 ± 13
Day-time heart rate (bpm)
69 ± 12
Night-time systolic ABP (mmHg)
151 ± 21
Night-time diastolic ABP (mmHg)
80 ± 13
Night-time heart rate (bpm)
62 ± 11
Systolic nocturnal dipping rate (%)
5.1 ± 11.0
Diastolic nocturnal dipping rate (%)
9.9 ± 10.1
Laboratory values
HbA1c (%)
6.6 ± 1.3
Creatinine (mg/dl)
1.2 ± 0.5
eGFR, CKD-Epi formula (mL/min/1.73 m²)
69.5 ± 24.7
Triglyceride
207.2 ± 117.5
Cholesterol (mg/dl)
196.0 ± 52.0
LDL-cholesterol (mg/dl)
131.5 ± 42.0
Hemoglobin (g/dl)
13.9 ± 1.6
Hematocrit (%)
40.6 ± 4.4
Antihypertensive medication
Number of antihypertensive medication, n (%)
6.2 ± 1.6
ACE inhibitors, n (%)
20 (35)
ARBs, n (%)
45 (78)
Direct renin inhibitors, n (%)
25 (43)
Betablockers, n (%)
46 (79)
Calcium-channel blockers, n (%)
49 (85)
Diuretics, n (%)
54 (93)
Table 1 (continued)
Aldosterone antagonists, n (%)
10 (17)
Vasodilators, n (%)
21 (36)
Centrally acting sympatholytics, n (%)
46 (79)
Data are presented as mean ± SD
Na Sodium, Na-MRI Na magnetic resonance imaging, BP blood
pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI
body mass index, LDL low density lipid, HbA1c glycated hemoglobin,
eGFR estimated glomerular filtration rate, CKD-EPI Chronic Kidney
Disease Epidemiology Collaboration, ACE angiotensin-converting
enzyme, ARB angiotensin receptor blocker
Table 2 Change of 24-h ambulatory and office blood pressure 6
months after renal denervation (n = 53)
BP change
6 months
Office systolic BP (mmHg)
−12.9 ± 21.1*
Office diastolic BP (mmHg)
−7.7 ± 12.5*
Office heart rate (bpm)
−3.2 ± 10.0*
24-h systolic ABP (mmHg)
−8.6 ± 14.0*
Day-time systolic ABP (mmHg)
−8.6 ± 14.3*
Night-time systolic ABP (mmHg)
−10.3 ± 22.6*
24-h diastolic ABP (mmHg)
−4.7 ± 9.5*
Day-time diastolic ABP (mmHg)
−4.7 ± 12.5*
Night-time diastolic ABP (mmHg)
−4.6 ± 12.5*
24-h ambulatory heart rate (bpm)
1.1 ± 6.1
Day-time ambulatory heart rate (bpm)
1.4 ± 6.7
Night-time ambulatory heart rate (bpm)
0.5 ± 6.5
Data are presented as mean ± SD
BP blood pressure, ABP ambulatory blood pressure, bpm beats per
minute
*p < 0.05 versus baseline Demographic data
Age (years)
61.6 ± 9.7
Male/female (n/n)
37/14
Body mass index (kg/m2)
30.2 ± 3.9
Weight (kg)
90.0 ± 15.6
Comorbidities
Diabetes mellitus, n (%)
32 (55)
Coronary artery disease, n (%)
20 (35)
Left ventricular hypertrophy, n (%)
15 (26)
Hyperlipidaemia, n (%)
24 (41)
History of stroke, TIA, n (%)
9 (16)
Current smoking, n (%)
9 (16)
Na-MRI
Sodium content in M. Correlation analysis triceps surae (AU)
20.6 ± 4.4
Sodium content in skin (AU)
20.9 ± 4.3
Office BP
Office systolic BP (mmHg)
158 ± 23
Office diastolic BP (mmHg)
87 ± 16
Office heart rate (bpm)
71 ± 15
24-h ABP
24-h systolic ABP (mmHg)
157 ± 16
24-h diastolic ABP (mmHg)
87 ± 13
24-h ambulatory heart rate (bpm)
67 ± 12
Day-time systolic ABP (mmHg)
159 ± 16
Day-time diastolic ABP (mmHg)
89 ± 13
Day-time heart rate (bpm)
69 ± 12
Night-time systolic ABP (mmHg)
151 ± 21
Night-time diastolic ABP (mmHg)
80 ± 13
Night-time heart rate (bpm)
62 ± 11
Systolic nocturnal dipping rate (%)
5.1 ± 11.0
Diastolic nocturnal dipping rate (%)
9.9 ± 10.1
Laboratory values
HbA1c (%)
6.6 ± 1.3
Creatinine (mg/dl)
1.2 ± 0.5
eGFR, CKD-Epi formula (mL/min/1.73 m²)
69.5 ± 24.7
Triglyceride
207.2 ± 117.5
Cholesterol (mg/dl)
196.0 ± 52.0
LDL-cholesterol (mg/dl)
131.5 ± 42.0
Hemoglobin (g/dl)
13.9 ± 1.6
Hematocrit (%)
40.6 ± 4.4
Antihypertensive medication
Number of antihypertensive medication, n (%)
6.2 ± 1.6
ACE inhibitors, n (%)
20 (35)
ARBs, n (%)
45 (78)
Direct renin inhibitors, n (%)
25 (43)
Betablockers, n (%)
46 (79)
Calcium-channel blockers, n (%)
49 (85)
Diuretics, n (%)
54 (93) Na Sodium, Na-MRI Na magnetic resonance imaging, BP blood
pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI
body mass index, LDL low density lipid, HbA1c glycated hemoglobin,
eGFR estimated glomerular filtration rate, CKD-EPI Chronic Kidney
Disease Epidemiology Collaboration, ACE angiotensin-converting
enzyme, ARB angiotensin receptor blocker Table 2 Change of 24-h ambulatory and office blood pressure 6
months after renal denervation (n = 53)
BP change
6 months
Office systolic BP (mmHg)
−12.9 ± 21.1*
Office diastolic BP (mmHg)
−7.7 ± 12.5*
Office heart rate (bpm)
−3.2 ± 10.0*
24-h systolic ABP (mmHg)
−8.6 ± 14.0*
Day-time systolic ABP (mmHg)
−8.6 ± 14.3*
Night-time systolic ABP (mmHg)
−10.3 ± 22.6*
24-h diastolic ABP (mmHg)
−4.7 ± 9.5*
Day-time diastolic ABP (mmHg)
−4.7 ± 12.5*
Night-time diastolic ABP (mmHg)
−4.6 ± 12.5*
24-h ambulatory heart rate (bpm)
1.1 ± 6.1
Day-time ambulatory heart rate (bpm)
1.4 ± 6.7
Night-time ambulatory heart rate (bpm)
0.5 ± 6.5
Data are presented as mean ± SD
BP blood pressure, ABP ambulatory blood pressure, bpm beats per
minute
*p < 0.05 versus baseline heart rate. Correlation analysis Statistical analysis was performed using SPSS Statistics
28.0 (IBM, Armonk, NY, USA) and data were expressed as
mean ± standard deviation (SD) in text and tables. Paired t-
test was applied for the comparison of 6 months follow-up
BP data versus baseline. Predictors of BP change were
assessed by comparing responders versus non-responders
defined by the median reduction of 24-h, day-time and
night-time systolic ABP at 6 month follow-up visit,
respectively. Bivariate correlation analyses were assessed We observed a correlation between baseline 24-h systolic
ABP and BP response 6 months after RDN (r = −0.394,
p = 0.003). In addition, we observed a correlation between
skin sodium content and BP response (r = 0.339, p = 0.013,
Fig. 1). Other than that, we did not observe any correlation
between BP response and other parameters (all p > 0.1). In
particular, no correlation was observed with gender, LDL-
cholesterol, aldosterone-antagonist medication or night-time M. Guenes-Altan et al. 364 heart rate. We also did not observe any correlation between
baseline
24-h
systolic
ABP
and
skin
Na
content
(r = −0.070, p = 0.601). Predictors of BP response
To identify any predictors of BP response after RDN we
splitted the study cohort according to the median systolic
24-h ABP reduction after 6 months into responders and
non-responders
(median ≤−10.0 mmHg
versus > −10.0
mmHg, Table 3). We identified patients with low skin Na,
high BMI, female patients, patients with high LDL-cho-
lesterol,
medication
with
aldosterone
antagonists
and
diuretics, and high night-time heart rate to be more likely to
respond to RDN with >10 mmHg fall in 24-h ABP after
6 months. After adjustment for baseline 24-h systolic ABP,
skin Na, gender, LDL-cholesterol, night-time heart rate and
aldosterone-antagonist medication remained significantly
different between responders and non responders (Table 3)
Table 1 Clinical characteristics
Demographic data
Age (years)
61.6 ± 9.7
Male/female (n/n)
37/14
Body mass index (kg/m2)
30.2 ± 3.9
Weight (kg)
90.0 ± 15.6
Comorbidities
Diabetes mellitus, n (%)
32 (55)
Coronary artery disease, n (%)
20 (35)
Left ventricular hypertrophy, n (%)
15 (26)
Hyperlipidaemia, n (%)
24 (41)
History of stroke, TIA, n (%)
9 (16)
Current smoking, n (%)
9 (16)
Na-MRI
Sodium content in M. Predictors of BP response To identify any predictors of BP response after RDN we
splitted the study cohort according to the median systolic
24-h ABP reduction after 6 months into responders and
non-responders
(median ≤−10.0 mmHg
versus > −10.0
mmHg, Table 3). We identified patients with low skin Na,
high BMI, female patients, patients with high LDL-cho-
lesterol,
medication
with
aldosterone
antagonists
and
diuretics, and high night-time heart rate to be more likely to
respond to RDN with >10 mmHg fall in 24-h ABP after
6 months. After adjustment for baseline 24-h systolic ABP,
skin Na, gender, LDL-cholesterol, night-time heart rate and
aldosterone-antagonist medication remained significantly
different between responders and non-responders. (Table 3) Skin sodium content as a predictor of blood pressure response to renal denervation 365 Fig. 1 Correlation between skin sodium content at baseline and 24-h systolic ambulatory blood pressure change 6 months after renal denervation skin sodium content at baseline and 24-h systolic ambulatory blood pressure change 6 months after renal denervation Fig. 1 Correlation between skin sodium content at baseline and 24-h systolic ambulatory blood pressure change 6 of initial value (Wilder’s principle) [24, 26–34]. In our
study we focused on the importance of skin Na content,
assessed by Na-MRI, in 58 patients with TRH as a potential
predictor of the BP response after RDN. The skin, like the
muscles, is an important storage of extracellular Na content
[16]. Na accumulation in tissue may exaggerate hyper-
trophy of the myocardial and vascular smooth muscle cells
and thereby augment HTN associated complications [9, 16]. It is known that patients with TRH achieve a greater
reduction in BP by restricting salt compared to other
hypertensive patients and that patients with TRH are more
sensitive to salt intake [35]. However, the effect of RDN on
sodium homeostasis is not well understood. Ott et al. per-
formed Na-MRI in 41 patients who had underwent RDN
and observed no change in tissue Na content at the second
examination after 6 months, in accordance with the results
of the current study [18]. In contrast, a post-hoc analysis of
137 patients showed an increase in urinary Na in patients
with TRH 6 months after RDN, but the reliability of the
measurements were questionable as they were based on the
Kawasaki formula and spot urine [36]. Predictors of BP response Experimental animal
studies have also shown an increased Na excretion in the
acute stage after RDN and a decrease in Na excretion after
renal sympathetic nerve stimulation [37–39]. We repeated the whole analysis by dividing patients into
2 groups based on the median of day-time and night-time BP
change after 6 months, respectively. Besides systolic ABP,
skin Na, aldosterone-antagonist medication, baseline 24-h,
day-time and night-time heart rate remained significantly
different between responders and non-responders (Tables 4
and 5). Age, body mass index (BMI) and renal function (i.e. eGFR) were not identified as determinants of BP response. Thus, lower skin sodium content (together with a non-
significant signal for muscle sodium content) emerged as a
BP independent predictor of BP fall after RDN. Discussion In our study we performed a post-hoc analysis of 58 patients
with uncontrolled TRH who underwent RDN and Na-MRI. We
observed
a
reduction
of
office
BP
by
−12.9/
−7.7 mmHg and 24-h ABP by −8.6/−4.7 mmHg 6 months
after RDN. Considering BP reduction, our results are con-
sistent with the results of the Global Symplicity Registry. In
this
worldwide
registry,
office
BP
decreased
by
−11.6/−4.3 mmHg and 24-h ABP decreased by −6.6/
−3.9 mmHg 6 months after RDN [25]. From a clinical perspective, it is important to identify
potential predictors for the efficacy of RDN procedure as
guidance to personalize treatment options in hypertension. Many studies have uniformly identified a high baseline
systolic ABP as a predictor for good BP response after
RDN, but this phenomenon is unspecific and known as law In this study we focused on a potential role of Na as a
predictor of the BP drop after RDN. We restricted our
analysis to ABP measurements following the current con-
sensus statement [11, 12]. According to the change of
systolic 24-h, day-time and night-time ABP 6 months
after RDN we splitted the study cohort into responders and 366 M. Guenes-Altan et al. non-responders by the respective median drop in ABP We
according to median decrease of the following BP para-
Table 3 Univariate analysis of responders versus non-responders at 6 months based on the median of the change of 24-h systolic ABP
Baseline
Median ≤−10 mmHg
Median > −10 mmHg
P value
P value*
Responder (n = 27)
Non-Responder (n = 26)
Change of systolic ABP at
6 months (mmHg)
−18.7 ± 8.4
0.2 ± 8.2
<0.001
<0.001
Baseline characteristics
BMI (kg/m2)
31.2 ± 4.2
28.8 ± 3.3
0.024
0.066
Gender (m/f)
17/10
23/3
0.031
0.027
Age (years)
60.4 ± 10.4
62.7 ± 8.4
0.386
0.389
Na-MRI
Sodium in M. Discussion triceps surae (AU)
19.5 ± 3.3
21.6 ± 5.3
0.095
0.111
Sodium in skin (AU)
19.7 ± 3.3
22.2 ± 5.1
0.042
0.040
Baseline office BP
Systolic BP (mmHg)
156.5 ± 16.9
152.2 ± 23.2
0.448
Diastolic BP (mmHg)
88.1 ± 16.6
83.6 ± 12.2
0.268
Heart rate (bpm)
73.1 ± 13.9
65.6 ± 14.0
0.059
0.086
Baseline ABP
24-h systolic BP (mmHg)
158.9 ± 12.5
151.8 ± 15.8
0.077
24-h diastolic BP (mmHg)
89.2 ± 12.9
83.7 ± 10.1
0.090
24-h heart rate (bpm)
69.2 ± 12.3
64.2 ± 9.8
0.121
0.124
Systolic day-time BP (mmHg)
161.1 ± 13.0
154.3 ± 15.5
0.091
Diastolic day-time BP (mmHg)
91.8 ± 12.9
86.2 ± 10.3
0.089
Heart rate day-time (bpm)
70.4 ± 11.7
65.8 ± 10.6
0.162
0.159
Systolic night-time BP (mmHg)
153.7 ± 17.1
145.2 ± 24.3
0.145
Diastolic night-time BP (mmHg)
83.1 ± 13.4
77.0 ± 12.7
0.099
Night-time heart rate (bpm)
65.3 ± 12.8
58.9 ± 8.0
0.045
0.047
Laboratory values
Hematocrit (%)
41.9 ± 3.8
39.6 ± 4.9
0.059
0.102
LDL-cholesterol (mg/dl)
150.6 ± 39.1
114.7 ± 37.0
0.001
<0.001
Antihypertensive medication
Diuretics, n (%)
23 (85.2)
26 (100)
0.041
0.053
Aldosterone-antagonist, n (%)
7 (25.9)
1 (3.8)
0.025
0.032
ACE inhibitors, n (%)
10 (37)
9 (35)
0.854
0.699
ARBs, n (%)
21(78)
21 (81)
0.788
0.691
Direct renin inhibitors, n (%)
14 (52)
8 (31)
0.119
0.182
Betablockers, n (%)
21 (78)
21 (81)
0.788
0.971
Calcium-channel blockers, n (%)
23 (85)
22 (85)
0.954
0.896
Vasodilators, n (%)
9 (33)
10 (38)
0.697
0.608
Centrally acting sympatholytics, n (%)
21 (78)
20 (77)
0.941
0.824
Data are presented as mean ± SD. Discussion Bold values represent p-values ≤0.05
Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker
*p = adjusted for baseline 24-h systolic BP ysis of responders versus non-responders at 6 months based on the median of the change of 24-h systolic ABP Table 3 Univariate analysis of responders versus non-responders at 6 months based on the median of the chang Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker
*p = adjusted for baseline 24-h systolic BP according to median decrease of the following BP para-
meters: 24-h ABP, day-time ABP and night-time ABP. With respect to muscle Na content, a lower content tended
to be related to better ABP response. Thus, baseline skin Na
content (and to lesser extent muscle sodium content) non-responders by the respective median drop in ABP. We
found patients with low skin Na content to be more likely to
respond to RDN than those with high baseline skin Na. This
result persisted after adjustment for baseline 24-h systolic
BP and were also found after separating the study cohort Skin sodium content as a predictor of blood pressure response to renal denervation 367 emerged as a predictor for systolic ABP response to RDN in
addition to and independent from baseline systolic ABP
populations, such as in patients with T2D, hypertension
Conn’s syndrome and CKD [17 23 40 41] We previously
Table 4 Univariate analysis of responders versus non-responders at 6 months based on the median of the change of day-time systolic ABP
Baseline
Median ≤−12 mmHg
Median > −12 mmHg
P value
P value*
Responder (n = 27)
Non-Responder (n = 26)
Change of day-time systolic BP at
6 months (mmHg)
−19.7 ± 7.6
1.8 ± 9.1
<0.001
<0.001
Baseline characteristics
BMI (kg/m2)
30.7 ± 4.2
29.3 ± 3.7
0.182
0.392
Gender (m/w)
0.389
0.256
Age (years)
59.5 ± 10.1
63.7 ± 8.2
0.106
0.089
Na-MRI
Sodium in M. Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker
*p = adjusted for baseline 24-h day-time systolic BP Data are presented as mean ± SD. Bold values represent p-values ≤0.05 Discussion triceps surae (AU)
19.5 ± 3.0
21.6 ± 5.5
0.074
0.081
Sodium in skin (AU)
19.6 ± 2.6
22.4 ± 5.4
0.021
0.015
Baseline office BP
Systolic BP (mmHg)
152.4 ± 17.4
156 ± 22.8
0.481
Diastolic BP (mmHg)
87.1 ± 16.3
84.7 ± 12.9
0.550
Heart rate (bpm)
73.2 ± 14.5
65.6 ± 13.3
0.056
0.150
Baseline ABP
24-h systolic BP (mmHg)
159.8 ± 13.5
150.8 ± 14.5
0.023
24-h diastolic BP (mmHg)
89.2 ± 12.1
83.6 ± 11.0
0.081
24-h heart rate (bpm)
70.5 ± 12.1
63.0 ± 9.2
0.019
0.025
Systolic day-time BP (mmHg)
162.5 ± 13.8
152.8 ± 13.9
0.014
Diastolic day-time BP (mmHg)
91.9 ± 12.1
86.1 ± 11.2
0.074
Heart rate day-time (bpm)
71.8 ± 12.0
64.5 ± 9.6
0.025
0.030
Systolic night-time BP (mmHg)
153.6 ± 19.8
145.3 ± 22.1
0.153
Diastolic night-time BP (mmHg)
82.6 ± 13.4
77.5 ± 13.0
0.159
Night-time heart rate (bpm)
66.1 ± 12.2
58.1 ± 8.2
0.010
0.012
Laboratory values
Hematocrit (%)
40.9 ± 4.1
40.7 ± 4.9
0.883
0.759
Triglyceride
233.6 ± 119.2
76.7 ± 104.7
0.071
0.071
Cholesterol (mg/dl)
204.3 ± 43.9
189.4 ± 58.7
0.299
0.286
LDL-cholesterol (mg/dl)
140.6 ± 36.0
125.0 ± 46.4
0.178
0.161
Antihypertensive medication
Diuretics, n (%)
24 (88.9)
25 (96.2)
0.317
0.432
Aldosterone-antagonist, n (%)
7 (25.9)
1 (3.8)
0.025
0.036
ACE inhibitors, n (%)
10 (37.0)
9 (34.6)
0.854
0.583
ARBs, n (%)
21 (77.8)
21 (80.8)
0.788
0.681
Direct renin inhibitors, n (%)
14 (51.9)
8 (30.8)
0.119
0.201
Betablockers, n (%)
20 (74.1)
22 (84.6)
0.344
0.596
Calcium-channel blockers, n (%)
23 (85.2)
22 (84.6)
0.954
0.961
Vasodilators, n (%)
11 (40.7)
8 (30.8)
0.449
0.619
Centrally acting sympatholytics, n (%)
21 (77.8)
20 (76.9)
0.941
0.746
Data are presented as mean ± SD. Bold values represent p-values ≤0.05
Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker
*p = adjusted for baseline 24-h day-time systolic BP of responders versus non-responders at 6 months based on the median of the change of day-time systolic ABP Table 4 Univariate analysis of responders versus non-responders at 6 months based on the median of the chang Data are presented as mean ± SD. Discussion Bold values represent p-values ≤0.05 Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker
*p = adjusted for baseline 24-h day-time systolic BP populations, such as in patients with T2D, hypertension
Conn’s syndrome and CKD [17, 23, 40, 41]. We previously
showed that high tissue sodium content is linked to emerged as a predictor for systolic ABP response to RDN in
addition to and independent from baseline systolic ABP. High tissue sodium was observed in several diseased 368 M. Guenes-Altan et al. Table 5 Univariate analysis of responders versus non-responders at 6 months based on the median of the change of night-time systolic ABP
Baseline
Median ≤−13 mmHg
Median > −13 mmHg
P value
P value*
Responder (n = 27)
Non-Responder (n = 26)
Change of night-time systolic BP at
6 months (mmHg)
−26.6 ± 11.1
3.2 ± 11.9
<0.001
<0.001
Baseline characteristics
BMI (kg/m2)
30.5 ± 4.6
29.5 ± 3.2
0.338
0.931
Gender (m/w)
18/9
22/4
0.129
0.150
Age (years)
60.7 ± 10.0
62.4 ± 8.9
0.524
0.351
Na-MRI
Sodium in M. Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker *p = adjusted for baseline 24-h night-time systolic BP Discussion triceps surae (AU)
19.8 ± 3.5
21.2 ± 5.3
0.257
0.251
Sodium in skin (AU)
19.7 ± 3.2
22.2 ± 5.1
0.038
0.016
Baseline office BP
Systolic BP (mmHg)
157.3 ± 21.1
151.4 ± 19.1
0.290
Diastolic BP (mmHg)
89.4 ± 13.5
82.3 ± 15.1
0.078
Heart rate(bpm)
71.3 ± 12.8
67.6 ± 15.9
0.350
0.133
Baseline ABP
24-h systolic BP (mmHg)
160.7 ± 13.2
149.9 ± 14.0
0.005
24-h diastolic BP (mmHg)
90.4 ± 11.9
82.4 ± 10.5
0.011
24-h heart rate (bpm)
69.9 ± 11.2
63.6 ± 10.6
0.053
0.004
Systolic day-time BP (mmHg)
160.4 ± 13.3
155.0 ± 15.6
0.183
Diastolic day-time BP (mmHg)
91.4 ± 11.9
86.7 ± 11.7
0.148
Heart rate day-time (bpm)
70.6 ± 10.9
65.5 ± 11.3
0.117
0.017
Systolic night-time BP (mmHg)
159.9 ± 17.8
138.8 ± 19.1
<0.001
Diastolic night-time BP (mmHg)
86.6 ± 12.5
73.4 ± 10.7
<0.001
Night-time heart rate (bpm)
65.7 ± 11.6
58.5 ± 9.3
0.021
0.003
Laboratory values
eGFR, CKD-EPI (ml/min/1.73 m²)
74.9 ± 25.3
62.8 ± 21.2
0.065
0.129
Hematocrit (%)
41.1 ± 3.5
40.4 ± 5.3
0.574
0.668
Triglyceride
218.8 ± 125.8
192.1 ± 103.0
0.403
0.291
Cholesterol (mg/dl)
201.9 ± 45.8
191.9 ± 57.8
0.487
0.265
LDL-cholesterol (mg/dl)
137.6 ± 34.7
128.2 ± 48.3
0.416
0.165
Antihypertensive medication
Diuretics, n (%)
24 (88.9)
25 (96.2)
0.317
0.303
Aldosterone-antagonist, n (%)
5 (18.5)
3 (11.5)
0.478
0.443
ACE inhibitors, n (%)
10 (37.0)
9 (34.6)
0.854
0.720
ARBs, n (%)
21 (77.8)
21 (80.8)
0.788
0.431
Direct renin inhibitors, n (%)
13 (48.1)
9 (34.6)
0.318
0.409
Betablockers, n (%)
19 (70.4)
23 (88.5)
0.104
0.124
Calcium-channel blockers, n (%)
22 (81.5)
23 (88.5)
0.478
0.747
Vasodilators, n (%)
8 (29.6)
11 (42.3)
0.336
0.243
Centrally acting sympatholytics, n (%)
22 (81.5)
19 (73.1)
0.465
0.841
Data are presented as mean ± SD. Bold values represent p-values ≤0.05
Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker
*p = adjusted for baseline 24-h night-time systolic BP of responders versus non-responders at 6 months based on the median of the change of night-time systolic ABP Table 5 Univariate analysis of responders versus non-responders at 6 months based on the median of the chang Data are presented as mean ± SD. *p = adjusted for baseline 24-h night-time systolic BP Data are presented as mean ± SD. Bold values represent p-values ≤0.05 night-time systolic BP *p = adjusted for baseline 24-h night-time systolic BP Discussion Bold values represent p-values ≤0.05 Na sodium, Na-MRI Na magnetic resonance imaging, BP blood pressure, bpm beats per minute, ABP ambulatory blood pressure, BMI body mass
index, LDL low density lipid, ACE angiotensin-converting enzyme, ARB angiotensin receptor blocker Skin sodium content as a predictor of blood pressure response to renal denervation 369 hypertrophic vascular remodeling and to left ventricular
hypertrophy [40, 42]. Thus, we assume that patients with a
higher skin Na content may be in a more advanced stage of
arterial hypertension with greater extent of hypertrophic
vascular remodeling and are therefore less likely to respond
to renal denervation. Despite BP reduction, RDN may have pleiotropic effects. We defined non-responders and responders only by their BP
change. However, there may be positive effects of RDN
beyond BP reduction also in patients defined as non-
responders. Our study has several limitations. It is a single-center and
post-hoc study with a small sample size with a limited
follow-up of 6 months. However, 24-h, day-time and night-
time systolic BP reduction demonstrated consistent results,
with low skin Na being related to greater BP reduction after
RDN. Though not significant, a similar signal has been
found for baseline muscle content even after adjustment for
baseline 24-h ABP. Nevertheless, only prospective studies
with
longer
follow-up
are
able
to
corroborate
our
observations. Our findings also suggest that gender, LDL cholesterol,
aldosterone-antagonist
medication,
24-h,
day-time
and
night-time heart rate may play a role in determining BP
response to RDN. Böhm et al. identified in the SPYRAL
HTN-OFF MED pivotal trial a high baseline heart rate to
determine the effectiveness of RDN [43]. In contrast, Esler
et al. found no correlation between HR and renal sympa-
thetic activation but only between HR and cardiac sympa-
thetic activation [44]. It was previously shown that RDN
reduces cardiac sympathetic activity [45]. We identified 24-
h, day-time and night-time HR to be a predictive parameter
for good BP response after RDN and this result remained
significant after adjustment for baseline 24-h systolic ABP. However, we could not identify any correlation between
24-h, day-time or night-time heart rate and BP response. In
addition, we identified patients with aldosterone-antagonist
medication use to have a better BP response to RDN. Interestingly aldosterone-antagonist medication has also
been identified as a predictor in the Symplicity HTN 3 trial
[27]. Data availability The datasets used and analyzed for this register are available
from the corresponding author on reasonable request. Acknowledgements We gratefully acknowledge the expert technical
assistance of Dorothea Bader-Schmieder, Ingrid Fleischmann, Kerstin
Fröhlich-Endreß, Ulrike Heinritz, Simone Pejkovic, Wiebke Maurer
and Theresa Federlein. Despite identifying that female patients were more likely
in the responder group, we did not find any correlation
between sex and BP response. Funding Open Access funding enabled and organized by Projekt
DEAL. Several studies also analyzed vascular parameters to
identify potential predictors for BP response after RDN. Weber et al. identified pulsatile hemodynamics such as
augmentation index, augmentation pressure and estimated
aortic pulse wave velocity to be potential predictors for BP
response after RDN [49]. In accordance, other studies
analyzed the influence of invasively measured pulse wave
velocity on BP response after RDN and showed that
patients with lower pulse wave velocity at baseline were
more likely to be responders to RDN [50, 51]. Additionally,
Fengler
et
al. identified
cardiac
magnetic
resonance
assessment of central and peripheral vascular function as a
potential predictor of RDN [52]. In accordance, we pre-
viously identified lower baseline central pulse pressure
(measured non-invasively with SphygmoCor™device),
indicative for the degree of arterial stiffening, to be a pre-
dictive value for better BP response after RDN [53]. Conclusion Our findings suggest that patients with low skin Na have a
greater BP reduction after RDN, independent of baseline
ABP. Utilizing Na-MRI prior to RDN might provide a
valuable tool in selecting patients with TRH that have a BP
reduction after RDN above the median and thereby being
particularly suitable for the procedure. Discussion In
accordance,
a
single-center
trial
identified
aldosterone-antagonist medication in patients with TRH to
be predictive for better BP response after RDN [46]. However, other studies could not identify any relationship
between
aldosterone-antagonist
medication
and
BP
response after RDN [47, 48]. We also assessed correlation
between aldosterone-antagonist medication use and BP
response but did not observe a correlation. References The SGLT2 inhibitor empagliflozin reduces tissue sodium
content in patients with chronic heart failure: results from a
placebo-controlled
randomised
trial. Clin
Res
Cardiol. 2023;112:134–44. 26. Vogel B, Kirchberger M, Zeier M, Stoll F, Meder B, Saure D,
et al. Renal sympathetic denervation therapy in the real world:
results
from
the
Heidelberg
registry. Clin
Res
Cardiol. 2014;103:117–24. 10. Dörr O, Ewen S, Liebetrau C, Möllmann H, Gaede L, Linz D,
et al. Neuropeptide Y as an indicator of successful alterations in
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Medical, Ablative Solutions. Speaker and Adviser bureau: Medtronic,
Recor Medical, Ablative Solutions. Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons
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adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate if
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Quantitative EEG as a biomarker in mild cognitive impairment with Lewy bodies
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Alzheimer's research & therapy
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Abstract Objectives: To investigate using quantitative EEG the (1) differences between patients with mild cognitive
impairment with Lewy bodies (MCI-LB) and MCI with Alzheimer’s disease (MCI-AD) and (2) its utility as a potential
biomarker for early differential diagnosis. Methods: We analyzed eyes-closed, resting-state, high-density EEG data from highly phenotyped participants (39
MCI-LB, 36 MCI-AD, and 31 healthy controls). EEG measures included spectral power in different frequency bands
(delta, theta, pre-alpha, alpha, and beta), theta/alpha ratio, dominant frequency, and dominant frequency variability. Receiver operating characteristic (ROC) analyses were performed to assess diagnostic accuracy. Results: There was a shift in power from beta and alpha frequency bands towards slower frequencies in the pre-
alpha and theta range in MCI-LB compared to healthy controls. Additionally, the dominant frequency was slower in
MCI-LB compared to controls. We found significantly increased pre-alpha power, decreased beta power, and slower
dominant frequency in MCI-LB compared to MCI-AD. EEG abnormalities were more apparent in MCI-LB cases with
more diagnostic features. There were no significant differences between MCI-AD and controls. In the ROC analysis
to distinguish MCI-LB from MCI-AD, beta power and dominant frequency showed the highest area under the curve
values of 0.71 and 0.70, respectively. While specificity was high for some measures (up to 0.97 for alpha power and
0.94 for theta/alpha ratio), sensitivity was generally much lower. Conclusions: Early EEG slowing is a specific feature of MCI-LB compared to MCI-AD. However, there is an overlap
between the two MCI groups which makes it difficult to distinguish between them based on EEG alone. Keywords: Dementia with Lewy bodies, Alzheimer’s disease, Quantitative electroencephalography, Biomarker Keywords: Dementia with Lewy bodies, Alzheimer’s disease, Quantitative electroencephalograp Quantitative EEG as a biomarker in mild
cognitive impairment with Lewy bodies Julia Schumacher1*
, John-Paul Taylor1, Calum A. Hamilton1, Michael Firbank1, Ruth A. Cromarty1,
Paul C. Donaghy1, Gemma Roberts1, Louise Allan1,3, Jim Lloyd2, Rory Durcan1, Nicola Barnett1, John T. O’Brien4 and
Alan J. Thomas1 * Correspondence: julia.schumacher@newcastle.ac.uk
1Translational and Clinical Research Institute, Faculty of Medical Sciences,
Newcastle University, Biomedical Research Building 3rd floor, Campus for
Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK
Full list of author information is available at the end of the article Background stratification for clinical trials and research studies and
for optimizing clinical care such as by highlighting the
need to carefully avoid using medication with anticholin-
ergic properties and encouraging active identification of
therapeutic targets such as orthostatic hypotension and
constipation [1]. The early diagnosis of dementia is becoming increasingly
important as it is likely that potential disease-modifying
treatments will have their greatest effect at this stage. Additionally, accurate and early differential diagnosis of
dementia
subtypes
is
crucial
for
correct
patient The intermediate stage between normal aging and de-
mentia in which cognitive decline is present, but inde-
pendence in activities of daily living is still preserved, is
referred to as mild cognitive impairment (MCI) [2, 3]. While much research has focused on MCI patients who * Correspondence: julia.schumacher@newcastle.ac.uk
1Translational and Clinical Research Institute, Faculty of Medical Sciences,
Newcastle University, Biomedical Research Building 3rd floor, Campus for
Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK
Full list of author information is available at the end of the article * Correspondence: julia.schumacher@newcastle.ac.uk
1Translational and Clinical Research Institute, Faculty of Medical Sciences,
Newcastle University, Biomedical Research Building 3rd floor, Campus for
Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK
Full list of author information is available at the end of the article Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82
https://doi.org/10.1186/s13195-020-00650-1 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82
https://doi.org/10.1186/s13195-020-00650-1 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 2 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 2 of 12 later develop Alzheimer’s disease (AD), MCI in the con-
text of dementia with Lewy bodies (DLB) has only been
characterized more recently with research criteria re-
cently published [4–6]. Differential diagnosis is often im-
peded by a substantial clinical overlap between AD and
DLB, which is especially pronounced in early stages,
highlighting the need for objective biomarkers. In DLB,
dopaminergic brain imaging and myocardial scintigraphy
are indicative biomarkers [7]; these can improve diag-
nostic accuracy at the MCI stage [8] and are included in
the new research criteria for MCI with Lewy bodies
(MCI-LB). EEG has been suggested as a non-invasive al-
ternative, and an abnormal EEG is listed as a supportive
biomarker in the DLB diagnostic criteria [7]. However,
its value for differential diagnosis at the MCI stage is less
certain. based on the clinical history and research assessments. In
addition to a detailed clinical assessment, participants had
already undergone dopaminergic imaging with
123I-N-
fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl)
single-
photon emission computed tomography (FP-CIT SPECT)
and 123iodine-metaiodobenzylguanidine (MIBG) myocar-
dial scintigraphy through their involvement in an ongoing
study investigating the diagnostic accuracy of imaging bio-
markers in MCI, and this information was used to apply
diagnostic criteria (see below). After the initial baseline visit, participants were followed
annually for up to 3 years with a mean follow-up time of
16.5 months (standard deviation = 6.5 months). Patients
who were taking dopaminergic medication were assessed
in the “ON” motor state. The aims of the present study were therefore to inves-
tigate changes in quantitative EEG measures in a large
cohort of well-characterized patients, all of whom had
biomarker assessments with both dopaminergic and car-
diac MIGB imaging, and to test the utility of EEG as a
biomarker for early differential diagnosis. Diagnosis Diagnoses were based on all information available at the
end of the recruitment period (December 2019) includ-
ing any baseline and follow-up visits. MCI diagnoses
were made independently by a consensus panel of three
experienced old-age psychiatrists (AJT, PCD, JPT) in ac-
cordance with NIA-AA criteria [3], i.e., subjective and
objective cognitive impairment with maintained inde-
pendence of function with minimal aids or assistance
and a CDR of 0 or 0.5. Patients with a diagnosis of
dementia were excluded from the study. Furthermore,
patients with possible contributing frontotemporal or
vascular etiologies or with a history of parkinsonism of
more than 1 year prior to the onset of cognitive impair-
ment were excluded. The presence or absence of the
core Lewy body symptoms was rated by the panel utiliz-
ing the rating scales and all information from the clinical
assessments [7]. Findings from the FP-CIT and MIBG
scans were used for diagnosis (see below), but the clin-
ical MCI diagnoses as well as the rating of the presence/
absence of core DLB symptoms were performed blind to
these imaging findings. The secondary aim was to better understand the rela-
tionship between EEG abnormalities in the MCI-LB
group and the core Lewy body symptoms. We hypothe-
sized that EEG abnormalities would be more severe in
patients with more core symptoms. Participants All participants in this study were over 60 years of age. Patients with a clinical diagnosis of MCI in memory ser-
vices in the north east of England were screened and
approached for this study if they reported additional
clinical symptoms suggestive of Lewy body disease (e.g.,
mood changes, sleep disturbances, or autonomic symp-
toms) or any core DLB features (visual hallucinations,
cognitive fluctuations, parkinsonism, and REM sleep
behavior disorder). After obtaining written informed
consent, participants underwent a baseline clinical as-
sessment including the Addenbrooke’s Cognitive Exam-
ination–Revised (ACE-R), from which the Mini-Mental
State Examination (MMSE) score was derived; the Uni-
fied Parkinson’s Disease Rating Scale motor sub-score
(UPDRS-III); the Epworth Sleepiness Scale (ESS); and
the Geriatric Depression Scale (GDS). Additionally, the
Instrumental Activities of Daily Living (IADL) Scale, the
Clinician Assessment of Fluctuations (CAF), the Dementia
Cognitive Fluctuations Scale (DCFS), the North-East Vis-
ual Hallucinations Interview (NEVHI), the Neuropsychi-
atric Inventory (NPI), and the Mayo Sleep Questionnaire
(MSQ) were administered to informants, and the Clinical
Dementia Rating (CDR) Scale and the Cumulative Illness
Rating Scale for Geriatrics (CIRS-G) were completed A diagnosis of MCI with probable Alzheimer’s disease
(MCI-AD) was given to patients who had no core Lewy
body symptoms, negative FP-CIT and MIBG findings,
and evidence of cognitive decline that was characteristic
of AD, i.e., they met the additional NIA-AA criterion for
“etiology of MCI consistent with AD pathophysiologic
process” [3]. Probable MCI with Lewy bodies (MCI-LB) was diag-
nosed if a patient had two or more core Lewy body
symptoms or one core symptom in addition to a positive
FP-CIT or MIBG scan [6]. Out of 103 MCI participants who were included in the
study, 20 had only one core Lewy body symptom with
negative FP-CIT and MIBG scans or no core symptoms
in addition to a positive FP-CIT or MIBG scan, i.e., they
did not meet the criteria for either MCI-AD or probable Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 3 of 12 Schumacher et al. Alzheimer's Research & Therapy Fig. 1 Study flowchart MCI-LB and were therefore not included in the present
analysis (Fig. 1). Additionally, eight MCI participants did
not have usable EEG data available. This study therefore
included 39 participants who were diagnosed with prob-
able MCI-LB and 36 who were diagnosed with MCI-AD. Participants Healthy control participants (N = 31) were recruited
from relatives and friends of patients and from a local
research register. Control participants had the same as-
sessment as the patients and had no history of psychi-
atric or neurological illness and no evidence of any
cognitive decline. They also had normal structural MR
imaging. The study was approved by the local ethics
committee, and written informed consent was obtained
from all participants. EEG technician during the recording to monitor adher-
ence to the protocol. The ground electrode was attached
to the right clavicle, and all EEG channels were refer-
enced to Fz during recording. Pre-processing of eyes-closed EEG data was performed
using the EEGLAB toolbox (version 14) in MATLAB
(R2017a) [9] and was blinded to group membership. First, EEG data were bandpass-filtered between 0.3 and
54 Hz using a second-order Butterworth filter and split
into non-overlapping epochs of 2 s. Subsequently, EEG
recordings were visually inspected to identify noisy
channels and noisy epochs which were excluded prior to
applying independent component analysis for further
artifact removal. The resulting components were visually
inspected, and components representing muscular, car-
diac, ocular, or electrical (50 Hz line noise) artifacts were
rejected. The previously excluded channels were then re-
placed using spherical spline interpolation, and data
were recomputed against the average reference. For each
participant, the first 45 2-s-long artifact-free epochs were
selected for further analysis. EEG acquisition and pre-processing Resting-state high-density EEG recordings were acquired
from all participants using Waveguard caps (ANT
Neuro, The Netherlands) comprising 128 sintered Ag/
AgCl electrodes that were placed according to the 10-5
system. Participants were seated during the recording
and instructed to remain awake. Electrode impedance
was kept below 5 kΩ, and continuous EEG data were re-
corded at a sampling frequency of 1024 Hz. Three hun-
dred seconds of eyes-closed data were recorded from
each participant. Participants were supervised by the Frequency analysis For each 2-s epoch, the power spectral density (PSD)
was estimated using Bartlett’s method in MATLAB
(R2017a) with a frequency resolution of 0.5 Hz and a Page 4 of 12 Page 4 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 the different EEG measures to distinguish between MCI-
AD and MCI-LB patients. The sensitivity/specificity cut-
off was determined using Youden’s index. Hamming window across the power spectrum from 2 to
30 Hz, for each electrode separately. To compensate for
inter-individual
variability
in
brain
neurophysiology,
anatomy, and physical tissue properties, the PSD was
normalized
by
the
total
power
across
the
power
spectrum [10]. To assess the association between the range of Lewy
body symptomatology and EEG abnormalities, we per-
formed an exploratory analysis in which we compared
quantitative EEG measures between MCI-LB patients
who had two core symptoms or one core symptom and
one abnormal biomarker (N = 13) with those patients
who had more than two core symptoms/abnormal bio-
markers (N = 26) using two-sample t tests. Furthermore,
Spearman’s correlations between symptom/biomarker
count (ranging from 2 to 6) and the different EEG mea-
sures were computed in the MCI-LB group. For each electrode separately, the mean power across all
included epochs was estimated for different standard EEG
frequency bands including delta (2–4 Hz), theta (4–5.5
Hz), pre-alpha (5.5–8 Hz), alpha (8–13 Hz), and beta (13–
30 Hz). Higher frequencies were excluded because these
are particularly affected by muscular artifacts [11]. The
dominant frequency was calculated as the frequency with
the highest power between 4 and 15 Hz (averaged across
epochs). Dominant frequency was calculated for all elec-
trodes as well as from occipital electrodes only (PO9,
PO7, POO9h, PO5, O1, PO3, POO3h, OI1h, POz, Oz,
PO4, POO4h, PO6, O2, OI2h, PO8, POO10h, PO10). Dominant frequency variability was defined as the stand-
ard deviation of dominant frequency across epochs [12]. The association between quantitative EEG measures
and overall cognitive impairment was assessed using
Spearman’s correlations, in the MCI-AD and MCI-LB
groups separately. p values were FDR-corrected for mul-
tiple comparisons. Additionally, we investigated the association between
EEG characteristics and the core Lewy body symptoms
of visual hallucinations and cognitive fluctuations which
have been shown to be related to EEG abnormalities in
dementia patients [10, 13, 14]. Frequency analysis To this end, two-sample t
tests were performed, dichotomizing the MCI-LB group
according to the presence/absence of visual hallucina-
tions and cognitive fluctuations. Statistics Statistical analyses were performed in SPSS and R
(https://www.r-project.org/). Relative power within the
different frequency bands was compared between the
three groups using a multivariate ANOVA with a
within-subject factor of frequency band and a between-
subject factor of diagnosis, followed by univariate ANO-
VAs and post hoc tests, Bonferroni-corrected for mul-
tiple
comparisons. Since
relative
power
was
not
normally distributed in all groups, the variables were
log-transformed before applying the ANOVA. Theta/
alpha ratio, dominant frequency, and dominant fre-
quency variability were compared between the groups
using univariate ANOVAs followed by post hoc tests
(Bonferroni-corrected). To account for differences in the
number of male and female participants in the three
groups, sex was included as a covariate in all analyses. Given previous reports of an effect of acetylcholin-
esterase inhibitors on the EEG signal [15, 16], a two-
sample t test was performed in the MCI-LB group to
compare the EEG characteristics between patients who
were taking acetylcholinesterase inhibitors and those pa-
tients not taking these medications. Demographics All groups were similar in age (Table 1). The proportion
of male participants was higher in the MCI-LB group
whereas more MCI-AD patients were female. MCI-LB
patients had significantly lower years of education com-
pared to controls, but there was no significant difference
between the two MCI groups in terms of education. More MCI-LB patients were taking cholinesterase inhib-
itors and Parkinson’s disease medication compared to
MCI-AD patients. The two MCI groups were matched
in terms of overall cognitive impairment. As expected,
the MCI-LB group had more parkinsonism, higher cog-
nitive fluctuation, and visual hallucination scores com-
pared to the MCI-AD group. To test the applicability of this analysis to a clinical
setting, the group comparison of relative power within
the different frequency bands was repeated only includ-
ing the 21 electrodes that are part of the 10-20 system
that is routinely used in clinical practice. Additionally, to test whether EEG changes have a differ-
ent spatial distribution in the different groups, we con-
ducted a supplementary analysis splitting the whole set of
electrodes into four macroscopic regions (frontal, central,
lateral, and posterior) and applying a repeated measures
ANOVA with region as the within-subject factor and
diagnosis as the between-subject factor [10]. If the inter-
action between region and diagnosis was significant, this
was followed up by post hoc univariate ANOVAs. EEG frequency analysis Alzheimer's Research & Therapy (2020) 12:82 Page 5 of 12 Table 1 Demographic and clinical variables, mean (standard deviation)
HC (N = 31)
MCI-AD (N = 36)
MCI-LB (N = 39)
Group differences
Male to female
22:9
15:21
35:4
χ2 = 20.0, p < 0.001a
p(HC,MCI-AD) = 0.02
p(HC,MCI-LB) = 0.045
p(MCI-AD,MCI-LB) < 0.001
Age
73.7 (7.3)
76.1 (7.7)
74.7 (6.4)
F(2, 103) = 1.0, p = 0.38b
AChEI
–
7 (19%)e
18 (46%)f
χ2 = 5.7, p = 0.02c
PD meds
–
0e
4 (10%)f
χ2 = 3.8, p = 0.052c
Years of education
14.7 (4.0)g
12.9 (3.4)h
12.1 (2.8)
F(2, 100) = 5.1, p = 0.008c
p(HC,MCI-AD) = 0.12
p(HC,MCI-LB) = 0.006
p(MCI-AD,MCI-LB) = 0.87
ACE-R
92.7 (4.2)
82.4 (8.5)
83.8 (9.2)
t73 = 0.7, p = 0.50d
MMSE
28.5 (1.1)
26.9 (2.1)
26.6 (2.5)
t73 = 0.7, p = 0.51d
UPDRS III
5.5 (4.4)
15.1 (13.8)
23.3 (14.2)
t73 = 2.5, p = 0.01d
DCFS
–
6.9 (1.9)i
8.5 (3.3)k
t61 = 2.4, p = 0.02d
CAF total
–
1.4 (2.7)i
3.7 (4.2)k
t61 = 2.5, p = 0.02d
NPI total
–
8.6 (9.3)i
16.4 (12.9)k
t61 = 2.7, p = 0.01d
NEVHI
–
0.8 (1.5)e
2.7 (4.1)
t71 = 2.6, p = 0.01d
GDS
1.3 (1.8)
3.5 (2.5)
5.1 (4.1)
F(2, 103) = 13.4, p < 0.001b
p(HC,MCI-AD) = 0.01
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.08
ACE-R Addenbrooke’s Cognitive Examination–Revised, AChEI number of patients taking acetylcholinesterase inhibitors, CAF total clinician assessment of fluctuation
total score, DCFS Dementia Cognitive Fluctuation Scale, GDS Geriatric Depression Scale, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s
disease, MCI-LB probable mild cognitive impairment with Lewy bodies, MMSE Mini-Mental State Examination, NEVHI North-East Visual Hallucinations Interview, NPI
Neuropsychiatric Inventory, PD meds number of patients taking dopaminergic medication for the management of Parkinson’s disease symptoms, UPDRS III Unified
Parkinson’s Disease Rating Scale III (motor subsection)
aChi-square test HC, MCI-AD, MCI-LB
bUnivariate ANOVA HC, MCI-AD, MCI-LB
cChi-square test MCI-AD, MCI-LB
dStudent’s t test MCI-AD, MCI-LB
eN = 34
f ACE-R Addenbrooke’s Cognitive Examination–Revised, AChEI number of patients taking acetylcholinesterase inhibitors, CAF total clinician assessment of fluctuation
total score, DCFS Dementia Cognitive Fluctuation Scale, GDS Geriatric Depression Scale, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s
disease, MCI-LB probable mild cognitive impairment with Lewy bodies, MMSE Mini-Mental State Examination, NEVHI North-East Visual Hallucinations Interview, NPI
Neuropsychiatric Inventory, PD meds number of patients taking dopaminergic medication for the management of Parkinson’s disease symptoms, UPDRS III Unified
Parkinson’s Disease Rating Scale III (motor subsection)
aChi-square test HC, MCI-AD, MCI-LB
bUnivariate ANOVA HC, MCI-AD, MCI-LB
cChi-square test MCI-AD, MCI-LB
dStudent’s t test MCI-AD, MCI-LB
eN = 34
fN = 38
gN = 29
hN = 35
iN = 27
kN = 36 Figs. EEG frequency analysis 2 and 3): There were no group differences in terms
of delta power. Theta power was increased in MCI-LB
(mean = 8.8, standard deviation (SD) = 4.8) compared to
controls (mean = 5.7, SD = 3.2) with no significant differ-
ence between controls and MCI-AD (mean = 7.0, SD =
3.1) or between the two MCI groups. Pre-alpha power
was increased in MCI-LB (mean = 28.8, SD = 14.2) and
MCI-AD (mean = 19.6, SD = 10.2) compared to controls
(mean = 13.3, SD = 9.2), and it was further increased in
MCI-LB compared to MCI-AD. Alpha power was de-
creased in MCI-LB (mean = 28.3, SD = 14.7) compared
to controls (mean = 39.9, SD = 15.8) with no difference
between controls and MCI-AD (mean = 35.6, SD = 12.7)
or between the two MCI groups. However, when the
alpha band was split further into low-alpha (8–10 Hz)
and high-alpha (10–13 Hz), it became evident that high-
alpha power was significantly reduced in MCI-LB com-
pared to both controls (p < 0.001) and MCI-AD (p < 0.001) with no difference between MCI-AD and controls
(p = 1.0) while there were no group differences in terms
of low-alpha power (F(2, 102) = 1.7, p = 0.2). Beta power
was reduced in MCI-LB (mean = 18.9, SD = 11.0) com-
pared to both controls (mean = 26.7, SD = 10.3) and
MCI-AD (mean = 25.4, SD = 8.7), but not different be-
tween MCI-AD and controls. Figs. 2 and 3): There were no group differences in terms
of delta power. Theta power was increased in MCI-LB
(mean = 8.8, standard deviation (SD) = 4.8) compared to
controls (mean = 5.7, SD = 3.2) with no significant differ-
ence between controls and MCI-AD (mean = 7.0, SD =
3.1) or between the two MCI groups. Pre-alpha power
was increased in MCI-LB (mean = 28.8, SD = 14.2) and
MCI-AD (mean = 19.6, SD = 10.2) compared to controls
(mean = 13.3, SD = 9.2), and it was further increased in
MCI-LB compared to MCI-AD. Alpha power was de-
creased in MCI-LB (mean = 28.3, SD = 14.7) compared
to controls (mean = 39.9, SD = 15.8) with no difference
between controls and MCI-AD (mean = 35.6, SD = 12.7)
or between the two MCI groups. EEG frequency analysis The multivariate ANOVA revealed an overall effect of
diagnosis: F(10, 196) = 5.1, p < 0.001. Follow-up univari-
ate ANOVAs showed the following (see Table 2 and A receiver operating characteristic (ROC) analysis was
conducted in R to assess the sensitivity and specificity of Schumacher et al. EEG frequency analysis Sex was included as a covariate
DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable
mild cognitive impairment with Lewy bodies of using all electrodes. Dominant frequency variability
was not significantly different between the groups. y
g
Area under the ROC curve (AUC) values, sensitivity,
and specificity for the different EEG measures for differ-
entiating between MCI-LB and MCI-AD are shown in
Table 3. Beta power and dominant frequency achieved
the highest AUC values of 0.71 and 0.70, respectively. Specificity was high for some measures (up to 0.97 for
alpha power); however, sensitivity was generally much
lower. The results did not change when restricting the ana-
lysis to the 21 electrodes that are part of the 10-20 sys-
tem (see Supplementary Table S1). The regional analysis showed very similar results
compared to the analysis where all electrodes were
combined (see Supplementary Table S2). The only
slight difference was that alpha power was signifi-
cantly reduced in MCI-LB compared to MCI-AD pa-
tients in posterior (p = 0.047) and lateral (p = 0.01)
regions whereas mean alpha power from all electrodes
was not significantly different between the two MCI
groups (p = 0.07). EEG frequency analysis Alzheimer's Research & Therapy (2020) 12:82 Page 6 of 12 Table 2 Group comparison of quantitative EEG characteristics
HC
MCI-AD
MCI-LB
Group comparison
Delta power
14.1 [11.3, 17.0]
14.1 [11.8, 16.3]
14.9 [12.7, 17.1]
F(2, 102) = 0.8, p = 0.47
Theta power
5.7 [4.6, 6.9]
7.0 [6.0, 8.1]
8.8 [7.2, 10.3]
F(2, 102) = 4.8, p = 0.01
p(HC,MCI-AD) = 0.19
p(HC,MCI-LB) = 0.004
p(MCI-AD,MCI-LB) = 0.46
Pre-alpha power
13.3 [9.9, 16.7]
19.6 [16.1, 23.1]
28.8 [24.2, 33.4]
F(2, 102) = 16.0, p < 0.001
p(HC,MCI-AD) = 0.006
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.02
Alpha power
39.9 [34.1, 45.6]
33.6 [29.3, 37.9]
28.3 [23.6, 33.1]
F(2, 102) = 5.5, p = 0.005
p(HC,MCI-AD) = 0.39
p(HC,MCI-LB) = 0.001
p(MCI-AD,MCI-LB) = 0.07
Beta power
26.7 [22.9, 30.4]
25.4 [22.4, 28.4]
18.9 [15.3, 22.5]
F(2, 102) = 8.1, p = 0.001
p(HC,MCI-AD) = 1.0
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.001
Theta/alpha ratio
0.34 [0.27, 0.40]
0.42 [0.36, 0.47]
0.51 [0.44, 0.57]
F(2, 102) = 7.5, p < 0.001
p(HC,MCI-AD) = 0.22
p(HC,MCI-LB) = 0.001
p(MCI-AD,MCI-LB) = 0.10
DF, all electrodes
8.4 [8.0, 8.9]
8.0 [7.6, 8.3]
7.2 [6.8, 7.6]
F(2, 102) = 8.7, p < 0.001
p(HC,MCI-AD) = 0.29
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.01
DF, occipital electrodes
8.5 [8.1, 9.0]
8.0 [7.6, 8.3]
7.3 [6.9, 7.7]
F(2, 102) = 8.7, p < 0.001
p(HC,MCI-AD) = 0.16
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.03
DFV, occipital electrodes
1.3 [0.9, 1.6]
1.4 [1.1, 1.7]
1.0 [0.8, 1.2]
F(2, 102) = 3.6, p = 0.03
p(HC,MCI-AD) = 0.54
p(HC,MCI-LB) = 1.0
p(MCI-AD,MCI-LB) = 0.17
Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc
tests, Bonferroni-corrected for multiple comparisons. EEG frequency analysis Sex was included as a covariate
DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable
mild cognitive impairment with Lewy bodies Table 2 Group comparison of quantitative EEG characteristics
HC
MCI-AD
MCI-LB
Group comparison
Delta power
14.1 [11.3, 17.0]
14.1 [11.8, 16.3]
14.9 [12.7, 17.1]
F(2, 102) = 0.8, p = 0.47
Theta power
5.7 [4.6, 6.9]
7.0 [6.0, 8.1]
8.8 [7.2, 10.3]
F(2, 102) = 4.8, p = 0.01
p(HC,MCI-AD) = 0.19
p(HC,MCI-LB) = 0.004
p(MCI-AD,MCI-LB) = 0.46
Pre-alpha power
13.3 [9.9, 16.7]
19.6 [16.1, 23.1]
28.8 [24.2, 33.4]
F(2, 102) = 16.0, p < 0.001
p(HC,MCI-AD) = 0.006
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.02
Alpha power
39.9 [34.1, 45.6]
33.6 [29.3, 37.9]
28.3 [23.6, 33.1]
F(2, 102) = 5.5, p = 0.005
p(HC,MCI-AD) = 0.39
p(HC,MCI-LB) = 0.001
p(MCI-AD,MCI-LB) = 0.07
Beta power
26.7 [22.9, 30.4]
25.4 [22.4, 28.4]
18.9 [15.3, 22.5]
F(2, 102) = 8.1, p = 0.001
p(HC,MCI-AD) = 1.0
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.001
Theta/alpha ratio
0.34 [0.27, 0.40]
0.42 [0.36, 0.47]
0.51 [0.44, 0.57]
F(2, 102) = 7.5, p < 0.001
p(HC,MCI-AD) = 0.22
p(HC,MCI-LB) = 0.001
p(MCI-AD,MCI-LB) = 0.10
DF, all electrodes
8.4 [8.0, 8.9]
8.0 [7.6, 8.3]
7.2 [6.8, 7.6]
F(2, 102) = 8.7, p < 0.001
p(HC,MCI-AD) = 0.29
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.01
DF, occipital electrodes
8.5 [8.1, 9.0]
8.0 [7.6, 8.3]
7.3 [6.9, 7.7]
F(2, 102) = 8.7, p < 0.001
p(HC,MCI-AD) = 0.16
p(HC,MCI-LB) < 0.001
p(MCI-AD,MCI-LB) = 0.03
DFV, occipital electrodes
1.3 [0.9, 1.6]
1.4 [1.1, 1.7]
1.0 [0.8, 1.2]
F(2, 102) = 3.6, p = 0.03
p(HC,MCI-AD) = 0.54
p(HC,MCI-LB) = 1.0
p(MCI-AD,MCI-LB) = 0.17
Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc
tests, Bonferroni-corrected for multiple comparisons. Sex was included as a covariate
DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable
mild cognitive impairment with Lewy bodies DFV, occipital electrodes
1.3 [0.9, 1.6]
1.4 [1.1, 1.7]
1.0 [0.8, 1.2] Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc
tests, Bonferroni-corrected for multiple comparisons. Mean [95% confidence interval] of different quantitative EEG characteristics. Group comparisons were performed using univariate ANOVAs followed by post hoc
tests, Bonferroni-corrected for multiple comparisons. Sex was included as a covariate
DF dominant frequency, DFV dominant frequency variability, HC healthy controls, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB probable
mild cognitive impairment with Lewy bodies EEG frequency analysis However, when the
alpha band was split further into low-alpha (8–10 Hz)
and high-alpha (10–13 Hz), it became evident that high-
alpha power was significantly reduced in MCI-LB com-
pared to both controls (p < 0.001) and MCI-AD (p < The
theta/alpha
ratio
was
increased
in
MCI-LB
(mean = 0.51, SD = 0.21) compared to controls (mean =
0.34, SD = 0.17), but not significantly different between
MCI-AD (mean = 0.42, SD = 0.16) and controls or be-
tween the two MCI groups. Dominant frequency was slower in MCI-LB (mean =
7.2, SD = 1.1) compared to both MCI-AD (mean = 8.0,
SD = 1.1) and controls (mean = 8.4, SD = 1.2), but there
was no significant difference between controls and MCI-
AD. These results did not change when calculating the
dominant frequency from the occipital electrodes instead Schumacher et al. Discussion In this study, we investigated the differences in quantita-
tive EEG measures between highly phenotyped patients
with MCI-AD and MCI-LB and in comparison with
similarly aged healthy controls. We showed that there
are significant differences between the two MCI groups
at the group level with more severe EEG abnormalities
in MCI-LB compared to MCI-AD and that these EEG
changes correlated with the burden of Lewy body fea-
tures, supporting their high specificity for Lewy body
disease. However, their diagnostic accuracy for differen-
tiating MCI-LB from MCI-AD is modest and not con-
sistent with previous studies [17]. Fig. 2 Mean power spectra for the three diagnostic groups. Shaded
areas indicate standard errors. HC, healthy controls; MCI-AD, mild
cognitive impairment with Alzheimer’s disease; MCI-LB, probable
mild cognitive impairment with Lewy bodies hallucinations (N = 30, mean (SD) = 31.1 (15.4); t37 = 2.5,
p = 0.02; Fig. 4a). Additionally, the theta/alpha ratio was
increased in patients with visual hallucinations (mean
(SD) = 0.61 (0.13)) compared to patients without visual
hallucinations (mean (SD) = 0.47 (0.22), t24 = 2.4, p =
0.02, Fig. 4b). Overall, the findings suggest a slowing of the EEG in
MCI-LB patients compared to healthy controls by a shift
in power from beta and alpha frequency bands towards
slower frequencies in the pre-alpha and theta range. This
was also reflected by a shift of the dominant frequency
towards slower frequencies. These findings are consist-
ent with the results from previous EEG studies at the de-
mentia stage which generally report an increase in slow-
wave activity and slowing of the dominant EEG rhythm
in DLB [10, 18–20]. A slowing of the dominant fre-
quency has also been reported in MCI-LB patients be-
fore [21]. None of the EEG measures showed a significant differ-
ence between those MCI-LB patients with cognitive fluc-
tuations (N = 21) compared to those without cognitive
fluctuations (N = 18) or between MCI-LB patients taking
cholinesterase inhibitors (N = 18) compared to those pa-
tients not taking cholinesterase inhibitors (N = 20, all
p > 0.1). Similarly, EEG measures were not different be-
tween MCI-AD patients taking cholinesterase inhibitors
(N = 7) compared to those MCI-AD patients not taking
these medications (N = 27). We did not observe any group differences in dominant
frequency variability which has previously been found to
be increased in DLB patients compared to controls [12,
17, 19]. Association with clinical symptoms There was a significant reduction in alpha power in
MCI-LB patients with visual hallucinations (N = 9, mean
(SD) = 19.1 (6.4)) compared to those without visual Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 7 of 12 Page 7 of 12 Schumacher et al. Alzheimer's Research & Therapy Fig. 2 Mean power spectra for the three diagnostic groups. Shaded
areas indicate standard errors. HC, healthy controls; MCI-AD, mild
cognitive impairment with Alzheimer’s disease; MCI-LB, probable
mild cognitive impairment with Lewy bodies in the MCI-AD group (Supplementary Table S4). In the
MCI-LB group, more severe cognitive impairment corre-
lated with higher theta power, higher pre-alpha power,
lower alpha power, higher theta/alpha ratio, and de-
creased dominant frequency (Supplementary Table S4). Discussion In each boxplot, the central line corresponds to the sample median; the upper and
lower border of the box represent the 25th and 75th percentile, respectively; and the length of the whiskers is 1.5 times the interquartile range. Corresponding results from statistical comparisons between the groups are presented in Table 2. DF, dominant frequency; DFV, dominant
frequency variability; HC, healthy controls; MCI-AD, mild cognitive impairment with Alzheimer’s disease; MCI-LB, probable mild cognitive
impairment with Lewy bodies In a previous EEG study in MCI patients, Bonanni
et al. [17] found that all MCI patients who converted to
DLB within a 3-year follow-up period had an abnormal
EEG whereas 93% of MCI patients who developed AD
within the follow-up period had normal EEGs. The high
specificity of EEG abnormalities for MCI-LB in this re-
port is in line with our findings whereas the high sensi-
tivity stands in contrast to the findings of the present
study. However, the sample in this previous study was
more selective by only including patients who developed
dementia within 3 years and might therefore be biased
towards MCI patients with more severe disease and thus
having a rapid progression to dementia. When also tak-
ing into account those MCI patients who did not con-
vert to dementia within 3 years, diagnostic accuracy was
reduced to an overall predictive value of 76% [17]. Fur-
thermore, in a multi-center study in dementia patients,
the diagnostic accuracy of EEG was also found to be
much lower [19], indicating that heterogeneity in EEG
acquisition and analysis protocols across different cen-
ters might be an important limiting factor. between more widespread Lewy body disease and more
severe EEG abnormalities and suggests that Lewy body
disease might affect electro-cortical activity in a dose-
dependent manner. While there were significant group-level differences in
EEG characteristics between the two MCI groups, it is
difficult to translate this, at present, into a measure for
use in early clinical diagnosis. Power in the beta fre-
quency range and dominant frequency had the highest
diagnostic accuracy; however, they only reached moder-
ate AUC values of around 0.7. Discussion However, other studies have failed to replicate
these findings [10, 18, 22], and our results further indi-
cate that an increase in dominant frequency variability
may not be a reliable feature of Lewy body disease. MCI-LB patients with more than two core symptoms/
abnormal biomarkers had significantly higher pre-alpha
power, lower alpha power, and a higher theta/alpha ratio
compared to MCI-LB patients with only two core symp-
toms or one core symptom and one abnormal biomarker
(Supplementary Table S3). Higher delta power, theta
power, pre-alpha power, and theta/alpha ratio correlated
with
higher
Lewy
body
symptom/biomarker
count
whereas higher alpha power, beta power, and dominant
frequency correlated with lower Lewy body symptom/
biomarker count (Supplementary Table S3). There were
no significant differences in overall cognition (ACE-R
scores) between MCI-LB patients with more than two
core symptoms/abnormal biomarkers compared to pa-
tients with two symptoms, and the Lewy body symptom/
biomarker count was not significantly correlated with
the ACE-R score (see Supplementary Table S3). The only difference between MCI-AD patients and
healthy controls was an increase in pre-alpha power,
suggesting only a small degree of EEG slowing. This is
again consistent with previous reports at the dementia
stage which have generally found less severe EEG abnor-
malities in AD compared to DLB [10, 12, 19]. Addition-
ally, we found significantly increased pre-alpha power,
decreased beta power, and slower dominant frequency
in the MCI-LB compared to the MCI-AD group, which
indicates more severe EEG slowing in MCI-LB than in
MCI-AD patients and mirrors the findings in dementia
patients. Furthermore, we found more severe EEG ab-
normalities in MCI-LB patients with more core Lewy
body symptoms and/or abnormal biomarkers and a
higher symptom count correlated with more severe EEG
slowing. This indicates that there is an association More severe cognitive impairment (as measured by the
ACE-R) was correlated with higher pre-alpha power,
lower alpha power, and decreased dominant frequency Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 8 of 12 Schumacher et al. Alzheimer's Research & Therapy Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page ) Fig. 3 (See legend on next page.) Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Page 9 of 12 (See figure on previous page.)
Fig. 3 Group comparison of quantitative EEG characteristics. a under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheime
e mild cognitive impairment with Lewy bodies The sensitivity/specificity cutoff was determined using Youden s index
AUC area under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB
probable mild cognitive impairment with Lewy bodies Discussion The specificity for MCI-
LB was very high for certain EEG measures (up to 0.97
for alpha power with a cutoff of < 20.5) which suggests
that if a substantial shift of power towards slower fre-
quencies is observed, i.e., in the case of a very abnormal
EEG, a diagnosis of MCI-LB over MCI-AD becomes
highly likely. The relationship between EEG slowing and
Lewy body symptom count (see above) suggests that
these might be the MCI-LB patients with more wide-
spread Lewy body disease and these are easier to distin-
guish from MCI-AD patients based on their more severe
EEG slowing. However, sensitivity was generally much
lower, i.e., in the case of a more normal EEG, differenti-
ating between MCI-AD and MCI-LB is difficult and
many MCI-LB cases would be missed by applying these
measures for diagnostic purposes. These findings thus
show that early changes in quantitative EEG characteris-
tics are specific, but not sensitive to Lewy body disease
which is in alignment with the diagnostic performance
of other biomarkers such as FP-CIT and MIBG in early
Lewy body disease [8]. Another previous EEG study that attempted to differ-
entiate MCI-AD from MCI-LB did not find any EEG
measures that reliably distinguished between the two
MCI subgroups [21]. However, this was a retrospective
study of selected MCI-AD and MCI-LB patients who
were recruited using clinical criteria which differed
across the several centers who supplied patient data. Furthermore, participants from the different centers
were also assessed using different EEG protocols which
again makes the comparison difficult [21]. Conclusions In conclusion, the present study suggests that early
EEG slowing is specific to MCI-LB and, from a diagnos-
tic point of view, a very abnormal EEG favors MCI-LB
over MCI-AD. However, the overlap between the two
MCI groups is large, and for patients with a more nor-
mal EEG, it is difficult to distinguish between the two
MCI groups based on EEG characteristics alone. Given
its specificity for Lewy body disease burden, EEG may
be a promising future diagnostic biomarker and may
have value as part of a panel of other biomarkers. To
investigate this further, there needs to be a consensus
regarding the standardization of acquisition protocols,
analysis approaches, and choice of EEG parameters. Work in this field will undoubtedly yield benefits simi-
lar to those seen with CSF biomarkers for example [23]. More complex EEG analyses such as functional con-
nectivity or source reconstruction methods may pro-
vide better sensitivity for detecting differences between
the two MCI groups as well as between MCI-AD pa-
tients and controls [21, 24]. Furthermore, multimodal
approaches including EEG features in addition to
MIBG/FP-CIT or structural MRI markers may be an-
other route by which it will be possible to achieve bet-
ter accuracy for the early distinction between AD and
DLB at the MCI stage. Fig. 4 Comparison of quantitative EEG characteristics between
patients with and without visual hallucinations. a Comparison of
alpha power and b theta/alpha ratio between MCI-LB patients with
visual hallucinations (VH+, N = 9) and without visual hallucinations
(VH−, N = 30). In each boxplot, the central line corresponds to the
sample median; the upper and lower border of the box represent
the 25th and 75th percentile, respectively; and the length of the
whiskers is 1.5 times the interquartile range We explored the influence of the presence of visual
hallucinations on quantitative EEG characteristics in
MCI-LB patients and found that early EEG slowing, both
in terms of a reduction of power in the alpha frequency
range and a shift of power towards slower frequencies,
might be more severe in patients who experience visual
hallucinations compared to those patients who do not
have visual hallucinations, a finding which has previously
been reported in dementia patients [14]. Conclusions However, the
number of patients experiencing visual hallucinations in
our cohort was small (N = 9) and these findings should
therefore be interpreted with caution, and replication in
a larger group of patients with a higher occurrence of
visual hallucinations is needed. Discussion Table 3 Results from receiver operating characteristic (ROC) analysis to distinguish MCI-AD from MCI-LB
EEG measure
AUC [95% CI]
Cutoff for MCI-LB
Sensitivity
Specificity
Delta power
0.54 [0.41, 0.67]
> 21.9
0.23
0.89
Theta power
0.60 [0.47, 0.73]
> 10.7
0.33
0.89
Pre-alpha power
0.68 [0.56, 0.81]
> 28.1
0.56
0.83
Alpha power
0.66 [0.53, 0.78]
< 20.5
0.41
0.97
Beta power
0.71 [0.59, 0.83]
< 19.0
0.61
0.81
Theta/alpha ratio
0.64 [0.51, 0.77]
> 0.56
0.49
0.83
DF, all electrodes
0.70 [0.58, 0.82]
< 7.1
0.51
0.86
DF, occipital electrodes
0.69 [0.57, 0.81]
< 7.1
0.51
0.86
The sensitivity/specificity cutoff was determined using Youden’s index
AUC area under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB
probable mild cognitive impairment with Lewy bodies AUC area under the receiver operating curve, CI confidence interval, DF dominant frequency, MCI-AD mild cognitive impairment with Alzheimer’s disease, MCI-LB
probable mild cognitive impairment with Lewy bodies Page 10 of 12 Page 10 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Schumacher et al. Alzheimer's Research & Therapy Fig. 4 Comparison of quantitative EEG characteristics between
patients with and without visual hallucinations. a Comparison of
alpha power and b theta/alpha ratio between MCI-LB patients with
visual hallucinations (VH+, N = 9) and without visual hallucinations
(VH−, N = 30). In each boxplot, the central line corresponds to the
sample median; the upper and lower border of the box represent
the 25th and 75th percentile, respectively; and the length of the
whiskers is 1.5 times the interquartile range MCI-LB patients who were taking acetylcholinesterase
inhibitors to those who were not, we did not find any
significant differences in any EEG measures. The same
was found in the MCI-AD patients although this result
should be interpreted with caution because of the small
number of MCI-AD patients who were taking acetyl-
cholinesterase inhibitors (N = 7). Furthermore, it would
be unethical to withdraw medication, and including
medicated patients is more reflective of clinical practice. Another potential limitation is the lack of AD bio-
markers in our study. Additional file 1: Table S1. Group comparison of quantitative EEG
characteristics, restricting the analysis to 21 electrodes from the 10-20 sys-
tem. Table S2. Group comparison of quantitative EEG characteristics,
splitting the set of electrodes into four macroscopic regions. Table S3.
Association between Lewy body symptom severity and EEG characteris-
tics in the MCI-LB group. Two-sample t-tests comparing EEG measures
between MCI-LB patients with two symptoms/biomarkers (N=13) and
MCI-LB patients with more than two symptoms/biomarkers (N=26) and
Spearman’s correlations between EEG characteristics and symptom/bio-
marker count (ranging from 2 to 6). P-values are FDR-corrected for mul-
tiple comparisons. Table S4. Association between the severity of overall
cognitive impairment (ACE-R scores) and EEG characteristics in the MCI-
LB and MCI-AD groups using Spearman’s correlations. P-values are FDR-
corrected for multiple comparisons. Limitations Strengths of the present study include consistent and ro-
bust clinical assessments, diagnoses by a consensus
panel, the use of two diagnostic biomarkers, and the
prospective longitudinal design of the study. A potential
limitation is the use of acetylcholinesterase inhibitors in
the MCI patients as more patients in the MCI-LB group
were taking these medications compared to the MCI-AD
group. In the north east of England, services implement
expert advice and thus have high prescription rates for
DLB/MCI-LB patients because neuropsychiatric symp-
toms are treated using anti-dementia drugs early on. It
has
been
shown
that
cholinergic
medication
can
normalize EEG measures [15, 16], and it is therefore
possible that differences between the two MCI groups
were occluded by the higher number of MCI-LB patients
taking these medications. However, when comparing Author details
1 Author details
1Translational and Clinical Research Institute, Faculty of Medical Sciences,
Newcastle University, Biomedical Research Building 3rd floor, Campus for
Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK. 2Nuclear Medicine
Department, Newcastle upon Tyne Hospitals NHS Foundation Trust,
Newcastle upon Tyne, UK. 3Institute of Health Research, University of Exeter,
Exeter, UK. 4Department of Psychiatry, University of Cambridge School of
Medicine, Cambridge CB2 0SP, UK. 16. Babiloni C, Del Percio C, Bordet R, Bourriez JL, Bentivoglio M, Payoux P, et al. Effects of acetylcholinesterase inhibitors and memantine on resting-state
electroencephalographic rhythms in Alzheimer’s disease patients. Clin
Neurophysiol. 2013;124:837–50. https://doi.org/10.1016/j.clinph.2012.09.017. 1Translational and Clinical Research Institute, Faculty of Medical Sciences,
Newcastle University, Biomedical Research Building 3rd floor, Campus for
Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK. 2Nuclear Medicine
Department, Newcastle upon Tyne Hospitals NHS Foundation Trust,
3 17. Bonanni L, Perfetti B, Bifolchetti S, Taylor J-P, Franciotti R, Parnetti L, et al. Quantitative electroencephalogram utility in predicting conversion of mild
cognitive impairment to dementia with Lewy bodies. Neurobiol Aging. 2015;36:434–45 Available from: http://linkinghub.elsevier.com/retrieve/pii/
S0197458014004783. Funding
h This research was funded by the Alzheimer’s Research UK (ARUK-PG2015-13)
and supported by the NIHR Newcastle Biomedical Research Centre (grant
numbers BH120812 and BH120878). GE Healthcare provided the FP-CIT radi-
oligand for this investigator-led study. 12. Bonanni L, Thomas A, Tiraboschi P, Perfetti B, Varanese S, Onofrj M. EEG
comparisons in early Alzheimer’s disease, dementia with Lewy bodies and
Parkinson’s disease with dementia patients with a 2-year follow-up. Brain. 2008;131:690–705 Available from: http://www.brain.oxfordjournals.org/cgi/
doi/10.1093/brain/awm322. Consent for publication Consent for publication
Not applicable Not applicable 15. Kai T, Asai Y, Sakuma K, Koeda T, Nakashima K. Quantitative
electroencephalogram analysis in dementia with Lewy bodies and
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linkinghub.elsevier.com/retrieve/pii/S0022510X05002005. 15. Kai T, Asai Y, Sakuma K, Koeda T, Nakashima K. Quantitative
electroencephalogram analysis in dementia with Lewy bodies and
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linkinghub.elsevier.com/retrieve/pii/S0022510X05002005. Competing interests p
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The authors declare that they have no competing interests. Received: 13 May 2020 Accepted: 2 July 2020 18. Peraza LR, Cromarty RA, Kobeleva X, Firbank MJ, Killen A, Graziadio S, et al. Electroencephalographic derived network differences in Lewy body
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y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s13195-020-00650-1. pp
y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s13195-020-00650-1. Additional file 1: Table S1. Group comparison of quantitative EEG
characteristics, restricting the analysis to 21 electrodes from the 10-20 sys-
tem. Table S2. Group comparison of quantitative EEG characteristics,
splitting the set of electrodes into four macroscopic regions. Table S3. Association between Lewy body symptom severity and EEG characteris-
tics in the MCI-LB group. Two-sample t-tests comparing EEG measures
between MCI-LB patients with two symptoms/biomarkers (N=13) and
MCI-LB patients with more than two symptoms/biomarkers (N=26) and
Spearman’s correlations between EEG characteristics and symptom/bio-
marker count (ranging from 2 to 6). P-values are FDR-corrected for mul-
tiple comparisons. Table S4. Association between the severity of overall
cognitive impairment (ACE-R scores) and EEG characteristics in the MCI-
LB and MCI-AD groups using Spearman’s correlations. P-values are FDR-
corrected for multiple comparisons. Page 11 of 12 Page 11 of 12 Page 11 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 Authors’ contributions 9. Delorme A, Makeig S. EEGLAB: an open source toolbox for analysis of
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wrote the manuscript. CAH, MF, RAC, GR, RD, and NB acquired the data and
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revised the manuscript. MF, LA, JL, and JTOB conceived the study,
participated in its design and coordination, and revised the manuscript. JPT
and AJT conceived the study, participated in its design and coordination,
diagnosed the patients, interpreted the data, and revised the manuscript. All
authors read and approved the final version of the manuscript. JS analyzed and interpreted the data, performed the statistical analyses, and
wrote the manuscript. CAH, MF, RAC, GR, RD, and NB acquired the data and
revised the manuscript. PCD acquired the data, diagnosed the patients, and
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and vascular dementia. Neurol Int. 2000;54:1616–25. https://doi.org/10.1212/
WNL.54.8.1616. Ethics approval and consent to participate Ethics approval and consent to participate
The study was approved by the Newcastle & North Tyneside 2 Research
Ethics Committee (reference number: 15/NE/0420). Written informed consent
was obtained from all participants prior to study participation. Ethics Committee (reference number: 15/NE/0420). Written informed consent
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disease: comparison with dementia with Lewy bodies. Neurobiol Aging. 2018;67:75–83 Available from: https://linkinghub.elsevier.com/retrieve/pii/
S0197458018300915. Abbreviations 3. Albert MS, DeKosky ST, Dickson D, Dubois B, Feldman HH, Fox NC, et al. The
diagnosis of mild cognitive impairment due to Alzheimer’s disease:
recommendations from the National Institute on Aging-Alzheimer’s
Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimer’s Dement. 2011;7:270–9. https://doi.org/10.1016/j.jalz.2011.03.008. ACE-R: Addenbrooke’s Cognitive Examination–Revised; AD: Alzheimer’s
disease; AUC: Area under the ROC curve; CAF: Clinician assessment of
fluctuations; CDR: Clinical Dementia Rating; CIRS-G: Cumulative Illness Rating
Scale for Geriatrics; DCFS: Dementia Cognitive Fluctuations Scale;
DLB: Dementia with Lewy bodies; EEG: Electroencephalography;
ESS: Epworth Sleepiness Scale; FDR: False discovery rate; FP-CIT: 123I-N-
fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) single-photon emission
computed tomography; GDS: Geriatric Depression Scale; IADL: Instrumental
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MSQ: Mayo Sleep Questionnaire; MMSE: Mini-Mental State Examination;
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et al. EEG markers of dementia with Lewy bodies: a multicenter cohort
study. J Alzheimers Dis. 2016;54:1649–57. 2. Petersen RC, Smith GE, Waring SC, Ivnik RJ, Tangalos EG, Kokmen E. Mild
cognitive impairment. Arch Neurol. 1999;56:303 Available from: http://
archneur.jamanetwork.com/article.aspx?doi=10.1001/archneur.56.3.303. 20. Cromarty RA, Elder GJ, Graziadio S, Baker M, Bonanni L, Onofrj M, et al. Neurophysiological biomarkers for Lewy body dementias. Clin Neurophysiol. Page 12 of 12 Schumacher et al. Alzheimer's Research & Therapy (2020) 12:82 (2020) 12:82 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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English
| null |
Evaluating the Genetics of Common Variable Immunodeficiency: Monogenetic Model and Beyond
|
Frontiers in immunology
| 2,018
|
cc-by
| 13,996
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Specialty section: Specialty section:
This article was submitted to
Primary Immunodeficiencies,
a section of the journal
Frontiers in Immunology Common variable immunodeficiency (CVID) is the most frequent symptomatic primary
immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and
poor response to vaccines. Its diagnosis is made based on clinical and immunological
criteria, after exclusion of other diseases that can cause similar phenotypes. Currently,
less than 20% of cases of CVID have a known underlying genetic cause. We have
analyzed whole-exome sequencing and copy number variants data of 36 children
and adolescents diagnosed with CVID and healthy relatives to estimate the propor-
tion of monogenic cases. We have replicated an association of CVID to p.C104R in
TNFRSF13B and reported the second case of homozygous patient to date. Our results
also identify five causative genetic variants in LRBA, CTLA4, NFKB1, and PIK3R1, as
well as other very likely causative variants in PRKCD, MAPK8, or DOCK8 among others. We experimentally validate the effect of the LRBA stop-gain mutation which abolishes
protein production and downregulates the expression of CTLA4, and of the frameshift
indel in CTLA4 producing expression downregulation of the protein. Our results indicate
a monogenic origin of at least 15–24% of the CVID cases included in the study. The pro-
portion of monogenic patients seems to be lower in CVID than in other PID that have also
been analyzed by whole exome or targeted gene panels sequencing. Regardless of the Common variable immunodeficiency (CVID) is the most frequent symptomatic primary
immunodeficiency characterized by recurrent infections, hypogammaglobulinemia and
poor response to vaccines. Its diagnosis is made based on clinical and immunological
criteria, after exclusion of other diseases that can cause similar phenotypes. Currently,
less than 20% of cases of CVID have a known underlying genetic cause. We have
analyzed whole-exome sequencing and copy number variants data of 36 children
and adolescents diagnosed with CVID and healthy relatives to estimate the propor-
tion of monogenic cases. We have replicated an association of CVID to p.C104R in
TNFRSF13B and reported the second case of homozygous patient to date. Our results
also identify five causative genetic variants in LRBA, CTLA4, NFKB1, and PIK3R1, as
well as other very likely causative variants in PRKCD, MAPK8, or DOCK8 among others. Original Research
published: 14 May 2018
doi: 10.3389/fimmu.2018.00636 Edited by:
Frédéric Rieux-Laucat,
INSERM UMR1163 Institut
Imagine, France Edited by:
Frédéric Rieux-Laucat,
INSERM UMR1163 Institut
Imagine, France Edited by:
Frédéric Rieux-Laucat,
INSERM UMR1163 Institut
Imagine, France Guillem de Valles-Ibáñez1†, Ana Esteve-Solé2,3‡, Mònica Piquer 2,3‡,
E. Azucena González-Navarro3,4, Jessica Hernandez-Rodriguez1, Hafid Laayouni1,5,
Eva González-Roca3,4, Ana María Plaza-Martin2,3, Ángela Deyà-Martínez2,3,
Andrea Martín-Nalda6,7, Mónica Martínez-Gallo7,8,9, Marina García-Prat6,7,
Lucía del Pino-Molina10, Ivón Cuscó11,12, Marta Codina-Solà11,12, Laura Batlle-Masó1,13,
Manuel Solís-Moruno1,13, Tomàs Marquès-Bonet1,14,15, Elena Bosch1,
Eduardo López-Granados10, Juan Ignacio Aróstegui3,4, Pere Soler-Palacín6,7,
Roger Colobran7,8,9, Jordi Yagüe3,4, Laia Alsina2,3, Manel Juan3,4* and Ferran Casals13* Reviewed by:
Markus G. Seidel,
Medizinische Universität
Graz, Austria
Yuval Itan,
Icahn School of Medicine at
Mount Sinai, United States Reviewed by:
Markus G. Specialty section: We experimentally validate the effect of the LRBA stop-gain mutation which abolishes
protein production and downregulates the expression of CTLA4, and of the frameshift
indel in CTLA4 producing expression downregulation of the protein. Our results indicate
a monogenic origin of at least 15–24% of the CVID cases included in the study. The pro-
portion of monogenic patients seems to be lower in CVID than in other PID that have also
been analyzed by whole exome or targeted gene panels sequencing. Regardless of the Received: 12 December 2017
Accepted: 14 March 2018
Published: 14 May 2018 Seidel,
Medizinische Universität
Graz, Austria
Yuval Itan,
Icahn School of Medicine at
Mount Sinai, United States *Correspondence:
Manel Juan
mjuan@clinic.cat;
Ferran Casals
ferran.casals@upf.edu *Correspondence:
Manel Juan
mjuan@clinic.cat;
Ferran Casals
ferran.casals@upf.edu 1 Institut de Biologia Evolutiva (UPF-CSIC), Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra,
Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain, 2 Allergy and Clinical Immunology Department, Hospital Sant
Joan de Déu, Institut de Recerca Pediàtrica Hospital Sant Joan de Déu, Barcelona, Spain, 3 Functional Unit of Clinical
Immunology Hospital Sant Joan de Déu-Hospital Clinic, Barcelona, Spain, 4 Servei d’Immunologia, Centre de Diagnòstic
Biomèdic, Hospital Clinic-IDIBAPS, Barcelona, Spain, 5 Bioinformatics Studies, ESCI-UPF, Barcelona, Spain, 6 Pediatric
Infectious Diseases and Immunodeficiencies Unit, Hospital Universitari Vall d’Hebron (HUVH), Vall d’Hebron Institut de
Recerca (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain, 7 Jeffrey Modell Diagnostic and Research Center for
Primary Immunodeficiencies, Barcelona, Spain, 8 Immunology Division, Department of Clinical and Molecular Genetics,
Hospital Universitari Vall d’Hebron (HUVH), Vall d’Hebron Research Institute (VHIR), Barcelona, Spain, 9 Department of Cell
Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain, 10 Clinical Immunology
Department, University Hospital La Paz and Physiopathology of Lymphocytes in Immunodeficiencies Group, IdiPAZ Institute
for Health Research, Madrid, Spain, 11 Department of Experimental and Health Sciences, Universitat Pompeu Fabra,
Barcelona, Spain, 12 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain, 13 Servei
de Genòmica, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Parc de Recerca Biomèdica
de Barcelona, Barcelona, Spain, 14 Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain,
15 CNAG-CRG, Centre for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Spain †Present address:
Guillem de Valles-Ibáñez,
Department of Paediatrics and
Child Health, University of Otago,
Wellington, New Zealand ‡These authors have contributed
equally to this work. INTRODUCTION we have analyzed high coverage whole-exome sequencing and
copy number variants data for 36 CVID pediatric patients. We
hypothesize that focusing on pediatric cases will allow us to esti-
mate the maximum proportion of monogenic CVID cases, based
on the higher incidence of infectious disease in childhood and
theoretical and molecular evidence of higher impact of inborn
single gene defects in childhood than in adults, which tend to
present more complex genetics of predisposition to infection
(15, 16). Because of the heterogeneity of CVID etiology and mani-
festations, we first examined the role of known genetic variants
and candidate genes for CVID, and then expanded the analysis to
interacting proteins and genes in the same pathway, and finally to
the rest of the genome. We propose single candidate genes for the
CVID patients according to different models of inheritance and
by considering both genetic variants properties such as the allele
frequency, bioinformatic predictions of the phenotypic effect or
evolutionary conservation rates, as well as gene features such as
haploinsufficiency and essentiality predictors. In addition, beyond
the estimation of the proportion of patients under a monogenic
model, we also propose exploring other possible disease models
such us the oligogenic or polygenic by considering the presence
of mutations in interacting proteins or the accumulation of func-
tional variants in immunological pathways, as well as the disease
association with rare functional genetic variants by comparison
to healthy controls (17). Common variable immunodeficiency (CVID) is the most
prevalent symptomatic primary humoral immunodeficiency
with a prevalence from 1:10,000 to 1:50,000 in North America
and Europe (1). The diagnosis criteria consist in low serum con-
centrations of IgG, IgA and/or IgM, recurrent bacterial infections
and poor antibody response to vaccines, in addition to the exclu-
sion of other known causes of hypogammaglobulinemia (1–4). Patients’ phenotypes are highly heterogeneous due to different
time onsets and to a high variety of related complications, such as
autoimmune manifestations, lymphoproliferation, enteropathy,
and lymphoid malignancies, suggesting that CVID could be a
common outcome of diverse immune system failures.h The clinical heterogeneity of CVID has hindered both the
diagnostic and the identification of the underlying genetic defect
of the disease, allowing a molecular characterization of the origin
in less than 20% of the patients, and usually in familiar forms of
the disease which constitute only a small fraction of the CVID
cases (1, 5–7). INTRODUCTION Despite that, mutations in the genes CR2, LRBA,
NFKB1, NFKB2, IL21, TNFRSF13B, TNFRSF13C, CD81, IKZF1,
PRKCD, MS4A1, and CD19 are listed in the OMIM database1 as
causative of disease, inducing reclassification of CVID in these
new diagnostics, and establishing new therapeutic approaches
based on the affected pathways that have markedly improved
affected patients’ prognoses (8). Specific variants in these genes as
well as in others not listed in the OMIM database (NOD2, MSH5,
TNFRSF13B, HLA) have been reported to confer susceptibility to
the disease or to originate similar phenotypes to CVID (CTLA4,
PLCG2, PIK3CD, PIK3R1), blurring even more the boundaries
that define this disorder. Furthermore, some of the mutations
have incomplete penetrance (9, 10) and many sporadic cases
remain unexplained after deep genetic analyzes, suggesting that
an important fraction of CVID cases might not follow a mono-
genic Mendelian pattern of inheritance (11). Citation: de Valles-Ibáñez G, Esteve-Solé A,
Piquer M, González-Navarro EA,
Hernandez-Rodriguez J, Laayouni H,
González-Roca E, Plaza-Martin AM,
Deyà-Martínez Á, Martín-Nalda A,
Martínez-Gallo M, García-Prat M,
del Pino-Molina L, Cuscó I,
Codina-Solà M, Batlle-Masó L,
Solís-Moruno M, Marquès-Bonet T,
Bosch E, López-Granados E,
Aróstegui JI, Soler-Palacín P,
Colobran R, Yagüe J, Alsina L,
Juan M and Casals F (2018)
Evaluating the Genetics of Common
Variable Immunodeficiency:
Monogenetic Model and Beyond. Front. Immunol. 9:636. doi: 10.3389/fimmu.2018.00636 May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 1 de Valles-Ibáñez et al. WES for CVID exact proportion of CVID monogenic cases, other genetic models have to be considered
for CVID. We propose that because of its prevalence and other features as intermediate
penetrancies and phenotypic variation within families, CVID could fit with other more
complex genetic scenarios. In particular, in this work, we explore the possibility of CVID
being originated by an oligogenic model with the presence of heterozygous mutations
in interacting proteins or by the accumulation of detrimental variants in particular immu-
nological pathways, as well as perform association tests to detect association with rare
genetic functional variation in the CVID cohort compared to healthy controls. Keywords: common variable immunodeficiency, primary immunodeficiency, exome sequencing, loss-of-function,
rare disease genetics Keywords: common variable immunodeficiency, primary immunodeficiency, exome sequencing, loss-of-function,
rare disease genetics Keywords: common variable immunodeficiency, primary immunodeficiency, exome sequencing, loss-of-function,
rare disease genetics 1 https://www.omim.org/ (Accessed: September, 2015). Frontiers in Immunology | www.frontiersin.org Individuals Included in the Studyh Individuals Included in the Study
This study includes 36 patients diagnosed with CVID, includ-
ing both sporadic and familiar cases, without any genetically
confirmed primary immunodeficiency (PID), and completing
the conventional criteria for CVID classification: (1) from 2 to
18 years old at the age of diagnosis; (2) lack of antibody produc-
tion after immunization of antigen exposure in at least two assays;
(3) 2 years post-diagnosis to exclude lymphoid malignancy; (4)
IgG levels 2.5th centile for age and low IgA or/and IgM levels. CVID patients presenting one of the following features were
excluded from the study: (a) well-known gene-identified PID
such as hyper IgM; CD19+ or CD20+ B cell deficiency; ICOS
or transmembrane activator and calcium-modulating cyclophilin
ligand interactor (TACI) gene mutation already diagnosed; (b)
secondary immunodeficiencies such as those due to complica-
tions such as associated tumors and lymphomas or from other In recent studies using whole-genome and exome sequencing
to study CVID, 15–30% of CVID patients have been proposed
to have a monogenic origin (12–14), with genetic variants both
at candidate or new genes for CVID, although not all of these
mutations have been functionally validated. In this work, we
aim to estimate the proportion of monogenic cases in CVID
and to explore other possible genetic models for CVID. For that, May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 2 WES for CVID de Valles-Ibáñez et al. used predicted haploinsufficiency (31), intolerance to functional
variation (32), and essentiality (33) scores to infer the possible
model of the disease and prioritize candidate genes in the differ-
ent patients.i therapies (side-effects following splenectomy, corticosteroid, or
immune suppressive therapies). Patients L283, L286, and N216
were reported to be consanguineous. In addition, parents and
siblings have also been included in the study, when available. Written informed consent for genetic analysis and research was
obtained from all participants and ethical approval for the project
was obtained from the institutional ethical committees.f We used the Fisher’s exact test to assess the statistical signifi-
cance of an excess of rare functional variants in cases compared
to controls, from two by two tables with the total number of rare
functional variants, and the total number of synonymous variants
in patients and controls. In both cases, variants present in more
than 10 individuals were excluded from the analysis to exclude
false positives produced by sequencing artifacts. Genetic Analyses The protein–protein interaction (PPI) data was obtained from
the Human Protein Reference Database (35) considering the
whole set of non-redundant interactions between two proteins. Gene lists for each pathway were extracted from the KEGG data-
base (36–38). We considered the 25 pathways shown in Table S1
in Supplementary Material. DNA was extracted from blood samples. CNV analysis was
performed with the CytoScanHD array (Affymetrix) according
to the manufacturer’s protocol. The CytoScanHD array contains
743,304 SNPs and 2,696,550 CNV markers. The obtained cychp
files were analyzed with Chromosome Analysis Suite v.2.1.0.16
software and NetAffx na33 annotation version. For CNV detec-
tion and to prevent false positives, we considered alterations
involving at least 25 markers and more than 150 Kb in length
for gains, and 35 markers and more than 75 Kb for losses. For
detection of loss of heterozygosity (LOH) regions, we considered
alterations of at least 50 markers in more than 5 Mb. Exome cap-
ture was performed with the Agilent SureSelect XT enrichment
system. DNA was sequenced in an Illumina HiSeq 2000 platform
in a 2 × 75 paired-end cycles run. PCR duplicates were removed
with Picard.2 Sequence reads were mapped to the human refer-
ence genome (hg19) using GEM (20). Variant calling was per-
formed using GATK (21) and SNP annotation with SnpEff (22)
and SnpSift (23). Candidate mutations were visually inspected
with the Integrative Genomics Viewer (24) and, when required,
validated by Sanger sequencing. Somatic variants analysis was
performed with VarScan2 (25), considering the high impact
variants predicted with SnpEff (22), P-value <0.05, present in less
than 40% of the reads and in a maximum of two patients. 2 http://www.picard.sourceforge.net (Accessed: January, 2016).
3 http://evs.gs.washington.edu/EVS/ (Accessed: April, 2016). Individuals Included in the Studyh We applied the
Li and Leal’s collapsing method (34) to detect an excess of CVID
patients with rare functional variation when compared to con-
trols. Statistical significance was also assessed using the Fisher’s
exact test. For these two analyses, only nucleotide substitutions
were considered. We used two different sets of controls: whole-exome sequences
from 36 individuals from a Spanish cohort diagnosed with
autism spectrum disorders (ASD) (18) and 267 whole-exome
sequences from healthy controls from a Spanish cohort (19). In
the case where no data were available for the 267 whole-exome
sequences, we retrieved data from the CIBERER Spanish Variant
Server (csvs.babelomics.org) and used data for individuals with
different syndromes not related to primary immunodeficiencies. Functional Validationsfi To assess the effect of specific gene alterations, additional
functional tests were performed. Mainly with peripheral blood
mononuclear cells (PBMCs) or Epstein–Barr transformed B cells
(EBV-B), including lymphocyte phenotyping and western-blot
Ficoll–Hypaque (Sigma-Aldrich, St. Louis, MO, USA) density
gradient centrifugation of heparinized blood was used for PBMC
isolation. Cells were cultured with complete medium [RPMI
(Gibco, Grand Island, NY, USA) supplemented with 10% heat-
inactivated fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA),
1 µg/ml penicillin and 1 µg/ml streptomycin (Invitrogen, Grand
Island, NY, USA)]. Viable cells were counted using a hemocytom-
eter in an inverted microscope. CTLA-4 expression detection was performed as described
elsewhere (39–42). Specifically, for Treg cell phenotyping and
CTLA-4 expression PBMCs were left with medium (resting) or
stimulated with PHA (5μg/ml, Sigma-Aldrich, St. Louis, MO,
USA) for 24 h. Treg intracellular staining was performed with
Treg Detection Kit (CD4/CD25/FoxP3) kit (Miltenyi Biotec,
Germany) following manufacturer’s instructions. CD3 BV421
(BD bioscience, San Jose, CA, USA), CD4 FITC, and CD25 APC
(Miltenyi) were used for extracellular staining and FoxP3 APC
(Miltenyi) and CTLA4 PE (BD biosciences) for intracellular
staining and then acquired with the cytometer (FACS Canto II,
BD biosciences).l Genetic Data and Statistical Analyses y
Only functional variants were considered, including missense,
stop-gain and stop-loss, splice donor or acceptor sites mutations,
and frameshift insertions and deletions. In addition to standard
filters for mapping and variant calling and annotation we also
discarded indels clustering within 10 base pairs of another indel
and for most of the analyses we excluded those variants present
in 10 or more individuals in our dataset. We used allele frequen-
cies from The 1000 Genomes Project (26) and the NHLBI and
Exome Sequencing Project.3 We used GERP (27, 28) to asses for
evolutionary conservation and Polyphen (29) and SIFT (30) to
predict the phenotypic impact of missense variants. We have also Lymphocyte stimulation capacity was assessed by flow cyto-
metric detection of activation markers. PBMCs were stimulated
for 7 days and then surface-stained with the following antibodies
against activation markers: CD62L, CD25, HLA-DR, CD69, and
CD40-L (BD Biosciences) and then acquired with the cytometer
(FACS Canto II, BD biosciences). Protein extraction and Western Blot: LRBA determination
was performed in EBV-B cells (43). EBV-B cells were lysed with
1% NP-40 buffer. Protein concentration was normalized between May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 3 WES for CVID de Valles-Ibáñez et al. Four of them were found in the CVID patients of this study
(Table 1). control and patient. Products were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and western blotting. A nitrocellulose membrane was blocked with a 2% milk TBS, then
incubated overnight with primary antibodies anti-LRBA (1:500,
polyclonal, Abcam, United Kingdom) and anti-GAPDH (1:1000,
polyclonal, Bio-Rad, United Kingdom) then the membrane was
washed with TTBS and incubated for 1,5 h with Goat Anti-
Rabbit IgG H&L (HRP) (1:5000, Abcam). It was then developed
with SuperSignal™ West Pico Chemiluminescent Substrate
(Thermo Scientific, Waltham, MA, USA) following the manu-
facturer’s instructions and acquired with ImageQuant LAS-4000
(GE Healthcare Life Sciences, Buckinghamshire, England, UK)
equipment. Two of the reported variants are included in the TNFRSF13B
gene (TACI), which is known to harbor functional mutations in
5–10% of patients diagnosed with CVID (48, 49). However, the
existence of healthy controls with heterozygous mutations in this
gene and the lack of a clear Mendelian pattern of inheritance in
families have led to consider some of the mutations at TNFRSF13B
as risk factors (9, 10) which could be determinant only in the
case of homozygous individuals (50). Genetic Data and Statistical Analyses Thus, TNFRSF13B would
be considered a modifier gene rather than a causal gene in
monogenic cases (51). The p.C104R variant is the most common
TNFRSF13B functional mutation found in CVID patients (51). Three of the patients in this study present this mutation, in one
case in homozygous state, being the second case found to date
(52). This mutation is significantly more frequent in our CVID
patients compared to the Spanish cohort controls (19) (P = 0.003,
Fisher’s exact test) and absent in the ASD controls (18) (Table 1). In the same gene, we report nine samples with the protein change
P251L, although in this case the proportion is not significantly
higher than in controls. In addition, a direct causal role for this
variant can probably be discarded because of its high frequency
in the reference populations (14% in the ExAc database, 11%
for the European population). On the other hand the P21R
variant of the TNFRSF13C gene found in four patients, and also
one healthy parent, shows a higher frequency when compared
to controls (P = 0.003, Fisher’s exact test). However, this vari-
ant (rs77874543) has also been found in non-CVID exomes in
homozygosity, and has a population frequency higher than 5%. Finally, we also detected two patients with the L85F substitution
in the MSH5 gene (47). The same aminoacid substitution was
also present in the mother of one these patients, not diagnosed
with CVID but with some of the clinical features described in the
patient. Nonetheless, this genetic variant has been found at lower
frequencies in CVID patients compared to controls, and has a
population frequency of 2% or higher in some populations (7%
in Africans), which suggests that it does not have a determinant
role in CVID. OMIM CVID-Causing Mutationsh g
The OMIM database4 includes known variants originating CVID
in 13 genes: ICOS, TNFRSF13B, TNFRSF13C, CD19, CR2, MS4A1,
CD81, IL21, LRBA, NFKB1, NFKB2, PRKCD, and IKZF1. There
is also evidence that defects in other genes (CTLA4, PLCG2) can
cause a similar phenotype or modify the severity of the disease
with comorbidities (MSH5). These genes are mainly related
to T-cell and B-cell defects leading to a deficiency in antibody
production. In these 16 genes, we found a total of 96 nucleotide
variants and 6 CNVs previously described to be putatively related
to CVID in the literature (Table S3 in Supplementary Material). RESULTS We generated whole-exome sequencing data for the 36 CVID
patients included in the study, as well as for eight relatives, with
an average coverage of 120×. In addition, we also generated
CNV data for all the samples except in one case where DNA
was not available. Table S2 in Supplementary Material shows
the number of functional genetic variants described in each
sample, classified in different annotation categories: missense,
stop-gain (or nonsense), start-gain, splice site, and inframe
and frameshift indels, with total numbers similar to what has
been previously reported (44). Table S2 in Supplementary
Material also contains the number of structural variants and
LOH regions detected in the genotyping analysis with the
CytoScanHD array. Loss-of-Function (LoF) Variants Furthermore, the expression of CTLA4 is downregulated in Treg
cells of the LRBA-deficient patient (Figure 1C), in agreement
with the previous description of CTLA4 detection in Treg cells
from LRBA-deficient patients (39).h in Supplementary Material shows the number of LoF variants
identified in each individual of the study. The number of LoF vari-
ants ranges from 78 to 153, similar to what has been previously
described (44, 53, 54). Applying different frequency thresholds
substantially reduces the number of LoF variants per individual
(54, 55). We established a permissive allele frequency threshold of
1%, and first focused the analysis on the LoF variants described in
candidate genes for CVID (Table 2). With this aim, we generated
a list of 97 candidate genes for CVID (Table S4 in Supplementary
Material), including genes in the OMIM database,5 genes defined
in a review by Bogaert and colleagues (51), and others from the
literature. Second, we also analyzed the presence of LoF variants
in proteins interacting with the proteins encoded by candidate
genes (see Materials and Methods) (Table 2). Finally, we also
report all the genes with LoF variants using a very low frequency
threshold (0.001) (Table S5 in Supplementary material). Eight patients harbor a LoF variant at a frequency less than
1% in CVID candidate genes (Table 2). Among them, L283
presents a new homozygous nonsense variant at the exon 4 of
the LRBA gene [chr4:151392836G > A (hg19)]. This stop codon
at LRBA (R2214*) is introduced at the beginning of the BEACH
domain (IPR000409 in InterPro), a highly conserved domain
with known crystal structure but unknown function (56). This i
The N211 patient presents a new LoF genetic variant located
at the CTLA4 gene, which has already been reported to harbor
causal heterozygous CVID variants (41, 42). The mutation
causes a frameshift deletion not previously described and absent
in the reference databases. We performed Sanger sequencing
of this mutation and confirmed that it is a de novo mutation
absent in the parents (Figure S1 in Supplementary Material)
and, therefore, a strong candidate to originate CVID. We per-
formed functional analyses to study the expression of CTLA4
in Treg cells and we found that it is downregulated before and
after stimulation with PHA. CTLA4 detection was lower than
in the case of the aforementioned LRBA-deficient patient after
PHA stimulation (Figure 1C). Finally, we also analyzed the
lymphocyte stimulation in the patient. Loss-of-Function (LoF) Variants Loss-of-Function variants include stop-gain and loss muta-
tions, splice-site mutations, and frameshift indels, which are
predicted to disrupt proteins and, therefore, could likely relate
to disease phenotypes, and in fact account for approximately
20% of the coding variants associated with disease (53). Table S2 4 https://omim.org/ (Accessed: September, 2015). Table 1 | Known common variable immunodeficiency (CVID) variants detected in CVID patients in this study. Gene
cDNA
Aa change
Genotypea
(reference)
hg19_pos
CVID (N = 36)b
Controls
(literature)
Controls
(Autism, N = 36)b
Controls (Spain,
N = 267)b
TNFRSF13B
c.752C > T
p.P251L
0/1 (10)
17:16842991
9
Yes
0
36 (3)
TNFRSF13B
c.310T > C
p.C104R
*/1 (45)
17:16852187
3 (1)
Yes
0
2
TNFRSF13C
c.62G > C
p.P21R
2*0/1 (46)
22:42322716
4
Yes
0
16c
MSH5
c.253C > T
p.L85F
2*0/1 (47)
6:31709045
2
Yes
0
55 (2)
a0/1 heterozygotes; 1/1 homozygotes; */1 heterozygotes and homozygotes; 2*0/1 compound heterozygotes. bHomozygous individuals are shown in brackets. cNo data available for the 267 controls. Instead, we used data from 578 whole-exome sequences at the CIBERER Spanish Variant Server (csvs.babelomics.org). yg
cNo data available for the 267 controls. Instead, we used data from 578 whole-exome sequences at the CIBERER Spanish Variant Server (csvs.babelomics.org). May 2018 | Volume 9 | Article 636 May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org de Valles-Ibáñez et al. WES for CVID mutation was validated by Sanger sequencing in the patient, and
also detected in heterozygosis in both parents and three healthy
siblings (Figure 1A). Copy number and SNP analyses confirmed
the existence of consanguinity in this patient. We estimated a
consanguinity index of 0.058 compatible with descendants from
third degree kinship marriages, based in the total of 174 Mb
included in LOH regions (57), with 10 LOH regions of more
than 5 Mb. We then performed assays with the patient cells to
test the effect of the variant on the protein. The western blot gel
electrophoresis separation (Figure 1B) shows that the cells of the
patient do not produce any detectable amount of LRBA protein,
thus validating the deleterious effect of the mutation abolishing
protein production probably through nonsense-mediated decay. PPI, protein–protein interaction. Loss-of-Function (LoF) Variants Figure 1 | (A) Chromatograms corresponding to the Sanger sequencing of the LRBA nonsense mutation region in L283 and five healthy relatives. (B) Western blot
analysis of LRBA and GAPDH for L283 patient (P) and a healthy control C+. LRBA protein is not detectable in the LRBA-deficient patient. (C) CTLA4 expression is
downregulated in LRBA- and CTLA4-deficient patients. CTLA4 expression was assessed in Treg cells (CD3+CD4+CD25hiFoxP3+ cells) in resting and in
PHA-stimulated cells (24 h). Bars represent mean values and error bars represent SE of the mean values for adult healthy controls (n = 5). Figure 1 | (A) Chromatograms corresponding to the Sanger sequencing of the LRBA nonsense mutation region in L283 and five healthy relatives. (B) Western blot
analysis of LRBA and GAPDH for L283 patient (P) and a healthy control C+. LRBA protein is not detectable in the LRBA-deficient patient. (C) CTLA4 expression is
downregulated in LRBA- and CTLA4-deficient patients. CTLA4 expression was assessed in Treg cells (CD3+CD4+CD25hiFoxP3+ cells) in resting and in
PHA-stimulated cells (24 h). Bars represent mean values and error bars represent SE of the mean values for adult healthy controls (n = 5). Loss-of-Function (LoF) Variants After 7 days stimulation
with PHA, the stimulation ratio of different lymphocyte stimula-
tion markers was increased in the patient compared to a healthy
control (Figure 2). Table 2 | Genes with Loss-of-Function (LoF) homozygous or heterozygous
variants in common variable immunodeficiency (CVID) candidate genes and
interacting proteins. Individual
CVID < 0.01
PPI < 0.01
L283
LRBA(hom)
L287
C7orf64(het), PDGFRB(hom), RIPK4(het)
L289
HDAC1(het)
L291
GP6(het)
L292
NOD2(het)
SLA2(het), ZNF655(het)
L297
NFKB1(het)
L298
MAPK8(het)
L299
FGFR3(het)
N202
PIK3R1(het)
FHOD1(het)
N204
HP(het), PLSCR1(het)
N205
HNF1A (comp_het)
N206
R PA 2(het)
N207
NFKB1(het)
N208
EEF1G(het)
N210
DERL3(het), HP(het), PDGFRB(het)
N211
CTLA4(het)
N213
IBTK(het), PDGFRB(het)
N216
CASP1(het), HCLS1(het), NCOR2(het)
N223
BCAP31(het), SLC6A8(het),
TNFRSF12A(het)
N224
BCAP31(hom), CASP1(het),
SLC6A8(hom), TNFRSF12A(het)
N227
BCAP31(het), SLC6A8(het)
N229
CR1(het), SPI1(het)
N231
PML(het), TNFRSF12A(het)
N232
TNFRSF12A(het)
N233
NOD2(het)
TNFRSF12A(het)
N234
IL10RA(het), NFKB1(het)
TNFRSF12A(het)
N235
C9(het), PIAS1(het), TRPV1(het)
PPI, protein–protein interaction. 5 http://omim.org (Accessed: September, 2015). N202 presents a heterozygous splicing variant in PIK3R1. This variant has been previously reported to originate an immu-
nodeficiency because of its dominant gain of function effect on
PI3K signaling (58) in agreement with its high haploinsufficiency
prediction value of 0.89 (31). For the remaining five patients
presenting a low frequency heterozygous LoF variant in a CVID
candidate gene (Table 2), three of them have a variant in NFKB1,
which has also been reported to harbor heterozygous mutations
originating CVID (7). Two of them share a start loss variant
affecting one of the transcripts, although its frequency of 0.002
makes it unlikely to have a causal (monogenic) role in the disease. By contrast, a new splice-site mutation in NFKB1 is described
in N234, being a good candidate to originate the disease. In addition, N227 presents a 13 MB heterozygous deletion (chr4:
94,135,868–107,295,574) not present in parents which includes
the NFKB1 gene among others (Table S2 in Supplementary
Material). Finally, although the variants described at NOD2
and IL10RA are not present in any database, no CVID cases
with heterozygous variants at these genes have been described,
in agreement with their low haploinsufficiency values (0.119
and 0.173, respectively). In addition, Table 2 also includes low
frequency LoF variants of genes interacting with candidate genes
related with CVID. May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 5 WES for CVID de Valles-Ibáñez et al. Functional Genetic Variation at Candidate
Genes for CVID Figure 2 | Stimulation ratio of different lymphocyte activation markers after
PHA stimulation (7 days). Stimulation ratio: mean fluorescence intensity of
PHA-stimulated/basal conditions. Figure 2 | Stimulation ratio of different lymphocyte activation markers after
PHA stimulation (7 days). Stimulation ratio: mean fluorescence intensity of
PHA-stimulated/basal conditions. We then explored the presence of functional variants, other than
LoF described above, in candidate genes for CVID. The final
number of variants with frequency less than 1% in each individual
is shown in Table S6 in Supplementary Material, differentiating
variants in candidate genes, variants in interacting proteins and
in other genes. We excluded from this, and subsequent analyses,
the two individuals with a functionally validated LoF candidate
(L283 and N211, see above), and the variants also present in
healthy relatives (when this information is available from exome
sequencing). We first analyzed the presence of single variants in
CVID genes that could originate the disease following a dominant Figure 2 | Stimulation ratio of different lymphocyte activation markers after
PHA stimulation (7 days). Stimulation ratio: mean fluorescence intensity of
PHA-stimulated/basal conditions. May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 6 de Valles-Ibáñez et al. WES for CVID gene names are shown in Table S7 in Supplementary Material. The number of genes per patient can be reduced using addi-
tional filters based in evolutionary conservation or predicted
phenotypic effect. We established a threshold of a GERP > 2
for the functional variants, since positions with values greater
than 2 are considered to be conserved among mammals and,
therefore, more to prone to be of functional importance (28). On the functional effect, we used the Polyphen prediction and
established a threshold value of 0.5 (60) (Table 5). Table S8 in
Supplementary Material also shows additional information on
gene properties which might aid the prioritization of candidate
genes. Four genes are detected as compound heterozygotes in
more than one patient (with GERP > 2 and Polyphen > 0.5):
SLC25A5 (eight), ACOT4 (7), KMT2C (two), and OR10X1
(two). However, SLC25A5 and OR10X1 are two genes which
have been recurrently reported in next-generation sequenc-
ing studies (61), probably because of being prone to mapping
artifacts and, thus, to accumulating false variants. On the other
hand, ACOT4 (with a function apparently not related to the
immune function), is also a paralog of ACOT1. Functional Genetic Variation at Candidate
Genes for CVID Finally, KMT2C
is also present in two patients, although one of them is N227
which harbors a large deletion encompassing NFKB1 among
other genes. model. Because the number of genes with one or more functional
variants is too high we applied stringent filters to produce a short
list of candidate genes. We selected the variants with a GERP
conservation score higher than 2 (28), a Polyphen score higher
than 0.5 (for nucleotide variants) and a frequency in the ExAC
and GMAF databases below 0.001. The nine variants at CVID
genes fulfilling these conditions are shown in Table 3. Two of
them are in frame indels and, therefore, less prone to have an
effect on the protein. Among the heterozygous missense variants,
PRKCD, CLEC16A, and DOCK8 (this latter absent in the healthy
sister N209) are the more interesting candidates, considering
their haploinsufficiency predictions (31) and essentiality values
estimated from network and evolutionary properties (59). We
then considered the recessive genetic model with the disease
being originated by two rare functional variants in the same gene. We analyzed the presence of homozygous variants or compound
heterozygotes in CVID candidate genes, at frequencies below 0.01
(Table 4). Interestingly, two candidate genes (CR2 and PLCG2)
are found as compound heterozygotes in patients N233 and
N212, respectively. Oligogenic Disease For the patients without a clear candidate gene for a monogenic
origin of the disease, we then considered an oligogenic model
of inheritance. In particular, we considered the digenic model. DIDA, a database of digenic diseases, included 44 diseases with
213 digenic combinations collected from the literature until June
2015 (62). This form of disease refers to both situations with a
primary and a secondary locus or cases where two loci contribute
to the disease with roughly the same importance (63). Modifier Table 3 | Functional heterozygous genetic variants with high predicted phenotypic effect at common variable immunodeficiency (CVID) candidate genes. Patient
chr
Position
Gene
Function
Polyphen
rs
GERP
esp5400_all
HI1
HI2
RVIS
Essent
L294
chr9
100,774,719
ANP32B
Inframe indel
–
–
–
0
0.808
0.655
–0.16 (41.25%)
0.89
N214
chr7
2,976,742
CARD11
Missense
0.654
–
2.18
0
0.181
0.517
–1.39 (4.33%)
0.81
N212
chr11
chr1
60,892,540
CD5
Missense
0.936
–
3.08
0
0.284
0.402
0.8 (87.66%)
0.666
N232
160,523,750
CD84
Missense
0.999
rs146076557
5.25
0.000279
0.132
0.488
0.04 (57.15%)
0.077
L292
chr16
11,073,195
CLEC16A
Missense
0.857
rs74163607
5.3
0.000201
NA
0.578
–1.01 (8.2%)
0.547
N201
chr1
207,651,294
CR2
Missense
0.659
rs146465618
5.59
0.000093
0.234
NA
0.06 (57.56%)
0.558
N210
chr9
chr1
377,046
DOCK8
Missense
0.868
rs148693111
5.71
0.000186
0.535
0.57
–1.94 (1.9%)
0.845
L287
234,744,945
IRF2BP2
Inframe indel
–
–
2.62
0
0.852
0.626
–
0.992
N213
chr1
234,744,945
IRF2BP2
Inframe indel
–
–
2.62
0
0.852
0.626
–
0.992
N216
chr1
234,744,945
IRF2BP2
Inframe indel
–
–
2.62
0
0.852
0.626
–
0.992
L288
chr3
53,218,928
PRKCD
Missense
0.733
–
5.91
0
0.636
0.553
–1.04 (7.77%)
0.966
HI1 and HI2 haploinsufficiency predictions (24); RVIS, residual variation score of genetic variation intolerance (25) with the percentile of intolerant human genes in parentheses;
Essent, essentiality index estimated from network and evolutionary properties (43). HI1 and HI2 haploinsufficiency predictions (24); RVIS, residual variation score of genetic variation intolerance (25) with the percentile of intolerant human genes in parentheses;
Essent, essentiality index estimated from network and evolutionary properties (43). Table 4 | Compound heterozygotes at common variable immunodeficiency (CVID) genes. Compound Heterozygotes
at Non-CVID Genes We expanded the analysis beyond the list of CVID candidate
genes to the rest of the genome. We based our approach on the
use of stringent filters (frequency, conservation, predicted effect)
and the consideration of predictors of the degree of essentiality
of the gene. This approach produces a list of new candidate genes
in each CVID patient which can be ranked using the different
variant and gene properties. We produced a list of genes harbor-
ing compound heterozygotes in each patient and applied two
different allele frequency thresholds of 0.01 and 0.001. Table 5
shows the number of compound heterozygotes per patient, and May 2018 | Volume 9 | Article 636 Oligogenic Disease Patient
CVID gene
Variants
Interacting protein
Variants
L288
PRKCD
1 het
RUNX2
1 hom
L293
DOCK8
1 het
CDC42
1 het
L299
STAT1
1 het
FGFR3
1 hom
L299
STAT1
1 het
FGFR4
1 hom, 1 het
N205
PIK3R1
1 het
AXL
1 hom
N205
PIK3R1
1 het
TYK2
1 het
N206
PIK3R1
1 het
AXL
1 hom
N207
PIK3CD
1 het
RALY
1 het
N208
PIK3CD
1 het
RALY
1 hom
N210
DOCK8
1 het
CDC42
1 het
N233
CR2
1 het
FHOD1
1 het
N234
RAD50
1 het
NBN
1 het
N234
NFKB1
1 het
NCOR2
2 het
considering a maximum frequency of 0.05. Interestingly, two
pairs of related patients (sisters N205 and N206, and brothers
N207 and N208) share the presence of variants at the interacting
proteins PIK3R1-AXL and PIK3CD-RALY, respectively. In four
more patients (Table 6), the CVID genes had already been sug-
gested as probably causal (Tables 2–4) following recessive (N233)
or dominant models (L288, N210, N234). considering a maximum frequency of 0.05. Interestingly, two
pairs of related patients (sisters N205 and N206, and brothers
N207 and N208) share the presence of variants at the interacting
proteins PIK3R1-AXL and PIK3CD-RALY, respectively. In four
more patients (Table 6), the CVID genes had already been sug-
gested as probably causal (Tables 2–4) following recessive (N233)
or dominant models (L288, N210, N234). We then expanded the analysis to a scenario where variants
in several genes of an individual might contribute to the disease. For this purpose, we assessed the presence of particular CVID
patients which compared to the rest of the patients in the study
harbors an excess of rare functional variants at any of 25 KEGG
pathways related to the immune function (36) (Figure 3). We
used a frequency threshold of 1% and estimated the ratios of
functional to synonymous variants in each sample, to correct for
possible differences in coverage across samples. We considered
as outliers those individuals departing from twice the SD of the
average number of rare functional variants (Figure 3). Table 7
shows the CVID patients with an excess of rare functional genetic
variants in a particular pathway. The presence of more than one
pathway in three of the patients is mostly due to the fact that these
patients have genetic variants in genes with a role in several path-
ways. Oligogenic Disease chr
Position
ref
alt
Polyphen
rs
GERP
esp5400 all
GMAF
Effect
Gene
idsample
Genotype
chr1
207643100
C
A
0.002
–
−3.25
–
–
Non-synonymous
CR2
N233
0/1
chr1
207648456
G
T
0.05
rs144572703
4.47
0.005763
0.0018
Non-synonymous
CR2
N233
0/1
chr16
81939089
T
C
0.598
rs187956469
5.18
0.002838
0.0032
Non-synonymous
PLCG2
N212
0/1
chr16
81942175
A
G
0.005
rs75472618
6.5
0.007067
0.0064
Non-synonymous
PLCG2
N212
0/1 7 WES for CVID de Valles-Ibáñez et al. Table 5 | Number of genes harboring compound heterozygotes mutations in
the patients included in this study. Sample
Genes
1%
Genes
0.1%
Genes 1% filtereda
Genes 0.1%
filtereda
L283
53
21
FAM186B, MYH11,
SLC25A5, SDK1
FAM186B
L287
26
11
CBS, TRIB3
0
L288
29
18
0
0
L289
39
19
FHL3, SLC25A5, FMN2
0
L290
31
15
0
0
L291
27
18
CAMPSAP3, VPS13C
CAMPSAP3
L292
26
16
PKHD1L1, PLEC, MLH1
PKHD1L1
L294
31
20
PRSS16
PRSS16
L295
34
15
0
0
L296
25
13
0
0
L297
30
17
SLC25A5
0
L298
28
7
BMP1
0
L299
27
14
ACOT4, GPR112, SLC25A5,
UNC13C
ACOT4,
GPR112
N201
33
23
ACOT4, SLC25A5, Z FYVE26
ACOT4
N202
27
13
ACOT4, WFS1, SLC25A5
ACOT4
N203
33
13
SLC25A5, SEPT1
0
N204
34
14
ACOT4
ACOT4
N205
28
15
0
0
N206
34
18
ACOT4
ACOT4
N207
29
17
0
0
N208
23
12
0
0
N210
33
18
EPPK1
0
N211
25
13
GLTSCR1
0
N212
25
11
ACOT4
ACOT4
N213
27
10
ACOT4, SLCS5A5
ACOT4
N214
19
6
SLC25A5
0
N216
75
21
PENK, NUP214, KDM4C
PENK
N223
35
19
OR10X1
0
N224
38
22
OR10X1
0
N227
37
25
KMT2C
0
N229
39
20
0
0
N231
35
20
BAI1, TTN, MTDH
BAI1
N232
18
12
KMT2C, PMFBP1
0
N233
35
19
0
0
N234
37
22
CMYA5
0
N235
42
22
0
0
aVariants with GERP > 2 and Polyphen > 0.5. Table 6 | Patients with rare functional variants (MAF < 0.01) and GERP > 2 in
a common variable immunodeficiency (CVID) candidate gene and interacting
proteins. Table 5 | Number of genes harboring compound heterozygotes mutations in
the patients included in this study. Table 6 | Patients with rare functional variants (MAF < 0.01) and GERP > 2 in
a common variable immunodeficiency (CVID) candidate gene and interacting
proteins. proteins. Oligogenic Disease In the case of patient N208, it shows an excess of variants
in five pathways that share the presence of three MAP Kinases
(MAPK14, MAP2K2, and MAP2K3). Of interest, we found four
patients with an excess of rare functional genetic variants in the
B cell signaling pathway and three in the T cell signaling pathway,
in addition to another two in the tumor necrosis factor and Fc
epsilon RI signaling pathways (Table 7). aVariants with GERP > 2 and Polyphen > 0.5. genes, affecting the severity of the disease, can also be considered
a type of digenic inheritance (64).h The case of TNFRSF13B, with several common variants related
to CVID but with reported healthy carriers, could fit with this
digenic model where additional variants would be needed to
develop the disease. We analyzed the two patients with variants
in this gene (Table 1), and searched for variants in genes inter-
acting with TNFRSF13B. Patient L297 harboring the C104R
change in homozygosis, also has a heterozygous missense variant
with a 2% frequency in TNFRSF13C, which directly interacts
with TNFRSF13B. No other variants in interacting proteins
were described in the patients with known CVID variants in
TNFRSF13B, TNFRSF13C, or MSH5 (Table 1). We expanded this
analysis by assessing the presence of heterozygous rare functional
variants in a CVID gene and in an interacting protein in the same
patient. Table 6 shows the 10 patients in which this situation has
been found, considering variants with GERP > 2 and below
0.01 frequency (see Table S9 in Supplementary Material) when Frontiers in Immunology | www.frontiersin.org Association to Rare Variants Table 9 | Common variable immunodeficiency (CVID) genes with an excess
of patients harboring rare functional genetic variants in patients compared to
controls. Table 7 | Pathways with an excess of genes with rare functional variants in
common variable immunodeficiency (CVID) patients. Table 7 | Pathways with an excess of genes with rare functional variants in
common variable immunodeficiency (CVID) patients. Patient
Pathway
Genea
L296
BCSP
CD79A, CR2, DAPP1, PLCG2
L297
TNFSP
CREB3, CREB3L1, NFKB1, PIK3CG, TNFRSF13B
N207
BCSP
PIK3CD, DAPP1, NFKB1, CD81, NFATC3
TCSP
PIK3CD, NFKB1, MAP14, NFATC3
N208
BCSP
PIK3CD, IKBKB, CD81, NFATC3, MAP2K2
FCERISP
PIK3CD, MAPK14, MAP2K3, MPA2K2
TCSP
PIK3CD, MAPK14, IKBKB, NFATC3, MAP2K2
TLRS
PIK3CD, MAPK14, IKBKB, IFNA14, MAP2K2, MAP2K3
TNFSP
PIK3CD, MAPK14, IKBKB, MAP2K3
N210
TCSP
CDC42, MAP3K8, PAK6, SOS2
N212
BCSP
PIK3CG, PLCG2(2), NFATC2, MAPK1, RAC2
FCERISP
PIK3CG, PLCG2(2), MAPK1, RAC2
NKCMC
PIK3CG, PTK2B, PTPN11, PLCG2(2), NFATC2, IFNAR1,
MAPK1, RAC2
NLRSP
CASP5, NLRP1(4), MAPK1
N216
MRP
MLH3, MSH2, PMS2
N229
PAP
PRKCZ, GUCY1B3, LCP2, ITPR2, ORAI1, PLA2G4B,
ROCK1, TBXA2R, PIK3R2
N231
IINIgA
HLA-DRB1(2), TNFRSF13B, ICOSLG
aNumber of genetic variants at this gene when is greater than one, indicated in
brackets. after applying Bonferroni’s multiple test correction. Table S10 in
Supplementary Material reports these functional variants and
their properties. Second, we used the Li and Leal’s collapsing method (34)
to detect an excess of CVID patients harboring rare functional
genetic variants. In this method, individuals with and without
at least one functional rare variant are compared between CVID
patients and controls. This test has been performed only for those
genes with similar lengths for the targeted regions to avoid false
positives with more functional variants because of a larger scanned
region in CVID patients. Table 9 presents six genes (PIK3CD,
ICOSLG, TNFRSF13B, PIK3R1, CD84, and PRKCD) showing a
statistically significant excess of individuals with rare functional
variants in CVID cases when compared to controls. Interestingly,
PRKCD showed also a significant excess of functional variation
in cases in the previous analysis (Table 8), although only PIK3CD
remains significant after Bonferroni’s correction. Genetic variants
in each gene are shown in Table S10 in Supplementary Material. Finally, we assessed a possible excess of functional variants in
the 25 KEGG pathways by comparing our CVID patients to a set of
controls (see Materials and Methods), by comparing the ratios of
rare (<1%) functional to synonymous variants in each sample. Association to Rare Variants Next, we assessed the association of rare functional genetic
variation to CVID. In this case, analyses are performed to detect
an excess of rare functional variation in a particular gene or
pathway in CVID patients compared to controls, rather than
the detection of the causal genetic variant(s) in particular indi-
viduals. To analyze the presence of genes harboring an excess
of rare functional variants in the CVID patients compared to
healthy controls, we first compared the ratio of rare functional
to synonymous variants for each gene in cases compared to May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 8 WES for CVID de Valles-Ibáñez et al. Table 8 | Excess of rare functional variants in common variable
immunodeficiency (CVID) patients. Gene
Rare Funct
CVID
Syn
CVID
Rare Funct
Controls
Syn
Controls
P-value
PRKCD
3
4
5
1693
2.36e−06
CLEC16A
6
11
8
321
1.30e−05
DOCK8
3
11
0
347
4.68e−05
PLCG2
4
14
18
1465
9.28e−05
Rare Funct, number of rare genetic variants; Syn, number of synonymous variants. Table 9 | Common variable immunodeficiency (CVID) genes with an excess
of patients harboring rare functional genetic variants in patients compared to
controls. Gene
Patients
functa
Patients
no functb
Controls
functc
Controls
no functd
P-value
PIK3CD
9
25
3
264
1.84E−07
ICOSLG
4
30
6
261
0.018
TNFRSF13B
3
31
4
263
0.034
PIK3R1
2
32
1
266
0.035
CD84
3
31
5
262
0.050
PRKCD
3
31
5
262
0.050
aNumber of patients with at least one rare functional genetic variant. bNumber of patients with no rare functional genetic variants. cNumber of controls with at least one rare functional genetic variant. dNumber of controls with no rare functional genetic variants. Table 8 | Excess of rare functional variants in common variable
immunodeficiency (CVID) patients. Gene
Rare Funct
CVID
Syn
CVID
Rare Funct
Controls
Syn
Controls
P-value
PRKCD
3
4
5
1693
2.36e−06
CLEC16A
6
11
8
321
1.30e−05
DOCK8
3
11
0
347
4.68e−05
PLCG2
4
14
18
1465
9.28e−05
Rare Funct, number of rare genetic variants; Syn, number of synonymous variants. Figure 3 | Number of functional genetic variants in common variable
immunodeficiency (CVID) patients and controls in immunological pathways. Abbreviations and the number of genes in each pathway are shown in the
Section “Materials and Methods.” Table 8 | Excess of rare functional variants in common variable
immunodeficiency (CVID) patients. DISCUSSION sisters (N205 and N206) harbor a new missense variant in PIK3R1. This variant has not been previously reported and is located in a
conserved nucleotide according to its GERP value (3.24), although
it is not predicted to be damaging using SIFT and Polyphen. On the
situations fitting a recessive model, for the PLCG2 gene, one of the
variants is predicted to be damaging with Polyphen and also shows
a very high level of evolutionary conservation, although for the
second variant both the evolutionary conservation and predicted
phenotypic effect are low. Similarly, only one of the variants at CR2
in patient N212 shows a high level of evolutionary conservation,
and none of the two variants is predicted to be damaging with
Polyphen, being therefore a less promising candidate to originate
CVID. Finally, KMT2C encodes for a nuclear methyltransferase
(MLL3) of the mixed-lineage leukemia family the genes of which
are among the more frequently mutated in cancer (74); somatic
mutations at MLL3 have been related to different types of cancer
(75), while in activated B-cells, deficiencies in the MLL3–MLL4
complex have been shown to manifest defective immunoglobulin
class switching (76).h In this work, we first approach the proportion of monogenic cases
in CVID by using deep whole-exome sequencing combined with
CNV analysis, in a cohort with mostly early diagnosis patients
(and all of them less than 18 years old), which is expected to
optimize the probability of including monogenic cases (15). We
propose candidate genetic variants and genes with different
levels of confidence (Figure 4). The higher confidence cases
are the five LoF variants very likely to originate CVID: one in
LRBA and CTLA4 (both functionally validated), two in NFKB1
(a large deletion and a new splice-site variant), and one in PIK3R1
(a known splice-site variant causing disease). Thus, a minimum of
15% of the 33 cases included in this study (the 36 patients include
three pairs of relatives) would have a monogenic origin of CVID. Among the LoF variants described in proteins interacting with
CVID candidate genes (Table 2), a new LoF variant in MAPK8
is also a good candidate variant. MAPK8 shows high essentiality
and haploinsufficiency prediction scores and is thought to play
a key role in T cell proliferation, apoptosis, and differentiation
(65–67). This stop gain variant is not found in genetic databases
although it affects a base with a very low GERP value. DISCUSSION We have
also described the presence of LoF variants in the genes CR1,
IBTK, and NCOR2 (Table 2) that have been related to B cell
development and activation (68), agammaglobulinemia (69), and
lymphoma (70, 71), respectively. However, CR1 and IBTK show
low predicted haploinsufficiency values and the cases described
at NCOR2 follow a recessive model for the disease. Thus, the proportion of CVID monogenic cases described in
this work would rank from 15 to 24% or higher (Figure 4), similar
to what has been described in previous studies (12–14) although
lower than the 40% proposed in a recent analysis of 278 PID families
including 20 CVID cases (77) (Table 10). However, these studies
follow differing filtering strategies and stringency criteria making
the results to be only roughly comparable between them. Overall,
the fraction of monogenic CVID cases seems to be slightly lower
to that described in other PID (78, 79), with some recent analyses
showing considerably higher detection rates of PID monogenic
cases (77, 80) which is especially high in a study of severe combined
immunodeficiency (SCID) (81) (Table 10). The higher percentage
of Mendelian patients described in some other PID (77, 80) and
especially SCID (81) is probably because of a higher severity
which is also expected to correlate with the number of Mendelian
cases (15). However, it is important to highlight that different
factors can contribute to an underestimation of the Mendelian
cases in CVID in comparison to other PID. First, because of the
clinical heterogeneity of CVID it is not recommended to apply the
standard exome sequencing strategy where candidate genes are In addition to these LoF mutations at candidate CVID genes and
interacting proteins, we propose other possible monogenic cases
produced by missense variants at CVID candidate genes follow-
ing a dominant (PRKCD, CLEC16A, DOCK8) or recessive models
(CR2, PLCG2), as well as in other genes not previously associated
with CVID (KMT2C). Of interest, missense variants and deletions
in PLCG2 with dominant inheritance have been related to PID in
previous studies (72, 73). RVIS scores are also negative in these three
genes which suggests a certain level of intolerance to mutations,
although in the case of immunological diseases this value seems to
be less indicative than for other diseases (32). Finally, two affected Figure 4 | Scheme of the approaches and main results of this study. Association to Rare Variants We
detected a significant excess of variants in two of the pathways in
CVID patients when compared to controls: Fc epsilon RI signaling
and cytokine–cytokine receptor interaction pathways (P < 0.001
and 0.002, respectively), plus two other marginally significant
pathways after applying multiple test correction: cytosolic-DNA
sensing, and NFKB signaling (P = 0.002 and 0.001, respectively). The four pathways also show a significant excess of functional vari-
ation in CVID patients when compared to the ASD controls set. aNumber of genetic variants at this gene when is greater than one, indicated in
brackets. healthy controls. Table 8 shows the results of the analysis for
the 60 genes analyzed (with at least one synonymous variant
in each cohort), for the 34 patients without a validated can-
didate gene for a monogenic origin of the disease. Four genes
(PRKCD, CLEC16A, DOCK8, and PLCG2) show a statistically
significant excess of rare functional variants in CVID patients, healthy controls. Table 8 shows the results of the analysis for
the 60 genes analyzed (with at least one synonymous variant
in each cohort), for the 34 patients without a validated can-
didate gene for a monogenic origin of the disease. Four genes
(PRKCD, CLEC16A, DOCK8, and PLCG2) show a statistically
significant excess of rare functional variants in CVID patients, May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 9 WES for CVID de Valles-Ibáñez et al. DISCUSSION And third, exome and even genome sequencing have
some limitations that may produce false negatives because of
the difficulties to detect structural variation. However, based on
our results, the contribution of CNVs to monogenic CVID cases
would be quite limited, in contrast to a more important role for
common CNVs proposed in previous studies (84, 85). We have
used one of the highest density array optimized for CNV detection
(86), and detected only a candidate CNV consisting of one big
deletion including, among others, the NFKB1 gene. Similarly, in
the recent whole-exome sequencing analysis of 278 PID families
CNV represented 8% of the likely causing mutations, but no causal
CNV was found among the 20 CVID patients (77). g
Considering the digenic model, we have combined exome
sequencing with PPI data, and described cases of patients with
rare functional variants in CVID candidate genes and an interact-
ing protein. Although promising, to date the number of reported
examples in the literature with pieces of evidence of digenic
inheritance remains quite low (62), probably because of difficul-
ties in statistical and mainly functional analyses to demonstrate
a real role in the disease (63). We have used a prudent approach
based on the existence of physical interactions between proteins,
to produce a reduced number of candidate interactions. Other
tools to identify related genes, as the human genome connectome
(92, 93) or GIANT (94) could also be used. However, since inter-
actions predicted by these tools are based both in physical and
functional associations, the number of candidate protein pairs
would be higher. Still at the individual level, we have considered
a polygenic model and hypothesized that CVID in a particular
patient might be produced by an accumulation of rare functional
genetic variants in genes related to the same function, producing
a list of patients with an excess of genes with functional variants
in the same immunological pathway. Finally, we have performed
tests of association of rare genetic variants to disease. In this
case, the goal is not proposing candidate gene(s) in a particular
patient but to detect genes enriched for rare functional varia-
tion in the cohort of CVID cases compared to healthy controls. DISCUSSION Candidate genes in bold are those with more evidences of being causal. heme of the approaches and main results of this study. Candidate genes in bold are those with more evidences of being causal. May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 10 WES for CVID de Valles-Ibáñez et al. Table 10 | NGS studies on common variable immunodeficiency (CVID) and other primary immunodeficiency (PID). Study
Syndrome
Approach (Coverage)
N
Mendelian casesa
Functional study
Maffucci et al. (13)
CVID
WES (NA), 269 genes(NA)
50
15
No
van Schouwenburg et al. (12)
CVID
WGS (27-40X)
34
NA
RNAseq
This work (2018)
CVID
WES (120X), CNV
36
5–8
CTLA4, LRBA
Gallo et al. (79)
PID
571 genes (580X), WES (>10X)
45
27, 18
10 Genes
Stoddard et al. (78)
PID
173 genes (305X)
120
18
No
Stray-Pedersen et al. (77)
PID, CVID
WES (>100X)
278, 20
110, 8
No
Al-Mouse et al. (80)
PID
162 genes (461X)
139
35
No
Yu et al. (81)
SCID
196 genes (1000X)
20
14
No
aReported in the original study. NA, not available. Table 10 | NGS studies on common variable immunodeficiency (CVID) and other primary immunodeficiency (PID). of many genes and environmental factors (90). Other features,
such as different penetrancies and severities or the phenotypic
variation in affected families, could also suggest an oligogenic
origin for CVID, where the disease is caused or modulated by a
few genes (91). Thus, we have performed different approaches to
explore the possibility of CVID cases being originated by genetic
variants in two or several genes. compared across patients to identify as causal the gene present in
several patients (82). Because of that, we have used a conservative
approach by mainly considering a list of candidate genes, and
used genetic variants characteristics (evolutionary conservation,
Polyphen values) and gene features (haploinsufficiency, essential-
ity or tolerance to functional variation) mainly to indicate but not
conclusively exclude a given candidate gene. For example, filtering
by genic intolerance to functional variation is more effective in
detecting false-positive rather than identifying the causal gene
since it is known that genes producing Mendelian diseases show
from medium to high intolerance values (83). Second, because of
the higher prevalence of CVID compared to other PID, the use of
too stringent frequency filters is not recommended, which hinders
the identification of causal genes by increasing the number of
candidates. Frontiers in Immunology | www.frontiersin.org FUNDING This study was funded by grants SAF2012-35025 and SAF2015-
68472-C2-2-R from the Ministerio de Economía y Competitividad
(Spain) and FEDER (EU) to FC; by Direcció General de Recerca,
Generalitat de Catalunya (2014SGR-866 and 2017SGR-702) to
FC and EB; to EB by grant BFU2016-77961-P from Ministerio de
Economía, Industria y Competitividad (Spain) AEI (Spain) and
FEDER (EU); by Instituto de Salud Carlos III, grant PI14/00405,
cofinanced by the European Regional Development Fund (ERDF)
to RC; partially funded by CERCA Programme/Generalitat de
Catalunya (JIA), and SAF2015-68472-C2-1-R grant from the
Spanish Ministry of Economy and Competitiveness co-financed
by European Regional Development Fund (ERDF) to JIA; GV-I
was supported by grant BES-2012-051794; JH-R was supported
by grant BES-2013-064333. TMB is supported by U01 MH106874
grant, Howard Hughes International Early Career, Obra Social “La
Caixa” and Secretaria d’Universitats i Recerca del Departament
d’Economia i Coneixement de la Generalitat de Catalunya. All
phases of this study were supported by the projects PI12/01990
and PI15/01094 to LA and PI13/00676 to MJ. This work was also
supported by the Jeffrey Modell Foundation. This study makes use
of data generated by the Medical Genome Project. A full list of the
investigators who contributed to the generation of the data is avail-
able from http://www.medicalgenomeproject.com/en. Funding
for the project was provided by the Spanish Ministry of Economy
and Competitiveness, projects I + D + i 2008, Subprograma de
actuaciones Científicas y Tecnológicas en Parques Científicos y
Tecnológicos (ACTEPARQ 2009) and ERFD. We think that CVID is a main example of rare disease where
it is possible to arrive at similar phenotypes by several different
genetic defects, either by mutations in different genes or by differ-
ent genetic mechanisms including from monogenic to epigenetic
scenarios. After the success of new sequencing technologies,
and in particular of whole-exome sequencing in unraveling the
molecular mechanisms of many rare syndromes, rare diseases
such as CVID that do not completely fit with a Mendelian model
represent a new challenge for medical genomics. In this manu-
script, we have proposed different approaches to the analysis
of CVID from whole-exome sequencing data, and have shown
its power and limitations as a diagnostic tool for the study of
these diseases. DISCUSSION Interestingly, most of the genes with significant results in these
analyses (Figure 4) are among the ones with more pieces of evi-
dence of being related to primary immunodeficiencies (51), thus
supporting their role in the etiology of CVID. However, the appli-
cation of these cohort approaches can be limited to syndromes
as CVID because of its genetic heterogeneity. Instead, the use
of higher levels of association such as pathways or functionally
related genes can reduce the genetic heterogeneity and increase
the detection power. Independently of the exact proportion of monogenic cases
in CVID, in an important percentage of patients the disorder
remains genetically uncharacterized, and it seems clear than
other possible models beyond the monogenic scenario should
be considered. A genome-wide association study performed on
363 CVID patients has revealed susceptibility factors in MHC
and ADAM, among others (84), but association with common
variation seems to be far from explaining all non-monogenic
situations. As has been proposed for complex disease, this CVID
missing heritability (87) must be hidden under other models that
have not been deeply explored, as oligogenic, accumulation of
rare functional variation, epigenetic (11, 88) or even somatic (89). In fact, the prevalence of CVID would fit with a model where the
disease is produced by mutations in two or in a few genes, an
intermediate scenario between the very rare disorders originated
by a single locus and common disease produced by the interaction The detection of somatic genetic variants from exome
sequencing data is not straightforward. The detection power May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 11 WES for CVID de Valles-Ibáñez et al. AUTHOR CONTRIBUTIONS FC and MJ conceived the project. LA, MJ, and FC designed the
study. GV-I coordinated the bioinformatic analysis. GV-I, AE-S,
and EAG-N performed the functional validation experiments. All the authors participated in the analysis of the data. GV-I,
AE-S, MP, PS-P, RC, LA, MJ, and FC wrote the manuscript. ETHICS STATEMENT All subjects gave written informed consent in accordance with
the Declaration of Helsinki. The protocol was approved by the
Comitè ètic d’Investigació Clínica-Parc de salut Mar (Barcelona). FUNDING Beyond the identification of the causal gene in
some patients, we hope that these kinds of studies can also be
used to help detect key pathways related to the development of
the disease, thus contributing to a better understanding of its
etiology. From our and previous results, we conclude that in an
important proportion of patients it will be essential to integrate
data from different omic approaches to solve the genetic origin
of the disease. ACKNOWLEDGMENTS We thank all participants in the study. Exome sequencing was
performed at the CNAG (Barcelona). ETHICS STATEMENT ultimately depends on the mutation frequency in the tissue,
which is conditioned by the cell populations affected by the
mutation and their relative abundance in blood, and will be,
therefore, practically undetectable if present in low-frequency
cell populations. On the other hand, high-frequency mutations
present in more than 40% of the reads cannot be differentiated
from germline mutations unless very high coverages are achieved. In addition to high coverages, the modification of standard
NGS data analysis pipelines, which by default discard genetic
variants in allelic imbalance, is required. We have tentatively
analyzed exome sequencing data generated in this study (with
120X is the higher for CVID produced to date) scanning for
low frequency variants with predicted high impact in our set of
candidate genes. Not one of the patients presented a candidate
somatic variant in any of the 97 CVID genes. A previous study
proposed no role for somatic CNV in CVID, based on the sta-
bility of the overall CNV burden over time (85). However, for
a proper analysis of the role of somatic variation much higher
sequencing coverage would be needed, and the possibility of
sequencing different cell populations or tissues with different
origin could be also considered since variant callers for somatic
variant calling are optimized for the comparison between
healthy and affected tissue (tumor). We also propose that, as
a change to the experimental design of our study, late onset
CVID cases should be included in a study targeting somatic
variation. Finally, epigenetics is also suspected to contribute to
CVID. Altered epigenetic profiles are known to be related both
to common and rare genetic disease (95). However, although
epigenetics is known to play an important role in B lymphocyte
differentiation and activation, there is less evidence of their
involvement in PID (96). Interestingly, it has been proposed
that the hypermethylation of important B lymphocyte genes
has a role in CVID, through the analysis of monozygotic
discordant twins (88). Thus, methylation could explain some
of the many cases of CVID with intermediate penetrances,
and also suggests an important role of mutations affecting
gene expression (mostly not detected in exome sequencing
approaches) in CVID. This study was carried out in accordance with the recommenda-
tions of the “Guidelines on the Informed Consent” of the Bioethics
Committee of Catalonia (Departament de Salut, Generalitat
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ncomms8335 Copyright © 2018 de Valles-Ibáñez, Esteve-Solé, Piquer, González-Navarro,
Hernandez-Rodriguez, Laayouni, González-Roca, Plaza-Martin, Deyà-Martínez,
Martín-Nalda, Martínez-Gallo, García-Prat, del Pino-Molina, Cuscó, Codina-Solà,
Batlle-Masó, Solís-Moruno, Marquès-Bonet, Bosch, López-Granados, Aróstegui,
Soler-Palacín, Colobran, Yagüe, Alsina, Juan and Casals. This is an open-access
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provided the original author(s) and the copyright owner are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms. 89. Niemela JE, Lu L, Fleisher TA, Davis J, Caminha I, Natter M, et al. Somatic
KRAS mutations associated with a human nonmalignant syndrome of auto-
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gb-2011-12-9-228 May 2018 | Volume 9 | Article 636 Frontiers in Immunology | www.frontiersin.org 15
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Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract
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⁎ Corresponding author.
E-mail address: rodrigoleite@ufgd.edu.br (R.S.R. Leite).
Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. a r t i c l e
i n f o Background: β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main
product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to
improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF)
using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Article history:
Received 18 February 2015
Accepted 15 May 2015
Available online 27 June 2015
Keywords:
Cellobiase
Cellulases and hemicellulases
Industrial enzymes
Microbial enzymes Article history:
Received 18 February 2015
Accepted 15 May 2015
Available online 27 June 2015
Keywords:
Cellobiase
Cellulases and hemicellulases
Industrial enzymes
Microbial enzymes g
g
y
p
p
y
Results: A high production of β-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by
cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The
enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The
optimal activity of β-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5–10.5. The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to
reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to
10% of ethanol. Conclusions: β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the
utility of this enzyme in diverse industrial processes. © 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. Production of β-glucosidase on solid-state fermentation by Lichtheimia
ramosa in agroindustrial residues: Characterization and catalytic
properties of the enzymatic extract Production of β-glucosidase on solid-state fermentation by Lichtheimia
ramosa in agroindustrial residues: Characterization and catalytic
properties of the enzymatic extract Nayara Fernanda Lisboa Garcia a, Flávia Regina da Silva Santos a, Fabiano Avelino Gonçalves b,
Marcelo Fossa da Paz a, Gustavo Graciano Fonseca b, Rodrigo Simões Ribeiro Leite a,⁎ a Laboratório de Enzimologia e Processos Fermentativos, Faculdade de Ciências Biológicas e Ambientais, Universidade Federal da Grande Dourad
b Laboratório de Bioengenharia, Faculdade de Engenharia, Universidade Federal da Grande Dourados, Dourados, MS, Brazil Electronic Journal of Biotechnology 18 (2015) 314–319 Electronic Journal of Biotechnology 18 (2015) 314–319 Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.ejbt.2015.05.007
0717-3458/© 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction enzymatic hydrolysis of cellulose in order to obtain fermentable
sugars and the production of functional foods derived from soy. The
enzyme is also used in the juice and beverage industry, where it can
improve the aromatic quality of wine and other grape derivatives [3]. The pronounced scarcity of fossil fuels related to environmental
problems resulting from their processing and consumption has
prompted the search for alternative sources of biofuels and renewable
energy. This in turn, has generated significant interest in the use of
cellulases and other enzymes to convert vegetal biomass into
fermentable sugars [1]. The obtainment of industrial enzymes in a sustainable and
economically viable manner requires the pursuit of renewable
raw materials and processes at low cost. The use of solid-state
fermentation (SSF) can reduce the environmental impact and
add value to the by-products of agroindustry [4]. The iterative
improvement and advantages of SSF have been described in several
reports,
which
studied
the
influence
of
different
cultivation
parameters on the production of microbial enzymes [5,6]. The
advantages of SSF include the simplicity of growth conditions, because
they are very similar to the environmental systems where many
microorganisms develop (especially filamentous fungi); the reduced
energy consumption, and that complex equipment or sophisticated
control systems are not required. The method also results in higher
levels of productivity and low catabolite repression, and favors
increased stability of the secreted enzymes [7]. Enzymatic hydrolysis of cellulose to glucose requires at least
three different enzymes including endo-glucanases (EC 3.2.1.4),
that internally hydrolyze cellulose chains, reducing its degree of
polymerization; exo-glucanases (EC 3.2.1.91) that attack the
non-reducing and reducing extremities of cellulose, releasing
cellobiose;
and
β-glucosidases
(EC
3.2.1.21)
that
hydrolyze
cellobiose and oligosaccharides, thereby releasing glucose [2]. The ability of β-glucosidase to utilize different glycosidic substrates
renders it suitable for several industrial processes, including the In general, the industrial applicability of an enzyme is closely related
to the cost of its production and physicochemical characteristics. The N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 315 2.6. Characterization of β-glucosidase produced by the fungus L. ramosa 2.6. Characterization of β-glucosidase produced by the fungus L. ramosa production costs can be reduced by screening hyper producer
strains, associated with the cultivation process optimized in low-cost
mediums [3]. Previous work conducted by our Research Group
revealed high β-glucosidase production by the fungus Lichtheimia
ramosa by SSF using several lignocellulosic materials [6,8]. 2.1. Microorganism The filamentous fungus L. ramosa was isolated from sugarcane
bagasse provided by São Fernando Açúcar e Álcool Ltda., Dourados,
MS, Brazil [8]. The microorganism was maintained on Sabouraud
Dextrose Agar medium; after growth at 28°C for 48 h, the strain was
stored at 4°C. 1. Introduction This study
aimed to optimize the β-glucosidase production by this fungus by SSF. The β-glucosidase produced was biochemically characterized and the
catalytic properties of the enzymatic extract were evaluated. 3.1. Production of β-glucosidase by solid-state fermentation The extraction of the enzyme from the fermented substrate was
carried out by adding 50 mL of distilled water, and constantly shaking
at 100 rpm for 1 h. The sample was filtered and centrifuged at
3000 × g for 5 min. The supernatant was considered the enzymatic
extract and was used in the following steps. 3.1.1. Selection of substrates for β-glucosidase production Among the tested substrates, the cultivation of the fungus L. ramosa
in wheat bran provided higher β-glucosidase production (162.2 U/g
or 16.22 U/mL) (Table 1). The wheat bran has suitable nutritional
composition as a substrate for microbial growth; it contains 2.3. Solid-state fermentation (SSF) The CMCase and xylanase activities were quantified using 3%
carboxymethylcellulose (Sigma C5678) and 1% xylan (Sigma
Birch-Wood), respectively. The reducing sugar released was quantified
by the DNS method [11]. The β-xylosidase activity was measured with
the synthetic substrate p-nitrophenyl-β-D-xylopyranoside (4 mM,
Sigma), following the methodology described in Section 2.5. The
potential to hydrolyze cellobiose was evaluated with a glucose-oxidase
kit (Glucose-PP Analisa). Specifically, 100 μL of the enzymatic extract
was added to 0.9 mL of 50 mM sodium acetate buffer containing 0.5%
cellobiose (Fluka). One unit of enzymatic activity was defined as the
amount of enzyme capable of producing 1 μmol of product per min of
reaction. The enzyme was produced by cultivating the fungus in 250 mL
Erlenmeyer flasks containing 5 g of substrate (wheat bran, soy bran,
corn cob, corn straw, rice peel, or sugarcane bagasse), previously
washed and dried at 60°C for 24 h. The optimal substrate for enzyme
production was used in subsequent steps to evaluate the effects of
varying the pH of cultivation medium, moisture content, temperature,
and time of cultivation. The parameter selected in each step was used
for further cultivation, in an iterative strategy designed to optimize
the fermentation process for β-glucosidase production. All material
was previously autoclaved for 20 min at 121°C, and the experiments
were performed in duplicate. 2.6.1. Effect of pH The optimum pH for β-glucosidase activity was determined by
measuring the activity at 50°C at different pH values (3.0–8.0), with
increments of 0.5, using 0.1 M citrate–phosphate buffer solution. The
pH stability was determined incubating the enzyme for 24 h at 25°C at
different pH values, appropriately diluted with buffer solutions: 0.1 M
citrate–phosphate (pH 3.0–8.0), 0.1 M Tris–HCl (pH 8.0–8.5), and
0.1 M glycine NaOH (pH 8.5–10.5), with increments of 0.5, adopting
as 100% the highest value of residual activity obtained after the
samples treatment. The residual activity was determined under
optimal conditions of pH and temperature. 2.6.3. Effect of glucose and ethanol on β-glucosidase activity .3. Effect of glucose and ethanol on β-glucosidase activity The enzymatic activity was quantified with the addition of glucose or
ethanol at different concentrations in the reaction mixture (0–200 mM
glucose or 0–30% of ethanol). The activities were measured under
optimal conditions of pH and temperature. 2.6.2. Effect of temperature The optimum temperature for β-glucosidase activity was obtained
by determining the enzymatic activity over a temperature range of
30°C–75°C, with increments of 5°C, at the respective optimum pH. Thermostability was determined by incubating the enzyme for 1 h at
different temperatures (30°C–70°C), with increments of 5°C, adopting
as 100% the highest value of residual activity obtained after the
samples treatment. The residual activities were measured under
optimal conditions of pH and temperature. 2.5. Determination of β-glucosidase activity Table 1
β-Glucosidase production in different agroindustrial substrates (75% of initial moisture)
by L. ramosa by solid state fermentation, at 96 h of incubation, under 28°C. Substrate
U/mL
U/g
Wheat bran
16.22 ± 0.42
162.2 ± 4.2
Soy bran
1.15 ± 0.07
11.5 ± 0.7
Corn cob
0.35 ± 0.04
3.5 ± 0.45
Corn straw
0.27 ± 0.02
2.7 ± 0.2
Rice peel
0.068 ± 0.00
0.68 ± 0.00
Sugarcane bagasse
1.11 ± 0.025
11.1 ± 0.25 2.4. Enzyme extraction 3.1. Production of β-glucosidase by solid-state fermentation 2.2. Inoculum The organism was cultivated in inclined 250 mL Erlenmeyer flasks
containing 40 mL of Sabouraud Dextrose Agar and maintained for
48°C h at 28°C. A fungal suspension was obtained by adding 25 mL of
nutrient solution and gently scraping the surface of the culture. The
nutrient solution was composed of 0.1% ammonium sulfate, 0.1%
magnesium sulfate heptahydrate, and 0.1% ammonium nitrate (w/v)
[9]. As inoculum, 5 mL of this suspension was transferred to each
250 mL Erlenmeyer flask containing lignocellulosic material as
substrates. Table 1 Table 1
β-Glucosidase production in different agroindustrial substrates (75% of initial moisture)
by L. ramosa by solid state fermentation, at 96 h of incubation, under 28°C. The β-glucosidase activity was determined with 50 μL of enzymatic
extract, 250 μL of sodium acetate buffer (0.1 M, pH 4.5), and 250 μL of
p-nitrophenyl-β-D-glucopyranoside (4 mM, pNPβG, Sigma) during a
10 min reaction at 50°C. The enzymatic reaction was stopped with
2 mL of sodium carbonate (2 M), and the liberated product was
spectrophotometrically quantified at 410 nm. One unit of enzyme
activity was defined as the amount of enzyme required to release
1 μmol of nitrophenol per minute of reaction [10]. N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 316 appropriate quantities of carbohydrates, proteins, fats, fiber and ashes
(Ca, Mg, P, K, S), favoring enzymes production [12]. medium, interfering with the gas transfer and temperature. On the
other hand, the low moisture content can decrease the solubility of
nutrients, disfavoring microbial metabolic activity [17]. High level β-glucosidase production can be achieved during the
culture of microorganisms by solid-state fermentation, using wheat
bran as either the main substrate, or as a substantial component of the
mixture [3,8,12,13]. Thus, wheat bran was selected for subsequent
assays in order to optimize the cultivation process for β-glucosidase
production. The ideal temperature for β-glucosidase production by L. ramosa was
35°C, about 249.0 U/g (24.9 U/mL) (Fig. 1c). Fig. 1c reveals that a higher
amount of enzyme was produced in cultures carried out at 30°C–40°C. This optimal temperature for enzyme production is higher than the
range most commonly considered optimal for the cultivation of
mesophilic microorganisms, which is usually between 28°C and 30°C
[13,18]. This characteristic favors the use of this strain in industrial
processes, where variations in process temperature are acceptable,
considering that the control of fermentation parameters on a large
scale is not as precise as in laboratory conditions. 3.1.2. Influence of fermentative parameters on β-glucosidase production
using wheat bran as substrate 3.1.2. Influence of fermentative parameters on β-glucosidase production
using wheat bran as substrate The greatest β-glucosidase production by the fungus was obtained
from the cultivation where the initial pH of the nutrient solution was
adjusted to 5.0 (Fig. 1a). However, the microorganism showed
considerable enzyme production for all pH values evaluated. Previous
reports indicate that filamentous fungi produce cellulases at pH values
below neutrality [14]. Table 1 Most filamentous fungi show optimal growth in
slightly acidic pH. In general, values of pH higher than 7.0 reduce
fungal growth and, thereby reducing the enzyme production [15]. A considerable reduction in enzyme production was evident
in cultures performed at 25°C and 45°C (Fig. 1c). Temperatures
that are significantly lower than the optimal for microbial growth
disfavor nutrient transport and the exchange of products between the
intracellular and extracellular environment. This is because low
temperatures reduce both the permeability of the plasma membrane
and the speed of the metabolic reactions. On the other hand, very high
temperatures cause the collapse of membranous structures and
denature structural proteins and enzymes. Accordingly, both cases
result in reduced enzyme production [12]. The pH was not controlled during the cultivation process due to the
heterogeneity of the process of solid-state fermentation. According
to Pandey et al. [16], the difficulty of monitoring and controlling
fermentation parameters in solid-state fermentation is perhaps,
the main drawback of this process. Variations of pH during
the fermentation process are due to the metabolic activity of the
microorganisms, and may be increased or decreased according to the
by-products released or the nutrients consumed during the process. Finally, the influence of cultivation time was investigated. The
highest enzyme production was obtained at 96 h of incubation at 35°C
using wheat bran as the substrate with 65% of moisture and pH 5.0
(Fig. 1d). The overall optimization of the process increased the
β-glucosidase production from 162.2 U/g (16.22 U/mL) to 274.0 U/g
(27.4 U/mL) (Table 1 and Fig. 1d, respectively). In addition to
increased enzyme production, the optimization permitted a reduction
in cultivation time to less than the duration used in preliminary assays
carried out by our research group [4,8]. Gonçalves et al. [8] reported
the production of 17.26 U/mL of β-glucosidase in cultivation of the
fungus L. ramosa for 120 h by solid-state fermentation. Our current
results reinforce the importance of optimizing the culture parameters,
as evidenced by a 68.9% increase in the β-glucosidase production
when compared to the initial values. Among the moisture values evaluated, the highest enzyme
production was obtained in wheat bran with 65% of initial moisture
(Fig. 1b). Values between 60 and 70% of moisture are often used
for cultivation of filamentous fungi when the aim is to produce
β-glucosidase. Leite et al. Table 1 [3] reported the β-glucosidase production
(70 U/g of substrate) by the cultivation of Thermoascus aurantiacus in
wheat bran with 60% of moisture. Brijwani et al. [13] obtained higher
production of β-glucosidase (10.71 U/g) using soybean peel and
wheat bran with 70% of moisture, during co-cultivation of Trichoderma
reesei and Aspergillus oryzae by solid-state fermentation. The moisture in solid-state fermentation can influence the synthesis
and secretion of extracellular enzymes. The presence of free water
between the particles of the substrate reduces the porosity of the The reduced cultivation time, achieved in the present work, is also a
key improvement for fermentation techniques that use L. ramosa, since
the cost of enzyme production is proportional to incubation time. A Fig. 1. Influence of fermentation parameters on β-glucosidase production by the fungus L. ramosa, by SSF in wheat bran. (a) Influence of initial cultivation pH; (b) Influence of initial
substrate moisture; (c) Influence of cultivation temperature; (d) Influence of cultivation time. Conditions: pH 5.0; moisture 65%; temperature 35°C. Fig. 1. Influence of fermentation parameters on β-glucosidase production by the fungus L. ramosa, by SSF in wheat bran. (a) Influence of initial cultivation pH; (b) Influence of initial
substrate moisture; (c) Influence of cultivation temperature; (d) Influence of cultivation time. Conditions: pH 5.0; moisture 65%; temperature 35°C. N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 317 the production of extremely stable β-glucosidase by mesophilic
strains [3]. reduced amount of enzyme was found in extracts obtained after 96 h of
cultivation (Fig. 1d). This is likely explained by the consumption of the
culture medium nutrients and the excretion of by-products by the
microorganism used for fermentation. Such by-products, which may
interfere with protein synthesis as well as enzymatic activity, include
proteases and substances that reduce macro and micronutrients, alter
the pH, and decrease water availability [19]. The β-glucosidase produced by the fungus L. ramosa showed
remarkable structural stability. The enzyme retained its original
activity after 24 h of incubation over a pH range of 3.5–10.5 (Fig. 2c). Regarding the thermal stability of the enzyme, about 90% of the
catalytic activity was recovered after 1 h at 55°C (Fig. 2d). The level of β-glucosidase production obtained during the current
study is significantly higher than that described by other groups. Leite
et al. [3] obtained 7.0 U/mL of β-glucosidase with 72 h of T. aurantiacus cultivation in wheat bran. Delabona et al. 3.2.2. Effect of glucose and ethanol on β-glucosidase activity Evaluation of the effect of ethanol on enzymatic activity is essential
in studies with β-glucosidases, since these enzymes are frequently
exposed to substantial concentrations of alcohol in many industrial
applications [29]. The enzymatic extract obtained, under optimal culture conditions,
was used in subsequent steps for the biochemical characterization of
β-glucosidase. Fig. 3a shows the effect of different concentrations of ethanol (0–
30%) on the enzymatic activity. Ethanol concentrations up to 5%
potentiated the enzymatic activity, and elicited an increase of up to
20% compared to the initial activity. Increasing ethanol concentration
to 15% dramatically reduced enzymatic activity to only 22% of
the original level. However, at a concentration of 10% of ethanol,
β-glucosidase retained a level of catalytic activity similar to the
control. Considering that the final ethanol concentration in fermented
broths obtained by traditional processes is around 10% [30], we infer
that the enzyme is sufficiently stable to be applied in industrial
fermentation processes containing ethanol. Table 1 [20] obtained
105.82 U/g of substrate by cultivation of the fungus Aspergillus
fumigatus in wheat bran for 96 h. Xin and Geng [21] obtained 61.6 U/g
of substrate with T. reesei cultivated at 26°C for 192 h on woodchips. Ng et al. [2], reported the production of 159.1 U/g of substrate by the
fungus Penicillium citrinum YS40-5 after cultivation in rice bran for
96 h. Zimbardi et al. [22] optimized the β-glucosidase production by
the fungus Colletotrichum graminicola in wheat bran, with a maximum
production substrate of 159.3 U/g, after 168 h. Silva et al. [6] reported
the production of 0.061 U/mL of β-glucosidase by the fungus L. ramosa
in pequi residue (typical fruit of the Cerrado vegetation), after 48 h in
solid-state fermentation. The results are more significant when compared with previously
published data. β-glucosidase produced by different species of
Penicillium showed stability from pH 4.0 to 6.0 [26,27]. The enzyme
produced by the fungus Trichoderma harzianum was stable at
temperatures below 55°C for 15 min, maintaining only 36% of
initial activity after 15 min at 60°C [28]. Delabona et al. [5] describe
the stability of β-glucosidase produced by the fungus A. fumigatus
P40M2 at temperatures from 40°C to 60°C and at pH 3.0 to 5.5. The β-glucosidase produced by the yeast Sporidiobolus pararoseus
maintained its catalytic activity for 1 h at 40°C; at higher temperatures,
only 30% of the initial activity was recovered [24]. 3.2. Characterization of β-glucosidase produced by the fungus L. ramosa 3.2. Characterization of β-glucosidase produced by the fungus L. ramosa 3.2.1. Effect of pH and temperature However, relatively little is known regarding the characteristics of
these enzymes, and thus further studies in this area are required. Moreover, there is the possibility to improve these enzymes
production in new works of culture optimization. Silva et al. [36]
reported the production of 60 U/mL of CMCase and 107 U/mL by the
fungus T. aurantiacus by solid-state fermentation, using corncob as
substrate. Delabona et al. [20] report the production of 160.1 U/g of
CMCase and 1055.62 U/g of xylanase by the fungus A. fumigatus
cultivated in agroindustrial residue products. Different concentrations of glucose were added to the reaction
mixture to evaluate the behavior of β-glucosidase in the presence
of this inhibitor. The enzyme was strongly inhibited by glucose,
maintaining approximately 30% of its original activity in the reaction
carried out with 100 mM glucose (Fig. 3b). The majority of microbial β-glucosidases are inhibited by glucose,
which is a major limitation of the use of these enzymes in industrial
processes [3]. High glucose concentrations can interfere directly or
indirectly with substrate binding to the enzyme active site, reducing
the reaction rate [33]. The inhibition of β-glucosidase produced by
L. ramosa was completely reversed when the substrate concentration
was increased to the same glucose concentration, indicating that the
interaction of the enzyme with the inhibitor is competitive. Another interesting aspect of the current study, shown in Table 2, is
the impressive potential of the enzyme to hydrolyze cellobiose, as it
yields values similar to those obtained with a synthetic substrate
(pNPG). The microbial β-glucosidases can be classified into three
major groups: (1) Aryl β-glucosidases, which exhibit high specificity
to hydrolyze aryl-glycosides substrates, (2) true cellobiases, which are
enzymes with that hydrolyze cellobiose with high specificity and (3)
enzymes with low specificity: enzymes that act on different types of
glycosides substrates [25,31]. Apparently, β-glucosidase expressed
from the fungus L. ramosa has low specificity; that is, it has the
potential to hydrolyze different glycosides substrates. However, to
confirm this hypothesis, further studies should be performed with the
purified enzyme. According to Bhatia et al. [25], most of microbial
β-glucosidases are classified in the third group. Competitive inhibition can be reversed by increasing substrate
concentration; the same fact is not observed in non-competitive
inhibition. In competitive inhibition, the inhibitor and the substrate
compete for the same binding site of the enzyme (the active site, in
this case). 3.2.1. Effect of pH and temperature 3.2.1. Effect of pH and temperature The β-glucosidase produced by the fungus L. ramosa showed
higher catalytic activity at pH 5.5 and temperature of 65°C (Fig. 2a and
Fig. 2b). Surprisingly, the enzyme showed higher catalytic activity at
temperatures above 50°C, not observed routinely in enzymes
produced by mesophilic microorganisms. Belancic et al. [23] obtained
the best activity at pH 5.0 and optimum temperature of 40°C for
β-glucosidase produced by Debaryomyces vanrijiae. Most fungal
β-glucosidases show optimum activity between 40°C and 50°C and at
pH between 4.0 and 6.0 [24,25]. However, some studies have reported The increase in the catalytic potential of β-glucosidases observed by
the ethanol addition is related to the glucosyltransferase activity [31]. Ethanol can increase the rate of reaction by acting as preferred
acceptor of glycosyl residues during enzymatic catalysis [32]. The Fig. 2. Effect of pH and temperature on the activity (A and B) and stability (C and D) of β-glucosidase produced by the fungus L. ramosa by SSF in wheat bran, with 65% of moisture and
initial pH 5.0, incubated for 96 h at 35°C. Fig. 2. Effect of pH and temperature on the activity (A and B) and stability (C and D) of β-glucosidase produced by the fungus L. ramosa by SSF in wheat bran, with 65% of moisture and
initial pH 5.0, incubated for 96 h at 35°C. N.F.L. Garcia et al. / Electronic Journal of Biotechnology 18 (2015) 314–319 318 J Fig. 3. (a) effect of ethanol, and (b) glucose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. Fig. 3. (a) effect of ethanol, and (b) glucose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. g. 3. (a) effect of ethanol, and (b) glucose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and ose, on the activity of β-glucosidase produced by L. ramosa by SSF in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35 hydrolysis and transglycosylation occur through the same biochemical
pathway, differing only in the nature of the final acceptor [25]. 3.2.1. Effect of pH and temperature Thus, increasing the substrate concentration to equal or
greater values than those of the inhibitor, favors the binding of the
enzyme to the substrate, which is reflected in the reversibility of
enzymatic inhibition [3]. The reversibility of inhibition by glucose and stability to ethanol
confirm the potential of this β-glucosidase for applications that
require simultaneous saccharification and fermentation processes [3],
where the monosaccharides released by enzymatic hydrolysis are
simultaneously converted to ethanol by fermenting microorganisms
[34,35]. Table 2 Table 2
Catalytic potential of the enzymatic extract obtained by solid-state fermentation by L. ramosa in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. Enzyme
Substrate
U/mL
U/g
CMCase
Carboxymethylcellulose
15.21
152.1
β-Glucosidase
p-nitrophenyl-β-D-glucopyranoside (pNPG)
23.47
237.7
β-Glucosidase
Cellobiose
23.45
234.5
Xylanase
Xylan
2.85
28.5
β-Xylosidase
Xylopyranoside p-nitrophenyl-β-D (pNPX)
11.57
115.7 Catalytic potential of the enzymatic extract obtained by solid-state fermentation by L. ramosa in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. Catalytic potential of the enzymatic extract obtained by solid-state fermentation by L. ramosa in wheat bran, with 65% of moisture and initial pH 5.0, incubated for 96 h at 35°C. 4. Conclusions L. ramosa has proven to be a remarkable fungus for use in
β-glucosidase production when cultivated by solid-state fermentation
using wheat bran as the substrate. This fungus is capable of producing
several enzymes, including CMCase and β-xylosidase. β-Glucosidase
was highly stable across a range of pH and temperatures and retained
its
original
activity
in
solutions
containing
10%
of
ethanol. Furthermore, the inhibitory effects of glucose were completely
reversed at high substrate concentrations. The enzyme can hydrolyze
different glycoside substrates; due to these characteristics, the
β-glucosidase produced by L. ramosa can be used for the production of
second-generation ethanol as well as for the improvement of food and
beverage quality. 3.3. Catalytic potential of the enzymatic extract 3.3. Catalytic potential of the enzymatic extract The production of other cellulases and also hemicellulases by
L. ramosa, in optimized culture conditions, was evaluated in this work
(Table 2). The enzymatic extract exhibited CMCase (152.1 U/g or
15.21 U/mL), xylanase (28.5 U/g or 2.85 U/mL), and β-xylosidase
(115.7 U/g or 11.57 U/mL) activity. The production of the CMCase and hemicellulases by L. ramosa is not
very impressive when compared with hyper-producing strains. Financial support The authors gratefully acknowledge the financial support of the
Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq
(444630/2014-7), the "Fundação de Apoio ao Desenvolvimento do
Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul-FUNDECT
(23/200.211/2014), and the Coordenação de Aperfeiçoamento Pessoal
de Nível Superior-CAPES. Enzyme
Substrate
U/mL
U/g
CMCase
Carboxymethylcellulose
15.21
152.1
β-Glucosidase
p-nitrophenyl-β-D-glucopyranoside (pNPG)
23.47
237.7
β-Glucosidase
Cellobiose
23.45
234.5
Xylanase
Xylan
2.85
28.5
β-Xylosidase
Xylopyranoside p-nitrophenyl-β-D (pNPX)
11.57
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0206599&type=printable
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English
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Quantification of avian hazards to military aircraft and implications for wildlife management
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PloS one
| 2,018
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public-domain
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Quantification of avian hazards to military
aircraft and implications for wildlife Morgan B. PfeifferID1,2*, Bradley F. Blackwell1, Travis L. DeVault1 Morgan B. PfeifferID1,2*, Bradley F. Blackwell1, Travis L. DeVault1
1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio,
United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela
University, George, South Africa 1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio,
United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela
University, George, South Africa 1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio,
United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela
University, George, South Africa 1 USDA, APHIS, Wildlife Services, National Wildlife Research Center, Ohio Field Station, Sandusky, Ohio,
United States of America, 2 School of Natural Resource Management, George Campus, Nelson Mandela
University, George, South Africa a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * morgan.b.pfeiffer@aphis.usda.gov * morgan.b.pfeiffer@aphis.usda.gov Editor: Ludek Bartos, Institute of Animal Science,
CZECH REPUBLIC Received: May 21, 2018
Accepted: October 16, 2018
Published: November 1, 2018 Copyright: This is an open access article, free of all
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or
otherwise used by anyone for any lawful purpose. The work is made available under the Creative
Commons CC0 public domain dedication. Data Availability Statement: All relevant data are
available from Dryad, DOI: https://doi.org/10.5061/
dryad.s0n4n05. Funding: This study was funded by the
Comannder, Navy Installations Command (CNIC)
Airfield Operations Program. No additional external
funding was received for this study. The funder had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. Abstract Collisions between birds and military aircraft are common and can have catastrophic effects. Knowledge of relative wildlife hazards to aircraft (the likelihood of aircraft damage when a
species is struck) is needed before estimating wildlife strike risk (combined frequency and
severity component) at military airfields. Despite annual reviews of wildlife strike trends with
civil aviation since the 1990s, little is known about wildlife strike trends for military aircraft. We hypothesized that species relative hazard scores would correlate positively with aircraft
type and avian body mass. Only strike records identified to species that occurred within the
U.S. (n = 36,979) and involved United States Navy or United States Air Force aircraft were
used to calculate relative hazard scores. The most hazardous species to military aircraft
was the snow goose (Anser caerulescens), followed by the common loon (Gavia immer),
and a tie between Canada goose (Branta canadensis) and black vulture (Coragyps atratus). We found an association between avian body mass and relative hazard score (r2 = 0.76) for
all military airframes. In general, relative hazard scores per species were higher for military
than civil airframes. An important consideration is that hazard scores can vary depending on
aircraft type. We found that avian body mass affected the probability of damage differentially
per airframe. In the development of an airfield wildlife management plan, and absent esti-
mates of species strike risk, airport wildlife biologists should prioritize management of spe-
cies with high relative hazard scores. RESEARCH ARTICLE OPEN ACCESS Citation: Pfeiffer MB, Blackwell BF, DeVault TL
(2018) Quantification of avian hazards to military
aircraft and implications for wildlife management. PLoS ONE 13(11): e0206599. https://doi.org/
10.1371/journal.pone.0206599 Citation: Pfeiffer MB, Blackwell BF, DeVault TL
(2018) Quantification of avian hazards to military
aircraft and implications for wildlife management. PLoS ONE 13(11): e0206599. https://doi.org/
10.1371/journal.pone.0206599 Editor: Ludek Bartos, Institute of Animal Science,
CZECH REPUBLIC
Received: May 21, 2018
Accepted: October 16, 2018
Published: November 1, 2018 Introduction One of the most ubiquitous human-wildlife conflicts involves wildlife colliding with transpor-
tation [1]. These collisions usually result in the death of the animal, damage to the vehicle, and
even injuries to humans or human fatalities [2]. Compared to other countries, the United
States of America (U.S.) has the most reported wildlife collisions (strikes) with aircraft, likely
because of numerous aircraft operations, mandatory strike reporting in some sectors, and an
abundance of large-bodied birds [3, 4]. Ranking of wildlife hazards to civil aircraft has been Competing interests: The authors have declared
that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 1 / 16 Military avian hazard scores conducted regularly since the 1990s [5–7], but rarely for military aviation. Furthermore, to our
knowledge, relative hazard scores (RHS) for military aircraft have not been calculated or pub-
lished [8]. According to the United States Air Force (USAF) wildlife strike database from
1985–1998, an average of $35 million (U.S.) in damage was attributed to wildlife strikes annu-
ally [8]. To mitigate this risk of wildlife strikes, most United States military bases with a flying
mission require a Bird/Wildlife/Animal Aircraft Strike Hazard program [9,10]. A necessary component of estimating strike risk (see below), and thus prioritizing resources
and time for mitigating wildlife strikes, is the ranking of wildlife by the hazard they pose to avi-
ation. Hazard is defined in terms of the likelihood of damage, substantial damage, or negative
effect-on-flight procedures when a strike event occurs [11]. This hazard, or severity, of a strike
with an animal is one component used to estimate strike risk, along with a measure of the fre-
quency of the event (i.e. how often strikes occur per species) [12]. Because RHS have never
been calculated for military aircraft, our objective was to calculate these scores as the first step
in estimating risk, which is dependent upon a number of local-level variables [12], in addition
to mission type. Wildlife hazard ranking has been conducted for civil aviation in a number of ways [6, 11,
13]. A previous study on USAF wildlife strikes ranked species groups (i.e. species that are
closely related phylogenetically) based on the number of strikes within each damage class (i.e. categories of monetary damage costs to aircraft) and found that vultures (Cathartidae), geese
(Anatidae), pelicans (Pelecanidae), blackbirds/starlings (Icteridae/Sturnidae), and buteo
hawks (Accipitridae) were ranked as most hazardous, in that order [8]. Introduction However, not all wild-
life strikes result in aircraft damage; therefore, damaging or negative effect-on-flight strikes
per species should be considered as a proportion of all strikes (including non-damaging
strikes) for that species or group [11]. In recent wildlife hazard rankings for civil aviation, a
composite hazard score was calculated that included the sum of the ranks of strikes with dam-
age, substantial damage, and strikes that had a negative effect-on-flight [11, 12, 14]. Replicating
the methods of the previous examples must be done with special consideration of the differ-
ences between civil and military aviation and the assumed variation in the bird strike risk. Military and civil operations differ in their mission types, including air speeds and maneu-
verability, which could affect wildlife hazard scores. During certain training procedures, mili-
tary aircraft travel at high speeds and close to the ground, which likely increases the strike
hazard compared to civil aircraft because of more time spent in bird rich altitudes [15]. Dam-
age to aircraft is a function of kinetic energy which is the product of the mass of the bird in
relation to the velocity of the aircraft [16]. However, birds with smaller body mass might still
pose a substantial hazard to military, as compared to civil aircraft because of the increased air-
craft speeds, particularly at lower altitudes, characteristic of military operations. In other
words, whereas civil strikes involving smaller birds generally pose less hazard, collisions of
small bird species with military aircraft can involve much higher airspeeds. Hence, a hazard
difference between civil and military aviation may be more noticeable with the smaller rather
than the larger birds. In addition, unlike civil aviation, military training operations can gener-
ally adjust their flight schedule if hazards, including the risk of bird collisions, is predicted to
be elevated [17]. Given these considerations, our purpose was to calculate avian RHS for the United States
Navy (USN), including the United States Marine Corp, and the USAF. We predicted that air-
craft type and body mass would influence RHS [11]. Because military airframes are designed
for a variety of purposes and generally only conduct certain maneuvers (e.g. fighter airframes
that accelerate rapidly and turn with high precision vs. cargo airframes designed to move
heavy payloads), we calculated RHS separately for grouped military airframe type. Materials and methods We used wildlife strike records from databases maintained by the USN and the USAF. The
USN wildlife strike database spanned 27 years (1990–2017) and contained 21,661 wildlife
strike records. The USAF dataset spanned 23 years (1994–2017) and contained 104,129 wildlife
strike records. Wildlife remains found on an aircraft after a strike event are required to be sent
to the Feather Identification Lab at the Smithsonian Institution, where personnel identify wild-
life based on feather characteristics and/or DNA [18]. Some species information for the USN
database was gleaned from the Smithsonian Institution’s internal database by matching the
event number. We filtered these databases to only include strikes that occurred within the U.S. where the
species involved in the strike was identified. We did not filter strikes by altitude (i.e. < 152 m),
which was done in similar studies with civil aviation to focus on the airport environment [11]. Military aircraft do not adhere to the 3˚ glide slope of ascent and decent of civil aircraft, from
which the threshold of 152 m is based [19], and routinely conduct low-level flights. If multiple
animals were involved in one strike event, they are entered as separate strikes, but with the
same report number. To prevent duplication, only one strike per event was used (i.e. duplicate
report numbers were removed). Duplicate report numbers were removed based on body size;
the larger of the two species was retained. Less than 12% of all records involved more than one
reported species, in which we kept the larger of the two species for the analysis. Only species
with more than 20 strikes were used in our analyses [12, 14]. To reduce complexity and facili-
tate management decisions, those species involved in fewer than 50 strikes were combined
into species groups (S1 Table) based on phylogeny [20]. Species groups (n = 17) were com-
posed of closely related species as per [20] and did not include any strikes in which guild was
identified but not species. A total of 12 species with more than 20 but less than 50 strikes
(noted in S1 Table) could not be grouped because they were not closely related to other species
and left as is in the analyses A similar species grouping scheme is accepted by the FAA in the
management of hazardous species at airports and this scheme allows us to compare to other
studies [12, 14]. Introduction We pre-
dicted that airframes with more sensitive components would experience higher RHS PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 2 / 16 Military avian hazard scores compared to robust airframes. Calculating hazard scores in this way can help prioritize spe-
cies-specific wildlife management plans for military airfields across the U.S. serving different
mission types. Based on previous work involving civil aircraft [6, 11], we predicted that body
mass and migration flyway (Central, Mississippi, Pacific, Atlantic) would contribute to pre-
dicting level of damage (see below) for military aircraft. Lastly, because of differing reporting
rates, we predicted that probability of damage would differ between the two military branches. Furthermore, significant relationships between body mass and RHS could be used to calculate
hazard scores for species that are not currently in our dataset (e.g. solve the equation for RHS
based on avian body mass). PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Materials and methods Damage to aircraft from a wildlife strike in the military databases is recorded as a damage
class (A/B/C/D/E/H, Table 1), based on monetary costs of repairs and human injuries rather
than a categorical definition like in civil aviation. The A damage class is the most severe and H
is the least [6, 8]. The USAF are required to report wildlife strikes under the Air Force Instruc-
tion 91–202 Mishap Prevention Program [9] and the Air Force Instruction 91–204 Safety
Investigations Reports [21]. The USN is required to report wildlife strikes under the Com-
mander, Navy Installations Command Instruction 3750.1 [10]. The military databases were filtered by their damage class. None of the defined damage cat-
egories are related to a strike in which no damage occurred, which is needed for the hazard
ranking [6]. Therefore, strikes for which class severity was unknown in the USAF dataset were 3 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Military avian hazard scores Table 1. Definitions of United States Navy (USN) and United States Air Force (USAF) damage classes in U.S. dol-
lars. Damage class ‘H’ was divided for the USN dataset based on internal discussions. Damage Class
Associated monetary Cost
A
> $2,000,000
B
$500,000 –$2,000,000
C
$50,000 –$500,000
D
$20,000 –$50,000
E
< $50,000
H (damaging)
> $55
H (non-damaging)
$55
https://doi.org/10.1371/journal.pone.0206599.t001 efinitions of United States Navy (USN) and United States Air Force (USAF) damage classes in U.S. dol-
age class ‘H’ was divided for the USN dataset based on internal discussions. https://doi.org/10.1371/journal.pone.0206599.t001 considered to be non-damaging (pers. comm. D. Sullivan, Chief, USAF BASH Team). In the
USN dataset, there were no records in which strike severity was unknown. After discussions
with USN personnel, we separated Class H strikes in the USN dataset into damaging (> $55 of
damage costs) or non-damaging ( $55 of damage costs) strikes. The $55 threshold is the aver-
age cost of collecting wildlife remains from an aircraft after a strike occurs, and generally does
not indicate damage to the aircraft. As these classifications differed from the civil database
(minor vs. substantial damage) and were much broader, we used the median damage repair
costs for civil aviation that had substantial damage to create a cutoff value between minor and
substantial damage for the military dataset [6]. Materials and methods We then calculated separately the percentage of
total strikes for each species group that resulted in damage and substantial damage [11, 14]. We were unable to use the effect-on-flight metric because these data only were recorded in the
USAF and not the USN database. To increase sample size for our across-branch analysis, we
combined the USN and USAF strike records, which operate similar aircraft types and are
located across the U.S. We ranked species groups as per Dolbeer et al. [6], minus the effect-on-flight metric. This
procedure involved determining the percentage of total strikes for each species group that
resulted in damage and substantial damage. The RHS was calculated by summing those per-
centages and scaling them to 100 by dividing the sum of percentages for that species by the
maximum percentage for any species. Next, the species groups were ordered from most to
least hazardous, including tied ranks. We then categorized airframe groups that were similar
in size and flight patterns (S2 Table). Airframe groups were suggested by military aviation
experts and included four groups: rotorcraft (e.g. helicopters), fighters, cargo, and stealth air-
frames. Some airframes could be classified in multiple categories (i.e. F-35 is considered a
stealth fighter). For our purposes we grouped airframe by mission type (quick maneuverabil-
ity, moving payloads, or surveillance missions), which may differ from other airframe group-
ings. We highlight these unique airframe groups in S2 Table. We calculated RHS for each
airframe group from the combined USAF and USN dataset. We investigated differences in rel-
ative hazard scores of species groups and airframes using contingency tables and the chi-
square test for goodness of fit [22]. As previous research identified avian body mass as an important predictor for RHS, we
examined the relationship between RHS and avian body mass using averaged avian body mass
within species [23]. For species groups, we weighted averaged body masses by the respective
number of strikes for each species in the group. The bird body masses were log-transformed to
normalize the data. We regressed species RHS against log-transformed avian body masses for
all aircraft and the four airframe groups separately via a quadratic function or linear regression
(based on best model fit). These relationships do not have biological ties, but rather relate to
the physics of the mass of the bird species and the rate and pattern of influence on the RHS. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Materials and methods 4 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Military avian hazard scores We assessed model fit by the coefficient of determination (r2 value) and p value (α = 0.05)
[24]. To emphasize the differences in airframe and flight demands that likely contribute to var-
iance in strikes between military and civil aircraft, we descriptively compared species RHS
between military and civil aircraft [12]. Lastly, we converted damage class into two binary variables for damage (1 = any level of
damage occurred, 0 = no damage occurred) and for substantial damage (1 = substantial dam-
age occurred, 0 = no substantial damage occurred) and used a binary logistic regression to
evaluate how log body mass, migration flyway, reporting military branch, and airframe influ-
enced the probability damage [14]. All variables were considered fixed effects and were
included based on a priori hypotheses. Log body mass was a continuous variable and the
remainder were categorical variables. Migration flyway included four levels: Atlantic, Central,
Mississippi, and Pacific. Reporting military branch included USAF or USN. We also included
interaction variables of the predictors. Candidate models were evaluated by their Akaike’s
Information Criterion (AIC) with the best model having the lowest AIC [25]. We also calcu-
lated the McFadden’s r2 value as an indication of fit 1- log likelihood of the model over the log
likelihood of the null model [26]. Some strikes involved more than one individual bird, and we
could account for this random effect for the specific strike event by using a generalized linear
mixed model approach with strike event as the random variable. However, with over 30,000
strikes, our models failed to converge. All database filtering and data analyses were conducted
in R ver. 3.4.3. [27] and the ‘lme4’ and ‘arm’ packages [28,29]. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Military avian hazard scores Table 2. Relative hazard scores (RHS) for 108 species groups from most to least hazardous for military aircraft within the United States. Results From 1990–2017 an average of $20 million in damage and human injuries was attributed to
wildlife strikes (n = 6,733) per annum for the USN in foreign and domestic operations. For the
USAF in the U.S., from 1994–2017, an average of $38 million per annum in damage and
human injuries was attributed to wildlife strikes (n = 104,129). After removing strikes that
occurred in other countries, not identified to avian species, and involved a species struck more
than 20 times, the combined dataset was reduced to 36,979 strikes. Of this 3,646 (10%) strike
records were from the USN and 33,333 strike records (90%) came from the USAF. The median cost for civil aviation from 2000–2015 with substantial damage was $87,570. The maximum cost associated with Class D/E/H strikes fell below $87,570, so only Class A/B/
C strikes were considered as comprising substantial damage ($50,000 to $2,000,000) in our
military dataset. For both military branches there were 923 strikes with substantial damage
(Class A/B/C). An additional 3,024 strikes reported minor damage (Class D/E/H), which
brought the total of damaging strikes to 3,947. Strikes reported with no damage (damage class
unknown) represented the majority of the strike records (n = 33,032). A total of 186 bird species was involved in 20 or more strikes with USN and USAF aircraft,
and 3 species were struck more than 2,000 times: horned lark (Eremophila alpestris), mourning
dove (Zenaida macroura), and barn swallow (Hirundo rustica). Birds were categorized into
108 groups. The percentage of strikes with some level of damage ranged from 1% (burrowing
owl [Athene cunicularia]) to 74% (snow goose [Anser caerulescens]). There were 40 species or
species groups involved in strikes with no substantial damage (Table 2). The species group
involved in the highest percentage of strikes with substantial damage was the snow goose
(42%). The top three species by their composite rank were snow goose, common loon (Gavia
immer), and a tie between Canada goose (Branta canadensis) and black vulture (Coragyps atra-
tus) (Table 2). Filtering the strike records to only fighter, cargo, stealth, and rotorcraft and to species or
species groups that were struck over 20 times reduced the total number of strike records to 5 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Species
% with
damage
Damage
rank
% with substantial
damage
Substantial damage
rank
Relative hazard
score
(RHS)
Composite
rank
Snow goose (Anser caerulescens)
74
1
42
1
100
1
Common loon (Gavia immer)
70
2
30
2
86
2
Black vulture (Coragyps atratus)
58
3
25
4
72
3
Canada goose (Branta canadensis)
56
4
29
3
74
3
Turkey vulture (Cathartes aura)
48
5
21
5
60
5
Northern pintail (Anas acuta)
44
8
16
7
51
6
Mallard (Anas platyrhynchos)
45
6
13
10
50
7
Swainson’s hawk (Buteo swainsoni)
41
9
14
8
47
8
Double-crested cormorant (Phalacrocorax
auritus)
44
7
13
11
48
9
Herring gull (Larus argentatus)
32
14
18
6
43
10
Red-tailed hawk (Buteo jamaicensis)
37
11
14
9
43
10
Bald eagle (Haliaeetus leucocephalus)
40
10
11
13
44
12
Other ducks
36
12
10
15
39
13
Pied-billed grebe (Podilymbus podiceps)
29
16
12
12
35
14
Great blue heron (Ardea herodias)
31
15
11
14
36
15
Osprey (Pandion haliaetus)
35
13
9
16
37
15
Other egrets
29
18
7
19
31
17
Other hawks
27
19
7
21
29
18
American coot (Fulica americana)
29
17
5
25
29
19
Ring-billed gull (Larus delawarensis)
19
25
8
18
23
20
Sprague’s pipit (Anthus spragueii)
17
28
8
17
22
21
Cattle egret (Bubulcus ibis)
18
26
6
22
21
22
White-winged dove (Zenaida asiatica)
20
23
5
26
21
23
Common grackle (Quiscalus quiscula)
18
27
6
24
21
24
Great horned owl (Bubo virginianus)
20
22
4
29
21
24
Other falcons
15
33
7
20
19
26
Mississippi kite (Ictinia mississippiensis)
23
20
3
35
22
27
Other gulls
16
30
4
28
18
28
American crow (Corvus brachyrhynchos)
13
37
6
23
16
29
Rock dove (Columba livia)
15
31
4
30
17
30
White-throated swift (Aeronautes saxatalis)
14
34
4
32
15
31
European starling (Sturnus vulgaris)
13
37
3
33
14
32
Yellow-billed Cuckoo (Coccyzus
americanus)
15
32
2
38
15
32
Vaux’s swift (Chaetura vauxi)
12
40
4
31
14
34
Barn owl (Tyto alba)
12
41
3
34
12
35
Baltimore oriole (Icterus galbula)
10
48
2
36
11
36
Other blackbirds
9
58
5
27
12
37
Black-bellied plover (Pluvialis squatarola)
13
36
1
50
12
38
Scarlet tanager (Piranga olivacea)
14
35
1
52
13
39
Other doves
11
44
2
44
11
40
Purple martin (Progne subis)
11
42
1
48
11
41
American robin (Turdus migratorius)
10
49
2
43
10
42
Other plovers
11
46
2
46
11
42
(Continued) ores (RHS) for 108 species groups from most to least hazardous for military aircraft within the United States. Military avian hazard scores Table 2. (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 6 / 16 (Continued)
Species
% with
damage
Damage
rank
% with substantial
damage
Substantial damage
rank
Relative hazard
score
(RHS)
Composite
rank
Yellow-bellied sapsucker (Sphyrapicus
varius)
9
60
2
37
10
44
Mourning dove (Zenaida macroura)
9
55
2
45
9
45
Brown-headed cowbird (Molothrus ater)
10
47
1
55
10
46
Northern flicker (Colaptes auratus)
20
21
0
82
18
47
Ovenbird (Seiurus aurocapillus)
8
63
2
40
9
47
Other shorebirds
9
57
1
47
9
49
Other thrushes
11
43
1
62
10
50
Brown thrasher (Toxostoma rufum)
20
24
0
82
17
51
Cave swallow (Petrochelidon fulva)
11
45
1
61
10
51
Cardinals, grosbeaks, and allies
10
51
1
60
10
53
Red-winged blackbird (Agelaius phoeniceus)
7
70
2
41
8
53
Wood thrush (Hylocichla mustelina)
17
29
0
82
14
53
Great crested flycatcher (Myiarchus crinitus)
8
65
1
48
8
56
Meadowlarks
7
71
2
42
7
56
American kestrel (Falco sparverius)
7
68
1
51
7
58
Gray catbird (Dumetella carolinensis)
10
50
0
70
9
59
Ruby-crowned kinglet (Regulus calendula)
7
67
1
53
7
59
Violet-green swallow (Tachycineta
thalassina)
12
39
0
82
10
61
Scissor-tailed flycatcher (Tyrannus
forficatus)
9
56
1
68
8
62
Sora (Porzana carolina)
10
52
0
82
8
63
Blue-gray gnatcatcher (Polioptila caerulea)
7
69
1
66
7
64
Semipalmated sandpiper (Calidris pusilla)
10
53
0
82
8
64
Northern mockingbird (Mimus polyglottos)
9
54
0
82
8
66
Killdeer (Charadrius vociferous)
6
81
1
59
6
67
Orchard oriole (Icterus spurius)
9
58
0
82
8
67
Other flycatchers
8
61
0
82
7
69
Other terns
4
104
2
39
5
69
Other wood warblers
6
75
0
69
6
71
Short-eared owl (Asio flammeus)
8
62
0
82
7
71
Red-eyed vireo (Vireo olivaceus)
6
74
0
71
6
73
Barn swallow (Hirundo rustica)
6
79
1
67
6
74
Upland sandpiper (Bartramia longicauda)
8
64
0
82
7
74
House wren (Troglodytes aedon)
5
92
1
55
5
76
Lapland longspur (Calcarius lapponicus)
6
82
1
65
5
76
Dunlin (Calidris alpine)
8
66
0
82
7
78
Hermit thrush (Catharus guttatus)
6
76
0
75
6
79
Indigo bunting (Passerina cyanea)
5
88
1
63
5
79
Horned lark (Eremophila alpestris)
5
95
1
58
5
81
American goldfinch (Spinus tristis)
6
72
0
82
6
82
Chimney swift (Chaetura pelagica)
6
77
0
78
6
83
Ruby-throated hummingbird (Archilochus
colubris)
6
73
0
82
6
83
American pipit (Anthus rubescens)
5
100
1
57
5
85
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 7 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Military avian hazard scores (
)
Species
% with
damage
Damage
rank
% with substantial
damage
Substantial damage
rank
Relative hazard
score
(RHS)
Composite
rank
Cliff swallow (Petrochelidon pyrrhonota)
6
78
0
79
5
85
Western kingbird (Tyrannus verticalis)
4
105
1
54
4
87
Common nighthawk (Chordeiles minor)
5
96
1
64
5
88
Cedar waxwing (Bombycilla cedrorum)
5
84
0
77
5
89
Other wrens
6
80
0
82
5
90
Common snipe (Gallinago gallinago)
6
83
0
82
5
91
Other sparrows
5
94
0
72
5
92
Golden-crowned kinglet (Regulus satrapa)
5
85
0
82
5
93
Bank swallow (Riparia riparia)
5
86
0
82
4
94
Other vireos
5
87
0
82
4
95
Lesser nighthawk (Chordeiles acutipennis)
5
89
0
82
4
96
Least sandpiper (Calidris minutilla)
5
90
0
82
4
97
Yellow-rumped warbler (Dendroica
coronate)
5
91
0
81
4
97
Common yellowthroat (Geothlypis trichas)
5
99
0
74
4
99
House finch (Haemorhous mexicanus)
5
92
0
82
4
100
Snow bunting (Plectrophenax nivalis)
5
97
0
82
4
101
Other longspurs
5
98
0
82
4
102
Savannah sparrow (Passerculus
sandwichensis)
4
107
0
73
4
102
Dark-eyed junco (Junco hyemalis)
4
106
0
75
4
104
Tree swallow (Tachycineta bicolor)
5
101
0
80
4
104
Bobolink (Dolichonyx oryzivorus)
4
102
0
82
4
106
Wilson’s Snipe (Gallinago delicate)
4
103
0
82
4
107
Burrowing owl (Athene cunicularia)
1
108
0
82
1
108 31,082. There were 9,535 strike records for military fighter airframes (65 species struck over 20
times, S3 Table), 20,174 records for cargo airframes (98 species struck over 20 times, S4 Table),
674 strike records for stealth airframes (12 species struck over 20 times, S5 Table), and 699
strike records for rotorcraft (16 species struck over 20 times, S6 Table). There were 64 species
groups that were similar for fighter and cargo airframes; however, only 6 species were repre-
sented across all 4 aircraft groups and the civil dataset. RHS for the 6 species and species
groups across airframe groups differed significantly (χ2
20 = 105.11 P < 0.001, Fig 1). Of the 6
species groups compared among airframe groups, all had higher RHS for the stealth than all
civil airframes. There was a strong positive quadratic relationship between RHS and avian body mass for
the species groups involved in bird strikes with all military aircraft (r2 = 0.76, Fig 2(D). The
quadratic curve was steeper for the fighter airframe with a higher y-intercept, compared to the
cargo trend (Fig 2). The best fit for rotorcraft and log avian body mass was a linear regression
p
g
p
See S1 Table for a list of species in each species group (i.e. Other ducks). Strike data are from separate databases maintained by the USN (1990–2017) and USAF (1994–
2017). https://doi.org/10.1371/journal.pone.0206599.t002
PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018
8 / 16 p
g
p
See S1 Table for a list of species in each species group (i.e. Other ducks). Strike data are from separate databases maintained by the USN (1990–2017) and USAF (1994–
2017). 31,082. There were 9,535 strike records for military fighter airframes (65 species struck over 20
times, S3 Table), 20,174 records for cargo airframes (98 species struck over 20 times, S4 Table),
674 strike records for stealth airframes (12 species struck over 20 times, S5 Table), and 699
strike records for rotorcraft (16 species struck over 20 times, S6 Table). There were 64 species
groups that were similar for fighter and cargo airframes; however, only 6 species were repre-
sented across all 4 aircraft groups and the civil dataset. RHS for the 6 species and species
groups across airframe groups differed significantly (χ2
20 = 105.11 P < 0.001, Fig 1). Of the 6
species groups compared among airframe groups, all had higher RHS for the stealth than all
civil airframes. There was a strong positive quadratic relationship between RHS and avian body mass for
the species groups involved in bird strikes with all military aircraft (r2 = 0.76, Fig 2(D). The
quadratic curve was steeper for the fighter airframe with a higher y-intercept, compared to the
cargo trend (Fig 2). The best fit for rotorcraft and log avian body mass was a linear regression 8 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Military avian hazard scores Fig 1. Comparison of relative hazard scores for 6 avian species groups for 4 military airframe groups and all civil airframes for 6 species. Relative hazard scores are calculated from bird strikes within the U.S. See S2 Table for airframe group compositions. Civil relative hazard scores
come from [11]. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 https://doi.org/10.1371/journal.pone.0206599.g002 https://doi.org/10.1371/journal.pone.0206599.g002 contributed to the probability of substantial damage. USAF aircraft were predicted to have a
greater probability of a strike with substantial damage than USN aircraft (Table 4). Species are ordered from left to right by ascending averaged body mass. https://doi.org/10.1371/journal.pone.0206599.g001 Fig 1. Comparison of relative hazard scores for 6 avian species groups for 4 military airframe groups and all civil airframes for 6 species. Relative hazard scores are calculated from bird strikes within the U.S. See S2 Table for airframe group compositions. Civil relative hazard scores
come from [11]. Species are ordered from left to right by ascending averaged body mass. https://doi.org/10.1371/journal.pone.0206599.g001 https://doi.org/10.1371/journal.pone.0206599.g001 (Fig 2(C), whereas the quadratic relationship was the best fit for fighter and cargo airframes
(Fig 2(A) and 2(B), S7 Table). Stealth airframes did not exhibit a relationship with avian body
mass regardless of equations used and is not included in Fig 2. The best logistic regression models predicting the probability of damage was the same for
any damage and substantial damage and included all predictor variables and the airframe ×
avian body mass interaction (S8 Table). Probability of any damage and substantial damage
increased with avian body mass. Within airframe type, fighters had the highest probability of
damage or substantial damage compared to cargo airframes (Tables 3 & 4). Probability of dam-
age or substantial damage was influenced by avian body mass, and this effect differed per air-
frame. These probabilities differed per airframe type for bird species with the same body mass
(Fig 3). Migration flyway, specifically the Mississippi flyway, had the lowest probability of sub-
stantial damage compared to the Atlantic flyway (Table 4). Reporting military branch also PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 9 / 16 Military avian hazard scores Fig 2. Relationship between avian body mass and relative hazard score for avian groups with military cargo (a), fighter (b), rotorcraft (c)
and all military airframes (d). Only strikes identified to species and occurred within the U.S. were included. Strike data are from separate
databases maintained by the U.S. Navy (1990–2017) and U.S. Air Force (1994–2017). Equations and coefficient of determination (r2 values) and
95% confidence intervals are displayed. Fig 2. Relationship between avian body mass and relative hazard score for avian groups with military cargo (a), fighter (b), rotorcraft (c)
and all military airframes (d). Only strikes identified to species and occurred within the U.S. were included. Strike data are from separate
databases maintained by the U.S. Navy (1990–2017) and U.S. Air Force (1994–2017). Equations and coefficient of determination (r2 values) and
95% confidence intervals are displayed. Military avian hazard scores Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables
include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and
th
i f
×
i
l
b d
i t
ti Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables
include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and
the airframe × avian log body mass interaction Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables
include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and
the airframe × avian log body mass interaction. Table 3. Binary logistic regression models predicting any level of damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.12. Predictor variables
include migration flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch (USN or USAF), and
the airframe × avian log body mass interaction. Discussion Ranking of wildlife hazards is an essential component of strike risk [12]. Although we did not
estimate strike risk in this study, these RHS can help guide future research in developing air-
frame-specific risk metrics for military airfields. We predicted that airframe and avian body
mass, migration flyway, and reporting military branch would influence the relative hazard
scores and the probability of damage. For the combined USAF and USN datasets, there was a strong quadratic trend between
avian body mass and RHS. As avian body mass increased, so too did RHS, as observed in simi-
lar studies [11]. This significant trend can be used to calculate RHS for species not included in PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 10 / 16 Reference categories include Atlantic flyway, cargo airframe, USAF branch, and the interaction term cargo× log mass. Reference categories include Atlantic flyway, cargo airframe, USAF branch, and the interaction term cargo× log mass. Confidence intervals
Parameter
Coefficient
SE
2.5%
97.5%
Intercept
-5.28
0.09
-5.45
-5.11
Central
0.05
0.05
-0.05
0.14
Mississippi
-0.06
0.05
-0.17
0.04
Pacific
-0.09
0.06
-0.21
0.03
Fighter
1.45
0.13
1.20
1.70
Rotorcraft
0.59
0.36
-0.14
1.27
Stealth
-0.01
0.39
-0.79
0.74
Log mass
0.70
0.02
0.66
0.73
USN
0.04
0.07
-0.10
0.18
Fighter × log mass
-0.24
0.02
-0.29
-0.20
Rotorcraft × log mass
-0.22
0.07
-0.35
-0.08
Stealth × log mass
0.00
0.08
-0.16
0.14
Reference categories include Atlantic flyway, cargo airframe, USAF branch, and the interaction term cargo × log mass. https://doi org/10 1371/journal pone 0206599 t003 our dataset (i.e. equations in Fig 2 can be solved for RHS with the given body mass of the bird
species). We caution the use of the linear equation identified for rotorcraft for predicting RHS
because of the low coefficient of determination (r2 value). The relationship between airframes
and RHS, however, would be useful for the few species that are not included in the dataset
because of the high coefficient of determination. g
The stealth airframes had higher relative hazard scores compared to civil airframe groups. Interestingly, for 6 species struck by both military and civil aircraft, all had higher RHS for Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables
include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body
mass interaction. Confidence intervals
Parameter
Coefficient
SE
2.5%
97.5%
Intercept
-8.54
0.21
-8.96
-8.13
Central
-0.18
0.09
-0.37
0.01
Mississippi
-0.31
0.10
-0.51
-0.10
Pacific
0.19
0.11
-0.02
0.40
Fighter
1.94
0.29
1.37
2.50
Rotorcraft
1.42
1.02
-0.83
3.26
Stealth
-1.47
1.05
-3.77
0.39
Log mass
0.98
0.03
0.91
1.04
USN
-1.73
0.25
-2.25
-1.28
Fighter × log mass
-0.26
0.05
-0.35
-0.17
Rotorcraft × log mass
-0.32
0.18
-0.67
0.06
Stealth × log mass
0.32
0.16
0.03
0.66 Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). s://doi.org/10.1371/journal.pone.0206599.t004 McFadden’s r2 value is 0.23. Predictor variables
include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables
include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body
i t
ti redicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
mation Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables
Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables
include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log body
mass interaction. Table 4. Binary logistic regression models predicting substantial damage to Navy (USN) and Air Force (USAF) within the United States. This model represents the
best as evaluated by the lowest Akaike’s Information Criterion (AIC) value (see S8 Table for other candidate models). McFadden’s r2 value is 0.23. Predictor variables
include flyway (Central, Mississippi, Pacific, Atlantic), airframe (cargo, rotorcraft, stealth, fighter), avian log body mass, military branch, and the airframe × avian log bod
mass interaction. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 11 / 16 Military avian hazard scores Fig 3. Interaction plot of airframes, avian log body mass, and predicted probability of damage (a) and substantial damage (b). Only
strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the United
States Navy (1990–2017) and Air Force (1994–2017). https://doi.org/10.1371/journal.pone.0206599.g003 Fig 3. PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Conclusion We found that avian hazards to military aviation differed from civil aviation. Specifically, spe-
cies RHS were higher for military than civil aircraft. Furthermore, we found differences in
RHS among airframe groups. As predicted, airframes that travel at greater speeds (fighters)
experienced greater damage from birds when struck, especially at the lower end of the body
mass scale. Although these results are based on strikes within and outside of the airfield envi-
ronment, the calculated hazard scores can be used in conjunction with a frequency component
to estimate risk. Military avian hazard scores high cruising altitudes [8, 15]. All airframes, except rotorcraft, exhibited an exponential rela-
tionship between avian body mass and RHS. This relationship indicates that below a certain
body mass, the RHS exhibits the opposite trend of increasing with decreasing body mass. We
suggest that this trend is not biologically relevant, but a byproduct of the mathematical analysis
such as from the numerous outliers. Furthermore, there are few RHS for birds with extremely
small body masses. Interestingly, the trend between body mass and RHS for rotorcraft was not
a strong relationship and the best fit was linear. Possible explanations for this result include
differences in strike reporting rates, flight patterns, or airframe components. For example,
strikes to wind screens, rotors, and tail rotors, components that are quite exposed, can result in
frequent, substantial damage [30] We acknowledge that these RHS are based on a degree of frequency (species only included
if struck 20 or more times) and thus influenced by differences in strike rates across species. Certain species are struck more frequently than others. Frequently struck birds use the airport
for foraging and roosting activities [3] and generally are more maneuverable [31]. However,
there is significant individual and species variability [32, 33] that influence avian responses to
oncoming aircraft. Given this caveat, our results were similar to other studies in that birds
associated with water were ranked as most hazardous [6, 8, 11, 14], as well as raptors [6, 8, 11]. Our results indicate that avian hazards to military aviation are airframe specific. Depending
on the airfield, hazard scores can be calculated per airframe type. For example, if an airfield is
primarily used for fighter missions/training, the fighter airframe RHS should be used, ensuring
that species on the airfield would be prioritized based on the severity of the strike specific to
that airframe. These results, coupled with data on species seasonal relative abundance on and
near airfields [30, 34], will be important in directing airfield management [12]. Ultimately, the
development of airframe-specific strike risk metrics will yield more accurate information on
airframe vulnerability to bird strikes by mission type. Supporting information S1 Table. Bird species (n = 186) involved in more than 20 strikes with military aircraft
grouped into 108 species groups. Species with fewer than 50 strikes were combined into spe-
cies groups based on phylogeny. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz-
ardous for fighter aircraft within the United States.
(DOCX) Interaction plot of airframes, avian log body mass, and predicted probability of damage (a) and substantial damage (b). Only
strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the United
States Navy (1990–2017) and Air Force (1994–2017). Fig 3. Interaction plot of airframes, avian log body mass, and predicted probability of damage (a) and substantial damage (b). Only
strikes identified to species and occurred within the U.S. were included. Strike data are from separate databases maintained by the United
States Navy (1990–2017) and Air Force (1994–2017). https://doi.org/10.1371/journal.pone.0206599.g003 https://doi.org/10.1371/journal.pone.0206599.g003 https://doi.org/10.1371/journal.pone.0206599.g003 stealth than civil airframes. The mourning dove had a military RHS of 100 for stealth, but a
score of 9 for civil aircraft [11]. We suspect that the materials composing stealth aircraft
“skin” and specialized aircraft components have elevated sensitivity to strikes. For example, a
strike in 2006 which involved a mourning dove (123 g) resulted in $129,787 damage to the
wing of a stealth bomber. Mourning doves are capable of causing similar amounts of damage
(> $130,000) for civil aircraft [7]; however, all of the reported mourning dove strikes from
2010–2015 involved the bird being ingesting into an engine, not external wing damage. Relative hazard scores for fighter airframes were generally higher than cargo and all civil
airframes. Furthermore, our logistic regression models indicated that fighter airframes experi-
enced a higher probability of damage with birds of a lower body mass compared to cargo air-
frames. It is likely that higher speeds and lower flight altitudes of fighter airframes versus cargo
aircraft contributed to higher RHS and probability of damage. Cargo airframes are likely simi-
lar to civil aviation in terms of design and flight patterns, with long ascents and descents and PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 12 / 16 S2 Table. Airframe groups (n = 189) involved in bird strikes with military aircraft grouped
into 8 airframe groups.
(DOCX) Results from the binomial generalized linear model of factors that influence the
probability of a bird strike to cause (a) any damage and the probability of a bird strike causing
(b) substantial damage with military aircraft. (DOCX) Acknowledgments We thank the Commander, Navy Installations Command (CNIC) Airfield Operations Pro-
gram for funding. We thank James E. Higgins, Dan Sullivan, Mike Begier, Jenny Washburn,
Paul Block, Maguel Brooks, and Mark Schmidt for providing the data. We thank Jason
Kougher, Eddie Earwood, and James Laughlin for their assistance and knowledge on the mili-
tary strike databases and airframe groups. We thank Brian Washburn, Richard Dolbeer, and
Thomas Seamans for reviewing earlier versions of this manuscript. Author Contributions Conceptualization: Bradley F. Blackwell, Travis L. DeVault. Conceptualization: Bradley F. Blackwell, Travis L. DeVault. Data curation: Morgan B. Pfeiffer, Travis L. DeVault. Methodology: Morgan B. Pfeiffer, Bradley F. Blackwell, Travis L. DeVault. Supervision: Travis L. DeVault. Visualization: Morgan B. Pfeiffer, Bradley F. Blackwell, Travis L. DeVault. Writing – original draft: Morgan B. Pfeiffer. Writing – original draft: Morgan B. Pfeiffer. Writing – review & editing: Morgan B. Pfeiffer, Bradley F. Blackwell, Travis L. DeVault. S2 Table. Airframe groups (n = 189) involved in bird strikes with military aircraft grouped
into 8 airframe groups.
(DOCX) S2 Table. Airframe groups (n = 189) involved in bird strikes with military aircraft grouped
into 8 airframe groups. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz-
ardous for fighter aircraft within the United States. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz-
ardous for fighter aircraft within the United States. (DOCX) S3 Table. Relative hazard scores (RHS) for 65 avian species groups from most to least haz-
ardous for fighter aircraft within the United States. (DOCX) 13 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0206599
November 1, 2018 Military avian hazard scores S4 Table. Relative hazard scores (RHS) for 65 species groups from most to least hazardous
for cargo aircraft within the United States. (DOCX)
S5 Table. Relative hazard scores (RHS) for 12 species groups from most to least hazardous
for stealth aircraft within the United States. (DOCX)
S6 Table. Relative hazard scores (RHS) for 16 species groups from most to least hazardous
for stealth aircraft within the United States. (DOCX)
S7 Table. Summary of quadratic and linear relationships with military airframe and avian
log body mass. No significant relationship was found for stealth airframes and avian log body
mass. Bold values indicate best fit by the coefficient of determination (r2 values). (DOCX)
S8 Table. Results from the binomial generalized linear model of factors that influence the
probability of a bird strike to cause (a) any damage and the probability of a bird strike causing
(b) substantial damage with military aircraft. (DOCX) S4 Table. Relative hazard scores (RHS) for 65 species groups from most to least hazardous
for cargo aircraft within the United States. (DOCX) S5 Table. Relative hazard scores (RHS) for 12 species groups from most to least hazardous
for stealth aircraft within the United States. (DOCX) S6 Table. Relative hazard scores (RHS) for 16 species groups from most to least hazardous
for stealth aircraft within the United States. (DOCX) S7 Table. Summary of quadratic and linear relationships with military airframe and avian
log body mass. No significant relationship was found for stealth airframes and avian log body
mass. Bold values indicate best fit by the coefficient of determination (r2 values). (DOCX) S8 Table. References 1. Conover MR. Resolving human-wildlife conflicts: the science of wildlife damage management. New
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https://revistaseletronicas.pucrs.br/ojs/index.php/iberoamericana/article/download/36317/19073
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Portuguese
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Sobre a Mulher Escrava no Rio Grande do Sul
|
Estudos ibero-americanos/Estudos Ibero-Americanos
| 1,990
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cc-by
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*
Autora
do livro RS: escravlsmo
e abolição,
Porto Alegre,
Mercado
Aberto,
1982.
Professora
do curso de Graduação
e Pôs-Graduação
de História
da Universidade
Federal do Rio Grande do Sul. 47 47 F.8tudos Ibero-Amerkanos.
PUCRS, XVI(1,2):47-56, jul. e dez., 1990 Sobre a Mulher Escrava no
Rio Grande do Sul Margaret M Bakos* Margaret M Bakos* A versão original desta comunicação foi elaborada a partir de um
convite da Secretaria Municipal de Cultura, na pessoa do organizador do
Curso, professor Mário Maestri, para falar sobre a mulher negra escrava
no Rio Grande do Sul. O tema proposto
instigou um questionamento
inicial: em quais
aspectos o escravismo seria diferente para o homem e para a mulher? As relações sociais escravistas coisificam os indivíduos, impedin-
do-os de se movimentarem
livremente,
embotando
e inibindo os seus
sentimentos e instintos. Homens e mulheres, na escravidão, tomam-se
seres de propriedade
de outrem, com vontade sujeita à autoridade
do
dono e seu trabalho ou serviços são obtidos através da coerção. Nesta linha de raciocínio, toma-se
difícil delinear
e conseqüen-
temente falar sobre a situação específica da mulher no cativeiro, pois a
base estrutural
da sua existência não está em ser fêmea. Está em ser
escrava. Será impossível identificarmos,
a nível estrutural,
uma posição
masculina ou feminina na escravidão? Toda diferença entre o papel do homem e da mulher no escravismo
será sempre conjuntural e relacionada
ou com a atividade que o negro
exerce, ou com o momento hist6rico que vivencia. Homens e mulheres
negros escravizados no Rio Grande
do Sul, ao longo de dois séculos, F.8tudos Ibero-Amerkanos. PUCRS, XVI(1,2):47-56, jul. e dez., 1990 Estudos Ibero-Americanos,
XVl( 1,2) - 1990 Estudos Ibero-Americanos,
XVl( 1,2) - 1990 48 sofreram, capitularam e reagiram ao cativeiro através de ações, mais ou
menos cooperativas
ou agressivas, independentemente
do seu sexo,
relacionadas basicamente com as variáveis apontadas. A minha proposta nesta apresentação
não é, portanto, estabelecer
um inventário,
no meu entender
forçadíssimo,
das diferenças
entre
homens e mulheres escravizados. A tendência, nesta perspectiva, é a de
situar aos primeiros
como heróis em potencial,
em constante
enfren-
tamento com o jugo senhoril, caracterizando
as segundas como vítimas
duplas, da passividade
inerente
ao sexo e da violência dos senhores,
conforme
as retrataram
inúmeras
vezes os historiadores
positivistas. Impõe-se uma nova visão, pois não é assim que vejo as mulheres escravas,
genericamente
falando. Acredito
que entre
os escravos encontramos
gente assumida, sensível e combativa, mas também homens e mulheres
entregues aos desmandos dos senhores, interna e profundamente
auto-
coisificados, independente
do seu sexo. Ao tentar resgatar aspectos do engajamento feminino na luta contra
a opressão, analiso três momentos definidos na relação escravista: I - No Trabalho A idéia desta abordagem originou-se a partir de um debate que se
estabeleceu,
ao final de uma palestra que proferi, em 1988, em Porto
Alegre. Eu falara sobre a mulher escrava na capital do Estado e, para
ilustrar o encontro, li alguns anúncios, publicados em jornais do século
passado, que ou solicitavam ou ofereciam escravas negras para serviços
domésticos. Muitos dos anúncios solicitavam que a serviçal fosse real-
mente habilitada como cozinheira. Neste momento, uma das assistentes,
estranhando o fato, espontaneamente,
comentou que sempre pensara que
as negras fossem "naturalmente"
boas cozinheiras e competentes domés-
ticas, de um modo geral. O questionamento
que me ocorreu em torno desta intervenção foi
sobre as origens históricas deste pensamento que, possivelmente, ainda se
haja presente na cosmovisão de muitos gaúchos. O escravo negro começou a ser introduzido no Rio Grande do Sul
nos finais do século XVIII, com vistas a atender
as necessidades
das
charqueadas
sulinas. Aos poucos começou-se a utilizar o escravo para as
atividades de peonagem e agricultura. Nestes primeiros tempos, o que
mais importava era possuir um escravo negro, fisicamente apto para estas Sobre a mulher escrava no ... 49 atividades. Foi com o desenvolvimento
dos centros urbanos,
a partir
principalmente da primeira metade do século XIX, que a mulher escrava
passou a ser uma peça cobiçada e fundamental para a sociedade gaúcha. Uma das poucas estatísticas
que temos, e que diferencia
a presença
numérica dos escravos homens e mulheres, data de 1859. Ela arrola 25
cidades sedes de municípios, salientando-se
como localidades que mais
possuem escravos as seguintes: atividades. Foi com o desenvolvimento
dos centros urbanos,
a partir
principalmente da primeira metade do século XIX, que a mulher escrava
passou a ser uma peça cobiçada e fundamental para a sociedade gaúcha. Uma das poucas estatísticas
que temos, e que diferencia
a presença
numérica dos escravos homens e mulheres, data de 1859. Ela arrola 25
cidades sedes de municípios, salientando-se
como localidades que mais
possuem escravos as seguintes: Localidade
Porto Alegre
Pelotas
Jaguarão
Rio Grande
Cruz Alta
Homens
4.556
3.095
2.929
2.503
2.315
Mulheres
3.861
1.693
2.127
1.866
1.704 Localidade
Porto Alegre
Pelotas
Jaguarão
Rio Grande
Cruz Alta Vemos assim que nestas localidades urbanas havia uma presença de
escravos
negros,
homens
e mulheres,
significativa,
o que revela
a
necessidade de ambos para diferentes trabalhos. I - No Trabalho Uma análise do seu valor de compra, no mesmo ano (1859), nos
permite
resgatar
que a diferença
de preço
também
não era muito
significativa: Preços de escravos negros no Rio Grande do Sul em 1859 Preços de escravos negros no Rio Grande do Sul em 1859
Para a agricultura
Homens
1.250$000
Mulheres
1.150$000
Para serviços domésticos
Homens
1.400$000
Mulheres
1.250$000 Para a agricultura
Homens
1.250$000 Para serviços domésticos
Homens
1.400$000 Para serviços domésticos Se as mulheres negras eram numericamente
importantes
e econo-
micamente valorizadas, resta perguntar
para que atividades eram des-
tinadas. Talvez uma das melhores
fontes para obter
esta resposta
seja
justamente a análise dos anúncios de jornais. O O Diário de Porto Alegre,
de 1827, é uma das mais antigas fontes para este tema e aponta cerca de
21 atividades para as quais o trabalho escravo era requisitado,
em Porto Estudos Ibero-Americanos, XVI( 1,2) - 1990 50 Alegre. As mulheres
eram
procuradas
para
trabalharem
como cos-
tureiras, doceiras, lavadeiras, engomadeiras,
cozinheiras, amas de leite,
arrumadeiras,
fiadeiras e refinadoras de açúcar. Com vistas a responder
ao questionamento
inicial, é importante
chamar atenção para alguns
ângulos sob os quais esta série de anúncios pode ser analisada: Alegre. As mulheres
eram
procuradas
para
trabalharem
como cos-
tureiras, doceiras, lavadeiras, engomadeiras,
cozinheiras, amas de leite,
arrumadeiras,
fiadeiras e refinadoras de açúcar. Com vistas a responder
ao questionamento
inicial, é importante
chamar atenção para alguns
ângulos sob os quais esta série de anúncios pode ser analisada: a) A mulher escrava foi utilizada como mão-de-obra em dezenas de
atividades designadas, para fins públicos, como domésticas, ligando-se a
estas
remotas origens uma série de preconceitos,
que pode ser resu-
mida no ditado popular machista e racista que diz ter a mulher negra um
lugar
garantido:
"no
palco,
na
cama
e na cozinha, sem fazer con-
curso público e com estabilidade assegurada". (O Tição, Ano 1, out. 80,
p.3) b) O que chama a atenção nesta coletânea de anúncios, em número
de 46, é que 13 relacionam-se
com a atividade de fazer comida. Assim,
não fica difícil entender por que foi tão profundamente
internalizada, nas
pessoas, a relação escrava negra \ cozinha. A ponto de alguém, no ano de
1988, ainda considerar
que todas as negras eram em princípio
boas
cozinheiras. Entretanto,
é justamente
aí que se pode buscar os aspectos
da resistênciavcoerçâo
ao trabalho, que, em última análise, assinalam a
semelhança
entre homemvmulher,
na escravidão. I - No Trabalho Explicando
melhor,
tanto faz, num regime de trabalho compulsório, se se é escravovescrava; a
diferença entre um e outra não se faz por aí. Ser senhor'(senhora
isto é o
que importa. Os pólos da relação escravista, independente
do seu sexo,
dominam ou ao chicote ou apenas ao seu próprio corpo. Sob esta ótica
repensei os dados e, em base às fontes que enfatizavam a preparação para
o trabalho
doméstico, que se exigia das escravas, tentei recuperar
o
processo de ensino'(aprendizagem
que muitas negras devem ter passado
entre o fogão e a despensa, desesperadas
por não saberem o que fazer
com panelas e víveres, que não conheciam, dos quais não gostavam e que
eram obrigadas a cheirar, cozinhar, servir e comer. O chicote, a senzala e
o terror foram certamente
os mestres mais eficientes do período e os
responsáveis pelo nossa preconceituosa
idéia de que todas as negras são
"naturalmente"
boas
cozinheiras. Até
este
momento,
não
se tem
uma análise exaustiva da documentação
que evidencie tais conclusões. Entretanto,
isto não justifica que se aceite a História que forma este-
reótipos,
tomando
como verdadeiras
afirmações
geradas
pela tradi-
ção
oral
e pela
leitura
simplista
dos
documentos,
sem críticas
e
questionamentos. A cozinheira,
a copeira e a babá permitiram
que ai Sobre a mulher escrava no ... 51 senhora se alimentasse e que a sua família se perfumasse, se banhasse,
namorasse nas janelas e saraus e procriasse novas sinhazinhas. É através
da análise do sistema escravista que se evidencia a formação, a ferro e
fogo, das habilidades
"naturais"
da mulher
negra,
cuja excelência
somente será questionada quando a mulher imigrante empobrecida vier a
disputar com a negra o trabalho doméstico, principalmente
o urbano. Entretanto,
até os dias de hoje, ainda são comuns os ditados populares,
que a tradição oral preservou, e que nos fazem olhar com simpatia ou
resistência seres humanos, com relação à sua capacidade
do trabalho. Quem nunca ouviu: "Negra de canela fina é que é boa trabalhadeira!"? 11- Na resistência Considero
como resistência
toda reação
de inconformidade
do
negro quanto à escravidão. Isto inclui desde tentativas de fugas, formação
de quilombos,
agressões
contra
senhores
e feitores,
assassinato
dos
próprios filhos, suicídios até o "corpo mole" inocente e preguiçoso, o
"dengue" doce que alicia e comove, influenciando o humor dos circun-
dantes. Esta
abrangência
se justifica
pela impossibilidade
de saber,
atualmente, se o sorriso de submissão não teria sido tão falso quanto o
famoso e decantado riso do palhaço de circo. Não há nenhum
trabalho
sistemático
sobre fugas, quilombos
e
criminalidade escrava no Rio Grande do Sul que permita uma compa-
ração estatística entre o número de mulheres e homens que fugiram. Este
estudo seria difícil pelas inúmeras variáveis que teriam de ser levadas em
conta. Resta, pois, exemplificar a resistência feminina, relendo fontes, até
agora, utilizadas para generalizações em torno da resistência escrava. Um dos documentos mais antigos sobre a revolta dos escravos no
Rio Grande do Sul refere-se a um episódio ocorrido na Real Feitoria do
Linho cânhamo, em 2 de agosto de 1822. Nesta ocasião, houve uma
denúncia de que havia pretos desta fazenda que costumavam roubar na
vizinhança, sendo destacado
um grupo de soldados para prendê-los,
porém eles nada puderam
fazer, pois os negros não se entregaram
à
prisão e, instigados por suas mulheres,
que gritavam
para
que eles
matassem
os soldados,
entraram
nas suas moradias,
armaram-se
e
atacaram a guarda que terminou voltando para os seus quartéis. (Feitoria
Nacional do Linho Cânhamo, lata 300 - AHRGS.)
~ Estudos !bero-Americanos, XVI(1,2) -1990 Estudos !bero-Americanos, XVI(1,2) -1990 52 Neste episódio, resgata-se a negra que grita e instiga, que nada teme
na luta contra o branco armado; mas não é preciso ficar apenas nesta
inferência, podemos ver, além disso,a companheira
de uma luta maior
contra
a escravidão,
que se expressa quando
ela incita uma atitude
agressiva. Leitura nesta direção pode ser feita sobre o significado da
participação
feminina na história do quilombismo gaúcho. No quilombo
do Negro Lucas, em Rio Grande,
em 1833, par-
ticipavam seis homens e quatro
mulheres,
que viviam em uma casa
grande,
onde
preparavam
sebo, graxa e charque
para
vender. Nos
quilombos de Rio Pardo foram indiciadas sete mulheres que, juntamente
com treze homens, viviam de roubos nas plantações das vizinhanças. 11- Na resistência Para
viver em um quilombo
era,
primeiramente,
no mínimo,
necessário que o escravo enfrentasse a problemática de sua fuga, que era
mais ou menos difícil, dependendo das atividades que ele exercia, do grau
de vigilância que sofria e, naturalmente,
da sua constituição física, que
devia ser a melhor
possível, pois que ele deveria
percorrer
longos
percursos,
alimentar-se
esporadicamente,
quando
encontrasse
o que
comer,
e sofrer
de muita ansiedade
com a ameaça
permanente
de
enfrentamento
aberto com autoridades
constituídas
-
como o fami-
gerado capitão de mato -
até chegar a um refúgio. Essa foi também a
saga da mulher
quilombola,
naturalmente
no caso de ela ter
ido
espontaneamente
para
o quilombo. Em tal caso, há necessidade
de
esboçar um quadro mais realista de um ato de resistência? Até mesmo os
representantes
do sistema,
com melhores
vestimentas,
montarias
e
esquemas de sobrevivência, amargavam na incumbência de perseguir os
negros fugidos, como é sobejamente ilustrado na documentação
oficial. Então, porque não valorizarmos ao máximo a ação quilombola como um
alto de extrema coragem e valentia? Se eles não tinham o objetivo de
transformar
a sociedade,
como afirma Gorender,
pelo menos, a hos-
tilizavam. Se buscavam recompor
a sociedade tribal africana, segundo
pensamento do mesmo autor, homens e mulheres partilhavam do mesmo
desejo. Naturezas femininas e masculinas, sob a escravidão, interagiam
com o medo, planejavam atingir o estado utópico, o nirvana de qualquer
cativo, em qualquer época histórica: a liberdade! Há alguns, verdadeiramente
não muitos, relatos de casos em que as
escravas resistiram ao assédio de negros, seus companheiros de cativeiro,
como o incidente que culminou no assassinato da esposa de um feitor Sobre a mulher escrava no ... 53 negro -
em fazenda do Alegrete
-
porque ela não cedeu a um escravo
que queria forçá-Ia "para fins libidinosos". (Relatório
do Presidente
da
Província do Rio Grande do Sul João Capistrano Miranda Castro, 1848,
Porto Alegre, Tipografia Porto Alegrense.) O ato que ficou registrado no
processo crime aberto contra o assassino nos permite,
agora, rever a
vítima no seu comportamento
corajoso, que a levou à morte, desmis-
tificando a idéia romântica da mulher por índole submissa. Em uma pesquisa sobre os cárceres de Porto Alegre, na primeira
metade do século XIX, encontrei mulheres negras aprisionadas ou pelo
simples arbítrio de seus senhores ou por terem cometido crimes. (Bakos,
1982: 209-221.) Apenas
na cadeia
de Justiça,
foram encontradas
13
escravas negras presas. 11- Na resistência Impossível saber quais os
sofrimentos físicos e mentais, as torturas, que elas sofreram na passagem
de um espaço para o outro, pois o cenário maior dos quadros continuava
a ser o escravismo. Havia dorvchicote, fedorvsujeira, em todos os locais
destinados
aos negros. Nos autos dos processos, ficou registrado:
um
machado foi desviado, da lenha para a senhora, que na visão da escrava,
foi umajuíza injusta dos seus atos. Um punhado de veneno para ratos foi
parar
nas baixelas
senhoris. Tudo
por
mãos de negras,
tudo
para
exteriorizar sentimentos de raiva, medo, ódio: pura emoção. Com esta
leitura, não pretendo
desculpar
ou minorar
um ato agressivo, muito
menos justificar
o emprego
da violência nas relações
humanas. Ao
contrário, vejo a capacidade
de agredir física ou mentalmente
ao seu
semelhante como a pior das características humanas. Por esta razão, por
ser constitutiva e essencial na luta pela vida, a violência pode ser reduzida
a um viés de análise histórica. Entender o porquê da violência, na visão de
quem a pratica e a sofre, pode ser mais um meio para o historiador
resgatar o sentido do jogo social pela vida. As respostas
de Felisberta
e Florinda
são muito
eloqüentes:
"agredi,
porque
fui agredi da sem motivo", "agredi,
mas não queria
machucar, só queria assustar". Poderíamos analisar dezenas de processos
como estes, mas não estão aí resumidos, apenas nestes dois, os princípios
básicos do ser humano no seu relacionamento
em sociedade? Para isto,
não foi necessário que as negras saissem às ruas, fazendo parte de grupos
armados, praticando violências planejadas. As cenas protagonizadas
por
Felisberta e Florinda aconteceram entre as paredes de casas, onde viviam
pessoas
ordeiras. Naturalmente
ali se reproduziam
as relações
de
trabalho escravistas, com muita crueza, pois na privacidade
do espaço
íntimo, as autoridades são mais presentes, despóticas, viciosas, impunes. Entretanto, no esquema rotineiro, uma 'coisa' saiu do seu lugar. Tanto faz
se era uma 'coisa' masculina ou feminina. Interessa que ela saiu; com isso,
mexeu com a ordem, mostrou que não era só 'coisa', era gente e igual a
toda gente: homem ou mulher. Felisberta e Florinda, protagonistas
da
mudança, nossos objetos de análise, conseguiram,
à época, com suas
atitudes, saírem do espaço privado da casa. Se elas queriam sair, nunca
saberemos. Mas o fato aí está para ser analisado: elas foram conduzidas a
um fórum, foram interrogadas,
ouvidas. Suas vozes ficaram registradas
nos autos dos processos, chegaram até nós. 11- Na resistência A seguir, discrimino dois crimes de que escravas foram acusadas, no
Rio Grande
do Sul, procurando
com isto apenas exemplificar o pen-
samento de que as mulheres reagiram, como os homens, à escravidão,
agredindo
os seus senhores
com as armas de que dispunham. Julgo
importante
apontar
a existência
de
centenas
de
processos
crimes
envolvendo negras escravas ou livres, nos arquivos públicos
do Rio
Grande
do Sul, os quais
ainda
não foram
analisados
ou o foram
precariamente. Importantes
referenciais
sobre o significado do ato de
agressão da cativa podem
e devem ser inferidos
das leituras
destes
processos,
que
se constituem,
fora
de dúvida,
na mais rica
fonte
disponível para a compreensão
das atitudes do escravo de ambos os
sexos, resgatando
para
a atualidade
elementos
constitucionais
das
naturezas feminina e masculina. Dados do Escravo
Ano
Motivo
Felisberta
1821
Castigada
S. Gabriel
sem motivo
Florinda
1828
queria só
P. Alegre
ameaçar 1828
P. Alegre Florinda 54 Estudos Ibero-Americanos, XVI(1,2) -1990 Estudos Ibero-Americanos, XVI(1,2) -1990 As respostas
de Felisberta
e Florinda
são muito
eloqüentes:
"agredi,
porque
fui agredi da sem motivo", "agredi,
mas não queria
machucar, só queria assustar". Poderíamos analisar dezenas de processos
como estes, mas não estão aí resumidos, apenas nestes dois, os princípios
básicos do ser humano no seu relacionamento
em sociedade? Para isto,
não foi necessário que as negras saissem às ruas, fazendo parte de grupos
armados, praticando violências planejadas. As cenas protagonizadas
por
Felisberta e Florinda aconteceram entre as paredes de casas, onde viviam
pessoas
ordeiras. Naturalmente
ali se reproduziam
as relações
de
trabalho escravistas, com muita crueza, pois na privacidade
do espaço
íntimo, as autoridades são mais presentes, despóticas, viciosas, impunes. Entretanto, no esquema rotineiro, uma 'coisa' saiu do seu lugar. Tanto faz
se era uma 'coisa' masculina ou feminina. Interessa que ela saiu; com isso,
mexeu com a ordem, mostrou que não era só 'coisa', era gente e igual a
toda gente: homem ou mulher. Felisberta e Florinda, protagonistas
da
mudança, nossos objetos de análise, conseguiram,
à época, com suas
atitudes, saírem do espaço privado da casa. Se elas queriam sair, nunca
saberemos. Mas o fato aí está para ser analisado: elas foram conduzidas a
um fórum, foram interrogadas,
ouvidas. Suas vozes ficaram registradas
nos autos dos processos, chegaram até nós. 11- Na resistência Impossível saber quais os
sofrimentos físicos e mentais, as torturas, que elas sofreram na passagem
de um espaço para o outro, pois o cenário maior dos quadros continuava
a ser o escravismo. Havia dorvchicote, fedorvsujeira, em todos os locais
destinados
aos negros. Nos autos dos processos, ficou registrado:
um
machado foi desviado, da lenha para a senhora, que na visão da escrava,
foi umajuíza injusta dos seus atos. Um punhado de veneno para ratos foi
parar
nas baixelas
senhoris. Tudo
por
mãos de negras,
tudo
para
exteriorizar sentimentos de raiva, medo, ódio: pura emoção. Com esta
leitura, não pretendo
desculpar
ou minorar
um ato agressivo, muito
menos justificar
o emprego
da violência nas relações
humanas. Ao
contrário, vejo a capacidade
de agredir física ou mentalmente
ao seu
semelhante como a pior das características humanas. Por esta razão, por
ser constitutiva e essencial na luta pela vida, a violência pode ser reduzida
a um viés de análise histórica. Entender o porquê da violência, na visão de
quem a pratica e a sofre, pode ser mais um meio para o historiador
resgatar o sentido do jogo social pela vida. 55 Sobre a mulher escrava no ... Ill- No engajamento social Uma outra instância em que a mulher escrava pode ser analisada é
no seu engajamento na sociedade. Um bom exemplo é a sua participação
na Confraria de Nossa Senhora do Rosário, fundada em Porto Alegre, em
1786, que
tinha
como principal
objetivo
a educação
religiosa
e a
benemerência,
mas que também funcionava como um local de agru-
pamento
dos negros e, muitas vezes, para
conspirações
ou ataques
espontâneos à ordem constituída. Assim, embora institucionalizadas,
as
confrarias eram muitas vezes perseguidas
e as suas procissões e festas
proibidas e os seus membros visados pela polícia local. Mesmo assim, a
presença da mulher negra livre ou escrava era uma constante, prevista,
inclusive no Regimento da Confraria, mas da qual ela participava com
todo o seu engajamento, conspirando também, como o homem ... Conclusão A submissão da mulher foi uma invenção de um mundo comandado
por homens que também inventaram a escravidão. (Mott, 1988: 21) Mas,
subjacente
a estes garrotes
ideológicos
e concretos,
a mulher, cons-
titucionalmente, tinha forças e meios para resistir, com garra e oportunis-
mo, tal como
o homem. Por
isto
ela não
merece
ser
registrada
historicamente
como dupla vítima do sistema. O que realmente lhe é
devido é uma revalorização
de seus atos, concedendo-lhes
a devida
dimensão e mérito. Isto apenas será possível se entendermos
que a
mulher escrava podia resistir ao jugo senhoril, de uma forma diferente
que o homem. Se aceitarmos como resistência coisas do cotidiano, muito
simples, como a indolência no trabalho, a dificuldade no aprendizado de
novas tarefas, o tratamento "dengoso" que dispensava ao senhor e à sua
família. Ao lado destas atitudes que a livravam dos excessos de trabalho e
dos castigos, a escrava negra, como vimos, também agrediu a sociedade
escravista: fugiu, furtou, matou, como todas no país. Foram protagonistas
da História da escravidão negra no Brasil, vítimas, mas também agres-
sores, todos os seres humanos vindos da África e os seus descendentes,
independentemente
do seu sexo: impossível fazer um recorte e falar sobre
a mulher escrava especificamente,
sem desenvolver uma argumentação
machista! Estudos Ibero-Americanos, XVI(1,2) -1990 Estudos Ibero-Americanos, XVI(1,2) -1990 56 Bibliografia citada BAKOS,
M. Os escravos negros
nos cárceres
de Porto
Alegre:
Sugestões
de uma
documentação
do século XIX. In Revista de Estudos
Ibero Americanos. PUCRS,
8(2):209-23, 1982. -. RS: Escravismo e Abolição. Porto Alegre, Mercado Aberto, 1982. CARDOSO, C. Escravo ou Camponês? São Paulo, Brasiliense, 1987. GIACOMINI, S. Mulher e Escrava. Rio de Janeiro, Vozes, 1988. GORENDER,
J. Questionamento
sobre a teoria econômica
do escravismo colonial. In
Estudos Econômicos. 13(1):7-41, 1983. MAESfRI,
M.J. O Escravo no Rio Grande do Sul. Caldas do Sul, Escola Superior São
Lourenço dos Brindes, 1984. MOTI, Mário Lúcio. Submissão e Resistência. São Paulo, Contexto, 1988. MOTIA,
F. Crime e rebeldia escrava no Rio Grande do Sul (1820-1845). Trabalho de
conclusão de bacharelado. UFRGS, jan. 1985.p.47. [Mimeo1 REIS, J.R. & SILVA, E. Negociação e Conflito. Rio de Janeiro, Cia. das Letras, 1989. SCHWARTZ, S. Mocambos, quilombos e palmares: a resistência escrava no Brasil Colonial. In Estudos Econômicos. 17(especial), 1987.
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English
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Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides
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BMC plant biology
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BioMed Central BioMed Central BioMed Central Research article Address: 1Department of Biotechnology, University of the Western Cape, Private Bag X17, Cape Town - Bellville 7535, South Africa, 2Department
of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch 7701, South Africa and 3South African National Bioinformatics
Institute, University of the Western Cape, Private Bag X17, Cape Town - Bellville 7535, South Africa mail: Stuart Meier - smeier@uwc.ac.za; René Bastian - rbastian@uwc.ac.za; Lara Donaldson - Lara@SCIENCE.uct.ac
hane Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za Email: Stuart Meier - smeier@uwc.ac.za; René Bastian - rbastian@uwc.ac.za; Lara Donaldson - Lara@SCIENCE.uct.ac.za;
Shane Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za
* Corresponding author mail: Stuart Meier - smeier@uwc.ac.za; René Bastian - rbastian@uwc.ac.za; Lara Donaldson - Lara@SCIENCE.uct.ac
hane Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za
C
di
th e Murray - shane.murray@cpgr.org.za; Vladimir Bajic - vlad@sanbi.ac.za; Chris Gehring* - cgehring@uwc.ac.za
responding author * Corresponding author Received: 10 September 2007
Accepted: 29 February 2008 Received: 10 September 2007
Accepted: 29 February 2008 Published: 29 February 2008 Published: 29 February 2008 BMC Plant Biology 2008, 8:24
doi:10.1186/1471-2229-8-24 This article is available from: http://www.biomedcentral.com/1471-2229/8/24 This article is available from: http://www.biomedcentral.com/1 © 2008 Meier et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Plant Biology Open Access Background tures respectively [15]. Further, irPNP rapidly and specifi-
cally induced a transient elevation of cGMP levels in the
conductive stele tissue of maize roots [6] and in stomatal
guard cell protoplasts [7] and recently recombinant
AtPNP-A was shown to stimulate protoplast swelling in a
cGMP dependent manner [18]. IrPNPs also modulate ion
fluxes across plant membranes [19] and recombinant
AtPNP-A induced spatially dependent H+, K+ and Na+
fluxes in A. thaliana roots [20]. Endogenous levels of
irPNP are increased in response to NaCl stress in whole-
plants and in Arabidopsis suspension culture cells exposed
to high salt or osmoticum [15]. Collectively, these studies
indicate that PNP-like molecules may function as extracel-
lular signalling molecules that directly affect water and
solute transport in response to stress. Based on biochemi-
cal and physiological data we propose mechanisms of
action for AtPNP-A at the cellular level as summarised in
Figure 1 (adapted from [21]). g
Natriuretic Peptide (NP) systems have been identified in
mammals, fish, amphibians, birds and reptiles. NPs and
their receptors are commonly associated with organs
involved in cardiac and osmoregulatory homeostasis. In
amphibians, birds and fish, NPs have been shown to play
a critical role in the regulation of blood fluid volume and
composition [1]. The first indication that NPs function in plants came from
radio-immuno assays on plant tissue extracts from Florida
beauty [2] and it was shown that the rate of transpiration,
solute flow and solute uptake in carnation and chrysan-
themum was rapidly and significantly increased after
exogenous application of synthetic human atrial NP
(ANP) [3]. Subsequently it was demonstrated that rat ANP
can induce stomatal opening in a concentration depend-
ent manner [4] and this effect appears to be dependent on
the intracellular second messenger cGMP (guanosine
3',5'-cyclic monophosphate) since it is inhibited by the
guanylate cyclase inhibitor LY 83583, but can be induced
by the synthetic cell permeant cGMP analogue 8-Br-cGMP
[5-7]. Binding experiments of ANP to isolated leaf mem-
branes provide evidence for specific receptor ligand inter-
actions [8]. Despite an increasing body of physiological and biochem-
ical data [21], the biological role of this systemically
mobile peptide has remained elusive. In order to infer a Model of AtPNP-A action at the cellular level
Figure 1
Model of AtPNP-A action at the cellular level. Background The
model proposes that AtPNP-A can dock to receptor-like
molecules that directly act as particulate guanylyl cyclases
(pGCs) or indirectly activate soluble GCs (sGCs). GCs cata-
lyse the reaction from GTP to cGMP. The latter acts as sec-
ond messenger affecting cytosolic Ca2+ levels, modulating ion
channels, activating phosphorylation through kinases and
influences the transcriptome. Phosphodiesterases (PDEs) in
turn metabolise cGMP to GMP (adapted from [21]). A plant NP (PNP) from A. thaliana (AtPNP-A) and several
closely related sequences in different species have since
been identified [9,10]. AtPNP-A, its most closely related
sequence AtPNP-B, and orthologues in other higher plant
species, share a family-45 glucosidase domain with the
cell wall loosening expansins [11] and are related to
expansins on the basis of this structural homology [9,12]. AtPNP-A (At2g18660) is a small protein of 126 amino
acids in length (MW: 14016 kD; pI: 9.22) that is encoded
by a gene with a single intron of 100 bp. The region most
conserved between PNPs from different plant species has
also been shown to be the key to its physiological activity
[13]. Evidence for systemic mobility of PNPs comes from
the structure and processing of the molecules [9]. The pro-
tein contains an N-terminal 24 amino acid signal peptide
(MW: 2249) that directs the molecule into the extracellu-
lar space and PNPs that are recognized by anti-human
atrial natriuretic polypeptide rabbit serum have been
localised in situ in conductive tissue [14] and were isolated
from xylem sap [15], and proteomics studies have identi-
fied the AtPNP-A protein in the apoplastic space in A. thal-
iana [16]. Model of AtPNP-A action at the cellular level
Figure 1
Model of AtPNP-A action at the cellular level. The
model proposes that AtPNP-A can dock to receptor-like
molecules that directly act as particulate guanylyl cyclases
(pGCs) or indirectly activate soluble GCs (sGCs). GCs cata-
lyse the reaction from GTP to cGMP. The latter acts as sec-
ond messenger affecting cytosolic Ca2+ levels, modulating ion
channels, activating phosphorylation through kinases and
influences the transcriptome. Phosphodiesterases (PDEs) in
turn metabolise cGMP to GMP (adapted from [21]). Model of AtPNP-A action at the cellular level
Figure 1
Model of AtPNP-A action at the cellular level. The
model proposes that AtPNP-A can dock to receptor-like
molecules that directly act as particulate guanylyl cyclases
(pGCs) or indirectly activate soluble GCs (sGCs). GCs cata-
lyse the reaction from GTP to cGMP. Abstract Background: Plant natriuretic peptides (PNPs) are a class of systemically mobile molecules
distantly related to expansins. While several physiological responses to PNPs have been reported,
their biological role has remained elusive. Here we use a combination of expression correlation
analysis, meta-analysis of gene expression profiles in response to specific stimuli and in selected
mutants, and promoter content analysis to infer the biological role of the Arabidopsis thaliana PNP,
AtPNP-A. Results: A gene ontology analysis of AtPNP-A and the 25 most expression correlated genes
revealed a significant over representation of genes annotated as part of the systemic acquired
resistance (SAR) pathway. Transcription of these genes is strongly induced in response to salicylic
acid (SA) and its functional synthetic analogue benzothiadiazole S-methylester (BTH), a number of
biotic and abiotic stresses including many SA-mediated SAR-inducing conditions, as well as in the
constitutive SAR expressing mutants cpr5 and mpk4 which have elevated SA levels. Furthermore,
the expression of AtPNP-A was determined to be significantly correlated with the SAR annotated
transcription factor, WRKY 70, and the promoters of AtPNP-A and the correlated genes contain an
enrichment in the core WRKY binding W-box cis-elements. In constitutively expressing WRKY 70
lines the expression of AtPNP-A and the correlated genes, including the SAR marker genes, PR-2 and
PR-5, were determined to be strongly induced. Conclusion: The co-expression analyses, both in wild type and mutants, provides compelling
evidence that suggests AtPNP-A may function as a component of plant defence responses and SAR
in particular. The presented evidence also suggests that the expression of AtPNP-A is controlled by
WRKY transcription factors and WRKY 70 in particular. AtPNP-A shares many characteristics with
PR proteins in that its transcription is strongly induced in response to pathogen challenges, it
contains an N-terminal signalling peptide and is secreted into the extracellular space and along with
PR-1, PR-2 and PR-5 proteins it has been isolated from the Arabidopsis apoplast. Based on these
findings we suggest that AtPNP-A could be classified as a newly identified PR protein. Page 1 of 12
(page number not for citation purposes) (page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Background The latter acts as sec-
ond messenger affecting cytosolic Ca2+ levels, modulating ion
channels, activating phosphorylation through kinases and
influences the transcriptome. Phosphodiesterases (PDEs) in
turn metabolise cGMP to GMP (adapted from [21]). A number of physiological and biochemical studies have
implicated AtPNP-A with a role in the regulation of ion
and solute homeostasis. Immuno-reactive PNP (irPNP)
extracts and recombinant AtPNP-A have been shown to
induce swelling in leaf mesophyll protoplasts [17] and in
protoplasts isolated from Arabidopsis cell suspension cul- Page 2 of 12
(page number not for citation purposes) Page 2 of 12
(page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 any bias in GO functional annotation terms in the corre-
lated list (list 1) compared to the remainder of the A. thal-
iana genome [see Additional file 1]. In the GO search
category of biological process there is a significant (Family
Wise Error Rate – FWER) adjusted p-value) enrichment in
genes involved in biotic defence responses at a number of
levels. The most notable bias being at level 8 with a signif-
icant (adjusted p-value 0.0000038) enrichment in genes
involved in SAR (Table 1). SAR is an inducible plant
defense response against local pathogen infection that
gives rise to a systemic long lasting resistance to a broad
range of virulent pathogens [25]. The SAR response is
characterised by the accumulation of endogenous salicylic
acid (SA) in infected tissues and later in distal uninfected
tissues with a subsequent induction of a select group of
pathogenesis-related genes (PR genes) [26]. biological role for AtPNP-A, we made use of the large
repositories of A. thaliana microarray data to study the
expression profiles of AtPNP-A and the 25 most expres-
sion correlated genes in response to various treatments as
well as in mutants. We further analysed the promoters of
these genes for known regulatory motifs. The results of
our study predict a function for AtPNP-A in plant abiotic
and biotic stress responses, and in particular in systemic
acquired resistance (SAR). Furthermore, we demonstrate
how computational analyses that link regulatory potential
as encoded by promoter elements and expression data can
provide novel insights into the function of a specific gene
as well as groups of genes. Results and Discussion Expression Correlation and GO Analyses Page 3 of 12
(page number not for citation purposes) SAR: Systemic acquired resistance; RBS: Responsive to biotic stress Expression Correlation and GO Analyses In the first step of the analyses we extracted and ranked the
25 genes whose expressions are most tightly correlated
with AtPNP-A (Table 1). The moderate correlation (r) val-
ues of the listed genes (maximum r = 0.73) may reflect
that the expression of AtPNP-A is subject to complex com-
binatorial control via multiple promoter motifs with com-
plex inputs from multiple, potentially antagonistic,
signalling pathways. The enrichment in SAR annotated genes in our list is par-
ticularly striking considering that in FatiGO the entire A. thaliana genome contains only 21 annotated SAR genes
and four of these are present in our list of correlated genes. The four correlated SAR genes include NIMIN1
(At1g02450; r = 0.61) that is involved in the transcrip-
tional regulation of PR genes [26], PR-1 (At2g14610; r =
0.63); PR-2 (At3g57260; r = 0.73) and PR-5 (At1g75040;
0.61) whose expression is commonly used as diagnostic
markers of the SA dependent SAR response [27]. An In order to identify a functional role of AtPNP-A, the cor-
related genes were analysed in FatiGO+ [22-24] to identify Table 1: List of genes that are expression correlated with AtPNP-A (At2g18660) Table 1: List of genes that are expression correlated with AtPNP-A (At2g18660)
Locus
r-value
Annotation
At3g57260 RBS, SAR
0.731
Pathogenesis-related protein 2 (PR-2)
At5g10760
0.681
Aspartyl protease family protein
At2g04450 RBS
0.676
Triphosphatase activity, stress response
At5g52760
0.661
Heavy-metal-assoc. domain-containing
At2g17040
0.659
No apical meristem (NAM) family protein
At5g55450 RBS
0.647
Protease inhibitor/lipid transfer protein
At1g21250
0.645
Wall-associated kinase 1 (WAK1)
At4g23610
0.641
Hin1 – role in hypersensitive response
At1g13470
0.634
Mitochondrial protein of unknown function
At4g14365
0.634
Zinc finger (C3HC4-type RING) family
At1g73800
0.630
Calmodulin binding protein
At3g56710
0.629
SigA-binding protein, plastid sigma factor
At4g04490
0.627
PK family, liposaccharide biosynthesis
At2g14560
0.626
Protein of unknown function (DUF 567)
At2g14610 RBS, SAR
0.626
Pathogenesis-related protein 1 (PR-1)
At1g21520
0.626
Expressed protein
At2g24850
0.626
Aminotransferase, resp. to wounding & JA
At4g23150
0.625
Protein kinase family protein
At3g60420
0.622
Phosphohistidine phosphatase activity
At2g32680
0.620
Disease resistance, leucine rich-repeats
At1g74440
0.614
Similar to YGL010w-like protein
At1g02450 RBS, SAR
0.613
NPR1/NIM1-interacting prot. Expression Correlation and GO Analyses 1 (NIMIN1)
At4g11890
0.606
Protein kinase family protein
At1g75040 RBS, SAR
0.604
Pathogenesis-related protein 5 (PR-5)
At1g08450
0.602
Calreticulin 3 precursor, Ca2+ binding
SAR: Systemic acquired resistance; RBS: Responsive to biotic stress Page 3 of 12
(page number not for citation purposes) Page 3 of 12
(page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Expression profile of AtPNP-A and correlated genes in
response to selected treatments
Figure 2
Expression profile of AtPNP-A and correlated genes in
response to selected treatments. The results presented
illustrate the fold change (log2) in expression of AtPNP-A,
WRKY 70 and WRKY 46 and the average fold change for the
25 correlated genes in response to abiotic stresses (A) and
biotic and chemical treatments (B). (A) The treatments were:
UV-B shoot 3 h (n = 2); O3 6 h (n = 3); Osmotic stress in the
shoot after 3 h (n = 2); K+ starvation in the shoot after 7 days
(n = 3); NaCl in the roots after 6 h (n = 2) and cold acclima-
tion after 14 days (n = 3). (B) The treatments were: Erysiphe
cichoracearum 3 days after inoculation (n = 4); Erysiphe orontii
3 days after inoculation (n = 2); Phytophthora infestans 1 day
after inoculation (n = 3); BTH after 8 h (n = 3); SA after 3 h
(n = 2) and cyclohexamide after 3 h (n = 2). Error bars repre-
sent standard errors of the mean. extended correlation analysis revealed that an additional
11 SAR annotated genes, including NPR1 (or NIM1;
At1g64280; r = 0.52), which is an essential key positive
regulator of signal transduction leading to the SAR
response and expression of PR proteins, are significantly
correlated (9 positive, 2 negative; p < 0.01; bivariate nor-
mal distribution) with the expression of AtPNP-A [see
Additional file 2]. Other correlated genes in list 1 anno-
tated to be involved in plant defence responses and
response to biotic stimuli include a disease resistance fam-
ily protein containing leucine rich-repeats (At2g32680), a
stress responsive gene with triphosphatase activity
(At2g04450) and a protease inhibitor (At5g55450). The
GO analysis for the cellular component and molecular
function category revealed no significant difference in bio-
logically relevant labels between the two lists. Expression
response t
Figure 2 p
p
g
p
g
Expression profile of AtPNP-A and correlated genes in
response to selected treatments. The results presented
illustrate the fold change (log2) in expression of AtPNP-A,
WRKY 70 and WRKY 46 and the average fold change for the
25 correlated genes in response to abiotic stresses (A) and
biotic and chemical treatments (B). (A) The treatments were:
UV-B shoot 3 h (n = 2); O3 6 h (n = 3); Osmotic stress in the
shoot after 3 h (n = 2); K+ starvation in the shoot after 7 days
(n = 3); NaCl in the roots after 6 h (n = 2) and cold acclima-
tion after 14 days (n = 3). (B) The treatments were: Erysiphe
cichoracearum 3 days after inoculation (n = 4); Erysiphe orontii
3 days after inoculation (n = 2); Phytophthora infestans 1 day
after inoculation (n = 3); BTH after 8 h (n = 3); SA after 3 h
(n = 2) and cyclohexamide after 3 h (n = 2). Error bars repre-
sent standard errors of the mean. The strong up-regulation of AtPNP-A and correlated genes
by SA and benzothiadiazole S-methylester (BTH), a syn-
thetic functional SA analogue [28], is a key indicator that
these genes are involved in plant defence and specifically
SAR since SA has been shown to be essential and sufficient
to induce the SAR response in plants [29] (Figure 2B). In
addition to AtPNP-A, all of the 25 correlated genes were
significantly (ANOVA p-value < 0.05) up-regulated by
more than two fold after 8 h and 24 h treatments with 60
µM BTH (Supplementary Table 3 in [28]) further linking
these genes to the SAR defense pathway. Expression of
AtPNP-A is also significantly correlated with the isochoris-
mate synthase-1 (ICS-1) gene (At1g74710; r = 0.50) that
is critical for SA biosynthesis [29]. expression and the correlated genes in response to these
pathogens is in accord with the literature informing that
SA-dependent defenses generally act against biotrophs in
contrast to jamonic acid- and ethylene-dependent
responses that counteract necrotrophs [30]. The large increases in gene expression induced by ozone
and UV-B (+6.86log2 and +8.09log2 respectively for AtPNP-
A) is consistent with these genes being part of the SAR
response since both these treatments have previously
been shown to stimulate SA production and induce the
expression of PR genes [31-34]. Expression Correlation and GO Analyses The results of the Swiss-Prot keyword search also identi-
fied a significant enrichment in genes annotated as PR
proteins (adjusted p value = 0.002), involved in signalling
(adjusted p value = 0.026) and associated with the apo-
plast (adjusted p value = 0.040) in list 1. It was noted that
along with PR-1, PR-2 and PR-5, AtPNP-A is one of six
genes in list 1 annotated as having signalling function. Microarray Expression Profiles The over representation of genes involved in defence
responses, and specifically SAR is consistent with the
observation that AtPNP-A and the correlated genes are
most highly expressed in microarray experiments where
defence responses are elicited. The treatments that induce
up-regulation of AtPNP-A and the correlated genes more
than two-fold include SA and other SAR inducing condi-
tions as well as a number of abiotic stresses (Figure 2). Expression
response t
Figure 2 The expression of Ca2+ sensing molecules is rap-
idly induced in response to biotic and abiotic stresses [51]
and functions to decode Ca2+signatures and/or relay sig-
nals to downstream targets, including kinases, which fur-
ther amplify the Ca2+ signal by inducing downstream
phosphorylation cascades [38]. The presence of three
kinases (At4g04490; r = 0.63, At4g23150; r = 0.63 and
At4g11890; r = 0.61) in amongst the correlated genes
(Table 1) is entirely consistent with such a signaling cas-
cade. Moreover, the expression correlation of three stress
responsive mitogen-activated protein kinase (MAP
kinase) genes MAPKK (At4g26070; r = 0.59), MPK 11
(At1g01560; r = 0.58) and MAPKK (At4g29810; r = 0.57)
to AtPNP-A also ties in with the proposed cascades [see
Additional file 2]. Activation of MAPKs has indeed been
reported after exposure to pathogens [52] as well as a
number of abiotic stresses [53]. K+ is the key inorganic ion required in high quantities by
plants while Na+ on the other hand is toxic at high concen-
trations [38]. Na+ is able to compete with K+ ions for
uptake and binding sites thus maintaining the correct
Na+/K+ ratio in plants is of the utmost importance [39]. Decreases in K+ might cause the plant to take up more Na+
in order to maintain adequate osmotic pressure [40]. Therefore either the increase in cytosolic Na+ or a decrease
in osmotic pressure as a consequence of K+ starvation or a
combination of both may cause AtPNP-A induction. Elevated expression of AtPNP-A and correlated genes, par-
ticularly defence genes and SAR annotated genes, by abi-
otic osmotic stresses as well as defence eliciting treatments
may well reflect that both types of challenges lead to com-
mon homeostatic disturbances which in turn transcrip-
tionally activate a set of common response genes. This
concept is supported by several studies that recognise a
role of SA in abiotic stresses such as drought, salinity and
temperature [41,42] and the accumulation of PR proteins
is in fact a common plant response to both abiotic and
biotic stresses further highlighting the overlap in biotic
and abiotic defence mechanisms [43]. While transcriptional responses to some stresses, includ-
ing the osmotic, salt, UV-B and some of the biotic treat-
ments, were measured over multiple time points, the data
presented here are generally the earliest time point that
induced the largest increase in AtPNP-A expression. Expression
response t
Figure 2 The biotic stresses that induced the largest increase in
expression of AtPNP-A include infection with the bio-
trophic pathogens Phytophthora infestans, Erysiphe cichora-
cearum and Erysiphe orontii that depend on living host
tissue for survival (Figure 2B). Activation of AtPNP-A Page 4 of 12
(page number not for citation purposes) Page 4 of 12
(page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 http://www.biomedcentral.com/1471-2229/8/24 The expression of AtPNP-A and the correlated genes is also
strongly modulated by a number of abiotic stresses
including K+ starvation, osmotic, and NaCl stress as well
as cold acclimation (Figure 2A). A common element of
abiotic stresses is that they decrease water potential [35]. It is noteworthy that the induction of AtPNP-A in response
to ion and osmotic stresses is tissue specific with the
response to high Na+ being specific to root tissue with lit-
tle change observed in shoots while both low K+ and high
osmolarity induced elevated transcription in shoots only
[36]. This is of interest since the AtPNP-A protein has been
shown to affect water movement in shoots [37] and pro-
toplasts [17] as well as ion fluxes in roots [20]. It is thus
tempting to speculate that AtPNP-A may have a role in
maintaining plant water and ion homeostasis under stress
conditions. cell membranes is also correlated with that of AtPNP-A (r
= 0.60) [see Additional file 2] since ion conductance in
these channels is regulated by cGMP as well as Ca2+ and
calmodulin. These channels have also been implicated in
regulating SA-dependent biotic defense responses [46]. AtPNP-A expression is also correlated with a number of
Ca2+ sensing/binding proteins including, the above men-
tioned CNGC20, calreticulin 3 (At1g08450; r = 0.60), two
calmodulin-binding proteins (At1g73800; r = 0.63 and
At1g73805; r = 0.588) with family members involved in
the induction of plant defense responses (NCBI sequence
viewer, pfam07887) and a Ca2+-binding EF hand domain
containing protein (At3g47480; r = 0.59). One of the ear-
liest responses to biotic and abiotic stresses is an increase
in cytosolic free Ca2+[47] that in turn plays a role in acti-
vating the oxidative burst after elicitor treatment [48,49]
and is also linked to signaling SA-induced PR gene expres-
sion [50]. Page 5 of 12
(page number not for citation purposes) Common Motifs in cis and Transcription Factors f
p
The common expression profiles of APNP-A and the 25
correlated genes in response to both biotic and abiotic
stresses suggests that these genes are under common regu-
latory control and are thus likely to share common cis-ele-
ments in their promoter regions. To reveal aspects of
common transcriptional activation we analyzed promoter
regions of these genes 1 kb upstream of the predicted tran-
scription start site (TSS) for the presence of known plant
cis-elements. in the A. thaliana genome suggesting that they are impor-
tant regulatory elements in these expression correlated
genes. In plants, W-box cis-elements are known to bind WRKY
TFs [60] indicating that these TFs may be important in reg-
ulating the expression of the correlated genes. This is com-
pletely consistent with our expression analysis results
since the WRKY family of TFs [60] have well established
roles in regulating disease responses in plants [61]. In
addition, they have also been documented to mediate abi-
otic plant responses to freezing [62], wounding [63], oxi-
dative stress [64], drought, salinity, cold, and heat [65-
67]. In our study, expression of AtPNP-A is moderately
correlated with the expression of WRKY 70 (At3g56400; r
= 0.60) and WRKY 46 (At2g46400; r = 0.56) [see Addi-
tional file 2]. When viewing the expression profiles of
WRKY 70 and WRKY 46 genes it is apparent that the vari-
ous treatments which induced large increases in the
expression of the correlated genes in Table 1 also induced
marked changes in the expression of the WRKY genes (Fig-
ure 2). This links the expression of AtPNP-A and the
WRKY genes to common biological responses and raises The analysis in POBO [58] indicated that the invariant
core TTGAC W-box motif was present in 25/26 of our cor-
related genes a total number of 78 times at an average of
2.99 copies/promoter compared to the average of 2.24
across all A. thaliana promoters (t-test p-value >0.0001)
(Figure 3 and [see Additional file 3]). The analysis in Ath-
ena [59] identified that the extended and more stringent
TTGAC(A/T) W-box motif was present in 22/26 genes a
total of 54 times at an average of 2.08 copies/promoter (p-
value = 0.0037; data not shown). Expression
response t
Figure 2 The
expression of AtPNP-A in some cases showed induction at
earlier time points than considered in this study, however,
in all cases the expression of AtPNP-A generally increased
over time and thus high transcript levels were sustained
for the duration of the stress, e.g. five days for E. orontii
and 24 h for osmotic, salt and UV-B treatments (data not
shown). The UV-B experiment can be distinguished from
the other experiments in that the stress was not main-
tained for the duration of the experiment. Rather, plants
were irradiated for 15 minutes before being transferred
back to the standard phytochamber conditions until har-
vest. The expression of AtPNP-A in shoots was elevated at
30 minutes (1.84log2; data not shown), peaked at 3 h
(8.09log2, > 250 fold) (Figure 2A) and remained elevated The generation of reactive oxygen species and changes in
ion fluxes have been identified as early responses to both
abiotic and biotic stresses, including an influx of H+ and
Ca2+ and an efflux of K+ and Cl- [35]. AtPNP-A has been
shown to modulate H+, Na+ and K+ fluxes [20] thus further
implicating AtPNP-A in plant stress responses as do stud-
ies which indicate that AtPNP-A signals via the intracellu-
lar second messenger cGMP [5,6,18] since cGMP has been
shown to be an important signaling molecule in pathogen
[44] and osmotic stress responses [45] in plants. It seems
particularly relevant that the expression of a gene encod-
ing a cyclic nucleotide-gated channel (CNGC20;
At3g17700), which has been shown to be involved in the
transport of Ca2+ and K+, and in some cases Na+, across Page 5 of 12
(page number not for citation purposes) Page 5 of 12
(page number not for citation purposes) BMC Plant Biology 2008, 8:24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Frequency of occurrence of the W-box (TTGAC) core motif
in artificial clusters generated in POBO for A. thaliana back-
ground promoters compared to the promoters of AtPNP-A
and the 25 most positively expression correlated genes
Figure 3
Frequency of occurrence of the W-box (TTGAC)
core motif in artificial clusters generated in POBO
for A. thaliana background promoters compared to
the promoters of AtPNP-A and the 25 most posi-
tively expression correlated genes. Expression
response t
Figure 2 The 1 kb upstream
promoter sequences of the 26 expression correlated genes
were analysed in POBO (see methods and [see Additional
file 3]) to determine the frequency of occurrence of the
TTGAC W-box core motif. The analysis determined that
compared to the A. thaliana background (2.24 copies/pro-
moter), there was a significant (t-test: p-value > 0.0001)
enrichment in the frequency of the TTGAC motif in our
dataset (2.99 copies/promoter). (6.46log2; data not shown) at 24 h after irradiation. This
documents that AtPNP-A expression remains very high
and sustained after the stress has been removed and thus
may indicate that the initial damage inflicted, and not the
actual presence of the stress itself, is the driving force for
the maintained transcriptional activation. The increase in expression of AtPNP-A (4.49log2) in
response to the protein synthesis inhibitor cycloheximide
(CHX) implies that transcription of AtPNP-A can occur
independently of de novo protein synthesis and that con-
curs with the definition of immediate early response genes
[54] that have been proposed to play important roles in
the early regulation of defence responses [55]. It has been
postulated that CHX induces gene expression via dual
mechanisms; by preventing synthesis or activation of a
short-lived transcriptional repressor or by removing spe-
cific labile transcript degrading enzymes [56]. There is evi-
dence that the induced expression of genes encoding
secreted proteins, such as AtPNP-A does not require de
novo protein synthesis [57]. The ability to rapidly induce
expression of AtPNP-A independently of de novo protein
synthesis thus implies both an important and early role
for this gene in response to specific elicitors. Frequency
in artificial
ground pro
and the 25
Figure 3 q
y
(
)
g
g
p
p
p
p
y
p
g
g
Frequency of occurrence of the W-box (TTGAC)
core motif in artificial clusters generated in POBO
for A. thaliana background promoters compared to
the promoters of AtPNP-A and the 25 most posi-
tively expression correlated genes. The 1 kb upstream
promoter sequences of the 26 expression correlated genes
were analysed in POBO (see methods and [see Additional
file 3]) to determine the frequency of occurrence of the
TTGAC W-box core motif. The analysis determined that
compared to the A. thaliana background (2.24 copies/pro-
moter), there was a significant (t-test: p-value > 0.0001)
enrichment in the frequency of the TTGAC motif in our
dataset (2.99 copies/promoter). q
y
(
)
g
g
p
p
p
p
y
p
g
g
Frequency of occurrence of the W-box (TTGAC)
core motif in artificial clusters generated in POBO
for A. thaliana background promoters compared to
the promoters of AtPNP-A and the 25 most posi-
tively expression correlated genes. The 1 kb upstream
promoter sequences of the 26 expression correlated genes
were analysed in POBO (see methods and [see Additional
file 3]) to determine the frequency of occurrence of the
TTGAC W-box core motif. The analysis determined that
compared to the A. thaliana background (2.24 copies/pro-
moter), there was a significant (t-test: p-value > 0.0001)
enrichment in the frequency of the TTGAC motif in our
dataset (2.99 copies/promoter). Page 6 of 12
(page number not for citation purposes) Expression
in selected
Figure 4 The expression correlation between AtPNP-A and the dis-
cussed WRKY genes and the overrepresentation of W-
boxes in the correlated genes prompted a manual analysis
of the promoter of AtPNP-A which revealed the presence
of four copies of the core TTGAC W-box motif and two
occurrences of more stringent TTGAC(C/T) motif clus-
tered in close proximity (starting at -738 and -775) rela-
tive to the predicated TSS. The result of the manual
inspection coincided with the results returned form Ath-
ena and POBO. Similar frequencies of these motifs were
observed in a study of 26 SAR regulated genes (termed PR-
1 regulon genes) in which only W-boxes were present in
the promoters of all 26 genes at an average of 4.3 copies
of the core and 2.1 copies of the more stringent W-box ele-
ments within 1100 bp upstream of the predicated TSS
[60]. These values represent a significant enrichment in
W-boxes since these authors determined that the statisti-
cal expectation for a randomly distributed pentamer
(TTGAC) was 2.1 copies and for the hexamer (TTGAC(C/
T)) 1.1 copies per 1100 bp of promoter [60]. Expression profile of AtPNP-A and selected corre-
lated genes in selected A. thaliana mutants. The
expression profiles of AtPNP-A and the correlated genes were
examined in a number of SA/SAR related mutants. The
expression of AtPNP-A and selected genes is greatly elevated
in WRKY 70 over-expresser lines and in mutants with ele-
vated SA levels such as cpr5 and mpk4. Conversely, in the SA
deficient mutant nahG, expression of the selected genes is
markedly reduced. Error bars represent standard errors of
the mean. Expression profile of AtPNP-A and selected corre-
lated genes in selected A. thaliana mutants. The
expression profiles of AtPNP-A and the correlated genes were
examined in a number of SA/SAR related mutants. The
expression of AtPNP-A and selected genes is greatly elevated
in WRKY 70 over-expresser lines and in mutants with ele-
vated SA levels such as cpr5 and mpk4. Conversely, in the SA
deficient mutant nahG, expression of the selected genes is
markedly reduced. Error bars represent standard errors of
the mean. WRKY 70 is a SAR annotated TF in FatiGO and has been
shown to be an essential factor in plant defense responses
necessary for the induction of PR gene expression in A. thaliana [28,69]. Expression
in selected
Figure 4 In a microarray study, a constitutive over-
expressor of WRKY 70 was shown to induce constitutive
expression of SA induced PR genes and five of our corre-
lated genes, including PR-2 and PR-5, which are widely
considered SAR marker genes, correspond to genes in this
study that were up-regulated > 2.5 fold compared to con-
trols [69]. While the 8K Affymetrix chip used for this study
did not contain an AtPNP-A representative sequence, in
an unpublished experiment using the 24K chip, AtPNP-A
was up-regulated over 50 fold in over-expressing WRKY
70 lines and was amongst the top 20 genes that are up-reg-
ulated in this study (Figure 4; Personal communication:
Gunter Brader, Faculty of Biosciences, University of Hel-
sinki). Additionally, a strong induction in expression of
the 25 correlated genes is also observed in this experiment
providing further evidence that indicates that WRKY 70
may positively regulate the expression of AtPNP-A and the
expression correlated genes. In summary, the presented evidence is entirely consistent
with transcription of AtPNP-A and the correlated genes
being positively regulated by WRKY TFs. The promoter of
AtPNP-A and the correlated genes contain an enrichment
of the core W-box motif and expression of AtPNP-A is cor-
related with two WRKY genes in response to various SAR
eliciting and biotic and abiotic stresses. The correlation of
WRKY 46 in response to ion and osmotic abiotc stresses
was both treatment and tissue specific. In the light of these
facts we suggest that the expression of AtPNP-A may be
closely regulated by WRKY TFs in response to SAR-induc-
ing and abiotic stresses. While transcription of WRKY 70 and WRKY 46 is gener-
ally strongly induced in response to SAR eliciting treat-
ments, only WRKY 46 is consistently co-expressed with
AtPNP-A in response to the abiotic (ion and osmotic)
stresses (Figure 2A and 2B). As previously described for
AtPNP-A and the correlated genes, the induced expression
of WRKY 46 is specific to shoots in response to K+ starva-
tion and osmotic stress and to roots in response to NaCl
stress. Common Motifs in cis and Transcription Factors Although in Athena this
p-value does not qualify the W-box motifs to be enriched
in our correlated genes according to the stringent enrich-
ment threshold of <10-4 (Bonferroni correction), it does
show that a very high percentage of our genes contain
multiple copies of the stringent W-box. Both these pro-
moter analysis methods indicate that multiple copies of
the W-box elements are present in a high percentage of
our correlated genes with the core TTGAC motifs being
significantly enriched compared to expected frequencies Page 6 of 12
(page number not for citation purposes) Page 6 of 12
(page number not for citation purposes) BMC Plant Biology 2008, 8:24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 Expression profile of AtPNP-A and selected correlated genes
in selected A. thaliana mutants
Figure 4
Expression profile of AtPNP-A and selected corre-
lated genes in selected A. thaliana mutants. The
expression profiles of AtPNP-A and the correlated genes were
examined in a number of SA/SAR related mutants. The
expression of AtPNP-A and selected genes is greatly elevated
in WRKY 70 over-expresser lines and in mutants with ele-
vated SA levels such as cpr5 and mpk4. Conversely, in the SA
deficient mutant nahG, expression of the selected genes is
markedly reduced. Error bars represent standard errors of
the mean. the possibility that WRKY 70 and WRKY 46 may positively
regulate AtPNP-A transcription. AtPNP-A as PR Protein Based on the above results we suggest that AtPNP-A could
be classified as a PR protein since it possesses many of the
criteria that define this class of proteins. The name "patho-
genesis related protein" is a collective term that encom-
passes all proteins that are present at almost undetectable
levels in healthy tissue but are induced at the protein level
following pathogen infection. The classification of these
proteins is based on their pathogen inducible expression
rather than defined functional roles in defence [43]. This
point is brought into focus when considering PR-1, which
is the quintessential marker of the SAR response yet its
biological role is largely unknown [80]. Although AtPNP-
A is yet to be proven to be induced at the protein level in
response to pathogens, elevated protein levels have been
shown as a result of abiotic stresses [15]. In addition, tran-
scription of AtPNP-A is low under control conditions but
strongly induced in response to biotic and abiotic stresses
and the protein has been identified and isolated from the
A. thaliana apoplast together PR-1, PR-2 and PR-5 proteins
[16]. AtPNP-A has other features characteristic of PR pro-
teins including an N-terminal signal peptide [43] that
directs the molecule into the extracellular space. Further,
induction of AtPNP-A at the transcript level appears to
occur independent of de novo protein synthesis character-
istic of genes encoding secreted proteins [28]. The evolu-
tionary history of AtPNP-A suggests that PNPs, like the
related expansins, derived from ancestral family-45
endoglucanases that have lost their hydrolytic activity and
have sub-functionalized into extracellular, systemically
mobile signalling molecules [9]. Conversely, the nahG mutant that is defective in SA pro-
duction and signalling, is the only experiment presented
in this study that documents a large reduction in the
expression of AtPNP-A (-4.6log2) and the correlated genes
(Figure 4). This experiment was performed in senescing
leaves [72] to identify SA-dependent global gene expres-
sion patterns during developmental senescence since SA
has previously been shown to be required for expression
of some senescence-induced genes [73]. In this study, and
in the ATGE developmental series of A. thaliana microar-
ray experiments [74] transcript levels of AtPNP-A were ele-
vated approximately 2.8 fold in senescing leaves
compared to adult green leaves (data not shown) indicat-
ing that AtPNP-A is a senescence enhanced gene. Insights from AtPNP-A Expression in Mutants In the cpr5 (constitutive expresser of pathogenesis related
genes) and mpk4 mutants that have elevated levels of SA
and display constitutive expression of PR genes [70,71],
the expression of AtPNP-A and the correlated genes was
markedly elevated (Figure 4). It is of particular interest
that the four listed mutants that displayed the largest
increase in the expression of AtPNP-A in Genevestigator The link between AtPNP-A, SA signalling and the WRKY
TFs is also supported by expression profiles of AtPNP-A
and the correlated genes in mutants including a WRKY 70
over-expresser and various SAR related mutants present in
the mutant surveyor in Genevestigator [68]. Page 7 of 12
(page number not for citation purposes) Page 7 of 12
(page number not for citation purposes) BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 http://www.biomedcentral.com/1471-2229/8/24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 BMC Plant Biology 2008, 8:24 http://www.biomedcentral.com/1471-2229/8/24 are all cpr5 mutants, being cpr5/scv1, cpr5/npr1, cpr5, cpr5/
npr1/svi1 (range +7.55 to +6.21log2) (data not shown). AtPNP-A as PR Protein Further-
more, since transcript levels of AtPNP-A were reduced
beyond detection limits in senescing leaves in the nahG
mutant this induction appears to be SA-dependent. This
pattern is completely consistent with other results since
premature senescence, including leaf yellowing and
necrosis can be induced by biotic and aboitic stresses that
stimulate SA production, including ozone [75] and UV-B
[76] which also induce large increases in expression of
AtPNP-A. Thus, there is evidence documenting induction
of AtPNP-A expression in SA-mediated natural develop-
mental and stress activated processes which both culmi-
nate in cell death indicating that AtPNP-A may be
involved in these processes. The mutant analyses further
enforce that the transcriptional regulation of AtPNP-A and
the correlated genes is largely controlled by SA. Future directions In order to determine the physiological role of AtPNP-A in
A. thaliana a T-DNA insertion mutant, that is available
from SALK, could be used. Phenotyping this mutant in
response to SA-inducing abiotic and biotic stresses as well
as during developmental senescence, will help character-
ise specific physiological processes in which AtPNP-A is
involved. If such a mutant demonstrated a compromised
SAR response, it would greatly strengthen the claim that
AtPNP-A is indeed involved in the SAR response pathway. Additionally, it will be interesting to look at the expres-
sion of the correlated genes in an AtPNP-A mutant in
response to SAR inducing conditions since this may ena-
ble us to determine a role for AtPNP-A in the context of a
SAR response pathway. The role of a TGA TF in PR gene expression
Additional evidence for co-regulation of AtPNP-A with
SAR annotated genes is provided by the observation that
expression of another SAR annotated TF, the TGA3 bZIP
TF (At1g22070), is correlated with that of AtPNP-A (r =
0.49). Upon induction of SAR, NIMIN1 and NPR1 form a
ternary complex with TGA factors in the nucleus which
enhances their binding to the positive regulatory as-1
(activator sequence 1) or as-1-like (TGACG) cis-elements
that are present in the promoter of several plant genes acti-
vated during defense, including A. thaliana PR-1 [77-79]. A manual inspection of the AtPNP-A promoter identified
two occurrences of the TGACG motif in close proximity to
the TSS (start at -94 and +24). The correlation in expres-
sion between AtPNP-A, NPR1, NIMIN1 and TGA3 (Table
1, and [see Additional file 2]) along with the identifica-
tion of TGA3 cis-elements in the promoter of AtPNP-A is
strong evidence that these two factors contribute to the
regulation of AtPNP-A expression. Page 8 of 12
(page number not for citation purposes) Promoter analysis The web-based Athena [59] and POBO [58]) applications
were used to analyse the promoters (-1 kb upstream of the
predicted TSS) of AtPNP- and the 25 top correlated genes. In POBO [58], the 1 kb promoter sequences were
uploaded and the analysis was run against A. thaliana
background (clean) searching for the TTGAC W-box core
motif using the default settings (number of sequences to
pick-out = 50, number of samples to generate = 1000,
sequence length = 1000 bps). A two-tailed p-value was cal-
culated in the linked online GraphPad web-site using the
generated t-value and degrees of freedom to determine the
statistical differences between input sequences and back-
ground. Identification of Correlated Genes Identification of Correlated Genes We downloaded (01/07/2005) A. thaliana gene expres-
sion levels for 1877 experiments from the NASCArrays
database [81], using the bulk data download option. Perl
scripts were used to calculate non-parametric correlation
coefficients (Spearman's rho) between the expression of
AtPNP-A (At2g18660) and each of the approximately
22,000 genes represented on the Affymetrix array that was
used to generate this data set. We ranked genes according
to the correlation coefficient and reported genes that were
most positively correlated with At2g18660. The p-values
were calculated using the bivariate normal distribution,
with p representing the probability of observing an equal
or larger positive or negative correlation by chance. Functional Classification and expression analysis of
Correlated Genes To characterise the correlated genes the web-based
'FatiGO+' program [22] was used to search for differential
distributions of gene ontology (GO) and biological terms
within this list. The search was conducted using AtPNP-A
(At2g18660) and the 25 most positively correlated genes
in Table 1 (list 1 = 26 genes). This list was compared to a
reference gene list that contained the remaining genes in
the entire A. thaliana genome (list 2 = 26147 genes). Sta-
tistical significance was determined using the Family Wise
Error Rate (FWER) to calculate the adjusted p-value. In Athena, the analysis was performed with the visualisa-
tion tool using the 26 correlated genes with settings of
1000 bp upstream and do not cut off at adjacent genes. The statistical significance of over-represented TF binding
sites is automatically calculated using a hypergeometric
probability model to calculate the p-value. A Bonferroni
correction was automatically used in Athena to account
for multiple hypotheses testing (up to 105 different TF
binding sites) and determined that the p-value threshold
for significant enrichment was < 10-4. The expression profiles of AtPNP-A and the positively cor-
related genes (Table 1) were initially examined using
Affymetrix public microarray data in the gene response
viewer tool (GRV) in Genevestigator [68]). The analysis
was performed using the ATH1: 22K array chip type and
included all of the available 2507 chip sources. For better
temporal and spatial response resolution we obtained
normalised microarray data from the following sites: http://www.biomedcentral.com/1471-2229/8/24 http://www.biomedcentral.com/1471-2229/8/24 NASCArrays, Ozone-26 (reference ID); P. infestans-123;
UV-B stress-144; Potassium starvation-105; BHT-392. TAIR (ATGenExpress): Salicylic acid-ME00364; E.orontii-
ME00354; Salt stress-ME00328; Osmotic stress-ME00327;
Cold acclimation-ME00369; Cyclohexamide-ME00361. GEO (NCBI): E. cichoracearum-GSE431. and abiotic stimuli and in mutant studies. The expression
of AtPNP-A is correlated with the ICS-1 gene that is
involved in SA biosynthesis and NPR1 and NIMIN1,
which are key positive regulators of the SAR response, and
two annotated SAR TFs TGA 3 and WRKY 70. Addition-
ally, like the PR genes, the promoter of AtPNP-A contains
as-1 and W-box cis-elements that correspond to binding
sites for the TGA 3 and WRKY TFs. Further, over expressing
WRKY 70 lines have been shown to cause a greater than
50-fold increase in the expression of AtPNP-A which is
consistent with this TF being a positive regulator of
AtPNP-A transcription. The induced expression of AtPNP-
A by SAR elicitors and its secretion into the apoplast is
similar to that of PR proteins and strongly implicates a
role for AtPNP-A in plant SAR defence responses which
may involve the modification of cellular ion and water
homeostasis during stress responses. In order to further reveal the relationship of AtPNP-A
expression with that of key genes involved in the SAR
response, the mutant surveyor in Genevestigator was used
to compare gene expression in different types of defence
related A. thaliana mutants. The genes investigated in this
study included AtPNP-A, the 25 correlated genes, and
WRKY 70 and WRKY 46. Normalised array data from the
mutant experiments were obtained from: TAIR-ME00373
for cpr5/npr1 mutants; NASCarray-52 for the nahG mutant
and array express (EBI) for the mpk4 mutant (E-MEXP-
173). For the WRKY 70 over expresser, data was obtained
through personal communication with Gunter Brader,
Faculty of Biosciences, University of Helsinki. Conclusion AtPNP-A is an annotated "signal" molecule that is
secreted into the apoplastic space and has been implicated
with a role in the control of ion and solute movements in
plants (Figure 1). The expression of AtPNP-A is signifi-
cantly correlated with that of genes involved in the SAR
defence response pathway in response to various biotic Page 8 of 12
(page number not for citation purposes) Page 8 of 12
(page number not for citation purposes) BMC Plant Biology 2008, 8:24 Additional file 3 Promoter analysis. AtPNP-A (At2g18660) and the expression correlated
genes were analysed in POBO and Athena for the presence of W-boxes. Click here for file p
p
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approved the final manuscript. J
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English
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Experiences of an earthquake during pregnancy, antenatal mental health and infants’ birthweight in Bhaktapur District, Nepal, 2015: a population-based cohort study
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BMC pregnancy and childbirth
| 2,020
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cc-by
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Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414
https://doi.org/10.1186/s12884-020-03086-5 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414
https://doi.org/10.1186/s12884-020-03086-5 (2020) 20:414 Open Access Experiences of an earthquake during
pregnancy, antenatal mental health and
infants’ birthweight in Bhaktapur District,
Nepal, 2015: a population-based cohort
study Experiences of an earthquake during
pregnancy, antenatal mental health and
infants’ birthweight in Bhaktapur District,
Nepal, 2015: a population-based cohort
study Goma Kumari Khatri1*
, Thach Duc Tran1, Sushil Baral2 and Jane Fisher1 Goma Kumari Khatri1*
, Thach Duc Tran1, Sushil Baral2 and Jane Fisher1 Abstract Background: Infant birthweight is an important public health indicator that is a proxy of maternal and baby’s
health. Earthquakes can cause acute distress, but can also contribute to chronic stress through long-term
disruptions to social, economic and domestic circumstances. The aims of this study were to examine the direct
effect of earthquake experiences on the birthweight of infants of women who experienced the 2015 Nepal
Earthquakes during pregnancy and whether mental health mediated this relationship. Methods: This is a prospective, population-based cohort study. Pregnant women in Bhaktapur District, Nepal who
had experienced the 2015 earthquakes after conception were recruited. Baseline data were collected in structured
individual interviews. Follow up was a short telephone interview two weeks after the baby’s birth. Infant
birthweight recorded on the hospital-issued birth certificate. The direct effect of earthquake experiences on
birthweight and the indirect effect via antenatal common mental disorders (CMDs) were tested using Sobel tests
simultaneously. Results: In total 497/498 women contributed baseline data and 469 (94.4%) women also provided the birth weight
of their infants. In total 6% of infants had low birth weight (< 2.5 kg). The earthquake experiences did not have a
significant direct effect on birthweight (p = 0.116). However, the effect of earthquake experiences had a significant
indirect effect on infant birthweight via CMDs. When 15 covariates were controlled, women who had moderate to
severe earthquake experiences had an increase of 1.58 scores of antenatal CMD symptoms that resulted in their
babies being 20.50 g lighter than those who had low earthquake experiences (p = 0.026). Conclusions: The findings from this study indicate that the relationship is mediated by the mental health of
women during pregnancy. Data demonstrate that the mental health of women who are pregnant should be
prioritised in post-disaster management not only because of the burden experienced by women but also because
of the risk for the growth and development of their babies. Keywords: Birthweight, Earthquake experiences, Antenatal, Common mental disorders * Correspondence: shikak2003@gmail.com; goma.khatri@monash.edu
1School of Public Health and Preventive Medicine, Monash University, Level
4, 553 St Kilda Rd, Melbourne, Victoria 3004, Australia
Full list of author information is available at the end of the article Background from ‘the United States’ and ‘non-United States coun-
tries’ and found that relative risk of LBW was signifi-
cantly
higher
among
women
who
had
‘antenatal
depression’ compared to those who had not. The study
also reported that LBW was significantly higher in ‘non-
United States’ countries compared to those in ‘the
United States’ [6]. g
Infant birthweight is an important public health indica-
tor. It reflects maternal health as well as a predictor of
the health and development of the baby [1]. Worldwide,
it is estimated that low birthweight (LBW), usually de-
fined as being born weighing less than 2500 g, contrib-
utes to up to 80% of all neonatal deaths [2]. It is
estimated that about 96.5% of infants born with LBW
are living in resource-constrained low-income countries
[2]. Finding from a national survey in Nepal revealed
that LBW of infants based on the reported birth weight
by mothers who had a live birth in the five years prior to
the survey was 12.3%. About 5 and 12% of the mothers
reported that their babies were small and very small at
birth respectively [1]. Babies with LBW who survive, are
at higher risk of compromised growth and development
in their subsequent lives [3]. There have been seven studies that reported that
women’s experiences of an earthquake might increase
the risk of giving birth to LBW of their infants. Among
them, six [15–20] were from middle-and high-income
countries and only one [21] from a low-income country. All were audits of birth records or secondary analyses of
existing data. None investigated directly, the relation-
ships among women’s experiences of the earthquake
while pregnant, their mental health and its relationship
with the birthweight of their infants. Multiple causative factors are associated with LBW, but
maternal health is a well-established determinant. While
poor maternal nutrition, and chronic or acute stresses
during pregnancy independently increase the likelihood of
LBW, these may interact with each other in ways that
amplify the risk of LBW [2, 4]. These causative factors are
highly prevalent in low-income settings [2, 5, 6]. There is to date no study examining the possible path-
ways of the effect of earthquakes on birth outcomes, but
this is of central importance for effective post-disaster re-
sponses. Antenatal mental health that can be compro-
mised by experiencing a natural disaster and might play a
mediating role in this relationship. Study design A prospective, population-based study of a cohort of
women who were pregnant and had experienced the
2015 Earthquakes since conceiving. It included a base-
line survey in late pregnancy and a follow-up interview
conducted two weeks after birth. g
y
g
Earthquakes cause acute distress, but can also contrib-
ute to chronic stress through long-term disruptions to so-
cial, economic and domestic circumstances [8–10]. Our
study [11] was conducted after the 2015 Nepal earth-
quakes and reported that 21.9% of participants had
Edinburgh Postnatal Depression Scale (EPDS) scores> 12,
and another 17.1% scored 10–12 indicating a high preva-
lence of clinically-significant common mental disorders
(CMDs) symptoms, which include anxiety, depressive, ad-
justment, and somatoform disorders [12]. This is a higher
prevalence than those reported in a systematic review and
meta-analysis (about 16%) from low and lower-middle-
income countries in general circumstances [5]. Another
study by Kane et al. [13] was conducted in the same earth-
quake context among the general population. The study
reported that 34.3% experienced depressive symptoms,
33.8% anxiety and 5.2% had PTSD symptoms. The study
found that women were more likely to experience depres-
sion, anxiety and PTSD then men [13]. Study setting Data were collected in Bhaktapur District from October
2015 to April 2016. Bhaktapur is one of the 14 of 77 dis-
tricts in Nepal that were severely affected by the April
2015 Earthquakes which were measured at 7.8 on the
Richter Scale [22]. Bhaktapur has six sub-administrative
divisions that include rural and urban communities. It
also includes a UNESCO-designated World Heritage Site
of major cultural, historical, and religious significance. Conceptual framework Conceptual framework in (Fig. 1) shows the proposed
direction of associations among study variables. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
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data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 12 Page 2 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Background The aims of this study
were to examine the direct effect of earthquake experi-
ences on the birthweight of infants of women who experi-
enced the 2015 Nepal Earthquakes during pregnancy and
whether mental health mediated this relationship. In Nepal, Sharma et al. [7] conducted a case-control study in
a tertiary hospital to identify determinants of LBW. They found
that women who had a history of premature birth, had not
been able to consume nutritious food during pregnancy, and
were of younger (< 20years) age had a significantly higher risk
of giving birth to a LBW infant [7]. We are not aware of any
study in Nepal that has examined and reported on associations
between pregnancy mental health and infant birthweight. Procedure Participants were recruited from all Bhaktapur sub-
administrative
divisions. Potential
participants
were
identified in three ways: (i) together, the district public
health office, district public health nurses, community
health centre staff and female community health volun-
teers (FCHVs) created a list of eligible pregnant women
(ii) The Women and Children Development district offi-
cers identified eligible women who had not been listed
(iii) formal and informal local community leaders, eld-
erly women and pregnant women were asked to identify
potential participants in their villages. With the address
given in the list of eligible women, we visited their home
to inform them about the study and invite to participate
in the study. All women were invited to participate in the study if they
met the following criteria: (1) to be aged at least 15 years,
(2) living in Bhaktapur District, and (3) pregnant during the
2015 Earthquakes. Pregnant women were excluded if they:
(1) had a multifetal pregnancy, or (2) had a cognitive dis-
ability, or (3) were local but outside Bhaktapur at the time
of the major 2015 earthquakes, or (4) moved (migrated) to
Bhaktapur to live after the major earthquakes. Those who contributed baseline data were invited again to
participate in the follow-up interview. Women who had a
stillbirth and those who did not measure the weight of their
babies at birth were excluded from analyses on birthweight. There were two waves of data collection: one during
pregnancy and the other two weeks after women had
given birth. Participants The sample size was calculated to test the indirect effect of
earthquake experiences on birthweight via antenatal CMD
symptoms with a study power of 80% and an alpha error of In general circumstances, antenatal CMDs appear to
increase the risk of LBW [6, 14]. Grote et al. [6] con-
ducted a systematic review of eleven studies reported Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 3 of 12 Fig. 1 Conceptual framework. Source: Khatri, GK (2019) [23]. Note. Arrow signs show the postulated direction of association among variables. Plus
(+) sign indicates positive and minus (−) sign indicates a negative association between variables Fig. 1 Conceptual framework. Source: Khatri, GK (2019) [23]. Note. Arrow signs show the postulated direction of association among variables. Plus
(+) sign indicates positive and minus (−) sign indicates a negative association between variables 0.05 (5% level of significance). In total 480 women were re-
quired to identify Cohen’s effect ≥0.14 of earthquake expe-
riences on CMD symptoms and ≥0.26 of CMD symptoms
on birthweight, allowing for attrition of 20% [24]. Public involvement Members of the local community were included in di-
verse ways. Local bilingual health experts contributed to
the translation of the interview schedules to Nepali, cul-
tural verification and back translation to English. Twelve
pregnant women are meeting inclusion criteria apart
from living in Bhaktapur pilot-tested the schedules for
acceptability
and
comprehensibility. Health workers,
community leaders, elderly women, female community
health volunteers, and staff of the Bhaktapur District
Women’s Development Office identified eligible preg-
nant women. HERD International, a local research or-
ganisation, partnered in conducting this study. Potential participants were informed about the study
by giving an explanatory statement. The statement was
read by a researcher or a family member to those who
could not read it. Participants were assured that their
privacy and confidentiality would be maintained strictly. Women who were willing to participate gave consent by
signing the form or providing a thumbprint. When a participant gave consent, she was invited to
complete an interview in person for the first wave of
data collection (baseline). All interviews were conducted
in Nepali in private rooms at local health centres or,
rarely, in a private space at their homes. Six trained and female interviewers who had experi-
ence in data collection for other research projects man-
aged by HERD International conducted the interviews
following training from and with supervision from bilin-
gual co-investigator (GKK). Primary data sources The primary data source was a structured interview
schedule, which included standardised tools and study-
specific questions (Table 1, Questionnaires). Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 4 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Table 1 Primary data sources and coding
Interview
time
Variables
Primary data sources and coding
birth outcome
Follow up
Birthweight
In Nepal, a birth certificate is prov
facility. It records the date and tim In Nepal, a birth certificate is provided to each mother who has given birth in a medical
facility. It records the date and time of the birth and the baby’s birthweight. Women
were asked to report the birth weight of their babies from the birth certificate. Women
who gave birth at home and did not have a birth certificate but where a skilled person
had measured birth weight using a standard scale were also asked to report the weight. Those who did not measure baby birth weight were not able to be included in analyses. The weight in grams was used as a continuous variable to examine an association
among covariates. Birth weight was also categorized into two groups: ≥2.5 kg deemed
to be normal and < 2.5 kg as low birth weight. Mediating variable
Baseline
Symptoms of Common Mental
Disorders (CMDs) during pregnancy The Edinburgh Postnatal Depression Scale-Nepali version (EPDS-N) comprises 10 items,
scored from 0 to 3 with a total score of 0 to 30. This version has been formally validated
against Diagnostic and Statistical Manual (DSM) IV diagnostic criteria among women
who had recently given birth in Nepal: Sensitivity 68.43%; Specificity 93.80%; Positive Pre-
dictive Value 65% and Negative Predictive Value 94.64 [31]. The EPDS total score was
used as a continuous variable to examine a mediation effect. We also reported propor-
tions of the scores: < 9, 10 to 12 and ≥13 to enable comparison between groups. Independent variables
Baseline
Earthquake experiences Study-specific questions adapted from previous studies [9, 20] collecting information
about 17 items: where they are living now (1 item); property damage (3 items); impact
on daily life including basic needs (6 items); being injured/trapped (2 items); witnessing
injury/people trapped/death (4 items), intensity of fear experienced during the
earthquake (1 item). Details of these items have reported elsewhere [11]. Each item was
scored 0 (not experienced) to 1 (experienced) and summed to create a total score. Data management and statistical analysis Data were collected on paper forms and double-entered
using Epidata version 3.1 computer software [25] at HERD
Nepal. Data analyses were performed using Stata version
14 [26]. Descriptive analyses were used to compare partic-
ipants’ characteristics in the group who provided complete
data (which could be included in the final analyses) with
those of participants who provided incomplete data
(which could not be included) and between groups with
babies of different birthweights. Fisher exact test for cat-
egorical and Mann-Whitney test for continuous variables
were used to test socio-demographic differences between
the groups were and were not included in the analyses. In order to examine the interaction effect between the
exposure (earthquake experiences) and mediator (depres-
sive symptoms) on birthweight and the confounding effect
of depressive symptoms on the effect of earthquake expe-
riences on birthweight, we conducted multivariate regres-
sion analyses stratified by having depressive symptoms or
not. The criterion for not having clinically significant de-
pressive symptoms was an EPDS score ≤12. The criterion
for having clinically significant depressive symptoms was
an EPDS score ≥13. These criteria were based on a formal
validation of the EPDS among women who had recently
given birth in Nepal [31]. The multivariate regression pre-
dicting birthweight included all covariates that had been
included in the mediation analysis. The direct effect of earthquake experiences on birth-
weight and the indirect effect via EPDS total score were
tested using simultaneous Sobel tests [27, 28]. According
to Pek and Hoyle [28], we assumed that women’s mental
health problems (EPDS total score) mediated the effect of
earthquake experiences on birthweights. In this analysis,
birth weight (in grams) was the primary outcome, the ex-
posure variable was earthquake experiences, and EPDS
total score was a mediating variable [28]. This analysis
controlled for 15 covariates that were selected based on
the results of bivariable analyses and previous studies. Primary data sources BMC Pregnancy and Childbirth (2020) 20:414 Table 1 Primary data sources and coding (Continued)
Interview
time
Variables
Primary data sources and coding
Violence Against Women Questionnaire, including lifetime experiences of controlling be-
haviour (7 items), emotional (4 items), physical (6 items), and sexual (3 items) violence by
the current intimate partner [43]. Any lifetime experiences of intimate partner violence
calculated combining any of four sub-types of violence. Baseline
Practical and emotional support specific to
the earthquakes
Study-specific questions adapted from previous studies [10, 20]. Participants were asked
whether they received any support including shelter, food, clothes, informal emotional
and professional psychological support and rescue from the hazardous place during/
after earthquakes by any individual or any governmental and non-governmental
organization. Violence Against Women Questionnaire, including lifetime experiences of controlling be-
haviour (7 items), emotional (4 items), physical (6 items), and sexual (3 items) violence by
the current intimate partner [43]. Any lifetime experiences of intimate partner violence
calculated combining any of four sub-types of violence. Study-specific questions adapted from previous studies [10, 20]. Participants were asked
whether they received any support including shelter, food, clothes, informal emotional
and professional psychological support and rescue from the hazardous place during/
after earthquakes by any individual or any governmental and non-governmental
organization. The second interview (follow-up) was a very short tele-
phone interview two weeks after childbirth to ask about
the date, place and mode of birth, and the baby’s birth-
weight. All these interviews were conducted by GKK. The second model predicted EPDS scores from earth-
quake experiences and other covariates. The final model
predicted birthweight from all variables including earth-
quake experiences, EPDS scores and other covariates. Fi-
nally, the test results were summarized including the
estimations and significant tests of the direct effect of
the earthquake experiences on birthweight; the indirect
effect of the earthquake experiences on birthweight via
antenatal CMD symptoms, and the total effect of the
earthquake experiences on birthweight. The second model predicted EPDS scores from earth-
quake experiences and other covariates. The final model
predicted birthweight from all variables including earth-
quake experiences, EPDS scores and other covariates. Fi-
nally, the test results were summarized including the
estimations and significant tests of the direct effect of
the earthquake experiences on birthweight; the indirect
effect of the earthquake experiences on birthweight via
antenatal CMD symptoms, and the total effect of the
earthquake experiences on birthweight. Results
Sample In total 497/498 eligible pregnant women were recruited
and contributed baseline data. All gave consent to be
followed up, and 492 women participated in the follow-
up interview. Altogether 469 (94.4%) were able to pro-
vide the baby’s weight at birth and were included in the
analyses (Fig. 2). Bivariate analyses were conducted using the chi-square
test or regression to select potential covariates that should
be included in the final model of the mediation analysis. Covariates were selected on the basis that there were sig-
nificant differences at p ≤0.1. However, some covariates
such as household wealth, intimate partner violence and
education levels were included in the final model even
though they were not significant because they are estab-
lished risk factors for low birthweight [29, 30]. Primary data sources The
score ranged from 0 to 17. The total score was grouped in tertle, and low tertile was
labelled as low, and middle and upper tertile labelled as middle/high earthquake
experiences. Covariates
Baseline and
follow up
Length of gestation at birth Date of the baby’s birth and the date of the last menstrual period were recorded to
calculate the length of gestation at birth. At baseline, we asked participants about their
date of last menstrual period. In the follow-up interview, date of baby birth was recorded. The last date of menstru-
ation was subtracted from date of baby birth and divided by seven to calculate the total
weeks as the length of gestation at birth. We used weeks as a continuous variable to
examine an association with other variables. Study-specific questions adapted from our prior research [39] to ascertain women’s age,
highest educational level, employment, consume chewing tobacco/smoking, and
alcohol, household economics, family structure, and husband’s education, employment,
consumption of chewing tobacco/smoking, and alcohol. Household economics was calculated based on household characteristics and durable
assets using the World Bank method [39, 40]. Woman’s weight was measured using a portable digital weighing scale. Height was
measured against the smooth but hard wall. Height and weight were used to calculate
body mass index using international formula (weight/height2). It was used as a
continuous variable to examine its associations with other covariates. Study-specific questions adapted from our prior research [39] about gravidity, parity,
prior spontaneous/induced abortions, pregnancy intention, taking iron tablets during
current pregnancy, foetal health, sex of index foetus and a number of children. Single yes/no question was used to assess if the participant had been diagnosed with or
treated for any psychiatric illness within last year. The Relationship Assessment Scale (RAS) comprises 7 items scored from 1 to 5 and total
scored from 7 to 35. Mean inter-item correlation of the scale is 0.49; test-retest reliability
(among undergraduate students in USA) is 0.85 and high correlation with Dyadic Adjust-
ment Scale [41] (0.86 to .88). Examples of items are: ‘How well does your partner meet
your needs? In general, how satisfied are you with your relationship? How much do you
love your partner?’ Higher scores indicate a better quality of the relationship [42]. Items from the World Health Organization Multi-Country Study on Women’s Health and Page 5 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Khatri et al. Birth weight Birthweight ranged from 1800 g to 5000 g. Mean birth-
weight was 3045.3 ± 447.8 g. About 6% (5.8%) women re-
ported
birthweight
less
than
2500 g. Most
women
reported a rounded birthweight: 13.65% reported 3000 g;
11.51% reported 3500 g; 9.81% reported 2500 g; 8.10%
reported 2700 g; 6.18% reported 2800 g and about 5% in
each weight category reported 2900 g, 3000 g, 3100 g and
3200 g. The multivariate regression analyses predicting birth-
weight stratified by having or not having clinically sig-
nificant depressive symptoms revealed that moderate to
high experiences of the earthquake had no significant ef-
fect on birthweight in the absence of depressive symp-
toms (adjusted coefficient = 52.26, p = 0.29) or in the
presence of depressive symptoms (adjusted coefficient =
171.94, p = 0.10). Participants’ characteristics A comparison of the characteristics of participants
whose data could and could not be included in the final
analyses is presented in Table 2. Compared to the group
who could be included, the group who were not in-
cluded had significantly younger age, lower body mass
index, no formal or primary education, more women
consumed
chewing
tobacco/smoking,
and
lower In the Sobel test, three different multiple linear regres-
sion models were performed simultaneously. The first
model predicted birthweight from earthquake experi-
ences and other covariates but without EPDS scores. Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 6 of 12 Fig. 2 Flow diagram of participants able to provide data about their baby’s birth weight. Note. n = number Fig. 2 Flow diagram of participants able to provide data about their baby’s birth weight. Note. n = number that earthquake experiences did not have a significant
direct effect on birth weight. Compared to low experi-
ences of the earthquakes, women who had middle or
high earthquake experiences had higher EPDS scores. Women who had higher EPDS scores had babies with
lower birthweights. household wealth. In addition, they were more likely to
have a no-formal educated partner, experienced emo-
tional and physical/sexual violence perpetrated by an in-
timate
partner,
had
an
inconvenient/unwanted
pregnancy, lower ANC check-ups and iron tablet intake. No women reported a history of psychiatric illness. Antenatal CMDs symptoms significantly mediated the
effect of earthquake experiences on baby birthweight
(Fig. 3). This means that while controlling for all covari-
ates, women who had moderate to high earthquake ex-
periences had an increase of 1.58 points in the EPDS
scores that resulted in a 20.50 g reduction in their in-
fants’ birth weight compared to those who had low
earthquake experiences. Association among earthquake experiences, antenatal
CMDs symptoms and birth weight The main findings of the Sobel test analysis are pre-
sented in Fig. 3, and more detailed findings are pre-
sented in Tables S1, S2, S3 and S4. The results showed Page 7 of 12 Khatri et al. Association among earthquake experiences, antenatal
CMDs symptoms and birth weight BMC Pregnancy and Childbirth (2020) 20:414 Page 8 of 12 Table 2 Characteristics of participants at the baseline interview (Continued) Table 2 Characteristics of participants at the baseline interview (Continued)
Characteristics
Included (n = 469)
Not included (n = 28)
p-value
Girl
207 (44.1)
9 (39.1)
Number of children, n %
0.6
None
246 (52.5)
13 (46.4)
One or more
223 (47.5)
15 (53.6)
Length of gestation at baby birth (weeks), mean (SD)
39.9 (1.5)
39.1 (2.5)
0.2
Earthquake experiences, mean (SD)
3.3 (1.3)
3.6 (1.3)
0.2
Having practical/emotional social support, n %
348 (74.2)
18 (64.2)
0.3
EPDS, mean (SD)
8.0 (5.1)
10.6 (6.7)
0.06
EPDS total score categories, n %
0.2
< 9 score
289 (61.6)
14 (50.0)
10 to 12 score
81 (17.3)
4 (14.3)
≥13 score
99 (21.1)
10 (35.7)
Note. ‘Included’ means data included for final analysis; ‘not included’ means data not included for final analysis (loss to follow up)
n = number; % = percentage; SD = standard deviation; EPDS = Edinburgh Postnatal Depression Scale Note. ‘Included’ means data included for final analysis; ‘not included’ means data not included for final analysis (loss to follow up)
n = number; % = percentage; SD = standard deviation; EPDS = Edinburgh Postnatal Depression Scale 20.50 g less compared to women who had low earthquake
experiences via antenatal CMD symptoms. Independent
of other covariates, women who had higher CMD symp-
toms gave birth to babies with significantly lower birth-
weight compared to women with lower CMD symptoms. Among the covariates, body mass index of women
during advanced pregnancy and length of gestational age
at birth were also associated consistently and independ-
ently with birthweight (Table S1, S3). There is no study examining the direct and indirect ef-
fect of earthquake experiences on birth weight simultan-
eously to compare our findings. However, the findings of
our study are in line with the findings of audits of hos-
pital records that babies born after earthquakes had
lower birthweight compared to those who born before
the earthquakes [16, 18–21] and indicate that experien-
cing an earthquake while pregnant can adversely affect Association among earthquake experiences, antenatal
CMDs symptoms and birth weight BMC Pregnancy and Childbirth (2020) 20:414 Table 2 Characteristics of participants at the baseline interview
Characteristics
Included (n = 469)
Not included (n = 28)
p-value
Socio-demographic characteristics
Age (years), mean (SD)
26.5 (4.8)
24.9 (5.2)
0.05
Body mass index during late pregnancy, mean (SD)
27.1 (3.8)
25.4 (3.8)
0.01
Education, n %
< 0.001
No formal/ primary education
86 (18.3)
17 (60.7)
Secondary and above education
383 (81.7)
11 (39.3)
Having income-generating work, n %
152 (32.4)
8 (28.6)
0.8
Consume chewing tobacco/smoking, n %
12 (2.6)
7 (25.0)
< 0.001
Alcohol consumption, n %
118 (25.2)
7 (25.0)
1.0
Family structure, n %
0.3
Joint/extended family
234 (49.9)
11 (39.3)
Nuclear family
235 (50.1)
17 (60.7)
Education of partners, n %
< 0.001
No formal/ primary education
51 (10.9)
13 (46.4)
secondary and above education
418 (89.1)
15 (53.6)
Partners with income-generating work, n %
437 (93.2)
27 (96.4)
1.0
Consume chewing tobacco/smoking by partner, n %
157 (33.5)
20 (71.4)
< 0.001
Alcohol consumption by partner, n %
239 (51.0)
19 (67.9)
0.1
Household wealth, mean (SD)
.09 (2.03)
−1.53 (1.66)
< 0.001
Intimate partner relationship
Quality of relationship with an intimate partner, n %
0.002
Least optimal (< 28 RAS score)
105 (22.4)
14 (50.0)
Optimal (> 28 RAS score)
364 (77.6)
14 (50.0)
Any lifetime experience of intimate partner controlling behaviour, n %
163 (34.8)
15 (50.0)
0.1
Any lifetime experience of intimate partner emotional violence, n %
191 (40.7)
17 (60.7)
0.05
Any lifetime experience of intimate partner physical and or sexual violence, n %
109 (23.2)
16 (57.1)
< 0.001
Any lifetime experiences of any intimate partner violence, n %
270 (57.6)
20 (71.4)
0.2
Reproductive characteristics
History of pregnancy, n %
Nulliparous
211 (45.0)
9 (32.1)
0.2
Two or more pregnancies
258 (55.0)
19 (67.9)
History of miscarriage/abortion, n %
85 (18.1)
7 (25.0)
0.3
Taking iron tablet during current pregnancy, n %
449 (95.7)
20 (71.4)
< 0.001
The good foetal health, n %
447 (95.3)
24 (85.7)
0.05
Unwelcome/inconvenient pregnancy, n %
61 (13.0)
10 (35.7)
0.003
Attended antenatal check-up for current pregnancy
466 (99.4)
25 (89.3)
0.003
Number of the antenatal visit for current pregnancy
< 0.001
none
3 (0.6)
3 (10.7)
once
8 (1.7)
2 (7.1)
twice
15 (3.2)
5 (17.9)
Three times
33 (7.0)
5 (17.9)
Four or more times
410 (87.4)
13 (46.4)
Sex of index foetus
0.7
Boy
262 (55.9)
14 (60.9) Khatri et al. Discussion One possible explanation for this is
that earthquake experiences may increase stress in preg-
nant women that negatively affect the growth and devel-
opment of their foetus rather than there being a direct
effect of earthquake experiences on the foetus [6, 32]. About 6% of women reported that their babies had
LBW (< 2500 g), but a further 10% of women reported a
rounded Fig. 2500 g. It is common practice for rounding
up to occur and so it is likely that almost 16% of babies
who had been exposed in utero to the earthquake were
of LBW. In contrast to the national scenario, only two partici-
pants had a low body mass index (< 18.5 kg/m2) in this
study. Most of the study participants reported that they
had received advice about nutrition and diet (96.2%) and
took iron tablets (94.4%) during the current pregnancy. This could be related to the unique circumstance of the
earthquake that this cohort of women may receive spe-
cial attention to protect their foetus. When we used
body mass index as a continuous variable to predict
birthweight, it showed that with the increment of body
mass index, birthweight of baby increased significantly
and independently. Our study reported a higher prevalence of LBW than
those of other studies conducted in an earthquake con-
text. For instance, Tan et al. [19] audited birthweight of
babies from hospital records before and after the 2008
Wenchuan earthquake in China. They found that 5.01%
of babies born after the earthquake had LBW (≤2500 g)
which was significantly higher than those were born be-
fore the earthquake (3.72%). China is a middle-income
country where the rate of LBW is much lower than in
Nepal in general circumstances too [2]. Women in
China may receive better care and services in post-
earthquake management than those of Nepali women
that may affect the difference of baby birthweight. y
In contrast to a previous study [29], our study did not
find household wealth to be a risk factor. The household
wealth of this sample was relatively homogeneous, and
there may have been insufficient variance to detect dif-
ferences in birthweight between households with higher
and lower wealth. A household wealth of women who
were ‘included’ and were ‘not included’ in final analyses
was significantly different. Discussion To our knowledge, this is the world’s first study in a low-
income country to examine the relationship between preg-
nant women’s experiences of an earthquake and the birth-
weight of their infants and whether antenatal CMD
symptoms mediate the effect of earthquake experiences
on baby birthweight. Women who had moderate or high
earthquake experiences gave birth to babies who weighed Fig. 3 Key findings of the Sobel test of the direct effect of earthquake experiences on birthweight and the indirect effect mediated by EPDS total
score. Note. Single-headed solid arrows show the direction of association among variables. EPDS = Edinburgh Postnatal Depression Scale; ß = linear
regression coefficient Fig. 3 Key findings of the Sobel test of the direct effect of earthquake experiences on birthweight and the indirect effect mediated by EPDS total
score. Note. Single-headed solid arrows show the direction of association among variables. EPDS = Edinburgh Postnatal Depression Scale; ß = linear
regression coefficient Fig. 3 Key findings of the Sobel test of the direct effect of earthquake experiences on birthweight and the indirect effect mediated by EPDS total
score. Note. Single-headed solid arrows show the direction of association among variables. EPDS = Edinburgh Postnatal Depression Scale; ß = linear
regression coefficient Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 9 of 12 Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 This practice applies to pregnant women too. Even
though there are different nutritional policies and pro-
grams to combat nutritional problems [1], there is no
policy or practise to ensure specific food security for
pregnant women in post-earthquake management in
Nepal [33]. It was reported that nutritious food was dis-
tributed to pregnant women immediately after the earth-
quake, but the report did not specify what nutritious
food and what number of women benefited. The report
did not provide details about who benefited from the
supply: all women or only women in urban or in rural
areas and how long the supplementation was sustained
for [34, 35]. birth outcomes. Further, our study findings are in line
with previous investigations of the relationship between
antenatal CMDs and baby birthweight in general cir-
cumstances, which have found a significant negative as-
sociation [6, 14]. Discussion This suggests that ‘not in-
cluded’ women were even more likely than those who
were included to have a baby with LBW and that our
findings were more likely to have been under- than
over-estimates of the impact of the earthquake on
birthweight. In Nepal context, our study reported a higher preva-
lence of LBW than that reported in the recent Nepal
Demographic and Health Survey (NDHS) 2016 (12%
baby had LBW < 2500 g) [1]. Our study site was in Bhak-
tapur district, which was relatively well resourced, with
more accessible health services than in other districts in
Nepal. The earthquake created a unique circumstance. There were anecdotal records from hospital staff that
women heard informally that earthquakes might have a
negative impact on foetal health and growth and ‘many
women’
sought
termination
of
their
pregnancies. Women who elected to continue their pregnancies were
reported to be cautious about foetal wellbeing, and they
presented for antenatal check-ups (Table 2) at a much
higher rate than the national average [1]. Despite this
healthcare, LBW was still very high. Our study reported a small number of women (4.1%)
had a preterm birth (< 37 weeks). This could be because
we missed the group at high risk of preterm birth or
who sought an abortion before we approached them
since the data collected six months after the major
earthquake. Because of the small number of preterm
birth, length of gestation at birth was used as a continu-
ous variable to predict birthweight. Consistent with pre-
vious studies [2, 7, 36], our data revealed that the higher
the length of gestation at birth, the higher the weight of
babies. This study confirms previous findings [2, 4] that ma-
ternal body mass index is associated significantly with
baby birthweight. While malnourishment among women
in reproductive age in Nepal is high, women rarely con-
sume additional nutritious food during pregnancy [1, 7]. According to NDHS 2016, 17.3% of women of repro-
ductive age had a low body mass index (< 18.5 kg/m2),
an indicator of undernutrition [1]. In Nepal, women,
generally feed other family members first and then they
consume any remaining food at the end of the meal. Implications and conclusions The study has brought to attention the public health im-
portance of the experiences of earthquakes for women
who are pregnant not only for their own health but also
because of the impact on their baby’s birthweight. These
findings indicate that antenatal CMDs among women
should be a focus of post-disaster interventions not only
because of the burden [5] imposed on women’s partici-
pation but also because of the risk for the growth and
development of their foetuses [3, 6]. In Nepal, menstruation is considered unclean, and it is
culturally prohibited for women to attend everyday reli-
gious observations or sacred ceremonies or to go into
the home kitchen or to prepare food when they are
menstruating. These are publicly observable circum-
stances and readily linked to specific ceremonies or
events, and so it is easy for women in Nepal to recall the
first day of the last menstrual period. It is generally be-
lieved that they rarely recall the date inaccurately. Nevertheless, we acknowledge that the date of the last
menstrual period was self-reported. Although we think
this is unlikely, it might not have been recalled precisely
and have led to an inaccurate estimation of the length of
gestation at birth. In our study, a very few women had a low body mass
index and preterm birth, which are well-established risk
factors of LBW. Despite that our study reported a high
prevalence of LBW. This indicates that women’s mental
health problems were a key factor for this high preva-
lence of LBW. While most participants reported that they received
antenatal check-ups, they had a high prevalence of
symptoms of antenatal CMDs [11], none of which were
detected or treated. Maternal and child health is a na-
tional priority in Nepal, and the country has made pro-
gress to reduce maternal and child morbidity and
mortality in recent decades. However, rates are still very
high compared to other countries [1]. In Nepal, there is
no specific programs and intervention for the mental
health of women in the perinatal period, but this could
assist in the reduction of maternal and child morbidity
and mortality. These data clearly indicate that specialised
consideration of perinatal mental health is a grave need
in the post-disaster context in Nepal. We acknowledge that mediation analysis is a sophisti-
cated statistical technique that requires assumptions. Strengths The study was a population-based prospective study. Population-based studies permit the findings to be gen-
eralised to equivalent settings. We were able to do a Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 10 of 12 Page 10 of 12 Page 10 of 12 validated against a gold standard comparator for use
among pregnant women [28]. postpartum follow-up as a prospective study. Prospective
investigations are able to ascertain causal relationships
including, in this study, between earthquake experiences,
pregnant women’s mental health and birthweight. This
is a robust design for mediation analyses [28]. The study
was adequately powered to detect the direct and indirect
effects of the earthquake on birthweight. The study was powered to detect within-group vari-
ation of earthquake experiences on birthweight, but as
the earthquake affected the entire country, it was not
possible to recruit a comparison group of women who
had no experience of the earthquakes. Data were col-
lected about six months after the earthquake. Therefore,
the sample did not include women who had already
given birth, including those who had premature births. We were told that some women had sought abortions
immediately after the earthquakes because they feared
that there might have been adverse effects on the foetus. This could lead to underestimates of the impact of the
earthquake on infant birthweight. Nevertheless, the
study included all eligible women who met inclusion cri-
teria during the study period. The strengths of this study
outweigh the limitations and can be generalised with
some confidence. There were very high recruitment and retention frac-
tions. Data were collected in individual interviews which
enabled women with low literacy to participate. We con-
trolled for other known risk and protective factors for
baby birthweight and antenatal CMD symptoms to con-
trol for potential confounding effects. Limitations We acknowledge the limitation of the study that baby
birthweight was based on the birth certificates provided
by health institutions rather than being measured by the
research team. Most birthweights were recorded on birth
certificates as figures that had been rounded up; it is
possible that the percentage of LBW was underesti-
mated. For this reason, we used birthweight as a con-
tinuous measure in outcome analyses. The earthquake
experience measurement scale was adapted from one
used in previous studies and was carefully pilot tested,
but it was not standardised or locally validated. Acknowledgements We are indebted to the research participants who contributed their time and
trusted us with their intimate experiences despite the major disruption of
the earthquakes. We are very grateful to the six HERD health researchers for
their courage and dedication to collecting high-quality data despite the ad-
verse circumstances in Nepal at the time of data collection. We would like to
acknowledge the important contributions of the Female Community Health
Volunteers (FCHV), the health staff of Bhaktapur district, the officers of the
Women and Children Development Office Bhaktapur, and members of the
community for their valuable support during data collection. We would like
to acknowledge the organizations that supported the authors by providing
scholarship and fellowships. 6. Grote NKP, Bridge JAP, Gavin ARP, Melville JLMD, Iyengar SP, Katon WJMD. A meta-analysis of depression during pregnancy and the risk of preterm
birth, low birth weight, and intrauterine growth restriction. Arch Gen
Psychiatry. 2010;67(10):1012. 6. Grote NKP, Bridge JAP, Gavin ARP, Melville JLMD, Iyengar SP, Katon WJMD. A meta-analysis of depression during pregnancy and the risk of preterm
birth, low birth weight, and intrauterine growth restriction. Arch Gen
Psychiatry. 2010;67(10):1012. 7. Sharma SR, Giri S, Timalsina U, Bhandari SS, Basyal B, Wagle K, Shrestha L:
Low birth weight at term and its determinants in a tertiary hospital of
Nepal: A case-control study. PLoS One 2015, 10(4). 8. Anwar J, Mpofu E, Matthews LR, Brock KE. Risk factors of posttraumatic
stress disorder after an earthquake disaster. J Nerv Ment Dis. 2013;201(12):
1045–52. 9. Qu Z, Tian D, Zhang Q, Wang X, He H, Zhang X, Huang L, Xu F. The impact
of the catastrophic earthquake in China's Sichuan province on the mental
health of pregnant women. J Affect Disord. 2012;136(1–2):117–23. Funding GKK is supported by an Australia Award PhD scholarship, TT is supported by
a National Health and Medical Research Council Early Career Fellowship, JF is
supported by Finkel Professorial Fellowship which receives funding from the
Finkel Family Foundation. The funders had no role in study design, data
collection, data analysis, data interpretation, or writing of the report. The
authors received no separate funding for this project. Abbreviations 4. Wadhwa PD, Entringer S, Buss C, Lu MC. The contribution of maternal stress
to preterm birth: issues and considerations. Clin Perinatol. 2011;38(3):351–84. CMD: Common mental disorder; EPDS: The Edinburgh Postnatal Depression
Scale; LBW: Low birthweight CMD: Common mental disorder; EPDS: The Edinburgh Postnatal Depression
Scale; LBW: Low birthweight 5. Fisher J, de Mello MC, Patel V, Rahman A, Tran T, Holton S, Holmes W. Prevalence and determinants of common perinatal mental disorders in
women in low-and lower-middle-income countries: a systematic review. Bull World Health Organ. 2012;90(2):139–49. Implications and conclusions The design we used in this study, a prospective cohort,
can help us to be confident that pregnancy mental
health is a mediator rather than a confounder or an
effect-modifier. Nevertheless, the associations between
exposure, mediator and the outcome can be confound-
ing or effect modification. Secondly, unmeasured vari-
ables including ‘post-traumatic growth’ may have a
counter effect on mental health problems that may influ-
ence the mediation effect of mental health problems on
birthweight. Also, there is the possibility of measurement
errors of earthquake experiences and pregnancy mental
health because the scales were not locally formally Even though it is challenging to conduct ethical, com-
prehensive and culturally-sensitive studies after a natural
disaster, it is essential to generate evidence to inform ef-
fective
post-disaster
interventions. These
data Khatri et al. BMC Pregnancy and Childbirth (2020) 20:414 Page 11 of 12 Page 11 of 12 demonstrate that it is feasible to generate robust evidence
in this situation. for data collection and assisted with data management. All authors (GKK, TT,
SB and JF) read and approved the manuscript. for data collection and assisted with data management. All authors (GKK, TT,
SB and JF) read and approved the manuscript. In conclusion, this study has addressed an important
knowledge gap. These findings provide evidence about
the importance of women’s antenatal mental health in
post-disaster management which is currently missing
from the World Health Organization’s guideline “Key
Steps for Maternal and Newborn Health Care in Hu-
manitarian Crisis” [37]. The data inform enhancements
to existing maternal and child health programs in Nepal
and will strengthen the country’s capacity to meet the
Sustainable Development Goals [38]. Author details
1
f Author details
1School of Public Health and Preventive Medicine, Monash University, Level
4, 553 St Kilda Rd, Melbourne, Victoria 3004, Australia. 2HERD International,
Kathmandu 44600, Nepal. Additional file 2 Supplementary Table 2 (S2) Model 2 in Sobel test
analysis. Multiple linear regression model predicting the symptoms of
CMDs from earthquake experiences and other covariates Additional file 2 Supplementary Table 2 (S2) Model 2 in Sobel test
analysis. Multiple linear regression model predicting the symptoms of
CMDs from earthquake experiences and other covariates Received: 10 July 2018 Accepted: 2 July 2020 Ethics approval and consent to participate Ethics approvals for this study were obtained from Monash University
Human Research Ethics Committee (CF15/2776–2015001130) on 19th August
2015 and Nepal Health Research Council (219/2015) on 4th October 2015. Written informed consent was obtained from all participants. References
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in Epi-Data and analysed the data under the supervision of JF and TT. GKK
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[http://www.who.int/maternal_child_adolescent/documents/keysteps.pdf]. 38. Sustainable Development Goals [http://www.un. sustainabledevelopment/health/]. 38. Sustainable Development Goals [http://www.un.org/
sustainabledevelopment/health/]. 39. Fisher J, Tran TD, Biggs B, Dang TH, Nguyen TT, Tran T. Intimate partner
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Industrial Wastes-Cum-Strength Enhancing Additives Incorporated Lightweight Aggregate Concrete (LWAC) for Energy Efficient Building: A Comprehensive Review
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Citation: Kumar, R.; Srivastava, A.;
Lakhani, R. Industrial Wastes-
Cum-Strength Enhancing Additives
Incorporated Lightweight Aggregate
Concrete (LWAC) for Energy Efficient
Building: A Comprehensive Review. Sustainability 2022, 14, 331. https://
doi.org/10.3390/su14010331 Academic Editors: Elena Michelini,
Daniele Ferretti and Syed Minhaj
Saleem Kazmi Keywords: lightweight aggregate concrete; industrial waste; thermal insulation; energy-efficient
building; sustainability Industrial Wastes-Cum-Strength Enhancing Additives
Incorporated Lightweight Aggregate Concrete (LWAC) for
Energy Efficient Building: A Comprehensive Review Rajesh Kumar Rajesh Kumar *
, Abhishek Srivastava and Rajni Lakhani Rajesh Kumar *
, Abhishek Srivastava and Rajni Lakhani Organic Building Materials (OBM) Group, CSIR-Central Building Research Institute,
Roorkee 247667, Uttarakhand, India; ashish27.srivastav@gmail.com (A.S.); rlakhani@cbri.res.in (R.L.)
* Correspondence: rajeshkumar@cbri.res.in or rk2896315@gmail.com Abstract: Lightweight aggregate concrete (LWAC) exhibits the advantages of thermal insulation,
reduces energy consumption building costs, improves building efficiency and easy construction. Furthermore, the utilization of industrial wastes in concrete is advantageous in terms of environ-
mental sustainability. In order to explore this, several researchers investigated the idea of integrating
industrial wastes in LWAC. However, the lack of knowledge regarding the performance of industrial
waste-based lightweight aggregate concrete hinders the adaptation of this concept and application of
LWAC in the construction sector. Therefore, this paper summarizes the research in relation to the
sustainable LWACs containing oil palm shell (OPS), lightweight expanded clay aggregate (LECA),
vermiculite, perlite, pumice and sintered fly ash as lightweight aggregate, along with industrial
wastes and strength-enhancing additives (viz. fibers, polymers, etc.). Firstly, desirable physical,
chemical, morphological and mineralogical characterization of different lightweight aggregates are
presented, and then a comprehensive overview on fresh, hardened, durability and thermal prop-
erties of LWAC incorporating industrial wastes are discussed in comparison with normal weight
concrete. The review also highlights the current challenges and suggests the research gaps for further
development of eco-friendly LWAC. It is concluded that vermiculite, perlite, pumice, OPS, sintered
fly ash and LECA with some suitable industrial waste materials have the potential to be used in
the construction sector. Moreover, LWAC with industrial waste has 50–65% lower carbon emission
(kg CO2 eq/m3) in the environment. The scientific contribution of this paper provides insights into
different LWACs and the knowledge base for future research and paradigm shift of using LWACs as
more common alternative building materials. sustainability sustainability sustainability Figure 2 Location-wise research trend
Figure 2. Location-wise research trend.
Figure 2. Location-wise research trend. Figure 2 Location-wise research trend
Figure 2. Location-wise research trend. Figure 2. Location-wise research trend. The use of lightweight aggregates (LWA) in concrete has the following advantages:
The use of lightweight aggregates (LWA) in concrete has the following advantages:
The use of lightweight aggregates (LWA) in concrete has the following advantages: The use of lightweight aggregates (LWA) in concrete has the following advantages:
The use of lightweight aggregates (LWA) in concrete has the following advantages
The use of lightweight aggregates (LWA) in concrete has the following advantages: The use of lightweight aggregates (LWA) in concrete has the followin
1
Less dead load structure stability as well as economic viability [2];
The use of lightweight aggregates (LWA) in concrete has the follow
1
Less dead load structure stability as well as economic viability [2];
1. Less dead load, structure stability as well as economic viability [2]; 1. Less dead load, structure stability as well as
2
Cost reduction [3];
1. Less dead load, structure stability as well as
2
Cost reduction [3];
2. Cost reduction [3]; . Cost reduction [3];
Relati ely lo
the
al o du ti ity [4 6]
2. Cost reduction [3];
3
Relatively low thermal conductivity [4 6];
3. Relatively low thermal conductivity [4–6]; 3. Relatively low thermal conductivity [4–6];
4
H
li
ti
i
t
d
t
hi h i
b ildi
t
[7 8]
3. Relatively low thermal conductivity [4–6];
4
Has application in prestressed concrete high rise buildings etc [7 8
4. Has application in prestressed concrete, high rise buildings, etc. [7,8] 4. Has application in prestressed concrete, high rise buildings, etc. [7,8];
5
I
th
k bilit
if
tt d
i
t
i
t [9]
4. Has application in prestressed concrete, high rise buildings, etc. [7,8]
5
Improves the workability if pre wetted prior to use in concrete [9]
5. Improves the workability if pre-wetted prior to use in concrete [9]. Has application in prestressed concrete, high rise buildings, etc. [7,8];
I
th
k bilit
if
tt d
i
t
i
t [9]
Has application in prestressed concrete, high rise buildings, etc. [7,8];
Improves the workability if pre wetted prior to use in concrete [9]
Improves the workability if pre-wetted prior to use in concrete [9]. 5. Improves the workability if pre-wetted prior to use in concrete [9]. Figure 2 Location-wise research trend
Figure 2. Location-wise research trend.
Figure 2. Location-wise research trend. However, lower absolute mechanical strength, higher water absorption, porosity,
etc., are some of the negative impacts. LWAC is an alternative solution to NWC, especially
when lightweight energy-efficient solutions are needed. Thus, to overcome the problem
of natural stone deterioration and for making energy-efficient buildings, different light-
weight concretes are used. Chart 1 shows the classification of LWAC. It can be ascertained
from the flow chart that LWAC can be used for load-bearing and non-load-bearing pur-
poses. Different LWAs commonly used in making LWAC are vermiculite, OPS, perlite,
LECA, pumice, scoria, tuff, cinder, lytag, etc. (Koksal et al. [1]). LWAs are a type of
coarse/fine aggregates used for the manufacturing of LWC products, and these products
are used in different structural work (Alengaram et al. [2]). LWAs have a cellular type
structure and were used for producing different types of masonry blocks, wall panels,
cladding and LWC. When they are used as fine aggregates, they function similar to active
5. Improves the workability if pre wetted prior to use in concrete [9]. However, lower absolute mechanical strength, higher water absorption, porosity,
etc., are some of the negative impacts. LWAC is an alternative solution to NWC, especially
when lightweight energy-efficient solutions are needed. Thus, to overcome the problem
of natural stone deterioration and for making energy-efficient buildings, different light-
weight concretes are used. Chart 1 shows the classification of LWAC. It can be ascertained
from the flow chart that LWAC can be used for load-bearing and non-load-bearing pur-
poses. Different LWAs commonly used in making LWAC are vermiculite, OPS, perlite,
LECA, pumice, scoria, tuff, cinder, lytag, etc. (Koksal et al. [1]). LWAs are a type of
coarse/fine aggregates used for the manufacturing of LWC products, and these products
are used in different structural work (Alengaram et al. [2]). LWAs have a cellular type
structure and were used for producing different types of masonry blocks, wall panels,
cladding and LWC. When they are used as fine aggregates, they function similar to active
However, lower absolute mechanical strength, higher water absorption, porosity, etc.,
are some of the negative impacts. LWAC is an alternative solution to NWC, especially
when lightweight energy-efficient solutions are needed. Thus, to overcome the problem of
natural stone deterioration and for making energy-efficient buildings, different lightweight
concretes are used. Chart 1 shows the classification of LWAC. 1. Introduction Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. In the 21st century, energy-efficient buildings are one of the important issues, which
include both techno-economic and sustainable environmental factors [1]. Energy-efficient
buildings are designed to use energy as little as possible. Many developing countries are
leading towards the construction of green buildings using cost-effective, durable concrete. The huge demand for concrete in the infrastructural development using normal-weight
aggregates (NWAs) has reduced the natural stone deposits, which causes irreplaceable
damage to our environment. As a result, the priority of searching for sustainable materials
has been enhanced worldwide. LWAC is one such alternative to normal weight aggregate
concrete (NWC) with various physical, mechanical, social and economic advantages [2]. Figures 1 and 2 show the current trend of research (year-wise and location-wise) in the
area of lightweight aggregate concrete (Source: https://www.scopus.com/; accessed on 12
September 2021). The trend shows that day by day, the practical use of LWACs is increasing
and thus requires the current status of the research conducted to date. Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/sustainability Sustainability 2022, 14, 331. https://doi.org/10.3390/su14010331 2 of 21
creas-
ncreas- Sustainability 2022, 14, 331 Figure 1. Year-wise research trend. Figure 2 Location wise research trend
Figure 1. Year-wise research trend. Figure 1. Year-wise research trend. Figure 2 Location-wise research trend
Figure 2. Location-wise research trend. Figure 1. Year-wise research trend. Figure 1. Year-wise research trend. Figure 1. Year-wise research trend. h
d 2. Types of LWAs In general, LWAs can be divided into two categories. Chart 1. Classification of lightweight concretes.
Chart 1. Classification of lightweight concretes. The cellular structure within the particles is formed at high temperatures, generally
at 1100 ◦C or higher. Due to the cellular structure of particular aggregate particles, the
aggregates are light in weight, and the specific gravity, as well as the unit weight of LWAs,
is lower than that of NWAs. The maximum size grading designations of LWAs generally
available are 19 mm, 13 mm or 10 mm. LWAs have more water absorption (up to 5 to
20%) and allow limited access for fresh cement paste into the open pores of the LWAs. Due
to their porous structure, LWAs require to be wet for 24 h before adding it into the mix. In the case of LWC, the bond between the aggregate and the matrix is stronger than the
normal concrete. LWC provides a reduction in dead loads and improves the thermal and
fire properties of buildings. It is necessary to limit the slump to improve the workability,
durability and avoid segregation. In LWC, excessive mixing should be avoided because it
tends to crack the LWA particles (ACI 213 [10]). p
The paper presents the wide classification of different kind of LWAs that are used to
develop LWACs and discuss their physio-chemical and morphological characterization. Additionally, fresh, hardened, functional and durable properties of sustainable LWAC are
presented along with the effect of different SCMs and fibers to improve the performance
of LWACs. Furthermore, ongoing research activities at CSIR-CBRI on the effect of marble
slurry and Class-F fly ash on LECA incorporated LWAC is also discussed. Figure 2 Location-wise research trend
Figure 2. Location-wise research trend.
Figure 2. Location-wise research trend. It can be ascertained from
the flow chart that LWAC can be used for load-bearing and non-load-bearing purposes. Different LWAs commonly used in making LWAC are vermiculite, OPS, perlite, LECA,
pumice, scoria, tuff, cinder, lytag, etc. (Koksal et al. [1]). LWAs are a type of coarse/fine
aggregates used for the manufacturing of LWC products, and these products are used in
different structural work (Alengaram et al. [2]). LWAs have a cellular type structure and
were used for producing different types of masonry blocks, wall panels, cladding and LWC. When they are used as fine aggregates, they function similar to active pozzolanic materials. It can be produced from the naturally available raw materials such as expanded clay, shale,
slate, etc., as well as from SCMs such as FA and slags, etc. 3 of 21
marble Sustainability 2022, 14, 331 Chart 1. Classification of lightweight concretes. Chart 1. Classification of lightweight concretes. Chart 1. Classification of lightweight concretes. Chart 1. Classification of lightweight concretes. 2.1. Natural Aggregate Natural LWAs are the materials that are available as natural resources. These are
naturally ready to use with mechanical treatment, i.e., crushing and sieving. Mostly, natural
LWAs are of volcanic origin, e.g., pumice and scoria [11,12]. Thus, they are only found
in a few areas of the world. Pumice is formed when the molten lava from the explosive
eruption of a volcano cools [13]. Quick cooling freezes the material existing at the molten Sustainability 2022, 14, 331 4 of 21 state, which does not have zero probability of a crystallization process. Scoria is darker
in color than pumice but has all the properties same as that of pumice. Volcanic tuffs are
a kind of volcanic rock, which develops pores through rapid cooling and hardening of
lava. The pore may vary from 10% to 60%. These tuffs are classified as rhyolite, dacite,
andesite, etc. Similarly, volcanic slag is also derived from lava, which is less vitreous and
more crystalline slag-like materials. These all-natural LWAs are used to produce LWC with
a density range from 1860 to 1988 kg/m3 [14–16]. 2.2. Manufactured/Artificial Aggregate These LWAs are classified as brick rubble, cinder, sintered cinder, blast-furnace slag,
LECA, etc. These types of aggregates are produced by thermal treatment of either naturally
occurring materials such as clay, shale, vermiculite, perlite and slate, etc., or industrial
byproducts/waste materials such as FA, municipal solid waste (MSW), waste of dredging,
blast furnace slag, etc. Cinder LWAs are produced as coal burnt residues in the industrial
boilers. The residue is melted and sintered to form cinders. Sintered fly ash is developed
at a temperature of 1100–1300 ◦C [17]. They have a hard, coarse red shell and fine pore
structures, which are commercially available in a size range of 7 to 30 mm. Similarly,
foamed blast-furnace slag aggregates are produced by sintering slag at a temperature of
1400–1600 ◦C. The size range lies between less than 3 mm and 20 mm, with a varying bulk
density of 300 to 700 kg/m3. LECA is produced using clay or shale after heating at the
point of incipient vitrification (at a temperature of about 1200 ◦C) [18]. After exposure,
clay/shale expands or bloats to seven times their original volume and forms a cellular
structure that remains stable even after cooling. In the market, different kind of LECA
is available by different names such as hydite, rocklite, lytag, aglite, keramzit, etc. In a
similar manner, expanded perlite is developed after exposing perlite at a temperature of
900–1100 ◦C, which leads to an increase in volume by 15–20 times [19]. Vermiculite is a type
of mica with high magnesium content, which is formed at a temperature of 900 ◦C [20]. 3. Different LWAs and Their Use to Develop Green Building Materials During the extracting process of oil from
the oil palm tree, OPS are generated, which is a waste product. In order to protect the
environment, action was taken to utilize OPS as LWAs. The dumped oil palm kernel shells
left at the factory yard are shown in Figure 3. Figure 4 shows that OPS aggregate has an
irregular flaky, angular shape. Sustainability 2022, 14, x FOR PEER REVIEW
6 of 22 Table 2. Chemical Composition of LWAs. Table 2. Chemical Composition of LWAs. Table 2. Chemical Composition of LWAs. Type of
Aggregate
References
Chemical Components (%)
Main
Minor
SiO2
Al2O3
Fe2O3
CaO
MgO
SO3
K2O
Na2O
LOI
OPS
Shafigh et al. [14];
Foong et al. [38]
18.47–21.32
4.27–6.20
2.06–3.62
64.09–65.41
2.08–2.43
4.25–5.50
0.28–0.73
0.21–0.25
1.41–1.80
LECA
Al-Bahar and
Bogahawatta [39];
Sajedi and
Shafigh [40];
Masoud et al. [41]
53.3–66.05
15.05–19.78
6.2–9.52
1.05–2.98
0.78–3.67
0.23–0.25
2.55–4.1
0.17–1.54
1.37–15.11
Vermiculite
Koksal et al. [1];
Abidi et al. [42];
Sayadi et al. [43]
36.9–46
10–17.7
5.50–11.2
1–3.5
16–35
0.02–0.10
1–6
0.13–0.2
7.5–9.2
Perlite
Turkmen and
Kantarci [44]
71–76
9.91–16
0.40–1.57
0.5–2.19
0.01–0.28
0.04–0.10
4–5
2.9–4
1.48–2.0
Pumice
Demirel and
Kelestemur [45];
Binici et al. [35];
Onoue et al. [46]
41.41–63.4
12.97–21.9
1.26–11.41
1.8–13.73
0.3–15
0.44–0.50
1.73–5.40
1.80–5.20
1.60–7.32
Sintered
Fly ash
Kayali [47];
Kockal and
Ozturan [15];
Guneyisi et al. [36]
56.2–64.60
19.58–28.5
4.0–7.23
0.54–4.24
0.66–4.64
0.30–0.69
0.01–5.95
0.32–2.06
0.49–5.10 3. Different LWAs and Their Use to Develop Green Building Materials There are different types of LWAs that are used in the construction of buildings [21]. Some vitally used LWAs are vermiculite, OPS, perlite, LECA, pumice and sintered fly ash. Tables 1 and 2 summarize the physical and chemical characterization results obtained by
several researchers. Table 1. Physical properties of LWAs. Type of Aggregate
References
Specific
Gravity
Water
Absorption
for 24 h (%)
Fineness
Modulus (FM)
Bulk Density
(Compacted)
(kg/m3)
OPS
Mannan and Ganapathy [22];
Teo et al. [4]; Shafigh et al. [14];
Sobuz et al. [23]; Eziefula et al. [21]
1.17–1.30
33.0–19.6
5.64–6.24
572–656
LECA
Maghsoudi et al. [5]; Real et al. [24];
Bogas and Cunha [11]; Shafigh et al. [25]
0.51–1.18
16.42–26.5
15.8–5.96
273–667
Vermiculite
Schackow et al. [26]; Divya et al. [27];
Arun et al. [28]
1.10–3.0
2.65
2.46
-
Perlite
Demirbog and Gul [29];
Karakoc and Demirboga [19];
Oktay et al. [30]; Zulkifeli and Saman [31]
0.12–0.42
82.5
-
200
Pumice
Hossain [32]; Sari and Pasamehmetoglu [33];
Gunduz and Ugur [34]; Binici et al. [35];
Kockal and Ozturan [15]
1.57–3.10
-
-
870
Sintered Fly ash
Guneyisi et al. [36]; Gomathi and Sivakumar [37]
2.10–2.25
0.14
6.24
- Table 1. Physical properties of LWAs. 5 of 21 Sustainability 2022, 14, 331 Table 2. Chemical Composition of LWAs. Type of
Aggregate
References
Chemical Components (%)
Main
Minor
SiO2
Al2O3
Fe2O3
CaO
MgO
SO3
K2O
Na2O
LOI
OPS
Shafigh et al. [14];
Foong et al. [38]
18.47–21.32
4.27–6.20
2.06–3.62
64.09–65.41
2.08–2.43
4.25–5.50
0.28–0.73
0.21–0.25
1.41–1.80
LECA
Al-Bahar and
Bogahawatta [39];
Sajedi and
Shafigh [40];
Masoud et al. [41]
53.3–66.05
15.05–19.78
6.2–9.52
1.05–2.98
0.78–3.67
0.23–0.25
2.55–4.1
0.17–1.54
1.37–15.11
Vermiculite
Koksal et al. [1];
Abidi et al. [42];
Sayadi et al. [43]
36.9–46
10–17.7
5.50–11.2
1–3.5
16–35
0.02–0.10
1–6
0.13–0.2
7.5–9.2
Perlite
Turkmen and
Kantarci [44]
71–76
9.91–16
0.40–1.57
0.5–2.19
0.01–0.28
0.04–0.10
4–5
2.9–4
1.48–2.0
Pumice
Demirel and
Kelestemur [45];
Binici et al. [35];
Onoue et al. [46]
41.41–63.4
12.97–21.9
1.26–11.41
1.8–13.73
0.3–15
0.44–0.50
1.73–5.40
1.80–5.20
1.60–7.32
Sintered
Fly ash
Kayali [47];
Kockal and
Ozturan [15];
Guneyisi et al. [36]
56.2–64.60
19.58–28.5
4.0–7.23
0.54–4.24
0.66–4.64
0.30–0.69
0.01–5.95
0.32–2.06
0.49–5.10
3.1. OPS Concrete (OPSC)
OPS are considered as agricultural waste and lighter in weight, which is found in
abundance all over the world. Malaysia is one of the largest oil palm producers in the
world, which produces 4 MT of OPS annually [2]. 3.1. OPS Concrete (OPSC) OPS are considered as agricultural waste and lighter in weight, which is found in
abundance all over the world. Malaysia is one of the largest oil palm producers in the
world, which produces 4 MT of OPS annually [2]. During the extracting process of oil from
the oil palm tree, OPS are generated, which is a waste product. In order to protect the
environment, action was taken to utilize OPS as LWAs. The dumped oil palm kernel shells
left at the factory yard are shown in Figure 3. Figure 4 shows that OPS aggregate has an
irregular flaky, angular shape. REVIEW
6 of 22 Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). Figure 3. OPS dumped at the factory yards [2]. (Reprinted with permission from Alengaram et al. (2013), 2021, Elsevier). 6 of 21
m et al Sustainability 2022, 14, 331 Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021,
Elsevier). Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021,
Elsevier). Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021,
Elsevier). Figure 4. OPS of different sizes [2]. (Reprinted with permission from Alengaram et al. (2013), 2021,
Elsevier). The concrete with compressive strength of more than 25 MPa was achieved using
OPS as a lightweight coarse aggregate. The construction of low-cost buildings can be per-
formed using OPS concrete. Mannan and Ganapathy [22] compared the different proper-
ties of OPS concrete with control concrete. It was found that the 28-day compressive
strength, flexural strength and splitting tensile strength of OPS concrete were between 20
and 24 MPa, 2.75 and 4.00 MPa and 1.78 and 2.41 MPa, respectively. The 28-day elasticity
modulus of OPS concrete was 0.70–0.76 × 104 MPa. OPS concrete showed 14% higher dry-
ing shrinkage than control concrete at 90 days, and it also had higher water absorption. Mannan and Ganapathy [48] studied the different properties of concrete using OPS as a
coarse aggregate and fly ash (FA). 3.1. OPS Concrete (OPSC) It was found that the OPS concrete containing FA
showed lower air content compared to control concrete without FA. The density of the
fresh concrete of OPS concrete lies between 1910 and 1958 kg/m3. The 28-day compressive
strength of samples was in the range of 20.1–24.2 MPa, which satisfies the requirement of
structural LWC. Olanipekun et al. [3] studied the strength characteristics as well as the
cost analysis of concrete made by using OPS and crushed granular coconut shells. The
level of replacement for LWA with coarse aggregate was in the range of 25 to 100%. The
results show that by increasing fractions of OPS, the uniaxial compressive strength of
LWA concrete was decreased. A cost reduction between 30% and 42% could be achieved
using OPS as a coarse aggregate in concrete production. The concrete with compressive strength of more than 25 MPa was achieved using OPS
as a lightweight coarse aggregate. The construction of low-cost buildings can be performed
using OPS concrete. Mannan and Ganapathy [22] compared the different properties of OPS
concrete with control concrete. It was found that the 28-day compressive strength, flexural
strength and splitting tensile strength of OPS concrete were between 20 and 24 MPa, 2.75
and 4.00 MPa and 1.78 and 2.41 MPa, respectively. The 28-day elasticity modulus of OPS
concrete was 0.70–0.76 × 104 MPa. OPS concrete showed 14% higher drying shrinkage
than control concrete at 90 days, and it also had higher water absorption. Mannan and
Ganapathy [48] studied the different properties of concrete using OPS as a coarse aggregate
and fly ash (FA). It was found that the OPS concrete containing FA showed lower air content
compared to control concrete without FA. The density of the fresh concrete of OPS concrete
lies between 1910 and 1958 kg/m3. The 28-day compressive strength of samples was in
the range of 20.1–24.2 MPa, which satisfies the requirement of structural LWC. Olanipekun
et al. [3] studied the strength characteristics as well as the cost analysis of concrete made
by using OPS and crushed granular coconut shells. The level of replacement for LWA
with coarse aggregate was in the range of 25 to 100%. The results show that by increasing
fractions of OPS, the uniaxial compressive strength of LWA concrete was decreased. A cost
reduction between 30% and 42% could be achieved using OPS as a coarse aggregate in
concrete production. 3.1. OPS Concrete (OPSC) g
gg
g
p
p
Teo et al. [4] investigated the properties of OPS-based LWAC and found that the 28-day
air-dry density and compressive strength were 1960 kg/m3 and 28.12 MPa, respectively. The 28-day tensile strength, flexural strength and elastic modulus were found as 2.02 MPa,
4.97 MPa and 5.31 GPa, respectively. The curing condition affects the durability of OPS
concrete in a significant manner. OPS concrete in water curing gave better durability
performance than normal weight concrete. Shafigh et al. [14] studied the different properties
such as air content, cube compressive strength, density and water absorption of OPS-based
high strength lightweight concrete (HSLC). Five types of curing conditions with different
OPS concrete mixtures were taken, and then their compressive strength was determined. The effect of partial replacement of limestone powder (as a filler material) by the fine
aggregate was determined. It was observed that the workability of the developed concrete
was decreased by increasing limestone powder content. Water absorption of high-strength
OPS concrete varies from low to high range, which is a good sign of strong concretes. Alengaram et al. [2] investigated that OPSC inherited high strength to density ratio than
that of NWC. It was also shown that OPSC could be used to produce medium to high
strength concrete. Liu et al. [49] studied the physico-mechanical and thermal properties of OPS foamed
geopolymer concrete (OPSFGC). OPSFGC mixtures were prepared to have densities of 1300, Sustainability 2022, 14, 331 7 of 21 7 of 21 1500 and 1700 kg/m3 in which an artificial foaming agent was used for casting. The control
OPS non-foamed geopolymer concrete (OPSNFGC) was cast for comparison studies. It
was found that the 28-day compressive strengths of OPSFGC of all three densities were
lower than the OPSNFGC. As the density decreases, the 28-day splitting tensile strength
and modulus of rupture also decreases. The thermal conductivity of the mix with a density
of 1300 and 1500 kg/m3 was 0.47 and 0.50 W/mK, respectively. Sobuz et al. [23] studied
the characteristics of concrete produced using OPS fractions ranging from 0 to 50% by
replacing conventional coarse aggregate. The compressive strength was reduced with
the increasing percentage of OPS aggregates. The structural requirement of LWC was
satisfactory. Mo et al. [50] described the effect of steel fiber on the hardness features such
as flexural toughness, compressive toughness and fracture parameters of steel fiber OPS
concrete (SFOPSC). 3.1. OPS Concrete (OPSC) The outcome of adding steel fiber in the range of 0.5 to 1.0% in SFOPSC
on the tensile strength, compressive toughness and flexural toughness was improved by
1.41 times, 6 times and 16 times, respectively. Mo et al. [51] made a comparison between
lightweight OPS concrete and NWC of 25, 35 and 45 MPa strength grades. It was concluded
that young modulus, indirect tensile strength and drying shrinkage of OPS concrete was
lower than that of NWC, while the bond strength of OPS concrete was 80% higher. g
g
Mo et al. [52] studied the durability properties of OPS concrete in which cement
and natural sand were replaced with ground granulated blast furnace slag (GGBS) and
manufactured sand, respectively. GGBS was also found to be of great use in improving the
long-term compressive strength gain as well as bringing down the strength reduction in
OPS concrete when exposed to heat. The use of GGBS as a partial cement substitution in
the OPS concrete resulted in an increase in compressive strength gain compared to OPS
concrete without GGBS. Eziefula et al. [21] performed comparative studies of different
mechanical properties of OPSC with periwinkle shell concrete. The mechanical strength
and bulk density of periwinkle shells and OPS-based concrete qualify for the requirements
of LWACs. However, the strength characteristics of OPS concrete were inferior to that of
the periwinkle shell-based concrete. Khankhaje et al. [53] investigated different properties
of OPSC and cockleshell concrete (CSC). It was found that CSC possesses better properties
compared to OPSC. It was due to the high water absorption capacity of OPS. Alengaram
et al. [54] studied microscopic analysis of OPS using scanning electron microscope as shown
in Figure 5, which shows micro pores (size: 16–24 µm) on the convex surface of OPS surface,
which causes high water absorption. REVIEW
8 of 22 Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011), 2021, Elsevier). Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011),
2021, Elsevier). Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011), 2021, Elsevier)
Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011),
2021, Elsevier). Figure 5. Micro-pores on outer surface [54]. (Reprinted with permission from Alengaram et al. (2011) 2021 Elsevier)
Figure 5. Micro-pores on outer surface [54]. 3.1. OPS Concrete (OPSC) (Reprinted with permission from Alengaram et al. (2011),
2021, Elsevier). 3 2 LECA C
t
3.2. LECA Concrete 3.2. LECA Concrete
LECA aggregates are manufactured from low-lime plastic clay. For aggregate prep-
aration clay is firstly heated and then dried After drying sintering is performed in spe
LECA aggregates are manufactured from low-lime plastic clay. For aggregate prepara-
tion, clay is firstly heated and then dried. After drying, sintering is performed in specific Sustainability 2022, 14, 331 8 of 21 kinds of rotary kilns at a temperature of 1100–1300 ◦C. In this process, the gas, i.e., released
after the heating process, becomes entrapped inside the pellets during cooling. LECA
aggregates have rounded irregular shapes [55] and incorporate many multi-separated
interconnected voids of different sizes, which make them lightweight. The morphology of
LECA is shown in Figure 6. LECA is mostly dark brown or reddish or brown-red or gray
in colors. These aggregates are also available in yellow or black color due to variation in
chemical composition and process of manufacturing. It has a pH value of 7 and is thus
inert in nature. It can be used as a replacement for both coarse and fine aggregates. cific kinds of rotary kilns at a temperature of 1100–1300 °C. In this process, the gas, i.e.,
released after the heating process, becomes entrapped inside the pellets during cooling. LECA aggregates have rounded irregular shapes [55] and incorporate many multi-sepa-
rated interconnected voids of different sizes, which make them lightweight. The morphol-
ogy of LECA is shown in Figure 6. LECA is mostly dark brown or reddish or brown-red
or gray in colors. These aggregates are also available in yellow or black color due to vari-
ation in chemical composition and process of manufacturing. It has a pH value of 7 and is
thus inert in nature. It can be used as a replacement for both coarse and fine aggregates. Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with per-
mission from Nkansah et al. (2012), 2021, Elsevier). Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with permis-
sion from Nkansah et al. (2012), 2021, Elsevier). Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with per-
mission from Nkansah et al. (2012), 2021, Elsevier). Figure 6. Surface image of a LECA particle under SEM showing pores [56]. (Reprinted with permis-
sion from Nkansah et al. (2012), 2021, Elsevier). Zach et al. 3 2 LECA C
t
3.2. LECA Concrete [5] studied the possible use of the non-stationary hot-wire method for de-
termining the thermal conductivity of the LECA concrete coefficient. It was found that the
thermal conductivity of LECA concrete ranged from 0.14 to 0.16 W/m.K. Maghsoudi et al. [57] developed self-compacting lightweight concrete (SCLC) using LECA. Various mix
designs of SCLC were cast to obtain the final standard SCLC, with a compressive strength
of 20.8–28.5 MPa at 28 days. Zohrabi et al. [58] investigated the mechanical properties of
LWC containing LECA with metakaolin, polypropylene (PP) and steel fibers. The PP fi-
bers have more effect on the energy absorption capability of LWC rather than on strength
Zach et al. [5] studied the possible use of the non-stationary hot-wire method for
determining the thermal conductivity of the LECA concrete coefficient. It was found that
the thermal conductivity of LECA concrete ranged from 0.14 to 0.16 W/m.K. Maghsoudi
et al. [57] developed self-compacting lightweight concrete (SCLC) using LECA. Various mix
designs of SCLC were cast to obtain the final standard SCLC, with a compressive strength
of 20.8–28.5 MPa at 28 days. Zohrabi et al. [58] investigated the mechanical properties
of LWC containing LECA with metakaolin, polypropylene (PP) and steel fibers. The PP
fibers have more effect on the energy absorption capability of LWC rather than on strength
properties. The addition of 1% steel fibers increased the compressive strength and flexural
strength by 36.3% and 52.6%, respectively. Grabois et al. [6] investigated the fresh and
hardened properties of steel fibers incorporated SCLC. The 28-day compressive strength
was found above 30 MPa for a density of 1700–1900 kg/m3. Concretes with coarse and
fine LWAs depicted higher drying shrinkage than those containing only coarse LWAs. The
results showed that the thermal conductivity decreased by 60% by using coarse and fine
LECA aggregates. gg
g
Bogas and Cunha [11] investigated the physico-mechanical behavior of non-structural
lightweight concrete (NSLWC), which was developed by volcanic scoria. The different
properties of various NSLWC fill solutions were analyzed. It was found that the concrete
incorporated with coarse LECA aggregate, with a diameter of 4–8 mm showing thermal
conductivity of 0.23 W/mK, while the tensile and compressive strength at 28 days were
found as 0.5 and 5.3 MPa, respectively. The 28-day modulus of elasticity was 4.9 GPa with
capillary absorption of 0.501 × 10−3 mm/min0.5. Vijayalakshmi and Ramanagopal [59]
reviewed the utilization of LECA to produce structural LWC. 3.3. Vermiculite Concrete Vermiculite LWA is formed due to weathering action or hydrothermal changes in
biotite or phlogopite. It is basically a hydrous phyllosilicate or hydrated magnesium
aluminum silicate mineral, which shows volumetric expansion after the heating process and
thus causes exfoliation. After exfoliation, vermiculite forms elongated concertina particles,
which have specific characteristics such as incombustible, lightweight, compressible, inert
and highly absorbent. It has an appearance similar to mica and is available in various
parts of the world such as India, the USA, Australia, Russia, South Africa, Bulgaria, etc. In construction practices, these aggregates impart good thermal resistance at a cheaper
cost. Sundhakumar [62] investigated the microstructure of expanded vermiculite (EV) and
inferred that the lower thermal conductivity of EV-containing cementitious products is
caused by a large number of air voids presented in it. The SEM micrograph of vermiculite
is shown in Figure 7. REVIEW
10 of 22 Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015),
2021, Elsevier). Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015),
2021, Elsevier). Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015),
2021, Elsevier). Figure 7. SEM micrograph of vermiculite [42]. (Reprinted with permission from Abidi et al. (2015),
2021, Elsevier). Lorenzon et al. [63] investigated the properties of cement–vermiculite mortar (CVM),
which showed similar physical characteristics to that of the wood in terms of thermal per-
formance. CVM absorbed more amount of water and lost water rapidly compared to the
pinewood manufactured box. As a result, it was obtained that the exfoliated vermiculite
mortars can be used as boxes for honeybees instead of pinewood. Al-Jabri et al. [64] uti-
lized vermiculite and polystyrene beads by replacing OPC cement and developed con-
crete blocks. It was found that polystyrene incorporated LWC blocks provide lower ther-
mal conductivity as compared to vermiculite and conventional concrete. Schackow et al. Lorenzon et al. [63] investigated the properties of cement–vermiculite mortar (CVM),
which showed similar physical characteristics to that of the wood in terms of thermal
performance. CVM absorbed more amount of water and lost water rapidly compared to the
pinewood manufactured box. As a result, it was obtained that the exfoliated vermiculite
mortars can be used as boxes for honeybees instead of pinewood. Al-Jabri et al. [64] utilized
vermiculite and polystyrene beads by replacing OPC cement and developed concrete
blocks. 3 2 LECA C
t
3.2. LECA Concrete Further, different fresh and
mechanical properties of the developed concrete using LECA aggregates were compared
with NWCs. Shafigh et al. [25] compared the engineering characteristics of NWCs with
concrete containing two types of LWAs, namely, LECA concrete and oil-palm-boiler-clinker
(OPBC) concrete. Natural coarse aggregates were replaced with these LWAs, and then fresh
and hardened properties were explored. It was found that the 28-day compressive strength Sustainability 2022, 14, 331 9 of 21 OPBC concrete was about 27% higher than the LECA concrete. While the modulus of
elasticity of the OPBC and LECA concretes was 59% and 37% of that of NWC, respectively. OPBC concrete was about 27% higher than the LECA concrete. While the modulus of
elasticity of the OPBC and LECA concretes was 59% and 37% of that of NWC, respectively. y
p
y
Heiza et al. [60] analyzed the effect of varying reinforcement ratios on reinforced
two-way slabs keeping the dimensions constant. LECA was used to produce a structural
LWC with low density and high self-compacting features. LECA was suggested to be
pre-wetted since it provides higher strength. Reddy et al. [61] studied the flexural strength
of conventional concrete with partial to complete substitution of coarse aggregate by LECA. Thus, the overall use of LECA fine and coarse aggregates can reduce the demand for
natural aggregates while designing concrete structures. The LECA concrete gives low
density compared to conventional concrete and provides better insulation against heat and
sound, while the cube’s compressive strength was reduced continuously with the increase
in percent substitution of natural aggregate by LECA. 3.3. Vermiculite Concrete It was found that polystyrene incorporated LWC blocks provide lower thermal
conductivity as compared to vermiculite and conventional concrete. Schackow et al. [26] Sustainability 2022, 14, 331 10 of 21 10 of 21 developed the LWC containing vermiculite and expanded polystyrene (EPS) along with a
water-reducing super plasticizer and air-entraining agent (AEA). After developing LWC,
different mechanical and thermal properties were compared. At a lower incorporation
percentage, both AEA and LWAs impart higher compressive strength for LWC. The density
observed for EPS LWC and vermiculite LWC ranged from 1.070 g/cm3 to 1.250 g/cm3 and
1.130 g/cm3 to 1.290 g/cm3, respectively. Vermiculite LWC had less thermal conductivity. Koksal et al. [1] compared the physico-mechanical, thermal and micro structural
properties of EV and silica fume (SF) incorporated cement-based mortars. At elevated
temperatures, the strength and durability attributes were improved using SF in vermiculite
containing lightweight mortars. The thermal conductivity of developed mortars depicted
a decrement up to 0.257 W/mK, which showed an increment of 58.2% in overall thermal
performance behavior. Abidi et al. [42] utilized vermiculite, perlite and cement to produce
novel lightweight composite material for building construction, and then various thermo-
mechanical properties were studied. It was found that thermal conductivity was decreased
with an increase in porosity rate. When the matrix was reinforced with 5 to 25 by wt% of
vermiculite, the thermal conductivity of the composite decreased from 0.50 to 0.45 W/mK. Gunasekaran et al. [65] studied the properties of mortar by replacing natural sand with
vermiculite in the range of 5 to 30% by wt. of sand. It was concluded that the use of
vermiculite mortar is quite economical and provides better compressive strength. Divya et al. [27] studied the different parameters such as compressive strength, flexural
strength and split tensile strength after using vermiculite aggregates as partial substitution
with 40 to 60% by weight of fine aggregate. The optimum strength was observed to be at a
50% substitution ratio. Sairam and Sailaja [66] replaced different percentages of cement and
fine aggregate with vermiculite and FA mineral admixtures. Then, different mechanical
properties of the M35 grade of LWC were investigated, and it was found that strength
decreased with an increasing amount of vermiculite content. Mo et al. [20] developed
cement mortar by partially replacing sand with EV. 3.3. Vermiculite Concrete It was found that the density and
strength of mortar were reduced, while the water absorption rate, thermal resistance and
stability of the developed mortar were increased after incorporating EV. 3.4. Perlite Concrete The later age strength is generally improved for the products made
using perlite aggregate due to the high water absorption capacity of expanded perlite. The
morphology of the perlite aggregate is shown in Figure 8. Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015),
2021, Elsevier). Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015), 2021,
Elsevier). Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015),
2021, Elsevier). Figure 8. SEM micrographs of perlite [42]. (Reprinted with permission from Abidi et al. (2015), 2021,
Elsevier). Karakoc and Demirboga [19] studied the properties of concrete mixtures with varia-
ble expanded perlite aggregate (EPA)–fine aggregate ratios. The mixtures have 0–30%
EPA. Under dry and wet curing conditions, compressive strength at 28 days ranged from
40 to 57 and 54 to 81 MPa, respectively. At 30% EPA content, thermal conductivity was
found minimum. Sengul et al. [67] investigated the mechanical and thermal properties of
LWC containing EPA. Mixtures were prepared by partially substituting natural aggregate
by EPA, and it was observed that the unit weight of LWC in fresh state varied between
700 and 2000 kg/m3. The uniaxial compressive strength and Young’s modulus of LWC
were reduced by increasing EPA amount. Water absorption and sorptivity coefficient in-
crease at high EPA content. From a thermal performance point of view, thermal conduc-
tivity was improved by adding EPA content. Gandage et al. [68] used class C FA and per-
lite as cement and fine aggregates, respectively. Thermal conductivity was measured at
five different temperatures readings. It was concluded that as the test temperature was
increased, the thermal conductivity decreased. Comparative high 28-day strengths were
obtained at 5% perlite content. Oktay et al. [30] investigated the properties of concrete containing SF, super plasticizer
(SP) and AEAs with a fixed w/c ratio. Normal aggregates were replaced by EPA at different
volume fractions. A reduction in bulk density and thermal performance was observed. Zulkifeli and Saman [31] studied the compressive and flexural strength of mortar using
EPA. It was found that the modulus of rupture and strength was increased at a temperature
of 200 ◦C. Karatas [12] studied the thermal resistance of self-compacting mortars (SCM)
that were developed by normal and LWAs such as EPA and pumice. SF and FA were
used as mineral additives. 3.4. Perlite Concrete Siliceous volcanic rocks are the naturally occurring resources, which are also known
as perlite. Perlite is formed naturally after the hydration of obsidian and has amorphous
nature. It has the unusual characteristic of largely expanding when heated, which is the
main cause of its lower density. The specific feature that differentiates perlite from other
volcanic glasses is that when rapidly heated at a temperature ranging between 900 ◦C and
1200 ◦C, it expands about 5 to 20 times of its original volume. This makes it lightweight and
insulating. It is suitable to produce LWC using expanded perlite due to its low density. The
major applications of perlite LWA are LWC blocks, masonry mortar, plasters, ceiling tiles,
etc. Globally, the largest producer of perlite is Turkey, with total reserves of about 4.5 billion
tons. The later age strength is generally improved for the products made using perlite
aggregate due to the high water absorption capacity of expanded perlite. The morphology
of the perlite aggregate is shown in Figure 8. p
gg
g
g
Karakoc and Demirboga [19] studied the properties of concrete mixtures with variable
expanded perlite aggregate (EPA)–fine aggregate ratios. The mixtures have 0–30% EPA. Under dry and wet curing conditions, compressive strength at 28 days ranged from 40 to
57 and 54 to 81 MPa, respectively. At 30% EPA content, thermal conductivity was found
minimum. Sengul et al. [67] investigated the mechanical and thermal properties of LWC
containing EPA. Mixtures were prepared by partially substituting natural aggregate by
EPA, and it was observed that the unit weight of LWC in fresh state varied between 700
and 2000 kg/m3. The uniaxial compressive strength and Young’s modulus of LWC were
reduced by increasing EPA amount. Water absorption and sorptivity coefficient increase at
high EPA content. From a thermal performance point of view, thermal conductivity was
improved by adding EPA content. Gandage et al. [68] used class C FA and perlite as cement Sustainability 2022, 14, 331 11 of 21
C and
tweight 11 of 21
C and
tweight and fine aggregates, respectively. Thermal conductivity was measured at five different
temperatures readings. It was concluded that as the test temperature was increased, the
thermal conductivity decreased. Comparative high 28-day strengths were obtained at 5%
perlite content. y
j
pp
p
y
p
ceiling tiles, etc. Globally, the largest producer of perlite is Turkey, with total reserves of
about 4.5 billion tons. 3.5. Pumice Concrete
3.5. Pumice Concrete Pumice, also known as pumicite in its fine powder form, is categorized as a volcanic
rock that contains highly vesicular volcanic glass and generally looks light in color. Due
to its durability and hardness, pumice has been used in LWC for years. Pumice aggregates
produce lightweight fire-resistant and sound insulating LWC blocks. The pumice can be
used to manufacture low-density LWC that can be used in high-rise buildings structures
with significantly improved functional properties. It does not increase the total load of the
structure; thereby, it is a very useful material for the repair of the old monuments. Fine
pumice is used to develop mortars and provides good thermal insulating properties. Gunduz and Ugur [34] developed the LWAC with fine pumice (FPA) and coarse pumice
Pumice, also known as pumicite in its fine powder form, is categorized as a volcanic
rock that contains highly vesicular volcanic glass and generally looks light in color. Due to
its durability and hardness, pumice has been used in LWC for years. Pumice aggregates
produce lightweight fire-resistant and sound insulating LWC blocks. The pumice can be
used to manufacture low-density LWC that can be used in high-rise buildings structures
with significantly improved functional properties. It does not increase the total load of
the structure; thereby, it is a very useful material for the repair of the old monuments. Fine pumice is used to develop mortars and provides good thermal insulating properties. Gunduz and Ugur [34] developed the LWAC with fine pumice (FPA) and coarse pumice
aggregates (CPA). The study showed that the pumice aggregate concrete possesses lower
thermal conductivity. It was also concluded by the authors that the major influencing
parameters for thermal performance were the cement/fines ratio and dry unit weight. p
p
y
g
Gunduz [73] studied different structural and functional properties of pumice ag-
gregate lightweight concrete (PALWC). Lower aggregate/cement ratio showed higher
compressive strength, modulus of elasticity and density, while on the other hand, higher ag-
gregate/cement ratio showed lower water absorption, shrinkage and thermal performance. Parhizkar et al. [13] investigated the properties of volcanic pumice. The results showed
that the volcanic pumice lightweight concretes fulfilled the requirements of lightweight
structural concrete. Tasdemir et al. [74] compared the properties of seven different LWC,
developed by using pumice aggregates, fine natural aggregate and coarse limestone ag-
gregate. 3.5. Pumice Concrete
3.5. Pumice Concrete The compressive strength of the concretes was compared, which showed that the
strengths of the expanded polystyrene beads were lower than the pumice or EPA. Kilincar-
slan et al. [75] used the pumice aggregates of four different kinds. The physico-mechanical
properties and thermal performance of the developed concretes were also investigated. The result showed that the Nevsehir kind of pumice aggregate was the most suitable type
of LWAs to be used in foam concrete. 3.4. Perlite Concrete Bulk density, porosity and water absorption results were
determined for the hardened SCM. Results of the experiments showed that the use of LWAs
increased the total water absorption and porosity of mortars. The compressive strength
of specimens (which were exposed to 300 ◦C) was increased due to the incorporation of
EPA up to 10%. Kotwica et al. [69] studied the methodology for utilizing waste EPA as
a valuable, high-performance pozzolanic supplementary building material. The results
showed that the addition of ground waste EPA leads to gains in strength up to 50%. Polat
et al. [70] developed lightweight mortar and concrete using EPA. It was concluded through
experimentation that as compared to controlled samples, strength, ultrasonic pulse velocity
(UPV) and shrinkage were reduced with increasing EPA content, while the degree of
hydration was increased with increment in EPA content. Wan et al. [71] used three LWAs,
i.e., EPA, scoria and polystyrene, to produce lightweight self-compacting concrete (LWSCC),
and then different fresh and hardened properties were determined. It was reported that all
LWAs imparted a negative effect on the workability and compressive strength of LWSCC. Demir and Baspinar [72] confirmed the inert behavior of expanded perlite aggregates for
all test conditions. No crystalline phase was formed around the perlite grains as shown in
Figure 9. 12 of 21
ehavior
formed 12 of 21
ehavior
formed Sustainability 2022, 14, 331 Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted
with permission from Demir and Baspinar (2008), 2021, Elsevier). Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted with
permission from Demir and Baspinar (2008), 2021, Elsevier). Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted
with permission from Demir and Baspinar (2008), 2021, Elsevier). Figure 9. The pore structure of the perlite in the fly ash–lime–gypsum mixture [72]. (Reprinted with
permission from Demir and Baspinar (2008), 2021, Elsevier). 3.6. Sintered Fly Ash Concrete Commercially, sintered FA aggregates are known as lytag. It is manufactured by
pyro-processing of fly ash, which is a waste product generated from thermal power plants. The main process to develop sintered FA is sintering and pelletization at a temperature
range from 1150 to 1350 ◦C. The sintered FA aggregates are found in brown color with a
black internal core (Figure 10). The pore sizes range between 10 and 200 µm, as depicted in
Figure 11. 13 of 21
wn color
µm, as
n color
µm, as 13 of 21
wn color
µm, as
n color
µm, as Sustainability 2022, 14, 331 Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per-
mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with permission
from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per-
mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per-
mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with permission
from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 10. Sintered FA aggregates (left) and their cross-section (right) [76]. (Reprinted with per-
mission from Nadesan and Dinakar (2017), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission
from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission
from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from
Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission
from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission
from Swamy and Lambert (1981), 2021, Elsevier). Figure 11. Microstructure of sintered fly ash aggregate (×320) [77]. (Reprinted with permission from
Swamy and Lambert (1981), 2021, Elsevier). Kockal and Ozturan [78] developed LWC using various kinds of sintered FA ag-
gregates. The results showed that low specific gravity of cold-bonded and sintered FA
aggregate was helpful for the production of LWC with densities within the range of
1860–1943 kg/m3. 3.6. Sintered Fly Ash Concrete Twenty-eight days uniaxial compressive strength and elastic modulus
ranged from 42.3 to 55.8 MPa and 22.4 and 28.6 MPa, respectively. Kockal and Oztu-
ran [15] investigated the effect of aggregate properties on the strength and durability of
lightweight FA aggregate concrete. In the development of LWAC, three different types
of LWA, i.e., sintered lightweight ash aggregates, cold bonded lightweight FA aggregate
and normal-weight aggregate, were used. Then, the effects of all three aggregates on
physico-mechanical and durability properties of developed concrete were determined. Sin-
tered FA aggregate concretes had more strength and elasticity modulus than cold-bonded
ash aggregate concretes. Nadesan and Dinakar [76] studied the utilization of sintered FA
aggregate to produce structural LWC. The results showed that the sintered FA aggregate
concrete satisfies the requirement of structural LWC. 4. The Comparisons among Different LWAs/LWACs on the Basis of
Various Parameters Researchers reported different properties of LWC, which are shown in Table 3. Majorly,
the investigated properties were uniaxial compressive strength, split tensile strength, modu-
lus of rupture or flexural strength, thermal conductivity, elastic modulus, water absorption,
etc. For comparison purposes of compressive strength, splitting tensile strength, density
and thermal conductivity, graphs containing six major LWAs are depicted in Figures 12–15. The control concrete (CC) showed the maximum compressive strength among all LWC, i.e.,
38 MPa, as presented in Figure 12. As the content of LWA in concrete increased, the compres-
sive strength decreased. Here, 50% pumice used in concrete gave maximum compressive
strength up to 36 MPa, while vermiculite LWC showed minimum compressive strength. The tensile strength of conventional concrete was varied in the range from 4 to 4.7 MPa, as
depicted in Figure 13. Adding LWA in the mix proportion showed a gradual decrement Sustainability 2022, 14, 331 14 of 21 14 of 21 in tensile strength. Forty percent FA sintered aggregate replacement showed maximum
tensile strength, while 40% OPS replacement provided minimum strength. Figure 14 shows
that perlite used as an aggregate showed a maximum density around 2300 kg/m3, while
pumice concrete showed a minimum density of 1300 kg/m3. The thermal conductivity of
LWC was lower than the NWCs. As shown in Figure 15, LECA concrete showed minimum
thermal conductivity when used as a whole in place of coarse aggregate while control
concrete showed maximum k value, i.e., 2.3 W/mK. concrete showed maximum k value, i.e., 2.3 W/mK. Table 3. Fresh, hardened and durability properties of LWAs, as per the literature. Aggregate
Type
References
Compressive
Strength
Splitting
Tensile
Strength
Flexural
Strength
Thermal
Conductivity
Modulus
of
Elasticity
Water
Absorption
OPS
Mannan and Ganapathy [22]
✓
✓
✓
✓
Sobuz et al. [23]
✓
✓
✓
✓
Mo et al. [51]
✓
✓
✓
Mo et al. [52]
✓
✓
LECA
Maghsoudi et al. [57]
✓
✓
Zohrabi et al. [58]
✓
✓
Kumar and Prakash [79]
✓
✓
Heiza et al. [60]
✓
✓
Reddy et al. [61]
✓
Al-Jabri et al. [64]
✓
✓
Vermiculite
Schackow et al. [26]
✓
Koksal et al. [1]
✓
✓
✓
✓
Divya et al. [27]
✓
✓
✓
Mo et al. [20]
✓
✓
Arun et al. [28]
✓
✓
✓
Karakoc and Demirboga [19]
✓
✓
Perlite
Sengul et al. [67]
✓
✓
✓
✓
Gandage et al. 4. The Comparisons among Different LWAs/LWACs on the Basis of
Various Parameters [68]
✓
✓
✓
Polat et al. [70]
✓
Wan et al. [71]
✓
✓
Gunduz and Ugur [34]
✓
✓
✓
✓
✓
Pumice
Gunduz [73]
✓
✓
✓
✓
Parhizkar et al. [13]
✓
✓
✓
Tasdemir et al. [74]
✓
✓
✓
Kilincarslan et al. [75]
✓
✓
Sintered fly
ash
Kockal and Ozturan [78]
✓
✓
✓
Kockal and Ozturan [15]
✓
✓
✓
✓
Nadesan and Dinakar [76]
✓
✓
✓
Sustainability 2022, 14, x FOR PEER REVIEW
15 of 22
Karakoc and Demirboga [19]
Perlite
Sengul et al. [67]
Gandage et al. [68]
Polat et al. [70]
Wan et al. [71]
Gunduz and Ugur [34]
Pumice
Gunduz [73]
Parhizkar et al. [13]
Tasdemir et al. [74]
Kilincarslan et al. [75]
Sintered fly
ash
Kockal and Ozturan [78]
Kockal and Ozturan [15]
Nadesan and Dinakar [76]
Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. Table 3. Fresh, hardened and durability properties of LWAs, as per the literature. Aggregate
Type
References
Compressive
Strength
Splitting
Tensile
Strength
Flexural
Strength
Thermal
Conductivity
Modulus
of
Elasticity
Water
Absorption
OPS
Mannan and Ganapathy [22]
✓
✓
✓
✓
Sobuz et al. [23]
✓
✓
✓
✓
Mo et al. [51]
✓
✓
✓
Mo et al. [52]
✓
✓
LECA
Maghsoudi et al. [57]
✓
✓
Zohrabi et al. [58]
✓
✓
Kumar and Prakash [79]
✓
✓
Heiza et al. [60]
✓
✓
Reddy et al. [61]
✓
Al-Jabri et al. [64]
✓
✓
Vermiculite
Schackow et al. [26]
✓
Koksal et al. [1]
✓
✓
✓
✓
Divya et al. [27]
✓
✓
✓
Mo et al. [20]
✓
✓
Arun et al. [28]
✓
✓
✓
Karakoc and Demirboga [19]
✓
✓
Perlite
Sengul et al. [67]
✓
✓
✓
✓
Gandage et al. [68]
✓
✓
✓
Polat et al. [70]
✓
Wan et al. [71]
✓
✓
Gunduz and Ugur [34]
✓
✓
✓
✓
✓
Pumice
Gunduz [73]
✓
✓
✓
✓
Parhizkar et al. [13]
✓
✓
✓
Tasdemir et al. [74]
✓
✓
✓
Kilincarslan et al. 4. The Comparisons among Different LWAs/LWACs on the Basis of
Various Parameters [75]
✓
✓
Sintered fly
ash
Kockal and Ozturan [78]
✓
✓
✓
Kockal and Ozturan [15]
✓
✓
✓
✓
Nadesan and Dinakar [76]
✓
✓
✓
Sustainability 2022, 14, x FOR PEER REVIEW
15 of 22
Karakoc and Demirboga [19]
Perlite
Sengul et al. [67]
Gandage et al. [68]
Polat et al. [70]
Wan et al. [71]
Gunduz and Ugur [34]
Pumice
Gunduz [73]
Parhizkar et al. [13]
Tasdemir et al. [74]
Kilincarslan et al. [75]
Sintered fly
ash
Kockal and Ozturan [78]
Kockal and Ozturan [15]
N d
d Di
k
[76]
Table 3. Fresh, hardened and durability properties of LWAs, as per the literature. Figure 12 Compressive strength of different LWACs Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. Figure 12. Compressive strength of different LWACs. 15 of 21 Sustainability 2022, 14, 331 Figure 12. Compressive strength of different LWACs. Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. ER REVIEW
16 of
Figure 14. Density of different LWACs. Figure 15. Thermal conductivity of different LWACs. h
h
l
d
h
d
d
Figure 14. Density of different LWACs. ER REVIEW
16 of
Figure 14. Density of different LWACs. Figure 15. Thermal conductivity of different LWACs. Figure 15. Thermal conductivity of different LWACs. R REVIEW
16 of Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. R REVIEW
16 of
Figure 14. Density of different LWACs. Figure 14 Density of different LWACs
R REVIEW
16 of Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. E IE
R REVIEW Figure 13. Tensile strength of different LWACs. Figure 13. Tensile strength of different LWACs. R REVIEW g
g
Figure 14 Density of different LWACs Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. Figure 14. Density of different LWACs. 5. Research and Development at CSIR-CBRI A systematic R&D work was conducted at CSIR-Central Building Research Institute,
Roorkee (Haridwar, India), on the development of LWACs using LECA and Class F FA
and dry marble slurry powder (MSP). In this study, optimization of LECA aggregates,
cement, w/c ratio was performed. LECA was used as a coarse and fine aggregate in three
different sizes: 0–2 mm, 2–8 mm and 8–15 mm. The FA was obtained from NTPC-Dadri
(UP), which was classified as class F based on ASTM: C618. The specific gravity of FA
was 2.24 and fineness 325 m2/kg. The marble slurry used in this study was obtained
from Udaipur, Rajasthan (India). The mean particle size of MS was 9.54 µm. According to
Blaine’s apparatus of specific surface area, the specific surface area of MS was 350 m2/kg. MS has a higher surface area than that of OPC and FA, which can fill capillary pores as well
as gel pores. In order to increase the workability of LWC, a polycarboxylate ether-based
superplasticizer (SP) is used. Ordinary tap water is used in all types of concrete mixes. REVIEW
17 of 22
apparatus of specific surface area, the specific surface area of MS was 350 m2/kg. MS has
a higher surface area than that of OPC and FA, which can fill capillary pores as well as gel
pores. In order to increase the workability of LWC, a polycarboxylate ether-based super-
plasticizer (SP) is used. Ordinary tap water is used in all types of concrete mixes. ff
h
h
l
f h d
l
d
l Different physico-mechanical properties of the developed concrete specimens along
with thermal conductivity were determined. According to ACI 213R-87, the minimum
compressive strength for structural LWC should be 17.2 MPa. Mix contains OPC: 400 kg/m3;
sand: 550 kg/m3; 15% MS + 15% FA by replacement of cement; LECA: 325 kg/m3; SP (%):
0.78 with w/c ratio of 0.40 satisfied the requirement of LWC, i.e., 20.4 MPa. According to
ASTM C330, a minimum splitting tensile strength of 2.0 MPa is a requirement for structural
grade lightweight aggregate concrete. The same mix shows the tensile strength of 2.8 MPa,
which satisfies the strength criteria of LWAC. The optimized mix showed the maximum
flexural strength out of all concrete mixes, i.e., 3.1 MPa. Thermal conductivity (k value)
of concrete was measured using the guarded hot plate method followed by IS: 3346–1980
(Figure 16). 4. The Comparisons among Different LWAs/LWACs on the Basis of
Various Parameters Fi
15 Th
l
d
i i
f diff
LWAC
gu e
e s y o
e e
Cs
Figure 14. Density of different LWACs. igu e 5. e
a co ducti ity o di e e t
ACs
Figure 14 shows that perlite used as an agg
Figure 15. Thermal conductivity of different LWACs. Figure 15. Thermal conductivity of different LWACs. Sustainability 2022, 14, 331 16 of 21 Figure 14 shows that perlite used as an aggregate showed a maximum density around
2300 kg/m3, while pumice concrete showed a minimum density of 1300 kg/m3. The
thermal conductivity of LWC was lower than the NWCs. As shown in Figure 15, LECA
concrete showed minimum thermal conductivity when used as a whole in place of coarse
aggregate while control concrete showed maximum k value, i.e., 2.3 W/mK. 5. Research and Development at CSIR-CBRI Specimens of dimensions 300 mm × 300 mm × 50 mm were cast for testing. The results revealed that the thermal conductivity of the LWC using 15% MS + 15% FA
along with LECA shows a lower k value, i.e., 0.21 W/mK. Different physico-mechanical properties of the developed concrete specimens along
with thermal conductivity were determined. According to ACI 213R-87, the minimum
compressive strength for structural LWC should be 17.2 MPa. Mix contains OPC: 400
kg/m3; sand: 550 kg/m3; 15% MS + 15% FA by replacement of cement; LECA: 325 kg/m3;
SP (%): 0.78 with w/c ratio of 0.40 satisfied the requirement of LWC, i.e., 20.4 MPa. Accord-
ing to ASTM C330, a minimum splitting tensile strength of 2.0 MPa is a requirement for
structural grade lightweight aggregate concrete. The same mix shows the tensile strength
of 2.8 MPa, which satisfies the strength criteria of LWAC. The optimized mix showed the
maximum flexural strength out of all concrete mixes, i.e., 3.1 MPa. Thermal conductivity
(k value) of concrete was measured using the guarded hot plate method followed by IS:
3346–1980 (Figure 16). Specimens of dimensions 300 mm × 300 mm × 50 mm were cast for
testing. The results revealed that the thermal conductivity of the LWC using 15% MS +
15% FA along with LECA shows a lower k value, i.e., 0.21 W/mK. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. Figure 16. Thermal conductivity test set up for LWAC containing fly ash and marble waste. The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC
mix showed denser structure as compared to control concrete with LECA only. The inclu-
sion of MS into concrete improved the microstructure due to the filler effect and accelera-
tion of hydration. Less crystalline phase was observed in the interfacial transition zone
(ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement
The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC
mix showed denser structure as compared to control concrete with LECA only. 5. Research and Development at CSIR-CBRI The inclu-
sion of MS into concrete improved the microstructure due to the filler effect and acceleration
of hydration. Less crystalline phase was observed in the interfacial transition zone (ITZ)
of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement of The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC
mix showed denser structure as compared to control concrete with LECA only. The inclu-
sion of MS into concrete improved the microstructure due to the filler effect and accelera-
tion of hydration. Less crystalline phase was observed in the interfacial transition zone
(ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement
f
i
d ITZ l
l
d f
h i
l
i
b
i
f li h
The SEM results revealed that the incorporation of 15% FA and 15% MS in the LWAC
mix showed denser structure as compared to control concrete with LECA only. The inclu-
sion of MS into concrete improved the microstructure due to the filler effect and acceleration
of hydration. Less crystalline phase was observed in the interfacial transition zone (ITZ)
of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement of Sustainability 2022, 14, 331 17 of 21
e inclu-
celera 17 of 21
e inclu-
celera microstructure and ITZ also resulted from the internal curing by pre-wetting of lightweight
aggregate (Figure 17a–d). (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement
of microstructure and ITZ also resulted from the internal curing by pre-wetting of light-
weight aggregate (Figure 17a–d). microstructure and ITZ also resulted from the internal curing by pre-wetting of lightweight
aggregate (Figure 17a–d). (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement
of microstructure and ITZ also resulted from the internal curing by pre-wetting of light-
weight aggregate (Figure 17a–d). microstructure and ITZ also resulted from the internal curing by pre-wetting of lightweight
aggregate (Figure 17a–d). (ITZ) of the optimized mix (containing 15% MS + 15% FA) at 28 days. The improvement
of microstructure and ITZ also resulted from the internal curing by pre-wetting of light-
weight aggregate (Figure 17a–d). (a)
(b)
REVIEW
18 of 22
(c)
(d)
Figure 17. SEM Results of laboratory prepared LWAC specimens. 6. Conclusions
6. Conclusions The current paper reviews the prior studies conducted on the properties and uses of
LWAs to develop sustainable concrete as green building materials. The utilization of sin-
tered FA, vermiculite, pumice, OPS, LECA and perlite as LWA to produce LWC in build-
ing construction was reviewed through the recent and past literature. The physical, me-
chanical, durability, functional and structural behaviors of LWACs were discussed. Now-
adays, LWACs have become an important low-cost green building material that imparts
high thermal resistance properties with a reduced dead load of the superstructure. As the
LWAs have different types of physical and mechanical characteristics, on the basis of end-
user applications, available LWACs are used for different construction purposes. The current paper reviews the prior studies conducted on the properties and uses
of LWAs to develop sustainable concrete as green building materials. The utilization of
sintered FA, vermiculite, pumice, OPS, LECA and perlite as LWA to produce LWC in
building construction was reviewed through the recent and past literature. The physical,
mechanical, durability, functional and structural behaviors of LWACs were discussed. Nowadays, LWACs have become an important low-cost green building material that
imparts high thermal resistance properties with a reduced dead load of the superstructure. As the LWAs have different types of physical and mechanical characteristics, on the basis
of end-user applications, available LWACs are used for different construction purposes. 5. Research and Development at CSIR-CBRI (a) Control concrete; (b) FA
30%; (c) MS 30%; (d) FA15% + MS15%. Figure 17. SEM Results of laboratory prepared LWAC specimens. (a) Control concrete; (b) FA 30%;
(c) MS 30%; (d) FA15% + MS15%. (a)
REVIEW (b)
18 of 22 (b) (a) (c) (d) (c) (d) Figure 17. SEM Results of laboratory prepared LWAC specimens. (a) Control concrete; (b) FA
30%; (c) MS 30%; (d) FA15% + MS15%. Figure 17. SEM Results of laboratory prepared LWAC specimens. (a) Control concrete; (b) FA 30%;
(c) MS 30%; (d) FA15% + MS15%. Thus, the usage of LECA with 15% FA + 15% MS as a cement replacement in LWC
shows better compressive, tensile, flexural strength and lower thermal conductivity, and
hence it can be proposed for structural purposes. Further research is in process in regard
to different durability aspects along with different functional properties such as fire re-
sistance and sound insulation properties. Thus, the usage of LECA with 15% FA + 15% MS as a cement replacement in LWC
shows better compressive, tensile, flexural strength and lower thermal conductivity, and
hence it can be proposed for structural purposes. Further research is in process in regard to
different durability aspects along with different functional properties such as fire resistance
and sound insulation properties. 7 Recommendations for the Further Research
7. Recommendations for the Further Research 7. Recommendations for the Further Research
On the basis of the existing literature a few recommendations are proposed below:
On the basis of the existing literature, a few recommendations are proposed below: On the basis of the existing literature, a few recommendations are proposed below:
1. Studies of hardened, durable and functional properties for different LWACs in vari-
ou I dia
li
ati
o ditio
1. Studies of hardened, durable and functional properties for different LWACs in various
Indian climatic conditions; ous Indian climatic conditions;
2. Comprehensive studies of different durability attributes of high-performance LWAC
t i i
diff
t SCM
fib
d
l
2. Comprehensive studies of different durability attributes of high-performance LWAC
containing different SCMs, fibers and polymers; containing different SCMs, fibers and polymers;
3. Statistical modeling for durability properties based on theoretical and empirical stud-
i
l
i h h i
lid
i
i
h fi ld
3. Statistical modeling for durability properties based on theoretical and empirical
studies along with their validation in the fields; ies along with their validation in the fields;
4. Field studied for fire resistance of LWA concrete incorporated with SCMs, fibers, fire-
resistant admixtures etc
4. Field studied for fire resistance of LWA concrete incorporated with SCMs, fibers,
fire-resistant admixtures, etc.; Sustainability 2022, 14, 331 18 of 21 18 of 21 5. Study investigating the toughness and fracture energy of LWACs (LECA, OPS, sintered
fly ash, pumice, perlite, vermiculite) is needed as these properties are necessary while
modeling their mechanical behavior. Thus, it is envisaged that LWACs, because of their techno-economic and environmental
advantages, are supposed to capture their major share in the building industry in the 21st
century. For this optimism, the most important additional motivating factor is the ever-
increasing importance of the need for sustainable development. This is the appropriate
time to utilize industrial byproducts such as fly ash, GGBS, silica fume, etc., by thinking
more futuristically and investing in R&D for the sake of our world, its environment and its
construction industry. Author Contributions: Conceptualization, R.K.; methodology, R.K.; validation, R.K., R.L.; formal
analysis, R.K., R.L.; investigation, R.K.; data curation, R.K.; writing—original draft preparation, R.K.,
A.S.; writing—review and editing, R.K., A.S.; supervision, R.K. All authors have read and agreed to
the published version of the manuscript. 7 Recommendations for the Further Research
7. Recommendations for the Further Research Funding: The authors are grateful to ‘Ministry of Environment, Forest and Climate Change, New
Delhi, Government of India’ (File Number: 19–44/2018/RE; Project No.: GAP0120) and ‘Indo-US
Science and Technology Forum (IUSSTF), New Delhi, India’ (Grant No. GAP: 806) for the sustained
financial support to the project. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not Applicable. Data Availability Statement: Not Applicable. Data Availability Statement: Not Applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Grabois, T.M.; Cordeiro, G.C.; Filho, R.D.T. Fresh and hardened-state properties of self-compacting lightweight concrete reinforced
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Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats
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Brazilian Journal of Pharmaceutical Sciences
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Influence of isoniazid on T lymphocytes,
cytokines, and macrophages in rats Bianchuan Cao1†, Qiuju Li1†, Zhe Huang2, Xiufang Huang1,
Yihong Zhu1, Ke Xiao1, Fuli Huang1 and Li Zhong iD 1* 1Department of Infectious Disease, The Affiliated Hospital of Southwest Medical
University, Luzhou, Sichuan, China, 2Department of Paediatrics, The Affiliated
Hospital of Southwest Medical University, Luzhou, Sichuan, China T lymphocytes, cytokines, and macrophages play important roles in the clearance of Mycobacterium
tuberculosis (Mtb) by the immune system. This study aimed to investigate the effects of isoniazid
on the functions of both innate and adaptive immune cells. Healthy rats were randomly divided into
experimental and control groups. Each group was randomly divided into three subgroups and named
according to the duration of drug feeding, 1, 3, and 3 months followed by drug withdrawal for 1
month. The experimental groups were fed with isoniazid (12 mg/mL) and the control groups with
normal saline. The percentage of CD4+ and CD8+T lymphocytes, level of interleukin (IL)-12 and
interferon (IFN)-γ, and function of macrophages were determined at these three time points. Isoniazid
significantly increased the percentage of CD4+T lymphocytes and the CD4+/CD8+T lymphocyte cell
ratio (P < 0.05). It transiently (<1 month) enhanced the functions of rat macrophages significantly (P
< 0.05). In summary, isoniazid could increase the percentage of CD4+T lymphocytes, CD4+/CD8+T
lymphocyte cell ratio, and enhance macrophage function in healthy rats. Keywords: Cytokines. Immune. Isoniazid. Macrophages. T lymphocytes. *Correspondence: L. Zhong, Department of Infectious Disease, The
Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street,
Luzhou, Sichuan, 646000, China. E-mail: zhongli620@163.com. †These
authors contributed equally to this work. *Correspondence: L. Zhong, Department of Infectious Disease, The
Affiliated Hospital of Southwest Medical University, No. 25 Taiping Street,
Luzhou, Sichuan, 646000, China. E-mail: zhongli620@163.com. †These
authors contributed equally to this work. Article Brazilian Journal of
Pharmaceutical Sciences Brazilian Journal of
Pharmaceutical Sciences http://dx.doi.org/10.1590/s2175-97902019000418547 INTRODUCTION incidence of drug-resistant TB, the severity of TB cases
has increased (Das, Satapathy, Murmu, 2016; Ebonyi et
al., 2016; Lima et al., 2017). Therefore, TB has become
a serious public health problem in China. Tuberculosis (TB) is an infectious disease caused
by Mycobacterium tuberculosis (Mtb) bacteria which
may damage multiple organ systems and poses a
serious harm to human health. According to the World
Health Organization’s report on the global tuberculosis
epidemic, about one-third of the world’s population was
infected with Mtb in 2015, 10.4 million new TB patients
were diagnosed, and more than 1.4 million patients
died of the disease (Sisay et al., 2016). China has a
high incidence of TB, ranking second in the world. At
present, due to the increase in human immunodeficiency
virus/acquired immunodeficiency syndrome, the use
of immunosuppressive and cytotoxic drugs, and the T lymphocytes, cytokines, and macrophages play
important roles in the clearance of Mtb-related antigens. Insufficient number or dysfunction of T lymphocytes
and macrophages, and the lack of related cytokines may
lead to decreased immunity to Mtb. Therefore, immune
status is closely related to the occurrence, development,
and therapeutic response to TB (Grover et al., 2014;
Mosquera-Restrepo et al., 2017). In recent years, it has
been found that some antibacterial drugs not only kill
pathogens but also regulate immune function during
treatment (Hamilton-Miller, 2001; Schultz, Speelman,
van der Poll, 2001; Tauber, Nau, 2008). Some b-lactam
antibiotics, such as cefodizime, can improve immune
function (Wang et al., 2015). Although anti-tuberculosis
drugs have been used for many years, studies on Page 1/9 Page 1/9 Braz. J. Pharm. Sci. 2020;56: e18547 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong their immune regulation are lacking. Therefore, it is
important to investigate the effects of anti-tuberculosis
drugs on immune function, especially those related to
the clearance of Mtb. powder was dissolved in saline at a concentration of
12 mg/mL (Sichuan Meida Kangjiale Pharmaceutical
Co. Ltd., Chengdu, China). Rats were administered
isoniazid solution (1 mL/100 g body weight, once every
other day) intragastrically. Isoniazid is one of the most important first-line
drugs for treating TB. It has strong antibacterial
effects; thus, it is used throughout the period of
reinforcement and consolidation. The side effects
of isoniazid, such as liver damage, may be related
to the high level of immune response (Usui et al.,
2017; Vinnard et al., 2017). Grouping A total of 60 rats were randomly divided into
experimental and control groups (30 rats in each group). Each group was randomly divided into three subgroups
(10 rats in each subgroup) and named according to the
time points of drug feeding 1, 3, and 3 months, followed
by drug withdrawal for 1 month. MATERIAL AND METHODS All the experimental animals and procedures were
approved by the animal experiment ethics committee
of the Southwest Medical University, China (approval
number: 201703192). Sample collection The rats were anesthetized with 3% pentobarbital
sodium solution (30 mg/kg). Next, 2 mL blood samples
were obtained from the hearts. Anticoagulated whole
blood (300 μL) was used to determine the percentage
of T lymphocytes. The serum of the remaining blood
was used to detect the levels of cytokines after letting it
stand and centrifugation. Detection of T lymphocyte
percentage by flow cytometry The anticoagulated whole blood (100 µL) was placed
in the test tube for flow cytometry. The samples were
mixed with 20 μL of Allophycocyanin (APC) Mouse
anti-Rat CD3 (BD, NJ, USA), 20 μL of Fluorescein
isothiocyanate (FITC) Mouse anti-Rat CD8a (BD),
and 20 μL of Phycoerythrin (PE) Mouse anti-Rat CD4
(BD). The samples (100 µL of anticoagulated whole
blood) in the isotype control group were mixed with
isotype control. All samples were analyzed to detect the
percentage and ratio of CD4+ and CD8+T lymphocytes
under a BD fluorescence-activated cell sorter (FACS)
Verse II flow cytometer (BD). INTRODUCTION At present, it is not clear
whether isoniazid has any effect on immune functions
related to the clearance of Mtb. Therefore, we explored
the effects of isoniazid on the immune functions in
healthy rats, and found that isoniazid can serve as a
potential immune booster. Control group: The rats were administered normal
saline (1 mL/100 g body weight, once every other day)
intragastrically. Feeding conditions were the same as
those in the experimental group. Detection of the levels of interleukin-12
and interferon-γ by enzyme-linked
immunosorbent assay The levels of serum interleukin (IL)-12 and
interferon (IFN)-γ were detected using a microplate
reader (Thermo Fisher Scientific Inc., MA, USA), IL-
12 enzyme-linked immunosorbent assay (ELISA)
kit (Invitrogen, CA, USA), and IFN-γ ELISA kit
(eBioscience, CA, USA). All the reagent samples and
standards were prepared as instructed. The plate was
washed, and the standard and sample were added,
followed by antibody. Then the stop solution was added, Animals Two-month-old male Sprague-Dawley rats of clean
grade were purchased from Chongqing Tengxin Bio-
Technology Co., Ltd. [Chongqing, China; production
license: SCXK (Yu) 2012-0008]. All the rats were fed
under the same standard conditions with a temperature
of 23 °C ± 3 °C and relative humidity of 50% ± 10%. RESULTS AND DISCUSSION Most people infected with Mtb develop a latent
TB infection without any clinical symptoms that is not
cleared by the immune system. Only a small number of
patients develop an active TB infection. When various
factors cause a decrease in the body’s immune function,
the latent Mtb can be activated, allowing it to develop
into active TB (Petruccioli et al., 2016; Riou et al.,
2016). Therefore, the inhibition and clearance of Mtb are
closely related to immune function. This study selected
normal healthy rats as subjects to avoid the effects of
other factors on the immune system. Detection of acid phosphatase activity in rat
peritoneal macrophages by Gomori assay Rats were injected intraperitoneally with 0.7 mL of
6% sterile starch broth, and 0.1-0.2 mL of abdominal
fluid was extracted after 3 days. The peritoneal fluid was
dripped on a precooled slide and fixed with 10% neutral
formalin. After adding acid phosphatase solution and
1% ammonium sulfide successively, the cells were
stained with Giemsa stain. The slides were examined
under ordinary optical microscope to observe brown
particles or plaques in macrophages (Anhe, Lima-
Oliveira, Azeredo-Oliveira, 2007). Drug delivery method Experimental group: Isoniazid tablets (Chengdu
Jinhua Pharmaceutical Co. Ltd., China) were ground into
a powder, and the equivalent dose ratio was calculated
according to human and rat body surface area (rats
received six times the adult human dosage). Isoniazid Page 2/9 Page 2/9 Braz. J. Pharm. Sci. 2020;56: e18547 Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats and the plate was read. (The detailed instructions on the
relevant ELISA kit was followed). using two independent-sample t test, and t’ test was
used to assess the heterogeneity of variance. The
results were expressed as mean ± standard deviation
(x ± s), and a P value < 0.05 was considered statistically
significant. Acid phosphatase test: At the same time
point, the experimental and control groups were
compared using the rank-sum test. The results were
expressed as mean rank, and a P value < 0.05 was
considered statistically significant. Test of carbon clearance capacity
and phagocytic capacity Diluted ink (0.1 mL/20 g body weight) was injected
into the tail vein of the rats. Blood samples (30 μL)
were obtained from the hearts after 5 (t1) and 15 (t2)
min. The optical density (OD) values of the samples
obtained at t1 (OD1) and t2 (OD2) were measured at
650 nm wavelength. The rats were sacrificed by cervical
dislocation, and the liver and spleen were weighed. The
carbon clearance index K was calculated according
to the formula K = (1 g ODl - l g OD2)/(t2 - t1). The
phagocytic index a was calculated according to the
formula a = body weight/(liver weight + spleen weight)
× K1/3 (Singh, Yadav, Noolvi, 2012). Comparison of CD4+T, CD8+T lymphocytes
and CD4+/CD8+T lymphocyte cell ratio After feeding drug for 1 and 3 months, the percentage
of CD4+T lymphocytes and the CD4+/CD8+T lymphocyte
cell ratio was higher in the experimental group than in
the control group, and the differences were statistically
significant (P < 0.05). Moreover, after feeding drug for
3 months followed by drug withdrawal for 1 month,
no statistically significant difference was found in the
CD4+T lymphocytes or CD4+/CD8+T lymphocyte cell
ratio between the experimental and control groups (P
> 0.05; Table I). No statistically significant difference
was found in the CD8+T lymphocytes between the
experimental and control groups at any time point
(Figure 1, Table I). Statistical analysis The experimental data were analyzed using
the SPSS 19.0 software (SPSS Inc., IL, USA). T
lymphocytes, cytokines test, carbon clearance capacity
and phagocytic capacity test: At the same time point,
the experimental and control groups were compared Page 3/9 Braz. J. Pharm. Sci. 2020;56: e18547 Page 3/9 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong FIGURE 1 - CD4+T lymphocytes in (A) experimental group after drug feeding for 1 month, (B) control group after drug feedi
or 1 month, (C) experimental group after drug feeding for 3 months, (D) control group after drug feeding for 3 months, (
xperimental group after drug feeding for 3 months followed by drug withdrawal for 1 month, (F) control group after dr
eeding for 3 months followed by drug withdrawal for 1 month. CD8+T lymphocytes in (G) experimental group after dr
eeding for 1 month, (H) control group after drug feeding for 1 month, (I) experimental group after drug feeding for 3 month
J) control group after drug feeding for 3 months, (K) experimental group after drug feeding for 3 months followed by dr FIGURE 1 - CD4+T lymphocytes in (A) experimental group after drug feeding for 1 month, (B) control group after drug feeding
for 1 month, (C) experimental group after drug feeding for 3 months, (D) control group after drug feeding for 3 months, (E)
experimental group after drug feeding for 3 months followed by drug withdrawal for 1 month, (F) control group after drug
feeding for 3 months followed by drug withdrawal for 1 month. CD8+T lymphocytes in (G) experimental group after drug
feeding for 1 month, (H) control group after drug feeding for 1 month, (I) experimental group after drug feeding for 3 months,
(J) control group after drug feeding for 3 months, (K) experimental group after drug feeding for 3 months followed by drug
withdrawal for 1 month, (L) control group after drug feeding for 3 months followed by drug withdrawal for 1 month. Page 4/9 Page 4/9 Braz. J. Pharm. Sci. Statistical analysis 2020;56: e18547 Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats TABLE I - Comparison of CD4+, CD8+T lymphocytes and CD4+/CD8+ ratio in the experimental and control groups at the three
time points
Time point
Experimental group(n=10)
Control group (n=10)
P-value
CD4+T lymphocytes (%)
Feeding drug for 1 month
48.332 ± 5.486
37.625 ± 4.192
0.000 a
Feeding drug for 3 months
42.169 ± 6.041
35.421 ± 7.882
0.045 a
Feeding drug for 3 months followed
by drug withdrawal for 1 month
36.656 ± 5.639
32.642 ± 5.325
0.119 a
CD8+T lymphocytes (%)
Feeding drug for 1 month
11.243 ± 1.572
13.642 ± 3.292
0.058 a
Feeding drug for 3 months
12.280 ± 4.398
15.611 ± 5.557
0.154 a
Feeding drug for 3 months followed
by drug withdrawal for 1 month
15.780 ± 6.048
15.963 ± 3.814
0.936 a
CD4+/CD8+ ratio
Feeding drug for 1 month
4.372 ± 0.780
2.880 ± 0.637
0.000 a
Feeding drug for 3 months
3.903 ± 1.579
2.552 ± 1.065
0.038 a
Feeding drug for 3 months followed
by drug withdrawal for 1 month
2.604 ± 0.869
2.185 ± 0.723
0.256 a
a = Two independent-sample t test. P < 0.05 denoted statistical significant difference TABLE I - Comparison of CD4+, CD8+T lymphocytes and CD4+/CD8+ ratio in the experimental and control groups at the three
time points TABLE I - Comparison of CD4+, CD8+T lymphocytes and CD4+/CD8+ ratio in the experimental and control groups at the three
time points a = Two independent-sample t test. P < 0.05 denoted statistical significant difference Significance of T lymphocytes in the Mtb clearance CD4+T lymphocytes and CD4+/CD8+T lymphocyte cell
ratio in the peripheral blood of rats, and the percentages
could be restored after isoniazid withdrawal. Moreover,
isoniazid had no significant effect on CD8+T lymphocytes
in the peripheral blood of rats. Mtb is an intracellular bacterium; CD4+ and
CD8+T lymphocytes play important roles in the immune
response against Mtb infection. Previous studies
found that for patients with TB, the number of CD4+T
lymphocytes decreased and the number of CD8+T
lymphocytes increased. The CD4+/CD8+T lymphocyte
cell ratio, which could be used to reflect the severity
of TB, decreased or even inverted (Arroyo et al., 2016;
Reiley et al., 2010; Scanga et al., 2000). Braz. J. Pharm. Sci. 2020;56: e18547 Significance of cytokines in the treatment of TB of IL-12 could be restored after isoniazid withdrawal. These results were not statistically significant, possibly
due to small sample size and short-term administration. Therefore, the effects of isoniazid on the level of IL-12
in the serum of rats may be further examined in future
studies by increasing the sample size and extending the
administration time. The data presented here suggest
that isoniazid may have no significant effect on IFN-γ
level in the serum of rats. Cytokines, such as IL-12 and IFN-γ, also play
important roles in the immune response to Mtb infection
(Chen et al., 2017; Mortaz et al., 2016). IL-12 can promote
the proliferation and activation of T lymphocytes
(Hamilton et al., 2016) and stimulate the production of
IFN-γ. IL-12 can also activate macrophages and enhance
the ability of anti-Mtb with IFN-γ (Thada et al., 2016). Recent studies showed that cytokine imbalance was a
key factor in the occurrence and development of TB, and
was closely related to TB prognosis (Anuradha et al.,
2016; Kreins et al., 2015). Comparison of IL-12 and IFN-γ level After feeding drug for 1, 3, and 3 months followed
by drug withdrawal for 1 month, no statistically
significant difference was found in the IL-12 and IFN-γ
level between the experimental and control groups (P >
0.05; Table II). The effects of isoniazid on T lymphocytes in healthy
male rats were investigated in this study. The results
showed that isoniazid could increase the percentage of Page 5/9 Page 5/9 Braz. J. Pharm. Sci. 2020;56: e18547 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong TABLE II - Comparison of IL-12 and IFN-γ level in the experimental and control groups at the three time points
Time point
Experimental group (n=10)
Control group (n=10)
P-value
IL-12 (pg/mL)
Feeding drug for 1 month
134.409 ± 25.128
131.752 ± 41.084
0.874 a
Feeding drug for 3 months
137.034 ± 65.937
96.211 ± 18.836
0.088 a
Feeding drug for 3 months followed
by drug withdrawal for 1 month
113.241 ± 38.698
96.019 ± 20.463
0.234 a
IFN-γ (pg/mL)
Feeding drug for 1 month
121.552 ± 21.847
107.609 ± 34.765
0.297 a
Feeding drug for 3 months
118.444 ± 8.158
127.379 ± 24.003
0.289 a
Feeding drug for 3 months followed
by drug withdrawal for 1 month
114.280 ± 18.796
105.517 ± 19.753
0.323 a
a = Two independent-sample t test. P < 0.05 denoted statistical significant difference a = Two independent-sample t test. P < 0.05 denoted statistical significant difference Comparison of carbon clearance index,
phagocytic index and acid phosphatase level P < 0.05 denoted statistical significant difference TABLE III - Comparison of carbon clearance, phagocytic index and acid phosphatase level in the experimental and control
groups at the three time points TABLE III - Comparison of carbon clearance, phagocytic index and acid phosphatase level in the experimental and control
groups at the three time points arbon clearance, phagocytic index and acid phosphatase level in the experimental and control
ts Braz. J. Pharm. Sci. 2020;56: e18547 Comparison of carbon clearance index,
phagocytic index and acid phosphatase level After 1 month of drug feeding, the carbon clearance
index, phagocytic index, and acid phosphatase level in
the experimental group was higher than in the control
group, and the difference was statistically significant (P
< 0.05). Moreover, after feeding drug for 3 and 3 months
followed by drug withdrawal for 1 month, no statistically
significant difference was found in the carbon clearance
index, phagocytic index, or acid phosphatase level
between the experimental and control groups (P > 0.05;
Table III). The effects of isoniazid on the levels of IL-12 and
IFN-γ in healthy male rats were investigated in this
study. The results showed that the level of IL-12 in
the serum of rats decreased as the rat’s age increased. However, the change in IL-12 level in the experimental
group was not the same as in the control group. It did
not decrease as the rat’s age increased, indicating that
isoniazid might increase the level of IL-12 in the serum
of rats, and this effect may become more significant
as the administration time is extended. Also, the level Page 6/9 Braz. J. Pharm. Sci. 2020;56: e18547 Page 6/9 Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats TABLE III - Comparison of carbon clearance, phagocytic index and acid phosphatase level in the experimental and control
groups at the three time points
Time point
Experimental group (n=10)
Control group (n=10)
P-value
Carbon clearance index
Feeding drug for 1 month
0.017 ± 0.004
0.006 ± 0.001
0.000 a
Feeding drug for 3 months
0.011 ± 0.001
0.012 ± 0.002
0.798 a
Feeding drug for 3 months followed
by drug withdrawal for 1 month
0.024 ± 0.009
0.029 ± 0.009
0.248 a
Phagocytic index
Feeding drug for 1 month
6.383 ± 0.602
4.637 ± 0.374
0.000 a
Feeding drug for 3 months
6.366 ± 0.578
6.377 ± 0.491
0.964 a
Feeding drug for 3 months followed
by drug withdrawal for 1 month
8.391 ± 1.526
8.142 ± 0.989
0.671 a
Acid phosphatase level (Mean rank)
Feeding drug for 1 month
13.40
7.60
0.029 b
Feeding drug for 3 months
10.00
11.00
0.739 b
Feeding drug for 3 months followed
by drug withdrawal for 1 month
11.25
9.75
0.579 b
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Ippolito G, et al. Modulation of interferon-gamma response
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T, et al. Selective reduction of IFN-gamma single positive
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individuals with latent tuberculosis infection. Tuberculosis
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resistance in an urban area of Odisha, India. SOURCES OF SUPPORT Kreins AY, Ciancanelli MJ, Okada S, Kong XF, Ramirez-Alejo
N, Kilic SS, et al. Human TYK2 deficiency: Mycobacterial
and viral infections without hyper-IgE syndrome. J Exp Med. 2015;212(10):1641-1662. This study was supported by the Sichuan Provincial
Health Department (No. 090209). Significance of macrophages in anti-tuberculosis not obvious. A possible explanation is that as innate
immune cells, macrophages often respond swiftly to
external stimuli then produce high levels of inflammatory
mediators. On the other hand, the inflammatory activity
of macrophages needs to be tightly regulated in order
to prevent the inflammation-induced tissue damage. Many anti-inflammatory cytokines are also produced
by activated macrophages to terminate their excessive
activation. In contrast, the effects of adaptive immune
cells, such as T lymphocytes, are generally persistent
over a relatively long time period. Therefore, isoniazid
treatment-induced enhancement of macrophage function
may be antagonized by negative feed-back mechanisms,
thus decreasing the long-term effect of isoniazid. Macrophages play a role in innate immunity,
and are critical cells in the immune response to Mtb
infection. Activated macrophages can inhibit or even
kill Mtb, which plays an important role in the control of
Mtb infection (Auricchio et al., 2003). Carbon clearance
capacity, phagocytic capacity, and acid phosphatase of
macrophages can reflect their function. The effects of isoniazid on macrophage function in
healthy male rats were explored in this study. Isoniazid
enhanced the carbon clearance capacity, phagocytic
capacity, and acid phosphatase activity of macrophages
for a short period of time, but the long-term effects were Page 7/9 Page 7/9 Bianchuan Cao, Qiuju Li, Zhe Huang, Xiufang Huang, Yihong Zhu, Ke Xiao, Fuli Huang, Li Zhong Hamilton CA, Mahan S, Entrican G, Hope JC. Interactions
between natural killer cells and dendritic cells favour
T helper1-type responses to BCG in calves. Vet Res. 2016;47(1):85. Hamilton CA, Mahan S, Entrican G, Hope JC. Interactions
between natural killer cells and dendritic cells favour
T helper1-type responses to BCG in calves. Vet Res. 2016;47(1):85. CONCLUSION In summary, isoniazid could increase the percentage
of CD4+T lymphocytes and CD4+/CD8+T lymphocyte
cell ratio in peripheral blood and enhance macrophage
function in healthy male rats. Hamilton-Miller JM. Immunopharmacology of antibiotics:
direct
and
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immunomodulation
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and HIV-1 co-infected Nigerian children being treated for
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inhibits Pseudomonas aeruginosa-induced tumour necrosis Page 8/9 Braz. J. Pharm. Sci. 2020;56: e18547 This is an open-access article distributed under the terms of the Creative Commons Attribution License. Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats factor-alpha production in human whole blood. J Antimicrob
Chemother. 2001;48(2):275-278. Usui T, Meng X, Saide K, Farrell J, Thomson P, Whitaker P,
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T-cell clones in patients with anti-tuberculosis drug-related
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activity of butanol fraction of Gentiana olivieri Griseb. on
Balb/C mice. Asian Pac J Trop Biomed. 2012;2(6):433-437. Vinnard C, Ravimohan S, Tamuhla N, Ivaturi V, Pasipanodya
J, Srivastava S, et al. Isoniazid clearance is impaired among
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Yi Xue Za Zhi. 2015;31(4):528-531. (in Chinese) Wang B, Bian R, Wei C, Sun S, Gao P. Cefodizime increases
peripheral blood CD4/CD8 and Th1/Th2 ratios in senile
patients with bacterial pneumonia. Xi Bao Yu Fen Zi Mian
Yi Xue Za Zhi. 2015;31(4):528-531. (in Chinese) Tauber SC, Nau R. Immunomodulatory properties of
antibiotics. Curr Mol Pharmacol. 2008;1(1):68-79. Thada S, Ponnana M, Sivangala R, Joshi L, Alasandagutti M,
Ansari MS, et al. Polymorphisms of IFN-gamma (+874A/T)
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household contacts in Hyderabad, India. Hum Immunol. 2016;77(7):559-565. Received for publication on 24th July 2018
Accepted for publication on 02nd January 2019 Received for publication on 24th July 2018
Accepted for publication on 02nd January 2019 Braz. J. Pharm. Sci. 2020;56: e18547 Page 9/9
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Plant Growth-Promoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes
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1. Introduction Salt deposits in the crop field are a result of the use of
saline underground irrigation water. Discharge of industrial
effluents into irrigation canals is also a potential source of salts
in agricultural soil. Saline water reduces the transpiration
rate of plants by disrupting the evapotranspiration system
thus reducing crop yield [6]. A high percentage of salt in
the root zone affects root density, root turgor pressure, and
water absorption, which eventually affects plant growth and
development. The okra plant is sensitive to salinity especially
in the early stage of its growth [7] where salinity affects water
and nutrient uptake of the plant, and ionic stress reduces
leaf expansion. Altered morphological traits in the canola
plant [8], reduced plant dry matter and leaf area in soybeans
[9], and reduced yield of canola [10] due to salinity have
been reported. In the root zone, high salt concentration
decreases soil water potential and water availability, which
causes dehydration at the cellular level, eventually leading to
osmotic stress [11]. Salt deposits in the crop field are a result of the use of
saline underground irrigation water. Discharge of industrial
effluents into irrigation canals is also a potential source of salts
in agricultural soil. Saline water reduces the transpiration
rate of plants by disrupting the evapotranspiration system
thus reducing crop yield [6]. A high percentage of salt in
the root zone affects root density, root turgor pressure, and
water absorption, which eventually affects plant growth and
development. The okra plant is sensitive to salinity especially
in the early stage of its growth [7] where salinity affects water
and nutrient uptake of the plant, and ionic stress reduces
leaf expansion. Altered morphological traits in the canola
plant [8], reduced plant dry matter and leaf area in soybeans
[9], and reduced yield of canola [10] due to salinity have
been reported. In the root zone, high salt concentration
decreases soil water potential and water availability, which
causes dehydration at the cellular level, eventually leading to
osmotic stress [11]. Soil salinity is a major problem in agriculture that limits
plant growth and causes significant loss of crop productivity
worldwide [1, 2]. Salinity affects up to 20% and 50% of the
total cultivated and irrigated land in the world, respectively
[3]. However, the use of saline water in agriculture is gradually
increasing owing to shortage of fresh water. Hindawi Publishing Corporation
BioMed Research International
Volume 2016, Article ID 6284547, 10 pages
http://dx.doi.org/10.1155/2016/6284547 Hindawi Publishing Corporation
BioMed Research International
Volume 2016, Article ID 6284547, 10 pages
http://dx.doi.org/10.1155/2016/6284547 Hindawi Publishing Corporation
BioMed Research International
Volume 2016, Article ID 6284547, 10 pages
http://dx.doi.org/10.1155/2016/6284547 Sheikh Hasna Habib,1,2 Hossain Kausar,3,4 and Halimi Mohd Saud1 1Department of Agricultural Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
2Oilseed Research Centre, Bangladesh Agricultural Research Institute (BARI), Gazipur 1701, Bangladesh
3Laboratory of Food Crops, Institute of Tropical Agriculture, Faculty of Agriculture, Universiti Putra Malaysia,
43400 Serdang, Selangor, Malaysia 4Department of Agroforestry and Environmental Science, Sher-E-Bangla Agricultural University, Dhaka, Banglade Correspondence should be addressed to Halimi Mohd Saud; halimi@upm.edu.my Received 10 September 2015; Revised 29 December 2015; Accepted 30 December 2015 Academic Editor: Qaisar Mahmood Copyright © 2016 Sheikh Hasna Habib et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. Salinity is a major environmental stress that limits crop production worldwide. In this study, we characterized plant growth-
promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and examined their effect on
salinity stress tolerance in okra through the induction of ROS-scavenging enzyme activity. PGPR inoculated okra plants exhibited
higher germination percentage, growth parameters, and chlorophyll content than control plants. Increased antioxidant enzyme
activities (SOD, APX, and CAT) and upregulation of ROS pathway genes (CAT, APX, GR, and DHAR) were observed in PGPR
inoculated okra plants under salinity stress. With some exceptions, inoculation with Enterobacter sp. UPMR18 had a significant
influence on all tested parameters under salt stress, as compared to other treatments. Thus, the ACC deaminase-containing PGPR
isolate Enterobacter sp. UPMR18 could be an effective bioresource for enhancing salt tolerance and growth of okra plants under
salinity stress. 2. Materials and Methods All plates were incubated for 48 h and
the optical density (OD) was measured at 600 nm with the
Thermo Scientific Multiskan™GO microplate spectropho-
tometer (Thermo Fisher Scientific Inc., USA).h (hi
,
)
The OD values of ACC- and (NH4)2SO4-containing
wells were compared with the MgSO4-containing wells to
determine the ability of PGPR isolates to utilize ACC for their
growth. The PGPR isolates were categorized into three groups
as isolates with higher, medium, and lower ACC utilizing
rate depending upon the ratio of their OD values at 600 nm
for ACC substrate as compared to (NH4)2SO4. Isolates with
higher ACC-utilization rate showed OD values for wells with
ACC substrate close to OD values for wells with (NH4)2SO4
in the initial 48 h of growth. Similarly, isolates with medium
ACC-utilization rate showed lower OD values for ACC wells
as compared to those for (NH4)2SO4 in the initial 48 h of
growth. Isolates with lower ACC-metabolic rate possessed the
lowest OD values for ACC wells (close to OD values of wells
with MgSO4) in the same time. f
The plant growth-promoting effects of the interactions of
Pseudomonas fluorescens YsS6, P. migulae 8R6, and their ACC
deaminase deficient mutants on the growth of tomato plants
were investigated under 165 mM and 185 mM of salt stress. The plants treated with ACC deaminase-containing bacterial
isolates exhibited higher fresh and dry biomass, higher
chlorophyll content, and a greater number of flowers and
buds than the ACC deaminase deficient bacteria and control
plants [27]. Similarly, Saravanakumar and Samiyappan [22]
reported that the ACC deaminase-containing P. fluorescens
strain TDK1 increased the vigor index of groundnut seedlings
significantly under 120 mM of salt stress condition as com-
pared to plants pretreated with a Pseudomonas strain lacking
ACC deaminase activity. ACC deaminase-containing Bacillus
subtilis also induced salinity stress tolerance in hydropon-
ically grown tomato plants [28]. Bacteria containing high
amounts of SOD and CAT play an important protective role
against the deleterious effect of ROS under stress conditions
[16, 29]. Moreover, induction of salt stress tolerance using
PGPR is an efficient and inexpensive method. However, there
are very few reports on PGPR induced salinity tolerance
in the okra plant caused by changes in ROS-scavenging
enzymes. Therefore, in this study, we characterized the ACC
deaminase-containing PGPR with respect to its effect on 2.3. Identification of PGPR Isolates. 2. Materials and Methods 2.1. Source of Plant Growth-Promoting Rhizobacteria. Fifteen
PGPR isolates were used in this study. All PGPR isolates
were initially isolated from crop fields of the Universiti
Putra Malaysia (UPM), Serdang, Selangor, and Semerak,
Pasir Puteh, Kelantan, Malaysia, by using the dilution plate
technique. All selected PGPR isolates possessed nitrogen
fixation, phosphate solubilization, indoleacetic acid (IAA)
synthesis, and salt tolerant properties [30]. Genetic engineering is an attractive approach that can
generate plants resistant to salt stress [18]. AtNHX1 overex-
pressing transgenic Brassica napus plants were found to grow,
flower, and produce seeds under 200 mM salt stress [19]. However, the transgenic approach is time-consuming and
expensive. ACC deaminase-containing PGPR can reduce the
deleterious effects of environmental stress and can enhance
stress tolerance of plants by a variety of mechanisms such
as the synthesis of phytohormones, mineral solubilization,
nutrient uptake, increased leaf area, increased chlorophyll
and soluble protein content, and antioxidant enzyme activi-
ties [20]. Ethylene is important for plant growth and devel-
opment, as well as in the fruit ripening process, but an
excess amount of ethylene might decrease seed germination
and root growth [21, 22]. It is also reported that ethylene
production increases under stress conditions and results in
inhibitory effects on plants [23]. However, ACC deaminase-
containing PGPR can hydrolyse ACC, the precursor of
ethylene, thereby reducing the excess ethylene and rescue
plants from inhibitory effects [24–26]. 2.2. ACC Deaminase Activity of Plant Growth-Promoting
Rhizobacteria. All 15 PGPR isolates were tested for ACC
deaminase activity following the method described by Jacob-
son et al. [31]. Bacterial isolates were grown on Nutrient Broth
(NB, Difco™) for 48 h at 28 ± 2∘C. The cultures were diluted
10-fold with sterile MgSO4 (0.1 M) solution. ACC (3 mM) was
filter sterilized with 0.2 𝜇m filter membrane and stored at
−20∘C until further use. In a 96-well plate, 120 𝜇L of minimal
salt medium (MSM) was added to each well. To the first 4
lanes, 15 𝜇L of MgSO4 (0.1 M) was added and, to the next 4
lanes, 15 𝜇L of (NH4)2SO4 (0.1 M) was added. Thawed ACC
(15 𝜇L) was added to the remaining 4 lanes. Each of the PGPR
isolates (15 𝜇L) was added into each well separately. For the
untreated control, 15 𝜇L of MgSO4 (0.1 M) was used instead
of the PGPR culture. 1. Introduction Consequently, on
one hand, salt-affected areas are constantly increasing, and,
on the other hand, a significant amount of arable land is being
abandoned every year because of salinity [4]. Okra (Abelmoschus esculentus L.) is an annual vegetable
crop cultivated in tropical and subtropical regions. It is
considered a high-value vegetable crop owing to its high
levels of vitamins, minerals, carbohydrates, and fats [5]. Although it has good nutritional value as well as high con-
sumer demand, the yield of okra per hectare is very low,
and this lower productivity arises mainly from soil salinity. 2 BioMed Research International growth, antioxidant enzyme activities, and expression profiles
of ROS pathway genes in the okra plant under high salt stress. Salinity stress generates reactive oxygen species (ROS),
namely, H2O2, O−2, and OH−that damage the DNA, RNA,
and proteins [12, 13]. These ROS compounds also cause
chlorophyll destruction and damage the root meristem activ-
ity [14]. Antioxidant enzymes such as superoxide dismutase
(SOD), catalase (CAT), and ascorbate peroxidase (APX) have
the ability to scavenge the ROS and maintain them at low
levels. Superoxide dismutase is a metalloenzyme that plays an
important role in protecting cells from oxidative damage, by
catalyzing the conversion of the superoxide radical to H2O2
[15, 16]. Ascorbate peroxidase has vital defensive role against
ROS [17] and can catalyze the breakdown of H2O2 that is
produced by SOD. Catalase reduces ROS levels by catalyzing
the breakdown of H2O2 into H2O and O2 [12, 13]. 2. Materials and Methods PGPR isolates, UPMR2
and UPMR18, with the highest ACC-metabolizing activities
were identified by 16S rRNA gene sequencing as Bacillus
megaterium and Enterobacter sp., respectively (Figure 1(a)). In brief, each PGPR isolate was grown at 28∘C for 48–72 h
in 5 mL of NB. Of each culture, 1 mL was transferred into
separate 1.5 mL microcentrifuge tubes. Bacterial cells were
pelleted by centrifugation at 12000 g for 5 min (Eppendorf
Centrifuge 5810 R, Hamburg, Germany). Bacterial genomic
DNA was extracted with the Genomic DNA Mini Kit BioMed Research International 3 1
2
M
1400bp
(a)
Bacillus nealsonii KC329823
Bacillus circulans strain KM349203
UPMR2
Bacillus megaterium KF956591
UPMR18
Enterobacter sp. GQ871449
Enterobacter sp. LC014954
Bacillus sp. AB425363
100
100
100
73
78
0.1
(b)
Figure 1: (a) Agarose gel electrophoresis of 16S rDNA PCR products of bacterial isolates. Lane M: 100 bP DNA ladder. Lanes 1 and 2: bacterial
isolates UPMR2 and UPMR18. (b) The neighbor-joining tree shows the phylogenetic relationship of the isolates UPMR2 and UPMR18 with
related isolates from the NCBI database. 1
2
M
1400bp
Bacillus nealsonii KC329823
Bacillus circulans strain KM349203
UPMR2
Bacillus megaterium KF956591
UPMR18
Enterobacter sp. GQ871449
Enterobacter sp. LC014954
Bacillus sp. AB425363
100
100
100
73
78
0.1 1400bp
Bacillus nealsonii KC329823
Bacillus circulans strain KM349203
UPMR2
Bacillus megaterium KF956591
UPMR18
Enterobacter sp. GQ871449
Enterobacter sp. LC014954
Bacillus sp. AB425363
100
100
100
73
78
0.1
(b) 1
2
M
(a) (b) (a) Figure 1: (a) Agarose gel electrophoresis of 16S rDNA PCR products of bacterial isolates. Lane M: 100 bP DNA ladder. Lanes 1 and 2: bacterial
isolates UPMR2 and UPMR18. (b) The neighbor-joining tree shows the phylogenetic relationship of the isolates UPMR2 and UPMR18 with
related isolates from the NCBI database. (25, 50, 75, and 100 mM) and kept at 25∘C in the dark. After three days, the emergence of the radicle from the seed
was considered as an index of germination. The germination
percentage was calculated as follows: (Yeastern Biotech Co., Ltd.) according to the manufacturer’s
protocol.h p
The universal primer pairs 27f (5-AGAGTTTGATCM-
TGGCTCAG-3) and 1492R (5-GGTTACCTTGTTACG-
ACTT-3) were used to amplify the 16S rRNA coding region. 2. Materials and Methods The soil surface was covered with black plastic to prevent
salt loss. Four treatment groups were defined as follows: T0:
noninoculated salt treated plants, T1: UPMR2 inoculated salt
treated plants, T2: UPMR18 inoculated salt treated plants, and
T3: both UPMR2 and UPMR18 inoculated salt treated plants. Each treatment group comprised 16 plants. Plants were grown
for 15 days under salt stress conditions. Growth parameters,
that is, plant height, root length, fresh weight of leaf, stem, and
root, and dry weight of leaf, stem, and root, were recorded at
15 days after salt treatment. 2.5. Evaluation of PGPR Isolates on Growth Promotion of
Okra under Salt Stress. Okra seeds were surface sterilized
and sown in Petri dishes. After emergence of the radicle
and plumule, the seedlings were planted in plastic pots (one
seedling/pot) filled with sterilized soil. The soil belongs to
Serdang soil series, which was sandy-loam in texture (order
Ultisols). The soil contained total N (0.78%), available P
(54.92 ppm), potassium (218 ppm), and total C (2.94%) and
had a pH of 5.32. Twenty-five milliliters of PGPR suspension
(108 cfu/mL) was applied to the roots of potted okra plants
at 15 days after transplantation (DAT) and 22 DAT. Control
plants were treated with the same amount of water. The
plants were subjected to salt stress two weeks after the second
inoculation by watering with 75 mM of NaCl solution. To
prevent the plants from undergoing osmotic shock, NaCl
concentration was imposed in 25 mM increments per day
until the final concentration was attained after three days [33]. The soil surface was covered with black plastic to prevent
salt loss. Four treatment groups were defined as follows: T0:
noninoculated salt treated plants, T1: UPMR2 inoculated salt
treated plants, T2: UPMR18 inoculated salt treated plants, and
T3: both UPMR2 and UPMR18 inoculated salt treated plants. Each treatment group comprised 16 plants. Plants were grown
for 15 days under salt stress conditions. Growth parameters,
that is, plant height, root length, fresh weight of leaf, stem, and
root, and dry weight of leaf, stem, and root, were recorded at
15 days after salt treatment. 2.4. Evaluation of PGPR Isolates on Okra Seed Germination
under Salt Stress. Two PGPR isolates UPMR2 and UPMR18
were selected based on their growth promotion, halotoler-
ance, and ACC deaminase activities and were investigated in
the context of seed germination in okra under different levels
of NaCl stress. 2. Materials and Methods The 25 𝜇L PCR reaction mixture consisted of the following
components: 1 𝜇L of each extracted PGPR genomic DNA,
0.15 𝜇M of each primer, 1x PCR reaction buffer (Fermentas,
USA), 0.2 mM of dNTP mix, 2.5 U of Taq polymerase
(Fermentas, USA), 25 mM of MgCl2, and nucleic acid-
free water to make up final volume. The thermal cycling
conditions were as follows: one cycle for 5 min at 94∘C,
30 cycles at 94∘C for 30 sec, 55∘C for 30 sec, and 72∘C for
1 min followed by an additional cycle at 72∘C for 5 min. The
amplified PCR products were purified using QIAquick Gel
Extraction Kit (QIAGEN Inc., USA) and sent for sequencing. The obtained sequences were analyzed and compared with
sequences obtained from the GenBank database using the
BLAST program to determine the percent similarity. A
molecular phylogenetic tree was constructed in Mega version
4 software following the neighbor-joining method [32]. The
BLASTX analysis showed that the isolates UPMR2 and
UPMR18 matched with B. megaterium and Enterobacter sp.,
respectively, with 99% similarity. Phylogenetic analysis of
the bacterial isolates was also performed on the basis of the
neighborhood joining method with 100 bootstrap sampling
replicates (Figure 1(b)). Germination % = (No. of seeds germinated
No. of seeds sown
) × 100. (1) Germination % = (No. of seeds germinated
No. of seeds sown
) × 100. (1) (1) 2.5. Evaluation of PGPR Isolates on Growth Promotion of
Okra under Salt Stress. Okra seeds were surface sterilized
and sown in Petri dishes. After emergence of the radicle
and plumule, the seedlings were planted in plastic pots (one
seedling/pot) filled with sterilized soil. The soil belongs to
Serdang soil series, which was sandy-loam in texture (order
Ultisols). The soil contained total N (0.78%), available P
(54.92 ppm), potassium (218 ppm), and total C (2.94%) and
had a pH of 5.32. Twenty-five milliliters of PGPR suspension
(108 cfu/mL) was applied to the roots of potted okra plants
at 15 days after transplantation (DAT) and 22 DAT. Control
plants were treated with the same amount of water. The
plants were subjected to salt stress two weeks after the second
inoculation by watering with 75 mM of NaCl solution. To
prevent the plants from undergoing osmotic shock, NaCl
concentration was imposed in 25 mM increments per day
until the final concentration was attained after three days [33]. 2. Materials and Methods patterns of genes of the ROS pathway (APX, CAT, DHAR,
and GR) were analyzed by semiquantitative RT-PCR using
the primer pairs [40] mentioned in Table 1 with actin as a
control. 2.5.2. Enzymatic Assay. Leaf protein was extracted from
frozen leaflet samples ground in liquid nitrogen using ice-
cold mortar and pestle. Protein was extracted in 3 mL of
extraction buffer containing 100 mM K-phosphate buffer
(pH 7.8), 0.1 mM EDTA, 14 mM 2-mercaptoethanol, and
0.1% (v/v) Triton X-100 for SOD (EC 1.15.1.1) activity or
50 mM K-phosphate buffer (pH 7.0), 2 mM EDTA, 20 mM
ascorbate, and 0.1% (v/v) Triton X-100 for CAT (EC 1.11.1.6)
and APX (EC 1.11.1.11) activities. The homogenate was than
centrifuged at 15000 ×g for 15 min at 4∘C. The supernatant
was used for total protein [36] and enzymatic assays. RT-PCR was performed using aliquots of 1 𝜇L cDNA
samples. The PCR conditions were set as follows: one cycle at
95∘C for 5 min, followed by 35 cycles at 95∘C for 30 sec, 52∘C
for 30 sec, and 72∘C for 15 sec followed by an additional cycle
of five minutes at 72∘C. The PCR products were separated on
1.5% agarose gel and were observed under UV light. 2.6. Statistical Analysis. All experiments were conducted
using completely randomized design (CRD) with four repli-
cates. The data were subjected to analysis of variance
(ANOVA) and tested for significance using the least signif-
icant difference (LSD) by PC-SAS software (SAS Institute,
Cary, NC, USA, 2001). 2.6. Statistical Analysis. All experiments were conducted
using completely randomized design (CRD) with four repli-
cates. The data were subjected to analysis of variance
(ANOVA) and tested for significance using the least signif-
icant difference (LSD) by PC-SAS software (SAS Institute,
Cary, NC, USA, 2001). SOD activity was determined according to the method
described by Giannopolitis and Ries [37]. The reaction
mixture contained 50 mM K-phosphate buffer (pH 7.8),
0.1 mM EDTA, 13 mM methionine, 75 𝜇M nitrobluetetra-
zolium (NBT), 2 𝜇M riboflavin, and 100 𝜇L enzyme extract. SOD activity was determined by the ability of the enzyme to
inhibit photochemical reduction of NBT on blue formazan,
followed by monitoring absorbance of the reaction mixture
at 560 nm.h 2. Materials and Methods Seeds of the local Malaysian okra variety five anchor
were used in this study. The seeds were surface sterilized
with 0.5% (v/v) sodium hypochlorite for 20 min followed by
repetitive washes with distilled water. The seeds were then
soaked in PGPR suspension (108 cfu/mL) or in distilled water
(control) for 24 h. A total of 10 seeds were sown in Petri dishes
(9 mm diameter) with two sheets of Whatman number 1 filter
papers. The seeds were moistened either with distilled water
(control) or with solutions of varying NaCl concentration 2.5.1. Leaf Chlorophyll Measurements. The total leaf chloro-
phyll and chlorophyll a (chla) and chlorophyll b (chlb)
content and the chlorophyll a/b ratio were determined at
15 days after salt treatment by using the method described
by Moran and Porath [34]. Frozen leaflet samples of 0.2 g
from each treatment were cut into small pieces and placed
in a glass vial containing 2 mL of N,N-dimethylformamide 4 BioMed Research International Table 1: Primer pairs used for RT-PCR in this study. NCBI
accession
number
Primer
name
Sequence (5-3)
X55749
Actin
F: CTGGTGGTGCAACAACCTTA
R: GAATGGAAGCAGCTGGAATC
AB041343
APX
F: ACCAATTGGCTGGTGTTGTT
R: TCACAAACACGTCCCTCAAA
AY442179
CAT
F: TGCCCTTCTATTGTGGTTCC
R: GATGAGCACACTTTGGAGGA
X76533
GR
F: GGATCCTCATACGGTGGATG
R: TTAGGCTTCGTTGGCAAATC
DQ512964
DHAR
F: AGGTGAACCCAGAAGGGAAA
R: TATTTTCGAGCCCACAGAGG Table 1: Primer pairs used for RT-PCR in this study. (DMF) and were covered with aluminum foil. The vials were
then incubated at 4∘C for 48 h. The absorbance readings of
chlorophyll a and b were taken at wave lengths of 663 nm and
645 nm, respectively, with a UV-visible spectrophotometer
(Genesys 10 UV, Thermo Fisher Scientific, USA) using DMF
as a blank. The concentrations of total chlorophyll, chla, and
chlb were calculated according to the following equations
[35]: Chlorophyll a = (0.0127𝐷663 −0.00269𝐷645) ,
Chlorophyll b = (0.0229𝐷645 −0.0468𝐷663) ,
Total chlorophyll = Chlorophyll a + Chlorophyll b,
(2) Chlorophyll a = (0.0127𝐷663 −0.00269𝐷645) ,
Chlorophyll b = (0.0229𝐷645 −0.0468𝐷663) ,
(2) Total chlorophyll = Chlorophyll a + Chlorophyll b, where 𝐷663 is absorbance at 663 nm wave length and 𝐷645 is
absorbance at 645 nm wave length. patterns of genes of the ROS pathway (APX, CAT, DHAR,
and GR) were analyzed by semiquantitative RT-PCR using
the primer pairs [40] mentioned in Table 1 with actin as a
control. 3. Results The total CAT activity in the leaf was assayed based on the
rate of H2O2 consumption at 240 nm [38]. The assay mixture
of 3 mL contained 100 mM phosphate buffer (pH 7.0), 0.1 mM
EDTA, 0.1% H2O2, and 20 𝜇L enzyme extract. After addition
of the enzyme extract to the reaction mixture, decrease in
H2O2 levels was determined by measuring the absorbance at
240 nm with a UV1000 spectrophotometer and quantified by
using the extinction coefficient (36 M21 cm21). 3.1. ACC Deaminase Activity. The ACC deaminase activity
of the PGPR isolates was determined qualitatively to char-
acterize the isolates for their ability to use ACC as the sole
nitrogen source. The PGPR isolates were categorized into
three groups, as isolates with higher (>0.7), medium (0.5–
0.69), and lower (<0.5) ACC metabolism rate depending on
their growth which was measured in terms of cell density
at OD600 (data not shown). On the basis of their high ACC
deaminase activity, two PGPR isolates B. megaterium UPMR2
and Enterobacter sp. UPMR18 were selected to assess their salt
tolerance potency in okra plants. fi
Total leaf APX activity was estimated at 290 nm by the
method described by Chen and Asada [39]. The 3 mL APX
assay mixture contained 50 mM K-phosphate buffer (pH
7.0), 0.1 mM H2O2, 0.5 mM ascorbate, and 20 𝜇L enzyme
extract. The amount of ascorbate oxidized was calculated
using extinction coefficient 𝐸= 2.8 mM−1 cm−1. 3.2. Effect of PGPR Isolates on Germination of Okra Seeds
under Salt Stress. The results showed that the germination
of okra seeds was significantly (𝑝≤0.05) affected by the
PGPR isolates under different salt concentrations (Table 2). Both the PGPR isolates either separately or in combination
showed 100% seed germination up to 75 mM NaCl con-
centrations. Seed germination declined to 80% at 100 mM
NaCl concentration. On the other hand in the noninoculated
group, seed germination percentage was 70% at 75 mM NaCl 2.5.3. Expression of ROS Pathway Genes. Total RNA was
extracted from approximately 0.1 g of frozen okra leaf sam-
ples using TRIzolⓇreagent (Invitrogen, USA). The first-
strand cDNA was synthesized from 1 𝜇g of total RNA
using QuantiTectⓇReverse Transcription Kit (Qiagen, USA)
according to the manufacturer’s instructions. The expression 5 BioMed Research International BioMed Research International Table 2: Effect of PGPR inoculation on germination of okra seeds
under salt stress. concentration while the lowest germination percentage (50%)
was recorded at 100 mM NaCl concentration. 3.3. Effect of PGPR Isolates on Growth of Okra Seedlings
under Salt Stress. The effects of PGPR inoculation on okra
plants were assessed at 75 mM NaCl concentration. The
results demonstrate that the application of PGPR isolates
B. megaterium UPMR2 and Enterobacter sp. UPMR18 sig-
nificantly (𝑝≤0.05) increased shoot and root growth of
okra as compared to noninoculated plants. Significantly (𝑝≤
0.05), the highest leaf fresh (4.04 g) and dry (0.469 g) weight
were obtained in Enterobacter sp. UPMR18 (T2) treated
plants compared to other treatments. As with leaf biomass,
significantly (𝑝≤0.05) highest shoot dry weight (0.28 g) and
root fresh weight (0.78 g) were also observed in the plants
receiving Enterobacter sp. UPMR18 (T2) under salt stress. In
addition, significantly (𝑝≤0.05) highest shoot fresh weight
and root dry weight were recorded both in Enterobacter sp. UPMR18 (T2) treatment and in the combined application of
B. megaterium UPMR2 and Enterobacter sp. UPMR18 (T3)
treated plants under salinity stress conditions (Table 3). 3. Results Treatments
Salinity levels
0 mM 25 mM 50 mM 75 mM 100 mM
Control
100
100
100
70b
50b
Bacillusmegaterium
(UPMR2)
100
100
100
100a
80a
Enterobacter sp. (UPMR18)
100
100
100
100a
80a
Coinoculation of
UPMR2 & UPMR18
100
100
100
100a
80a
Means within columns with the same letters are not significantly different at
𝑝< 0.05. Table 2: Effect of PGPR inoculation on germination of okra seeds
under salt stress. Table 2: Effect of PGPR inoculation on germination of okra seeds
under salt stress. inoculated with Enterobacter sp. UPMR18 (T2), combined
PGPR (T3) application, and B. megaterium UPMR2 (T1),
respectively, as compared to control plants under salt stress. Similarly, the activity of SOD was also increased significantly
(𝑝≤0.05) (approximately 2 and 1.5 times higher) in the
plants inoculated with Enterobacter sp. UPMR18 (T2) and
with the combined application of both PGPR (T3), compared
to the noninoculated stressed plants. 3.6. Expression Analysis of ROS Pathway Genes by RT-PCR. The ROS-scavenging enzymes, CAT, SOD, and APX, were
assessed in okra plants after salinity stress, after inocu-
lation, or without inoculation with PGPR B. megaterium
UPMR2 and Enterobacter sp. UPMR18. The PGPR inoculated
salinized plants exhibited the maximum percent increase
in ROS-scavenging enzymes with respect to the noninocu-
lated salinized control plants. APX activity was significantly
increased in B. megaterium UPMR2 inoculated salinized
plants and was approximately 13 times higher than that
of noninoculated salinized plants (Figure 4). Significantly
increased CAT activity was also observed in all PGPR inoc-
ulated salinized plants than that of control (noninoculated
salinized plants). CAT activity was recorded as approxi-
mately 3.4, 5.4, and 4.8 times higher in plants inoculated
with B. megaterium UPMR2, Enterobacter sp. UPMR18,
and combined application of PGPR isolates, respectively,
compared to that of noninoculated plants under the same
stress conditions (Figure 4). Similarly, SOD activity was also
increased significantly (approximately 2 and 1.5 times higher)
in plants inoculated with Enterobacter sp. UPMR18 and with
combined application of PGPR, respectively, growing under
stress condition compared to noninoculated salt stressed
plants (Figure 4). 4. Discussion Application of ACC deaminase-containing PGPR as a soil
amendment resulted in enhanced seed germination, chloro-
phyll content, and growth of okra plants under salinity stress
by maintaining low stress ethylene levels and increasing the
ROS-scavenging enzymes. Ethylene, a plant growth regulator,
is involved in various physiological responses [41]. However,
it is regarded as a stress hormone since it is synthesized at
a rapid rate under stress [42]. Stress ethylene decreases seed
germination and root development and eventually hinders
plant growth [21, 22]. Microorganisms synthesizing the ACC
deaminase enzyme can cleave ACC to 𝛼-ketobutyrate and
ammonia, thereby decreasing ethylene stress in plants [43–
45]. In this study, two PGPR B. megaterium UPMR2 and
Enterobacter sp. UPMR18 that possessed characteristics of
ACC deaminase activity demonstrated their effectiveness in
inducing salt tolerance and consequent improvement in the
growth of okra plants under salt stress. 3.4. Determination of Okra Leaf Chlorophyll Content. The
data obtained for plant pigments showed that chlorophyll a
was significantly (𝑝≤0.05) highest in plants treated with
Enterobacter sp. UPMR18 (T2) and the combined application
of both the PGPR (T3) compared to other treatments. Chlorophyll b and chlorophyll a/b ratio were increased
significantly (𝑝≤0.05) in all inoculated plants compared
to noninoculated salinized plants. Similarly, all the PGPR
treated plants showed significantly (𝑝≤0.05) higher values
for total chlorophyll content than the control (Figure 2). 3.5. Determination of Activities of ROS-Scavenging Enzymes. All PGPR inoculated plants exhibited higher percent increase
in ROS-scavenging enzymes in comparison to the noninocu-
lated control plants (Figure 3). APX activity was significantly
(𝑝≤0.05) highest in B. megaterium UPMR2 (T1) inoculated
salinized plants where it was approximately 13 times higher
than the noninoculated plants. Significantly (𝑝≤0.05) higher
CAT activity was also observed in all PGPR inoculated plants
as compared to control. CAT activity was recorded to be
approximately 5.4, 4.8, and 3.4 times higher in the plants Okra is a salt sensitive crop, especially in its early growth
stage [46]. Seed germination of okra was reduced at a higher
rate with increasing level of salinity in the noninoculated
group (Table 2). Salinity increases the osmotic potential
of growth medium and, as a result, seeds require more
energy to absorb water, resulting in decreased germination BioMed Research International 6 Table 3: Effect of PGPR inoculation on growth attributes of okra plants under salinity stress. 4. Discussion Treatment
LFW/plant
LDW/plant
SFW/plant
SDW/plant
RFW/plant
RDW/plant
(g)
(g)
(g)
(g)
(g)
(g)
T0
2.63 ± 0.13c
0.26 ± 0.03c
1.57 ± 0.14c
0.14 ± 0.02c
0.34 ± 0.03d
0.04 ± 0.01b
T1
2.83 ± 0.15c
0.28 ± 0.03c
1.90 ± 0.08b
0.16 ± 0.02c
0.47 ± 0.05c
0.03 ± 0.00b
T2
3.92 ± 0.17a
0.46 ± 0.01a
2.56 ± 0.29a
0.28 ± 0.01a
0.79 ± 0.08a
0.07 ± 0.02a
T3
3.28 ± 0.25b
0.37 ± 0.01b
2.65 ± 0.07a
0.22 ± 0.01b
0.65 ± 0.06b
0.06 ± 0.00a
T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: UPMR2 and UPMR18
inoculated salt treated plants. LFW: leaf fresh weight, LDW: leaf dry weight, SFW: shoot fresh weight, SDW: shoot dry weight, RFW: root fresh weight, and
RDW: root dry weight. Means within columns with the same letters are not significantly different at 𝑝< 0.05. Table 3: Effect of PGPR inoculation on growth attributes of okra plants under salinity stress. Treatment
LFW/plant
LDW/plant
SFW/plant
SDW/plant
RFW/plant
RDW/plant
(g)
(g)
(g)
(g)
(g)
(g)
T0
2.63 ± 0.13c
0.26 ± 0.03c
1.57 ± 0.14c
0.14 ± 0.02c
0.34 ± 0.03d
0.04 ± 0.01b
T1
2.83 ± 0.15c
0.28 ± 0.03c
1.90 ± 0.08b
0.16 ± 0.02c
0.47 ± 0.05c
0.03 ± 0.00b
T2
3.92 ± 0.17a
0.46 ± 0.01a
2.56 ± 0.29a
0.28 ± 0.01a
0.79 ± 0.08a
0.07 ± 0.02a
T3
3.28 ± 0.25b
0.37 ± 0.01b
2.65 ± 0.07a
0.22 ± 0.01b
0.65 ± 0.06b
0.06 ± 0.00a
T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: UPMR2 and UPMR18
inoculated salt treated plants. LFW: leaf fresh weight, LDW: leaf dry weight, SFW: shoot fresh weight, SDW: shoot dry weight, RFW: root fresh weight, and
RDW: root dry weight. Means within columns with the same letters are not significantly different at 𝑝< 0.05. T0: noninoculated salt treated plants, T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: UPMR2 and UPMR18
inoculated salt treated plants. LFW: leaf fresh weight, LDW: leaf dry weight, SFW: shoot fresh weight, SDW: shoot dry weight, RFW: root fresh weight, and
RDW: root dry weight. Means within columns with the same letters are not significantly different at 𝑝< 0.05. 4. Discussion Treatments
T0
T1
T2
T3
c
b
a
a
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Chlorophyll a mg/g fresh leaf
Treatments
T0
T1
T2
T3
b
a
a
a
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Chlorophyll b mg/g fresh leaf Treatments
T0
T1
T2
T3
c
b
a
a
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Chlorophyll a mg/g fresh leaf
Treatments
T0
T1
T2
T3
b
a
a
a
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Chlorophyll b mg/g fresh leaf
Treatments
T0
T1
T2
T3
c
b
a
a
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
Chlorophyll a/b mg/g fresh leaf
Treatments
T0
T1
T2
T3
c
b
a
a
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Total chlorophyll mg/g fresh leaf
Figure 2: Effect of PGPR inoculation on chlorophyll content of okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2
inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3; both UPMR2 and UPMR18 inoculated salt treated plants. Error bars refer to standard error of means of four replicates. Means within columns with the same letters are not significantly different at
𝑝< 0.05. Treatments
T0
T1
T2
T3
b
a
a
a
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Chlorophyll b mg/g fresh leaf Treatments
Treatments
Treatments
T0
T1
T2
T3
c
b
a
a
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
Chlorophyll a/b mg/g fresh leaf
Treatments
T0
T1
T2
T3
c
b
a
a
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Total chlorophyll mg/g fresh leaf Treatments
T0
T1
T2
T3
c
b
a
a
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
Total chlorophyll mg/g fresh leaf Figure 2: Effect of PGPR inoculation on chlorophyll content of okra leaves under salt stress. T0: noninoculated salt treated plants, T1: UPMR2
inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3; both UPMR2 and UPMR18 inoculated salt treated plants. Error bars refer to standard error of means of four replicates. Means within columns with the same letters are not significantly different at
𝑝< 0.05. [47, 48]. Our results were consistent with the results of
previous studies [21, 22]. These studies demonstrated that
increased salt concentrations decrease seed germination and
root growth in dicotyledonous plants. 4. Discussion BioMed Research International 7 Treatments
T0
T1
T2
T3
c
d
b
a
CAT activity
0
300
600
900
1200
(𝜇mol min−1 mg protein−1) Treatments
T0
T1
T2
T3
c
d
b
a
APX activity
0
100
200
300
400
500
600
Treatments
T0
T1
T2
T3
c
d
b
a
CAT activity
0
300
600
900
1200
(𝜇mol min−1 mg protein−1)
(𝜇mol min−1 mg protein−1) Treatments
T0
T1
T2
T3
c
c
b
a
SOD activity
0
10
20
30
40
50
60
70
80
(𝜇mol min−1 mg protein−1) Treatments
T0
T1
T2
T3
c
c
b
a
SOD activity
0
10
20
30
40
50
60
70
80
(𝜇mol min−1 mg protein−1) Treatments Figure 3: Effect of PGPR inoculation on antioxidant enzymes activities in okra leaves under salt stress. T0: noninoculated salt treated plants,
T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt
treated plants. Error bars refer to standard error of means of four replicates. APX: ascorbate peroxidase, CAT: catalase, and SOD: superoxide
dismutase. Means within columns with the same letters are not significantly different at 𝑝< 0.05. observed that inoculation of plants with wild-type P. flu-
orescens YsS6 and P. migulae 8R6 significantly increased
the total chlorophyll content of tomato plants compared
with their ACC deaminase deficient mutants and control
plants under salt stress. Higher chlorophyll content was also
reported in ACC deaminase-containing PGPR inoculated
salt stressed rice [59] and cucumber [60] compared to
noninoculated plants. [52]. Cucumber plants inoculated with wild-type P. putida
UW4 and Gigaspora rosea BEG9 showed significantly higher
root and shoot fresh biomass than the plants that received
ACC deaminase deficient bacteria and untreated control
plants under 72 mM salt stress [53]. When canola seeds were
inoculated with ACC deaminase-containing salt tolerant
bacteria under the 150 mM NaCl condition, the biomass of
treated plants increased by up to 47% of the control plants
[54]. Red pepper seedlings inoculated with ACC deaminase-
containing salt tolerant bacteria reduced 57% stress ethylene
production and produced similar amounts of biomass as to
those in no salt treatment control plant [55]. Salinity stress leads to the formation of ROS, namely,
superoxide (O2
−), singlet oxygen (O2), hydroxyl (OH−), and
hydrogen peroxide (H2O2), which cause severe damage to
cell structures by exerting oxidation of cell membranes in
a process known as oxidative stress [17, 61]. 4. Discussion In addition, ger-
mination of Limonium stocksii and Suaeda fruticosa seeds
was inhibited with increasing NaCl concentrations [49]. In
contrast, when the seeds were inoculated with the PGPR
suspension, seed germination was reduced at a lower rate
despite increasing salinity. This may indicate that the ACC
deaminase-containing PGPR isolates, B. megaterium UPMR2
and Enterobacter sp. UPMR18, are able to ameliorate the effect
of NaCl on growth medium. The PGPR got attached to the
seed surface and synthesized phytohormones in response
to amino acids produced by the seeds perhaps alleviating the salinity stress [50]. Our results were also supported by the
findings of Jalili et al. [51] who demonstrated that the rate
of germinating canola seeds (Brassica napus L.) inoculated
with ACC deaminase-containing plant growth-promoting
P. fluorescens and P. putida was significantly higher under
salinity stress. y
ACC deaminase-containing PGPR inoculated okra plants
showed higher root and shoot biomass than noninoculated
plants under salinity stress. This might be due to the
presence of PGPR isolates in the growth medium, which
alleviate the effects of salinity on okra plants by producing
ACC deaminase. ACC deaminase-containing plant growth-
promoting bacteria have been documented to facilitate the
growth of a variety of plants under high salinity conditions BioMed Research International
7
Treatments
T0
T1
T2
T3
c
d
b
a
APX activity
0
100
200
300
400
500
600
Treatments
T0
T1
T2
T3
c
d
b
a
CAT activity
0
300
600
900
1200
Treatments
T0
T1
T2
T3
c
c
b
a
SOD activity
0
10
20
30
40
50
60
70
80
(𝜇mol min−1 mg protein−1)
(𝜇mol min−1 mg protein−1)
(𝜇mol min−1 mg protein−1)
Figure 3: Effect of PGPR inoculation on antioxidant enzymes activities in okra leaves under salt stress. T0: noninoculated salt treated plants,
T1: UPMR2 inoculated salt treated plants, T2: UPMR18 inoculated salt treated plants, and T3: both UPMR2 and UPMR18 inoculated salt
treated plants. Error bars refer to standard error of means of four replicates. APX: ascorbate peroxidase, CAT: catalase, and SOD: superoxide
dismutase. Means within columns with the same letters are not significantly different at 𝑝< 0.05. References [1] S. I. Allakhverdiev, A. Sakamoto, Y. Nishiyama, M. Inaba,
and N. Murata, “Ionic and osmotic effects of NaCl-induced
inactivation of photosystems I and II in Synechococcussp.,” Plant
Physiology, vol. 123, no. 3, pp. 1047–1056, 2000. Figure 4: Effect of PGPR inoculation on DHAR, GR, APX, and CAT
transcript levels by semiquantitative RT-PCR analysis of okra leaves
under salt stress. T0: noninoculated salt treated plants, T1: UPMR2
inoculated salt treated plants, T2: UPMR18 inoculated salt treated
plants, and T3: both UPMR2 and UPMR18 inoculated salt treated
plants. Actin: positive control, APX: ascorbate peroxidase, CAT:
catalase, GR: glutathione reductase, and DHAR: dehydroascorbate
reductase. [2] R. Munns, “Comparative physiology of salt and water stress,”
Plant, Cell & Environment, vol. 25, no. 2, pp. 239–250, 2002. [3] Z. Cheng, O. Z. Woody, B. J. McConkey, and B. R. Glick,
“Combined effects of the plant growth-promoting bacterium
Pseudomonas putida UW4 and salinity stress on the Brassica
napus proteome,” Applied Soil Ecology, vol. 61, pp. 255–263, 2012. [4] W. B. Frommer, U. Ludewig, and D. Rentsch, “Taking transgenic
plants with a pinch of salt,” Science, vol. 285, no. 5431, pp. 1222–
1223, 1999. inoculated plants were adapted to saline conditions by elim-
inating ROS through APX, CAT, and SOD activities. Our
results are also supported by the findings of Gururani et
al. [63], who reported enhanced activities of different ROS-
scavenging enzymes in PGPR inoculated potato plants under
stress [63]. Moreover, in our study semiquantitative RT-PCR
results (Figure 4) revealed that expression levels of different
ROS pathway genes encoding CAT, APX, GR, and DHAR
were increased in the ACC deaminase-containing PGPR
treated salinized plants compared to the untreated controls. This result confirmed that the plants acquired protection
from salt challenge as a consequence of PGPR inoculation. Our results were in agreement with the findings of Gururani
et al. [63] who reported enhanced mRNA expression of
different ROS pathway genes under salt, drought, and heavy-
metal stress in PGPR inoculated potato plants. [5] F. N. Emuh, A. E. Ofuoku, and E. Oyefia, “Effect of intercrop-
ping okra (Hibiscus esclentus)with pumpkin (Curcubita maxima
Dutch ex Lam) on some growth parameters and economic yield
of maize (Zea mays) and maximization of land use in a fadama
soil,” Research Journal of Biological Sciences, vol. 1, no. 1–4, pp. 50–54, 2006. [6] L. M. Dudley, A. Ben-Gal, and U. References Shani, “Influence of plant, soil,
and water on the leaching fraction,” Vadose Zone Journal, vol. 7,
no. 2, pp. 420–425, 2008. [7] A. Cerd´a, J. Pardines, M. A. Botella, and V. Martinez, “Effect
of potassium on growth, water relations, and the inorganic and
organic solute contents for two maize cultivars grown under
saline conditions,” Journal of Plant Nutrition, vol. 18, no. 4, pp. 839–851, 1995. [8] H. M. Zadeh and M. B. Naeini, “Effects of salinity stress on the
morphology and yield of two cultivars of canola (Brassica napus
L.),” Journal of Agronomy, vol. 6, no. 3, pp. 409–414, 2007.f Acknowledgment APX The authors thank the Universiti Putra Malaysia for financial
and technical support through the research Grant “Geran
(GP-IBT/2013/9407000) Universiti Putra Malaysia.” CAT 4. Discussion However, a
defensive system called the antioxidant enzyme system is
also activated under stress conditions. This system consists of
several ROS-scavenging enzymes such as superoxide dismu-
tase (SOD), peroxidase (POD), glutathione reductase (GR),
monohydroascorbate reductase (MDHAR), ascorbate perox-
idase (APX), and catalase (CAT). These antioxidant enzymes
have the ability to remove the free radicals produced during
abiotic stress conditions in the cell [13, 58, 62]. The okra plants
inoculated with ACC deaminase-containing PGPR exhibited
significant elevation of antioxidant enzyme activities (APX,
CAT, and SOD) compared to noninoculated plants under
saline conditions (Figure 3), thus confirming that PGPR Leaf chlorophyll concentration is an indicator of salt
tolerance and responds to increasing salinity [56]. Chloro-
phyll content was significantly higher in okra plants receiving
bacterial suspension compared with control plant. In control
plants, chlorophyll is destroyed due to excessive amount of
salts, ions (Na and Cl), or reactive oxygen species (ROS)
which disturb the cellular metabolism and result in the
degeneration of cell organelles in the leaf tissue [57, 58]. On the other hand, the inoculated salt stressed okra plants
exhibited higher chlorophyll content and dark green leaves
owing to the presence of ACC deaminase-containing PGPR
isolates that maintain the photosynthetic efficiency of plants
by reducing ethylene biosynthesis. Ali and colleagues [27] BioMed Research International 8 Actin
DHAR
GR
APX
CAT
T3
T2
T1
T0
Figure 4: Effect of PGPR inoculation on DHAR, GR, APX, and CAT
transcript levels by semiquantitative RT-PCR analysis of okra leaves
under salt stress. T0: noninoculated salt treated plants, T1: UPMR2
inoculated salt treated plants, T2: UPMR18 inoculated salt treated
plants, and T3: both UPMR2 and UPMR18 inoculated salt treated
plants. Actin: positive control, APX: ascorbate peroxidase, CAT:
catalase, GR: glutathione reductase, and DHAR: dehydroascorbate
reductase. Actin
T3
T2
T1
T0 in areas where salinity is a major constraint. However, further
research is required to validate the effectiveness of this
PGPR isolate, Enterobacter sp. UPMR18, in field conditions
before recommendation for large scale okra cultivation at the
agricultural level. DHAR Conflict of Interests The authors declare that there is no conflict of interests
regarding the publication of this paper. GR 5. Conclusion [30] S. H. Habib, H. M. Saud, M. R. Ismail, and H. Kausar,
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composition, proline content, antioxidant enzymes of soybean,”
American Journal of Plant Physiology, vol. 5, no. 6, pp. 350–360,
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due to ACC deaminase-containing PGPR. The current study
showed that ACC deaminase-containing Enterobacter sp. UPMR18 emerged as the best treatment agent for enhancing
seed germination and growth of okra seedlings under salinity
stress. This may perhaps be due to a reduction in the
growth inhibitory effect of salt on okra plants through the
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“Isoenzymes of superoxide dismutase in nodules of Phaseolus
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oxygen species produced by NADPH oxidase regulate plant cell
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acterization of plant growth promoting rhizobacteria isolated
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help plants tolerate abiotic stress,” Trends in Plant Science, vol. 14, no. 1, pp. 1–4, 2009. [39] G.-X. Chen and K. Asada, “Ascorbate peroxidase in tea leaves:
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matic and molecular properties,” Plant and Cell Physiology, vol. 30, no. 7, pp. 987–998, 1989. [24] S. Mayak, T. Tirosh, and B. R. Glick, “Effect of wild-type and
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analysis of enhanced tolerance in transgenic potato plants
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“1-Aminocyclopropane-1-carboxylic acid deaminase mutants
of the plant growth promoting rhizobacterium Pseudomonas
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of plant ethylene concentrations by plant growth-promoting
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salinity stress damage by plant growth-promoting bacterial BioMed Research International 10 [44] S. Rashid, T. C. Charles, and B. R. Glick, “Isolation and char-
acterization of new plant growth-promoting bacterial endo-
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“Germination and growth response of three wheat cultivars to
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Tuning anhydrous proton conduction in single-ion polymers by crystalline ion channels
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Nature communications
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ARTICLE OPEN ARTICLE Results g
p
y
The design of proton-conducting polymers in the form of so-
called single-ion polymers would resolve the above issue, since
the current is carried only by protons, and no anion depletion
should theoretically be observed.28 However, compared to con-
ventional polymer electrolytes, where both protons and counter
anions exhibit mobility, single ion-polymers exhibit lower con-
ductivities by orders of magnitude.17–19,29,30 With this in mind,
most studies on single-ion polymers typically aim at expediting
the relaxation of polymer chains and ions.30,31 The presence of
crystalline phases in such materials, commonly formed by clus-
tered ions (dimers, quadrupoles, and so on), is avoided because it
tends to slow down polymer chain relaxation. Glass transition temperature (Tg) of PSS:Im in PSS:Im-b-PMB
was 40 °C, which decreased to 34 °C, 29 °C, and 15 °C with the
addition of ZImS, ZImS/Im, and ZImS2/Im, respectively (Sup-
plementary Fig. 1). The low Tg value is the main reason for
employing low molecular weight polymer in this study to achieve
thermodynamic equilibrium without extensive thermal annealing. However, one needs to ask the question of whether the above
reasoning is always correct. The apparent advantage of crystalline
electrolytes is the gain of molecularly arranged ion-binding
sites, which may favor the structural diffusion of protons over
vehicular diffusion.32 Therefore, the formation of well-organized
crystalline protogenic channels from single-ion
conducting
polymers should allow the fabrication of highly efficient proton-
conducting materials featuring long-range hydrogen bonding
networks.33 Structural analysis of PSS:Im-b-PMB comprising additives. Neat ZImS and neutral Im are commonly obtained as monoclinic
crystals of the P21/c space group.34,35 We found that the co-
presence of ZImS and Im results in the formation of different
crystal structures. For example, ZImS/Im afforded monoclinic
crystals of the P1m1 space group (Supplementary Fig. 2 and
Supplementary Table 1). The crystallization behavior of ZImS and Im was remarkably
altered in the presence of polymer sulfonate groups. ZImS in PSS:
Im phases did not crystallize when incorporated into PSS:Im-b-
PMB, as confirmed by the wide-angle X-ray scattering (WAXS)
profile shown in Fig. 2a. A typical amorphous halo, lacking the
evidence of crystalline phases, was clearly revealed. Results Synthesis of single-ion conducting polymers. Poly(styrene-b-
methylbutylene) (PS-b-PMB, 1.0-b-1.0 kg mol–1) block copoly-
mer was synthesized as a precursor polymer. Degree of poly-
merization (N) of PS and PMB were 10 and 14, respectively. 20
mol.% of styrene units were randomly sulfonated to yield poly
(styrene sulfonate-b-methylbutylene) (PSS-b-PMB, 1.2-b-1.0 kg
mol–1) that was subsequently doped with one equivalent of imi-
dazole (Im) relative to the quantity of sulfonic acid groups. The
above sample was denoted as PSS:Im-b-PMB, and its chemical
structure is shown in Fig. 1. PSS homopolymer with N = 10 and a
sulfonation level of 20 mol.% was synthesized and doped by Im to
prepare PSS:Im as a control sample. In addition to these efforts, much attention has been paid to
the development of anhydrous proton conductors in view of the
increased importance of achieving high proton conductivity at
elevated temperatures.17–22 As part of this standpoint, much
effort
has
been
directed
at
the
investigation
of
ionic
liquid–containing polymers, which have been identified as pro-
mising next-generation ion conductors owing to the good ther-
mal/chemical stability and high anhydrous conductivity at
elevated temperatures.17–26 Although these studies have altered the stream of research on
proton-conducting materials, the prevention of anion diffusion in
ionic liquid–containing polymers remains a major challenge.27
Likewise, the same concern has been raised for other types of
ionic conductors, i.e., metal-organic frameworks and covalent
organic frameworks, owing to the substantial quantities of
counter anions contained therein.10,11 Thus, although various
high-conductivity materials have been developed, cases of their
successful applications remain rare given that the actual current
values observed during device operation are very low.11,27 In proton-conducting polymers such as PSS:Im-b-PMB and
PSS:Im, the covalent attachment of anions to the polymer
backbone eliminates anion migration. Considering the fact that
most single-ion polymers suffer from low ionic conductivity, PSS:
Im-b-PMB and PSS:Im were additionally modified by the
introduction of a zwitterion, 3-(1-methyl-3-imidazolium) propa-
nesulfonate (ZImS), and neutral Im to allow the modulation of
ion dissociation degree and charge number density. The chemical
similarities of Im and ZImS with PSS:Im ensure good thermo-
dynamic compatibility with polymers. As denoted in Fig. 1, ZImS:
Im molar ratio was varied as 1:0, 1:1, and 2:1 (per mole of –SO3–-
Im+ moieties in the polymer), which were referred to as ZImS,
ZImS/Im, and ZImS2/Im, respectively. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 2
S
O
HN
NH
m
n
N
N
N
SO3
–
+
NH
+
+
O
O
8
14
Fig. 1 Molecular structures. Molecular structures of PSS:Im-b-PMB samples
containing different molar ratios of ZImS:Im (1:0, 1:1, and 2:1 per mole of
–SO3–-Im+ moieties in the polymer) I I
n the past few decades, much effort has been devoted to the
fabrication of proton-conducting materials1,2 ranging from
protogenic polymers,3−6 liquid crystals,7,8 to porous organic
frameworks.9–11 Although these studies were focused on different
substances, they shared the same goal of achieving high proton
conductivity via the creation of well-defined less tortuous ionic
channels in mechanically stable supports.2,12,13 In addition, the
suppression of anion diffusion in such materials has become
increasingly important, since this diffusion is related to device
polarization under applied dc voltage.14 The formation of well-defined ionic channels in mechanical
supports can be achieved by covalent linking of ionophilic
polymers to an ionophobic matrix,15,16 which results in sponta-
neous nanometer-scale self-assembly with adjustable geometry. Several studies have dealt with the design of microphase-
separated ion-containing polymers of various architectures13,17
to establish morphology-transport relationship. Fig. 1 Molecular structures. Molecular structures of PSS:Im-b-PMB samples
containing different molar ratios of ZImS:Im (1:0, 1:1, and 2:1 per mole of
–SO3–-Im+ moieties in the polymer) Tuning anhydrous proton conduction in single-ion
polymers by crystalline ion channels Onnuri Kim
1, Kyoungwook Kim
2, U. Hyeok Choi3 & Moon Jeong Park
1,2 The synthesis of high-conductivity solid-state electrolyte materials with eliminated polar-
ization loss is a great challenge. Here we show a promising potential of single-ion block
copolymers with crystalline protogenic channels as efficient proton conductors. Through the
self-organization of zwitterion, imidazole, and polystyrene sulfonate with controlled dipolar
interactions therein, the distance between neighboring proton donors and acceptors in ionic
crystals, as well as the dipolar orientation in nanoscale ionic phases was precisely tuned. This
allowed a markedly high static dielectric constant comparable to water and fast structural
diffusion of protons with a low potential barrier for single-ion polymers. The optimized
sample exhibited a high proton diffusion coefficient of 2.4 × 10–6 cm2 s–1 under anhydrous
conditions at 90 °C. 1 Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. 2 Division of Advanced Materials Science,
Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea. 3 Department of of Polymer Engineering, Pukyong National University,
Busan 608-737, Korea. These authors contributed equally: Onnuri Kim, Kyoungwook Kim. Correspondence and requests for materials should be addressed
to M.J.P. (email: moonpark@postech.ac.kr) NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 1 1 Results Distances between the neighboring Im+ and Im were marked in each crystal ZImS
Scattering intensity (a.u.)
q (nm–1)
ZImS2/Im
ZImS/Im
(002)
(020)
(–1–13)
(1–21)
(021 )
(020)
(200)
(002)
(121)
(–211)
(–1–21)
(1–31)
(–121)
(211)
(220)
(021)
a
(1–20)
(102)
(130)
(0–12)
(0–22)
(–111)
(111)
(210)
10
12
14
16
18
(–102)
(–121) q (nm–1)
10
12
14
16
18
ZImS/Im
ZImS2/Im
b b Scattering intensity (a.u.) c c
3
2
w/ZImS/Im
1
z
y
1: 5.113 Å
2: 6.060 Å
3: 5.998 Å
C
H
N
O
S w/ZImS2/Im
1
2
3
z
x
1: 7.972 Å
C
H
N
O
S
2: 6.548 Å
3: 5.372 Å Fig. 2 Self-organization of charged/dipolar moieties. a WAXS profiles of PSS:Im-b-PMB comprising various additives, measured at 25 °C, indexed with
triclinic P1 space group (ZImS/Im) and monoclinic P2 1/c space group (ZImS2/Im). b Le Bail refinement of the WAXS patterns of PSS:Im-b-PMB with
ZImS/Im and ZImS2/Im. c Molecular arrangements of SS:Im with ZImS/Im and ZImS2/Im. C, H, N, O, and S atoms are shown in black, pink, blue, red, and
yellow, respectively. Distances between the neighboring Im+ and Im were marked in each crystal Fig. 3). Dissimilar dipolar orientations were expected for the
samples depending on the molar ratio of ZImS:Im. Based on these
structures and refined lattice parameters, the molecular arrange-
ments of SS:Im, ZImS, and Im with different molar ratios are
depicted in Fig. 2c. Table 1 Ionic crystals formed in PSS:Im-b-PMB upon the
addition of ZImS/Im and ZImS2/Im
PSS:Im-b-PMB
w/ ZImS/Im
w/ ZImS2/Im
Crystal structure
Triclinic
Monoclinic
Space group
P 1
P 2 1/c
a (Å) (α)
5.70 (62.5°)
9.82 (90.0°)
b (Å) (β)
11.93 (104.7°)
11.78 (94.9°)
c (Å) (γ)
12.66 (99.6°)
8.02 (90.0°) Table 1 Ionic crystals formed in PSS:Im-b-PMB upon the
addition of ZImS/Im and ZImS2/Im While both crystals displayed an analogous distance of ~ 2.7 Å
between Im+ and –SO3– moieties in unit cells, the distances
between the neighboring Im+/Im moieties were noticeably
different, which is of importance for determining the structural
diffusion of protons. For the sample comprising ZImS/Im, the
short distances of 5.11 Å, 6.06 Å, and 5.99 Å were obtained in the
a-, b-, and c-directions of the triclinic lattice. On the contrary, this
distance increased to 5.37 Å, 7.97 Å, and 6.55 Å for the sample
with ZImS2/Im showing the monoclinic lattice. Results On the
contrary, the addition of ZImS/Im or ZImS2/Im resulted in the
appearance of a series of diffraction peaks indexed to triclinic
crystal of the P1 space group or monoclinic crystal of the P2 1/c Herein, we report a peculiar type of proton conductors based
on single-ion conducting block copolymers, where anion migra-
tion is fundamentally prevented. Notably, the self-organization of
dipolar moieties in the above conductors results in the formation
of ionic crystals confined in the nanoscale ionic phases of block
copolymers, which allows us to control the distance between
neighboring proton donors and acceptors, as well as the dipolar
orientation in ionic phases to increase the rate of structural dif-
fusion of protons along protogenic channels. NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 ZImS
Scattering intensity (a.u.)
q (nm–1)
q (nm–1)
ZImS2/Im
ZImS/Im
(002)
(020)
(–1–13)
(1–21)
(021 )
(020)
(200)
(002)
(121)
(–211)
(–1–21)
(1–31)
(–121)
(211)
(220)
(021)
a
(1–20)
c
(102)
(130)
(0–12)
(0–22)
(–111)
(111)
(210)
10
12
14
16
18
10
12
14
16
18
(–102)
(–121)
ZImS/Im
3
2
w/ZImS/Im
1
z
y
1: 5.113 Å
2: 6.060 Å
3: 5.998 Å
ZImS2/Im
C
H
N
O
S
w/ZImS2/Im
1
2
3
z
x
1: 7.972 Å
C
H
N
O
S
2: 6.548 Å
3: 5.372 Å
b
Fig. 2 Self-organization of charged/dipolar moieties. a WAXS profiles of PSS:Im-b-PMB comprising various additives, measured at 25 °C, indexed with
triclinic P1 space group (ZImS/Im) and monoclinic P2 1/c space group (ZImS2/Im). b Le Bail refinement of the WAXS patterns of PSS:Im-b-PMB with
ZImS/Im and ZImS2/Im. c Molecular arrangements of SS:Im with ZImS/Im and ZImS2/Im. C, H, N, O, and S atoms are shown in black, pink, blue, red, and
yellow, respectively. NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 PSS:Im-b-PMB displayed lamellar morphology, as shown by the
Bragg peaks (inverted black triangles) at 1q* and 2q* (q* = 2π/
d100 and d100 = 6.4 nm). The presence of ZImS in PSS:Im phases
of PSS:Im-b-PMB destabilized the microphase-separated mor-
phology, which was attributed to the weakening of interactions
between Im+ and SO3- units of polymer by the presence of neighboring ZImS moieties. The well-defined lamellar structure
was restored upon the introduction of ZImS/Im and ZImS2/Im,
and both of these samples were shown to contain ionic crystals. The addition of ZImS, ZImS/Im, and ZImS2/Im into PSS:Im-b-
PMB progressively increased the sample domain size to 6.9, 7.1,
and 7.7 nm, respectively. The swelling of PSS:Im phases with
additives can be envisaged by the reductions in the Tg value
(Supplementary Fig. 1). Scattering intensity (a.u.)
ZImS2/Im
ZImS/Im
ZImS
7 nm
103
102
101
100
10–1
0.5
1.0
1.5
2.0
2.5
Neat
q (nm–1)
Fig. 3 Effects of additives on self-assembly of PSS:Im-b-PMB. SAXS profiles
of PSS:Im-b-PMB comprising various additives, measured at 25 °C. Bragg
peaks at 1q*, 2q* (q* = 2π/d100) indicate lamellar morphology with ca. 7 nm
domain size, as schematically depicted in the inset Scattering intensity (a.u.)
ZImS2/Im
ZImS/Im
ZImS
7 nm
103
102
101
100
10–1
0.5
1.0
1.5
2.0
2.5
Neat
q (nm–1) The size and location of ionic crystals were further examined
by transmission electron microscopy (TEM) imaging. Figure 4a
and Fig. 4b display representative bright-field TEM and high-
resolution
TEM
micrographs
of
ZImS/Im–
and
ZImS2/
Im–containing PSS:Im-b-PMB, respectively, revealing that 2–3
nm-sized crystalline structures were selectively confined within
PSS:Im phases (darkened area) of lamellae for both samples. For
clarity, ionic crystals are marked by circles in each high-resolution
TEM image. The lattice parameters of ionic crystals obtained by
fast Fourier transform (FFT) analysis were in good agreement
with WAXS results. Figure 4c schematically depicts the formation of ionic crystal in
ionic phases of lamellar grains for ZImS/Im–containing PSS:Im-
b-PMB, as a representative example. PS moieties are not shown
for clarity. The crystal is formed by the co-organization of SS:Im,
ZImS, and Im molecules in amorphous polymer matrix with
controlled dipolar interactions therein. Proton transport properties. Is the formation of nano-confined
ionic crystals with long-range hydrogen-bonding networks truly
beneficial for the enhancement of structural diffusion of proton in
single-ion conducting polymers? Results Therefore, it was
concluded that long-range proton conduction networks can be
easily formed in PSS:Im phases comprising ZImS/Im. space group, respectively. Miller indices of the reflection planes
(hkl) of each crystal were obtained by Mercury DSD 3.9 software,
as given in the figure. Refinement of lattice parameters and structure factor ampli-
tudes were obtained by Le Bail refinement of WAXS intensities
and (hkl) Miller indices using a JANA2006 crystallographic
program.36 Fig. 2b shows the refinement of data for ZImS/
Im–containing and ZImS2/Im–containing PSS:Im-b-PMB and
the results are summarized in Table 1. It is apparent that the
lattice parameters of these crystals were broadly variable,
depending on the ZImS:Im molar ratio. y
g
It should be noted here that no evidence of crystal formation
was observed for PSS:Im homopolymers with embedded ZImS,
ZImS/Im, and ZImS2/Im (Supplementary Fig. 4), contrary to the
cases of block copolymers described above. Thus, the confine-
ment of dipolar moieties in nanoscale PSS domains was
concluded to play a key role in facilitating crystal formation. The incorporation of ZImS and/or Im into PSS:Im-b-PMB
affected its self-assembled morphology. Figure 3 shows repre-
sentative small angle X-ray scattering (SAXS) profiles of the
investigated samples, which were measured at room temperature
and remained unchanged over the temperature range 25–130 °C. To delineate how PSS:Im, ZImS, and neutral Im combined into
crystalline phases, the conformations of Im-doped styrene
sulfonate (SS:Im) with incorporated ZImS and neutral Im were
optimized by ab initio calculations based on the density
functional
theory
(DFT,
B3LYP/6-31 G(d))
(Supplementary 3 3 ARTICLE a Proton diffusion coefficients, b ionic conductivities, obtained for PSS:Im-b-PMB comprising additives, measured at
different temperatures. In a and b the role of ionic crystals in determining the ion transport properties of single-ion polymers was clearly shown. c
Schematics depicting dominant proton hopping mechanisms in PSS:Im-b-PMB containing ZImS/Im 2.7
a
b
1
8
101
101
100
10–1
150
120
90
60
30
T-Tg (K)
10–1
10–2
100
6
4
2
0
DH+ × 1010 (filled symbols, m2 s–1)
Conductivity (mS cm–1)
DH+/DC–H (open symbols)
2.8
2.9
3.0
1000/T (K–1)
1000/T (K–1)
3.1
2.2
2.4
2.6
2.8
3.0
w/ZImS
w/ZImS/Im
w/ZImS2/Im
ZImS
ZImS/Im
ZImS2/Im 2.7
a
1
8
6
4
2
0
DH+ × 1010 (filled symbols, m2 s–1)
DH+/DC–H (open symbols)
2.8
2.9
3.0
1000/T (K–1)
3.1
w/ZImS
w/ZImS/Im
w/ZImS2/Im b
101
101
100
10–1
150
120
90
60
30
T-Tg (K)
10–1
10–2
100
Conductivity (mS cm–1)
1000/T (K–1)
2.2
2.4
2.6
2.8
3.0
ZImS
ZImS/Im
ZImS2/Im b c
Ionic phases
Nonionic phases
H+ hopping
PSS:Im-b-PMB
Fig. 5 Proton transport properties. a Proton diffusion coefficients, b ionic conductivities, obtained for PSS:Im-b-PMB comprising additives, measured at
different temperatures. In a and b the role of ionic crystals in determining the ion transport properties of single-ion polymers was clearly shown. c
Schematics depicting dominant proton hopping mechanisms in PSS:Im-b-PMB containing ZImS/Im c
Ionic phases
Nonionic phases
PSS:Im-b-PMB c Ionic phases
Nonionic phases
PSS:Im-b-PMB PSS:Im-b-PMB Fig. 5 Proton transport properties. a Proton diffusion coefficients, b ionic conductivities, obtained for PSS:Im-b-PMB comprising additives, measured at
different temperatures. In a and b the role of ionic crystals in determining the ion transport properties of single-ion polymers was clearly shown. c
Schematics depicting dominant proton hopping mechanisms in PSS:Im-b-PMB containing ZImS/Im Tammann equation (solid lines in Fig. 5b) further suggested
dissimilar activation barriers to ion conduction, i.e., 1127 K for
ZImS, 692 K for ZImS/Im, and 990 K for ZImS2/Im. The higher
normalized conductivity and lower activation barrier obtained
with ZImS/Im are notable. spin-echo (PGSE) NMR spectroscopy. Proton diffusion coeffi-
cient (DH+) was then calculated based on proton dissociation
degree (x) of the samples, determined as 52% (with ZImS), 65%
(with ZImS/Im), and 54% (with ZImS2/Im) by confocal Raman
spectroscopy (Supplementary Fig. 5). The considerably high x
value observed for PSS:Im-b-PMB with ZImS/Im is particularly
noteworthy. Based on proton diffusion coefficients in Fig. 5a and ionic
conductivities in Fig. ARTICLE To answer this question, self-
diffusion coefficients associated with N–H (DN–H) and C–H
(DC–H) protons of Im+ and neutral Im were measured for PSS:
Im-b-PMB with different additives using 1H pulsed-field gradient Fig. 3 Effects of additives on self-assembly of PSS:Im-b-PMB. SAXS profiles
of PSS:Im-b-PMB comprising various additives, measured at 25 °C. Bragg
peaks at 1q*, 2q* (q* = 2π/d100) indicate lamellar morphology with ca. 7 nm
domain size, as schematically depicted in the inset 0.21 nm
(002)
(020)
(002)
= 96°
(020)
(022)
(022)
(022)
(022)
c
b
(022)
(002)
(020)
(002)
0.24 nm
(020)
(022)
= 61°
a ZImS/Im
ZImS2/Im
Non-ionic
Non-ionic
Non-ionic
Ionic
Ionic
Ionic
Fig. 4 Ionic crystals in self-assembled lamellar grains. Bright-field TEM, high-resolution TEM micrographs, and FFT patterns obtained from PSS:Im-b-PMB
with the addition of a ZImS/Im and b ZImS2/Im. The darkened area of lamellae indicates PSS:Im phases for both samples and ionic crystals are marked by
circles in each TEM image for clarity. Scale bars in bright-field TEM and high-resolution TEM images are 20 nm and 2 nm, respectively. c Schematic
illustration of the formation of ionic crystal in ionic phases of lamellar grains for PSS:Im-b-PMB with ZImS/Im (022)
(002)
(020)
(002)
0.24 nm
(020)
(022)
= 61°
a ZImS/Im a c Non-ionic
Non-ionic
Non-ionic
Ionic
Ionic
Ionic 0.21 nm Fig. 4 Ionic crystals in self-assembled lamellar grains. Bright-field TEM, high-resolution TEM micrographs, and FFT patterns obtained from PSS:Im-b-PMB
with the addition of a ZImS/Im and b ZImS2/Im. The darkened area of lamellae indicates PSS:Im phases for both samples and ionic crystals are marked by
circles in each TEM image for clarity. Scale bars in bright-field TEM and high-resolution TEM images are 20 nm and 2 nm, respectively. c Schematic
illustration of the formation of ionic crystal in ionic phases of lamellar grains for PSS:Im-b-PMB with ZImS/Im NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 2.7
c
a
b
1
8
101
101
100
10–1
150
120
90
60
30
T-Tg (K)
10–1
10–2
100
6
4
2
0
DH+ × 1010 (filled symbols, m2 s–1)
Conductivity (mS cm–1)
DH+/DC–H (open symbols)
2.8
2.9
3.0
1000/T (K–1)
Ionic phases
Nonionic phases
H+ hopping
PSS:Im-b-PMB
1000/T (K–1)
3.1
2.2
2.4
2.6
2.8
3.0
w/ZImS
w/ZImS/Im
w/ZImS2/Im
ZImS
ZImS/Im
ZImS2/Im
Fig. 5 Proton transport properties. ARTICLE 5b, Haven ratios were calculated for each
sample at 60 °C and 90 °C. This is to quantify the dissimilar ion
dissociation behavior of each sample by evaluating how much it
deviates from unity. At 60 °C, the Haven ratios were 7.7, 7.1, and
5.2 for PSS:Im-b-PMB samples with ZImS, ZImS/Im and ZImS2/
Im, respectively. Upon heating the samples to 90 °C, at the
temperature well-above the Tg values, the Haven ratios decreased
to 6.2 (ZImS), 2.3 (ZImS/Im), and 1.2 (ZImS2/Im). Significant
reductions in the Haven ratios seen only for crystal-forming
samples are particularly noteworthy, indicating high dissociation
degree of ions bound in the crystal structure to contribute to
efficient proton transport, as schematically illustrated in Fig. 5c. Note in passing that the grain size of lamellar structures decreased
in the order of ZImS2/Im > ZImS/Im > ZImS. This would also
have an impact on the Haven ratio, but the concrete under-
pinning remains as a future study. y
Figure 5a shows the DH+ values and DH+ / DC-H ratios of
investigated samples, clearly displaying the role of ionic crystals in
determining the proton diffusion behavior in single-ion polymers. Note that the accessible temperature window of the NMR
spectrometer was 25–90 °C because it was calibrated using
ethylene glycol standard in water. Overall, DH+ values obtained
for the sample comprising ZImS were lower than those obtained
with ZImS/Im and ZImS2/Im by factors of 2–5. Notably, the DH+
/ DC-H ratio was as high as ~ 7.6 for the sample comprising ZImS/
Im, whereas much lower values of 4.5 and 1.9 were obtained for
ZImS2/Im-containing and ZImS-containing analogs, respectively. This finding suggests that proton hopping was most pronounced
in the sample comprising ZImS/Im because of the shortest
distances between protogenic moieties therein (Fig. 2c). Figure 5b shows the temperature-dependent ionic conductivity
of PSS:Im-b-PMB with different additives, revealing that this
quantity decreased in the order of ZImS2/Im > ZImS/Im > ZImS,
in good agreement with the results of diffusivity measurements. With excluded influence of Tg values of the samples on
conductivity, as shown in inset plot of Fig. 5b, proton transport
efficiency was highest for the ZImS/Im-containing sample if the
sample temperature was lower than 90 °C. The lowest normalized
conductivity was seen for the ZImS-embedded one for entire
temperature window examined. NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications Discussion
W
l We explored the dielectric relaxation behavior of the above-
mentioned samples. Figure 6a shows static dielectric constant (εs)
of the samples, measured at different temperatures (dielectric
permittivity spectra are given in Supplementary Fig. 7). ZImS/
Im–containing PSS:Im-b-PMB exhibited high εs values over 78
while the lowest εs value of 13 was observed with the incor-
poration of ZImS. It should be noted that εs value of neat PSS:Im-
b-PMB was as low as 5. The sample with ZImS2/Im exhibited g ≈1 and Vp/Vm > 1,
ascribed to the increased density of dipoles, resulting in a sig-
nificant dipole overlap to restrict dipolar correlation. This leads us
to conclude that the dipole correlation affects the structure
(amorphous
vs. crystalline)
and
proton
hopping
distance
(ordered dipolar moieties reduce the distance). The origin of the differences in εs values was elucidated by
analyzing local directional correlations between neighboring
dipoles for each sample. Based on DFT calculations, we deter-
mined three major dipole moments of ion pair (SO3--Im+, 7 D),
ZImS (17.5 D), and neutral pair (Im-ZImS, 19.3 D), as listed in
Supplementary Table 2. This computational method also enabled
us to estimate the optimized dipole structures, as provided in
Supplementary Fig. 3. The Kirkwood-Fröhlich g-factor was cal-
culated by equation (1)30 using the number density of dipolar
moieties (vi) and the dipole moment (mi) (listed in Supplemen-
tary Table 2). The overlap of dipole polarizability volumes,
defined as the ratio of Debye’s polarizability volume Vp to
molecular volume Vm, was calculated by equation (2). Repre-
sentative Vp/Vm values of each sample obtained at 60 °C are listed
in Supplementary Table 2. It is worth noting that no substantial enhancement of εs was
observed for PSS:Im homopolymers with various additives
(Supplementary Fig. 8). This finding implies that synergistic
dipole alignments occurred only in samples containing nano-
confined ionic crystals, the formation of which is thus of key
importance for improving proton transport properties. g
We would like to conclude this paper by commenting on the
single-ion properties of PSS:Im-b-PMB comprising additives. Because zwitterion is composed of covalently connected cation
and anion, it is electroneutral. Nevertheless, the excessive use of
zwitterion in polymer electrolytes may result in charge depletion
at electrolyte/electrode interfaces at a given voltage. However, this
was avoided in our polymers because the ZImS is packed in ionic
crystals and further confined within PSS:Im domains, as con-
firmed by polarization experiments (Supplementary Fig. ARTICLE Model fits by Vogel-Fulcher- It is worth noting that we confirmed no substantial effects of
the
molecular
weight
of
PSS:Im-b-PMB
on
ionic
crystal
formation and conductivity enhancement. Supplementary Fig. 6
shows scattering profiles and temperature-dependent conductiv-
ity data obtained from PSS:Im-b-PMB (20–39 kg mol−1, a
sulfonation level of 25 mol.%, lamellae, d100=56 nm) with
additives. Ionic crystals were again formed only with ZImS/Im
and ZImS2/Im, leading to markedly enhanced ionic conductivity,
compared with amorphous sample comprising ZImS. This NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 90
a
b
2.0
1.5
g factor
1.0
0.5
0.0
60
s
30
0
2.6
2.8
3.0
1000/T (K–1)
3.2
w/ZImS
w/ZImS/Im
w/ZImS2/Im
w/ZImS
w/ZImS/Im
w/ZImS2/Im
3.4
2.6
2.8
3.0
1000/T (K–1)
3.2
Fig. 6 Dielectric properties. a Static dielectric constant and b Kirkwood-Fröhlich g factor obtained for PSS:Im-b-PMB comprising additives, measured at
different temperatures 90
a
60
s
30
0
2.6
2.8
3.0
1000/T (K–1)
3.2
w/ZImS
w/ZImS/Im
w/ZImS2/Im
3.4 b
2.0
1.5
g factor
1.0
0.5
0.0
w/ZImS
w/ZImS/Im
w/ZImS2/Im
4
2.6
2.8
3.0
1000/T (K–1)
3.2
hlich g factor obtained for PSS:Im-b-PMB comprising additives measured at b tatic dielectric constant and b Kirkwood-Fröhlich g factor obtained for PSS:Im-b-PMB comprising additives, measured Fig. 6 Dielectric properties. a Static dielectric constant and b Kirkwood-Fröhlich g factor obtained for PSS:Im-b-PMB comprising additives, measured at
different temperatures samples owing to the presence of SO3––Im+ ion pairs. With
ZImS addition, the low g-factor of < 1 and Vp/Vm < 1 were
obtained, which indicated that the ion pair (SO3––Im+) and
ZImS tend to form a quadrupole (i.e., two dipoles are antiparallel
each other), leading to offset of dipole moment by dipole-dipole
attraction. This is consistent with the lowest εs value of PSS:Im-b-
PMB containing ZImS (Fig. 6a). finding suggests the crucial role of co-organization of neutral Im
and ZImS in developing the unique crystalline structures in PSS:
Im phases of PSS:Im-b-PMB, which is intimately related to the
improved ion transport properties. Recent work of Winey and coworkers37 may be relevant to our
results given that ionic moieties (–SO3-–H3O+) are confined by
surrounding crystalline matrix (polyethylene backbones). How-
ever, the fundamental difference lies in the fact that PSS:Im-b-
PMB block copolymers have essentially amorphous PS backbones
and PSS chains directly involved in the ionic crystal formation for
participation in proton transport. ARTICLE On the contrary, the highest g-factor of ~ 2 was obtained for
PSS:Im-b-PMB comprising ZImS/Im to enhance the εs value. This
can be rationalized by the DFT calculations (Supplementary
Fig. 3), displaying that neutral Im breaks the quadrupole to make
surrounding dipoles are arranged parallelly, thereby providing the
system with a net dipole moment. The net dipole moment is
intimately connected to close packing of neighboring dipolar
moieties and the dipole-dipole interaction acts as an enthalpic
contribution to the ionic crystal formation. This intriguing
dipolar orientation was further envisaged by the Vp/Vm ratio of
~1 (Supplementary Table 2), signaling that ion pairs were forced
to strongly interact at the overlap point.30 NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications References 1. Jiménez-García, L., Kaltbeitzel, A., Pisula, W., Gutmann, J. S., Klapper, M. &
Müllen, K. Phosphonated hexaphenylbenzene: a crystalline proton conductor. Angew. Chem. Int. Ed. 48, 9951 (2009). 2. Braun, A. & Chen, Q. Experimental neutron scattering evidence for proton
polaron in hydrated metal oxide proton conductors. Nat. Commun. 8, 15830
(2017). Preparation of single-ion conducting polymers. Inhibitor-free anhydrous THF
( ≥99.9%) and anhydrous methanol (99.8%) were purchased from Sigma-Aldrich
and used without further purification. Predetermined quantities of PSS:Im-b-PMB
(or PSS:Im), ZImS, and neutral Im were dissolved into 80/20 vol.% methanol/THF
mixtures to prepare ca. 5 wt% solutions using the balance (Mettler Toledo, ME 204)
inside the argon-filled glove box. Under argon atmosphere, solvents were slowly
evaporated at room temperature for 2 days and were further exposed to vacuum at
70 °C for 7 days. 3. Kreuer, K. D. On the development of proton conducting polymer membranes
for hydrogen and methanol fuel cells. J. Membr. Sci. 185, 29–39 (2001). 4. Hickner, M. A., Ghassemi, H., Kim, Y. S., Einsla, B. R. & McGrath, J. E. Alternative polymer systems for proton exchange membranes (PEMs). Chem. Rev. 104, 4587–4612 (2004). 5. Çelik, S. Ü., Bozkurt, A. & Hosseini, S. S. Alternatives toward proton
conductive anhydrous membranes for fuel cells: Heterocyclic protogenic
solvents comprising polymer electrolytes. Prog. Polym. Sci. 37, 1265–1291
(2012). X-ray scattering (SAXS and WAXS) experiments. Synchrotron SAXS and
WAXS measurements on the single-ion polymers were performed using the PLS-II
4 C and 9 A beamlines at the Pohang Accelerator Laboratory (PAL), equipped with
temperature-controlled sample stage and two-dimensional detector. The samples
were laminated into an air-tight cell inside argon-filled glove box to avoid the issue
of water contamination. Three different sample-to-detector distances of 2.0 m, 0.2
m, and 0.1 m were used to cover a wide scattering wave vector q (q = 4π sin(θ/2)/λ,
where θ is the scattering angle). The scattering data were azimuthally averaged to
obtain intensity versus q. 6. Kusoglu, A. & Weber, A. Z. New insights into perfluorinated sulfonic-acid
ionomers. Chem. Rev. 117, 987–1104 (2017). 7. Soberats, B., Yoshio, M., Ichikawa, T., Taguchi, S., Ohno, H. & Kato, T. 3D
anhydrous proton-transporting nanochannels formed by self-assembly of
liquid crystals composed of a sulfobetaine and a sulfonic Acid. J. Am. Chem. Soc. 135, 15286–15289 (2013). 8. Kobayashi, T., Ichikawa, T., Kato, T. & Ohno, H. Development of glassy
bicontinuous cubic liquid crystals for solid proton-conductive materials. Adv. Method
S
th
i Synthesis of sulfonated polymers. A poly(styrene-b-methylbutylene) (PS-b-
PMB, 1.0-b-1.0 kg mol-1) precursor block copolymer was synthesized by sequential
anionic polymerization of styrene (ReagentPlus®, 99.9%, Sigma-Aldrich) and iso-
prene ( ≥99%, Sigma-Aldrich) using sec-butyllithium initiator in cyclohexane,
followed by selective hydrogenation of isoprene units using Ni-Al catalyst. A
polystyrene (PS, 1.0 kg mol-1) homopolymer was also prepared by anionic poly-
merization as a control sample. The absolute molecular weights of PS-b-PMB block
copolymer and PS homopolymer were characterized by end-group analysis using
1H Nuclear Magnetic Resonance (1H-NMR, Bruker AVB-300) spectroscopy in
CDCl3. Size exclusion chromatography (SEC, Waters Breeze 2 HPLC) was used to
characterize polydispersity indices of PS-b-PMB and PS, which were determined to
be 1.03 and 1.02, respectively, with PS standards and tetrahydrofuran (THF) eluent. Subsequent sulfonation reaction of PS-b-PMB block copolymer and PS homo-
polymer using acetic sulfate in anhydrous dichloromethane ( ≥99.8%, Sigma-
Aldrich) yielded PSS-b-PMB block copolymer and PSS homopolymer. The sulfo-
nation level was determined to be 20 mol.% by 1H-NMR in acetone-d6. The PSS-b-
PMB and PSS were doped with equivalent imidazole ( ≥99%, Sigma-Aldrich) to
the mole of sulfonic acid group of polymer using methanol to yield PSS:Im-b-PMB
block copolymer and PSS:Im homopolymer. Dielectric relaxation spectroscopy. The dielectric permittivity spectra of the
samples (the same samples employed for impedance spectroscopy measurements)
were acquired using a Novocontrol GmbH Concept 40 broadband dielectric
spectrometer, equipped with Novocontrol sample chamber, under a sinusoidal
voltage of 0.1 V in a frequency range of 10–107 Hz. The sample cell consists of
two polished brass electrodes (10 mm-diameter top electrode and 30 mm
diameter bottom electrode) and a silica spacer. Samples were annealed at 120 °C
for 1 h prior to the measurements to achieve good contact of the sample with
electrodes. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-07503-4 ARTICLE interactions and effective confinements of ionic moieties in
polymer matrix can inhibit leaching of constituents over time, our
polymers have potential advantages over conventional ionic
liquid-containing polymer electrolytes. Our approach should find
wide applicability in various electrochemical applications. Raman spectroscope (WITec, Ulm, Germany), equipped with a HeNe laser, under
low laser excitation power below 3 mW. The spatial resolution of the spectrometer
was 250 nm. The samples were placed on dimple-patterned slide glasses inside
glovebox and covered with a window glass to avoid the contact of samples with air. Self-diffusion coefficient measurements. Pulsed gradient spin echo (PGSE) 1H-
NMR experiments on PSS:Im-b-PMB block copolymers comprising various
additives were performed using a Bruker AVB-300 spectrometer. The samples were
loaded into 4 mm (o.d.) NMR microtubes in an argon-filled glovebox and were
sealed with caps. Time interval of the field gradient and duration time between two
gradient pulses were in the range 1–10 ms and 0.1–0.3 s, respectively. The samples
were equilibrated for 1 h at each temperature prior to the measurements, which was
calibrated using ethylene glycol standard. In summary, we demonstrate that proton transport rate in
single-ion conducting polymers can be enhanced by creating
high-dielectric-constant crystalline protogenic channels in an
amorphous polymer matrix. Specifically, the strong dipolar
interactions in nanoscale ionic phases of block copolymers
resulted in the formation of 2–3 nm-sized ionic crystals packed
within. The developed approach allowed the realization of
dominant structural diffusion of protons, improved ion dis-
sociation degree, and significantly enhanced dielectric constant of
the single-ion polymers. Ionic conductivity measurements. The through-plane conductivities of PSS:Im-b-
PMB comprising additives were measured by impedance spectroscopy using
PARSTAT 2273 and Solartron 1260 A. The dimension of solvent-cast membrane
was 0.8 cm × 0.8 cm × 200 μm, which was placed inside engraved counter Pt elec-
trode and covered by Pt working electrode with Kapton spacers inside the glove
box. Samples were annealed for 30 min at each temperature and data were collected
in a wide frequency range of 10–105 Hz. Received: 6 August 2018 Accepted: 5 November 2018 Received: 6 August 2018 Accepted: 5 November 2018 Synthesis of zwitterion. 3-(1-methyl-3-imidazolium) propanesulfonate (ZImS)
was synthesized by mixing 1-methylimidazole (ReagentPlus®, 99.9%, Sigma-
Aldrich) and 1,3-propanesultone ( ≥99%, Sigma-Aldrich) in acetone ( ≥99.5%,
Sigma-Aldrich) at room temperature. After 5 days of stirring, the mixtures were
purified by repeated filtration and precipitation. White powder was recovered by
vacuum drying at 60 °C and the successful synthesis of ZImS was confirmed by
combining 1H-NMR and Fourier transform Infrared (FT-IR, a Spectrum Two,
PerkinElmer, USA) spectroscopy experiments. Data availability The data supporting the findings of this study are available within the paper and its
Supplementary Information. Other datasets analyzed for the present study are
available from the corresponding author on reasonable request. Received: 6 August 2018 Accepted: 5 November 2018 Discussion
W
l 9). Note
that the content of ZImS is low as 13 wt% for PSS:Im-b-PMB
containing ZImS/Im (neutral Im content is 4 wt%). g ¼ 9ε0kT
P
i vim2
i
εs ε1
ð
Þ 2εs þ ε1
ð
Þ
εs ε1 þ 2
ð
Þ2
ð1Þ
Vp
Vm
¼
X
i
vim2
i
12πε0kT
ð2Þ ð1Þ ð2Þ Although a large number of ion-containing polymers have
been reported in literature, cases of developing well-defined
protogenic crystals with in-depth underpinning of conductivity-
structure relationship remains rare. Given that strong dipolar Figure 6b illustrates the temperature dependence of the g-fac-
tor, showing that ionic orientational polarization is present in all 6 Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ 25. Wang, S. W., Liu, W. & Colby, R. H. Counterion dynamics in polyurethane-
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Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. 29. Ryu, S.-W. et al. Effect of counter ion placement on conductivity in single-ion
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8130–8137 (2008). Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant
funded by the Korea government (MEST) (No. NRF-2017R1A2B3004763) and by the
Korea government (MSIT) (No. NRF-2017R1A5A1015365). We also acknowledge the
Creative Materials Discovery Program through the NRF funded by Ministry of Science
and ICT (2018M3D1A1058624) and the Global Frontier R&D program on Center for
Multiscale Energy System through the NRF funded by the Ministry of Education, Science
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crystalline structures analysis. K.K performed Le Bail refinement and charge-flipping
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conductors. Nat. Commun. 8, 15893 (2017). Additional information q
23. Ueki, T. & Watanabe, M. Macromolecules in ionic liquids: progress,
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3750–3756 (2010). Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. Origin of fast ion diffusion in super-ionic
conductors. Nat. Commun. 8, 15893 (2017). © The Author(s) 2018 © The Author(s) 2018 NATURE COMMUNICATIONS | (2018) 9:5029 | DOI: 10.1038/s41467-018-07503-4 | www.nature.com/naturecommunications 8 8
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https://openalex.org/W4384282410
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https://www.researchsquare.com/article/rs-3156608/latest.pdf
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English
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Out-of-hospital cardiac arrest in children: An epidemiological study based on the German Resuscitation Registry identifying modifiable factors for return of spontaneous circulation
|
Research Square (Research Square)
| 2,023
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cc-by
| 8,550
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Out-of-hospital cardiac arrest in ch
epidemiological study based on the
Resuscitation Registry identifying m
for return of spontaneous circulatio
Stephan Katzenschlager
(
stephan.katzenschlager@med.uni-heide
Heidelberg University Hospital
Inga K. Kelpanides
Oslo University Hospital
Patrick Ristau
University Hospital Schleswig-Holstein
Matthias Huck
Heidelberg University Hospital
Stephan Seewald
University Hospital Schleswig-Holstein
Sebastian Brenner
University Clinic Carl Gustav Carus
Florian Hoffmann
Dr. von Hauner Children's Hospital, Ludwig- Maximilians-University
Jan Wnent
University Hospital Schleswig-Holstein
Jo Kramer-Johansen
University of Oslo
Ingvild B. M. Tjelmeland
University of Oslo
Markus A. Weigand
Heidelberg University Hospital
Jan-Thorsten Gräsner
University Hospital Schleswig-Holstein
Erik Popp
Heidelberg University Hospital Out-of-hospital cardiac arrest in children: An
epidemiological study based on the German
Resuscitation Registry identifying modifiable factors
for return of spontaneous circulation Matthias Huck
Heidelberg University Hospital Stephan Seewald
University Hospital Schleswig-Holstein Sebastian Brenner
University Clinic Carl Gustav Carus Florian Hoffmann Research Article Research Article Page 1/24 Keywords: Out of hospital cardiac arrest, resuscitation, pediatric cardiac arrest, emergency medical service,
epidemiology Keywords: Out of hospital cardiac arrest, resuscitation, pediatric cardiac arrest, emergency medical service,
epidemiology Results OHCA in children aged < 1 year had the highest incidence per 100 000 inhabitants, with 23.42, accounting
for 42.0% of all included cases. Overall, hypoxia was the leading presumed cause of OHCA; whereas trauma
and drowning accounted for a high proportion in children aged > 1 year. Bystander-witnessed OHCA and
bystander CPR rate were highest in children aged 1–4 years, with 43.9% and 62.3%, respectively. In
reference to EMS-started CPR, bystander CPR with ventilation were associated with an increased odds ratio
for ROSC at hospital admission after adjusting for age, sex, year of OHCA and location of OHCA. Conclusion This study provides an epidemiological overview of OHCA in children in Germany and identifies bystander
CPR with ventilation as one primary factor for survival. Abstract This work provides an epidemiological overview of out-of-hospital cardiac arrest (OHCA) in children in
Germany between 2007 and 2021. We wanted to identify modifiable factors associated with survival. Methods Data from the German Resuscitation Registry (GRR) was used, and we included patients registered between
1st January 2007 and 31st December 2021. We included children aged > 7 days and < 18 years, where
cardiopulmonary resuscitation (CPR) was started and treatment was continued by emergency medical
services (EMS). Incidences and descriptive analyses are presented for the overall cohort and each age
group. Multivariate binary logistic regression was performed on the whole cohort to determine the influence
of (1) CPR with/without ventilation started by bystander, (2) OHCA witnessed status, and (3) nighttime on
the outcome hospital admission with ROSC. Posted Date: July 14th, 2023 Posted Date: July 14th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3156608/v1 DOI: https://doi.org/10.21203/rs.3.rs-3156608/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read
Full License Version of Record: A version of this preprint was published at Critical Care on September 7th, 2023. See the
published version at https://doi.org/10.1186/s13054-023-04630-3. Page 2/24 Trial registrations German Clinical Trial Register: DRKS00030989, December 28th 2022. German Clinical Trial Register: DRKS00030989, December 28th 2022. Study design In this retrospective cross-sectional cohort study, patients under the age of 18 with an OHCA from January
1st 2007 until December 31st 2021 were included. This analysis was performed with data from GRR and
followed the Strengthening the Reporting of Observational studies in Epidemiology (STROBE)
guidelines [16]. Introduction Out-of-hospital cardiac arrest (OHCA) in children is a rare event with a high burden for the victim, caregivers,
rescue personnel, and society. Globally, the incidence of OHCA in children remains low, with around
8/100,000 inhabitants per year [1–3]; however, incidence in < 1 year-olds is substantially higher [3–5]. The Page 3/24 low incidence compared to OHCA in adults, combined with ethical issues, has resulted in a lack of
interventional studies on OHCA in children [6]. Survival rates have increased for in-hospital cardiac arrests,
but this success did not show higher rates of survival with good neurologic outcome [7]. Emergency medical
service (EMS) crews are challenged by the heterogeneity of different age groups, facing wide anatomical
and physiological ranges. Aetiologies of OHCA in children differ from those in adults, as hypoxia is one of
the leading presumed causes [8, 9]. However, the aetiology varies across the paediatric OHCA population,
with an increase in trauma in the adolescent group and a decrease in respiratory failure and hypoxemia. Due to those differences, compared to adults, treatment changes and the focus lies more on ventilation and
oxygenation rather than early defibrillation [10]. As opposed to the assumption of a cardiac cause in adult
OHCA without any other obious causes, an universal presumed cause of cardiac arrest in children has not
been identified; leaving caregivers and researchers with another challenge in the endeavour to improve the
treatment of OHCA in children. Although an Utstein recommendation on reporting OHCA in children exists [11], comparability between
different studies is limited due to the different age groups reported. It is crucial to differentiate between
respiratory and cardiopulmonary arrest; however, this is not always possible, especially in retrospective
cohort studies. While there is an increase in the analyses of specific treatment strategies [2, 12, 13], recent
large epidemiological studies across a whole nation or a continent are lacking. These can help EMS
systems to tailor their pediatric advanced life support (PALS) training and provide the best possible care. This paper is, to our knowledge, the first to give an epidemiologic overview of OHCA in children, including
patients in Germany between 2007 and 2021, and analyse modifiable factors for the occurrence sustained
ROSC at hospital admission. The German Resuscitation Registry (GRR) is one of Europe biggest cardiac
arrest registries [14, 15], therefore, significantly contributing to the scarce population data and literature on
OHCA in children. EMS system in Germany The EMS system is a two-tier system with ambulance crews consisting of at least a paramedic (German:
'Notfallsanitäter') and an emergency medical technician (EMT) (German: 'Rettungssanitäter'). The second
tier that is always dispatched to suspected cardiac arrests, is a physician response unit staffed with an
emergency physician and a paramedic. The physician response unit can either be a rapid response car or an
air ambulance. In addition, some regions have implemented first responder systems. Paramedics are trained
in advanced life support (ALS) treatment of OHCA in adults and paediatrics. Ultimately, the emergency
physician makes the treatment decisions on scene. German Resuscitation Registry (GRR) GRR covered approximately 37.4% (31.1 million) of the population of Germany by the end of 2021. Out of
the proportion covered within GRR, 16.7% (5.2 million) were under the age of 18 years. This increased from
only 1.3 million children covered within the registry in 2007 (Supplement Table 1a). To date, about 100 EMS
organisations are participating in GRR on a voluntarily basis. In addition to sites in Germany, some Austrian
and Swiss EMS systems choose to participate in the GRR. For the acquisition of pre-hospital data, a dataset
with 118 variables in concordance with the Utstein recommendations exists [17]. This dataset focuses on Page 4/24 Page 4/24 time intervals, pre-hospital interventions, patient specific factors such as age, known diseases, pre-
emergency state according to the American Society of Anesthesiology physical status classification (ASA
classification) [18], and patient status at hospital admission. ASA classification ranged from 1 to 5 and was
determined by the treating physician according to the information available on scene. Preexisting
conditions were entered into GRR by the treating physician on scene. Due to the structure of the database,
up to three existing conditions could be entered. ‘Reference centers’ are EMS systems that provide high data
quality for both, pre- and in-hospital data [19]. All pre-hospital data are collected and entered into GRR by the
treating emergency physician. Inclusion and exclusion criteria We included all patients >7 days and <18 years of age suffering an OHCA with resuscitation attempts and
continued treatment by EMS in Germany. No restrictions for sex or the aetiology of OHCA were applied. Data from participants outside of Germany and data on patients with an incorrect or missing age were
excluded. Furthermore, cases where the patient was declared deceased on arrival (DoA) by the EMS team
without resuscitation attempts by neither a bystander nor EMS were excluded. Descriptive baseline
characteristics of patients <=7 days and children declared DoA are presented in the supplementary material
(Supplement Table 2 and 3). EMS time intervals The following time intervals were calculated based on the times reported by the EMS system: The following time intervals were calculated based on the times reported by The following time intervals were calculated based on the times reported by the EMS system: - Response time, defined as the interval from the emergency call started until the first EMS team arrived at
the destination defined by dispatch - On-scene time, defined as the interval from the first EMS team on scene until transport to the hospital was
either started or the patient was declared dead - Transport time, defined as the interval from the start of the transport by EMS until arrival at the hospital Page 5/24
- EMS treatment time, defined as the interval from the arrival of the first EMS team until arrival at the
hospital (= On scene interval + transport interval). When the patient diseased and was not transported, EMS
treatment duration was defined as the on-scene interval. Page 5/24
- EMS treatment time, defined as the interval from the arrival of the first EMS team until arrival at the
hospital (= On scene interval + transport interval). When the patient diseased and was not transported, EMS
treatment duration was defined as the on-scene interval. - Duration to first shock, defined as the interval from the emergency call until anyone delivered the first
shock in patients with shockable rhythm Data synthesis and statistical analysis All analyses were performed using SPSS (Statistical Product and Services Solutions, Version 28, SPSS Inc.,
Chicago, IL, USA). Baseline and demographic characteristics are presented as incidence per 100,000
children and frequencies for the overall cohort and as frequencies only for the age-specific subgroups. Continuous variables are summarised as means with standard deviations (SD) or medians and interquartile
ranges (Q1-Q3). Incidence was calculated based on the official population calculations from the German Federal Statistical
Office [20]. We assumed that the proportion of children is equally distributed across Germany. The complete
data for incidence calculation is available in the supplementary material (Supplement Table 1b). The four
age groups were categorised according to the latest Utstein recommendation, with >7 days to <1 year
(henceforth named <1 year), 1 to 4 years, 5 to 12 years, and 13 to <18 years [11]. Treatment before EMS
arrival was defined as interventions performed either by bystanders or first responders. Nighttime was
defined between 10pm to 6am. First rhythm assessed was defined as the first rhythm when CPR was
commenced. Bradycardia was defined as a heartrate below 60 beats per minute; due to the structure of
GRR, this is only possible in patients below 14 years of age. EMS time intervals were analysed using Mann-Whitney-U due to the non-normal distribution of the data. The
effect size was calculated as the differences between pseudo medians with nonparametric 95% confidence
intervals (CI) using the Hodges-Lehmann method [21]. Multivariate binary regression analysis was used to calculate the adjusted odds ratio (aOR) for the factors
'chest compression started by', 'chest compression and ventilation started by', 'OHCA witnessed by', and
'nighttime' for the outcome 'ROSC at hospital admission'. Regression analysis was adjustmented for age
(per year, continuous variable), sex, year of OHCA (continuous variable) and location of OHCA. Missing data were not imputed. A two-sided level of significance < 0.05 was considered statistically
significant. Steering committee and ethical approval According to the regulations of the GRR, a study protocol was drafted and approved by the scientific
advisory board before the beginning of the data analysis (Ref. number: 2022-06). This study was approved by the University Heidelberg ethics committee with reference number S-085/2022,
and registered at the German Clinical Trial Register (www.drks.de—study number DRKS00030989). Demographic overview Demographic overview Page 6/24 Over the 15 years, 235,537 OHCAs were recorded in GRR. The complete list of excluded cases with reasons
is available in Figure 1. Figure 1 – Study flowchart demonstrating included cases and incidences per age group; *1 case with an
unknown immediate outcome. Abbreviations: OHCA = out-of-hospital cardiac arrest; GRR = German
Resuscitation Registry; CPR = cardiopulmonary resuscitation The main reason for exclusion was age ≥18 years (n = 233,142; 98.9%). Furthermore, 476 cases where no
CPR was performed due to obvious signs of death and 97 cases with age ≤7 days were excluded. (Supplement Table 2 and 3). In total, 1,740 datasets of children suffering from OHCA and receiving CPR
were included in this study. This results in an overall incidence across all age groups of 3.08/100,000
children. There was slight variance within the incidence per year with a minimum of 1.68 in 2010 and a
maximum of 4.26 in 2019. Incidence was highest in children aged <1 year, with 23.42/100,000 (Figure 1). Non shockable rhythms had the highest overall incidence, with 2.46/100,000 for initial rhythm. In contrast,
the incidence of ventricular fibrillation (VF) was 0.29/100,000. The highest proportion of VF as initial rhythm
was found in children aged 13 to <18 years, with 21.4% (n=74) (Figure 2a, Table 1). Across all age groups, "Home" was the most common location of OHCA with 1.91/100,000. In the age group
13 to <18 years, "Home" and "Public place" had proportions of 49.5% (n=163) and 41.3% (n=136),
respectively (Figure 2b). The most common presumed cause of OCHA was hypoxia and cardiac with the
overall incidences of 0.91 and 0.43/100,000, respectively. Presumed cause of OHCA differed between the
age groups. For children <1 year, hypoxia and sudden infant death syndrome (SIDS) were the leading
presumed causes of OHCA. In older children, trauma, drowning and cardiac causes are increasing (Table 1). Across all age groups, "Home" was the most common location of OHCA with 1.91/100,000. In the age group
13 to <18 years, "Home" and "Public place" had proportions of 49.5% (n=163) and 41.3% (n=136), 13 to 18 years, Home and Public place had proportions of 49.5% (n 163) and 41.3% (n 136),
respectively (Figure 2b). The most common presumed cause of OCHA was hypoxia and cardiac with the
overall incidences of 0.91 and 0.43/100,000, respectively. Presumed cause of OHCA differed between the
age groups. Demographic overview For children <1 year, hypoxia and sudden infant death syndrome (SIDS) were the leading
presumed causes of OHCA. In older children, trauma, drowning and cardiac causes are increasing (Table 1). respectively (Figure 2b). The most common presumed cause of OCHA was hypoxia and cardiac with the
overall incidences of 0.91 and 0.43/100,000, respectively. Presumed cause of OHCA differed between the
age groups. For children <1 year, hypoxia and sudden infant death syndrome (SIDS) were the leading
presumed causes of OHCA. In older children, trauma, drowning and cardiac causes are increasing (Table 1). Across the entire time span, OHCAs were mainly unwitnessed (1.80/100,000); however, this declined from
2007 (2.19) to 2021 (1.88). Bystander-witnessed OHCAs increased from an incidence of 0.51 in 2007 to
1.11 in 2021. This is equally distributed across all age groups. Across the entire time span, OHCAs were mainly unwitnessed (1.80/100,000); however, this declined from
2007 (2.19) to 2021 (1.88). Bystander-witnessed OHCAs increased from an incidence of 0.51 in 2007 to
1.11 in 2021. This is equally distributed across all age groups. Figure 2 – (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age
group; Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing Figure 2 – (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age
group; Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing The full demographic results including pre-existing conditions and pre emergency status are presented in
Table 1. The full demographic results including pre-existing conditions and pre emergency status are presented in
Table 1. Demographic overview # Location of OHCA: 110 cases are
missing. Respective case numbers are: <1 Year: 702, 1-4 Years: 336, 5-12 Years: 263, 13-<18 Years: 329. ##
Nighttime: 51 cases are missing. Respective case numbers are: 0-<1 Year: 712, 1-4 Years: 356, 5-12 Years
273, 13-<18 Years: 348. ### First rhythm assessed: 49 cases are missing. Respective case numbers are: 0-
<1 Year: 712, 1-4 Years: 358, 5-12 Years: 277, 13-<18 Years: 344.* SIDS was only recorded in children up to
the age of 2 years. Abbreviations: OHCA = out of hospital cardiac arrest; SIDS = sudden infant death
syndrome; EMS = emergency medical service; CPR = cardiopulmonary resuscitation; ASA = American
Society of Anaesthesiologists Classification; RACA = Return of spontaneous circulation after cardiac arrest Percentages were rounded, wherefore they might not add up to 100%. # Location of OHCA: 110 cases are
missing. Respective case numbers are: <1 Year: 702, 1-4 Years: 336, 5-12 Years: 263, 13-<18 Years: 329. ##
Nighttime: 51 cases are missing. Respective case numbers are: 0-<1 Year: 712, 1-4 Years: 356, 5-12 Years
273, 13-<18 Years: 348. ### First rhythm assessed: 49 cases are missing. Respective case numbers are: 0-
<1 Year: 712, 1-4 Years: 358, 5-12 Years: 277, 13-<18 Years: 344.* SIDS was only recorded in children up to
the age of 2 years. Abbreviations: OHCA = out of hospital cardiac arrest; SIDS = sudden infant death
syndrome; EMS = emergency medical service; CPR = cardiopulmonary resuscitation; ASA = American
Society of Anaesthesiologists Classification; RACA = Return of spontaneous circulation after cardiac arrest Percentages were rounded, wherefore they might not add up to 100%. # Location of OHCA: 110 cases are
missing. Respective case numbers are: <1 Year: 702, 1-4 Years: 336, 5-12 Years: 263, 13-<18 Years: 329. ##
Nighttime: 51 cases are missing. Respective case numbers are: 0-<1 Year: 712, 1-4 Years: 356, 5-12 Years
273, 13-<18 Years: 348. ### First rhythm assessed: 49 cases are missing. Respective case numbers are: 0-
<1 Year: 712, 1-4 Years: 358, 5-12 Years: 277, 13-<18 Years: 344.* SIDS was only recorded in children up to
the age of 2 years. Abbreviations: OHCA = out of hospital cardiac arrest; SIDS = sudden infant death
syndrome; EMS = emergency medical service; CPR = cardiopulmonary resuscitation; ASA = American
Society of Anaesthesiologists Classification; RACA = Return of spontaneous circulation after cardiac arrest Demographic overview Table 1 – Baseline Characteristics Table 1 – Baseline Characteristics Page 7/24 Characteristic
Incidence/100,000
Overall
N = 1,740
3.08/100,000
<1 Year
n = 731
23.42/100,000
1 – 4 Years
n = 369
2.98/100,000
5 – 12 Years
n = 283
1.16/100,000
13 – <18
Years
n = 357
2.24/100,000
Age, median (Q1
to Q3)
1.8 years (0.3
to 10.9)
93 days (40 to
171)
2.2 years (1.5
to 3.2)
8.7 years (6.6
to 11.1)
16.2 years
(14.9 to 17.1)
Sex, n female (%)
686 (39.4%)
288 (39.4%)
160 (43.4%)
107 (37.8%)
131 (36.7%)
Location of OHCA,
n (%) / Incidence#
Healthcare Facility
outside hospital
Public Place
Home
Educational
Institution /
Workplace
Not recorded
45 (3%) /
0.07
378 (23.2%)
/ 0.66
1111 (68.2%)
/ 1.91
18 (1%) /
0.03
78 (5%) /
0.23
25 (4%)
59 (8%)
587 (83.6%)
3 (0.4%)
28 (4%)
8 (2%)
86 (25.6%)
218 (64.9%)
5 (2%)
19 (6%)
6 (2%)
97 (36.9%)
143 (54.4%)
3 (1%)
14 (5%)
6 (2%)
136 (41.3%)
163 (49.5%)
7 (2%)
17 (5%)
Presumed cause,
n (%) / Incidence
Cardiac
Trauma
Drowning
Hypoxia
Intoxication
SIDS
Other
Unknown
241 (13.9%)
/ 0.43
193 (11.1%)
/ 0.37
134 (8%) /
0.22
540 (31.0%)
/ 0.91
24 (1%) /
0.04
211 (12.1%)
/ 0.38
20 (3%)
22 (3%)
7 (1%)
229 (31.3%)
3 (0.4%)
202 (27.6%)
51 (7%)
111 (15.2%)
29 (8%)
38 (10.3%)
72 (19.5%)
143 (38.7%)
2 (0.5%)
9 (2%)*
37 (10.0%)
39 (10.5%)
43 (15.2%)
45 (15.9%)
46 (16.2%)
83 (29.3%)
1 (0.4%)
-
34 (12.0%)
31 (11.0%)
63 (17.6%)
88 (24.6%)
9 (3%)
85 (23.8%)
18 (5%)
-
34 (9%)
60 (16.8%) Page 8/24
Incidence/100,000
,
3.08/100,000
23.42/100,000
2.98/100,000
1.16/100,000
n = 357
2.24/100,000
Age, median (Q1
to Q3)
1.8 years (0.3
to 10.9)
93 days (40 to
171)
2.2 years (1.5
to 3.2)
8.7 years (6.6
to 11.1)
16.2 years
(14.9 to 17.1)
Sex, n female (%)
686 (39.4%)
288 (39.4%)
160 (43.4%)
107 (37.8%)
131 (36.7%)
Location of OHCA,
n (%) / Incidence#
Healthcare Facility
outside hospital
Public Place
Home
Educational
Institution /
Workplace
Not recorded
45 (3%) /
0.07
378 (23.2%)
/ 0.66
1111 (68.2%)
/ 1.91
18 (1%) /
0.03
78 (5%) /
0.23
25 (4%)
59 (8%)
587 (83.6%)
3 (0.4%)
28 (4%)
8 (2%)
86 (25.6%)
218 (64.9%)
5 (2%)
19 (6%)
6 (2%)
97 (36.9%)
143 (54.4%)
3 (1%)
14 (5%)
6 (2%)
136 (41.3%)
163 (49.5%)
7 (2%)
17 (5%)
Presumed cause,
n (%) / Incidence
Cardiac
Trauma
Drowning
Hypoxia
Intoxication
SIDS
Other
Unknown
241 (13.9%)
/ 0.43
193 (11.1%)
/ 0.37
134 (8%) /
0.22
540 (31.0%)
/ 0.91
24 (1%) /
0.04
211 (12.1%)
/ 0.38
156 (9%) /
0.31
241 (13.9%)
/ 0.43
20 (3%)
22 (3%)
7 (1%)
229 (31.3%)
3 (0.4%)
202 (27.6%)
51 (7%)
111 (15.2%)
29 (8%)
38 (10.3%)
72 (19.5%)
143 (38.7%)
2 (0.5%)
9 (2%)*
37 (10.0%)
39 (10.5%)
43 (15.2%)
45 (15.9%)
46 (16.2%)
83 (29.3%)
1 (0.4%)
-
34 (12.0%)
31 (11.0%)
63 (17.6%)
88 (24.6%)
9 (3%)
85 (23.8%)
18 (5%)
-
34 (9%)
60 (16.8%)
Relation to sports
activity, n (%)
40 (2%)
5 (1%)
1 (0.3%)
8 (3%)
26 (7%)
Nighttime (10pm
to 6pm), n (%)##
369 (21.2%)
166 (23.3%)
63 (17.7%)
55 (20.1%)
85 (24.4%) Characteristic
Incidence/100,000
Overall
N = 1,740
3.08/100,000
<1 Year
n = 731
23.42/100,000
1 – 4 Years
n = 369
2.98/100,000
5 – 12 Years
n = 283
1.16/100,000
13 – <18
Years
n = 357
2.24/100,000
Witnessed by, n
(%) / Incidence
Bystander
First Responder
EMS
Unwitnessed
632 (36.3%)
/ 1.07
22 (1%) /
0.04
113 (7%) /
0.19
973 (55.9%)
/1.80
214 (29.3%)
11 (2%)
46 (6%)
460 (62.9%)
162 (43.9%)
5 (1%)
16 (4%)
186 (50.4%)
109 (38.5%)
3 (1%)
22 (8%)
149 (52.7%)
147 (41.2%)
3 (1%)
29 (8%)
178 (49.9%)
Bystander CPR, n
(%)
911 (52.3%)
368 (50.3%)
230 (62.3%)
149 (52.7%)
164 (45.9%)
Telephone
assisted CPR, n
(%)
444 (25.5%)
210 (28.7%)
109 (29.5%)
55 (19.4%)
70 (19.6%)
First assessed
rhythm, n (%) /
Incidence###
Asystole
PEA
Ventricular
Fibrillation
Bradycardia
1155 (68.3%)
/ 2.05
245 (14.5%)
/ 0.41
153 (9%) /
0.29
138 (8%) /
0.23
529 (74.3%)
87 (12.3%)
33 (5%)
63 (9%)
240 (67.0%)
64 (17.9%)
10 (3%)
44 (12.3%)
186 (67.1%)
32 (11.6%)
36 (13.0%)
23 (8%)
200 (58.1%)
62 (18.1%)
74 (21.4%)
8 (2%)
Pre Emergency
Status, n (%) /
Incidence
ASA I
ASA II
ASA III
ASA IV
ASA V
690 (39.7%)
/ 1.02
145 (8%) /
0.21
209 (12.0%)
/ 0.31
135 (8%) /
0.19
304 (41.6%)
53 (7%)
97 (13.3%)
37 (5%)
1 (0.1%)
239 (32.7%)
145 (39.3%)
27 (7%)
49 (13.3%)
33 (9%)
1 (0.3%)
114 (30.9%)
102 (36.0%)
21 (7%)
39 (13.8%)
36 (12.7%)
-
85 (30.0%)
139 (38.9%)
44 (12.3%)
24 (7%)
29 (8%)
1 (0.3%)
120 (33.6%) Page 9/24 Page 9/24 Characteristic
Incidence/100,000
Overall
N = 1,740
3.08/100,000
<1 Year
n = 731
23.42/100,000
1 – 4 Years
n = 369
2.98/100,000
5 – 12 Years
n = 283
1.16/100,000
13 – <18
Years
n = 357
2.24/100,000 Pre-existing
conditions, n (%)/
Incidence
Cardiac
Pulmonary
Metabolic
Malignancy
Neurologic
Immunodeficiency
181 (10.4%)
/ 0.27
120 (7%) /
0.20
80 (5%) /
0.12
19 (1%) /
0.03
204 (11.7%)
/ 0.33
21 (1%) /
0.03
99 (13.5%)
56 (8%)
35 (5%)
10 (1%)
46 (6%)
5 (1%)
34 (9%)
29 (8%)
16 (4%)
4 (1%)
56 (15.2%)
6 (2%)
29 (10.2%)
25 (9%)
14 (5%)
3 (1%)
54 (19.1%)
6 (2%)
19 (5%)
18 (5%)
15 (4%)
2 (1%)
48 (13.4%)
4 (1%) Percentages were rounded, wherefore they might not add up to 100%. Pre-hospital treatment Referring to the first link in the chainmail of survival [22], bystanders performed chest compression only
(CCO) in 20.0% (n=348) of all cases. Chest compression and ventilation (CCV) were performed in 18.6%
(n=324), with a higher proportion compared to CCO in 1 to 4 and 5 to 12 years (27.6% vs 20.1%, and 19.1%
vs 17.3%, respectively). Out of 235 performed defibrillations, EMS performed 97.5% (n=229). In total six
defibrillations were performed before EMS arrival, four of those by first responders. Refractory VF, defined as
>3 shocks, was stated in 21.3% (n=50) of all cases, with the largest proportion in the 13 to <18 years group
(26.5%, n=26). A similar proportion was found in the <1 year age group with 23.4% (n=15). Endotracheal
intubation (ETI) was the most frequently used strategy for airway management in all age groups. In
comparison, bag-mask-ventilation during CPR only was used in 3.8% (n=67) of all cases. Any type of
advanced airway management during the course of prehospital treatment achieved higher aOR for hospital
admission with ROSC (Figure 3, Supplement Table 4). Regarding supraglottic airway devices, laryngeal
tubes (LT) were used in 54.7% (175/320) (Supplement Table 5c). Although an airway management device Page 10/24 Page 10/24 was used in most cases, end-tidal CO2 (etCO2) at hospital admission was not routinely reported. With an
increase over the study period (Supplement Table 1b). Administration of adrenaline using intraosseous (i.o.)
access was highest in the youngest age group (<1 year) with 42.1% and declined to 21.0% in the oldest age
group (13 to <18 years). Full details on pre-hospital treatment are presented in Table 2 and Supplement
Table 6. was used in most cases, end-tidal CO2 (etCO2) at hospital admission was not routinely reported. With an
increase over the study period (Supplement Table 1b). Administration of adrenaline using intraosseous (i.o.)
access was highest in the youngest age group (<1 year) with 42.1% and declined to 21.0% in the oldest age
group (13 to <18 years). Full details on pre-hospital treatment are presented in Table 2 and Supplement
Table 6. Pre-hospital treatment * Percentage calculated from the total
of defibrillations (n = 235). ** Percentage calculated from the total cases where advanced
airwaymanagement with SGA or ETI was performed (n = 1390). *** Percentage calculated from the total
number of the administered drug (N). Abbreviations: BMV = bag-mask ventilation; SGA = supraglottic airway
device; SD = standard deviation; CPR = cardiopulmonary resuscitation; Percentages were rounded, wherefore they might not add up to 100%. * Percentage calculated from the total
of defibrillations (n = 235). ** Percentage calculated from the total cases where advanced
airwaymanagement with SGA or ETI was performed (n = 1390). *** Percentage calculated from the total
number of the administered drug (N). Abbreviations: BMV = bag-mask ventilation; SGA = supraglottic airway
device; SD = standard deviation; CPR = cardiopulmonary resuscitation; Pre-hospital treatment Table 2 - Pre-hospital Treatment Page 11/24 Overall
N = 1,740
<1 Year
n = 731
1 – 4
Years
n = 369
5 – 12
Years
n = 283
13 – <18
Years
n = 357
Treatment before EMS arrival,
n (%)
Chest compression only
Ventilation only
Chest compression and
ventilation
Defibrillation*
362 (20.8%)
35 (2%)
355 (20.4%)
6 (0.3%)
145
(19.8%)
11 (2%)
150
(20.5%)
1 (0.1%)
76
(20.6%)
10 (3%)
108
(29.3%)
-
54 (19.1%)
3 (1%)
60 (21.2%)
2 (1%)
87 (24.4%)
11 (3%)
39 (10.9%)
3 (1%)
Airway management, n (%)
BMV only
67 (4%)
26 (4%)
8 (2%)
8 (3%)
25 (7%)
SGA**
320 (23%)
150
(26%)
59 (21%)
34 (15%)
77 (25%)
Endotracheal Intubation**
End-tidal CO2 at admission **
1070 (77%)
425 (31%)
420
(74%)
119
(21%)
221
(79%)
103
(37%)
193 (85%)
85 (37%)
236 (75%)
118 (38%)
Medication
Adrenaline, N
i.v. n (%)***
i.o. n (%)***
1579
873 (55%)
668 (42%)
673
344
(51%)
308
(46%)
360
177
(49%)
173
(48%)
269
156 (58%)
112 (42%)
277
196 (71%)
75 (21%)
Atropine, N
i.v. n (%)***
i.o. n (%)***
176
106 (60%)
65 (37%)
52
28 (54%)
23 (44%)
56
31 (55%)
23 (41%)
37
23 (62%)
14 (38%)
31
24 (77%)
5 (16%)
Amiodarone, N
i.v. n (%)***
i.o. n (%)***
70
61 (87%)
9 (13%)
17
17
(100%)
-
4
2 (50%)
2 (50%)
10
7 (70%)
3 (30%)
39
35 (90%)
4 (10%)
Crystalloid Fluids, n (%)
964 (55.4%)
352
(48.2%)
194
(52.6%)
189
(66.8%)
229 (64.1%)
Thrombolysis, n (%)
19 (1%)
8 (1%)
-
-
10 (3%) Overall
N = 1,740
<1 Year
n = 731
1 – 4
Years
n = 369
5 – 12
Years
n = 283
13 – <18
Years
n = 357
1 Shock, n (%)*
84 (35.7%)
21
(32.8%)
12
(48.0%)
20 (41.7%)
31 (31.6%)
2-3 Shocks, n (%)*
48 (20.4%)
4 (6%)
3 (12.0%)
10 (20.8%)
31 (31.6%)
>3 Shocks, n (%)*
50 (21.3%)
15
(23.4%)
2 (8%)
7 (14.6%)
26 (26.5%)
unknown, n (%)*
50 (21.3%)
24
(37.5%)
8 (32.0%)
11 (22.9%)
10 (10.2%)
Mechanical CPR, n (%)
47 (3%)
-
-
-
47 (13.2%) Percentages were rounded, wherefore they might not add up to 100%. * Percentage calculated from the total Percentages were rounded, wherefore they might not add up to 100%. Bystander influencing immediate outcome Chest compressions and ventilation started by bystanders had an adjusted odds ratio (aOR) for hospital
admission with ROSC of 2.78 (95% CI 2.12 to 3.65), compared to CCV started by EMS. When only chest
compressions were started by bystanders, aOR was also significant, with 1.72 (95% CI 1.38 to 2.14). OHCA
witnessed by bystanders or EMS had increased aOR for hospital admission with ROSC compared to an
unwitnessed OHCA. In addition to witness status and bystander CPR, the time of OHCA was analysed. Odds
ratio were decresead during nighttime for admission with ROSC (Figure 3, Supplement Table 9). Figure 3 – Forest plot with adjusted odds ratio for hospital admission with ROSC. Abbreviations: CCV =
chest compression and ventilation; CCO = chest compression only; EMS = emergency medical service; ROSC
= return of spontaneous circulation; CI = confidence interval Outcome Of 1,740 cases, the main proportion achieved no pre-hospital ROSC across all age groups. Of 1,740 cases, the main proportion achieved no pre-hospital ROSC across all age groups. The incidence of children declared dead on scene after unsuccessful resuscitation was 1.20/100,000;
similar to those admitted with ROSC to hospital admission (1.13/100,000). In 46.9% (n=343) in the group <1
year, children were declared dead on scene, compared to 28.2% (n=104) in 1 to 4 years, 31.8% (n=90) in 5 to
12 years and 35.0% (n=125) in 13 to <18 years Immediate and short-term outcomes for all age groups are shown in Table 3. In total, 994 children did not
achieve ROSC prehospitally; with the majority being unwitnessed arrests and presenting with asystole as
initial rhythm (Supplement Table 7). Long term outcome, where available, is presented in Supplement Table
8. EMS time intervals did not differ between any short-term outcome. An overall median response time of
seven minutes (Q1 to Q3 5 to 9) was observed. Univariate analysis showed no difference for any ROSC
when EMS response time was below 7 minutes compared to above 7 minutes (OR 0.86 [95% CI 0.70 to
1.04]) with a similar rate of bystander CPR (62.4% vs. 62.3%; p = 1.0). Across different outcomes, EMS time
intervals did not differ (Supplement Table 9, Supplement Figure 1). Table 3 - Short term outcomes Page 13/24 Overall
N = 1,740
0 – <1
Year
n = 731
1 – 4
Years
n = 369
5 – 12
Years
n = 283
13 – <18
Years
n = 357
Status at hospital admission, n (%)
/ Incidence
no admission, dead on scene
662 (38.0%) /
1.20
343
(46.9%)
104
(28.2%)
90
(31.8%)
125
(35.0%)
admitted with ongoing CPR
427 (24.5%) /
0.75
170
(23.3%)
108
(29.3%)
74
(26.1%)
75 (21.0%)
admitted with ROSC
650 (37.4%) /
1.13
218
(29.8%)
157
(42.5%)
118
(41.7%)
157
(44.0%)
Unknown
1 (0.1%)
-
-
1 (0.4%)
-
Abbreviations: CPR = cardiopulmonary resuscitation; ROSC = return of spontaneous circulation Abbreviations: CPR = cardiopulmonary resuscitation; ROSC = return of spontaneous circulation Discussion This study identified 1,740 children with OHCA and CPR in Germany from 2007 until 2021. Children aged >7
days to <1 year had the highest incidence of OHCA with 23.42/100,000. While 83.6% of OHCA occurred at
home in the youngest age group, occurrence of OHCA in public places had a significant proportion in school
children (5 to 12 years) and adolescents (13 to <18 years) with 37% and 41%, respectively. Trauma and
drowning constituted a significant proportion of presumed causes in children above 1 year, while cardiac
causes accounted for 17.6% in the oldest age group. The overall incidence of any ROSC was low at
1.27/100,000. Adolescents were most frequently admitted with ROSC (44.0%; 157/357). Admittance with
ongoing CPR was equally distributed across all age groups. Page 14/24
Bystander CCV and CCO had significantly higher aOR for hospital admission with ROSC. Confidence
intervals of CCO and CCV for hospital admission with ROSC barely overlap, indicating a possible significant
difference in favour of CCV [23]. To conclude, this points out the relevance of bystander-initiated ventilation Page 14/24 Page 14/24 in children suffering from OHCA even if survival to hospital discharge has not yet improved [24,25]. One
confounder might be that bystanders who perform CCV are better trained and provide more sufficient CPR
than those who provide chest compressions "only". This finding has also been identified within adults and
should be translated into future first aid courses and can also be implemented in childbirth preparation
courses [23]. Although the group of first responders was small, it is important to differentiate between
bystanders and first responders [26]. Bystanders will perform CPR by chance, whereas first responers
perform CPR by system. In this cohort advanced airway management (AAM) had higher aOR of hospital
admission with ROSC, whereas it was not associated with survival in other systems (4.6% vs 5.1%) [2,27]. These findings have been repreated throughout different systems. Due to the low incidence, recruitement of
a significant sample size is associated with a very high effort, needing nation wide participation in a
prospective trial. Therefore, prospective studies on AAM in children are likely to fail. The herein-presented data on immediate outcome compares to different ROSC rate at hospital admission,
ranging from 8.1% [5] to 38.7% [28] in various systems. As awareness for recognition of cardiac arrest has
increased over the last couple of years, different study periods might partially explain these differences. Discussion Age groups have not been uniformly defined across recent studies; however, children aged <1 year
consistently have the highest proportion of OHCA [29]. Based on this study, future EMS training should focus on children <1 year in general and on the other age
groups on treating trauma, drowning, and hypoxia as reversible causes. In adults, when no obvious cause of
OHCA is present, ‘cardiac’ is stated as the presumed cause; in children an uniformly presumed cause in the
absence of any clear reason for OHCA is undefined. This is mirrored in a combined share of 20.5%
"unknown" and "other causes" within this study which corroborates a 30.0% of unknown causes in a recent
study by Holgersen et al. [28]. Although one does not influence the timing of OHCA, the decreased aOR for
nighttime indicates there is a continuous need to evaluate monitoring devices, such as surveillance
matresses, for a potential role in aiding the early detection of cardiac or respiratory detoriation, alerting
caregivers to prevent or recognise cardiac arrests immediately [30]. To our knowledge there has not been a
study showing the benefit of such devices, however cardiac arrest registries could record the presence of
this potentially preventive device. Always keeping the parent stress levels in mind. The high number (>80%) of unknown outcomes at 24 hours, 30 days, and discharge suggests mandatory
participation for hospitals receiving children with OHCA. Other registries were able to determine reliable 30-
day outcomes [28,31], enabled due to the linkage of different registries through a personal identification
number. Data protection regulations should emphazise the usage of anonymized or pseudonymized data to
gain a better understanding of scarce patient populations. Page 15/24
Although this study presents previously scarcely reported data from one of the largest cardiac arrest
registries in Europe, there are some limitations. (1) The latest Utstein recommendation on reporting OHCA in
the paediatric population from 1995 proposes a reporting flowchart [11]. However, as current registry
variables do not support the proposed format, such a chart was not applied in our study. (2) Due to the
retrospective nature of this study, it was not possible to assess causation for different interventions like
airway management [2,27] or dosage of adrenaline [13]. (3) The lack of long-term outcome data limits the significance of hospital admission with ROSC. Discussion (4) Due to the anonymised data, it was impossible to
contact the participating sites to clarify discrepancies or collect missing data. (5) Any information on
bystanders is missing. Therefore, it is impossible to differentiate bystanders with and without medical
backgrounds. (6) Determining the cause of death and providing this information to EMS providers can
improve patient care. Even when a coroner evaluates the cause of death, 11.2% (49/474) remain
undetermined, displaying high complexity and uncertainty [32]. Future studies should attempt to establish global collaborations to provide a complete picture of OHCA in
the paediatric population. Existing registries should try to collect prospective information on treatment
decisions in order to get a better understanding of the rationale behind those decisions in this rare event. Conclusion This study reports the incidence of OHCA in the paediatric population over a 15-year period in Germany. Chest compressions and ventilation by bystander are identidified to have a higher impact on immediate
survival compared with chest compression only. This study corroborates the findings of an increased
incidence in <1 year old and a low survival rate. Acknowledgements We thank all participants from the German Resuscitation Registry for their contribution to this study. We
also thank Alicja Suliman, graphic designer, for creating the visual abstract. We thank all participants from the German Resuscitation Registry for their c
also thank Alicja Suliman, graphic designer, for creating the visual abstract. Authors' contributions SK, IKK, IT, JW, JKJ, JTG and EP made substantial contributions to the conception of this work. SK, IKK, IT,
JKJ and JTG designed the work. SK and PR performed the data acquisition, MH significantly contributed to
the data acquistion. SK performed the data analysis, PR and SS contributed to the data analysis. SB, FH
and EP contributed to the interpretation of data. SK drafted the manuscript and all authors have
substantively revised it. The final version of this manuscript is read and approved by all authors. Funding This study was done without funding Ethics approval and consent to participate Ethics approval and consent to participate This study was approved by the University Heidelberg ethics committee with reference number S-085/2022
on March 23rd 2022. Due to anonymous data, patients consent was waived. Abbreviations Page 16/24 AAM
Advanced airway management
ALS
Advanced life support
aOR
Adjusted Odds ratio
ASA
American Society of Anaesthesiologists Classification
CCO
Chest compression only
CCV
Chest compression and ventilation
CPC
Cerebral performance category
CPR
Cardiopulmonary resusctiation
DoA
Deceased on arrival
EMS
Emergency medical service
EMT
Emergency medical technician
ETI
Endotracheal intubation
EuReCa
European Registriy of Cardiac arrest
GRR
German Resuscitation Registry
i.o. Intraosseous access
i.v. Intravenous access
LT
Laryngeal tube
OHCA
Out of hospital cardiac arrest
PALS
Paediatric advanced life support
PEA
Pulseless electrical activity
RACA
Return of spontaneous circulation after cardiac arrest
ROSC
Return of spontaneous circulation
SD
Standard deviation
SIDS
Sudden infant death syndrome
STROBE
Strengthening the Reporting of Observational studies in Epidemiology
uaOR
Unadjusted Odds ratio
VF
Ventricular fibrillation
VT
Ventricular tachycardia AAM
Advanced airway management
ALS
Advanced life support
aOR
Adjusted Odds ratio
ASA
American Society of Anaesthesiologists Classification
CCO
Chest compression only
CCV
Chest compression and ventilation
CPC
Cerebral performance category
CPR
Cardiopulmonary resusctiation
DoA
Deceased on arrival
EMS
Emergency medical service
EMT
Emergency medical technician
ETI
Endotracheal intubation
EuReCa
European Registriy of Cardiac arrest
GRR
German Resuscitation Registry
i.o. Intraosseous access
i.v. Intravenous access
LT
Laryngeal tube
OHCA
Out of hospital cardiac arrest
PALS
Paediatric advanced life support
PEA
Pulseless electrical activity
RACA
Return of spontaneous circulation after cardiac arrest
ROSC
Return of spontaneous circulation
SD
Standard deviation
SIDS
Sudden infant death syndrome
STROBE
Strengthening the Reporting of Observational studies in Epidemiology
uaOR
Unadjusted Odds ratio
VF
Ventricular fibrillation Declarations Consent for publication Availability of data and materials An aggregated dataset used during the current study is available from the corresponding author on
reasonable request. JKJ has no COI for the current research work, but has received unrestricted grant from Laerdal foundation
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of Pediatric Out-of-Hospital Cardiac Arrest Admitted to the Emergency Department. Sci Rep 2019;9:1–9. https://doi.org/10.1038/s41598-019-43020-0. 32. Ong MEH, Stiell I, Osmond MH, Nesbitt L, Gerein R, Campbell S, et al. Etiology of pediatric out-of-
hospital cardiac arrest by coroner’s diagnosis. Resuscitation 2006;68:335–42. https://doi.org/10.1016/j.resuscitation.2005.05.026. Figures Page 21/24 Figure 2 (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age group;
Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing (a) Proportion of the first rhythm assessed by age group, (b) Location of arrest according to age group;
Abbreviations: PEA = Pulsless electric activity, N/A = not available or missing Page 23/24 Figure 1 Study flowchart demonstrating included cases and incidences per age group; *1 case with an unknown
immediate outcome. Abbreviations: OHCA = out-of-hospital cardiac arrest; GRR = German Resuscitation
Registry; CPR = cardiopulmonary resuscitation Study flowchart demonstrating included cases and incidences per age group; *1 case with an unknown
immediate outcome. Abbreviations: OHCA = out-of-hospital cardiac arrest; GRR = German Resuscitation
Registry; CPR = cardiopulmonary resuscitation Page 22/24 Figure 2 Figure 2 Figure 3 Forest plot with adjusted odds ratio for hospital admission with ROSC. Abbreviations: CCV = chest
compression and ventilation; CCO = chest compression only; EMS = emergency medical service; ROSC =
return of spontaneous circulation; CI = confidence interval Forest plot with adjusted odds ratio for hospital admission with ROSC. Abbreviations: CCV = chest
compression and ventilation; CCO = chest compression only; EMS = emergency medical service; ROSC =
return of spontaneous circulation; CI = confidence interval Page 23/24 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to dow PedsOHCAGerCARSupplements20230710.docx Page 24/24
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RESEARCH ARTICLE a1111111111
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Citation: Bergeaud A, Potiron Y, Raimbault J
(2017) Classifying patents based on their semantic
content. PLoS ONE 12(4): e0176310. https://doi.
org/10.1371/journal.pone.0176310 OPEN ACCESS
Citation: Bergeaud A, Potiron Y, Raimbault J
(2017) Classifying patents based on their semantic
content. PLoS ONE 12(4): e0176310. https://doi. org/10.1371/journal.pone.0176310 Editor: Zhong-Ke Gao, Tianjin University, CHINA
Received: December 26, 2016
Accepted: April 7, 2017
Published: April 26, 2017 Copyright: © 2017 Bergeaud et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Antonin Bergeaud1☯, Yoann Potiron2☯, Juste Raimbault3,4☯* Antonin Bergeaud1☯, Yoann Potiron2☯, Juste Raimbault3,4☯* 1 Paris School of Economics - EHESS and Bank of France, Paris, France, 2 Faculty of Business and
Commerce, Keio University, Tokyo, Japan, 3 UMR CNRS 8504 Ge´ographie-cite´s, Universite´ Paris VII, Paris,
France, 4 UMR-T 9403 IFSTTAR LVMT, Ecole Nationale des Ponts et Chausse´es, Champs-sur-Marne,
France ☯These authors contributed equally to this work. * juste.raimbault@polytechnique.edu a1111111111
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a1111111111 Abstract In this paper, we extend some usual techniques of classification resulting from a large-scale
data-mining and network approach. This new technology, which in particular is designed to
be suitable to big data, is used to construct an open consolidated database from raw data on
4 million patents taken from the US patent office from 1976 onward. To build the pattern net-
work, not only do we look at each patent title, but we also examine their full abstract and
extract the relevant keywords accordingly. We refer to this classification as semantic
approach in contrast with the more common technological approach which consists in taking
the topology when considering US Patent office technological classes. Moreover, we docu-
ment that both approaches have highly different topological measures and strong statistical
evidence that they feature a different model. This suggests that our method is a useful tool
to extract endogenous information. OPEN ACCESS
Citation: Bergeaud A, Potiron Y, Raimbault J
(2017) Classifying patents based on their semantic
content. PLoS ONE 12(4): e0176310. https://doi. org/10.1371/journal.pone.0176310
Editor: Zhong-Ke Gao, Tianjin University, CHINA
Received: December 26, 2016
Accepted: April 7, 2017
Published: April 26, 2017
Copyright: © 2017 Bergeaud et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. OPEN ACCESS
Citation: Bergeaud A, Potiron Y, Raimbault J
(2017) Classifying patents based on their semantic
content. PLoS ONE 12(4): e0176310. https://doi. org/10.1371/journal.pone.0176310
Editor: Zhong-Ke Gao, Tianjin University, CHINA
Received: December 26, 2016
Accepted: April 7, 2017
Published: April 26, 2017
Copyright: © 2017 Bergeaud et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Classifying patents based on their semantic content show that the laser/ink-jet printer technology resulted from the recombination of two differ-
ent existing technologies. public, commercial, or not-for-profit sectors for
this research. public, commercial, or not-for-profit sectors for
this research. Consequently, technological classification combined with other features of patents can be a
valuable tool for researchers interested in studying technologies throughout history and to pre-
dict future innovations by looking at past knowledge and interaction across sectors and tech-
nologies. But it is also crucial for firms that face an ever changing demand structure and need
to anticipate future technological trends and convergence (see, e.g., [11]) to adapt to the result-
ing increase in competition discussed in [12] and to maintain market share. Curiously, and in
spite of the large number of studies that analyze interactions across technologies [13], little is
known about the underlying “innovation network” (e.g. [14]). Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. In this monograph, we propose an alternative classification based on semantic network
analysis from patent abstracts and explore the new information emerging from it. In contrast
with the regular technological classification which results from the choice of the patent
reviewer, semantic classification is carried automatically based on the content of the patent
abstract. Although patent officers are experts in their fields, the relevance of the existing classi-
fication is limited by the fact that it is based on the state of technology at the time the patent
was granted and cannot anticipate the birth of new fields. To correct for this, the USPTO regu-
larly make changes in its classification in order to adapt to technological change (for example,
the “nanotechnology” class (977) was established in 2004 and retroactively to all relevant previ-
ously granted patents). In contrast we don’t face this issue with the semantic approach. The
semantic links can be clues of one technology taking inspiration from another and good pre-
dictors of future technology convergence (e.g. [15] study semantic similarities from the whole
text of 326 US-patents on phytosterols and show that semantic analysis have a good predicting
power of future technology convergence). One can for instance consider the case of the word
optic. Introduction Innovation and technological change have been described by many scholars as the main
drivers of economic growth as in [1] and [2]. [3] advertised the use of patents as an economic
indicator and as a good proxy for innovation. Subsequently, the easier availability of compre-
hensive databases on patent details and the increasing number of studies allowing a more
efficient use of these data (e.g. [4]) have opened the way to a very wide range of analysis. Most of the statistics derived from the patent databases relied on a few key features: the iden-
tity of the inventor, the type and identity of the rights owner, the citations made by the patent
to prior art and the technological classes assigned by the patent office post patent’s content
review. Combining this information is particularly relevant when trying to capture the diffu-
sion of knowledge and the interaction between technological fields as studied in [5]. With
methods such as citation dynamics modeling discussed in [6] or co-authorship networks
analysis in [7], a large body of the literature such as [8] or [9] has studied patents citation net-
work to understand processes driving technological innovation, diffusion and the birth of
technological clusters. Finally, [10] look at the dynamics of citations from different classes to Data Availability Statement: All data files are
available from the Dataverse Repository, at URLs
http://dx.doi.org/10.7910/DVN/BW3ACK and http://
dx.doi.org/10.7910/DVN/ZULMOY. All source code
and results are available on the GitHub repository
of the project at https://github.com/
JusteRaimbault/PatentsMining. Funding: Antonin Bergeaud received no specific
grant from any funding agency in the public,
commercial, or not-for-profit sectors for this
research. Yoann Potiron received private funding
from Keio University. Juste Raimbault received no
specific grant from any funding agency in the PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 1 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Background In our analysis, we will consider all utility patents granted in the United States Patent and
Trademark Office (USPTO) from 1976 to 2013. A clearer definition of utility patent is given in
S1 Text. Also, additional information on how to correctly exploit patent data can be found in
[4] and [23]. An existing classification: The USPC system Each USPTO patent is associated with a non-empty set of technological classes and subclasses. There are currently around 440 classes and over 150,000 subclasses constituting the United
State Patent Classification (USPC) system. While a technological class corresponds to the tech-
nological field covered by the patent, a subclass stands for a specific technology or method
used in this invention. A patent can have multiple technological classes, on average in our data
a patent has 1.8 different classes and 3.9 pairs of class/subclass. At this stage, two features of
this system are worth mentioning: (i) classes and subclasses are not chosen by the inventors of
the patent but by the examiner during the granting process based on the content of the patent;
(ii) the classification has evolved in time and continues to change in order to adapt to new
technologies by creating or editing classes. When a change occurs, the USPTO reviews all the
previous patents so as to create a consistent classification. Classifying patents based on their semantic content Furthermore, most of the existing studies rely on a subsample of patent data, whereas we
implement it on the full US Patent database from 1976 to 2013. This way, a general structure
of technological innovation can be studied. We draw from this application promising qualita-
tive stylized facts, such as a qualitative regime shift around the end of the 1990s, and a signifi-
cant improvement of citation modularity for the semantic classification when comparing to
the technological classification. These thematic conclusions validate our method as a useful
tool to extract endogenous information, in a complementary way to the technological
classification. Finally, the statistical model introduced in Section Statistical Model seems to indicate that
patents tend to cite more similar patents in the semantic network when fitted to data. In partic-
ular, this propensity is shown to be significantly bigger than the corresponding propensity for
technological classes, and this seems to be consistent over time. On the account of this infor-
mation, we believe that patent officers could benefit very much from looking at the semantic
network when considering potential citation candidates of a patent in review. The paper is organized as follows. Section Background presents the patent data, the existing
classification and provide details about the data collection process. Section Semantic Classifi-
cation Construction explains the construction of the semantic classes. Section Potential Results
tests their relevance by providing exploratory results. Finally, section Conclusion discusses
potential further developments and conclude. More details, including robustness checking,
figures and technical derivations can be found in S2, S3 and S4 Text. Until more recently, this word was often associated with technologies such as photogra-
phy or eye surgery, while it is now almost exclusively used in a context of semi-transistor
design and electro-optic. This semantic shift did not happen by chance but contains informa-
tion on the fact that modern electronic extensively uses technologies that were initially devel-
oped in optic. Previous research has already proposed to use semantic networks to study technological
domains and detect novelty. [16] was one of the first to enhance this approach with the idea of
visualizing keywords network illustrated on a small technological domain. The same approach
can be used to help companies identifying the state of the art in their field and avoid patent
infringement as in [17] and [18]. More closely related to our methodology, [19] develop a
method based on patent semantic analysis of patent to vindicate the view that this approach
outperform others in the monitoring of technology and in the identification of novelty innova-
tion. Semantic analysis has already proven its efficiency in various fields, such as in technology
studies (e.g. [20] and [21]) and in political science (e.g. [22]). Building on such previous research, we make several contributions by fulfilling some short-
comings of existing studies, such as for example the use of frequency-selected single keywords. First of all, we develop and implement a novel fully-automatized methodology to classify pat-
ents according to their semantic abstract content, which is to the best of our knowledge the
first of its type. This includes the following refinements for which details can be found in Sec-
tion Semantic Classification Construction: (i) use of multi-stems as potential keywords; (ii) fil-
tering of keywords based on a second-order (co-occurrences) relevance measure and on an
external independent measure (technological dispersion); (iii) multi-objective optimization of
semantic network modularity and size. The use of all this techniques in the context of semantic
classification is new and essential from a practical perspective. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 2 / 22 Data collection and basic description Each patent contains an abstract and a core text which describe the invention. To see what a
patent looks like in practice, one can refer to the USPTO patent full-text database http://patft. uspto.gov/netahtml/PTO/index.html or to Google patent which publishes USPTO patents in
pdf format at https://patents.google.com. Although including the full core texts would be natu-
ral and probably very useful in a systematic text-mining approach as done in [26], they are too
long to be included and thus we consider only the abstracts for the analysis. Indeed, the seman-
tic analysis counts more than 4 million patents, with corresponding abstracts with an average
length of 120.8 words (and a standard deviation of 62.4), a size that is already challenging in
terms of computational burden and data size. In addition, abstracts are aimed at synthesizing
purpose and content of patents and must therefore be a relevant object of study (see [27]). The
USPTO defines a guidance stating that an abstract should be “a summary of the disclosure as
contained in the description, the claims, and any drawings; the summary shall indicate the
technical field to which the invention pertains and shall be drafted in a way which allows the
clear understanding of the technical problem, the gist of the solution of that problem through
the invention, and the principal use or uses of the invention” (PCT Rule 8). We construct from raw data a unified database. Data is collected from USPTO patent red-
book bulk downloads, that provides as raw data (specific dat or xml formats) full patent
information, starting from 1976. Detailed procedure of data collection, parsing and consolida-
tion are available in S2 Text. The latest dump of the database in Mongodb format is available
at http://dx.doi.org/10.7910/DVN/BW3ACK. Collection and homogenization of the database
into a directly usable database with basic information and abstracts was an important task as
USPTO raw data formats are involved and change frequently. We count 4,666,365 utility patents with an abstract granted from 1976 to 2013. A very small
number of patents have a missing abstract, these are patents that have been withdrawn and we
do not consider them in the analysis. The number of patents granted each year increases from
around 70,000 in 1976 to about 278,000 in 2013. When distributed by the year of application,
the picture is slightly different. Classifying patents based on their semantic content information of all citations made by each patent (backward citations) and all citations received
by each patent as of the end of 2013 (forward citations). We can thus build a complete network
of citations that we will use later on in the analysis. Turning to the structure of the lag between the citing and the cited patent in terms of appli-
cation date, we see that the mean of this lag is 8.5 years and the median is 7 years. This distribu-
tion is highly skewed, the 95th percentile is 21 years. We also report 164,000 citations with a
negative time lag. This is due to the fact that some citations can be added during the examina-
tion process and some patents require more time to be granted than others. In what follows, we choose to restrict attention to pairs of citations with a lag no larger than
5 years. We impose this restriction for two reasons. First, the number of citations received
peaks 4–5 years after application. Second, the structure of the citation lag is necessarily biased
by the truncation of our sample: the more recent patents mechanically receive less citations
than the older ones. As we are restricting to citations received no later than 5 years after the
application date, this effect will only affect patents with an application date after 2007. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 A bibliographical network between patents: Citations As with scientific publications, patents must give reference to all the previous patents which
correspond to related prior art. They therefore indicate the past knowledge which relates to
the patented invention. Yet, contrary to scientific citations, they also have an important legal
role as they are used to delimit the scope of the property rights awarded by the patent. One can
consult [24] for more details about this. Failing to refer to prior art can lead to the invalidation
of the patent (e.g. [25]). Another crucial difference is that the majority of the citations are actu-
ally chosen by the examiners and not by the inventors themselves. From the USPTO, we gather PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 3 / 22 Classifying patents based on their semantic content following application, 10% of patents receive only one citation and 1% of them receive more
than 100 citations. A within class citation is defined as a citation between two patents sharing
at least one common technological class. Following this definition, 84% of the citations are
within class citations. 14% of the citations are between two patents that share the exact same
set of technological classes. Towards a complementary classification Potentialities of text-mining techniques as an alternative way to analyze and classify patents
are documented in [26]. The author’s main argument, in support of an automatic classification
tool for patent, is to reduce the considerable amount of human effort needed to classify all the
applications. The work conducted in the field of natural language processing and/or text analy-
sis has been developed in order to improve search performance in patent databases, build tech-
nology map or investigate the potential infringement risks prior to developing a new
technology (see [28] for a review). Text-mining of patent documents is also widely used as a
tool to build networks which carry additional information to the simplistic bibliographic con-
nections model as argued in [16]. As far as the authors know, the use of text-mining as a way
to build a global classification of patents remains however largely unexplored. One notable
exception can be found in [15] where semantic-based classification is shown to outperform the
standard classification in predicting the convergence of technologies even in small samples. Semantic analysis reveals itself to be more flexible and more quickly adaptable to the appari-
tion of new clusters of technologies. Indeed, as argued in [15], before two distinct technologies
start to clearly converge, one should expect similar words to be used in patents from both
technologies. Finally, a semantic classification where patents are gathered based on the fact that they
share similar significant keywords has the advantage of including a network feature that can-
not be found in the USPC case, namely that each patent is associated with a vector of probabil-
ity to belong to each of the semantic classes (more details on this feature can be found in
Section 1). Using co-occurrence of keywords, it is then possible to construct a network of pat-
ents and to study the influence of some key topological features. As reviewed previously, the
use of co-occurrences is the usual way to construct a semantic network. Other hybrid tech-
nique such as bipartite semantic/authors networks, do not have the nice feature of relying
solely on endogenous semantic information contained in data. Semantic classification construction In this section, we describe methods and empirical analysis leading to the construction of
semantic network and the corresponding classification. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Data collection and basic description The number of patents steadily increase from 1976 to 2000 and
remains constant around 200,000 per year from 2000 to 2007. Restricting our sample to patent
with application date ranging from 1976 to 2007, we are left with 3,949,615 patents. These pat-
ents cite 38,756,292 other patents with the empirical lag distribution that has been extensively
analyzed in [4]. Conditioned on being cited at least once, a patent receives on average 13.5 cita-
tions within a five-year window. 270,877 patents receive no citation during the next five years PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 4 / 22 Keywords relevance estimation Relevance definition. Following the heuristic in [29], we estimate relevance score in
order to filter multi-stem. The choice of the total number of keywords to be extracted, which
we shall denote Kw, is important, too small a value would yield similar network structures but
including less information whereas very large values tend to include too many irrelevant key-
words. We choose to set this parameter to Kw = 100,000. We first consider the filtration of k
Kw (with k = 4) to keep a large set of potential keywords but still have a reasonable number of
co-occurrences to be computed. This step has only very marginal effects on the nature of the
final keywords but is necessary for computational purposes. The filtration is done on the unit-
hood ui, defined for keyword i as ui = fi log(1 + li) where fi is the multi-stem’s number of appa-
ritions over the whole corpus and li its length in words. A second filtration of Kw keywords is
done on the termhood ti, where the formal definition can be found in Eq (1). It is computed as
a chi-squared score on the distribution of the stem’s co-occurrences and then compared to a
uniform distribution within the whole corpus. Intuitively, uniformly distributed terms will be
identified as plain language and they are thus not relevant for the classification. More precisely,
we compute the co-occurrence matrix (Mij), where Mij is defined as the number of patents
where stems i and j appear together. The termhood score ti is defined as ti ¼
X
j6¼i
Mij P
kMik
P
kMjk
2
P
kMik
P
kMjk
:
ð1Þ ð1Þ Moving window estimation. The previous scores are estimated on a moving window
with fixed time length following the idea that the present relevance is given by the most recent
context and thus that the influence vanishes when going further into the past. Consequently,
the co-occurrence matrix is chosen to be constructed at year t restricting to patent which
applied during the time window [t −T0; t]. Note that the causal property of the window is cru-
cial as the future cannot play any role in the current state of keywords and patents. This way,
we will obtain semantic classes which are exploitable on a T0 time span. Keywords extraction Let P be the set of patents, we first assign to a patent p 2 P a set of potentially significant key-
words K(p) from its text AðpÞ (that corresponds to the concatenation of its own title and
abstract). K(p) are extracted through a similar procedure as the one detailed in [29]: 1. Text parsing and Tokenization: we transform raw texts into a set of words and sentences,
reading it (parsing) and splitting it into elementary entities (words organized in sentences). 2. Part-of-speech tagging: attribution of a grammatical function to each of the tokens defined
previously. 5 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Classifying patents based on their semantic content 3. Stem extraction: families of words are generally derived from a unique root called stem (for
example compute, computer, computation all yield the same stem comput) that we
extract from tokens. At this point the abstract text is reduced to a set of stems and their
grammatical functions. 4. Multi-stems construction: these are the basic semantic units used in further analysis. They
are constructed as groups of successive stems in a sentence which satisfies a simple gram-
matical function rule. The length of the group is between 1 and 3 and its elements are either
nouns, attributive verbs or adjectives. We choose to extract the semantics from such nomi-
nal groups in view of the technical nature of texts, which is not likely to contain subtle
nuances in combinations of verbs and nominal groups. Text processing operations are implemented in python in order to use built-in functions
nltk library [30] for most of above operations. This library supports most of state-of-the-art
natural language processing operations. Source code is openly available on the repository of
the project at https://github.com/JusteRaimbault/PatentsMining. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Construction of the semantic network We keep the set of most relevant keywords KW and obtain their co-occurrence matrix as
defined in Section 1. This matrix can be directly interpreted as the weighted adjacency matrix
of the semantic network. At this stage, the topology of raw networks does not allow the extrac-
tion of clear communities. This is partly due to the presence of hubs that correspond to fre-
quent terms common to many fields (e.g. method, apparat) which are wrongly filtered as
relevant. We therefore introduce an additional measure to correct the network topology: the
concentration of keywords across technological classes, defined as: ctechðsÞ ¼
X
NðtecÞ
j¼1
kjðsÞ
2
P
ikiðsÞ
2 ; where kj(s) is the number of occurrences of the sth keyword in each of the jth technological
class taken from one of the N(tec) USPC classes. The higher ctech, the more specific to a techno-
logical class the node is. For example, the terms semiconductoris widely used in electron-
ics and does not contain any significant information in this field. We use a threshold
parameter, defined as θc, and keep nodes with ctech(s) > θc. Likewise, edges with low weights
correspond to rare co-occurrences and are considered to be noise. To account for this we
define the threshold parameter for edges θw, and we filter edges with a weight below θw, follow-
ing the rationale that two keywords are not linked “by chance” if they appear simultaneously a
minimal number of time. To control for size effect, we normalize by taking yw ¼ yð0Þ
w NP
where NP is the number of patents in the corpus (NP ¼ jPj). yð0Þ
w is thus a varying parameter
interpreted as a noise threshold per patent. Communities are then extracted using a standard
modularity maximization procedure as described in [31] to which we add the two constraints
captured by θw and θc, namely that edges must have a weight greater than θw and nodes a con-
centration greater than θc. At this stage, both parameters θc and yð0Þ
w are unconstrained and
their choice is not straightforward. Indeed, many optimization objectives are possible, such as
the modularity, network size or number of communities. We find that modularity is maxi-
mized at a roughly stable value of θw across different θc for each year, corresponding to a stable
yð0Þ
w across years, which leads us to choose yð0Þ
w ¼ 4:1 10 5. Classifying patents based on their semantic content choice of maximum time lag for citations made in Section 1. Accordingly, the sensitivity analy-
sis for T0 = 2 can be found in Appendix S3 Text. choice of maximum time lag for citations made in Section 1. Accordingly, the sensitivity analy-
sis for T0 = 2 can be found in Appendix S3 Text. Construction of the semantic network There is a priori no reason to choose any specific point among the dif-
ferent optimums. Consequently, we have tried the analysis with all the candidate values for θc
and found that the results are the most reasonable when taking θc = 0.06 (see Fig 1). We show
in Fig 2 an example of semantic network visualization. Construction of the semantic network Then for the choice of θc, different
candidates points lie on a Pareto front for the bi-objective optimization on number of commu-
nities and network size. There is a priori no reason to choose any specific point among the dif-
ferent optimums. Consequently, we have tried the analysis with all the candidate values for θc
and found that the results are the most reasonable when taking θc = 0.06 (see Fig 1). We show
in Fig 2 an example of semantic network visualization. where kj(s) is the number of occurrences of the sth keyword in each of the jth technological
class taken from one of the N(tec) USPC classes. The higher ctech, the more specific to a techno-
logical class the node is. For example, the terms semiconductoris widely used in electron-
ics and does not contain any significant information in this field. We use a threshold
parameter, defined as θc, and keep nodes with ctech(s) > θc. Likewise, edges with low weights
correspond to rare co-occurrences and are considered to be noise. To account for this we
define the threshold parameter for edges θw, and we filter edges with a weight below θw, follow-
ing the rationale that two keywords are not linked “by chance” if they appear simultaneously a
minimal number of time. To control for size effect, we normalize by taking yw ¼ yð0Þ
w NP
where NP is the number of patents in the corpus (NP ¼ jPj). yð0Þ
w is thus a varying parameter
interpreted as a noise threshold per patent. Communities are then extracted using a standard
modularity maximization procedure as described in [31] to which we add the two constraints
captured by θw and θc, namely that edges must have a weight greater than θw and nodes a con-
centration greater than θc. At this stage, both parameters θc and yð0Þ
w are unconstrained and
their choice is not straightforward. Indeed, many optimization objectives are possible, such as
the modularity, network size or number of communities. We find that modularity is maxi-
mized at a roughly stable value of θw across different θc for each year, corresponding to a stable
yð0Þ
w across years, which leads us to choose yð0Þ
w ¼ 4:1 10 5. Then for the choice of θc, different
candidates points lie on a Pareto front for the bi-objective optimization on number of commu-
nities and network size. Keywords relevance estimation For example, this
enables us to compute the modularity of classes in the citation network as in section 1. In the
following, we take T0 = 4 (which corresponds to a five year window) consistently with the PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 6 / 22 Characteristics of semantic classes For each year t, we define as N
ðsemÞ
t
the number of semantic classes which have been computed
by clustering keywords from patents appeared during the period [t −T0, t] (we recall that
we have chosen T0 = 4). Each semantic class k ¼ 1; . . . ; N
ðsemÞ
t
is characterized by a set of key-
words K(k, t) which is a subset of KW selected as described in previous sections. The cardinal
of K(k, t) distribution across each semantic class k is highly skewed with a few semantic classes
containing over 1,000 keywords, most of them with roughly the same number of keywords. In contrast, there are also many semantic classes with only two keywords. There are around PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 7 / 22 Classifying patents based on their semantic content Fig 1. Sensitivity analysis of network community structure to filtering parameters. We consider a specific window 2000–2004 and the
obtained plots are typical. (Left panel) We plot the number of communities as a function of the edge threshold parameter θw for different
values of the node threshold parameter θc. The maximum is roughly stable across θc (dashed red line). (Right panel) To choose θc, we do a
Pareto optimization on communities and network size: the compromise point (red overline) on the Pareto front (purple overline: possible
choices after having fixed yð0Þ
w ; blue level gives modularity) corresponds to θc = 0.06. https://doi.org/10.1371/journal.pone.0176310.g001 Fig 1. Sensitivity analysis of network community structure to filtering parameters. We consider a specific window 2000–2004 and the
obtained plots are typical. (Left panel) We plot the number of communities as a function of the edge threshold parameter θw for different
values of the node threshold parameter θc. The maximum is roughly stable across θc (dashed red line). (Right panel) To choose θc, we do a
Pareto optimization on communities and network size: the compromise point (red overline) on the Pareto front (purple overline: possible
choices after having fixed yð0Þ
w ; blue level gives modularity) corresponds to θc = 0.06. https://doi.org/10.1371/journal.pone.0176310.g001 https://doi.org/10.1371/journal.pone.0176310.g001 30 keywords by semantic class on average and the median is 2 for any t. Fig 3 shows that the
average number of keywords is relatively stable from 1976 to 1992 and then picks around 1996
prior to going down. Title of semantic classes. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Characteristics of semantic classes USPC technological classes are defined by a title and a highly
accurate definition which help retrieve patents easily. The title can be a single word (e.g.: class
101: “Printing”) or more complex (e.g.: class 218: “High-voltage switches with arc preventing
or extinguishing devices”). As our goal is to release a comprehensive database in which each
patent is associated with a set of semantic classes, it is necessary to give an insight on what
these classes represent by associating a short description or a title as in [26]. In our case, such
description is taken as a subset of keywords taken from K(k, t). For the vast majority of seman-
tic classes that have less than 5 keywords, we decide to keep all of theses keywords as a descrip-
tion. For the remaining classes which feature around 50 keywords on average, we rely on the
topological properties of the semantic network. [32] suggest to retain only the most frequently
used terms in K(k, t). Another possibility is to select 5 keywords based on their network cen-
trality with the idea that very central keywords are the best candidates to describe the overall
idea captured by a community. For example, the largest semantic class in 2003–2007 is charac-
terized by the keywords: Support Packet; Tree Network; Network Wide; Voic
Stream; Code Symbol Reader. Size of technological and semantic classes. We consider a specific window of observa-
tions (for example 2000–2004), and we define Z the number of patents which appeared during
that time window. For each patent i = 1, , Z we associate a vector of probability where each
component p
ðsemÞ
ij
2 ½0; 1, with j = 1, , N(sem) and where X
NðsemÞ
j¼1
p
ðsemÞ
ij
¼ 1 X
NðsemÞ
j¼1
p
ðsemÞ
ij
¼ 1 (when there is no room for confusion, we drop the subscript t in N
ðsemÞ
t
). On average across all
time windows, a patent is associated to 1.8 semantic classes with a positive probability. Next 8 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Classifying patents based on their semantic content Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters
θc = 0.06 and yw ¼ yð0Þ
w NP ¼ 4:5e 5 9:1e5. Characteristics of semantic classes For-
mally, we define the number of technological classes as N(tec) (which is not time dependent
contrary to the semantic case) and for j = 1, , N(tec) the corresponding matrix of probability
is defined as USPTO and consists in dividing equally the patents between all the classes they belong to. For-
mally, we define the number of technological classes as N(tec) (which is not time dependent
contrary to the semantic case) and for j = 1, , N(tec) the corresponding matrix of probability
is defined as p
ðtecÞ
ij
¼
Bij
X
NðtecÞ
k¼1
Bik
; p
ðtecÞ
ij
¼
Bij
X
NðtecÞ
k¼1
Bik
; where Bij equals 1 if the ith patent belongs to the jth technological class and 0 if not. When
there is no room for confusion, we will drop the exponent part and write only pij when refer-
ring to either the technological or semantic matrix. Empirically, we find that both classes
exhibit a similar hierarchical structure in the sense of a power-law type of distribution of class
sizes as shown in Fig 4. This feature is important, it suggests that a classification based on the
text content of patents has some separating power in the sense that it does not divide up all the
patents in one or two communities. where Bij equals 1 if the ith patent belongs to the jth technological class and 0 if not. When
there is no room for confusion, we will drop the exponent part and write only pij when refer-
ring to either the technological or semantic matrix. Empirically, we find that both classes
exhibit a similar hierarchical structure in the sense of a power-law type of distribution of class
sizes as shown in Fig 4. This feature is important, it suggests that a classification based on the
text content of patents has some separating power in the sense that it does not divide up all the
patents in one or two communities. Characteristics of semantic classes The corresponding file in a vector format (.svg), that can be zoomed and explored, is available
as S1 File. https://doi.org/10.1371/journal.pone.0176310.g002 Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters
θc = 0.06 and yw ¼ yð0Þ
w NP ¼ 4:5e 5 9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available
as S1 File Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters
θc = 0.06 and yw ¼ yð0Þ
w NP ¼ 4:5e 5 9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available
as S1 File. Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters
θc = 0.06 and yw ¼ yð0Þ
w NP ¼ 4:5e 5 9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available
as S1 File. https://doi.org/10.1371/journal.pone.0176310.g002 Fig 2. An example of semantic network visualization. We show the network obtained for the window 2000–2004, with parameters
θc = 0.06 and yw ¼ yð0Þ
w NP ¼ 4:5e 5 9:1e5. The corresponding file in a vector format (.svg), that can be zoomed and explored, is available
as S1 File. https://doi.org/10.1371/journal.pone.0176310.g002 we define the size of a semantic class as we define the size of a semantic class as we define the size of a semantic class as S
ðsemÞ
j
¼
X
Z
i¼1
p
ðsemÞ
ij
: Correspondingly, we aim to provide a consistent definition for technological classes. For that
purpose, we follow the so-called “fractional count” method, which was introduced by the PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 9 / 22 Classifying patents based on their semantic content Fig 3. This figure plots the average number of keywords by semantic class for each time window [t −4; t] from t = 1980 to t = 2007. https://doi.org/10.1371/journal.pone.0176310.g003 Fig 3. This figure plots the average number of keywords by semantic class for each time window [t −4; t] from t = 1980 to t = 2007. https://doi.org/10.1371/journal.pone.0176310.g003 USPTO and consists in dividing equally the patents between all the classes they belong to. Potential refinements of the method Our semantic classification method could be refined by combining it with other techniques
such as Latent Dirichlet Allocation which is a widely used topic detection method (e.g. [33]),
already used on patent data as in [34] where it provides a measure of idea novelty and the
counter-intuitive stylized facts that breakthrough invention are likely to come out of local
search in a field rather than distant technological recombination. Using this approach should
first help further evaluate the robustness of our qualitative conclusions (external validation). Also, depending on the level of orthogonality with our classification, it can potentially bring an PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 10 / 22 Classifying patents based on their semantic content Fig 4. Sizes of classes. Yearly from t = 1980 to t = 2007, we plot the size of semantic classes (left-side) and technological classes (right-
side) for the corresponding time window [t −4, t], from the biggest to the smallest. The formal definition of size can be found in Section
Characteristics of Semantic Classes. Each color corresponds to one specific year. Yearly semantic classes and technological classes
present a similar hierarchical structure which confirms the comparability of the two classifications. This feature is crucial for the statistical
analysis in Section Statistical Model. Over time, curves are translated and levels of hierarchy stays roughly constant. https://doi.org/10.1371/journal.pone.0176310.g004 Fig 4. Sizes of classes. Yearly from t = 1980 to t = 2007, we plot the size of semantic classes (left-side) and technological classes (right-
side) for the corresponding time window [t −4, t], from the biggest to the smallest. The formal definition of size can be found in Section
Characteristics of Semantic Classes. Each color corresponds to one specific year. Yearly semantic classes and technological classes
present a similar hierarchical structure which confirms the comparability of the two classifications. This feature is crucial for the statistical
analysis in Section Statistical Model. Over time, curves are translated and levels of hierarchy stays roughly constant. https://doi.org/10.1371/journal.pone.0176310.g004 https://doi.org/10.1371/journal.pone.0176310.g004 additional feature to characterize patents, in the spirit of multi-modeling techniques where
neighbor models are combined to take advantage of each point of view on a system. Our use of network analysis can also be extended using newly developed techniques of
hyper-network analysis. Potential refinements of the method Indeed, patents and keywords can for example be nodes of a bipartite
network, or patents be links of an hyper-network, in the sense of multiple layers with different
classification links and citation links. The combination of citation network modeling by Sto-
chastic Block Modeling with topic modeling was studied for scientific papers by [35], outper-
forming previous link prediction algorithms. [36] provide a method to compare macroscopic
structures of the different layers in a multilayer network that could be applied as a refinement
of the overlap, modularity and statistical modeling studied in this paper. Furthermore, is has
recently been shown that measures of multilayer network projections induce a significant loss
of information compared to the generalized corresponding measure [37], which confirms the
relevance of such development that we left for further research. An other potential research development would be to further exploit the temporal structure
of our dataset. Indeed, large progress have recently been made in complex network analysis of
time-series data (see [38] for a review). For example, [39] develops a method to construct mul-
tiscale network from time series, which could in our case be a solution to identify structures in
patents trajectories at different levels, and be an alternative to the single scale modularity anal-
ysis we use. Classifying patents based on their semantic content several measures derived from this semantic classification at the patent level: Diversity, Origi-
nality, Generality (Section Patent Level Measures) and Overlapping (Section Classes overlaps). We then show that the two classifications show highly different topological measures and
strong statistical evidence that they feature a different model (Sections Citation Modularity
and Statistical Model). Results In this section, we present some key features of our resulting semantic classification showing
both complementary and differences with the technological classification. We first present 11 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Classifying patents based on their semantic content around 1996. This can be seen whether or not we include patents with zero diversity. The
diversity of complicated patents stabilizes after a constant decrease, and the overall diversity
begins to strongly decrease. This means that on the one hand the number of single class patents
begins to increase and on the other hand complicated patents do not change in diversity. It
can be interpreted as a change in the regime of specialization, the new regime being caused by
more single-class patents. More commonly used in the literature are the measures of originality and generality. These
measures follow the same idea than the above-defined diversity in quantifying the diversity of
classes (whether technological or semantic) associated with a patent. But instead of looking at
the patent’s classes, they consider the classes of the patents that are cited or citing. Formally,
the originality Oi and the generality Gi of a patent i are defined as O
ðzÞ
i
¼ 1 X
NðzÞ
j¼1
X
i02Ii
pi0j
X
NðzÞ
k¼1
X
i02Ii
pi0k
0
B
B
B
B
B
@
1
C
C
C
C
C
A
2
and G
ðzÞ
i
¼ 1 X
NðzÞ
j¼1
X
i02~I i
pi0j
X
NðzÞ
k¼1
X
i02~I i
pi0k
0
B
B
B
B
B
@
1
C
C
C
C
C
A
2
; O
ðzÞ
i
¼ 1 X
NðzÞ
j¼1
X
i02Ii
pi0j
X
NðzÞ
k¼1
X
i02Ii
pi0k
0
B
B
B
B
B
@
1
C
C
C
C
C
A
2
and G
ðzÞ
i
¼ 1 X
NðzÞ
j¼1
X
i02~I i
pi0j
X
NðzÞ
k¼1
X
i02~I i
pi0k
0
B
B
B
B
B
@
1
C
C
C
C
C
A
2
; O
ðzÞ
i
¼ 1 X
NðzÞ
j¼1
X
i02Ii
pi0j
X
NðzÞ
k¼1
X
i02Ii
pi0k
0
B
B
B
B
B
@
1
C
C
C
C
C
A
2
and G
ðzÞ
i
¼ 1 X
NðzÞ
j¼1
X
i02~I i
pi0j
X
NðzÞ
k¼1
X
i02~I i
pi0k
0
B
B
B
B
B
@
1
C
C
C
C
C
A
2
; where z 2 {tec, sem}, Ii denotes the set of patents that are cited by the ith patent within a five
year window (i.e. if the ith patent appears at year t, then we consider patents on [t −T0, t])
when considering the originality and ~I i the set of patents that cite patent i after less than five
years (i.e. we consider patents on [t, t+T0]) in the case of generality. Note that the measure of
generality is forward looking in the sense that G
ðzÞ
i
used information that will only be available
5 years after patent applications. Both measures are lower on average based on semantic classi-
fication than on technological classification. Fig 6 plots the mean value of O
ðsemÞ
i
, O
ðtecÞ
i
, G
ðsemÞ
i
and G
ðtecÞ
i
. where z 2 {tec, sem}, Ii denotes the set of patents that are cited by the ith patent within a five
year window (i.e. if the ith patent appears at year t, then we consider patents on [t −T0, t])
when considering the originality and ~I i the set of patents that cite patent i after less than five
years (i.e. we consider patents on [t, t+T0]) in the case of generality. Note that the measure of
generality is forward looking in the sense that G
ðzÞ
i
used information that will only be available
5 years after patent applications. Both measures are lower on average based on semantic classi-
fication than on technological classification. Fig 6 plots the mean value of O
ðsemÞ
i
, O
ðtecÞ
i
, G
ðsemÞ
i
and G
ðtecÞ
i
. Patent level measures Given a classification system (technological or semantic classes), and the associated probabili-
ties pij for each patent i to belong to class j (that were defined in Section Characteristics of
Semantic Classes), one can define a patent-level diversity measure as one minus the Herfindhal
concentration index on pij by D
ðzÞ
i
¼ 1 X
NðzÞ
j¼1
p2
ij; with z 2 ftec; semg: We show in Fig 5 the distribution over time of semantic and technological diversity with
the corresponding mean time-series. This is carried with two different settings, namely includ-
ing/not including patents with zero diversity (i.e. single class patents). We call other patents
“complicated patents” in the following. First of all, the presence of mass in small probabilities
for semantic but not technological diversity confirms that the semantic classification contains
patent spread over a larger number of classes. More interestingly, a general decrease of diver-
sity for complicated patents, both for semantic and technological classification systems, can be
interpreted as an increase in invention specialization. This is a well-known stylized fact as doc-
umented in [40]. Furthermore, a qualitative regime shift on semantic classification occurs Fig 5. Patent level diversities. Distributions of diversities (Left column) and corresponding mean time-series (Right column) for t = 1980 to
t = 2007 (with the corresponding time window [t −4, t]). The first row includes all classified patents, whereas the second row includes only
patents with more than one class (i.e. patents with diversity greater than 0). https://doi org/10 1371/journal pone 0176310 g005 Fig 5. Patent level diversities. Distributions of diversities (Left column) and corresponding mean time-series (Right column) for t = 1980 to
t = 2007 (with the corresponding time window [t −4, t]). The first row includes all classified patents, whereas the second row includes only
patents with more than one class (i.e. patents with diversity greater than 0). PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 12 / 22 Classes overlaps A proximity measure between two classes can be defined by their overlap in terms of patents. Such measures could for example be used to construct a metrics between semantic classes. Intuitively, highly overlapping classes are very close in terms of technological content and one
can use them to measure distance between two firms in terms of technology as done in [41]. Formally, recalling the definition of (pij) as the probability for the ith patent to belong to the Fig 6. Patent level originality (left hand side) and generality (right hand side) for t = 1980 to t = 2007 (with the corresponding time
window [t −4, t]) as defined in subsection Patent Level Measures. https://doi org/10 1371/journal pone 0176310 g006 Fig 6. Patent level originality (left hand side) and generality (right hand side) for t = 1980 to t = 2007 (with the corresponding time
window [t −4, t]) as defined in subsection Patent Level Measures. Fig 6. Patent level originality (left hand side) and generality (right hand side) for t = 1980 to t = 2007 (with the corresponding time
window [t −4, t]) as defined in subsection Patent Level Measures. https://doi.org/10.1371/journal.pone.0176310.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 13 / 22 Classifying patents based on their semantic content jth class and NP as the number of patents it writes jth class and NP as the number of patents it writes Overlapjk ¼ 1
NP
X
NP
i¼1
pijpik:
ð2Þ ð2Þ The overlap is normalized by patent count to account for the effect of corpus size: by con-
vention, we assume the overlap to be maximal when there is only one class in the corpus. A
corresponding relative overlap is computed as a set similarity measure in the number of pat-
ents common to two classes A and B, given by oðA; BÞ ¼ 2 jA\Bj
jAjþjBj. Intra-classification overlaps. The study of distributions of overlaps inside each classifica-
tion, i.e. between technological classes and between semantic classes separately, reveals the
structural difference between the two classification methods, suggesting their complementary
nature. Their evolution in time can furthermore give insights into trends of specialization. We
show in Fig 7 distributions and mean time-series of overlaps for the two classifications. The
technological classification globally always follow a decreasing trend, corresponding to more
and more isolated classes, i.e. specialized inventions, confirming the stylized fact obtained in
previous subsection. Classes overlaps For semantic classes, the dynamic is somehow more intriguing and sup-
ports the story of a qualitative regime shift suggested before. Although globally decreasing as
technological overlap, normalized (resp. relative) mean overlap exhibits a peak (clearer for
normalized overlap) culminating in 1996 (resp. 1999). Looking at normalized overlaps, classifi-
cation structure was somewhat stable until 1990, then strongly increased to peak in 1996 and
then decrease at a similar pace up to now. Technologies began to share more and more until a
breakpoint when increasing isolation became the rule again. An evolutionary perspective on
technological innovation [42] could shed light on possible interpretations of this regime shift:
as species evolve, the fitness landscape first would have been locally favorable to cross- Fig 7. Intra-classification overlaps. (Left column) Distribution of overlaps Oij for all i 6¼ j (zero values are removed because of the log-
scale). Right column) Corresponding mean time-series. (First row) Normalized overlaps. (Second row) Relative overlaps. https://doi.org/10.1371/journal.pone.0176310.g007 Fig 7. Intra-classification overlaps. (Left column) Distribution of overlaps Oij for all i 6¼ j (zero values are removed because of the log-
scale). Right column) Corresponding mean time-series. (First row) Normalized overlaps. (Second row) Relative overlaps. https://doi.org/10.1371/journal.pone.0176310.g007 Fig 7. Intra-classification overlaps. (Left column) Distribution of overlaps Oij for all i 6¼ j (zero values are removed because of the log-
scale). Right column) Corresponding mean time-series. (First row) Normalized overlaps. (Second row) Relative overlaps. https://doi.org/10.1371/journal.pone.0176310.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 14 / 22 Classifying patents based on their semantic content Fig 8. Distribution of relative overlaps between classifications. (Left) Distribution of overlaps at all time steps; (Right) Corresponding
mean time-series. The decreasing trend starting around 1996 confirms a qualitative regime shift in that period. https://doi.org/10.1371/journal.pone.0176310.g008 Fig 8. Distribution of relative overlaps between classifications. (Left) Distribution of overlaps at all time steps; (Right) Corresponding
mean time-series. The decreasing trend starting around 1996 confirms a qualitative regime shift in that period. https://doi.org/10.1371/journal.pone.0176310.g008 https://doi.org/10.1371/journal.pone.0176310.g008 insemination, until each fitness reaches a threshold above which auto-specialization becomes
the optimal path. It is very comparable to the establishment of an ecological niche [43], the
strong interdependency originating here during the mutual insemination resulting in a highly
path-dependent final situation. Inter-classification overlaps. Overlaps between classifications are defined as in (Classes
overlaps), but with j standing for the jth technological class and k for the kth semantic class: pij
are technological probabilities and pik semantic probabilities. Classes overlaps They describe the relative corre-
spondence between the two classifications and are a good indicator to spot relative changes, as
shown in Fig 8. Mean inter-classification overlap clearly exhibits two linear trends, the first
one being constant from 1980 to 1996, followed by a constant decrease. Although difficult to
interpret directly, this stylized fact clearly unveils a change in the nature of inventions, or at
least in the relation between content of inventions and technological classification. As the tip-
ping point is at the same time as the ones observed in the previous section and since the two
statistics are different, it is unlikely that this is a mere coincidence. Thus, these observations
could be markers of a hidden underlying structural changes in processes. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 Classifying patents based on their semantic content Fig 9. Temporal evolution of semantic and technological modularities of the citation network. (Left) Simple directed modularity,
computed with patent main classes (main technological class and semantic class with larger probability). (Right) Multi-class modularity,
computed following [44]. Fig 9. Temporal evolution of semantic and technological modularities of the citation network. (Left) Simple directed modularity,
computed with patent main classes (main technological class and semantic class with larger probability). (Right) Multi-class modularity,
computed following [44]. https://doi.org/10.1371/journal.pone.0176310.g009 https://doi.org/10.1371/journal.pone.0176310.g009 (i.e. the number of citations made by the ith patent to others and the number of citations
received by the ith patent). Qd can be defined for each of the two classification systems: z 2
{tec, sem}. If z = tec, ci is defined as the main patent class, which is taken as the first class
whereas if z = sem, ci is the class with the largest probability. Multi-class modularity in turns is given by QðzÞ
ov ¼ 1
NP
X
NðzÞ
c¼1
X
1i;jNP
Fðpic; pjcÞAij bout
i;c kout
i bin
j;ckin
j
NP
"
#
; where bout
i;c ¼ 1
NP
X
j
Fðpic; pjcÞ and bin
j;c ¼ 1
NP
X
i
Fðpic; pjcÞ: We take F(pic, pjc) = pic pjc as suggested in [44]. Modularity is an aggregated measure of how
the network deviates from a null model where links would be randomly made according to
node degree. In other words it captures the propensity for links to be inside the classes. Over-
lapping modularity naturally extends simple modularity by taking into account the fact that
nodes can belong simultaneously to many classes. We document in Fig 9 both simple and
multi-class modularities over time. For simple modularity, Q
ðtecÞ
d
is low and stable across the
years whereas Q
ðsemÞ
d
is slightly greater and increasing. These values are however low and sug-
gest that single classes are not sufficient to capture citation homophily. Multi-class modulari-
ties tell a different story. First of all, both classification modularities have a clear increasing
trend, meaning that they become more and more adequate with citation network. The speciali-
zations revealed by both patent level diversities and classes overlap is a candidate explanation
for this growing modularities. Secondly, semantic modularity dominates technological modu-
larity by an order of magnitude (e.g. 0.0094 for technological against 0.0853 for semantic in
2007) at each time. This discrepancy has a strong qualitative significance. Our semantic classi-
fication fits better the citation network when using multiple classes. As technologies can be
seen as a combination of different components as shown by [5], this heterogeneous nature is
most likely better taken into account by our multi-class semantic classification. https://doi.org/10.1371/journal.pone.0176310.g009 Citation modularity An exogenous source of information on relevance of classifications is the citation network
described in Section A bibliographical network between patents: citations. The correspon-
dence between citation links and classes should provide a measure of accuracy of classifica-
tions, in the sense of an external validation since it is well-known that citation homophily is
expected to be quite high (see, e.g, [14]). This section studies empirically modularities of the
citation network regarding the different classifications. To corroborate the obtained results, we
propose to look at a more rigorous framework in Section Statistical Model. Modularity is a
simple measure of how communities in a network are well clustered (see [31] for the accurate
definition). Although initially designed for single-class classifications, this measure can be
extended to the case where nodes can belong to several classes at the same time, in our case
with different probabilities as introduced in [44]. The simple directed modularity is given in
our case by Q
ðzÞ
d ¼ 1
NP
X
1i;jNP
Aij kin
i kout
j
NP
dðci; cjÞ; with Aij the citation adjacency matrix (i.e. Aij = 1 if there is a citation from the ith patent to the
jth patent, and Aij = 0 if not), kin
i ¼ jIij (resp. kout
i
¼ j~I ij) in-degree (resp. out-degree) of patents with Aij the citation adjacency matrix (i.e. Aij = 1 if there is a citation from the ith patent to the
jth patent, and Aij = 0 if not), kin
i ¼ jIij (resp. kout
i
¼ j~I ij) in-degree (resp. out-degree) of patents PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 15 / 22 Classifying patents based on their semantic content obtained in Section Citation Modularity. The mere difference between this approach and the
citation modularity approach lies in the choice of the underlying model, and the according
quantities of interest. In addition for the semantic approach, we want to see if when restricting
to patents with higher probabilities to belong to a class, we obtain better results. To do that, we
choose to look at within class citations proportion (for both technological and semantic
approaches). We provide two obvious reasons why we choose this. First, the citations are com-
monly used as a proxy for performance as mentioned in Section Citation Modularity. Second,
this choice is “statistically fair” in the sense that both approaches have focused on various goals
and not on maximizing directly the within class proportion. Nonetheless, the within class pro- obtained in Section Citation Modularity. The mere difference between this approach and the
citation modularity approach lies in the choice of the underlying model, and the according
quantities of interest. In addition for the semantic approach, we want to see if when restricting
to patents with higher probabilities to belong to a class, we obtain better results. To do that, we
choose to look at within class citations proportion (for both technological and semantic
approaches). We provide two obvious reasons why we choose this. First, the citations are com-
monly used as a proxy for performance as mentioned in Section Citation Modularity. Second,
this choice is “statistically fair” in the sense that both approaches have focused on various goals
and not on maximizing directly the within class proportion. Nonetheless, the within class pro-
portion is too sensitive to the distribution of the shape of classes. For example, a dataset where
patents for each class account for 10% of the total number of patents will mechanically have a
better within class proportion than if each class accounts for only 1%. Consequently, an ade-
quate statistical model, which treats datasets fairly regardless of their distribution in classes, is
needed. This effort ressembles to the previous study of citation modularity, but is complemen-
tary since the model presented here can be understood as an elementary model of citation net-
work growth. Furthermore, the parameters fitted here can have a direct interpretation as a
citation probability. We need to introduce and recall some notations. We consider a specific window of observa-
tions [t −T0, t], and we define Z the number of patents which appeared during that time win-
dow. We let t1, , tZ their corresponding appearance date by chronological order, which for
simplicity are assumed to be such that t1 < < tZ. For each patent i = 1, , Z we consider Ci
the number of distinctive couples {cited patent, cited patent’s class} made by the ith patent (for
instance if the ith patent has only made one citation and that the cited patent is associated with
three classes, then Ci = 3). Let z 2 {tec, sem}, we define N
ðzÞ
i
the number of patents associated to
at least one of the ith classes at time ti −1. For l = 1, , Ci we consider the variables Bl, i, which
equal 1 if the cited patent’s class is also common to the ith patent. We assume that Bl, i are inde-
pendent of each other and conditioned on the past follow Bernoulli variables B min 1; N
ðzÞ
i
i 1 þ yðzÞ
(
)
! ; where the parameter 0 θ(z) 1 indicates the propensity for any patent to cite patents of its
own technological or semantic class. When θ(z) = 0, the probability of citing patents from its
own class is simply N
ðzÞ
i ði 1Þ
1, which corresponds to the observed proportion of patents
which belong to at least one of the ith patent’s classes. Thus this corresponds to the estimated
probability of citing one patent if we assume that the probability of citing any patent k = 1, ,
i −1 is uniformly distributed, which could be a reasonable assumption if classes were assigned
randomly and independently from patent abstract contents. Conversely if θ(z) = 1, we are in
the case of a model where there are 100% of within class citations. A reasonable choice of θ(z)
lies between those two extreme values. Finally, we assume that the number of distinctive cou-
ples Ci are a sequence of independent and identically distributed random variables following
the discrete distribution C, and also independent from the other quantities. ( ) We estimate θ(z) via maximum likelihood, and obtain the corresponding maximum likeli-
hood estimator (MLE) ^yðzÞ. Statistical model In this section, we develop a statistical model aimed at quantifying performance of both tech-
nological and semantic classification systems. In particular, we aim at corroborating findings PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 16 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 https://doi.org/10.1371/journal.pone.0176310.t001 The likelihood function, along with the standard deviation expres-
sion and details about the test, can be found in S4 Text. The fitted values, standard errors and
p-values corresponding to the statistical test θ(sem) = θ(tec) (with corresponding alternative
hypothesis θ(sem) > θ(tec)) on non-overlapping blocks from the period 1980–2007 are reported
on Table 1. Note that the estimation included patents up until 2010 in the period 2006–2007 PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
April 26, 2017 17 / 22 Classifying patents based on their semantic content and not the patents from 1980 in the period 1980–1985 for homogeneity in size with other
periods. This doesn’t affect the significativity of the results. Semantic values are reported for
four different chosen thresholds p−= .04, .06, .08, .1. It means that we restricted to the couples
(ith patent, jth class) such that pij p−. The choice of considering non-overlapping blocks (instead of overlapping blocks) is merely
statistical. Ultimately, our interest is in the significance of the test over the whole period 1980–
2007. Thus, we want to compute a global p-value. This can be done considering the local p-val-
ues (by local, we mean for instance computed on the period 2001–2005) assuming indepen-
dence between them. This assumption is reasonable only if the blocks are non-overlapping. All
of this can be found in S4 Text. Finally, note that from a statistical perspective, including over-
lapping blocks wouldn’t yield more information. The values reported in Table 1 are overwhelmingly against the null hypothesis. The global
estimates of θ(sem) are significantly bigger than the estimate of θ(tec) for all the considered
thresholds. Although the corresponding p-values (which are also very close to 0) are not
reported, it is also quite clear that the bigger the threshold, the higher the corresponding θ(sem)
is estimated. This is consistently seen for any period, and significant for the global period. This
seems to indicate that when restricting to the couples (patent, class) with high semantic proba-
bility, the propension to cite patents from its own class θ(sem) is increasing. We believe that this
might provide extra information to patent officers when making their choice of citations. Indeed, they could look first to patents which belong to the same semantic class, especially
when patents have high probability semantic values. Note that the introduced model can be seen as a simple model of citations network growth
conditional to a classification, which can be expressed as a stochastic block model (e.g. [45],
[46]). The parameters are estimated computing the corresponding MLE. In view of [47], this
can be thought as equivalent to maximizing modularity measures. Classifying patents based on their semantic content Table 1. Estimated values of θ(tec) and θ(sem) and corresponding standard errors obtained from a Maxi-
mum Likelihood estimator as presented in section Statistical Model. Approach
Estimated Value
st. er. p-value
1980–1985 period
technological
.664
.008
semantic p−= .04
.741
.047
.053
semantic p−= .06
.799
.081
.049
semantic p−= .08
.828
.126
.097
semantic p−= .10
.834
.166
.153
1986–1990 period
technological
.634
.007
semantic p−= .04
.703
.022
.001
semantic p−= .06
.768
.040
.0004
semantic p−= .08
.804
.069
.007
semantic p−= .10
.832
.114
.041
1991–1995 period
technological
.619
.006
semantic p−= .04
.655
.009
.0004
semantic p−= .06
.713
.017
9e-08
semantic p−= .08
.731
.025
7e-06
semantic p−= .10
.750
.037
9e-06
1996–2000 period
technological
.551
.003
semantic p−= .04
.585
.002
0
semantic p−= .06
.638
.004
0
semantic p−= .08
.660
.006
0
semantic p−= .10
.686
.008
0
2001–2005 period
technological
.567
.003
semantic p−= .04
.621
.004
0
semantic p−= .06
.676
.007
0
semantic p−= .08
.701
.010
0
semantic p−= .10
.710
.013
0
2006–2007 period
technological
.600
.007
semantic p−= .04
.683
.016
1e-06
semantic p−= .06
.732
.025
2e-07
semantic p−= .08
.760
.036
6e-06
semantic p−= .10
.782
.048
9e-05
1980–2007 global period
technological
.606
.002
semantic p−= .04
.665
.009
8e-11
semantic p−= .06
.721
.017
9e-12
semantic p−= .08
.747
.025
9e-09
semantic p−= .10
.782
.035
3e-07
https://doi.org/10.1371/journal.pone.0176310.t001 Table 1. Estimated values of θ(tec) and θ(sem) and corresponding standard errors obtained from a Maxi-
mum Likelihood estimator as presented in section Statistical Model. 18 / 22 Classifying patents based on their semantic content Regarding firm innovative strategy, a second extension could be to study trajectories of
firms in the two networks: technological and semantic. Merging these information with data
on the market value of firms can give a lot of insight about the more efficient innovative strate-
gies, about the importance of technology convergence or about acquisition of small innovative
firms. It will also allow to observe innovation pattern over a firm life cycle and how this differ
across technology field. A third extension would be to use dig further into the history of innovation. USPTO patent
data have been digitized from the first patent in July 1790. However, not all of them contain a
text that is directly exploitable. We consider that the quality of patent’s images is good enough
to rely on Optical Character Recognition techniques to retrieve plain text from at least 1920. With such data, we would be able to extend our analysis further back in time and to study how
technological progress occurs and combines in time. [50] conduct a similar work by looking at
recombination and apparition of technological subclasses. Using the fact that communities are
constructed yearly, one can construct a measure of proximity between two successive classes. This could give clear view on how technologies converged over the year and when others
became obsolete and replaced by new methods. S2 Text. Data collection procedure. Detailed description of data collection.
(PDF) S2 Text. Data collection procedure. Detailed description of data collection. (PDF) S3 Text. Network sensitivity analysis. Extended figures for Network Sensitivity Analysis. (PDF) S3 Text. Network sensitivity analysis. Extended figures for Network Sensitivity Analysis. (PDF) S4 Text. Statistical definitions and derivations. Extended definitions and derivations for the
statistical model. (PDF) S4 Text. Statistical definitions and derivations. Extended definitions and derivations for the
statistical model. (PDF) S1 File. Semantic network visualization. Vector file of the semantic network (Fig 2). (SVG) Supporting information S1 Text. Definition of utility patent. Describes with more details the definition of patents
and context. (PDF) Conclusion The main contribution of this study was twofold. First we have defined how we built a network
of patents based on a classification that uses semantic information from abstracts. We have
shown that this classification share some similarities with the traditional technological classifi-
cation, but also have distinct features. Second, we provide researchers with materials resulting
from our analysis, which includes: (i) a database linking each patent with its set of semantic
classes and the associated probabilities; (ii) a list of these semantic classes with a description
based on the most relevant keywords; (iii) a list of patent with their topological properties in
the semantic network (centrality, frequency, degree, etc.). The availability of this data suggests
new avenues for further research. Linking our dataset with existing open ones can lead to vari-
ous powerful developments. For example, using it together with the disambiguated inventor
database provided by [48] could be a way to study semantic profiles of inventors, or of cities as
inventor addresses are provided. The investigation of spatial diffusion of innovation between
cities, which is a key component of Pumain’s Evolutive Urban Theory [49], would be made
possible. A first potential application is to use the patents’ topological measures inherited from their
relevant keywords. The fact that these measures are backward-looking and immediately avail-
able after the publication of the patent information is an important asset. It would for example
be very interesting to test their predicting power to assess the quality of an innovation, using
the number of forward citations received by a patent, and subsequently the future effect on the
firm’s market value. PLOS ONE | https://doi.org/10.1371/journal.pone.0176310
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Pressure-Assisted Development and Characterization of Al-Fe Interface for Bimetallic Composite Castings: An Experimental and Statistical Investigation for a Low-Pressure Regime
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Metals
| 2,021
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cc-by
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Pressure Assisted Development and
Characterization of Al-Fe Interface for bi-metallic
Composite Castings: An Experimental and
Statistical Investigation for Low Pressure Regime Tayyiba Rashid
University of Engineering & Technology Lahore
Muhammad Qaiser Saleem
University of Engineering & Technology Lahore
Nadeem Ahmad Mufti
University of Engineering & Technology Lahore
Noman Asif
University of Engineering & Technology Lahore
Kashif Ishfaq
University of Engineering & Technology Lahore
Maham Naqvi ( mn446@njit.edu )
New Jersey Institute of Technology Tayyiba Rashid
University of Engineering & Technology Lahore
Muhammad Qaiser Saleem
University of Engineering & Technology Lahore
Nadeem Ahmad Mufti
University of Engineering & Technology Lahore
Noman Asif
University of Engineering & Technology Lahore
Kashif Ishfaq
University of Engineering & Technology Lahore
Maham Naqvi ( mn446@njit.edu )
New Jersey Institute of Technology Pressure assisted development and characterization of Al-Fe interface for
bi-metallic composite castings: An experimental and statistical
investigation for low pressure regime Tayyiba Rashid1,2, Muhammad Qaiser Saleem2, Nadeem Ahmad Mufti2, Noman Asif2,
Kashif Ishfaq2, Maham Naqvi3* Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engineering, University of
ngineering & Technology Lahore, 54890, Pakistan Institute of Quality & Technology Management, University the Punjab Lahore, 54000, Pakistan Research Article Posted Date: April 8th, 2021 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Metals on October 23rd, 2021. See the
published version at https://doi.org/10.3390/met11111687. Keywords: Al-Fe interface, composite castings, interfacial properties, pressure-assisted
casting, cooling rate, grey relational analysis g
g
gy
Department of Mechanical & Industrial Engineering, New Jersey Institute of Technology, Newark NJ 071
SA g
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gy
3 Department of Mechanical & Industrial Engineering, New Jersey Institute of Technology, Newark NJ 07102,
USA 2 Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engineering, University of
Engineering & Technology Lahore, 54890, Pakistan Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engineering
Engineering & Technology Lahore, 54890, Pakistan Q
y
gy
g
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2 Department of Industrial & Manufacturing Engineering, Faculty of Mechanical Engin 1. Introduction: The continual strive for high strength cast components has garnered a renewed research
interest in the development of metal reinforced castings (alternatively termed as dissimilar
metal castings or metallic composite castings) with reportages of promising results [1–3]. In
this context, a mix metal structure of steel and aluminum alloy poses itself to be a viable
tradeoff between cost and strength. Interestingly, the studies on the so-called 'die soldering'
phenomenon involving iron and aluminum alloys already indicate towards the affinity
existing between these two materials [4, 5]. Aluminum's high affinity for Iron causes a strong
reaction when Al melt comes into contact with ferrous material. This reaction produces iron-
aluminum-silicon intermetallic compounds, which cause cast metal to stick to it. Various
studies in this regard have primarily focused on exploring the factors that are responsible for
promoting interface development between the two materials. The soldering is found to be
highly dependent on temperature, pre-heat temperature, die surface conditions
(topography/coatings) [5] and the chemistry of alloys [6]. A review of some of the available
studies in this regard justifies that a strong rationale exists for developing Iron reinforced
aluminum composites via casting route. Han et al. [7] studied the soldering for both pure Aluminum as well as Aluminum alloy 380
when fabricated inside a steel mold. In this work, a soldering critical temperature is suggested
at which Iron (Fe) begins to react with Aluminum (Al) and results in the formation of an Al-
rich liquid phase and solid intermetallic compounds. When the temperature at the die surface
is higher than this critical temperature, the aluminum-rich phase is liquid and joins the die
with the casting during the subsequent solidification. They purported that the critical
temperature for soldering is either the solidus temperature or the coherency temperature
between the two metals. In another research, Han et al.[8] reported liquid fraction of the
solidifying metal (Al) to be influencing soldering tendency and mentioned surface roughness
and placement angle of the steel die to be important. A higher liquid fraction, rougher surface
and placement at a certain anglewere reported to be favoring the soldering phenomenon. Kim
et al. [4], in their study, reported on the effect of Si concentration (in Al alloy) on the
formation of Al-Si-Fe intermetallic layer on different grades of steel (H13 and 1045 steel). They concluded a higher percentage of Si to be resulting in increased thickness of the
intermetallic layer. Abstract Review of the available literature indicates that development of metal reinforced castings
present intriguing prospects but carry inherent challenges owing to differences in thermal
coefficients, chemical affinities, diffusion issues and varying nature of intermetallic
compounds. It is supported that pressure application during solidification may favorably
influence the dynamics of the aforementioned issues, nevertheless, not only certain
limitations have been cited but also some pressure and process regimes have not been found
to be investigated and optimized. This work employs the pressure-assisted approach for bi-
metallic steel reinforced aluminum composite castings at low-pressure regime and thoroughly
investigates the role of three process parameters namely pouring temperature (800°C-900°C),
pressure (10-20 bars) and holding time (10-20 sec) for producing sound interfaces. Taguchi
L9 orthogonal array has been employed as DOE while dominant factors have been
determined via ANOVA and Grey relational analysis multi-objective optimization technique. Supplementary analysis through optical micrographs, SEM and EDS has been relied upon to
quantify interfacial layer thicknesses and to study microstructural and compositional aspects
of the interface. Nano-indentation tests under static and dynamic loading have also been
performed for mechanical strength characterization. It has been found that uniform interfaces
with verifiable diffusion are obtainable with pouring temperature being the most influential
parameter (PCR 92.84%) in this pressure regime. Optimum parameters determined from the
work, yield ~328% thicker interface layer, 19.42% better nano-hardness and 19.10%
improved cooling rate when compared to the process conditions with least parametric levels. Keywords: Al-Fe interface, composite castings, interfacial properties, pressure-assisted
casting, cooling rate, grey relational analysis Keywords: Al-Fe interface, composite castings, interfacial properties, pressure-assisted
casting, cooling rate, grey relational analysis Corresponding Author: Maham Naqvi; mn446@njit.edu 1 1 1. Introduction: They were able to achieve 28µm thick interface with 9% wt Si while
keeping the dipping temperature constant at 680ºC. It is important to note that the
aforementioned studies and most of the other available literature such as [9–12] have been 2 2 limited to hot dipping and aluminizing methodologies to produce the interfaces; these
methods may not be representative of the state-of-the-art procedures employed for producing
commercial castings. Work may also be found that employs contemporary methods (namely
gravity die casting, high pressure die casting, diffusion-treatment and sand casting) to
produce commercial castings for various material combinations including Al-Steel [13–17]. Incidentally, researchers have highlighted some issues in multi material castings such as
lower diffusion of one element into the other and non-uniform thickness of the interface layer
[15] thus necessitating a thorough investigation for Al-Steel interfaces produced through
specific casting processes; this should enable the production of sound multi-material castings
to meet industrial demands. Additionally, aspects like cooling rate of the casting and
mechanical characterization of the developed interface are found to have received relatively
less attention in reported studies. Correspondingly, the dynamics of interface development in
a real life scenario with the role of process-specific parameters may need further attention. For the context, the few studies that have used mainstream casting routes while developing
the interfaces may be cited not only to mention some positive aspects but to also highlight
some process specific issues as reported. Viala et al. [16] used the gravity die molding
method and reported on the interfacial chemistry of aluminum and steel. By achieving a
continuous metallurgical bond between steel and aluminum, they successfully replaced a
165g cogwheel shaped insert with a 105g simple one. This helped in significant weight
reduction of the cast component. In another study, Bhagat et al. [17] employed squeeze
casting process for producing aluminum alloy-stainless steel metal composites while
experimenting with various fiber volume fractions when stainless steel was used as a mesh in
aluminum alloy matrix. They demonstrated the potential effectiveness of squeeze casting
process for producing such composites while mentioning 850°C metal pouring temperature,
550°C die pre-heating temperature, and 110 MPa pressure to be suitable parameters. Nevertheless, their work highlighted some issues as encountered. 1. Introduction: Although 40% volume
fraction was reported to be resulting in higher tensile strength of the composite in comparison
to that reported for aluminum alloy (without reinforcement) but the composites were still
reported to have lower strength (in general) to what had been predicted by the rule of
mixtures. They attributed it to the generation of a weak interface as a result of 'more than
desirable' reaction between fiber and matrix materials in the interface. They also cited
limitations in generating uniform interface thickness and observance of large scatter in the
values. The need for squeeze casting process parameters optimization was also cited to
overcome the issues faced in interface development. Understandably, the higher-pressure 3 3 values employed in their work were those that are typical of a squeeze casting process and
follow-up work of any sort that employed pressure assistance in lower value regime(s) for Al-
Fe interface hasn’t been found in the available literature as yet. The recent work of Kashif et
al. [18] also made use of the squeeze pressure but for producing Al overcast joints; since their
work targeted only one type of material (Al) therefore the differences in thermal coefficients
and affinity issues were supposedly neither much concerning issues nor were they thoroughly
studied. Nevertheless, their work indicates the renewed interest in light of the potential
feasibility of pressure-assisted approach for the multi-material components. The potential of the pressure driven casting process and its specific issues as highlighted for
previous work on aluminum steel interfaces requires that a lower pressure regime may be
investigated to evaluate if the so-called 'more than desirable' reactions may be avoided and
better interfaces are developed. Additionally, the limitations as stated for the previous studies
on the Al-Fe interfaces, call for a structured parametric investigation for evaluating an
extended range of interface properties while correlating the results with important process
dynamics, such as thermal/cooling aspects and pressure ratings. Further useful insight and
potential benefits could be revealed when study of this sort is supplemented with superior
optimization algorithms such as Grey relational analysis (GRA) which is a multi-response
optimization technique used to identify the best quality characteristics through the
determination of the optimum conditions of input parameters [19]. This work employs pressure-assisted casting route for producing bi-metallic steel reinforced
aluminum composite castings. 1. Introduction: A low-pressure regime has been employed and the role of
three process parameters, namely pressure, pouring temperature and time for application of
pressure (holding time) has been thoroughly investigated for producing sound interfaces. An
Al-Si alloy has been used with mild steel reinforcement. Interfacial layer thicknesses have
been quantified alongwith the study of its microstructural, compositional and nano-hardness
aspects. The evaluation of nano-hardness is given consideration, realizing that in view of the
intermetallics formed, interface may be better characterized by nano-scale hardness
parameter than microhardness. Discussions are supplemented with analysis of cooling rates
as calculated for the experiments to better understand and explain the ensuing process
dynamics. Multi-objective parametric optimization (through GRA) has been done for the role
of input factors for the targeted outputs. 4 4 2.1 Experimental details A cylindrical shaped Al-5% Si alloy specimen (Փ60 x L=70 mm) reinforced by three equally
spaced vertical mild steel rods placed at 120° apart (when viewed in cross-section) was cast
using an 80-ton vertical hydraulic press. Instead of using a mesh structure, rods were
employed for reinforcement so as to focus on interface development at different experimental
conditions while preventing any potential adversities arising from complicated flow patterns
(had mesh been used). Six K-type thermocouples interfaced with the data acquisition system4
were inserted into pre-defined locations to capture thermal profiles along radius and height. The thermocouples were connected to a Data Logger5 and ultimately linked to a computer
system. Prior to placement in die (made of D2), the mild steel rods (Փ5 x L= 50 mm) were
pickled with 90% hydrochloric acid solution followed by immersion in iso-propyle alcohol to
clean rust (if present) on the surface as cleaning treatment used by others [20, 21]. For the
pouring, melt was prepared in pit furnace. The die was pre-heated to 500°C using an external
heating system; upon pouring, pressure was applied and maintained for a specified time
(holding time). The bimetal casting was allowed to cool naturally at room temperature
afterwards. Three settings of each input parameters, namely pouring temperature, applied
pressure and holding time were used for experimentation. The values of input parameters
were chosen on the basis of trial experiments conducted prior to actual experimentation. Taguchi L9 orthogonal array design was used as DOE. A total of nine experiments were
performed. Fig. 1 provides the schematic of the specimen as placed in the die providing
essential details such as locations of thermocouple placements and size of the specimen
produced etc. The actual images for experimental setup & die are given in Fig. 2. Table 1
presents the details of variables and constants. For post-experimentation analysis, samples for
microstructure, SEM analysis and mechanical characterization were extracted from the
castings. Fig. 3 shows the schematic (3a & 3b) and actual cast component (3c) with the
locations marked from where specimens for post-experimental analysis were taken. 2.1 Experimental details Table 1 Design of experiment
Variables
Level 1
Level 2
Level 3
Pouring temperature (°C)
800
850
900
Applied pressure (Bar)
10
15
20
Holding time (Sec)
10
15
20 5 5 Constants
Reinforcement type
( RoundMS rod)
Melt Mass
1.1kg
Matrix
Aluminum-5% Silicon
Die Temperature
(500°C)
Fig.1 Schematic of the die
Fig.2 Experimental setup
Fig.3 a & b) Schematics of the part showing the length & location of the pins c) actual cast
component Constants Matrix
Aluminum-5% Silicon Melt Mass
1.1kg Die Temperature
(500°C) Fig.1 Schematic of the die
Fig.2 Experimental setup
Fig.3 a & b) Schematics of the part showing the length & location of the pins c) actual cast
component Fig.1 Schematic of the die Fig.1 Schematic of the die
Fig.2 Experimental setup Fig.2 Experimental setup Fig.2 Experimental setup Fig.3 a & b) Schematics of the part showing the length & location of the pins c) actual cast
component 6 Fig.4 Research methodology Fig.4 Research methodology
Optical micrographs6 were taken at a magnification of 500X to examine the microstructure
and to evaluate the thickness of the diffusion layer. Interface SEM7 images were taken at a
magnification of 3000X for in-depth examination of compositional and microstructural
details. Samples for microstructure and SEM were prepared using standard metallographic
procedures. For mechanical characterization nanoindentation testing8 was done for the
interface. The loading and unloading rate of 200mN/min and the acquisition rate of 10Hz
were utilized. A record of penetration depth was made over a complete loading cycle with a
maximum load of 100mN using depth sensing indentation. Three samples were tested in
each testing condition to ensure repeatability and the average is reported herein. The
complete research methodology is highlighted in Fig. 4. Fig.4 Research methodology Optical micrographs6 were taken at a magnification of 500X to examine the microstructure
and to evaluate the thickness of the diffusion layer. Interface SEM7 images were taken at a
magnification of 3000X for in-depth examination of compositional and microstructural
details. Samples for microstructure and SEM were prepared using standard metallographic
procedures. For mechanical characterization nanoindentation testing8 was done for the
interface. The loading and unloading rate of 200mN/min and the acquisition rate of 10Hz
were utilized. A record of penetration depth was made over a complete loading cycle with a
maximum load of 100mN using depth sensing indentation. 8 NHTX S/N: 01-2569 nano-indentation machine, Austria p
y
7 Inspect S-50 of Thermo Fisher Scientific USA 6 Leica DMI 500 microscopic machine, Germany 2.2.1 Data pre-processing GRA begins with the grey relational generationthrough the normalization of data gathered
through experimentation. For GRA, data pre-processing is needed as there may be a
difference of range and units in different data sequences or the scatter range of the sequence
is large. Therefore, in data pre-processing, the original sequence is transferred into a
comparable sequence. The range for normalization is kept between 0.00 to 1.00. The best
normalized results must be equal to 1.00 [23]. Equation 1 is used for this purpose. 𝑥𝑖(𝑘) =
𝑦𝑖(𝑘) −𝑚𝑖𝑛𝑦𝑖(𝑘)
𝑚𝑎𝑥𝑦𝑖(𝑘) −𝑚𝑖𝑛𝑦𝑖(𝑘)
(1) (1) where 𝑥𝑖(𝑘) is the normalized value for kth observation after data pre-processing, 𝑦𝑖(𝑘) is the
original sequence while 𝑚𝑖𝑛 𝑦𝑖(𝑘) and 𝑚𝑎𝑥 𝑦𝑖(𝑘) are the smallest and largest experimental
values of particular responses from the design matrix, respectively. i is the data number
(observations) which in our design matrix is from 1 to 9. 2.2 Statistical analysis Statistical analysis was carried out for the obtained results using statistical software Minitab
19.1. The Analysis of Variance (ANOVA) was performed at a confidence level of 95% in
order to find out the significant input parameters and their percentage contribution (PCR) for
selected responses. Additionally, Grey Relational Analysis (GRA) was employed for multi-
objective optimization of casting parameters. This analysis is a suitable multi-step method
[22] used to measure the degree of approximation (among sequences) through Grey
Relational Grade (GRG).The main objectives takenfor this work were the maximization of
Interface thickness (IT), nano-level hardness (H) and the cooling rate (Rc). Correspondingly,
"larger-the-better" was taken as the criteria. 2.1 Experimental details Three samples were tested in
each testing condition to ensure repeatability and the average is reported herein. The
complete research methodology is highlighted in Fig. 4. 7 7 3 Results, analysis and discussions The experimentation results for the 9 experiment runs are provided in Table 2. The
succeeding sections contain the detailed analysis and discussions. 2.2.2 Gray relational coefficient and gray relational grade After the data normalization, grey relational coefficients (GRC) are calculated using
normalized values to highlight the relation between desired/ideal and actual normalized
results. The GRC highlights the relationship between desirable and real experimental
normalized results. The GRG is computed by averaging the GRC. This GRG value gives a
single grade for response variables [24]. Equation 2 is used for GRC calculations. GRG
values range from 0 to 1. µ𝑖(k) = δ𝑚𝑖𝑛+ mδ𝑚𝑎𝑥
δ0𝑖(k) + 𝑚δ𝑚𝑎𝑥)
(2) (2) 8 8 δ0𝑖(k) = ‖𝑥0(𝑘) −𝑥𝑖(𝑘)‖
(3) (3) (3) µ𝑖(k) is the grey relational coefficient, m is the identification/distinguishing coefficient that is
usually set to 0.5 [25]. δ𝑚𝑖𝑛 and δ𝑚𝑎𝑥 are the minimum and maximum deviations of each
response variable. δ0𝑖(k) is called deviation sequence which is calculated by using Equation
3. This determines the deviation of normalized data from the ideal value of 1.00. On the other
hand, Equation 4 is used for GRG calculation by taking the average of GRCs. 𝐺𝑅𝐺𝑖= 1
𝑛∑𝑤𝑖
𝑛
𝑘=1
µ𝑖 ∑𝑤𝑖=1
𝑛
𝑘=1
(4) (4) GRG values range from 0 to 1. The highest value of GRG infers the ultimate combination of
eminent casting process characteristics. GRG values range from 0 to 1. The highest value of GRG infers the ultimate combination of
eminent casting process characteristics. 3.1.1 Microscopic and compositional analysis The thickness of the interface where materials diffuse into each other has been measured for
all the experiments via micrographs. The representative micrographs for all the nine
experiment conditions are presented in Fig. 5 for comparison purposes. As can be seen from
the micrographs, interface thickness increases as the higher values of parameters (pressure
and pouring temperature) are used. An average interface thickness of ~42microns achieved at
higher values of above mentioned parameters is around 328% more than ~9.8microns
obtained at the least values of process parameters employed herein. In essence, the improved
interface thickness values are indicative of the increased diffusion as temperature and
pressure values increase. Table 2 Experimentation results
Exp. No
Pouring
Temperature
(°C)
Applied
Pressure
(bar)
Holding
Time
(Sec)
IT
(µm)
H
(GPa)
Rc
(°C/sec)
1
800
10
10
9.8
9.01
1.78
2
800
15
15
11.0
9.25
1.94
3
800
20
20
13.8
9.58
2.09 Table 2 Experimentation results 9 4
850
10
15
18.9
9.96
1.86
5
850
15
20
28.5
10.03
1.98
6
850
20
10
30.7
10.14
2.09
7
900
10
20
38.0
10.27
1.93
8
900
15
10
40.9
10.45
2.07
9
900
20
15
42.0
10.76
2.12
Fig.5 Microstructures of the interface region of bimetals made at different temperature and
pressure
10bar, 10sec
15bar, 15sec
20bar, 20sec
Pouring Temp. (800°C)
10bar, 15sec
15bar, 20sec
20bar, 10sec
Pouring Temp. (850°C)
10bar, 20sec
15bar, 10sec
20bar, 15sec
Pouring Temp. (900°C) 4
850
10
15
18.9
9.96
1.86
5
850
15
20
28.5
10.03
1.98
6
850
20
10
30.7
10.14
2.09
7
900
10
20
38.0
10.27
1.93
8
900
15
10
40.9
10.45
2.07
9
900
20
15
42.0
10.76
2.12
Fig.5 Microstructures of the interface region of bimetals made at different temperature and
pressure
10bar, 10sec
15bar, 15sec
20bar, 20sec
Pouring Temp. (800°C)
10bar, 15sec
15bar, 20sec
20bar, 10sec
Pouring Temp. (850°C)
10bar, 20sec
15bar, 10sec
20bar, 15sec
Pouring Temp. (900°C) 20bar, 10sec 20bar, 10sec Pouring Temp. (850°C)
10bar, 20sec 15bar, 10sec 20bar, 15sec 20bar, 15sec 10bar, 20sec 15bar, 10sec Pouring Temp. (900°C) Pouring Temp. (900°C) Fig.5 Microstructures of the interface region of bimetals made at different temperature and
pressure These results wherein higher temperature has been seen to cause more interface thickness in
general, are effectively supported by Fick’s law for diffusion [26] when reviewed in
conjunction with the diffusivity relation. 3.1.1 Microscopic and compositional analysis It can thus be shown that the diffusionis a function
of temperature. At higher temperatures, atoms/molecules gain more mobility in liquids as
well as solids. A higher initial concentration of a particular element may cause its diffusion
into other solid. Fick’s first law has expressed this diffusional flow mathematically as shown
in Equation 5. in Equation 5. 10 𝑱𝒙= −𝑫𝛅𝐂
𝛅𝐗
(5)
𝛅𝐂 (5) 𝑱𝒙= −𝑫𝛅𝐂
𝛅𝐗 Where Jx is the flow rate of the diffusing species in the direction X based on the gradient (
𝛅𝐂
𝛅𝐗). Here D is termed as the diffusion coefficient (also known as diffusivity). The following
expression (Equation 6) is used to calculate diffusivity D. Where Jx is the flow rate of the diffusing species in the direction X based on the gradient (
𝛅𝐂
𝛅𝐗). Here D is termed as the diffusion coefficient (also known as diffusivity). The following
expression (Equation 6) is used to calculate diffusivity D. 𝐷= 𝐷𝑜𝑒−𝑄
𝑅𝑇
⁄ (6) 𝐷
𝐷𝑜𝑒
( )
Do, is the pre-exponential coefficient, Q is the activation energy per mole of diffusing
species, and R is the universal gas constant. The T is the absolute temperature. This equation
shows that the Diffusion coefficient is based on temperature and as the reciprocal of
temperature (1/T) decreases, diffusion increases. The increase in temperature triggers
diffusion to progress more swiftly, resulting in thicker diffusion layers at the interface. Correspondingly, a higher temperature is seen to result in better values. In terms of pressure, the results arein general conformance to the other available literature
[27, 28], which shows the role of pressure to be favorable as well. Here too, for a pouring
temperature of 900°C, the thickness at 20 bar is around 10% higher compared to the one
obtained at 10 bar even when a relatively lesser holding time has been employed. An
additional and visible advantage of the pressure-assisted process is the generation of a nearly
uniform interface for each case as can be seen in Fig. 5. More specifically, when compared
with other reported work such as by Chen et al. [29] who employed nearly 200 times higher
pressure in compound forging process to fabricate Al-Steel bimetal, it is observed that the
achieved interface here is much thicker than the already reported work. In their work the
interface thickness of 7.9µm was achieved when the initial temperature of the steel ball was
kept at 900°C & the applied pressure was 503MPa. Incidentally, the average interface
thickness achieved in current study ranges from 9.8µm to 42.0µm which is much higher that
of achieved by the compound forging. in Equation 5. The possible reason for higher interface obtained
herein is that, a higher pressure employed in the reported work would increase rate of heat
transfer which would result in quicker heat loss from the components, thus not allowing
much time for the constituents to diffuse; this is not the case herein where the lesser values of
pressure would inevitably be resulting into relatively lesser rate of heat loss which is expected
to give reasonable time for the constituents to diffuse up to a certain distance. Additionally,
the forging process is a solid state process for which the rate of diffusion would be lesser than
the current process where aluminum alloy has been poured in liquid state. Furthermore when compared with reported work [30] on compound casting wherein the Furthermore, when compared with reported work [30] on compound casting wherein the
applied force is presumably much lesser than the pressure employed in the current work, 11 much lower interface thicknesses has been observed. The maximum interface thickness of
around 30µm is reported for Al-steel bimetallic which is ~28.57% less than what has been
achieved with the pressure assisted approach herein. Even for the case of other systems such as Al-Cu, it is deduced that very high pressure may
not necessarily be resulting in the very thick interfaces. Liu et al. [31] while producing bi-
metal component (Al-Cu) through squeeze casting process, observed increase in the thickness
of the reaction layer as the temperature was increased (due to the higher diffusion at elevated
temperature) however, increase in squeeze pressure resulted in decreasing trend of the overall
thickness. The highest value obtained for thickness was about 80µm at 30MPa pressure
which further decreased when pressure was increased to 110MPa. As stated earlier, the
higher rate of heat transfer during solidification associated with higher pressure values is the
most likely reason. Above analysis indicates the importance of the lower pressure regime in
producing an appropriate level of interface thickness enabled by the balance between pressure
and available time for diffusion to occur. Fig. 6 gives the summary of results for interface
thickness in graphical form. Fig.6 Interface thickness obtained for 9 experimental conditions Fig.6 Interface thickness obtained for 9 experimental conditions To confirm the diffusion of materials at the interface and to compare the effects of input
parameters on interface constituents, an EDS analysis was performed. Fig. 3.1.2 Quantification for the role of parameters for interface thickness In order to understand the role of different parameters, the main effect plots for interface
thickness are provided in Fig. 8. The results are supplemented by analysis of variance
(ANOVA) (Table 3) for their percentage contribution (PCR) to the outputs. Here, the
pouring temperature comes out to be the only significant parameter at 95% confidence level
with a percentage contribution of 92.84%. As can be seen from the plots, both pressure and
pouring temperature positively correlate with the thickness whereas holding time in the
investigated regime does not seem to be much influential. in Equation 5. 7 presents the
elemental profile plots/line scan for all the parametric conditions. Here, the EDS scans of the
interface validate the presence of primary constituents of the two metals involved in this
composite. This line scan also highlights the percentage variation of Al, Si and Fe at the
interface region. Within the interface, the concentration of primary element diminishes as we
move to the opposite side of the base metal/alloy to which that constituent belongs. Another
interesting observation is that more Al diffuses into the interface in comparison to steel that
could be attributed to the smaller size of Al atoms as compared to the Fe atoms [26]. It is 12 pertinent to specify that the percentage of constituents is also influenced by the experimental
conditions. Higher pressure and temperature values are seen to favor more aluminum
diffusion into the interface than Fe. Correspondingly, at higher values of these parameters,
(say experiment 9) the percentage of aluminum increases with the decrease in percentage of
Fe. The possible reason for this is the larger dissemination of molten aluminum into steel at a
higher temperature and pressure resulting in a rather compact and uniform interface
thickness. 3.1.3 Nano-hardness The nano-hardness values obtained for all 9 experiments have been shown in Fig. 9. It can be
seen that maximum hardness (10.76 GPa) is obtained at the interface in experiment 9 which
is in line with the results obtained for interface thickness. If compared to Experiment 1
conditions, where we have least values of all processing parameters, an improvement of
19.42% is highlighted. Another important aspect is that, the range of hardness achieved at
interface is higher than the hardness of individual Al Alloy (1.50GPa) and steel (2.50GPa). It
is not uncommon for the interface to develop higher nano-hardness than the base materials. The work by Jiang et al. [32] where magnesium/steel bimetal was developed, reported higher
nano-hardness values (11.1GPa) of the interface layer as compared to the hardness of
magnesium (1.1GPa) and steel matrixes (4.2GPa). It is important to mention that, Al melt has
a capability to spread homogeneously over the iron insert thus initiating the diffusion of
elements (Fe, Al & Si) at the interface. The higher values of hardness for the developed
interface may be associated to the presence of intermetallics such as, Fe2Al5,τ10-Al9Fe4Si3
FeAl3, Al12Mg17, Al12Mg17. It is obvious that the types of intermetallic layers produced, and
their thicknesses play critical role in obtaining materials with desirable performances. In
another work [27] an interaction of solid iron rod with molten Al was investigated. 13 Intermetallic such as FeAl3 and Fe2Al5 were identified through XRD analysis within the
interface layer. Also the Fe2Al5 layer thickness was found to be larger than the FeAl3 layer. Additionally, Jian et al. [28] also observed intermetallic such as Fe2Al5, τ10-Al9Fe4Si3, FeAl3,
τ5-Al8Fe2Si, τ6-Al4.5FeSi which could be the reason for the higher nano-hardness (10.49GPa)
for the interface reported in their wok. Literature survey further reveals that the intermetallics
such as Fe2Al5 & FeAl3 formed in the interface layer are brittle [33]. The in general inverse
relationship of brittle materials and hardness is already established which explains the higher
nano-hardness values within the interface. Vendra and Rabiei [34] investigated the diffusion
of iron and Aluminum, and observed the presence of brittle Fe2Al7Si phase. They also
conducted nano-hardness test while citing it adequate approach in view of the small size of
needle shaped light gray/dark phase which could render regular microhardness (MH) testing
method as inadequate for the estimation of their hardness. 3.1.3 Nano-hardness A comprehensive load-displacement curve at the interface developed at all experimental
conditions is shown in Fig. 10. It is evident from the graph that with the increase in hardness
of the interface, the penetration of the indenter is reduced, indicating the increasing strength
of the interface. It can be seen that at the same load conditions, the interface developed at
more favorable conditions is able to resist deformation more indicating a harder interface. The least indenter penetration (0.95µm) has been achieved for interface produce at highest
level of parameters which is 26.92% less when compared to the maximum penetration
(1.3µm) achieved with least parametric levels. 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate 14 Fig.7 SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic
composite (for all experiments) Fig.7 SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic
composite (for all experiments) Fig.8 Main effect plots for interface thickness Fig.8 Main effect plots for interface thickness 15 Table 3 ANOVA for interface thickness
Source
DF
Seq SS
Adj SS
Adj
MS
F-
Value
P-
Value
Contribution
T(°C)
2 1241.31 1241.31 620.654 135.48
0.007
92.84%
P(bar)
2
68.55
68.55
34.274
7.48
0.118
5.13%
Ht(Sec)
2
18.00
18.00
9.001
1.96
0.337
1.35%
Error
2
9.16
9.16
4.581
0.69%
Total
8 1337.02
100.00%
R-sq: 99.31% R-sq(adj): 97.26%
Fig.9 Hardness value obtained for all 9 experiments Table 3 ANOVA for interface thickness Source
DF
Seq SS
Adj SS
Adj
MS
F-
Value
P-
Value
Contribution
T(°C)
2 1241.31 1241.31 620.654 135.48
0.007
92.84%
P(bar)
2
68.55
68.55
34.274
7.48
0.118
5.13%
Ht(Sec)
2
18.00
18.00
9.001
1.96
0.337
1.35%
Error
2
9.16
9.16
4.581
0.69%
Total
8 1337.02
100.00%
R-sq: 99.31% R-sq(adj): 97.26% Fig.9 Hardness value obtained for all 9 experiments Fig.9 Hardness value obtained for all 9 experiments Fig.9 Hardness value obtained for all 9 experiments Fig.10 Load vs displacement graph
-50
0
50
100
150
200
250
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Force (mN)
Displacement (µm)
Load Vs Displacement
Experiment 1
Experiment 2
Experiment 3
Experiment 4
Experiment 5
Experiment 6
Experiment 7
Experiment 8
Experiment 9 Load Vs Displacement Fig.10 Load vs displacement graph 16 Fig.11 Main effects plot for nano-hardness
Table 4 ANOVA for nano-hardness
Source
DF
Seq SS
Adj SS
Adj
MS
F-
Value
P-
Value
Contribution
T(°C)
2 2.25398 2.25398 1.12699 116.18
0.009
88.28%
P(bar)
2 0.25579 0.25579 0.12789
13.18
0.071
10.02%
Ht(Sec)
2 0.02400 0.02400 0.01200
1.24
0.447
0.94%
Error
2 0.01940 0.01940 0.00970
0.76%
Total
8 2.55317
100.00%
R-sq: 99.24 % R-sq(adj): 96.96 % Fig.11 Main effects plot for nano-hardness Fig.11 Main effects plot for nano-hardness Fig.11 Main effects plot for nano-hardness Table 4 ANOVA for nano-hardness Table 4 ANOVA for nano-hardness
Source
DF
Seq SS
Adj SS
Adj
MS
F-
Value
P-
Value
Contribution
T(°C)
2 2.25398 2.25398 1.12699 116.18
0.009
88.28%
P(bar)
2 0.25579 0.25579 0.12789
13.18
0.071
10.02%
Ht(Sec)
2 0.02400 0.02400 0.01200
1.24
0.447
0.94%
Error
2 0.01940 0.01940 0.00970
0.76%
Total
8 2.55317
100.00%
R-sq: 99.24 % R-sq(adj): 96.96 % The results for nano-hardness are best explained when supplemented by cooling rates
calculated for the experiments. 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate Fig. 11 shows the main effect plots for nano level hardness wherein an increasing trend for
hardness values with an increased temperature. At higher temperatures, the diffusion of Al
into steel is increased and improves the formation of intermetallic, resulting in increased
hardness values. The increase in pressure and holding time has also shown a higher hardness
value. The ANOVA (Table 4) shows that with a p-value of 0.009, the temperature is the most
significant parameter and has the highest PCR value (88.28%). 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate It was observed that experiment 9, wherein pressure and
temperature were at their maximum values (of the investigated set), resulted into the highest
cooling rate. Refer to Fig. 12 which shows values of different cooling rates calculated for all
9 experiments. The cooling rate obtained for experiment 9 is around 20% higher than
experiment 1 where pressure and temperature were at their least. In experiment 9, the higher
available temperature gradient has been made possible by the higher pouring temperature
along with the applied pressure, which in its own independent capacity has been reported to
increase the interfacial heat transfer coefficient during solidification [35–37]. All this results
into accelerated heat transfer from the casting. Correspondingly, the microstructure which is
reported to be a function of cooling rate [38–40] would also be refined which in turn results
in the betterment of mechanical properties. The interdependency of mechanical propertiesand
microstructure onthe cooling rate has been found in literature [41]. The same could be
inferred here wherein higher values of nano-indentation have been achieved at these
conditions. Importantly, as can be seen from Fig. 12, the three highest cooling rates
(Experiment 9, 6 and 3) are all obtained when the applied pressure was at the highest values 17 (20bar), not only supporting the given explanation but also hinting towards the role of
pressure in developing accelerated cooling rates.If we compare this work with reported
literature (where higher values of pressure have been used), with the application of pressure,
increase in cooling rate has been reported [40]. This is further confirmed when ANOVA is performed for the cooling rates (Table 5). Here,
the pressure is the most contributing parameter with PCR value (81.98%) and is statistically
significant at 95% confidence level. It is also important to note that the cooling rates pattern
doesn't necessarily match entirely with the pattern obtained for nano-hardness results. For
example, the two cooling rates (for experiments 3 and 6) that immediately lag the best
cooling rate of experiment 9 do not result in the second and third highest nano-hardness
values. This can also be explained; at higher cooling rates a possibly adverse effect could be
that a very quick temperature drop may not allow enough time for diffusion to properly take
place. 3.1.4 Quantification for the role of parameters for nano-hardness and cooling rate Interestingly both of these experiment conditions (3 and 6) though having a higher-
pressure value (which is statistically significantparameter for cooling rate) but the
corresponding pouring temperatures are lower (800 and850°C). The temperature has been
determined to be a statistically significant parameter for interface thickness. Accordingly, at
these lower pouring temperatures despite getting relatively higher cooling rates, relatively
lesser interface thickness and nano-indentation hardness have been achieved. Experiment 9
has both the parameters at their favorable values so not only that highest cooling rate has
been the outcome but also interface thickness and nano-indentation are also at their best. Fig.12 Cooling rate at different experimental conditions
Table 5 ANOVA for cooling rate
Source
DF
Seq SS
Adj SS
Adj MS F-Value
P-Value
Contribution
T(°C)
2
0.015268
0.015268
0.007634
5.38
0.157
14.29%
P(bar)
2
0.087589
0.087589
0.043794
30.89
0.031
81.98% 3.4 Multi-objective optimization using GRA The optimization has been carried out for development of sound interface. Table 6 contains
the normalizing & deviation sequence, GRC and GRG. The highest grade shows the optimal
parametric setting for the process. The highest GRG value is obtained for experiment number
9, where temperature is 900ºC, pressure is 20bar, while holding time is 15sec. Table 6 Normalizing sequence & deviation sequence Table 6 Normalizing sequence & deviation sequence
Normalizing Sequence
Deviation Sequence
Grey Relational
Coefficient
GRG
Exp. No
IT
H
Rc
IT
H
Rc
IT
H
Rc
-
1
0.0000
0.0000 0.0000 1.0000
1.0000 1.0000
0.3333
0.3333
0.3333
0.3333
2
0.0373
0.1342 0.4693 0.9627
0.8658 0.5307
0.3418
0.3661
0.4851
0.3977
3
0.1242
0.3251 0.9102 0.8758
0.6749 0.0898
0.3634
0.4256
0.8477
0.5456
4
0.2826
0.5432 0.2202 0.7174
0.4568 0.7798
0.4107
0.5226
0.3907
0.4413
5
0.5807
0.5823 0.5873 0.4193
0.4177 0.4127
0.5439
0.5449
0.5478
0.5455
6
0.6491
0.6438 0.9220 0.3509
0.3562 0.0780
0.5876
0.5840
0.8651
0.6789
7
0.8758
0.7205 0.4372 0.1242
0.2795 0.5628
0.8010
0.6414
0.4704
0.6376
8
0.9658
0.8253 0.8459 0.0342
0.1747 0.1541
0.9360
0.7411
0.7645
0.8139
9
1.0000
1.0000 1.0000 0.0000
0.0000 0.0000
1.0000
1.0000
1.0000
1.0000 18 Ht(Sec)
2
0.001154
0.001154
0.000577
0.41
0.711
1.08%
Error
2
0.002836
0.002836
0.001418
2.65%
Total
8
0.106845
100.00%
R-sq:97.35% R-sq(adj): 89.38% 3.4.1 ANOVA for GRA After ranking, an ANOVA is also performed for GRG values to find the most significant
parameter(s). It can be seen from Table 7 that temperature and pressure, with a p-value less
than 0.05, are significant for GRG. The percentage contribution for temperature is 88.57%,
while for pressure it is 31.07%. The main effect plot for GRG is provided in Fig. 14. The
response table (Table 8) shows the levels resulting in the highest GRG values. Temperature
and pressure at level 3 while holding time at level 2 shows the highest GRG. GRG for all the
experimental conditions along with the rank are shown in Fig. 15. 19 Fig.13 Main effects plots for GRG
Table 7 ANOVA for GRG
Source
DF
Seq SS
Adj SS
Adj MS F-Value
P-Value
Contribution
T(°C)
2
0.238796
0.238796
0.119398
52.78
0.019
66.98%
P(bar)
2
0.110781
0.110781
0.055390
24.49
0.039
31.07%
Ht(Sec)
2
0.002422
0.002422
0.001211
0.54
0.651
0.68%
Error
2
0.004524
0.004524
0.002262
1.27%
Total
8
0.356523
100.00%
R-sq: 98.73% R-sq(adj): 94.92%
Table 8 Response table for GRG
Level
1
2
3
Delta
Rank
T(°C)
0.4255 0.5552 0.8172 0.3916
1
P(bar)
0.4708 0.5857 0.6130 0.2707
2
Ht(Sec) 0.6087 0.6130 0.5762 0.0368
3
Fig.14 Gray relational grade for all experiments Fig.13 Main effects plots for GRG Fig.13 Main effects plots for GRG Fig.13 Main effects plots for GRG Fig.14 Gray relational grade for all experiments 20 3.4.2 Confirmation tests A confirmatory experiment was performed for interface thickness, nano-hardness & cooling
rate based on optimization done herein. The response table for means (Table 8), highlights
levels at which all three parameters will give optimal results. From this table, the levels with
highest mean value are selected thus the optimal parametric levels are a temperature of
900ºC, the pressure of 20 bar, and a holding time of 15sec. Based on this suggested
parametric setting, confirmatory experimentation was carried out. The results from the initial
experimental matrix and the confirmatory experiment are highlighted in Table 9. The values
obtained from the confirmatory experiments validated the optimized levels. The confirmatory
run not only validated the reliability of the optimization step (results within 5% of the
predicted value) but also repeatability was ensured. Table 9 Confirmation experiment
Performance
characteristics
Optimal values
from (design
matrix)
Predicted
values
Confirmatory
experiment values
%age error of
confirmatory values
IT (µm)
42.0
41.20
43.2
4.85
H(GPa)
10.76
10.76
10.84
0.74
Cooling Rate
2.12
2.14
2.14
- Table 9 Confirmation experiment Table 9 Confirmation experiment The samples from the optimum combination of input values were further tested for nano-
cyclic loading conditions with a maximum applied load of 200mN. The purpose of applying
cyclic load at the interface was to evaluate the interface developed for deformation under
dynamic loading conditions. When cyclic load is applied on the metallic materials in plastic
range then the tendency to resist deformation is different as compared to continuous load
[42]. It can be seen from Fig. 15 that the sample at the optimum condition restricted
penetration much more than the other with the least experimental conditions. Due to the
increase in pouring temperature and applied pressure, a uniform interface and associated
properties resulted in higher hardness at the interface and correspondingly minimum
penetration of the indenter. 21 Fig.15 Load vs displacement graph for cyclic loading
0
50
100
150
200
250
0.00
1.00
2.00
3.00
4.00
Load (mN)
Displacement(µm)
Load vs Displacement (CL)
A(Pouring Temp
900 °C, Squeeze
Pressure 20bar,
Holding Time
15Sec)
B(Pouring Temp
800 °C, Squeeze
Pressure 10bar,
Holding Time
10Sec) Load vs Displacement (CL) Load vs Displacement (CL) Load vs Displacement (CL) Fig.15 Load vs displacement graph for cyclic loading Not Applicable Not Applicable Acknowledgment This work has been facilitated by University of Engineering & Technology (UET), Lahore,
Pakistan as part of the post graduate PhD research work at the Department of Industrial &
Manufacturing Engineering (UET) Lahore, Pakistan. 4 Conclusions This study while focusing within the context of multi-material castings, developed steel
reinforced Al, bi-metallic composite casting using pressure assisted approach and optimized
the process parameters for interface thickness, nano-mechanical properties (nano-hardness)
and cooling rate. Taguchi L9 orthogonal array was used for experimentation (with pressure,
pouring temperature and holding time as input parameters) supplemented by statistical
analysis of ANOVA and GRA. It has been found that It is possible to generate interfaces of uniform thicknesses within the investigated
pressure regime of 10-20 bars. Pouring temperature has come out to be the most
significant factor in this regime in general. Micrographs and EDS analysis confirms
generation of uniform interface along with diffusion of micro-constituents. Highest interface thickness was achieved at the experiment conditions of temperature
900ºC, pressure 20bar and holding time 15sec where the interface was ~328% higher
than the conditions of 800ºC, 10bar, & 10sec. ANOVA reveals that temperature is the
most significant factor with PCR of 92.84% for interface thickness. Pressure has a
meager contribution of ~5% for interface thickness. The nano-indentation tests revealed 19.42% increase in nano-hardness while load
displacement graph showed 26.92% reduced indenter penetration at experimental
conditions of 900 ºC, 20bar and 15sec when compared to the experimental conditions 22 with least levels (800ºC, 10bar, & 10sec). For this output, temperature was found to
be the most significant parameter with PCR of 88.28%. The results are well supplemented by cooling rate calculations for which pressure is
the most significant factor. The results are well supplemented by cooling rate calculations for which pressure is
the most significant factor. The multi-objective optimization using GRA shows highest grey relational grade
(GRG) for experimental condition 9 (temperature 900ºC, pressure 20bar and holding
time 20sec). Further, the ANOVA for GRG shows the temperature to be the most
significant parameter with a p-value of 0.023. The optimized parameters have been
seen to exhibit higher nano-hardness also in dynamic loading regime. An indirect comparison of the presented work with other published literature indicates
generation of sound interfaces at considerably less harsh pressure conditions. Uniform
interfaces are developed with comparable nano-hardness and thickness (which in
certain cases are found to be even thicker). Authors Contributions Tayyiba Rashid conducted the experiments, performed measurements and analysis. Muhammad Qaiser Saleem helped conceptualize the research plan, supervised the work and
assisted in article write-up. Nadeem Ahmad Mufti modified the article structure and
contributed to analysis and discussion. Noman Asif and Kashif Ishfaq helped in statistical
analysis and data interpretation phases. Maham Naqvi helped in analysis and discussion
phases. Competing Interests Competing Interests
The authors declare that they have no conflict of interest p
g
The authors declare that they have no conflict of interest p
g
The authors declare that they have no conflict of interest Consent to Publish Consent to Publish
All authors give consent to publish this manuscript in The International Journal of Advanced
Manufacturing Technology Consent to Publish
All authors give consent to publish this manuscript in The International Journal of Advanced
Manufacturing Technology Availability of data and materials
Not Applicable Ethical Approval
The research does not involve human participants or animals and the authors warrant that the Ethical Approval
The research does not involve human participants or animals and the authors warrant that the 23 paper fulfills the ethical standards of the journal. This manuscript has not been published or
presented elsewhere in part or in entirety and is not under consideration by another journal paper fulfills the ethical standards of the journal. This manuscript has not been published or
presented elsewhere in part or in entirety and is not under consideration by another journal Consent to Participate p
The research does not involve human participants or animals and the authors warrant that the
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https://doi.org/10.1007/s10853-006-0480-2 28 Figures Figure 1 Figure 1 Schematic of the die Figure 2
Experimental setup
Figure 3
a & b) Schematics of the part showing the length & location of the pins c) actual cast component Figure 2
Experimental setup
Figure 3
a & b) Schematics of the part showing the length & location of the pins c) actual cast component Figure 2 Experimental setup Experimental setup p
p a & b) Schematics of the part showing the length & location of the pins c) actual cast component a & b) Schematics of the part showing the length & location of the pins c) actual cast component Figure 4 Figure 4 Research methodology Research methodology Figure 5 Microstructures of the interface region of bimetals made at different temperature and pressure Microstructures of the interface region of bimetals made at different temperature and pressure Figure 6
Interface thickness obtained for 9 experimental conditions Figure 6 Interface thickness obtained for 9 experimental conditions Interface thickness obtained for 9 experimental conditions Figure 7
SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite (
experiments) Figure 7 SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite (for all
experiments) SEM micrographs and EDS analysis of the interface of the aluminum/steel bi metallic composite (for all
experiments) Figure 8
Main effect plots for interface thickness Figure 8
Main effect plots for interface thickness Figure 8
Main effect plots for interface thickness Figure 8
Main effect plots for interface thickness Figure 8
Main effect plots for interface thickness Figure 8 Figure 8 Main effect plots for interface thickness Main effect plots for interface thickness Figure 9 Hardness value obtained for all 9 experiments Hardness value obtained for all 9 experiments Hardness value obtained for all 9 experiments Figure 10 Figure 10 Load vs displacement graph
Figure 11
Main effects plot for nano-hardness Load vs displacement graph Figure 11
Main effects plot for nano-hardness Figure 11 Main effects plot for nano-hardness Main effects plot for nano-hardness Figure 12
Cooling rate at different experimental conditions Figure 12 Figure 12 Cooling rate at different experimental conditions Figure 13
Main effects plots for GRG Figure 13
Main effects plots for GRG Figure 13 Main effects plots for GRG Main effects plots for GRG Main effects plots for GRG Figure 14 Gray relational grade for all experiments Figure 15 Figure 15 Figure 15 Load vs displacement graph for cyclic loading Load vs displacement graph for cyclic loading
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https://openalex.org/W2324903489
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https://actavet.vfu.cz/media/pdf/avb_1998067040207.pdf
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English
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Cellular Immune Response In Rainbow Trout (Oncorhynchus mykiss): Flow Cytometric Study
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Acta veterinaria Brno
| 1,998
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cc-by
| 4,249
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Address for correspondence:
Dr. Stefan Chilmoncz .... k
INRA. Unitt! de VirologiC et Immunlogie Molcculaires
78J5:! lou)" en Josas France. ACTA VET. BRNO 1998,67: 207-213 ACTA VET. BRNO 1998,67: 207-213 CHILMONCZYK S .. MONGE D.
INRA Unite de Virologie et lmmunologie Moleculaires. Jouy en Josas. France Received September 15.1998
Accepted September 21.1998 Received September 15.1998
Accepted September 21.1998 Abstract Chilmonczyk. S .. D. Monge: Cellular Immune Response in Rainbow Trout (Oncorhynchus
mykiss): Flow Cytometric Study. Acta vet. Brno 1998.67: 207-213. Chilmonczyk. S .. D. Monge: Cellular Immune Response in Rainbow Trout (Oncorhynchus
mykiss): Flow Cytometric Study. Acta vet. Brno 1998.67: 207-213. Assays devoted to the study of non-specific cellular defense mechanisms of rainbow trout:
phagocytosis. oxidative burst and nonspecitic cytotoxicity. were developed. Compared to
conventional methodologies flow cytometry provides a rapid. suitable and reproducible method
for analysing fish cellular immune functions. In addition to the assessment of the defense
mechanisms interesting data are directly provided from the observation of unstained cells. Flow
cytometric profiles of cells are representative of the organ from which the leukocytes originated
and each lymphoid organ displays a characteristic cytogram pattern. Flow cytometry emerges as
a useful tool to study immune reactions and pathogenic processes. Flow cytometr),. rainbow trout. leukocytes. phagocytosis. oxidative burst. nonspecific cytotoxic cell Flow cytometry (FCM) allows a cell by cell qualitative and quantitative analysis of cell
functions and cell activities. FCM offers great potential for investigations in the areas of fish
haematology, pathology and immunology because multiparameter measurements of single
cells are easily and rapidly performed. Immunologists routinely used FCM to characterize phenotypically the leukocyte
subpopulations. So far, in fish, only a few number of antibodies directed against surface
markers are avalaible. The panel of monoclonal antibodies which could identify different
subpopulations offish leukocytes is limited (Deluca et al. 1983; Koumans et al. 1994;
S liere ndrec h t et al. 1995) and unfortunately the existing markers display no cross reactivity
between fish species. Even though now cytometric immunophenotyping offish cells presents
a limited interest, FCM offers real advantages over traditional methods. FCM measurements
are made on the individual cell and not on the whole population and morphologic, biochemical
and functional characteristics can be analyzed simultaneously. Thus it is possible to correlate
the cell function or activity with one subpopulation. y
p p
While in mammals FCM has been used for long, in fish, only a few such studies have been
performed. Recently a battery of assays have been developed to monitor different aspects of
the rainbow trout (RBT) cellular immune response (Voccia eta1.1994; Chilmonczyk et
al. 1995; 1997). The present paper describes some FCM methods we have developed for the
investigation of functional aspects of the RBT cell defense mechanisms. Phone: +33 (Oil 34652476
Fax: +33 lOiI 34652591
E-mail: stephan@biorec.jouy.inra.fr CELLULAR IMMUNE RESPONSE IN RAINBOW TROUT
(Oncorhynchus mykiss): FLOW CYTOMETRIC STUDY CHILMONCZYK S .. MONGE D. CHILMONCZYK S .. MONGE D. INRA Unite de Virologie et lmmunologie Moleculaires. Jouy en Josas. France Oxidative burst Oxidative burst activity was quantified using flow cytometry as a measure of intracellular hydrogen peroxide
production as previously described (Chilmonczyk et aJ. I 997).The assay depends upon the cell incorporation
of2' -7' -dichlorofluore'icin diacetate (DCFH-DA). which is a stable nonfluorescent molecule hvdrolvzed to DCFH
by cytosolic enzymes, Then by the action of HoO" produced in leukocytes stimulated with' Phorbol Myristate
Acetate (PMA). DCFH is oxidised to the fluoresceni dichlorofluorescein (DCF). Briefly. isolated PKL (1.5 x lO6
Iml) were resuspended in I ml MEM and incubated for 15 min at room temperature with DCFH-DA. Cellular
baseline of fluorescence (DCF) was determined at this zero time and PMA (10 ng/ml. final concentration) was
immediately added and the level of intracellular fluorescence was measured in unstimulated and PMA stimulated
cells by flow cytometry. A typical kinetic experiment included the analysis of lO 000 individual cells analysed from
the same tube sample 5. 15,30.45 and 60 min over the reaction period, At each sampling time. a stimulation index
was determined as the ratio of fluorescence of PMA stimulated cells to that of unstimulated cells. Nonspecific Cytotoxic Cells (NCC) We have adapted to RBT the method of Chang et al. (1993) using the mouse lymphoma YAC-I cell line
(Ki e s s lin g et aJ. 1975) as target cells, The assay is based on the simultaneous expression of:2 fluorescent signals
allowing the discrimination between a) unstained effector cells. b) green fluorescent viable target cells c) red plus
green fluorescent dead target cells. Target cells are labeled with a green fluorescent dye: 3. 3'-
dioctadecyloxacarbocyanin perchlorate (DIO-C 18). a lipophilic carbocyanine membrane dye, Target cells are
incubated with RBT leukocytes at a effector - target ratio of 50: 1 for 2h at 20°C in presence of propidium iodide
which permeates only through the membrane of dead cells and stains the nucleic acids emiting a red fluorescence. FCM acquisitions are performed on the base of green and red fluorescence parameters displayed by target cells:
dead and damaged target cells appear doubly stained. Only the target cells. which are easily distinguished from an
abundance of effectors by their distinct green stable fluorescence. are considered in the aquisition of data. Phagocytosis
h
i agocytos s
Phagocytosis a"ays were performed il1l'itro as previously described (Chilmonczyk et aJ. 1995), Briefly. leukocyte suspensions (1.5 x 106 cells/mll\IEM) were incubated for 18 h at 16°C with fluoresceinated (FITC)
latex beads (Polysciences). Leukocytes were incubated with FITC-beads in a proportion of I cell for 100 FlTC-
beads. Non-ingested beads were separated from the phagocytes by centrifuging (lOa g x lO min) mer a cushion of
3% BSA in PBS. The cell pellet was then resuspended in MEM for flow cytometric measurement. The fluorescence
histograms of cell number l'eJ'SUS fluorescence intensity were analysed and phagocytic activity was expressed as
the percentage of cells that had ingested at least 3 FlTC-Iatex beads. Oxidative burst Cell subpopulations Cell subpopulations p p
The auwtluorescence emitted by unstained li\'ing cells allows the distinction between leukocyte subpopulations
according to the siLe (forward light scatter: FSC parameter) and the cytosolic complexity (side light scatter: SSC
parameter) of the cells. The autofluorescence scattering signals are sufficient on their own to discriminate cells with
different morphology: lymphocytes (101\ FSC I SSC) are accurately differentiated from granulocytes (higher FSC
I SSC). Quantification of dead cells \\as determined by adding pmpidium iodide (PI) (Sigma) at a final
concentration of 5fjg/ml (Dei tc h et aJ. 198~). Phagocytosis
Ph
i Fish Leukocytes were collected from healthy (control) or experimentally infected rainbow trout (Oncorhynchus
.lIlykiss - RBT). Fish (5- IO g) were subjected to waterborne Viral Haemorrhagic Septicaemia Virus (VHSV) while
PKX parasite infections (intraperitoneal injection) were performed on 30 g RBT. Control and infected fish were
reared in recirculated filtered water therrnoregulated at IO I °C (VHSV infections) or 161°C (PKX infections). Phone: +33 (Oil 34652476
Fax: +33 lOiI 34652591
E-mail: stephan@biorec.jouy.inra.fr 208 Leukocyte collection y
Freshl)' collected blood anticoagu]Qted \\ ith heparin was diluted (1:20) with !\1inimum Essential Medium
(MEM). Cells from pronephros. spleen and thymus \\ ere obtained by forcing the tissues through a stainless steel
mesh in cold serum-free !\1EM. Then leukocvtes were isolated throu~h a Ficoll cushion. The leukoevte-rich
interphase \\ as collected. washed in PBS and resuspended in ME!\1. ~
, Flow cytometric analysis y
y
Flow cytometric anal)ses were can-ied out using a standard fluorescence activated cell analyser (FACScan. Becton Dickinson I. For each sample I () 000 or 20 nOll individual celb \\ ere recorded using a dot plot combination
of low an~le forward scattered (FSC) and ri~ht an21e scattered (SSC) laser li2ht. Data \\'ere analvsed with the
program LYSIS]] \ersion 2.0. ~
~
~
, Table I Table I
Variability occurring in the aspect of the FCM cytograms of pronephros leukocytes. Compared
to the regular pattern displayed by control RBT. fish subjected to pathogens (VHSV or PKX)
or to a crowding stress presented highly modified cytogram patterns. PN Leukocytes
Cytogram pattern
from
regular
modified
Control
88 % (128/145)
12 % (171145)
VHSV infected
60 % (25/42)
40 % (17142)
PKX infected
6 % (6/92)
94 % (86/92)
Stressed
41 % (10124)
59 % (14/24) riability occurring in the aspect of the FCM cytograms of pronephros leukocytes. Compared
the regular pattern displayed by control RBT. fish subjected to pathogens (VHSV or PKX)
or to a crowding stress presented highly modified cytogram patterns. or to a crowding stress presented highly modified cytogram patterns. PN Leukocytes
Cytogram pattern
from
regular
modified
Control
88 % (128/145)
12 % (171145)
VHSV infected
60 % (25/42)
40 % (17142)
PKX infected
6 % (6/92)
94 % (86/92)
Stressed
41 % (10124)
59 % (14/24) Phagocytosis FCM assessment of phagocytic activity is based on the determination of cells engulfing
fluorescent (FITC)-latex beads. This method allows a clear delineation between phagocytic
and non-phagocytic cell populations on the basis of fluorescence parameters. p
g
y
p p
p
Phagocytosis assays were carried out on leukocytes collected from the main RBT
lymphoid organs including the thymus. In RBT the pronephros is the main phagocytic organ. A slight but not negligible number of active phagocytes occur in the thymus (Table 2). Our
data show that the phagocytosis ability may differ greatly between fish (Table 3). Table 2 Table 2
FCM assessment of phagocytosis in leukocyte populations of 8 RBT fingerlings. % ± SD of cells having
ingested 3 or more FITC-latex beads. Pronephros
Blood
Spleen
Thymus
12.2 ±5.1
6.1 ±3.1
6.7± 3.9
1.1 ± 0.5
Table 3
FCM assessment of phagocytosis (% of phagocytes having ingested 3 and more FITC-latex beads)
and oxidative burst (increase of mean of fluorescence intensity after PMA activation). Both assays were performed on pronephros cells from the same trout and % of granulocytes
was recorded from unstained pronephros leukocyte population. RBT
Pronephros
Phagocytic
Ox. Burst
granulo. (%)
activity ('7c)
(15 mm PMA)
I
60.4
20.3
77.8
2
32.9
12.6
52.9
3
51.6
19.1
65.4
4
58.6
15.6
85.1
5
20.4
9.1
13.9
6
37.9
16.1
49.7 Table 2
FCM assessment of phagocytosis in leukocyte populations of 8 RBT fingerlings. Results FCM characterization of leukocyte subpopulations Using the autofluorescence of unstained cells, FCM allows a clear delineation of cell
subpopulations. In RBT the flow cytometric profiles of cell populations, designed by a two-
parameter cytogram using FSC versus SSC (dot plot representation), are representative of
the organ from which the leukocytes originated. Each lymphoid organ displays
a characteristic cytogram pattern. Spleen and thymus cell populations are for the most part
composed of lymphocytes while pronephros leukocytes display 3 populations
(lymphocytes, monocytes and granulocytes). In blood the situation is less obvious, blood 209 leukocytes are generally characterized by 2 major populations (lymphocytes and
granulocytes), however, blood cytograms may differ greatly between fish. Depending upon
experimental and/or pathological conditions, strong modifications may occur and the aspect
of the dotplot profiles diverges from that of the characteristic cytogram representative of
each lymphoid organ (Table I). During the acquisition of the data cell viability is easily
recorded using propidium iodide which stains specifically the DNA of dead cells. g p
p
p
y
Electronic gating allows the selection of populations of interest, or the exclusion from
aquisition of unwanted population, cell debris or dead cells. Table I % ± SD of cells having
ingested 3 or more FITC-latex beads. Pronephros
Blood
Spleen
Thymus
12.2 ±5.1
6.1 ±3.1
6.7± 3.9
1.1 ± 0.5 Table 2
FCM assessment of phagocytosis in leukocyte populations of 8 RBT fingerlings. % ± SD of cells having
ingested 3 or more FITC-latex beads. Table 3 Table 3
FCM assessment of phagocytosis (% of phagocytes having ingested 3 and more FITC-latex
and oxidative burst (increase of mean of fluorescence intensity after PMA activation)
Both assays were performed on pronephros cells from the same trout and % of granuloc
was recorded from unstained pronephros leukocyte population. RBT
Pronephros
Phagocytic
Ox. Burst
granulo. (%)
activity ('7c)
(15 mm PMA)
I
60.4
20.3
77.8
2
32.9
12.6
52.9
3
51.6
19.1
65.4
4
58.6
15.6
85.1
5
20.4
9.1
13.9
6
37.9
16.1
49.7 M assessment of phagocytosis (% of phagocytes having ingested 3 and more FITC-latex beads
and oxidative burst (increase of mean of fluorescence intensity after PMA activation). oth assays were performed on pronephros cells from the same trout and % of granulocytes
was recorded from unstained pronephros leukocyte population. 210 Oxidative burst Nonspecific Cytotoxic Cells % ± SD of target cell lysis occurring in pronephros (PN)
and spleen was compared between control and PKX infected trout sampled at different time during the
course of the disease. A = beginning of the infection; B = acute phase of the disease; C = recovering fish Control
PKX infected RBT
(n=12)
A (n=8)
B(n=10)
C (n=8)
PN
23.4
21.8
21.2
27.6
± 11.5
± 8.8
±15.1
± 13.1
Spleen
15.4
9.3
21.4
13.4
±8.6
±4.9
±7.3
±3.6 Nonspecific Cytotoxic Cells Nonspecific Cytotoxic Cells p
y
NCC activity was demonstrated in the main lymphoid tissues of RBT (Table 4). Cytotoxic
effector cells have been mostly demonstrated in pronephros than in blood and spleen which both
displayed similar level of killing activity. A significant activity was also measured in the thymus. The modulation of NCC activity under pathologic conditions was assayed in trout
experimentally infected with PKX the parasite causative of the Proliferative Kidney
Disease. FCM evaluation ofNCC activity was compared between healthy control trout and
PKX parasited fish. Infected trout were sampled at 3 different periods during the course of
the disease: a) at the beginning, b) in the acute phase, c) in recovering fish (Table 5). Compared to controls, infected trout displayed neither impairment nor enhancement ofNCC
activity. Tables 4 and 5 show that percentage of cytotoxicity may vary considerably between
individual fish. This large inter-fish variability causes difficulties in the interpretation of the
data. Table 4
FCM assessment ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood and lymphoid
organs of 8 RBT. Blood
Pronephros
Spleen
Thymus
12.4 ± 6.2
25.3 ± 7.9
15.8±5.6
11.2 ±6.2
Table 5
Modulation of NCC activity in PKX infected fish. % ± SD of target cell lysis occurring in pronephros (PN)
and spleen was compared between control and PKX infected trout sampled at different time during the
course of the disease. A = beginning of the infection; B = acute phase of the disease; C = recovering fish
Control
PKX infected RBT
(n=12)
A (n=8)
B(n=10)
C (n=8)
PN
23.4
21.8
21.2
27.6
± 11.5
± 8.8
±15.1
± 13.1
Spleen
15.4
9.3
21.4
13.4
±8.6
±4.9
±7.3
±3.6
Discussion
Immunologists routinely used FCM to characterize phenotypically the leukocyte
subpopulations. Unfortunately, in trout hitherto only a few number of antibodies directed Table 4
FCM assessment ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood and lymphoid
organs of 8 RBT. Blood
Pronephros
Spleen
Thymus
12.4 ± 6.2
25.3 ± 7.9
15.8±5.6
11.2 ±6.2 Table 4
FCM assessment ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood
organs of 8 RBT. Table 4
ofNCC activity: % ± SD of target cells (YAC·l) lysis occurring in blood and lymphoid
organs of 8 RBT. Table 5 Table 5
Modulation of NCC activity in PKX infected fish. Oxidative burst The assay allows the estimation of intracellular production of hydrogen peroxide in PMA
stimulated cells. The fluorescence measured by the flow cytometer is proportional to the amount
of HP2 produced by the cells. A typical experiment includes the kinetic analysis of 104
individual cells at 5, 15 and 45 min post PMA stimulation.The results are expressed as the
difference between the mean fluorescence produced by PMA stimulated cells and unstimulated
cells. As the assay requires a small amount of cells, comparative analysis of phagocytosis and
oxidative burst activity are easily performed. In addition. the data recorded from the analysis of
cell functions can be correlated with one cell subpopulation. Table 3 shows that pronephros in
which the granulocytes constitute the major fraction of the leukocytes are characterized by
a high phagocytic activity and a strong oxidative burst (RBT 1 and 4). Inversely a minor fraction
of granulocytes display a lower phagocytosis and a very weak oxidative burst (RBT 5). Nonspecific Cytotoxic Cells Discussion Immunologists routinely used FCM to characterize phenotypically the leukocyte
subpopulations. Unfortunately, in trout hitherto only a few number of antibodies directed 211 against surface markers are avalaible (Deluca et al. 1983; Slierendrecht et al. 1995). Because of the lack of phenotypic markers the study of the fish cellular immune response
involves the screening of morphological characteristics and functional mechanisms. Such
combined analysis is easily performed using a flow cytometer. FCM offers a real advantage
over conventional techniques because useful informations related to the leukocyte
subpopulations are easily provided by the flow cytometer. In spite of the paucity of cell
markers, interesting data are obtained from the direct study of non-labeled cells, on the basis
of cell autofluorescence. These morphometric informations are very useful. Thus the
heterogeneity occurring in the cytogram profiles is correlated with the health status of the
fish. This heterogeneity could represent a significant part of the inter-fish variability
occurring in the results. g
Phagocytosis has been described to playa major role in fish defense mechanisms. The
phagocytic system, mechanisms of phagocytosis and killing activity have been described
and comprehensively reviewed (MacArthur et al. 1985; Secombes and Fletcher
1992). In mammals, assessment of phagocytosis by FCM has been used for long (Dun and
Tyrer 1981; Bjerknes and Bassoe 1983). y
j
)
More recently in fish, FCM in vitro phagocytosis assays have been developed to assess
the modulation of immune response by pathogens (Chilmonczyk 1995), xenobiotics
(Voccia et al. 1994; 1997) or both (Chilmonczyk et al. 1997). It is possible to run
a phagocytosis assay with a small number of cells (2x 105 ). This is a real advantage allowing
in vitro kinetic studies and sampling of small fish. p
g
Oxidative mechanisms represent an important part of the intracellular microbicidial
activity involving a variety of toxic oxygen species produced by activated phagocytes. The
associated activity which results from the activation of NADPH oxidases is known as the
oxidative or respiratory burst.The FCM technique we have developed, allows an accurate
estimation of the capacity of cells to undergo an oxidative burst in response to a variety of
stimuli. Oxidative burst assays are commonly used in immunotoxicology and
immunopathology to study the modulations of cell defense mechanisms by pollutants,
toxicants or pathogens (Chilmonczyk et al. 1997; Voccia et al. 1994; 1997). As in
phagocytosis assays, only a number small of cells is needed to assess the oxidative burst. Bunecna imunitni odpoved' u pstruha duhoveho (Oncorhynchus mykiss):
stu die pomocl prutokove cytometrie Pozornost byla venovana vyvoji metod vhodnych pro studium nespecifickych bunecnych
obrannych mechanismu u pstruha duhoveho: fagocytoza, aktivita respiracniho vzplanuti a
nespecificka cytotoxicita. Pfi srovnani s konvencnimi metod ami je prutokova cytometrie
rychlou, vhodnou a reprodukovatelnou metodou pro analyzu rybich bunecnych imunitnich
funkci. Vedle hodnoceni obrannych mechanismu jsou prezentovany i udaje z pozorovani
nebarvenych bunek. Prutokove cytometricke profily bunekjsou reprezentativni pro organ,
z nehoz leukocyty pochazeji. Kazdy Iymfoidni organ ma svuj charakteristicky cytogram. Prutokova cytometrie se ukazuje jako vhodny nastroj ke studiu imunitnich reakci a
patologickych procesu. Discussion This allows the possibility of performing both assays in parallel on the same cell sample
(Table 3). NCC are considered as the fish equi valent of mammalian natural killer (NK) cells (E van s
and J aso-Friedmann 1992). NCC activity has been demonstrated in several fish species
including catfish (Evans etal.1984)shark(McKinney etaI.1986),RBT(Hayden and
Lall x 1985; Green lee et al. 1991; R i stow et al. 1995) or carp (S u z u m ura et al. 1994). NCC activity was demonstrated in the main lymphoid tissues ofRBT (Table 4). The activity
measured in the thymus confirms previous observation (Hayden and Laux 1985). This
wide-spread cytotoxic activity suggests that a substantial part of the nonspecific defense
mechanisms could be due to NCe. NCC flow cytometric assay has the advantage of being able to detect dead target cells
before lysis, thus the incubation time required to detect killing is shorterthan in conventional
radioactive assay. Our data show that levels of cytotoxicity varied considerably between
individual fish. In this particular case the large inter-fish variability could be due, in part, to
non-adequate (because of mammalian origin) target cells. The use of fish target cell should
improve the accuracy of the NCC assay. So far, in fish, only a few FCM studies have been performed even though FCM offers great
potential for the study of the fish immune cells. The functional assays we have developed to
assess phagocytosis, oxidative burst and NCC activity, have proven to be accurate and 212 reliable tests. Compared to conventional methods, the use of FCM presents obvious
advantages. FCM is a very sensitive method that allows rapid multiparameter analysis of
single cells and evaluation of cell functions. FCM assays require much less cells per analysis
and are more rapid than conventional assays, thousands of cells can be analyzed within a few
seconds. A large number of fluorescent dyes used in mammalian models are suitable for
investigations in fish. Thus flow cytometer emerges as a useful tool to study haematopoietic
cells and cellular activity. References BASSOE, C. F .. LAERUM, O. D., SOLBERG. C. 0., HANEBERG, B. 1983: Phagocytosis of bacteria by human
leukocytes measured by flow cytometry. Proc. Soc. Exp. BioI. Med. 174: 182-186 BJERKNES. R.. BASSOE, C. F. 1983: Human leukocyte phagocytosis of zymosan particles measured by flow
cytometry. Acta. Pathol. Microbiol. Immune Scand. 91: 341-348 CHANG. L.. GUSEWITCH. G. A.. CHRITTON, D. B. W., FOLZ, J. c., LEBECK, L. K., NEHLSEN-
CANNARELLA, S. L. 1993: Rapid flow cytometric assay for the assessment of natural killer cell activity. J. Immunol. Methods 166: 45-54 CHILMONCZYK. S., VOCCIA, I.. MONGE. D. 1995: Pathogenesis of viral haemorrhagic septicemia virus:
cellular aspects. Vet. Res. 26: 501-511 CHILMONCZYK, S., VOCCIA, I., TARAZONA, J. V., MONGE, D. 1997: Flow cytometric analysis of fish
leucocyte populations exposed to pollutants and pathogens: modulatory effects induced by experimental
procedures. In: Zelikoff, J. T. (ed.) Ecotoxicology: Responses, Biomarkers and Risk Assessment. OECD by
SOS Publications, Fair Haven, N1, pp. 171-184 pp
DEITCH, A. D .. LAW, H., DEVERE-WHITE. R. 1982: A stable propidium iodide staining procedure for flow
cytometry. J. Histochem. Cytochem. 30: 967-972 DELUCA. D .. WILSON, M .. W ARR, G. W. 1983: Lymphocyte heterogeneity in the trout, Salmo gairdneri. defined with monoclonal antibodies to IgM. Eur. J. Immunol.13: 546-551 DUNN, P. A .. TYRER. H. W. 1981: Quantification of neutrophile phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry. J. Lab. Clin. Med. 98: 374-381 YRER. H. W. 1981: Quantification of neutrophile phagocytosis, using fluorescent latex beads. f
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d fl
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M d 98 374 381 EVANS, D. L.. GRAVES, S. S., COBB, D., DA WE, D. L. 1984: Nonspecific cytotoxic cells of fish Uctalurus
punctatus). II. Parametre of target cell lysis and specificity. Dev. Compo Immunol. 8: 303-312 GRAVES, S. S., COBB, D., DA WE, D. L. 1984: Nonspecific cytotoxic cells of fish Uctalurus
I. Parametre of target cell lysis and specificity. Dev. Compo Immunol. 8: 303-312 s). II. Parametre of target cell lysis and specificity. Dev. Compo Immunol. 8: 303-312 EVANS. D. L..JASO- FRIEDMANN, L. 1992: Nonspecific cytotoxic cells as effectors of immunity in fish. Annual
rev. of Fish Diseases. 2: 109-121 GREENLEE. A. R., BROWN. R. A., RISTOW, S. S. 1991: Nonspecific cytotoxic cells of rainbow trout
(Oncorhynchus mykiss) kill Y AC-I targets by both necrotic and apoptotic mechanisms. Dev. Compo Immunol. 15: 153-164 HA YDEN. B. 1.. LAUX, D. C. References 1985: Cell mediated lysis of murine target cells by nonimmune salmonid lymphoid
preparations. Dev. Compo Immunol. 9: 627-639 KIESSLING. R .. KLEIN. E., WIGZELL, H. 1975: Cytotoxic cells with specificity for mouse Maloney leukemia
cells. Specificity and distribution according to genotype. Eur. J. Immunol. 5: 112-117 213 KOUMANS-VAN DIEPEN, 1. C. E., TAVERNE-THIELE, 1. 1.. VAN RENS, B. T. T. M., ROMBOUT, 1. H. W. M. 1994: Immunocytochemical and flow cytometric analysis of B cells and plasma cells in Carp (Cyprinus
carpio L.); an ontogenetic study. Fish Shellfish Immunol. 4: 19-28 p
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MACARTHUR, 1. I., FLETCHER, T. C. 1985: Phagocytosis in fish. In: Manning, M. 1., Tatner, M. F. (eds) Fish
immunology. Academic Press, London, pp. 29-46 MCKINNEY, E. C., HA YNES,L.. DROESE,A. L. 1986: Macrophage-like effector of spontaneous cytotoxicity
from the shark. Dev. Compo Immunol. 10: 497-508 p
RISTOW, S. S., GRABOWSKI, L. D., WHEELER, P. A., PRIEUR, D. T., THORGAARD, G. H. 1995: Arlee line
of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity. Dev. Compo
Immunol. 19: 497-505
. SECOMBES, C. 1., FLETCHER, T. C. 1992: The role ofphagocytes in the protective mechanisms offish. Ann. Rev. Fish Diseases. 2: 53-71 SLlERENDRECHT, W. 1., LORENZEN, N., GLAMAN, 1., KOCH, c., ROMBOUT, 1. H. W. M. 1995:
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English
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Dietary valine requirement of tambaqui (Colossoma macropomum) with different body weights
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Ciência e Agrotecnologia
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cc-by
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Exigência de valina dietética para tambaqui (Colossoma macropomum)
com diferente peso corporal Jakeline Veras da Silva1
, Felipe Barbosa Ribeiro2*
, Jefferson Costa de Siqueira2
Marcos Antonio Delmondes Bomfim2
, Dáphinne Cardoso Nagib do Nascimento1
, Rafael Si Jakeline Veras da Silva1
, Felipe Barbosa Ribeiro2*
, Jefferson Costa de Siqueira2
,
Marcos Antonio Delmondes Bomfim2
, Dáphinne Cardoso Nagib do Nascimento1
, Rafael Silva Marchão3 1Universidade Federal do Maranhão/UFMA, Programa de Pós-graduação em Ciência Animal/PPGCA, Chapadinha, MA, Brasil
2Universidade Federal do Maranhão/UFMA, Centro de Ciências Agrárias e Ambientais/CCAA, Chapadinha, MA, Brasil
3Universidade Federal do Vale do São Francisco/UNIVASF, Programa de Pós-Graduação em Ciência Animal, Petrolina, PE, Brasil
*Corresponding author: felipebribeiro@yahoo.com
Received in January 31, 2022 and approved in May 3, 2022 ABSTRACT Tambaqui (Colossoma macropomum) is one of the highly produced freshwater fish in Brazilian fish farming. However, its production
efficiency associated with the refinement of diet formulations is limited due to a lack of information on nutritional requirements
for certain essential amino acids such as valine. This study was designed to estimate the valine requirement of tambaqui having
different body weights. A total of 720 juveniles were used in the feed trials. The first trial consisted of 360 fish with an average
initial weight of 33.28 ±0.19 g, and the second trial comprised 360 fish (121.19 ±1.29 g). The trials consisted of dose–response
experiments with five levels of valine (5.0, 7.0, 9.0, 11.0, and 13.0 g/kg diet) and four replicate tanks per treatment. The control
diet was obtained by adding L-valine to the diet containing 5.0 g/kg diet (the first level of valine tested) to ensure it reached the
diet containing 7.0 g/kg diet, corresponding to the second level tested. Analysis of variance, Dunnett’s test for comparison with the
control diet, and regression analysis were performed. The results showed that the valine intake, weight gain, specific growth rate,
and feed conversion ratio were significantly improved by the dietary valine (P < 0.05) in both trials. Dietary levels of valine exerted
quadratic effects on body protein, body fat, body protein deposition, and nitrogen retention efficiency of tambaqui with 33 g and
121 g as body fat deposition was affected by the dietary valine level only in tambaqui weighing 33 g. Quadratic regression analysis
of body protein deposition data revealed the optimum dietary valine requirement of tambaqui (33.0 g–83.0 g) and (121.0 g–277.0
g) as 11.9 g/kg diet and 9.1 g/kg diet, respectively. Index terms: Amazon fish; branched-chain amino acid; essential amino acid; fish nutrition. 2022 | Lavras | Editora UFLA | www.editora.ufla.br | www.scielo.br/cagro
All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution BY. Ciência e Agrotecnologia, 46:e002122, 2022
http://dx.doi.org/10.1590/1413-7054202246002122 eISSN 1981-1829
Animal Science and Veterinary 2022 | Lavras | Editora UFLA | www.editora.ufla.br | www.scielo.br/cagro
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b 2022 | Lavras | Editora UFLA | www.editora.ufla.br | www.scielo.br/cagro INTRODUCTION of valine for other fish species has also been determined
(Abidi; Khan, 2004; Castillo; Gatlin, 2018; Dong et al.,
2013; Ren et al., 2015; Xiao et al., 2018; Zehra; Khan,
2014; Zhou et al., 2020). However, the optimal valine
requirements have not been established for tambaqui,
especially requirements at different body weights and
using the diet dilution technique (Fisher; Morris, 1970). In addition to being the most expensive component
of aquatic feeds, proteins are an important source of essential
amino acids (EAAs) and non-essential amino acids (non-
EAAs) and play a crucial role in the growth and physiological
functions of fish (National Research Council - NRC, 2011). However, fish do not have metabolic requirements for
protein, necessitating the need for an adequate balance of
EAAs and non-EAAs (Costa et al., 2021; Wilson, 2002). Diets formulated solely based on crude protein content
may not meet the needs of all amino acids (AA), especially
essential ones, thereby compromising performance and
increasing the release of nitrogen compounds into the
environment due to over-supplementation of nitrogen
(Marchão et al., 2020; Silva et al., 2018). Therefore, considering the lack of information
about the valine requirement for the tambaqui diet and
the importance of specific nutrition for the species, we
estimated the valine requirement for tambaqui with
different body weights. RESUMO O tambaqui (Colossoma macropomum) é um dos peixes de água doce mais produzidos na piscicultura brasileira. No entanto,
sua eficiência produtiva associada ao refinamento das formulações de dietas é limitada devido à falta de informações sobre as
exigências nutricionais de determinados aminoácidos essenciais, como a valina. Objetivou-se estimar a exigência de valina para o
tambaqui com diferentes pesos corporais. Um total de 720 juvenis foi utilizado nos experimentos, o primeiro com 360 peixes com
peso médio inicial de 33,28 ±0,19 g e o segundo com 360 peixes (121,19 ±1,29 g). Os experimentos foram conduzidos em dose-
resposta com cinco níveis de valina (5,0; 7,0; 9,0; 11,0 e 13,0 g/kg de dieta) e quatro repetições por tratamento. A dieta controle foi
obtida pela adição de L-valina à dieta contendo 5,0 g/kg de dieta (primeiro nível de valina testado), de modo que atingiu a dieta
contendo 7,0 g/kg de dieta, correspondente ao segundo nível testado. Foram realizados análise de variância dos dados, teste de
Dunnett’s para comparação com dieta controle e análise de regressão. Os resultados para a ingestão de valina, ganho de peso,
taxa de crescimento específico, taxa de conversão alimentar foram significativamente melhorados pela valina dietética (P<0,05) em
ambos os experimentos. Os níveis dietéticos de valina exerceram efeitos quadráticos sobre a proteína corporal, gordura corporal,
deposição de proteína corporal e eficiência de retenção de nitrogênio do tambaqui com 33 g e 121 g, sendo a deposição de gordura
corporal afetada pelo nível de valina somente no tambaqui com 33 g. Com base na análise de regressão quadrática dos dados de
deposição de proteína corporal, as exigências dietéticas de valina de tambaqui (33,0 g-83,0 g) e (121,0 g-277,0 g) foram estimadas
em 11,9 g/kg da dieta e 9,1 g/kg da dieta, respectivamente. rmos para indexação: Peixe da Amazônia; aminoácido de cadeia ramificada; aminoácido essencial; nutriç 2 SILVA, J. V. et al. Ciência e Agrotecnologia, 46:e002122, 2022 aDry matter soybean meal: 95.20% and corn: 93.90%. Experimental diets Valine is an EAA and potentially limiting in
conventional fish feeds (Rodrigues et al., 2020). Oliveira
et al. (2021), in their study with pacu fish, reported dietary
valine, lysine, and arginine as the most limiting EAAs, and
signified the importance of the composition of these AAs
during the preparation of diets for this species. In juvenile
Nile tilapia, methionine, threonine, and valine are the most
limiting dietary EAAs (Diógenes et al., 2016). To formulate the experimental diets, we first
analyzed the total AA contents of corn and soybean
meal. Next, we converted these into digestible AAs using
digestibility coefficients of ingredients (Table 1). The
total AA contents of corn and soybean meal used in the
experimental diets were obtained using high-performance
liquid chromatography (HPLC). Valine majorly participates in the synthesis of
body proteins, in addition to performing other important
physiological functions, including the repair and growth
of muscle tissue and the maintenance of nitrogen balance
in the body (Suryawan et al., 2011). In addition, valine
has been reported to increase fish growth by increasing
digestive and absorption capacity, thus, influencing the
balance of intestinal microflora (Dong et al., 2013). Therefore, it is necessary to understand valine requirements
according to fish species and their development stages. Table 1: Total and digestible amino acid compositions
of corn and soybean meal used in experimental diets
(based on natural matter). Amino acid (%)
Corna
Soybean
meala
TAAb
DAAc
TAAb
DAAc
Lysine
0.27
0.25
2.76
2.61
Methionine
0.12
0.12
0.50
0.48
Methionine + Cystine
0.38
0.22
1.58
0.99
Threonine
0.25
0.25
1.71
1.68
Tryptophan
0.23
0.18
0.59
0.56
Isoleucine
0.24
0.24
2.09
2.03
Valine
0.35
0.35
2.10
2.04
Leucine
0.67
0.66
3.30
3.21
Arginine
0.40
0.39
3.31
3.28
Crude protein
7.61
46.66
aDry matter soybean meal: 95.20% and corn: 93.90%. bTotal amino acids, determined using high-performance liquid
chromatography (HPLC) by the CBO laboratory, Valinhos, SP. cDigestible amino acids, calculated based on the digestibility
coefficients presented by NASCIMENTO et al. (2020) for
tambaqui (Colossoma macropomum). Table 1: Total and digestible amino acid compositions
of corn and soybean meal used in experimental diets
(based on natural matter). Experimental diets Amino acid (%)
Corna
Soybean
meala
TAAb
DAAc
TAAb
DAAc
Lysine
0.27
0.25
2.76
2.61
Methionine
0.12
0.12
0.50
0.48
Methionine + Cystine
0.38
0.22
1.58
0.99
Threonine
0.25
0.25
1.71
1.68
Tryptophan
0.23
0.18
0.59
0.56
Isoleucine
0.24
0.24
2.09
2.03
Valine
0.35
0.35
2.10
2.04
Leucine
0.67
0.66
3.30
3.21
Arginine
0.40
0.39
3.31
3.28
Crude protein
7.61
46.66
aDry matter soybean meal: 95 20% and corn: 93 90% The tambaqui (Colossoma macropomum), produced
in 25 of the 26 Brazilian states, is considered one of the
most important species for Brazilian aquaculture (Instituto
Brasileiro de Geografia e Estatística - IBGE, 2019). It
is particularly known for the flavor of its meat in the
consumer market. The tambaqui is an omnivorous species
of freshwater from the Amazon and Orinoco rivers, which
feeds on fruits, seeds, and small organisms. It displays
desirable characteristics for fish farming and can easily
adapt to conditions and rearing systems. However, high
nutritional value feeds are required when raised in captivity
to maximize production (Woynárovich; Van Anrooy, 2019). Th
i
f EAA f
b
i h
b aDry matter soybean meal: 95.20% and corn: 93.90%. bTotal amino acids, determined using high-performance liquid
chromatography (HPLC) by the CBO laboratory, Valinhos, SP. cDigestible amino acids, calculated based on the digestibility
coefficients presented by NASCIMENTO et al. (2020) for
tambaqui (Colossoma macropomum). The requirement of EAAs for tambaqui has been
studied for lysine (Marchão et al., 2020), methionine plus
cysteine (Souza et al., 2019), threonine (Firmo et al., 2018),
and tryptophan (Bomfim et al., 2020). The requirement Ciência e Agrotecnologia, 46:e002122, 2022 3 Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights Diets were formulated using the dilution technique to
obtain increased levels of digestible valine (Fisher; Morris,
1970). The dilution technique, an alternative technique
for formulating experimental diets, was recently used by
Nascimento et al. (2020) in a study on determining the
optimum dietary EAA profile of Nile tilapia and Marchão et
al. (2020) in a study on the requirement for digestible lysine
for tambaqui juveniles. This technique mixes two diets,
one containing a high content of the tested AA and another
protein-free diet (PFD), to obtain a diet with intermediate
levels of AAs tested and reduced levels of AAs. Crude protein levels ranged from (110.4 to 287.0
g/kg diet) because the diets were formulated using the
dilution technique. Experimental diets In this technique, the AA balance of the
treatments remained constant because it is based on the use
of a high-protein diet deficient in the evaluated AA. The
evaluated AA is diluted in a protein-free isoenergetic diet,
resulting in its intermediate levels (NRC, 2011; Siqueira et
al., 2013). To confirm valine as the first-limiting nutrient
and that the responses obtained were a function of valine
and not crude protein in the diet, a sixth diet (control
diet) was used. The control diet was obtained by adding
synthetic valine (L-valine 98.5%) to the diet containing
the first level of digestible valine (5.0 g/kg diet) to reach a
concentration of 7.0 g/kg diet, corresponding to the second
level tested, as proposed by Nonis and Gous (2008). Initially, a reference diet (RD) was formulated
containing a 287.0 g/kg diet of crude protein (CP) and a
13.0 g/kg diet of digestible valine (77% digestible valine:
lysine ratio) based on corn and soybean meal (Table 2). Subsequently, this diet was diluted with another PFD
based on corn starch, containing the same levels of energy,
vitamins, and minerals, to obtain diets with increasing
dietary valine concentrations (5.0, 7.0, 9.0, 11.0, and 13.0
g/kg diet) (Table 2). Initially, the ingredients of experimental
diets were ground (Trf 60, Trapp), sifted, and mixed
(Horizontal Mixer 300 kg, Branorte). To obtain
intermediate diets (5.0, 7.0, 9.0, 11.0, and 13.0 g/kg
diet), mixtures were diluted (Table 2). Subsequently,
they were extruded in an equipment with a 3–5 mm
sieve (Extruder model MX 40, Inbramaq, Laboratório
de Nutrição e Alimentação de Organismos Aquáticos
do Maranhão, Chapadinha, Brazil). Other AAs exceeded the ideal ratios to digestible
lysine recommended by the NRC (2011) for Nile tilapia
by at least two percentage points to prevent another AA
from becoming limiting. Table 2: Diet formulation and chemical composition of the protein-free diet (PFD), reference diet (RD), control
diet (CD), and experimental diets (g/kg natural matter bases). Table 2: Diet formulation and chemical composition of the protein-free diet (PFD), reference diet (RD), control
diet (CD), and experimental diets (g/kg natural matter bases). Table 2: Diet formulation and chemical composition of the protein-free diet (PFD), reference diet (RD), control
diet (CD), and experimental diets (g/kg natural matter bases). Table 2: Diet formulation and chemical composition of the protein free diet (PFD), reference diet (RD), control
diet (CD), and experimental diets (g/kg natural matter bases). Ciência e Agrotecnologia, 46:e002122, 2022 Experimental diets Ingredients (g/kg)
Digestible valine Levels
PFD
5.0
7.0
9.0
11.0
13.0(RD)
CD
Soybean meal
0.00
206.9
289.7
372.5
455.3
538.1
206.9
Corn
0.00
136.5
191.1
245.7
300.3
354.9
136.5
Corn starch
802.9
494.1
370.5
247.0
123.5
0.00
494.1
Soybean oil
103.2
80.6
71.6
62.5
53.5
44.5
80.6
Cellulose
37.5
23.1
17.3
11.5
5.8
0.00
23.1
Lysine-HCl (78.4%)
0.0
0.9
1.3
1.7
2.0
2.4
0.9
DL-Methionine (99%)
0.0
2.0
2.8
3.6
4.5
5.3
2.0
L-Threonine (98.5%)
0.0
0.9
1.3
1.7
2.1
2.4
0.9
L-Tryptophan (98%)
0.0
0.1
0.1
0.2
0.2
0.3
0.1
L-Valine (99%)
0.0
0.3
0.4
0.6
0.7
0.8
2.3
L-Leucine (99%)
0.0
0.6
0.8
1.0
1.2
1.4
0.6
Calcitic limestone
7.0
7.2
7.3
7.4
7.5
7.6
7.2
Dicalcium phosphate
38.2
35.7
34.7
33.6
32.6
31.6
35.7
Vitamin and mineral premixf
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Vitamin Ce
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Continue... SILVA, J. V. et al. SILVA, J. V. et al. 4 Table 2: Continuation. Ingredients (g/kg)
Digestible valine Levels
PFD
5.0
7.0
9.0
11.0
13.0(RD)
CD
Salt
5.5
5.4
5.3
5.2
5.2
5.1
5.4
Antioxidant (BHT)
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Dilution (%)
D1
D2
D3
D4
D5*
**
Protein-free diet
100.00
61.54
46.15
30.77
15.38
0.00
Reference diet
0.00
38.46
53.85
69.23
84.62
100.00
Chemical composition (g/kg in the natural matter)
Crude protein
0.00
110.4
154.6
198.7
242.9
287.0
110.4
Digestible energy (kcal kg–1)d
3,000
3,000
3,000
3,000
3,000
3,000
3,000
Crude fibera
34.7
34.7
34.7
34.7
34.7
34.7
34.7
Total Caa
12.0
12.0
12.0
12.0
12.0
12.0
12.0
P availablea
7.0
7.0
7.0
7.0
7.0
7.0
7.0
Total Naa
2.2
2.2
2.2
2.2
2.2
2.2
2.2
Digestible Valineb
0.00
5.0
7.0
9.0
11.0
13.0
7.0
Digestible Lysineb
0.00
6.5
9.0
11.6
14.2
16.8
6.5
Digestible Met. Ciência e Agrotecnologia, 46:e002122, 2022 * Reference diet (13.0 g digestible valine/kg of dry diet). f Vitamin and mineral supplement, amounts supplied per kg diet: Vit. A, 6,000 IU; Vit. D3, 1,000 IU; Vit. E, 60.0 mg; Vit. K3,
12.0 mg; Vit. B1, 24.00 mg; Vit. B2, 24.00 mg; Vit. B6, 24.00 mg; Vit. B12, 24.00 mg; Vit. C, 24.00 mg; folic acid, 6.00 mg; Ca
pantothenate, 60.00 mg; biotin, 0.24 mg; choline chloride, 108 g; niacin, 100.00 mg; Fe, 250.00 mg; Cu, 15.0 mg; Mn, 100.00 mg;
Zn, 150.00 mg; I, 0.5 mg; Co, 0.05 mg; Se, 0.5 mg. Experimental diets + Cistb
0.00
4.3
6.1
7.8
9.5
11.3
4.3
Digestible Threonineb
0.00
4.7
6.6
8.5
10.4
12.3
4.7
Digestible Tryptophanb
0.00
1.4
1.9
2.5
3.1
3.6
1.4
Digestible Arginineb
0.00
7.3
10.3
13.2
16.1
19.1
7.3
Digestible Leucineb
0.00
8.1
11.3
14.5
17.8
21.0
8.1
Relationships
Digestible Met + Cys/Lys (0.63)c
0.00
0.66
0.67
0.67
0.67
0.67
0.66
Digestible Thr/Lys (0.69)c
0.00
0.72
0.73
0.73
0.73
0.73
0.72
Digestible Typ/Lys (0.19)c
0.00
0.21
0.21
0.21
0.21
0.21
0.21
Digestible Arg/Lys (0.75)c
0.00
1.12
1.12
1.12
1.12
1.12
1.12
Digestible Val/Lys (0.94)c
0.00
0.77
0.77
0.77
0.77
0.77
1.07
Digestible Leu/Lys(1.19)c
0.00
1.25
1.25
1.25
1.25
1.25
1.25
Table 2: Continuation. * Reference diet (13.0 g digestible valine/kg of dry diet). ** Control diet = D1 + 0.20% L-valine. a Based on the values proposed by Rostagno et al. (2011).fi a Based on the values proposed by Rostagno et al. (2011).fi b Based on the digestibility coefficients proposed by Nascimento et al. (2020). y
d Based on the digestibility coefficients proposed by Furuya et al. (2010). d Based on the digestibility coefficients proposed by Furuya et al. (2010). e Vit. C: ascorbic acid 2-monophosphate calcium salt, 42% active ingredient. f Vitamin and mineral supplement, amounts supplied per kg diet: Vit. A, 6,000 IU; Vit. D3, 1,000 IU; Vit. E, 60.0 mg; Vit. K3,
12.0 mg; Vit. B1, 24.00 mg; Vit. B2, 24.00 mg; Vit. B6, 24.00 mg; Vit. B12, 24.00 mg; Vit. C, 24.00 mg; folic acid, 6.00 mg; Ca
pantothenate, 60.00 mg; biotin, 0.24 mg; choline chloride, 108 g; niacin, 100.00 mg; Fe, 250.00 mg; Cu, 15.0 mg; Mn, 100.00 mg;
Zn, 150.00 mg; I, 0.5 mg; Co, 0.05 mg; Se, 0.5 mg. Ciência e Agrotecnologia, 46:e002122, 2022 5 Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights The experimental diets were provided daily as six
meals (08:00, 10:00, 12:00, 14:00, 16:00, and 18:00 h) until
satiation. At each meal, they were supplied in small quantities
with successive passes, allowing the maximum intake. (dry) in an oven with forced-air circulation, dried for 72 h at
65 °C, and then ground in a micro mill (IKA A11 basic) and
stored in plastic containers at -20 °C until body composition
was analyzed. Experimental diets The samples were analyzed for whole-body
composition (dry matter, moisture content, crude protein,
fat, and ash) according to the (Association of Official
Analytical Chemists - AOAC, 2019) standard procedures. • FCR (g− 1) = feed intake (g)/weight gain (g). • FBA = final body ash, IBA = initial body ash, IW = initial
weight, FW = final weight. • NRE (%) = [FBN (%) × FW(g)]−[IBN (%) × IW(g)]/
[feed intake (g) × N level in ratio (%)]/100i • NRE (%) = [FBN (%) × FW(g)]−[IBN (%) × IW(g)]/
[feed intake (g) × N level in ratio (%)]/100i Sample collection and analysis • FBN = final body nitrogen, IBN = initial body nitrogen,
IW = initial weight, FW = final weight. • FBN = final body nitrogen, IBN = initial body nitrogen,
IW = initial weight, FW = final weight. • FBN = final body nitrogen, IBN = initial body nitrogen,
IW = initial weight, FW = final weight. At the beginning of the experiment, 20 fish were
sampled, euthanized by subjecting them to the benzocaine
solution (500 mg L–1), and stored at −20 °C to determine the
whole-body composition. At the end of each trial period, fish
were not fed for 24 h before sampling. All fish from each
experimental unit were individually weighted, euthanized
with a benzocaine solution (500 mg L–1), and frozen in
a freezer at −20 °C. Subsequently, the fish samples were
thawed, and the whole fish was crushed and dehydrated • FCR (g− 1) = feed intake (g)/weight gain (g). • FCR (g− 1) = feed intake (g)/weight gain (g). • FCR (g− 1) = feed intake (g)/weight gain (g). Based on the body composition (moisture, ash,
protein, and fat), the daily deposition rates of body protein
(BPD), body fat (BFD), and body ash (BAD), as well as
the nitrogen retention efficiency (NRE), were calculated
according to the equations given below: During the trials, the fish were kept in polyethylene
boxes with a capacity of 1000 L each, equipped with
individual water supplies, drainage systems, and aeration
systems. The water supply of boxes was derived from an
artesian well, with a flow rate of 30 L h–1 per box. • BPD (mg day− 1) = {[FBP (%) × FW (mg)] − [IBP (%) ×
IW (mg)]/100}/experimental period (days)i The temperature of the water was measured daily
at 7:30, and 17:30 h using a mercury bulb thermometer
graduated from 0 to 50 °C. Controls for pH, the content of
dissolved oxygen, and ammonia in the water were measured
every 3 days using a pH meter (HI 8424, Hanna), oximeter
(HI 9146, Hanna), and a commercial kit (Arcor) for toxic
ammonia test, respectively. The temperature of water ranged
from 24.7 ±0.6 °C to 27.1 ±0.2 °C (first trial) and 26.4 ±0.7 °C
to 27.5 ±0.1 °C (second trial). The pH was kept at 7.02 ±0.3,
dissolved oxygen was maintained above 10.5 ±0.4 mg/L, and
ammonia nitrogen was kept below 1.0 ppm. • IBP = initial body protein, FBP = final body protein, IW
= initial weight, FW = final weight. • BFD (mg day− 1) = {[FBF (%) × FW (mg)]−[IBF (%) ×
IW (mg)]/100}/experimental period (days)i • BFD (mg day− 1) = {[FBF (%) × FW (mg)]−[IBF (%) ×
IW (mg)]/100}/experimental period (days)
• FBW = final body fat, IBW = initial body fat, IW = initial
weight, FW = final weight. • FBW = final body fat, IBW = initial body fat, IW = initial
weight, FW = final weight. • BAD (mg day− 1) = {[FBA (%) × FW (mg)]−[IBA (%) ×
IW (mg)]/100} experimental period (days)i • BAD (mg day− 1) = {[FBA (%) × FW (mg)]−[IBA (%) ×
IW (mg)]/100} experimental period (days)
• FBA = final body ash, IBA = initial body ash, IW = initial
weight, FW = final weight. Ciência e Agrotecnologia, 46:e002122, 2022 Ethics Statement, experimental facility, fish, and water
quality parameters The study was conducted at the Laboratory of
Nutrition and Food for Aquatic Organisms of Maranhão,
located at the Center of Agrarian and Environmental
Sciences of the Federal University of Maranhão,
Chapadinha, in Brazil (03◦ 44′ 33′ ′ S, 43◦ 21′ 21′ ′ W;
altitude 105 m) and lasted for 50 days for each trial. It was
conducted using experimental procedures approved by the
Animal Use Ethics Committee of the Federal University of
Maranhão (Protocol N° 23115.031736/2020–83). At the end of each trial, the following performance
and feed efficiency indexes were evaluated: feed intake
(FI), digestible valine intake (DVI), weight gain (WG),
specific growth rate (SGR), and feed conversion ratio
(FCR), according to the equations given below: • FI (g) = dry feed consumed during the experimental
period • FI (g) = dry feed consumed during the experimental
period • DVI (g) = [feed intake (g) × digestible valine level in
feed (%)]/100i The study was divided into two trials. In total, 720
tambaqui (C. macropomum) were used, being 360 fish, with
an average initial weight of 33.3 ±0.2 g, in the first trial and
360 fish, with an average initial weight of 121.2 ±1.3 g, in the
second trial. In each trial, the fish were randomly distributed
into 24 experimental units, with 15 fish in each unit. i • WG (g) = final mean weight (g)−initial mean weight (g)
• SGR (% day–1) = [In final weight (g) −In initial weight
(g)] × 100]/experimental period (days) Statistical analysis The data were submitted to the Shapiro–Wilk and
Levene tests to test for normality and homoscedasticity. Once the assumptions met, the data were then submitted
to analysis of variance. The variables affected by valine
levels were subjected to regression analysis performed Ciência e Agrotecnologia, 46:e002122, 2022 6 SILVA, J. V. et al. using linear and quadratic models. To compare the effects
of the CD (1.125%) with other treatments, Dunnett’s test
was used. All statistical analyses were performed using
the SAS 9.0 software (Statistical Analysis System - SAS,
2002), with a value of P < 0.05 indicating significance. In both trials, the fish raised on the control diet (CD)
with 7.0 g/kg valine in the diet showed superior responses to
those raised on a diet containing 5.0 g/kg valine in the diet,
with emphasis on body protein deposition, feed conversion
ratio, and nitrogen retention efficiency. The experimental
diets in our study were formulated using the dilution
technique (Fisher; Morris, 1970), and despite the variations
in protein intake among treatments, the results obtained in
animals submitted to the CD confirmed that valine was
the first limiting AA in experimental diets. In addition, the
results were related only to variations in the valine intake
and not to the crude protein content of the diets. Sep-Pooled standard error of the mean.
*Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). p
*Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). P > F – Significance of the “F” test of the analysis of variance. (
)
Significance of the “F” test of the analysis of variance.
l d t
d
d
f th Sep-Pooled standard error of the mean.f P > F – Significance of the “F” test of the analysis of variance. he mean.
ffer from the control diet (CD) by the Dunnett test (P < 0.05). y
tandard error of the mean.
e same column differ from the control diet (CD) by the Dunnett test (P < 0.05). CD – Control diet (D1 + 0.20% L-valine).i he F test of the analysis of variance.
ror of the mean.
umn differ from the control diet (CD) by the Dunnett test (P < 0.05). he “F” test of the analysis of variance.
ror of the mean. i
d standard error of the mean.
n the same column differ from the control diet (CD) by the Dunnett test (P < 0 05) RESULTS AND DISCUSSION Variables
Body
weight (g)
Digestible
valine levels
(g/kg diet)
FI
(g fish –1)
DVI
(g fish –1)
WG
(g)
SGR
(% day–1)
FCR
(g g–1)
33 g
CD
103.74 ±9.18
257.20 ±8.77
47.08 ±1.76
4.21 ±0.02
2,23 ±0.25
5.0
116.31 ±4.17
193.84 ±6.94*
36.57 ±2.50
4.07 ±0.04*
3.21 ±0.14 *
7.0
113.22 ±3.61
264.19 ±8.43
47.13 ±3.23
4.21 ±0.04
2.42 ±0.10
9.0
110.87 ±4.61
332.60 ±13.84*
56.02 ±5.66
4.31 ±0.06
2.02 ±0.13
11.0
108.45 ±2.85
397.63 ±10.44*
59.12 ±0.97 *
4.35 ±0.01
1.84 ±0.02
13.0
114.75 ±5.69
497.26 ±24.66*
63.61 ±1.95*
4.40 ±0.02*
1.80 ±0.06
P>F1
0.7167
<0.0001
0.0003
0.0002
<0.0001
Linear effect
0.5699
<0.0001
<0.0001
<0.0001
<0.0001
Quadratic effect
0.2621
0.3200
0.1636
0.0900
0.0004
121 g
SEp
10.84
27.14
6.14
0.072
0.27
CD
224.28 ±2.53
341.11 ±4.62
98.16 ±1.36
4.10 ±0.00
2.40 ±0.15
5.0
220.12 ±1.89
244.58 ±2.10*
79.84 ±2.24
4.03 ±0.01
2.76 ±0.09
7.0
220.24 ±2.04
342.60 ±3.17
106.9 ±15.13
4.09 ±0.05
2.15 ±0.28
9.0
222.14 ±4.61
444.29 ±9.22*
124.84 ±8.51
4.19 ±0.03
1.81 ±0.14
11.0
220.40 ±1.99
538.76 ±4.87*
102.77 ±9.22
4.11 ±0.03
2.19 ±0.18
13.0
220.13 ±0.52
635.92 ±1.49*
91.92 ±3.63
4.08 ±0.01
2.41 ±0.11
P>F1
0.9760
<0.0001
0.0147
0.0316
0.0094
Linear effect
0.9844
<0.0001
0.4442
0.2289
0.2002
Quadratic effect
0.6524
0.6404
0.0017
0.0068
0.0012
SEp
5.14
0.054
14.35
0.054
0.30
CD – Control diet (D1 + 0.20% L-valine).i Ciência e Agrotecnologia, 46:e002122, 2022 Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights 7 reflects lower growth rates, possibly due to the more rapid
assimilation of crystalline AAs than AAs present in the
diet as a component of protein. The use of the diet dilution technique provides
more accurate results with respect to the performance of
animals than the supplementation technique because the
dilution technique provides constant levels of AAs in all
treatments, uses a lower concentration of synthetic AAs,
and provides better efficiency in the use of dietary nutrients
(Siqueira et al., 2013). This formulating diet approach
avoided the need to include large quantities of crystalline
AAs in the diet, similar to ours during the supplementation
technique. RESULTS AND DISCUSSION No dead fish were observed during the 50 days of
each trial. No evidence of outward pathological signs was
noted in fish, considering the low levels of dietary valine. The growth performance, body composition, and body
deposition are shown in Tables 3, 5, and 7, respectively. Table 3: Mean values ±standard error of feed intake (FI), digestible valine intake (DVI), weight gain (WG), specific
growth rate (SGR), and feed conversion ratio (FCR) of tambaqui with different body weights as a function of
digestible valine levels in the diet. Table 3: Mean values ±standard error of feed intake (FI), digestible valine intake (DVI), weight gain (WG), specific
growth rate (SGR), and feed conversion ratio (FCR) of tambaqui with different body weights as a function of
digestible valine levels in the diet. g
g
g
pi
growth rate (SGR), and feed conversion ratio (FCR) of tambaqui with different body weights as a function of
digestible valine levels in the diet. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION Therefore, comparisons
between digestible valine values reported in the literature
and those described in our study are difficult, even among
species considered omnivorous. In this study, improvements in weight gain, specific
growth rate, feed conversion ratio, body protein, body
protein deposition, and nitrogen retention efficiency of
tambaqui fed with increasing dietary valine levels indicated
the importance of valine for the productive performance
of this species and showed its ability to assimilate the
valine crystal for growth. Previous reports on feeding trials
with different dietary valine levels showed that growth
performances in fish were improved by supplementation
of dietary valine (Ahmad; Ahmed; Dar, 2021; Dong et al.,
2013; Rollin et al., 2006; Xiao et al., 2018). For body composition and body deposition in
tambaqui with an initial body weight of 33 g, the increased
dietary valine levels influenced the body protein (%),
body protein deposition, and nitrogen retention efficiency
in a quadratic way (P < 0.05), which increased up to the
levels of dietary valine of 10.7, 11.9, and 9.5 g/kg diet,
respectively. In addition, body fat and body fat deposition
showed a quadratic response (P < 0.05), which decreased
until the dietary valine levels of 10.7 and 9.4 g/kg diet,
respectively (Tables 5, 6, 7 and 8 and Figure 2). Xiao et al. (2018) conducted a study with juvenile
Nile tilapia (initial weight = 6.48 g) and estimated the
optimum valine requirement in 11.5 g/kg diet for weight
gain, which was higher than the dietary valine values
reported in our study for weight gain in fish with an initial
weight of 121 g. Although Nile tilapia is also an omnivorous
species, fingerlings are expected to have a higher metabolic
rate and higher AA requirements than post-juvenile fish. Values higher than those presented in the present study have
also been reported by Dong et al. (2013), which estimated
the valine requirement in 13.7 g/kg diet for improving the
specific growth rate of juvenile Jian carp, characterized by
being the first artificial breed Chinese carp, which grows
30% faster than the common carp (Dong; Yuan, 2002). In trial two (with an initial weight of 121 g), the
levels of dietary valine influenced body protein (%), body
protein deposition, and nitrogen retention efficiency in a
quadratic manner (P < 0.05), which increased up to 9.0,
9.1, and 8.2 g/kg diet, respectively. RESULTS AND DISCUSSION (2010) reported that comparisons
should not be made between AA requirement values in
species with distinct feeding behaviors, except when they
are expressed as dietary proteins. Similar to trial one, the tambaqui with an initial
body weight of 121 g (trial two) showed an increase in
the valine intake with valine supplementation (P < 0.05)
and a quadratic reduction in feed conversion rate up to
the valine level of 9.3 g/kg diet. However, the weight gain
and specific growth rate of trial one differed by showing
a quadratic response, which increased up to the levels of
dietary valine of 9.2 and 9.4 g/kg diet, respectively, and
gradually decreased thereafter (Tables 3 and 4 and Figure 1). The higher level determined to optimize the feed
conversion ratio of tambaqui with 33 g as compared to the
level determined for tambaqui with 121 g of body weight
can be explained by a change in AA requirements during
the growth, which decreases throughout an animal’s life
due to the reduction in the growth rate (NRC, 2011). The
improvement in feed conversion ratio was provided by
a higher concentration of valine because no feed intake
differences were observed in any of the trials. The optimal valine requirement to maximize the
weight gain and specific growth rate of tambaqui with an
initial weight of 121 g was recorded in 9.2 and 9.4 g/kg
of dry diet, respectively; these values were lower than the
valine requirements of tambaqui with an initial weight of 33
g, which showed a linear increase in weight gain and specific
growth rate with the supplementation of dietary valine. As
different stages of growth have metabolic or physiological
requirements for specific AAs, the requirement for AAs may
change during life stages, as observed in the present study
(Wilson; Poe; Robinson, 1985).i Abidi and Khan (2004) and Xiao et al. (2018)
reported an improved feed conversion ratio with an
increase in the dietary concentration of valine up to 15.0
and 12.7 g/kg diet, respectively. These values were lower
than those mentioned in our study. Several factors, such
as fish size, developmental stages, type of diet, dietary
protein and energy concentrations, feeding frequency, and
environmental conditions, interfere with AA requirements
(Abidi; Khan, 2004; Abimorad et al., 2010; Yue et al.,
2014; Zehra; Khan, 2014). RESULTS AND DISCUSSION Moreover, according to Cowey (1995), the
presence of large amounts of crystalline AAs in fish diets Regarding the tambaqui with a bodyweight of 33 g
(trial one), the feed intake was not influenced (P > 0.05) by
the increasing valine levels in the diets, whereas the valine
intake, weight gain, and specific growth rate increased by an
increment in the valine level from 5.0 to 13.0 g/kg diet (P <
0.05). The feed conversion ratio was reduced in a quadratic
manner, estimating the optimum valine requirement at 11.7
g/kg diet (Tables 3 and 4 and Figure 1). Table 4: Adjusted regression equations, coefficients of determination and requirement values for the variables,
digestible valine intake (DVI), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) of
tambaqui with different body weights as a function of digestible valine levels in the diet. Body
weight
(g)
Variable
Model
Equation
R2
P>F
Level
(g/kg diet)
33 g
DVI (g)
Linear
Ŷ = 37.014× + 3.9789
0.99
<0.0001
-
WG (g)
Linear
Ŷ = 3.3043× + 22.752
0.94
0.0003
-
SGR (% day–1)
Linear
Ŷ = 0.04× + 3.908
0.93
0.0002
-
FCR (g g–1)
Quadratic
Ŷ = 0.030893 × 2 – 0.72657× + 6.0457
0.99
<0.0001
11.7
121 g
DVI (g)
Linear
Ŷ = 48.941× + 0.7547
0.99
<0.0001
-
WG (%)
Quadratic
Ŷ =-2.0702 × 2 + 38.261× – 58.831
0.87
0.00017
9.2
SGR (% day–1)
Quadratic
Ŷ =-0.00639 × 2 + 0.12061× + 3.5828
0.79
0.0068
9.4
FCR
Quadratic
Ŷ = 0.042604 × 2 – 0.80044× + 5.6765
0.90
0.0012
9.3
P > F-Significance of the “F” test of the analysis of variance. P > F-Significance of the “F” test of the analysis of variance. P > F-Significance of the “F” test of the analysis of variance. Figure 1: Graphic representation of feed conversion ratio of 33 g (solid line) and 121 g (dotted line) tambaqui fed
with diets containing different levels of digestible valine. Figure 1: Graphic representation of feed conversion ratio of 33 g (solid line) and 121 g (dotted line) tambaqui fed
with diets containing different levels of digestible valine. 8 SILVA, J. V. et al. maximize weight gain (45.3 g/kg of dietary protein). The
data here were also expressed in g/kg of dietary protein
because the red sea bream Pagrus major is a strictly
carnivorous species and tambaqui is omnivorous. In
addition, Abimorad et al. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION For body fat (%), a
decrease was observed until the valine level of 10 g/kg of
the diet with a subsequent increase in fat and no effect of
valine supplementation on body fat deposition (P > 0.05). For dry matter, a linear reduction was observed (P < 0.05)
in this trial (Tables 5, 6, 7 and 8 and Figure 2). Rahimnejad and Lee (2013) evaluated the digestible
valine requirement of juvenile red sea bream Pagrus major
and estimated 9.0 g/kg diet for the maximum weight gain,
equivalent to 20.0 g/kg of dietary protein, which was lower
than the dietary valine values mentioned in our study to Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights 9 9 Table 5: Mean values ±standard error of dry matter, protein, fat, moisture, and body ash contents of tambaqui
with different weights as a function of digestible valine levels in the diet. Table 5: Mean values ±standard error of dry matter, protein, fat, moisture, and body ash contents of tambaqui
with different weights as a function of digestible valine levels in the diet. Table 5: Mean values ±standard error of dry matter, protein, fat, moisture, and body ash contents of tambaqui
with different weights as a function of digestible valine levels in the diet. RESULTS AND DISCUSSION Variable
Body
weight (g)
Digestible valine levels
(g/kg diet)
Dry matter (%)
Protein (%)
Fat (%)
Moisture (%)
Ash (%)
33 g
CD
30.00 ± 0.67
16.33 ± 0.18
10.69 ± 0.15
70.00 ± 0.67
3.23 ± 0.18
5.0
28.13 ± 1.21
12.42 ± 0.32 *
13.02 ± 0.41*
71.87 ± 1.21
2.69 ± 0.16
7.0
30.09 ± 1.27
16.04 ± 0.25
11.20 ± 0.25
69.91 ± 1.27
3.22 ± 0.30
9.0
26.50 ± 1.23
15.05 ± 0.41*
8.83 ± 0.47*
73.50 ± 1.23
2.86 ± 0.13
11.0
28.75 ± 0.27
18.21 ± 0.26*
7.51 ± 0.30 *
71.25 ± 0.27
3.03 ± 0.15
13.0
26.21 ± 0.62
15.92 ± 0.19
9.57 ± 0.13
73.79 ± 0.62
3.22 ± 0.17
P > F1
0.0803
<0.0001
<0.0001
0.8303
0.2598
Linear effect
0.1235
<0.0001
<0.0001
0.1235
0.1681
Quadratic effect
0.4134
<0.0001
<0.0001
0.4134
0.8300
SEp
1.91
0.56
0.62
1.91
0.38
121 g
CD
39.95 ± 0.79
16.28 ± 0.46
19.08 ± 0.58
60.05 ± 0.79
4.59 ± 0.06
5.0
40.63 ± 0.82
15.74 ± 0.49
19.93 ± 0.66
59.37 ± 0.82
4.97 ± 0.25
7.0
40.50 ± 0.74
16.81 ± 0.23
18.93 ± 0.49
59.51 ± 0.74
4.75 ± 0.13
9.0
37.60 ± 1.08
19.11 ± 0.52*
13.50 ± 0.50*
62.41 ± 1.08
4.99 ± 0.33
11.0
37.61 ± 0.82
16.96 ± 0.41
15.49 ± 0.55*
62.39 ± 0.82
5.16 ± 0.30
13.0
37.89 ± 1.11
15.73 ± 0.45
17.08 ± 0.64
62.12 ± 1.11
5.08 ± 0.31
P>F1
0.0587
0.0004
<0.0001
0.0587
0.8589
Linear effect
0.0119
0.9300
0.0001
0.0119
0.4649
Quadratic effect
0.2983
<0.0001
<0.0001
0.2983
0.8406
SEp
1.80
0.87
1.14
1.80
0.49
CD – Control diet (D1+0.20% L-valine). P > F – Significance of the “F” test of the analysis of variance. Sep-Pooled standard error of the mean Table 6: Regression equations, coefficients of determination, and requirement values for variables fat, protein,
and dry matter in tambaqui with different body weights as a function of the levels of digestible valine in the diet. Table 6: Regression equations, coefficients of determination, and requirement values for variables fat, protein,
and dry matter in tambaqui with different body weights as a function of the levels of digestible valine in the diet. P > F-Significance of the “F” test of the analysis of variance. (
)
P > F – Significance of the “F” test of the analysis of variance. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION Body weight (g)
Variable
Model
Equation
R2
P>F
Level (g/kg
diet)
33 g
Protein (%)
Quadratic
Ŷ =-0.1369 × 2 + 2.9239x+1.4031
0.72
<0.0001
10.7
Fat (%)
Quadratic
Ŷ = 0.15732 × 2 + 3.3613x+26.276
0.91
<0.0001
10.7
121 g
Protein (%)
Quadratic
Ŷ = –0.16194 × 2 +2.9208x+4.9953
0.77
<0.0001
9.0
Fat (%)
Quadratic
Ŷ = 0.225 × 2 + 5.4067x + 37.519
0.73
<0.0001
10.0
Dry matter (g g–1)
Linear
Ŷ =-0.4189x + 42.613
0.70
0.0119
-
P > F-Significance of the “F” test of the analysis of variance. P > F-Significance of the “F” test of the analysis of variance. Ciência e Agrotecnologia, 46:e002122, 2022 10 SILVA, J. V. et al. Table 7: Mean values ±standard error of body protein (BPD), fat (BFD), and ash (BAD) deposition and nitrogen
retention efficiency (NRE) in tambaqui with different body weights as a function of digestible valine levels in the diet. retention efficiency (NRE) in tambaqui with different body weights as a function of digestible valine levels in the diet. RESULTS AND DISCUSSION Variable
Body
weights (g)
Digestible valine
BPD
BFD
BAD
NRE
levels (g/kg diet)
(mg day–1)
(mg day–1)
(mg day–1)
(%)
33 g
CD
162.99 ± 6.82
155.68 ± 14.76
26.17 ± 3.41
40.24 ± 1.83
5.0
64.22 ± 7.91*
154.32 ± 7.45
10.19 ± 2.89
22.41 ± 2.39*
7.0
158.05 ± 13.56
153.13 ± 8.18
26.50 ± 7.44
40.46 ± 2.45
9.0
168.70 ± 12.70
128.76 ± 17.37
25.50 ± 5.29
34.34 ± 1.24
11.0
245.37 ± 6.87*
107.03 ± 7.12*
30.77 ± 3.52
41.93 ± 0.77*
13.0
213.96 ± 4.91*
158.80 ± 2.92
37.62 ± 2.27
29.43 ± 1.41*
P>F1
<0.0001
0.0090
0.0120
<0.0001
Linear effect
<0.0001
0.2511
0.0012
0.0148
Quadratic effect
<0.0001
0.0099
0.4810
<0.0001
SEp
18.71
21.62
8.97
3.57
CD
281.66 ± 36.85
395.79 ± 13.06
55.82 ± 9.72
36.35 ± 1.00
121 g
5.0
151.70 ± 15.93*
314.42 ± 35.30
42.64 ± 9.02
28.06 ± 2.84*
7.0
266.90 ± 63.00
343.99 ± 76.32
53.07 ± 21.64
36.06 ± 2.32
9.0
492.54 ± 38.02*
160.66 ± 39.64*
95.43 ± 27.85
50.25 ± 2.33*
11.0
295.07 ± 48.42
193.20 ± 26.84*
78.43 ± 18.38
24.64 ± 1.54*
13.0
193.38 ± 27.90
232.33 ± 38.40
61.70 ± 16.56
13.77 ± 1.98*
P>F1
0.0004
0.0626
0.3794
<0.0001
Linear effect
0.4135
0.0592
0.3238
<0.0001
Quadratic effect
<0.0001
0.1974
0.1435
<0.0001
Sep
82.25
85.66
36.80
4.18
CD – Control diet (D1 + 0.20% L-valine). P > F – Significance of the “F” test of the analysis of variance. Sep-Pooled standard error of the mean. *Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). CD – Control diet (D1 + 0.20% L-valine).i CD Control diet (D1 0.20% L valine). P > F – Significance of the “F” test of the analysis of variance. gi
Sep-Pooled standard error of the mean. f Table 8: Adjusted regression equations, determination coefficients, and requirement values for variables body
protein deposition (BPD), body fat deposition (BFD), and body ash deposition (BAD), and nitrogen retention
efficiency (NRE) in tambaqui with different body weights as a function of the level of digestible valine in the diet. (
)
P > F – Significance of the “F” test of the analysis of variance.
l d
d
d
f h Sep
oo ed sta da d e
o o t e
ea .
*Means in the same column differ from the control diet (CD) by the Dunnett test (P < 0.05). gi
y
Sep-Pooled standard error of the mean. f Significance of the F test of the analysis of variance.
ooled standard error of the mean. f ror of the mean.
umn differ from the control diet (CD) by the Dunnett test (P < 0.05). “F” test of the analysis of variance. RESULTS AND DISCUSSION Body
Weight (g)
Variable
Model
Equation
R2
P>F
Level (g/
kg diet)
33 g
BPD (mg day–1)
Quadratic
Ŷ =-3.2942 × 2 + 78.634x – 244.47
0.92
<0.0001
11.9
BFD (mg day1)
Quadratic
Ŷ = 1.9384 × 2 – 36.748x + 298.62
0.50
0.0099
9.4
BAD (mg day–1)
Linear
Ŷ = 2.957x – 0.49
0.86
0.0012
-
NRE (%)
Quadratic
Ŷ =-0.8465 × 2 + 16.012x – 35.06
0.71
<0.0001
9.5
121 g
BPD (mg day–1)
Quadratic
Ŷ =-15.302 × 2 + 281.01x – 887.31
0.77
<0.0001
9.1
NRE (%)
Quadratic
Ŷ =-1.3845 × 2 + 22.921x – 52.511
0.79
<0.0001
8.2
P > F-Significance of the “F” test of the analysis of variance. Dietary valine requirement of Tambaqui (Colossoma macropomum) with different body weights 11 Figure 2: Graphic representation of body protein deposition of tambaqui with 33 g (solid line) and 121 g (dotted
line) fed diets containing different levels of digestible valine. Figure 2: Graphic representation of body protein deposition of tambaqui with 33 g (solid line) and 121 g (dotted
line) fed diets containing different levels of digestible valine. Regarding body composition, the responses of BP,
body protein deposition, and nitrogen retention efficiency
to the increased dietary valine levels in tambaqui with
an initial body weight of 33 g were a little higher than
those reported for tambaqui with an initial weight of 121
g due to different growth rates, which is directly related
to different depositions of muscle tissue. The optimum
valine requirement for tambaqui (33 g and 121 g) in our
study was lower than the dietary valine values reported for
juvenile hybrid grouper (15.8 g/kg diet; Zhou et al., 2020),
juvenile tilapia (12.7 g/kg diet; Xiao et al., 2018), Indian
major carp (15.8 g/kg diet; Abidi; Khan, 2004). Despite
differences in valine levels to maximize body protein
deposition, studies have shown an improvement in protein
deposition with dietary valine supplementation, suggesting
that a better dietary AA balance probably prevents the
selective catabolism of AAs and consequently increases
protein synthesis (Tantikitti; Chimsung, 2001). increased. This result is consistent with previous reports of
feeding trials with red sea bream (Rahimnejad; Lee, 2013)
and Jian carp (Dong et al., 2013). The body fat deposition
for tambaqui weighing 121 g was not affected by increased
dietary valine; however, the highest body fat deposition
was reported at the two lowest dietary valine levels. Ciência e Agrotecnologia, 46:e002122, 2022 RESULTS AND DISCUSSION Only the tambaqui weighing 33 g showed a
significant increase in body ash deposition with an increase
in the dietary valine level. As previously discussed, a better
balance of AAs improves the body’s protein deposition,
leading to better use of dietary minerals for the formation
of osseous tissues to support muscle tissues (Pianesso et
al., 2015). A close functional relationship exists between
osseous, and muscle tissues, Bomfim et al. (2020) evaluated
the growth curve of tambaqui (C. macropomum) and
reported that the maximum development of total length was
reached at 243.4 days, whereas the maximum development
of body weight was reached at 333.9 days, suggesting an
earlier bone development than muscle tissue because that
fish weight gain is associated with the accretion of proteins.i In addition, the quadratic nitrogen retention
efficiency response to increasing levels of valine in the
diet showed that at excessive levels, the percentage of
retained nitrogen decreased, consequently increasing the
excretion of nitrogen compounds into the environment
and contributing to contamination and eutrophication of
the aquatic environment (Furuya et al., 2013). The AA requirement for fish has been determined
by the level that optimizes performance variables, mainly
weight gain (NRC, 2011). However, weight gain consists
of increases in proteins, lipids, minerals, and water (NRC,
2011), and there is no metabolic demand for AAs to
maintain lipid and mineral reserves (Marchão et al., 2020). In this sense, body protein deposition is the most suitable
variable to determine the AA requirement for fish (Bomfim
et al., 2020; Lee et al., 2020; Silva et al., 2018; Costa Sousa
et al., 2021), especially valine because it is used in greater
proportion for protein synthesis (Rodrigues et al., 2018). A better balance of AAs in the diet can contribute
to an increase in protein synthesis, with a concomitant
decrease in the accumulation of lipid reserves (Tantikitti;
Chimsung 2001), as demonstrated in our studies, in
which the whole-body fat composition decreased with
supplementation of dietary valine, and subsequently Ciência e Agrotecnologia, 46:e002122, 2022 12 SILVA, J. V. et al. AUTHOR CONTRIBUTION COWEY, C. B. Protein and amino acid requirements: A critique of
method. Journal of Applied Ichthyology, 11(3-4):199-204, 1995. Conceptual idea: Silva, J.V.; Ribeiro, F.B.; Siqueira,
J.C.; Bomfim, M.A.D.; Methodology design: Silva, J.V;
Ribeiro, F.B; Siqueira, J.C.; Bomfim, M.A.D.; Marchão, R.S.;
Data collect: Silva, J.V; Nascimento, D.C.N.; Marchão, R.S.;
Data analysis and interpretation: Siqueira, J.C.; Silva, J.V;
Ribeiro, F.B.; Writing and editing: Silva, J.V; Ribeiro, F.B.;
Siqueira, J.C.; Nascimento, D.C.N.; Marchão, R.S. DIÓGENES, A. F. et al. Establishing the optimal essential amino acid
ratios in juveniles of Nile tilapia (Oreochromis niloticus) by the
deletion method. Aquaculture Nutrition, 22(2):435-443, 2016. DONG, M., et al. Growth, body composition, intestinal enzyme
activities and microflora of juvenile Jian carp (Cyprinus
carpio var. Jian) fed graded levels of dietary valine. Aquaculture Nutrition, 19(1):1-14, 2013. CONCLUSIONS BOMFIM, M. A. D. et al. Digestible tryptophan requirement for
tambaqui (Colossoma macropomum) fingerlings. Revista
Ciência Agronômica, 51(2):e20196724, 2020. The results of our study demonstrated that dietary
valine provides optimal growth and increases the body
protein of tambaqui with different body weights. Using a
quadratic regression analysis of body protein deposition
data, the optimum dietary valine requirement of tambaqui
(33.0 g–83.0 g) and (121.0 g–277.0 g) was estimated
to be 11.9 g/kg diet and 9.1 g/kg diet, respectively,
corresponding to 4.53% of dietary crude protein. These
values are within the range of valine requirements of
similar fish species reported by the NRC, 2011. CASTILLO, S.; GATLIN, D. M. III. Dietary requirements for
leucine, isoleucine and valine (branched-chain amino
acids) by juvenile red drum Sciaenops ocellatus. Aquaculture
Nutrition, 24:1056-1065, 2018. COSTA SOUSA, M. et al. Lysine requirements of tambatinga
(♀Colossoma macropomum × ♂Piaractus brachypomus)
fingerlings using different diet formulation techniques. Aquaculture Nutrition, 27(6):1825-1836, 2021. ACKNOWLEDGMENTS DONG, Z. J.; YUAN, X. H. The utilizations of heterosis in common
carp in China. Aquaculture Asia, 12:14-15, 2002. The Fundação de Amparo à Pesquisa e ao
Desenvolvimento Científico e Tecnológico do Maranhão
(FAPEMA) for project financing. This study was financed
in part by Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES)-Brazil, Finance code 001. FIRMO, D. S. et al. Threonine to lysine ratio in diets of tambaqui
juveniles (Colossoma macropomum). Semina: Ciências
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HIGH-TOOL – a strategic assessment tool for evaluating EU transport policies
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© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made. HIGH-TOOL – a strategic assessment tool
for evaluating EU transport policies E. Szimba1*
, J. Ihrig2, M. Kraft2, K. Mitusch2, M. Chen3, M. Chahim3, J. van Meijeren3, J. Kiel4, B. Mandel5, A. Ulied6,
E. Larrea6, G. De Ceuster7, R. Van Grol8, Z. Berki9, A. Székely9 and R. Smith10 * Correspondence: szimba@kit.edu
1Karlsruhe Institute of Technology
(KIT), Network Economics, Institute
of Economics (ECON), Waldhornstr. 27, Bldg. 01.96, 76131 Karlsruhe,
Germany Abstract In this paper the strategic transport policy assessment instrument HIGH-TOOL is
presented. The model has been developed for the European Commission, allowing
policy-makers to identify the most advantageous transport policies and to
strategically evaluate the impacts of transport policies on transport, environment and
economy. Full list of author information is
available at the end of the article The main innovation of this policy assessment tool lies in the integration of originally
independently functioning models – i.e. passenger and freight demand,
demography, and vehicle stock models, as well as economic, environmental and
safety assessment models. With its traffic zones at the regional level of NUTS-2 and
its aggregated view on the transport system, the instrument has a relatively lean
structure avoiding runtime problems, without losing the spatial dimension. What distinguishes HIGH-TOOL from all other European transport policy assessment
instruments: the model is an open source tool, it is freely available and does not
require any commercial software to be run. In combination with its modular
structure the HIGH-TOOL model can relatively easily be adjusted to other modelling
methodologies or data. It can also comparatively easily be made responsive to “new”
policies which are not in the scope of the current model version. Thus the HIGH-
TOOL model lays the foundation for further innovations in the assessment of
transport policies and mobility concepts. Keywords: Assessment tool, Transport policy, Transport demand modelling,
Transport model, Impact assessment, European Union (2018) 3:11 (2018) 3:11 Szimba et al. Journal of Shipping and Trade
https://doi.org/10.1186/s41072-018-0037-y Journal of Shipping
and Trade Journal of Shipping
and Trade Open Access Full list of author information is
available at the end of the article Introduction and policy context Decisions on transport policy measures proposed by the European Union (EU) as ad-
dressed by the White Paper on Transport (European Commission 2011a) have
long-term and important impacts on economy, environment and society. Transport
policy measures can lock up capital for decades and cause manifold external effects
and indirect effects in many sectors – thus, policy measures may have a tremendous
scope, especially if proposed at the European level. Various authors, such as Sieber et al. (2013), Nilsson et al. (2008) or McIntosh et al. (2011), emphasize the increasing importance of impact assessment tools as decision
support instruments for policy making, allowing policy-makers to analyze relationships
within a complex system and to reach decisions based on quantitative information. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 2 of 30 Page 2 of 30 In this context, the strategic assessment model “HIGH-TOOL” has been devel-
oped to compute economic, environmental and social impacts of transport pol-
icies,
allowing
European
policy-makers
to
identify
the
most
advantageous
transport policies and to carry out strategic assessments of transport policies. HIGH-TOOL, which stands for “high-level strategic transport model”, addresses
EU transport policies in the scope of the European Commission’s White Paper on
Transport. The tool’s output indicators are largely aligned with assessment indica-
tors of EU policy documents such as the EU’s Impact Assessment Guidelines
(European Commission, 2009). The main objective of the HIGH-TOOL project has been the development of an open
strategic transport policy assessment instrument for the European Commission, which
is largely based on already existing tools, which has a low runtime and which provides
a high level of user-friendliness. Within this setting, the HIGH-TOOL model integrates
originally independently functioning models. This paper provides an overview of structure, modelling approach and application as-
pects of the HIGH-TOOL model. Thus, the paper has a “synopsis” character, providing
an overview perspective rather than a description of the modelling or validation ap-
proach at a highly detailed level. The paper is structured as follows: Chapter 2 provides an overview of general tool
features, structure of the model, and of the methodology behind the individual model-
ling entities (modules). Chapter 4 summarizes the testing and validation approach. Chapter 5 addresses the scope of transport policy measures considered, the approach
chosen to operationalize these policies, and presents the “hypernet” conception. Modelling philosophy Reflecting one of the key goals of the HIGH-TOOL project – i.e. the development of
an assessment instrument that is largely based on already existing tools –, the
HIGH-TOOL model integrates originally independently functioning models – i.e. pas-
senger and freight demand, demography, and vehicle stock models, as well as eco-
nomic, environmental and safety assessment models. This requires that already existing
European state-of-the-art transport demand and assessment models are integrated by a
common platform and are connected to a common database, relying on a common
foundation, such as a common business-as-usual scenario, geographical scope, spatial
entities, and demand segmentation. The contents of this chapter are partly based on Szimba et al. (2017), as well as on
project reports by Szimba (2016), Mandel et al. (2016), and Van Grol et al. (2016). Introduction and policy context Chap-
ter 6 presents a case study. The paper closes with chapter 7, the conclusions. General scope The HIGH-TOOL model has a global scope. However, the main focus is attached to
Europe, and particularly to the Member States of the European Union. The spatial
scope is the level of NUTS-21 for all EU Member States (EU28), Norway and
Switzerland, NUTS-0 for EU neighboring countries, and country bundles for intercon-
tinental transport. In total 314 modelling zones are considered. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 3 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 3 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 The tool’s timeline are 5-years steps from 2010 to 2050. The year 2010 is the base
year of the HIGH-TOOL model. The HIGH-TOOL baseline or business-as-usual sce-
nario is aligned with the EU Reference Scenario 2013 (European Commission 2013). Thus the forecasts of the HIGH-TOOL baseline are largely consistent with those of the
EU Reference Scenario 2013. Passenger demand is differentiated by following modes: air, rail, road (passenger car
and powered 2-wheelers), and long-distance coach. The urban demand sub-module
additionally considers urban bus, urban tram/metro, cycling and walking. The demand
differentiation by trip purpose covers business, private, vacation, and commuting trips. The freight transport is modelled by multi-modal transport chains, considering the
transport modes air, rail, road, inland waterways, and maritime transport. The demand
is considered for NST-2 commodities (52 commodity groups). The vehicle fleet is dis-
tinguished by 60 vehicle types and 17 fuel types. The HIGH-TOOL model was largely developed in Java, thus ensuring platform inde-
pendence. The User Interface was programmed as a stand-alone online application
based on AngularJS and SailsJS, both free and open source software components pro-
grammed in JavaScript. The HIGH-TOOL Database that stores input, output data as
well as modelling parameters of all modules, is realized as a PostgreSQL database with
PostGIS extension (Kiel et al. 2016b). Model structure The HIGH-TOOL model consists of three main elements: Core modules that represent
the modelling framework; the Database that facilitates the exchange of data; and the
User Interface for application of the model and providing access to the assessment re-
sults. The basic structure follows the natural architecture common for most Decision
Support Systems (DSS). The core modules are assembled such that a wide spectrum of
specific transport topics can be addressed by the model while minimizing interdepend-
encies in favor of short model runtime. In consequence, the number of core modules
was finally condensed to seven and their internal structure was designed to allow for
fast processing avoiding loops, while reflecting essential relations consistently. The core
modules of the HIGH-TOOL model are constrained to these design features over de-
tailed implementation of complex interdependencies, as the tool is outlined to provide
decision support by testing policy scenarios for the European Union on a strategic level
before the most promising ones are implemented in more detailed (network-based)
models such as TRANSTOOLS or Vaclav. The core modules are as follows: Demography (DEM), Economy & Resources (ECR),
Passenger Demand (PAD), Freight Demand (FRD), Vehicle Stock (VES), Environment
(ENV), and Safety (SAF). In the center of the model are the passenger and freight de-
mand modules (PAD and FRD). Since transport demand is driven (among others) by
demographic and economic developments, DEM and ECR provide inputs for the de-
mand modules. Based on transport demand, environmental and safety impacts are
computed by ENV and SAF. To estimate economic impacts of transport policies, PAD
and FRD provide demand data to ECR. As the composition of the vehicle fleets effects
both transport costs and emission factors, VES provides inputs to the demand modules
PAD and FRD, as well as to ENV. Figure 1 displays the detailed structure of the model, Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Page 4 of 30 Page 4 of 30 also depicting the structure within the core modules. The model structure is explained in
detail, including the variables which are exchanged between the modules, by Mandel et al. (2016). The core modules interact sequentially with each other (see section “Module
Interaction”). Fig. 1 Detailed structure of the HIGH-TOOL model Fig. 1 Detailed structure of the HIGH-TOOL model also depicting the structure within the core modules. Model structure The model structure is explained in
detail, including the variables which are exchanged between the modules, by Mandel et al. (2016). The core modules interact sequentially with each other (see section “Module
Interaction”). Demography module The Demography module (DEM) calculates the regional population and labor force in
the 28 EU Member States as well as in Norway and Switzerland. UN projections
(United Nations 2014) are used for other countries worldwide and are adapted to the
geographic zoning system used in HIGH-TOOL. The population and labor force are derived from the Europop assumptions on demo-
graphic trends (European Commission 2011b) at country level for the EU 28, Norway
and Switzerland. These trends are aligned with the EU Reference Scenario 2013’s as-
sumptions concerning fertility rates, life expectancies at birth and net migration Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 5 of 30 Page 5 of 30 (European Commission 2013). Net migration is not modelled explicitly; it is taken
exogenously from the EU Reference Scenario. The projected population values
from the EU Reference Scenario by country are disaggregated to geographic zones
based on historical demographic trends concerning population development. This
is done by using a demographic model which calculates future population by zone,
based upon fertility rates and life expectancies. The population and labor force
values from Europop are then disaggregated based upon the results from the
demographic model. This allows consistent results with the Europop population
trends. Kiel et al. (2016b) and Van Grol et al. (2016) contain a more detailed speci-
fication of the demographic model and the calibrated values of the parameters and
coefficients. Regional disaggregation of the population excluding migration is based on the 2010
historical regional distribution. Net migration is regionally distributed using a distribu-
tion proxy based on income and employment rate. Labor force is estimated from the
labor force percentage defined in the EU Reference Scenario and underlying
assumptions. Being calibrated to the EU Reference Scenario 2013 and the projections by the
United Nations (UN), the results of the Demography module are aligned with the
expected economic development in Europe. However, the demographic develop-
ment is an exogenous variable in HIGH-TOOL, i.e. the module is not sensitive to
any policy. Figure 2 provides an overview of the structure of the Demography module. The Demography module’s most relevant data interchanges with other modules are
population data at regional scale (NUTS-2), differentiated by age and gender cohort,
which are input to ECR, VES and PAD. Furthermore, regional labor force data are pro-
vided to ECR and VES. Fig. 2 Structure of the Demography module Fig. Demography module 2 Structure of the Demography module Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 6 of 30 Szimba et al. Journal of Shipping and Trade Economy & Resources Module The Economy & Resources module (ECR) comprises two components: Economy and
Resources, as well as of the outputs GDP, Trade, Energy, Resources, and Production/
Distribution (see Fig. 3). The Economy & Resources module (ECR) comprises two components: Economy and
Resources, as well as of the outputs GDP, Trade, Energy, Resources, and Production/
Distribution (see Fig. 3). The Economy sub-module estimates total output, capital stock and labor use in the
economy, for which the general drivers (Gross Domestic Product (GDP), household in-
come per capita and population) are – for an initial run without any policies – exogen-
ously defined by the EU Reference Scenario 2013 (European Commission 2013). These
drivers are disaggregated from country to zone based on ETISplus data (regional GDP, re-
gional population, and labor force). The combined component (GDP, Trade, Energy, Re-
sources, Production/ Distribution) estimates employment, trade, resource consumption,
and purchasing power. The resources component calculates environmental indicators
(without combustion) using the EXIOBASE database (Wood et al. 2015) for CO2, NOx,
SOx, PM, biomass, fossil fuel use, metal use, mineral use, wood use, and water use. The methodology chosen for the Economy & Resources module is driven by a set of
sequential algebraic equations, based on the first-order conditions for utility and profit
maximization. The Economic module follows the algebraic framework of a (comput-
able) general equilibrium model. The total output per region is generated using a
Cobb-Douglas equation using total wages and total value of capital stock by region as
inputs. Future trends in terms of growth in Gross Domestic Product and development
of sectoral shares in Gross Value Added are obtained from the EU Reference Scenario
2013. The growth paths of economic and environmental indicators (e.g., intermediate
inputs, CO2 emissions, water use or wood use) are driven by the total sectoral output
trajectory. Moreover, projected demand is calculated as the sum of final demand of Fig. 3 Structure of the Economy & Resources module Fig. 3 Structure of the Economy & Resources module Fig. 3 Structure of the Economy & Resources module Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 7 of 30 Page 7 of 30 households and the intermediate demand of sectors (under the assumption of fixed
Leontief technology). For modelling trade flows the following method is applied: For the base year, trade
flows by country pairs in the base year are derived from the ETISplus database. Economy & Resources Module ETIS-
plus estimates NUTS-2 inter-regional trade using national data from Eurostat statistics
with its known limitation such as less detailed or missing data at regional level. Subse-
quently, sectoral Gross Value Added data at NUTS-2 level (source: Eurostat) are ap-
plied to scale down the country-level trade to the regional level. This creates different
trade shares per commodity for each region. In this way the heterogeneity of each re-
gion in a country is incorporated, i.e. not all regions export the same product mix. On
the import side, the regional GDP share is applied to disaggregate the trade flows. In
order to compute the share of intra-regional trade with respect to outgoing
inter-regional trade, we combine RHOMOLO data (see Ivanova et al. 2011) on
intra-regional trade (i.e. trade between NUTS-2 regions within in a country) with ETIS-
plus inter-regional trade (i.e. trade between NUTS-2 regions of different countries) for
regions where both data is available. The average commodity share of inter-regional trade is used to estimate the
intra-regional trade for the remaining regions. By doing so, we implicitly assume that the
freight handling factor (i.e. the frequency of lifts of the tons in the supply chain from the
place of production to the place of consumption) for intra-regional trade is at least equal
to the inter-regional trade. For intra-regional trade we assume that the share of a com-
modity is equal to the average exporting share of a commodity to the different regions. For the assessment of economic impacts of a policy scenario the following effects are
captured by the ECR module. The changes in monetary costs of freight transport (i.e. the cost differences between
the policy scenario and the baseline), differentiated by mode and commodity, are trans-
lated into changes in consumer prices. The consumer prices include both transport and
trade margins as well as consumption taxes and subsidies. A decrease in transportation cost improves regional accessibility, which has a positive
impact on the sector output at the regional level through provision of better access to
labor markets and more varieties of intermediate goods. Next, the projected demand for goods and services in a region are calculated as
the sum of the final demand of households and the intermediate demand of sec-
tors. Economy & Resources Module We calculate regional intermediate demand as the sum of intermediate de-
mand over all the sectors located in the region (under the assumption that
intermediate goods and services are used in the production according to fixed
Leontief technology). The regional intermediate demand is recalculated using the
spending on the purchase of new vehicles in each sector and time period, new
transport investment in Research and Development (RTD) and new infrastructure
investments (these variables drive direct change in intermediate demand due to
transport related investments). Finally, we assume that the household sector is driven by a Cobb-Douglas utility
function (see, e.g. Ivanova 2014). Changes in the total costs of passenger transport have
an impact on the level of disposable income of the households. Increases in passenger
transportation costs reduce the amount of money available for purchasing of other
goods and services, affecting also the household sector’s consumption of cars. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 8 of 30 Page 8 of 30 Changes in trade are modelled by the change in regional sectoral output between the
baseline trajectory and the policy scenario (i.e. growth of sectoral output is applied to
exports). Finally, the (new) Gross Domestic Product is derived at regional level on the basis of
the variables calculated above as the sum of the value added of individual sectors plus
taxes on final demand. The ECR module’s most relevant input data are regional population and labor force
data provided by DEM, transport costs and transport demand by PAD and FRD, as well
as vehicle tax revenue and vehicle stock data (purchase costs of new vehicles, vehicle
stock by mode and fuel type) by VES. ECR’s main output to other modules are regional
GDP per capital values for VES, trade flows in monetary values to FRD, and regional
GDP, GVA and employment data for PAD. Vehicle stock module Journal of Shipping and Trade (2018) 3:11 Page 9 of 30 Page 9 of 30 (2018) 3:11 Szimba et al. Journal of Shipping and Trade Fig. 4 Structure of the Vehicle Stock module Fig. 4 Structure of the Vehicle Stock module Vehicle stock module The Vehicle Stock module (VES) converts passenger and freight demand to vehicle
fleet size, which is disaggregated to vehicle type and vehicle age cohort for calculation
of emissions and energy use. Vehicle types include propulsion and fuel technologies,
and the module embraces 61 road and 12 non-road vehicle types. The vehicle age co-
horts range from 0 to 29 years. Fleet stock forecasts are provided at country and region for each of the 28 EU Mem-
ber States and for each period (5-year intervals) up to 2050. The module also delivers
forecasts of average fixed (e. g., purchase costs, vehicle technology related costs) and
variable costs (e.g., fuel costs, taxes, maintenance/repair costs, insurances, crew costs,
cargo handling costs) for each vehicle type, and total tax revenue per country. Taking into account the transport demand and the vehicle stock in the previous
period, as well as the vehicles that survived in current period, the demand for new vehi-
cles and the average mileage per vehicle are calculated. The logit and the stock dynamic model inside the Vehicle Stock module use the cal-
culated average generalized costs and behavioral aspects to define the shares of the dif-
ferent types of new vehicles entering the market as well as their numbers. This
calculation produces the detailed existing vehicle stock in the current period. The methodology underlying the Vehicle Stock module is aligned with TRACCS
(Papadimitrio et al. 2013) and TREMOVE (De Ceuster et al. 2007). The structure of
the module is shown by Fig. 4. There is no explicit distinction between company cars and private cars, but the effect
is implicitly included in the decreasing annual mileage with increasing age, as well as
by the different annual mileage for different vehicle types (e.g. higher annual mileage
for diesel as these are more likely to be company cars). The module’s most important input data are population data by DEM, GDP per
capita data by ECR, as well as demand data (vehicle-km, load factors/ car occupancy
rates) by PAD and FRD. The VES module’s main outputs to other modules are vehicle
tax revenue and vehicle stock data (purchase costs of new vehicles, vehicle stock by
mode and fuel type) to ECR, vehicle stock by mode, fuel type and vehicle technology to
ENV, as well as generalized fixed and variable costs per vehicle-km (differentiated by
mode and fuel type) to PAD and FRD. Szimba et al. Passenger demand module The core Passenger Demand (PAD) module computes the number of passenger trips
per year by transport mode for each origin/destination (O/D) relation at NUTS-2 level. It distinguishes the four main transport modes, rail, road, air, coach. PAD largely fol-
lows the classical four-step approach in transport demand modelling of generation, dis-
tribution, modal split and assignment (Ortúzar and Willumsen 2011). However, instead
of the assignment step, the module translates the number of trips into transport per-
formance indicators by the conversion step. The methodology of PAD is outlined by Fig. 5. It relies on a rather complex mathem-
atical framework encompassing 44 equations that are described in detail by Van Grol et
al. (2016). Considerably simplified, the computation of the O/D trip matrices (Tijm) car-
ried out by the generation, distribution and the modal split models can be outlined as
follows: Tijm ¼ T i
Aij
X
j
Aij
Sijm Tijm ¼ T i
Aij
X
j
Aij
Sijm where: Tijm Number of trips per year from origin i to destination j by transport mode m Ti Generated passenger trips in zone i Aij Accessibility of opportunities in destination j from origin i Sijm Market share of transport mode m of those trips from zone i to zone j. In the first step, the trip demand Ti is generated for each origin based on the popula-
tion and specific trip rate factors. These trip rate factors are distinguished by year and Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 10 of 30 Szimba et al. Journal of Shipping and Trade Fig. 5 Structure of the Passenger Demand module Fig. 5 Structure of the Passenger Demand module by country under consideration of four trip purposes and eight age groups. They are
computed by a regression approach that takes economic and demographic changes in
European countries into account with the regression approach being calibrated for the
first year 2010 based on ETISplus data (Szimba et al. 2013). For the forecast years and
for the policy scenarios trip rate factors are dependent on changes of the explanatory
variables GDP, employment, and income level. PAD is therefore sensitive to economic
and demographic changes. In an intermediate step, four indicators are subsequently computed in order to inte-
grate the trip distribution and the modal split model. Passenger demand module First, Generalized Travel Times
(GTTijm) are computed by the cost functions based on travel impedance indicators,
such as travel time, travel distance and level of service. All inter-zonal public transport
modes (rail, air, coach) are considered by the GTT calculation as trip chains consisting
of access mode, main mode and egress mode. GTTs follow the concept of travel time
in which the cost unit refers to minutes rather than to monetary terms. Second, based
on GTTs, Expected Minimum Costs (EMCij) and market shares of transport modes
(Sijm) are computed by a Nested Logit model following the concept of logsums (De
Jong et al. 2007). Third, Deterrence Values (DVij) are calculated by the deterrence
model and in the last step, Accessibilities (Aij) are computed based on EMCs and DVs. Finally, Aij are used by the trip distribution model for the computation of the O/D trip
matrix, and Sijm by the modal split model in order to divide the matrix by transport
mode. The last modelling step is the conversion stage. In this step, transport performance
indicators (passenger-kilometers and vehicle-kilometers) are derived, based on the O/D
trip matrices, as well as travel distances and occupancy rates from ETISplus. In
addition, road trips are split by car and powered two-wheelers, under the assumption
of country-specific shares and motorization levels. Page 11 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 The core PAD module is complemented by two additional modules, the urban pas-
senger demand, and the intercontinental air passenger sub-module. The urban passenger demand sub-module computes the number of urban trips per
year at country level. It distinguishes six transport modes: Car, motorcycle, tram/
metro, bus, cycling, walking. Given that urban trips by car and motorcycle are a subset
of the intra-zonal NUTS-2 trip demand modelled by the core PAD module, the urban
sub-module is linked to the generation step of the core PAD module in order to ensure
the overall consistency. The urban sub-module follows a generic, elasticity-based approach and consists of a
generation, a modal split, and a conversion model. In the first step, the number of
urban trips is generated for each NUTS-2 region based on “urban share factors” and
trip rate factors. Passenger demand module The “urban share factors” contain the share of the urban population
which are pre-calculated for each NUTS-2 region on the basis of GIS data. Also the
trip rate factors are pre-calculated for each NUTS-2 region. They rely on trip
purpose-specific trip rates derived from household surveys. In the second step, the
market shares for each urban transport mode is estimated under application of a logit
model for the three transport mode categories: private road, public transport and slow
modes. Finally, the conversion model disaggregates the three mode categories into the
six transport modes and computes aggregated demand indicators at country level. The intercontinental air passenger sub-module uses a regression-based approach to
estimate the number of flights respectively demand between European regions and
intercontinental destinations based on socio-economic data, regional attractors and
network impedances. In general air transportation impedances consider cost structures
such as CO2 taxation, national air transport charges or expenditures calculated by air-
lines in respect of passenger rights. The split between European and intercontinental
air transport mirrors the necessity to deal with the different regional granulation under-
lying the assessment model’s purpose: while Europe is in the focus and modeled at re-
gional level (NUTS-2), the other regions of the world are considered through a less
detailed approach, i.e. 19 aggregated zones such as Far East, USA, Russia, or
South-America. In order to avoid biased model results because of a highly different size
of assumed traffic cells, air demand modeling has been split. The PAD module’s most important input data are population and labor force data at
regional level by DEM, regional GDP, GVA and employment by ECR, as well as the ve-
hicle stock and generalized fixed and variable costs per vehicle-km (differentiated by
country, mode and fuel type) by VES. The module’s main outputs to other modules are transport demand (vehicle mileage,
passenger-kilometers, number of passengers) by mode and region, as well as costs by
region and origin/destination relation to ECR, transport demand and vehicle occupancy
rates to VES, transport demand (vehicle mileage by O/D, origin and mode, and differ-
entiated by urban and non-urban) to ENV and SAF. Figure 5 displays the structural overview of the Passenger Demand module. Freight demand module The Freight Demand (FRD) module uses national and international freight transport
chains as a base. FRD consists of four components: trade conversion, distribution, Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 12 of 30 mode split and conversion (see Fig. 6). Together with the Economy & Resources mod-
ule, it follows an approach analogue to the classical four-step methodology of gener-
ation, distribution, modal split and assignment. The latter is replaced by calculation of
performance indicators in the conversion component. The module delivers trade in value by production-consumption location (P/C), which
is converted to volumes, applying volume density assumptions per O/D (related to legs
in a chain between production and consumption location) and commodity (assumed
constant over time) extracted from ETISplus (Szimba et al. 2013). The trade projections are produced by the Economy & Resources module (in Euro,
2010 constant values). The input from the Economy & Resources module is defined by
sectors that (apart from transport sectors) match commodity types c. The trade projec-
tions are used to calculate growth factors. As such the freight model can be regarded as
a marginal model. The FRD applies the value/volume conversion method from
NEAC-10 to ETISplus data to estimate the volumes in tons per year (Tt,i,j,c) by com-
modity type c and production-consumption relation ij as follows: Tt;i: j:c ¼ aci∋i;cj∋j;c bci∋i;cj∋j;c cci∋i;cj∋j;c V t;i;j;c where: Tit,i,j,c Freight trade flow of commodity c between production and consumption loca-
tion i and j in time period t [tonnes] aci,cj,c Value-volume ratio for trade from production country ci to consumption coun-
try cj per commodity type c [Euro] aci,cj,c Value-volume ratio for trade from production country ci to consumption coun-
try cj per commodity type c [Euro] bci,cj,c Logistics factor for domestic trade when country ci is country c bci,cj,c Logistics factor for domestic trade when country ci is country cj cci,cj,c Logistics factor for intra-zonal trade when zone i is zone j. Fig. 6 Structure of the Freight Demand module Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 13 of 30 Page 13 of 30 Kiel et al. (2016a, 2016b) and Van Grol et al. (2016a) contain a more detailed meth-
odology description of the freight demand model and the calibrated values of the model
parameters. The distribution of mode chains follows a multinomial logit model and distributes
the freight flow across multi-modal transport chains (routed through transshipment
points) collected from the ETISplus database. In HIGH-TOOL a mode chain consti-
tutes a multimodal routing from production location to consumption location through
up to two transshipment points. The ETISplus database provides multimodal freight
flows (in tons) per production-consumption location pair ij in the form of mode chains
r using at most two transshipment points. The probability (Pt,I,j,c,r) of using a specific
route r is calculated as follows: Pt;i;j;c;r ¼
eTCt;i; j;c;r
X
m∈M
eTCt;i; j;c;r in which in which TCt;i;j;c;r ¼ β0 min costt;i;j;c;r TCt;i;j;c;r ¼ β0 min costt;i;j;c;r where: where: Pt,i,j,c,r|m Probability of choosing route r between consumption location i and produc-
tion location j for commodity c in time period t give mode m [%] TCt,I,j,c,r Minimal total costs for route r between production location i and consump-
tion location j for commodity c at time period t min_costt,i,j,r,c Minimum total cost of route r between consumption location i and pro-
duction location j for commodity c in time period t β0 Constant β0 Constant r The route for a production-consumption location pair given the set of mode(s) m
used to traverse it. The specific set of modes used in a route allows it to be differentiated from another
route going through the same transshipment points. The modal combination with low-
est costs for a certain route is used as a proxy for the attractiveness of all routes con-
necting the production-consumption location pair ij. Here, the probability that a specific route is selected for a P/C pair ij is calculated by
comparing the estimated costs for all available routes. The number of total tons as pro-
vided in the previous step is applied on these routes to obtain route flows (Tt,i,j,c,r): Tt;i; j;c;r ¼ Tt;i; j;c; Pt;i;j;c;r where: Tt,i,j,c,r Freight flow for commodity c from production location i to consumption loca-
tion j in time period t using route r [tons] Pt,i,j,c,r Probability choosing route r between production location i to consumption lo-
cation j for commodity c in time period t [%] Tt,i,j,c Freight flow for commodity c from production location i to consumption loca-
tion j in time period t [tons]. The end product of this step is for each P/C pair ij a list of chains with allocated tons. Van Grol et al. (2016) provides a detailed description of the distribution of chains. The Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 14 of 30 mode split calculation is based on the approach applied in ETISplus and the NEAC-10
model (Newton et al. 2015). The cost functions and utilities in the modal split compo-
nent rely on TRANSTOOLS v2. The modal split calculation estimates a modal share
for each leg of the chains produced by the distribution step. For each of these legs the
total leg costs are the summation of the costs encountered in each country traversed
along the leg. Each origin and destination is connected by the transport routes extracted from the
ETISplus database. These transport routes are part of transport chains that form a set
of up to three legs that connect an origin and destination through up to two transship-
ment regions. For each of the legs the modal split calculation is applied. The modal
split component considers various cost elements such as fixed costs, variable costs, en-
ergy costs, waiting costs, and loading/unloading costs. These are influenced by the VES
module that can be affected by policy measures to compute generalized cost per avail-
able mode connecting an origin and destination of a leg through a multinomial logit
function according to TRANSTOOLS (Burgess et al. 2008; NEA 2007). Van Grol
et al. (2016) provides a detailed description of the mode split model. where: The mode split model uses the following logit approach: Pmjcdij ¼
eV mjcij
X
m∈M
eV mjcij Pmjcdij ¼
eV mjcij
X
m∈M
eV mjcij with V mjcij ¼ αcmcmcij þ βcm where: Pm|cij Probability of choosing mode m given commodity group c, origin i and destin-
ation j Pm|cij Probability of choosing mode m given commodity group c, origin i and destin-
ation j Vm|cij Utility of mode m for commodity group c and O/D relation ij Vm|cij Utility of mode m for commodity group c and O/D relation ij Cmcij Costs for mode m given commodity group c and O/D relation ij αcm Logit parameter for commodity group c and mode m βcm Constant for commodity c and mode m m Set of available modes (road, rail, inland shipping, sea, other). Subsequently based on total generalized costs for routes connecting the trade rela-
tion’s origin and destination, demand is distributed across the route’s chains connecting
origin and destination through transshipment regions in the route choice component. As the model uses a hyper network connecting the different zones, the route choice is
performed at an aggregated level. The changes in route choice are expressed as changes
in time and/or distance which are translated into fixed, variable and energy costs. The translation of the chain matrices into unimodal demand matrices is fairly straight
forward. Each chain comprises up to a maximum of three legs. Each leg contains an
origin and destination. The origin or destination can be the production or consumption
location, but they can also be the transshipment points. The chain matrix is split into
different leg-matrices by commodity group and mode. This results in mode specific
matrices by commodity group that can be used to calculate the ton-kilometers by mode
and commodity. Szimba et al. Journal of Shipping and Trade (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 15 of 30 Page 15 of 30 The translation of the unimodal demand matrices in tons into trip matrices is done
by using assumptions on the development of the average load per mode or vehicle and
the share of empty trips. This has been calibrated for the base year, in order to achieve
consistency with the amount of trips reported by statistics. The conversion step, i.e. where: the calculation of transport demand performance indicators
(ton-kilometer and vehicle-kilometer), is conducted both for the origin region and re-
lated to the “on the territory” perspective. The latter is calculated by applying the shares
of distances of a leg per country, using data from ETISplus. Finally, assumptions on share of only-cargo flights and capacity of air freight trans-
port are applied to extract air freight transport by only-cargo aircraft from the total de-
mand for air transport. The module’s most important input data are regional trade data (differentiated by
commodities) from ECR, and generalized fixed and variable costs per vehicle-km (dif-
ferentiated by country, mode and fuel type) from VES. The module’s main outputs to other modules are transport demand by mode and
commodity (vehicle mileage, ton-kilometers, tons carried) as well as transport costs by
region and origin/destination relation to ECR, transport demand and vehicle occupancy
rates to VES, transport demand (vehicle mileage by O/D, mode and commodity) to
ENV and vehicle mileage by country and mode on the basis of the “on the territory”
principle to SAF. Environment module The Environment (ENV) module calculates wheel-to-tank fuel consumption and emis-
sions for each vehicle type. The key variables in this calculation are fuel consumption
or fuel intensity, and emission factors or emission index. These factors are divided into
technologies that are represented in the module by age cohort or vintage. The module produces estimates of CO2 emissions and five other pollutants: CO,
VOC, NOx, SO2 and PM2.5. Fuel consumption and emissions are calculated per
origin country and disaggregated to zones based on the share of transport demand
in each zone. The module comprises of two parts (see Fig. 7). Firstly, the predicted transport de-
mand segmented by country, mode and fuel type is disaggregated by vehicle type and
vehicle technology (represented by the vehicle age cohort). Secondly, fuel consumption
and emissions are derived and calculated for each mode, vehicle type, fuel, and age co-
hort (technology) using the previously disaggregated transport demand, fuel consump-
tion and emission factors. Data on fuel consumption and emission factors for all vehicle age cohorts (technol-
ogy) are available for the year 2010. For each period in the remaining simulation period
(2015–2050), only factors of the new vehicles (vehicles between 0 and 4 years old) are
available in the dataset. These factors are modifiable to enable policy simulation, such
as introduction of new emission standards in a specific time or simulation period. Fuel consumption and emission factors of older vehicles (vehicles more than 4 years
old) are derived from the dataset for the previous period. The ENV module uses vehicle mileage data (differentiated by O/D and mode) from
PAD and FRD. The vehicle mileage used by PAD differentiates between urban and Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Page 16 of 30 non-urban transport, while the FRD data differentiate by commodity. Furthermore,
VES provides the vehicle fleet by country, mode, fuel type, vehicle type and vehicle
technology. There are no data flows from ENV to any other core module. Fig. 7 Structure of the Environment module Fig. 7 Structure of the Environment module non-urban transport, while the FRD data differentiate by commodity. Furthermore,
VES provides the vehicle fleet by country, mode, fuel type, vehicle type and vehicle
technology. There are no data flows from ENV to any other core module. Safety module The Safety module (SAF) assesses the impact of transport policy measures on safety,
and yields predictions of the number of fatalities and injuries, and associated social
costs (see Fig. 8). The required input includes historical mobility data from the Data Stock, predicted
mobility (from the Passenger and Freight Demand modules), and user input changes to
safety risk and safety risk causal factors. Risk is defined as the number of occurrences
(fatalities, injuries) per unit of mobility (in vehicle-kilometer or number of trips). The module distinguishes between road and non-road modes, that are subsequently
dealt with at different levels of detail. Road safety is treated in the most detail and pre-
dicts fatalities as well as serious and minor injuries. Road is further split into car, truck,
powered two-wheelers, public transport, bike, and pedestrians. Non-road modes in-
clude rail, air, short sea shipping, and inland waterways. The results are computed per country and time period. p
p
y
p
For
each
transport
mode,
there
are
two
components. The
first
is
the
Business-as-Usual (BAU), which calculates safety risks and makes predictions based on
risk trend lines (from historical mobility and safety data) and mobility predictions (from
the Passenger and Freight Demand modules). The second is the scenario component
that adapts the BAU risks according to the anticipated effect of safety measures mod-
elled. The effect is derived from changes in accident causal factors (which are the policy
inputs) and the elasticities and equations relating these to changes in risk. Safety pre-
dictions for the scenario follow from these scenario risks and mobility predictions. Road fatalities, serious and minor injuries are predicted. For the other transport modes, Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 17 of 30 (2018) 3:11 Szimba et al. Journal of Shipping and Trade Fig. 8 Structure of the Safety module the focus is on fatalities. For all modes the social costs are calculated. The general ap-
proach of adjusting risk trends based on changes in accident causal factors is based on
the European Road Safety Action Program (ERSAP) (Delhaye et al. 2010). The SAF module uses vehicle mileage by country and mode on the basis of the “on
the territory” principle from PAD and FRD. Furthermore, passenger demand data (pas-
senger-kilometers, number of trips) per country, age group and gender, differentiated
by urban and non-urban transport, are used from PAD. Safety module The module does not provide
any data to other core modules. Module interaction The core modules interact sequentially with each other. The sequential solution reduces the
computation loops, as results for a period t are passed to computations in t + 1. An iterative
process would be much more time consuming as the modules would interact, re-compute,
store and read data several times until the results for a certain time period become available
and the model can move forward to the next time period. The sequence starts with DEM to
produce demographic outputs for all forecast years 2015–2050. Subsequently ECR is run,
fed by DEM results of time step t and by VES, PAD and FRD outputs of time step t-1. Afterwards VES is activated, on the basis of DEM/ECR (step t), and PAD/FRD (step t-1)
outputs. Subsequently, PAD and FRD are run, using results from DEM/ECR/VES, and
ECR/VES, respectively. Finally, results by PAD, FRD and VES are delivered for all years to
ENV for the computation of the environmental impacts and by PAD and FRD to SAF for
the computation of the safety impacts. The tool’s base year is 2010. Thus, the first time step
2015 is partly driven by 2010 results, and 2020 by 2015 results etc. The interaction scheme is shown by Fig. 9. Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Page 18 of 30 Fig. 9 Model execution order Fig. 9 Model execution order Page 19 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Model validation The HIGH-TOOL model was subjected to an extensive validation and testing ap-
proach, consisting of: robustness tests to ensure that the model is capable of discover-
ing invalid inputs; check of coherence of model outputs with the EU Reference
Scenario 2013 (European Commission 2013); comparison of model outputs with the
ASTRA-EC model; sensitivity tests to test the plausibility of the modules’ reactions on
changes in input variables; and the conduction of case studies (see van Meijeren et al.,
2016; Kiel et al. (2016a)). Since the results of the HIGH-TOOL baseline required consistency with the forecasts
of the EU Reference Scenario 2013 (EU Ref), significant effort was spent to validate the
HIGH-TOOL modules against this reference. The validation approach embraced both
the validation of each module as stand-alone module and the validation of the inte-
grated tool, in which all modules are interconnected with each other. As an example of validation outcomes, Table 1 compares the modelled rail passenger
transport demand for the years 2010, 2020, 2030 and 2050 with the forecasts by the EU
Reference Scenario 2013, while Table 2 shows the development of the total freight
transport demand. Overall the validation analyses have revealed a high level of consistency between
HIGH-TOOL results and the EU Reference Scenario 2013. Any major differences,
which occur for a limited number of results, can be well explained, largely by differ-
ences in data definition. Moreover, the differences between forecasts by HIGH-TOOL
and the EU Reference Scenario are explained by the pattern that no information was
accessible concerning how the policy measures underlying the EU Reference Scenario
2013 have been operationalized into the PRIMES model for the generation of the fore-
casts. Thus also differences how the policies underlying the EU Reference Scenario
2013 have been operationalized by HIGH-TOOL and PRIMES, explain differences
among the results. More details on validation approach and results are provided by van
Meijeren et al. (2016). Transport policy measures
Operationalization Transport Policy Measures (TPMs) are implemented in HIGH-TOOL by a specific set
of policy levers (or input parameters of the HIGH-TOOL model) and associated values. To arrive at the set and the values for the policy levers several sources have been
reviewed. These sources differ in the strength of their underpinning and the scalability
of parameter values to the spatial level that is considered in HIGH-TOOL. The follow-
ing sources have been reviewed (see Van Grol et al., 2016): Table 1 HIGH-TOOL results vs. EU Ref, rail passenger transport demand (million pkm)
2010
HIGH-TOOL
2010
EU Ref
2030
HIGH-TOOL
2030
EU Ref
2050
HIGH-TOOL
2050
EU Ref
EU28
417,213
405,505
581,036
606,485
708,525
770,552
EU15
371,151
359,537
499,760
520,224
602,662
656,002
EU13
46,063
45,967
81,276
86,260
105,863
114,551 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 20 of 30 Table 2 HIGH-TOOL results vs. EU Ref, EU28 + 2 total freight transport demand (million tkm)
2010
HIGH-TOOL
2010
EU Ref
2030
HIGH-TOOL
2030
EU Ref
2050
HIGH-TOOL
2050
EU Ref
EU28
2,239,665
2,312,415
3,175,986
3,199,693
4,102,472
3,652,618
EU15
1,663,948
1,718,291
2,279,797
2,305,708
2,871,405
2,616,397
EU13
575,717
594,124
896,189
898,386
1,231,067
1,042,883 Table 2 HIGH-TOOL results vs. EU Ref, EU28 + 2 total freight transport demand (million tkm) (1) Large-scale (European) model applications – TPMs that have been modelled in
models such as SUMMA, EXPEDITE, and TRANSTOOLS. (1) Large-scale (European) model applications – TPMs that have been modelled in
models such as SUMMA, EXPEDITE, and TRANSTOOLS. (2) Examples from practice – TPMs that have already been implemented in practice
and are described in the literature. (3) Research publications – TPMs for which useful information was found in research
publications. Most of these studies describe model simulations, field tests, surveys,
or expert judgement. (4) Qualitative guidelines – TPMs for which a qualitative description of policy impacts
is adapted from the ASSIST project. Under consideration of this review, one or more variables of the HIGH-TOOL model
have been selected for each TPM, which translate a TPM’s effect on the transport sec-
tor. For a majority of the TPMs, mode-specific user cost variables, i.e. elements of the
generalized cost functions, have been selected to operationalize a TPM. Detailed information on how the different policy measures are operationalized is pro-
vided by Mandel et al. (2016). Transport policy measures
Operationalization In this project report each transport policy measure is
categorized, and its general impact mechanism is drafted. Furthermore, the report pro-
vides a documentation on which specific model variable(s) is (are) altered in order to
operationalize a transport policy measure. Policy selection Instead of embracing a dedicated transport supply module, the HIGH-TOOL instrument’s
supply parameters can be adjusted by the user either indirectly by the selection of transport
policy measures or bundles, or directly by changing specific policy levers. Furthermore, the
experienced user may conduct any change in the tool’s database to simulate specific policies. The HIGH-TOOL instrument offers 30 pre-defined Transport Policy Measures
(TPM), which can be selected either individually or in combinations. The scope of the
pre-defined Transport Policy Measures is shown by Table 3. Policies can be specified by the user in terms of intensity, temporal effectiveness
(2015 to 2050) and geographical distribution (countries and regions in Europe). Also combinations of pre-defined TPMs can be applied. All combinations of TPMs
have been analyzed in terms of interdependencies (see section “Consideration of policy
bundles”). While the majority of the policies have been revealed to be additive, the user
is informed by the system on the existence of interdependencies, if interdependent pol-
icy combinations are chosen. Furthermore, customized policy packages can be defined using any combination of
policy levers. The policy levers are organized per module. The number of levers per
module are shown in Table 4. Page 21 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Szimba et al. Policy selection Journal of Shipping and Trade Table 3 Transport Policy Measures covered by HIGH-TOOL
Category
Single Pre-Defined Transport Policy Measures
Efficiency standards and flanking measures
Improving local public transport
Deployment of efficient vehicles
Replacement of inefficient LDVs and buses
HDV limitation for urban areas
LDV speed limit
Diffusion of H2 fuel cell cars
Diffusion of electro cars
Replacement of inefficient cars
Pricing
CO2 feebates for road transport
CO2 certificate system for road transport
Circulation tax for cars
Internalization of external costs
HDV infrastructure change
Urban road charging
Research and innovation
Intelligent road vehicles
Dynamic traffic management for road
Intelligent traffic information system for road
Road vehicle safety technology protecting other transport users
Safety systems for road vehicle users
Internal market
Acceleration of TEN-T implementation
River information system
European Rail Traffic Management System
Harmonized handling of dangerous goods
Harmonization of rail safety
Harmonized social rules for truck drivers
Opening the internal IWW market
Enhance service quality at ports
Maritime traffic management system
Freight corridor management
Single rail vehicle authorization and certification Finally, using the Expert Mode the user can edit input tables and/or the hypernet to
control the impedances used in the model. The Expert Mode is an optional feature for
advanced editing of the database values before running the model. Consideration of policy bundles When TPMs are combined their dependency is analyzed by reviewing a possible
overlap in policy levers. If the sets of policy levers do not overlap, the TPMs are
independent and their result additive. If the sets of policy levers do overlap, special
care is needed in the implementation of policy lever values because these TPMs
may not be completely independent. Depending on the nature of the involved
TPMs, the combined effect of their policy levers might be as follows: Page 22 of 30 Page 22 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Table 4 Number of policy levers per module for the Customised Policy package interface
Module
Number of individual levers
Economy and Resources
3
Vehicle Stock
430
Passenger Demand
100
Freight Demand
79
Environment
127
Safety
60 (1) Reduced – when TPMs are conflicting and cancel each other out; (2) Maximized – the effect of the combined TPMs equals the effect of the strongest
TPM; (3) Limited – combining the TPM has some added effect, but this added effect is
limited, and (4) Maximum – although the TPMs share the same policy levers they are independent
and thus additive. (4) Maximum – although the TPMs share the same policy levers they are independent
and thus additive. There are no pairs of TPMs that fall into the first group, while pairs in the last group
can be freely combined by adding up their policy lever values. However, six groups of
TPMs have been identified that share the same policy levers and are considered to be
partly or largely overlapping. For the shared policy levers of these groups it has been
defined how to model them simultaneously (see Van Grol et al., 2016). The hypernet approach With its requirement to provide assessment results quickly and at a strategic level, the
HIGH-TOOL model is not a network-based transport model such as TRANSTOOLS. Nevertheless, the passenger and freight demand modules are based on transportation
impedances at the
level of NUTS-2 O/D relations. The impedances for the
HIGH-TOOL base year 2010 are founded on ETISplus impedances, while the baseline’s
impedances for the forecast years have been computed by the network-based transport
model Vaclav for the infrastructure configuration underlying the EU Reference Sce-
nario, and aligned with the impedances for the year 2010. This
approach
allows
the
modelling
of
infrastructure
policies
by
modifying
mode-specific travel impedances generically for transport in the whole EU, selected
countries or selected NUTS-2 regions, which however does not allow the consideration
of network effects of infrastructure policies. Therefore, in order to allow the consideration of network effects in the strategic as-
sessment of transport infrastructure policies, a “hypernet” approach was developed. The hypernet approach allows the change of travel impedances on a virtual link be-
tween two NUTS-2 regions. After running a shortest-path algorithm new impedance
matrices are obtained, thus covering the network dimension of the infrastructure pol-
icy. The hypernet is an optional submodule covering road and rail and is linked to the
passenger demand module. For the creation of the hypernet, regions have been connected in the hypernet if: ei-
ther the zones are bordering each other and have an ETISplus network link that Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 23 of 30 connects both zones, or they are connected by a ferry link in the ETISplus network. A
representative NUTS-3 zone, i.e. the NUTS-3 zone containing the largest city/ capital
city within the NUTS-2 zone, was chosen for each NUTS-2 zone. Each hypernet node
is allocated an access/ egress impedance to model travel impedances to access the
inter-regional hypernet links. The intra-zonal impedances are based on inter-NUTS-3
impedance values from ETISplus and can be influenced by policies. Also the
inter-zonal impedances are based on the ETISplus impedance matrices. Future travel
impedances up to the model’s forecast horizon 2050 were calculated from enhanced
ETISplus networks, including all infrastructure projects planned on the TEN-T core
and comprehensive networks (see European Union, 2013). The hypernet approach Figure 10 displays the constructed hyper-network links for road (ETISplus road net-
work (red), Zone centroids (green) and constructed HIGH-TOOL road hypernet (blue)
for the base year 2010). Case study description This case study examines the application of the hypernet facility of the HIGH-TOOL
instrument. The assumption is made that rail passenger travel times will further de-
crease by 10% along the “Magistrale” corridor Paris–Strasbourg–Karlsruhe–Munich–
Vienna–Bratislava (see Fig. 11). The travel time decrease is assumed to be on top of the
time savings due to TEN-T/CEF policies already in the baseline scenario. Thus, the in-
vestment assumptions do not refer to concrete rail infrastructure projects, but are
hypothetical. The infrastructure improvements are assumed to become effective in the
year 2030. Further details on this case study are provided by Kiel et al. (2016b). Fig. 10 Scope of the hypernet (road) (2018) 3:11 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 24 of 30 Fig. 11 Map showing the hypernet rail links of the Magistrale corridor (blue), other hypernet rail links (red)
and NUTS-2 zone centroids (green) Fig. 11 Map showing the hypernet rail links of the Magistrale corridor (blue), other hypernet rail links (red)
and NUTS-2 zone centroids (green) Model results The model predicts an increase in rail passenger demand while the demand of Ac-
knowledgements modes (road – i.e. private passenger cars –, coach and air) is expected
to decrease (see Table 5). The results do not only reveal a mode shift effect, but also re-
veal
that
the
increase
in
rail
passenger-kilometers
exceeds
the
loss
of
passenger-kilometers by competing mode. Thus, the assumed improvement of rail
level-of-service is expected to generate induced traffic that explains almost half of the
additional rail traffic demand. This pattern is largely in line with the observations made
for the French high-speed rail line Paris-Lyon (Vickerman 1996) and can be explained
by direct and indirect effects, such as changes in travelers’ mobility behavior and the
emerging of new activities due to the reduced travel time (Cascetta and Coppola 2013). The percentage changes in relation to the total passenger transport demand are rela-
tively limited, which is explained by the limited geographical scope of the measures, as
well as by the pattern that the assumed infrastructure improvements relate to inter-zonal
passenger transport flows at the level of NUTS-2, which represent only a small share of
the overall market. In 2050, the rail passenger demand is expected to increase by 0.02%
for EU28 + 2, representing 4.4 million passenger-kilometers per day (1.6 Gpkm per year). The results of our rather hypothetical case study are largely in line with the findings of
the “Magistrale corridor study” predicting an increase of about 14 million rail passenger
trips per year due to upgrading this railway corridor (IWW et al. 2001). Regarding impacts on demand by mode of transport for 2050 at country level, the
strongest impacts in absolute terms are expected for Germany, followed by France, Table 5 Case study – Total passenger-kilometres 2010–2050, by modes (in million), EU28 + 2,
difference Scenario-Baseline
2030
2035
2040
2045
2050
Air
−45
−55
−53
−55
−55
Rail
1306
1569
1534
1575
1608
Coach
−35
−42
−43
−45
−47
Road
−605
−743
−713
−734
−742 Table 5 Case study – Total passenger-kilometres 2010–2050, by modes (in million), EU28 + 2,
diff
S
i
B
li Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 25 of 30 Page 25 of 30 Austria and Slovakia (see Fig. 12). These countries are the key beneficiaries of the as-
sumed infrastructure investments. Model results Due to the network effects, which are covered by
the hypernet approach, also the demand structures of other countries, which are not
directly concerned by the investments – such as the Czech Republic, Italy and Romania
– reveal slight impacts in favor of rail. The modal shift from road and air to rail leads to a decrease in fuel consumption,
CO2 emissions and the emission of air pollutants (see Table 6). Furthermore, the modal
shift results in a slight reduction in the number of road accidents (see Table 7). Finally,
the HIGH-TOOL model predicts moderate economic impacts (see Table 8): the de-
crease in rail passenger travel times results in savings in generalized costs and, thus,
improves regional accessibility, which has a positive impact on Gross Value Added
through provision of better access to labor markets and lower costs of intermediate
goods (second order effects). The economic impacts do not embrace any first order ef-
fects (direct or multiplier effects of the infrastructure investments), as the underlying
investment amount has not been specified for the purposes of the case study. Conclusions The HIGH-TOOL model is an open source instrument, is both publicly and freely
available and does not require any commercial software products to be run. These fea-
tures clearly distinguish the HIGH-TOOL model from any other European transport
demand model or policy assessment instrument. The model’s openness ensures thor-
ough transparency of computations, and allows the experienced user to modify calcula-
tion methodologies, data or model parameters. To develop the HIGH-TOOL model, originally independently functioning models
have been integrated on a common platform. A key enabler of the development work
in HIGH-TOOL was the European reference database ETISplus, covering a large share
of data sets relevant for the models which were integrated in HIGH-TOOL. In this re-
spect, HIGH-TOOL can be regarded as a logical consequence of the European Union’s
strategy to provide not only publicly available data for transport policy and modelling –
as accomplished by EU-funded projects such as ETIS-BASE and ETISplus –, but also
to establish a publicly available open source tool for strategic policy assessment. Fig. 12 Impact on passenger transport demand by transport mode per country (year 2030, changes in
millions pkm) Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 26 of 30 Table 6 Case study – Impact on emissions and fuel consumption p.a. (in tons), EU28 + 2,
difference Scenario-Baseline Table 6 Case study – Impact on emissions and fuel consumption p.a. (in tons), EU28 + 2,
difference Scenario-Baseline
Year
Fuel consumption
CO2
NOx
PM
SO2
2030
−9963
−35,105
−54
−3
−1
2035
−9411
−33,449
−60
−3
0
2040
−9956
−34,320
−62
−3
0
2045
−10,253
−35,044
−65
−3
−1
2050
−8934
−31,284
−62
−3
−1 Also the existence of the EU Reference Scenario, which outlines long-term projec-
tions until 2050 and covers aspects such as transport demand, energy consumption and
vehicle fleet, was a substantial support for the calibration of the HIGH-TOOL model. On the other hand, it is becoming a broadly accepted view that there will be large
structural changes in the (near) future in many economic sectors, also within transport
and mobility (see e.g., Chen et al. 2016; Brynjolfsson and McAfee 2014; Blasi et al. 2013). The National Research Council of the USA (2010) states: “All forecasting meth-
odologies depend to some degree on the inspection of historical data. Conclusions (million €), EU28 + 2, difference
Scenario-Baseline
Year
Primary
Secondary
Tertiary
2030
0
0
0
2035
0.1
6.5
3.7
2040
0.1
7.0
4.1
2045
0.2
7.3
4.4
2050
0.1
7.4
4.6 Table 8 Case study – Impact on Gross Value Added by sector p.a. (million €), EU28 + 2, difference
Scenario-Baseline application and improve the spatial representation of infrastructure-related policies. In-
creasing the regional level of detail of traffic cells and connecting the HIGH-TOOL
model with a network-based model however, imply unfavourable impacts on model run
time, which needs to be avoided as far as possible by smart data handling and process-
ing methodology. application and improve the spatial representation of infrastructure-related policies. In-
creasing the regional level of detail of traffic cells and connecting the HIGH-TOOL
model with a network-based model however, imply unfavourable impacts on model run
time, which needs to be avoided as far as possible by smart data handling and process-
ing methodology. In combination with an enhanced approach to model network effects of transport
policies, a dedicated transport supply module could be implemented to consolidate the
representation of the supply side (including the operationalization of policies) in one
entity. Moreover, the increasing relevance of Sharing Economy concepts in transport (e.g.,
car/bike sharing; ride sharing) calls for a more sophisticated and explicit consideration
of these schemes by transport demand and policy assessment tools. Further mega trends in transport are electrification and autonomous driving. The
electrification of the road transport sector indicates the requirement to connect
transport demand and energy modelling more closely, in order to obtain a better
understanding of interdependencies between these sectors and to explore the po-
tential of electrification of transport for decentral energy supply concepts (e.g., by
using electric vehicles as mobile energy storage). Autonomous driving will result in
tremendous impacts on the transport sector by enhancing access to mobility, im-
prove safety, and add the potential to alleviate congestion, reduce travel time and
reduce environmental impacts (see e.g., Harper et al. 2016; Fagnant and Kockelman
2014; Hars 2014). Though it remains unclear how much of the potential benefits
will actually be exploited, since vehicle mileage is expected to increase (Szimba and
Orschiedt 2017). Conclusions Supported by further research on travel behaviour, traffic engin-
eering and the scope of business models expected from autonomous driving, an
extended version of the HIGH-TOOL model could be used to estimate the impacts
of autonomous driving at European scale. Finally, the consideration of future modes of transport such as drones or the Hyper-
loop concept may provide avenues for model enhancement. With its high degree of openness and its modular structure, the HIGH-TOOL model
provides the basis for an efficient further development of the model in the indicated di-
rections and beyond, and thus lays the foundation for further innovations in the realm
of assessment of transport policies and mobility concepts. Conclusions However, exclu-
sive reliance on historical data inevitably leads to an overemphasis on evolutionary
innovation and leaves the user vulnerable to surprise from rapid or nonlinear develop-
ments. […] A methodology that can forecast disruptive technologies must overcome
the evolutionary bias and be capable of identifying unprecedented change”. Also
the currently instable political and geo-political situation in many world regions,
wars and migration cause a high level of uncertainty in terms of future develop-
ment of demographic, social, societal and socio-economic patterns which substan-
tially influence transport demand. However, the official national and EU forecasts
currently tend to suffer from a lack of adapted methodology to be able to antici-
pate for the future in which direct and indirect impacts of disruptive technologies
will play a major role. Thus, further research is needed to develop a generally ac-
cepted, trusted, transparent and repeatable approach that does not solely rely on
historical developments, but which allows to deal with breaks in trends and derived
developments. The current version of the HIGH-TOOL model offers various possibilities for further
developments: for instance, enhancing the zoning system of transport demand model-
ling from the spatial level of NUTS-2 to NUTS-3 will significantly reduce the share of
intra-zonal transport demand and increase the accuracy of transport demand model-
ling. Also, a closer link to a network-based model, which goes beyond the currently im-
plemented hypernet approach for passenger transport, will enhance the tool’s scope of Table 7 Case study – Impact on road accidents p.a. (number of injured persons), EU28 + 2,
difference Scenario-Baseline Table 7 Case study – Impact on road accidents p.a. (number of injured persons), EU28 + 2,
difference Scenario-Baseline
Year
Serious injuries
Slight injuries
2030
−9
−67
2035
−5
−77
2040
−9
−69
2045
−11
−82
2050
−8
−73 Table 7 Case study – Impact on road accidents p.a. (number of injured persons), EU28 + 2,
difference Scenario-Baseline
Year
Serious injuries
Slight injuries
2030
−9
−67
2035
−5
−77
2040
−9
−69
2045
−11
−82
2050
−8
−73 Szimba et al. Journal of Shipping and Trade (2018) 3:11 Page 27 of 30 Page 27 of 30 Table 8 Case study – Impact on Gross Value Added by sector p.a. 1NUTS: Nomenclature of Units for Territorial Statistics Competing interests The authors declare that they have no competing interests. Funding The research has received funding from the European Union’s Seventh Framework Programme for research,
technological development and demonstration under grant agreement no° 321624. Declaration I confirm that I have read SpringerOpen’s guidance on competing interests and have included a statement indicating
that none of the authors have any competing interests in the manuscript. Abbreviations AngularJS: A client framework to build web applications in JavaScript; BAU: Business as usual; CEF: Connecting Europe
Facility; CO2: Carbon Dioxide; DEM: Demography module of the HIGH-TOOL model; DV: Deterrence Value;
ECR: Economy & Resources module of the HIGH-TOOL model; EMC: Expected Minimum Cost; ENV: Environment
module of the HIGH-TOOL model; ERSAP: European Road Safety Action Program; EU Ref: EU Reference Scenario 2013;
EU: European Union; EU28 + 2: 28 Member States of the European Union plus Norway and Switzerland; EU28: 28
Member States of the European Union; Eurostat: Statistical Office of the European Union; FRD: Freight Demand
module of the HIGH-TOOL model; GDP: Gross Domestic Product; Gpkm: Giga passenger-kilometer; GVA: Gross Value
Added; HDV: Heavy Duty Vehicle; LDV: Light Duty Vehicle; NOx: Nitric oxide and nitrogen dioxide; NST: Eurostat’s
Standard Goods Classification for Transport Statistics; NUTS: Nomenclature of Territorial Units for Statistics; A number
assigned to it defines the level of granularity, such as 0 for the country level; O/D: Origin/ Destination; P/C: Production/
Consumption; PAD: Passenger Demand module of the HIGH-TOOL model; PostGIS: An open source software program
that adds support for geographic objects to the PostgreSQL object-relational database; PostgreSQL: An object-
relational database; RTD: Research and Development; SAF: Safety module of the HIGH-TOOL model; Sails.js: A server
framework to build Node.js applications; SOx: Sulfur oxide; TEN-T: Trans-European Networks for Transport;
TPM: Transport Policy Measures Acknowledgements
Th
h
ld lik The authors would like to thank Hettie Boonman, Tina Laparidou and Cristian Lorenzo Martínez for their dedication for
contributing to the development of the tool. The authors are also grateful to the anonymous reviewers for their
valuable comments and suggestions to improve the quality of the paper. Endnotes 1NUTS: Nomenclature of Units for Territorial Statistics Page 28 of 30 Szimba et al. Journal of Shipping and Trade (2018) 3:11 (2018) 3:11 Availability of data and materials y
The (co-)author(s) declare that the tool presented in this paper (and the involved data) is publicly available and can be
requested from the HIGH-TOOL homepage (www.high-tool.eu). Authors’ contributions All authors have contributed to the conception and design of the presented model, the development of the tool and
involved data collection, analysis and interpretation. Under coordination by the corresponding author, all authors have
been involved in the drafting of the article, and the critical revision of the paper. All authors have approved the
version to be published. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 15 January 2018 Accepted: 14 October 2018 Received: 15 January 2018 Accepted: 14 October 2018 Received: 15 January 2018 Accepted: 14 October 2018 Author details
1 1Karlsruhe Institute of Technology (KIT), Network Economics, Institute of Economics (ECON), Waldhornstr. 27, Bldg. 01.96, 76131 Karlsruhe, Germany. 2Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. 3Netherlands
Organisation for Applied Scientific Research TNO, The Hague, The Netherlands. 4Panteia, Zoetermeer, The Netherlands. 5MKmetric Gesellschaft für Systemplanung mbH, Karlsruhe, Germany. 6MCRIT S.L., Barcelona, Spain. 7Transport &
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De novo sequencing of the transcriptome reveals regulators of the floral transition in Fargesia macclureana (Poaceae)
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De novo sequencing of the transcriptome reveals
regulators of the floral transition in Fargesia
macclureana (Poaceae) Ying Li
International Center for Bamboo and Rattan
https://orcid.org/0000
Chunxia Zhang
Nanjing Forestry University
Kebin Yang
International Center for Bamboo and Rattan
Jingjing Shi
International Center for Bamboo and Rattan
Yulong Ding
Nanjing Forestry University
Zhimin Gao
(
gaozhimin@icbr.ac.cn
)
https://orcid.org/0000-0003-4464-7159 Ying Li
International Center for Bamboo and Rattan
h
Chunxia Zhang
Nanjing Forestry University
Kebin Yang
International Center for Bamboo and Rattan
Jingjing Shi
International Center for Bamboo and Rattan
Yulong Ding
Nanjing Forestry University
Zhimin Gao
(
gaozhimin@icbr.ac.cn
)
https://orcid.org/0000-0003-4464-7159 Research article Keywords: Transcriptome, Floral transition, Bamboo, Qinghai–Tibet Plateau
Posted Date: January 3rd, 2020
DOI: https://doi.org/10.21203/rs.2.10521/v4
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full
License License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full
License Version of Record: A version of this preprint was published on December 30th, 2019. See the published version
at https://doi.org/10.1186/s12864-019-6418-2. Page 1/27 Abstract Background: Fargesia macclureana (Poaceae) is a woody bamboo species found on the Qinghai–Tibet Plateau
(QTP) approximately 2,000 ~ 3,800 m above sea level. It rarely blossoms in the QTP, but it flowered 20 days
after growing in our lab, which is in a low-altitude area outside the QTP. To date, little is known regarding the
molecular mechanism of bamboo flowering, and no studies of flowering have been conducted on wild bamboo
plants growing in extreme environments. Here, we report the first de novo transcriptome sequence for F. macclureana to investigate the putative mechanisms underlying the flowering time control used by F. macclureana to adapt to its environment. Results: Illumina deep sequencing of the F. macclureana
transcriptome generated 140.94 Gb of data, assembled into 99,056 unigenes. A comprehensive analysis of the
broadly, specifically and differentially expressed unigenes (BEUs, SEUs and DEUs) indicated that they were
mostly involved in metabolism and signal transduction, as well as DNA repair and plant-pathogen interactions,
which may be of adaptive importance. In addition, comparison analysis between non-flowering and flowering
tissues revealed that expressions of FmFT and FmHd3a, two putative F. macclureana orthologs, were
differently regulated in NF- vs F- leaves, and carbohydrate metabolism and signal transduction were two major
KEGG pathways that DEUs were enriched in. Finally, we detected 9,296 simple sequence repeats (SSRs) that
may be useful for further molecular marker-assisted breeding. Conclusions: F. macclureana may have evolved
specific reproductive strategies for flowering-related pathways in response to photoperiodic cues to ensure long
vegetation growing period. Our findings will provide new insights to future investigations into the mechanisms
of flowering time control and adaptive evolution in plants growing at high altitudes. Background The flowering time is of crucial importance to ensure the reproductive success of flowering plants. Previous
results have indicated that the floral transition is orchestrated by several parallel and interactive genetic
pathways that are regulated by a variety of environmental and endogenous signals [1]. Many key genes and
regulatory networks have been identified in herbaceous annual plants such as Arabidopsis [2, 3], rice [4], gourds
[5], potato [6] and sorghum [7]. However, much less is known about such regulation in perennial plants. Despite
the increasing attention on perennial dicotyledonous woody plants such as poplar [8, 9], eucalyptus [10] and
citrus [11] species, to date, the molecular mechanism underlying floral regulation in monocotyledonous woody
plants remains elusive. Furthermore, previous studies investigated flowering mainly by artificially altering the
external signals (e.g. photoperiod and light intensity) and did not assess the impact of the original environment
on the adaptive evolution of species-specific reproductive strategies. Bamboo plants are an important group in the Bambusoideae subfamily of the monocotyledonous Poaceae. They exhibit a wide degree of variation in the timing (1-120 years) and nature (sporadic vs. gregarious) of
flowering among species [12]. Sporadic flowering involves flowering in only a few isolated clumps, which set
little or no seed and usually remain alive afterward [13]. In contrast, gregarious flowering involves all individuals
of a species regardless of age and/or location within and among the populations at the same time, which is
usually followed by death and seed setting [14]. And the simultaneous death of many individuals triggers
serious ecological consequences, including changes in the population dynamics of neighboring plants,
differences in soil properties, various effects on endangered animals that depend on bamboo [15], and the
knock-on effects on human economies in many parts of the world [16]. Therefore, dissecting the regulators that Page 2/27 Page 2/27 control the unique life history of bamboo may be of use for plant ecology and human society. However, to date,
little is known regarding the molecular mechanisms of bamboo flowering, in part because of the sporadic
occurrence of these flowering episodes and the long intervals between events. control the unique life history of bamboo may be of use for plant ecology and human society. However, to date,
little is known regarding the molecular mechanisms of bamboo flowering, in part because of the sporadic
occurrence of these flowering episodes and the long intervals between events. Background Many genes have been identified as regulators of reproductive development in different bamboo species,
including the MADS-box transcription factors [17-19], CONSTANS (CO) [20] and FLOWERING LOCUS T (FT) [21],
among others. In addition, studies of sequenced transcriptomes have identified microRNAs related to floral
development [22-24]. However, samples collected in these analyses were limited to mature spikelets or to
different spikelets at different development stages. Thus, it is likely that dynamic changes in genes occurring at
different development stages may be missing. In addition, the specific response of particular tissues to internal
and external cues and how plants integrate these signals to regulate different phases of reproductive
development (including the floral transition, florigen transport, and floral organ specification) has not yet been
elucidated in bamboo. Furthermore, no studies of flowering have been conducted on wild bamboo plants
growing in extreme environments. Here, we took advantage of an unexpected flowering event in highland arrow bamboo, Fargesia macclureana
[25], and performed the first de novo transcriptome analysis. This transcriptome includes data from six
different tissues collected at different development stages, including inflorescences in the initial and peak
flower stage (I- and P- spikelets), branchlets, and leaves from both flowering and non-flowering bamboo plants
(F/NF-branchlets and F/NF -leaves). F. macclureana is a woody bamboo species found in areas 2,000 ~ 3,800
m above sea level on the Qinghai–Tibet Plateau (QTP) (Fig. 1), which is the highest and largest plateau in the
world. The growth environment of the QTP is characterized by low temperature and low oxygen availability,
reduced pathogen incidence, and intense radiation [26]. F. macclureana rarely blossoms in the QTP, but it
flowered 20 days after growing in our lab, which is in a low-altitude area outside the QTP. Our goal is to use the
transcriptomic data to gain a deeper understanding of the mechanisms underlying the control of flowering time
and the adaptation of F. macclureana to the complex extreme conditions of the QTP. On one hand, we expect to
detect regulatory hubs involved in the flowering mechanisms. On the other hand, we aim to discover signs of
the adaptive evolutionary changes in F. macclureana in response to the harsh environmental conditions in the
QTP, which may, in turn, provide a broader insight into the adaptive mechanisms for plants that grow at high
altitudes. De novo transcriptome assembly yielded 99,056 unigenes Illumina deep sequencing of the F. macclureana transcriptome generated 140.94 Gb of data, including
471,537,304 clean reads in 18 unique samples (Additional file 1: Table S1). The average Q20 (sequencing error
rate less than 1%) and Q30 (sequencing error rate less than 0.1%) percentages were 95.64% and 89.95%
respectively. The GC content of all samples ranged from 53.78% to 55.86%, with an average of 54.81%. Sample
data were assembled into 289,122 transcript scaffolds, with an N50 and average length of 1,765 bp and 1,183
bp, respectively. The final de novo assembly included 99,056 unigenes, with an N50 and average length of
1,587 bp and 926 bp, respectively. Among these unigenes, 71.02% (70,354) were shorter than 1,000 bp and
12.06% (11,950) were longer than 2,000 bp (Table 1). Page 3/27 Page 3/27 Page 3/27 Page 3/27 Most unigenes were functionally annotated and classified A total of 47,306 unigenes were annotated (Additional file 2: Table S2). Of these, 45,516 (96.22%) unigenes
were found to encode products that showed significant similarity to characterized proteins in the non-redundant
protein sequence database (Nr) at an E-value threshold of 10-5 (Table 2). We also found that 7,027 (15.45 %)
unigenes showed similarity to genes found in rice, 11.33% were similar to those found in Brachypodium
distachyon, and we also found a significant proportion of the unigenes that were similar to those found in
Setaria italica, Oryza brachyantha, and Zea mays (Fig. 2a). We identified 24,847 (52.52%), 28,317 (59.86%) and
43,909 (92.82%) unigenes that showed significant matches to entries in the Swiss-Prot, Pfam, and eggnog
databases, respectively (Table 2). Many unigenes expressed in the F. macclureana transcriptome were
functionally annotated as regulators of plant responses to evolutionarily important phenotypes, including
membrane stabilization, heat stress response and pathogen defense (Additional file 2: Table S2). Most BEUs were involved in genetic information processing, environmental adaptation and signal transduction As shown in the Venn diagram (Fig. 4a), we found nearly equal numbers of unigenes that were broadly and
specifically expressed in I-spikelets, P-spikelets, F-branchlets, and F-leaves. COG analysis indicated that most
BEUs were clustered in signal transduction mechanisms (T), replication, recombination and repair (L), and
transcription (K), besides general function prediction only (R). GO enrichment analysis for these BEUs indicated
that they were also mainly involved in reproduction, environmental adaptation and signal transduction, which
was largely similar with that for all predicted unigenes (Additional file 4: Table S4-a). KEGG enrichment analysis also indicated that these BEUs were mainly enriched in pathways related to
environmental adaptation (including circadian rhythm, endocytosis, and plant-pathogen interactions), signal
transduction (including plant hormone signal transduction, phosphatidylinositol signaling system, and inositol
phosphate metabolism) and genetic information processing (including spliceosome, mRNA surveillance, and
RNA transport and degradation; Additional file 4: Table S4-b). Functional annotation indicated that many unigenes were involved in metabolism and genetic information
processing We were able to annotate 13,128 unigenes (27.75% of the total) in 25 different categories of the COG (clusters
of orthologous groups) classification database (Fig. 2b). Of these, the cluster for “General function prediction
only” (3,277, representing 24.96% of the 13,128 unigenes annotated by this database) was the largest group,
followed by “Replication, recombination and repair” (2,202, 16.77%), “Transcription” (1,571, 11.97%), and
“Translation, ribosomal structure and biogenesis” (1,429, 10.88%). The “Signal transduction mechanisms”,
“posttranslational modification, protein turnover, chaperones”, “carbohydrate and amino acid transport and
metabolism” and “transport and metabolism” categories also contained a significant proportion of the
annotated unigenes. GO enrichment analysis indicated that these predicted unigenes were categorized into three main categories—
i.e. biological process (BP), cellular component (CC), and molecular function (MF). As shown in Fig. 2c, for
unigenes that were enriched in the BP category, they were mainly involved in biological processes related to
reproduction, posttranslational modification and signal transduction; as for those in the CC category, they were
mainly involved in cellular components related to membrane, ubiquitin ligase complex, mitochondrion,
chloroplast and etc.; while for those in the MF category, they were mainly involved in molecular functions
related to signaling transduction (e.g. “ATP binding”, “zinc ion binding”, “protein kinase activity”, and etc.)
(Additional file 3: Table S3). We also mapped 14,307 unigenes (representing 30.24% of the total) to six different KEGG subsystems,
including metabolism, genetic information processing, environmental information processing, cellular
processes, and organismal systems. As shown in Fig. 3, the majority of these unigenes (7,922, representing
66.17% of the 14,307 unigenes classified using KEGG annotations) were assigned to metabolic pathways,
including carbohydrate metabolism, energy metabolism, and others. In addition, 4,024 unigenes (28.13%) were
assigned to genetic information processing, including transcription, translation, and folding, and 474 unigenes
(3.31%) were found to be related to membrane transport and signal transduction. We also found 707 genes
(4.94%) that were related to transport and catabolism and 377 genes (2.64%) related to environmental
adaptation. Page 4/27 The SEUs were mostly involved in carbohydrate metabolism, energy metabolism, and environmental adaptation As shown in Fig. 4a, we identified 10,653 unigenes that were specifically expressed in spikelets, including 5,528
and 5,025 unigenes in I- and P-spikelets, respectively. We also found 9,067 and 7,437 unigenes that were
specifically expressed in F-branchlets and F-leaves, respectively. COG annotation indicated that the distribution
patterns of SEUs among the 26 terms were similar, with the number of SEUs within each term varying among
the three tissues (Fig. 4b). The GO enrichment analysis indicated that these SEUs not only shared some common GO terms, but also had
some particular ones. As shown in Fig. 4c and Additional file 4: Table S4-c, for those SEUs that were enriched in
the BP category, they were broadly involved in several important biological processes, including “protein
phosphorylation”, “regulation of flower development”, “protein ubiquitination”, “regulation of transcription, DNA-
templated”, “reciprocal meiotic recombination” and “meiotic chromosome segregation”. In addition, SEUs in I-
and P- spikelets were also involved in some processes related to reproduction; and those in F-branchlets were
mainly involved in processes related to posttranslational modification; while those in F-leaves were mainly
involved in processes related to plant-pathogen interaction. As for those in the CC category, they were broadly
involved in several important cellular components, including “mitochondrion”, “plasma membrane” and
“plastid”. In addition, SEUs in I- and P-spikelets were also involved in ribosome and mitochondria; and those in
F-branchlets were mainly involved in endoplasmic reticulum and proteasome; and those in F-leaves were
mostly involved in chloroplast. As for those in the MF category, they were broadly involved in several molecular
functions, including “ATP binding”, “ubiquitin-protein transferase activity” and “protein tyrosine kinase activity”. In addition, SEUs in I- and P-spikelets were also involved in DNA and microtubule binding; those in F-branchlets
were also enriched in oxidoreductases activities; and those in F-leaves were also enriched in enzymes involved
in carbohydrate metabolism. As shown in Additional file 5: Fig. S1, KEGG pathway analysis indicated that SEUs in I- and F-spikelets mainly
mapped to the ribosome pathway, with those in F-branchlets mainly mapped to the ribosome, amino acid
biosynthesis, and carbon metabolism pathways, and those in F-leaves mainly mapped to KEGG pathways
related to energy metabolism (including oxidative phosphorylation, fatty acid metabolism, and Page 5/27 Page 5/27 photosynthesis), environmental adaptation (e.g. proteasomes), genetic information processing, and various
unrelated metabolic pathways (e.g. tryptophan metabolism, beta-alanine metabolism, and N-glycan
biosynthesis). DEUs were mostly involved in carbohydrate and energy metabolism, signal transition and environmental
adaptation As shown in Table 3, many unigenes showed differential expressions across all 15 groups sampled. The
number of DEUs in each sample pair ranged from 970 between I- vs P-spikelets to 13,577 in NF-leaves vs I-
spikelets. For most pairwise comparisons, the number of up- and down-regulated DEUs was approximately the
same, except for four groups, including I- vs P-spikelets, F-branchlets vs both I- and P- spikelets, and F-leaves vs
P-spikelets. The Venn diagram of DEU sets shows that 5,494 unigenes were differentially expressed in F-branchlets/F-
leaves vs I- and P-spikelets. For those DEUs that were up-regulated in spikelets, they are mainly mapped to
KEGG pathways related to carbohydrate metabolism, plant-pathogen interactions and DNA repair (Fig. 5a). Notably, among the 970 DEUs identified between I- and P-spikelets, 916 up-regulated DEUs were mapped to
KEGG pathways related to metabolic activity (Additional file 6: Table S5). A total of 5,494 unigenes were differentially expressed in the DEU sets of spikelets/F-leaves vs F- branchlets. Upregulated DEUs in F-branchlets were mapped to KEGG pathways including phenylalanine metabolism,
phenylpropanoid biosynthesis, ABC transporters, and flavone and flavonol biosynthesis (Fig. 5b). Those that
were upregulated in F- and NF-leaves vs F- branchlets were mainly mapped to plant hormone signal
transduction, homologous recombination, base excision repair, and mismatch repair (Additional file 6: Table
S5). Notably, 3,275 (50.20% of the total) DEUs found between NF- and F-branchlets were upregulated; these
were mainly mapped to KEGG pathways related to replication and recombination (Additional file 6: Table S5). Those that were downregulated were mainly mapped to carbon fixation and photosynthesis (Additional file 6:
Table S5). We also found that 6,966 (43.69% of the total) DEUs found in spikelets/F-branchlets vs F-leaves were up-
regulated, and were mainly mapped to KEGG pathways related to carbohydrate metabolism (Fig. 5c). 2,492
(49.52%) DEUs in NF-vs F-leaves were up-regulated, and these were mainly mapped to starch and sucrose
metabolism (Additional file 6: Table S5). In contrast, downregulated DEUs were mainly mapped to KEGG
pathways related to photosynthesis (Additional file 6: Table S5). Two putative FT orthologs were regulated differently in the circadian rhythm–p Among the 5,032 DEUs identified between NF- and F-leaves, 70 were mapped to the circadian rhythm–plant
KEGG pathway (Additional file 7: Fig. S2) and 10 of them showed differential expressions (Additional file 8:
Table S6). Notably, c109220.graph_c0 and c110963.graph_c4 were both annotated as FT orthologs: the former
was a putative bamboo ortholog of Heading date 3a (Swissprot: PE=1 SV=1), and the latter was another
ortholog of rice FT; we designated them as FmHd3a and FmFT, respectively. As shown in Fig. 6a, protein sequence alignment indicated that both FmFT and FmHd3a had high amino acid
sequence similarities (77.14%) with the known FT/TFL1 proteins and had the critical amino acids of FT/Hd3a Page 6/27 Page 6/27 proteins. For example, they both carry a conserved phosphatidylethanolamine-binding protein (PEBP) domain,
D-P-D-x-P and G-x-H-R motifs, and the invariant histidine (asterisk), all of which are relevant to the ability of
PEBP proteins to bind phosphoryl ligands, hence interfering with certain kinases and effectors [27-28]. Furthermore, all five proteins carry tyrosine-139 and tryptophan-143 (triangles) in the PEBP domain, two
conserved sites in FT homologs acting as flowering inducers [29]. Whereas, our comparison also revealed that
there were differences in amino acid sequences between FTs and Hd3as. proteins. For example, they both carry a conserved phosphatidylethanolamine-binding protein (PEBP) domain,
D-P-D-x-P and G-x-H-R motifs, and the invariant histidine (asterisk), all of which are relevant to the ability of
PEBP proteins to bind phosphoryl ligands, hence interfering with certain kinases and effectors [27-28]. Furthermore, all five proteins carry tyrosine-139 and tryptophan-143 (triangles) in the PEBP domain, two
conserved sites in FT homologs acting as flowering inducers [29]. Whereas, our comparison also revealed that
there were differences in amino acid sequences between FTs and Hd3as. Additionally, phylogenetic analysis indicated that these ten proteins were subdivided into two distinct
subgroups (Fig. 6b). FT and Hd3a proteins from O. sativa (OsFT, OsRFT1 and OsHd3a), F. macclureana (FmFT
and FmHd3a) and A. thaliana (AtFT) were clustered in a branch. Furthermore, OsFT and FmFT were clustered
together, just like OsHd3a and FmHd3a; they were both closer to each other than they were to AtFT. Interestingly, two FT orthologs were regulated differently in NF- vs F- leaves. FmHd3a was significantly
upregulated in F-leaves (FDR = 4.23, log2FC = 5.55), while FmFT was significantly downregulated in F-leaves
(FDR = 4.25E-07, log2FC = -4.81). WGCNA results identified gene modules related to specific tissues WGCNA results showed that unigenes expressed in the six different tissues of flowering and nonflowering
plants tested here clustered into 18 branches representing 18 different genetic modules (Additional file 9: Fig. S3a). Unigenes within each module were highly co-expressed, while those in different modules were co-
expressed to a lower degree (Additional file 9: Fig. S3b). In six of the samples collected, we identified nine
significant gene modules including 1,344 unigenes. Here, correlation coefficient of a module with a related trait
> 0.7 was used as a threshold of significance (Additional file 9: Fig. S3c). Notably, these six tissues were more
strongly divided into clades according to whether they were flowered or not rather than by the differences
among tissues (Additional file 9: Fig. S3d). In addition, the unigenes in gene modules relating to I - and P- spikelets were most strongly enriched in KEGG
pathways related to carbohydrate metabolism, genetic information processing, and environmental information
processing. In contrast, those related to F- and NF- branchlets were mostly enriched in KEGG pathways related
to metabolism, plant hormone signal transduction, and genetic information processing. The gene modules
related to F-leaves were enriched in pathways related to plant hormone signal transduction and protein
processing, while the gene modules related to NF-leaves were enriched in KEGG pathways related to oxidative
phosphorylation (Additional file 10: Table S7). Two putative FT orthologs were regulated differently in the circadian rhythm–p RT-qPCR analysis also showed that FmHd3a was significantly more highly
expressed in I- /P- spikelets and F-leaves than in NF-leaves or NF- branchlets (Fig. 6c). Identification of SSRs We detected a total of 9,296 SSRs in 7,668 unigenes longer than 1,000 bp (Additional file 11: Table S8). 1,628
(21.23%) unigenes contained more than one SSR. Mono-nucleotide repeats were the most common (46.28% of
all SSRs) at a density of 71 SSRs per Mb, followed by tri- (26.32%) and di- (22.06%) nucleotide repeats, with
densities of 40 and 32 SSRs per Mb, respectively (Fig. 7). Activated Hd3a expression probably accelerates flowering in F. macclureana FT is a key floral regulator that controls the timing of flowering and seasonal growth cessation in response to
light and the circadian clock in many plant species [8, 10, 30]. In this study, FmHd3a, a bamboo ortholog of rice
FT, was significantly expressed only in tissue samples collected from flowering plants. In rice, Hd3a functions
as a major photoperiodic flowering regulator and participates in the OsGI–Hd1–Hd3a module, which is similar
to the GI-CO-FT module in Arabidopsis [31]. Hd1 activates and suppresses Hd3a expression by promoting
heading under the short day (SD) and long day (LD) conditions, respectively [32-33]. As F. macclureana rarely
blossoms on the QTP, which experiences a long photoperiod with a low ratio of red to far-red light, it may have
evolved specific reproductive strategies involving flowering-related pathways in response to photoperiodic cues
to ensure long vegetation growing period. It is probably that the weak light intensity with a low proportion of
blue light might activate Hd3a expression even in the LD conditions, thereby accelerating flowering. Notably,
reproduction pathways play an important role in the mechanisms of plant adaptation to extreme environments. Previous studies showed that the phytochrome and flowering time regulatory protein 1 (PFT1) from
Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the QTP, showed signs of
positive selection for adaptive divergence [34]. We presume that the function of Hd3a in promoting flowering is
likely to be conserved between bamboo and rice, because both of them belong to the Poaceae. Interestingly, FmFT and FmHd3a were regulated differently in NF- vs F- leaves, although they were both
annotated as FT regulators in the circadian rhythm-plant pathway. Basically, FT sub-family protein acts as
florigen; the diverse functions of the FT gene family in flowering regulation had been demonstrated in many
different plant species [29]. For example, sugar beet BvFT1 repressed flowering and the divergence within three
amino acids of an external loop in PEBP domain was demonstrated to be the major cause [35]. Similarly, a
single amino acid exchange in PEBP domain was sufficient to convert FT to TFL1, an activator and a repressor
[36-37]. In our study, five proteins carry invariant amino acids in positions where variation occurred; however,
there were many differences in predicted amino acid sequences between them in PEBP domain. Especially for
some positions, amino acids of AtFT and FmHd3a are the same, but different from that of FmFT (Fig. Discussion Page 7/27 Page 7/27 Carbohydrate metabolism and signal transport may be major factors in floral transition, organogenesis, and
death after flowering Bamboo exhibits excellent flexibility and fracture toughness, and so far, the presence of fibers within the
bamboo culm was thought responsible for these remarkable mechanical properties [38]. And the development
of plant fibers is accompanied by the of carbohydrate metabolism [39]. Perhaps this is the reason why many
DEUs were involved in carbohydrate metabolism pathway. Interestingly, our results indicated that starch and
sucrose metabolism was a major enriched KEGG pathway for the DEUs from several combination pairs,
including NF- vs F-leaves, branchlets/leaves vs I- & P- spikelets. Transcripts and metabolic signatures of maize
leaves have shown that the balance between transitory starch and sucrose is associated with the autonomous
floral transition [40]. And in Lilium, carbohydrates have been found to be transported from the vascular bundles
to floral organs during reproduction [41]. Yang et al., (2017) also reported that the deficiency in the resources in
male flowers reduced pollen viability in Tapiscia sinensis due to biased carbohydrate transport toward the
female flowers [42]. Therefore, we suspect there may be a correlation between DEUs related to starch and
sucrose metabolism and arrow bamboo floral organ development. In rice, excessive uridine 5´-diphosphoglucose-glucose (UDPG) can result in programmed cell death,
accumulation of reactive oxygen species and an increase in the caspase-like activity [43] and inactivate starch
synthase disrupted normal male reproduction by delaying programmed cell death in cotton [44], suggestive of a
correlation between starch and sucrose metabolism and death. Bamboo flowering, especially in masting
species, often causes plants to wilt and die after setting seed. It is possible that increased starch and sucrose
metabolism might trigger the excessive accumulation of reactive oxygen species and result in the altered
activity of key enzymes in important biological pathways. In the present study, unigenes related to the signal transduction pathways were significantly upregulated in the
tissues of flowering arrow bamboo plants. The transcriptomic profiles of Posidonia oceanica also showed a
strong metabolic activation of hormones in the heat stress-induced flowering plants [45]. Signal transduction-
related genes were also found to have undergone both significant positive selection and expansion events on
the adaptive evolution of Crucihimalaya himalaica [34] and cyanobacterium Trichormus sp. NMC-1 [46] on the
QTP. In the present study, unigenes related to the signal transduction pathways were significantly upregulated
in the tissues of flowering arrow bamboo plants. Activated Hd3a expression probably accelerates flowering in F. macclureana 6a). Possibly, FmFT acts as a repressor FT due to the conversion of certain amino acids in PEBP domain, while
FmHd3a acts as an inducer one. Thus, we suspect that the floral transition of F. macclureana is regulated by a
complex regulatory network in which at least two unique FT orthologs interact with the circadian clock
pathways. However, how these circadian clock pathways mediate the activation of FmHd3a and FmFT in
response to light signalling remains to be elucidated by future research. Notably, we detected FmHd3a expressions in all four tissues collected from the flowering plants, but not in NF-
leaves or NF-branchlets. We also noticed that all plants were sampled from the same provenance and grown in
chambers under the same conditions after transplantation, but all non-flowering plants each had a longer
section of rhizome than the flowering ones. Given that rhizome can provide nutrients for plants to maintain
normal growth, thus, we hypothesize that the broken balance between vegetative and reproductive growth after
transplantation result in the floral transition. Additionally, the DEUs upregulated in F-leaves relative to NF-leaves
were mainly enriched in KEGG pathways related to starch, sucrose, and galactose metabolism, and
corresponding down-regulated DEUs were mapped to the light and carbon fixation, plant circadian rhythm, and
photosynthesis pathways. Therefore, we speculate that bamboo FT orthologs might be regulated by upstream
regulators involved in carbohydrate metabolism. corresponding down-regulated DEUs were mapped to the light and carbon fixation, plant circadian rhythm, and
photosynthesis pathways. Therefore, we speculate that bamboo FT orthologs might be regulated by upstream
regulators involved in carbohydrate metabolism. Page 8/27 Carbohydrate metabolism and signal transport may be major factors in floral transition, organogenesis, and
death after flowering We suspect that this may be due to the long distance transport
of the FT proteins, which ensures floral promotion at the shoot apex [47], or the phytohormone signaling and
calcium signaling, which play diverse roles in the specification of flower organs during arrow bamboo
reproductive development [48-49]. Conclusions In the present study, we constructed a novel de novo transcriptome analysis for F. macclureana. Based on two
major KEGG pathways of carbohydrate metabolism and signal transduction that DEUs were enriched in, as well
as the different regulation of FmFT and FmHd3a in NF- vs F- leaves, we speculate that both environmental
signals and physiological status have effect on floral transition in F. macclureana. Significant expressions of
unigenes enriched in DNA repair and plant-pathogen interaction pathways may reflect the adaptation of F. macclureana to its high radiation and pathogen-specific environment on the QTP. We identified both similarities
and differences in adaptive mechanisms (e.g., disease-resistance and DNA repair pathways) and stress-
induced flowering mechanisms (e.g., carbohydrate metabolism and signal transduction pathways) among
plants that grow at high altitudes. Although further experimental verification is needed, our results provide
insight into the regulation of flowering time in highland bamboo as well as how this species adapts to harsh
and extreme environments. F. macclureana has presumably evolved an integrated mechanism to adapt to the harsh environment of the
QTP We detected the broad expressions of unigenes encoding putative Hsp proteins, such as heat shock protein 70
(Hsp70) and heat shock protein 90 (Hsp90). Both Hsp70 and Hsp90 are important for maintaining cellular
protein homeostasis under stress conditions and they function by activating other targets [50-52]. It is likely
that the sudden exposure to the higher temperature of the lab (i.e. outside the QTP) triggered the expressions of
these unigenes. Warmer temperatures can greatly reduce flowering synchrony among individuals from 72
woody and herbaceous species [53]. It was also reported that P. oceanica, a highly clonal and long-lived Page 9/27 Page 9/27 species, massively bloomed after a simulated heatwave [45]. Given the cold temperatures present at the high-
altitude regions of the QTP [26], it is reasonable to presume that F. macclureana has developed into a heat-
sensitive but not heat-tolerant bamboo species and flowering is probably a stress-induced response to the
higher temperature in the lab. DNA repair and disease-resistance pathways have been found to play a crucial role in the highland adaptation
of Tibetan highland plants [46, 54]. In the present study, we detected significant expressions of many unigenes
related to pathogen response that contained either a nucleotide binding (NB)-ARC domain or a leucine-rich
repeat (LRR) domain, which were present in most resistance (R) proteins [55-57]. We also identified many
differentially expressed unigenes that were significantly enriched in the DNA repair pathways. Since relatively
fewer species of pathogenic microorganisms and intense UV radiation exist on the QTP [58], it is reasonable to
presume that F. macclureana has evolved a relatively narrow range of pathogen specificity and specific DNA-
repair mechanisms. Sudden exposure to the lab environment, which contains a heavier load of pathogens and
weak light intensity, may have induced an innate defensive response of F. macclureana, and those that were
enriched in the plant-pathogen interaction and DNA repair response pathways may be important for F. macclureana to cope with the new environment present in the lab. Although further studies are needed to
investigate the molecular mechanisms responsible for the putative adaptive evolutionary changes, this study
provides insights into how plants adapt to harsh and extreme environments. Tissues collection The studied plant species is highland arrow bamboo (Fargesia macclureana), and it grows mainly as an
underbrush of coniferous forest or coniferous and broad-leaved mixed forest, and sometimes forms a pure
population in the QTP at an altitude of approximately 2,000 ~ 3800 m above sea level (Fig. 1). F. macclureana
was formally identified by Stapleton in 1993 [25] and detailed explanations are provided in the volume 22 of
Flora of China (http://foc.iplant.cn/) [59]. A voucher specimen of this material has been deposited in the
Bamboo Research Institute of Nanjing Forestry University. A total of six seedlings of F. macclureana were Page 10/27 Page 10/27 sampled from the wild with the permission of the local forestry department and collected from the Bayi District,
Linzhi City, Tibet, China (29˚46′ 0.95″ N, 94̊ 44′46.36″ E, altitude: ~2,200 m). They were dig out of the ground,
each with a section of rhizome, which can provide nutrients for plants. Then they were all transferred to
individual pots at the State Forestry Administration Key Open Laboratory at the International Center for Bamboo
and Rattan in Beijing (N: 39˚59′ 17.52″, E: 116̊ 28′46.06″, altitude ~34 m). During growth, the plants were
maintained at 28 ± 1 °C and 50-55% relative humidity under a 16/8 h (light/dark) photoperiod regimen with a
light intensity of 200 μmol · m-2 · s-1. All the seedlings were watered with a 1/3 B5 macronutrient nutrient
solution three times a week. After twenty days, four seedlings which had shorter rhizomes flowered, while the
other two didn't blossom until the time of sampling. One month later, we collected samples of six tissues for
further de novo sequencing, including inflorescences in the initial flower stage (I-spikelets), inflorescences at the
peak flower stage (P-spikelets), branchlets of the flowering plants (F-branchlets), leaves of the flowering plants
(F-leaves), branchlets of the non-flowering plants (NF-branchlets) and leaves of the non-flowering plants (NF-
leaves). We collected three independent replicates of each tissue type. RNA extraction, quantification, and qualification Total RNA was extracted from each of the six unique tissues mentioned above using a RNeasy plant RNA
extraction kit (Qiagen, Dusseldorf, Germany), and the extraction procedure was performed according to the
manufacturer’s instructions. RNA degradation and contamination were monitored using 1% agarose gels. RNA
purity was checked using a NanoPhotometer® spectrophotometer (Implen GmbH, Munich, Germany). RNA
concentration was measured using a Qubit® RNA Assay Kit and a Qubit®2.0 Fluorometer (Life Technologies,
CA, USA). RNA integrity was assessed using an RNA Nano 6000 Assay Kit run on an Agilent Bioanalyzer 2100
system (Agilent Technologies, CA, USA). Library preparation for transcriptome sequencing Library construction and RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). A total of 3 μg RNA per sample was used for RNA preparation. Briefly, mRNA was purified from total RNA using
poly-T oligo-attached magnetic beads, followed by fragmentation carried out using divalent cations at elevated
temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using
random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was
subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into
blunt ends via the exonuclease and polymerase activities. Next, the 3´ ends of the DNA fragments were
adenylated and ligated to the NEBNext adaptors with hairpin loop structures to prepare samples for
hybridization, this was to select cDNA fragments that are 150-200 bp in length. Library fragments were then
purified using an Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA), and 3 μl USER enzyme (New
England Biolabs, Ipswich, MA, USA) was added to the size-selected, adaptor-ligated cDNA at 37°C for 15 min
followed by five min at 95°C before PCR. PCR was performed using Phusion High-Fidelity DNA polymerase
(Thermo Fisher, Waltham, MA, USA), universal PCR primers, and the Index (X) Primer. Finally, the PCR products
were purified using the AMPure XP system and library quality was assessed on the Agilent Bioanalyzer 2100. Clustering and sequencing Clustering and sequencing Expression analysis of broadly and specifically expressed unigenes For all unigenes, those that were expressed in all six tissues were defined as broadly expressed unigenes
(BEUs). Similarly, unigenes that were specifically expressed in only one tissue were defined as specifically
expressed unigenes (SEUs). The identification of BEUs and SEUs was conducted by using tools on the
BMKCloud platform (http://www.biocloud.net). Clustering and sequencing Page 11/27 The clustering of index-coded samples was performed using a cBot Cluster Generation System and a TruSeq
PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), and all experimental procedures were performed
according to the manufacturer’s instructions. After that, library preparations were sequenced on an Illumina
Hiseq 2000 platform and paired-end reads were generated. The clustering of index-coded samples was performed using a cBot Cluster Generation System and a TruSeq
PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), and all experimental procedures were performed
according to the manufacturer’s instructions. After that, library preparations were sequenced on an Illumina
Hiseq 2000 platform and paired-end reads were generated. De novo transcriptome assembly Raw data in fastq format were first processed using in-house perl scripts. Clean data were obtained by
removing low-quality reads and reads that contain the adapters or poly-N sequences. Meanwhile, we checked
the quality of our unassembled read dataset by examining various measures including Q20, Q30, GC-content,
and sequence duplication. All the downstream analyses were performed using high-quality clean data. The transcriptome was assembled using clean reads from all libraries and samples. The assembly was
produced using Trinity [60] with min_kmer_cov set to 2 and all other parameters set to their respective default
values. Functional annotation of the transcriptome Gene function was annotated using the following databases: Nr (NCBI non-redundant protein sequences), Pfam
(Protein family), KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins), Swiss-Prot (a database of
manually annotated and reviewed protein sequences), KEGG (the Kyoto Encyclopedia of Genes and Genomes),
and the GO (Gene Ontology) database. Quantification of gene expression levels Gene expression levels were estimated using RSEM [61] for each sample: clean data were mapped back onto
the assembled transcriptome, followed by a read count for each gene. The expression levels of unigenes were
calculated and normalized using FPKM (fragments per kb per million fragments) [62]. Weighted gene co-expression network analysis (WGCNA) WGCNA was performed on all unigenes identified using the WCGNA R package. We calculated the adjacency
matrices and performed the topological overlap measures (TOMs), which show the degree of overlap in shared
neighbors between pairs of genes in the network to define gene clusters in our transcriptome dataset. 1 − TOM
was used as a dissimilarity measure for hierarchical clustering and module detection. Modules of the clustered
genes were then selected using the Dynamic Tree Cut algorithm as implemented by WGCNA. To identify
modules that are significantly related to particular tissues, expression profiles of each module were
summarized by a module eigengene (ME) and the correlations between the modules and corresponding tissues
were calculated. Page 12/27 Page 12/27 Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) p To understand the higher-level functions of the observed unigenes, we performed GO term annotation and
KEGG pathway enrichment analysis using BMKCloud (http://www.biocloud.net/; [64]). We used KOBAS 2.0 [65]
to test the statistical enrichment of differentially expressed genes in KEGG pathways. Pathways with P values <
0.05 were considered significantly enriched. Protein-protein interactions (PPIs) The DEU and SEU sequences were queried using BLASTX against the related species to predict PPIs that the
DEUs and SEUs may be involved in. This search procedure was capable of identifying PPIs that may be similar
to any others found in the STRING database (http://string-db.org/). These PPIs were then visualized using
Cytoscape [66]. Detection of SSRs Picard-tools version 1.41 and samtools version 0.1.18 were used to sort data, remove duplicated reads, and
merge the bam alignment results of each sample. SSRs were identified using MISA (https://webblast.ipk-
gatersleben.de/misa/). Expression analysis of differently expressed unigenes (DEUs) Before analysis, we conducted a principal component analysis (PCA) and removed one replicate that showed
an inconsistent expression pattern in the NF-branchlets and NF-leaves to ensure consistency in the expression
patterns of unigenes between replicates (Additional file 12: Figure S4). Expression analysis of the DEUs between pairs of tissues/groups was performed using the DESeq package
(1.10.1) in R. DESeq provides statistical routines for identifying differential expression in the digital gene
expression data using a model based on the negative binomial distribution. The resulting P values were
adjusted using the Benjamini-Hochberg method for controlling the false discovery rate [63]. Here, uni-transcripts
with an absolute value of log2 ratio ≥ 2, an FDR significance score < 0.01, and an adjusted P-value < 0.05 were
deemed to be differentially expressed. Funding This work was supported by the Special Funds for Fundamental Scientific Research on Professional Work
Supported by International Center for Bamboo and Rattan (No. 1632019008) and the Sub-Project of National
Science and Technology Support Plan of the Twelfth Five-Year in China (No. 2015BAD04B01). The funder is the
corresponding author of this manuscript and he played an important role in the study design, data collection
and analysis, decision to publish, or preparation of the manuscript. Consent for publication Not applicable. Abbreviations BEUs: Broadly expressed unigenes; BP: Biological process; CC: Cellular component; COG: Clusters of
orthologous groups; DEUs: Differentially expressed unigenes; F-branchlets: Branchlets of the flowering plants; F-
leaves: Leaves of the flowering plants; FPKM: Fragments per kb per million fragments; GO: Gene ontology; I-
spikelets: Inflorescences in the initial flower stage; KEGG: Kyoto Encyclopedia of Genes and Genomes; ME:
Module eigengene; MF: Molecular function; NF-branchlets: Branchlets of the non-flowering plants; NF-leaves:
Leaves of the non-flowering plants; Nr: NCBI non-redundant protein sequences; PCA: Principal component
analysis; Pfam: Protein family; PPIs: Protein-protein interactions. P-spikelets: Inflorescences at the peak flower
stage; QTP: Qinghai–Tibet Plateau; SEUs: Specifically expressed unigenes; SSRs: Simple sequence repeats;
TOMs: topological overlap measures. WGCNA: Weighted gene co-expression network analysis. Authors’ contributions Conceived and designed the experiments: ZM G. Performed the experiments: KB Y and JJ S. Analyzed the data:
CX Z, YL D and Y L. Interpreted the results and wrote the paper: Y L. All authors have read and approved the
final manuscript. Availability of data and materials The RNA sequencing dataset generated during the current study have been submitted to NCBI Sequence Read
Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra) with the accession number PRJNA544133. Validation of FmHd3a transcript levels by RT-qPCR To verify the expression pattern of the FmHd3a, we used RT-qPCR to assess the expressions of FmHd3a in six
distinct tissues. First-strand cDNA was synthesized from total RNA extracted by using a reverse transcription
system (Promega, Madison, WI, USA) following the manufacturer’s instructions. Each RT-qPCR amplification
was performed at least three times, and NTB and TIP41 were used as internal controls [67]. Primers for these
genes are listed in Additional file 13: Table S9. The relative expression levels of FmHd3a in different tissues
were calculated using the 2-ΔΔCT method [68]. The statistical significance of differences in the mean levels of
expression was tested using a one-way ANOVA. Significant differences in transcript abundance between
different tissues were then compared using Duncan’s multiple range tests as implemented by SPSS version
17.0 (IBM SPSS, Chicago, USA). We considered mean differences at P < 0.05 and P < 0.01 to be statistically
significant and highly statistically significant, respectively. Page 13/27 Page 13/27 Page 13/27 Page 13/27 Ethics approval and consent to participate Not applicable. Competing interests The authors declare that they have no competing interests. Declarations Ethics approval and consent to participate Acknowledgements Page 14/27 Page 14/27 Page 14/27 Not applicable. References 1. Putterill J, Varkonyi-Gasic E. FT and florigen long-distance flowering control in plants. Curr Opin Plant Biol. 2016;33:77-82. 2. Wigge PA, Kim MC, Jaeger KE, et al. Integration of spatial and temporal information during floral induction
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12. 57. Takken FL, Albrecht M, Tameling WI. Resistance proteins: molecular switches of plant defence. Table 1 Length range of transcripts and unigenes identified in the transcriptome of F.
macclureana. Table 1 Length range of transcripts and unigenes identified in the transcriptome of F. macclureana. Page 18/27 Page 18/27 Length Range
Transcripts
Unigenes
200-300
36,390 (12.59%)
25,291 (25.53%)
300-500
47,515 (16.43%)
21,257 (21.46%)
500-1,000
78,453 (27.13%)
23,806 (24.03%)
1,000-2,000
77,456 (26.79%)
16,752 (16.91%)
2,000+
49,308 (17.05%)
11,950 (12.06%)
Total number
289,122
99,056
Total length
341,956,623
91,685,618
N50 length
1,765
1,587
Mean length
1,182.74
925.59
Total number
289,122
99,056
Total length
341,956,623
91,685,618
N50 length
1,765
1,587
Mean length
1,182.74
925.59
Table 2 Statistics of annotation
analysis of unigenes Anno_Database
Annotated_Number
percentage
300<=length<1,000
length>=1,00
COG_Annotation
13,128
27.75
3,261
7,515
GO_Annotation
34,055
71.99
10,659
17,855
KEGG_Annotation
14,307
30.24
4,550
7,397
KOG_Annotation
23,492
49.66
6,863
12,779
Pfam_Annotation
28,317
59.86
7,823
16,896
Swissprot_Annotation
24,847
52.52
7,450
14,500
eggNOG_Annotation
43,909
92.82
14,040
21,568
Nr_Annotation
45,516
96.22
15,031
22,271
All_Annotated
47,306
100.00
15,602
22,437 Table 3. Differentially expressed unigenes (DEUs; Fold change > 2; FDR < 0.01) among tissues
of F. macclureana. DEUs_total: the total number of DEUs; DEUs_up (%): the number (and
percentage) of up-regulated DEUs; DEUs_down (%): the number (and percentage) of down-
regulated DEUs). Table 3. Differentially expressed unigenes (DEUs; Fold change > 2; FDR < 0.01) among tissues
of F. macclureana. DEUs_total: the total number of DEUs; DEUs_up (%): the number (and
percentage) of up-regulated DEUs; DEUs_down (%): the number (and percentage) of down-
regulated DEUs). Table 3. Differentially expressed unigenes (DEUs; Fold change > 2; FDR < 0.01) among tissues
of F. macclureana. DEUs_total: the total number of DEUs; DEUs_up (%): the number (and
percentage) of up-regulated DEUs; DEUs_down (%): the number (and percentage) of down-
regulated DEUs). Page 19/27 Number
Group
DEUs_total
DEUs_up (%)
DEUs_down (%)
1
I-spikelets vs P-spikelets
970
916 (94.43)
54 (5.57)
2
F-branchlets vs I-spikelets
4,970
3,046 (61.29)
1,924 (38.71)
3
F-branchlets vs P-spikelets
5,124
3,338 (65.14)
1,786 (34.86)
4
F-branchlets vs F-leaves
8,467
3,967 (46.85)
4,500 (53.15)
5
F-leaves vs I-spikelets
12,829
6,791 (52.93)
6,038 (47.07)
6
F-leaves vs P-spikelets
10,791
6,625 (61.39)
4,166 (38.61)
7
NF-branchlets vs I-spikelets
11,628
6,135 (52.76)
5,493 (47.24)
8
NF-branchlets vs P-spikelets
10,809
5,893 (54.52)
4,916 (45.48)
9
NF-branchlets vs F-branchlets
6,524
3,275 (50.20)
3,249 (49.80)
10
NF-branchlets vs F-leaves
11,670
5,902 (50.57)
5,768 (49.43)
11
NF-branchlets vs NF-leaves
3,853
1,946 (50.51)
1,907 (49.49)
12
NF-leaves vs I-spikelets
13,577
6,921 (50.98)
6,656 (49.02)
13
NF-leaves vs P-spikelets
11,718
6,130 (52.31)
5,588 (47.69)
14
NF-leaves vs F-branchlets
11,659
5,606 (48.08)
6,053 (51.92)
15
NF-leaves vs F-leaves
5,032
2,492 (49.52)
2,540 (50.48) Supplementary File Legends Additional file 1: Table S1. Statistic of sequencing and assembly data. Additional file 1: Table S1. Statistic of sequencing and assembly data. Additional file 2: Table S2. 47,306 unigenes were annotated and their predicted functions. Additional file 4: Table S4. The most enriched GO terms and KEGG pathways for the specially expressed
unigenes (SEUs) and broadly expressed unigenes (BEUs) in all tissues collected from flowering plants. Additional file 4: Table S4. The most enriched GO terms and KEGG pathways for the specially expressed
unigenes (SEUs) and broadly expressed unigenes (BEUs) in all tissues collected from flowering plants. Additional file 5: Figure S1. KEGG annotation of unigenes that were specifically expressed in P-spikelets (a), F-
branchlets (b) and F-leaves (c) of arrow bamboo flowering plants. The size of dots is proportional to the
number of unigenes. Additional file 6: Table S5. KEGG enrichment of differentially expressed unigenes (DEUs) between different
tissues. Additional file 7: Figure S2. Hub unigenes in regulatory networks of flowering identified based on analysis of
DEUs among tissues. Unigenes c109220.graph_c0 and c110963.graph_c4, showing differential expressions
between NF-leaves and F-leaves, are both bamboo orthologs of FLOWERING LOCUS T (FT), which was marked
with a red square; while unigenes down-regulated were marked with green squares. Page 20/27 Additional file 8: Table S6. Expressions of 10 unigenes that were differentially expressed between F-leaves vs
NF-leaves and mapping into the circadian phythm-plant KEGG pathway. Additional file 9: Figure S3. Weighted gene co-expression network analysis (WGCNA) of all unigenes identified
in the transcriptome of F. macclureana. (a) The phylogenetic tree diagram and the heat map related to the
traits. This diagram is divided into three parts: the cluster tree of gene system, the module color of
corresponding genes, and the correlation between genes related to each trait in tested samples and its module. The redder the color, the more positive the correlation; conversely, blue is negatively correlated. (b) Gene co-
expression network heatmaps drawn by randomly selected 1,500 genes, in which the left and the upper sides
are the symmetrical system clustering tree of gene network/module, and the lower right area indicates the
dissimilarity between genes, and the smaller the value is, the darker the color is. (c) Module and trait correlation
heat map showing the relationship between a module and a given trait. Additional file 11: Table S8. 9,296 SSRs identified from 7,668 unigenes. Additional file 11: Table S8. 9,296 SSRs identified from 7,668 unigenes Additional file 12: Figure S4. Principal component analysis (PCA) of unigenes expressions for 18 samples
collected from inflorescences in the initial and peak flower stage (I- and P- spikelets), branchlets and leaves of
flowering and non-flowering bamboo plants (F/NF-branchlets and F/NF-leaves). Additional file 13: Table S9. Primer pairs used for RT-qPCR. Seedlings of Fargesia macclureana flowered shortly after being transferred from the Qinghai–Tibet Plateau
(QTP) approximately 2,000 ~ 3,800 m above sea level to a low altitude lab. (a-b) Floret and spikelet of a
flowering seedling maintained at the low altitude lab outside the QTP. (c-d) The seedling and shoot of plants
growing on the QTP. (e) The original growing environment of F. macclureana. Supplementary File Legends The closer the correlation between a
shape and a module is to the absolute value of 1, it is likely that this trait is related to the module gene work. (d)
Systematic clustering tree of samples based on unigenes expressions. Additional file 10: Table S7. KEGG enrichment of unigenes in nine significant gene module relating to P-
spikelets. Figure 1 Seedlings of Fargesia macclureana flowered shortly after being transferred from the Qinghai–Tibet Plateau
(QTP) approximately 2,000 ~ 3,800 m above sea level to a low altitude lab. (a-b) Floret and spikelet of a
flowering seedling maintained at the low altitude lab outside the QTP. (c-d) The seedling and shoot of plants
growing on the QTP. (e) The original growing environment of F. macclureana. Page 21/27 Figure 2 Fi
2 Figure 2 Figure 2 Function annotation and classification of unigenes identified from the transcriptome of F. macclureana. (a) Nr
annotation. (b) Clusters of orthologous groups (COG) annotation. Out of 45,516 Nr hits, 13,128 unigenes had a
COG classification. A: RNA processing and modification B: Chromatin structure and dynamics C: Energy
production and conversion D: Cell cycle control, cell division, chromosome partitioning E: Amino acid transport
and metabolism F: Nucleotide transport and metabolism G: Carbohydrate transport and metabolism H:
Coenzyme transport and metabolism I: Lipid transport and metabolism J: Translation, ribosomal structure and
biogenesis K: Transcription L: Replication, recombination and repair M: Cell wall/membrane/envelope
biogenesis N: Cell mobility O: Posttranslational modification, protein turnover, chaperones P: Inorganic ion
transport and metabolism Q: Secondary metabolites biosynthesis, transport and metabolism R: General
function prediction only S: Function unknown T: Signal transduction mechanism U: Intracellular trafficking,
secretion, and vesicular transport V: Defense mechanisms W: Extracellular structures Y: Nuclear structure Z:
Cytoskeleton. (c) GO annotation. Results were summarized in three main categories: biological process, cellular Function annotation and classification of unigenes identified from the transcriptome of F. macclureana. (a) Nr
annotation. (b) Clusters of orthologous groups (COG) annotation. Out of 45,516 Nr hits, 13,128 unigenes had a
COG classification. A: RNA processing and modification B: Chromatin structure and dynamics C: Energy
production and conversion D: Cell cycle control, cell division, chromosome partitioning E: Amino acid transport
and metabolism F: Nucleotide transport and metabolism G: Carbohydrate transport and metabolism H:
Coenzyme transport and metabolism I: Lipid transport and metabolism J: Translation, ribosomal structure and
biogenesis K: Transcription L: Replication, recombination and repair M: Cell wall/membrane/envelope
biogenesis N: Cell mobility O: Posttranslational modification, protein turnover, chaperones P: Inorganic ion
transport and metabolism Q: Secondary metabolites biosynthesis, transport and metabolism R: General
function prediction only S: Function unknown T: Signal transduction mechanism U: Intracellular trafficking,
secretion, and vesicular transport V: Defense mechanisms W: Extracellular structures Y: Nuclear structure Z:
Cytoskeleton. (c) GO annotation. Results were summarized in three main categories: biological process, cellular Page 22/27 component and molecular function. The right and left y-axes indicated the number and percentage of unigenes
in a certain category, respectively. Figure 3
KEGG annotation of unigenes in the transcriptome of F. macclureana. The x-axis indicated the number of
unigenes in a certain category. The right y-axis showed the main clusters of KEGG pathways. Figure 3 KEGG annotation of unigenes in the transcriptome of F. macclureana. The x-axis indicated the number of
unigenes in a certain category. The right y-axis showed the main clusters of KEGG pathways. Page 23/27 Page 23/27 Figure 4 Unigenes that were specifically expressed in different tissues collected from flowering plants of F. macclureana. (a) Venn diagram of unigenes expressed in spikelets in the initial flower stage (I-spikelets, A) and the peak
flower stage (P-spikelets, B), branchlets (F-branchlets, C) and leaves (F-leaves, D) of flowering plants. (b) COG
annotation of unigenes that were specifically expressed in I-spikelets, P-spikelets, F-branchlets and F-leaves. (c)
GO enrichment of unigenes that were specifically expressed in I- & P- spikelets, F-branchlets and F-leaves. BP:
biological process; CC: cellular component; MF: molecular function. Page 24/27 Page 24/27 Figure 5
KEGG annotation of unigenes that were specifically expressed in P-spikelets (a), F-branchlets (b) and F-leaves
(c) of arrow bamboo flowering plants. The size of dots is proportional to the number of unigenes. Figure 5 KEGG annotation of unigenes that were specifically expressed in P-spikelets (a), F-branchlets (b) and F-leaves
(c) of arrow bamboo flowering plants. The size of dots is proportional to the number of unigenes. Page 25/27
Figure 6 Figure 6 Alignment (a) and phylogenetic analyses (b) of FT/TFL1 proteins from F. macclureana and other plants, as well
as the expression pattern of FmHd3a across six different tissues collected by RT-qPCR (c). The involving
sequences and their accession numbers are: three FT/TERMINAL FLOWER 1 (TFL1) proteins from A. thaliana:
AtFT (AT1G65480.1), AtTFL1 (NP_196004.1) and BROTHER OF FT AND TFL1 (AtBFT, AT5G62040.1); and four
FT/TFL1 proteins from Oryza sativa: OsFT (XP_015619436.1), OsRFT1 (rice RICE FLOWERING LOCUS T 1
(RFT1), BAO03220.1), OsHd3a (XP_015611892.1) and two RCNs (putative TFL1/CENTRORADIALIS (CEN)
orthologs): OsRCN1 (AAD42895.1) and OsRCN3 (AAD42896.1). Amino acid sequences with double underline
and single underline indicate the critical motifs in PEBP proteins [27]; asterisk and triangles indicate the
invariant histidine in the PEBP domain [28] and two conserved sites in inducer FTs [29], respectively. Hollow
circles indicate the position where the critical amino acid variation occurred between Arabidopsis TFL1 and FT,
an activator and a repressor of flowering [36]. Relative expression levels were calculated using the 2−ΔΔCT
method to reflect expressions relatively more veritably. The statistical significance was tested by one-way
ANOVA, considering P < 0.05 and P < 0.01 as statistically significant and extremely significant, respectively. Significant differences in transcript abundance between different tissues were then compared by Duncan’s
multiple range test using SPSS 17.0 (SPSS Inc., Chicago, USA). Figure 7
Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats,
respectively. Figure 7 Figure 7 Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats,
respectively. Densities of different SSR types. c and p1-6 represent mono-, di-, tri-, tetrad-, penta- and hexa-nucleotide repeats,
respectively. Page 26/27 Page 26/27 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. TableS5.xlsx
TableS6.xlsx
FigureS6.docx
FigureS1.tif
FigureS2.tif
FigureS4.tif
TableS4.xlsx
TableS3.xlsx
TableS7.xlsx
FigureS3.tif
TableS2.xlsx
TableS1.docx
TableS8.xlsx
FigureS5.docx
TableS9.xlsx TableS5.xlsx TableS6.xlsx FigureS6.docx FigureS5.docx TableS9.xlsx Page 27/27
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From Connectivity to Advanced Internet Services: A Comprehensive Review of Small Satellites Communications and Networks
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Wireless communications and mobile computing
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Hindawi
Wireless Communications and Mobile Computing
Volume 2019, Article ID 6243505, 17 pages
https://doi.org/10.1155/2019/6243505 Hindawi
Wireless Communications and Mobile Computing
Volume 2019, Article ID 6243505, 17 pages
https://doi.org/10.1155/2019/6243505 Scott C. Burleigh,1 Tomaso De Cola
,2 Simone Morosi
,3 Sara Jayousi,3
Ernestina Cianca
,4 and Christian Fuchs
2 Ernestina Cianca
, and Christian Fuchs
1Jet Propulsion Laboratory, California Institute of Technology, USA
2German Aerospace Center (DLR), Institute of Communications and Navigation, 82234 Oberpfaffenhofen, Germany
3Information Engineering Department, University of Florence, 50139 Florence, Italy
4Department of Electronic Engineering, University of Rome Tor Vergata, Italy 1Jet Propulsion Laboratory, California Institute of Technology, USA
2German Aerospace Center (DLR), Institute of Communications and Navigation, 82234 Oberpfaffenhofen, Germa
3Information Engineering Department, University of Florence, 50139 Florence, Italy
4Department of Electronic Engineering, University of Rome Tor Vergata, Italy Correspondence should be addressed to Simone Morosi; simone.morosi@unifi.it Received 27 December 2018; Accepted 18 March 2019; Published 2 May 2019 Academic Editor: Pham Tien Dat Academic Editor: Pham Tien Dat Copyright © 2019 Scott C. Burleigh et al. Thisis an openaccessarticledistributed under the CreativeCommons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recently, the availability of innovative and affordable COTS (Commercial Off-The-Shelf) technological solutions and the ever-
improving results of microelectronics and microsystems technologies have enabled the design of ever smaller yet ever more
powerful satellites. The emergence of very capable small satellites heralds an era of new opportunities in the commercial space
market. Initially applied only to scientific missions, Earth observation and remote sensing, small satellites are now being deployed
to support telecommunications services. This review paper examines the operational features of small satellites that contribute to
their success. An overview of recent advances and development trends in the field of small satellites is provided, with a special focus
on telecommunication aspects such as the use of higher frequency bands, optical communications, new protocols, and advanced
architectures. Review Article
From Connectivity to Advanced Internet Services: A
Comprehensive Review of Small Satellites Communications
and Networks Scott C. Burleigh,1 Tomaso De Cola
,2 Simone Morosi
,3 Sara Jayousi,3
Ernestina Cianca
,4 and Christian Fuchs
2
1Jet Propulsion Laboratory, California Institute of Technology, USA
2German Aerospace Center (DLR), Institute of Communications and Navigation, 82234 Oberpfaffenhofen, Germany
3Information Engineering Department, University of Florence, 50139 Florence, Italy
4Department of Electronic Engineering, University of Rome Tor Vergata, Italy 1. Introduction Figure 1: INTELSAT, EUTELSAT, and INMARSAT annual revenues (left). Annual sales of VSAT terminals by LSAT, EUTELSAT, and INMARSAT annual revenues (left). Annual sales of VSAT terminals by type (right) [7]. a matter of fact, in the last of few decades, amazing and
unexpected progresses and changes have been obtained in
the diverse fields of broadcasting, mobile communications,
Earth observation and remote sensing, interplanetary explo-
ration, transport, and remote monitoring, so encompassing
commercial, civil, and military applications [7]. However, it
is worth stressing that starting from the postwar times to the
today scenarios, satellite systems have undergone themselves
a radical and systemic evolution which proves the fact that
their abilities perfectly adapt to the ever-changing needs of
both the society and the market; particularly, while in the first
decades, governments and national agencies were the main
players in the start of space race, in the design of satellite
missions, and in the development of satellite-based systems,
more recently private companies have largely increased their
role in this strategic industry [8].h the telecommunication aspects such as the use of higher-
frequency bands and optical communications, protocols, and
architectures. It is worth outlining that some surveys about small
satellites have been published recently [3–6]: whereas [5]
focuses on the evolution of the antennas for small sats, [6]
concentrates on intersatellite link and related communica-
tion protocols for small sat constellations. On the other
hand, [4] reviews the history of small satellite development
and summarizes its capabilities and applications. A rather
comprehensive review is provided in [3], which deals with
many aspects, from hardware components and structures, to
network topology and communication protocols; moreover,
[3] focuses on Cubesat class of small sats. With respect to pre-
vious surveys, this paper provides a more extensive overview
on telecommunication aspects and aims at describing this
rapid evolving field, giving more insights into new protocols,
architectures, and technology developments.h This trend has also been enforced by the privatization
of the main international satellite organizations which has
taken place at the end of the last century and produced
high revenues as shown by Figure 1 [7]. As far as the
VSAT and broadband satellite systems are concerned, the
same trends of deregulation and stimulation of the market
forces have been experienced from their launch to the
final successful spreading as reported by the graphs in
Figure 1 [7]. 1. Introduction gy
p
The paper is organized as follows: in Section 2, a brief
history of the evolution of the small satellites is provided,
trying to unveil the commercial reasons of their success. An overview of the services and applications which are
enabled by the small satellite is given in Section 3. Sec-
tion 4 is devoted to a description of the evolution of the
payloads, focusing on the used frequencies and the Soft-
ware Defined Radio (SDR) concept. New telecommunication
architectures and the suitable protocols for small satellites
based systems are described in Sections 5 and 6, respec-
tively. Finally, the perspectives and the open challenges are
discussed in Section 7 and the conclusions are drawn in
Section 8. On the other hand, the end of the twentieth century has
also seen the birth and the first steps of a new paradigm that
is based on the exploitation of the so-called small satellites,
whose size and weight are much smaller than the huge
geostationary orbit (GEO) or the big medium Earth orbit
(MEO) and low Earth orbit (LEO) ones. These new systems
are identified as micro-, nano-, and picosatellites according
to their dimensions [3]. The early missions of small satellites
were mainly organized and performed by research groups
of Universities and Research Organizations with the goals
of enabling a technology demonstration or an application
validation [9]. 1. Introduction technological achievements with hundreds of small satellites
being launched in the last few years and even more envisaged
to be commissioned in the near future. In the short span of the first two decades of the new
millennium, a revolution has taken place in the field of
satellite systems: the availability of innovative and affordable
COTS technological solutions and the ever-improving results
that are produced by microelectronics and microsystems
technologies have paved the way to a process of size reduction
for the satellite components and to the design of smaller and
smaller satellites that have been defined as small satellites
(whose weight is less or equal to 1000 kg), microsatellites
(from 10 to 100 kg), nanosatellites (1 −10 kg), and picosatel-
lites (0.1 −0.99 kg) [1].h So far, the main drivers of small satellites develop-
ments have been Earth observation and remote sensing,
as they may greatly contribute to filling the gap of data
poverty in many industry verticals (e.g., agriculture, disas-
ter management, forestry, and wildlife). Nevertheless, new
investments in developing mega-constellations (hundreds)
of pico/nanosatellites [1] for providing global communica-
tions, the increased role of satellites in Machine-to-Machine
(M2M) communications [2], and the interest in taking
advantage of one of the main possibilities enabled by small
satellites, which is the development of distributed systems
with interconnected satellites, are moving the attention also
towards the telecommunication aspects. Therefore, this paper
provides an overview of recent advances and development
trends in the field of small satellites, with a special focus on These technological trends have allowed new oppor-
tunities in the space market and the implementation of
long-awaited projects that have been postponed or sup-
pressed for years due to high inherent costs. More impor-
tantly, a new space rush has been originated by these Wireless Communications and Mobile Computing 2 2 4500
4000
3500
3000
2500
2000
1500
1000
500
0
Revenue ($million)
1964
1966
1968
1970
1972
1974
1976
1978
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
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2008
Year
INMARSAT
EUTELSAT
INTELSAT
900,000
800,000
700,000
600,000
500,000
400,000
300,000
200,000
100,000
0
1985
1986
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1989
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1995
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2006
2007
2008
Consumer Internet
SME Broadband
enterprise
Figure 1: INTELSAT, EUTELSAT, and INMARSAT annual revenues (left). Annual sales of VSAT terminals by type (right) [7]. Wireless Communications and Mobile Computing Wireless Communications and Mobile Computing Specific prices into SSO for two launcher categories
Source : Euroconsult’s 2016 Prospects for the Small Satellite Market
k$/kg
80
70
60
50
40
30
20
10
0 Falcon 9
Soyuz Dnepr Rockot
PSLV Vega
Firefly
Rocket Lab
Virgin
Dedicated smallsat launches
Shared smallsat launches
Figure 2: Specific prices for two launcher categories [10]. Specific prices into SSO for two launcher categories
Source : Euroconsult’s 2016 Prospects for the Small Satellite Market
k$/kg
80
70
60
50
40
30
20
10
0 Falcon 9
Soyuz Dnepr Rockot
PSLV Vega
Firefly
Rocket Lab
Virgin
Dedicated smallsat launches
Shared smallsat launches
Figure 2: Specific prices for two launcher categories [10]. Moreover, three constellations for satcom and Earth
observation accounted for 38% of the total and this share
should grow up to 68% in the next 5 years driven by several
large projects. As a result, this analysis unveils the main
booster of the massive development of the small satellites: the
ability to relatively easily build a constellation. This peculiar
aspect will be considered in the following sections with a
specific focus on the intersatellite communications. pi
Finally, it is worth introducing the main player of the
massive increase of the number of small satellites: the Cube-
Sat. The CubeSat has been designed as the goal of a Stanford
University program which was started in 1999 to obtain a very
low-cost/weight satellite which could be quickly developed
and used for educational purposes [3]. Together with the
California Polytechnic State University, Stanford University
developed CubeSat specifications with the goal to obtain a
customizable satellite, but with standard shape and weight,
in order to simplify launch and deployment operations. As
it is known, a CubeSat is made by one (1U) or more (nU)
10 cm X 10 cm X 10 cm units, with a mass of up to 1.33 kg per
unit [11]. The nature of CubeSats has enabled the standardised
production of subsystems that can even be purchased as a
COTS product from online shops, so keeping the mission cost
very low [9].h Figure 2: Specific prices for two launcher categories [10]. Wireless Communications and Mobile Computing Smallsat <500kg demand between 2010 2020
OneWeb
Cubesat constellations
Single cubesat
Single satellite mission >10 kg
Large EO
constellation >20#
Other constellation
Source : Euroconsult’s 2016 Prospects
for the Small Satellite Market
400
350
300
250
200
150
100
50
0
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
Figure 3: Number of small satellites in the last 15 years. [10]. Smallsat <500kg demand between 2010 2020 These peculiar features of the CubeSat solution have been
very important in the fast increase of the small satellite
missions and in the huge development of companies whose
main core is in the new space market, such as Terra Bella
(formerly Skybox Imaging), Spire, Planet Labs, and OneWeb,
who are developing mega-constellations of small spacecrafts
in LEO orbit [9]. 3. Overview of Services and Applications Around the year 2000, the SmallSats were able to properly
exploit innovative COTS technological solutions (hardware
and software), achieving the ability to compete effectively and
to make profit. The successful growth of the modern Small-
Sats services, encompassing a large variety of application
contexts, shall be analysed also based on a new management
approach the small satellite organizations started to adopt: the
agile methodology. This paradigm comes from the IT indus-
try and it is based on a highly iterative design technique: well-
defined objectives, missions and requirements, incremental
changes to the design for a continuous improvement of the
system performance, short timescale, and reduced cost. Agile
approaches and the exploitation of the latest off-the-shelf
technologies represent the two main drivers of the New Space
Age [4]. More interestingly, these early attempts have paved the
way to a new philosophy which has been aimed at implement-
ing and exploiting very-low-cost satellites [10]. As reported
in [3], the estimated production and launch cost for a single
small satellite can be approximately assumed to span from
100, 000 to 200, 000 USD: particularly, in the case of shared
small satellite, the launch unitary costs per 𝑘𝑔can lower
down to few kEuros, as sketched in Figure 2. As a result, these unprecedented features of small satellites
have been favourably considered by market forces which have
been largely stimulated in the last thirty years and pushed
to start a new gold rush in space, with original objectives
and well-targeted applications such as Earth observation
and communications, in the civil, military, and commercial
dominions. Since the possible applications will be reviewed
in the following section, it is now important to provide some
rough numbers that can give the idea of how strong the new
race to the space exploitation is. In the following, a brief overview of the main applications
and services of the SmallSats is provided. Since some of
the following acronyms may be unknown to the reader, a
comprehensive list is provided in Table 1. Overall, 551 satellites (<
400𝑘𝑔) have been launched
between 2010 and 2015, while the expectations for the succes-
sive five years are targeted on other 1380 launches [10]: these
trends are shown in Figure 3. These numbers are confirmed
by the 300 launches of nano/microsatellites which have been
recorded in 2017 [1]. (1) Earth Observation and Remote Sensing. 2. A Brief History of Small Sats Evolution From the dawn of the space era to their latest developments,
satellite communications have been one of the most reli-
able indicators of the technical and societal evolution: as 3 3. Overview of Services and Applications AcronymDefinition
Acronym
Definition
ACM
Adaptive Coding and modulation
ADC
Analog to Digital Converter
BP
Bundle Protocol
CCSDS
Consultative Committee for Space Data Systems
COTS
Commercial Off the Shelf
DLR
German Aerospace Center
DSA
Dynamic Spectrum Access
DSP
Digital Signal Processor
DTN
Delay Tolerant Network
DVB-S2
Digital Video Broadcasting - Satellite 2nd generation
EDRS
European Data Relay System
ELaNa
Educational Launch of Nanosatellites
EO
Earth observation
ESA
European Space Agency
FEC
Forward Error Correction
FPGA
Field Programmable Gate Array
FSK
Frequency Shift Keying
GEO
Geostationary Orbit
GPP
General Purpose Processor
GRACE
Gravity Recovery and Climate Experiment
GSTP
General Support Technology Programme HTS
High Throughput Satellite
HTTP
Hypertext Transfer Protocol
HW
Hardware
ICN
Information-centric networking
IOT
Internet of things
IP
Internet Protocol
ISS
International Space Station
IT
Information technology
JPL
Jet Propulsion Lab
LDPC
Low Density Parity Check
LEO
low Earth Orbit
LTP
Licklider Transmission Protocol
LUCE
LUnar Cubesats for Exploration
LUMIO Lunar Meteoroid Impacts Observer
MAC
Mean Access Control
MarCO Mars Cube One
MEC
Multi-Access Edge Computing
MEO
medium Earth Orbit
MMIC
Monolithic Microwave Integrated Circuit
M2M
Machine-to-Machine
NASA
National Aeronautics and Space Administration
NC
Network Coding
NEA Scout Near-Earth Asteroid Scout
NFV
Network function Virtualization
OPALS
Optical Payload for Lasercomm Science
OSIRIS Optical Space Infrared Downlink
PICASSO
Pico-Satellite for Atmospheric and Space Science Observations
PRETTYPassive REflecTomeTrY
QARMAN QubeSat for Aerothermodynamic Research and Measurements on Ablation
QKD
Quantum Key Distribution (QKD)
QoS
Quality of Service
QoE
Quality of Experience
SCAN
Space Communications and Navigation
SDN
Software Defined Networking
SDLS
Space Data Link Security
SDR
Software Defined Radio
SOTA
Small Optical Transponder
SW
Software
TBIRD
Terabyte Infrared Delivery
TCP
Transmission Control Protocol
TTL
Time-To-Live
UHF
Ultra High Frequency
USD
US Dollar
USLP
Unified Space Link Protocol
VHF
Very High Frequency
VLC
Visible Light Communications
VMMO
Volatile and Mineralogy Mapping Orbiter ESA CubeSat missions have been funded under the In-
Orbit Demonstration part of the General Support Tech-
nology Programme (GSTP): GOMX-3 and GOMX-4B for
demonstrating new capabilities of nanosatellites, QARMAN
(QubeSat for Aerothermodynamic Research and Measure-
ments on Ablation) for demonstrating re-entry technologies,
PICASSO (Pico-Satellite for Atmospheric and Space Science
Observations) for the analysis of the ozone distribution in the
stratosphere, the temperature profile up to the mesosphere
and the electronic plasma characterization in the ionosphere,
RadCube for real-time monitoring of the cosmic radiation
and space weather environment, and PRETTY (Passive
REflecTomeTrY), a nanosatellite to measure and register ice
on the glaciers or on the poles and wave movements of the
oceans. 3. Overview of Services and Applications So far, the primary
use for nano/microsatellites has been Earth observation (EO)
and remote sensing. The implementation of large satellite
constellations allows performing many simultaneous and
distributed measurements or observations (Earth resources Wireless Communications and Mobile Computing 4 Table 1: List of the acronyms. 3. Overview of Services and Applications monitoring, weather monitoring, and disaster monitoring)
with an increased temporal resolution of collected data (i.e.,
shorter revisit times) [12]. A more extensive use of small satellites for EO and remote
sensing calls for higher and higher data rate links to download
the acquired information in a short time. (2) Science and Technology Demonstration Missions. Micro-
and nanosatellites enable a wider access to space and rep-
resent an affordable test for young engineers and scientists
to prove prototype systems and experience the idea of
a future satellite. To this aim, NASA created the NASA
Educational Launch of Nanosatellites (ELaNa), an initia-
tive oriented to students of several disciplines (science,
technology, engineering, and mathematics). A number of Wireless Communications and Mobile Computing 5 58%
26%
12%
4%
50%
16%
10%
22%
3%
Historical
(2013 – 2017)
Communications
EO/RS
Scientific
Future
(2018 – 2022)
Technology
Novel Applications
EO/RS
Scientific
Technology
Communications
Figure 4: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Figure 4: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Fleet Space, and Myriota are some of the main commu-
nications operators offering IOT/M2M and data relaying
services. (3) Interplanetary Exploration Missions. Small Satellite Plat-
forms have led to a new era of space exploration espe-
cially thanks to new enabling technologies and new highly
capable launch vehicles, which open many opportunities for
future lunar and planetary exploration. National Aeronautics
and Space Administration (NASA) and European Space
Agency (ESA) have adopted the Interplanetary CubeSat
Model, supporting missions and studies, ranging from Mars
and Lunar observation to the study of meteoroids and
asteroids. Some of them are MarCO (Mars Cube One)
[13], NEA Scout (Near-Earth Asteroid Scout) [14], LUCE
(LUnar Cubesats for Exploration), LUMIO (Lunar Mete-
oroid Impacts Observer), VMMO (Volatile and Mineralogy
Mapping Orbiter), Lunar Flashlight [15], and Arkyd series
[16]. An overview of the nano/microsatellite trends by appli-
cation in the near term is provided in Figure 4 [1]. Although the analysis highlights that the primary use
for nano/microsatellites will remain Earth observation and
remote sensing, an increase of communications constella-
tions is expected. SpaceWorks estimates that about 700 com-
munications nano/microsatellites will require launch over the
next 5 years. (5) Commercial, Civil, and Military Applications. 3. Overview of Services and Applications Transport,
smart environments (including remote monitoring), quality
of life, safety, and security represent the main application con-
texts of the adoption of small, micro-, and nanosatellites [17]. As examples of commercial constellation of nanosatellites,
Aerial & Maritime and Sky & Space Global are two GomSpace
commercial missions: the former is oriented to aircraft and
vessel tracking for situational awareness, while the latter
will provide a global communication infrastructure in space. Moreover, Astrocast has a project for offering global M2M
services as remote monitoring, geolocalization, intelligent
data collection, and predictive maintenance [18]. (4) Communications Services. Small micro- and nanosatellites
organized in constellations can be used for providing data
distribution (broadcasting applications) and data exchange
(Internet of things and Machine-to-Machine paradigm) and
also for extending the Internet access to the entire Earth [11]. According to Space Works Market outlook, in the next years,
communication constellations of micro- and nanosatellites,
which are now in the technology demonstration phase,
will be used to serve and support the rapidly growing
Internet of things (IOT) and Machine-to-Machine (M2M)
market. Sky & Space Global, Kepler Communications, Hiber
(Magnitude Space), Helios Wire, Astrocast, Blink Astro, Figure 5 shows the SpaceWorks analysis on the Nano/
Microsatellite Operator Trends: Military Operators (aiming
to support national defense activities), Commercial Opera-
tors (whose purpose is profit revenue generating activities), 6 Wireless Communications and Mobile Computing 0%
25%
50%
75%
100%
Historical (2013 - 2017)
Future (2018 - 2022)
Percentage of Satellites
Civil
Military
Commercial
Commercial operators
are expected to
encompass over 70%
of nano/microsatellites
launched in the next 5
years
Despite rapid
commercial market
share growth, civil and
military operator
demand is expected to
remain consistent over
the next 5 years
Figure 5: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Figure 5: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWorks Enterprises, Inc. (SEI), 2018. Figure 5: Nano/microsatellite market forecast, 8th edition, approved for public release, SpaceWo and Civil Operators (nonmilitary or non-profit activities) are
considered than S-band, the efficiency of solid-state high-power ampli-
fiers drops from 80% (at UHF) to 30%. At such frequencies,
most common antennas are still wire antennas or planar
antennas, such as patch and slot antennas. Patch antennas
have gained special attention for CubeSats, as they are
relatively easy to fabricate. A variety of patch antenna designs
have been investigated at S bands. 3. Overview of Services and Applications Downlinks on S-Band
would be expected to be able to implement data rates from
100 kbps to 1 Mbps. Larger data rates require the use of higher-
frequency bands such as Ku-, K-, and Ka-band, which are the
state of the art for large spacecrafts, but they are still young
technologies in the small satellite world. A Ka-band trans-
mitter on a CubeSat began orbital operations in 2015 [21]. At
such higher frequencies, it is possible to implement also high-
gain reflector antennas which can meet the strict size and
weight requirements of a small satellite. Reflectarray antennas
are also very suitable, as they provide high gain and can be
easily integrated with the CubeSat structure. Their structure
consists of flat panels, which can be folded and stowed on
the CubeSat [5]. In [22], the development of a reflectarray for
the Mars Cube One (MarCO) is described. MarCO is the first
CubeSat mission designed for Mars operation. The frequency
of operation in this case was 8.425 GHz, with a measured gain
of 29.2dB. 4. Payload Evolution In the early implementation of small satellites, mainly used
as a platform for university and technology development
projects [19], the payload was supposed to perform very
simple operations such as transmission of a beacon, storing
data or transmitting data collected by simple sensors at very
low data rate (1 to 9.6kbps). Amateur frequencies at UHF were
mainly used and operated via the standard AX.25 [20]. At
such low frequencies, wire antennas (dipoles, monopoles, and
helical) are especially common as the wavelength is long and
achieving good radiation efficiency within a small volume is
challenging. A considerable number of the CubeSats that are
currently in space use wire antennas for their simplicity of
implementation. Moreover, the omnidirectionality of dipoles
makes them viable candidates for intersatellite communi-
cations. Emerging applications and the associate need for
transmitting at higher data rate or performing more complex
tasks, keeping low mass and weight, have raised the need for
larger bandwidths and higher-frequency bands, an increasing
request of digital implementation and SW control. As a matter of fact, small satellites also represent a viable
and cost-effective way to test new frequency bands for satellite
communications (both in terms of HW components and
propagation channel), such as W-band [23]. The investigation
of such high-frequency bands is mainly motivated by the
need of bandwidths in High Throughput Satellites [24]. On
the other hand, those frequencies could be an interesting
option for intersatellite links of small sats [25]. At Q/V and
W-band, horn antennas can be a viable option for small sats
as they provide good gain and could be fabricated also for uni-
versity experiments [26]. A potential horn design that could 4.1. Evolution in the Frequency Bands and Antennas. In
recent years, the use of higher frequencies than the common
VHF/UHF bands such as S-Band (mainly for telemetry) and
X-Band (for data transmission) has become more widely
available thanks to the advent of commercially available
Monolithic Microwave Integrated Circuits (MMICs). The
shift towards higher-frequency bands implies other require-
ments on the spacecraft design, mainly on the power system
and the antennas. For instance, already at frequencies higher 7 Wireless Communications and Mobile Computing Figure 6: Left: CAD-model of OSIRIS4Cubesat terminal. Right: Artist’s impression of terminal integration in 1U Cubesat [35]. CAD-model of OSIRIS4Cubesat terminal. Right: Artist’s impression of terminal integration in 1U Cubesat [35]. 4. Payload Evolution be considered for future Ka-band CubeSat communication is
discussed in [27, 28].h the order of 300 g, consumes 8 W of electrical power, and
requires only 0.3U of space within the CubeSat. It reaches a
data rate of 100 Mbps. OSIRIS4CubeSat will be offered on the
market by DLR’s commercialization partner Tesat Spacecom
under the market name CubeL. The need of higher data rates, low cost, and small size
has also moved the attention towards FSO communications,
especially for intersatellite links as presented in the next
section. An important challenge in optical satellite-to-ground
communications is the limited availability due to clouds. This
can be overcome by employing a world-wide network of
optical ground stations. By using a sufficient buffer memory
onboard the satellites, this enables overcoming the issues
due to limited availability of the space-to-ground link [36]. Although most optical ground stations available to date have
been developed mainly for research purposes, both new and
established ground segment operators have expressed strong
interest in building up the required infrastructure. Thus, it
is only a matter of time until optical links can be used in an
operational manner, even in small-satellite applications. 4.2. Laser Communication Terminals for Small Satellites and
CubeSats. In recent years, free-space optical communica-
tions have become a mature alternative to traditional RF com-
munication systems. With the use of laser communication
terminals in systems like the European Data Relay System
(EDRS) [29] for intersatellite links, the technology has passed
the barrier from research to the operational application. Concerning downlinks from satellite to Earth, a number of
demonstrations have been performed in recent years, like
the Small Optical Transponder (SOTA) experiment [30] of
the National Institute of Information and Communications
Technology, which used a dedicated satellite, or the Optical
Payload for Lasercomm Science (OPALS) experiment [31] of
the NASA’s Jet Propulsion Lab (NASA-JPL), which demon-
strated optical downlinks from the International Space Sta-
tion (ISS). The Aerospace Corporation demonstrated an
optical downlink from a 1.5U-CubeSat [32]. Even optical links
from the Moon to Earth have been demonstrated [33]. 4.3. Towards SDR Payloads. Since the early development
of small satellites, one trend in the payload design can
be identified: privileging the use of low-cost COTS and
in general HW components and moving towards a digi-
tal implementation. Wireless Communications and Mobile Computing 8 to Digital Converters (ADCs), General Purpose Processors
(GPPs), Digital Signal Processors (DSPs), and FPGAs. SDR
payloads are considered as a needed technological step in
traditional satellite systems for assuring a longer lifetime and
a more efficient resource utilization [37], even if so far, few
SDR payloads have flown on big satellites. For small satellites,
which are designed with few years of lifetime in mind, the
reason for moving towards SDR payloads is mainly related to
the offered flexibility to adapt to new science opportunities
and potentially reducing development cost and risk through
reuse of common space platforms to meet specific mission
requirements. SDR can be used to support multiple signals,
increase data rates over reliable intersatellite and ground links
to Earth, and also help in facing the shortage of available
frequencies for communications in the more crowded bands. As a matter of fact, the use of an SDR approach also allows
implementing Dynamic Spectrum Access (DSA) techniques
and hence a more efficient spectrum utilization. To date, no
satellite application of DSA is in use, although companies
such as Tethers Unlimited (U.S.), with funding from NASA,
are looking at upgrading SDR platforms with advanced
cognitive radio.h (i) They are increasingly used to form application-
focused segments of the infrastructure supporting
existing communication architectures, notably the
Internet. (ii) They also form and/or utilize altogether new, distinct
communication architectures. (ii) They also form and/or utilize altogether new, distinct
communication architectures. 5.1. As Supporting Infrastructure. The use of Earth-orbiting
satellites to conduct Internet traffic is of course not new. From TELSTAR in 1962 through Iridium, Globalstar, ViaSat,
and EchoStar, the market for relaying data via radio links to
satellites has grown rapidly. But historically, those satellites
have been large and expensive, whether operating in LEO
or GEO orbits. What is new is the use of large numbers of
small satellites for this purpose. The field has grown rapidly
in recent years as new concepts are proposed; many of them
highly ambitious: (i) The OneWeb constellation is initially expected to
comprise 882 small Internet service delivery satellites
in LEO orbit, potentially growing to 2620 satellites
[42]. The challenge of this more digital approach is related
to one of the strong limitations of a small satellite: power
consumption. Wireless Communications and Mobile Computing For this reason, FPGA has been preferred so
far, especially for higher data rates in the X- and Ka-band,
as they allow performing compute intensive tasks in parallel
and use more efficiently every clock cycle [38]. Additionally,
modern FPGAs have embedded processing systems, such
as ARM cores, integrated inside the FPGA. Few SDRs
have already flown in small satellites and other are under
development, e.g., AstroSDR, NanoDock SDR, GAMALINK,
and STI-PRX-01. It is definitely a hot topic of research and
development, and there is growing interest in developing and
testing new solutions. Pinto et al. [39] exploited SDR in small
satellite systems to design an intersatellite communication
model which could be easily reconfigured to support any
encoding/decoding, modulation, and other signal processing
schemes. In [40], a novel SDR architecture on an embedded
system is proposed, whose potential applications are the
ground station for multisatellite communications, deployable
mobile ground station network, and can be further extended
to distributed satellite system. (ii) Samsung has proposed a 4600-satellite constellation,
projected to be able to carry one billion terabytes of
Internet data per month [43]. (iii) The SpaceX corporation’s Starlink constellation is
envisioned to comprise up to 12,000 small satellites in
LEO orbit, with the capacity to carry up to 10% of local
Internet traffic in densely populated areas [44]. 5.2. As Participants in New Architectures. In addition to
supporting the propagation of traffic within the Internet,
however, small satellites require new, increasingly capable
telecommunication architectures to sustain their own oper-
ations. Coordination among satellites in LEO orbit relies
on cross-links between satellites, relay services provided
by ground stations (typically via the terrestrial Internet),
or a combination of both. This capability is critical for
constellations such as the GRACE (Gravity Recovery and
Climate Experiment) mission and the QB-50 project.t 5.2. As Participants in New Architectures. In addition to
supporting the propagation of traffic within the Internet,
however, small satellites require new, increasingly capable
telecommunication architectures to sustain their own oper-
ations. Coordination among satellites in LEO orbit relies
on cross-links between satellites, relay services provided
by ground stations (typically via the terrestrial Internet),
or a combination of both. Wireless Communications and Mobile Computing This capability is critical for
constellations such as the GRACE (Gravity Recovery and
Climate Experiment) mission and the QB-50 project.t Moving farther, the twin MARCO spacecrafts (each a 6U
CubeSat) accompanying the InSight spacecraft on its mission
to Mars will primarily serve to relay information from the
InSight lander to its mission operations center on Earth, while
the lander is engaged in entering the atmosphere of Mars,
descending to the surface, and landing. As shown in Figure 7,
the link from InSight to each MARCO orbiter will be in
the UHF band, while the MARCO vehicles communication
with Earth will be by X-band radio transmission. Each
MARCO can use only one of these links at a time, so
the communication architecture will be very different from
the continuous end-to-end connectivity that characterizes
Internet traffic.fi A new generation of SDR technologies have been inte-
grated in the SCAN Testbed (Space Communications and
Navigation Testbed), which is an advanced integrated com-
munications system and laboratory facility to be installed on
the International Space Station (ISS), to develop, test, and
demonstrate new communications, networking, and naviga-
tion capabilities in the space environment [41]. The SCAN
Testbed consists of reconfigurable and reprogrammable SDR
transceivers/transponders operating at S-band, Ka-band, and
L-band, along with the required RF/antenna systems neces-
sary for communications. fi
Projecting that deviation from the Internet traffic model
back to high-volume terrestrial communications, a satellite
communications architecture that is designed to tolerate the
associated delays in end-to-end communication on a large
scale has been proposed. The “Ring Road” architecture [45] 4. Payload Evolution Thanks to the availability of modern
high-speed and low-power digital signal processors and
high speed memories, the trade-off between the HW/SW
implementation is moving more and more towards the SW
implementation and the concept of SDR. SDR is an evolution
of flexible and reconfigurable payloads. An early adopter
of reconfigurable technology for space applications was the
Australian FedSat microsatellite communications payload
launched in 2002. The FedSat communications payload uti-
lized Field Programmable Gate Array (FPGA) components
for baseband digital signal processing and included a code
upload mode allowing it to be reprogrammed while in orbit
[9]. The evolution from reconfigurable and reprogrammable
devices to SDR has been driven by the demand for flexible
and reconfigurable radio communications in support of
military and public safety operations and it has been pushed
by advances in the enabling technologies such as Analog A number of further demonstration missions are cur-
rently planned, such as NASA’s Terabyte Infrared Delivery
(TBIRD) mission, aiming at demonstrating a 100 Gbps link
from a CubeSat to ground [34], or within the Optical Space
Infrared Downlink (OSIRIS) programme of the German
Aerospace Center (DLR), which aims at demonstrating opti-
cal downlinks from small satellites and CubeSats to Earth
[35]. Practical implementations of current optical commu-
nication systems for small satellite applications may reach
data rates of about 10 Gbps with a terminal weight in the
order of 5 kg and a power consumption of about 50 W. For
applications on CubeSats, Figure 6 shows OSIRIS4 CubeSat
implementation as an example. The terminal weighs in Wireless Communications and Mobile Computing The constellation operates as follows: The constellation operates as follows: The constellation operates as follows: (i) A user at a cold spot node issues data in a bundle (such
as an email message or an HTTP proxy query). The
node queues the bundle up for transmission to the
next courier that flies overhead [48]. (ii) Eventually, a courier flies over the cold spot. The
courier’s orbit is well known, so the contact between
the courier and the cold spot can be scheduled far in
advance. The courier and cold spot begin communi-
cations using BP over LTP (Licklider Transmission
Protocol [49]; see more details in the next section)
over whatever radio frequencies are available. Bundles
from elsewhere that are destined for this cold spot
called “forward traffic” are transmitted from the
courier to the cold spot node for forwarding within
the local network, if any. Bundles issued from the
cold spot called “return traffic” are transmitted to the
courier and queued on-board for future transmission. Figure 7: The MARCO communication architecture. Image credit:
NASA/JPL-CalTech. D
B
E
A
2
3
4
1
“cold spot” (no Internet connectivity)
“hot spot” (connected to Internet)
“courier” (nanosatellite, a “data mule”)
C
non-RingRoad node on Internet
Y
non-RingRoad node on local area net
X
Figure 8: The Ring Road network architecture [45]. D
B
E
A
2
3
4
1
C
Y
X (iii) The courier computes a route for each bundle it
receives from the cold spot. It knows about its own
future scheduled contacts, so any bundle that is
destined for some other cold spot that the courier will
reach before the bundle’s TTL (Time-To-Live) expires
is queued for future transmission to that cold spot. All
other bundles are queued for transmission to the next
hot spot the courier will fly over. (iv) When the courier flies over a hot spot, the queued
bundles are transmitted to the hot spot and the
courier receives bundles that the hot spot node has
queued for transmission to that courier. (v) When a hot spot node receives bundles from a courier,
it computes a route for each bundle. If the bundle’s
destination endpoint is directly reachable via the
Internet (e.g., a database server in Montreal), then
the hot spot uses BP over TCP/IP to send the bundle
immediately to that endpoint. 5. New Telecommunication Architectures Small satellites are playing an increasingly important role in
telecommunication architectures in two main ways: 9 Wireless Communications and Mobile Computing Figure 7: The MARCO communication architecture. Image credit:
NASA/JPL-CalTech. (i) Router satellites, called “courier” nodes, in polar orbit (ii) Nodes residing in computers that are attached to the
Internet, called “hot spots” (iii) Nodes residing in computers that are highly isolated,
with no electronic connectivity, called “cold spots.” Wireless Communications and Mobile Computing As more satellites are added, the frequency
of coverage of any given cold spot increases and N
drops, which increases the carrying capacity of the
network as a whole (the aggregate storage capacity
of all the couriers), so that the number of cold spots
supported can increase. Adding more hot spots on
the ground would also incrementally increase the
carrying capacity of the network, by enabling earlier
drainage of the return-traffic bundles in couriers’ on-
board storage and thereby making room for more
bundles, which would further increase the number of
supportable cold spots you could support. (i) Unlike a crosslink-based routing-fabric constellation,
there is no need to orbit the whole constellation all
at once in order to get data moving. The network
could begin with one hot spot, one cold spot, and
one courier. In that case, the round-trip time for
the cold spot would be very long as there would
be only one contact per N orbits of the satellite,
where N is however many orbits would be needed
to bring the cold spot back into the satellites ground
track. Nonetheless, bidirectional data flow between
the cold spot and any point on the Internet would
be reliably supported, albeit at very low effective data
rates. As more satellites are added, the frequency
of coverage of any given cold spot increases and N
drops, which increases the carrying capacity of the
network as a whole (the aggregate storage capacity
of all the couriers), so that the number of cold spots
supported can increase. Adding more hot spots on
the ground would also incrementally increase the
carrying capacity of the network, by enabling earlier
drainage of the return-traffic bundles in couriers’ on-
board storage and thereby making room for more
bundles, which would further increase the number of
supportable cold spots you could support. Still related to the objective of distributing small satellite
data across the Internet is providing the network architecture
with content-oriented functions in order to differentiate
QoS management and routing functions applied to the data
objects obtained from the small satellite systems. The constellation operates as follows: Otherwise, the hot spot
consults the contact schedule to determine which
courier has the earliest scheduled contact with the
destination cold spot and then reconsults the contact
schedule to determine which hot spot has the earliest
scheduled contact with that courier. If the first hot
spot that will see that courier is the local hot spot itself,
then the hot spot simply queues the bundle locally for
future transmission to that courier; otherwise, it uses
BP over TCP/IP to send the bundle immediately to
that computed best-way-forward hot spot. (v) When a hot spot node receives bundles from a courier,
it computes a route for each bundle. If the bundle’s
destination endpoint is directly reachable via the
Internet (e.g., a database server in Montreal), then
the hot spot uses BP over TCP/IP to send the bundle
immediately to that endpoint. Otherwise, the hot spot
consults the contact schedule to determine which
courier has the earliest scheduled contact with the
destination cold spot and then reconsults the contact
schedule to determine which hot spot has the earliest
scheduled contact with that courier. If the first hot
spot that will see that courier is the local hot spot itself,
then the hot spot simply queues the bundle locally for
future transmission to that courier; otherwise, it uses
BP over TCP/IP to send the bundle immediately to
that computed best-way-forward hot spot. “cold spot” (no Internet connectivity)
“hot spot” (connected to Internet)
“courier” (nanosatellite, a “data mule”)
non-RingRoad node on Internet
non-RingRoad node on local area net
Figure 8: The Ring Road network architecture [45]. is based on the use of delay-tolerant networking (DTN)
protocols [46], discussed later. The basic idea is to deploy,
gradually, one satellite at a time, a constellation of DTN
Bundle Protocol (BP) [47] routers in LEO orbit. As shown
in Figure 8, the network encompasses three classes of DTN
nodes: (vi) When a hot spot node receives bundles from some
node in the Internet (possibly another hot spot), it
computes a route for each bundle as above. When a
courier flies overhead, it exchanges bundles with the Wireless Communications and Mobile Computing Wireless Communications and Mobile Computing 10 authorities, and single users for different applications (e.g.,
space data exploitation, education purposes, surveillance and
monitoring, etc.). In this context, data retrieval will likely
happen over Internet terrestrial infrastructure, hence calling
for proper integration strategies to be deployed between
the ground segment of the small satellite system and the
core terrestrial network. This integration task can be easily
considered in the broader plan of converging satellite and
5G networks [50] (and papers included in that special issue),
which has recently become a hot topic for the satellite
industry. Without entering the details of the architecture
proposals [51] elaborated to meet this goal, it is of pivotal
importance to provide a flexible integrated architecture. Network flexibility is indeed recommended in order to
ensure proper coexistence of existing Internet flows and
small satellite data retrieval, which may be regarded in terms
of different network slices, each characterised by diverse
QoS/QoE characteristics. To this end, the implementation
of proper SDN (Software Defined Networking) and NFV
(Network Function Virtualization) solutions is desirable, so
as to achieve also the “softwarisation” of the satellite network,
whose understanding is however still not complete and will
deserve additional studies for the case of small satellite
constellations. courier. When the courier subsequently flies over a
cold spot, it exchanges bundles with it in the same
way, and so on. courier. When the courier subsequently flies over a
cold spot, it exchanges bundles with it in the same
way, and so on. The concept offers a number of advantages: (i) Unlike a crosslink-based routing-fabric constellation,
there is no need to orbit the whole constellation all
at once in order to get data moving. The network
could begin with one hot spot, one cold spot, and
one courier. In that case, the round-trip time for
the cold spot would be very long as there would
be only one contact per N orbits of the satellite,
where N is however many orbits would be needed
to bring the cold spot back into the satellites ground
track. Nonetheless, bidirectional data flow between
the cold spot and any point on the Internet would
be reliably supported, albeit at very low effective data
rates. Wireless Communications and Mobile Computing This may
suggest the application of the existing Information Cen-
tric Networking architectures [52], whose baseline concept
should be however adapted in order to meet the content
characteristics of the data objects retrieved from the satellite
systems and to interface with the network architecture (e.g.,
DTN-based) proposed for the satellite network (as illustrated
in the previous subsection). (ii) While the routing is somewhat complex, it takes place
in potentially powerful ground-based computers at
hot spots, not in the courier satellites. This means
that small, mass-produced satellites can be suitable as
couriers. (iii) All elements of the architecture are, therefore, rela-
tively inexpensive. In more detail, ICN-based architectures build on publish-
subscribe (pub-sub) paradigms, so that users subscribe to
content distributions services and accordingly contents are
distributed upon request reception. One of the main peculiar-
ities of ICN networks is in that contents are explicitly mapped
to object names, which enable more advanced content-aware
routing and security schemes. Moreover, this approach helps
implement a content-centric networking approach, hence
superseding the typically employed host-centric approach
(i.e., as implemented in IP-based systems), where locations
and content descriptions are mapped into a unique iden-
tifier (e.g., IP address), hence posing some limitations on
implementing content-based networking functions. Another
intrinsic key advantage of ICN networks is to implement
distributed caching functionalities throughout the entire
network, hence possibly simplifying the integration of MEC
(Multi-Access Edge Computing) and Cloud Computing func-
tionalities, which are pivotal building blocks in the modern
communication networks. As a conclusion, this SmallSat-based architecture could
enable very widely available network data service at low cost,
starting with a very modest initial investment. 5.3. Integration with Terrestrial Architectures. The potentials
offered by Small- and CubeSats constellations from a service
point of view have to be analysed from a wider angle
in order to consider the data availability from different
stakeholders. In the case of processing centres placed nearby
control centres or in any case directly connected to them via
dedicated terrestrial infrastructure, the architecture design
may essentially consist in the extension of the exemplary one
illustrated in the previous subsections. This can be achieved
by terminating the proposed DTN architecture directly at the
processing centres or by making use of specialised gateways
capable of interfacing native DTN architectures with non-
DTN aware counterpart (i.e., in the case of legacy networks
building on pure TCP/IP protocol architectures). Wireless Communications and Mobile Computing ICN functionalities are typically supported by spe-
cialised networking elements, i.e., ICN routers, which can
be deployed not only in terrestrial networks, but also in the
space counterpart, provided that satellites offer the necessary On the other hand, the increasing interest towards the
service provided by small satellite constellations may result in
distributing data to enterprises, universities, schools, public Wireless Communications and Mobile Computing 11 11 End-Users
MEC/Cloud Computing
Satellite constellation
Message Formats
Message Formats
Message Formats
Message Formats
Message Formats
Content
request
Content
retrieval
5G Network
ICN/DTN
terrestrial
router
ICN/DTN
space router
Figure 9: Integrated satellite-5G network based on ICN/DTN concepts for content delivery [52]. Satellite constellation ICN/DTN
space router 5G Network Content
request Figure 9: Integrated satellite-5G network based on ICN/DTN concepts for content delivery [52]. capabilities are provided at multiple layers of the protocol
stack. storage and computing capabilities. As a matter of fact, the
coexistence of DTN- and ICN-based protocol architectures in
the same network deployment is possible in order to exploit
the main advantage offered by the two with respect to disrup-
tion resilience and caching, although specific adaptations of
the protocol interfaces are necessary (not treated in this paper
as beyond the scope). 6.1. Physical Layer. Originally, the only communication links
supported for CubeSat satellites were UHF links operated
via AX.25. Given the low requirements in terms of data rate
of most of the original, mainly scientific, missions, simple
modulation schemes have been used, such as binary-FSK [53,
54]. It is also worth outlining that the AX.25 protocol allows
detecting errors but not correcting them. The emerging
need for transmitting at higher data rate and keeping low
mass and weight is pushing to use larger bandwidths and
higher-frequency bands, as reported in Section 4, but also
to use more efficiently the available bandwidths through
more advanced modulations schemes. Moreover, the shift
towards SDR payload and ground stations, made possible by
the rapid evolution of digital electronics, opens the opportu-
nity to implement more advanced communication protocols
and modulation schemes, including error correction capa-
bilities and dynamic adaptation of modulation parameters In general, the overall network architecture encompass-
ing 5G and satellite segments, building on ICN/DTN archi-
tectures, and interacting with MEC and cloud computing
elements is exemplified in Figure 9, where the case of a
satellite constellation complementing a 5G access network to
boost the content delivery is sketched. Wireless Communications and Mobile Computing 12 depending on the current link conditions [55]. This has
motivated some theoretical studies on the choice of the most
appropriate modulations [56, 57]. However, there are already
some innovative transceiver designed for CubeSat and Small
Satellites using higher-frequency bands such as X-band up to
Ka-band, implementing Variable and Adaptive Coding and
Modulation (VCM, ACM) capabilities [58, 59]. For instance,
RADIOSAT is an innovative transceiver developed by ESA,
working at a Ka-band and integrated with a DVB-S2 modem,
overall characterized by low power consumption. On the
design of intersatellite link, it is worth mentioning the recent
studies on the use of Visible Light Communications (VLCs),
which can provide higher data rates with smaller, light-
weight nodes, while avoiding the usual interference problems
associated with RF, as well as the apparent radio spectrum
scarcity below the 6 GHz band. Furthermore, the electronics
required for achieving precision pointing accuracy for laser
communication systems will be avoided. With approximately
300 THz of free bandwidth available for VLC, high capacity
data transmission rates could be provided over short dis-
tances using arrays of LEDs [60]. central fact in both circumstances is the potential inability
of each network node to request timely assistance from
any other, for any purpose, and at any given moment. The
unifying principle in the design of the features of DTN, then,
is recognition of this fact. Nodes must be able to make their
own operational decisions locally, on their own, with global
information that may well be stale or incomplete, and the
network must be able to continue to operate at some useful
level even when these decisions are flawed. The core protocol
of DTN is BP [47], a network-layer protocol that functions
as the DTN analog to the Internet Protocol. BP is similar to
IP in that a BP node receives data issued by an application
entity, stores the data in some medium, and forwards the data
through the network toward the node serving the application
entity that is the destination of the data. It principally differs
from IP in that a forwarding node does not immediately
discard data items (called “bundles”) for which no onward
communication link is currently available; instead, it may
store bundles for a lengthy period of time, waiting for a
link to become available. The DTN analog to the Internet’s
TCP is the LTP [49]. Wireless Communications and Mobile Computing An LTP “engine” divides an outbound
bundle into small “segments” and transmits the segments to
the LTP engine serving the BP node that BP has determined
to be the best next proximate destination for the bundle
the next step on the bundle’s end-to-end path. Both LTP
and TCP account for transmitted data, detecting data loss
and automatically recovering from that loss by retransmitting
segments as necessary. The principal difference between LTP
and TCP is this: 6.2. Link Layer. While operators of Earth-orbiting CubeSats
initially had few options beyond AX.25, new and more
capable protocols that are suited for space flight operations
at Earth and beyond Earth orbit are becoming available. The new CCSDS Unified Space Link Protocol (USLP) [61]
is designed to be adaptable to a very wide range of space
data transmission conditions. It includes a “virtual channels”
concept that enables a single physical link to be transparently
shared among multiple data streams at higher layer, together
with further multiplexing accommodation multiplexer access
points that enable multiple data services to share a single
virtual channel. It also provides mechanisms for aggregating
small service data units and segmenting those aggregations,
for extensive control over the sizes of protocol data units. CCSDS has also defined a security service at the link
layer, called Space Data Link Security (SDLS). Security is
rapidly becoming an urgent concern of space flight mission
designers, as security breaches at ground stations and mission
operations centers served by the Internet grow ever more
troublesome. SDLS provides a security standard for simple
space flight missions, where a single spacecraft is in contact
with its control center through a ground station. It includes
data origin authentication, connection and connectionless
confidentiality, connection integrity with and without recov-
ery, and connectionless integrity. (i) In TCP, the entity that discovers and reports data
loss is the TCP instance serving the application entity
that is the destination of the data, and the data
loss is reported to the TCP instance that serves the
application entity that was the source of the data. That
is, retransmission is “end-to-end” and TCP is situated
above IP in the Internet protocol stack. (ii) In a space flight mission scenario, end-to-end retrans-
missions could result in extremely lengthy delays in
data delivery because the source and destination of
data might be on different planets separated by many
light minutes of propagation latency. 6. Advances in Communications and
Network Protocols New protocols for communication with and among small
satellites have emerged rapidly in the past decade. The new Wireless Communications and Mobile Computing Wireless Communications and Mobile Computing In LTP, data loss
is instead reported to the LTP instance at the prox-
imate source of the data (the immediately prior BP
node on the end-to-end path), which retransmits the
lost segments as early as possible. LTP retransmission
is “point-to-point” within the network, and LTP is
situated below BP in the DTN protocol stack. 6.3. Network Layer. DTN concepts date back to the early
days of the Interplanetary Networking Research Group of the
Internet Research Task Force. DTN is a network architecture
that is aimed at eking as much data communication as
possible out of inhospitable networks—in particular, those
where link interruptions (whether anticipated or not) are
frequent and significant and/or where signal propagation
latency is high. The effects of high delay and of disconnection
are in fact similar in many ways, and the network architecture
features developed for DTN serve to mitigate both. The 6.3. Network Layer. DTN concepts date back to the early
days of the Interplanetary Networking Research Group of the
Internet Research Task Force. DTN is a network architecture
that is aimed at eking as much data communication as
possible out of inhospitable networks—in particular, those
where link interruptions (whether anticipated or not) are
frequent and significant and/or where signal propagation
latency is high. The effects of high delay and of disconnection
are in fact similar in many ways, and the network architecture
features developed for DTN serve to mitigate both. The Complementary to the use of DTN protocol solution is
the exploitation of network coding (NC) [62] for improving
the robustness of data transmission as well as optimised use
of the available network resources (i.e., bandwidth). Taking
as reference the case of Ring Road network model for small
satellite constellations, network coding can be applied on all
the network nodes (i.e., on the space and ground segments)
[63]. In this case, network coding functionalities would
actually consist in online (on fly) encoding and decoding
functions. In more detail, each NC-enabled node will be in 13 Wireless Communications and Mobile Computing entire network, but only limited to the network legs exhibiting
more challenges from a communication reliability point of
view. As such, no re-encoding functionalities are necessary
(as those made possible by random linear network coding)
and on the contrary classical packet layer FEC solutions
can be considered, i.e., based on LDPC or Reed-Solomon
codes. Wireless Communications and Mobile Computing In this respect, some proposals have been already
worked out by CCSDS with reference to the case of erasure
codes applied space downlinks [67], where the potential of
LDPC-based erasure codes was exploited especially for the
case of free-space optical link communications. Although in
this case, network coding is implemented only on specific
links, the node capability to implement encoding/decoding
functionalities as well as to store data prior to processing
functions is certainly an important requirement to be taken
into account in the system design phase in the light of the
typically resources-constrained implementations of nodes in
space. Other activities looking into implementation of net-
work coding for intersatellite links have been also considered,
although the aforementioned constraints coming from the
space segments were not completely taken into account,
hence requiring additional study for a deeper understanding
of all underlying implications and requirements. charge of collecting a given number of information packets
and to encode them so as to generate a certain number
of redundancy packets, where the overall network coding
configuration plays an important role in what concerns
both the specific number of input information and output
redundancy packets as well as the adopted coding strategy
[62]. In this respect, the use of random linear network coding
has gained quite some popularity in the last two decades,
so that it is nowadays considered on the most appealing
approach to implement NC in real network deployments. In
particular, the application on random linear network coding
of data chunks to be dumped to ground stations would
help increase the reliability of data exchange against sporadic
fluctuations of the transmission channel quality. Moreover,
the network coding can be also exploited to transmit a
reduced number of data packets, hence improving the actual
bandwidth utilisation. This advantage can be particularly
relevant if multicast data communications are exploited
[64, 65], so that the performance advantages recognised for
network coding can be fully exploited. g
y
p
On the other hand, in spite of the aforementioned
advantages, it is also worth considering the complexity
implications arising from the implementation of network
coding on the space segment [66]. As a matter of fact,
network coding implementation requires some dedicated
computation capability for online coding functions as well as
specific on-board storage to keep temporary copies of the data
chunks being subject to encoding or decoding procedures. Wireless Communications and Mobile Computing Moreover, some attention has to be also paid to the protocol
layer wherein network coding is being applied, so that often
either (i) layered or (ii) integrated approaches are considered
[67]. In the former, NC is implemented as a dedicated shim
layer placed in between existing protocol layers in order to
have a limited increase in the overall system implementa-
tion. In the latter, instead, NC functionalities have to be
incorporated in an existing protocol, hence increasing the
overall implementation complexity. Another point relates
to the actual position of NC functionalities in a protocol
stack, for which no specific consensus has been reached
yet. On the one hand, it would be desirable to keep NC
implementation as much closer as possible to the lower layers
of the protocol stack (i.e., datalink) in order to have a more
efficient recovery of possible packet losses. On the other hand,
implementing NC in the upper layers of the protocol stack
would help matching more precisely the characteristics of
data services and eventually also meet the corresponding QoS
requirements. In this respect, a good compromise could be
to implement NC functionalities directly within the bundle
protocol or immediately beneath it as part of any of the
convergence layers (i.e., UDP or LTP) considered for that
specific mission design. As such, it is immediate to see that
all these requirements have to be properly taken into account
in the full system design, with respect to the capabilities
offered by existing satellite payloads and the actual service
requirements to be targeted by the considered system. 7. Perspectives and Open Challenges The paper has reviewed the state of the art of small satel-
lite systems, highlighting the distinctive features enabling
novel applications and focusing on telecommunication
services.h The provision of advanced Internet services through
mega-constellations of pico/nanosatellites is going to become
reality in the near future. However, several challenges must be
faced yet, which are summarized in the following. (i) Physical Layer. (a) The use of frequency bands higher than Ka-
band and the use of free space optical (FSO)
communications for Earth-satellite links (i.e.,
not only for intersatellite links), as reported in
Section 4, raise one important challenge: the
propagation channel can be strongly attenuated. Both for high frequency RF transmission and
for FSO, this issue could be overcome by pro-
viding a ground network with a high number
of ground stations at highly diverse sites. The
concept of site diversity has been extensively
studied in the field of High Throughput Satel-
lite (HTS), and recent works have highlighted
the fact that SDN paradigm could provide the
gateways implementing the concept of Smart
Diversity, a high level of reconfigurability that
could allow efficient resources allocation during
traffic switching events [68]. (b) Besides the few theoretical studies mentioned in
Section 6.1, and some transceiver implementing
ACM techniques, much more work is needed
to design optimized modulation and coding (b) Besides the few theoretical studies mentioned in
Section 6.1, and some transceiver implementing
ACM techniques, much more work is needed
to design optimized modulation and coding Another interesting point related to the use of network
coding in the proposed network architecture is about their
use in the form of [67] for mitigating packet losses. In this
case, network coding is not implemented throughout the 14 Wireless Communications and Mobile Computing 14 schemes able to satisfy strict requirements in
terms of mass, weight, size, and power con-
sumption. which show the feasibility of ground-to-space quan-
tum key distribution (QKD) [75–78]. QKD uses
individual light quanta in quantum superposition
states to guarantee unconditional communication
security between distant parties. Satellite-based QKD
promises to establish a global-scale quantum network
by exploiting the negligible photon loss and decoher-
ence in the empty outer space. No eavesdropping can
take place as the distribution of entangled photons
between the ground and the satellite is used to
certify the quantum nature of the link. By placing the
entangled photon source on the ground, the space
segments contain “only” the less complex detection
system, enabling its implementation in a compact
enclosure, compatible with the 12U CubeSat standard
[75]. (vi) Adoption of the SDN/NFV. It is clear that SDN/NFV paradigms will play a key
role in the integration of satellite systems with 5G. However, the use of SDN/NFV in a network of small
satellites has yet to be investigated; as discussed in
Section 6, it could be important. Indeed, small satellite
network deployments could accelerate the infusion of
SDN concepts into satellite systems. For instance, on-
board SDN-compatible routers could be developed
and operated on small satellites as router functions
migrate into software. It is worth mentioning that 3GPP Service and system
Aspects (SA) activities have identified satellite systems both
as a possible solution for stand-alone infrastructure and as
complements to terrestrial networks [79]. In this framework,
HTS systems could play a key role in some of the 5G
application scenarios once they will be able to provide
extremely high data rate. However, in many other 5G applications scenarios that
focus on M2M communications or require extremely low
latency, only small satellite constellations can really provide
an effective complement to terrestrial systems. It is crucial to
effectively face the challenges discussed above in order not to
miss the opportunities offered by the 5G ecosystem. The challenge is still open. Interesting works are
ongoing on the use of physical layer approaches
to security in satellite communications [73, 74]. No
specific work on application of physical layer security
to CubeSat can be found; even this could open novel
solutions to overcome the challenges of security in the
small satellite framework. (i) Physical Layer. In [76], a LEO satellite has been developed
and launched to implement decoy-state QKD with
over kHz key rate from the satellite to ground over
a distance up to 1200 km, which is up to orders of
magnitudes more efficient than that expected using an
optical fiber (with 0.2 dB/km loss) of the same length. In [78], it was demonstrated that a 4 kg CubeSat can
generate a quantum-secure key, which has so far only
been shown by a much larger 600 kg satellite mission. (ii) MAC Layer. In view of emerging system constraints, the imple-
mentation in small satellites of the scheduled and
random-access MAC protocols adopted in existing
satellite networks needs further investigation. (iii) Upper Layers. Definition is needed for interoperable application-
layer protocols to be employed on top of the lower
layer satellite protocols, addressing a wide range of
application scenarios and traffic data configurations. (iv) Routing over Time. Due to the frequent topology changes in a CubeSat
network, successful data delivery will require ample
long-term storage at intermediate nodes to deal with
satellite link disruptions. (v) Security Issues in LEO Satellite Networks. v) Security Issues in LEO Satellite Networks. Telemetry, command and control messages, and mis-
sion specific data are sent through radio links. There-
fore, security concerns arise. CubeSats are susceptible
to Denial of Service (DoS) attacks as well as eaves-
dropping and data can be accessed by unauthorized
user. The attacker could send spurious commands
causing excessive resources consumption, data loss, or
mission failure. Security challenges are exasperated by
the use of SDR payload which opens the possibility of
placing new software on the SDR unit through unau-
thorized and potentially malicious software installed
on the platform [69]. Another security concern that
has been raised recently is related to the use of small
satellites that have propulsion systems and they could
be hacked and endangering other satellites [70]. As
also reported in Section 6, communication protocols
currently implemented for CubeSat have almost no
security features. Security mechanisms developed for
conventional terrestrial networks, characterized by
lengthy handshake exchanges and substantial com-
putational effort, can hardly be directly applied to
networks of small satellites. Power, space, and weight
constraints related to CubeSat pose challenges in
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The effect of <i>Cassia alata</i> L. leaf extract on egg hatching and survival of <i>Pangasianodon hypophthalmus</i> larvae
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The effect of Cassia alata L. leaf extract on egg hatching and
survival of Pangasianodon hypophthalmus larvae Iko Imelda Arisa1,3,4*, Sri Agustina2,3,5, and Lisa Handayani1 1 Departement of Aquaculture, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia
2 Departement of Marine Science, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia
3 Research Center for Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia 1 Departement of Aquaculture, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia
2 Departement of Marine Science, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia
3 Research Center for Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia
4Animal Histology and Fish Nutrition Laboratory, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111
I d
i y
y
3 Research Center for Marine and Fisheries, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111Indonesia
4Animal Histology and Fish Nutrition Laboratory, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111
Indonesia ,
y
,
,
,
4Animal Histology and Fish Nutrition Laboratory, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111
Indonesia 5Laboratory of Marine Chemistry and Biotechnology of Fisheries, Marine and Fisheries Faculty, Universitas Syiah Kuala, Banda
Aceh, Aceh, 23111 Indonesia Abstract. This study aims to determine the effect of using Cassia alata L. leaf extract on the prevention of
fungus on fish eggs Pangasianodon hypophthalmus This research was conducted at the Blangpidie Fish
Seed Center. Aceh Barat Daya from July to August 2020. Statistical analysis used a completely randomized
design (CRD) method with 7 treatment levels and 3 replications. The treatments were conducted by soaking
the fish eggs in the leaf extract for 20 minutes. Those treatments were A (control), B (2.5 ppm), C (5 ppm),
D (7.5 ppm) E (10 ppm), F (12.5 ppm), and G (15 ppm). ANOVA test results showed that the addition of
leaf extract C. alata had a significant (P <0.05) effect on hatchability and survival of P. hypophthalmus
larvae. A concentration of 15 ppm is the best with a hatchability value of 85.33% and a concentration of 10
ppm is the most optimum for the survival value of P. hypophthalmus larvae, namely 92.99%. 2 Material and methods Pangasianodon hypophthalmus is one of the potential
aquaculture commodities to be developed. However,
according to Muhlis et al. [1], P. hypophthalmus
hatchery activities encountered several obstacles,
including disease attacks in the egg phase caused by
fungi. Fungal infection at the time of hatching eggs
affects the damage to the eggs and causes a decrease in
hatchability. Furthermore Larasati et al. [2] also
mentions that the problem that is often faced in catfish
farming is the low degree of egg hatching. Low egg
hatchability will have an impact on the supply of catfish
seeds which are low, so that aquaculture production
activities are also low. The research was carried out in July to August 2020 at
the Blang Pidie Fish Seeds Center, Aceh Barat Daya
District, Aceh Indonesia. The research design used was
a completely randomized design (CRD) consisting 7
treatments and 3 replications. As for the types of
treatments tested were as follows: A=immersion
without C. alata leaves extract (0 ppm); B= immersion
with C.alata leaves extract at a dose of 2.5 ppm; C=
immersion with C.alata leaves extract at a dose of 5
ppm; D= immersion with C.alata leaves extract dose of
7.5 ppm; E= immersion with C.alata leaves extract at a
dose of 10 ppm; F= immersion with C.alata leaves
extract at a dose of 12.5 ppm; G= immersion with C. alata leaves extract at a dose of 15 ppm. Efforts to control disease in cultured fish eggs due to
pathogenic infections that use chemicals or antibiotics
in the long term can have negative impacts, among
others, can cause bacterial and fungal resistance and can
pollute the environment [3,4]. So the disease requires
alternative treatments such as the use of natural
ingredients. One of the natural ingredients that has the
potential to inhibit the growth of pathogens in fish is the
use of Cassia alata leaves. This plant is known by
different names in different countries, namely as
Gelenggang (Malaysia), Ketepeng Badak or Ketepeng
Cina (Indonesia) and bush ringworm (Australia) and has
been used as as traditional medicine to treat various
diseases [5,6]. E3S Web of Conferences 339, 01003 (2022)
10thICMR-2ndINSAEF 2021 E3S Web of Conferences 339, 01003 (2022)
10thICMR-2ndINSAEF 2021 https://doi.org/10.1051/e3sconf/202233901003 *Corresponding author: ikoimeldaarisa@unsyiah.ac.id © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/). DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
ses/by/4 0/) 2.3.2 Hatching rate (HR) Calculation of egg hatching rate parameter in research
based on Arfiah et al. [9]: =
100% =
100% The data from this study showed that the highest
hatchability of fish eggs was found in the 15 ppm
treatment, which was 85.33%. This indicates that the
antifungal content of Cassia alata leaf extract with a
concentration of 15 ppm is optimal in inhibiting the
fungus Saprolegnia sp. to infect P. Hypophthalmus fish
eggs. C. alata is a traditional medicine that has excellent
antibacterial and antifungal properties [20-21]. Previous
research Bowo et al. [22] stated that a concentration of
60 ppm C. alata leaf extract showed the best results in
inhibiting the growth of the fungus Saprolegnia sp,
while for egg hatchability and survival of gouramy
larvae the highest was obtained at a concentration of 10
ppm. Where: HR = Hatching Rate (%) 2.3.1 Eggs condition Observation of eggs started after soaking using C. alata
leaf extract until the eggs hatched. Eggs infected with
fungi will show clinical symptoms in the form of threads
like cotton covering the eggs. Observations of eggs that
recovered due to fungal infection after soaking were
observed every 1 hour interval using a microscope. 2.1 Cassia alata L. leaves extract Cassia alata leaves were washed with clean water and
dried under the sun light until the leaves were easily
crushed. The dried leaves are ground into flour. A total
of ±500 g of C. alata leaves powder was soaked in 3 L
96% PA absolute ethanol for 24 hours. The results of the
immersion are filtered (Whattman No.41), the results
are extracted using a rotary evaporator, then the extract
is stored at -5 °C until it is used for testing. E3S Web of Conferences 339, 01003 (2022)
10thICMR-2ndINSAEF 2021 https://doi.org/10.1051/e3sconf/202233901003 2.3.3 Survival rate Calculation of survival rate parameter in research based
on Effendie [10]: ]
=
100
(2) =
100 (2) Where: SR = Survival Rate (%); Nt = number of test
dead fish that died during rearing (ind); No =the total
number of test fish observed (ind). 2.3.4 Water quality parameters Several water quality parameters measured during the
study were pH, temperature, and dissolved oxygen. pH
parameter measurement using a pH meter (PCSTestrTM
35), temperature parameter measurement was carried
out using a thermometer (PCSTestrTM 35) and dissolved
oxygen parameter measurement using a DO meter
(Thermo Scientific Eutech DO 6+). Water quality
parameters measurements are carried out every day. Tabel 1. Hatching rate and survival rate of P. hypophthalmus Tabel 1. Hatching rate and survival rate of P. hypophthalmus
Treatments
Parameters
Hatching Rate
(%)
Survival Rate
(%)
A (0 ppm)
22.00 ± 4.0a
55.32±16.4a
B (2.5 ppm)
37.33 ± 6.1b
74.45±12.3ab
C (5 ppm)
45.33 ± 5.0b
81.75±10.7ab
D (7.5 ppm)
61.33 ± 7.6c
90.56±9.4b
E (10 ppm)
67.33 ± 8.1cd
92.99±8.7b
F (12.5 ppm)
75.33 ± 3.0de
90.29±3.9b
G (15 ppm)
85.33 ± 5.0e
92.73±1.3b
Note:writing different superscripts in the column indicates a
significant difference between treatments (P<0,05). hypophthalmus
Treatments
Parameters
Hatching Rate
(%)
Survival Rate
(%)
A (0 ppm)
22.00 ± 4.0a
55.32±16.4a
B (2.5 ppm)
37.33 ± 6.1b
74.45±12.3ab
C (5 ppm)
45.33 ± 5.0b
81.75±10.7ab
D (7.5 ppm)
61.33 ± 7.6c
90.56±9.4b
E (10 ppm)
67.33 ± 8.1cd
92.99±8.7b
F (12.5 ppm)
75.33 ± 3.0de
90.29±3.9b
G (15 ppm)
85.33 ± 5.0e
92.73±1.3b 3. Results and Discussion Fish eggs Pangasianodon hypophthalmus 50 healthy
eggs that have been fertilized are put into each treatment
container. Futhermore, as many as 25 fish eggs that have
been infected with fungi and put into each of these
containers and then left for 6 hours. After the healthy
eggs were infected with the fungus, they were
transferred to each container which was given a solution
of C. alata leaf extract in accordance with the provisions
of the treatment concentration. The eggs were soaked in
a solution of C. alata leaf extract for 20 minutes. Cassia alata extract has enormous pharmacological
value in supporting the use of traditional medicine in
curing several important diseases, including antioxidant,
anti-fungal and anti-bacterial properties [11]. Based on
the results of the study, it was found that the use of C. alata leaf extract could increase egg hatchability and
survival of P. hypophthalmus (Table 1), where the
higher the dose of extract used in the treatment will also
increase the hatchability of fish eggs. Fatmawati et al. [6] stated that C. alata leaf extract contains chemical
compounds that can be used as antimicrobials. Furthermore, Makinde et al [12] said that the
antibacterial and antifungal activity of the aqueous and
methanolic extracts of C. alata leaves found that they
had more antifungal properties than antibacterial. Phytochemical analysis of C. alata extract including
flavonoids, tannins, steroids, phenols and alkaloids [13,
14, 15]. Then Roshida et al. [16] stated that tannins are
antibacterial by presipitating protein. The antimicrobial
effect of tannins through reactions with cell membrane
inctivation of enzymes, destruction or inactivation of
functions. Meanwhile, flavonoids have antimicrobial
properties can form complex compounds with dissolved
extracellular proteins and microbial cell walls. Falvonoids also have antibacterial and antioxidant
properties that work by increasing the immune system,
where leukocytes as antigen-eaters are produced faster
and activate the lymphoid system more quickly [17-19]. Note:writing different superscripts in the column indicates a 2.4 Data analysis Research parameter data such as egg hatchability and
survival rate of fish were analyzed using Analysis of
Variance (ANOVA), if the results were influential then
Duncan's test was performed with a 95% confidence
level. While the water quality parameter measurement
data is presented descriptively. 2 E3S Web of Conferences 339, 01003 (2022)
10thICMR-2ndINSAEF 2021 https://doi.org/10.1051/e3sconf/202233901003 Tabel 2 Water quality parameters measurement data
Treatments
Temperature
(oC)
pH
DO
(mgL-1)
A (0 ppm)
26.4 – 26.8
6,7 – 6.5
5.3 – 5.5
B (2.5 ppm)
26.5 – 26.9
6.6
5.4 – 5.6
C (5 ppm)
26.5 – 26.9
6.5 – 6.7
5.4 – 5.5
D (7.5 ppm)
26.6 – 26.8
6.5 – 6.7
5.4 – 5.5
E (10 ppm)
26.6 – 26.9
6.6 – 6.7
5.4 – 5.5
F (12.5 ppm)
26.6 – 26.9
6.5 – 6.7
5.4 – 5.6
G (15 ppm)
26.5 – 26.9
6.5 – 6.7
5.4 – 5.5 5. Y.Y. Ling, P.S. Fun, A. Yeop, M.M. Yusoff, J. Gimbun, Material Todays: Proceedings 19 (2019) 6. S. Fatmawati, Yuliana, A.S. Purnomo, M.F.A. Bakar, Helion 6 (2020) 7. M. Bahi., R. Mutia., E. Mustanir, Lukitaningsih, J
Natural 14, 1 (2014) 8. S. Saena, S. yanto, A. Amirah, J Pendidikan
Teknologi Pertanian 6, 1 (2020) 9. H. Arfiah, Muftucha, O. Caman, J Akuakultuur
Indonesia 5 (2006) The results of the analysis of variance in use of C. alata leaf extract had an effect on survival of catfish
larvae (P<0.05). During the study, the observation of the
survival rate of P. hypophthalmus larvae was
categorized as good. It is suspected that the chemical
content in leaf extractof C. alata was able to inhibit the
growth of fungi in P. hypophthalmus eggs. 10. M.I. Effendie, Metode Biologi Perikanan (Yayasan
Dewi Sri, Bogor, 1979) 11. O.T. Tcheghebe, V.R.S. Tala, F.N. Tatong, G.K. Tchuente, Medicine and Medical Sciences
(LRJMSS) 4, 2 (2017) 12. A.A. Makinde, J.O. Igoli, L. TA’Ama, S.J. Shaibu,
A. Garba, African Journal of Biotechnology 6, 13
(2007) g
g
yp p
gg
Good water quality management can inhibit the
spread of fungi in hatchery activities both at time of
hatching eggs and rearing post larvae in aquaculture
activities. Good management of aquaculture media
water quality will suppress the spread of fungal diseases
through water management is carried out for all water
quality parameters, both chemical, physical and
biological. 4. Conclusion 19. R.A. Nugroho, R. Aryani, W.D. Anggraini, E.H. Hardi, R. Rudianto, E. Kusumawati, S. Sudrajat,
F.M. Nur, H. Manurung. IOP Conference Series:
Earth and Environmental Science 348, 1(2019) The use of Cassia alata L. leaf extract make an impact
to hatchability of fish eggs and the survival of
Pangasianodon hypophthalmus fish larvae. The highest
fish egg hatchability value of 85.33% was obtained by
soaking C. alata leaf extract at a dose of 15 ppm and the
survival value the highest fish larvae of 92.99% was
obtained at a dose of 10 ppm. 20. A. Happy, M.Soumya, S.V. Kumar, S. Rajeshkumar, R.D. Sheba, T. Lakshmi, V.D. Nallaswamy, Biochemistry and Biophysics Reports
17 (2019) 21. S.A. Gaddam, V.S. Kotakadi, D.V.R.S. Gopal,
Y.S. Rao, A.V. Reddy, J of Nanostructure
Chemistry 4 (2014) 2.4 Data analysis During the research, it was found that the
water quality parameters measurement value data still
within the tolerance range of P. hypophthalmus (Table
2). Based on the Indonesian National Standard [23], the
optimal pH value for catfish rearing ranges from 6.5 -
8.5. The value of dissolved oxygen in the media for
hatching eggs and rearing P. hypophthalmus larvae is >
5 mgL-1. Furthermore, the research of Putra et al. [24]
states that the water quality for hatching and rearing
catfish larvae is a temperature of 26.2 – 27.90 and
dissolved oxygen 4.7 – 5.5 mgL-1. 13. R.P.S. Kumar, V. Malayaman, S. Shinduja, J of
Chemical and Pharmaceutical Research 5, 11
(2013) 14. R. Dewi, Y. Firza, M. Nashiry, F.S. Al-Suede,
A.S.A. Majid, Australian Herbal Insight 2 (2019) 15. J.M.N. Abille, A Review on The Medicinal Plant
Cassia alata (L.) ROXB. (FABACEAE)
(Biological Sciences Department, College of
Science & Computer Studies De La Salle
University – Dasmarinas, Cavite, Philippines pp 1-
12, 2019) 16. Rosidah, W.M. Afizia J Akuatika 3, 1 (2012) 17. R.S. Hardhini, Amir, D.N. Setyowati J Perikanan
8, 1 (2018) 18. F.M. Nur, R.A. Nugroho, S. Fachmy. AIP
Conference Proceedings 1813, (2017) Reference 22. A.T. Bowo, Sunarto, Rachimi, J Ruaya 1, 1 (2014) 1. Muhlis, M. Ghofur, M. Sugihartono, J Akuakultur
Sungai dan Danau 4, 1 (2019) 23. Standar Nasional Indonesia,Produksi Benih Ikan
Patin Siam (Pangasius hyphthalamus) Kelas
Benih Sebar (SNI : 01- 6483.4 – 2000) 2. S. Larasati, F. Basuki, T. Yuniarti, J of
Aquaculture Management and Technology 6, 4
(2017) 24. P.L. Putra, D. Jubaedah, M. Syaifudin, J
Akuakultur Rawa Indonesia 8, 1 (2020) 3. Noga, Pestisida Nabati untuk Penyakit Ikan
(Penebar Swadaya, Jakarta, 2010) 3. Noga, Pestisida Nabati untuk Penyakit Ikan
(Penebar Swadaya, Jakarta, 2010) 4. D.Q. Pham, H.T. Pham, J.W. Han, T.H. Nguyen,
H.T. Nguyen, T.D. Nguyen, T.T.T. Nguyen, C.T. Ho, H.M. Pham, H.D. Vu, G.J. Choi, Q.L. Dang,
Industrial Crops & Products 166 (2021) 4. D.Q. Pham, H.T. Pham, J.W. Han, T.H. Nguyen,
H.T. Nguyen, T.D. Nguyen, T.T.T. Nguyen, C.T. Ho, H.M. Pham, H.D. Vu, G.J. Choi, Q.L. Dang,
Industrial Crops & Products 166 (2021) 3
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Department of Error
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Lancet
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*Charles H King, Alison P Galvani
chk@cwru.edu Cognitive deficits and educational loss in
children with schistosome infection—a
systematic review and meta-analysis. PLoS Negl Trop Dis 2018; published online
Jan 12. DOI:10.1371/journal.pntd.0005524. Correspondence Correspondence Correspondence this in three parts. First, we thank the
commentator for the suggestion to
consider adding genital lesions as an
outcome of schistosomiasis. We plan to
review the associated literature on this
outcome in a subsequent iteration of
the GBD Study. We also will review the
new evidence on the cognitive deficits
associated with schistosomiasis, as
presented in their referenced article. Until now, our assessment has been
that the evidence for a causal link
between schistosomiasis infection
and loss of cognition was not strong
enough to warrant inclusion in the GBD
studies. Second, anaemia is a sequela
of schistosomiasis that has been
included since GBD 2010. Finally, the
“minimal disability weights” assigned
to schistosomiasis infection are derived
from large-scale population surveys. Until new evidence emerges that
challenges the values of these disability
weights, we will continue to use the
current values. used a boosted regression tree method
to generate environmental suitability
maps for schistosomiasis that used
georeferenced data and geospatial
covariates (including vegetation,
precipitation, and population). Geotagged epidemiological infor
mation was paired with several
hypothesised environmental drivers of
schistosomiasis presence to define the
environment that best characterises
the reported occurrences of the
parasite. We produced species-specific
maps and determined the optimal
thresholds for transmission using the
geopositioned dataset. Species-specific
maps were combined into one all-
species schistosomiasis map to show
areas that are environmentally suitable
for transmission of schistosomiasis. We overlaid this map with population
data at a scale of 25 km² to generate
a national estimate of the total
population living within areas that
are suitable for schistosomiasis
transmission. We used DisMod-MR
2.1, the Bayesian meta-regression tool
developed for GBD 2016 estimations,
to estimate prevalence within this PAR,
such that environmental factors and
localised data were both used to inform
our prevalence estimates. CHK reports a grant from the Schistosomiasis
Consortium for Operational Research and
Evaluation. APG declares no competing interests. Institute for Health Metrics and Evaluation, Seattle,
WA 98121, USA Charles King and Alison Galvani
cite three specific concerns in their
commentary regarding the Global
Burden of Disease (GBD) Study1 2016
and we respond briefly to them. 1
GBD 2016 Disease and Injury Incidence and
Prevalence Collaborators. Global, regional, and
national incidence, prevalence, and years lived
with disability for 328 diseases and injuries for
195 countries, 1990–2016: a systematic
analysis for the Global Burden of Disease Study
2016. Lancet 2017; 390: 1211–59. pl
y
King and Galvani state that environ
mental factors were not considered
within the revised Bayesian meta-
regression of the GBD Study 2016. However, we did use environmental
factors in our model. The schistoso
miasis prevalence model in the GBD
Study 2016 underwent a substantial
revision that included a systematic
review and literature data extraction
and required developing models of
both environmental suitability for
schistosomiasis transmission (used as
a proxy for identifying populations at
risk [PAR]) and prevalence within those
at-risk populations. To estimate PAR, we *Charles H King, Alison P Galvani
chk@cwru.edu Center for Global Health and Diseases, Case Western
Reserve University School of Medicine, Cleveland,
OH 44106, USA (CHK); and Center for Infectious
Disease Modeling and Analysis (CIDMA), Yale
School of Public Health, New Haven, CT, USA (APG) 1
GBD 2016 Disease and Injury Incidence and
Prevalence Collaborators. Global, regional, and
national incidence, prevalence, and years lived
with disability for 328 diseases and injuries for
195 countries, 1990-2016: a systematic
analysis for the Global Burden of Disease Study
2016. Lancet 2017; 390: 1211–59. 1
GBD 2016 Disease and Injury Incidence and
Prevalence Collaborators. Global, regional, and
national incidence, prevalence, and years lived
with disability for 328 diseases and injuries for
195 countries, 1990-2016: a systematic
analysis for the Global Burden of Disease Study
2016. Lancet 2017; 390: 1211–59. 2
GBD 2015 Mortality and Causes of Death
Collaborators. Global, regional, and national
life expectancy, all-cause mortality, and cause-
specific mortality for 249 causes of death,
1980–2015: a systematic analysis for the
Global Burden of Disease Study 2015. Lancet 2016; 388: 1459–544. 2
GBD 2015 Mortality and Causes of Death
Collaborators. Global, regional, and national
life expectancy, all-cause mortality, and cause-
specific mortality for 249 causes of death,
1980–2015: a systematic analysis for the
Global Burden of Disease Study 2015. Lancet 2016; 388: 1459–544. 3
Steinmann P, Keiser J, Bos R, Tanner M,
Utzinger J. Schistosomiasis and water
resources development: systematic review,
meta-analysis, and estimates of people at risk. Lancet Infect Dis 2006; 6: 411–25. 3
Steinmann P, Keiser J, Bos R, Tanner M,
Utzinger J. Schistosomiasis and water
resources development: systematic review,
meta-analysis, and estimates of people at risk. Lancet Infect Dis 2006; 6: 411–25. 4
Kjetland EF, Ndhlovu PD, Kurewa EN, et al. Prevention of gynecologic contact bleeding
and genital sandy patches by childhood anti-
schistosomal treatment. Am J Trop Med Hyg
2008; 79: 79–83. 4
Kjetland EF, Ndhlovu PD, Kurewa EN, et al. Prevention of gynecologic contact bleeding
and genital sandy patches by childhood anti-
schistosomal treatment. Am J Trop Med Hyg
2008; 79: 79–83. JS reports grants from Merck outside the submitted
work. All other authors declare no competing
interests. 5
Ezeamama AE, Bustinduy AL, Nkwata AK, et al. Cognitive deficits and educational loss in
children with schistosome infection—a
systematic review and meta-analysis. PLoS Negl Trop Dis 2018; published online
Jan 12. DOI:10.1371/journal.pntd.0005524. 5
Ezeamama AE, Bustinduy AL, Nkwata AK, et al. Authors’ reply King and Galvani state that “many of
the case count data remain inferred”. Although the prevalence model infers
prevalence information from countries
in similar regions, this prevalence
model is representative of the PAR
and only uses data in those at risk. These prevalence estimates were
then scaled to represent prevalence
at the GBD location level through
the PAR estimates extracted from
the environmental suitability maps,
which used the association between
environmental factors and more than
12 000 geolocated records. Institute for Health Metrics and Evaluation, Seattle,
WA 98121, USA Department of Error Sinharay R, Gong J, Barratt B, et al. Respiratory and
cardiovascular responses to walking down a traffic-
polluted road compared with walking in a traffic-free
area in participants older than 60 years with chronic
lung or heart disease and age-matched healthy
controls: a randomised, crossover study. Lancet
2017; 391: 339–49—In this Article (published
online first on Dec 5, 2017), the corresponding
author has been corrected, the middle initial for
Frank Kelly has been added, the role of the
funding source has been updated, and author
initials have been updated throughout. These
corrections have been made to the online
version as of Jan 25, 2018, and the printed
Article is correct. King and Galvani also state that
“severe and yet common clinical
outcomes of schistosomiasis were not
integrated into the disability weights”
and “serious clinical manifestations
of intestinal schistosomiasis were
not incorporated”. We will address 308 www.thelancet.com Vol 391 January 27, 2018
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Aldosterone-to-renin ratio is related to arterial stiffness when the screening criteria of primary aldosteronism are not met
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Aldosterone‑to‑renin ratio
is related to arterial stiffness
when the screening criteria
of primary aldosteronism are
not met
OPEN Eeva Kokko1, Pasi I. Nevalainen1,2, Manoj Kumar Choudhary1, Jenni Koskela1,2,
Antti Tikkakoski1,3, Heini Huhtala4, Onni Niemelä1,5, Marianna Viukari6, Jukka Mustonen1,2,
Niina Matikainen6 & Ilkka Pörsti1,2* Aldosterone-to-renin ratio (ARR) is a screening tool for primary aldosteronism (PA), but the
significance of ARR when the PA criteria are not met remains largely unknown. In this cross-sectional
study we investigated the association of ARR with haemodynamic variables in 545 normotensive
and never-medicated hypertensive subjects (267 men, 278 women, age range 19–72 years) without
suspicion of PA. Supine haemodynamic data was recorded using whole-body impedance cardiography
and radial tonometric pulse wave analysis. In sex-adjusted quartiles of ARR, determined as serum
aldosterone to plasma renin activity ratio, the mean values were 282, 504, 744 and 1467 pmol/
µg of angiotensin I/h, respectively. The only difference in haemodynamic variables between the
ARR quartiles was higher pulse wave velocity (PWV) in the highest quartile versus other quartiles
(p = 0.004), while no differences in blood pressure (BP), heart rate, wave reflections, cardiac output or
systemic vascular resistance were observed between the quartiles. In linear regression analysis with
stepwise elimination, ARR was an independent explanatory factor for PWV (β = 0.146, p < 0.001, R2
of the model 0.634). In conclusion, ARR was directly and independently associated with large arterial
stiffness in individuals without clinical suspicion of PA. Therefore, ARR could serve as a clinical marker
of cardiovascular risk. Trial registration: ClinicalTrails.gov: NCT01742702. Primary aldosteronism (PA) is the most common form of secondary hypertension1, yet most cases remain
undiagnosed2,3. The estimated prevalence of PA has conventionally ranged from 5 to 15% among hypertensive
patients4–6, but according to a recent study, the prevalence might be as high as 16% to 22%7. It is known that
PA is associated with significantly higher risk of cardiovascular events and target organ damage than essential
hypertension at comparable levels of blood pressure5,8,9. Aldosterone excess increases the accumulation of growth
factors and collagen fibres in arterial wall10,11 and it has been shown that PA patients have an increased arterial
wall stiffness in comparison with patients who have essential hypertension12,13.h f
p
p
yp
Screening of PA is based on the aldosterone-to-renin ratio (ARR). www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2020) 10:19804 Methods
St d
b Study subjects. The recruitment of the study subjects has previously been described in detail21–23. All
subjects were examined by a physician and routine laboratory analyses for elevated blood pressure (BP) were
taken24. The medical history, lifestyle behaviour and family history were documented. Alcohol use was evalu-
ated as standard drinks (~ 12 g of absolute alcohol) per week, and smoking amount was estimated in pack-years. The exclusion criteria were history of coronary artery disease, stroke, cardiac insufficiency, valvular heart dis-
ease, chronic kidney disease25, secondary hypertension, alcohol or substance abuse26, psychiatric illnesses other
than mild to moderate depression or anxiety, heart rhythm other than sinus rhythm, use of antihypertensive
or uric-acid-lowering medications, and ongoing pregnancy. Altogether 606 subjects, aged 19–72 years, were
eligible for the study. Data of ARR was missing in 48 subjects while 13 subjects met the screening criteria of PA
(serum aldosterone > 550 pmol/l and ARR > 750 pmol/µg of angiotensin I/h, plasma potassium concentration
< 3.3 mmol/l, BP > 150/100 mmHg) and were therefore excluded. The Endocrine Society screening for PA is
based on BP > 150/100 mmHg on three different days1,5. In the present study the BP criteria were applied on the
5-min laboratory BP measurements (see below), and the results were confirmed by applying the criteria for the
office BP measurements on a single occasion. The final study population consisted of 545 subjects.h fi
ghi
y p p
j
Signed informed consent was obtained from all participants. The study complies with the declaration of
Helsinki and was approved by the Ethics Committee of the Pirkanmaa Hospital District (study code R06086M). Laboratory analyses and indexes glucose tolerance. Blood and urine samples were taken after 12 h
of fasting. Plasma renin activity (GammaCoat Plasma Renin Activity 125-I RIA Kit, DiaSorin, Saluggia, Italy)
and aldosterone concentration (Active Aldosterone RIA, Beckman Coulter, Fullerton, CA, USA) were deter-
mined using commercial kits. Plasma C-reactive protein (CRP), sodium, potassium, glucose, creatinine, cystatin
C, uric acid, triglyceride, and total, HDL and LDL (high- and low-density lipoprotein, respectively) cholesterol
concentrations were determined using Cobas Integra 800 (F. Hoffmann-LaRoche Ltd, Basel, Switzerland). Insu-
lin and parathyroid hormone (PTH) were determined using electrochemiluminescence immunoassay (Cobas
e411, Roche Diagnostics). Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney
Disease – Epidemiology collaboration (CKD-EPI) cystatin C equation27. www.nature.com/scientificreports/ events at a young age (< 40 years), hypertension together with sleep apnoea, and all first-degree relatives of
patients with PA1,5.hi p
The role of ARR for screening of PA is robust, but there is a scarcity of data evaluating the significance of
ARR in subjects who remain below the screening threshold for PA. Previously, ARR has been associated with the
development and severity of hypertension, even in patients without excessive aldosterone levels14–18. A Japanese
long-term observational analysis demonstrated that elevated ARR is associated with an increased incidence of
cardiovascular disease in patients with essential hypertension19. In 60 healthy adults with a mean age 43 years,
Shapiro et al. reported a direct correlation (rP = 0.298) between ARR and pulse wave velocity (PWV)20. In 2000
participants of the Framingham study, Lieb et al. found that ARR was a significant independent correlate of PWV
(β = 0.20). However, the participants were not screened for the presence of PA, 33% were ingesting antihyper-
tensive medications, and 12% had diabetes mellitus18. ,
In this cross-sectional study we analysed the association of ARR with several cardiovascular variables in nor-
motensive subjects and previously undiagnosed never-medicated hypertensive patients. We tested the hypothesis
whether ARR correlates with haemodynamic parameters in subjects who are not fulfilling the clinical criteria
for the screening of PA. Aldosterone‑to‑renin ratio
is related to arterial stiffness
when the screening criteria
of primary aldosteronism are
not met
OPEN The Endocrine Society recommends the
screening in the following cases with clinical suspicion pf PA: Blood pressure > 150/100 mmHg on three different
days, drug resistant hypertension that is uncontrolled with ≥ three antihypertensive drugs, controlled hyperten-
sion with ≥ four antihypertensive drugs, hypertension with spontaneous or diuretic-induced hypokalaemia,
hypertension with adrenal incidentaloma, hypertension with a family history of hypertension or cerebrovascular 1Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland. 2Department of Internal
Medicine, Tampere University Hospital, Tampere, Finland. 3Department of Clinical Physiology, Tampere
University Hospital, Tampere, Finland. 4Faculty of Social Sciences, Tampere University, Tampere, Finland. 5Clinical
Laboratory and Medical Research Unit, Seinäjoki Central Hospital, Seinäjoki, Finland. 6Endocrinology, Helsinki
University Hospital and Research Programs Unit, Clinical and Molecular Medicine, University of Helsinki, Helsinki,
Finland. *email: ilkka.porsti@tuni.fi | https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 www.nature.com/scientificreports/ Methods
St d
b Insulin sensitivity was evaluated by the
quantitative insulin sensitivity check index (QUICKI)28, and homeostatic model assessment of insulin resistance
(HOMA-IR)29. In addition, the subjects were invited to participate in a standard 75-g oral glucose tolerance test
(OGTT) for the determination of the Matsuda index30. Experimental protocol. Haemodynamic recordings were performed in a noiseless, temperature-controlled
laboratory by research nurses21,22,31. Products containing caffeine, smoking or heavy meal were not allowed for
≥ 4 h, and alcohol consumption was not allowed for ≥ 24 h before the investigation. The subjects rested supine
on the examination table with impedance cardiography electrodes placed on body surface, tonometric sensor for
pulse wave analysis on left radial pulsation, and oscillometric brachial cuff for BP calibration to the right upper
arm. The left arm with the tonometric sensor was stabilized to 90° in a support, which held the measurement
probe at the heart level.h p
The measurement consisted of a 5-min period, during which haemodynamic data was captured continuously. For the statistical analyses, the mean values of each 1-min period of recording were calculated. The repeatability
and reproducibility of the protocol have been demonstrated to be good21,22,31. Pulse wave analysis. Radial BP and pulse wave were recorded by a tonometric sensor (Colin BP-508 T,
Colin Medical Instruments Corp., USA) fixed on the radial pulse21,22. The radial BP signal was calibrated approx-
imately every 2.5 min by brachial BP measurements. Aortic BP was derived using the SphygmoCor pulse wave
monitoring system (SphygmoCor PWMx, AtCor medical, Australia)32. Aortic pulse pressure, augmentation
index (AIx, augmented pressure/pulse pressure * 100), and AIx adjusted to heart rate 75/min (AIx@75) were
also determined. The central forward wave amplitude was defined as the difference between waveform foot and
first systolic inflection point pressure in the aortic waveform33,34. Whole‑body impedance cardiography. We used the CircMon device (JR Medical Ltd., Tallinn, Estonia)
to assess changes in body electrical impedance during cardiac cycles to record heart rate, stroke volume, cardiac Scientific Reports | (2020) 10:19804 | https://doi.org/10.1038/s41598-020-76718-7 www.nature.com/scientificreports/ Table 1. Results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio: demographic
data and clinical characteristics. BP, blood pressure; mean (standard deviation) or median [25th–75th
percentile]. *P < 0.05 versus Q1; †P < 0.05 versus Q2. a Office BP was available from 123–129 participants per
group. Methods
St d
b b Haemodynamic measurement systolic/diastolic BP values were 12.7–15.6/16.0–17.3 mmHg, and heart
rate 2.8–4.5 beats/min, lower than in the office with no significant differences between the quartiles. Quartile 1 (n = 135)
Quartile 2 (n = 137)
Quartile 3 (n = 137)
Quartile 4 (n = 136)
Male/female (n)
66/69
67/70
67/70
67/69
Number of female hormone users
16
12
21
21
Number of oestrogen users
11
10
18
16
Age (years)
41.9 (11.4)
44.8 (11.5)
46.0 (11.5)*
49.5 (10.9)*†
Weight (kg)
78.7 (15.8)
79.6 (15.6)
80.0 (14.8)
81.8 (15.0)
Height (cm)
173.2 (9.5)
172.2 (9.6)
173.8 (8.8)
172.7 (9.2)
Body mass index (kg/m2)
26.1 (4.2)
26.8 (4.6)
26.4 (4.1)
27.3 (3.9)
Alcohol (standard drinks/week)
3 [0–5]
3 [1–6]
3 [1–6]
2 [0–4]
Current smokers (n)
22
18
11
13
Office measurementsa
Systolic BP (mmHg)
135.3 (20.0)
137.5 (20.2)
139.7 (18.6)
144.1 (21.0)*
Diastolic BP (mmHg)
86.2 (13.1)
87.6 (12.2)
88.2 (10.7)
91.8 (11.6)*†
Heart rate (bpm)
67.5 (9.6)
67.0 (9.3)
65.2 (9.6)
67.8 (9.1)
Normotensive/hypertensive (n)
75 / 60
71 / 66
67 / 70
49 / 87*
Haemodynamic measurementsb
Systolic BP (mmHg)
127.4 (18.1)
129.0 (18.1)
129.1 (17.8)
132.0 (18.3)
Diastolic BP (mmHg)
72.5 (11.9)
73.4 (12.0)
73.4 (12.0)
76.1 (12.8)
Heart rate (bpm)
63.9 (10.2)
63.8 (10.3)
62.1 (9.3)
63.6 (8.1)
Normotensive/hypertensive (n)
107 / 28
103 / 34
100 / 37
95 / 41 Table 1. Results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio: demographic
data and clinical characteristics. BP, blood pressure; mean (standard deviation) or median [25th–75th
percentile]. *P < 0.05 versus Q1; †P < 0.05 versus Q2. a Office BP was available from 123–129 participants per
group. b Haemodynamic measurement systolic/diastolic BP values were 12.7–15.6/16.0–17.3 mmHg, and heart
rate 2.8–4.5 beats/min, lower than in the office with no significant differences between the quartiles. output, and PWV35–37. Systemic vascular resistance was calculated from radial BP and cardiac output measured
by CircMon. Stroke volume, cardiac output and systemic vascular resistance were presented as indexes related
to body surface area calculated using the DuBois equation38. The method and electrode configuration has been
previously reported36,37. p
y
p
With CircMon the recorded stroke volume and cardiac output are in good agreement with values obtained
utilizing 3-dimensional echocardiography31 and the thermodilution and direct oxygen Fick methods35,36, and the
PWV values show very good correlation with values measured using ultrasound or the tonometric method37,39. Statistics. Methods
St d
b To illustrate the influence of ARR on the haemodynamic variables, the study participants were
divided into quartiles of ARR. As serum aldosterone concentration was slightly higher in women than in men
(see ‘Results’), the ARR quartiles 1–4 were constructed separately for women and men. q
p
y
Analyses of normally distributed data were performed using analysis of variance (ANOVA) and ANOVA
for repeated measurements, and non-normally distributed data using Kruskal–Wallis test with Mann–Whitney
U-test in the post-hoc analyses. The Bonferroni correction was applied in all post-hoc analyses. The ANOVA for
repeated measures analyses were adjusted for sex, age, BMI, and eGFR, as appropriate, by using these variables
as covariates in the analyses. Normally distributed variables in the tables were presented as means and stand-
ard deviations and non-normally distributed variables as medians and 25th–75th percentiles. The figures were
depicted as means and standard errors of the mean (SEM). Pearson’s correlations were calculated for normally
distributed and Spearman’s correlations for non-normally distributed variables. The values of the haemodynamic
variables for the statistics in Table 1 and regression analysis were calculated as averages from the minutes 3–5 of
the recordings when the signal was most stable. P < 0.05 was considered significant. g
g
gi
To investigate factors independently associated with PWV in the whole study population, linear regression
analysis with stepwise elimination was applied. The covariates in the analysis were age, sex, BMI, categorised
smoking status (never, present, previous), categorised alcohol consumption (low, moderate, high); plasma aldos-
terone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid, calci-
triol, PTH, QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. Coefficients (B) and standard
coefficients (Beta) of regression were calculated, and assumptions of linearity were confirmed by the analysis of
residuals. IBM SPSS Statistics Version 26 (IBM Corporation, Armonk, NY, USA) was used for statistics. Results
S
d Study population and laboratory values. The proportion of male subjects was 49%. Plasma renin activ-
ity and ARR did not significantly differ between women versus men [0.99 (0.90) vs. 0.97 (0.95) ng of angiotensin
I/ml/h; 779 (527) vs. 719 (531) pmol/µg of angiotensin I/h, respectively; mean (SD)]. However, serum aldoster-
one concentration was higher in women than in men (561 (481) vs. 449 (208) pmol/l, respectively, p < 0.001). Therefore, the ARR results were examined in sex-adjusted quartiles (Q) (Table 1). https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ Table 2. Laboratory results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio. Results shown as mean (standard deviation) or median [25th–75th percentile]; GFR, estimated glomerular
filtration rate from plasma cystatin C using the CKD-EPI formula27. HDL, high-density lipoprotein; LDL,
low-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; QUICKI, quantitative
insulin sensitivity check index. *P < 0.05 versus Q1; †P < 0.05 versus Q2; ‡P < 0.05 versus Q3. a n = 101–118 for
24-h urine excretion results. b n = 88–98 for Matsuda index results in each quartile. Results
S
d Quartile 1 (n = 135)
Quartile 2 (n = 137)
Quartile 3 (n = 137)
Quartile 4 (n = 136)
Aldosterone-to-renin ratio (pmol/µg of
Ang I/h)
287 [227–342]
497 [451–560]*
740 [674–799]*†
1302 [1085–1614]*†‡
Serum aldosterone (pmol/l)
419 [297–552]
448 [324–599]
431 [336–636]
427 [323–575]
Plasma renin activity (ng of Ang I/ml/h)
1.47 [1.12–2.17]
0.89 [0.67–1.21]*
0.58 [0.46–0.88]*†
0.29 [0.20–0.44]*†‡
Sodium (mmol/l)
140.4 (1.8)
139.9 (1.8)
140.1 (2.1)
140.7 (1.9)†
24-h urine sodium excretion (mmol)a
153 (62)
155 (61)
154 (65)
148 (57)
Potassium (mmol/l)
3.8 (0.2)
3.8 (0.3)
3.8 (0.3)
3.8 (0.3)
24-h urine potassium excretion (mmol)a
85 (30)
87 (30)
86 (27)
79 (22)
Calcium (mmol/l)
2.30 (0.09)
2.29 (0.10)
2.30 (0.10)
2.29 (0.10)
Parathyroid hormone (pmol/l)
4.37 (1.59)
4.37 (1.41)
4.43 (1.49)
4.71 (1.59)
25OH-D3 (nmol/l)
74.2 (33.5)
66.1 (28.3)
74.9 (45.4)
70.7 (30.0)
1,25(OH)2-D3 (pmol/l)
110.2 (31.4)
109.3 (36.3)
107.6 (33.8)
106.7 (34.9)
C-reactive protein (mg/l)
0.7 [0.5–1.5]
1.0 [0.5–2.2]
0.5 [0.4–1.3]†
1.0 [0.5–2.1]‡
Creatinine (µmol/l)
74 (13)
72 (13)
75 (14)
73 (14)
Cystatin C
0.81 (0.14)
0.83 (0.14)
0.85 (0.15)
0.86 (0.15)
Estimated GFR (ml/min/1.73 m2)
104.7 (18.1)
100.9 (17.8)
98.3 (18.0)*
95.3 (17.1)*
Total cholesterol (mmol/l)
5.0 (1.0)
5.1 (1.0)
5.0 (1.0)
5.3 (1.0)*
Triglycerides (mmol/l)
1.11 [0.67–1.38]
1.22 [0.74–1.43]
1.21 [0.67–1.46]
1.27 [0.87–1.54]
HDL cholesterol (mmol/l)
1.6 (0.4)
1.6 (0.5)
1.6 (0.4)
1.5 (0.4)
LDL cholesterol (mmol/l)
2.9 (0.9)
3.0 (1.0)
2.9 (0.9)
3.2 (0.9)
Uric acid (µmol/l)
302 (71)
297 (65)
292 (71)
303 (86)
Glucose (mmol/l)
5.3 (0.5)
5.5 (0.5)
5.4 (0.5)
5.6 (0.8)*
Insulin (mU/l)
6.8 [5.0–10.4]
6.8 [5.3–9.7]
5.9 [4.3–8.1]
7.0 [4.7–9.4]
Matsuda indexb
5.74 [3.74–8.18]
5.94 [3.50–8.78]
7.62 [4.44–9.69]
5.80 [3.89–7.72]
HOMA-IR
1.56 [1.14–2.61]
1.70 [1.23–2.37]
1.43 [1.02–2.11]
1.69 [1.13–2.40]
QUICKI
0.357 [0.331–0.375]
0.352 [0.335–0.371]
0.362 [0.341–0.382]
0.353 [0.335–0.376] Table 2. Laboratory results of the study participants in sex adjusted quartiles of aldosterone-to-renin ratio. Results shown as mean (standard deviation) or median [25th–75th percentile]; GFR, estimated glomerular
filtration rate from plasma cystatin C using the CKD-EPI formula27. HDL, high-density lipoprotein; LDL,
low-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; QUICKI, quantitative
insulin sensitivity check index. *P < 0.05 versus Q1; †P < 0.05 versus Q2; ‡P < 0.05 versus Q3. a n = 101–118 for
24-h urine excretion results. b n = 88–98 for Matsuda index results in each quartile. None of the subjects used anti-hypertensive medicines. Results
S
d The proportions of female hormone users did not dif-
fer between the ARR quartiles. Mean participant age was 46 years, with an age range of 19–72 years in male and
21–72 years in female subjects. Participant age was higher in Q3 versus Q1, and in Q4 versus Q1 and Q2. Mean
BMI was 26.6 kg/m2 with no differences between the quartiles. Average alcohol use was moderate 4.3 standard
drinks/week, and the prevalence of present and previous smokers was 12% and 31%, respectively. Alcohol intake
and the prevalence of smokers were corresponding in all quartiles (Table 1).fif p
p
g
q
(
)
When applying the office cut-off values of hypertension (systolic BP ≥ 140 or diastolic BP ≥ 90)24, the number
of never-medicated hypertensive subjects was 283 (51.9%). Office systolic BP was higher in Q4 than in Q1, and
office diastolic BP in Q4 than in Q1 and Q2. The proportion of hypertensive subjects in the office measurements
was higher in Q4 than in Q1. g
During the haemodynamic measurements, systolic and diastolic BP values were 13–16 and 16–17 mmHg
lower, respectively, and heart rate 3–5 beats/min lower, than in the office measurements, with no significant
differences between the quartiles. With the office cut-off values of hypertension, no significant differences were
found in the proportion of hypertensive subjects between the quartiles during the haemodynamics measure-
ments (Table 1). In sex adjusted quartiles of ARR, the mean values were 282, 504, 744 and 1467 pmol/µg of angiotensin I/h,
respectively (Table 2). The differences in ARR were explained by variations in plasma renin activity, as aldoster-
one concentrations did not differ between the quartiles. Plasma renin activity was different in all quartiles with
highest values in Q1 and lowest values in Q4. In our study, age was inversely correlated both with plasma renin
activity and serum aldosterone concentration (rS = − 0.369 and rS = − 0.253, respectively, p < 0.01 for both), while
age and ARR were directly correlated (rS = 0.238, p < 0.01). g
y
( S
p
)
Average measures of plasma electrolytes, PTH, CRP, uric acid, glucose metabolism and renal function
were within the normal range in all quartiles (Table 2). In seven participants fasting plasma glucose ranged
7.0–10.3 mmol/l, i.e. in the diabetic range, but none of them presented with glucosuria. Altogether 53 participants
had impaired fasting plasma glucose concentration ranging 6.2–6.9 mmol/l. Scientific Reports | (2020) 10:19804 | Results
S
d Plasma sodium concentration was
minimally higher in Q4 than in Q2. No differences were observed in 24-h excretion of sodium or potassium
to the urine, plasma potassium, calcium, PTH, creatinine, uric acid, triglycerides, HDL cholesterol or LDL https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ Figure 1. Radial systolic (a) and diastolic (b) blood pressure calibrated from brachial blood pressure
measurements, and aortic systolic (c) and diastolic (d) blood pressure in quartiles (n = 135–137) of aldosterone-
to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM,
analysis of variance for repeated measurements results are depicted as mean and standard error of the mean Figure 1. Radial systolic (a) and diastolic (b) blood pressure calibrated from brachial blood pressure
measurements, and aortic systolic (c) and diastolic (d) blood pressure in quartiles (n = 135–137) of aldosterone-
to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM,
analysis of variance for repeated measurements, results are depicted as mean and standard error of the mean. Figure 1. Radial systolic (a) and diastolic (b) blood pressure calibrated from brachial blood pressure
measurements, and aortic systolic (c) and diastolic (d) blood pressure in quartiles (n = 135–137) of aldosterone-
to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM,
analysis of variance for repeated measurements, results are depicted as mean and standard error of the mean. cholesterol. However, total cholesterol was somewhat higher in Q4 when compared to Q1. Estimated GFR was
lower in Q3 and Q4 when compared to Q1. Although fasting plasma glucose concentration was higher in Q4
than in Q1, all the evaluated insulin sensitivity indices (QUICKI, HOMA-IR, Matsuda index) were invariable
between the ARR quartiles (Table 2). Haemodynamic measurements. ARR and haemodynamics. The results representing the haemodynamic
variables in the sex-, age-, and eGFR-adjusted quartiles of ARR are shown in Figs. 1, 2 and 3. No differences in
radial or aortic BP were observed between the quartiles (Fig. 1). Heart rate, stroke index, cardiac index and sys-
temic vascular resistance index were also similar in all quartiles (Fig. 2). No differences were found in forward
wave amplitude, augmentation index or extracellular water balance between the quartiles, either. However, in
the absence of differences in central BP, aortic to popliteal PWV was clearly higher in Q4 versus other quartiles
(p = 0.004) (Fig. 3). Results
S
d Among the study subjects, the Spearman correlation between ARR and PWV was 0.219
(p < 0.01). p
Aldosterone and haemodynamics. When examined in sex-adjusted quartiles of serum aldosterone con-
centration, the quartiles presented with differences in age and BMI (not shown). In the sex-, age- and BMI-
adjusted quartiles of serum aldosterone concentration, no differences in the haemodynamic variables were
detected between the quartiles except for PWV, the value of which was lower in Q1 versus Q4 (8.12 ± 0.12 vs. 8.58 ± 0.12 m/s, p = 0.045). p
Renin activity and haemodynamics. In sex-adjusted quartiles of plasma renin activity, no additional adjust-
ments were needed (not shown). Corresponding to the results in quartiles of aldosterone concentration, all
haemodynamic variables in quartiles of renin activity were similar except for PWV. However, the difference in
PWV was observed between Q1 and Q2 (8.65 ± 0.13 vs. 8.08 ± 0.12 m/s, p = 0.006), while PWV in Q1, Q3 and
Q4 was corresponding (ranging from 8.37 ± 0.12 to 8.65 ± 0.12 m/s, p = 0.559–1.000). Determinants of large arterial stiffness. The results of the linear regression analyses with stepwise
elimination are presented in Table 3. The independent explanatory factors for PWV were age, ejection duration, Scientific Reports | (2020) 10:19804 | https://doi.org/10.1038/s41598-020-76718-7 www.nature.com/scientificreports/ Figure 2. Heart rate (a), stroke index (b), cardiac index (c) and systemic vascular resistance index (d) in
quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated
glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, mean and standard
error of the mean. Figure 2. Heart rate (a), stroke index (b), cardiac index (c) and systemic vascular resistance index (d) in
quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were adjusted for sex, age, and estimated
glomerular filtration rate; ANOVA-RM, analysis of variance for repeated measurements, mean and standard
error of the mean. uric acid concentration, mean aortic pressure, ARR, BMI, low alcohol consumption, and heart rate (R2 = 0.634). The variables in the model included both aldosterone concentration and renin activity, yet neither of them
turned out as independent predictors for PWV. QUICKI was used as an insulin sensitivity variable in the model,
as the Matsuda index was not available from all participants (Table 2). Results
S
d In our study population the correla-
tion between QUICKI and Matsuda index was 0.884 (p < 0.001) and between QUICKI and HOMA-IR-0.720
(p < 0.001). p
When the office cut-off for hypertension (BP ≥ 140/90 mmHg) was applied for the laboratory measurements24,
ARR was an independent explanatory factor for PWV in both the normotensive (p < 0.001, R2 = 0.580) and
hypertensive (p = 0.012, R2 = 0.523) subgroups. The result did not change when applying the home BP measure-
ment cut-off (BP ≥ 135/85 mmHg).fi f
If the office BP measurements were applied for the exclusion of participants with potential PA, 499 subjects
were eligible for the statistical analyses. Also, in this analysis ARR was an independent explanatory factor for
PWV (p < 0.001), while the other explanatory factors were age, mean aortic pressure, heart rate, and plasma
concentrations of HDL cholesterol, uric acid, and triglycerides (R2 = 0.572 of the model). Discussion In this study we investigated the association of ARR with several cardiovascular variables in normotensive sub-
jects and never-medicated hypertensive patients without clinical suspicion of PA and without cardiovascular
or renal comorbidities, and cardiovascular medications. In analyses adjusted for confounding factors, ARR was
significantly associated with PWV but not with any other haemodynamic variable. The linear regression analyses
confirmed that ARR was an independent explanatory factor for PWV, an acknowledged marker of large arterial
stiffness40,41. As increased arterial stiffness is a strong independent predictor of cardiovascular events41, while
higher ARR is associated with an increased incidence of cardiovascular disease19 and predicts future stroke in
hypertensive patients42, a higher ARR may predispose to the future development of cardiovascular diseases.if yp
p
g
y p
p
p
Age is the most significant explanatory factor for large arterial stiffness43,44. In the present study, subjects in
the highest ARR quartile were older than in the other quartiles. Consequently, the Figs. 1, 2 and 3 were adjusted https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ Figure 3. Forward wave amplitude (a), augmentation index (b), extracellular water balance (c) and aortic-to-
popliteal pulse wave velocity (d) in quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were
adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated
measurements, mean and standard error of the mean. Figure 3. Forward wave amplitude (a), augmentation index (b), extracellular water balance (c) and aortic-to-
popliteal pulse wave velocity (d) in quartiles (n = 135–137) of aldosterone-to-renin ratio (ARR); analyses were
adjusted for sex, age, and estimated glomerular filtration rate; ANOVA-RM, analysis of variance for repeated
measurements, mean and standard error of the mean. Table 3. Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression
analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity
with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma
aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid,
calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density
lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR,
estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI formula27. Table 3. Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression
analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity
with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma
aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid,
calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density
lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR,
estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI formula27. www.nature.com/scientificreports/ for differences in age in addition to sex and estimated GFR. Moreover, despite the presence of age in the regres-
sion model, ARR was an independent explanatory factor for PWV. The inverse correlation between age and
plasma renin is known from previous studies45. High sodium intake would also lower renin1,5, but this was not
the cause for lower renin in the ARR quartiles 2–4 versus quartile 1, as 24-h sodium excretion to the urine was
similar in all quartiles.h q
The screening positive cases with putative PA were excluded from our study population that was predomi-
nantly normotensive during the haemodynamic measurements. We found that ARR was directly and indepen-
dently associated with large arterial stiffness, while the linear regression model displayed no direct association
between serum aldosterone concentration or plasma renin activity and PWV. This finding may reflect higher
renin-independent aldosterone release in the participants with high ARR. Indeed, a continuum of renin-inde-
pendent aldosteronism in normotensive subjects has been previously identified46,47. High aldosterone levels
related to prevailing plasma renin activity may predispose to the chronic adverse effects of aldosterone in the
vascular system already prior to the clinical diagnosis of PA or hypertension46. The molecular basis for this
continuum of aldosterone secretion may be explained by the aldosterone-producing cell clusters that have been
discovered in the zona fasciculata of morphologically normal non-neoplastic adrenal glands: these clusters may
be found in > 50% of normal adrenal glands, leading to mild autonomous aldosterone secretion and increased
ARR with increasing age far more commonly than previously perceived48,49.f g g
y
p
y p
Some former studies have suggested a direct association between ARR and arterial stiffness. Shapiro et al. reported a significant association of ARR with PWV in 60 normotensive subjects, but in contrast to our study,
their approach did not provide evidence of ARR as an independent explanatory factor of PWV20. In the Framing-
ham study, Lieb et al. found that ARR was directly associated with PWV and four other measures of vascular
pathophysiology18. As a major difference to our study, medicated hypertensive subjects and patients with known
diabetes were not excluded from their analyses. Also, there was a 3-year interval between the laboratory exami-
nations and haemodynamic measurements, whereas in our study these procedures were performed within a
median period of 8 days from one another (25th to 75th percentile 2–17 days). www.nature.com/scientificreports/ A small study in 24 patients with
essential hypertension by Mahmud and Feely found no correlation between ARR and PWV, but showed that
aldosterone antagonist-induced decrease in systolic BP correlated with pre-treatment ARR50. However, in 102
patients with confirmed PA, no correlation was found between ARR and baseline PWV, or between ARR and
the reduction in PWV following adrenalectomy51. Yet, the subsequent increase in plasma renin activity after
adrenalectomy was an explanatory factor for the reduction in PWV during 6 months of follow-up51. Altogether,
the effects of aldosterone excess on large arterial stiffness may be manifested early in the course of the disease,
and the beneficial changes in the vasculature following surgical treatment do directly correlate with the plasma
concentrations of aldosterone or ARR. Noteworthy, in the present study the systolic and diastolic BP values were 13–16 and 16–17 mmHg lower,
respectively, during the haemodynamic laboratory measurements than in the office measurements. This resem-
bles the white-coat effect in hypertension, which has been related to large arterial stiffness in both untreated52
and treated hypertensive patients53. Out-of-office BP monitoring provides better assessment of overall BP and
response to treatment in patients with white-coat hypertension54. The average difference between the office and
laboratory BP in our study was rather large, whereby haemodynamic measurements under standard conditions
can also result in clearly lower BP when compared with the office measurements. The correlations between office
and laboratory measurements were 0.70 and 0.65 for systolic and diastolic BP, respectively. We used the algo-
rithm recommended by The Endocrine Society for the screening of PA patients1,5, in order to exclude potential
PA patients from the study population. Regardless of whether office BP > 150/100 mmHg measured on a single
occasion, or average BP > 150/100 mmHg during haemodynamic measurements, was applied for the exclusion
of participants, ARR was an independent explanatory factor for PWV. Of note, the results were corresponding
when studied in normotensive and hypertensive subjects with two different cut-off values for hypertension
(140/90 or 135/85 mmHg). g
Seven of the present participants had fasting plasma glucose in the diabetic range, 53 participants had
impaired fasting plasma glucose, while 89% of the participants presented with glucose values within the normal
range. PA has been linked to abnormalities in glucose metabolism, especially to insulin resistance and impaired
glucose tolerance55–57. Discussion Pulse wave velocity (m/s)
B
Beta
P
R2 = 0.634
(constant)
5.046
Age
0.071
0.488
< 0.001
Ejection duration
− 0.016
− 0.185
< 0.001
Uric acid
0.005
0.199
< 0.001
Mean aortic pressure
0.018
0.148
< 0.001
Aldosterone-to-renin ratio
4.68 × 10–4
0.146
< 0.001
Body mass index
0.041
0.103
0.001
Heart rate
0.017
0.098
0.010
Low alcohol consumption category
− 0.220
− 0.062
0.025 Pulse wave velocity (m/s)
B
Beta
P
R2 = 0.634
(constant)
5.046
Age
0.071
0.488
< 0.001
Ejection duration
− 0.016
− 0.185
< 0.001
Uric acid
0.005
0.199
< 0.001
Mean aortic pressure
0.018
0.148
< 0.001
Aldosterone-to-renin ratio
4.68 × 10–4
0.146
< 0.001
Body mass index
0.041
0.103
0.001
Heart rate
0.017
0.098
0.010
Low alcohol consumption category
− 0.220
− 0.062
0.025 Pulse wave velocity (m/s)
B
Beta
P
R2 = 0.634
(constant)
5.046
Age
0.071
0.488
< 0.001
Ejection duration
− 0.016
− 0.185
< 0.001
Uric acid
0.005
0.199
< 0.001
Mean aortic pressure
0.018
0.148
< 0.001
Aldosterone-to-renin ratio
4.68 × 10–4
0.146
< 0.001
Body mass index
0.041
0.103
0.001
Heart rate
0.017
0.098
0.010
Low alcohol consumption category
− 0.220
− 0.062
0.025 Table 3. Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression
analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity
with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma
aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid,
calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density
lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR,
estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI formula27. Table 3. Significant explanatory variables for aortic to popliteal pulse wave velocity in linear regression
analysis with stepwise elimination. Variables included in the model correlated with pulse wave velocity
with a p-value < 0.05: age, sex, BMI, categorised smoking status, categorised alcohol consumption; plasma
aldosterone, renin, aldosterone-to-renin ratio, HDL cholesterol, LDL cholesterol, triglycerides, CRP, uric acid,
calcitriol, PTH; QUICKI, eGFR, ejection duration, heart rate, and mean aortic pressure. HDL, high-density
lipoprotein; LDL, low-density lipoprotein; QUICKI, quantitative insulin sensitivity check index; eGFR,
estimated glomerular filtration rate from plasma cystatin C using the CKD-EPI formula27. https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ However, the approach to examine central haemodynamics instead of plainly focusing on
brachial artery pressure, may be better related with the level of cardiovascular risk64,65. y p
y
In conclusion, a direct association between ARR and PWV was observed in 545 normotensive and never-
treated hypertensive subjects when the screening criteria of PA were not met. Therefore, our results indicate
that ARR is related to arterial stiffness in individuals without clinical suspicion of PA according to the prevail-
ing guidelines. Altogether, increased ARR in the absence of stage II and more severe hypertension should be
recognized as an indicator of increased cardiovascular risk. Data availability Analyses and generated datasets during the current study are not available publicly as our clinical database
contains several indirect identifiers and the informed consent obtained does not allow publication of individual
patient data. The datasets are available from the corresponding author on reasonable request. Received: 3 August 2020; Accepted: 2 November 2020 www.nature.com/scientificreports/ Even in the absence of PA, plasma aldosterone concentration was related to insulin resist-
ance in 251 male African American subjects58. Higher ARR was also associated with insulin resistance in 483
young adult African Americans without cardiovascular or renal disease59. In our study, no differences in insulin
sensitivity between the ARR quartiles were observed as the indices QUICKI, HOMA-IR and Matsuda did not
deviate. Therefore, the differences in PWV between the ARR quartiles could not be explained by variations in
insulin sensitivity. Also the plasma concentrations of uric acid, the levels of which are usually elevated in insulin
resistance and metabolic syndrome23, were similar in quartiles of ARR.l Changes in calcium metabolism may influence BP60, and induce alterations in the components of the renin-
angiotensin system in the vasculature61. Moreover, vitamin D receptor activation downregulates the synthesis of
renin in the juxtaglomerular cells and influences the expression of other components of the renin-angiotensin
system in the kidney62,63. In the present study, the plasma concentrations of calcium and vitamin D metabolites
were similar in the ARR quartiles. Therefore, putative changes in the metabolism of calcium and vitamin D were
not the explanations for the differences in arterial stiffness between the ARR quartiles.h ff
Our study has limitations. The cross-sectional design cannot substantiate causality. A potential selection bias
caused by the recruitment of voluntary subjects and the exclusion protocol must be acknowledged. The haemo-
dynamic recordings lasted for five minutes and the values of the last three minutes were used for the analyses,
which gives a rather short window of observation. Still, the analyses were based on average on 190 cardiac cycles
in each subject (mean HR in the study population was 63.3 beats/min). In addition, we applied indirect non-
invasive methods requiring mathematical processing to derive PWV, stroke volume and cardiac output from Scientific Reports | (2020) 10:19804 | https://doi.org/10.1038/s41598-020-76718-7 www.nature.com/scientificreports/ the bioimpedance signal36, and central aortic BP waveform from the applanation tonometry signal32. Therefore,
the results must be interpreted with caution, albeit the methods have been validated against direct or invasive
measurements31,35,37. However, the approach to examine central haemodynamics instead of plainly focusing on
brachial artery pressure, may be better related with the level of cardiovascular risk64,65. the bioimpedance signal36, and central aortic BP waveform from the applanation tonometry signal32. Therefore,
the results must be interpreted with caution, albeit the methods have been validated against direct or invasive
measurements31,35,37. References p
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0. Laurent, S. et al. Expert consensus document on arterial stiffness: methodological issues and clinical applications. Eur. Heart J. 27
2588–2605 (2006).f 1. Vlachopoulos, C., Aznaouridis, K. & Stefanadis, C. Prediction of cardiovascular events and all-cause mortality with arterial stiff-
ness. J. Am. Coll. Cardiol. 55, 1318–1327 (2010). 42. Satoh, M. et al. Aldosterone-to-renin ratio as a predictor of stroke under conditions of high sodium intake: the Ohasama study. Am. J. Hypertens. 25, 777–783 (2012).hf yp
43. Safar, M. E. & London, G. M. Therapeutic studies and arterial stiffness in hypertension: recommendations of the European Society
of Hypertension. J. Hypertens. 18, 1227–1535 (2000).ffl yp
yp
4. McEniery, C. M. et al. Normal vascular aging: differential effects on wave reflection and aortic pulse wave velocity: the Anglo-
Cardiff Collaborative Trial (ACCT). J. Am. Coll. Cardiol. 46, 1753–1760 (2005). f
45. Drayer, J. I., Weber, M. A., Laragh, J. H. & Sealey, J. E. Renin subgroups in essential hypertension. Clin. Exp. Hypertens. A 4,
1817–1834 (1982). 46. Acknowledgementsh g
The authors are deeply grateful to Paula Erkkilä, RN and Reeta Kulmala, RN for invaluable contribution to the
haemodynamic measurements. The authors wish to acknowledge CSC – IT Center for Science, Finland, for
computational resources. The study was supported by the Competitive State Research Financing of the Expert
Responsibility Area of Tampere University Hospital, Finnish Foundation for Cardiovascular Research, Päivikki
and Sakari Sohlberg Foundation, Pirkanmaa Regional Fund of the Finnish Cultural Foundation, Emil Aaltonen
Foundation, and Ida Montin Foundation. References Koskela, J. K. et al. Association of resting heart rate with cardiovascular function: a cross-sectional study in 522 Finnish subjects. BMC Cardiovasc. Disord. 13, 102 (2013). https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | Competing interests h Competing interests
The authors declare no competing interests. p
g
The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to I.P. Author contributions E.K. and I.P. reviewed the literature and wrote the original version of the manuscript. I.P., E.K. and H.H. per-
formed the statistical analyses. P.N., J.K., A.T. and I.P. carried out the clinical examinations of patients. E.K., P.N.,
M.K.C., J.K., A.T., J.M., M.V., N.M. and I.P. participated in the design of the technical details and methodology
of the study. O.N. was responsible for hormonal laboratory analyses. All authors contributed to the discussion https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ and editing the manuscript. I.P. was responsible for designing and conducting the study. All authors take the
responsibility for the contents of the manuscript. and editing the manuscript. I.P. was responsible for designing and conducting the study. All authors take the
responsibility for the contents of the manuscript. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 https://doi.org/10.1038/s41598-020-76718-7 Scientific Reports | (2020) 10:19804 |
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The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice
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Frontiers in immunology
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cc-by
| 9,101
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The GABAA Receptor Influences
Pressure Overload-Induced
Heart Failure by Modulating
Macrophages in Mice Jin Bu 1†, Shiyuan Huang 2†, Jue Wang 3, Tong Xia 2, Hui Liu 2, Ya You 2,
Zhaohui Wang 2* and Kun Liu 4* 1 Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China,
2 Department of Geriatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
China, 3 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, China, 4 Institution of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, China Keywords: GABAA receptor, amphiregulin, macrophage, monocyte, pressure-overload hypertrophy Edited by:
Guochang Hu,
University of Illinois at Chicago,
United States Background: Myocardial macrophages have key roles in cardiac remodeling and
dysfunction. The gamma-aminobutyric acid subtype A (GABAA) receptor was recently
found to be distributed in macrophages, allowing regulation of inflammatory responses to
various diseases. This study aimed to clarify the role of GABAA receptor-mediated
macrophage responses in pressure overload-induced heart failure. Reviewed by:
Adam R. Wende,
University of Alabama at Birmingham,
United States
Robert Morris Blanton,
Tufts Medical Center, United States
*Correspondence:
Zhaohui Wang
zhaohuiwang@hust.edu.cn
Kun Liu
liukun@hust.edu.cn
†These authors share first authorship Reviewed by:
Adam R. Wende,
University of Alabama at Birmingham,
United States
Robert Morris Blanton,
Tufts Medical Center, United States Reviewed by:
Adam R. Wende,
University of Alabama at Birmingham,
United States
Robert Morris Blanton,
Tufts Medical Center, United States Methods and Results: C57BL/6J mice underwent transverse aortic constriction for
pressure-overload hypertrophy (POH) and were intraperitoneally treated with a specific
GABAA receptor agonist (topiramate) or antagonist (bicuculline). Echocardiography,
histology, and flow cytometry were performed to evaluate the causes and effects of
myocardial hypertrophy and fibrosis. Activation of the GABAA receptor by topiramate
reduced ejection fraction and fractional shortening, enlarged the end-diastolic and end-
systolic left ventricular internal diameter, aggravated myocardial hypertrophy and fibrosis,
and accelerated heart failure in response to pressure overload. Mechanistically,
topiramate increased the number of Ly6Clow macrophages in the heart during POH
and circulating Ly6Chigh classic monocyte infiltration in late-phase POH. Further,
topiramate drove Ly6Clow macrophages toward MHCIIhigh macrophage polarization. As
a result, Ly6Clow macrophages activated the amphiregulin-induced AKT/mTOR signaling
pathway, and Ly6ClowMHCIIhigh macrophage polarization increased expression levels of
osteopontin and TGF-b, which led to myocardial hypertrophy and fibrosis. Conversely,
GABAA receptor blockage with bicuculline reversed these effects. *Correspondence:
Zhaohui Wang
zhaohuiwang@hust.edu.cn
Kun Liu
liukun@hust.edu.cn
†These authors share first authorship *Correspondence:
Zhaohui Wang
zhaohuiwang@hust.edu.cn
Kun Liu
liukun@hust.edu.cn
†These authors share first authorship Specialty section:
This article was submitted to
Inflammation,
a section of the journal
Frontiers in Immunology Specialty section:
This article was submitted to
Inflammation,
a section of the journal
Frontiers in Immunology
Received: 20 February 2021
Accepted: 20 April 2021
Published: 31 May 2021 Received: 20 February 2021
Accepted: 20 April 2021
Published: 31 May 2021 ORIGINAL RESEARCH
published: 31 May 2021
doi: 10.3389/fimmu.2021.670153 Animals and Treatments Six- to 8-week-old male C57BL/6J mice, weighing 22 to 24 g,
were housed at room temperature under a 12-h light/dark cycle. Food and water were provided freely. The surgical and
experimental procedures were approved by Animal Care and
Use Committee of Tongji Medical College, Huazhong University
of Science and Technology. p
p
Mechanical pressure-overload hypertrophy (POH) results in
HF progression from EF preservation to EF reduction with
myocardial hypertrophy and fibrosis. Recently, It was shown
that macrophages acted as key innate immune cells with
important roles in the initiation and development of cardiac
remodeling and dysfunction in mice with POH (5, 6). Macrophages can be constitutively present within the
myocardium like kupffer cells in the liver and derived from
blood monocytes as well (7, 8). Macrophages of different origins
exert distinct effects on either early or late phases of POH. The
proliferation of cardiac resident Ly6Clow macrophages as a
mechanism of self-renewal occurs within early-phase POH,
and infiltrating macrophages arise from circulating classic
Ly6Chigh monocytes in late-phase POH (9). Macrophages are
sensitive to their surroundings and alter their physiology in
response to environmental factors (10). A previous study
showed that infiltration of microRNA-155–expressing
macrophages in response to pressure overload promoted
cardiac inflammation, hypertrophy, and failure (11). By
contrast, depletion of macrophages attenuated left ventricle
(LV) remodeling and dysfunction in POH mice (12). After 1 week of adaptive feeding, mice were assigned into either a
sham surgery group or a transverse aortic constriction (TAC)
group. Subsequently, mice in the TAC group were randomly
assigned to one of the following treatments: (1) Sodium chloride
(NaCl)-treated group (vehicle), NaCl at 8 ml/kg; (2) topiramate-
treated group, topiramate at 35 mg/kg; (3) bicuculline-treated
group, bicuculline at 2 mg/kg (provided by Sigma-Aldrich, USA). The four groups were named as follows: sham, vehicle, topiramate-
treated TAC group, and bicuculline-treated TAC group. Starting 6 h
after TAC surgery, mice were intraperitoneally (i.p.) injected once a
day consecutively for 3 weeks. At the end of the study, the hearts
were harvested and weighed to compare the heart weight/body
weight (HW/BW, mg/g). Then, LV tissues were collected for
further experiments. Neutralizing Antibody Administration
Mice were intraperitoneally administered five micrograms of
affinity-purified anti-mouse-AREG polyclonal antibody (AF989,
R&D systems) or IgG immediately before TAC and every 2 days
after TAC. INTRODUCTION In a transverse aortic constriction (TAC) model used to assess
POH, we treated mice with a specific GABAA receptor agonist
(topiramate) or antagonist (bicuculline). We demonstrated that
activation of the GABAA receptor by topiramate accelerated HF. Conversely, blockade of the GABAA receptor by bicuculline
protected POH mice against LV enlargement and EF reduction. The data indicated that GABAA receptor modulation would
become a new strategy in the treatment of patients with
pressure overload-induced HF. Heart failure (HF) is a leading cause of morbidity and mortality
and has a poor prognosis (1). Chronic pressure overload, as seen
in persistent hypertension or aortic stenosis, is a major risk factor
for HF (2). The pressure-overload heart experiences the
transitions from compensated hypertrophic remodeling with
preserved contractile function to eccentric hypertrophy with
contractile dysfunction (3). Notwithstanding the fact that
current drugs capable of inhibiting neuroendocrine signaling
can promote hypertrophy regression, HF progression and its
poor prognosis remain unavoidable. Recent clinical evidence
indicates that the progression from HFpEF to HFrEF has a
worse prognosis than HFrEF and president HFpEF that is not
accompanied by ejection fraction (EF) declines during follow-up
(4). This underscores the need to unravel the mechanism of
compensation to decompensation HF transition. Surgical Protocol TAC model was performed as described previously (17). In brief,
mice were anaesthetized by isoflurane and artificially ventilated at
a rate of 120 strokes per minute using a rodent ventilator with a
mixture of O2 and air (1:2 vol/vol). Then, the aorta was constricted
between the innominate and left common carotid arteries by
placing a 5-0 nylon suture against a blunted 26-gauge needle. Blood pressure gradient across ligation site was confirmed by
echography. Mice in sham group were subjected to identical
operation without reducing aorta diameter to 26-gauge. Animals and Treatments Gamma-aminobutyric acid subtype A (GABAA) receptors are
the principal neurotransmitter receptors of the central nervous
system(CNS), and they are also found to be distributed in
immune cells, such as neutrophils, monocytes, and
macrophages (13). Recently, we published a study showing that
activation or inhibition of the GABAA receptor exerted a
pronounced effect on post-infarction ventricular remodeling by
modulating monocyte/macrophage subsets (14). The GABAA
receptor has also been shown to affect macrophage recruitment
and activity in tumor microenvironments and stress-induced
colon inflammation (15, 16). Thus, the GABAA receptor can
influence pathological processes in various diseases via the
modulation of macrophages. However, it remains unclear
whether macrophage modulation affects pressure overload-
induced cardiac remodeling and dysfunction, which have
increasingly been associated with macrophage-mediated
inflammation in POH. Citation: Bu J, Huang S, Wang J, Xia T, Liu H,
You Y, Wang Z and Liu K (2021) The
GABAA Receptor Influences Pressure
Overload-Induced Heart Failure by
Modulating Macrophages in Mice. Front. Immunol. 12:670153. doi: 10.3389/fimmu.2021.670153 Conclusions: Control of the GABAA receptor activity in monocytes/macrophages plays
an important role in myocardial hypertrophy and fibrosis after POH. Blockade of the
GABAA receptor has the potential to improve pressure overload-induced heart failure. May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 1 The GABAA Receptor and Macrophage Bu et al. Frontiers in Immunology | www.frontiersin.org Echocardiography A Vevo1100 high-resolution in vivo imaging system (Fujifilm
Visualsionics) was used for echocardiographic analysis. Mice
were anesthetized with isoflurane. The LV B-mode and M-mode Here, we sought to address the role of the GABAA receptor in
pressure overload-induced cardiac remodeling and dysfunction. May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 2 The GABAA Receptor and Macrophage Bu et al. neubauer chamber (BD Bioscience, USA) with Trypan blue
(Sigma-Aldrich, USA). Cell suspension were incubated with
antibodies against CD45 (eBioscience, USA), Ly-6G
(eBioscience, USA), Ly-6C (eBioscience, USA), and CD11b
(eBioscience, USA) for 30 min at 4°C, and washed twice for
flow cytometry (FCM) analysis. images were captured from parasternal short-axis or long-axis
views at the level of the papillary muscles (18). The LV end-
diastolic diameter (LVIDd) and LV end-systolic diameter
(LVIDs) were measured from M-mode tracings of parasternal
short-axis views at the mid and apical levels. The ejection
fraction (EF) was calculated as EF (%) = 100 × [(LV Vol;d −
LV Vol;s)/LV Vol;d]. LV Mass was calculated as LV mass (mg) =
1.053 × [(LVID;d + LVPW;d + IVS;d)3 −LVID;d3]. The
fractional shortening (FS) was calculated as FS(%) = 100 ×
[(LVID;d −LVID;s)/LVID;d]. y
y
y
To analyze macrophages and their subsets, heart macrophages
were collected as described previously (14). The right atria, left
atria, and atrioventricular valves were removed after the whole
heart tissue was excised. Then, LV was cut into small pieces in PBS
and digested with collagenase B. The cell suspension was filtered
and resuspended to get single cell suspension. After total viable
cells were calculated, they were conjugated with the following
antibodies for the identification defined as Ly-6Chigh and Ly-6Clow
macrophages according to Ly-6C (eBioscience, USA), CD45
(eBioscience, USA), F4/80 (eBioscience, USA), and CD11b
(eBioscience, USA). After incubated with these antibodies for
30 min at 4°C, the cells were washed twice. To analyze the
subsets of Ly-6Clow macrophages, cardiac single cell suspension
was got according to the above. Cell suspension was incubated
with antibodies against CD45 (eBioscience, USA), Ly-6C
(eBioscience, USA), F4/80 (eBioscience, USA), MHCII
(eBioscience, USA), and CD11b (eBioscience, USA) for 30 min
at 4°C, and washed twice for FCM analysis. Similarly, to analyze
the expression of Ki-67 protein, CD45 (eBioscience, USA), Ly-6C
(eBioscience, USA), F4/80 (eBioscience, USA), Ki-67 (eBioscience,
USA), and CD11b (eBioscience, USA) were incubated for
FCM analysis. EdU Incorporation Assay To assess DNA synthesis in cardiac macrophages, 200 µg of EdU
(5-ethynyl-2′-deoxyuridine) was injected into mice (i.p). Two
hours after the injection, the heart was excised, and cell
suspension was dispersed from the heart tissue and analyzed
for incorporated EdU using FCM. For co-immunostaining of F4/80 and amphiregulin (Areg),
antigen retrieval was performed by using boiling citrate buffered
saline for 2 min after de-waxing and washing the sections in PBS. The heart slides were blocked for 1 h with 10% normal donkey
serum. The tissue sections were incubated overnight with anti-
Areg (A12680, ABclonal) and anti-F4/80 (Ab6640, Abcam)
antibodies. Next day, the sections were washed and incubated
for 2 h with Alexa-Fluor-488–conjugated anti-goat or Alexa-
Fluor-594–conjugated anti-rat secondary antibodies (Invitrogen). DAPI was used to stain the cell nuclei. The stained samples were
viewed using a confocal laser-scanning microscope. Western Blot Analysis Western blot was performed as described previously (21). LV
tissue was homogenized in radioimmunoprecipitation buffer
containing 1% protease inhibitor cocktail, and the concentration
of the supernatant was measured by means of BCA. After the
target proteins were loaded onto an SDS polyacrylamide gel,
proteins were transferred from the gel to a PVDF membrane. Subsequently, the membranes were incubated overnight with
Areg, mTOR (A2445, ABclonal), AKT (A18120, ABclonal), P-
AKT (AP0655, ABclonal), or P-mTOR (AP0094, ABclonal)
antibody after being blocked for 1 h. After washed in TBST, the
membranes were incubated with HRP-linked secondary antibody
for 60 min at room temperature. The protein bands were detected
using an enhanced chemiluminescence agent (ECL reagents)
and analyzed. Flow Cytometry To determine monocytes and their subsets or neutrophils,
peripheral blood was collected in 50 mM EDTA (Sigma-
Aldrich, USA) as anticoagulant at the day of sacrifice. Spleens
and femurs were excised after vascular perfusion with sterile PBS. Spleens were triturated in PBS with the end of a 1-ml syringe,
and filtered through 40 mm nylon mesh. Flushed bone marrow
was also strained through the nylon mesh. Then, the cell
suspension was centrifuged and resuspended to get single cell
suspension. The total viable cell numbers were determined using Histological Analysis and
Immunofluorescence The cross-sectional area of cardiomyocytes (CMs) was assessed
on the basis of wheat germ agglutinin (WGA) staining. For that,
5 µm thick paraffin-embedded sections from mice hearts were
deparaffinized, rinsed in PBS and subsequently stained with
Alexa Fluor 488-conjugated WGA according to the
manufacturer-provided protocol. For the quantification of the
cross-sectional area, slides were imaged by a fluorescence
microscope and only CMs that were aligned transversely were
considered. Cell area was calculated in more than 200 cells. To determine collagen deposition, paraffin sections (5 µm
thick) of hearts were stained with Masson’s trichrome as
described previously (19). Perivascular fibrosis was calculated
as the perivascular collagen area relative to the vessel area, and
interstitial fibrosis was the percentage of the Azan’s trichrome-
stained area per total area of cardiac tissue. The blinded
measurements were made by two independent observers using
ImagePro Plus 6.0 (Media Cybernetics, USA). To further detect
collagen content, sections were stained in Sirius Red solution as
described previously (20). Type I collagen fibrosis was
determined as the ratio of red/yellow-stained area to total
collagen-stained area. Statistical Analysis All values were expressed as the mean ± SEM and multiple group
comparisons were analyzed by one-way analysis of variance
(ANOVA), followed by Bonferroni’s test. The survival rate was
analyzed by Kaplan–Meier survival analysis and compared by
the log-rank test. A value of P < 0.05 was regarded to be
statistically significant. The statistical analysis was performed
with SPSS 17.0 and GraphPad Prism 7.0. Activation or Blockade of the GABAA
Receptor Promotes or Alleviates Pressure
Overload–Induced Myocardial Remodeling Activation or Blockade of the GABAA
Receptor Promotes or Alleviates Pressure
Overload–Induced Myocardial Remodeling
Consistent with findings in cardiac function, topiramate-treated
TAC mice showed more severe myocardial hypertrophy, and
interstitial or perivascular fibrosis, compared with vehicle. As
shown in Figures 2A–C, F, cross-sectional area of individual
cardiomyocyte measured respectively by WGA or Masson
trichrome staining increased as well as visible heart
enlargement did in topiramate-treated TAC mice, compared
with vehicle. Bicuculline-treated TAC mice had a variance
effect. In addition, Masson trichrome and Sirius Red staining
showed that areas of interstitial or perivascular fibrosis and I type
collagen ratio after TAC were significantly increased by
topiramate treatment (Figures 2D, E, G–I). However, cardiac
fibrosis was significantly less prominent in mice treated with
bicuculline after TAC than in vehicle. Furthermore, the GABAA
receptor agonist or antagonist had no effect on the structure of
the aorta, mesenteric arteries, and kidneys (Supplementary 1). GAPDH-F
CAGTGGCAAAGTGGAGATTGTTG
GAPDH-
R
TCGCTCCTGGAAGATGGTGAT GAPDH-F
CAGTGGCAAAGTGGAGATTGTTG
GAPDH-
R
TCGCTCCTGGAAGATGGTGAT GAPDH-F
CAGTGGCAAAGTGGAGATTGTTG
GAPDH-
R
TCGCTCCTGGAAGATGGTGAT C c l 2 - F
C A G G T C C C T G T C A T G C T T C T
C c l 2 -
R
CCCATTCCTTCTTGGGGTCA Cxcl 12-F
CCTTCAGATTGTTGCACGGC
Cxcl 12-
R
TTACAAAGCGCCAGAGCAGA osteopontin-F TTGCTTGGGTTTGCAGTCTTC
osteopontin-
R TATAGGATCTGGGTGCAGGCT osteopontin-F TTGCTTGGGTTTGCAGTCTTC
osteopontin-
R TATAGGATCTGGGTGCAGGCT TGF-B1-F
AGGGCTACCATGCCAACTTC
TGF-B1-
R
CCACGTAGTAGACGATGGGC Activation or Blockade of the GABAA
Receptor Selectively Increases or
Reduces Accumulation of Ly6Clow
Macrophages in the Heart p
g
To assess the effect of the GABAA receptor on macrophage
polarization after TAC, we analyzed macrophage subpopulations
in the heart by FCM at days 1, 3, 7, 14, 21, and 28 post-TAC. We
used a Ly-6C marker to identify two subsets of macrophages with
distinct functions, namely Ly6Chigh and Ly6Clow macrophages. As shown in Figures 3A, B, compared with vehicle, topiramate
resulted in the expansion of CD45+CD11b+F4/80+Ly6Clow
macrophages (hereafter referred to as Ly6Clow macrophages) in
the myocardium at days 3, 7, 14, 21, and 28 post-TAC, whereas
bicuculline treatment exhibited opposite effects. Mice treated
with either topiramate or bicuculline after TAC did not have
significantly altered CD45+CD11b+F4/80+Ly6Chigh macrophage
populations (hereafter referred to as Ly6Chigh macrophages),
compared with vehicle at days 1, 3, 7, 14, and 21 post-TAC
(Figures 3A, B and Supplementary 2). However, topiramate
treatment markedly induced the expansion of Ly6Chigh
macrophages at day 28 post-TAC (late-phase POH) compared
to vehicle (Figures 3A, B). RNA Extraction and Quantitative
Real-Time PCR (RT-PCR) Analysis (
)
y
The total RNA of the LV were isolated using TRIzol®Reagent,
and then, cDNA was synthesized using Prime Script™RT May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 3 Bu et al. The GABAA Receptor and Macrophage was significantly higher in bicuculline-treated than that in
topiramate-treated TAC mice and vehicle (Figure 1H). was significantly higher in bicuculline-treated than that in
topiramate-treated TAC mice and vehicle (Figure 1H). Master Mix(Takara, Japan). The internal control was
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All
reactions were performed in a 10-ml volume-containing 1 ml
cDNA, 5 ml SYBR-Green reaction mix, 0.2 ml sense primer, 0.2
ml anti-sense primer (both from TSINGKE), 0.4 ml ROX, and
3.2 ml ddH2O twice. All reactions were performed in a Applied
Biosystems. Relative changes were determined using the 2−DDCt
method. The primer sequences were as follows: Activation or Blockade of the GABAA
Receptor Aggravates or Attenuates
Pressure Overload-Induced Cardiac
Dysfunction To assess the effect of the GABAA receptor on TAC-induced
cardiac function, echocardiography was performed in
topiramate- or bicuculline-treated TAC mice. Four weeks after
TAC, the transition from concentric to dilated hypertrophy in
the LV was accelerated by topiramate, while the development of
HF was attenuated by bicuculline. At day 28 post-TAC,
topiramate-treated TAC mice showed significantly reduced
cardiac systolic function, expressed as fractional shortening
(FS) or ejection fraction (EF) in comparison to vehicle. By
contrast, cardiac systolic function in bicuculline-treated TAC
mice was significantly improved compared with vehicle (Figures
1B, C and Supplementary Table 1). Four weeks after pressure
overload, cardiac hypertrophy parameters including end-
diastolic and end-systolic left ventricular internal diameter
(LVIDd and LVIDs), LV mass, and heart-to-body weight, were
significantly aggravated by topiramate, and attenuated by
bicuculline (Figures 1A, D–G and Supplementary Table 1). The survival rate was observed up to 30 days post-TAC, which Recent studies have shown that cardiac Ly6Clow macrophages
were independent of monocyte infiltration in early-phase POH. We hypothesized that activation of the GABAA receptor
increased levels of Ly6Clow macrophages by stimulating local
proliferation in early-phase POH. Indeed, we found that
activation of the GABAA receptor led to the local proliferation
of macrophages, as demonstrated by 5-ethynyl-2′-deoxyuridine
(EdU) staining at day 7, but not day 28 post-TAC (Figure 3C
and Supplementary 2). In parallel, activation of the GABAA
receptor significantly induced the expression of the Ki-67 protein, May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org The GABAA Receptor and Macrophage Bu et al. A
B
D
E
F
G
H
C
FIGURE 1 | Activation or blockade of the GABAA receptor aggravates or attenuates cardiac dysfunction in pressure-overload hypertrophy mice. Mice underwent
sham or transverse aortic constriction (TAC) operation; mice in the TAC group were treated with intraperitoneal injections once a day of NaCl, topiramate or
bicuculline for 3 weeks. (A) Representative left ventricular echocardiographic recordings at day 28 post-TAC. Short-axis M-mode (left) and long-axis B-mode (right). Arrows and lines marked (left ventricle internal dimension) LVID in systole (dashed) and diastole (firm). (B) Ejection fraction (%EF), (C) fractional shortening (%FS),
(D) left-ventricular mass (LV mass), (E) left ventricle internal dimension in systole (LVIDs), and (F) in diastole (LVIDd) in TAC- and sham-operated mice at day 28 post-
TAC. (G) Heart weight/body weight (HW/BW) in indicated groups at day 28 post-TAC. Activation or Blockade of the GABAA
Receptor Aggravates or Attenuates
Pressure Overload-Induced Cardiac
Dysfunction Data showed the mean ± SEM of %FS, %EF, LV mass, LVIDd, LVIDs and
HW/BW for each experimental group, by one-way ANOVA with Bonferroni’s multiple comparison test. (H) Compared with vehicle, the survival rate increased in
bicuculline-treated TAC group during 30 days follow-up post-TAC (n = 20 in each group). For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment,
#P < 0.05 vs. vehicle. NaCl-treated TAC mice (for simplicity, vehicle). (n = 4–6 in sham group, n = 10 in TAC-operated group). D E F
G H FIGURE 1 | Activation or blockade of the GABAA receptor aggravates or attenuates cardiac dysfunction in pressure-overload hypertrophy mice. Mice underwent
sham or transverse aortic constriction (TAC) operation; mice in the TAC group were treated with intraperitoneal injections once a day of NaCl, topiramate or
bicuculline for 3 weeks. (A) Representative left ventricular echocardiographic recordings at day 28 post-TAC. Short-axis M-mode (left) and long-axis B-mode (right). Arrows and lines marked (left ventricle internal dimension) LVID in systole (dashed) and diastole (firm). (B) Ejection fraction (%EF), (C) fractional shortening (%FS),
(D) left-ventricular mass (LV mass), (E) left ventricle internal dimension in systole (LVIDs), and (F) in diastole (LVIDd) in TAC- and sham-operated mice at day 28 post-
TAC. (G) Heart weight/body weight (HW/BW) in indicated groups at day 28 post-TAC. Data showed the mean ± SEM of %FS, %EF, LV mass, LVIDd, LVIDs and
HW/BW for each experimental group, by one-way ANOVA with Bonferroni’s multiple comparison test. (H) Compared with vehicle, the survival rate increased in
bicuculline-treated TAC group during 30 days follow-up post-TAC (n = 20 in each group). For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment,
#P < 0.05 vs. vehicle. NaCl-treated TAC mice (for simplicity, vehicle). (n = 4–6 in sham group, n = 10 in TAC-operated group). of monocyte subsets (CD45+Ly6G-CD11b+Ly6Chigh or
CD45+Ly6G-CD11b+Ly6Clow) in the peripheral blood and
spleen, and monocytes (CD45+Ly6G-CD11b+Ly6C+) in
marrow bone by FCM at days 1, 7, 14, 21, and 28. The results
showed that the GABAA receptor exerted no influence on
monocytes in the peripheral blood, spleen, and marrow bone
at days 1, 7, 14, and 21 post-TAC (Figures 4D–F and
Supplementary 3–5). However, treatment with either
topiramate or bicuculline markedly affected Ly6Chigh monocyte
counts in the peripheral blood and spleen not in marrow bone at
day 28 post-TAC (late-phase POH) (Figures 4A–F). Activation or Blockade of the GABAA
Receptor Aggravates or Attenuates
Pressure Overload-Induced Cardiac
Dysfunction For
example, topiramate treatment resulted in Ly6Chigh monocyte a nuclear proliferation marker, in Ly6Clow macrophages at day 7
post-TAC (Figures 3D, E). Collectively, these results suggest that
activation of the GABAA receptor increases the number of Ly6Clow
macrophages by stimulating local proliferation of macrophages in
early-phase POH. Activation or Blockade of the GABAA
Receptor Promotes or Reduces Monocyte
Recruitment in Late-Phase POH To further explore the effect of the GABAA receptor on systemic
innate immunity in mice after TAC, we analyzed the number May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org The GABAA Receptor and Macrophage Bu et al. Bu et al. A
B
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FIGURE 2 | Activation or blockade of the GABAA receptor promotes or alleviates myocardial remodeling in pressure-overload hypertrophy mice. (A) Representative
visible images of hearts (top) and Masson trichrome staining of heart vertical section (bottom) at day 28 post-TAC. Scale bar = 2.5 mm. (B, C) Cardiomyocytes by
wheat germ agglutinin (WGA) staining (B), and Masson trichrome staining (C) at day 28 post-TAC. scale bar = 50 mm. (D, E) Cardiac fibrosis by Masson trichrome
staining (D), and Sirius Red staining (E) at day 28 post-TAC. (top, perivascular fibrosis; bottom, interstitial fibrosis). (F–I) Quantified analysis of cardiomyocytes size
(F), the perivascular collagen area relative to the vessel area (G), the percentage of interstitial fibrosis area in each picture (H), and the ratio of type I collagen fibrosis
to total collagen-stained area (I). Sections were analyzed with Image-Pro Plus 6.3 software. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s
multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6–10). D D A B E E C C C I I
H G G F H G FIGURE 2 | Activation or blockade of the GABAA receptor promotes or alleviates myocardial remodeling in pressure-overload hypertrophy mice. (A) Representative
visible images of hearts (top) and Masson trichrome staining of heart vertical section (bottom) at day 28 post-TAC. Scale bar = 2.5 mm. (B, C) Cardiomyocytes by
wheat germ agglutinin (WGA) staining (B), and Masson trichrome staining (C) at day 28 post-TAC. scale bar = 50 mm. (D, E) Cardiac fibrosis by Masson trichrome
staining (D), and Sirius Red staining (E) at day 28 post-TAC. (top, perivascular fibrosis; bottom, interstitial fibrosis). (F–I) Quantified analysis of cardiomyocytes size
(F), the perivascular collagen area relative to the vessel area (G), the percentage of interstitial fibrosis area in each picture (H), and the ratio of type I collagen fibrosis
to total collagen-stained area (I). Sections were analyzed with Image-Pro Plus 6.3 software. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s
multiple comparison test. Activation or Blockade of the GABAA
Receptor Promotes or Reduces Monocyte
Recruitment in Late-Phase POH For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6–10). TAC (Supplementary 7). However, in topiramate/bicuculline-
treated TAC mice, chemokine C-X-C motif ligand-12 (Cxcl12)
levels were unaltered at days 7 and 28 post-TAC, which aligned
well with unaltered blood neutrophil densities (Figure 4H and
Supplementary 6). These data demonstrate that activation of the
GABAA receptor induces circulating Ly6Chigh classic monocyte
infiltration in late-phase POH via CCL2/CCR2-dependent
monocyte migration. counts in the peripheral blood and spleen to increase from 0.81 ±
0.07 × 104 to 1.78 ± 0.37 × 104, and 0.01 ± 0.01 × 104 to 0.03 ±
0.01 × 104, respectively. Furthermore, blood neutrophil counts
were evaluated at days 7 and 28 post-TAC, revealing that counts
in topiramate/bicuculline-treated TAC mice did not differ from
those in vehicle (Supplementary 6). To determine whether the accumulation of myocardial
macrophages depends on the recruitment of monocytes from
blood in late-phase POH, we measured chemokine expression in
myocardial tissues at days 7 and 28 post-TAC. Intriguingly,
chemokine C-C motif ligand-2 (CCL2) levels were elevated in
topiramate-treated TAC mice versus vehicle at day 28, but not
day 7 post-TAC (Figure 4G), whereas CCL2 levels in
bicuculline-treated TAC mice were lower than those in vehicle. We also found that topiramate treatment increased the
percentage of CCR2+ macrophages in the heart at day 28 post- Frontiers in Immunology | www.frontiersin.org GABAA Receptor-Mediated Myocardial
Hypertrophy Is Associated With Areg-
Induced Akt/mTOR Signaling To elucidate the mechanism by which the GABAA receptor affects
cardiac macrophages and thus, induces myocardial hypertrophy, May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 6 The GABAA Receptor and Macrophage Bu et al. B B A
B
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E
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FIGURE 3 | Activation or blockade of the GABAA receptor selectively increases or reduces the number of Ly6Clow macrophages in the hearts of pressure-overload
hypertrophy mice. Macrophage (CD45+F4/80+CD11b+) subpopulations were respectively defined as Ly6Chigh or Ly6Clow macrophages according to Ly-6C
expression levels. (A) Representative images of Ly6Chigh and Ly6Clow macrophages at days 7, 14, and 28 post-TAC. The representative images of 1, 3, and 21 days
after TAC were shown in supplementary materials. (B) The number of Ly6Clow macrophages or Ly6Chigh macrophages (per mg heart tissue) among the total number
of live cells isolated from hearts at the indicated time points after TAC. (C) The number of CD45+CD11b+F4/80+EdU+cell (per mg heart tissue) among the total
number of live cells isolated from hearts at the indicated time points after TAC. (D) Representative images of Ki-67+ expression in Ly6Clow macrophages at day 7
post-TAC. (E) The percentage of Ki-67+ expression in Ly6Clow macrophages. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple
comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for
all other groups). A B C C C C D E E D
E E FIGURE 3 | Activation or blockade of the GABAA receptor selectively increases or reduces the number of Ly6Clow macrophages in the hearts of pressure-overload
hypertrophy mice. Macrophage (CD45+F4/80+CD11b+) subpopulations were respectively defined as Ly6Chigh or Ly6Clow macrophages according to Ly-6C
expression levels. (A) Representative images of Ly6Chigh and Ly6Clow macrophages at days 7, 14, and 28 post-TAC. The representative images of 1, 3, and 21 days
after TAC were shown in supplementary materials. (B) The number of Ly6Clow macrophages or Ly6Chigh macrophages (per mg heart tissue) among the total number
of live cells isolated from hearts at the indicated time points after TAC. (C) The number of CD45+CD11b+F4/80+EdU+cell (per mg heart tissue) among the total
number of live cells isolated from hearts at the indicated time points after TAC. (D) Representative images of Ki-67+ expression in Ly6Clow macrophages at day 7
post-TAC. GABAA Receptor-Mediated Myocardial
Hypertrophy Is Associated With Areg-
Induced Akt/mTOR Signaling (E) The percentage of Ki-67+ expression in Ly6Clow macrophages. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple
comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for
all other groups). The GABAA Receptor Affects Fibrosis by
Regulating MHCIIlow/MHCIIhigh Ratios in
Subpopulations of Ly6Clow Macrophages
To understand how the GABAA receptor affects fibrosis, we
assessed the crosstalk that occurred between macrophages and
fibroblasts. As reported, Ly6Clow macrophages can be further
classified into two subpopulations based on expression levels of
major histocompatibility complex (MHC) class II proteins. We
found that topiramate treatment reduced MHCIIlow/MHCIIhigh
ratios in subpopulations of Ly6Clow macrophages, and bicuculline
increased the ratios of MHCIIlow/MHCIIhigh at days 7 and 28 post-
TAC, compared with vehicle (Figures 7A, B). Furthermore, RT-
PCR revealed that topiramate treatment increased osteopontin
(OPN) and TGF-b transcript levels at days 7 and 28 post-TAC,
whereas bicuculline treatment reduced their expression levels,
compared with vehicle (Figures 7C, D). These data suggest that
activation of the GABAA receptor polarizes macrophages toward we tested whether humoral factors produced from macrophages
affected cardiomyocytes. Areg has recently been shown to induce
cardiomyocyte hypertrophy (22). We found that topiramate
increased the areas of regions that co-stained positive for Areg
and F4/80 and upregulated Areg protein levels in the heart. Conversely, bicuculline treatment significantly reduced the area
of co-stained regions and levels of Areg protein expression
(Figures 5A–C). Furthermore, increased cardiomyocyte size in
topiramate-treated mice was accompanied by increasing levels of
phosphorylation of AKT and mTOR, while bicuculline reduced
phosphorylation levels of both proteins relative to those which
were observed in vehicle (Figures 5D–F and Supplementary 8). The capability of the GABAA receptor activation to induce Akt
and mTOR phosphorylation was blocked by a neutralizing
antibody specific for Areg (Figure 6). These data suggest that
Areg-induced Akt/mTOR signaling pathway may be essential for
GABAA receptor-mediated cardiac hypertrophy. we tested whether humoral factors produced from macrophages
affected cardiomyocytes. Areg has recently been shown to induce
cardiomyocyte hypertrophy (22). We found that topiramate
increased the areas of regions that co-stained positive for Areg
and F4/80 and upregulated Areg protein levels in the heart. Conversely, bicuculline treatment significantly reduced the area
of co-stained regions and levels of Areg protein expression
(Figures 5A–C). GABAA Receptor-Mediated Myocardial
Hypertrophy Is Associated With Areg-
Induced Akt/mTOR Signaling Furthermore, increased cardiomyocyte size in
topiramate-treated mice was accompanied by increasing levels of
phosphorylation of AKT and mTOR, while bicuculline reduced
phosphorylation levels of both proteins relative to those which
were observed in vehicle (Figures 5D–F and Supplementary 8). The capability of the GABAA receptor activation to induce Akt
and mTOR phosphorylation was blocked by a neutralizing
antibody specific for Areg (Figure 6). These data suggest that
Areg-induced Akt/mTOR signaling pathway may be essential for
GABAA receptor-mediated cardiac hypertrophy. May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 7 The GABAA Receptor and Macrophage Bu et al. A
B
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FIGURE 4 | Activation or blockade of the GABAA receptor promotes or reduces monocyte recruitment in pressure-overload hypertrophy mice. (A, B) Representative
images for flow cytometric analysis of CD45+Ly6G-CD11b+Ly6Chigh and CD45+Ly6G-CD11b+Ly6Clow monocytes in the blood (A), and spleen (B) at day 28 post-TAC. (C) Representative images for flow cytometric analysis of monocytes (CD45+Ly6G-CD11b+Ly6C+) in bone marrow at day 28 post-TAC. The representative images of other
time points are shown in supplementary materials. (D) The number of Ly6Chigh or Ly6Clow monocytes (per ml blood) among the total number of live cells isolated from blood at
the indicated time points. (E) The number of Ly6Chigh or Ly6Clow monocytes (per mg spleen) among the total number of live cells isolated from spleen at the indicated time
points. (F) The number of monocytes (per mg marrow bone) among the total number of live cells isolated from marrow bone at the indicated time points. (G, H) Expression of
Ccl2 and Cxcl12 marker genes at days 7 and 28 post-TAC. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate
treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for all other groups). D D D A B E E E C F H F
G
H G G FIGURE 4 | Activation or blockade of the GABAA receptor promotes or reduces monocyte recruitment in pressure-overload hypertrophy mice. (A, B) Representative
images for flow cytometric analysis of CD45+Ly6G-CD11b+Ly6Chigh and CD45+Ly6G-CD11b+Ly6Clow monocytes in the blood (A), and spleen (B) at day 28 post-TAC. (C) Representative images for flow cytometric analysis of monocytes (CD45+Ly6G-CD11b+Ly6C+) in bone marrow at day 28 post-TAC. GABAA Receptor-Mediated Myocardial
Hypertrophy Is Associated With Areg-
Induced Akt/mTOR Signaling The representative images of other
time points are shown in supplementary materials. (D) The number of Ly6Chigh or Ly6Clow monocytes (per ml blood) among the total number of live cells isolated from blood at
the indicated time points. (E) The number of Ly6Chigh or Ly6Clow monocytes (per mg spleen) among the total number of live cells isolated from spleen at the indicated time
points. (F) The number of monocytes (per mg marrow bone) among the total number of live cells isolated from marrow bone at the indicated time points. (G, H) Expression of
Ccl2 and Cxcl12 marker genes at days 7 and 28 post-TAC. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate
treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (n = 3 mice for sham group, n = 6–8 mice for all other groups). Ly6ClowMHCIIhigh subpopulations after pressure overload in
the heart. which were identified as Ly6Clow macrophages during early-phase
POH, and promoted bloodborne monocyte recruitment and
macrophage polarization in late-phase POH. We also found that
topiramate promoted the polarization of Ly6Clow macrophages
labeled with MHCIIhigh. As a consequence of Ly6Clow macrophage
polarization by topiramate treatment, the Areg-induced Akt/
mTOR signaling pathway was activated, which resulted in
myocardial hypertrophy. With a decrease in the ratios of
MHCIIlow/MHCIIhigh subpopulations of Ly6Clow macrophages,
OPN and TGF-B levels increased, thereby contributing to
fibrosis. As the antagonist, bicuculline exhibited the opposite effect. Frontiers in Immunology | www.frontiersin.org DISCUSSION In this study, we identified the role of GABAA receptors
in pressure overload-induced HF. Using the GABAA-specific
receptor agonist or antagonist, we found that activation of the
GABAA receptor by topiramate resulted in cardiac dysfunction, as
shown by worsened FS & EF, enlarged LVIDd & LVIDs, and
aggravation of cardiac hypertrophy and fibrosis, in comparison to
vehicle. In contrast, blockade of the GABAA receptor by bicuculline
reversed this effect in mice with POH, protecting them against
cardiac dysfunction and hypertrophy. Mechanistically, topiramate
stimulated the local proliferation of cardiac resident macrophages, In this study, we identified the role of GABAA receptors
in pressure overload-induced HF. Using the GABAA-specific
receptor agonist or antagonist, we found that activation of the
GABAA receptor by topiramate resulted in cardiac dysfunction, as
shown by worsened FS & EF, enlarged LVIDd & LVIDs, and
aggravation of cardiac hypertrophy and fibrosis, in comparison to
vehicle. In contrast, blockade of the GABAA receptor by bicuculline
reversed this effect in mice with POH, protecting them against
cardiac dysfunction and hypertrophy. Mechanistically, topiramate
stimulated the local proliferation of cardiac resident macrophages, TAC is a classic model that presents dynamic changes of
pressure overload-induced hypertrophy and HF (23). In response
to TAC, as it is commonly held, the compensatory cardiac
hypertrophy occurs within the initial 2 weeks for the sake of
preserved contractile function (24). In a period ranging from 2 to
4 weeks after TAC, decompensation and HF manifest (9). Here, we May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 8 The GABAA Receptor and Macrophage Bu et al. Bu et al. A
B
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FIGURE 5 | Areg expression and Akt/mTOR signaling are involved in the GABAA receptor-mediated cardiac hypertrophy. (A) Representative micrographs showing
immunofluorescent staining of Areg (red) and a macrophage marker, F4/80 (green) in a sample of heart tissue. Nuclei were counterstained with DAPI (blue). Scale bar =
50 µm. (B) Representative images of Areg protein at day 7 post-TAC in heart. (D) Representative images of P-Akt and P-mTOR protein at day 7 post-TAC in heart. (C, E, F) Quantitative analysis of Areg protein (C), P-Akt protein (E), and P-mTOR protein (F) at day 7 post-TAC in heart. GAPDH mAb (middle) was used as a loading
control. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. DISCUSSION For bicuculline treatment,
#P < 0.05 vs. vehicle. (each group, n = 6). A
B
C B A B C C E
F D D E FIGURE 5 | Areg expression and Akt/mTOR signaling are involved in the GABAA receptor-mediated cardiac hypertrophy. (A) Representative micrographs showing
immunofluorescent staining of Areg (red) and a macrophage marker, F4/80 (green) in a sample of heart tissue. Nuclei were counterstained with DAPI (blue). Scale bar =
50 µm. (B) Representative images of Areg protein at day 7 post-TAC in heart. (D) Representative images of P-Akt and P-mTOR protein at day 7 post-TAC in heart. (C, E, F) Quantitative analysis of Areg protein (C), P-Akt protein (E), and P-mTOR protein (F) at day 7 post-TAC in heart. GAPDH mAb (middle) was used as a loading
control. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment,
#P < 0.05 vs. vehicle. (each group, n = 6). activation of the GABAA receptor A3 subunit has been shown to
impair colon barrier function by upregulating macrophage activity
in early-life stress mice (16). In comparison, inhibition of the
GABAA receptor reduced neuroinflammation in the hippocampus
and CNS (26, 27). These diverse effects might be due to GABAA
receptor activation in different pathological situations. present findings indicating that macrophages are a potential
pharmacological target against pressure overload-induced HF. Intriguingly, bicuculline, a GABAA receptor antagonist that is
clinically used as an analgesic, produced novel effect and
emerged as a chemical candidate for the reversal of cardiac
dysfunction due to pressure overload. It exerted a significant
influence on myocardial hypertrophy as well as fibrosis,
contrasted by topiramate. By affecting both the pathological
changes underlying pressure overload-induced HF, the process
of POH can be modulated. Therefore, during an initial or early
phase, blockade of the GABAA receptor made it possible to
manipulate cardiac resident macrophages to achieve the goal of
reversing POH. p
p
g
In early-phase POH, GABAA receptor activation or blockade
did not affect neutrophils and monocytes, whereas it promoted
or suppressed local proliferation of cardiac resident macrophages
marked with Ly6Clow as shown in Figures 3 and 4. These results
are in accordance with those of Liao X et al. who showed that
cardiac residence in macrophages is a mechanism of self-
renewal, which occurred within early-phase POH. Frontiers in Immunology | www.frontiersin.org DISCUSSION Increasing
evidences support that GABAergic drugs can cause CNS or
myocardial pathologic changes through activation or blockade
of the GABAA receptor in macrophages independent of CNS (14,
26). A recent study shows that Ly6Clow macrophages can induce
a hypertrophic response in cardiomyocytes for myocardial
adaptive response to pressure overload (22). Evidence indicates
that cardiac resident macrophages have tissue-repairing
capacities after injuries (28). In mice with myocardial Recently, increasing evidence has indicated that the GABAA
receptor is enriched in immune cells, where it exhibits anti- or pro-
inflammatory effects. Previous findings showed that activation of
the GABAA receptor could potently inhibit post-infarction
inflammation by acting upon bloodborne or cardiac resident
macrophages (14). Moreover, Kim et al. suggested that
GABAergic activation promoted macrophage antimicrobial
responses against mycobacterial infection (25). However, May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 9 The GABAA Receptor and Macrophage Bu et al. Bu et al. A
B
D
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FIGURE 6 | Blocking of Areg activity suppresses topiramate-mediated effect on Akt/mTOR signaling. (A) Representative images of Areg, P-Akt, and P-mTOR proteins at
day 7 post-TAC in heart. T: topiramate; I: IgG; A: Anti-Areg Ab + IgG + topiramate. (B–D) Quantitative analysis of Areg protein (B), P-Akt protein (C), and P-mTOR
protein (D) at day 7 post-TAC in heart. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05 vs. TAC + Topiramate+ IgG. (each group, n = 6). A B D C C D FIGURE 6 | Blocking of Areg activity suppresses topiramate-mediated effect on Akt/mTOR signaling. (A) Representative images of Areg, P-Akt, and P-mTOR proteins at
day 7 post-TAC in heart. T: topiramate; I: IgG; A: Anti-Areg Ab + IgG + topiramate. (B–D) Quantitative analysis of Areg protein (B), P-Akt protein (C), and P-mTOR
protein (D) at day 7 post-TAC in heart. Data are mean ± SEM, by one-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05 vs. TAC + Topiramate+ IgG. (each group, n = 6). macrophages may induce cardiac fibrosis, which leads to diastolic
dysfunction in mice with hypertension or advanced age (31). Our
data showed that activation of the GABAA receptor increased the
ratios of MHCIIhigh/MHCIIlow. Due to the shift toward MHCII high
macrophages, TGF-B or OPN expression was upregulated, thereby
aggravating cardiac fibrosis. Frontiers in Immunology | www.frontiersin.org DISCUSSION infarction (MI), proliferation of cardiac resident macrophages
together with monocyte-derived Ly6Clow macrophages activated
by topiramate attenuated ventricular remodeling by promoting
neovascularization and fibrosis instead of infarcts. However,
activation of the GABAA receptor compelled cardiac resident
macrophages to play a new role in ventricular remodeling in
response to pressure overload. These players contribute to
cardiac hypertrophy and fibrosis by producing effectors. For
instance, Areg, a humoral factor specifically produced by
Ly6Clow macrophages, induces a hypertrophic response and
activates AKT/mTOR signaling which is proportional to rate of
eccentric hypertrophy progression under pressure overload
(29, 30). In late-phase POH, which was accompanied by EF reduction
and enlargement of LV, Ly6Chigh monocytes infiltrated the
myocardium and were derived into macrophages, which
participated in myocardial injury and repair responses. Thus,
macrophages acted similar to MI. The mechanism of GABAergic drugs on cultured peritoneal
macrophages and bone marrow-derived macrophages was ever
investigated (13, 25). However, cardiac resident macrophages
emerge in recent years with little knowledge of their nature in
vivo or vitro and their subtypes (7, 32). Thus, it is imperative to
gain more knowledge in future. Similar to monocyte-derived Ly6Clow macrophages, cardiac
resident macrophages can also promote fibrosis. As reported
recently, these Ly6Clow macrophages can be further classified into
MHCIIhigh or MHCIIlow macrophages based on MHCII expression
levels (28). MHCIIlow macrophages favor matrix breakdown,
whereas MHCIIhigh macrophages display enrichment in a gene
set that regulates fibrosis. The high ratios of MHCIIhigh/MHCIIlow In conclusion, our study provides novel insights into the role of
cardiac resident macrophages and GABAA receptor in
pressure overload-induced HF. The pros and cons of our results May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 10 The GABAA Receptor and Macrophage Bu et al. B A
B
D
C
FIGURE 7 | The GABAA receptor affects fibrosis by regulating MHCIIlow/MHCIIhigh ratios in subpopulations of Ly6Clow macrophages. Ly6Clow macrophages could
be further classified into two subpopulations based on MHC II proteins. (A) Representative images of MHC II high and MHC II low macrophages at days 7 and 28
post-TAC in heart. (B) Quantified analysis of the percentage of MHC II low and MHC II high macrophages at days 7 and 28 post-TAC. (C, D) Relative mRNA
expression of OPN (C), and TGF-B marker genes (D) at days 7 and 28 post-TAC in the heart. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s
multiple comparison test. DISCUSSION For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6). A C D
C D D FIGURE 7 | The GABAA receptor affects fibrosis by regulating MHCIIlow/MHCIIhigh ratios in subpopulations of Ly6Clow macrophages. Ly6Clow macrophages could
be further classified into two subpopulations based on MHC II proteins. (A) Representative images of MHC II high and MHC II low macrophages at days 7 and 28
post-TAC in heart. (B) Quantified analysis of the percentage of MHC II low and MHC II high macrophages at days 7 and 28 post-TAC. (C, D) Relative mRNA
expression of OPN (C), and TGF-B marker genes (D) at days 7 and 28 post-TAC in the heart. Data show the mean ± SEM, by one-way ANOVA with Bonferroni’s
multiple comparison test. For topiramate treatment, *P < 0.05 vs. vehicle. For bicuculline treatment, #P < 0.05 vs. vehicle. (each group, n = 6). AUTHOR CONTRIBUTIONS show that cardiac resident macrophages can probably act as key
players in the progression from HFpEF to HFrEF. Thus, targeting
the GABAA receptor in cardiac resident macrophages will likely
benefit the early treatment of HFpEF. And the antagonist or blocker
emerges as a promising therapeutical chemical. JB and ZW conceived the study. JB, YY, TX, HL, and JW
performed the experiments and data analyses. KL and SH
provided intellectual inputs. JB and KL wrote the manuscript. All authors edited and approved the manuscript. All authors
contributed to the article and approved the submitted version. JB and ZW conceived the study. JB, YY, TX, HL, and JW
performed the experiments and data analyses. KL and SH
provided intellectual inputs. JB and KL wrote the manuscript. All authors edited and approved the manuscript. All authors
contributed to the article and approved the submitted version. FUNDING This work was supported by the National Natural Science
Foundation of China (81901429 to SH and 82070514 to KL). DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can be
directed to the corresponding authors. REFERENCES Inflammation in Mice. Hypertension (2017) 70(4):759–69. doi: 10.1161/
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Natriuretic Factor Transgene in an In Vivo Murine Model of Cardiac
Hypertrophy. Proc Natl Acad Sci USA (1991) 88(18):8277–81. doi: 10.1073/
pnas.88.18.8277 Copyright © 2021 Bu, Huang, Wang, Xia, Liu, You, Wang and Liu. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply with
these terms. 18. Wu L, Zhao F, Dai M, Li H, Chen C, Nie J, et al. P2y12 Receptor Promotes
Pressure Overload-Induced Cardiac Remodeling Via Platelet-Driven May 2021 | Volume 12 | Article 670153 Frontiers in Immunology | www.frontiersin.org 12
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Edge Intelligence in Private Mobile Networks for Next-Generation Railway Systems
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Frontiers in communications and networks
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Edge Intelligence in Private Mobile
Networks for Next-Generation
Railway Systems
Syed Muhammad Asad 1,2*, Ahsen Tahir 3, Rao Naveed Bin Rais 4, Shuja Ansari 1,
Attai Ibrahim Abubakar 1, Sajjad Hussain 1, Qammer H. Abbasi 1 and Muhammad Ali Imran 1,5
1James Watt School of Engineering, University of Glasgow, Glasgow, United Kingdom, 2Transport for London (TfL), London,
United Kingdom, 3Electrical Engineering, University of Engineering and Technology, Lahore, Pakistan, 4Electrical and Computer
Engineering, Ajman University, Ajman, United Arab Emirates, 5Artificial Intelligence Research Center (AIRC), Ajman University,
Ajman, United Arab Emirates The integration of Private Mobile Networks (PMN) with edge intelligence is expected to play
an instrumental role in realizing the next generation of industry applications. This
combination collectively termed Intelligent Private Networks (IPN) deployed within the
scope of specific industries such as transport systems can unlock several use cases and
critical applications that in turn can address rising business demands. This article presents
a conceptual IPN that hosts intelligence at the network edge employing emerging
technologies that satisfy a number of Next-Generation Railway System (NGRS)
applications. NGRS use cases along with their applications and respective beyond 5G
(B5G) enabling technologies have been discussed along with possible future research and
development directions that will allow these promising technologies to be used and
implemented widely. Edited by:
Akram Alomainy,
Queen Mary University of London,
United Kingdom Edited by:
Akram Alomainy,
Queen Mary University of London,
United Kingdom Reviewed by:
Syed Aziz Shah,
Coventry University, United Kingdom
Xiaodong Yang,
Xidian University, China
Aifeng Ren,
Xidian University, China Reviewed by:
Syed Aziz Shah,
Coventry University, United Kingdom
Xiaodong Yang,
Xidian University, China
Aifeng Ren,
Xidian University, China Keywords: B5G, edge intelligence, NB-IoT, intelligent transport systems, intelligent private network, next generation
transport systems, 6G *Correspondence:
Syed Muhammad Asad
s.asad.1@research.gla.ac.uk *Correspondence:
Syed Muhammad Asad
s.asad.1@research.gla.ac.uk 1 INTRODUCTION Connectivity is now found in almost all facets of modern lives. What started with the simple
provision of connecting humans paved the way for connected processes and developments that
changed the outlook of what now is a global economy. Over the past two decades, the number of
connected devices and subsequent processes have risen immensely. The advantages this connectivity
brings into our lives are not only limited to convenience and globalization but also provide the basis
of use cases and applications that substantially contribute toward the socioeconomic welfare of our
society. Specialty section:
This article was submitted to
IoT and Sensor Networks,
a section of the journal
Frontiers in Communications
and Networks
Received: 01 September 2021
Accepted: 20 September 2021
Published: 14 October 2021
Citation:
Asad SM, Tahir A, Rais RNB, Ansari S,
Abubakar AI, Hussain S, Abbasi QH
and Imran MA (2021) Edge Intelligence
in Private Mobile Networks for Next-
Generation Railway Systems. Front. Comms. Net 2:769299. doi: 10.3389/frcmn.2021.769299 Specialty section:
This article was submitted to
IoT and Sensor Networks,
a section of the journal
Frontiers in Communications
and Networks
Received: 01 September 2021
Accepted: 20 September 2021
Published: 14 October 2021 Specialty section:
This article was submitted to
IoT and Sensor Networks,
a section of the journal
Frontiers in Communications
and Networks Due to the rise in capacity, security, and critical communication needs Asad et al. (2019), major
industries are now seeking to establish their own Private Mobile Networks (PMN). These PMN are
private wide-area multiple access wireless networks that are highly scalable, provide an adequate
range, are and consistent Quality of Service (QoS) in comparison to license-exempt solutions such as
WiFi. The privatization of mobile networks was made possible with the advent of Long-Term
Evolution (LTE) due to its simplicity and an all-IP nature. Such networks consist of an Evolved
Packet Core (EPC) usually integrated with IP Multimedia System (IPMS) for handling voice calls and
a typical Radio Access Network (RAN) (Katsalis et al., 2017). Another key enabler for PMN is the
virtualization of computing and network resources where an entire core network and parts of access
network can be hosted in the cloud while the cognitive radio at the edge of the network can make ORIGINAL RESEARCH
published: 14 October 2021
doi: 10.3389/frcmn.2021.769299 Citation: Asad SM, Tahir A, Rais RNB, Ansari S,
Abubakar AI, Hussain S, Abbasi QH
and Imran MA (2021) Edge Intelligence
in Private Mobile Networks for Next-
Generation Railway Systems. Front. Comms. Net 2:769299. doi: 10.3389/frcmn.2021.769299 October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org Asad et al. IPN Architecture efficient use of unused spectrum to provide seamless end-to-end
connectivity. Improvements in the LTE technologies in terms of
added intelligence and improved capacity, both in core and access
networks, have paved the way for 5G mobile networks (Zhang,
2020). We perceive that these improvements along with
application-centric intelligence can further shape PMN for
individual industries. Current railway systems are facing many
challenges due to the deficiency of advanced 5G functions and
capabilities to support various use cases, services, and scenarios
(Dat et al., 2016). The 5G networking model use cases that
support railway systems architecture are amazing speed; great
service in a crowd; ubiquitous device communications; super real-
time and reliable connections; and the best user experience. All of
these use cases are challenges to railway networks which in turn
become a necessity due to high rise data rates and capacity needs. In this article, we present IPN for the Next-Generation
Railway System (NGRS) and outline enabling technologies and
frameworks that will be the key enablers in the scope of intelligent
mobility. This article is structured in such a way that we start by
outlining edge intelligence in the context of mobile networks in
Section 2 and then a discussion on key challenges in NGRS is
included in Section 3. Section 4 then includes mobile edge
technologies and use cases that can potentially address the
challenges outlined in Section 3. Section 5 presents the IPN
architecture used to integrate edge intelligence and PMN in the
context of addressing NGRS challenges via 5G or B5G
technologies. Finally, Section 6 presents the conclusion and
some future research directions. management, and service provisioning management. In Zhang
(2020), several Internet-of-Things (IoT) and 5G sensor networks
in railway communication have been exploited along with the
previous industry challenges and their forthcoming resolutions. In Zoha et al. (2020), a summary of self-healing block in self-
organizing 5G networks which presents a framework for
autonomous outage detection and coverage optimization in an
LTE has been presented. Hussain et al. 2.1 User-Centric Edge Intelligence 2.1 User Centric Edge Intelligence
The QoS of any network is a key metric in evaluating its
performance as it correlates to the users’ level of satisfaction. In a similar pursuit, added intelligence in the network make
them more user-centric by including users’ experience in the
feedback loop. Beyond 5G (B5G) networks will be able to
assess the users’ demands by running various distributed
federated learning algorithms at the edge of the network. With
the
inference
capability
derived
from
federated
learning in hand, the edge nodes performing user sentiment
analysis can subsequently inform the network of required
changes (Wang et al., 2019). 2 EDGE INTELLIGENCE IN MOBILE
NETWORKS The exponential increase in computing resources and substantial
improvements in Artificial Intelligence (AI) algorithms over the
past decade has paved the way for intelligence to be integrated
into the day-to-day operations of mobile networks. Several
studies, Niyato (2020), Li et al. (2017), Zhang (2020), Zoha
et al. (2020), and Hussain et al. (2020), have presented the use
of AI-based frameworks to enable the imminent and future
demands of ever-expanding mobile networks. For instance, in
Niyato (2020), artificial intelligence- (AI-) based applications and
services and their challenges in a comprehensive overview have
been provided. It also highlights AI services and their mechanism
as to how they are being applied to the network edge near the data
sources and demonstrates how AI and edge computing can be
mutually beneficial. To do so, it introduces and discusses edge
intelligence and intelligent edge and their implementation
methods and enabling technologies, namely, AI training and
inference in the customized edge-computing framework. In Li
et al. (2017), the standardization of 5G cellular networks is being
expedited to provide insight into the candidate techniques as a
whole and examine the design philosophy behind them. It
highlights one of the most fundamental features among the
revolutionary techniques in the 5G era; i.e., there emerges
initial intelligence in nearly every important aspect of cellular
networks, including radio resource management, mobility Citation: (2020) presented a split in
the 5G network into several 100-cell regions, each monitored by
an edge server, and proposed a framework that preprocesses raw
call detail records having user activities to create an image-like
volume, fed to a machine learning (ML) model. Yang et al. (2019)
presented the application of tactile Internet in healthcare by
considering the diabetic ketoacidosis (DKA) as a 5G use case
to monitor life-threatening complications of diabetes mellitus by
using the C-Band sensing technique. Reducing the computational
distribution and load in a dense deployment will require edge
intelligence. With edge intelligence and dense deployment of base
stations, the applications will be numerous. To explain the
intelligence presented in future IPN for NGRS, we classify
these
operations
into
user-centric,
network-centric,
and
application-centric
edge
intelligence. Although
these
will
operate hand in hand in utter harmony, it is important to
understand the perspective of intelligence from these different
angles as shown in Figure 1. efficient use of unused spectrum to provide seamless end-to-end
connectivity. Improvements in the LTE technologies in terms of
added intelligence and improved capacity, both in core and access
networks, have paved the way for 5G mobile networks (Zhang,
2020). We perceive that these improvements along with
application-centric intelligence can further shape PMN for
individual industries. Current railway systems are facing many
challenges due to the deficiency of advanced 5G functions and
capabilities to support various use cases, services, and scenarios
(Dat et al., 2016). The 5G networking model use cases that
support railway systems architecture are amazing speed; great
service in a crowd; ubiquitous device communications; super real-
time and reliable connections; and the best user experience. All of
these use cases are challenges to railway networks which in turn
become a necessity due to high rise data rates and capacity needs. In this article, we present IPN for the Next-Generation
Railway System (NGRS) and outline enabling technologies and
frameworks that will be the key enablers in the scope of intelligent
mobility. This article is structured in such a way that we start by
outlining edge intelligence in the context of mobile networks in
Section 2 and then a discussion on key challenges in NGRS is
included in Section 3. Section 4 then includes mobile edge
technologies and use cases that can potentially address the
challenges outlined in Section 3. Citation: Section 5 presents the IPN
architecture used to integrate edge intelligence and PMN in the
context of addressing NGRS challenges via 5G or B5G
technologies. Finally, Section 6 presents the conclusion and
some future research directions. 2.2 Network-Centric Edge Intelligence 2.2 Network Centric Edge Intelligence
The efficient utilization of network resources can make the
network more sustainable in terms of lower Operational and
Capital Expenditure (OPEX/CAPEX). With that in mind,
self-organizing and self-optimizing algorithms deployed at
the network edge can enable timely inference and quick
decisions for effective radio resource management, user
mobility management, network orchestration, and service
provisioning. 2.3 Application-Centric Edge Intelligence 2.3 Application-Centric Edge Intelligence
In B5G networks, several applications are expected to be hosted at
the edge of the network. This will require virtualization of
application containers and related computational resources at
the base station. Since applications have varying demands, the
application intelligence employed along with the deployment
strategy
will
vary
from
case
to
case;
for
example,
AI
algorithms used in train automation will have a different October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 2 Asad et al. IPN Architecture FIGURE 1 | Intelligent Private Network (IPN) with user, network, and application-centric edge intelligence for Next-Generation Railway Systems (NGRSs). FIGURE 1 | Intelligent Private Network (IPN) with user, network, and application-centric edge intelligence for Next-Generation Railway Systems (NGRSs). deployment strategy compared to AI campaigns that elevate
passenger travel experience. that would address all the outstanding issues. This lack of
literature
motivates this article
where we have gathered
evidence to identify the challenges and opportunities faced
within the current railway transport networks. This will help
us understand how the edge intelligence in B5G private networks
holds significant importance while prioritizing relevant solutions. 3 ANTICIPATED CHALLENGES IN NGRS The advancement in wireless networks along with the technical
developments in the transport sector has acquainted the industry
with an entirely new approach to public safety, travel efficiency,
and information dissemination. Intelligent Transport Systems
(ITS) refer to a new set of information and communication
technologies that allow transport entities to exchange data
with each other or the infrastructure to support several
transport-related applications. However, predominantly, the
focus within ITS has been toward road users; hence in this
article, we move that focus to intelligent railway systems. In
the context of IPN, we discuss the key challenges and their use
cases enabled by edge intelligence in railway transport networks. Frontiers in Communications and Networks | www.frontiersin.org 3.1 Safety and Security Public panic situations in the current underground train network
can become severely adverse if prompt monitoring and response
are not provided. Safety and security measures are highly essential
for diffusing such situations in the interest of public safety. There
are also concerns about crime and disorder that act as a barrier to
passengers’ travel. An evolving focus on such issues is critical to
reduce crime and improve passengers’ confidence. Public safety
applications utilizing intelligent solutions at the network edge can
help in the predicament of daily travel against the threat of
terrorism, financial constraints, and monitoring passenger
commutes. Currently, the underground transportation system is facing
key challenges with the passengers’ movement information and
pathways, bidirectional communication between passenger flows
on transit and information centers, and collection and processing
of “big data” coming from a range of Internet-of-Things (IoT)
devices. These challenges are mainly due to the maintenance,
reliability, connectivity, and logistics issues while innovation
management, cost management, deployment of IoT devices,
durability,
and
sustainability
of
these
devices
also
pose
concerns. The methodologies prevailing in ITS where railway
networks are concerned have not attained the level of research 3.2 Traffic and Crowd Management
Congestion due to traffic rise causes impatience, anger, and
frustration within passengers which in turn influence their
travel
behavior
causing
the
journeys
to
become
slow,
unpredictable, and stagnant (Asad et al., 2019). For transport
businesses, any form of disruption or congestion in their services
directly or indirectly affects their CAPEX and OPEX while
causing harm not only to the business operations but also to
the reputation of the company. If these travel delay issues are not October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 3 Asad et al. IPN Architecture addressed, on average, a passenger can waste up to two and a half
days per year waiting for a congested train. Identification of
passenger movement in underground train networks is also a
persisting challenge that contributes to high energy consumption
and subsequent failure of effective optimization measures for
mobile networks (Asad et al., 2020a; Asad et al., 2020b). AI can
fuel numerous traffic management applications residing at the
edge of the PMN while keeping a closer look at the traffic patterns
and inferring decisions in smarter ways. climate. The transport system must be resilient to extreme
weather conditions and must have the capability of self-
healing against climate adversities. 3.1 Safety and Security Disruption to the trains
and overall transport network could be caused by either
rainfall, heatwaves, high winds, or snow. With a distributed
private network employing intelligence at many facets, it can
provide useful information to the control center in identifying
weather patterns with future predictions allowing informed
decision-making. 4.1.2 Contact-Tracing System The novel Coronavirus 2019 (COVID-19) proved its viral
infectious nature and consequently forced the entire world
into a lockdown. For transport operators, such an outbreak
placed an enormous challenge in continuing their services in a
safe manner (Asad et al., 2020c). Train passengers are liable to
face viral health issues while traveling in congested trains where
all the travelers are in proximity to one another without social 4.1 Use Cases and Applications Evolution toward B5G networks for transport systems aims to
address challenges in essentially all businesses that come under
the umbrella of ITS. Edge intelligence is expected to remodel the
existing trends in terrestrial networks to accommodate the mobile
nature of sensor networks, train signaling, security, and overall
logistics within underground train networks. Although there are
numerous use cases, we enlist a few that we believe will address
the
current
challenges
around
underground
railway
environments. 3.4 Latency Real-time mission-critical applications and quick emergency
response operations require the satisfaction of specific latency
criteria without considering existing operational technology for
train networks. However, the current wideband radio spectrum
shared among various services pose challenges and obstacles in
effectively serving the critical transport entity operations. Similarly, anomalies from existing passenger traffic flows are
difficult to predict for such a demand critical consortium. For
instance, if a criminal has to be put into custody by chasing them
within a massive traffic flow, the real-time crowd information can
be highly vital in providing the emergency services (ES) a safe and
less disrupting passage to the criminal. 4.1.1 Intelligent Train-to-Train (IT2T) Communications
The wireless industry is developing processes and methods that
will
achieve
sustainable
solutions
to
support
IT2T
Communications. Most of the approaches and state-of-the-art
works regarding direct communications fall short in determining
the low-level requirements, use cases, and their applications to the
best of our knowledge. IT2T communications will be able to offer
low-latency intelligent train networks, autonomous trains, fast
adaptive management of congestion control, onboard Internet
and infotainment services, broadcasting of real-time fault
incidents,
real-time
fault
detection
and
recovery,
and
teleoperated trains ensuring fast and safe control against
hazards. Some of these use cases are now possible by utilizing
small-sized antenna arrays within underground trains and
tunnels operating in the sub-6 GHz and/or mmWave band. The
vulnerability
due
to
shadowing
and
poor
isotropic
propagation loss in the underground environment would need
to be controlled and managed effectively within these use cases
(Ford et al., 2017). 4 ADDRESSING NGRS CHALLENGES WITH
IPN EDGE INTELLIGENCE Common transport systems are designed based on the principle
of “reaction for any action.” While this has proven to work in
many cases, with the current increase in quick response
requirements, other modes of reactive operations are now
being sought. With the reactive approach, actions operate after
an event has occurred. For time-critical applications, this results
in a waste of resources and user QoS. Therefore, there is a need to
address these issues with proactiveness that can administer the
dynamics of the transport specifically in an underground train
network where the conditions are rapidly changing. This
proactive nature can be achieved by having intelligence
deployed at the edge of the network where the application can
predict the future network state well in advance. In the following subsections, we first discuss the use cases of
NGRS, and then we describe IPN technologies that we believe will
play an instrumental role in the realization of NGRS. 3.5 Planning and Costing Existing technologies within underground train networks are
extremely beneficial; however, they bring in deployment and
sustainability-related costs. Several strategies and techniques
have been developed and deployed for the planning and
costing of densely populated networks and energy-efficient
systems, but they fail to address many challenges. Planning
regulations are required to be in compliance with the active
development procedures and regulations. We believe that
network intelligence at the edge can provide a scalable and
cost-effective solution in the shape of a real-time informed
decision-making framework suitable for planning and costing
related activities of underground train networks. 4.1.7 Intelligent Wireless Audio Today, emergency services in the underground environment use
wireless audio links through existing technologies that offer
highly specialized RF communication solutions. Due to the
scarce spectrum and shared resources, in-use emergency audio
devices are facing challenges within the dynamic underground
environment. Besides this fact, the multioperational emergency
services
are
demanding
an
improvement
in
the
quality
(increment in quantity) of their radio spectrum in order to
provide effective safety to their staff and passengers against
antisocial behaviors. An innovative edge intelligence agent
employed within the IPN can address such needs with the use
of
ultrareliable
low-latency
communications
(URLLC). Integration of URLLC in IWA can be seen in current
automation industries (Pilz et al., 2018) where there are high
demands on communication for automation-related use cases but
it has scarcely been used as a use case in the application of the
transport industry specifically underground train networks where
high reliability and low-latency services would benefit the IWA
infrastructure. 4.1.4 Transport Hub Crowd Management p
g
Predicting and managing major transport hubs’ capacity in both
businesses as usual (BAU) and exceptional circumstances, e.g.,
rush hour, travel disruption, and planned events by using
Machine Learning (ML), is one of the challenging aspects for
Transport Hub Crowd Management (THCM). Inspired by fixed
monitoring systems, THCM could employ fixed sensors (Asad
et al., 2020a; Ansari et al., 2018) or 5G mobile networks (Li et al.,
2017) to cope with the limitations it currently faces. This is
stressed out by the exponential spread of uncertainties enabled by
a high passenger density and frequent footprint in transport hubs. Sensors in the hubs would use a triangulation method to detect
the location of passengers that would be echoed from their 5G
handsets, called echolocation. Digital tools such as Real-Time
Location Systems (RTLS) or Intelligent Positioning Systems (IPS)
hosted at the network edge will be important for precise
echolocation gathering of real-time crowd information. 4.1.3 Intelligent Drones System 4.1.3 Intelligent Drones System
Encouraged by the promising 5G capabilities, we envisage
surveying,
information
capturing,
and
live
surveillance
streaming
through Intelligent
Drones System (IDS). The
functional application of the IDS will be a key enabler to
address
overcrowding
situations
in
places
such
as
train
terminals. The other two functions for IDS can be the
provisioning
of
communication
services
through
network
surveillance
using
low
bandwidth
control
signals
and/or
streaming live camera surveillance using high bandwidth data
signals. The operation of IDS is expected to be a mixture of
multiple technologies to achieve highly versatile services. Drones
within IDS will be able to communicate with the IPN utilizing
their respective network slice in order to collate multiple
communication services into one back-end cloud network
(Solomitckii et al., 2018). This is achieved by integrating a
Centralized Intelligent Multiplexer (CIM) at the edge of the
network that receives multiple signals from different drones
and
combines
them
to
establish
the
accurate
content
(multipath propagation) for data analytics (Solomitckii et al.,
2018; Paulo Klaine, 2018). 4.1.6 Intelligent Monitoring Services
The rapid growth of connected wireless devices drives the
purpose of the Internet of Things (IoT) through mMTC
within 5G and beyond networks. IoT within the underground
environment requires the capability of interconnecting diverse
services via the wireless network utilizing narrow bandwidth and
consuming low power while providing maximum penetration in
the train tunnels. In a complex underground ecology, the number
of applications for such a use case suited to Narrow Band IoT
(NB-IoT) (Li et al., 2017) is, but not limited to, underground
tunnels gas and water metering, pressurized fuel detection, smoke
alarms detection, fire alarms monitoring, and event detectors. AI
within IPN is a key enabler for many isolated and distributed
devices to function under one centralized cloud. Additionally,
with such an IPN framework, broadcasting to and from all the
connected devices at one time is also an essential component. For
the implementation of broadcasting, it would require Intelligent
B5G-Xcast (IBX) by leveraging the findings of point to point
(PTP), point to multipoint (PTM), and 5G-Xcast project (Banchs
et al., 2019). The use of laser-based IoT devices to monitor and
subsequently recommend safe distances from the toxic gases
found
in
underground
confined
areas
also
has
utmost
importance for safety purposes. 3.6 Resilience The impact of weather on the transport network is another main
challenge that requires daily/hourly weather forecasting and
understanding of the anticipated impact of change in the October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 4 Asad et al. IPN Architecture distancing measures. Contact-tracing and passenger distancing
will be highly necessary and will act as one of the crucial types of
mediation to avoid further spread. In such circumstances, an
intelligent Contact-Tracing technique potentially hosted at the
edge of the network integrated within the transport infrastructure
will become an essential need for transport operators. able to provide robust remote connectivity from the location of
the fault within underground ecology to where the experts are. In
this case, a central control center with highly skilled workers
would be available to support multiple remote operations
simultaneously without physically going to the fault location. In other words, within the NGRS, the staff will be empowered to
complete tasks by remotely connecting with skilled specialists
while supporting a “paper-free” maintenance log, moving to fully
digital infrastructure for secure instructions. The use of Intelligent
Extended Reality (IXR) will be able to enhance a mixture of
Augmented Reality (AR), Virtual Reality (VR), and Tactile
Services (TS) for enhanced remote operations. Use cases Use cases
Intelligent Train-to-Train
(IT2T) Communication
Contact-Tracing
System (CTS)
Intelligent Drones
System (IDS)
Transport Hub Crowd
Management (THCM)
Disaster Scenario
Response (DSR)
Intelligent Monitoring
Services (IMS)
Intelligent Wireless
Audio (IWA)
Emergency Services (ES)
Intelligent Depots 4.0
(ID 4.0) Train signaling, train-t-train communication,
driverless trains, etc. Passengers/staff who tested positive against the
COVID-19 would be tracked (on a high level) by
using 5G network and 5G enabled devices
Overcrowding stations monitoring 5G drones Positioning, resource allocation, and management RTLS, IPS, cMTC, ICCTV, and 5G cellular and 5G
private network
5G Intelligent Extended Reality (IXR) portable
devices with HD cameras Remote monitoring, detection, and providing
suggestions for onsite incidents team equipped
with visualization cameras to see faults and hidden
services Sensor-based cMTC, smartphone, gadget
monitoring application, and LPWAN technology
such as NB-IoT. Laser-based 5G technology to
monitor toxic gases
5G handsets with URLLC connectivity Sensor-based cMTC, smartphone, gadget
monitoring application, and LPWAN technology
such as NB-IoT. Laser-based 5G technology to
monitor toxic gases
5G handsets with URLLC connectivity Underground tunnels gas metering, water
metering, pressurized fuel detection, smoke alarms
detection, fire alarms monitoring, and other alarms
and event detectors 5G handsets with MCPTT Sensor-based cMTC, gadget monitoring
application, CAI, and LPWAN technology such as
NB-IoT Sensor-based cMTC, gadget monitoring
application, CAI, and LPWAN technology such as
NB-IoT Sensor-based cMTC, gadget monitoring
application, CAI, and LPWAN technology such as
NB-IoT Diverse bandwidth and wireless
automation optimizing
logistics. Furthermore,
train
parking,
monitoring trains movement by Train Access Controller
(TAC)
through
Radio-frequency
identification
(RFID)
sensors,
identification
of
electrified
and
nonelectrified
tracks
by
using
AR
devices,
self-automated
track
components
between
the
running
rails,
use
of
Conversational AI (CAI) between moving trains and TAC
when accessing live tracks, intelligence in infringing over the
platform edge, automation in stopping a train in an
emergency through the use of specialized
low-latency
devices,
and
real-time
visibility
of
occupied
and
unoccupied
train
depot
tracks
are
the
hallmarks
of
Industry 4.0 that will feed into the ID 4.0. 4.2 IPN Technologies for NGRS 4.2 IPN Technologies for NGRS
ITS-focused AI is a key enabler that can offer a solution to the
complex train network in the form of an IPN in proximity to
an operational environment. The demand for ubiquity,
privacy, low latency, low-cost computing, and resource
management
requires
an
integration
of
heterogeneous
systems and edge compatible applications into one smart
network, presented as an IPN. With such a use case,
isolated and disparate edge services will provide sufficient
security by having multiple Virtual Private Networks (VPN)
to safeguard against malicious attacks. The motivation of IPN
comes from a combination of private mobile networks and
capabilities of B5G networks in lieu of ITS and more
specifically future railway systems which are summarized in 4.1.5 Disaster Scenario Response Edge intelligence within B5G networks will introduce an
assortment of services that will operate on high throughput
and low latency to address critical events that require a timely
response. In the same way, the underground train network is a
highly deserving environment to adopt such technology into
portable devices leveraging the benefits of Massive Machine-
Type Communications (mMTC) (Zhang, 2020). New portable
lightweight
devices
equipped
with
intelligent
computing
applications and storage enabled via the IPN cloud would be October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 5 IPN Architecture Asad et al. TABLE 1 | Summary of NGRS use cases and applications. Use cases
Applications
Enabling technologies
Research challenges
Intelligent Train-to-Train
(IT2T) Communication
Train signaling, train-t-train communication,
driverless trains, etc. 5G NR, mmWave, and Ultrareliable Low-Latency
Communication (URLLC)
High bandwidth and low latency
Contact-Tracing
System (CTS)
Passengers/staff who tested positive against the
COVID-19 would be tracked (on a high level) by
using 5G network and 5G enabled devices
5G UWB (ultrawideband) monitoring short-range
devices
Data security and privacy and low-
power communication
Intelligent Drones
System (IDS)
Overcrowding stations monitoring
5G drones
Real-time low-latency
communication and high-resolution
image processing
Transport Hub Crowd
Management (THCM)
Positioning, resource allocation, and management
RTLS, IPS, cMTC, ICCTV, and 5G cellular and 5G
private network
Diverse bandwidth and low latency
Disaster Scenario
Response (DSR)
Remote monitoring, detection, and providing
suggestions for onsite incidents team equipped
with visualization cameras to see faults and hidden
services
5G Intelligent Extended Reality (IXR) portable
devices with HD cameras
High bandwidth, latency, and
reliability
Intelligent Monitoring
Services (IMS)
Underground tunnels gas metering, water
metering, pressurized fuel detection, smoke alarms
detection, fire alarms monitoring, and other alarms
and event detectors
Sensor-based cMTC, smartphone, gadget
monitoring application, and LPWAN technology
such as NB-IoT. Laser-based 5G technology to
monitor toxic gases
Longer battery life and low latency
for critical alarms
Intelligent Wireless
Audio (IWA)
Emergency departments such as police,
detectives, special forces, and Accident and
Emergency (A&E)
5G handsets with URLLC connectivity
Low latency and reliability
Emergency Services (ES)
Ambulance, firefighters, first-aid staff, mission-
critical fault detection teams, paramedic
services, etc. 5G handsets with MCPTT
Diverse bandwidth, low latency, and
reliability
Intelligent Depots 4.0
(ID 4.0)
Depot services
Sensor-based cMTC, gadget monitoring
application, CAI, and LPWAN technology such as
NB-IoT
Diverse bandwidth and wireless
automation TABLE 1 | Summary of NGRS use cases and applications. 4.1.8 Emergency Services One of the great achievements the Third-Generation Partnership
Project (3GPP) has obtained is standardizing the Mission-Critical
Push-To-Talk (MCPTT) (Sanchoyerto et al., 2019) service as a
use case. This ES use case can be used to act against anything
abnormal such as fare evasion, suicide attempts, and incidents. MCPTT empowered with Enhanced Mobile Broadband (eMBB)
can be an essential focus in ITS when considering the
underground
train
environment. This
will
require
an
application that employs high bandwidth internet access, video
streaming, and virtual reality with latency-sensitive devices. At
the same time, utilizing network and radio slicing techniques,
several
high
bandwidth
sessions
for
separate
emergency
departments such as ambulance, firefighters, first-aid staff,
mission-critical fault detection teams, or other paramedic
services can be established within the IPN. 4.1.9 Intelligent Depots 4.0 (ID 4.0) AI-based industrial automation powered by IPN is making a
tremendous revolution toward B5G technologies. In the field
of supply chain, this transposes toward the edge intelligence
of logistics 4.0 that advances beyond process automation by
using intelligent collaboration and interconnection between
connected systems providing transformative applications
(Wang et al., 2017). ID in the train network is an
important use case that will push communications to
another
level
where
train
depots
will
use
automated
coordination
between
objects
and
autonomous
self- October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 6 Asad et al. IPN Architecture offerings from 410 to 7,125 MHz. The latter one introduces
frequency bands from 24.25 to 52.6 GHz with offerings of even
higher bandwidth than the first ranges. An important aim in
the development of the 5G NR within NGRS has been to ensure
dynamic
bandwidth
communications
depending
on
the
requirement. In the presented IPN, NR is introduced at the
interface of trains communication to the centralized smart
architecture ensuring a high degree of forward compatibility in
the radio interface design. In the context of NGRS, either IT2T
communication or Train Signaling radio interface allows for
substantial future evolution in terms of introducing new
technology
and
enabling
new
services. For
IT2T
communication or Train Signaling radio interface, NR
would support a massive number (e.g., ≥64) of steerable
antenna elements for both transmission and reception. At
higher frequency bands, the large number of antenna
elements are primarily used for beamforming to achieve
coverage, while at lower frequency bands they enable full-
dimensional multiple-input multiple-output (MIMO) and
interference avoidance by spatial filtering (Parkvall et al.,
2017). Table 1 along with their applications, enabling technologies,
and challenges. The idea of running several applications within
a
single
network
environment
consolidates
with
IPN’s
scalability, security, flexibility, and coverage expansion. This
will evolve the existing private mobile networks toward smart
transport digital facilities with the use of Machine Learning
(ML) (Asad et al., 2020a; Zhang, 2020; Klaine et al., 2017; Liu
et al., 2018; Ren et al., 2020; Haider et al., 2019; Fioranelli et al.,
2019), AI, and cognitive analytics that is fully primed for
existence at the edge establishing ground toward B5G
networks. IPN main features are as follows: • Scalability: massive connectivity and capacity in a cost-
effective manner for business expansion and growth. 1https://www.nokia.com/networks/portfolio/mobile-transport/. 4.2.3 Mission-Critical Push to Talk 4.2.3 Mission-Critical Push to Talk
MCPTT originates from old-fashioned Push-to-talk Over
Cellular (POC) services that integrate with mobile phone
networks
with
a
world
of
opportunities. The
service
supports pairs and group users for communication in the
specific time slotting procedure. Each user will be able to
converse both with audio (using low bandwidth) and with
video (using higher bandwidth). MCPTT system allows
participants
to
request
a
token
to
speak. A
priority
mechanism to arbitrate the participation of the users is
provided
in
the
communication
of
critical
services
(Sanchoyerto et al., 2019). Traditionally, the choice of
which user requests to depend on their token time frames
and numbers when using the service simultaneously. IPN
presents
intelligent
MCPTT
on
the
edge
devices
where simultaneous users on their critical missions would
be
addressed
without
delays. IPN-MCPTT
connectivity
works
on
a
principle
of
separated
Control
and
Data
Architecture (CDSA) where mission-critical intelligence is
handled on the edge without consulting IPN in every
communication leaving IPN to monitor activities that each
MCPTT device performs. Following are the components that we envisage will enable the
IPN framework. 4.2.4 Intelligent CCTVs g
Intelligent CCTVs integrated with edge intelligence will
guard assets and processes in real-time by employing AI
and ML algorithms for quick detection (Akbar and Azhar,
2018). The high-quality video stream from the CCTV
multiplexed with edge intelligence will even allow the
prediction of hazardous or emergency situations. The
system can also grant access, monitor, and log people’s
entry/exit into a building allowing a much-centralized
control of railway systems. ICCTVs hold the local cache to
record real-time events on the edge without the need of IPN
core
which
makes
the
system
intelligent,
locally 4.2.1 Artificially Intelligent Anyhaul y
g
y
Due to the overwhelming data growth experienced by mobile
network operators, they are compelled to further densify,
centralize, and/or cloudify their networks. This has led to
the provisioning of centralized streams and edge intelligent
IoT/M2M devices used in the NGRS which are the key drivers
to provide quick and robust responses. Through the use of
AIA, transport network would be cost-effectively driven by
reaching edge intelligent devices in no time from the
centralized core1. Centralized IPN would only be consulted
for control signaling when required, leaving edge devices to
perform faster responses on their own in their locally present
intelligent compute storage. With this approach, the IPN
architecture presented would be exploited for delivering
high
capacity,
ultralow
latency,
and
highly
accurate
synchronization distribution needed to boost the overall
performance of IPN while taking advantage of virtualized
edge intelligent resources (Hilt, 2019). 4.1.9 Intelligent Depots 4.0 (ID 4.0) • Security: block unauthorized access, maintain end-to-end
data
confidentiality,
and
provide
encryption
with
a
complete on-demand solution. • Flexibility: adaptive tethering and real-time adjustment with
the environmental processes. • Reliability: stable continuous connectivity and safe service
continuity for business-critical operations. • Evolution to B5G: future proof intelligence to support
existing
deployed
solutions
with
investment
opportunities
to
support
additional
use
cases
with
stricter requirements. 2Ultra-Wideband (UWB) Ofcom document can be found at: https://www.ofcom.
org.uk/__data/assets/pdf_file/0015/25152/uwb.pdf. 4.2.7 Critical Machine-Type Communication The cMTC is the interaction of machines for the actuation of
data collection for necessary and time-critical actions (Zhang,
2020). The network of machines such as sensors, RFID tags,
and collection of smart antennas in massive numbers with no
intervention of humans would be secure, robust, reliable, and
consistently scalable to cope with high capacity and low-
latency demands. The cMTC can also function by using
multiple varying frequency bands where NGRS have been
postulated as a potential solution offering a single IPN
platform for the “ecosystem” to several connected IoT
devices. cMTC
provides
coverage
in
challenging
environments such as underground train tunnels where
coverage penetration is difficult. It also employs faster
responses
through
the
use
of
faster
data
throughput,
without limitations on the usage. The presented architecture 4.2.2 New Radio 4.2.2 New Radio
The 3GPP standard for the 5G radio access technologies (RAT)
known as 5G New Radio (NR) supports a substantial
expansion unlike traditional technologies with two new
ranges of frequency bands, FR1 and FR2 (Pilz et al., 2018). The first range includes sub-6 GHz in addition to bandwidth October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 7 Asad et al. IPN Architecture has cMTC driven connected devices employing hybrid
configuration with a control center to provide centralized
control
through
IPN
with
edge
intelligence
for
the
connected devices. administrative,
and
less
overhead
intensive
with
only
required frames per second to be sent to the centralized
IPN in every recording. 4.2.8 Conversational Artificial Intelligence UAVs, more commonly known as drones, have a controller,
usually ground-based with a strong communication link between
the two. UAVs flight have various degrees of autonomy
operation (Solomitckii et al., 2018; Sambo et al., 2019)
either under remote control by a human operator or
autonomously by onboard computers. Drones can extend
network coverage and capacity to reach remote or highly
crowded areas. Either individually or as a swarm controlled
by IDS, they can also function as robotic equipment that offers
detection,
traction,
interference
mitigation,
and/or
optimization
of
systems
avoiding
costly
human
interventions. UAVs can be utilized to extend network
coverage in highly crowded areas such as train terminus. In
addition, UAVs can also be utilized in emergency coverage
in situations of local infrastructure malfunction or disaster. Intelligent UAVs have local automation to take important
decisions
at
the
edge
with
control
functions
to
be
monitored
from
IPN. This
network-centric
intelligence
provides a bridge that integrates UAVs into the IPN
infrastructure as access points. 4.2.8 Conversational Artificial Intelligence
CAI is an AI variant that enables people to communicate with
machines
such
as
applications
and
devices
with
the
functionality of human-like natural language via voice, text,
touch,
or
gesture. It
helps
in
achieving
tasks
with
proactiveness and personalized engagement (Dashtipour,
2016). CAI integrated with the rest of the network entities
will feature the IPN as a living entity that interacts with all its
users employing several social AI features hosted at the
network edge. At first, when a user tries to say something,
Natural
Language
Understanding
(NLU)
deciphers
its
meaning in the user’s understandable words regardless of
how words have been stated. Even among user’s grammatical
mistakes, the CAI is able to understand the user’s intent with
NLU. It even works with user’s idiosyncrasies and shortcuts
and remembers the context from one statement to another,
comprehending
what
is
being
said
throughout
the
conversation. At the second stage, CAI determines the
right response based on its intelligence using ML and by
understanding the user’s intent. Finally, using natural
language generation, the CAI generates a user-friendly
response for easy comprehension. 4.2.9 Ultrawideband 4.2.9 Ultrawideband
UWB is a radio technology that uses a wide frequency
spectrum to locate and track people in real-time when
short-range links are paramount. UWB has an unlicensed
frequency
range
from
3.1
to
10.6 GHz. Private
networks utilizing these bands within their premises can
reduce the risk of interference among their applications or
technologies. UWB is a potential alternative to other local area
wireless
technologies,
such
as
WiFi,
Bluetooth,
and
other
WLAN
technologies. The principal advantage of
UWB which distinguishes its features from others is its
offering of faster data transfer rates (100 Mbits/s up to
1 Gbits/s) in comparison. Extended battery and durability
give UWB an edge over prominent wireless technologies
deployed to date2. 4.2.6 Intelligent Network Management The automation technology designed for the management of
automated planning, automated configuration, automated
healing, and automated optimization is called Intelligent
Network
Management
(INM). The
functionality
and
behavior of the INM are standardized by the 3GPP and
NGMNs (Next-Generation Mobile Networks) consortium
while undergoing continuous improvements. AI will play
an instrumental role in making the private networks
sustainable as it requires almost none physical or remote
interventions. INM supports a real-time reconfigurable
managed
ecosystem
with
virtualization
for
fast
and
ultrafast services between centralized IPN and devices on
the edge through AIA. 5.2 Edge Caching technologies provided at the edge under the light of network,
application, and user-centric intelligence as shown in Figure 2. The
presented
architecture
would
also
exploit
service
continuity as a PMN where an edge-computing solution
will handle passenger’s mobility. IPN link is used for
backup and restore operation and for major reporting
purposes. Intelligent actions are done at the edge with
locally captured data. g
g
Under edge intelligence, edge caching is a simplistic method to
effectively improve the performance of the IPN where it
administers the incoming distributed data coming toward the
edge devices from end-users and its surrounding environment
(Zhang et al., 2018; Deng et al., 2020). This property exploits edge
caching with virtually no setup overhead apart from sending
control signals in the form of reporting to the IPN. Along with
this property, the data generated by edge devices itself fall under
edge caching. In our presented IPN, the data generated in IT2T
communication, CTS, IDS, gadgets used in DSR, IMS, IWA, and
MCPTT in ES is stored locally without the need to send every
instance data back to centralized IPN. Also, the sensors-based
cMTC such as RFID, NB-IoT, CAI, and mobile (accelerometer,
gyroscope, and magnetometer) gather environment data which is
processed and stored at reasonable places and is used by AI
algorithms to provide services to users. 5.1 IPN Connectivity y
The presented IPN is envisaged to have numerous edge devices in
its network with profound connectivity links. It integrates edge
devices and edge compatible applications into one smart network,
thus forming a heterogeneous system environment (Niyato,
2020). IPN controls the functions of all connected links and
devices by preprocessing through software-defined tools. The
training of edge devices is a lightweight task that mainly relies
on the edge servers performed by IPN in the initial setup. Once
edge devices are trained, they take their intelligent decisions
locally without IPN consultation on every step which reduces
the overhead, time, and cost. IPN also allows direct connectivity
to local cloud resource and offload major tasks to an on-premise
server. The
on-premise
server
then
takes
care
of
synchronization with the centralized cloud for reporting
purposes. 5 IPN-DRIVEN EDGE INTELLIGENT NGRS
ARCHITECTURE In the following subsections, we discuss IPN-driven edge
intelligence
architecture
comprised
of
centralized
IPN
connectivity and local edge decisions for NGRS (Niyato,
2020; Deng et al., 2020; Zhou et al., 2019; Dey et al., 2019;
Dai et al., 2020). This integrates our use cases with the October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 8 Asad et al. IPN Architecture FIGURE 2 | IPN-driven edge intelligent NGRS architecture. FIGURE 2 | IPN-driven edge intelligent NGRS architecture. 3Narrowband-IoT Vodafone document can be found at: https://www.vodafone.
com/business/iot/managed-iot-connectivity/nb-iot. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in
the article/Supplementary Material; further inquiries can be
directed to the corresponding author. • Low-latency communication: an important parameter for
use cases like IT2T communication, Intelligent Drone 5.4 Edge Inference • Low-power communication: for use cases like Contact-Tracing
Systems and Intelligent Monitoring Services (approx. 15 years
of battery life3), it will be crucial to establish low-power
communications for sustainable NGRS solutions. Edge Inference is a process that holds responsibility for evaluating
performance once the ML model is trained by datasets based on
learnt computational outputs on edge devices (Niyato, 2020). The
ML model is constructed and trained by powerful Central
Processing Units (CPUs) and Graphical Processing Units
(GPUs). These units support the fast processing of numerous
functions such as face image recognition and remote assistance
with data-hungry devices. Once deployed on the edge devices,
they maintain their performance and resilience when intelligently
designed to accelerate edge inference. • Reliability: to ensure communication reliability in scenarios
like Disaster Scenario Response and Emergency Services,
there would be an important future research direction in the
context of NGRS. Furthermore, as we move toward a net zero carbon footprint,
research on energy-efficient Heterogeneous Networks (HetNets)
will also be required to address the energy consumption of NGRS
IPN. Other challenges that would need to be addressed in order to
make IPN open up for general public use will be to enable neutral
hosting, security measures through blockchain, and dynamic
spectrum
sharing
among
mobile
network
operators
for
adaptive capacity protocols. 5.5 Edge Offloading Under edge intelligence, edge offloading is another critical feature
that actively offloads its tasks to other edge devices in a network (Dey
et al., 2019). Similarly, the presented IPN uses edge offloading
characteristics in its edge devices for offloading necessary tasks
like edge training, edge caching, or edge inference in a distributed
computing paradigm. This property exploits an intelligent ecosystem
created by edge devices. Edge offloading is of utmost importance due
to its resilient-like strategy by independent edge devices compared to
edge
caching,
edge
training,
and
edge
inference. Effective
implementation of IPN gives edge intelligence a structured way
to available resources in the edge environment. 5.3 Edge Training Traditionally the training of AI models works on a principle of
centralized deployment architecture which carries heavy traffic in
both communication directions (Kukreja et al., 2019; Zhou et al.,
2019), eventually creating overhead. In our presented IPN, ML-
driven federated learning technique also known as collaborative
learning is used to train the AI model with an aim to port onto edge
devices. The AI model runs on a powerful central server using its October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 9 Asad et al. IPN Architecture Systems, and Intelligent Wireless Audio. The role of
URLLC
and
mmWave
will
be
critical
toward
the
realization of low-latency communications. compatible edge software tools and runtime environment. With this
approach, controlling, retraining, and deployment of models to edge
devices using IPN cloud connectivity proves to be the most reliable
and effective method for edge training. Once all the edge devices are
intelligently trained, the communication link to the IPN would only
be used for backup and restore operation or for reporting purposes
where edge devices take their own decisions locally. • Communication with Diverse Bandwidth Requirements: to
meet the challenges of variable data rate services like in the
case of Transport Hub Crowd Management, Emergency
Services, and Intelligent Depots, it will be important to
develop systems that are capable of providing diverse data
rates while meeting task completion deadlines. AUTHOR CONTRIBUTIONS The NGRS will play an active role with edge intelligence integrated
within the IPN domain. NGRS should be constructed using a
variety of technologies and network configurations discussed in
this article and other related researches and will also depend on
geographic areas, deployment models, and application scenarios in
the underground train environments. We presented several use
cases, applications, required technologies, capacity needs, and
measures
in
this
article
that
we
believe
will
provide
opportunities for transport businesses to put B5G mobile
networks on their private trail. Most of the modern researches
have addressed or are currently investigating the traditional
challenges of ITS. However, there seems to be a gap between
5G/B5G use cases and their applications for ITS. It is believed that
these research gaps will drive future research to unlock the true
potentials of ITS through edge intelligence. Below we list few
critical parameters that will be important research themes in the
context of NGRS realization through edge intelligent IPN. Conceptualization, SMA, SA, and SH; data curation, SMA and
SA; formal analysis, SMA, SA, RR, and AA; funding acquisition,
RR and MI; methodology, SMA and SH; project administration,
SH, RR, QA, and MI; software, SA and SMA; supervision, SH, RR,
QA, and MI; validation, QA and MI; visualization, SMA, SA, and
QA; writing–original draft, SMA; writing–review and editing, SA
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with partial funds by Deanship of Graduate Studies and Research
(DGSR), Ajman University, under the grant number 2020-IRG-
ENIT-10. • Low-latency communication: an important parameter for
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Communications. doi:10.3390/books978-3-03928-149-7 October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 11 Asad et al. Zhou, Z., Chen, X., Li, E., Zeng, L., Luo, K., and Zhang, J. (2019). Edge Intelligence:
Paving the Last Mile of Artificial Intelligence with Edge Computing. Proc. IEEE
107, 1738–1762. doi:10.1109/JPROC.2019.2918951 Frontiers in Communications and Networks | www.frontiersin.org October 2021 | Volume 2 | Article 769299 REFERENCES IPN Architecture Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations or those of
the publisher, the editors, and the reviewers. Any product that may be evaluated in
this article or claim that may be made by its manufacturer is not guaranteed or
endorsed by the publisher. Zhou, Z., Chen, X., Li, E., Zeng, L., Luo, K., and Zhang, J. (2019). Edge Intelligence:
Paving the Last Mile of Artificial Intelligence with Edge Computing. Proc. IEEE
107, 1738–1762. doi:10.1109/JPROC.2019.2918951 Zoha, A., Abbasi, Q. H., and Imran, M. A. (2020). Anomaly Detection and Self-
Healing in Industrial Wireless Networks. John Wiley & Sons, 109–139. doi:10.1002/9781119552635.ch6 Copyright © 2021 Asad, Tahir, Rais, Ansari, Abubakar, Hussain, Abbasi and Imran. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums is
permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms. Conflict of Interest: The author SMA is employed by the company Transport for
London (TfL) as a Senior Network and Telecoms Architect. The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest. October 2021 | Volume 2 | Article 769299 Frontiers in Communications and Networks | www.frontiersin.org 12
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0–3 éves korú csecsemők és kisdedek táplálkozási szokásainak felmérése, különös tekintettel a makro- és mikronutriens-bevitelre
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Erdélyi-Sipos Alíz1 ■ Badacsonyiné dr. Kassai Krisztina1 ■ Kubányi Jolán1
Szűcs Zsuzsanna1 ■ Biró Lajos dr.2 ■ Raposa L. Bence dr.3 p
Következtetés: Bár a makro- és mikronutrienseknek a WHO/FAO/UNU által 2007-ben megadott beviteli értékein
alapuló, 2013-as EFSA-ajánlás meghatározza az ajánlott beviteli mennyiségeket, a mintában felmért eredmények
annak teljes körű megbízhatóságát nem támasztották alá. Orv Hetil 2019; 160(50): 1990–1998 Kulcsszavak: csecsemő, táplálkozás, gyermek, fehérjebevitel, testtömegindex Erdélyi-Sipos Alíz1 ■ Badacsonyiné dr. Kassai Krisztina1 ■ Kubányi Jolán1
Szűcs Zsuzsanna1 ■ Biró Lajos dr.2 ■ Raposa L. Bence dr.3 1Magyar Dietetikusok Országos Szövetsége, Budapest
2NutriComp Bt., Budapest
3Pécsi Tudományegyetem, Egészségtudományi Kar, Ápolástudományi,
Alapozó Egészségtudományi és Védőnői Intézet, Pécs Bevezetés: Az elmúlt időszakban megjelent kutatási eredmények alátámasztják azt a feltételezést, hogy a felnőttkori
krónikus betegségek kialakulását jelentősen befolyásolja a méhen belüli tápanyag-ellátottság és a kisdedkorban folyta
tott táplálás és táplálkozás. p
p
Célkitűzés: A vizsgálat célja a hazai 0–3 éves csecsemők, kisdedek táplálkozási szokásainak felmérése, illetve a csecse
mő- és kisdedkorú gyermekek táplálkozását a leginkább jellemző problémák feltárása, a táplálási/táplálkozási ajánlá
sok módosításához, korszerűsítéséhez szükséges adatok megismerése, szolgáltatása volt. Módszer: A vizsgálat a Magyar Dietetikusok Országos Szövetségének szakmai koordinálásával, iparági kutatás kereté
ben valósult meg 2015. június és augusztus között, Budapest, Debrecen, Győr, Szeged és Pécs városainak 0–3 éves
populációja körében. A felmérés antropometriai mérésekkel és validált háromnapos táplálkozási naplóval történt. Módszer: A vizsgálat a Magyar Dietetikusok Országos Szövetségének szakmai koordinálásával, iparági kutatás kereté
ben valósult meg 2015. június és augusztus között, Budapest, Debrecen, Győr, Szeged és Pécs városainak 0–3 éves
populációja körében. A felmérés antropometriai mérésekkel és validált háromnapos táplálkozási naplóval történt. Eredmények: A 4–12 hónapos (n = 220) csecsemők 18,6%-a 10 percentilis alatti értékkel rendelkezett, 10% 85–97
ili kö é
í 3%
h l d
97
ili
A 12 24 hó
(
227)
k k 15%
10 Módszer: A vizsgálat a Magyar Dietetikusok Országos Szövetségének szakmai koordinálásával, iparági kutatás kereté
ben valósult meg 2015. június és augusztus között, Budapest, Debrecen, Győr, Szeged és Pécs városainak 0–3 éves
populációja körében. A felmérés antropometriai mérésekkel és validált háromnapos táplálkozási naplóval történt. Eredmények: A 4–12 hónapos (n = 220) csecsemők 18,6%-a 10 percentilis alatti értékkel rendelkezett, 10% 85–97
percentilis közé esett, míg 3% meghaladta a 97 percentilist. A 12–24 hónapos (n = 227) gyermekek 15%-a 10 percen
tilis alatti testtömegindexszel (BMI) rendelkezett (sovány), 14% BMI-je 85–97 percentilis közé esett (túlsúlyos), és
2,6% a 97 percentilist meghaladó BMI-vel rendelkezett (elhízott). A 25–36 hónapos gyermekek (n = 184) 70%-ának
normál-BMI-je volt, 4%-uk túlsúlyos, 2%-uk elhízott, 24%-uk sovány volt. Míg a hazai referenciaérték alapján a fel
mérésben részt vevő 4–12 hónapos gyerekek 10,9%-a, az 1–2 évesek 20%-a, a 2–3 évesek 47%-a a magas fehérjebe
viteli tartományba esett, addig a 2013-ban kiadott EFSA- (Európai Élelmiszerbiztonsági Hatóság) ajánlás referencia
értékeihez viszonyítva viszont a vizsgált gyerekek 100%-a a magas fehérjebeviteli csoportba tartozik minden
korcsoportban. EREDETI KÖZLEMÉNY EREDETI KÖZLEMÉNY 0–3 éves korú csecsemők és kisdedek
táplálkozási szokásainak felmérése,
különös tekintettel
a makro- és mikronutriens-bevitelre Erdélyi-Sipos Alíz1 ■ Badacsonyiné dr. Kassai Krisztina1 ■ Kubányi Jolán1
Szűcs Zsuzsanna1 ■ Biró Lajos dr.2 ■ Raposa L. Bence dr.3 Rövidítések kezményének tekinthetők. Az említett jelenség ugyanak
kor bármely életkorban számos lehetőséget ad a beavat
kozásra [4, 5]. BMI = (body mass index) testtömegindex; EFSA = (European
Food Safety Authority) Európai Élelmiszerbiztonsági Hatóság;
FAO = (Food and Agriculture Organization of the United
Nations) Élelmezésügyi és Mezőgazdasági Világszervezet;
ICP = (infancy childhood puberty) csecsemő-, gyermek-, ser
dülőkori modell; IGF1 = (insulin-like growth factor 1) inzulin
szerű növekedési faktor-1; RÉ = referenciaérték; TGI = (Target
Group Index) Célcsoport Index; UNU = (United Nations
University) Egyesült Nemzetek Egyeteme; UVB = (ultraviolet
B) ibolyántúli B, közepes hullámhosszú sugárzás; WHO =
(World Health Organization) Egészségügyi Világszervezet [ ,
]
A gyermek születési testtömege, illetve hossza megha
tározó nemcsak a pillanatnyi morbiditás, mortalitás
szempontjából, de hosszú távon is befolyásolja az egész
ségügyi kilátásokat. Példaként említhető az intrauterin
növekedés elmaradása (kis súlyú magzat) és a szívkoszo
rúér-betegségek, a stroke, a diabetes és a magas vérnyo
más magasabb rizikója közötti összefüggés. Ugyanígy a
magas születési testtömeg (ún. macrosomia), valamint a
diabetes, illetve a szív- és érrendszeri betegségek, az
emlő- és egyéb daganatok emelkedett kockázata közötti
korreláció is megfigyelhető [6–11]. A felnőttkori króni
kus betegségek kialakulását igen jelentősen befolyásolja
az élet fogantatástól számított első 1000 nap időszaká
ban a táplálás módja, annak mennyisége és minősége
[12]. Ennek ismeretében a táplálás kiemelt jelentőséggel
kezelendő már a magzati élet kezdetétől. A tudományos
figyelem korábban a méhen belüli alultápláltság és a
csökkent születési testtömeg káros hatásaira irányult. Az
elhízás járványszerű terjedésével azonban napjainkban
egyre inkább a várandósság és a szoptatás alatt bekövet
kező anyai túltáplálkozásnak az utód hosszú távú egész
ségi állapotát érintő hatására tevődik át a hangsúly [13]. Az étrend minősége minden életszakaszban meghatáro
zó eleme az egyén egészségi állapotának. Szerepe a kró
nikus, nem fertőző betegségek kialakulásában ma már
kellően bizonyított, éppen ezért a táplálkozás területén
megvalósított intervenció nélkülözhetetlen eleme a pre
venciónak. A krónikus betegségek előfordulása napjaink
ban világszerte egyre szélesebb tömegeket érint. Ma már
azt is tudjuk, hogy ezen kórállapotok többsége ugyan
azon kockázati tényezőkre, köztük a helytelen táplálko
zásra vezethető vissza. Felmerül ugyanakkor, ha a meg
betegedések önmagukban nem fertőzőek is, mégis annak
tekinthetők a kockázati tényezők szintjén, hiszen az élet
módszokások öröklődnek egyik generációról, populáció
ról a másikra, illetve a globalizációnak köszönhetően
sokszor az országhatárokat is átlépik [1–3]. A kora csecsemőkori élet környezetének jelentősége is
egyre jobban felértékelődik, hiszen a korai gyors súlygya
rapodáshoz későbbi elhízás társulhat, a méhen belüli
gyarapodástól függetlenül. Nutrition assessment of 0–3-year-old infants and toddlers with particular focus
on macro- and micronutrient intake Introduction: Recent research findings support the assumption that the development of chronic diseases in adults is
greatly influenced by the supply of nutrients in the uterus and the nutrition, nourishment of the early, toddler ages. Aim: The aim of the present study was to evaluate the nutritional habits of infants and toddlers aged 0–3 in Hungary,
and to identify the most typical problems of their nutrition, to get to know and provide the necessary data for the
modification and modernization of feeding/nutrition recommendations for infants and young children in Hungary. Method: The study was carried out with the professional coordination of the Hungarian Dietetic Association
(MDOSZ) in the framework of industry research between June and August 2015, in the 0–3-year-old population, in 2019 ■ 160. évfolyam, 50. szám ■ 1990–1998. 1990 DOI: 10.1556/650.2019.31585 ■ © Szerző(k) EREDETI KÖZLEMÉNY the cities Budapest, Debrecen, Győr, Szeged and Pécs. The survey was conducted with anthropometric measure
ments and validated by three-day dietary log templates. cities Budapest, Debrecen, Győr, Szeged and Pécs. The survey was conducted with anthropometric measure
ts and validated by three-day dietary log templates. the cities Budapest, Debrecen, Győr, Szeged and Pécs. The survey was conducted with anthropometric measure
ments and validated by three-day dietary log templates. Results: 18.6% of infants aged 4 to 12 months (n = 220) had values below 10th percentile, 10% were between 85–
97th percentiles and 3% were above 97th percentile. 15% of children aged 12–24 months (n = 227) had a body mass
index (BMI) below 10th percentile (underweight), 14% were between 85–97th percentile (overweight) and 2.6%
had BMI over the 97th percentile (obese). 70% of 25–36-month-old children (n = 184) had normal BMI, 4% were
overweight, 2% obese, 24% underweight. Based on the Hungarian reference value, 10.9% of the 4–12-month-old
children, 20% of the 1–2-year-olds, 47% of the 2–3-year-olds were in high protein intake group. However, compared
to the 2013’s reference values of the EFSA (European Food Safety Authority) recommendation, 100% of the chil
dren belong to the high protein intake group in all age groups. y
y
y
g
p
Results: 18.6% of infants aged 4 to 12 months (n = 220) had values below 10th percentile, 10% were between 85–
97th percentiles and 3% were above 97th percentile. Nutrition assessment of 0–3-year-old infants and toddlers with particular focus
on macro- and micronutrient intake 15% of children aged 12–24 months (n = 227) had a body mass
index (BMI) below 10th percentile (underweight), 14% were between 85–97th percentile (overweight) and 2.6%
had BMI over the 97th percentile (obese). 70% of 25–36-month-old children (n = 184) had normal BMI, 4% were
overweight, 2% obese, 24% underweight. Based on the Hungarian reference value, 10.9% of the 4–12-month-old
children, 20% of the 1–2-year-olds, 47% of the 2–3-year-olds were in high protein intake group. However, compared
to the 2013’s reference values of the EFSA (European Food Safety Authority) recommendation, 100% of the chil
dren belong to the high protein intake group in all age groups. Conclusion: Although the EFSA recommendation – based on the WHO/FAO/UNU macro- and micronutrient
intake values in 2007 – defines the recommended intake quantities, the results in the sample did not support its
overall reliability. Keywords: infant, nutrition, child, protein intake, body mass index Erdélyi-Sipos A, Badacsonyiné Kassai K, Kubányi J, Szűcs Zs, Biró L, Raposa LB. [Nutrition assessment of 0–3-year-
old infants and toddlers with particular focus on macro- and micronutrient intake]. Orv Hetil. 2019; 160(50):
1990–1998. (Beérkezett: 2019. július 18.; elfogadva: 2019. augusztus 20.) Rövidítések Meglátása szerint a gyermek növekedése három jól
elkülöníthető szakaszban zajlik, melyek folyamán három
szabályozó tényező egymást átfedő hatásokat fejt ki. Gyermekkorban a növekedést a növekedési hormon sza
bályozza, melynek hatását az inzulinszerű növekedési
faktor, az IGF1 termelése közvetíti. A serdülőkori növe
kedési megugrást a nemi hormonok hozzátevődő ana
bolikus hatása okozza. A csecsemőkori gyors növekedést
ezzel ellentétben azoknak a tápanyagoknak a bevitele
serkenti, amelyek a növekedési faktor, az inzulin és az
IGF1 termelését stimulálják. Ez lehet a magyarázata an
nak, hogy az anyatejjel táplált csecsemőkhöz viszonyítva
a tápszerrel tápláltaknál előálló magasabb fehérjebevitel
megemelheti a vérplazmában és a szövetekben az inzu
lin- és IGF1-szintet, ilyen módon fokozva a korai súly
gyarapodást és később az elhízást [16]. A csecsemők fe
hérjebevitelével foglalkozó kutatás és klinikai érdeklődés
középpontjában korábban a hiány kockázatai, ezek meg
előzése és kezelése állt. Mivel a szükséglettől elmaradó
fehérjebevitel egyértelműen károsan érinti a növekedést,
fejlődést, csecsemőkorban hagyományosan bőséges fe
hérjebevitelt szorgalmaznak [17]. A koraifehérje-hipoté
zis azonban, vagyis annak a feltevésnek a tudományos
megfogalmazása, miszerint az első életévben nyújtott
feleslegesen nagy fehérjebevitelhez a csecsemő gyors
súlygyarapodása társul, ami a későbbi életkorokban az
elhízás és a vele rokon állapotok kockázatának növekedé
sével is jár, ezt az irányvonalat kérdőjelezi meg [18, 19]. Ezen folyamat legjobb megelőzési eszköze az anyatejes
táplálás, hisz tudjuk, hogy az anyatejjel táplált csecsemők
körében a túlsúly vagy elhízás előfordulása alacsonyabb,
mint tápszeres társaiknál. Amennyiben nem lehetséges a
szoptatás, vagy nem áll rendelkezésre elegendő anyatej,
akkor viszont kiemelt jelentőségű, hogy az anyatej fehér
je-összetételéhez legközelebb álló tápszerrel történjen a
táplálás [20]. g
Kutatásunkban feldolgozásra kerültek az egészséges,
időre született, 0–6 hónapos, nem kizárólag anyatejjel
táplált (anyatej, anyatej és/vagy tápszer) csecsemőknek
(n = 97), a 4–12 hónapos, a hozzátáplálást megkezdett
csecsemőknek (n = 220) és a 12–36 hónapos kisdedek
nek (n = 411) a táplálkozására, tápláltságára vonatkozó
adatok egyaránt. A kérdőívek (n = 728) 1,2%-a kizárásra
került azonosítási problémák, valamint a szélsőségesen
csekély vagy nagy energiafelvétel miatt, biztosítva ezzel a
szélsőértékek kizárását. A jelen cikkben a 0–6 hónapos
korosztályra vonatkozó mérési eredményeket nem mu
tatjuk be, a kizárólagos anyatejes táplálás preferálása
miatt. A felmérés ,,háromnapos táplálkozási napló” és kérdő
ív segítségével történt, a nemzetközi ajánlásnak megfele
lően, majd a visszakérdezett és pontosított adatokat die
tetikus szakemberek rögzítették a ,,NutriComp Étrend”
szoftverben, amellyel energiatartalom- és tápanyagszá
mítás történt. Módszer A nagyvárosi reprezentativitás biztosítása érdekében
több területről (Budapest, Debrecen, Szeged, Pécs,
Győr) kerültek bevonásra résztvevők a mintába. A min
tavétel alapját egyrészről a TNS Hofmann Kft. által biz
tosított Target Group Index (TGI) nagymintás kutatása,
másrészről a célzott adatfelvétel adta. A reprezentativitás
biztosítása érdekében a TGI kutatási mintájából megha
tározásra került a célcsoport nagysága, valamint a minta
demográfiai háttere, összetétele (korcsoport, lakóhely,
iskolai végzettség) egyaránt. A vizsgálatban részt vevő
édesanyák megkeresése kérdezőbiztos által történt, vé
letlenszerű, random mintaválasztással és ,,snow-balling”
módszerrel 2015. június és augusztus között. Kizárási
kritérium: az önkéntesség feltételén kívül, a normál, szá
jon át való táplálást akadályozó betegség, valamint a lá
zas állapot, fertőző betegség. A validált kérdőívek, táplál
kozási naplók adatainak statisztikai feldolgozását az Rövidítések Az energia- és tápanyagfelvételi adatokat a hazai aján
lások referenciaértékei (RÉ) alapján kategóriákba sorol
tuk (kritikus határérték alatti – a RÉ <70%-a; megfelelő
felvétel – a RÉ 70–130%-a; határérték feletti – a RÉ
>130%-a ), majd összehasonlítottuk. A szoftver adatbázi
sa lehetővé tette a speciális elemzéseket, mivel a korcso
port-specifikus, csecsemők és kisgyermekek számára ké
szített élelmiszereket, ételeket, valamint az esetlegesen
alkalmazott étrend-kiegészítőket is tartalmazta. A kutatásban részt vevő gyermekek antropometriai
paraméterei – mint a testtömeg, a testhossz és a testma
gasság – validált műszerekkel kerültek felmérésre. A
mért adatokból testtömegindexet (BMI) számoltunk,
majd a Cole-féle módszer alapján kategóriákat alkot
tunk; s mivel a gyermekek kétéves kora alatt nem áll ren
delkezésre megbízható BMI-határérték-táblázat, szük
ségszerűen a hazai BMI-percentilis-táblázatok határérté
kei alapján meghatározott kategóriák kerültek kialakí
tásra [21–24]. Rövidítések A megfigyeléseken alapuló
tanulmányok után most ennek a területnek a kutatása a
programozás hátterében álló molekuláris mechanizmu
sok jobb megismerésére törekszik [14, 15]. Többek kö
zött ezért is kiemelt jelentőségű a csecsemőtáplálás. Az
1980-as években Karlberg az emberi növekedés modulá
torainak modelljét terjesztette elő, a csecsemő-, gyer Szintén bizonyítottnak tekinthető, hogy a krónikus
megbetegedések rizikója már a magzati életben megjele
nik és az időskorig elkíséri az egyént. Ennélfogva a fel
nőttkorban manifesztálódó betegségek az egész életen át
tartó környezeti hatások (fizikai és szociális), valamint
életmódszokások (például táplálkozás) halmazati követ 1991 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP EREDETI KÖZLEMÉNY Excel 2016 (Microsoft Corporation, Redmond, WA,
Amerikai Egyesült Államok [USA]), valamint az SPSS
24.0 (IBM Corporation, Armonk, NY, USA) progra
mokkal végeztük el. mek-, serdülőkori modellt (infancy childhood puberty,
ICP). Meglátása szerint a gyermek növekedése három jól
elkülöníthető szakaszban zajlik, melyek folyamán három
szabályozó tényező egymást átfedő hatásokat fejt ki. Gyermekkorban a növekedést a növekedési hormon sza
bályozza, melynek hatását az inzulinszerű növekedési
faktor, az IGF1 termelése közvetíti. A serdülőkori növe
kedési megugrást a nemi hormonok hozzátevődő ana
bolikus hatása okozza. A csecsemőkori gyors növekedést
ezzel ellentétben azoknak a tápanyagoknak a bevitele
serkenti, amelyek a növekedési faktor, az inzulin és az
IGF1 termelését stimulálják. Ez lehet a magyarázata an
nak, hogy az anyatejjel táplált csecsemőkhöz viszonyítva
a tápszerrel tápláltaknál előálló magasabb fehérjebevitel
megemelheti a vérplazmában és a szövetekben az inzu
lin- és IGF1-szintet, ilyen módon fokozva a korai súly
gyarapodást és később az elhízást [16]. A csecsemők fe
hérjebevitelével foglalkozó kutatás és klinikai érdeklődés
középpontjában korábban a hiány kockázatai, ezek meg
előzése és kezelése állt. Mivel a szükséglettől elmaradó
fehérjebevitel egyértelműen károsan érinti a növekedést,
fejlődést, csecsemőkorban hagyományosan bőséges fe
hérjebevitelt szorgalmaznak [17]. A koraifehérje-hipoté
zis azonban, vagyis annak a feltevésnek a tudományos
megfogalmazása, miszerint az első életévben nyújtott
feleslegesen nagy fehérjebevitelhez a csecsemő gyors
súlygyarapodása társul, ami a későbbi életkorokban az
elhízás és a vele rokon állapotok kockázatának növekedé
sével is jár, ezt az irányvonalat kérdőjelezi meg [18, 19]. Ezen folyamat legjobb megelőzési eszköze az anyatejes
táplálás, hisz tudjuk, hogy az anyatejjel táplált csecsemők
körében a túlsúly vagy elhízás előfordulása alacsonyabb,
mint tápszeres társaiknál. Amennyiben nem lehetséges a
szoptatás, vagy nem áll rendelkezésre elegendő anyatej,
akkor viszont kiemelt jelentőségű, hogy az anyatej fehér
je-összetételéhez legközelebb álló tápszerrel történjen a
táplálás [20]. mek-, serdülőkori modellt (infancy childhood puberty,
ICP). 2019 ■ 160. évfolyam, 50. szám Tápláltsági állapot, energiabevitel táblázat
A 4–36 hónapos korosztály tápláltsági állapota Tápláltsági állapot, energiabevitel Az összes energiabevitelt megvizsgálva elmondható,
hogy a 4–12 hónapos korcsoport fele a határérték feletti,
magas energiabevitellel rendelkezik. Ez az arány az 1–2
évesek körében közel 85%, míg a 2–3 éves kisdedek ese
tében megközelíti a 76%-ot. A kutatásban felmért 4–36
hónapos korosztály energiabeviteli adatainak megoszlá
sát az 1. ábra szemlélteti. Megemlítendő továbbá, hogy az energiabeviteli ada
tok mellett a testtömegadatok kiértékelésekor is körülte
kintésre volt szükség. Az adatok helyes interpretációjá 1992 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP EREDETI KÖZLEMÉNY
51
15
24
49
85
76
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
Energiabevitel
1. ábra
A 4–36 hónapos korcsoport napi energiabevitele a referencia-beviteliérték arányában EREDETI KÖZLEMÉNY
51
15
24
49
85
76
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
Energiabevitel
1. ábra
A 4–36 hónapos korcsoport napi energiabevitele a referencia-beviteliérték arányában EREDETI KÖZLEMÉNY ónapos korcsoport napi energiabevitele a referencia-beviteliérték arányáb 1. ábra
A 4–36 hónapos korcsoport napi energiabevitele a referencia-beviteliérték arányában 1. ábra delkezik, potenciálisan sovány, 10–14,1%-uk potenciáli
san túlsúlyos (BMI: 85–97 percentilis), 2,6–3,5%-uk po
tenciálisan elhízott (BMI >97 percentilis) (1. táblázat)
[23]. hoz a hazai és a nemzetközi ajánlások felnőttkori
BMI-táblázatai mellett figyelembe kellett vennünk és
mérlegelnünk a vizsgált csoportok egyedi sajátosságait is. Ennek ismeretében elmondható, hogy a túlsúly és az el
hízás a 2–3 éves korosztály 6,5%-át érintette. A mért ada
tokat az 1. táblázat prezentálja. Ezen adatok ismeretében fontos felhívnunk a szakem
berek és a lakosság figyelmét, hogy egészséges gyermekek
esetében az első két évben (0–24 hó) nem kívánatos a
gyors testtömeg-gyarapodás. A percentilisek határán el
helyezkedő BMI pedig célzott beavatkozást igényel [25]. A hazai BMI-percentilis-táblázatok alapján (korcso
portok szerint szeparálva) a kétéves vagy annál fiatalabb
gyermekek 15–18,6%-a 10 percentilis alatti BMI-vel ren
1. táblázat
A 4–36 hónapos korosztály tápláltsági állapota
Hazai percentilisértékek, kategóriák
BMI-átlag
(kg/m2)
<10 pct
10–84,9 pct 85–96,9 pct
≥97 pct
4–12 hó
16,5
18,6
68,2
10,0
3,2
12–24 hó
16,5
15,0
68,3
14,1
2,6
Cole-kategóriák
„Sovány”
„Normál”
„Túlsúlyos” „Elhízott”
25–36 hó
15,8
23,9
69,6
4,3
2,2 1. táblázat
A 4–36 hónapos korosztály tápláltsági állapota 1. táblázat
A 4–36 hónapos korosztály tápláltsági állapota 1. Fehérjebevitel A testtömegkilogrammra meghatározott fehérjebevitel a
4–12 hónaposak esetében 3,1 g/ttkg (EFSA-ajánlás:
1,48–1,65 g/ttkg), míg a 12–36 hónapos gyermekeké
4,1 g/ttkg (EFSA-ajánlás: 1,13–1,26 g/ttkg) volt. Fél–
egy éves kor körüli időszaktól kezdődően a gyermekek
fehérjebevitele jelentősen meghaladja a testtömeg
arányos ajánlásokat. A mért fehérjebeviteli értékeket a
2. ábra mutatja be. A hazai BMI-percentilis-táblázatok alapján (korcso
portok szerint szeparálva) a kétéves vagy annál fiatalabb
gyermekek 15–18,6%-a 10 percentilis alatti BMI-vel ren A hazai BMI-percentilis-táblázatok alapján (korcso
portok szerint szeparálva) a kétéves vagy annál fiatalabb
gyermekek 15–18,6%-a 10 percentilis alatti BMI-vel ren 25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
2
98
100
100
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Fehérjebevitel
2. ábra
A 4–36 hónapos korcsoport fehérjebevitele 25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
2
98
100
100
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Fehérjebevitel
2. ábra
A 4–36 hónapos korcsoport fehérjebevitele 2019 ■ 160. évfolyam, 50. szám 1993 ORVOSI HETILAP EREDETI KÖZLEMÉNY
25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
45
65
68
43
27
24
12
8
8
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Zsírbevitel
3 áb
A 4 36 hó
k
í b
i
l EREDETI KÖZLEMÉNY
25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
45
65
68
43
27
24
12
8
8
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Zsírbevitel
3. ábra
A 4–36 hónapos korcsoport zsírbevitele EREDETI KÖZLEMÉNY 3. ábra
A 4–36 hónapos korcsoport zsírbevitele Zsírbevitel A 4–12 hónapos (hozzátáplált) csecsemők jelentős há
nyada kedvezőtlenül magas bevitelt mutat koleszterin
(40,9%) esetén. Ez igaz az 1–3 éves gyermekek felére is
(50,1%). A koleszterinbevitel szempontjából pozitív ös�
szefüggés van a tejek, tejtermékek, tojás, húsok, majd a
hús- és májkészítmények esetében. A zsírok felvételének tekintetében e korcsoportban kevés
bé jellemző a határérték feletti bevitel. A 4–12 hónaposak
43%-ának, az 1–2 évesek 27%-ának és a 2–3 éves kisdedek
24%-ának megfelelő a zsírbevitele. Az alacsony zsír
fogyasztás a leginkább az 1 éves feletti korcsoportot jel
lemzi (12–24 hónaposak: 65%, 25–36 hónaposak: 68%)
(3. ábra). Amennyiben a hazai referenciaérték-tarto
mányhoz (Rodler Imre [szerk.] Új tápanyagtáblázat) vi
szonyítjuk, akkor nagyobb százalékban szerepelnek a cse
csemők és a kisdedek a normáltartományban, és kevesebb
az alacsony zsírbevitelűek aránya. Az egyes tápanyagok
élelmiszercsoportonként való megoszlásának (százalékos
tápanyagprofil) közvetlen elemzésére nem volt lehetőség,
azonban vizsgálható az egyes tápanyagok bevitele és az
élelmiszercsoportok fogyasztásának összefüggése. A
4–12 hónapos gyermekek (n = 220) napi átlagos zsírfo
gyasztása 30,6 g/fő, amelyből állati eredetű 14,6 g, nö
vényi eredetű 6,7 g. 12–24 hónaposaknál (n = 259) a
napi zsírfogyasztáson (45,9 g) belül ez az arány 25,3 g és
16,6 g, 25–36 hónapos korban (n = 152) a napi 53,9 g
zsírbevitelből 30,5 g állati eredetű, míg 22,7 g növényi. Szénhidrát és hozzáadott cukor bevitele A 4–12 hónapos korcsoport közel felének határérték fe
letti, míg az 1–3 évesek többségének megfelelő (12–24
hónaposak: 77%, 25–36 hónaposak: 82%) a szénhidrát
bevitele (4. ábra). A hozzáadott cukor 4–12 hónapos korban fordított
összefüggést mutat a tej és tejtermékek fogyasztásával,
pozitív összefüggést pedig a kész és félkész ételek, bébi
ételek fogyasztásával. A hozzáadott cukor napi bevitele a
4–12 hónapos korcsoportban 11,5 g, az 1–2 éveseké
27,5 g, és a 2–3 éveseké 45,3 g. A szénhidrátokon belül
a hozzáadott cukorból származó energia a 12–36 hóna
pos gyermekek esetében közel 10%, csecsemők esetén
3–5% körüli. 2019 ■ 160. évfolyam, 50. szám
ORVOSI HETILAP
1994
25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
5
11
9
47
77
82
48
12
9
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Szénhidrátbevitel
4. ábra
A 4–36 hónapos korcsoport napi szénhidrátbevitele 25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
4–12 hónaposak (n = 220)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013
Alacsony
Normál
Magas
5
11
9
47
77
82
48
12
9
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Szénhidrátbevitel ORVOSI HETILAP 1994 2019 ■ 160. évfolyam, 50. szám EREDETI KÖZLEMÉNY 2. táblázat
Ásványi anyagok átlagos napi bevitele (4–36 hónapos gyerme
kek körében)
Korcsoport
(hó)
Nátrium
(mg)
Kálium
(mg)
Kalcium
(mg)
Magnézium
(mg)
Vas
(mg)
4–12
(n = 220)
623
1378,7
465,7
128,71
6
12–36
(n = 411)
1236
1949,9
632,57
228,62
7,4
12–24
(n = 259)
1704
1823,4
590,2
210,1
7,19
25–36
(n = 152)
2198,5
2165,4
704,6
260
7,76 2. táblázat
Ásványi anyagok átlagos napi bevitele (4–36 hónapos gyerme
kek körében) húskészítményt (átlagosan 33,6 g/fő/nap) és 18%-uk
belsőséget (átlagosan 9,1 g/fő/nap). A 12–36 hónapo
sak 99%-a fogyaszt húst és húskészítményeket (átlagosan
71,2 g/fő/nap), 33%-uk belsőséget (átlagosan 9,7 g/
fő/nap). /
p)
A kalciumbevitel a 4–12 hónapos (hozzátáplált) cse
csemők 22,1%-ánál és az 1–3 éves gyermekek 41,8%-
ánál hiányosnak tekinthető. Az életkor növekedésével
jelentősen nő az alacsony kalciumbevitellel rendelkezők
száma. A 4–12 hónapos csecsemők 79%-a kap tejet és
tejterméket (átlagosan 362,1 g/fő/nap). A 12–36 hó
naposak 100%-a kap ebből az élelmiszercsoportból (átla
gosan 320,7 g/fő/nap), vagyis az életkor előrehalad
tával csökken a tej és tejtermékek fogyasztásának
mennyisége. Ásványi anyagok és vitaminok bevitele Ásványi anyagok és vitaminok bevitele
A 4–12 hónapos (hozzátáplált) csecsemők jelentős há
nyadának (71%) kedvezőtlenül magas a nátriumbevitele,
míg az egyéb ásványi anyagok és vitaminok többségének
felvétele jónak mondható; ugyanakkor fontos megemlí
teni a jelentős prevalenciájú (22,1%) elégtelen kalcium
felvételt, valamint a nagyarányú vas- (43%) és D-vitamin-
hiányt (79%). Az 1–3 éves gyermekek száz százaléka
magas bevitelt mutat a nátrium esetén, és foszforból ez
az arány 67%; hiányosnak tekinthető a kalcium (41,8%),
a vas (25%) és a D-vitamin (87,1%) bevitele. E korcso
port kevésbé használ D-vitaminos étrend-kiegészítőt, így
étrend-kiegészítővel ez az arány csak 65%-ra csökken. A korcsoportokra lebontott ásványianyag-bevitelek átla
gát a 2. táblázat tartalmazza. Szénhidrát és hozzáadott cukor bevitele Az eredményt tovább rontja, hogy az 1–3
éves korosztály 31,6%-a kedvezőtlenül magas bevitelt
mutat a foszfor tekintetében, így még inkább eltolódik
az ajánlott 1,4 : 1 – 1,9 : 1 kalcium : foszfor arány. Ez az
intenzív csontfejlődés szakaszában igen nagy problémát
jelent. Az adatok a későbbi életkorban sem alakulnak
kedvezően [26]. Ásványi anyagok és vitaminok bevitele Vitaminok bevitele A tudományos publikáció formai követelményei nem te
szik lehetővé a teljes vizsgálati anyag részletes ismerteté
sét, ugyanakkor az egyik legfontosabb vitamin beviteli
értékei mellett nem mehetünk el szó nélkül, annak kor
csoport-specifikus fontossága miatt. p
pi
g
A D-vitamin-bevitel az elvárt szintnél alacsonyabb
értékeket mutatott mind a 12–24 hónapos kisdedek
(83%), mind a 25–36 hónapos gyermekek esetében
(94%). Étrend-kiegészítőket is figyelembe véve ezek az
értékek 58%-ra, illetve 76%-ra csökkentek. A táplálkozási
naplók elemzésekor az érintettek táplálékkal, illetve ét
rend-kiegészítővel történő D-vitamin-felvételének vizs
gálatára volt elsősorban lehetőségünk. Ugyanakkor a
vizsgálat során kitöltött kérdőívből kiderült, hogy a gyer
mek növekedésével egyenes arányban nő a szabad leve
gőn töltött idő hossza. Az 1 éves kor alatti gyermekekkel
tipikusan 1–2 órát, míg az idősebbekkel a legtöbben 2–4
órát töltenek a szabadban. Ez a bőrt érő napsugárzásnak,
illetve a napsugarak UVB-spektrumának az endogén-D-
vitamin-szintézisben betöltött szerepe miatt nagy jelen
tőségű [27]. A többlet-nátriumbevitel a 4–12 hónapos és az 1–3
éves korcsoportban elsősorban a kenyérfélék, a húské
szítmények és az ízesítőanyagok, fűszerek, egyéb élelmi
szercsoportok fogyasztásából származik. Az ételkészítési
szokásokkal és az élelmiszer-választásban megfigyelhető
sajátosságokkal magyarázható a kedvezőtlenül magas
nátriumbevitel. A vas nélkülözhetetlen a pszichomotoros és kognitív
funkciók fejlődéséhez, hiányában ezekben a funkciókban
zavarok keletkezhetnek. A kor előrehaladtával javuló
tendenciát mutat az elégtelen vasbevitel, hiszen a 4–12
hónapos (hozzátáplált) csecsemőknél talált nagyarányú
vashiány 42,3%-ról 1–3 éves gyermekeknél 25,3%-ra
csökkent. Fontos kiemelni, hogy megfelelő hozzátáplá
lással, étkeztetéssel ezek az arányok jelentősen javíthatók
lennének. A 4–12 hónapos csecsemők 70%-a kap húst és A felmért, részletes vitaminbeviteli átlagokat a 3. táb
lázat tartalmazza. 1995
ORVOSI HETILAP
2019 ■ 160. évfolyam, 50. szám
3. táblázat
Vitaminok átlagos napi bevitele (4–36 hónapos gyermekek körében)
Korcsoport
(hó)
A-vitamin
(µg)
β-Karotin
(mg)
B1-vitamin
(µg)
B2-vitamin
(µg)
B6-vitamin
(µg)
B12-vitamin
(µg)
C-vitamin
(mg)
D-vitamin
(µg)
Folsav
(µg)
4–12 (n = 220)
574,8
3,76
643,15
682,7
775,4
1,19
80,8
4,35
72,8
12–36 (n = 411)
419,9
2,47
726
1020,9
1115,5
2,51
86,37
3,36
109,1
12–24 (n = 259)
435,3
2,53
711,9
947,76
1048,7
2,00
78,9
3,93
102,4
25–36 (n = 152)
393,6
2,37
750,2
1145,7
1229,4
3,31
99
2,4
120,5 3. táblázat
Vitaminok átlagos napi bevitele (4–36 hónapos gyermekek körében) 2019 ■ 160. évfolyam, 50. Egyéb tápanyagok g
g
Vegyes táplálkozás mellett a 12–24 hónapos korcsoport
felének megfelelő az élelmirost-bevitele, csupán 4%-uk
nak alacsonyabb a referenciaértékhez hasonlítva. Az 1–3
évesek 59%-ára magasabb rostbevitel jellemző, 35%-uk
megfelelő mennyiségű élelmi rostot fogyaszt, és csak 6%-
nak elégtelen az élelmirost-bevitele (5. ábra). Vitaminok bevitele szám ORVOSI HETILAP EREDETI KÖZLEMÉNY
25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013 (12 hónapos kor alatt
Alacsony
Normál
Magas
4
6
51
35
45
59
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Élelmirost-bevitel EREDETI KÖZLEMÉNY
25–36 hónaposak (n = 152)
12–24 hónaposak (n = 259)
A referenciaérték forrása:
EFSA-ajánlás, EFSA Journal
2013 (12 hónapos kor alatt
nincs beviteli ajánlás)
Alacsony
Normál
Magas
4
6
51
35
45
59
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Élelmirost-bevitel
5. ábra
A 12–36 hónapos korcsoport élelmirost-bevitele 5. ábra
A 12–36 hónapos korcsoport élelmirost-bevitele 5. ábra
A 12–36 hónapos korcsoport élelmirost-bevitele Az EFSA által meghatározott, 0–3 éves gyermekekre
vonatkozó referencia-beviteliértékek szerint magas ener
giabevitelű a 12–24 hónapos gyermekek 84,6%-a; 25–36
hónapos korban ez az érték 75,7%. Míg a hazai referen
ciaérték alapján a felmérésben a 4–12 hónapos gyerekek
10,9%-a, az 1–2 évesek 20%-a, a 2–3 évesek 47%-a a ma
gas fehérjebeviteli tartományba esett, addig a 2013-ban
kiadott – a makro- és mikronutrienseknek a WHO/
FAO/UNU által 2007-ben megadott beviteli értékein
alapuló – EFSA-ajánlás referenciaértékeihez viszonyítva
a vizsgált gyerekek 100%-a a magas fehérjebeviteli cso
portba tartozik minden korcsoportban [31–32]. A Ma
gyar Gyermekorvosok Társaságának koordinálásában
2010-ben az 1–3 éves gyermekek komplex táplálkozási
vizsgálata történt meg hasonló módszerekkel, igaz, ki
sebb elemszámmal (92 fő 1–2 éves és 102 fő 2–3 éves)
[29]. Ezért a jelen vizsgálat néhány értékét összevetettük
az öt évvel ezelőtt kapott adatokkal is. Az 1–3 éves gyer
mekek esetében az energiabevitel szempontjából jelen
tős különbség nincs a két vizsgálat (2010 vs. 2015) ered
ménye között, de a magas energiatartományba mintegy
6%-kal több gyermek tartozik 2015-ben. A makrotáp
anyagok tekintetében közel megháromszorozódott a
magas zsírbevitelű gyermekek aránya (8,2% vs. 24,1%). Vizsgálatunkban az energiát szolgáltató makrotápanya
gok (zsír és szénhidrát) a 4–12 hónapos korcsoportban
közel normálmegoszlást mutattak, a kor növekedésével
azonban az EFSA-ajánlás szerint magas az alacsony zsír
bevitellel rendelkezők aránya (mintegy 65%) – a gyakor
latilag megfelelő, többségében normál- (mintegy 80%)
szénhidrátbevitel mellett [32]. 4–12 hónapos korban
pozitív összefüggés van a BMI és a napi energiabevitel,
valamint a makrotápanyagok (fehérje, zsír, szénhidrát)
napi bevitt mennyisége között. Az energiamegoszláson
belül a zsírból származó energiával pozitív, a szénhidrát
energia-%-kal negatív összefüggés mutatható ki. 12–36
hónapos korban a táplálkozás szerkezetének jelentős vál
tozása, valamint a szomatikus fejlődés dinamizmusa mi
att a csoport nem tekinthető homogénnek; ez megmu Irodalom [1] Kimokoti RW, Millen BE. Nutrition for the prevention of chron
ic diseases. Med Clin North Am. 2016; 100: 1185–1198. [2] Chizuru N, Ricardo U, Shiriki K, et al. The Joint WHO/FAO
Expert Consultation on diet, nutrition and the prevention of
chronic diseases: process, product and policy implications. Public
Health Nutr. 2004; 7: 245–250. Egy 2014-ben 4–10 éves budapesti és kecskeméti
gyermekekre reprezentatív táplálkozási felmérésben (n =
799) a kalcium átlagos napi felvétele korcsoportonként
kissé változó mértékben, de szintén elmaradt az ajánlott
napi 800 mg értéktől, és a gyermekek 35%-a kritikus,
határérték alatti mennyiségben fogyasztotta ezt az ásvá
nyi anyagot. Összességében a 4–12 hónapos gyermekek
22%-ánál, a 12–36 hónaposak 42%-ánál volt detektálha
tó az elégtelen kalciumbevitel. Itt is megfigyelhető, hogy
a napi foszforfelvétel átlaga meghaladja az ajánlott 620
mg értéket, így kedvezőtlen a kalcium és a foszfor hánya
dosa is. Az elégtelen kalciumbevitelhez vezető egyik té
nyező, hogy a kalcium fő élelmiszerforrásául szolgáló tej
és tejtermék fogyasztása a 4–6 éveseknél összességében
314 g/nap, míg a 7–10 éveseké 290 g/nap, s ez elmarad
a kívánatos mintegy 450–500 g/nap mennyiségtől [33]. [3] Brennan P, Perola M, Jan van Ommen G, et al. Chronic disease
research in Europe and the need for integrated population co
horts. Eur J Epidemiol. 2017; 32: 741–749. [4] Gluckman PD, Hanson MA, Cooper C, et al. Effect of in utero
and early-life conditions on adult health and disease. N Engl J
Med. 2008; 359: 61–73. [5] Barker DJ. In utero programming of chronic disease. Clin Sci
(Lond). 1998; 95: 115–128. [6] Osmond C, Barker DJ, Winter PD, et al. Early growth and death
from cardiovascular disease in women. BMJ 1993; 307: 1519–
1524. [7] Hales CN, Barker DJ. Type 2 (non-insulin-dependent) diabetes
mellitus: the thrifty phenotype hypothesis. Diabetologia 1992;
35: 595–601. [8] Cooper C, Fall C, Egger P, et al. Growth in infancy and bone
mass in later life. Ann Rheum Dis. 1997; 56: 17–21. [9] Rich-Edwards JW, Stampfer MJ, Manson JE, et al. Birth weight
and risk of cardiovascular disease in a cohort of women followed
up since 1976. BMJ 1997; 315: 396–400. A nátriumbevitel szempontjából is aggasztóak az ered
mények. Egy hazai vizsgálat megállapításai szerint 6 hó
napos kor felett minden korcsoportban magasabb a nát
riumbevitel: 400 mg [34]. [10] Charalambous M, da Rocha ST, Ferguson-Smith AC. Genomic
imprinting, growth control and the allocation of nutritional re
sources: consequences for postnatal life. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Megbeszélés 2015-ben végzett kutatásunk célcsoportját 0–3 éves kor
közötti csecsemők és kisdedek képezték. A vizsgálati
mintát önként jelentkező édesanyák és gyermekeik al
kották. A csecsemő- és kisdedkorban jellemző etetési és
táplálkozási szokások felmérése az esetleges intervenciók
tervezése, megvalósítása szempontjából ideális időszak,
mivel a helyes étkezési mintákat ekkor sajátíthatja el a
gyermek, és a hibák korrekciójára még lehetősége van a
családnak. Az epigenetika, a korai anyagcsere-programozás rávi
lágít a legfontosabb környezeti tényezők – kiemelten a
táplálkozás – hatására, a csecsemők hosszú távú egészsé
gére vagy éppen betegségeik kialakulásának kockázatára,
vagyis a csecsemő és kisded táplálása nemcsak az adott
időszakban befolyásolja a gyermek fejlődését, hanem a
felnőttkori nem fertőző betegségek kialakulására is ha
tással van [28]. A társadalom egészségfejlesztését, vala
mint a civilizációs betegségek prevencióját ennek okán
már gyermekkorban szükséges megkezdeni, ugyanis egy
2010-ben végzett felmérés szerint a vizsgált 1–3 éves
korcsoportú gyermekek táplálkozásában, illetve egy
2014-ben 4–10 éves gyermekek körében végzett vizsgá
lat adatainak elemzése alapján a felnőtt lakosságra is jel
lemző táplálkozási hibák nyomai már felfedezhetők [29,
30]. A csecsemő- vagy kisdedkorú gyermekek fejlődésük
azon periódusában vannak, amikor érdemi beavatkozás
tehető a kedvező táplálkozási szokások és preferenciák
kialakításáért, melyek a felnőttkori egészségi állapotot is
befolyásolhatják [12]. 1996 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP EREDETI KÖZLEMÉNY Szerzői munkamegosztás: K. J., B. K. K.: A vizsgálat ter
vezése és lefolytatása. Sz. Zs., E.-S. A., R. L. B.: Iroda
lomkutatás. B. L.: Statisztikai elemzések. E.-S. A., B. K. K., Sz. Zs., K. J., R. L. B.: Az eredmények interpretá
ciója. E.-S. A., B. K. K., Sz. Zs., R. L. B.: A kézirat meg
szövegezése. A cikk végleges változatát valamennyi szer
ző elolvasta és jóváhagyta. tatkozik abban is, hogy a nagy mintaszám ellenére sem
mutatható ki összefüggés a tápanyagkomponensek és a
BMI között. A mikrotápanyagok tekintetében kedvezőtlen változás
következett be: nőtt az alacsony kalciumbevitelűek ará
nya, javult azonban a megfelelő vas- és cinkbevitelűek
hányada. A 2010-ben 1–3 éves gyerekek (n = 211) között vég
zett felméréshez képest pozitívnak mondható, hogy
2015-re 10%-kal csökkent az alacsony, 3%-kal nőtt a nor
mál- és 7%-kal a magas vasbevitellel rendelkező 1–3 éves
gyermekek (n = 411) száma, de az 1–3 éves korosztály
25%-a még mindig nem jut megfelelő mennyiségű vas
hoz [29]. Megbeszélés A 2010-ben 1–3 éves gyerekek (n = 211) között vég
zett felméréshez képest pozitívnak mondható, hogy
2015-re 10%-kal csökkent az alacsony, 3%-kal nőtt a nor
mál- és 7%-kal a magas vasbevitellel rendelkező 1–3 éves
gyermekek (n = 411) száma, de az 1–3 éves korosztály
25%-a még mindig nem jut megfelelő mennyiségű vas
hoz [29]. Irodalom Nutrition in early life and the programming
of adult disease: a review. J Hum Nutr Diet. 2015; 28: 1–14. [29] Biró L, Szabó L. Complex nutritional examination of 1–3-year-
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melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye,
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Akadémiai Kiadó Zrt. A rendezvények és kongresszusok híranyagának leadása
a lap megjelenése előtt legalább 40 nappal lehetséges, a 6 hetes nyomdai átfutás miatt. Kérjük megrendelőink szíves megértését. A híranyagokat a következő címre kérjük:
Orvosi Hetilap titkársága: edit.budai@akademiai.hu
Akadémiai Kiadó Zrt. A rendezvények és kongresszusok híranyagának leadása A híranyagokat a következő címre kérjük:
Orvosi Hetilap titkársága: edit.budai@akademiai.hu
Akadémiai Kiadó Zrt. A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény,
melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye,
illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) 1998 2019 ■ 160. évfolyam, 50. szám ORVOSI HETILAP
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Organization of Lifelong Learning in the System of Professional Training of Teachers
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Organization of Lifelong Learning in the System of
Professional Training of Teachers Burlakova*(a), Anastasiya S. Budnik (b), Elizaveta S.Burlakova (c)
(a) Moscow State University of Psychology and Education, Moscow, Russia
(b) Moscow City Pedagogical University, Moscow, Russia
(c) Academy of Social Management, Moscow, Russia * Corresponding author. E-mail address: iiburlakova@mail.ru © 2019 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova
This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Published by Kazan Federal University and peer-reviewed under responsibility of IFTE-2019 (V
International Forum on Teacher Education) Proceedings IFTE-2019, 57-67
doi:10.3897/ap.1.e0051 V International Forum on Teacher Education V International Forum on Teacher Education Organization of Lifelong Learning in the System of
Professional Training of Teachers
Irina I. Burlakova*(a), Anastasiya S. Budnik (b), Elizaveta S.Burlakova (c)
(a) Moscow State University of Psychology and Education, Moscow, Russia
(b) Moscow City Pedagogical University, Moscow, Russia
(c) Academy of Social Management, Moscow, Russia Introduction Development of continuous education in EU countries In the context of globalization, most countries have reformed their professional training systems in line
with the “Lisbon strategy” adopted by the leaders of EU states, the goal of which was to create the most
competitive and dynamic knowledge-based economy in the world by 2010 (Kok, 2004). The European Commission, assessing the results of educational reforms in 2008, pointed to the need to
develop national strategies for lifelong learning. The principles of reorganization of the existing
educational systems and construction of lifelong learning are universal. The most significant of them are: - orientation of the educational system to the individual, unique personality and basic needs, among
which the leading place is the need for lifelong self-improvement, self-development and self-realization; - democratization of the educational system, accessibility and openness of any level and form of
education to each individual, regardless of social status, nationality, race, physical condition; - flexibility of the educational system, rapid response to the educational needs of the population, special
interests, styles and rates of potential students learning; - variability of educational services and the realization of the right of every person to choose their own
strategy for further education after graduation; - integration of formal and non-formal types of education, creation of a holistic educational segment
that turns the adult population of the country into “students”; - use of information technologies for educational purposes at any stage of their life (home, work,
recreation, transportation, etc.); - development of an integrated system of information support; - development of an integrated system of information support; - the existence of an extensive and constantly updated legal and regulatory framework for lifelong
learning. In European countries, lifelong learning is essential for two reasons: In European countries, lifelong learning is essential for two reasons: 1) the population of the European Union lives in a complex socio-political and economic environment,
where the full development of the person is impossible without mastering the ability to adapt quickly to
cultural, linguistic and ethnic diversity; 2) knowledge-centrality is the basis of modern European society. This means that the knowledge,
information and motivation to update it constantly and the skills, necessary to do this, become decisive
factors for the competitiveness of the individual and the effectiveness of the human potential in a market
economy (Commission of the European Communities, 2000). Abstract The relevance of the present issue is caused by the need for carrying out lifelong learning of teachers as an
essential condition of their professional development. The aim of the article is to prove the necessity of
integrating formal and informal professional and optional development of educators. The main methods of
the research are analysis and synthesis of the data obtained. The authors hold an analysis of the lifelong
learning development in the EU countries and in Russia, which can be formal and informal. The latest
represents an entirely new phenomenon in the social and educational practice. Its content bases on their
own principles, carries out specific functions and solves existing problems in an innovative way. The
article also views the strategies and models of lifelong professional education of teachers being under a
significant pressure of informational and communicational technologies and relying on informal
mechanisms of giving knowledge and forming competences. In the research part of the article the objective
and subjective difficulties of teachers and their professional needs are revealed. Lifelong learning is bound
to overcome these difficulties and satisfy the necessity of forming and developing new professional
competences. The analysis of the documents of educational institutions shows the dominance of informal
education. It proves the need for defining the status of informal education as a constituent part of
professional development of an educator. Keywords: lifelong learning of teachers, informal education, personality-oriented trajectory of continuous education,
models and strategies of lifelong learning, professional development of an educator. © 2019 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova
This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited. Published by Kazan Federal University and peer-reviewed under responsibility of IFTE-2019 (V
International Forum on Teacher Education) * Corresponding author. E-mail address: iiburlakova@mail.ru 58 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 Introduction In such circumstances, education is designed to help people to cope successfully with adaptation to new
conditions of life. These reasons for the development of lifelong learning are closely interrelated and
determine the two main goals: the active position of citizens and the competitiveness of a professional in
the labor market. It must be noted that the governments of European countries have come to a common
understanding of the need to develop lifelong learning as a combination of formal, non-formal and
informal education. Taking into account this factor, the modern national systems of lifelong learning
develop nowadays in the European Union. The concept of “lifelong learning” appeared in science in 1929, and in 1968 – in the materials of
UNESCO. In the terminology of UNESCO from 1997 lifelong learning is defined as “formal, non-formal,
non-institutional (informal)”, i.e. it is possible in any organizational form (UNESCO, 1995-2012). The scientific literature uses several concepts of lifelong learning, differing from - education as lifelong learning (life-long education); Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 59 - lifelong education for adults; - lifelong education for adults; - lifelong professional education. - lifelong professional education. The European Union defines formal education in the 2000 Memorandum as the process leading to
the issuance of a state diploma or certificate (Commission of the European Communities, 2000). According to the International standard classification (2004), formal education is provided through
formal institutions (College / University). Formal education is a system of hierarchically structured and
inherent to the majority of States including Russia (International Labour Organization). Non-formal education is a qualitatively new phenomenon in social and educational practice, the content
of which is based on its own principles, performs certain functions, solving many old problems in a new
way. Non-formal education is a flexible educational system focused on specific needs and interests of
students. Non-formal education has the characteristic of «complementarity» of the acquired knowledge in
relation to the professional education and experience. A study of non-formal education shows that it is
neither a simple addition to or continuation of traditional education nor an alternative to traditional
education. According to Klimov, non-formal education, located between the poles of formal and informal
education, is conscious in contrast to the latest, organized and managed, focused on the specific
educational needs of different professional groups (Klimov, 1998). Introduction In 1987, the Decision of the CPSU Central Committee and the USSR Council of Ministers “On
measures to improve the training and use of scientific-pedagogical and scientific personnel” sets the task of
restructuring the training of scientific-pedagogical and scientific personnel, presenting it as an integral and
mandatory part of the unified system of lifelong education in the country. In 1989, and then in 2010, the
concepts of lifelong learning were discussed and adopted, but they were not implemented. A new stage in the development of lifelong learning began with the adoption of a new Federal Law
“Оn education in the Russian Federation”. This law introduces such concepts as: “lifelong learning”, “e-
learning”, “modular principle of presenting the content of programs and building curricula”, “network
forms of program implementation”. The drafters of the education act define different ways of getting
education: - through the organization of credit-modular system of educational process; - through networking in the implementation of educational programs; - through the use of distance learning technologies in the educational process; - through the organization of training with the help of integrated educational programs; - through the use of information and educational resources in the educational process, etc. (Ministry of
Education and Science, 2012). The priority direction of the state policy in the sphere of education is the development of lifelong
learning, which includes various forms of education throughout the life of a person. In the Federal Program
of education development for 2016-2020 years, it is noted that due to the increase in the requirements for
teaching staff in connection with the adoption of the professional standard of the teacher, a fundamental
change in the socio-cultural educational environment, the need for highly qualified teaching staff capable
of effectively solving the problems of education modernization at all levels gets stronger (Ministry of
Education and Science of the Russian Federation, 2015). The government has set a difficult task to design a new image of the integrated education system as a
branch of the national economy, creating favorable conditions for both personal and professional
development of employees. In June 2015, the Ministry of Education and Science of the Russian Federation presented for discussion
the Concept of lifelong learning development of adults in the Russian Federation for the period up to 2025. Introduction McConnell and Brue (1992) highlighted the general characteristics of all types of non-formal
education: - continues throughout life; - focuses on active participation in the training of students; - emphasizes the importance of the problems and needs of people as the fundamental points for the
organization of training; - carried out within certain communities (professional communities); - uses the methods and contexts of non-formal education, the impact of direct life experience - the absence of coercive based on other people's motivation; - high personal meaning of learning; - high personal meaning of learning; - internal responsibility of students for the result of educational activities; - flexibility in organization and methods of teaching; - high level of activity of students; - self-assessment of students' results on the basis of their relevant criteria. The increasing role of the teacher in the modernization of education has led to the fact that in developed
European countries the concept of “lifelong professional development of teachers” appeared. M. Fullan, a
researcher of the effects of school reforms carried out in the United States and Canada at the end of the
twentieth century, believes that the professional development of teachers – a set of formal and non-formal
education throughout the career (Fullan, 2010). Based on the design of the educational program, it is possible to trace the difference between formal
and non-formal education. In this sense, formal education can be seen as “top-down” education. Accordingly, almost all teacher-training programmers fall into this category. In turn, non-formal education
is organized exclusively on request and for the benefit of students, and students themselves participate in
the planning of programs, i.e. such education is carried out, largely, “from the bottom to up”. Lifelong learning in the normative acts of the Russian Federation The concept of “lifelong learning” appeared in the USSR in 1986 in the Decision of the CPSU Central
Committee and the USSR Council of Ministers “On measures to improve the quality of training and use of Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 60 specialists with higher education in the national economy”. qualified personnel. The answer to the ever-increasing need for updating knowledge and skills (competencies) is the active
introduction of modern technologies into the lifelong learning process, including e-learning, open
education, remote technologies. The Concept of development of lifelong learning in the Russian Federation for the period up to 2025 is
based on the following principles: The Concept of development of lifelong learning in the Russian Federation for the period up to 2025 is
based on the following principles: - the right of every citizen to lifelong learning; esponsibility of the citizen for his / her own professional growth and personal development; - individual character of educational trajectories and variability of forms and methods of education; - internationalization of adult lifelong learning; - information openness of the sphere of lifelong learning. The main directions of the Concept implementation are: 1. Ensuring the implementation of individual educational trajectories (routes). 2. Development of infrastructure and mechanisms for independent assessment and recognition of non-
formal learning qualifications and outcomes. 3. Creation of conditions for on-the-job training, formation of corporate training systems 4. Development and updating of educational programs in accordance with the requirements of
professional standards, taking into account the best national and international experience. 5. Formation of the system of quality assurance of lifelong learning for adults. 6. Development of personnel potential of the system of lifelong learning for adults. Educational organizations and organizations engaged in educational activities, implementing basic and
additional educational programs develop programs for the development of lifelong learning, taking into
account the principles of this Concept. As a result, of the Concept implementation the following effects will be provided: - strengthening of social stability of Russian society through the development of mechanisms for
recognition of qualifications and results of non-formal education, socialization and integration of citizens; - improving the competitiveness of the teaching staff of educational organizations through the
formation of professional competencies to meet the requirements of the modern labor market; - ensuring high quality and constant updating and variability of programs of continuous education
through the creation of a competitive environment; - attracting qualified personnel, combination of state control and independent quality assessment tools,
self-regulation of lifelong learning; - development of effective mechanisms to stimulate and support the lifelong professional development
of teaching and management personnel in the field of lifelong learning for adults (Ministry of Education
and Science of the Russian Federation, 2015). Introduction The Concept considers the priorities of state policy, principles and content that provide conditions for the
realization of the right of the adult population to receive education throughout life. Adult education is
offered in the following areas: - formal education (development of educational programs in organizations engaged in educational
activities); - non-formal education (training outside organizations engaged in educational activities, at the place of
work (in the form of mentoring, training, coaching, training, exchange of experience, etc.), education in
public and socially oriented organizations); - information (spontaneous education through the organization of individual cognitive activity or self-
education) (Ministry of Education and Science, 2015). It should be noted that the role of “corporate education” institutions is currently being strengthened
through the activities of personnel services of organizations that organize on-the-job training with the help
of tutors, mentors, instructors, organize professional conferences and seminars to exchange experience. In
general, their activities are aimed at the implementation of comprehensive training programs for highly Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 61 qualified personnel. qualified personnel. qualified personnel. Models and strategies of lifelong professional education of teachers Dynamic changes in all spheres of life and numerous educational reforms introduced in European
countries and the USA at the end of the 20th century have led to the appearance of a new model of
professional development in education called ‘post-technocratic’. It is an alternative of the existing model
of discrete formal education or advanced training. The post-technocratic model includes non-formal and
informal education and provides continuous professional development of teachers. The model implies:
- constant professional educational needs; - including ability of professional development in plans of developing educational institutions; 62 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 - combining needs for personal development with needs of educational institution; - regular monitoring and assessment of professional competences of teachers and heads of educational
institutions (Chechel, 2014). - regular monitoring and assessment of professional competences of teachers and heads of educational
institutions (Chechel, 2014). The teacher’s work in terms of reformation and constant modernization of the educational system leads
to the increase of tension and shortage of competences. Consequently, there is need for organizing proper
education in order to develop necessary knowledge and skills. P. Jackson called it the ‘model of deficit’. Its
point is that teachers are to be provided with knowledge and skills that are missing at the moment, but
become essential in specific educational conditions. This model has long remained dominant in many
countries (including Russia) which were members of the Organization of Economic Cooperation and
Development (OECD). The author of the model assumed that the teacher’s professionalism increases in a
discreet way – from one deficit to another, from one level of education to the next one, from one advanced
training course to another (The World Bank, 2003). This thesis is connected to such usual formal practice
of teachers’ work as the demand to take advanced training course at least once in three years. Although
everyone understands that daily teacher’s experience and abilities to master new forms of professional
activities are the main components of the teacher’s professionalism, this approach is seldom used in the
practice of teachers’ advanced training. A wider application in the sphere of professional education and advanced teachers’ training has been
given to the idea of ‘mentoring’ – consulting by more experienced colleagues or experts during the
working process. qualified personnel. This idea has features of traditional tutoring, but it means structural and informative
collaboration in order to develop professionalism. The accent is put on additional training of specialists
according to the specific character of their activity and without interfering with their work. The idea also
implies further constant access to informational resources of continuous professional development
including: - programs of preparation and discussing the needs of young teachers who have Bachelor’s degree to
continue their education on the next level; - creating conditions for continuous professional development and further statement of acquired
competences during non-formal and informal education; - availability of equal opportunities of continuous professional development of teachers from all regions
(Gracheva, 2012). There are also models of lineal, parallel and simultaneous inclusion of non-formal education into
formal one. These models are realized through such educational technologies as online discussions,
webinars, net interaction, scientifically methodological and practice-oriented seminars, attendance of
authorities and educational institutions, retraining, conferences, corporative training in the form of tutoring,
internship, experience exchange, etc. (Korshunov & Gaponova, 2017). In England there are so called ‘lighthouse’ schools which have been created to spread advanced
experience in the fields of methodology, technologies, pedagogy and psychology. Fixed competences
preceding professional pedagogical career are replaced with teachers’ immersion into the area of constantly
changing competences (Dachkovskaya, 2001). Non-formal and informal education is often realized through professional associations of teachers. The
main task of such societies is the exchange of practical experience gained during professional educational
activity. Fullan calls this process ‘interactive professional development’ singling it out as a key source of
realizing educational reforms (Fullan, 1991). Network interaction provides with such advantages as
constant professional development of teachers, decrease of isolation, finding of collective solutions of Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 63 common professional problems and experience exchange. An association is a platform for informal
communication which is created for meetings of professionals, connection with international professional
societies, discussion of project ideas, assessment of their results and spread of experience and results both
inside and outside the region. The infrastructure of such networks is worked out by the virtual variant of a
methodological study, which has become the meeting point of project groups, informal communication of
teachers, storage of methodological works of teachers and a reading hall at the same time. qualified personnel. This informative
resource centre is aimed at creating conditions for non-formal and informal education of future teachers. The symbiosis of formal (getting education, advanced training courses), non-formal (societies,
associations) and informal (informal professional communication) sides holds wide educational
opportunities (Tyunnikov & Maznichenko, 2014). Thus, the structure of professional development contains the networks of formal, non-formal and
informal education represented in the form of Euler diagram. Education is realized in terms of combining
formal (lectures, presentations, texts) and informal communication which is followed by discussions,
reflection and experimental part. Unfortunately, it must be stated that in Russia not a great number of
teachers are members of professional societies. However, a significant amount of useful information
motivates local subject methodological communities to form teachers’ associations. This practice in Russia
does not have either normative base or traditions. In terms of modernization of higher education there is a widespread practice of teaching at schools
when students and young teachers observe, assist and teach under the guidance of senior mentor. The
problem is that the mechanism of mentoring isn’t worked out properly and only a few teachers are ready to
have students during their practice period. However, in several regions there are networks of schools of
teachers’ professional development, which provide interschool advanced training courses for teachers. One of the innovations of education is group work of students and teachers. Classes in pedagogical
colleges, universities and advanced training courses often contain collective (group) forms of work. In
modern conditions the accent is made on projecting and realizing of professional development not only of
an individual teacher, but the whole group including teachers of a difficult form, the same school or those
working on a specific problem. It is necessary to state that the change of professional education strategies and development is
influenced by informational and communicative technologies. They add such innovative elements as new
network services and extra informational resources to professional education and advanced training of
teachers. During the development of informational communicative technologies the system of education
has absorbed them in full rate. For example, network libraries formed outside education are used actively
by students and teachers. Recently a wide spread in the educational process have been received by interactive technologies which
are an alternative of traditional pedagogical interaction. (Burlakova, 2014). The leading role of the teacher in interactive methods is a facilitator, a helper in organizing mental
activity, creativity and reflexive activity of students. The activeness of students is gained by combining
common educative forms of pedagogical interaction: frontal, group, pair work and individual. Practically
each interactive method implies interconnection and combination of all these forms (Romashina & Mayer,
2011). It is essential to cover the experience of European countries while reforming school inspections into
agencies of professional development. In fact, inspection gradually turns into a kind of consulting which in
its turn is closely connected to the teachers’ professional development. There is an interesting example of
Scottish inspectional agency, which declares not to assess schools, but to teach them self-assessment. Modern strategies of teachers’ professional development like traditional approaches face the problem of
assessing the results of professional education. In the past the dominant role was given to fixating the fact
of education itself when students or trainees got certificates of finishing the advanced training courses or
took exams. It was considered that this way could show the degree of acquiring knowledge and skills in the
context of the course taken, but not in the real educational environment. Such assessments lead to
formalization of curriculums. Nowadays the solution of this problem is realized in two directions: on the one hand, by improving the
ways of laboratory certification and testing of teachers who are not linked to specific courses, but they put
certain demands to those who project the courses. During such examination procedures an attempt is made
to imitate real contexts of teacher’s actions. On the other hand, there has been a wide spread of authentic
assessment, which implies teachers’ observing how students solves tasks in real situations. An instrument
of the authentic assessment can also be realized in the form of teacher’s portfolio, which presents data of
their pedagogical activity results. (Tyunnikov & Maznichenko, 2014). In general, it can be stated that new strategies of teachers’ professional development rely on non-formal
mechanisms of giving knowledge and forming competences. qualified personnel. The priority of interactive pedagogical process is
given to such characteristics as procedural forms, activity, communication, dialogue, opportunity of self-
expression, creativity, reflection, etc. Traditional pedagogical impact as an attribute of the authoritarian
pedagogical process is aimed, first of all, at formal implementation of compulsory curriculum. During the
realization of interactive methods it is learning, not teaching dominates. The teacher in this method is not a
translator of readymade information, but they organize autonomous activity of students directed at
producing knowledge about the reality, they encourage the search, research of phenomena and processes
and autonomous solution of problems. A teacher is merely one of the sources of information. Their
function is to create conditions for students’ activeness, initiative in activities, primarily the cognitive one 64 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 (Burlakova, 2014). Issue study In the process of research the following methods were used: theoretical (analysis; synthesis;
concretization; generalization); diagnostic (questioning; interviewing); empirical (study of the experience
of educational organizations, normative and educational documentation; pedagogical observation). The subject of this research is revealing students’ needs for continuing their professional education. The
concept of lifelong learning is based not on the offers from the system of professional education, but on the
demands of the labour market. Thus, it is crucial to state and analyze the needs of educational institutions
and their employees for the programs of further education and development. Some researchers from the Institute of Foreign Languages of Moscow City Teachers’ Training
University have conducted a research to reveal professional difficulties and needs of teachers. The research
was aimed at analyzing and preventing difficulties of future teachers in terms of working out various
modules of Master programs. During the research in 2017-2018 the following subjective professional
difficulties were revealed: - creation of curriculums connected to forming students’ universal educational skills; - use of new pedagogical technologies; - methodology of using ICT in educational process; - creation of upbringing and socialization programs for students; 65 Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 - forming students’ interdisciplinary skills; - working with talented students; - criterial assessment of students’ educational achievements; - organization of extra-curricular classes; - working with poor students; - planning and predicting final results of education; - motivating students to master a subject and prepare successfully for the state exams. Objective difficulties: - insufficient technical equipment of educational process, lack of computers, activity books, smart
boards; - difficulties in creating of school development program, the basic educational program of the
educational organization and subject programs; - not all teachers understand the ideology of new time and use old-fashioned soviet metho - incompetence in organizing lessons with more than 35 students. - incompetence in organizing lessons with more than 35 students. - unwillingness and inability to work with students with special physical and mental deve - unwillingness and inability to work with students with special physical and mental development;
During the research there has been singled out a wide range of teachers’ needs which can be divided
into basic and specific. During the research there has been singled out a wide range of teachers’ needs which can be divided
nto basic and specific. Issue study Basic needs include: Basic needs include: - enhancing the level of professional competence; - effective use of modern interactive pedagogical technologies; - mastering methodologies of organizing students’ project and research activities; - studying methods of alternative education; - knowledge of peculiarities of group work technology; - studying personality-oriented and operational approaches and their possible use in educational
process; - learning about methodology of giving integrative and binary lessons; - use of different forms of lessons, methods, techniques and means of education; - ability to form students’ motivation in mastering a subject; - getting information about projecting modern lessons; - organizing extra-curricular classes; - organizing pay educational services in order to raise non-budget resources for the educational
institution. Specific needs of teachers of different subjects are the following: Specific needs of teachers of different subjects are the following: -
knowledge of monitoring students’ skills in various lessons; -
mastering technology based on volunteering (equal educates equal);
enhancing professional competence; -
mastering technology based on volunteering (equal educates equal); -
enhancing professional competence; -
getting information about projecting modern lessons; -
understanding the structure of school reforms; -
ability to hold double lessons, massive events in various types of school curriculum Analysis of teacher’s needs for continuing their professional education of optional education shows the
contradiction between the things teachers know and can do and those they have to know and be able to do. Continuing professional education on the next level or optional professional education is not to be regarded
as correcting mistakes. Education implies revealing and satisfying the needs for forming and developing Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 66 new competences essential to conduct professional functions on a qualitatively new level. Results The result of analyzing documents (expert conclusion application) presented for attestation shows that
teachers of schools, optional education and pre-school educational institutions, etc. participate in non-
formal advanced training by doing the work stated. The teacher’s portfolio includes preparation and giving
a report at a pedagogical council, seminar or conference, work and presentations at school methodological
association, «open» classes or being a jury in contests, etc. If we compare the number of teachers finishing advanced training in formal and non-formal conditions
(according to the survey of teachers of Moscow and Moscow Region) we can see that non-formal
education prevails. It proves the necessity of posing the status of non-formal education as a constituent part
of teachers’ professional development. Discussions Today it is necessary to create a network of organizations that will be able to carry out quality
educational activities. It should be borne in mind that education is a licensed activity. Licensing, in turn,
involves the presence of an educational organization of a certain regulatory material base that meets the
requirements for educational activities, and human resources that can carry out this activity. This ability
must be confirmed by the relevant documents recognized by the licensing authorities. It follows that in the
system of continuing education there should be enough organizations to carry out educational activities on
the required scale: - educational institutions should have a sufficient number of places for those wishing to take a course of
study; - educational institutions should have a sufficient number of places for those wishing to take a course of
study; - educational institutions should have a sufficient number of qualified teachers and other specialists to
train everyone in accordance with their needs; - educational institutions should have a sufficient number of qualified teachers and other specialists to
train everyone in accordance with their needs; ain everyone in accordance with their needs; - availability of variable programs, teaching materials, textbooks, teaching AIDS and other resources
necessary for the implementation of educational activities. - availability of variable programs, teaching materials, textbooks, teaching AIDS and other resources
necessary for the implementation of educational activities. Conclusion Thus, we came to the conclusion that it is necessary to integrate formal and informal professional and
additional education in the process of continuous professional development of teachers. This should be
done at the legislative level of the Ministry of Education and Science of the Russian Federation in order to
take into account these forms of education in the certification of teachers, which does not always happen
on the ground. Continuing education should be both formal and non-formal. The latter is a qualitatively
new phenomenon in social and educational practice, the content of which is based on its own principles,
performs certain functions, solving many old problems in a new way. Modern strategies and models
considered in the article are under the significant influence of information and communication technologies
and are based on informal mechanisms of knowledge transfer and competence formation. Continuing
education of teachers will overcome difficulties and meet the needs for the formation and development of
new professional competencies. References Burlakova, I. I. (2014). Use of interactive technologies in the system of professional training of students. Irina I. Burlakova, Anastasiya S. Budnik, Elizaveta S.Burlakova / Proceedings IFTE-2019 67 Scientific notes of Orlov State University, 5, 335-339. Chechel, I. D. (2014). Post-technocratic model of continuous professional development of heads of
educational institutions. Standards and Monitoring in Education, 2, 47-56. Commission of the European Communities. (2000). A Memorandum on Lifelong Learning. Retrieved from
https://www.hsu-hh.de/wb/wp-content/uploads/sites/647/2017/10/Anthology-
Memorandum_on_Lifelong_Learning-EU-2000.pdf Commission of the European Communities. (2000). A Memorandum on Lifelong Learning. Retrieved from
https://www.hsu-hh.de/wb/wp-content/uploads/sites/647/2017/10/Anthology- Dashkovskaya, O. (2001). Lessons of a successful reform. Journal of School Management, 12. Retrieved
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base/evaluation_studies_and_reports/evaluation_studies_and_reports_2004/the_lisbon_strategy_f
or_growth_and_employment__report_from_the_high_level_group.pdf Korshunov, I. A., & Gaponova, O. S. (2017). Continuous education of adults in the context of economical
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2974 Ministry of Education and Science of Russian Federation. (2015). Concept of development of continuous
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ocx.pdf Ministry of Education and Science of Russian Federation. (2015). On the federal targeted programme of
developing education during 2016-2020. Retrieved from https://xn--80abucjiibhv9a.xn-
p1ai/%D0%B4%D0%BE%D0%BA%D1%83%D0%BC%D0%B5%D0%BD%D1%82%D1%8B/
5930/%D1%84%D0%B0%D0%B9%D0%BB/4787/FCPRO_na_2016-2020_gody.pdf Romashina, S. Y., & Mayer, A. A. (2011). Facilitative pedagogy. Barnaul: Azbuka. The World Bank. (2003). Lifelong Learning in the Global Knowledge Economy: Challenges for
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Neutralizing activity against Omicron BA.5 after tixagevimab/cilgavimab administration comparable to those after Omicron BA.1/BA.2 breakthrough infections
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Frontiers in immunology
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TYPE Original Research
PUBLISHED 02 March 2023
DOI 10.3389/fimmu.2023.1139980 OPEN ACCESS OPEN ACCESS
EDITED BY
Greta Forlani,
University of Insubria, Italy
REVIEWED BY
Luuk Hilbrands,
Radboud University, Netherlands
Daniela Dalla Gasperina,
University of Insubria, Italy
*CORRESPONDENCE
Yoo-kyung Lee
leeykyoung@korea.kr
Jae-Hoon Ko
jaehoon.ko@gmail.com
†These authors have contributed
equally to this work and share
first authorship
‡These authors have contributed equally to
this work
SPECIALTY SECTION
This article was submitted to
Vaccines and Molecular Therapeutics,
a section of the journal
Frontiers in Immunology
RECEIVED 10 January 2023
ACCEPTED 20 February 2023
PUBLISHED 02 March 2023
CITATION
Yang J, Won G, Baek JY, Lee YH, Kim H,
Huh K, Cho SY, Kang C-I, Chung DR,
Peck KR, Lee KW, Park JB, Yoon SE,
Kim SJ, Kim WS, Yim MS, Kim K, Hyeon S,
Kim BC, Lee Y-k and Ko J-H (2023)
Neutralizing activity against Omicron BA.5
after tixagevimab/cilgavimab administration
comparable to those after Omicron BA.1/
BA.2 breakthrough infections. Front. Immunol. 14:1139980. OPEN ACCESS doi: 10.3389/fimmu.2023.1139980
COPYRIGHT OPEN ACCESS
EDITED BY
Greta Forlani,
University of Insubria, Italy
REVIEWED BY
Luuk Hilbrands,
Radboud University, Netherlands
Daniela Dalla Gasperina,
University of Insubria, Italy
*CORRESPONDENCE
Yoo-kyung Lee
leeykyoung@korea.kr
Jae-Hoon Ko
jaehoon.ko@gmail.com
†These authors have contributed
equally to this work and share
first authorship
‡These authors have contributed equally to
this work Jinyoung Yang 1†, Gunho Won 2†, Jin Yang Baek 1,3†,
Young Ho Lee 1, Haein Kim 1, Kyungmin Huh 1, Sun Young Cho 1,
Cheol-In Kang 1, Doo Ryeon Chung 1, Kyong Ran Peck 1,
Kyo Won Lee 4, Jae Berm Park 4, Sang Eun Yoon 5,
Seok Jin Kim 5, Won Seog Kim 5, Min Su Yim 2, Kwangwook Kim 2,
Seokhwan Hyeon 2, Byung Chul Kim 2, Yoo-kyung Lee 2*‡
and Jae-Hoon Ko 1*‡ 1Division of Infectious Diseases, Department of Medicine, Samsung Medical Center, Sungkyunkwan
University School of Medicine, Seoul, Republic of Korea, 2Division of Vaccine Development
Coordination, Center for Vaccine Research, National Institute of Infectious Diseases, National
Institute of Health, Korea Disease Control and Prevention Agency, Cheongju, Republic of Korea, 3Asia
Pacific Foundation for Infectious Diseases (APFID), Seoul, Republic of Korea, 4Division of
Transplantation Surgery, Department of Surgery, Samsung Medical Center, Sungkyunkwan University
School of Medicine, Seoul, Republic of Korea, 5Division of Hematology-Oncology, Department of
Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine,
Seoul, Republic of Korea CITATION
Yang J, Won G, Baek JY, Lee YH, Kim H,
Huh K, Cho SY, Kang C-I, Chung DR,
Peck KR, Lee KW, Park JB, Yoon SE,
Kim SJ, Kim WS, Yim MS, Kim K, Hyeon S,
Kim BC, Lee Y-k and Ko J-H (2023)
Neutralizing activity against Omicron BA.5
after tixagevimab/cilgavimab administration
comparable to those after Omicron BA.1/
BA.2 breakthrough infections. Front. Immunol. 14:1139980. doi: 10.3389/fimmu.2023.1139980 Introduction: The effect of tixagevimab/cilgavimab (Evusheld™; AstraZeneca,
UK) should be evaluated in the context of concurrent outbreak situations. Methods: For serologic investigation of tixagevimab/cilgavimab during the BA.5
outbreak period, sera of immunocompromised (IC) hosts sampled before and
one month after tixagevimab/cilgavimab administration and those of healthcare
workers (HCWs) sampled one month after a 3rd shot of COVID-19 vaccines, five
months after BA.1/BA.2 breakthrough infection (BI), and one month after BA.5 BI
were investigated. Semi-quantitative anti-spike protein antibody (Sab) test and
plaque reduction neutralizing test (PRNT) against BA.5 were performed. Domestic outbreak situations and healthy
adult sera for immunity level in population Passive immunization using convalescent plasma or
monoclonal antibodies has been widely studied since the
coronavirus disease 2019 (COVID-19) pandemic began, but the
narrow administration time window (effective only at the early
course of illness), short half-life, and rapidly emerging severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2) variants limited
its clinical use (1–3). Tixagevimab/cilgavimab (Evusheld™;
AstraZeneca, Cambridge, England, UK) was designed as a
combination of two monoclonal antibodies that simultaneously
bind to distinct non-overlapping epitopes of the viral spike
protein and therefore exhibited broad coverage to variants (4–7). In addition, by reengineering Fc variant amino acids, the half-life of
tixagevimab/cilgavimab was extended to approximately 90 days and
the effectiveness in both pre-exposure prophylaxis and early
outpatient treatment was demonstrated (5–7). Although the
Omicron (B.1.1.529) variant and its subvariants, which contain
numerous mutations at the spike protein regions, dominated the
COVID-19 global outbreak in 2022, in vitro studies have
demonstrated that tixagevimab/cilgavimab retains decreased, but
still active neutralizing activity against the Omicron subvariants
including BA.1, BA.2, BA.4, and BA.5 (4, 8–10). In
South Korea, tixagevimab/cilgavimab has been prescribed for
immunocompromised (IC) hosts who may not obtain sufficient
vaccine-induced immunity from August 2022, when BA.5 became a
dominant strain after a large outbreak wave of BA.1/BA.2. To
estimate the clinical significance of tixagevimab/cilgavimab
administration in the context of concurrent outbreak situations,
we investigated serologic responses induced by tixagevimab/
cilgavimab administration in IC hosts in comparison with those
obtained from three-dose vaccinations, past infections from BA.1/
BA.2, and recent infections from BA.5 among healthy adults. Information about domestic outbreak situation including the
proportion of circulating SARS-CoV-2 variants and number of
confirmed cases was collected from the weekly report of the Korea
Disease Control and Prevention Agency (11). When a variant
occupied more than half of the domestic strains, the variant was
defined as a dominant strain. Accordingly, the BA.1/BA.2 outbreak
period was corresponding to week 3 (January 2022) to week 29 (July
2022) (Figure 1). The transition from BA.1 to BA.2 occurred at
week 12 (March 2022), but it was considered as a single BA.1/BA.2
outbreak period as the outbreak continued as one large wave (the
5th domestic outbreak wave). BA.5 dominance was observed from
week 30 (July 2022) and continued with two distinct outbreak waves
(the 6th and 7th waves). During the 7th wave, increase of the BA.2.75
sublineage (mainly BN.1) was observed from week 44 (November
2022). COPYRIGHT
© 2023 Y The ND50 of one month after BA.5 BI (ND50 1,272.5) was highest among
tested groups, but statistical difference was not noticed with tixagevimab/
cilgavimab 600 mg. Conclusion: Tixagevimab/cilgavimab provided a comparable neutralizing activity
against the BA.5 with a healthy adult population who were vaccinated with a 3rd
shot and experienced BA.1/BA.2 BI. KEYWORDS
COVID-19, SARS-CoV-2, Evusheld, neutralizing antibody, BA.5 variant COVID-19, SARS-CoV-2, Evusheld, neutralizing antibody, BA.5 variant COPYRIGHT
© 2023 Y COPYRIGHT
© 2023 Yang, Won, Baek, Lee, Kim, Huh,
Cho, Kang, Chung, Peck, Lee, Park, Yoon,
Kim, Kim, Yim, Kim, Hyeon, Kim, Lee and Ko. This is an open-access article distributed
under the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms. Results: A total of 19 IC hosts (five received tixagevimab/cilgavimab 300 mg and
14 received 600 mg) and 41 HCWs (21 experienced BA.1/BA.2 BI and 20
experienced BA.5 BI) were evaluated. Baseline characteristics did not differ
significantly between IC hosts and HCWs except for age and hypertension. Sab
significantly increased after tixagevimab/cilgavimab administration (median
130.2 BAU/mL before tixagevimab/cilgavimab, 5,665.8 BAU/mL after 300 mg,
and 10,217 BAU/mL after 600 mg; both P < 0.001). Sab of one month after the 3rd
shot (12,144.2 BAU/mL) or five months after BA.1/BA.2 BI (10,455.8 BAU/mL)
were comparable with that of tixagevimab/cilgavimab 600 mg, while Sab of one Frontiers in Immunology 01 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 month after BA.5 BI were significantly higher (22,216.0 BAU/mL; P < 0.001). BA.5
PRNT ND50 significantly increased after tixagevimab/cilgavimab administration
(median ND50 29.6 before tixagevimab/cilgavimab, 170.8 after 300 mg, and
298.5 after 600 mg; both P < 0.001). The ND50 after tixagevimab/cilgavimab 600
mg was comparable to those of five months after BA.1 BI (ND50 200.9) while
ND50 of one month after the 3rd shot was significantly lower (ND50 107.6; P =
0.019). The ND50 of one month after BA.5 BI (ND50 1,272.5) was highest among
tested groups, but statistical difference was not noticed with tixagevimab/
cilgavimab 600 mg. month after BA.5 BI were significantly higher (22,216.0 BAU/mL; P < 0.001). BA.5
PRNT ND50 significantly increased after tixagevimab/cilgavimab administration
(median ND50 29.6 before tixagevimab/cilgavimab, 170.8 after 300 mg, and
298.5 after 600 mg; both P < 0.001). The ND50 after tixagevimab/cilgavimab 600
mg was comparable to those of five months after BA.1 BI (ND50 200.9) while
ND50 of one month after the 3rd shot was significantly lower (ND50 107.6; P =
0.019). Domestic outbreak situations and healthy
adult sera for immunity level in population BA.5 sublineages such as BQ.1, BQ.1.1, and BF.7 were
detected from week 41 (November 2022), but did not exceed 5%
by November 2022. For the evaluation of immunity level in healthy adult population
when tixagevimab/cilgavimab was introduced in Korea, sera
obtained from a heterologous booster shot cohort of healthcare
workers (HCWs) were utilized (12). HCWs in this booster shot
cohort received a 3rd dose of mRNA COVID-19 vaccine
(BNT162b2; Comirnaty®, Pfizer, NY, USA) after two standard
doses of adenovirus vector (Adv) vaccines (ChAdOx1;
Vaxzevria®, AstraZeneca, Oxford, UK). In the booster shot
cohort, 21 HCWs who experienced breakthrough infections (BI)
during the BA.1/BA.2 outbreak period were included in the present
analysis. Sera sampled one month after the 3rd shot (mid-December Frontiers in Immunology 02 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 FIGURE 1
Schematic flow of sampling points presented with variant proportions and the number of domestic cases of COVID-19 per week in 2022, South
Korea *Most of the BA.2.75 sublineages are reported to be BN.1. †Twenty-one HCWs from a pre-existing booster shot cohort and 20 newly-
recruited HCWs who experienced BA.5 BI. HCW, healthcare worker; IC, immunocompromised; BI, breakthrough infection; COVID-19, coronavirus
disease 2019. Schematic flow of sampling points presented with variant proportions and the number of domestic cases of COVID-19 per week in 2022, South
Korea *Most of the BA.2.75 sublineages are reported to be BN.1. †Twenty-one HCWs from a pre-existing booster shot cohort and 20 newly-
recruited HCWs who experienced BA.5 BI. HCW, healthcare worker; IC, immunocompromised; BI, breakthrough infection; COVID-19, coronavirus
disease 2019. cell transplant or chimeric antigen receptor (CAR)-T cell
therapy within six months, 3) those who have received a solid
organ transplant within one year, 4) those who have human
immunodeficiency virus infection and are not expected to have
CD4 T cell ≥200/mm2 with sufficient treatment, and 5) those
who have primary immunodeficiency. IC hosts who met the
above criteria for tixagevimab/cilgavimab administration and
exhibited poor antibody response either to three doses of
COVID-19 vaccinations or to SARS-CoV-2 infection were
prospectively recruited. Poor antibody response was defined as
anti-spike protein antibody (Sab) response < 3,719.0 BAU/mL,
which is the lowest level of the booster shot HCW cohort
measured one month after the 3rd shot (n = 43, median
12,918.0 BAU/mL, IQR 8,989.0–17,304.0 BAU/mL, and range
3719.0–38,544.0 BAU/mL). The determination to receive 300 mg
(one vial) or 600 mg (two vials) of tixagevimab/cilgavimab was
solely the patient’s discretion. Domestic outbreak situations and healthy
adult sera for immunity level in population Blood sampling was conducted
before and one month after tixagevimab/cilgavimab
administration. Informed consent was obtained from all the
participants and this study was approved by our local
institutional review board (SMC 2020-03-113, 2021-01-165,
and 2021-11-0506). 2021) and five months after BA.1/BA.2 BI (late July 2022) were
used. Follow-up sera from HCWs of the booster shot cohort who
did not experience BI were not included in the present comparison,
because the antibody titers and neutralizing activities would have
waned and would be significantly lower than those of the sera
collected one month after the 3rd shot (12). Since a limited number
of HCWs in the booster shot cohort experienced BI during the BA.5
outbreak period, 20 HCWs who received three doses of vaccines
(adv/adv/mRNA) and experienced BA.5 BI (after week 32 when
BA.5 accounted for more than 90% of domestic cases) were
additionally recruited. Sampling was performed one month after
BA.5 BI (early October 2022). Baseline characteristics of the
study population Data on the baseline characteristics of age, sex, height,
weight, body mass index (BMI), body surface area (BSA),
underlying disease, date and type of COVID-19 vaccination,
and history of SARS-CoV-2 infection were collected. The
diagnosis of SARS-CoV-2 infection was based on the positive
result of either reverse transcription-polymerase chain reaction
or antigen testing of respiratory specimens. For the semi-
quantitative measurement of Sab, an electrochemiluminescence
immunoassay kit (Elecsys Anti-SARS-CoV-2 S, Roche
Diagnostics, Basel, Switzerland) was utilized. Ant-SARS-CoV-2
S antibody concentration ≥0.8 U/mL was considered positive,
and the linear range was 0.4–250 U/mL (13). Automated dilution
was performed for up to a 1:50 dilution in the cobas e analyzer,
and an additional manual dilution of up to 1:200 was applied for
the saturated specimens. A linear titer-correlation of the kit and
plaque reduction neutralization test (PRNT) was presented in
previous publications (14). To standardize binding assay results
to the binding antibody units (BAU) recommended by the
World Health Organization, a correction factor of 0.972
provided by the manufacturer was multiplied to the result (15,
16). To the evaluate neutralizing activity against BA.5, PRNT
was conducted for the selected specimens (12, 17, 18). Considering the capacity of biosafety level-3 laboratory and
time required for PRNT, 45 specimen were planned for PRNT. As binding assays were tested earlier, specimens with sufficient
remaining volume were selected preferentially. SARS-CoV-2
dilutions to 40–50 PFU/well (BA.5 sublineage, Incheon, GRA
clade, NCCP No. 43426) were prepared. Vero E6 cells were
inoculated with serum and virus mixtures on a 12-well plate and
incubated at 37°C, 5% CO2 for one hour. After the inoculums
were removed, the cells were overlaid with 1 ml of modified
Eagle’s medium (Gibco, Gaithersburg, MD, USA) containing
0.75% agarose and 2% fetal bovine serum (Gibco). The plates
were incubated at 37°C with 5% CO2 for two or three days. Stain
solution (0.07% crystal violet, 10% formaldehyde, and 5%
ethanol) was then added to the cells, and the visualized
plaques were counted. The 50% neutralizing dose (ND50) titer
was calculated using the Karber formula (19). For the serologic investigation of tixagevimab/cilgavimab
administration during the BA.5 outbreak period, a total of 19 IC
hosts (five received tixagevimab/cilgavimab 300 mg and 14 received
600 mg) and 41 HCWs (21 who had experienced BA.1/BA.2 BI and
20 who had experienced BA.5 BI) were evaluated (Figure 1). Table 1
shows the baseline characteristics of the patients. IC cohort with poor antibody response The indications for tixagevimab/cilgavimab administration
in South Korea were those who have not been confirmed to be
SARS-CoV-2 positive within seven days, who are ≥12 years of
age, weigh ≥40 kg, and meet one of the following conditions: 1)
those receiving immunosuppressive therapy or high-intensity
chemotherapy, 2) those who have received hematopoietic stem Frontiers in Immunology frontiersin.org 03 Yang et al. 10.3389/fimmu.2023.1139980 Baseline characteristics of the
study population The average age of
the IC hosts (53.9 ± 13.8 years) was older than the HCWs with
BA.1/BA.2 BI (43.3 ± 9.1 years) or HCWs with BA.5 BI (43.2 ± 10.0
years). The sex distribution, BMI, and BSA were not significantly
different between the groups. More patients in the IC hosts
exhibited hypertension (36.8%) compared to the HCWs with
BA.1/BA.2 BI (4.8%), while other underlying diseases except for
the main causes of IC did not differ between the groups. All the
HCWs received three doses of COVID-19 vaccine with Adv/Adv/
mRNA vaccine schedules. Among the IC hosts, 13 patients (68.4%)
received two or more doses of COVID-19 vaccines (two patients
received four doses of vaccines, eight patients received three doses
of vaccines, and three patients received two doses of vaccines), while
six patients did not receive any vaccinations (31.6%). All non-
vaccinated and two-dose-vaccinated IC hosts experienced SARS-
CoV-2 infections. Poor antibody responses were determined by the
samples taken either one month after three doses of vaccines or at
the convalescent status of SARS-CoV-2 infection (< 3,719.0
BAU/mL). Serologic response after tixagevimab/
cilgavimab, three-dose vaccination, and BI Table 2 and Figure 2 present the serologic response after
tixagevimab/cilgavimab, in comparison with three-dose
vaccination and BI. Sab was measured in all the collected
specimens. All the tixagevimab/cilgavimab groups exhibited a
statistically significant increase in Sab titers compared to before
administration (median 130.2 BAU/mL before tixagevimab/
cilgavimab, 5,665.8 BAU/mL after tixagevimab/cilgavimab 300
mg, 10,217 BAU/mL after tixagevimab/cilgavimab 600 mg, both
P < 0.001; Figure 2A), and the titers significantly increased more
when 600 mg was administered versus 300 mg (P = 0.002). Compared with tixagevimab/cilgavimab 600 mg administration,
there was no statistically significant difference in the Sab titers
one month after the 3rd shot (12,144.2 BAU/mL, P = 0.175) or five
months after BA.1 BI (10,455.8 BAU/mL, P = 0.778). In contrast,
the titers measured one month after BA.5 BI (22,216.0 BAU/mL)
were significantly higher than those after tixagevimab/cilgavimab
600 mg administration (P < 0.001). When compared with
tixagevimab/cilgavimab 300 mg administration, Sab titers were
significantly higher one month after the 3rd shot, five months
after BA.1 BI, and one month after BA.5 BI (P = 0.008, P = 0.019,
and P < 0.001, respectively). Statistical analysis For comparative analysis of baseline characteristics, analysis of
variance (ANOVA) and Tukey’s multiple comparisons test were
used for continuous variables, and chi-square or Fisher’s exact tests
were used for categorical variables. For the comparison of serologic
tests, the Mann-Whitney U test was utilized. In the investigation of
correlation between physical measurement values and antibody
titers, a linear regression model was used. All P values were two-
sided tests, and those < 0.050 were considered statistically
significant. GraphPad Prism version 9.4.1 (GraphPad Software,
San Diego, CA, USA) were used for all statistical analyses. Frontiers in Immunology 04 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 A total of 45 specimens underwent PRNT against BA.5. Both
i
i
b/ il
i
b
d 6
i d
d
i i
ll
cilgavimab 600 mg, both P < 0.001; Figure 2B). A statistically
i
ifi
diff
i
d b
i
i
b/
TABLE 1 Baseline characteristics of the study population. Frontiers in Immunology Statistical analysis IC hosts with poor antibody response
(n = 19)
HCWs with BA.1/BA.2 BI
(n = 21)
HCWs with BA.5 BI
(n = 20)
P value
Demographics
Age, year
53.9 ± 13.8
43.3 ± 9.1*
43.2 ± 10.0*
0.003
Male sex
13 (68.4)
9 (42.9)
8 (40.0)
0.149
BMI, kg/m2
23.2 ± 2.9
21.8 ± 2.4
23.6 ± 3.1
0.098
BSA‡, m2
1.5 ± 0.3
1.4 ± 0.3
1.6 ± 0.4
0.361
Underlying disease
Hypertension
7 (36.8)
1 (4.8)*
3 (15.0)
0.029
Diabetes mellitus
1 (5.3)
0 (0.0)
0 (0.0)
0.334
Dyslipidemia
1 (5.3)
0 (0.0)
1 (5.0)
0.572
Chronic heart disease
3 (15.8)
0 (0.0)
1 (5.0)
0.127
Thyroid cancer
1 (5.3)
0 (0.0)
1 (5.0)
0.572
Diseases causing IC condition
B-cell malignancy
9 (47.4)
none
none
NA
Lymphoma
8 (42.1)
none
none
NA
ALL
1 (5.3)
none
none
NA
Organ transplantation
9 (47.4)
none
none
NA
Kidney
8 (42.1)
none
none
NA
Heart
1 (5.3)
none
none
NA
Good’s syndrome
1 (5.3)
none
none
NA
Vaccination status
Four doses
Adv/Adv/mRNA/mRNA
2 (10.5)
0 (0.0)
0 (0.0)
0.107
Three doses
8 (42.1)
21 (100.0)*
20 (100.0)*
0.020
Adv/Adv/mRNA
1 (5.3)
21 (100.0)*
20 (100.0)*
<0.001
Adv/mRNA/mRNA
2 (10.5)
0 (0.0)
0 (0.0)
0.107
mRNA/mRNA/mRNA
5 (26.3)
0 (0.0)*
0 (0.0)*
0.003
Two doses
mRNA/mRNA
3 (15.8)
0 (0.0)
0 (0.0)
0.033
None†
6 (31.6)
0 (0.0)*
0 (0.0)*
0.001
SARS-CoV-2 infection
10 (52.6)
21 (100.0)*
20 (100.0)*
<0.001
BA.1/BA.2 period
10 (52.6)
21 (100.0)*
0 (0.0)*
<0.001
BA.5 period
0 (0.0)
0 (0.0)
20 (100.0)*
<0.001
Data are expressed as the number (%) of patients or mean ± standard deviation. *Significantly different in a two-group comparison with IC hosts. †All non-vaccinated hosts experienced SARS-
CoV-2 infection and exhibited poor antibody responses after convalescence. ‡BSA was calculated based on the Mosteller method. IC, immunocompromised; HCW, healthcare worker; BI, breakthrough infection; BMI, body mass index; ALL, acute lymphoblastic leukemia; NA, not applicable; Adv, adenovirus vector vaccines;
mRNA, messenger ribonucleic acid vaccines; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. TABLE 1 Baseline characteristics of the study population. Data are expressed as the number (%) of patients or mean ± standard deviation. *Significantly different in a two-group comparison with IC hosts. †All non-vaccinated hosts experienced SARS-
CoV-2 infection and exhibited poor antibody responses after convalescence. ‡BSA was calculated based on the Mosteller method. Statistical analysis BI, breakthrough infection; Sab, anti-spike protein antibody; BAU, binding antibody unit; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; IC, immunocompromised;
tixa./cilga., tixagevimab/cilgavimab; HCW, healthcare worker; ab, antibody; mo, month; IQR, interquartile range. BA.1 BI (ND50 200.9, P = 0.514), while ND50 of one month after the
3rd shot was significantly lower (ND50 107.6, P = 0.019). The ND50
of one month after BA.5 BI (ND50 1,272.5) was highest among
tested groups, but statistical difference was not noticed with
tixagevimab/cilgavimab 600 mg (P = 0.151). When compared
with tixagevimab/cilgavimab 300 mg administration, BA.5 PRNT
ND50 titers were not significantly different with those measured one
month after the 3rd shot, five months after BA.1 BI, and one month
after BA.5 BI (P = 0.222, P = 0.691, P = 0.151, respectively). A A
B
FIGURE 2
(A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing
activity against BA.5, presented as PRNT ND50. Binding and neutralizing
antibody responses after tixagevimab/cilgavimab in comparison with
those to vaccination and BI *Statistically significant difference was
noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./
cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization
test; ND50, 50% neutralization dose; ns, not significant. A To investigate the effect of physical characteristics on the effect
of tixagevimab/cilgavimab, we calculated the correlation of Sab
titers measured after the administration of tixagevimab/cilgavimab
600 mg with BMI, BSA, height, and weight (Figure 3). All physical
characteristics demonstrated statistically significant reverse
correlations with Sab (all P < 0.050), and the highest R2 value was
noticed with BSA (R2 = 0.595). The BA.5 PRNT ND50 values also
exhibited a reverse trend with physical measurement values, but
statistical significances were not observed. B
FIGURE 2
(A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing
activity against BA.5, presented as PRNT ND50. Binding and neutralizing
antibody responses after tixagevimab/cilgavimab in comparison with
those to vaccination and BI *Statistically significant difference was
noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./
cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization
test; ND50, 50% neutralization dose; ns, not significant. B
FIGURE 2 B Statistical analysis IC, immunocompromised; HCW, healthcare worker; BI, breakthrough infection; BMI, body mass index; ALL, acute lymphoblastic leukemia; NA, not applicable; Adv, adenovirus vector vaccines;
mRNA, messenger ribonucleic acid vaccines; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. A total of 45 specimens underwent PRNT against BA.5. Both
tixagevimab/cilgavimab 300 mg and 600 mg induced a statistically
significant increase in BA.5 PRNT ND50 than before administration
(median ND50 29.6 before tixagevimab/cilgavimab, 170.8 after
tixagevimab/cilgavimab 300 mg, and 298.5 after tixagevimab/ cilgavimab 600 mg, both P < 0.001; Figure 2B). A statistically
significant difference was not noticed between tixagevimab/
cilgavimab 300 mg and 600 mg (P = 0.310). Compared with
tixagevimab/cilgavimab 600 mg administration, there was no
statistically significant difference in the values five months after Frontiers in Immunology 05 frontiersin.org Yang et al. 10.3389/fimmu.2023.1139980 ologic responses to tixagevimab/cilgavimab in comparison with those to vaccination and BI. Groups
Sampling point
Sab, BAU/mL
BA.5 PRNT, ND50
IC hosts with poor antibody response
(n = 19)
Before tixa./cilga. Binding ab test, n = 19
PRNT, n = 15
130.2
(0.4–1,165.4)
29.6
(17.0–41.2)
After tixa./cilga. 300 mg
Binding ab test, n = 5
PRNT, n = 5
5,665.8
(3,162.9–8,105.0)
170.8
(124.2–379.0)
After tixa./cilga. 600 mg
Binding ab test, n = 14
PRNT, n = 10
10,216.7
(8,602.2–11,274.2)
298.5
(166.9–540.8)
HCWs with BA.1/BA.2 BI
(n = 21)
1 mo after 3rd shot
Binding ab test, n = 21
PRNT, n = 5
12,144.2
(8,386.9–16,562.9)
107.6
(83.3–202.6)
5 mo after BA.1/BA.2 BI Binding ab test, n = 21
PRNT, n = 5
10,455.8
(7,679.8–19,418.6)
200.9
(118.9–1,144.4)
HCWs with BA.5 BI
(n = 20)
1 mo after BA.5 BI
Binding ab test, n = 20
PRNT, n = 5
22,216.0
(15,272.1–33,148.1)
1,272.5
(415.3–2,448.7)
Data are expressed as median (IQR). BI, breakthrough infection; Sab, anti-spike protein antibody; BAU, binding antibody unit; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; IC, immunocompromised;
tixa./cilga., tixagevimab/cilgavimab; HCW, healthcare worker; ab, antibody; mo, month; IQR, interquartile range. Data are expressed as median (IQR). BI, breakthrough infection; Sab, anti-spike protein antibody; BAU, binding antibody unit; PRNT, plaque reduction neutralization test; ND50, 50% neutralization dose; IC, immunocompromised;
tixa./cilga., tixagevimab/cilgavimab; HCW, healthcare worker; ab, antibody; mo, month; IQR, interquartile range. Data are expressed as median (IQR). Discussion This result is in line with other study which suggested
tixagevimab/cilgavimab maintained the neutralization activity
against BA.5 in spite of a 30.7 fold reduction compared to against
the ancestral strain (10). To date, several real-world studies
suggested that pre-exposure tixagevimab/cilgavimab
administration would be effective in IC hosts during the early
Omicron period, but these studies did not reflect the emergence
and outbreak of the BA.5 subvariant (21, 22). Our serologic
investigation supports the continued use of tixagevimab/
cilgavimab during BA.5 predominance, and additional cohort
study would be required to evaluate the clinical effects at this period. In another study by Benotmane et al., evaluating serologic effect
of tixagevimab/cilgavimab administration among kidney transplant
recipients, only 9.5% obtained neutralizing activity against the BA.1
subvariant after the administration of tixagevimab/cilgavimab 300
mg, and there was no neutralizing activity when Sab titers < 2,500
BAU/ml after tixagevimab/cilgavimab administration (23). In the
present study, only one patient exhibited low Sab titer of 2145.2
BAU/mL and showed the lowest PRNT titer of ND50 99.4. and higher than those vaccinated with the 3rd dose. Those who
received tixagevimab/cilgavimab 300 mg also exhibited comparable
neutralizing activity with these comparators. PRNT ND50 of 118.25
has been suggested as the 50% protective neutralizing titer in our
previous study (12). Since most patients exhibited BA.5 PRNT
ND50 over 118.25 after tixagevimab/cilgavimab administration
except for one (ND50 99.4), the protective role of tixagevimab/
cilgavimab is anticipated during the BA.5 dominant outbreak
period. This result is in line with other study which suggested
tixagevimab/cilgavimab maintained the neutralization activity
against BA.5 in spite of a 30.7 fold reduction compared to against
the ancestral strain (10). To date, several real-world studies
suggested that pre-exposure tixagevimab/cilgavimab
administration would be effective in IC hosts during the early
Omicron period, but these studies did not reflect the emergence
and outbreak of the BA.5 subvariant (21, 22). Our serologic
investigation supports the continued use of tixagevimab/
cilgavimab during BA.5 predominance, and additional cohort
study would be required to evaluate the clinical effects at this period. Benotmane et al. suggested that low tixagevimab/cilgavimab-
induced antibody titers would be associated with high BMI. In
the present study, we also investigated the correlation of
tixagevimab/cilgavimab-induced antibody titers with physical
characteristics and noticed that BSA exhibited the best reverse
correlation with antibody titers. Discussion After the emergence of the Omicron variant with high
transmissibility, approximately a half of the South Korean
population experienced SARS-CoV-2 infection and IC hosts
continue to be at risk of exposure to the virus (11, 20). As
subvariants of Omicron succeeded the following outbreak waves,
it became necessary to investigate the serologic status of the
community together when estimating the serologic impact of
monoclonal antibody agents to a circulating variant. FIGURE 2
(A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing
activity against BA.5, presented as PRNT ND50. Binding and neutralizing
antibody responses after tixagevimab/cilgavimab in comparison with
those to vaccination and BI *Statistically significant difference was
noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./
cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization
test; ND50, 50% neutralization dose; ns, not significant. GU
(A) Anti-spike protein antibody, presented as BAU/mL. (B) Neutralizing
activity against BA.5, presented as PRNT ND50. Binding and neutralizing
antibody responses after tixagevimab/cilgavimab in comparison with
those to vaccination and BI *Statistically significant difference was
noticed. BI, breakthrough infection; BAU, binding antibody unit; tixa./
cilga., tixagevimab/cilgavimab; PRNT, plaque reduction neutralization
test; ND50, 50% neutralization dose; ns, not significant. Of note, the administration of tixagevimab/cilgavimab 600 mg
exhibited neutralizing activity against the BA.5 subvariant
comparable with healthy adults who experienced BA.1/BA.2 BI, Frontiers in Immunology frontiersin.org 06 Yang et al. 10.3389/fimmu.2023.1139980 A
B
D
C
FIGURE 3
Correlation of Sab titers with physical measurement values after the administration of tixagevimab/cilgavimab 600 mg. Correlation with (A) BMI, (B)
BSA, (C) height, and (D) weight is presented, respectively. Sab, anti-spike protein antibody; BMI, body mass index; BSA, body surface area; BAU,
binding antibody unit. B A B C D D C FIGURE 3
Correlation of Sab titers with physical measurement values after the administration of tixagevimab/cilgavimab 600 mg. Correlation with (A) BMI, (B)
BSA, (C) height, and (D) weight is presented, respectively. Sab, anti-spike protein antibody; BMI, body mass index; BSA, body surface area; BAU,
binding antibody unit. and higher than those vaccinated with the 3rd dose. Those who
received tixagevimab/cilgavimab 300 mg also exhibited comparable
neutralizing activity with these comparators. PRNT ND50 of 118.25
has been suggested as the 50% protective neutralizing titer in our
previous study (12). Since most patients exhibited BA.5 PRNT
ND50 over 118.25 after tixagevimab/cilgavimab administration
except for one (ND50 99.4), the protective role of tixagevimab/
cilgavimab is anticipated during the BA.5 dominant outbreak
period. Ethics statement All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. The studies involving human participants were reviewed and
approved by the Institutional Review Board of Samsung Medical
Center. The patients/participants provided their written informed
consent to participate in this study. Discussion This finding may explain the
finding of the present analysis that BA.5 PRNT ND50 titers
obtained by tixagevimab/cilgavimab 300 mg administration were
not significantly different from those after tixagevimab/cilgavimab
600 mg, as patients who received 300 mg had lower average BSA
(1.4 ± 0.2 m2) than those who received 600 mg (1.6 ± 0.3 m2). Additional studies about the optimal dosage of monoclonal
antibody agents according to body measurements should
be conducted. This study has several limitations. First, the present study was
conducted with a relatively small cohort, which likely affected
statistical significance. However, we tried to enroll a typical
population that may represent the immune status of the
community at the time period of tixagevimab/cilgavimab
administration, and the present study may provide important
insight into the serologic implications of tixagevimab/cilgavimab
administration during the BA.5 outbreak period. Second, the study
focused on serologic analysis and actual preventive effect of
tixagevimab/cilgavimab was not evaluated. Since the dominant
SARS-CoV-2 variant of each outbreak wave changes rapidly and
the evaluation of preventive effect for the changing variant requires
a considerable time, it would be important to investigate In another study by Benotmane et al., evaluating serologic effect
of tixagevimab/cilgavimab administration among kidney transplant
recipients, only 9.5% obtained neutralizing activity against the BA.1
subvariant after the administration of tixagevimab/cilgavimab 300
mg, and there was no neutralizing activity when Sab titers < 2,500
BAU/ml after tixagevimab/cilgavimab administration (23). In the
present study, only one patient exhibited low Sab titer of 2145.2
BAU/mL and showed the lowest PRNT titer of ND50 99.4. Frontiers in Immunology frontiersin.org 07 Yang et al. 10.3389/fimmu.2023.1139980 Funding This study was supported by a research program fund (#2021-
ER2303-00) from the Korea Disease Control and Prevention
Agency and a Samsung Medical Center Grant (#SMO1230121). Author contributions neutralizing activity of a monoclonal antibody agent against the
dominant variant in comparison with the immune status of the
community. In this context, our serologic study findings supported
the administration of tixagevimab/cilgavimab during the BA.5
outbreak period in South Korea. Third, the present study does
not provide information about how tixagevimab/cilgavimab would
provide preventive effect for severe disease. Unlike active
vaccination or natural infection that induce T cell immunity and
B cell memory (24, 25), the role of passive immunization in
preventing severe disease is still uncertain. To answer this
question, clinical cohort study need to be conducted for IC hosts
who were infected after tixagevimab/cilgavimab administration in
comparison with those infected without tixagevimab/cilgavimab. Finally, since the measurement of antibody titers was only
performed one month after tixagevimab/cilgavimab
administration and was limited to neutralization of the BA.5
variant, additional studies evaluating waning time points and
newly emerging variants such as BA.5 subvariants (e.g. BQ.1,
BQ.1.1, or BF.7) and BA.2.75 subvariants (e.g. BN.1) or
recombinants (e.g. XBB) are necessary. JY, BK, Y-kL, and J-HK were involved in the design of this
study. JY, YL, HK, KH, SC, C-IK, DC, KP, KL, JP, SY, SK, WK, and
J-HK enrolled the participants and collected specimens. GW, JB,
MY, KK, and SH performed the experiments. JY, GW, JB, and J-HK
assembled the data. JY, GW, KP, Y-kL, and J-HK were involved in
the writing. All authors contributed to the article and approved the
submitted version. Data availability statement The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. The original contributions presented in the study are included
in the article/supplementary material. Further inquiries can be
directed to the corresponding authors. 8. Dejnirattisai W, Huo J, Zhou D, Zahradnı́k J, Supasa P, Liu C, et al. SARS-CoV-2
omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.
Cell (2022) 185:467–484.e415. doi: 10.1016/j.cell.2021.12.046 Acknowledgments In conclusion, tixagevimab/cilgavimab provided a comparable
neutralizing activity against the BA.5 subvariant with those in a
healthy adult population who had previous experiences with BA.1/
BA.2 BI after three-dose vaccinations. The authors would like to express their gratitude to all the
participants who participated voluntarily in this study. 5. Levin MJ, Ustianowski A, De Wit S, Launay O, Avila M, Templeton A, et al.
Intramuscular AZD7442 (Tixagevimab–cilgavimab) for prevention of covid-19. New
Engl J Med (2022) 386:2188–200. doi: 10.1056/NEJMoa2116620 7. Montgomery H, Hobbs F, Padilla F, Arbetter D, Templeton A, Seegobin S, et al.
Efficacy and safety of intramuscular administration of tixagevimab-cilgavimab for early
outpatient treatment of COVID-19 (TACKLE): A phase 3, randomised, double-blind,
placebo-controlled trial. Lancet Respir Med (2022) 10:985–96. doi: 10.1016/S2213-2600(22)
00180-1 3. Sullivan DJ, Gebo KA, Shoham S, Bloch EM, Lau B, Shenoy AG, et al. Early
outpatient treatment for covid-19 with convalescent plasma. New Engl J Med (2022)
386:1700–11. doi: 10.1056/NEJMoa2119657 4. Bruel T, Hadjadj J, Maes P, Planas D, Seve A, Staropoli I, et al. Serum
neutralization of SARS-CoV-2 omicron sublineages BA.1 and BA.2 in patients
receiving monoclonal antibodies. Nat Med (2022) 28:1297–302. doi: 10.1038/s41591-
022-01792-5 6. Loo YM, Mctamney PM, Arends RH, Abram ME, Aksyuk AA, Diallo S, et al. The
SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman
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6. Loo YM, Mctamney PM, Arends RH, Abram ME, Aksyuk AA, Diallo S, et al. The
SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman
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10.1126/scitranslmed.abl8124
7. Montgomery H, Hobbs F, Padilla F, Arbetter D, Templeton A, Seegobin S, et al.
Efficacy and safety of intramuscular administration of tixagevimab-cilgavimab for early
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placebo-controlled trial. Lancet Respir Med (2022) 10:985–96. doi: 10.1016/S2213-2600(22)
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the rapid high-throughput automated electrochemiluminescence immunoassay
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j.jcv.2021.104820 25. Yang J, Lee KW, Baek JY, Bae S, Lee YH, Kim H, et al. Augmented humoral and
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10.1016/j.ajt.2022.12.022 09 09 Frontiers in Immunology frontiersin.org
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Individualizing Follow-Up Strategies in High-Grade Soft Tissue Sarcoma with Flexible Parametric Competing Risk Regression Models
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Individualizing follow-up strategies in high-grade soft tissue sarcoma
with flexible parametric competing risk regression models
Smolle, M.A.; Sande, M. van de; Callegaro, D.; Wunder, J.; Hayes, A.; Leitner, L.; ... ;
Szkandera, J. Citation
Smolle, M. A., Sande, M. van de, Callegaro, D., Wunder, J., Hayes, A., Leitner, L., …
Szkandera, J. (2020). Individualizing follow-up strategies in high-grade soft tissue sarcoma
with flexible parametric competing risk regression models. Cancers, 12(1). doi:10.3390/cancers12010047 Citation Version:
Publisher's Version
License:
Creative Commons CC BY 4.0 license
Downloaded from:
https://hdl.handle.net/1887/3182044 License: Note: To cite this publication please use the final published version (if applicable). Note: To cite this publication please use the final published version (if applica cancers cancers Individualizing Follow-Up Strategies in High
Soft Tissue Sarcoma with Flexible Parametric
Competing Risk Regression Models Haas 16,17,*,‡ and Joanna Szkandera 12,*,‡ 1
Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria 1
Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria
2
Department of Orthopaedic Surgery, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands
3
Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy 1
Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria
2
Department of Orthopaedic Surgery, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands
3
Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy
4
University Musculoskeletal Oncology Unit, Mount Sinai Hospital, University of Toronto,
Toronto, ON M5G IX5, Canada
5 1
Department of Orthopaedics and Trauma, Medical University of Graz, 8036 Graz, Austria
2
Department of Orthopaedic Surgery, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands
3
Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy
4
University Musculoskeletal Oncology Unit, Mount Sinai Hospital, University of Toronto,
Toronto, ON M5G IX5, Canada
5
f
l
d
l
d
d 5
Department of Surgery, Royal Marsden Hospital NHS Foundation Trust, London SW3 6JJ, UK
6 6
Sarcoma Centre, HELIOS-Klinikum Berlin-Buch, 13125 Berlin, Germany 7
Institute of Mathematics, Leiden University Medical Centre, 2333 ZA Leiden, T 7
Institute of Mathematics, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands
8
M di
l St ti ti
D
t
t f Bi
di
l D t S i
L id
U i
it
M di
l C
t 7
Institute of Mathematics, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands
8
Medical Statistics, Department of Biomedical Data Science, Leiden University Medical Centre,
2333 ZA Leiden, The Netherlands 9
Princess Máxima Center for Pediatric Oncology, Trial and Data Center, 3584 CT Utrecht, The Nethe 9
Princess Máxima Center for Pediatric Oncology, Trial and Data Center, 3584 CT Utrecht, The Netherlands
10
Department of Orthopaedics and Traumatology, Medical University of Vienna, 1090 Vienna, Austria
11
Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
12
Division of Clinical Oncology, Department of Medicine, Medical University of Graz, 8036 Graz, Austria
13
Institute of Pathology, Medical University of Graz, 8010 Graz, Austria Princess Máxima Center for Pediatric Oncology, Trial and Data Center, 3584 CT Utrecht, The Netherla
10
Department of Orthopaedics and Traumatology, Medical University of Vienna, 1090 Vienna, Austria p
p
gy
y
11
Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
12
Division of Clinical Oncology Department of Medicine Medical University of Graz 8036 Graz Austria 11
Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands 11
Department of Surgery, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
12
Division of Clinical Oncology, Department of Medicine, Medical University of Graz, 8036 Graz, Austria
13
Institute of Pathology, Medical University of Graz, 8010 Graz, Austria Division of Clinical Oncology, Department of Medicine, Medical University of Graz, 8036 Graz, Austria
13
Institute of Pathology, Medical University of Graz, 8010 Graz, Austria 13
Institute of Pathology, Medical University of Graz, 8010 Graz, Austria 14
Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz,
8036 Graz, Austria 15
Sarcoma Centre, HELIOS Klinikum Bad Saarow, 15526 Bad Saarow, Germany 16
Department of Radiotherapy, Leiden University Medical Centre, 2333 ZA Leiden, The Netherlands 17
Department of Radiotherapy, The Netherlands Cancer Institute Amsterdam,
1
CX A
d
Th N
h
l
d 17
Department of Radiotherapy, The Netherlands Cancer Institute Amsterdam,
1066 CX Amsterdam, The Netherlands *
Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkandera@medunigraz.at (J.S.);
Tel.: +31-20-5122135 (R.L.H.); +43-316-385-13115 (J.S.) *
Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkandera@medunigraz.at (J.S.);
T l
31 20 5122135 (R L H )
43 316 385 13115 (J S ) *
Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkand
Tel.: +31-20-5122135 (R.L.H.); +43-316-385-13115 (J.S.) *
Correspondence: r.l.m.haas@lumc.nl (R.L.H.); Joanna.szkandera@medunigraz.at (J.S.); †
These authors share the first authorship. Individualizing Follow-Up Strategies in High
Soft Tissue Sarcoma with Flexible Parametric
Competing Risk Regression Models Maria Anna Smolle 1,†
, Michiel van de Sande 2,†
, Dario Callegaro 3, Jay Wunder 4,
Andrew Hayes 5, Lukas Leitner 1
, Marko Bergovec 1, Per-Ulf Tunn 6, Veroniek van Praag 2,
Marta Fiocco 7,8,9, Joannis Panotopoulos 10
, Madeleine Willegger 10, Reinhard Windhager 10,
Sander P. D. Dijkstra 2, Winan J. van Houdt 11, Jakob M. Riedl 12
, Michael Stotz 12,
Armin Gerger 12, Martin Pichler 12, Herbert Stöger 12, Bernadette Liegl-Atzwanger 13,
Josef Smolle 14, Dimosthenis Andreou 15, Andreas Leithner 1, Alessandro Gronchi 3,
Rick L. Individualizing Follow-Up Strategies in High
Soft Tissue Sarcoma with Flexible Parametric
Competing Risk Regression Models ‡
These authors share the last authorship. Received: 1 December 2019; Accepted: 19 December 2019; Published: 21 December 2019 Received: 1 December 2019; Accepted: 19 December 2019; Published: 21 December 2019 Abstract: Currently, patients with extremity soft tissue sarcoma (eSTS) who have undergone curative
resection are followed up by a heuristic approach, not covering individual patient risks. The aim
of this study was to develop two flexible parametric competing risk regression models (FPCRRMs)
for local recurrence (LR) and distant metastasis (DM), aiming at providing guidance on how to
individually follow-up patients. Three thousand sixteen patients (1931 test, 1085 validation cohort)
with high-grade eSTS were included in this retrospective, multicenter study. Histology (9 categories),
grading (time-varying covariate), gender, age, tumor size, margins, (neo)adjuvant radiotherapy
(RTX), and neoadjuvant chemotherapy (CTX) were used in the FPCRRMs and performance tested
with Harrell-C-index. Median follow-up was 50 months (interquartile range: 23.3–95 months). Two hundred forty-two (12.5%) and 603 (31.2%) of test cohort patients developed LR and DM. Factors Cancers 2020, 12, 47; doi:10.3390/cancers12010047 www.mdpi.com/journal/cancers www.mdpi.com/journal/cancers 2 of 13 Cancers 2020, 12, 47 significantly associated with LR were gender, size, histology, neo- and adjuvant RTX, and margins. Parameters associated with DM were margins, grading, gender, size, histology, and neoadjuvant
RTX. C-statistics was computed for internal (C-index for LR: 0.705, for DM: 0.723) and external
cohort (C-index for LR: 0.683, for DM: 0.772). Depending on clinical, pathological, and patient-related
parameters, LR- and DM-risks vary. With the present model, implemented in the updated Personalised
Sarcoma Care (PERSARC)-app, more individualized prediction of LR/DM-risks is made possible. Keywords: soft tissue sarcoma; follow-up; flexible parametric competing risk regression model; local
recurrence; distant metastasis Keywords: soft tissue sarcoma; follow-up; flexible parametric competing risk regression model; local
recurrence; distant metastasis 1. Introduction Patients with high-grade extremity soft tissue sarcoma (eSTS) are at risk of developing local
recurrences (LR) and even more so of developing distant metastases (DM) after having undergone surgical
resection of the primary tumor [1–5]. These rates differ substantially per size, grade, and subtype [6]. Close follow-up regimens are currently used in order to detect LR and DM at stages where they are still
potentially treatable by re-resection or metastasectomy, respectively [7]. There is no clear consensus
when, by what means, and how often to perform follow-up in eSTS patients, with many centers
and guidelines having introduced a heuristic approach: for the first 3 years after surgery, patients
would be checked three or four times a year, then biannually for the following two years and annually
thereafter [8,9]. Imposing all eSTS patients on these strict follow-up regimens has raised public, scientific, and health
economic concerns over the last years. Numerous factors interact with the risk of developing LR or DM,
such as histological STS-subtypes, surgical margins, tumor size, grade, administration of neo(adjuvant)
radiotherapy (RTX) or chemotherapy (CTX), and patient-derived factors [1–4,10–12]. Consequently,
the current approach of “one-size-fits-all” may not account for the unequal risk of recurrence in the
heterogeneous eSTS population, involving an excessive number of surveillance imaging, possibly
leading to unnecessary delivery of imaging-induced radiation exposure, and the inherent burden for
radiology departments, as well as inappropriately refraining from it, a high number of outpatient visits
and financial costs and emotional stress for each individual patient [13]. However, an evaluation of
prognostic factors for LR and DM taking into consideration the time-varying rate for the occurrence
of events in a multicenter cohort, including important patient-(i.e., age, gender), tumor-(e.g., size,
grade, histological subtype), and treatment-related features (e.g., margins, (neo)adjuvant CTX/RTX),
is currently missing. Therefore, the aim of the present study was to estimate and validate two models predicting
risks of LR and DM over the first 5 years of follow-up by applying flexible parametric competing
risk regression modeling in a large, multicenter cohort of patients with primary localized high-grade
eSTS. The results have been implemented into the Personalised Sarcoma Care (PERSARC)-app [14] for
Individualized Sarcoma Care and follow-up. 2. Results Patients had undergone surgery with curative intent between January 1994 and October 2014 for
the test cohort and between January 2000 and December 2013 for the validation cohort, respectively. There was a slight male predominance (n = 1038; 53.8%) and the median patient age was 59 years
(interquartile range (IQR): 44.7–70 years). With 55.8%, 17.9%, and 13.9%, most tumors in the test cohort
were located in the thigh (n = 1078), upper arm (n = 346), and lower leg (n = 268), while the lower
arm (n = 142), the foot or toes (n = 65), and the hand or fingers (n = 32) were affected in 7.3%, 3.4%,
and 1.7%, respectively. Further clinicopathological features for both the test and validation cohort are
listed in Table 1. 3 of 13 Cancers 2020, 12, 47 Table 1. Patient characteristics and clinical, pathological, and treatment-related parameters. Table 1. Patient characteristics and clinical, pathological, and treatment-related parameters. Variables
Test Cohort (n = 1931)
Validation Cohort (n = 1085)
N (%)
Missing (%)
N (%)
Missing (%)
p-Value *
Age (continuous; years; median + IQR)
59 (44.7–70)
45 (2.3)
61 (47–74)
0 (0.0)
<0.0001
Gender
Male
1038 (53.8)
0 (0.0)
615 (56.7)
0 (0.0)
0.121
Female
893 (46.2)
470 (43.3)
Tumor
Location
Upper Extremity
520 (26.9)
1 (0.05)
312 (28.8)
0 (0.0)
0.285
Lower Extremity
1410 (73.1)
773 (71.2)
Depth
Epifascial
518 (26.9)
2 (0.1)
291 (26.8)
0 (0.0)
0.984
Subfascial
1411 (73.1)
794 (73.2)
Tumor Size (continuous; cm; median + IQR)
7 (4–11)
30 (1.6)
7.5 (5–12)
5 (0.5)
0.026
Histology
Myxoid liposarcoma
222 (11.6)
16 (0.8)
111 (10.2)
0 (0.0)
<0.0001
MPNST
83 (4.3)
43 (4.0)
Myxofibrosarcoma
451(23.6)
104 (9.6)
Synovial Sarcoma
174 (9.1)
79 (7.3)
UPS
325 (17.0)
375 (34.6)
Angiosarcoma/Vascular Sarcoma
22 (1.1)
20 (1.8)
Dedifferentiated/Pleomorphic
Liposarcoma
141 (7.4)
85 (7.8)
Leiomyosarcoma
221 (11.5)
118 (10.9)
Others
276 (14.4)
150 (13.8)
Grading
G2
719 (37.8)
30 (1.6)
382 (35.8)
19 (1.8)
0.282
G3
1182 (62.2)
684 (64.2)
Margins
R0
1494 (77.4)
0 (0.0)
768 (70.8)
0 (0.0)
<0.0001
R1/2
437 (22.6)
317 (29.2)
CTX
No
1408 (73.0)
1 (0. 2. Results 05)
1039
(95.8)
0 (0.0)
<0.0001
Neoadjuvant
262 (13.6)
40 (3.7)
Adjuvant
209 (10.8)
6 (0.6)
Neoadjuvant + Adjuvant
51 (2.6)
0 (0.0)
RTX
No
619 (32.9)
50 (2.6)
335 (30.9)
0 (0.0)
<0.0001
Neoadjuvant
303 (16.1)
460 (42.4)
Adjuvant
956 (50.8)
275 (25.4)
Neoadjuvant + Adjuvant
3 (0.2)
15 (1.4)
Follow-up (continuous; months; median + IQR)
50 (23.3–95)
11 (0.6)
56 (21–91)
0 (0.0)
0.254
* p-values calculated with Wilcoxon rank sum test for continuous variables, with chi2-test for binary and categorical
variables. p-values in bold are considered statistically significant. Abbreviations: CTX = chemotherapy. IQR =
interquartile range. MPNST = Malignant Peripheral Nerve Sheat Tumor. RTX = radiotherapy. UPS = Undifferentiated
pleiomorphic sarcoma. * p-values calculated with Wilcoxon rank sum test for continuous variables, with chi2-test for binary and categorical
variables. p-values in bold are considered statistically significant. Abbreviations: CTX = chemotherapy. IQR =
interquartile range. MPNST = Malignant Peripheral Nerve Sheat Tumor. RTX = radiotherapy. UPS = Undifferentiated
pleiomorphic sarcoma. Five- and 10-year overall survival (OS) was 73.6% (95%CI: 71.3–75.7) and 62.7% (95%CI: 59.8–65.5) in
the test cohort. In the validation cohort, 5- and 10-year OS were 64.9% (95%CI: 61.8–67.8) and 52.9% (95%CI:
48.9–56.8), respectively. Gender, tumor size, histological subtype (except for angiosarcoma/vascular
sarcoma (p = 0.127) and dedifferentiated/pleomorphic liposarcoma (p = 0.254), margins, neoadjuvant
and adjuvant RTX, as well as adjuvant CTX (all p < 0.05) had a significant influence on risk of LR in the
stepwise backward selection of the Fine and Gray model. Grading as a time-dependent effect was
kept in the model (p = 0.108), while age (p = 0.082) and neoadjuvant CTX (p = 0.214) were excluded. Consequently, gender, grading, tumor size, neoadjuvant and adjuvant RTX, histological subtype,
and adjuvant CTX were included in the flexible parametric competing risk regression model (Table 2). The subdistribution hazard and cumulative incidence functions for LR using ten clinical examples
are shown in Figure 1A,B (definition of these examples found in Table S1, together with estimated
conditional risks of LR). As an example, a male patient with a G2 myxofibrosarcoma sized 10 cm,
with contaminated resection margins (R1/2), no neoadjuvant or adjuvant RTX, and no adjuvant CTX,
has a significantly increased risk of developing LR, especially within the first 15 months of follow-up
(=clinical example IX). 2. Results On the other hand, a male patient with a 6 cm large, G3 synovial sarcoma,
resected with clear margins (R0), without adjuvant CTX or (neo-)adjuvant RTX, has a moderate LR risk
during the first 15 months, and an estimated low risk thereafter (=clinical example VIII). Five- and 10-year overall survival (OS) was 73.6% (95%CI: 71.3–75.7) and 62.7% (95%CI: 59.8–65.5) in
the test cohort. In the validation cohort, 5- and 10-year OS were 64.9% (95%CI: 61.8–67.8) and 52.9% (95%CI:
48.9–56.8), respectively. Gender, tumor size, histological subtype (except for angiosarcoma/vascular
sarcoma (p = 0.127) and dedifferentiated/pleomorphic liposarcoma (p = 0.254), margins, neoadjuvant
and adjuvant RTX, as well as adjuvant CTX (all p < 0.05) had a significant influence on risk of LR in the
stepwise backward selection of the Fine and Gray model. Grading as a time-dependent effect was
kept in the model (p = 0.108), while age (p = 0.082) and neoadjuvant CTX (p = 0.214) were excluded. Consequently, gender, grading, tumor size, neoadjuvant and adjuvant RTX, histological subtype,
and adjuvant CTX were included in the flexible parametric competing risk regression model (Table 2). The subdistribution hazard and cumulative incidence functions for LR using ten clinical examples
are shown in Figure 1A,B (definition of these examples found in Table S1, together with estimated
conditional risks of LR). As an example, a male patient with a G2 myxofibrosarcoma sized 10 cm,
with contaminated resection margins (R1/2), no neoadjuvant or adjuvant RTX, and no adjuvant CTX,
has a significantly increased risk of developing LR, especially within the first 15 months of follow-up
(=clinical example IX). On the other hand, a male patient with a 6 cm large, G3 synovial sarcoma,
resected with clear margins (R0), without adjuvant CTX or (neo-)adjuvant RTX, has a moderate LR risk
during the first 15 months, and an estimated low risk thereafter (=clinical example VIII). 4 of 13 Cancers 2020, 12, 47 Table 2. Estimated coefficients along with their 95% confidence intervals for local recurrence. Table 2. Estimated coefficients along with their 95% confidence intervals for local recurrence. 2. Results Variables
Coefficient
95%-CI
p-Value
Lower
Upper
Local Recurrence
Gender
Male
1
0.011
Female
0.698
0.529
0.921
Grading
G2
1
0.199
G3
0.816
0.598
1.113
Tumor size
1.026
1.004
1.049
0.019
Margins
R0
1
<0.001
R1/R2
2.761
2.021
3.774
Histology
Myxoid Liposarcoma
1
MPNST
4.227
1.837
9.729
0.001
Myxofibrosarcoma
4.156
2.056
8.400
<0.001
Synovial Sarcoma
3.116
1.429
7.014
0.005
UPS
3.373
1.620
7.025
0.001
Angiosarcoma/Vascular Sarcoma
3.316
0.981
12.341
0.074
Dedifferentiated/Pleomorphic
Liposarcoma
1.727
0.719
4.143
0.221
Leiomyosarcoma
2.779
1.294
5.966
0.009
Others
2.385
1.123
5.065
0.024
Neoadjuvant
RTX
No
1
<0.001
Yes
0.298
0.178
0.494
Adjuvant RTX
No
1
0.001
Yes
0.603
0.447
0.814
Adjuvant CTX
No
1
0.008
Yes
1.711
1.154
2.538
Restricted cubic spline 1
2.104
1.851
2.392
<0.001
Restricted cubic spline 2
1.332
1.230
1.442
<0.001
Restricted cubic spline 3
0.980
0.937
1.026
0.391
Restricted cubic spline for time-dependent effect of grading
0.944
0.813
1.096
0.449
Constant
0.048
0.024
0.097
<0.001
Death
Gender
Male
1
0.005
Female
0.736
0.595
0.910
Grading
G2
1
<0.001
G3
2.215
1.655
2.964
Tumor size
1.065
1.048
1.081
<0.001
Margins
R0
1
0.296
R1/R2
1.153
0.883
1.057
Histology
Myxoid Liposarcoma
1
MPNST
1.205
0.664
2.187
0.540
Myxofibrosarcoma
1.208
0.795
1.836
0.375
Synovial Sarcoma
1.461
0.888
2.404
0.136
UPS
1.150
0.753
1.758
0.517
Angiosarcoma/Vascular Sarcoma
4.729
2.335
9.580
<0.001
Dedifferentiated/Pleomorphic
Liposarcoma
1.420
0.863
2.338
0.167
Leiomyosarcoma
2.154
1.402
3.309
<0.001
Others
1.516
0.975
2.356
0.065
Neoadjuvant
RTX
No
1
0.007
Yes
1.543
1.127
2.111
Adjuvant RTX
No
1
0.296
Yes
1.145
0.888
1.476
Adjuvant CTX
No
1
0.022
Yes
0.679
0.488
0.946
Restricted cubic spline 1
4.220
3.393
5.250
<0.001
Restricted cubic spline 2
1.487
1.329
1.663
<0.001
Restricted cubic spline 3
0.965
0.921
1.011
0.139
Restricted cubic spline for time-dependent effect of grading
0.714
0.580
0.889
0.002
Constant
0.050
0.032
0.078
<0.001
p-values in bold are considered statistically significant. CI = Confidence interval. RTX = radiotherapy. CTX =
chemotherapy. UPS = undifferentiated pleomorphic sarcoma. MPNST = Malignant Peripheral Nerve Sheat Tumour. 5 of 13
RTX,
inical 5 of 13
RTX,
inical Cancers 2020, 12, 47
synovial sarcom
has a moderate Figure 1. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for local recurrence using ten clinical examples
(constellation of parameters shown in Tables S1 and S2). A
B
Figure 1. 2. Results Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for local recurrence using ten clinical examples (constellation
of parameters shown in Tables S1 and S2). A B B Figure 1. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for local recurrence using ten clinical examples
(constellation of parameters shown in Tables S1 and S2). Figure 1. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for local recurrence using ten clinical examples (constellation
of parameters shown in Tables S1 and S2). In the stepwise backward selection of the Fine and Gray model for distant metastasis (DM)
histological subtype (except for myxofibrosarcoma (p = 0.641), angiosarcoma/vascular sarcoma (p =
0.067) and dedifferentiated/pleomorphic liposarcoma (p = 0.592), grading, tumor size, gender,
margins, and neoadjuvant RTX (all p < 0.05) were significantly associated with development of
metastases. Age (p = 0.852), adjuvant RTX (p = 0.116), neoadjuvant CTX (p = 0.095), and adjuvant CTX
(p = 0.536) were excluded via stepwise backward selection. Thus, histological subtype, grading,
tumor size, margins, gender, and neoadjuvant RTX were included in the flexible parametric
competing risk regression model (Table 3). In the stepwise backward selection of the Fine and Gray model for distant metastasis (DM)
histological subtype (except for myxofibrosarcoma (p = 0.641), angiosarcoma/vascular sarcoma
(p = 0.067) and dedifferentiated/pleomorphic liposarcoma (p = 0.592), grading, tumor size, gender,
margins, and neoadjuvant RTX (all p < 0.05) were significantly associated with development of
metastases. Age (p = 0.852), adjuvant RTX (p = 0.116), neoadjuvant CTX (p = 0.095), and adjuvant CTX
(p = 0.536) were excluded via stepwise backward selection. Thus, histological subtype, grading, tumor
size, margins, gender, and neoadjuvant RTX were included in the flexible parametric competing risk
regression model (Table 3). In Figure 2A,B, subdistribution hazard and cumulative incidence functions for DM (using the
same ten clinical examples as in Figure 1A,B) are shown. Once again referring to clinical examples IX
(male, myxofibrosarcoma, G2, 10 cm, R1/2-margins, no neoadjuvant RTX) and VIII (male, synovial
sarcoma, G3, 6cm, R0-margins, no neoadjuvant RTX), risk of DM is lower in clinical example IX in
comparison to clinical example VI, while LR-risks are just the opposite, highlighting the importance of
an individualized follow-up strategy. 2. Results The conditional risks of these ten clinical examples changing over time estimated based on the
models presented above are provided in Table S1 for LR and Table S2 for DM. Conditional risks for
all possible combinations of prognostic factors may be estimated and have been implemented in the
updated version of the PERSARC app. The Harrell C index for LR was equal to 0.705 and 0.683 for the internal and external cohort,
respectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for the
external cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that the
LR model tended to underestimate the actual patient risk, especially in the validation cohort. On the
other hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very good
model calibration. 6 of 13 Cancers 2020, 12, 47 Table 3. Estimated coefficients along with their 95% confidence intervals for distant metastasis. Table 3. Estimated coefficients along with their 95% confidence intervals for distant metastasis. 2. Results Coefficient
95%-CI
p-Value
Lower
Upper
Distant Metastasis
Gender
Male
1
<0.001
Female
0.720
0.605
0.857
Grading
G2
1
<0.001
G3
1.737
1.412
2.136
Tumor size
1.069
1.056
1.083
<0.001
Margins
R0
1
0.006
R1/R2
1.347
1.087
1.669
Histology
Myxoid Liposarcoma
1
MPNST
1.825
1.158
2.875
0.009
Myxofibrosarcoma
1.064
0.750
1.508
0.729
Synovial Sarcoma
1.986
1.343
3.976
0.001
UPS
1.445
1.033
2.022
0.032
Angiosarcoma/Vascular Sarcoma
2.016
1.022
3.797
0.043
Dedifferentiated/Pleomorphic Liposarcoma
1.209
0.786
1.861
0.387
Leiomyosarcoma
2.689
1.900
3.797
<0.001
Other
1.835
1.293
2.604
0.001
Neoadjuvant
RTX
No
1
0.005
Yes
1.351
1.097
1.663
Restricted cubic spline 1
2.928
2.591
3.308
<0.001
Restricted cubic spline 2
1.458
1.374
1.547
<0.001
Restricted cubic spline 3
0.965
0.926
1.006
0.096
Restricted cubic spline 4
1.040
1.020
1.062
<0.001
Restricted cubic spline 5
0.995
0.982
1.008
0.427
Restricted cubic spline for time-dependent effect of grading
0.723
0.640
0.817
<0.001
Constant
0.108
0.078
0.149
<0.001
Death
Gender
Male
1
0.864
Female
0.968
0.666
1.407
Grading
G2
1
0.018
G3
1.873
1.116
3.145
Tumor size
1.023
0.997
1.050
0.087
Margins
R0
1
0.198
R1/R2
1.378
0.846
2.244
Histology
Myxoid Liposarcoma
1
MPNST
2.506
0.844
7.442
0.098
Myxofibrosarcoma
3.136
1.325
7.418
0.009
Synovial Sarcoma
0.600
0.150
2.416
0.472
UPS
1.781
0.714
4.443
0.216
Angiosarcoma/Vascular Sarcoma
11.165 *
3.507 *
35.542 *
<0.001 *
Dedifferentiated/Pleomorphic Liposarcoma
3.331
1.259
8.812
0.015
Leiomyosarcoma
1.798
0.675
4.782
0.241
Other
2.408
0.963
4.782
0.060
Neoadjuvant
RTX
No
1
0.048
Yes
0.541
0.295
0.993
Restricted cubic spline 1
3.604
2.494
5.211
<0.001
Restricted cubic spline 2
1.270
1.060
1.523
0.010
Restricted cubic spline 3
0.952
0.863
1.049
0.316
Restricted cubic spline 4
0.953
0.908
1.001
0.057
Restricted cubic spline 5
0.974
0.569
1.199
0.097
Restricted cubic spline for time-dependent effect of grading
0.826
0.569
1.199
0.314
Constant
0.010
0.004
0.025
<0.001
p-values in bold are considered statistically significant; CI = Confidence interval. RTX = radiotherapy. CTX =
chemotherapy UPS = undifferentiated pleomorphic sarcoma * too few events 7 of 13
in the 7 of 13
in the Cancers 2020, 12, 47
all possible com
d
d Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for distant metastasis using the same ten clinical
examples as in Figures 1A and B (constellation of parameters shown in Tables S1 and S2). A
B
Figure 2. 2. Results Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for distant metastasis using the same ten clinical examples
as in Figure 1A,B (constellation of parameters shown in Tables S1 and S2). Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for distant metastasis using the same ten clinical
examples as in Figures 1A and B (constellation of parameters shown in Tables S1 and S2). The Harrell C index for LR was equal to 0.705 and 0.683 for the internal and external cohort,
respectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for the
external cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that the
LR model tended to underestimate the actual patient risk, especially in the validation cohort. On the
other hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very good A
Figure 2. Subdistribution hazard function (A) an
parametric competing risk regression model fo
examples as in Figures 1A and B (constellation of
The Harrell C index for LR was equal to 0.7
ti
l
F
DM H
ll C t ti ti B
d cumulative incidence function (B) of the flexible
or distant metastasis using the same ten clinical
parameters shown in Tables S1 and S2). 705 and 0.683 for the internal and external cohort,
ual to 0 723 fo the i te
al oho t a d 0 772 fo the B Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for distant metastasis using the same ten clinical
examples as in Figures 1A and B (constellation of parameters shown in Tables S1 and S2). Figure 2. Subdistribution hazard function (A) and cumulative incidence function (B) of the flexible
parametric competing risk regression model for distant metastasis using the same ten clinical examples
as in Figure 1A,B (constellation of parameters shown in Tables S1 and S2). pectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for
ernal cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that
model tended to underestimate the actual patient risk, especially in the validation cohort. 2. Results On
her hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very go The Harrell C index for LR was equal to 0.705 and 0.683 for the internal and external cohort,
respectively. For DM, Harrell C statistics was equal to 0.723 for the internal cohort and 0.772 for the
external cohort. Calibration plots for LR (Figure 3A) using test and validation cohort showed that the
LR model tended to underestimate the actual patient risk, especially in the validation cohort. On the
other hand, calibration plots for DM with test and validation cohort (Figure 3B) showed very good
model calibration. Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding
local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding local
recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). Di
i The Harrell C index for LR was equal to 0.70
respectively. For DM, Harrell C statistics was equa
external cohort. Calibration plots for LR (Figure 3A
LR model tended to underestimate the actual patie
other hand, calibration plots for DM with test and
model calibration. 05 and 0.683 for the internal and external cohort
al to 0.723 for the internal cohort and 0.772 for the
) using test and validation cohort showed that the
nt risk, especially in the validation cohort. On the
validation cohort (Figure 3B) showed very good espe l to Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding
local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding local
recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). 3. Discussion
3. Discussion Figure 3. Calibration plots for the flexible parametric competing risk regression model regarding
local recurrence (A) and distant metastasis (B) for the test (top) and validation cohort (bottom). 3. Discussion
In the present retrospective multicenter cohort study, flexible parametric competing risk
regression modeling was applied in order to estimate individual three-to-six-month risks for local
In the present retrospective multicenter cohort study, flexible parametric competing risk
regression modeling was applied in order to estimate individual three-to-six-month risks for local
In the present retrospective multicenter cohort study, flexible parametric competing risk regression
modeling was applied in order to estimate individual three-to-six-month risks for local recurrence and
distant metastasis during the first 5 years of follow-up in patients undergoing curative resection for
high-grade extremity soft tissue sarcoma. It offers an evidence-based opportunity to individually schedule
follow-up visits instead of adhering to calendar-based guidelines [8,9]. The number of radiological
investigations for assessing disease status, especially after R0 resections and taking histological subtype
into account, could be significantly restricted, reducing patient- and healthcare burden. The advantage
of using flexible parametric competing risk regression models to estimate LR- and DM-risks in
eSTS-patients is based on the fact that these rates strongly vary upon time (i.e., they do not constantly
increase or decrease). To overcome this issue, flexible parametric competing risk regression models
represent the baseline distribution function as a restricted cubic spline function of log time instead of
a linear function of log time [15]. Moreover, it allows smooth estimation of both the cause-specific
hazard rates and cumulative incidence functions. Both models performed well at internal and external
calibration, with c-indexes comparable to previously published studies [14,16]. One of the limitations of the present study is its retrospective design, resulting in possible selection
biases regarding diagnosis, treatment, and follow-up of patients included, due to slightly differing
policies at the respective centers. By incorporating these factors in the statistical models, we aimed
at reducing this bias. Moreover, during the study period, several histological STS-subtypes were Cancers 2020, 12, 47 8 of 13 reclassified (i.e., malignant fibrous histiocytoma to undifferentiated pleomorphic sarcoma). At some,
but not all, participating centers, all histological diagnoses had been reevaluated by pathologists and,
if applicable, reclassified according to the current classification systems. In order to limit the impact of
this limitation, we only included patients treated in tertiary reference sarcoma centers with experienced
and dedicated sarcoma pathologists. 3. Discussion
3. Discussion p
g
Another limitation of the present study is that the models were developed based on patient
cohorts from experienced, tertiary tumor centers. This implies that generalizability of the predicted
risks to patients not treated at such centers has to be questioned. Moreover, considering that we did
only include patients with high-grade (G2/3), primary eSTS who had undergone surgery with curative
intent, the risks estimated are not applicable to patients with low-grade disease or metastases at initial
presentation. Furthermore, estimated risks of the current models should be applied with caution after
patients have already developed an event (i.e., LR or DM) during follow-up. Due to the retrospective
design of the study, not all variables could be ascertained in every patient, thus potentially reducing
the statistical power. However, it can be assumed that in this large patient collective, missing data may
have little to no bias to the conclusions made, wherefore cases with missing information on clinical
and/or pathological variables were not excluded [17]. As outlined in the introduction, current follow-up strategies follow a heuristic approach, with 3-
to 4-months intervals for the first three years, followed by biannual check-ups until the end of the 5th
year and annual appointments thereafter [8,9]. In clinical practice, it is not only of interest to estimate a patient’s cumulative risk after a specific
period of time but also to know about the conditional risks from one follow-up appointment to the
next, in case no event had occurred. We addressed this question by calculating conditional risks for
LR and DM depending on different, clinically relevant, examples. Notably, the present model allows
risk prediction at any constellation of variables, which are at the moment included in the updated
PERSARC app. This app allows the patient’s individualized risk of LR and DM to be estimated
by entering relevant prognostic parameters, such as histological subtype, tumor size, and resection
margins. With the estimated event-risk in time, physicians and patients may decide together when
the next follow-up examination should be scheduled. In light of the heterogeneity of eSTS with part
significantly differing outcomes, estimated event risks would facilitate planning of an individualized
follow-up protocol for each patient. 3. Discussion
3. Discussion Arbitrary thresholds of 4% for LR and 2% for DM were chosen in the present study to be of clinical
“relevance”, considering that LR is usually detected during clinical examination or even noticed by
patients themselves, while DM (most commonly to the lungs) require visualization by chest x-ray or
thoraci computed tomography (CT) scan [18,19]. However, thresholds should be changed on patients’
preference and clinical significance. Previously published studies have well-investigated risks of LR, DM, and overall survival (OS) in
large, retrospective cohorts of patients with eSTS [14,16,20,21]. The nomogram for OS by Kattan et
al. [21] in 2002 and the two more recent nomograms for DM and OS by Callegaro et al. [16] published
in 2016 added significant value to predict individual patient risks. Both studies used Cox-regression
models as the basis for their nomograms. In comparison to Cox-regression models, flexible parametric
competing risk regression models have a major advantage; while the Cox-regression models only
estimate the relative effects (i.e., hazard rates), flexible parametric competing risk regression models
estimate the baseline hazard using restricted cubic splines [22]. The cumulative incidence functions of
LR and DM predicted from flexible parametric competing risk regression models demonstrate the clear
variance in event rates. By applying a flexible parametric competing risk regression model, we aimed
at incorporating non-constant hazards, time-varying covariates, and death as the competing event in
order to obtain a robust, comprehensible, and accurate prediction of individual patient risks. Moreover,
with the clinical examples provided, the risk peaks during the first year of follow-up is clearly visible. Although appointments may be safely skipped in some patients due to very low risks of LR and/or
DM, others would benefit from closer follow-up intervals. Cancers 2020, 12, 47 9 of 13 Cancers 2020, 12, 47 Potentially due to the application of the present statistical model, interesting results emerged:
Female gender was independently associated with a significantly lower risk of LR and DM. An
association between gender and overall survival (OS) has been observed by Maretty-Nielsen et al. and
Wu et al. [2,23]. However, an association between gender and LR-free as well as DM-free survival has
not been described thus far [24]. Moreover, tumor grading, which is a well-known prognostic factor of
LR, was not significantly associated with an altered risk in the current flexible parametric competing
risk regression model. 3. Discussion
3. Discussion This may be explained by the fact that patients with usually fast-growing,
highly-aggressive G3 tumors will present with LR at early time points, while in those with relatively
slower-growing G2 tumors, LR is most probably detected at a later date. This hypothesis is corroborated
by the fact that grading did not meet the proportional hazards assumption, wherefore it was treated as
a time-varying covariate. On the other hand, another recently published multicenter study for grade III
eSTS did not incorporate grade II in the multivariate model for OS [14]. The current model has broader
applicability as it also incorporates patients with grade II eSTS. Additionally, margins as classified in
the current study only divide “clear” from “contaminated” margins, not taking into consideration that
histological subtypes with infiltrative growth pattern as undifferentiated pleiomorphic sarcoma (UPS)
and myxofibrosarcoma potentially require broader margins to markedly reduce LR-risk [25]. Thus,
unsurprisingly, also in the current flexible parametric competing risk regression model for LR, these
histological subtypes showed significantly higher LR-risks in comparison to other histologies. Statistical Analysis We focused on the first five years of follow-up to predict the conditional risk at the usually
scheduled follow-up times (every 3 months from 1st to 3rd year; every 6 months in 4th and 5th year),
i.e., the risk of experiencing an event at X + Y, given that the patient has not developed an event before X
months. The variables age and tumor size were centered at their mean value in order to allow prediction
at the average in case variables were not specified upon calculation. We used the Royston and Parmar
approach to fit a flexible parametric competing risk regression model in order to estimate the risk of LR
and DM, with death as the competing event [30]. In this model, the baseline distribution is modeled as
a restricted cubic spline function of log time [15,22]. Splines constitute flexible mathematical functions
defined by piecewise polynomials together with distinct constraints, ensuring that the overall curve is
smooth [22]. A feature of restricted cubic splines as used in the present model is that the fitted function
is forced to be linear before the first and after the last knot [31]. As automatic stepwise backward
selection of variables is currently not available for the flexible parametric competing risk regression
model, variable selection for the LR and DM models was based on a stepwise backward procedure
using a multivariable Fine and Gray model [32]. Variables with a p-value < 0.05 were excluded from
the model, except for histology, where all subtypes were kept in the analyses. The LR and DM models
were fit on the log cumulative subdistribution hazard scale, directly modeling the cause-specific
cumulative incidence function. Grading was incorporated in the model as a time-dependent effect
for LR and DM, as it did not meet the proportional hazards assumption. The number of knots of
the flexible parametric competing risk regression model for LR and DM was chosen based on the
lowest AIC (=Akaike Information Criterion) after fitting several models with knots from 0 to 5. For the
flexible parametric competing risk regression model estimating the risk of LR, two knots turned out as
most accurate, while for the model predicting the risk of DM, four knots were used (with no internal
knots for grading as a time-dependent covariate). Cumulative incidence functions were estimated
based on the defined models. 4. Materials and Methods In this retrospective multicenter study, 1931 consecutive patients with primary nonmetastatic
high-grade (G2/3) eSTS managed with surgery at a curative intent were included in the test cohort,
with patient information deriving from prospectively maintained STS databases at 5 participating
tertiary sarcoma referral centers. Patients with missing information on oncological follow-up (i.e.,
development of LR/DM) had to be excluded (n = 42). Extremity STS were defined as tumors from
the shoulder to the fingers (=upper limb) and from the pelvic girdle, excluding intrapelvic STS, to the
foot (=lower limb). The validation cohort consisted of 1085 patients with identical inclusion criteria
as for the test cohort from two independent tertiary sarcoma referral centers. As described above,
patient monitoring after surgery usually followed a standardized approach with clinical examination,
radiography using chest X-ray (CXR) or chest CT-scan (chest-CT) for control of DM and sonography or
magnetic resonance imaging (MRI) for control of LR. Demographic variables (patient age at diagnosis, gender), tumor-related parameters (tumor
size, depth, location, grading, histological subtype), treatment (histological margins, (neo)adjuvant
CTX/RTX), and outcome variables (date of LR or DM, date of death/last follow-up) were reported. Histological resection margins were divided into “clear” margins (=R0) and “contaminated” margins
(=R1/2), as classification and definition of margin status have changed over time [26–28]. Histological
subtypes were classified into 9 categories, with myxoid liposarcoma as the reference, compliant with
previous studies and the current World Health Organisation (WHO) classification (Table 1) [6,16]. The FNCLCC grading system (Fédération Française des Centres de Lutte Contre le Cancer/French
Federation of Centres for the Fight against Cancer) was used to categorize tumors into either intermediate
(=G2) or high-grade (G3) [29]. (Neo-)adjuvant RTX and CTX had been administered in case a high risk
of LR or DM had been anticipated by the multidisciplinary tumor board, according to locally preferred
guidelines, LR was defined as a radiologically and/or histologically confirmed tumor recurrence. DM must have been confirmed by radiography (sonography, MRI, CXR, chest-CT) and/or histologically. In the case of pulmonary nodules without subsequent surgical exploration, an increase in size of the
suspected metastasis must have been present. Patient, tumor, and treatment-related factors were
ascertained using medical records, histological reports, and prospectively maintained databases at the
respective centers. 10 of 13 Cancers 2020, 12, 47 Ethics approval was obtained in each participating center. 4. Materials and Methods The study was performed according to
the Declaration of Helsinki and approved by the Ethics Committee of the Medical University of Graz,
Austria (IRB-approval-number: 31-046 ex 18/19; date of approval: 24 May 2019). Statistical Analysis Conditional risks at the 3–6-months intervals were calculated based
on the cumulative incidence functions of the flexible parametric competing risk regression model. Threshold was set to 4% for LR, considering that they are often palpable and diagnosed during the
clinical examination or by patients themselves [18]. On the other hand, a 2% threshold for DM was
chosen, as DM (and most commonly lung metastases) can only safely be diagnosed by chest X-ray or
CT-scans of the thorax [19]. Model discrimination was tested using the Harrell C index, estimating the
probability of concordance between observed and predicted outcomes. A value of 0.5 indicates no
predictive discrimination, while a value of 1.0 indicates a perfect separation of patients with different
outcomes [33]. Furthermore, calibration plots were compiled to assess model calibration in the test
and validation cohort. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/1/47/s1,
Table S1: Conditional Risks for Local Recurrence–Threshold 4%; Table S2: Conditional Risks for Distant Metastasis–
Threshold 2%. References 1. Italiano, A.; Le Cesne, A.; Mendiboure, J.; Blay, J.Y.; Piperno-Neumann, S.; Chevreau, C.; Delcambre, C.;
Penel, N.; Terrier, P.; Ranchere-Vince, D.; et al. Prognostic factors and impact of adjuvant treatments on local
and metastatic relapse of soft-tissue sarcoma patients in the competing risks setting. Cancer 2014, 120, 3361–3369. [CrossRef] [PubMed] 2. Maretty-Nielsen, K.; Aggerholm-Pedersen, N.; Safwat, A.; Jorgensen, P.H.; Hansen, B.H.; Baerentzen, S.;
Pedersen, A.B.; Keller, J. Prognostic factors for local recurrence and mortality in adult soft tissue sarcoma of
the extremities and trunk wall: A cohort study of 922 consecutive patients. Acta Orthop. 2014, 85, 323–332. [CrossRef] [PubMed] 2. Maretty-Nielsen, K.; Aggerholm-Pedersen, N.; Safwat, A.; Jorgensen, P.H.; Hansen, B.H.; Baerentzen, S.;
Pedersen, A.B.; Keller, J. Prognostic factors for local recurrence and mortality in adult soft tissue sarcoma of
the extremities and trunk wall: A cohort study of 922 consecutive patients. Acta Orthop. 2014, 85, 323–332. [CrossRef] [PubMed] 3. Novais, E.N.; Demiralp, B.; Alderete, J.; Larson, M.C.; Rose, P.S.; Sim, F.H. Do surgical margin and local
recurrence influence survival in soft tissue sarcomas? Clin. Orthop. Relat. Res. 2010, 468, 3003–3011. [CrossRef] [PubMed] 3. Novais, E.N.; Demiralp, B.; Alderete, J.; Larson, M.C.; Rose, P.S.; Sim, F.H. Do surgical margin and local
recurrence influence survival in soft tissue sarcomas? Clin. Orthop. Relat. Res. 2010, 468, 3003–3011. [CrossRef] [PubMed] 4. Willeumier, J.; Fiocco, M.; Nout, R.; Dijkstra, S.; Aston, W.; Pollock, R.; Hartgrink, H.; Bovee, J.; van de Sande, M. High-grade soft tissue sarcomas of the extremities: Surgical margins influence only local recurrence not
overall survival. Int. Orthop. 2015, 39, 935–941. [CrossRef] [PubMed] 4. Willeumier, J.; Fiocco, M.; Nout, R.; Dijkstra, S.; Aston, W.; Pollock, R.; Hartgrink, H.; Bovee, J.; van de Sande, M. High-grade soft tissue sarcomas of the extremities: Surgical margins influence only local recurrence not
overall survival. Int. Orthop. 2015, 39, 935–941. [CrossRef] [PubMed] p
5. Singer, S.; Demetri, G.D.; Baldini, E.H.; Fletcher, C.D. Management of soft-tissue sarcomas: An overview and
update. Lancet Oncol. 2000, 1, 75–85. [CrossRef] 6. Fletcher, C.D.; Bridge, J.A.; Hodgendoorn, P.C.W.; Mertens, F. WHO Classification of Tumours of Soft Tissue and
Bone; IARC: Lyon, France, 2013. 7. Smolle, M.A.; van Praag, V.M.; Posch, F.; Bergovec, M.; Leitner, L.; Friesenbichler, J.; Heregger, R.; Riedl, J.M.;
Pichler, M.; Gerger, A.; et al. Surgery for metachronous metastasis of soft tissue sarcoma—A magnitude of
benefit analysis using propensity score methods. Eur. J. Surg. Oncol. 2018, 45, 242–248. [CrossRef] 8. 5. Conclusions In conclusion, the present study provides a model to individually predict patient’s LR and DM risks
during follow-up, applying a flexible parametric competing risk regression approach. These models
are at the moment being included in the updated version of the PERSARC app for Individualized
Sarcoma Care and follow-up. Although a risk-threshold of 4% for LR and 2% for DM was chosen in
the present study, the “optimal” threshold upon which an individual patient should undergo imaging
with MRI, chest-CT, or CXR, is still subjected to experts’ opinion and should be further discussed with
patients concerned. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/1/47/s1,
Table S1: Conditional Risks for Local Recurrence–Threshold 4%; Table S2: Conditional Risks for Distant Metastasis–
Threshold 2%. 11 of 13 11 of 13 Cancers 2020, 12, 47 Author Contributions: Conceptualization, M.A.S., A.L., R.L.H., and J.S. (Joanna Szkandera); data curation, M.A.S.,
D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., M.F., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger),
M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi); formal analysis, J.S. (Josef Smolle); investigation, D.C.,
J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.D., W.J.v.H., J.M.R., M.S., A.G. (Alessandro Gronchi), M.P.,
H.S., B.L.-A., and A.G. (Armin Gerger); methodology, M.v.d.S., M.F., S.D., J.S. (Josef Smolle), R.L.H., and J.S. (Joanna
Szkandera); project administration, M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); supervision, M.v.d.S., D.A.,
and R.L.H.; validation, V.v.P., M.F., D.A. and J.S. (Joanna Szkandera); visualization, M.A.S. and J.S. (Josef Smolle);
writing—Original draft, M.A.S., M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); writing—Review and editing,
D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger),
M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi). All authors have read and agreed to the published
version of the manuscript. Author Contributions: Conceptualization, M.A.S., A.L., R.L.H., and J.S. (Joanna Szkandera); data curation, M.A.S.,
D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., M.F., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger),
M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi); formal analysis, J.S. (Josef Smolle); investigation, D.C.,
J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.D., W.J.v.H., J.M.R., M.S., A.G. (Alessandro Gronchi), M.P.,
H.S., B.L.-A., and A.G. (Armin Gerger); methodology, M.v.d.S., M.F., S.D., J.S. (Josef Smolle), R.L.H., and J.S. (Joanna
Szkandera); project administration, M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); supervision, M.v.d.S., D.A.,
and R.L.H.; validation, V.v.P., M.F., D.A. and J.S. (Joanna Szkandera); visualization, M.A.S. and J.S. 5. Conclusions (Josef Smolle);
writing—Original draft, M.A.S., M.v.d.S., A.L., R.L.H., and J.S. (Joanna Szkandera); writing—Review and editing,
D.C., J.W., A.H., L.L., M.B., P.-U.T., V.v.P., J.P., M.W., R.W., S.P.D.D., W.J.v.H., J.M.R., M.S., A.G. (Armin Gerger),
M.P., H.S., B.L.-A., D.A., and A.G. (Alessandro Gronchi). All authors have read and agreed to the published
version of the manuscript. Funding: This work was supported by the Dutch Cancer Society (DCS)—KWF Kankerbestrijding [UL2015-8028]. The funding source had no role in the design of this study; execution, analyses, and interpretation of the data;
report writing; or decision to submit the article for publication. Conflicts of Interest: Author van de Sande reports grants from Daiichi Sankyo, outside the submitted work. The remaining authors (Maria A Smolle, Dario Callegaro, Jay Wunder, Andrew J. Hayes, Lukas Leitner, Marko
Bergovec, Per-Ulf Tunn, Veroniek van Praag, Marta Fiocco, Joannis Panotopoulos, Madeleine Willegger, Reinhard
Windhager, Sander Djikstra, Winan J van Houdt, Jakob M Riedl, Michael Stotz, Armin Gerger, Martin Pichler,
Herbert Stöger, Bernadette Liegl-Atzwanger, Josef Smolle, Dimosthenis Andreou, Andreas Leithner, Alessandro
Gronchi, Rick L. Haas, and Joanna Szkandera) have no conflicts of interest to declare. References Casali, P.G.; Abecassis, N.; Bauer, S.; Biagini, R.; Bielack, S.; Bonvalot, S.; Boukovinas, I.; Bovee, J.; Brodowicz, T.;
Broto, J.M.; et al. Soft tissue and visceral sarcomas: ESMO-EURACAN Clinical Practice Guidelines for
diagnosis, treatment and follow-up. Ann. Oncol. 2018, 29, iv51–iv67. [CrossRef] 9. von Mehren, M.; Randall, R.L.; Benjamin, R.S.; Boles, S.; Bui, M.M.; Ganjoo, K.N.; George, S.; Gonzalez, R.J.;
Heslin, M.J.; Kane, J.M., 3rd; et al. Soft Tissue Sarcoma, Version 2.2018, NCCN Clinical Practice Guidelines in
Oncology. J. Natl. Compr. Cancer Netw. 2018, 16, 536–563. [CrossRef] 10. Gingrich, A.A.; Bateni, S.B.; Monjazeb, A.M.; Darrow, M.A.; Thorpe, S.W.; Kirane, A.R.; Bold, R.J.;
Canter, R.J. Neoadjuvant Radiotherapy is Associated with R0 Resection and Improved Survival for Patients
with Extremity Soft Tissue Sarcoma Undergoing Surgery: A National Cancer Database Analysis. Ann. Surg. Oncol. 2017, 24, 3252–3263. [CrossRef] 12 of 13 Cancers 2020, 12, 47 11. Gronchi, A.; Ferrari, S.; Quagliuolo, V.; Broto, J.M.; Pousa, A.L.; Grignani, G.; Basso, U.; Blay, J.Y.; Tendero, O.;
Beveridge, R.D.; et al. Histotype-tailored neoadjuvant chemotherapy versus standard chemotherapy in
patients with high-risk soft-tissue sarcomas (ISG-STS 1001): An international, open-label, randomised,
controlled, phase 3, multicentre trial. Lancet Oncol. 2017, 18, 812–822. [CrossRef] 12. Posch, F.; Partl, R.; Doller, C.; Riedl, J.M.; Smolle, M.; Leitner, L.; Bergovec, M.; Liegl-Atzwanger, B.; Stotz, M.;
Bezan, A.; et al. Benefit of Adjuvant Radiotherapy for Local Control, Distant Metastasis, and Survival
Outcomes in Patients with Localized Soft Tissue Sarcoma: Comparative Effectiveness Analysis of an
Observational Cohort Study. Ann. Surg. Oncol. 2018, 25, 776–783. [CrossRef] [PubMed] 13. Royce, T.J.; Punglia, R.S.; Chen, A.B.; Patel, S.A.; Thornton, K.A.; Raut, C.P.; Baldini, E.H. Cost-Effectiveness of
Surveillance for Distant Recurrence in Extremity Soft Tissue Sarcoma. Ann. Surg. Oncol. 2017, 24, 3264–3270. [CrossRef] [PubMed] 14. van Praag, V.M.; Rueten-Budde, A.J.; Jeys, L.M.; Laitinen, M.K.; Pollock, R.; Aston, W.; van der Hage, J.A.;
Dijkstra, P.D.S.; Ferguson, P.C.; Griffin, A.M.; et al. A prediction model for treatment decisions in high-grade
extremity soft-tissue sarcomas: Personalised sarcoma care (PERSARC). Eur. J. Cancer 2017, 83, 313–323. [CrossRef] [PubMed] 15. Royston, P.; Lambert, P.C. Flexible Parametric Survival Analysis Using Stata: Beyond the Cox Model; Stata Press:
College Statoin, TX, USA, 2011. 16. Callegaro, D.; Miceli, R.; Bonvalot, S.; Ferguson, P.; Strauss, D.C.; Levy, A.; Griffin, A.; Hayes, A.J.;
Stacchiotti, S.; Pechoux, C.L.; et al. References Development and external validation of two nomograms to predict
overall survival and occurrence of distant metastases in adults after surgical resection of localised soft-tissue
sarcomas of the extremities: A retrospective analysis. Lancet Oncol. 2016, 17, 671–680. [CrossRef] 17. Kang, H. The prevention and handling of the missing data. Korean J. Anesthesiol. 2013, 64, 402–406. [CrossRef]
[PubMed] 18. Puri, A.; Ranganathan, P.; Gulia, A.; Crasto, S.; Hawaldar, R.; Badwe, R.A. Does a less intensive surveillance
protocol affect the survival of patients after treatment of a sarcoma of the limb? updated results of the
randomized TOSS study. Bone Jt. J. 2018, 100-B, 262–268. [CrossRef] 19. Hovgaard, T.B.; Nymark, T.; Skov, O.; Petersen, M.M. Follow-up after initial surgical treatment of soft tissue
sarcomas in the extremities and trunk wall. Acta Oncol. 2017, 56, 1004–1012. [CrossRef] 20. Rueten-Budde, A.J.; van Praag, V.M.; PERSARC studygroup; van de Sande, M.A.J.; Fiocco, M. Dynamic
prediction of overall survival for patients with high-grade extremity soft tissue sarcoma. Surg. Oncol. 2018,
27, 695–701. [CrossRef] 21. Kattan, M.W.; Leung, D.H.; Brennan, M.F. Postoperative nomogram for 12-year sarcoma-specific death. J. Clin. Oncol. 2002, 20, 791–796. [CrossRef] 22. Lambert, P.C.; Royston, P. Further development of flexible parametric models for survival analysis. Stata J. 2009, 9, 265–290. [CrossRef] 23. Wu, J.; Qian, S.; Jin, L. Prognostic factors of patients with extremity myxoid liposarcomas after surgery. J. Orthop. Surg. Res. 2019, 14, 90. [CrossRef] [PubMed] 24. Trovik, C.S.; Bauer, H.C.; Alvegard, T.A.; Anderson, H.; Blomqvist, C.; Berlin, O.; Gustafson, P.; Saeter, G.;
Walloe, A. Surgical margins, local recurrence and metastasis in soft tissue sarcomas: 559 surgically-treated
patients from the Scandinavian Sarcoma Group Register. Eur. J. Cancer 2000, 36, 710–716. [CrossRef] 5. Fujiwara, T.; Stevenson, J.; Parry, M.; Tsuda, Y.; Tsoi, K.; Jeys, L. What is an adequate margin for infiltra
soft-tissue sarcomas? Eur. J. Surg. Oncol. 2019. [CrossRef] 26. Kainhofer, V.; Smolle, M.A.; Szkandera, J.; Liegl-Atzwanger, B.; Maurer-Ertl, W.; Gerger, A.; Riedl, J.;
Leithner, A. The width of resection margins influences local recurrence in soft tissue sarcoma patients. Eur. J. Surg. Oncol. 2016, 42, 899–906. [CrossRef] [PubMed] 27. Tunn, P.U.; Kettelhack, C.; Durr, H.R. Standardized approach to the treatment of adult soft tissue sarcoma of
the extremities. Recent Results Cancer Res. 2009, 179, 211–228. 28. Wittekind, C.; Compton, C.C.; Greene, F.L.; Sobin, L.H. TNM residual tumor classification revisited. Cancer
2002, 94, 2511–2516. [CrossRef] 29. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Trojani, M.; Contesso, G.; Coindre, J.M.; Rouesse, J.; Bui, N.B.; de Mascarel, A.; Goussot, J.F.; David, M.;
Bonichon, F.; Lagarde, C. Soft-tissue sarcomas of adults; study of pathological prognostic variables and
definition of a histopathological grading system. Int. J. Cancer 1984, 33, 37–42. [CrossRef] 13 of 13 13 of 13 Cancers 2020, 12, 47 30. Mozumder, S.I.; Rutherford, M.J.; Lambert, P.C. stpm2cr: A flexible parametric competing risks model using
a direct likelihood approach for the cause-specific cumulative incidence function. Stata J. 2017, 17, 462–489. [CrossRef] 31. Durrleman, S.; Simon, R. Flexible regression models with cubic splines. Stat. Med. 1989, 8, 551–561. [CrossRef]
32. Fine, J.P.; Gray, R.J. A proportional hazards model for the subdistribution of a competing risk. J. Am. Stat. Assoc. 1999, 94, 496–509. [CrossRef] 32. Fine, J.P.; Gray, R.J. A proportional hazards model for the subdistribution of a competing risk. J. Am. Stat. Assoc. 1999, 94, 496–509. [CrossRef] 33. Harrell, F.E., Jr.; Lee, K.L.; Mark, D.B. Multivariable prognostic models: Issues in developing models,
evaluating assumptions and adequacy, and measuring and reducing errors. Stat. Med. 1996, 15, 361–387. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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https://aacr.figshare.com/articles/journal_contribution/Data_Supplement_from_High-Throughput_Time-Resolved_FRET_Reveals_Akt_PKB_Activation_as_a_Poor_Prognostic_Marker_in_Breast_Cancer/22402640/1/files/39848441.pdf
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Data Supplement from High-Throughput Time-Resolved FRET Reveals Akt/PKB Activation as a Poor Prognostic Marker in Breast Cancer
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pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer Supplementary Figure 1: Specificity of human anti-Akt (panAkt, pT308)
antibodies assessed by quantitative Western blot analysis in SKBR3 cells. Supplementary Figure 1: Specificity of human anti-Akt (panAkt, pT308)
antibodies assessed by quantitative Western blot analysis in SKBR3 cells. SKBR3 cells starved for 3h followed by pretreatment with 50µM LY294002 for 30
min prior to 100ng/ml EGF stimulation for 5min as indicated. Western blot analysis
of (A) phospho pAkt (pT308) and (B) total Akt (panAkt) was performed using the LI-
COR/Odyssey system, with (C) merged. (D) Quantification of Akt phosphorylation
(pT308) normalized to the amount of total Akt (panAkt) from three independent
experiments (***, p<0.0001). Supplementary Figure 2: Localization of endogenous Akt and pAkt (pT308) in
SKBR3 cells and FFPE human breast tumor tissues using TSA amplified
immunofluorescence. (A) SKBR3 cells were pretreated with LY294002 (50µM) for 30 min prior to EGF
stimulation (100ng/ml) for 5 min as indicated. Confocal images show the localization
of endogenous panAkt and pT308 using specific primary antibodies (as described in
Supporting Information Fig 1). Labeled Fab fragments were used as secondary
antibodies: anti-mouse Fab-ORG488 (green) and anti-rabbit Fab-ALX594 (red). (B)
SKBR3 cells were treated as in (A). However the signals were detected using a Fab
fragment-HRP based TSA immunofluorescence assay as described in Materials and
Methods. Confocal microscopy images show the localization of endogenous panAkt
(green) using an anti-mouse Fab-ORG488, and pT308 (red) using an anti-rabbit Fab- 1 1 pAkt is a marker for poor prognosis in breast cancer
HRP and TSA-ALX594. (C) FFPE human breast tumor tissue was labeled with
panAkt and pT308 for TSA amplified immunofluorescence, as in (B) pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer HRP and TSA-ALX594. (C) FFPE human breast tumor tissue was labeled with
panAkt and pT308 for TSA amplified immunofluorescence, as in (B) Supplementary Figure 3: Optimization of TSA amplification in MCF7 cells using
TSA amplified immunofluorescence assay. Supplementary Figure 3: Optimization of TSA amplification in MCF7 cells using
TSA amplified immunofluorescence assay. MCF7 cells were stimulated with EGF (100ng/ml) for 5 min. Confocal images show
the localization of endogenous panAkt (green, white arrow) and pT308 (red, white
arrow). Labeled Fab fragments were used as secondary antibodies: anti-mouse Fab-
ORG488 and anti-rabbit Fab-HRP/TSA-ALX594. The TSA amplification reaction
was allowed to develop for 5, 15, 20 or 30 min as indicated. The bottom panel shows
the control, with no primary antibody included. Cells were counter stained with DAPI
(blue) for DNA. Supplementary Figure 4: Quantification of endogenous pAkt (pT308) using
whole IgG secondary antibodies in fixed FFPE human breast tumor by amplified
FRET assay. (A) Intensity images and lifetime maps (scales are relative and in pseudo-color) of
donor (panAkt) and donor+acceptor (panAkt+pT308) for FFPE human breast tumor
tissues. panAkt was detected using an anti-mouse IgG(whole)-ORG488 and pT308
using a secondary anti-rabbit IgG(whole)-HRP and TSA-ALX594. Box and whiskers
plot shows quantification of FRET efficiency of 3 independent experiments, each
experiment consisting of measurements from at least 10 different regions of the same 2 2 pAkt is a marker for poor prognosis in breast cancer tissue section. (B) The same tissue sections prepared for (A) were imaged by confocal
microscopy. Supplementary Figure 5: Molecular heterogeneity of Akt activation in human
colon TMA revealed by amplified FRET. Supplementary Figure 5: Molecular heterogeneity of Akt activation in human
colon TMA revealed by amplified FRET. (A) H&E staining of TMAs prepared from colon tumor biopsies obtained from 7
patients. For each patient, 4 cores (circles 1 to 4) were selected from different regions
within each biopsy. In total 28 tumor cores (4 x 7 patients) were spotted to make the
TMA. The lower image shows an expanded view of one core divided into 4 sectors
for further analysis. The panels on the right show 3 different magnifications of sector
4. Intensity images and lifetime maps (scales are relative and in pseudo-color) of the
TMAs stained with (B) donor alone
(panAkt) or (C) donor+acceptor
(panAkt+pT308), followed by TSA amplification. TMAs were mapped and images
automatically acquired using our FRET/FLIM platform. (D) For each patient, the
maximum FRET efficiency of each core is shown; (inset) for each patient the mean
FRET efficiency for the 4 cores is shown. Bars represent mean. Supplementary Figure 6: Distribution of Akt activation revealed by amplified
FRET for all breast carcinoma patients (ER-/ER+). Supplementary Figure 6: Distribution of Akt activation revealed by amplified
FRET for all breast carcinoma patients (ER-/ER+). (A) Mean FRET efficiency for the patients grouped by ER status (***, p<0.0001). (B)
Mean intensity ratio for the patients grouped by ER status (ns, not significant). (C)
Population distribution of patients according to FRET quartiles, grouped by ER status 3 pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer pAkt is a marker for poor prognosis in breast cancer (black, high FRET quartile; light gray, low FRET quartile). (D) FRET efficiencies are
shown as box and whiskers plots representing the age distribution of patients
according to FRET tertiles (ns, not significant). Supplementary Figure 7: Quantification of endogenous pAkt (pT308) in fixed
FFPE human normal and tumor breast tissue using amplified FRET. Supplementary Figure 7: Quantification of endogenous pAkt (pT308) in fixed
FFPE human normal and tumor breast tissue using amplified FRET. (A)
Intensity images and lifetime maps (scales are relative and in pseudo-color) of
FFPE human breast tissues from two different normal (normal 1, normal 2) tissues
and one tumor tissue labeled with donor alone (panAkt) or donor+acceptor
(panAkt+pT308). FRET efficiencies are shown as box and whiskers plots
representing the mean ±SEM for at least 10 different regions from the same tissue
section (****, p<0.0001). The increased FRET efficiency represents the
phosphorylation status of endogenous Akt and shows the differential activation of
pT308 in normal and tumor tissues. 4 4
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Design para Acessibilidade e Inclusão: Design Positivo e DOP no desenvolvimento de projetos em saúde.
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Blucher Design Proceedings
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Keywords: Positive Design, Health, Accessibility, Inclusion. RESUMO Design e Psicologia sempre caminharam juntos, ambos com mesmo objetivo
de proporcionar bem-estar ao indivíduo; no caso do design, por meio de
soluções às necessidades e desejos do homem e no caso da psicologia por
meio de tratamento de ‘doenças’ ou situações relacionados a mente. Nesse
sentido buscamos firmar a compreensão de bem-estar relacionado à saúde,
como já o fez a Psicologia Positiva ao adotá-la como último elemento do
modelo PERMAH que é utilizado como estratégia para mensurar o índice de
bem-estar de uma pessoa. Dessa forma, o presente artigo propõe investigar a
Psicologia Positiva e o Design Positivo, aplicado a projetos na área da saúde
pela perspectiva do DOP – Design Orientado para Possibilidades – com atenção
para as premissas de acessibilidade e inclusão, evidenciadas como
particularidades a serem adotadas no desenvolvimento de qualquer projeto de
design. Palavras-chave: Design Positivo, Saúde, Acessibilidade, Inclusão. Design para Acessibilidade e Inclusão: Design Positivo e
DOP no desenvolvimento de projetos em saúde. Design for Accessibility and Inclusion: Positive Design and DOP in the
development of health projects. 1. Introdução A relação entre Design e Psicologia aponta na direção das primeiras instituições para formação
de desenhistas industriais, a exemplo das escolas Bauhaus e Ulm, que sofreram grande
influência da Psicologia por meio da Teoria da Gestalt. Nesse contexto, há evidências da
influência da psicologia no campo do Design e do Design através de elementos da psicologia
em projetos na área da saúde, o que podemos constatar ao analisarmos alguns projetos
destacados na presente pesquisa. Projetos na área da saúde, por sua própria natureza, buscam atender necessidades e requisitos
de acessibilidade e inclusão, contribuindo diretamente para o desenvolvimento de políticas
públicas. De frente a essa posição o designer pode ver-se provocado a promover e difundir
propostas metodológicas e possíveis soluções que atendam demandas na área da saúde se
valendo de sua capacidade de interpretação e criatividade. Desse modo, o designer se posiciona como um possível agente de transformação social, por
estender sua atuação a relações com outras áreas propondo projetos com implicações tão
especificas e importantes, no que tange ao bem-estar individual com consequências sociais
significativas. Na virada dos anos 2000 surge a Psicologia Positiva com a proposta de expandir o foco das
pesquisas e práticas clínicas da área da psicologia para além do diagnóstico e tratamento de
psicopatologias, olhando também para a construção de qualidades positivas. (MANDELLI et al,
2019, p.41). A atividade do design, segundo Desmet e Hassenzahl (2012, p.1.2) é entendida como uma
atividade centrada na remoção de problemas (ou seja, para tornar algo mais fácil, mais limpo,
mais barato, mais seguro ou mais pequeno), muitas vezes motivada por discrepâncias muito
concretas entre a forma atual e uma forma aparentemente ideal de fazer algo. A aspiração é
tornar o mundo um lugar melhor através da resolução dos seus problemas. [...] A concepção
orientada para problemas centra-se na "cura de doenças", ou seja, na eliminação de
problemas predominantes, em vez de se concentrar diretamente naquilo que nos faz felizes. 1. Introdução Para a Psicologia Positiva e o Design Positivo abordagens tradicionais, a exemplo do design
como solução de problemas são limitantes e não geram experiências, diferente do que propõe
o Design Positivo pela abordagem do Design Orientado para Possibilidade (DOP) que busca
contribuir para geração de experiências positivas e melhor qualidade de vida das pessoas Portanto, este artigo propõe uma reflexão entre Psicologia e Design, com foco no Design
Positivo pela abordagem DOP e as possibilidades de aplicações dessa abordagem em projetos
na área da saúde preconizando os princípios da acessibilidade e de inclusão, com o objetivo de
contribuir para geração de experiências e o bem-estar dos envolvidos. Abstract Design and Psychology have always gone together, both with the same goal of
providing well-being to the individual, in the case of design, through solutions
to the needs and desires of man, and in the case of psychology through the
treatment of 'diseases' or situations related to the mind. In this sense, we seek
to establish the understanding of well-being related to health, as Positive
Psychology has already done by adopting it as the last element of the PERMAH
model, which is used as a strategy to measure a person's well-being index. Thus, this article proposes to investigate Positive Psychology and Positive
Design, applied to projects in the health area from the perspective of PDO -
Possibility-Oriented Design - with attention to the premises of accessibility and
inclusion, evidenced as particularities to be adopted in the development of any
design project. Keywords: Positive Design, Health, Accessibility, Inclusion. 3. Psicologia Positiva Em 1998 a psicologia, na pessoa do Dr Martin Seligman (2012), à época presidente da
Associação Americana de Psicologia, sofria a intervenção de um modelo não convencional de
atenção à saúde dos pacientes, a saber, a Psicologia Positiva, defendida e justificada por ele
como ‘um movimento científico e profissional’, em seu livro FLORESCER: Uma nova
compreensão sobre a natureza da felicidade e do bem-estar. (SELIGMAN, 2012, p.9) Seligman é considerado, precursor desse movimento, que em um primeiro momento,
contrariando os métodos tradicionais da Psicologia, abordava a temática da felicidade como
objetivo principal, o que se tornaria a primeira teoria da Psicologia Positiva: a Felicidade
Autêntica. Seligman (2012, p.9) afirma que o objetivo da Psicologia, em seus moldes tradicionais era
aliviar o sofrimento e eliminar as condições debilitantes da vida, de acordo com o autor, um
dos objetivos é romper com o Monismo1 Aristotélico de que: [...] todo conhecimento e todo trabalho visa a algum bem, quais
afirmamos ser os objetivos da ciência política e qual é o mais alto de
todos os bens que se podem alcançar pela ação. Verbalmente, quase
todos estão de acordo, pois tanto o vulgo como os homens de cultura
superior dizem ser esse fim a felicidade e identificam o bem viver e o
bem agir como o ser feliz. Diferem, porém, quanto ao que seja a
felicidade, [...]. (ARISTOTELES, 322 a.C.) Para Seligman, (2012, p.13), o objetivo dessa ciência é explorar aquilo que faz a vida valer a
pena e produzir condições para isso, afirmando que, o intuito de compreender o bem-estar e
produzir condições capacitadoras de vida de modo algum se iguala ao de compreender o
sofrimento e desfazer as condições debilitantes da vida, proposta da psicologia tradicional. No ano de 2002, Seligman estabelece a Teoria da Felicidade Autêntica e em 2005, durante
disciplina ministrada por ele na Universidade Huntsman Hall, Filadélfia, EUA, ao abordar a
Teoria da Felicidade Autêntica, questionado por sua aluna Senia Maymin, o autor repensa sua
primeira teoria e sugere a Teoria do Bem-estar, concluindo que a felicidade não poderia ser o
objetivo da psicologia positiva. (SELIGMAN, 2012, p.14) A teoria do Bem-estar, proposta por Seligman (2012, p.16), é sustentada a partir da análise de
cinco elementos que se afastam do monismo da felicidade - emoção positiva, engajamento,
sentido, relacionamentos positivos e realização. 2. Referencial Teórico Para o desenvolvimento da pesquisa adotamos para discorrer sobre Psicologia Positiva,
SELIGMAN (2012). Para abordamos Design, definições e finalidade, utilizaremos como
literatura (CARDOSO (2008); MORAES, 2010; VERGANTI (2012), MANDELLI et al (2019). Design
Positivo será discutido a partir de DESMET (2013); POLHMEYER (2013); CAETANO e VAN DER
LINDEM (2018), PIMENTEL (2020); ROSA E TONETTO (2020); COTRIN (2021), e por fim, sobre Design e Saúde FREIRE (2016), BRITO (2018); ULBRICHT et al (2017), AMPHILÓQUIO E SOBRAL
(2018). Design e Saúde FREIRE (2016), BRITO (2018); ULBRICHT et al (2017), AMPHILÓQUIO E SOBRAL
(2018). 3. Psicologia Positiva E ainda por se tratar de uma teoria de livre
escolha cujos elementos possuem três importantes propriedades: contribuir para a formação
do bem-estar; a busca pelo elemento em si e não por satisfação pessoal; e, deve ser definido e
mensurado independente dos outros elementos. Portanto, o objetivo da Psicologia Positiva é o Florescimento Humano, principal critério para a
avaliação do Bem-Estar. Segundo Seligman: A teoria da felicidade autêntica é uma tentativa de explicar uma coisa
real - a felicidade -, definida pela satisfação com a vida, (...). A teoria 1 [Filosofia] Sistema filosófico segundo o qual a realidade (tudo o que existe) se reduz somente em um
único princípio, estando os seres condicionados a ele. do bem-estar nega que o tema da psicologia positiva seja uma coisa
real; ele é, antes, um construto - o bem-estar -, que por sua vez tem
diversos elementos mensuráveis, cada um deles uma coisa real e
cada um deles contribuindo para formar o bem-estar, mas nenhum
deles o definindo. (SELIGMAN, 2012, p.15) do bem-estar nega que o tema da psicologia positiva seja uma coisa
real; ele é, antes, um construto - o bem-estar -, que por sua vez tem
diversos elementos mensuráveis, cada um deles uma coisa real e
cada um deles contribuindo para formar o bem-estar, mas nenhum
deles o definindo. (SELIGMAN, 2012, p.15) A partir de então, a Psicologia Positiva passa a aplicar diversos elementos no desenvolvimento
de sua própria abordagem no tratamento dos pacientes assistidos por essa alternativa,
contrariando os métodos tradicionais com foco no tratamento da ‘doença’ ou, na linguagem
do design, na solução de problemas. Portanto, a Psicologia Positiva, pelos moldes da Teoria do bem-estar se propõe não apenas a
romper com o Monismo de Aristóteles, bem como contribuir para o bem-estar das pessoas e
melhor qualidade de vida. 3 Missão DIOPD - https://diopd.org/about-us/mission/ 03 de março de 2022. – Interpretação livre 3.1 Design Positivo Segundo Caetano e Van Der Lindem (2018, p.56), o debate do papel do bem-estar no Design se
inicia com Desmet (2011), ao propor que o bem-estar individual dos usuários poderia ser
melhorado através do uso de produtos sugerindo que, os produtos poderiam dar suporte em
atividades que contribuíssem para o bem-estar dos usuários através do desfrute de prazeres,
favorecimento de conexões sociais e ajuda no seu crescimento pessoal. No entanto, constatou
que os produtos não eram a fonte direta do bem-estar dos participantes de sua pesquisa, mas
sim davam suporte em atividades que promovessem o seu bem-estar. Ainda de acordo com Pohlmeyer (2013, 540-541), em anos anteriores a Assembleia Geral das
Nações Unidas, sobre temática da felicidade para políticas públicas, no ano de 2011, notou-se,
um interesse crescente acerca da ciência da felicidade com ênfase na Psicologia Positiva;
porém, pouco foi explorado, por parte do design, no tocante ao seu potencial, relevância e
aplicabilidade, e das percepções e resultados da referida teoria para criar e melhorar produtos
e serviços para o bem-estar subjetivo e florescimento humano Nesse contexto, Jimenez, et al (2015, p.607) afirma que: O designer ainda dá pouca importância a Psicologia Positiva como
atributo fundamental para o desenvolvimento de sua atividade,
ainda há alguns equívocos por parte dos designers, como exemplo,
muitos consideram que o designer precisa ‘apenas’ de ferramentas e
métodos, que é necessário considerar mudanças, tendências e
diferenças sociais (ignorando as suas origens) e se limitam apenas a
‘conversar’ com potenciais usuários, não se aprofundando no que as
pessoas sentem e porque sentem, nem em que características
despertam tais sentimentos. (Idem) O Design Positivo parte da abordagem do Design Orientado para Possibilidades, contrariando a
abordagem tradicional do Design concentrada na solução do problema, propondo a geração
de experiências e o bem-estar. De acordo com Cotrin (2021, p.45) enquanto alternativa a essa abordagem tradicional,
diversos pesquisadores, entre eles, Jordan (2000), Desmet (2002,2007), Norman (2004),
McCarthy & Wright (2004), Hassenzahl (2010), Gaver (2002), Dunne (2006) e Zimmerman
(2009), têm impulsionado essa corrente de pensamento: Para os autores tanto da abordagem pelo viés do problema, quanto
pelo viés da possibilidade, buscam a modificação do estado atual
para um estado futuro. Ambas reconhecem a existência de um
problema/questão da pesquisa, porém a diferença está no tipo de
reação ao problema. 3.1 Design Positivo A ONU – Organização das Nações Unidas – em 2011, convocada pelo Butão, país que adotou o
Felicidade Interna Bruta (FIB) como índice de avaliação para medir o desenvolvimento de sua
população (GOMES, 2013, p.77-73), realizou a Assembleia Geral das Nações Unidas,
convocando os 193 países participantes a incluir a felicidade e o bem-estar na promoção de
políticas públicas. No mesmo ano surge o Delf Institute of Positive Design (DIOPD) formado por pesquisadores,
designers e psicólogos, que compreendem a responsabilidade de gerar conhecimento que
permitam aos designers formular estratégias eficazes de forma a contribuir para a felicidade
das pessoas. Esse conhecimento não deve apenas ajudá-los em suas tentativas de projetar
deliberadamente relações significativas entre produto e usuário, mas, em última análise,
projetar produtos que contribuam para uma sociedade saudável, para tornar o mundo um
lugar melhor2. Em 2013, os pesquisadores Pieter Desmet e Anna Pohlmeyer propuseram o Design Positivo
com objetivo principal de responder “Como o design pode contribuir para o bem-estar dos
usuários? ”, sugerindo que futuros estudos no design deveriam fomentar atividades que
apoiassem a busca de uma vida mais gratificante para os usuários de produtos e
consequentemente favorecessem seu bem-estar. No mesmo ano, Pohlmeyer propôs o
Manifesto do Design Positivo, com propósito de englobar estudos, pesquisas e intenções em
Design Segundo os pesquisadores, Design Positivo é um termo para todas as formas de Design,
pesquisa de Design e intenção de Design que prestam atenção explícita ao impacto do Design
no bem-estar subjetivo de indivíduos e comunidades, que cria possibilidades, promove
florescimento humano, permite atividades significativas, abraça a complexidade humana,
aceita a responsabilidade e apoia todas as partes interessadas3. 3 Missão DIOPD - https://diopd.org/about-us/mission/ 03 de março de 2022. – Interpretação livre Segundo Caetano e Van Der Lindem (2018, p.56), o debate do papel do bem-estar no Design se
inicia com Desmet (2011), ao propor que o bem-estar individual dos usuários poderia ser
melhorado através do uso de produtos sugerindo que, os produtos poderiam dar suporte em
atividades que contribuíssem para o bem-estar dos usuários através do desfrute de prazeres,
favorecimento de conexões sociais e ajuda no seu crescimento pessoal. No entanto, constatou
que os produtos não eram a fonte direta do bem-estar dos participantes de sua pesquisa, mas
sim davam suporte em atividades que promovessem o seu bem-estar. 3.1 Design Positivo Enquanto o design orientado para solução de
problemas foca no presente, o design orientado para possibilidades
foca no futuro, nas potencialidades; o primeiro contém um caráter de
busca de uma solução, sendo ela singular e excludente, enquanto o
segundo lida com multiplicidade de natureza expandida e inclusiva. (COTRIN, 2021, p.45.46) É importante notarmos que, a fala de Cotrin (2021), motivada por importantes pesquisadores -
psicólogos e designers - argumentam a favor do compromisso do design com o
desenvolvimento de abordagens que rompam com os métodos tradicionais do design, que
tem por fundamento a solução de problemas, semelhante ao que propõe a Psicologia Positiva em relação aos métodos da Psicologia Tradicional ao tratar o paciente simplesmente pela cura
da doença. em relação aos métodos da Psicologia Tradicional ao tratar o paciente simplesmente pela cura
da doença. em relação aos métodos da Psicologia Tradicional ao tratar o paciente simplesmente pela cura
da doença. Portanto, o Design Positivo provoca pesquisadores e designers a pensarmos desenvolvimento
de projetos na área da saúde pela perspectiva do Design Orientado para Possibilidades, de
forma a contribuirmos para o maior bem-estar das pessoas, por meio da geração de
experiências positivas e não apenas da solução de problemas. Anna Pohlmeyer (2018) observou que, segundo a Organização Mundial da Saúde, a saúde é
mais do que a ausência de doença – é também uma questão de bem-estar mental e social e
que a saúde física vem sendo abordada pelo design por décadas e mais recentemente o design
para o bem-estar psicológico mereceu atenção no campo do design. Tal observação foi
apresentada em palestra realizada em 1º de junho de 2018 na Conferência Saúde Apoiada pela
Tecnologia, organizada pelo Centro de Pesquisa em Health & Wellbeing da Universidade de
Twente, localizada em Enschede, nos Países Baixos, com o objetivo de discutir o progresso e
desenvolvimento de intervenções em saúde e introduzir novos métodos e modelos para
mudança de comportamento usando tecnologias de monitoramento. A pesquisadora e psicóloga também abordou as oportunidades para o design, a exemplo de
tecnologias de intervenções comportamentais na forma de aplicativos pessoais que apoiam
pessoas com treinamentos e serviços objetivando melhorar a saúde e bem-estar. 3.1 Design Positivo Porém, a
pesquisadora afirma que, apesar de haver evidências cientificas sobre a eficácia dessas
intervenções, estas ainda enfrentam grandes desafios, a exemplo do alcance limitado e que o
Design pode ter impactos no bem-estar das pessoas através de rotinas já existentes no dia a
dia4. 3.2 Design Orientado para Possibilidade Como ocorreu com a Psicologia Positiva surgida na década de 1990, com uma nova abordagem
para assuntos tratados pelos métodos tradicionais, o Design Positivo também emerge, a partir
do Design Orientado para Possibilidades, como alternativa aos métodos tradicionais do design. Enquanto a Psicologia Positiva propõe uma nova abordagem para o tratamento de pessoas,
como alternativa ao método tradicional, o Design Positivo, por meio da abordagem do Design
Orientado para Possibilidade propõe ao Design tradicional, uma proposta de intervenção em
Design por meio da geração de soluções em projetos cujo foco não seja o problema em si, mas
o usuário, de maneira a promover experiências positivas para este. O Design Positivo surge como, uma alternativa à abordagem do design orientado para problemas, e
pode contribuir criando experiências positivas (a vida prazerosa/hedonismo), mas também
estimulando a consciência das pessoas, das suas capacidades, desafiando ou inspirando o usuário a
agir ou pensar de uma forma diferente (a boa vida/eudaimonia). DESMET E HASSENZAHL (2012, p.1-
11) Desmet (2010, p.2) descreve o design orientado a problemas como mera tentativa de “manter
os demônios dormindo”, uma metáfora em alusão às preocupações, valores e necessidades
das pessoas, despertadas apenas quando a situação representa uma ameaça à sua realização. O design precisa de um ponto de partida, uma ideia, uma semente
para nutrir e crescer. Enquanto uma abordagem orientada para o 4 https://diopd.org/brave-new-tech-at-health-supported-by-technology/ 06 de março de 2022 problema toma um problema como um começo, uma abordagem
orientada para a possibilidade procura uma possibilidade. É
importante ressaltar que essa possibilidade deve estar enraizada em
nosso conhecimento da felicidade, da prática humana e nas
necessidades humanas. Muito facilmente, uma tecnologia por si só,
pode ser entendida como uma possibilidade. (DESMET &
HASSENZAHL, 2012, p.11) Segundo Jimenez et al, (2014, p.607), falamos de "um problema" quando alguém quer alcançar
um objetivo e os meios para o fazer não são imediatamente óbvios. A resolução de problemas
é o processo de pensamento, em que aqueles meios são procurados intencionalmente. Há
numerosos exemplos que ilustram esta visão tradicional orientada para o problema do design. [...]. Esta forma tradicional de resolução de problemas é relevante uma vez que aumenta a
qualidade e eficácia dos produtos e serviços que todos nós usamos, procura minimizar
situações problemáticas que pode ameaçar o nosso bem-estar, e geralmente torna nosso viver
'mais fácil' e 'melhor’. 3.2 Design Orientado para Possibilidade No entanto, neutralizando o negativo ao resolver os nossos problemas
cotidianos não significa necessariamente proporcionando uma experiência positiva e valiosa
(Desmet e Hassenzahl, 2012). Focando o lado positivo da solução espectro com uma
abordagem orientada para as possibilidades promete um uma nova perspectiva sobre o papel
do design. A visão orientada para a possibilidade sobre o design foi recentemente proposta na
comunidade de pesquisa em design. Nas palavras de Hekkert e van Dijk (2011, p. 120), o
trabalho de um designer é procurar possibilidades e futuros possíveis, em vez de
simplesmente resolver problemas atuais. O design, portanto, pode contribuir para o bem-estar ao criar e mediar experiências positivas
ao estimular a consciência das pessoas sobre suas capacidades e, pela perspectiva do DOP,
pode criar ou apoiar novas possibilidades, considerando que o processo da abordagem do
design para possibilidade não é tão evidente quanto a abordagem tradicional, apresentando
desafios interessantes à pesquisa, à atuação do designer e ao ensino em design. 4. Design e Saúde Os tópicos anteriores trouxeram propostas de conceitos e teorias desenvolvidas em torno do
Design Positivo e da Psicologia Positiva. Tais referências interessam na medida em que,
possibilitam abrir espaço de diálogo para a relação entre Design e Saúde, vislumbrado novos
caminhos para que o designer possa expandir sua atuação em projetos voltados para a área da
saúde, com foco em acessibilidade e inclusão, favorecendo e contribuindo para o
desenvolvimento de políticas públicas. O Design é uma área de atuação profissional de grande complexidade, com características que
possibilitam ao profissional dessa área, o designer, propor soluções para demandas das mais
diversas áreas. A definição do design, segundo Verganti (2012, p.22), é algo fluido e escorregadio, por haver várias
discussões em torno do seu significado e, por consequência, a falta de convergência. O design compreendido pela definição funcionalista do século XX de que ‘a forma segue a função’, conceito
aplicado pelo arquiteto americano Louis Sullivan (18965) é considerado por Verganti como limitante. compreendido pela definição funcionalista do século XX de que ‘a forma segue a função’, conceito
aplicado pelo arquiteto americano Louis Sullivan (18965) é considerado por Verganti como limitante. compreendido pela definição funcionalista do século XX de que ‘a forma segue a função’, conceito
aplicado pelo arquiteto americano Louis Sullivan (18965) é considerado por Verganti como limitante. No entanto, o autor sugere como definição, a proposta por Thomas Maldonado (1963), adotada
pela International Council of Societies of Industrial Design (Conselho Internacional das
Sociedade de Desenho Industrial) em 1969 e que passou por alterações, sendo inserido o
design de serviços, processos e sistemas: Design é a atividade criativa cujo objetivo é estabelecer as
multifacetadas qualidades de objetos, processos, serviços e seus
sistemas no ciclo de vida completo. O design é, portanto, fator
central para humanizar as inovações tecnológicas e um fator crucial
de intercâmbio cultural e econômico. (VERGANTI, 2012, p.24) Design é a atividade criativa cujo objetivo é estabelecer as
multifacetadas qualidades de objetos, processos, serviços e seus
sistemas no ciclo de vida completo. O design é, portanto, fator
central para humanizar as inovações tecnológicas e um fator crucial
de intercâmbio cultural e econômico. (VERGANTI, 2012, p.24) Segundo Verganti (2012, p. 5 A frase "a forma segue a função" foi cunhada pelo arquiteto Louis H. Sullivan em seu ensaio de 1896
"The Tall Office Building Artistically Considered". "The Tall Office Building Artistically Considered". 4. Design e Saúde Se todos adquirirem alguma consciência do tamanho e do intricado das relações que
regem o mundo hoje, será possível caminhar coletivamente em direção a um objetivo, seja
qual for. Do ponto de vista histórico o autor afirma que: O grande trabalho do design tem sido ajustar conexões entres coisas
que antes eram desconexas, hoje entendido por projetar interfaces. Trata-se, contudo, de um processo bem maior e mais abrangente do
que imagina o projetista sentado à sua estação de trabalho. A parte
de cada um é entender a sua parte no todo. (Idem). Em relação a Cardoso (2013), podemos compreender que, de fato, o designer possui um papel
significativo no tocante ao desenvolvimento de soluções que, atendam às necessidades do
mundo, enquanto sistema complexo, e é nessa perspectiva que esse campo tende a colaborar
cada vez mais com o desenvolvimento de outras áreas, a exemplo de projetos que possibilitem
sua intervenção, inclusive propondo novas metodologias, abordagens e reflexões. Portanto, o design não apenas pode propor novas abordagens metodológicas, a exemplo do
Design Orientado para Possibilidade, como sugere o Design Positivo, como também prever sua
atuação mais abrangente, no que corresponde a busca por novas possibilidades de sua
atuação. Já a relação do design com a área da saúde não é necessariamente direta, mas pode ser
sentida em projetos que não foram pensados inicialmente para saúde, mas possuem
implicações e resultados positivos ou negativos, gerando de fato algum tipo de experiência ao
usuário, como veremos a seguir em alguns exemplos. Verganti (2012, p.63) cita como referência o projeto do vídeo game Wii da fabricante Nitendo,
lançado em 2006, considerado um projeto de produto revolucionário, pelo fato de que, no
contexto de sua abordagem, este propôs uma mudança radical em significado, quando
comparado aos seus concorrentes, passando de uma imersão passiva virtual acessível somente
para poucos para um entretenimento que inclui atividade física real para todos,
posteriormente, vindo a ser utilizado na prática de atividades físicas, como terapia e até
mesmo em processo de reabilitação de pessoas acometidas pelo Acidente Vascular Encefálico
(AVE), tornando-se elemento de socialização. É importante considerarmos que o design, de forma direta ou indireta, possui forte influência
em aspectos relacionados ao bem-estar e, consequentemente, à saúde do indivíduo, já que
estes dois elementos estão intrinsicamente relacionados. 4. Design e Saúde 37), o design engloba todas as profissões criativas que modificam
seu ambiente: engenharia, medicina, administração, arquitetura e pintura estão preocupados
não apenas com o que é essencial, mas com todas as possibilidades – não como as coisas são,
mas como podem vir a ser, resumindo, com o design. Em sintonia e para além do que aponta Verganti (2012) “o design pela sua capacidade
multidisciplinar e transversal, fornece rápidas respostas por meio de produtos, imagens e
novas possibilidades de interação”, de acordo com MORAES (2010, p. 10). Nesse âmbito,
segundo esse autor, o design como uma disciplina transversal (e mesmo ‘atravessável’), ao
aceitar e propor interações multidisciplinares, amplia ainda o seu diálogo com disciplinas
tecnológicas, econômicas e humanas, bem como com as do âmbito da gestão, da semiótica e
da comunicação. É nessa diversidade presente na atividade do designer que compreendemos a
responsabilidade, enquanto projetistas, ao propormos soluções para demandas de projetos
existentes nas mais diversas áreas, inclusive na área da saúde, que exige por parte deste uma
percepção maior de consequências futuras que um projeto mal resolvido possa provocar ou os
tipos de experiências que um projeto nessa área, geralmente para atender uma necessidade
ou demanda especifica, possa causar. Ao trabalhar o Design sob a ótica transversal e considerá-lo um campo político, que possui
responsabilidade social e desenvolve soluções para as necessidades dos usuários, observa-se a
importância do diálogo entre várias áreas, a fim de suprir as carências dos impactados. (PIMENTEL et al, 2020, p.178) CARDOSO (2008, p.225), ao referir-se ao Design e a importância de examinar a paisagem
material que nos cerca, afirma que nos deparamos com problemas de Design crônicos em
áreas como transportes, saúde, equipamentos urbanos e uma infinidade de outras instancias
do cotidiano. Para este autor, mesmo fora do âmbito dos serviços públicos, em um setor privado voltado por definição para questões de competitividade e dinamismo, percebe-se um
desconcerto frequente entre as preocupações da profissão e aquelas da própria sociedade. privado voltado por definição para questões de competitividade e dinamismo, percebe-se um
desconcerto frequente entre as preocupações da profissão e aquelas da própria sociedade. privado voltado por definição para questões de competitividade e dinamismo, percebe-se um
desconcerto frequente entre as preocupações da profissão e aquelas da própria sociedade. CARDOSO (2013, p.22) considera que, reconhecer a complexidade do sistema já é um grande
avanço. 4. Design e Saúde O designer pode não ter pensado nas
implicações da solução ou resposta a que se chegou para determinada demanda, necessidade
ou oportunidade, mas possivelmente a aplicação e posterior utilização dessa resposta dada
pelo design terá consequências na vida de determinado usuário ou conjunto de usuários,
sendo possível e cabível ao designer, nesse contexto, prever o futuro ao analisar o que o uso
de determinado design pode acarretar a comunidade. Um exemplo do impacto negativo do design na área da saúde, é o que ocorreu em 2003 com o
modelo de carro Fox, da Volkswagen. Considerado resultado de um projeto inovador, um
modelo compacto, estiloso e mais espaçoso que seus concorrentes, porém em 2004 começou a apresentar um problema recorrente que teve por consequência, diversos casos registrad a apresentar um problema recorrente que teve por consequência, diversos casos registrados
de pessoas que tiveram dedos decepados pelo sistema de rebatimento do banco traseiro6. a apresentar um problema recorrente que teve por consequência, diversos casos registrados
de pessoas que tiveram dedos decepados pelo sistema de rebatimento do banco traseiro6. O Wii, de forma positiva, e o Modelo Fox da Volkswagen, de forma negativa, nos advertem a
respeito do impacto do design na sociedade e a grande responsabilidade no desenvolvimento
de projetos independentes de qual área estejamos atuando. Porém, é importante notarmos que, só recentemente, a própria Psicologia Positiva, teria
inserido a Saúde como novo elemento no Modelo PERMA, proposto inicialmente por
Seligman, sendo Emiliya Zhivotovskaya, portanto, considerada responsável por, expandir o
modelo PERMA, passando a ser PERMAH (Health) ou PERMA – V (Vitality). O acrônimo7 PERMAH ou PERMA-V significa: P = Positive Emotions (Emoções Positivas); E =
Engajement (Engajamento); R = Relashionships (Relacionamentos); M = Meaning (Significado);
A = Accomplishment (Realização); H = health (Saúde) ou V = Vitality (Vitalidade). O Conceito de saúde mais aceito foi desenvolvido em 1947 e definido pela Organização Mundial da
Saúde (OMS) como "um estado de completa saúde física, mental, e bem-estar social e não apenas a
ausência de doença ou enfermidade". Este conceito da OMS está, em parte, alinhado com o modelo
holístico, e reforça as conotações positivas de saúde. 4. Design e Saúde (PAHO, 2018, p.4) Freire (2016, p.5) quanto ao Design voltado para projetos em saúde, afirma ser a principal
contribuição do Design reduzir a complexidade do conjunto de atividades que caracterizam tal
processo, sejam criando plataformas capazes de suportar essas interações, seja auxiliando o
usuário como ele pode estar utilizando determinado sistema. Segundo a autora: [...] as experiências dos usuários não podem ser projetadas. Por
consequência, os designers podem apenas analisar como elas se formam
e, partindo disso, criar alternativas para os elementos que as definem. [...]
uma vez entendida a experiência, o papel do designer é conduzir o
processo de projeto de maneira a desenvolver interfaces, processos ou
interações que carreguem não apenas as experiências estéticas, mas
também os significados esperados pelos usuários nessa interação. (Ibidem, p.8) De acordo com Amphilóquio e Sobral (2018, p.171) o designer, enquanto agente de inovação
social pode também provocar rupturas significativas na cultura, inclusive criar novas
necessidades e comportamentos. O Design, no decorrer da história, sofreu importantes mudanças
que alteraram o seu modo de projetar e de entender a sociedade, a preocupação deixou de ser
apenas industrial e passou a ganhar novas modelagens, como o design participativo, o design
thinking, o design sustentável e o design centrado no humano, que eles consideram serem
aberturas significativas para a promoção de uma sociedade inclusiva. Segundo Brito (2018, p.23) o Design se insere na perspectiva da saúde como uma área do saber
capaz de contribuir na obtenção dos melhores resultados em prol da promoção da saúde a 6 https://jornaldocarro.estadao.com.br/fanaticos/defeitos-famosos-o-vw-fox-que-decepava-dedos/ 14
de fevereiro de 2022, 12h45 Tenho dúvida sobre essa fonte. 7 Diz-se de ou palavra formada pela inicial ou por mais de uma letra de cada um dos segmentos
sucessivos de uma locução, ou pela maioria dessas partes; acrossílabo; 7 Diz-se de ou palavra formada pela inicial ou por mais de uma letra de cada um dos segmentos
sucessivos de uma locução, ou pela maioria dessas partes; acrossílabo; partir de uma série de atuações, seja no campo do desenvolvimento de produtos,
comunicação, elementos gráficos, ambientes ou serviços. partir de uma série de atuações, seja no campo do desenvolvimento de produtos,
comunicação, elementos gráficos, ambientes ou serviços. partir de uma série de atuações, seja no campo do desenvolvimento de produtos,
comunicação, elementos gráficos, ambientes ou serviços. 4. Design e Saúde Ao desenvolver sua pesquisa em Design de Moda com o objetivo de minimizar medo e estresse
provocados pelo processo de tratamento de crianças com câncer, por meio de uma proposta lúdica,
Pimentel (2020) aproxima o design da área da saúde partindo de princípios essenciais ao design e a
saúde para compor estratégias apresentadas como resultados em sua pesquisa, a exemplo do Mapa
de Empatia e da Neurociência. A autora afirma que: A realidade da criança com câncer é múltipla e complexa. O contexto
que envolve o diagnóstico e o tratamento da doença ultrapassa os
limites farmacológicos e medicinais, [...]. As dificuldades se estendem
para além da doença do corpo: atingem as emoções, as relações de
afeto e aprendizagem e a forma de se relacionar com o mundo e com
o futuro. Esses obstáculos permeiam também a relação da criança
com o vestuário, trazendo novas necessidades. O campo do Design
de Moda, aliado à Neurociência e à Psicologia, abre possibilidades
para o vestuário, que pode tornar-se ferramenta de auxílio à criança
com câncer potencializando a minimização dos sofrimentos. (Idem,
p.179) Cotrin (2021, p.46) ao avaliar as contribuições do design positivo no tratamento de crianças
com Transtorno de Espectro Autista, afirma que, pensar um projeto do ponto de vista das
possibilidades para o desenvolvimento do potencial humano, pode contribuir para uma
perspectiva ampliada. Cotrin (2021, p.46) ao avaliar as contribuições do design positivo no tratamento de crianças
com Transtorno de Espectro Autista, afirma que, pensar um projeto do ponto de vista das
possibilidades para o desenvolvimento do potencial humano, pode contribuir para uma
perspectiva ampliada. Desmet e Hassenzahl (2012), acerca da abordagem do Design Orientado para Possibilidade,
afirma sobre o projeto da prótese de pernas cheetah-foot da empresa Össur: Tradicionalmente, as próteses são desenvolvidas dentro do "modelo
de descontração" da tecnologia: ter duas pernas humanas é visto
como a situação ideal, não as tendo como um "problema". Como
consequência, as próteses visam muitas vezes imitar completamente
a função e especialmente a aparência da situação ideal - pernas reais. Deixando de lado esta abordagem focada no problema, contudo,
permitiu a Össur desenvolver membros revolucionários de fibra de
carbono, Cheetah Flex-Foot, que não imitam pernas humanas e que
se tornaram famosos por atletas internacionais como Oscar Pistorius. Fonte: Google imagens (2021). Nesse sentido, o Design aplicado a projetos na área da saúde, por meio da abordagem DOP
propõe novas perspectivas para esse campo de atuação, com a possibilidade de revisão dessa
literatura, aperfeiçoando e intensificando o uso dessa metodologia como alternativa a
metodologia tradicional e, contribuindo com a sociedade e o desenvolvimento, também, de
novos modelos em políticas públicas. 8 Também conhecido como design sem barreiras e relacionado ao design universal e ao design inclusivo
(LIDWELL et al, 2010). 4. Design e Saúde (DESMET E HASSENZAHL, 2012, p.5) Os autores explicam que, em vez de entenderem a ausência de pernas como um problema a
resolver, os desenhadores utilizaram uma situação aparentemente problemática como uma
possibilidade de explorar material e tecnologia para criar um novo tipo de pernas. Figura 1 – Prótese
de pernas cheetah-
foot da empresa
Össur de pernas cheetah-
Össur de pernas cheetah-
Össur de pernas cheetah-
Össur Figura 1 – Prótese
foot da empresa Figura 1 – Prótese
foot da empresa Fonte: Google imagens (2021). 5. Acessibilidade e Inclusão Ao tratarmos de acessibilidade e inclusão não limitamos o tema à área da saúde, são muito mais
amplas estas definições e é preciso que seja pensado dessa maneira ao nos referirmos a estes
assuntos, a começar por não os distinguir, mas aproximá-los, com o objetivo de explorarmos ainda
mais a aplicação do design à saúde, que não se trata apenas dos aspectos físicos. Lidwell et al (2010, p.16) afirmam que objetos e ambientes devem ser projetados para serem
utilizados, sem modificação, pelo maior número de pessoas8, pelo princípio da acessibilidade. Segundo os autores, a história nos mostra a acessibilidade concentrada em acomodar usuários com
deficiências, porém, à medida que o conhecimento e a experiência com o design acessível
aumentaram, ficou cada vez mais claro que muitas “adaptações” obrigatórias poderiam ser
projetadas para beneficiar todos os usuários, sendo necessário compreender o design acessível a
partir de suas quatro características: Perceptibilidade – Obtida quando o design é percebido por
todos, independentemente de suas capacidades sensoriais; Operabilidade – Obtida quando todos
conseguem utilizar o design independentemente de suas capacidades físicas; Simplicidade – Obtida
quando todos conseguem compreender e utilizar o design sem dificuldade, independentemente dos
níveis de experiência, alfabetização e concentração; e, Condescendência – Obtida quando os designs
diminuem a ocorrência e as consequências dos erros. (LIDWELL et al, 2010, p.16) Cohen et al (2012, p.17) ao abordarem a acessibilidade relacionado a museus, afirmam que há uma
dimensão política da acessibilidade, que está estreitamente relacionada ao exercício da cidadania, a
qual, para contemplar adequadamente a pluralidade e a diversidade dos modos de ser e de estar no
mundo, que caracterizam o conjunto de cidadãos, implica a adoção de uma visão ampliada do
conceito de acessibilidade. Na fala dos autores, fica claro que, acessibilidade deve incluir todos, não apenas pessoas com
deficiências, porém devem ser pensadas à luz dessas pessoas, sendo possível a partir dessa
compreensão adentrarmos ao conceito de inclusão. No que trata do desenvolvimento de projetos, tanto no aspecto de acessibilidade quanto de
inclusão, talvez uma premissa básica sugerida por Caya e Neto (2016, p. 5. Acessibilidade e Inclusão 55-56), que deva ser
considerada e difundida, é o entendimento de dois conceitos: 1 - Adaptabilidade, que consiste na
propriedade de um produto/sistema mudar ou ser mudado a fim de se adequar ou trabalhar
melhor em alguma situação para algum propósito, ou seja, que permite que o usuário altere
explicitamente certas características do mesmo, para adequá-los às suas vontades e necessidades. 2- Adaptatividade, que consiste na propriedade que um produto/sistema tem de se adaptar ao
usuário, ou seja, apresenta uma capacidade para adaptação. O sistema se ‘adapta’ de acordo com
sua percepção do usuário, e o usuário não solicita alterações no sistema, diferente do que ocorre
com a adaptabilidade. (Ulbricht et al, 2009, p.34) Por fim, projetos em Design na área da saúde devem ter como premissas acessibilidade e inclusão. Estes projetos, como afirmam, Mandelli e Tonetto (2019, p.5) podem resultar em artefatos, serviços
ou até dinâmicas que despertem experiências afetivas especificas, emoções, estados de humor,
bem-estar subjetivo ou até mesmo empatia. 6. Considerações Finais Como principal abordagem metodológica, partimos do Design Orientado pela Possibilidade,
fundamentada em pesquisa bibliográfica e revisão literária acerca da relevância do Design
Positivo como alternativa à metodologia do design tradicional. Nesse sentido, a utilização
dessa metodologia busca possibilitar o desenvolvimento de projetos em Design que, atendam
à área da saúde proporcionando aos usuários bem-estar, principalmente, por meio de
experiências positivas com foco nas premissas de acessibilidade e inclusão. Ao discorrermos sobre a relação do design e sua aplicabilidade a projetos que visam atender às
necessidades na área da saúde, compreendemos que há grande potencial nessa relação e
grandes possibilidades de obtermos resultados cada vez mais mensuráveis e validadas que
possam ser úteis à expansão dessa literatura. Ao investigarmos a Psicologia Positiva e consequentemente o Design Positivo pela abordagem
DOP, concluímos que a relação entre Design e Psicologia é histórica e pode ser positiva para
ambos os campos de pesquisa, já que eles têm por objetivo proporcionar o alcance do bem-
estar humano e social. Por fim, outro importante aspecto nessa relação são os princípios de acessibilidade e inclusão
aqui analisados, porém de forma mais objetiva, ainda havendo muito a se investigar. Nesse
sentido, o design é pensado de forma mais ampla, além do conceito estabelecido como
característica fundamental do design da primeira modernidade, de caráter funcionalista,
proposto por Louis Sullivan, de que ‘a forma segue a função’, sugerindo a partir dessa
colocação, uma revisão na maneira como pensamos design, psicologia, saúde, bem-estar,
acessibilidade e inclusão. 7. Referências Bibliográficas AMPHILÓQUIO, Willian; SOBRAL, João Eduardo C. Design e sociedade: uma reflexão sobre
acessibilidade, interação e inclusão. HFD, v.7, n.13. Fev/jul. 2018, p.171. ARISTÓTELES, Ética à Nicômaco. Texto integral. Brasília, Editora Martin Claret, 2003, p.5. BRITO, Talita. Contribuições para o cuidado da doença de Alzheimer e outras demências. AMPHILÓQUIO, Willian; SOBRAL, João Eduardo C. Design e sociedade: uma reflexão sobre
acessibilidade, interação e inclusão. HFD, v.7, n.13. Fev/jul. 2018, p.171. AMPHILÓQUIO, Willian; SOBRAL, João Eduardo C. Design e sociedade: uma reflexão sobre
acessibilidade, interação e inclusão. HFD, v.7, n.13. Fev/jul. 2018, p.171. ARISTÓTELES, Ética à Nicômaco. Texto integral. Brasília, Editora Martin Claret, 2003, p.5. BRITO, Talita. Contribuições para o cuidado da doença de Alzheimer e outras demências. 2018, 115 p. Dissertação (Mestrado em Design) - Universidade de Brasília, 2018. CAETANO, Ulisses.; VAN DER LINDEN, Júlio. Além do Design Positivo. 2018, Cap. 3, p. 55-76. CARDOSO, Rafael. Uma Introdução a história do Design. São Paulo: Editora Edgard Blücher,
2008, p.225. CAETANO, Ulisses.; VAN DER LINDEN, Júlio. Além do Design Positivo. 2018, Cap. 3, p. 55-76. CARDOSO, Rafael. Uma Introdução a história do Design. São Paulo: Editora Edgard Blücher,
2008, p.225. CARDOSO, Rafael. Uma Introdução a história do Design. São Paulo: Editora Edgard Blücher,
2008, p.225. CARDOSO, Rafael. Design para um mundo complexo. São Paulo: Editora Cosac Naify, 2013. CASAIS, M., MUGGE, R. & DESMET, PMA. Using symbolic meaning as a means to design for
happiness: The development of a card set for designers. 2016, p.1. COHEN, Regina; DUARTE, Cristiane R. de S.; BRASILEIRO, Alice de Barros H. Cadernos
Museológicos: Acessibilidades a museus. Instituto Brasileiro de Museus. Brasília, 2012, p. 17. COTRIN, Michelle de A. Design e bem-estar: Contribuições do Design Positivo para crianças
dentro do Transtorno do Espectro Autista. 2021, 169 p. Tese (Doutorado em Design) - Escola
de Design da Universidade de Minas Gerais, Belo Horizonte. Delft Institute of Positive Design. Nossa Missão. DIOPD. Disponível em:
<https://diopd.org/about-us/mission/>. Acesso em: 22/06/2021. DESMET, Pieter & HEKKERT, Paul. Special Issue Editorial: Design & Emotion. International
Journal of Design, 2009, p.1. DESMET, Pieter & HASSENZAHL, Marc. Towards happiness: Possibility-driven design. 2012, p. 2.8. DESMET, Pieter. Design for Mood: Twenty Activity-Based Opportunities to Design for Mood
Regulation. International Journal of Design Vol. 9 Nº. 2, 2015, p. 0-19. FREIRE, Karine de M.; DAMAZIO, Vera M. Design para serviços: uma intervenção em uma
Unidade Básica de Saúde do Sistema Único de Saúde Brasileiro. Estudos em Design. 7. Referências Bibliográficas Revista
(online). Rio de Janeiro, v.24. n.2. 2016, p.5.8. GOMES, Paulo Sérgio F. O Butão na ordem mundial do reconhecimento da sua identidade
nacional à influência nas relações externas. 2013, 313 p. Tese (Doutorado em Teoria Jurídico-
Política e Relações Internacionais) – Universidade de Évora, Portugal, 2013. JIMENEZ, Simon; POHLMEYER, Anna; DESMET, Pieter; HUZEN, Gerjan. Learning from the
positive: A Structured Approach to Possibility-Oriented Design. Em J. Salamanca, P. Desmet,
A. Burbano, G. Ludden, J. Maya (Eds.). Proceedings of Colors of Care: The 9th International
Conference on Design & Emotion, 2014, 607-615. LIDWELL, Willian.; HOLDEN, Kritina.; BUTLER, Jill.; Princípios Universais do Design. 2010, p.16 MANDELLI, Roberta R.; TONETTO, Leandro M. Design para empatia: brinquedos e
brincadeiras como oportunidade para promover o desenvolvimento emocional. Porto Alegre. Design Culture Symposium, 2019, p.41. MANDELLI, Roberta R.; ROSA, Valentina M.; TONETTO, Leandro M. Da emoção à felicidade:
uma introdução ao bem-estar com foco no positive design. Rio de Janeiro. 2019, p.5. MORAES, Dijon De. Metaprojeto: O design do design. São Paulo: Blucher, 2010. P.4. Pan American Health Organization. Health Indicators. Conceptual and operational
considerations. Washington, D.C.: PAHO; 2018, p.4. PIMENTEL, Marina C. Design de moda e ludicidade: minimização do medo e estresse de
crianças em tratamento de câncer. Dissertação (Mestrado em Design) – Universidade
Anhembi Morumbi. São Paulo, 2020, p. 18. POHLMEYER, Anne. Positive Design: New Challenges, Opportunities, and Responsibilities for
Design. In: A. Marcus (Ed.): Design, User Experience, and Usability, Part III, LNCS 8014. Berlin,
Germany: Springer-Verlag, 2013, p. 540. ROSA, Valentina M.; TONETTO, Leandro M. Modelo Conceitual de Design para o Bem-estar
para pacientes pediátricos. Colóquio Internacional de Design. Edição, 2020, p.2. SELIGMAN, Martin E. P. Florescer: uma nova compreensão sobre a natureza da felicidade e
do bem-estar. Ed. Objetiva, 2012. O carro que decepava dedos. Disponivel em: <https://jornaldocarro.estadao.com.br/fanaticos/defeitos-famosos-o-vw-fox-que-decepava-
dedos/>. Acesso em: 07/03/2022. Pan American Health Organization. Health Indicators. Conceptual and operational
considerations. Washington, D.C.: PAHO; 2018, p.4. SELIGMAN, Martin E. P. Florescer: uma nova compreensão sobre a natureza da felicidade e
do bem-estar. Ed. Objetiva, 2012. ULBRICHT, Vania R.; FADEL, Luciane M.; BATISTA, Claudia R. Design para acessibilidade e
inclusão. Ed. Blucher. São Paulo, 2017, p.34. VERGANTI, Roberto. Driven Inovation. Mudando as regras da competição: A inovação do
significado de produtos. São Paulo: Editora canal Certo, 2012, p. 22.37.63 Vitalidade – O bem estar físico da psicologia positiva. 2021. Disponivel em:
<https://bemestarnaessencia.com.br/vitalidade-o-bem-estar-fisico-do-modelo-perma-v/>
Acesso em 14/08/2022.
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Testicularis adrenalis rest tumor a congenitalis adrenalis hyperplasia hátterében
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ESETISMERTETÉS ESETISMERTETÉS Testicularis adrenalis rest tumor
a congenitalis adrenalis hyperplasia
hátterében Brzózka Ádám dr.1 ■ Kuthi Levente dr.2 ■ Hajdú Erzsébet dr.3
Magony Sándor dr.4 ■ Király István Előd dr.3
Kincses Zsigmond Tamás dr.1 ■ Szatmári Angelika5
Szalma Emese oh. ■ Palkó András dr.1 ■ Fejes Zsuzsanna dr.1, 6 1Szegedi Tudományegyetem, Általános Orvostudományi Kar, Radiológiai Klinika, Szeged
2Szegedi Tudományegyetem, Általános Orvostudományi Kar, Pathologiai Intézet, Szeged
3Szegedi Tudományegyetem, Általános Orvostudományi Kar, Urológiai Klinika, Szeged
4Szegedi Tudományegyetem, Általános Orvostudományi Kar, I. Belgyógyászati Klinika, Szeged
5Szegedi Tudományegyetem, Egészségtudományi és Szociális Képzési Kar, Szeged
6Affidea Diagnosztika, Szeged A congenitalis adrenalis hyperplasia különféle megjelenési formáiban a testicularis adrenalis rest tumorok prevalenci
ája eltérő. A tapintható, általában kétoldali hereelváltozás az esetek 90–95%-ában a 21-hidroxiláz enzim hiánya okoz
ta mellékvesekéreg-hyperplasia következményeként jelenik meg. Az adrenalis rest tumorok, bár jól ismert elváltozá
sok, diagnózisuk és kezelésük multidiszciplináris megközelítést igényel. Az endokrinológiai, urológiai, patológiai és
radiológiai szakmák együttműködése kiemelkedően fontos. Amennyiben korai stádiumban felismerésre kerül, adek
vát szteroidhormon-emelés segíthet a térfoglalás méretének csökkentésében. Késői felismerés esetén azonban irreve
zibilis károsodás jön létre, mely infertilitáshoz vezet. Mivel a kezelés az egyéb jó- és rosszindulatú hereelváltozások
hoz képest jelentősen különbözik, fontos a korai és pontos diagnózis felállítása. Esetismertetésünk kapcsán a
testicularis adrenalis rest tumorok differenciáldiagnoszikai és kezelési nehézségeit multidiszciplináris szemszögből
mutatjuk be. j
Orv Hetil. 2020; 161(16): 623–631. Kulcsszavak: testicularis adrenalis rest tumor, férfieredetű meddőség (Beérkezett: 2019. november 30.; elfogadva: 2020. január 18.) Brzózka Á, Kuthi L, Hajdú E, Magony S, Király IE, Kincses ZsT, Szatmári A, Szalma E, Palkó A, Fejes Zs. [Testicular
adrenal rest tumor in the background of congenital adrenal hyperplasia]. Orv Hetil. 2020; 161(16): 623–631. Rövidítések 4Dion = androszténdion; ACTH = (adrenocorticotropic hor
mone) adrenokortikotrop hormon; anti-TPO = thyreoidea-
peroxidáz elleni antitest; AR = androgénreceptor; CAH = con
genitalis adrenalis hyperplasia; CRH = (corticotropin-releasing
hormone) kortikotropinfelszabadító hormon; FSH = folli
culusstimuláló hormon; LH = luteinizáló hormon; LST =
Leydig-sejtes tumor; MR = (magnetic resonance) mágneses
rezonancia; TART = testicularis adrenalis rest tumor; UH =
ultrahang A jelen dolgozatban, egy eseten keresztül, több szak
ma szempontjából szeretnénk bemutatni a testicularis
adrenalis rest tumor (TART) felismerésének és differen
ciáldiagnosztizálásának a buktatóit, valamint az inter
diszciplináris kivizsgálásának részleteit. Az Orvosi Heti
lapban egy hasonló, de más módon felderített eset is
mertetését találtuk [2]. A scrotum ultrahang (UH)-vizsgálata könnyen, gyorsan,
költséghatékonyan hozzáférhető eljárás, melynek során a
térfoglaló folyamatok jól karakterizálhatók, továbbá a lá
tottak a meddőségi állapothoz is fontos információt
szolgáltatnak [1]. Mivel a páciensek nagy része meddő
ségi kivizsgálás keretében érkezik, az így felismert eltéré
sek állítják a leginkább kihívás elé az andrológiában és
UH-diagnosztikában dolgozó szakembereket. A heré
ben lévő tumorok a fiatal, fertilis korosztályt érintik, de a Testicular adrenal rest tumor in the background of congenital adrenal
hyperplasia The prevalence of testicular adrenal rest tumours varies in different forms of congenital adrenal hyperplasia. Patients
with 21-hydroxilase deficiency usually have bilateral and palpable testicular nodules. Although adrenal rest tumours
are well documented in the literature, the diagnosis and management require a multidisciplinary approach: the coop
erative work of endocrinologists, urologists, pathologists and radiologists is essential. In the case of an early diagnosis,
appropriately increased corticosteroid treatment may reduce the tumour mass. In advanced stages, tumours can lead
to irreversible parenchymal damage causing infertility. The importance of an early and accurate diagnosis cannot be
emphasized enough, since the therapy differs significantly from other benign or malignant testicular neoplasia. A case
of a testicular adrenal rest tumour is presented along with the multidisciplinary perspectives of the diagnosis and
management of these lesions. Keywords: testicular adrenal rest tumour, male infertility Brzózka Á, Kuthi L, Hajdú E, Magony S, Király IE, Kincses ZsT, Szatmári A, Szalma E, Palkó A, Fejes Zs. [Testicular
adrenal rest tumor in the background of congenital adrenal hyperplasia]. Orv Hetil. 2020; 161(16): 623–631. (Beérkezett: 2019. november 30.; elfogadva: 2020. január 18.) 2020 ■ 161. évfolyam, 16. szám ■ 623–631. DOI: 10.1556/650.2020.31696 ■ © Szerző(k) 623 Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS meddőségi kivizsgálás során találhatunk olyan intratesti
cularis térfoglalást is, mely az esetek egy kis részében be
nignus. Ezen esetek nagy körültekintést igényelnek a
megfelelő terápia megválasztása és a fertilitás biztosítása
érdekében. Esetismertetés A 24 éves férfit újszülöttkora óta congenitalis adrenalis
hyperplasiával (CAH) gondozzák. Három hónappal a
születése után, hányások miatt kezdődő kivizsgálás során
került sor a 21-hidroxiláz enzim hiányának kimutatására,
melynek következtében klasszikus sóvesztő CAH alakult 1. táblázat
A beteg laborértékei és gyógyszeres kezelése a bemutatott időszakban
Gyógyszeres
kezelés
17-OH-
progesz
teron,
nmol/l
(0,6–2,5)
Andro
szténdion,
nmol/l
(2,9–9,5)
DHEA,
nmol/l
(8,5–36,0)
Teszto
szteron,
nmol/l
(9,90–
28,00)
SHBG,
nmol/l
(14,5–
48,4)
DHEA-S,
μmol/l
(5,73–
13,40)
LH,
IU/l
(1,7–8,6)
FSH,
IU/l
(1,5–12,4)
ACTH,
pmol/l
(1,60–
13,90)
Első észlelés
Hidrokortizon:
15–10–10 mg
Fludrokortizon:
0,05 mg
358
>35
4,7
18,71
37,8
1,60
4,0
8,5
91,56
Terápia
módosítás
Hidrokortizon:
10–5–0 mg
Fludrokortizon:
2 × 0,025 mg
Prednizolon:
5 mg
114
16,5
4,2
14,69
42,7
1,19
5,8
15,7
17,04
I. kontroll
Hidrokortizon:
10–5–0 mg
Fludrokortizon:
2 × 0,025 mg
Prednizolon:
7,5 mg
<0,3
–
3,9
16,92
69,5
4,4
10,8
12,07
II. kontroll
Hidrokortizon:
10–5–0 mg
Fludrokortizon:
2 × 0,025 mg
Prednizolon:
2 × 7,5 mg
>75,0
>35,0
3,3
11,21
69,2
1,90
2,8
7,8
64,79
III. kontroll
Műtét
>75,0
>35,0
4,1
8,66
62,3
1,51
5,8
10,5
16,39
Műtét utáni
kontroll
Hidrokortizon:
10–5–0 mg
Fludrokortizon:
2 × 0,025 mg
Prednizolon:
2 × 7,5 mg
12,8
–
4,9
7,03
45
0,82
15,5
15,6
0,92
ACTH = adrenokortikotrop hormon; DHEA = dehidroepiandroszteron; DHEA-S = dehidroepiandroszteron-szulfát; FSH = folliculusstimuláló
hormon; LH = luteinizáló hormon; SHBG = nemihormon-kötő globulin 1. táblázat
A beteg laborértékei és gyógyszeres kezelése a bemutatott időszakban ACTH = adrenokortikotrop hormon; DHEA = dehidroepiandroszteron; DHEA-S = dehidroepiandroszteron-szulfát; FSH = folliculusstimuláló
hormon; LH = luteinizáló hormon; SHBG = nemihormon-kötő globulin 2020 ■ 161. évfolyam, 16. szám ORVOSI HETILAP 624 Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UT ESETISMERTETÉS ki. A beteg a diagnózis felállítása óta folyamatos minera
lo- és glükokortikoid-szteroid kezelésben részesült gyer
mekgyógyászaton. Kórelőzményében még adenotomia
és appendectomia szerepel. A beteg gyermek- és serdü
lőkori kontrollvizsgálatokra rendszeresen eljárt, gyógy
szerelését igény szerint módosították. ki. A beteg a diagnózis felállítása óta folyamatos minera
lo- és glükokortikoid-szteroid kezelésben részesült gyer
mekgyógyászaton. Kórelőzményében még adenotomia
és appendectomia szerepel. A beteg gyermek- és serdü
lőkori kontrollvizsgálatokra rendszeresen eljárt, gyógy
szerelését igény szerint módosították. att további gyógyszermódosítás történt: este 7,5 mg
prednizolon szedését javasoltuk, egyéb gyógyszereinek
meghagyásával. A következő reguláris kontroll során eredményei jó
terápiás effektust igazoltak, a vizsgálati laborértékek a
normáltartományon belülre kerültek. Ezen eredmények
alapján a beteg kezelését változatlanul folytattuk. A beteget 21 éves korában irányították a felnőttendo
krinológiára. Belszervi panaszt első észlelésekor, illetve a
további kontrollok során sem említett, rákérdezésre sze
xuális diszfunkciót sem. Ekkor gyógyszeres kezelése a
következő volt: reggel 15 mg, délben 10 mg, este 10 mg
hidrokortizon per os, valamint reggel 0,05 mg fludrokor
tizon volt; emellett tachycardia miatt 5 mg nebivololt is
szedett. Vérvétel történt, melyből a terápia és a betegkö
vetés szempontjából legfontosabb értékeket táblázatos
formában rögzítettük (1. táblázat). Az alap-laborered
ményekből kiemelendő a normáltartományban lévő nát
rium- és káliumszint: nátrium 141 mmol/l (136–145),
kálium 4,3 mmol/l (3,5–5,1). A kalcium- és parathor
monszint nem mutatott eltérést, a pajzsmirigyfunkció
euthyreosist igazolt, az anti-TPO a normáltartományon
belül volt. A hasi, kismedencei UH-vizsgálat kórosat
nem mutatott a mellékvesék területén. A magas ACTH-,
17-OH-progeszteron- és 4Dion-érték miatt gyógyszeres
kezelésének módosítását javasoltuk az alábbiak szerint:
10–5–0 mg hidrokortizon per os, 2 × 0,025 mg fludro
kortizon, este 5 mg prednizolon per os, kiegészítve napi
600 mg kálium-klorid per os adásával. Soron következő kontrolljakor a hormonmérések a
korábbiakhoz képest rosszabbul kezelt állapotot jeleztek:
mind a 17-OH-progeszteron, mind a férfi-, valamint az
adrenokortikotrop hormon kikerült a normáltartomány
ból. Ezek az aktuálisan nem a céltartományban lévő
eredmények – összehasonlítva a korábban céltartomány
ban álló eredményekkel – felvetették a compliance-prob
léma lehetőségét is, ezért a beteg és családja ismételt
részletes felvilágosítást kapott a betegségről és annak ke
zeléséről; emellett a prednizolon dózisát 2 × 5 mg-ra
emeltük, egyéb gyógyszereinek változatlan dózisban tör
ténő szedése mellett. Az ismételt ellenőrzés során az emelt dózisú glüko
kortikoidkezelés ellenére az eredmények továbbra sem
voltak a céltartományban. Urológiai vizsgálatot javasoltunk, amelynek során jö
vőbeli fertilitási igény kivizsgálásakor azoospermia igazo
lódott, ezért here-UH-vizsgálat történt. Korábban here-
UH-vizsgálat nem volt. Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UT Az UH-vizsgálatot GE LOGIQ
S8 (General Electric, Boston, MA, Amerikai Egyesült
Államok) ultrahangkészülékkel végeztük. A vizsgálat so
rán normális mérettartományban lévő heréket észlel
tünk, melyeken belül inhomogén echogenitású, helyen
ként részben piszkos hangárnyékot adó összefüggő
képlet volt látható mindkét oldalon. A képletek főként a
mediastinum testist érintették (1. ábra). A jobb here fel
ső pólusán egy, nagyjából 35 × 25 × 30 mm-es, durván Ennek hatására hormonértékei a következő vizsgálat
során javulást mutattak. Mind a 17-OH-progeszteron,
az androszténdion és az adrenokortikotrop hormon
szintje csökkent ugyan, de nem kerültek a normáltarto
mányba. Így a csökkenő tendenciát mutató, de céltarto
mány feletti ACTH- és 17-OH-progeszteron-érték mi 1. ábra
Az ultrahangvizsgálat lineáris (7–14 MHz) fejjel történt; jól látható a kétoldali intratesticularis térfoglalás, mely echószegényebb a környező állomány
nál, a jobb oldalon a here felső pólusára lokalizálódik elsősorban, bal oldalon testicularis adrenalis rest tumorra (TART) jellegzetes képet mutat, a
mediastinum testisre lokalizálódik Az ultrahangvizsgálat lineáris (7–14 MHz) fejjel történt; jól látható a kétoldali intratesticularis térfoglalás, mely echószegényebb a környező állomány
nál, a jobb oldalon a here felső pólusára lokalizálódik elsősorban, bal oldalon testicularis adrenalis rest tumorra (TART) jellegzetes képet mutat, a
mediastinum testisre lokalizálódik 20 ■ 161. évfolyam, 16. szám 625 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Az intraoperatív mikroszkó
pos képen látható a vékony hereparenchyma alatt a porckemény tapintatú terime. Jelentősen tágult heretubulus nem volt
megfigyelhető (b)
a) b) ESETISMERTETÉS b) a) 4. ábra Intraoperatívan a heretok intaktnak bizonyult. Egy szürkésfehér, porckemény szövet tűnt elő (a). Az intraoperatív mikroszkó
pos képen látható a vékony hereparenchyma alatt a porckemény tapintatú terime. Jelentősen tágult heretubulus nem volt
megfigyelhető (b) citoplazmájuk eozinofil, melyben helyenként lipidcsep
pek voltak megfigyelhetők. Reinke-kristályok nem ábrá
zolódtak. A citológiai atípia alacsonynak bizonyult, sejt
osztódás nem volt jelen (5. ábra). Immunhisztokémiailag
a sejtek diffúz CD56- és MelanA-pozitivitást, valamint
részleges szinaptofizin- és androgénreceptor-pozitivitást
mutattak. A látottak alapján a patológus a laesiót TART-
nak véleményezte. A környező hereállományban súlyos
parenchymakárosodás állt fenn. Ép spermogenezis sem a
tumor mellett, sem pedig attól távolabb nem volt jelen. A tubulusok jórészt hialinizálódtak, egy-egy tubulusban
pedig csupán Sertoli-sejtek voltak jelen (Johnsen-score
4/10) (6. ábra). Ez a szöveti megjelenés lényegében
összhangban állt az obstruktív azoospermia klinikai di
agnózisával. A beavatkozás során ép hímivarsejtet nem
sikerült izolálni. sebészeti herebiopsziát végeztünk, melynek kapcsán – a
páciens kérését is figyelembe véve – a térfoglaló folyama
tokból is történt mintavétel. A műtét során a heretok mindkét oldalon intakt volt. A jobb herének körülbelül a kétharmadát igen tömött ta
pintatú terime töltötte ki, a kicsiny ép szövet alatt egy
porckemény, sárga színű elváltozás tűnt elő. A bal oldalon
egy hasonló morfológiájú képlet mutatkozott (4. ábra). Az intraoperatív szövettani vizsgálat malignitást nem
igazolt. A paraffinba ágyazott anyag szövettani elemzésé
ből kiderült, hogy a megjelenés a két mintában egyezett. A kötőszövetes állományban egy-, illetve kétsejtes kö
tegeket, valamint szolid fészkeket formáló celluláris pro
liferáció volt kivehető. A laesionalis sejtek kicsik voltak, A műtét során a heretok mindkét oldalon intakt volt. A jobb herének körülbelül a kétharmadát igen tömött ta
pintatú terime töltötte ki, a kicsiny ép szövet alatt egy
porckemény, sárga színű elváltozás tűnt elő. A bal oldalon
egy hasonló morfológiájú képlet mutatkozott (4. ábra). Az intraoperatív szövettani vizsgálat malignitást nem
igazolt. A paraffinba ágyazott anyag szövettani elemzésé
ből kiderült, hogy a megjelenés a két mintában egyezett. A kötőszövetes állományban egy-, illetve kétsejtes kö
tegeket, valamint szolid fészkeket formáló celluláris pro
liferáció volt kivehető. A laesionalis sejtek kicsik voltak, 5. ábra
Kötegekbe, illetve szolid fészkekbe rendeződő sejtszaporulat,
melynek környezetében kifejezett fokú kötőszövet-képződés
észlelhető (hematoxilin-eozin festés, 100×-os nagyítás) (a). A
laesionalis sejtek kicsik, a citoplazma vakuolizált. Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS 2. ábra
Az ultrahangvizsgálat lineáris (7–14 MHz) volt, a B-módú vizsgálatot a gyér keringés kimutatására is érzékeny ’power’ Doppler-technikával egészítet
tük ki. Jól látható, hogy a terimében a keringés igen gyér Az ultrahangvizsgálat lineáris (7–14 MHz) volt, a B-módú vizsgálatot a gyér keringés kimutatására is érzékeny ’power’ Doppler-technikával egészítet
tük ki. Jól látható, hogy a terimében a keringés igen gyér 2. ábra jelzett, az ellenoldalon pedig egy 19 × 39 × 18 mm-es,
szintén a mediastinum testis mellett elhelyezkedő, ha
sonló megjelenésű képlet ábrázolódott (3. ábra). Mesze
sedésre, necrosisra utaló jelek egyik oldalon sem voltak
megfigyelhetők. A hasi MR-vizsgálat során csupán a mel
lékvesék fokozott halmozása került leírásra, kóros nyi
rokcsomó vagy metastasisra típusos eltérés nem volt ész
lelhető. Radiológiai és urológiai konzílium történt. inhomogén terület, a bal oldalon pedig hasonló megjele
nésű, 40 × 15 × 20 mm-es képlet került leírásra. Dopp
ler-vizsgálattal fokozott vascularisatio nem volt látható
az elváltozásokon belül (2. ábra). Annak ellenére, hogy
a herékben lévő két eltérés ultrahangos megjelenése nem
volt teljesen egyforma, a radiológiai vélemény, endokri
nológussal konzultálva, elsősorban a TART lehetőségét
vetette fel. Testicularis és hasi-kismedencei MR- (GE
Discovery MR750w 3T; General Electric) vizsgálat is
történt, mely a jobb herében egy 22 × 28 × 31 mm-es,
T1-, T2-jel-szegény, a mediastinum testis magasságában
elhelyezkedő, kissé lobulált kontúrú, éles szélű képletet Urológiai szakvizsgálat alkalmával ép külső genitáliák
mellett normál nagyságú, azonban mindkét oldalon
porckemény tapintatú herék voltak észlelhetők. Azoo
spermia miatt sebészi spermiumnyerés céljából mikro 3. ábra
Jól látható a B-módú ultrahangvizsgálat képein a bal herében a TART-ra jellegzetes térfoglalás (a). Az MRI T2-súlyozott képein jól látható a medias
tinum testisben lévő jellegzetes lobulált, a hereparenchymához képest jelszegény elváltozás (b)
MRI
mágnesesrezonancia képalkotás; TART
testicularis adrenalis rest tumor
a) b) b) a) ható a B-módú ultrahangvizsgálat képein a bal herében a TART-ra jellegzetes térfoglalás (a). Az MRI T2-súlyozott képein jól látható a medias
testisben lévő jellegzetes lobulált, a hereparenchymához képest jelszegény elváltozás (b) 3. ábra MRI = mágnesesrezonancia-képalkotás; TART = testicularis adrenalis rest tumor ORVOSI HETILAP 626 2020 ■ 161. évfolyam, 16. szám Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS
Intraoperatívan a heretok intaktnak bizonyult. Egy szürkésfehér, porckemény szövet tűnt elő (a). Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC A citológiai atí
pia minimális, sejtosztódás vagy Reinke-kristály nem látható (he
matoxilin-eozin festés, 400×-os nagyítás) (b) A beteg további endokrinológiai kezelése, valamint
rendszeres képalkotó vizsgálattal történő követése folya
matban van. 6. ábra
A herecsatornák jó részében csupán spermatogoniumok és
Leydig-sejtek látszanak. Elvétve korai spermiocyták még jelen
vannak, de kiérő spermiogenezis nem észlelhető. Az intersztíci
umban kötőszövet-felszaporodás és enyhe fokú Leydig-sejt-
hyperplasia figyelhető meg (hematoxilin-eozin festés, 200×-os
nagyítás) A herecsatornák jó részében csupán spermatogoniumok és
Leydig-sejtek látszanak. Elvétve korai spermiocyták még jelen
vannak, de kiérő spermiogenezis nem észlelhető. Az intersztíci
umban kötőszövet-felszaporodás és enyhe fokú Leydig-sejt-
hyperplasia figyelhető meg (hematoxilin-eozin festés, 200×-os
nagyítás) Kötegekbe, illetve szolid fészkekbe rendeződő sejtszaporulat,
melynek környezetében kifejezett fokú kötőszövet-képződés
észlelhető (hematoxilin-eozin festés, 100×-os nagyítás) (a). A
laesionalis sejtek kicsik, a citoplazma vakuolizált. A citológiai atí
pia minimális, sejtosztódás vagy Reinke-kristály nem látható (he
matoxilin-eozin festés, 400×-os nagyítás) (b) 5. ábra 2020 ■ 161. évfolyam, 16. szám 627 ORVOSI HETILAP ORVOSI HETILAP Patológiai vonatkozások A TART elkülönítése a here csírasejtes tumoraitól, a
lymphomától vagy a lágyrész-daganatoktól nem okoz
diagnosztikai nehézséget, a TART differenciáldiagnosz
tikájában viszont a legfontosabb entitás a Leydig-sejtes
tumor (LST). A TART-ot felépítő sejtek egyszerre hor
dozzák a mellékvesekéreg és a Leydig-sejtek jellemvoná
sait, ami arra utal, hogy ezek a sejtek totipotens embrio
nális őssejt eredetűek lehetnek [7]. Az LST-knek csupán
a 3%-a bilaterális, ezzel szemben TART esetén a kétol
daliság 83%-os. Míg az előbbi tumorok 10%-a rosszin
dulatú, ezzel szemben az utóbbiak mindig jóindulatúak. Itt fontos hangsúlyozni, hogy a TART a neve ellenére
hiperplasztikus folyamatnak és inkább pszeudotumor
nak tekintendő, az LST viszont valódi neoplasiának felel
meg. A két daganatféleség makroszkóposan is különbö
zik, a TART ugyanis jellemzően egygócú, fekete laesió
ként látható a here állományában, míg az LST gyakran
többgócú, sárga tumorok képében jelentkezik. Jelentős
fokú fibrosis, zsírszöveti metaplasia, lymphoid sejtes
aggregátumok, emelkedett citológiai atípia és kifejezett
lipokrómpigment-felhalmozódás gyakran megfigyelhe
tő jelenségek TART esetén [8]. Továbbá az ilyen tumo
rokban sejtosztódások egyáltalán nem vagy csak hosszas
keresgélés után fedezhetők fel, jóllehet ezek az LST-ese
tek 69%-ában jelen vannak. Habár a Reinke-kristályok
ról úgy tartják, hogy patognomikusak a Leydig-sejtekre,
ez a jellegzetesség az LST-knek csupán a 40%-ában mu
tatható ki, és a TART-okban sosincs jelen, tehát a Rein
ke-kristályok hiánya önmagában nem segíti a helyes di
agnózis felállítását [6, 8]. Immunhisztokémiailag a
legfontosabb marker a szinaptofizin, mely a TART-ese
tek 88%-ában erős pozitív reakciót ad, ezzel szemben az
LST-knek csupán a 8%-ában figyelhető meg hasonló in
tenzitású pozitivitás. További fontos marker még a
CD56, mely jellemzően pozitív a neuroendokrin tumo
rokban, továbbá az androgénreceptor (AR), mely az
előbbi daganat kapcsán általában negatív, az utóbbiban
viszont pozitív reakciót ad. Nem segíti viszont a két en
titás elkülönítését az inhibin-α, ez ugyanis mindkét tu
morban expresszálódik. Az evidenciák alapján a klinikai
adatok, a hormonszintek és a szövettani jellegzetességek
mellett a szinaptofizint, CD56-ot és AR-t tartalmazó
immunhisztokémiai panel segíti a helyes diagnózis felál
lítását [9]. A környező hereállomány a folyamat kiter
jedtségétől függően károsodott, a spermiogenezis kez
detben kismértékben, majd pedig fokozatosan csökken. Előrehaladott esetekben csupán hialinizálódott vagy
csak Sertoli-sejteket tartalmazó tubulusok vehetők ki a
peritumoralis hereparenchymában. A spermiogramban a
folyamat súlyosságától függően oligospermia vagy azoo
spermia észlelhető. A TART és az LST közötti hasonló
ságokat és különbségeket Wang és mtsai foglalták össze
[10] Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS Patológiai vonatkozások Posztoperatív hormonális eredményei változatlan
gyógyszeres kezelés mellett jó kezelési eredményt mutat
tak, így gyógyszeres kezelését továbbra is változatlanul
javasoltuk folytatni. A TART elkülönítése a here csírasejtes tumoraitól, a
lymphomától vagy a lágyrész-daganatoktól nem okoz
diagnosztikai nehézséget, a TART differenciáldiagnosz
tikájában viszont a legfontosabb entitás a Leydig-sejtes
tumor (LST). A TART-ot felépítő sejtek egyszerre hor
dozzák a mellékvesekéreg és a Leydig-sejtek jellemvoná
sait, ami arra utal, hogy ezek a sejtek totipotens embrio
nális őssejt eredetűek lehetnek [7]. Az LST-knek csupán
a 3%-a bilaterális, ezzel szemben TART esetén a kétol
daliság 83%-os. Míg az előbbi tumorok 10%-a rosszin
dulatú, ezzel szemben az utóbbiak mindig jóindulatúak. Itt fontos hangsúlyozni, hogy a TART a neve ellenére
hiperplasztikus folyamatnak és inkább pszeudotumor
nak tekintendő, az LST viszont valódi neoplasiának felel
meg. A két daganatféleség makroszkóposan is különbö
zik, a TART ugyanis jellemzően egygócú, fekete laesió
ként látható a here állományában, míg az LST gyakran
többgócú, sárga tumorok képében jelentkezik. Jelentős
fokú fibrosis, zsírszöveti metaplasia, lymphoid sejtes
aggregátumok, emelkedett citológiai atípia és kifejezett
lipokrómpigment-felhalmozódás gyakran megfigyelhe
tő jelenségek TART esetén [8]. Továbbá az ilyen tumo
rokban sejtosztódások egyáltalán nem vagy csak hosszas
keresgélés után fedezhetők fel, jóllehet ezek az LST-ese
tek 69%-ában jelen vannak. Habár a Reinke-kristályok
ról úgy tartják, hogy patognomikusak a Leydig-sejtekre,
ez a jellegzetesség az LST-knek csupán a 40%-ában mu
tatható ki, és a TART-okban sosincs jelen, tehát a Rein
ke-kristályok hiánya önmagában nem segíti a helyes di
agnózis felállítását [6, 8]. Immunhisztokémiailag a
legfontosabb marker a szinaptofizin, mely a TART-ese
tek 88%-ában erős pozitív reakciót ad, ezzel szemben az
LST-knek csupán a 8%-ában figyelhető meg hasonló in
tenzitású pozitivitás. További fontos marker még a
CD56, mely jellemzően pozitív a neuroendokrin tumo
rokban, továbbá az androgénreceptor (AR), mely az
előbbi daganat kapcsán általában negatív, az utóbbiban
viszont pozitív reakciót ad. Nem segíti viszont a két en
titás elkülönítését az inhibin-α, ez ugyanis mindkét tu
morban expresszálódik. Az evidenciák alapján a klinikai
adatok, a hormonszintek és a szövettani jellegzetességek
mellett a szinaptofizint, CD56-ot és AR-t tartalmazó
immunhisztokémiai panel segíti a helyes diagnózis felál
lítását [9]. A környező hereállomány a folyamat kiter
jedtségétől függően károsodott, a spermiogenezis kez
detben kismértékben, majd pedig fokozatosan csökken. Előrehaladott esetekben csupán hialinizálódott vagy
csak Sertoli-sejteket tartalmazó tubulusok vehetők ki a
peritumoralis hereparenchymában. A spermiogramban a
folyamat súlyosságától függően oligospermia vagy azoo
spermia észlelhető. A TART és az LST közötti hasonló
ságokat és különbségeket Wang és mtsai foglalták össze
[10]. Képalkotás – differenciáldiagnosztika bevérzést vagy meszesedést, továbbá a mediastinum tes
tis mentén helyezkedik el [18]. Erezettsége a környeze
ténél kissé csökkentebb. A lymphomától való elkülöní
tésben nagyon fontos a color-Doppler-módban végzett
vizsgálat, mivel a lymphoma vascularisatiója a környe
zeténél fokozottabb, valamint a lymphoma ezen meg
jelenése elsősorban nem a fiatal korosztályra jellemző
megbetegedés. A primer testicularis lymphoma a non-
Hodgkin-lymphomának egy viszonylag ritka formája,
amelynél az átlagéletkor 66–68 év; a here malignus meg
betegedéseinek kevesebb mint 5%-át teszi ki. A TART-
nál ritkábban, csupán 6–10%-a kétoldali [13]. A herék térfoglaló folyamatai között a kétoldaliság gya
korisága igen változatos megoszlást mutat. Gyermekkor
ban az akut lymphoblastos leukaemia az esetek 5%-ában
okozhat kétoldali eltérést [11]. Az elsősorban a fiatal
populációt érintő, csírasejtes tumorok ritkán (1,8%-ban)
válnak kétoldalivá [12]. Az idősebb populációban a csí
rasejtes tumoroknál ritkábban előforduló lymphoma
vagy az áttétek lehetnek kétoldaliak. A testicularis lym
phoma a csírasejtes tumorokhoz képest sokkal ritkábban
fordul elő [13]. Hereáttétek ismert tumoros betegekben
nagyon ritkán, csupán 0,04%-ban fordulnak elő a heré
ben, leggyakrabban a prosztatarák (35%), amelyet a
tüdő-, a vese- és a vastagbélrák, valamint a melanoma
malignum előrehaladott stádiumban adott áttétei követ
nek. A metastasisok megjelenési gyakorisága 55 éves
életkorra tehető. Az áttétek terjedése a lymphovascularis
rendszeren keresztül történik, de a vese-, illetve a prosz
tatarák áttéteinek kialakulásában fontos szerepe van a
plexus pampiniformisban lévő vénatágulatnak és a vénás
elégtelenség miatt kialakult ’reverse flow’-nak is. A ritkán
előforduló intratesticularis metastasis fő feltételezett oka
a vér–here-gát védőhatása, más régebbi elképzelések sze
rint a herében lévő alacsonyabb hőmérséklet sem kedvez
a daganatszövet növekedésének [14]. A korábbi térfog
laló folyamatokkal szemben a TART jellemzően kétolda
li elváltozást okoz. A differenciáldiagnózis megkönnyítésére Nuffer és
mtsai döntési folyamatot dolgoztak ki. Ha az anemnesz
tikus adatok alapján felmerül a TART, az UH-képen
típusos lokalizációban echószegény, lobulált eltérés
azonosítható akkor egyoldaliság esetén elsősorban a me
szesedés jelenlétét kell kizárni, valamint a kontúrok meg
jelenését kell jól megítélni. Amennyiben a kontúrok el
mosódottak, vagy a vizsgált eltérésben meszesedés
észlelhető, felmerül a tumoros eredet. Kétoldali eltérés
esetén különösen figyelni kell a fokozott vascularisatióra,
mely lymphomát jelezhet. A TART ultrahangos megjele
nése a környező hereparenchymánál gyérebb vascularisa
tiót mutat [18]. A TART-betegek MR-vizsgálatáról már a 2000-es
évek előtt is jelentek meg cikkek, melyek leírták, hogy a
TART a T1-szekvenciákon a normális hereállományhoz
képest izo/hiperintenzív, a T2-szekvenciákon azonban
hipointenzív megjelenésű [19, 20]. Az elváltozás mind
két alapszekvencián homogén, tokkal nem határolt. Képalkotás – differenciáldiagnosztika Kontrasztanyag adása után az esetek 85%-ában volt meg
figyelhető halmozás. Egy másik tanulmány szerint az
összes TART homogén kontrasztanyag-halmozást mutat
[21]. A cikkek szerzői egyetértenek abban, hogy az MR-
vizsgálat (az alapszekvenciákat figyelembe véve) és az
UH-vizsgálat diagnosztikus értéke megegyezik. Amen�
nyiben egy beteg esetében szóba jön a részleges orchiec
tomia, úgy az MR-vizsgálat már indokolttá válhat, mivel
ez a műtéti tervezést segíti. Az eddig megjelent cikkek
leginkább az alap-MR-szekvenciákat tanulmányozták
TART esetében. Az MR-spektroszkópia hasznos lehet a
hereállomány funkciójának megítélésében, mivel károso
dott here esetében a colin csúcs csökkenést mutat, már a
morfológiai elváltozások megjelenése előtt [22, 23]. A TART diagnózisának felállításában az UH és az MR
képalkotó vizsgálatok nyújthatnak segítséget. Az elsődle
ges szerep a jó elérhetőség és a költséghatékonyság miatt
az UH-vizsgálatoké. A herék UH-vizsgálatának diagnosztikus hasznát
már az 1970-es évektől kezdődően kutatták. 1977-ben
Miskin az akkoriban csúcstechnológiás, 5 MHz-es
transzducerrel végzett here-UH-vizsgálatok klinikai
hasznát emelte ki [15]. A technika rohamosan fejlődött,
és néhány éven belül megjelent az első közlemény, mely
az UH-vizsgálatoknak a TART diagnózisában és köve
tésében betöltött szerepéről szólt [16]. A szerzők szerint
a CAH- és a primer mellékvese-elégtelenségben szen
vedő betegek körében a herékben bilaterálisan, excentri
kusan elhelyezkedő nodulusok – melyekben esetlegesen
akusztikus árnyékolás is megfigyelhető – elsősorban
TART-nak véleményezhetők. A TART ultrahangos megjelenése viszonylag típusos:
a kezdeti stádiumban kis, multifokális, a környező here
parenchymához viszonyítva echószegény képletek jelen
nek meg a mediastinum testis területén; később ezek,
összeolvadva, nagyobb echószegény területet képeznek,
melyben hiperreflektív területek is lehetnek [17]. Az
echódús megjelenés ritka. Éles szegélyű, lobulált, a here
kontúrjait viszonylag kevéssé torzítja, rajta előboltosulást
nem okoz. Differenciáldiagnosztikai kihívást jelent elkü
löníteni a nem csírasejtes tumoroktól, úgymint a Leydig-
sejtes vagy a Sertoli-sejtes tumortól. Fontos támpont le
het, hogy a TART általában nem tartalmaz necrosist, Endokrinológiai háttér A 21-hidroxiláz-deficientia az egyik leggyakoribb mono
génes anyagcsere-betegség, melynek magyarországi gya
korisága 1 : 14 000 [3]. A CAH autoszomális recesszív módon öröklődő be
tegségek összefogaló neve, melyeket a mellékvese sztero
idhormon-szintézisének zavara jellemez. A különböző
megjelenési formákban a mineralo-, glükokortikoid-,
valamint a szexuálszteroidok bioszintézise károsodott. A 21-hidroxiláz enzim a mellékvesekéreg sejtjeinek sima
felszínű endoplazmatikus reticulumában katalizálja a
progeszteron és a 17-α-hidroxi-progeszteron hidroxilá
lását. A 21-hidroxiláz enzim defektusának következté
ben nem termelődik elég kortizol, ami miatt a hypotha
lamusban, valamint a hypophysisben fokozódik a
kortikotropinfelszabadító hormon (CRH) és az adreno
kortikotrop hormon (ACTH) termelése. Ennek hatására
egyrészt CAH alakul ki, másrészt pedig a szteroidszinté
zis az androgének és ösztrogének felé tolódik. A megvál
tozott hormontermelés következménye a lánygyerme
kek virilisatiója, valamint a mindkét nemben megfigyel
hető felgyorsult növekedés és csontosodás (klasszikus
virilisatiós forma). Az enzimdefektus miatt az aldoszte
ron szintézise is csökken, ami a betegek mintegy 75%-
ában sóvesztéshez vezet (klasszikus sóvesztő forma), és
korai, súlyos hyponatraemiás hypovolaemia kialakulását
okozhatja. A 21-hidroxiláz enzim defektusának enyhébb
formájában (nem klasszikus forma) a kortizol és az al
doszteron szintézise normális tartományban van, az and
rogének szintje azonban kissé emelkedett. Ezek a bete
gek enyhébb tünetekkel jelentkezhetnek, mint például
pubertas praecox, akné vagy fertilitási problémák [4].i A férfi CAH-betegek körében infertilitás alakulhat ki,
melynek oka kettős: egyrészt a hypogonadismus, más
részt a TART megjelenése, mely gyakori szövődménynek
számít [3]. A TART kialakulásának patomechanizmusa
nem teljesen ismert, feltehetőleg a magzati fejlődés során
a here állományába került adrenocorticalis sejtekből
alakul ki. Mivel a tumorok térfogata nagy dózisú kor
tikoszteroidkezelésre általában csökken, valamint megje
lenésének idején az ACTH-szint emelkedett, az valószí
nűsíthető, hogy a magas szérum-ACTH-szint és luteini
zálóhormon-hatás felelős a kialakulásukért. Egyes tanul
mányok szerint a tumorsejteken ACTH-receptorok is
megfigyelhetők, ami megerősíti, hogy embrionális kor
ban mellékvese-eredetű sejtcsoportok szálltak le a herék
kel együtt [5, 6]. A TART jellemzően a mediastinum
testis területén jelenik meg, eredendően nem malignus, a
mediastinum területén azonban fibrosist indukál, majd
pedig obstruktív azoospermiához és meddőséghez vezet. 2020 ■ 161. évfolyam, 16. szám 628 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS Urológiai-andrológiai szempontok A CAH az infertilitás gyakori okozója. A testicularis ad
renalis rest tumorok megjelenése jól ismert jelenség, és
fontos komplikációnak számít, amely a betegek kb. 30–
94%-ában alakul ki. Tipikus megjelenési helye a medias
tinum testis, ami miatt a heretubulusok elzáródhatnak,
emellett az elváltozás a környező szövetek tartós károso
dását okozza, ami következményes azoospermiához, így
infertilitáshoz vezet [24]. A mechanikus obstrukció/
destrukció mellett a mellékvesekéreg-szteroidhormonok 2020 ■ 161. évfolyam, 16. szám 629 Megbeszélés és azok metabolitjainak lokális toxikus hatása is hozzájá
rul a Sertoli- és/vagy a csírasejtek károsodásához [24,
25]. A nagy mennyiségben termelődő androgének pedig
az LH és az FSH szuppresszióján keresztül tovább ront
hatják a fertilitást [2]. A here térfoglaló folyamatainak hátterében döntően
urológiai megbetegedések állnak, így a kivizsgálás és a
kezelési terv is döntően urológiai szemléletet tükröz. Olyan esetekben viszont, amikor szisztémás társbetegség
is fennáll, a multidiszciplináris megközelítés megkerül
hetetlen. Korai stádiumban a daganattól távolabb lévő here
parenchymából nyert szövettani minta még sok esetben
nem mutat eltérést. Ennek lehetséges magyarázata a ká
rosodás progresszív volta, mely a tumor körüli szövetek
ben kezdődik, és a távolabbi területek csak később válnak
érintetté. Ez alapján érthető, hogy nemcsak a daganatok
lokalizációja, hanem azok mérete, illetve az obstrukció
időtartama is fontos tényező a gonadális diszfunkció ki
alakulása szempontjából [26]. Az elváltozás növekedésé
vel a herében irreverzibilis károsodás alakul ki, mely az
endokrin és az exokrin funkciót egyaránt érinti [24, 27]. Számos tanulmány igazolta, hogy az elváltozások ki
mutatására here-UH-vizsgálat elvégzése a legalkalma
sabb, mivel ezzel a még nem tapintható (<2 cm) laesiók
is jól detektálhatók [24]. A 21-hidroxiláz-deficientia az egyik leggyakoribb mo
nogénes anyagcsere-betegség; férfi betegek esetében in
fertilitás elsősorban TART jelenléte, gonadotropin
szuppresszió és herekárosodás miatt jöhet létre. Mivel a
TART előfordulása CAH-ban 10 éves kor után növek
szik, fontos a páciensek rendszeres, 1-2 évenkénti ultra
hangos szűrése. A TART már a második stádiumtól
könnyen jól elkülöníthető a mediastinum testis területén
látható kicsi, jól körülhatárolt, echószegény eltérésként,
mely gyakran kétoldali. Az időben megkezdett glüko
kortikoidkezeléssel, valamint az ACTH-szintek szupp
ressziójával a TART volumene jelentősen csökkenthető,
kialakulása megelőzhető, így a fertilitás megtartható. Amennyiben a TART nem reagál a terápiára, a termé
kenység megtartására a spermiumnyerés és -tárolás
megfontolandó [29]. Amennyiben a TART felfedezése a
fiatal felnőtt korra tolódik ki, fontos a pontos differenci
áldiagnózis, hogy a beteget a feleslegesen végzett szemi
kasztrációtól megóvjuk, amennyiben pedig azoospermia
igazolódik, mielőbbi mikrosebészeti herebiopszia elvég
zése javasolt a sikeres gyermekvállalás reményében. A si
keres kezelés az endokrinológus, a képalkotást végző
szakember, a patológus és az urológus/andrológus szo
ros együttműködésén és a jó kommunikáción múlik. Számos tanulmány igazolta, hogy az elváltozások ki
mutatására here-UH-vizsgálat elvégzése a legalkalma
sabb, mivel ezzel a még nem tapintható (<2 cm) laesiók
is jól detektálhatók [24]. Claahsen-van der Grinten és munkacsoportja a TART-ot
5 stádiumba sorolja, figyelembe véve a klinikai és a szö
vettani megjelenést, valamint a tumor növekedését. Megbeszélés Az
első stádiumban az elváltozás képalkotással még nem
mutatható ki, az adrenalis rest sejtek a rete testis terüle
tén helyezkednek el. Az UH-vizsgálatnak a második stá
diumtól van jelentősége, amelynél a TART már kimutat
ható, a mediastinum testis területén elhelyezkedő kicsi,
echószegény elváltozás formájában. A harmadik stádi
umtól a rete testis fokozatos kompressziója alakul ki,
melyhez a négyes stádiumban már fibrosis társul. Az ötö
dik stádiumban megjelenik a hereparenchyma károsodá
sa a tömeges TART mellett [27]. A TART kezelését illetően a korai stádiumban végzett
nagy dózisú hormonkezelés vagy a műtéti kezelés csök
kenti a tumor méretét, eliminálja az obstrukciót, meg
előzheti a további károsodást. Jóllehet a műtétet követő
en a TART kiújulását nem figyelték meg, a beavatkozás a
már kialakult károsodást nem javítja [2, 26, 28]. Anyagi támogatás: A közlemény megírása anyagi támo
gatásban nem részesült. Szerzői munkamegosztás: B. Á.: A vizsgálati szempontok
kidolgozása a képalkotás szemszögéből, szakirodalmi
másodelemzés, adatgyűjtés, a kézirat szövegezése. K. L.:
A vizsgálati szempontok kidolgozása a patológus szem
szögéből, szakirodalmi másodelemzés, adatgyűjtés, a
kézirat szövegezése. H. E.: A vizsgálati szempontok ki
dolgozása az urológus szemszögéből, szakirodalmi má
sodelemzés, adatgyűjtés, a kézirat szövegezése. M. S.:
A vizsgálati szempontok kidolgozása az endokrinológus
szemszögéből, szakirodalmi másodelemzés, adatgyűjtés,
a kézirat szövegezése. K. Zs. T.: A kézirat szövegezése. Sz. A., Sz. E.: A kézirat szövegezése, rendszerezés, szak
irodalmi másodelemzés. K. I. E.: Az andrológiai szem
pontok bemutatása, szakirodalmi elemzés. P. A., F. Zs.:
A kézirat szövegezése. A cikk végleges változatát vala
mennyi szerző elolvasta és jóváhagyta. Szerzői munkamegosztás: B. Á.: A vizsgálati szempontok
kidolgozása a képalkotás szemszögéből, szakirodalmi
másodelemzés, adatgyűjtés, a kézirat szövegezése. K. L.:
A vizsgálati szempontok kidolgozása a patológus szem
szögéből, szakirodalmi másodelemzés, adatgyűjtés, a
kézirat szövegezése. H. E.: A vizsgálati szempontok ki
dolgozása az urológus szemszögéből, szakirodalmi má
sodelemzés, adatgyűjtés, a kézirat szövegezése. M. S.:
A vizsgálati szempontok kidolgozása az endokrinológus
szemszögéből, szakirodalmi másodelemzés, adatgyűjtés,
a kézirat szövegezése. K. Zs. T.: A kézirat szövegezése. Sz. A., Sz. E.: A kézirat szövegezése, rendszerezés, szak
irodalmi másodelemzés. K. I. E.: Az andrológiai szem
pontok bemutatása, szakirodalmi elemzés. P. A., F. Zs.:
A kézirat szövegezése. A cikk végleges változatát vala
mennyi szerző elolvasta és jóváhagyta. Azon betegeknél, akiknél a kétoldali TART hosszú
ideje fennáll, a szervmegtartó sebészi kezelésnek nincs
hatása a gonadális funkcióra, a spermaparaméterekben
sem találtak javulást, továbbá a műtét okozta további ká
rosodások veszélye sem zárható ki [24, 25, 27]. ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS 2020 ■ 161. évfolyam, 16. szám Irodalom [16] Seidenwurm D, Smathers RL, Kan P, et al. Intratesticular adrenal
rests diagnosed by ultrasound. Radiology 1985; 155: 479–481. [1] Fejes Zs, Pásztor N, Karczagi L. The role of ultrasonography in
the investigation of male infertility. [Az ultrahangmódszerek
szerepe a férfimeddőség kivizsgálásában.] Orv Hetil. 2018; 159:
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renal rest tumours in postpubertal males with congenital adrenal
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ticular adrenal rest tumors. Am J Sonogr. 2018; 1: 1–4. g
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(H1-MRS) of the testis in men with semen analysis altered. Reprod Syst Sex Disord. 2016; 5: 182. [6] Bonaccorsi AC, Adler I, Figueiredo JG. Male infertility due to
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nal hyperplasia: lesions with both adrenocortical and Leydig cell
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ticular adrenal rest tumors in patients with congenital adrenal
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genital syndrome: from physiopathology to therapy. Presse Med. 2017; 46: 572–578. [27] Claahsen-van der Grinten HL, Hermus AR, Otten BJ. Testicular
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nophenotypic features of testicular tumour of the adrenogenital
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phoma. World J Urol. 1995; 13: 230–232. [29] Speiser PW, Arlt W, Auchus RJ, et al. Congenital adrenal hyper
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Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018; 103: 4043–4088. [Correction: J Clin Endocrinol Metab. 2019; 104: 39–40.] [12] Zequi Sde C, Da Costa WH, Santana TB, et al. Bilateral testicu
lar germ cell tumours: a systematic review. BJU Int. 2012; 110:
1102–1109. [13] Cheah CY, Wirth A, Seymour JF. Primary testicular lymphoma. Blood 2014; 123: 486–493. [14] Bart J, Groen, HJ, van der Graaf WT, et al. An oncological view
on the blood–testis barrier. Lancet Oncol. 2002; 3: 357–363. (Fejes Zsuzsanna dr.,
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scrotal masses. J Urol. 1977; 117: 185–188. Megbeszélés Ezért
ezekben az esetekben a sebészi kezelés már csak fájdalom
és a diszkomfort csökkentése céljából indokolt [27]. A fertilitási ráta igen alacsony. Korai stádiumban, a tu
mor eltávolításával egy ülésben végzett sebészi spermi
umnyerés, illetve az intracitoplazmatikus spermiumin
jekció lehetséges megoldást jelent [24, 26, 28]. A fertilitás biztosítása és a további irreverzibilis here
károsodás megelőzése céljából a minél korábban, a pu
bertás kezdetén, illetve a pubertáskorban végzett here-
UH-vizsgálat, a sebészi spermiumnyerés és a páciensek
szoros követése elengedhetetlen. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. 2020 ■ 161. évfolyam, 16. szám 630 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 08/24/22 12:22 PM UTC ESETISMERTETÉS Irodalom A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény,
melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye,
illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) Irodalom 2020 ■ 161. évfolyam, 16. szám 631 ORVOSI HETILAP
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English
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Virtual Analog Models of the Lockhart and Serge Wavefolders
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Applied sciences
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Virtual Analog Model of the Lockhart and Serge Wave Folders Citation for published version:
Esqueda, F, Pontynen, H, Parker, J & Bilbao, S 2017, 'Virtual Analog Model of the Lockhart and Serge
Wave Folders', Applied Sciences, vol. 7, no. 12. https://doi.org/10.3390/app7121328 Citation for published version: General rights
C
i h f
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Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)
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abide by the legal requirements associated with these rights. Digital Object Identifier (DOI):
10.3390/app7121328 Link:
Link to publication record in Edinburgh Research Explorer Edinburgh Research Explorer Article
Virtual Analog Models of the Lockhart and
Serge Wavefolders † Fabián Esqueda 1,*, Henri Pöntynen 1, Julian D. Parker 2 and Stefan Bilbao 3 Fabián Esqueda
, Henri Pöntynen , Julian D. Parker
and Stefan Bilbao
1
Aalto Acoustics Lab, Aalto University, 02150 Espoo, Finland; henri.pontynen@aalto.fi
2
Native Instruments GmbH, D-10997 Berlin, Germany; julian.parker@native-instruments.de
3
Acoustics and Audio Group, University of Edinburgh, Edinburgh EH9 3FD, Scotland, UK; s.bilbao@ed.ac.uk
*
Correspondence: fabian.esqueda@aalto.fi; Tel.: +358-50-4646-041
†
This paper is an extended version of our paper published in the 14th Sound and Music Computing
Conference (SMC-17), Espoo, Finland, 5–8 July 2017. q
,
y
, J
1
Aalto Acoustics Lab, Aalto University, 02150 Espoo, Finland; henri.pontynen@aalto.fi
2
Native Instruments GmbH, D-10997 Berlin, Germany; julian.parker@native-instruments.de
3
Acoustics and Audio Group, University of Edinburgh, Edinburgh EH9 3FD, Scotland, UK; s.bilbao@ed.ac.uk
*
Correspondence: fabian.esqueda@aalto.fi; Tel.: +358-50-4646-041
†
This paper is an extended version of our paper published in the 14th Sound and Music Computing
Conference (SMC-17), Espoo, Finland, 5–8 July 2017. †
This paper is an extended version of our paper published in the 14th Sound and Music Computing
Conference (SMC-17), Espoo, Finland, 5–8 July 2017. Academic Editor: Stefania Serafin
Received: 12 October 2017; Accepted: 13 December 2017; Published: 20 December 2017 Received: 12 October 2017; Accepted: 13 December 2017; Published: 20 December 2017 Abstract: Wavefolders are a particular class of nonlinear waveshaping circuits, and a staple of the
“West Coast” tradition of analog sound synthesis. In this paper, we present analyses of two popular
wavefolding circuits—the Lockhart and Serge wavefolders—and show that they achieve a very
similar audio effect. We digitally model the input–output relationship of both circuits using the
Lambert-W function, and examine their time- and frequency-domain behavior. To ameliorate the
issue of aliasing distortion introduced by the nonlinear nature of wavefolding, we propose the use
of the first-order antiderivative method. This method allows us to implement the proposed digital
models in real-time without having to resort to high oversampling factors. The practical synthesis
usage of both circuits is discussed by considering the case of multiple wavefolder stages arranged
in series. Keywords: acoustic signal processing; circuit modeling; nonlinear waveshaping; antialiasing;
synthesis; music Take down policy Take down policy
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investigate your claim. Download date: 24. Oct. 2024 applied
sciences Appl. Sci. 2017, 7, 1328; doi:10.3390/app7121328 1. Introduction In recent years, West Coast synthesis has become increasingly
popular, with contemporary manufacturers such as Make Noise and Verbos Electronics releasing their
own takes on classic Buchla circuits. This study presents virtual analog (VA) models for two analog synthesizer circuits: the Lockhart
wavefolder and the wavefolder used in the middle section of the Serge Wave Multipliers. Wavefolding
is a type of nonlinear waveshaping common in West Coast synthesis where portions of the input signal
that exceed certain threshold are inverted or “folded back”, hence the name of the effect. The two
circuits considered in this study were chosen because of the strong similarities between their general
behavior. In a similar way to guitar distortion pedals, both wavefolders exploit the saturating behavior
of semiconductor p–n junctions (i.e., transistors/diodes) to implement a folding function. Wavefolders are amongst the most emblematic building blocks of West Coast synthesis. In spite
of that, they have been mostly overlooked by both VA and digital waveshaping research. We have
recently begun to fill this research gap in [17], which presents a VA model of the wavefolder circuit
in the seminal Buchla 259 module. Previous work on circuit-based VA modeling has researched
the filters found in vintage synthesizers such as those produced by Moog [19–22], Electronic Music
Studios (EMS) [23,24], Korg [25,26] and Buchla [16]. Extensive work has also been done on modeling
guitar distortion pedals [13,27], tube amplifiers [11,28,29], modulation effects [30–33] and the Roland
TR-808 drum machine [34,35]. Measurement-based VA modeling, commonly known as “black-box
modeling”, has also been thoroughly studied within the context of guitar amplifiers and pedals [36–38]. This approach is particularly useful when the original circuit schematics are not available. A major challenge in VA modeling of nonlinear circuits, and digital waveshaping in general, is
aliasing suppression. Early research on waveshaping synthesis addressed this issue by using low-order
polynomial transfer functions, which not only allowed full parametric control of the produced spectrum
but also ensured that the output waveform was bandlimited [4]. In VA modeling, high oversampling
factors are usually necessary to prevent harmonics introduced by nonlinearities from reflecting into
the baseband as aliases [13]. Oversampling increases the computational requirements of the model, by
introducing additional filtering stages and scaling the number operations required to compute each
output sample. 1. Introduction Nonlinear waveshaping is a technique used in sound synthesis to generate complex harmonic
spectra. It consists of processing a signal with low harmonic content (typically a sinusoid) using
a nonlinear mapping function designed to introduce harmonic overtones to the output signal [1]. The first documented use of waveshaping in the digital domain can be traced back to 1969, when
Jean-Claude Risset emulated the sound of a clarinet by distorting a sinusoid with a clipping function [2]. Waveshaping techniques were extensively researched within the context of computer music in the
1970s, with several authors exploring the use of Chebyshev polynomials in particular, as an accurate
and computationally cheap alternative to additive synthesis [1,3–5]. The underlying principles behind
waveshaping synthesis are closely related to other well-known synthesis techniques, such as frequency
modulation (FM) and phase distortion (PD) synthesis [6,7]. These two techniques rely on distorting
the frequency and phase, respectively, of sinusoidal oscillators. Recent research on the topic of
distortion-based synthesis has explored the use of logic operators in lieu of traditional polynomial
waveshapers [8], and proposed extensions to both FM and PD synthesis [9,10]. The use of waveshaping in the analog domain began in the 1950s, when guitar players started
deliberately overdriving their tube amplifiers to alter the timbre of their instrument [11]. In 1961,
Gibson released the “Maestro FZ-1 Fuzz Tone”, the first commercially available fuzz distortion pedal,
which exploited the saturating behavior of transistors to introduce harmonic distortion [12]. Most guitar Appl. Sci. 2017, 7, 1328; doi:10.3390/app7121328 www.mdpi.com/journal/applsci www.mdpi.com/journal/applsci Appl. Sci. 2017, 7, 1328 2 of 23 distortion pedals, including popular designs such as the Ibanez Tube Screamer and Electro-Harmonix
Big Muff Pi, operate under this same basic principle [13,14]. distortion pedals, including popular designs such as the Ibanez Tube Screamer and Electro-Harmonix
Big Muff Pi, operate under this same basic principle [13,14]. In analog synthesizers, the use of distortion-based methods is one of the cornerstones of
“West Coast” synthesis, a paradigm pioneered by California-native Don Buchla during the 1960s. Buchla’s instruments focused on timbre manipulation at oscillator level by employing a variety of
techniques such as nonlinear waveshaping, oscillator synchronization and pitch modulation [15–17]. This approach to sound synthesis contrasts that of traditional subtractive synthesis, where timbre
is typically controlled by filtering harmonically-rich oscillator waveforms, like sawtooth and square
waves, using resonant filters [18]. 1. Introduction For VA models that require evaluating transcendental functions, as is the case with
the proposed Lockhart and Serge models, these added costs could compromise the integration of the
system within a larger, real-time computer music system. A sizable portion of VA research has concentrated on designing efficient algorithms to generate
alias-free geometric waveforms like those used in analog subtractive synthesizers, the so-called classic
analog waveforms. Well-known techniques include the bandlimited impulse train (BLIT) family of
methods, which involves the use of bandlimited basis functions and their integrated forms [39–41],
and the use of differentiated polynomial waveforms (DPW) [42–44]. Moreover, Välimäki and Franck
have applied the antialiasing principle behind the DPW algorithm to tackle aliasing in wavetable
oscillators [45]. Recent work on antialiasing techniques has extended the use of the bandlimited ramp
(BLAMP) method, originally proposed to antialias triangular oscillators in [25,41], to special cases of
linear piecewise nonlinearities such as signal rectification, and inverse/hard clipping [17,46,47]. In this work, we propose the use of the antiderivative antialiasing method introduced in [48,49]. This approach can be used to reduce the aliasing caused by arbitrary nonlinear waveshaping functions
and is applicable to the proposed wavefolder models. In its first-order form, the method can be Appl. Sci. 2017, 7, 1328 3 of 23 derived by analytically convolving a linear continuous-time representation of the input signal with a
rectangular lowpass kernel [48]. As shown in this work, the use of the antiderivative method reduces
the oversampling requirements of the proposed wavefolder models. A VA model of the Lockhart wavefolder was originally presented in [50]. This paper extends that
work by introducing a second wavefolding circuit and studying the similarities between both systems. Additionally, we present a different treatment of the required Lambert-W function and an extended
evaluation of the proposed antialiasing method in terms of computational costs. This paper is organized as follows. Sections 2 and 3 describe the model derivation of the Lockhart
and Serge wavefolders, respectively. Time-domain simulations of the circuits are also presented in these
two sections. Section 4 deals with two implications of VA wavefolding in the digital domain, namely
aliasing suppression and evaluation of the Lambert-W function. Section 5 presents frequency-domain
results of the Lockhart and Serge wavefolders, as well as an evaluation of the proposed antialiasing
method in terms of perceived sound quality and computational costs. 1. Introduction Section 6 discusses the practical
synthesis usage of both circuits and compares the behavior of the middle Serge Wave Multiplier with a
recommended four-stage topology built around the Lockhart wavefolder. Concluding remarks and
perspectives appear in Section 7. 2.1. Circuit Analysis The Lockhart wavefolder consists of an NPN and a PNP bipolar junction transistors connected at
their base and collector terminals. In order to model the large-signal behavior of the circuit, we replace
transistors Q1 and Q2 with their corresponding Ebers-Moll large-signal models [23,24]. Figure 1b
shows the large-signal equivalent circuit of the Lockhart wavefolder. We use a double subscript
notation to distinguish the voltages and currents in transistor Q1 from those in Q2. For example, IED,2
denotes the current through the base-emitter diode in Q2. Component values for the circuit have been
compiled in Table 1. Table 1. Component values for the Lockhart wavefolder circuit. Component
Value (kΩ)
Component
Value (V)
R
15
VCC
15
RL
1–50
VEE
−15 Table 1. Component values for the Lockhart wavefolder circuit. Component
Value (kΩ)
Component
Value (V)
R
15
VCC
15
RL
1–50
VEE
−15 We begin the analysis of the circuit by assuming that the supply voltages are equal but opposite in
sign (i.e., VCC = −VEE), and that |Vin| ≪VCC. This assumption means that the base-emitter junctions
of both Q1 and Q2 will always be forward-biased and their voltage drops will remain approximately
constant for all expected values of Vin. In Ken Stone’s version of the circuit, Vin is assumed to be
bounded between approximately ±1.2 V [53]. Applying KVL around both input–emitter loops gives us Vin
=
VCC −RIE,1 −VBE,1
(1)
Vin
=
VBE,2 + RIE,2 + VEE,
(2) (1) (1)
(2) (2) where IE,1 and IE,2 are the emitter currents, and VBE,1 and VBE,2 are the voltages across the base–emitter
junctions of Q1 and Q2, respectively. Solving Equations (1) and (2) for IE,1 and IE,2 gives us: where IE,1 and IE,2 are the emitter currents, and VBE,1 and VBE,2 are the voltages across the base–emitter
junctions of Q1 and Q2, respectively. Solving Equations (1) and (2) for IE,1 and IE,2 gives us: IE,1
=
VCC −VBE,1 −Vin
R
(3)
IE,2
=
Vin −VBE,2 −VEE
R
. (4) IE,1
=
VCC −VBE,1 −Vin
R
(3)
IE,2
=
Vin −VBE,2 −VEE
R
. (4) (3) (4) Next, we apply KCL at the collector node, which gives us Next, we apply KCL at the collector node, which gives us Next, we apply KCL at the collector node, which gives us Iout = IC,1 −IC,2,
(5)
where
IC,1
=
αFIED,1 −ICD,1
(6)
IC,2
=
αFIED,2 −ICD,2. 2. The Lockhart Wavefolder Figure 1a shows a simplified circuit diagram of the Lockhart wavefolder. This circuit was designed
in 1973 by R. Lockhart Jr., who intended it to be used as a general-purpose frequency tripler [51]. Following its publication in Bernie Hutchin’s Electronotes [52], Lockhart’s design was repurposed as
a wavefolder by Ken Stone, who realized its potential as a simple yet interesting waveshaper [53,54]. The Lockhart wavefolder has become ubiquitous in the music synthesizer do-it-yourself (DIY)
community. For example, it is the core processor in Yves Usson’s “Metalizer” module [55]. (a)
(a)
IE,2
IE,2
IED,2
IED,2
↵RICD,2
↵RICD,2
↵FIED,2
↵FIED,2
IE,1
IE,1
IC,2
IC,2
IC,1
IC,1
ICD,1
ICD,1
VBC,2
VBC,2
VBC,1
VBC,1
IED,1
IED,1
VBE,1
VBE,1
VBE,2
VBE,2
Iout
Iout
RL
RL
Vout
Vout
R
R
Vin
Vin
Q1
Q1
Q2
Q2
VCC
VCC
VEE
VEE
↵RICD,1
↵RICD,1
↵FIED,1
↵FIED,1
(b)
(b)
Vin
Vin
Q2
Q2
Vout
Vout
RL
RL
R
R
VCC
VCC
VEE
VEE
Q1
Q1
PNP
PNP
NPN
NPN
VBE,1
VBE,1
VBE,2
VBE,2
ICD,2
ICD,2
Figure 1. (a) Simplified schematic of the Lockhart wavefolder circuit (adapted from [53]); and (b) its
Ebers–Moll large-signal equivalent model. VBE,1
VBE,1 ↵RICD,1
↵RICD,1 VBC,1
VBC,1 ↵FIED,1
↵FIED,1 ↵FIED,2
↵FIED,2 (a)
(a) (b)
(b) Figure 1. (a) Simplified schematic of the Lockhart wavefolder circuit (adapted from [53]); and (b) its
Ebers–Moll large-signal equivalent model. Appl. Sci. 2017, 7, 1328 4 of 23 The main modifications made by Stone to Lockhart’s original design were the addition of an
input potentiometer to attenuate the amplitude of the input waveform, and an inverting amplifier
at the output of the circuit [54]. For the sake of simplifying the analysis, these are not shown in
Figure 1a. The inverting stage at the output is reintroduced in Section 2.3. In our treatment of the
circuit, we introduce the load resistance RL as an additional parameter which can be used to further
control the timbre of the folded waveform. 2.1. Circuit Analysis (7) Iout = IC,1 −IC,2,
(5) (5) where IC,1
=
αFIED,1 −ICD,1
(6)
IC,2
=
αFIED,2 −ICD,2. (7) (6) (6)
(7) (7) If we then assume that the contributions of the reverse currents αRICD1 and αRICD2 to the total
currents associated with the emitter nodes are negligible (i.e., αR ≈0), we can establish that 28
5 of 23 Appl. Sci. 2017, 7, 1328 5 of 23 IED,1
≈
IE,1
(8)
IED,2
≈
IE,2. (9) (8) (8)
(9) (9) Assuming αF = 1, as suggested in [23], and inserting Equations (8) and (9) into Equations (6) and
(7), respectively, yields a new expression for the total output current of the circuit: Assuming αF = 1, as suggested in [23], and inserting Equations (8) and (9) into Equations (6) and
(7), respectively, yields a new expression for the total output current of the circuit: Iout = IE,1 −IE,2 −ICD,1 + ICD,2. (10) (10) We then combine Equations (3) and (4) to derive an expression for the difference between
emitter currents:
V
+ V
2V + V
V We then combine Equations (3) and (4) to derive an expression for the difference between
emitter currents: IE,1 −IE,2 = VCC + VEE −2Vin + VBE,2 −VBE,1
R
. (11) (11) Since VCC + VEE = 0, and voltage drops VBE,1 and VBE,2 are assumed to be constant and equal,
their contribution to this expression disappears. Therefore, we can simplify this result as: Since VCC + VEE = 0, and voltage drops VBE,1 and VBE,2 are assumed to be constant and equal,
their contribution to this expression disappears. Therefore, we can simplify this result as: IE,1 −IE,2 = −2
RVin. (12) (12) Substituting Equation (12) into Equation (10) produces an expression for the total output current
Iout in terms of the input voltage and the currents through the collector diodes: Substituting Equation (12) into Equation (10) produces an expression for the total output current
Iout in terms of the input voltage and the currents through the collector diodes: Iout = −2
RVin −ICD,1 + ICD,2. 2.1. Circuit Analysis (13) (13) The current–voltage (I–V) relationship of diodes can be modeled using Shockley’s ideal diode
equation, defined as
The current–voltage (I–V) relationship of diodes can be modeled using Shockley’s ideal diode
equation, defined as
ID = Is
exp
VD
ηVT
−1
,
(14) (14) where ID is the current through the diode, Is is the reverse bias saturation current, VD is the voltage
across the diode, VT is thermal voltage and η is the ideality factor of the diode [56]. For the p–n
junctions inside transistors we can assume a reverse saturation current value Is = 10−17 A and an
ideality factor η = 1. A thermal voltage value VT = 25.864 mV is used throughout this study. where ID is the current through the diode, Is is the reverse bias saturation current, VD is the voltage
across the diode, VT is thermal voltage and η is the ideality factor of the diode [56]. For the p–n
junctions inside transistors we can assume a reverse saturation current value Is = 10−17 A and an
ideality factor η = 1. A thermal voltage value VT = 25.864 mV is used throughout this study. pplying Shockley’s diode equation to the collector diodes and substituting into Equation (13) Applying Shockley’s diode equation to the collector diodes and substituting into Equation (13)
gives us: Applying Shockley’s diode equation to the collector diodes and substituting into Equati
gives us: Iout = −2Vin
R
−Is
exp
VCD,1
ηVT
−1
|
{z
}
+ Is
exp
VCD,2
ηVT
−1
|
{z
}
. (15) (15) {z
ICD,2 {z
ICD,1
{z
ICD,2 ICD,1 Next, we use KVL to derive expressions for VCD,1 and VCD,2 in terms of Vin and Vout: Next, we use KVL to derive expressions for VCD,1 and VCD,2 in terms of Vin and Vout: VCD,1
=
Vout −Vin
(16)
VCD,2
=
Vin −Vout. (17) (16)
(17) (16)
(17) (16)
(17) (17) Now, the collector diodes in the large-signal model are antiparallel. Therefore, we can make
the further assumption that only one of them will conduct at a time depending on the polarity of
Vin. A similar treatment is presented in [57] for the case of diode pairs in guitar distortion circuits. This means that ICD,1 ≈0
for
Vin ≥0
and
ICD,2 ≈0
for
Vin < 0. 2.1. Circuit Analysis By combining these new assumptions with Equations (15)–(17) we arrive at the piecewise expres Iout = −2
RVin + λIs
exp
λ (Vin −Vout)
ηVT
−1
,
(18) (18) Appl. Sci. 2017, 7, 1328 6 of 23 where λ = sgn (Vin) and sgn () is the signum function where λ = sgn (Vin) and sgn () is the signum function where λ = sgn (Vin) and sgn () is the signum function where λ = sgn (Vin) and sgn () is the signum function sgn(x) :=
−1
if
x < 0
0
if
x = 0
1
if
x > 0. (19) (19) Equation (18) can be further simplified if we consider that the independent constant factor λIs that
results from its expansion will be very small (±10−17 A) and can therefore be neglected. This gives us: Equation (18) can be further simplified if we consider that the independent constant factor λIs that
results from its expansion will be very small (±10−17 A) and can therefore be neglected. This gives us: Iout = −2
RVin + λIs exp
λ (Vin −Vout)
ηVT
. (20) (20) Finally, we multiply both sides of this expression by RL to derive an input–output voltage
relationship for the Lockhart wavefolder: Finally, we multiply both sides of this expression by RL to derive an input–output voltage
relationship for the Lockhart wavefolder: Vout = −2RL
R Vin + λRLIs exp
λ (Vin −Vout)
ηVT
. (21) (21) Table 2. Parameter values for the Lockhart wavefolder described by Equation (26). Name
Value
Name
Value
Name
Value
R
15 kΩ
Is
10−17 A
VT
25.864 mV
RL
1–50 kΩ
η
1
–
– Table 2 summarizes all parameter values for the proposed Lockhart wavefolder model. Table 2 summarizes all parameter values for the proposed Lockhart wavefolder model. Table 2 summarizes all parameter values for the proposed Lockhart wavefolder mode 2.3. Model Discretization and Evaluation Model Discretization and Evaluation 2.3. Model Discretization and Evaluation
Th
l
d
h L
kh
f ld
d
d
Th
f
d
b
h The voltages inside the Lockhart wavefolder are time-dependent. Therefore, we can describe the
continuous-time model defined by Equation (26) as being of the form Vout(t) = f (Vin(t)),
(27) (27) where f () is the transfer function of the system and t is time. In the synthesis literature, the term
“transfer function” is commonly used to denote the waveshaping function [4]. It should not be confused
with the s- and z-domain transfer functions used in linear system analysis. As previously mentioned, Ken Stone’s circuit features an inverting stage before the output which
can be modeled by inverting the polarity of the right-hand side of Equation (26): Vout = αVin −ληVTW (∆exp (λβVin)) . (28) (28) Vout = αVin −ληVTW (∆exp (λβVin)) . While including this step is not strictly necessary, we have chosen to do so, as it will facilitate the
evaluation of the model. Now, given the form described by Equation (27), the Lockhart model can be
discretized trivially by replacing all continuous-time signals with their discrete-time equivalents, i.e., (29) Vout[n] = f (Vin[n]),
(29) where n is the sample index. 2.2. Explicit Formulation Equation (21) describes an implicit relationship between the input and output voltages of the
circuit; it cannot be solved algebraically. Instead, a closed-form solution for Vout can be derived with
the help of the Lambert-W function. The use of the Lambert-W function W() has been previously
researched within the context of VA modeling. Several authors have used it to solve the implicit I–V
relationship of diodes [56,58,59]. Parker and D’Angelo used W() to model the Buchla Lowpass-Gate,
a synthesizer circuit that employs a resistive opto-isolator (also known as a vactrol) in its control
path [16]. Strictly speaking, W() is multivalued; however, in this work, we only utilize the upper
branch of the function. This branch is known as W0() in the literature [56,60]. The Lambert-W function is used to solve equations of the form (A + Bx) exp (Cx) = D,
(22) (22) which have the explicit solution which have the explicit solution x = 1
CW
CD
B exp
AC
B
−A
B ,
(23) (23) where A, B, C and D ∈R [58]. where A, B, C and D ∈R [58]. Equation (21) can be arranged in the form described by Equation (22) by first rewriting it as Vout + 2RL
R Vin = λRLIs exp
λVin
ηVT
exp
−λVout
ηVT
,
(24) (24) and dividing both sides by exp (−λVout/ηVT), which gives us and dividing both sides by exp (−λVout/ηVT), which gives us and dividing both sides by exp (−λVout/ηVT), which gives us and dividing both sides by exp (−λVout/ηVT), which gives us
Vout + 2RL
R Vin
exp
λVout
ηVT
= λRLIs exp
λVin
ηVT
. (25) (25) Solving for Vout as defined in Equation (23) yields an explicit model for the Lockhart wavefolder Solving for Vout as defined in Equation (23) yields an explicit model for the Lockhart wavefolder Vout = ληVTW (∆exp (λβVin)) −αVin, Vout = ληVTW (∆exp (λβVin)) −αVin,
(26) (26) where where
α = 2RL
R ,
β = 2RL + R
ηVTR
and
∆= RLIs
ηVT
. α = 2RL
R ,
β = 2RL + R
ηVTR
and
∆= RLIs
ηVT
. 7 of 23 Appl. Sci. 2017, 7, 1328 where n is the sample index. p
The time-domain behavior of the proposed circuit model was validated by comparing it against a
reference simulation obtained using the SPICE (Simulation Program with Integrated Circuit Emphasis)
software LTspice (Version IV, Linear Technology, Milpitas, CA, USA, 2016) [61]. The results of this
simulation are shown in Figure 2a for values of Vin between −1.5 and 1.5 V. Figure 2b shows the
transfer function of the proposed model implemented in MATLAB (Version R2017a, MathWorks,
Natick, MA, USA, 2017) using Equation (28) and MATLAB’s native “lambertw” function. In both
cases, four different values of RL were simulated: 1, 5, 10 and 50 kΩ. From these figures, we can
observe the general behavior of the Lockhart wavefolder. At low input values, the system behaves
linearly, whereas for high input values the circuit inverts the slope of the driving signal. The transition
between non-folded and folded portions of the signal is gradual, which responds to the characteristic
soft saturating behavior of p–n junctions. The region where the transfer function folds the input signal
is indicated with a blue arrow in Figure 2b for the case when RL = 50 kΩ. As shown in these Figures,
increasing the value of RL sharpens the shape of the transfer function. The curves shown in Figure 2a,b indicate a good match between the SPICE simulations and the
proposed digital model. Figure 3 shows the absolute value (in mV) of the difference between both
simulations. From this plot, we can observe that the difference between the curves is indeed very small,
below 1 mV for all values of Vin measured. These small differences are perceptually insignificant and
can be attributed to the simplifications made during the analysis of the circuit and to the way in which
SPICE computes currents flowing through semiconductor devices. For example, SPICE introduces a
small fictitious conductance in parallel with each p–n junction in order to aid the convergence of its
iterative solvers. Additionally, the SPICE diode model will account for the small reverse current that
flows when the voltage across the diode is negative [61]. Appl. Sci. 2017, 7, 1328 8 of 23 8 of 23 1k
5k
10k
50k
Fold
Fold
Figure 2. Transfer function of the Lockhart wavefolder simulated using: (a) SPICE (Simulation Program
with Integrated Circuit Emphasis); and (b) the proposed virtual analog (VA) model. Different colors
indicate different values of RL. Fold
Fold 1k
5k
10k
50k Figure 2. where n is the sample index. Transfer function of the Lockhart wavefolder simulated using: (a) SPICE (Simulation Program
with Integrated Circuit Emphasis); and (b) the proposed virtual analog (VA) model. Different colors
indicate different values of RL. 1k
5k
10k
50k
Figure 3. Absolute value of the difference between a SPICE simulation of the Lockhart wavefolder and
its proposed VA model. Figure 3. Absolute value of the difference between a SPICE simulation of the Lockhart wavefolder and
its proposed VA model. Figure 4 shows a time-domain view of the output of the proposed model when driven by a 500-Hz
sinusoidal waveform with a peak amplitude of 1 V for two different load resistance values, RL = 10
and 50 kΩ. A sampling rate Fs = 44.1 kHz was used to generate these figures, which are plotted
against their corresponding SPICE simulations. From these results, we can once again observe the
effect of wavefolding and the impact of RL on the overall shape of the output. For high values of RL
the transition region between folded and non-folded values becomes very small and the resulting
waveform is almost discontinuous (see Figure 4b). In the frequency domain, this will translate to
higher harmonic content, similar to that of a square wave oscillator. A more detailed frequency-domain
analysis of the Lockhart circuit is presented in Section 5 of this study. Figure 4. Time-domain view of the proposed Lockhart wavefolder model plotted against its SPICE
simulation for a 500-Hz sinusoidal input (peak amplitude 1 V) with load resistance: (a) RL = 10 kΩ;
and (b) RL = 50 kΩ. Figure 4. Time-domain view of the proposed Lockhart wavefolder model plotted against its SPICE
simulation for a 500-Hz sinusoidal input (peak amplitude 1 V) with load resistance: (a) RL = 10 kΩ;
and (b) RL = 50 kΩ. 3. The Serge Middle Wave Multiplier The second circuit considered in this study is the middle section of the Serge Wave Multipliers
(often abbreviated as the Serge VCM). The Serge VCM is a synthesizer module designed in 1977 by
West Coast designer Serge Tcherepnin, founder of Serge Modular Music Systems. It offered three
separate and independent analog sound processors, namely the “top”, “middle” and “bottom” sections. As described in an original 1980 Serge product catalog, “The middle section generates a sweep of the
odd harmonics (1, 3, 5, 7, 9, 11 and 13th) when a triangle wave is applied to its input... This module can
be used to explore timbral areas beyond the range of ring modulation because there are more varied
harmonics than the sum and difference tones” [62]. Appl. Sci. 2017, 7, 1328 9 of 23 The middle Serge VCM is essentially a waveshaping circuit consisting of six identical wavefolding
stages arranged in series. An amplifier at the input of the circuit is used to modulate the gain of
the input waveform and control the amount of folds introduced [63]. In this section we focus on
the analysis of a single folding stage. The transfer function and frequency-domain behavior of the
complete system are presented in Section 6. Figure 5 shows the schematic of a single wavefolding
stage in the circuit. Component information is given in Table 3. -
+
Vin
Vin
Vout
Vout
R1
R1
R2
R2
Vx
Vx
R3
R3
Figure 5. Schematic of a single folding cell in the middle section of the Serge Wave Multipliers (VCM). Figure adapted from [63]. Figure 5. Schematic of a single folding cell in the middle section of the Serge Wave Multipliers (VCM). Figure adapted from [63]. Table 3. Component information for the Serge wavefolder circuit shown in Figure 5. Table 3. Component information for the Serge wavefolder circuit shown in Figure 5. Table 3. Component information for the Serge wavefolder circuit shown in Figure 5 Component
Value (kΩ)
Component
Description
R1
33
Diodes
1N4148 or similar
R2
100
Op-Amp
TL072 or similar
R3
100
–
– To derive the transfer function for the Serge wavefolding circuit, we first assume ideal op-amp
behavior and derive an expression for Vout in terms of Vin and Vx, the voltage at the non-inverting
input of the amplifier. This gives us: Vout = Vx −R3
R2
(Vin −Vx) . 3. The Serge Middle Wave Multiplier (30) (30) Since in this case R3 = R2, we can further simplify this result as: Since in this case R3 = R2, we can further simplify this result as: Since in this case R3 = R2, we can further simplify this result as: (31) Vout = 2Vx −Vin. (31) Next, we derive an expression for Vx by considering the subcircuit shown in Figure 6, which is
essentially a diode pair similar to those found in guitar distortion circuits [13,57,58]. Vin
Vin
Vx
Vx
IF
IF
IR
IR
R1
R1
I Figure 6. Equivalent view of the diode saturator at the non-inverting input of the op-amp in Figure 5. Figure 6. Equivalent view of the diode saturator at the non-inverting input of the op-amp in Figure 5. gure 6. Equivalent view of the diode saturator at the non-inverting input of the op-amp in Figure 5. Applying KVL around the outer loop of the circuit yields the relation Applying KVL around the outer loop of the circuit yields the relation Vin = R1I + Vx,
(32) (32) Vin = R1I + Vx, Appl. Sci. 2017, 7, 1328 10 of 23 where I is the current through resistor R1. Then, we apply KCL at the output node of the circuit, which
gives us I = IF −IR. (33) I = IF −IR. (33) ombining Equation (32) with Equation (33) and applying Shockley’s diode equation gives us uation (32) with Equation (33) and applying Shockley’s diode equation gives us Vin −Vx
R1
= Is
exp
Vx
ηVT
−1
|
{z
}
IF
−Is
exp
−Vx
ηVT
−1
|
{z
}
IR
. (34) (34) As before, we assume the diodes will not conduct simultaneously and arrive at the piecewise
relationship As before, we assume the diodes will not conduct simultaneously and arrive at the piecewise
relationship
λV
Vin −Vx = λR1Is
exp
λVx
ηVT
−1
,
(35) (35) where once again λ = sgn(Vin). To further simplify this expression we neglect the constant factor
λR1Is that results from its expansion. This gives us: Vin −Vx = λR1Is exp
λVx
ηVT
. (36) (36) Next, we rearrange this equation in the Lambert-W form described by Equation (22) by dividing
both sides by exp (λVx/ηVT). 3. The Serge Middle Wave Multiplier This yields Next, we rearrange this equation in the Lambert-W form described by Equation (22) by dividing
both sides by exp (λVx/ηVT). This yields (Vin −Vx) exp
−λVx
ηVT
= λR1Is,
(37) (37) which can be solved for Vx as: which can be solved for Vx as: Vx = Vin −ληVTW
R1Is
ηVT
exp
λVin
ηVT
. (38) (38) As a final step, we insert Equation (38) into Equation (31) to derive a complete expression for the
transfer function of a single wavefolding stage in the Serge middle VCM: As a final step, we insert Equation (38) into Equation (31) to derive a complete expression for the
transfer function of a single wavefolding stage in the Serge middle VCM: Vout = Vin −2ληVTW
R1Is
ηVT
exp
λVin
ηVT
. (39) (39) Figure 7 shows the transfer function of the circuit, evaluated in MATLAB for values of Vin between
−1.5 and 1.5 V. As before, the model was discretized trivially and is presented against its corresponding
SPICE simulation. Parameter values used in this simulation are compiled in Table 4. The value of
parameters Is and η for the 1N4148 diode were matched to those of its corresponding SPICE model [61]. Figure 7b shows the absolute difference between both simulations. These results indicate a good match
between the models, as the maximum difference was once again found to be below 1 mV. -1.5
-1
-0.5
0
0.5
1
1.5
-1
-0.5
0
0.5
1
SPICE
Model
-1.5
-1
-0.5
0
0.5
1
1.5
0
0.1
0.2
Figure 7. (a) Transfer function of a single wavefolding stage in the Serge middle VCM measured using
SPICE and the proposed model; and (b) the absolute difference between these two curves. -1.5
-1
-0.5
0
0.5
1
1.5
0
0.1
0.2 -1.5
-1
-0.5
0
0.5
1
1.5
-1
-0.5
0
0.5
1
SPICE
Model Figure 7. (a) Transfer function of a single wavefolding stage in the Serge middle VCM measured using
SPICE and the proposed model; and (b) the absolute difference between these two curves. 11 of 23 Appl. Sci. 2017, 7, 1328 Table 4. Simulation parameters for a single folding stage in the middle Serge Wave Multiplier. Name
Value
Name
Value
Is
2.52 nA
R1
33 kΩ
η
1.752
VT
25.864 mV Finally, Figure 8 shows the output of the Serge wavefolder for a 500-Hz sinusoidal input. 3. The Serge Middle Wave Multiplier As expected, the circuit behaves as a wavefolder, folding portions of the input waveform whose
absolute value exceeds approximately 0.3 V. This behavior is similar to that of the Lockhart wavefolder
(cf. Figure 4a). Figure 8. Time-domain view of the Serge wavefolder model plotted against its SPICE simulation for a
500-Hz sinusoidal input with peak amplitude if 1 V. Figure 8. Time-domain view of the Serge wavefolder model plotted against its SPICE simulation for a
500-Hz sinusoidal input with peak amplitude if 1 V. 3.1. Model Equivalence Equation (39) shares a close resemblance with Equation (28), the proposed Lockhart wavefolder
model. In fact, both expressions have the same form, which consists of the difference between a portion
of the input signal and an input-dependent nonlinear element. In the case of the Lockhart wavefolder,
when the RL = R/2 Equation (28) simplifies to Vout = Vin −ληVTW
RLIs
ηVT
exp
λVin
ηVT
,
(40) (40) which is remarkably close to Equation (39), with the only difference being the missing factor of two
outside the Lambert-W function. This factor accounts for the difference between physical parameters
Is and η in each circuit. Figure 9 shows a comparison of the transfer functions for the Lockhart
(RL = 7.5 kΩ) and Serge wavefolders implemented using the parameter values in Tables 2 and 4,
respectively. From this figure, we can observe that the only significant difference between both
transfer functions is in their sharpness at the folding points. This means the Lockhart wavefolder will
introduce sharper folds which will translate into brighter sounds at the output. From this analysis,
it is clear that both circuits result in a similar audio effect, even though they are produced using
different architectures. Figure 9. Transfer functions for the proposed Serge and Lockhart (RL = 7.5 kΩ) wavefolder models. Figure 9. Transfer functions for the proposed Serge and Lockhart (RL = 7.5 kΩ) wavefolder models. 4.1. Evaluating the Lambert-W Function A particular challenge of using the Lambert-W function in VA modeling, where real-time operation
is paramount, is that of computational efficiency. Optimizing the evaluation of W() is an active
research topic (see, e.g., [64]). For the case of guitar distortion circuits, Paiva et al. proposed the use
of a simplified iterative method which relies on a lookup table for its initial guess [57]. In this work,
we propose approximating the value of W() directly using Fritsch’s iteration, as suggested in [60]. In order to compute wm, an approximation to W(x), where x ∈R>0, we iterate over wm+1 = wm(1 + εm),
(41) (41) where where where εm
=
rm
1 + wm
qm −rm
qm −2rm
(42)
rm
=
ln
x
wm
−wm
(43)
qm
=
2 (1 + wm)
1 + wm + 2
3rm
,
(44) εm
=
rm
1 + wm
qm −rm
qm −2rm
(42) (42) rm
=
ln
x
wm
−wm
(43) (43) qm
=
2 (1 + wm)
1 + wm + 2
3rm
,
(44) (44) and m = 0, 1, 2, . . . , M −1. The value w0 is an initial guess and M is the number of iterations required
for εm to approximate zero within machine-size floating point precision. The special case W(0) = 0 is
defined separately. The efficiency of Fritsch’s iteration will depend on the choice of initial guess. As explained in [60],
an initial guess within 10−4 of the solution will yield an approximation to W() accurate to within 10−16
in just one iteration. Figure 10 shows the approximate times required to compute W(x) for a set of
values of x between 10−24 and 10300 using Fritsch’s iteration and the previously-proposed Halley’s
method [50]. This range was chosen as it covers all values of interest. For instance, when Vin = 5 V,
the argument of W() in the Serge wavefolder model will be approximately 1.52 × 1044. All times
were computed by averaging the result of 30 iterations implemented under identical circumstances. A piecewise approximation was used to compute the initial guess, as described in [60]. From this plot,
we can observe that Fritsch’s iteration outperforms Halley’s method by up to approximately 11 times. 4.1. Evaluating the Lambert-W Function A MATLAB implementation of the Lambert-W function used to perform these measurements can be
found in the accompanying website for this article. Halley
Fritsch
Figure 10. Averaged processing times required to compute W(x) using Halley’s method and Fritsch’s iteration. ure 10. Averaged processing times required to compute W(x) using Halley’s method and Fritsch’s iteration 4. Wavefolding in the Digital Domain In the previous sections, the time-domain behavior of the Lockhart and Serge wavefolder models
was examined via trivial discretization of their characteristic transfer functions. In this section, we move Appl. Sci. 2017, 7, 1328 12 of 23 on to consider two implications of virtual analog wavefolding: evaluation of the Lambert-W function
and aliasing. on to consider two implications of virtual analog wavefolding: evaluation of the Lambert-W function
and aliasing. 5. Results This section examines the frequency-domain behavior of the Lockhart and Serge wavefolders
and their proposed antialiased forms. Next, we evaluate the computational costs of the antiderivative
method with respect to oversampling for the case of the Lockhart model. 4.2. Aliasing Considerations when |Vin[n] −Vin[n −1]| is smaller than a predetermined threshold [48]. This special case simply
bypasses the antialiased form while compensating for the half-sample latency of the method. 4.2. Aliasing Considerations 4.2. Aliasing Considerations As discussed in Section 1, nonlinear waveshaping in the digital domain is susceptible to aliasing
distortion due to its frequency-expanding nature. Wavefolding is no exception to this problem. As an
arbitrary input waveform is folded, new harmonic overtones will be added to the frequency spectrum. Harmonics at frequencies exceeding half of the sampling rate, or the Nyquist limit, will be reflected into Appl. Sci. 2017, 7, 1328 13 of 23 the audio baseband as aliases. Aliasing is known to cause unpleasant artifacts—such as beating and
inharmonicity—that cannot be tolerated in a music computing scenario. Oversampling is commonly
employed to mitigate this issue; however, this approach increases the computational requirements of
the system by introducing additional operations. We propose the use of the first-order antialiasing method presented in [48,49]. This method is
designed to reduce aliasing caused by memoryless waveshaping functions with the form described by
Equation (29). The antialiased output of the waveshaping function is defined as Vout[n] = F(Vin[n]) −F(Vin[n −1])
Vin[n] −Vin[n −1]
,
(45) (45) where F() is the antiderivative of f (), the original transfer function. For the case of the Lockhart
wavefolder defined by Equation (28), the integrated transfer function is defined as F(Vin) = α
2V2
in −ηVT
2β [Ψ1(Ψ1 + 2)] ,
(46) (46) where Ψ1 = W (∆exp (λβVin)) , Ψ1 = W (∆exp (λβVin)) ,
(47) (47) and α, β and ∆remain as before. This result showcases an advantageous property of the Lambert-W
function W(); its antiderivative is defined in terms of W() itself. Therefore, computing F() does not
pose a major increase in computational costs with respect to evaluating simply f (). For the case of the
Serge wavefolder defined by Equation (39), the required antiderivative is given by F(Vin) = V2
in
2 −(ηVT)2 [Ψ2(Ψ2 + 2)] ,
(48) (48) where where Ψ2 = W
R1Is
ηVT
exp
λVin
ηVT
. (49) (49) When Vin[n] ≈Vin[n −1], Equation (45) can become ill-conditioned. This is avoided by defining
the special case When Vin[n] ≈Vin[n −1], Equation (45) can become ill-conditioned. This is avoided by defining
the special case Vout[n] = f
Vin[n] + Vin[n −1]
2
,
(50) (50) when |Vin[n] −Vin[n −1]| is smaller than a predetermined threshold [48]. This special case simply
bypasses the antialiased form while compensating for the half-sample latency of the method. 5.1. Frequency-Domain Behavior The spectrogram in Figure 11 shows the effect of increasing the value of RL in the Lockhart
wavefolder model for a constant 150-Hz sinusoidal input. As expected, the level of harmonic distortion
introduced by the circuit is proportional to the value of this resistance. Therefore, this parameter can
be used for additional timbral control. It should be noted that due to the antisymmetric nature of
the folding function, the system introduces odd harmonics only for input signals centered around
zero. Since the level of harmonics introduced by the Lockhart wavefolder depends on the choice
of RL, we consider the highest recommended case RL = 50 kΩas a worst-case scenario in terms of
aliasing distortion. pl. Sci. 2017, 7, 1328
14 o
Frequency (Hz)
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0
Figure 11. Spectrogram of the Lockhart wavefolder under 150-Hz sinusoidal input for values of RL
between 1 and 50 kΩ. 14 of 23 Appl. Sci. 2017, 7, 1328 Frequency (Hz)
-80
-60
-40
-20
0 Figure 11. Spectrogram of the Lockhart wavefolder under 150-Hz sinusoidal input for values of RL
between 1 and 50 kΩ. Figure 12 shows the spectrograms of a linear sweep from 20 Hz–5 kHz with peak amplitude of 1 V
processed by the proposed Lockhart and Serge wavefolder models. This frequency range was chosen
as it covers all fundamental frequencies of musical interest. A sample rate Fs = 1 MHz was used to
generate these figures in order to simulate an ideal alias-free continuous-time behavior. These results
will be used as a reference when evaluating the performance of the proposed antialiased forms. From
these spectrograms we can observe how the Lockhart wavefolder is capable of generating brighter
sounds. This perceptual attribute can be varied by changing the value of RL. Frequency (Hz)
-80
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-20
0
Frequency (Hz)
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-40
-20
0
Figure 12. Spectogram for a linear sweep from 20 Hz to 5 kHz processed using: (a) the proposed
Lockhart wavefolder model (RL = 50 kΩ); and (b) the proposed Serge wavefolder model. A sample
rate Fs = 1 MHz was used to simulate analog behavior. -80
-60
-40
-20
0 Figure 12. Spectogram for a linear sweep from 20 Hz to 5 kHz processed using: (a) the proposed
Lockhart wavefolder model (RL = 50 kΩ); and (b) the proposed Serge wavefolder model. 5.1. Frequency-Domain Behavior A sample
rate Fs = 1 MHz was used to simulate analog behavior. Figure 13a shows the result of processing the same linear sweep using the trivial (i.e., non-antialiased)
Lockhart model at a standard audio rate of Fs = 44.1kHz. When compared to Figure 12a, we can clearly
observe the high levels of aliasing distortion introduced by the model. This is somewhat ameliorated
in Figure 13b, where the sweep has been processed at a sample rate of Fs = 88.2kHz (i.e., two-times
oversampling). Figure 13c,d shows the result of processing the sweep using the proposed antialiasing
method at audio rate and with two-times oversampling, respectively. As shown in these spectrograms,
there is a significant reduction in aliasing, particularly below the fundamental frequency. This behavior is
advantageous in music applications because at low frequencies the audibility of aliasing distortion
is only limited by the hearing threshold. On the other end of the spectrum, the masking effects of
harmonics will help suppress the audible effects of high-frequency aliases [65]. The spectrograms in Figure 14a,b show the outcome of processing the 1 V linear sweep with the
proposed Serge wavefolder model at audio rate and with two-times oversampling, respectively. When
compared with Figure 13a,b it is evident that the Serge wavefolder model is less susceptible to aliasing
distortion. This can be attributed to the fact that its transfer function is not as sharp as that of the
Lockhart, particularly when RL = 50 kΩ. Figure 14c,d shows the result of processing the linear sweep
using the antiderivative method at audio rate and with oversampling by two. In this case, operating at
audio rate yields very effective results as there are very few visible aliases left below the fundamental. When combined with oversampling by two the antiderivative method produces a nearly alias-free
spectrum for the measured frequency range. Appl. Sci. 2017, 7, 1328 15 of 23 15 of 23 Frequency (Hz)
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0
Frequency (Hz)
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0
Frequency (Hz)
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0
Frequency (Hz)
-80
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-40
-20
0
Figure 13. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Lockhart
wavefolder (RL = 50 kΩ) model: (a) at audio rate; (b) using two times oversampling; (c) with
antialiasing at audio rate; and (d) with antialiasing and oversampling by two. 5.1. Frequency-Domain Behavior 2017, 7, 1328 16 of 23 16 of 23 signal quality obtained by the proposed antialiasing method when applied to the Lockhart wavefolder,
even when operating at audio rate. Moreover, these measurements show that the performance of the
proposed method, when combined with two-times oversampling, is on par with oversampling by a
factor of 8. For all fundamental frequencies below approximately 4.2 kHz, the ANMR lies below the
−10 dB line. This range can be regarded as sufficient for musical applications if we consider that the
highest fundamental frequency on a standard grand piano is 4186.01 Hz (MIDI note C8). Figure 15. Measured A-weighted noise-to-mask ratios (ANMRs) for a range of sinusoidal waveforms
processed: (a) using the Lockhart wavefolder model (RL = 50 kΩ) under six different sampling rates;
and (b) using the Serge wavefolder model under two different sampling rates, with and without the
proposed antialiasing method. Values below the −10 dB threshold indicate lack of perceivable aliasing. Figure 15. Measured A-weighted noise-to-mask ratios (ANMRs) for a range of sinusoidal waveforms
processed: (a) using the Lockhart wavefolder model (RL = 50 kΩ) under six different sampling rates;
and (b) using the Serge wavefolder model under two different sampling rates, with and without the
proposed antialiasing method. Values below the −10 dB threshold indicate lack of perceivable aliasing. Figure 15b shows the measured ANMRs for the Serge wavefolder. When implemented at audio
rate, the output is free from perceivable aliasing for fundamental frequencies up to approximately
2 kHz. These measurements go in accordance with the Spectrogram in Figure 14a, which shows
aliasing is significantly more evident above this frequency. The use of the antiderivative method
yields results comparable to those of oversampling by a factor of two, with all measured fundamental
frequencies below approximately 4.6 kHz lying below the −10 dB aliasing threshold. Overall, these
results indicate the proposed Serge wavefolder model can operate at audio rate with the help of the
antiderivative method, therefore avoiding the need for oversampling. 5.1. Frequency-Domain Behavior Frequency (Hz)
-80
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-20
0
Frequency (Hz)
-80
-60
-40
-20
0 Figure 13. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Lockhart
wavefolder (RL = 50 kΩ) model: (a) at audio rate; (b) using two times oversampling; (c) with
antialiasing at audio rate; and (d) with antialiasing and oversampling by two. Frequency (Hz)
-80
-60
-40
-20
0
Frequency (Hz)
-80
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-40
-20
0
Frequency (Hz)
-80
-60
-40
-20
0
Frequency (Hz)
-80
-60
-40
-20
0
Figure 14. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Serge
wavefolder model: (a) at audio rate; (b) using two times oversampling; (c) with antialiasing at audio
rate; and (d) with antialiasing and oversampling by two. Figure 14. Spectrogram for a 1 V linear sweep from 20 Hz–5 kHz processed with the proposed Serge
wavefolder model: (a) at audio rate; (b) using two times oversampling; (c) with antialiasing at audio
rate; and (d) with antialiasing and oversampling by two. The performance of the proposed antialiasing method was further evaluated by computing
the A-weighted noise-to-mask ratio (ANMR) for a set of sinusoidal input signals processed by both
wavefolder models. The ANMR has been previously researched as a perceptually-informed measure
to evaluate the audibility of aliasing distortion [41,65]. The algorithm computes the power ratio in
decibels between the wanted harmonics and aliased components, but takes into account the masking
effects of the former. An A-weighting filter is applied to all signals prior to the evaluation of the ANMR
in order to account for the frequency-dependent sensitivity of hearing for low-level sounds. Signals
with an ANMR value below −10 dB are considered to be completely free from perceivable aliasing. A detailed account of this method can be found in [65]. Figure 15a compares the measured ANMRs for a set of sinusoidal inputs with fundamental
frequencies between 1 and 5 kHz processed by the Lockhart model at different sampling rates. The ideal alias-free signals required to compute these values were synthesized using Fourier analysis
and additive synthesis, as suggested in [41]. All signals were downsampled back to audio rate
(i.e., 44.1 kHz) prior to evaluation. A dashed horizontal line has been used to indicate the −10 dB
hearing threshold for aliasing distortion. In Figure 15a we can observe the significant increase in Appl. Sci. 5.2. Computational Costs The computational costs of the antialiased Lockhart wavefolder model were measured by porting
the algorithms into C code using the 128-bit long double data type. Table 5 shows the computation
times for a 1-s 100-Hz sinusoidal input processed using the proposed model for different peak
amplitude values. These results were computed by averaging the processing times of one hundred
implementations. All tests were performed under identical circumstances, using a fixed resistance
value of RL = 50 kΩ, the highest recommended value. From these results we can observe that
the complexity of the model does not depend on the input and that the overhead of implementing
the antialiasing method is minimal. When operating at audio rate, the added computation time is
approximately 1 ms for a 1-second simulation. Moreover, these time measurements show that the
antialiased Lockhart model, implemented at a sample rate Fs = 88.2 kHz, is approximately 3.5 times
faster than oversampling by a factor of 8 (i.e., Fs = 352.8 kHz) and nearly twice as fast as oversampling
by a factor of 4. Changing the value of RL did not affect the execution times of the algorithms. 17 of 23 Appl. Sci. 2017, 7, 1328 Table 5. Averaged computation times (in milliseconds) for the proposed Lockhart wavefolder model
(RL = 50 kΩ) implemented in C for a 1-s 100-Hz sinusoidal input sampled at different oversampling
(OS) rates and with different peak amplitude levels. Amplitude (V)
Audio Rate
Audio Rate
OSx2
OSx4
OSx8
OSx2
(ms)
w/Antialiasing (ms)
(ms)
(ms)
(ms)
w/ Antialiasing (ms)
1
11.5
12.5
23.4
46.6
92.9
25.4
5
11.6
12.6
23.3
46.8
92.7
25.3
10
11.5
12.6
23.7
46.7
92.9
25.5
15
11.5
12.7
23.5
46.5
92.9
25.5 For high values of RL, long double representation is necessary to account for the large values that
will result at the argument of the Lambert-W function. For smaller values, 64-bit precision will be
sufficient to accommodate most input levels of interest. For instance, when RL = 7.5 kΩthe signal at
the input of the proposed Lockhart wavefolder model can have a peak amplitude of up to 9 V. The measurements in Table 5 were conducted by synthesizing all input signals at the target rates. In practical implementations, oversampling will require additional pre- and post-filtering stages that
will further increase the computational costs of the system. 5.2. Computational Costs The complexity and costs of these filtering
stages will be directly proportional to the required oversampling factor. This constitutes another
advantage of the proposed antialiasing method. 6. Practical Synthesis Usage For simplicity, in this section, we assume the range of Vin to be fixed at
±1 V; therefore, all gain modulation is done using GS only. Figure 17. (a) Transfer function of the proposed Serge middle VCM; and (b) its output when driven by
a 100-Hz sinewave for GS = 6 and zero dc offset. Figure 17. (a) Transfer function of the proposed Serge middle VCM; and (b) its output when driven by
a 100-Hz sinewave for GS = 6 and zero dc offset. The spectrogram in Figure 18a shows the effect of increasing GS from 0 to 6 for a 150-Hz sinusoidal
input. This plot effectively depicts the rich harmonic patterns introduced by the system, which are
far more complex than those introduce by traditional waveshaping methods and offer a wide timbral
palette for sound synthesis. The fluctuations in energy at the fundamental and first few harmonics
indicate the gain values at which each new fold is introduced. Figure 17b shows the effect of introducing
a dc offset at the input of the system for a constant 200-Hz sinusoidal input. This result shows how
the use of a dc offset can extend the timbral possibilities of the system even further, by introducing
complex patterns consisting of both even and odd harmonics. Frequency (Hz)
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-50
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-10
0
(a)
(a)
Frequency (Hz)
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-20
-10
0
0
3
1
2
dc o↵set (V)
dc o↵set (V)
(b)
(b)
Figure 18. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed
Serge middle VCM with varying gain GS from 0–6; and (b) a 200-Hz sinewave processed with varying
gain GS from 0–3 and dc offset from 0–3 V. Figure 18. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed
Serge middle VCM with varying gain GS from 0–6; and (b) a 200-Hz sinewave processed with varying
gain GS from 0–3 and dc offset from 0–3 V. Now, although the Lockhart wavefolder was originally designed to operate as a standalone
unit, it can be adapted into a series topology with relative ease. Here, we propose using the
wavefolding structure shown in Figure 19 to expand the synthesis capabilities of the Lockhart
wavefolder. 6. Practical Synthesis Usage In practical sound synthesis applications, a single folding stage is rarely used, as the timbral variety
it can produce is quite limited. Most analog designs, for example the Intellijel µFold and the
aforementioned Yusynth Metalizer, employ several wavefolding stages arranged in series. The number
of stages varies according to the design, but typically cascades of two to six stages are used. As mentioned in Section 3, the Serge middle VCM utilizes six identical folding stages. Figure 16
shows a simplified block diagram representation of the Serge middle VCM based on the original
design [63]. Blocks labeled “SWF” correspond to the proposed Serge wavefolder model. An ad hoc
gain factor of four, not present in the original circuit, has been added to compensate for the scaling of
the signal introduced by the cascade of wavefolders. Vin[n]
Vin[n]
SWF
SWF
SWF
SWF
GS
GS
SWF
SWF
dc o↵set
dc o↵set
SWF
SWF
SWF
SWF
SWF
SWF
Vout[n]
Vout[n]
4
Figure 16. Block diagram representation of the Serge middle VCM. Blocks labeled “SWF” indicate the
Serge wavefolder model. Figure 16. Block diagram representation of the Serge middle VCM. Blocks labeled “SWF” indicate the
Serge wavefolder model. In cascaded wavefolder structures like the one shown in Figure 16, timbral control can be achieved
in two manners. The first is by adjusting the gain of the input waveform (using GS in this case). This parameter controls the amount of folds introduced, allowing the overall brightness of the sound
to be varied. It can be modulated in real-time to provide articulation to a sound similar to filtering
in subtractive synthesis or modulation index in FM synthesis. The second way to control timbre
is by adding a dc offset to the input of the wavefolder. This breaks the aforementioned symmetry
of the folding function and introduces even harmonics. When modulated by using, for example, a
low-frequency oscillator, this parameter provides an effect reminiscent of pulse-width modulation. Figure 17a shows the transfer function of the Serge middle VCM model for the case of zero dc offset. This plot was generated by defining Vin to have a constant value of 1 V and sweeping through values
of GS between −8 and 8. Figure 17b shows the output of the Serge middle VCM when driven by a Appl. Sci. 2017, 7, 1328 18 of 23 18 of 23 100-Hz sinusoid with GS = 6. 6. Practical Synthesis Usage This design, while not based on any existing circuit, is comparable to that of the Yusynth
Metalizer which also utilizes four Lockhart circuits in series [55]. The following paragraphs describe
the sections of this proposed topology. Its frequency-domain behavior is then examined and compared
with that of the Serge middle VCM. The blocks labeled “LWF” in Figure 19 correspond to the proposed Lockhart wavefolder model. In order for this cascade of Lockhart wavefolders to behave as expected, we need to make sure that the
individual folding stages satisfy two criteria. Firstly, the individual folders must provide approximately
unity gain for small input values, i.e., below the folding point, and approximately negative unity gain Appl. Sci. 2017, 7, 1328 19 of 23 19 of 23 beyond the folding point. Secondly, each stage should start folding at the same point with respect to
its individual input. beyond the folding point. Secondly, each stage should start folding at the same point with respect to
its individual input. tanh()
tanh()
LPF
LPF
Vin[n]
Vin[n]
Vout[n]
Vout[n]
1/3
1/3
3
fc
fc
RL
RL
LWF
LWF
LWF
LWF
LWF
LWF
LWF
LWF
GL
GL
dc o↵set
dc o↵set
Figure 19. Block diagram for the proposed VA cascaded Lockhart wavefolder topology. Blocks labeled
“LWF” and “LPF” indicate the Lockart wavefolder model and lowpass filtering, respectively. Figure 19. Block diagram for the proposed VA cascaded Lockhart wavefolder topology. Blocks labeled
“LWF” and “LPF” indicate the Lockart wavefolder model and lowpass filtering, respectively. We can meet these criteria with the proposed Lockhart model by selecting an appropriate value
for RL and adding static gain stages before and after the folding stages. These gain blocks will also
help compensate for the attenuation introduced by the folding operation. First, we choose a value of
RL for which the Lockhart wavefolder exhibits unity gain for small input values. Having found this
resistance value, the pre- and post-gain stages can be determined by measuring the value of |Vout|
at exactly the folding point. The pre-gain is taken to be approximately this value, and the post-gain
is taken to be its inverse. In Section 3.1, it was shown that for RL = 7.5 kΩthe Lockhart wavefolder
exhibits approximately unity gain below the folding point. This value leads to pre- and post-gains of
approximately 1/3 and 3, respectively. 6. Practical Synthesis Usage Figure 20a shows the transfer function of the proposed structure measured at the output of the
post-gain block. We can observe how the folds introduced by this structure are evenly distributed,
unlike those in Figure 17a. As with the Serge middle VCM, timbral control is achieved by modulating
the value of GL and by adding a dc offset. The static gain blocks ensure the amplitude of the folded
output is bounded between approximately ±1 V for values of GL between −10 and 10 (assuming once
more that Vin has a peak amplitude of 1 V). Figure 20b shows the time-domain result of processing a
100-Hz sinusoidal input with the proposed structure for GL = 10 and zero dc offset. In this particular
design, additional timbral control can be achieved by modulating the value of RL. Figure 20. (a) Transfer function of the proposed cascaded Lockhart wavefolder structure measured
after the post-gain block; and (b) its output when driven by a 100-Hz sinusoidal input for GL = 10 and
zero dc offset. Figure 20. (a) Transfer function of the proposed cascaded Lockhart wavefolder structure measured
after the post-gain block; and (b) its output when driven by a 100-Hz sinusoidal input for GL = 10 and
zero dc offset. Lastly, we add two optional blocks. The first is a tanh() function after the post-gain block to
model the behavior of an output buffering stage and to limit the range of the output waveform. This tanh() block can also be antialiased using the antiderivative method described by Equation (45). The antiderivative of the tanh() function is given by log(cosh()) [48]. The second optional block is a
static one-pole lowpass filter with a cutoff at fc = 1.3 kHz whose purpose is to act as a simple tone
control. A similar static filtering stage can be found at the output of the Buchla 259 wavefolder [17]. The s-domain transfer function of this filtering stage is defined as H(s) =
wc
s + wc
,
(51) H(s) =
wc
s + wc
, (51) where wc = 2π fc. where wc = 2π fc. Appl. Sci. 2017, 7, 1328 20 of 23 20 of 23 Finally, we examine the time-varying behavior of the proposed structure by considering the case
of a 150-Hz input sinewave with variable gain GL and dc offset. Figure 21 shows the spectrogram that
results from varying GL from 0 to 15. 6. Practical Synthesis Usage As expected, the system introduces complex harmonic patterns
similar to those shown in Figure 18a. Likewise, Figure 18b demonstrates the effect of varying GL from
0 to 10 while simultaneously increasing the level of dc offset from 0 to 5 V. This response is comparable
to that of the Serge middle VCM (see Figure 18b). Frequency (Hz)
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-50
-40
-30
-20
-10
0
0
1
2
3
4
5
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-60
-50
-40
-30
-20
-10
0
dc o↵set (V)
dc o↵set (V)
(b)
(b)
Figure 21. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed
cascaded Lockhart topology with varying gain GL from 0 to15; and (b) a 200-Hz sinewave processed
with varying gain GS from 0 to 0 and dc offset from 0 to 5 V. -80
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-30
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0
0
1
2
3
4
5
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0
dc o↵set (V)
dc o↵set (V)
(b)
(b) Figure 21. Spectrogram of: (a) a 150-Hz sinewave with peak amplitude 1 V processed by the proposed
cascaded Lockhart topology with varying gain GL from 0 to15; and (b) a 200-Hz sinewave processed
with varying gain GS from 0 to 0 and dc offset from 0 to 5 V. A real-time demo of the proposed Lockhart wavefolder topology implemented using Max/MSP
and Gen is available at http://research.spa.aalto.fi/publications/papers/smc17-wavefolder. A real-time demo of the proposed Lockhart wavefolder topology implemented using Max/MSP
and Gen is available at http://research.spa.aalto.fi/publications/papers/smc17-wavefolder. Supplementary Materials: The following are available online at http://research.spa.aalto.fi/publications/
papers/smc17-wavefolder:
a real-time Max/MSP demo of the proposed Lockhart wavefolder topology
implemented using Gen~and a MATLAB implementation of the Lambert-W function using Fritsch’s iteration. 7. Conclusions In this work, we have explored the behavior of two West Coast synthesizer circuits: the Lockhart
and Serge wavefolders. By means of circuit analysis, we have derived closed-form expressions for the
characteristic transfer functions of both systems. These transfer functions were validated against SPICE
simulations implemented using LTspice. The results obtained indicate a good match between the
proposed models and their corresponding SPICE simulations. In addition to this, we observed that the
behavior of both circuits is very similar, despite the fact that their designs are fundamentally different. The issue of aliasing caused by wavefolding in the digital domain was treated by incorporating
the first-order antiderivative method. Within the context of the Lockhart wavefolder, it was shown
that the proposed antialiased model is perceptually free from the effects of aliasing distortion when
implemented at a sampling rate of Fs = 88.2 kHz. A thorough evaluation of the proposed Lockhart
model indicates that this configuration yields a signal quality equivalent to that of oversampling by
a factor of eight (i.e., Fs = 352.8 kHz) at nearly a fourth of the computational expenses. For the case
of the Serge wavefolder, the use of the antiderivative method produces an increase in signal quality
equivalent to that of oversampling by a factor of two (i.e., Fs = 88.2 kHz). Furthermore, a recommended synthesis topology built around the Lockhart model consisting of
four cascaded wavefolding stages, a saturator and a lowpass filter was presented. This topology
was compared against a model of the Serge middle VCM built using six wavefolding stages. These structures illustrate the capabilities of wavefolding in a synthesis environment. However,
it should be noted that the discussed topologies are not unique, as they can be modified according to
the needs of the particular application. This effectively showcases the flexibility of VA models. Supplementary Materials: The following are available online at http://research.spa.aalto.fi/publications/
papers/smc17-wavefolder:
a real-time Max/MSP demo of the proposed Lockhart wavefolder topology
implemented using Gen~and a MATLAB implementation of the Lambert-W function using Fritsch’s iteration. 21 of 23 Appl. Sci. 2017, 7, 1328 21 of 23 Acknowledgments: The authors would like to thank Ken Stone for the valuable correspondence about the origins
of the Lockhart circuit and Geoffrey Gormond for fruitful discussions on the behavior of the Serge VCM. Special
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implications for oscillator algorithm design. J. Acoust. Soc. Am. 2012, 132, 2721–2733. c⃝2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Prevalence, risk factors and health outcomes associated with polypharmacy among urban community-dwelling older adults in multi-ethnic Malaysia
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RESEARCH ARTICLE Abstract Citation: Lim LM, McStea M, Chung WW, Nor
Azmi N, Abdul Aziz SA, Alwi S, et al. (2017)
Prevalence, risk factors and health outcomes
associated with polypharmacy among urban
community-dwelling older adults in multi-ethnic
Malaysia. PLoS ONE 12(3): e0173466. doi:10.1371/journal.pone.0173466 Objectives The aim of this study was to determine the prevalence, risk factors and health outcomes
associated with polypharmacy in a cohort of urban community-dwelling older adults receiv-
ing chronic medications in Malaysia. Received: November 24, 2016
Accepted: February 22, 2017
Published: March 8, 2017 Prevalence, risk factors and health outcomes
associated with polypharmacy among urban
community-dwelling older adults in multi-
ethnic Malaysia Li Min Lim1, Megan McStea2,3, Wen Wei Chung4, Nuruljannah Nor Azmi3, Siti Azdiah Abdul
Aziz3,5, Syireen Alwi1, Adeeba Kamarulzaman3,6, Shahrul Bahyah Kamaruzzaman2,6, Siew
Siang Chua1, Reena Rajasuriar1,3,7* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 2 The
Malaysian Elders Longitudinal Research (MELOR) Group, Faculty of Medicine, University of Malaya, Kuala
Lumpur, Malaysia, 3 Centre of Excellence for Research in AIDS (CERiA), University of Malaya, Kuala
Lumpur, Malaysia, 4 Pharmacy Department, University Malaya Medical Centre, Kuala Lumpur, Malaysia,
5 Faculty of Pharmacy, National University of Malaysia (UKM), Kuala Lumpur, Malaysia, 6 Department of
Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 7 Peter Doherty Institute for
Infection and Immunity, Melbourne University, Melbourne, Australia Methods Copyright: © 2017 Lim et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited. This was a baseline study in the Malaysian Elders Longitudinal Research cohort. The inclu-
sion criteria were individuals aged 55years and taking at least one medication chronically
(3 months). Participants were interviewed using a structured questionnaire during home
visits where medications taken were reviewed. Health outcomes assessed were frequency
of falls, functional disability, potential inappropriate medication use (PIMs), potential drug-
drug interactions (PDDIs), healthcare utilisation and quality of life (QoL). Risk factors and
health outcomes associated with polypharmacy (5 medications including dietary supple-
ments) were determined using multivariate regression models. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This work was supported by the
University of Malaya (UM) High Impact Research
(HIR) Grant from the Department of Higher
Education, Ministry of Education, Malaysia [UM.C/
625/1/HIR/MOHE/ASH/02] to SBK and the
University Malaya Research Grant from UM
[UMRG RP029-14HTM] to RR. Background Polypharmacy has been associated with increased morbidity and mortality in the older
population. Editor: Alessandra Marengoni, University of
Brescia, ITALY * reena@um.edu.my Polypharmacy among community-dwelling older adults 56.9% of the cohort. The risk factors associated with increasing medication use were
increasing age, Indian ethnicity, male, having a higher number of comorbidities specifically
those diagnosed with cardiovascular, endocrine and gastrointestinal disorders, as well as
supplement use. Health outcomes significantly associated with polypharmacy were PIMS,
PDDIs and increased healthcare utilisation. Competing interests: The authors have declared
that no competing interests exist. Conclusion A significant proportion of older adults on chronic medications were exposed to polyphar-
macy and use of dietary supplements contributed significantly to this. Medication reviews
are warranted to reduce significant polypharmacy related issues in the older population. Introduction The older population aged 60 years and above is expected to grow by 56% worldwide between
2015 and 2030, with the fastest growth in urban settings of developing regions [1]. In Malaysia,
older populations have been projected to grow from 9.2% in year 2015 to almost a quarter of the
total population (23.6%) by the year 2050 and this has been anticipated to add considerable
demands on the countries’ health services [1, 2]. Among others, age related multi-morbidity and
disability in older adults will increase the use of medications [3, 4] and subsequently the risk of
polypharmacy which may be conceptually defined as the use of more medications than is medically
necessary, though there is currently no universal definition for this in the literature [5]. Polyphar-
macy is a concern especially in older adults as they are prone to age- and disease-related pharmaco-
kinetic and pharmacodynamic changes, and are thus more sensitive to drug therapy [6]. Polypharmacy in older individuals is strongly associated with multiple negative health out-
comes which include increased healthcare costs [7], functional impairment, falls and fractures
[8], harmful drug interactions [9] and adverse drug reactions [10, 11]. Intervention studies
which focused on reducing polypharmacy among older persons either with the involvement of
a pharmacist or a multidisciplinary team, have largely reported significant improvements in
the quality of medication use [12–15]. Few studies have extended such observation to include
health outcomes, but the findings have not been consistent [15, 16]. Despite a growing aging population and the known risks of increased medication related
issues in this group, studies on polypharmacy among the older population in Malaysia have been
limited to hospital settings and institutions of aged care [17–19]. Thus far, no studies have been
found on older community dwelling individuals. In addition, only a few studies have included
the use of dietary supplements in its assessment of polypharmacy despite their potential to cause
clinically significant drug interactions and adverse drug events [9]. In multi-ethnic Malaysia, the
use of herbal and dietary supplements are highly prevalent as reported in other Asian settings
[20, 21] and these are often utilised as adjunct therapies for chronic health conditions [22–25]. Therefore, the present study aimed to determine the prevalence, risk factors and health outcomes
associated with polypharmacy among urban community-dwelling older adults receiving chronic
medications in Malaysia and further examined the role of dietary supplements in this context. Materials and methods Study design, setting and population Results A total of 1256 participants were included with a median (interquartile range) age of 69(63–
74) years. The prevalence of polypharmacy was 45.9% while supplement users made up 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Data collection Participants’ baseline socio-demographic data and medical history were extracted from the
MELoR database. Information on all medications used including dietary supplements (brand
names and dosages) were verified by reviewing prescriptions and the actual medications dur-
ing the home visits. Medications were classified according to the WHO Anatomical Therapeu-
tic Chemical (ATC) classification system [27]. Dietary supplements which could not be
classified according to the ATC system were coded as “others”. Two researchers independently
classified the medications and any discrepancies were resolved by a third researcher, with the
consensus of the initial two researchers. Polypharmacy among community-dwelling older adults aspects of aging faced by the urban community-dwelling older adults in Malaysia. Details of
this cohort have been published previously [26]. Briefly, participants were individuals ran-
domly identified through the Malaysian electoral roll at three representative urban parliamen-
tary constituencies (Petaling Jaya South, Petaling Jaya North and Lembah Pantai), aged 55
years and above in 2013 and stratified by the three main ethnicities in Malaysia; Malays, Chi-
nese and Indians. 8769 invitations were sent to the main ethnic groups and volunteers were
invited through aged focused community groups. A total of 5815 participants with contactable
addresses were identified through these efforts. Of these, only 3334 were eligible for the study (cri-
teria below), had not moved to a new address or passed away. An age cut-off of 55 years was cho-
sen because this age range enabled the analysis of issues in older individuals as they approached
retirement. In Malaysia, the voluntary retirement age is 55 years and mandatory retirement age is
60 years. Individuals who were bed-bound, unable to be transported to the research centre for
assessment and unable to communicate or answer questions due to advanced dementia or severe
speech impediments, were excluded from the MELoR study. In addition, only individuals who
were using at least one chronic medication, whether prescription or non-prescription and dietary
supplements, were included in this study. Dietary supplements were defined as vitamins, miner-
als, amino acids, herbs and other botanicals as described previously [22]. Following written informed consent to participate in the study, all participants answered a
questionnaire (either in English, Malay, Chinese or Tamil language) executed in the setting of
their homes by trained interviewers. Subsequently, participants attended a study clinic in the
University Malaya Medical Centre (UMMC) where further health assessments and biochemi-
cal screenings were performed. The questionnaire covered aspects of socio-demographic, gen-
eral health, medical and medication history, healthcare utilisation and a series of validated
questionnaires to assess falls, functional disability and quality of life (QoL). Recruitment for
MELoR is ongoing and participants for the present study were selected among participants
recruited consecutively from October 2013 till June 2015. Study design, setting and population This was a baseline study conducted in the Malaysian Elders Longitudinal Research (MELoR)
cohort. MELoR is a multi-disciplinary study conducted to explore the multi-dimensional 2 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Polypharmacy among community-dwelling older adults used as it provides improved guidance on drug issues related to drug-drug interactions and
medications to be avoided in older individuals with impaired renal function. Two researchers
independently assessed participants’ medications for PIMs use and any discrepancies were
resolved by a third researcher with their consensus. Assessment of kidney function were based
on laboratory results obtained during the participants’ visit to UMMC for biochemical screen-
ing. As assessments for PIM use were done retrospectively, some assumptions were made
when medical or medication history were incomplete for specific assessments. These assump-
tions are detailed in S1 File. Potential drug-drug interactions (PDDIs) were assessed using the Thompson Microme-
dex1 2.0 interaction database (Truven Health Analytics Inc., Greenwood Village, CO, USA). Only interactions listed as major or contraindicated with clinically relevant and potentially
serious consequences were included in the present study. PDDIs were assessed using this inter-
action database as it included potential interactions with dietary supplements which were not
captured in the updated Beers Criteria [29]. Falls were defined based on self-reported responses to the question “Have you fallen in the
past 12 months?” Functional disability was assessed using the Modified Katz Index of Independence in Activ-
ities of Daily Living (ADL) [30] and Lawton Instrumental Activities of Daily Living (IADL)
[31]. For ADL, participants were classified as dependent if they reported needing help with
one or more daily living activities: walking, bathing, personal grooming, dressing, eating, get-
ting from bed to chair and using the toilet. For IADL, participants who reported low function
in one or more of the following activities were classified as dependent: using telephone, shop-
ping, preparing food, doing housework, travelling, taking medicine and managing money. Healthcare utilisation was assessed based on the participants’ reported number of visits to
any of the following health facilities in the past 12 months: public hospital or clinic, private
hospital or clinic and traditional or alternative medical health practitioners. Quality of life (QoL) was assessed using 12 items which originated from the CASP-19 (Con-
trol, Autonomy, Self-realisation and Pleasure) questionnaire [32]. CASP-12 as previously
described [33] is based on 12 Likert-like scale items which assessed four theoretically derived
dimensions of QoL in older adults: control, autonomy, self-realisation and pleasure. Each item
was numerically coded so that the most positive response was scored as 3 while the most nega-
tive response as 0. This outcome was analysed as a continuous variable (score range of 0–36),
with lower scores associated with poorer QoL. Chronic health problems were assessed based on self-reported diseases by participants and
verified by the medications taken. Payment method for healthcare services was assessed based on participants reported mode
of payment when they utilized any healthcare facility in the preceding 12 months. Study definitions and outcome measures Polypharmacy was defined as the concurrent use of five or more medications [28]. These
included prescribed, non-prescribed and over-the counter (OTC) medications including die-
tary supplements such as vitamins, minerals, herbals and other botanicals. Combined formula-
tions (multiple active ingredients in a preparation) were considered as a single medication. This definition for polypharmacy is among the most widely used in the literature, is objective
and allows comparability across different studies. Potential inappropriate medications (PIMs) use was defined according to the American
Geriatrics Society 2015 Updated Beers Criteria [29]. This guideline lists medications to be
avoided in older adults in all settings except in hospice and palliative care, with the aim to
reduce the risk of adverse drug events in older individuals. The updated Beers Criteria was 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Characteristics of participants The median (interquartile range, IQR) age of the participants was 69 (63–74) years, ranging
from 55 to 97 years. The participants were almost evenly distributed among the three main
ethnic groups in Malaysia that is, Indian (35.6%), Chinese (33.3%) and Malay (30.5%). The
median (IQR) number of comorbidities were 3 (2–4) and the maximum reported was 11. A
summary of the participants’ characteristics is shown in Table 1. The three most common
chronic health conditions reported were cardiovascular disorders (1002 participants; 79.8%),
endocrine disorders (472; 37.6%), bone and joint disorders (359; 28.6%). The three most com-
monly used medication classes were those that act on the cardiovascular system (1030; 82.0%),
alimentary tract and metabolism (860; 68.5%), blood and blood forming organs (371; 29.5%). A complete list of medication classes used are detailed in S1 Table. Polypharmacy among community-dwelling older adults produced a p<0.250 in the univariate analysis were included in the final multivariate model. For the analysis of health outcomes associated with polypharmacy, models were adjusted for
the number of comorbidities, age, ethnicity and gender. All statistical analyses were performed
using the Statistical Package for Social Science (SPSS) version 20.0 (SPSS Inc., Chicago, IL,
USA) and Stata Statistical Software: Release 14 (StataCorp LP, College Station, TX). Statistical
significance was defined as p<0.050. Results A total of 1504 participants aged 55 years and above were recruited into the MELoR study
from October 2013 through June 2015. 55% of eligible and contactable participants declined to
participate either due to time constraints or inability to attend the research clinic. There was
no difference in terms of gender and ethnicity (p>0.05 for all on chi-square analysis) among
those who declined to participate compared to those included in the study. Of the 1504
recruited, 248 individuals did not receive any medications and hence, only 1256 participants
were included in the present study. Ethics and sample size calculation The study protocol was approved by the Medical Ethics Committee of University Malaya Med-
ical Centre (MEC 943.6) and all participants provided informed written consent. Based on pre-
vious reported rates of polypharmacy in Malaysia in a hospital setting (46%), we estimated a
minimum sample size of 400 would be required for this study using the binomial exact method
with a 95% level of confidence and a 90% power. Statistical analysis Graphical tests were used to assess the normality of the data distribution. Parameters which
were missing in 1% of participants or less were considered as negligible [34]. The number of
medications taken as a count variable was used as a surrogate measure of polypharmacy in the
analysis of risk factors and health outcomes associated with polypharmacy. Multivariate Pois-
son regression analysis was performed to identify risk factors associated with polypharmacy. Ordinal logistic regression was used to assess the health outcomes associated with polyphar-
macy specifically falls, functional disability, PIMs and PDDIs while the association of poly-
pharmacy with healthcare utilisation and QoL were assessed using multivariate linear
regression. In the analysis of risk factors associated with polypharmacy, only variables that 4 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Prevalence of polypharmacy and supplement use A total of 5804 medications (prescribed or non-prescribed medications and dietary supple-
ments) were used, with a median of 4 (2–6) medications per person and a maximum of 20. The prevalence of polypharmacy was 45.9% with 576 individuals using at least five medica-
tions. Among those with polypharmacy exposure, 499 (86.6%) received 5 to 9 medications, 65
(11.3%) received 10 to 14 medications while 12 (2.0%) received 15 or more. The percentage of participants using 5 medications or more increased with age (Table 1). Polypharmacy was also more prevalent among Indian participants compared to the Malay and
Chinese. In terms of chronic health conditions, more than 70.0% of the participants who
reported gastrointestinal, renal and psychiatric disorders were also taking 5 medications or
more. In addition, 76.0% of the participants with PDDIs were classified in the polypharmacy
cohort and also 67.8% of those with PIMs. A majority of the participants (54.4%) who reported 5 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Polypharmacy among community-dwelling older adults Table 1. Characteristics of all participants included in the study (n = 1256) as well as those with polypharmacy (n = 576) and dietary supplement
users (n = 715). Characteristics
Total, n(column %) or
median (IQR)
Polypharmacy, n(row %) or
median (IQR)
Dietary supplement users, n(row %)
or median (IQR)
Total
1256 (100.0)
576 (45.9)
715 (56.9)
Age (n = 1256)
69 (63–74)
71 (66–76)
70 (64–75)
55–59
124 (9.9)
39 (31.5)
76 (61.3)
60–64
243 (19.4)
79 (32.5)
126 (51.9)
65–69
279 (22.2)
122(43.7)
160 (57.3)
70–74
300 (23.9)
151 (50.3)
178 (59.3)
75–79
202 (16.1)
118 (58.4)
119 (58.9)
80+
108 (8.6)
67 (62.0)
65 (60.2)
Gender (n = 1256)
Male
532 (42.3)
259 (48.7)
269 (50.6)
Female
724 (57.7)
317 (43.8)
446 (61.6)
Ethnic (n = 1255)
Malay
383 (30.5)
137 (35.8)
133 (34.7)
Chinese
418 (33.3)
170 (40.7)
294 (70.3)
Indian
447 (35.6)
265 (59.3)
284 (63.5)
Others
7 (0.6)
4 (50.0)
4 (50.0)
Highest education level (n = 1252)
Primary or No Formal
326 (26.0)
133 (40.8)
121 (37.1)
Secondary
538 (43.0)
232 (43.1)
317 (58.9)
Tertiary
388 (31.0)
210 (54.1)
275 (70.9)
Employment status (n = 1255)
No
1021 (81.4)
483 (47.3)
572 (56.0)
Yes
234 (18.6)
93 (39.7)
142 (60.7)
Payment method for healthcare service in the last
12 months† (n = 1145)
Used free service
56 (4.9)
32 (57.1)
34 (60.7)
Self/Out of pocket
591 (51.6)
252 (42.6)
332 (56.2)
Employer
292 (25.5)
156 (52.7)
156 (53.4)
Relatives
127 (11.1)
64 (50.4)
67 (52.8)
Welfare
112 (9.8)
62 (55.4)
67 (59.8)
Insurance
13 (1.1)
4 (30.8)
10 (76.9)
Non-governmental/Religious organizations
4 (0.4)
3 (75.0)
4 (100)
Chronic health problems†
(n = 1256)
Cardiovascular disorders
1002 (79.8)
607 (50.6)
519 (51.8)
Endocrine disorders
472 (37.6)
296 (62.7)
229 (48. Polypharmacy among community-dwelling older adults Table 1. (Continued)
Characteristics
Total, n(column %) or
median (IQR)
Polypharmacy, n(row %) or
median (IQR)
Dietary supplement users, n(row %)
or median (IQR)
Katz ADL
44 (3.5)
25 (56.8)
27 (61.4)
Lawton IADL
388 (30.9)
210 (54.1)
192 (49.5)
PIMs use (n = 1256)
400 (31.8)
271 (67.8)
177 (44.3)
PDDIs (n = 1256)
281 (22.4)
215 (76.5)
141 (50.2)
Smoking status (n = 1236)
Never
985 (79.7)
446 (45.3)
584 (59.3)
Current smoker
96 (7.8)
36 (37.5)
40 (41.7)
Ex-smoker
155 (12.5)
87 (56.1)
78 (50.3)
Alcohol consumption (n = 1250)
Never
889 (71.1)
388 (43.6)
467 (52.5)
Yes
280 (22.4)
147 (52.5)
204 (72.9)
Used to
81 (6.5)
37 (45.7)
40 (49.4)
More than 1 comorbidity
979 (78.0)
521 (53.2)
636 (65.0)
Using dietary supplements
715 (56.9)
401 (56.1)
-
Quality of life (QoL) (n = 124), CASP 12 score
28 (24–31)
28 (24–31)
28 (25–31)
Number of visits to any healthcare facility in the last
12 months† (n = 1202)
4 (2–6)
4 (2–7)
3 (1–6)
Number of comorbidities (n = 1256)
3 (2–4)
3 (2–5)
3 (1–4)
Number of prescribed and non-prescribed drugs
(n = 1256)
3 (1–5)
5 (3–6)
2 (1–4)
Number of dietary supplements (n = 1256)
1 (0–2)
2 (0–3)
2 (1–3)
Total number of medications (n = 1256)
4 (2–6)
6 (5–8)
5 (3–7)
IQR: interquartile range; ADL: activities of daily living; IADL: instrumental activities of daily living; PIMs: potential inappropriate medications; PDDIs:
potential drug-drug interactions
†More than one choice can be chosen. d i 10 1371/j
l
0173466 001 IQR: interquartile range; ADL: activities of daily living; IADL: instrumental activities of daily living; PIMs: potential inappropriate medications; PDDIs:
potential drug-drug interactions
†More than one choice can be chosen. having functional disability, assessed using either Katz ADL or Lawton IADL, and almost half
of those who reported having falls, were categorised in the polypharmacy group. More than half of the participants (56.9%) reported the use of at least one dietary supple-
ment, with the median (IQR) of 1 (0–2) in the total cohort and 2 (0–3) in the polypharmacy
group. Dietary supplements made up a total of 1782 or 30.7% of the total medications used
(details in S2 Table) and were utilised by 401 participants (69.6%) who had polypharmacy
exposure. When analysed by ethnic background, the proportion of Chinese who reported the
use of dietary supplements was almost double that of the Malays (Table 1). The chronic condi-
tions associated with the highest usage of dietary supplements were oncologic disorders, fol-
lowed by bone and joint disorders, and gastrointestinal disorders. Among participants with
PDDIs and PIMs, 50.2% and 44.3% respectively, reported the use of supplements. 5)
Bone and joint disorders
359 (28.6)
211 (58.8)
246 (68.5)
Urologic disorders
321 (25.6)
170 (53.0)
182 (56.7)
Ophthalmic disorders
237 (18.9)
130 (54.9)
134 (56.5)
Respiratory disorders
104 (8.3)
58 (55.8)
53 (51.0)
Oncologic disorders
84 (6.7)
47 (56.0)
72 (85.7)
Gastrointestinal disorders
64 (5.1)
45 (70.3)
43 (67.2)
Renal disorders
39 (3.2)
30 (76.9)
21 (53.8)
Neurologic disorders
22 (1.8)
14 (63.6)
12 (54.5)
Falls in the last 12 months (n = 1256)
294 (23.4)
146 (49.7)
151 (51.4)
Functional disability
(Continued) nts included in the study (n = 1256) as well as those with polypharmacy (n = 576) and dietary supplement (Continued) PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 6 / 18 vities of daily living; IADL: instrumental activities of daily living; PIMs: potential inappropriate medications; PDDIs: Polypharmacy among community-dwelling older adults variables were constant. Similarly, when the other variables were held constant in the model,
male participants had an incidence rate ratio (for the number of medications taken) of 1.09 times
that of female participants (an increase of 9%). As participants increased the number of dietary
supplements taken, they had a 19% increase in the total number of medications they were taking
(IRR 1.19; 95% CI: 1.15, 1.23, p<0.001). Similarly, as the number of comorbidities increased,
there was also an increased use of medications (IRR 1.14; 95% CI: 1.11, 1.17, p<0.001). Specifi-
cally, participants who had cardiovascular disorders (IRR 1.23; 95% CI: 1.14, 1.33, p<0.001),
endocrine disorders (IRR 1.47; 95% CI: 1.31, 1.64, p<0.001) and gastrointestinal disorders (IRR
1.13; 95% CI: 1.03, 1.24, p = 0.010) had a higher incidence rate ratio for the number of medica-
tions taken compared to those without these disorders. However, those with urologic disorders
were associated with a lower number of medication use. Health outcomes associated with polypharmacy Multivariate logistic analysis found that PIMs (OR 1.27; 95% CI: 1.20, 1.34, p<0.001) and
PDDIs (OR 1.34; 95% CI: 1.26, 1.42, p<0.001) were significantly associated with the increase
in number of medications used after adjusting for age, gender, ethnicity and the number of
comorbidities but not whether falls occurred in the past 12 months or functional impairment
existed (Table 3). Multivariate linear regression models found a significant association between
the increase in number of medications used and healthcare utilisation (β = 0.44; 95% CI: 0.30,
0.59, p <0.001) but not the quality of life scores. In a similar analysis performed for prescription/non-prescription drugs and dietary supple-
ments separately, it was found that increased supplement use remained significantly associated
with PDDIs but quality of life scores also improved (data not shown). The health impact asso-
ciated with prescription/non-prescription polypharmacy were similar to that found when the
total medication used was analysed. Risk factors associated with polypharmacy Risk factors associated with increasing medication use as a surrogate of polypharmacy are
summarised in Table 2. Multivariate Poisson regression analysis identified an increasing num-
ber of medications use as age increased compared to those aged 55–64 (p<0.001) when
adjusted for the other variables in the model. Malays (incidence rate ratio, IRR 0.89; 95% confi-
dence interval, CI: 0.30, 0.94,) and Chinese (IRR 0.83; 95% CI: 0.78, 0.88) had a lower inci-
dence rate ratio of medication use when compared to Indians (p<0.001) whilst all the other 7 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Discussion This study assessed the prevalence, risk factors and health outcomes associated with polyphar-
macy, including the contributory role of dietary supplement use among older individuals tak-
ing chronic medications in the community setting in Malaysia. The study found a high
prevalence of polypharmacy (46%) with dietary supplements constituting a third of the total
number of medications used in older individuals. Risk factors independently associated with
increased medication use were advanced age, being male, increasing number of comorbidities
specifically cardiovascular, endocrine, and gastrointestinal disorders, and dietary supplement
use. As for ethnic groups, Chinese and Malays were associated with lower risk of polyphar-
macy compared to Indians as well as individuals diagnosed with urologic disorders. The health
outcomes associated with increased medication use were potential inappropriate medication
use (PIMs), potential drug-drug interactions (PDDIs) and increased healthcare utilisation
while increased medication use was not found to be associated with falls, functional disability
and quality of life in this cohort. Increased use of dietary supplement was independently asso-
ciated with increased PDDIs and improved quality of life scores after adjusting for age, gender,
ethnicity and number of comorbidities. The prevalence of polypharmacy in older individuals varied between 39% and 89% in the
Asian regions, depending on the definition of polypharmacy used, the age range of the partici-
pants and the research settings [28, 35–39]. Prior studies on polypharmacy among the Malay-
sian elderly people, reported rates between 39% and 55% in the hospital setting [17, 19] but
currently, no data is available for community dwelling individuals. Studies assessing polyphar-
macy in the community are logistically harder to conduct and reports from other countries 8 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Polypharmacy among community-dwelling older adults Table 2. Risk factors associated with polypharmacy among urban community-dwelling older adults. Variables
Univariate Poisson regression
Multivariate Poisson regression
Crude IRR (95%CI)
p-value
Adjusted IRR (95%CI)
p-value
Number of dietary supplements
1.13 (1.11–1.15)
<0.001
1.19 (1.15–1.23)
<0.001
Number of comorbidities
1.15 (1.13–1.16)
<0.001
1.14 (1.11–1.17)
<0.001
Ethnic
<0.001
<0.001
Indian (reference)
1.00
1.00
Malay
0.73 (0.67–0.79)
0.89 (0.30–0.94)
Chinese
0.77 (0.71–0.83)
0.83 (0.78–0.88)
Age
<0.001
<0.001
55–59 (reference)
1.00
1.00
60–64
1.05 (0.93–1.21)
1.05 (0.95–1.15)
65–69
1.27 (1.11–1.44)
1.11 (1.01–1.22)
70–74
1.47 (1.29–1.67)
1.23 (1.12–1.34)
75–79
1.55 (1.36–1.77)
1.28 (1.15–1.41)
80+
1.63 (1.40–1.90)
1.29 (1.13–1.46)
Chronic health problems†
Cardiovascular disorders
1.42 (1.28–1.58)
<0.001
1.23 (1.14–1.33)
<0.001
Endocrine disorders
1.48 (1.38–1.58)
<0.001
1.47 (1.31–1.64)
<0.001
Bone and joint disorders
1.26 (1.17–1.35)
<0.001
_
Urologic disorders
1.12 (1.03–1.21)
0.006
0.91 (0.86–0.97)
0.002
Ophthalmic disorders
1.15 (1.06–1.25)
0.001
–
Respiratory disorders
1.13 (1.00–1.28)
0.051
_
Oncologic disorders
1.12 (1.03–1.32)
0.014
_
Gastrointestinal disorders
1.48 (1.29–1.68)
<0.001
1.13 (1.03–1.24)
0.010
Renal disorders
1.59 (1.36–1.85)
<0.001
–
Neurologic disorders
1.31 (0.97–1.77)
0.077
–
Psychiatric disorders
1.08 (0.71–1.64)
0.714
–
Smoke cigarettes
0.002
No (reference)
1.00
–
_
Current smoker
0.86 (0.76–0.97)
0.013
_
Ex-smoker
1.125 (1.01–1.23)
0.031
_
Payment method for healthcare service in last 12 months†
Used Free Service
1.14 1.01–1.28)
0.044
–
Self/Out of pocket
0.90 (0.84–0.97)
0.004
–
Employer
1.14 (1.05–1.23)
0.001
–
Relative
1.09(0.98–1.23)
0.123
–
Welfare
1.14 (1.02–1.28)
0.024
_
Insurance
0.73 (0.55–0.97)
0.028
_
Non-governmental/ religious organization
3.59 (0.37–34.56
0.270
_
Alcohol consumption history
0.01
No (reference)
1.00
–
Yes
1.10 (1.02–1.19)
0.013
-
Used to
1.17 (1.01–1.35)
0.037
Employment†
0.88 (0.81–0.97)
0.009
–
Highest education level
<0.001
Primary or below
1.00
_
_
Secondary
1.06(0.98–1.15)
0.170
_
Tertiary
1.22 (1.12–1.33)
<0.001
_
(Continued) PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 9 / 18 Notably, 1 in 3 older
individuals who were taking chronic medications in the community setting used five or more
medications concurrently, with 1 in 2 reporting dietary supplement use. Additionally, more
than 30% of the cohort experienced PIMs similar to a prior study among elderly nursing home
residents in Malaysia [42] while one fifth were exposed to clinically significant PDDIs. These
data highlight high rates of medication related issues among the community-dwelling older
individuals in Malaysia, which currently does not have a national programme for medication
review in the elderly population, unlike many other countries [43–45]. Given that Malaysia
will experience a sizable increase in their older population over the next decade, the introduc-
tion of such a programme is timely and the findings from this study would provide baseline
data to monitor the changes over time. Table 3. Health outcomes associated with polypharmacy among urban-community dwelling older adults. Health outcomes associated with polypharmacy
Categorical variables
Crude OR (95%CI)
p-value
†Adjusted OR (95%CI)
p-value
PIMs*
1.31 (1.25–1.37)
<0.001
1.27 (1.20–1.34)
<0.001
PDDIs*
1.38 (1.31–1.46)
<0.001
1.34 (1.26–1.42)
<0.001
Fall in last 12 months
1.04 (0.99–1.09)
0.060
–
–
Katz ADL
1.04 (0.94–1.14)
0.473
–
–
Lawton IADL
1.09 (1.05–1.14)
<0.001
–
–
Continuous variables
Crude β (95%CI)
p-value
†Adjusted β (95%CI)
p-value
Healthcare utilisation
0.46 (0.32–0.60)
<0.001
0.44 (0.30–0.59)
<0.001
CASP 12 QoL
-0.11 (-2.08 -,0.01)
0.040
-
-
OR: odd ratio; CI: confidence interval; PIMs: potential inappropriate medications; PDDIs: potential drug-drug interactions; ADL: activities of daily living;
IADL: instrumental activities of daily living
†Adjusted for age, gender, number of comorbidities and ethnicity
Healthcare utilisation unadjusted model:, R2 = 0.003; adjusted model: R2 = 0.038
CASP 12 QoL unadjusted model:, R2 = 0.003;
β: regression coefficient; CI: confidence interval; R2: coefficient of determination; CASP 12 QoL: control, autonomy, self realisation and pleasure measure
of quality of life Table 3. Health outcomes associated with polypharmacy among urban-community dwelling older adults. doi:10.1371/journal.pone.0173466.t003 Polypharmacy among community-dwelling older adults Table 2. (Continued)
Variables
Univariate Poisson regression
Multivariate Poisson regression
Crude IRR (95%CI)
p-value
Adjusted IRR (95%CI)
p-value
Gender
Male
1.06 (0.99–1.14)
0.085
1.09 (1.03–1.14)
0.001
IRR:incidence rate ratio; CI: confidence interval
†”No” is the reference group
Multicollinearity Overdispersion and interaction terms were checked and not found. Interactions were between the number of supplements and the number of comorbidities and the presence of endocrine disorders and number of
comorbidities
Area under the receiver operating characteristic (ROC) curve (79.9%) were applied to check the goodness of qfit
doi:10.1371/journal.pone.0173466.t002 often utilized data from health insurance claims [40] or telephone interviews [41] to estimate
the rates of polypharmacy in the community. These approaches may be inaccurate due to
underreporting and the omission of out of pocket purchases of non-prescription drugs includ-
ing dietary supplements. In the present study, medication use was reported by the study partic-
ipants and verified against the actual medications taken and prescriptions during home visits
which provided a more accurate estimate of the total medications taken. Notably, 1 in 3 older
individuals who were taking chronic medications in the community setting used five or more
medications concurrently, with 1 in 2 reporting dietary supplement use. Additionally, more
than 30% of the cohort experienced PIMs similar to a prior study among elderly nursing home
residents in Malaysia [42] while one fifth were exposed to clinically significant PDDIs. These
data highlight high rates of medication related issues among the community-dwelling older
individuals in Malaysia, which currently does not have a national programme for medication
review in the elderly population, unlike many other countries [43–45]. Given that Malaysia
will experience a sizable increase in their older population over the next decade, the introduc-
tion of such a programme is timely and the findings from this study would provide baseline
data to monitor the changes over time. often utilized data from health insurance claims [40] or telephone interviews [41] to estimate
the rates of polypharmacy in the community. These approaches may be inaccurate due to
underreporting and the omission of out of pocket purchases of non-prescription drugs includ-
ing dietary supplements. In the present study, medication use was reported by the study partic-
ipants and verified against the actual medications taken and prescriptions during home visits
which provided a more accurate estimate of the total medications taken. Polypharmacy among community-dwelling older adults Among the risk factors associated with polypharmacy, advanced age and increasing num-
ber of comorbidities were associated with increased medication use, similar to that reported in
other studies [17, 36, 46, 47]. Specifically, the diagnoses with cardiovascular, endocrine or gas-
trointestinal disorders were associated with a significant increase in medication use compared
to other disease conditions as previously described [17, 38, 46, 48]. Prior studies in the general
population in Malaysia which assessed medication related issues in individuals diagnosed with
cardiovascular and endocrine disorders had similarly highlighted a profile of multiple concur-
rent medication use in the management of these conditions [49, 50]. In hypertension and dia-
betes, which are among the most common cardiovascular and endocrine disorders in older
people, numerous studies had found that the overzealous prescribing of medications to reach
specific treatment targets may have only marginal benefits compared to the harms associated
with hypoglycemia and hypotension in this age group. These may lead to falls, fractures and
head injuries which could further impact physical function and quality of life [51–54]. There-
fore, many guidelines including those in Malaysia recommend a less intensified approach for
blood glucose and blood pressure management among the elderly patients [55–58] though this
may not be widely practised [59]. Additionally, Malaysia does not have a national health insur-
ance scheme and thus patients are not managed by a single medical practitioner but are free to
visit any medical establishment to seek treatment. This system encourages multi-clinic visits
and over-prescription [60] which invariably leads to polypharmacy. The lack of separation
between prescribing and dispensing roles between doctors and pharmacists in Malaysia fur-
ther complicates this situation. This study found ethnic groups, Malay and Chinese were less likely to be associated with
polypharmacy compared to the Indians when all other covariates were held constant. The rea-
sons for this are not entirely clear but polypharmacy disparities among different ethnicities
have been previously reported [47, 61] and may be associated with different underlying socio-
economic status [47, 62]. The present study did not have detailed information on the socio-
economic status of the study participants but similar to a prior study [62], the univariate analy-
sis showed that individuals paying from health insurance or paying out of pocket for health
services were less likely to experience polypharmacy compared to individuals receiving free or
subsidised healthcare services. Health outcomes associated with polypharmacy
Categorical variables
Crude OR (95%CI)
p-value
†Adjusted OR (95%CI)
p-value
PIMs*
1.31 (1.25–1.37)
<0.001
1.27 (1.20–1.34)
<0.001
PDDIs*
1.38 (1.31–1.46)
<0.001
1.34 (1.26–1.42)
<0.001
Fall in last 12 months
1.04 (0.99–1.09)
0.060
–
–
Katz ADL
1.04 (0.94–1.14)
0.473
–
–
Lawton IADL
1.09 (1.05–1.14)
<0.001
–
–
Continuous variables
Crude β (95%CI)
p-value
†Adjusted β (95%CI)
p-value
Healthcare utilisation
0.46 (0.32–0.60)
<0.001
0.44 (0.30–0.59)
<0.001
CASP 12 QoL
-0.11 (-2.08 -,0.01)
0.040
-
- Table 3. Health outcomes associated with polypharmacy among urban-community dwelling older adults. OR: odd ratio; CI: confidence interval; PIMs: potential inappropriate medications; PDDIs: potential drug-drug interactions; ADL: activities of daily living;
IADL: instrumental activities of daily living j
β: regression coefficient; CI: confidence interval; R2: coefficient of determination; CASP 12 QoL: control, autonomy, self realisation and pleasure measure
of quality of life 10 / 18 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Polypharmacy among community-dwelling older adults negative impact on quality of life [71], depending on the disease setting. In the present study,
an increase in supplement use in the elderly was associated with better quality of life scores. It
is important to note that the CASP-12 tool used to measure QoL in this study only encom-
passed the psychosocial constructs of quality of life including domains of control, autonomy,
pleasure and self-realization but not physical aspects. However, no association was found
between an increase in supplement use and measures of physical functioning including ADL
and IADL. Therefore, the association between supplement use and improved quality of life
should be interpreted with caution as other factors including socio-economic status which
could also impact QoL were not assessed and adjusted in the present model. j
Thus far, few studies have assessed health outcomes associated with polypharmacy in the
community dwelling elderly (reviewed in [8]). In the multivariate analysis, it was found that
increasing medication use (prescribed, non-prescribed and supplement use) was associated
with PDDIs and PIMS after adjusting for age, sex, ethnicity and the number of comorbidities,
as previously described [9, 72, 73]. In the present study, only drug interactions which were
considered clinically significant and could potentially lead to serious consequences were
assessed while PIMS identified the use of medications that had a high propensity to cause
adverse events in the elderly population. Both of these outcomes have significant morbidity
and mortality implications in the elderly population and are frequent causes of preventable
medication related hospitalisations [54, 74, 75]. In addition, it was found that increasing medi-
cation use was independently associated with increased healthcare visits to any healthcare
facility in the prior 12 months though the reasons for these visits were not explored in this
study. The use of multiple drugs is sometimes unavoidable in individuals with multiple comor-
bidities. However, this needs to be weighed against the potential harms of increasing treatment
burden in the elderly population. The findings in the present study highlight an urgent need to
introduce medication reconciliation programmes among community-dwelling elderly popula-
tion and increase educational initiatives among healthcare practitioners with regards to pre-
scribing issues in older individuals. Approaches which have been shown to reduce adverse
drug events and hospitalisation associated with polypharmacy should be considered [76–79]. PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 Future studies should explore the interaction between ethnicity
and socio-economic status on polypharmacy and medication related issues in Malaysia. In the multivariate analysis, an increase in the use of dietary supplements was associated
with almost double the incident risk ratio of polypharmacy (15%) compared to the increase in
the number of comorbidities (8%). This highlights the important contributory role of supple-
ment use to polypharmacy in the present study setting. A market survey on the profile of regu-
lar supplement users in Malaysia found older individuals often considered taking dietary
supplements as ‘nutritional insurance’ because they were concerned about their health follow-
ing the diagnosis of chronic conditions [63]. More than two-thirds of the participants who
reported using dietary supplement in the present study, had two or more comorbidities. Sup-
plement use was also highest among elderly individuals diagnosed with oncologic disorders, as
previously reported [64–66] though scientific evidence on the safety and efficacy of supple-
ments in cancer has so far been inconclusive (reviewed in [67]). In the present study, a signifi-
cant association between supplement use and potential drug-drug interactions was found and
this remained significant after adjusting for age, gender, ethnicity and multiple comorbidities. Thus, the high use of supplements among elderly people is a major concern, particularly given
that a majority of patients in our setting have previously reported not to disclose information
on their supplement intake to their healthcare practitioners [64, 65]. Association between supplement use and quality of life scores has not been consistent in
the literature. Some studies reported a positive association [68–70] while others reported a PLOS ONE | DOI:10.1371/journal.pone.0173466
March 8, 2017 11 / 18 Polypharmacy among community-dwelling older adults often omitted in studies which assessed polypharmacy. The sample size was large and represen-
tative of the urban community setting in Malaysia. Conclusions A number of significant health related issues specifically, PIMS, PDDIs and increased health-
care utilisation were found to be associated with polypharmacy among older urban commu-
nity dwelling individuals who were taking chronic medications. Dietary supplement use was
also high among older individuals and was significantly associated with PDDIs. Individuals
diagnosed with cardiovascular, endocrine and gastrointestinal disorders had a high propensity
to receive multiple medications which contributed significantly to polypharmacy. The present
study adds to the growing evidence that a significant proportion of older adults are exposed to
multiple medication related issues. More stringent guidelines with regards to the appropriate-
ness and therapeutic need of dietary supplements among older adults is also crucial, given its
contribution to polypharmacy in the present study. Healthcare providers should be vigilant of
such usage to prevent any interactions with prescribed medications. Pharmacists, especially
those in the community setting should provide targeted medication reviews for older individu-
als despite the lack of a national programme to reduce the potential morbidity associated with
polypharmacy in this age group. S2 File. Supplementary data 3: Relevant sections of the questionnaire used for the study
(English and Malay versions).
(PDF) (English and Malay versions). (PDF)
S3 File. Supplementary data 4: Minimal dataset for the study. (XLS)
S1 Table. Supplementary Table 1: Prevalence of medication classes used according to the
Anatomical Therapeutic Chemical (ATC) Classification System (ATC first level) among
1256 urban-community dwelling older adults. (PDF)
S2 Table. Supplementary Table 2. Details of the products taken by the 715 dietary supple-
ments users. (PDF) Supporting information S1 File. Supplementary data 1: Assumptions made during assessments of potential inap-
propriate medications (PIMs) use according to the American Geriatrics Society 2015
Updated Beers Criteria. (PDF) Updated Beers Criteria. (PDF)
S2 File. Supplementary data 3: Relevant sections of the questionnaire used for the study
(English and Malay versions). (PDF)
S3 File. Supplementary data 4: Minimal dataset for the study. (XLS)
S1 Table. Supplementary Table 1: Prevalence of medication classes used according to the
Anatomical Therapeutic Chemical (ATC) Classification System (ATC first level) among
1256 urban-community dwelling older adults. (PDF)
S2 Table. Supplementary Table 2. Details of the products taken by the 715 dietary supple-
ments users. (PDF)
Acknowledgments
We would like to acknowledge the contribution of the staff and participants of the MELoR
study. This work was supported by the University of Malaya (UM) High Impact Research
(HIR) Grant from the Department of Higher Education, Ministry of Education, Malaysia
[UM.C/625/1/HIR/MOHE/ASH/02] and the University Malaya Research Grant from UM
[UMRG RP029-14HTM]. Polypharmacy was not associated with falls, physical function (measured by ADL and IADL)
nor quality of life scores in this study though these associations were significant in the univari-
ate model and a model which did not include comorbidities as a covariate. This implies that
the presence of multiple comorbidities has a stronger modulating effect on these outcomes. Additionally, recent studies including those in the present study setting found that the con-
sumption of falls-risk increasing drugs, rather than polypharmacy per say were more impor-
tant in predicting the risk of falls in the elderly population [18, 80]. There were a number of limitations in the present study. Firstly, participants’ adherence to
the medications in their possession was not assessed and this could have affected the other vari-
ables. Secondly, there were some intrinsic limitations to the use of the 2015 Beers criteria to
assess PIMS as many of the medications listed in Beers were not available in the present study
setting. The Beers criteria was used instead of STOPP/START, the other commonly used tool to
measure PIMS in the elderly population. This was because the participants’ medical notes were
not available and hence, assessing the appropriateness of medications prescribed as required by
the latter tool could not be conducted. The present study could not exclude the possibility that
safer alternatives to the inappropriate medications listed in Beers had not already been tried in
the past or that it was the participants’ choice to continue using these medications despite the
inherent risks. The strengths of the present study include the study approach which involved
home visits that allowed a realistic estimate of the number of medications taken by the partici-
pants. In addition, the use of dietary supplements could be documented in the cohort as this is PLOS ONE | DOI:10.1371/journal.pone.0173466
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(HIR) Grant from the Department of Higher Education, Ministry of Education, Malaysia
[UM.C/625/1/HIR/MOHE/ASH/02] and the University Malaya Research Grant from UM
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Mitochondria preserve an autarkic one-carbon cycle to confer growth-independent cancer cell migration and metastasis
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Nature communications
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cc-by
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Mitochondrial One-Carbon Flux has a Growth-
Independent Role in Promoting Breast Cancer
Metastasis
Nicole Kiweler
Luxembourg Institute of Health
Catherine Delbrouck
Luxembourg Institute of Health
Laura Neises
Luxembourg Institute of Health
Vitaly Pozdeev
University of Luxembourg
Leticia Soriano-Baguet
Luxembourg Institute of Health https://orcid.org/0000-0002-4822-624X
Feng Xiang
Luxembourg Institute of Health
Mohaned Benzarti
Luxembourg Institute of Health
Lara Haase
University of Luxembourg
Maryse Schmoetten
University of Luxembourg
Christian Jaeger
University of Luxembourg
Muhammad Noman
Luxembourg Institute of Health
Alexei Vazquez
German Aerospace Center (DLR) https://orcid.org/0000-0003-2764-3244
Bassam Janji Mitochondrial One-Carbon Flux has a G
Independent Role in Promoting Breast
Metastasis
Nicole Kiweler
Luxembourg Institute of Health
Catherine Delbrouck
Luxembourg Institute of Health
Laura Neises
Luxembourg Institute of Health
Vitaly Pozdeev
University of Luxembourg
Leticia Soriano-Baguet
Luxembourg Institute of Health https://orcid.org/0000-0002-4822-624X
Feng Xiang
Luxembourg Institute of Health
Mohaned Benzarti
Luxembourg Institute of Health
Lara Haase
University of Luxembourg
Maryse Schmoetten
University of Luxembourg
Christian Jaeger
University of Luxembourg
Muhammad Noman
Luxembourg Institute of Health
Alexei Vazquez
German Aerospace Center (DLR) https://orcid.org/0000-0003-2764-3244
Bassam Janji
Luxembourg Institute of Health
Gunnar Dittmar
Luxembourg Institute of Health https://orcid.org/0000-0003-3647-8623
Dirk Brenner
Luxembourg Institute of Health https://orcid.org/0000-0001-8979-1045
Elisabeth Letellier Nicole Kiweler Laura Neises
Luxembourg Institute of Health Vitaly Pozdeev
University of Luxembourg Vitaly Pozdeev
University of Luxembourg University of Luxembourg
Johannes Meiser ( Johannes.Meiser@lih.lu )
Luxembourg Institute of Health
Article
Keywords: One-Carbon Metabolism, Formate Over§ow, Cancer Metastasis, Metabolism, Serine, Migration,
Antifolate, Methotrexate
Posted Date: July 12th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-639444/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Version of Record: A version of this preprint was published at Nature Communications on May 16th, University of Luxembourg
Johannes Meiser ( Johannes.Meiser@lih.lu )
Luxembourg Institute of Health
Article
Keywords: One-Carbon Metabolism, Formate Over§ow, Cancer Metastasis, Metabolism, Serine, Migration,
Antifolate, Methotrexate
Posted Date: July 12th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-639444/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Article License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Nature Communications on May 16th,
2022. See the published version at https://doi.org/10.1038/s41467-022-30363-y. Mitochondrial One-Carbon Flux has a Growth-Independent Role in Promoting Breast Cancer
1
Metastasis
2
Nicole Kiweler1, Catherine Delbrouck1,2, Laura Neises1, Vitaly I. Pozdeev3, Leticia Soriano-Baguet2,4,5, Feng
3
Xian6, Mohaned Benzarti1,2, Lara Haase1,2,5, Maryse Schmoetten3, Christian Jaeger5, Muhammad Zaeem
4
Noman1, Alexei Vazquez7, Bassam Janji1, Gunnar Dittmar2,6, Dirk Brenner4,5,8, Elisabeth Letellier3, Johannes
5
Meiser1*
6
7
1Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg
8
2Faculty of Science, Technology and Medicine, University of Luxembourg, 2 avenue de Université, Esch-sur-Alzette,
9
Luxembourg
10
3Faculty of Science, Technology and Medicine, Department of Life Sciences and Medicine, Molecular Disease
11
Mechanisms Group, University of Luxembourg, Esch-sur-Alzette, Luxembourg
12
4Experimental & Molecular Immunology, Department of Infection and Immunity, Luxembourg Institute of Health,
13
Luxembourg, Luxembourg
14
5Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg
15
6Proteomics of Cellular Signaling, Quantitative Biology Unit, Luxembourg Institute of Health, Luxembourg, Luxembourg
16
7Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom
17
8Odense Research Center for Anaphylaxis, Department of Dermatology and Allergy Center, Odense University Hospital,
18
University of Southern Denmark, Odense, Denmark
19
20
*Correspondence:
21
Email: johannes.meiser@lih.lu
22
Twitter: @JohannesMeiser
23 Mitochondrial One-Carbon Flux has a Growth-Independent Role in Promoting Breast Cancer
1
Metastasis
2 24
25 Graphical Abstract 26
27
28 One-Carbon Metabolism, Formate Overflow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate,
48
Methotrexate
49 Abstract
29 In summary, we identified mitochondrial serine catabolism as a targetable, growth-
44
independent metabolic vulnerability to limit metastatic progression. 45
46
Keywords
47
One-Carbon Metabolism, Formate Overflow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate,
48
Methotrexate
49
50 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients
30
with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer
31
cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify
32
metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients
30
with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer
31
cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify
32
metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33
Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did
34
not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35
Prolonged treatment even selected for more motile cancer subpopulations. We found that cytosolic inhibition
36
of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly
37
active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate
38
overflow are increased, suggesting that mitochondria provide a protective environment that allows serine
39
catabolism to support cellular motility during nucleotide synthesis inhibition. 40
Enhanced motility of growth-arrested cells was reduced by inhibition of PHGDH-dependent de novo serine
41
synthesis and genetic silencing of mitochondrial one-carbon cycle. In vivo targeting of mitochondrial one-
42
carbon cycle and formate overflow strongly and significantly reduced lung metastasis formation in an orthotopic
43
breast cancer model. In summary, we identified mitochondrial serine catabolism as a targetable, growth-
44
independent metabolic vulnerability to limit metastatic progression. 45
46
Keywords
47
One-Carbon Metabolism, Formate Overflow, Cancer Metastasis, Metabolism, Serine, Migration, Antifolate,
48
Methotrexate
49
50 Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did
34
not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35
Prolonged treatment even selected for more motile cancer subpopulations. Abstract
29 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients
30
with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer
31
cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify
32
metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33
Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did
34
not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35
Prolonged treatment even selected for more motile cancer subpopulations. We found that cytosolic inhibition
36
of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly
37
active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate
38
overflow are increased, suggesting that mitochondria provide a protective environment that allows serine
39
catabolism to support cellular motility during nucleotide synthesis inhibition. 40 Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients
30
with minimal treatment options available. Canonical drugs mainly target the proliferative capacity of cancer
31
cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Thus, we aimed to identify
32
metabolic determinants that enable cell plasticity and foster treatment resistance and tumor escape. 33
Using a panel of anti-cancer drugs, we uncovered that antifolates, despite inducing strong growth arrest, did
34
not impact the cancer cell’s motility potential, indicating that nucleotide synthesis is dispensable for cell motility. 35
Prolonged treatment even selected for more motile cancer subpopulations. We found that cytosolic inhibition
36
of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly
37
active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate
38
overflow are increased, suggesting that mitochondria provide a protective environment that allows serine
39
catabolism to support cellular motility during nucleotide synthesis inhibition. 40
Enhanced motility of growth-arrested cells was reduced by inhibition of PHGDH-dependent de novo serine
41
synthesis and genetic silencing of mitochondrial one-carbon cycle. In vivo targeting of mitochondrial one-
42
carbon cycle and formate overflow strongly and significantly reduced lung metastasis formation in an orthotopic
43
breast cancer model. Introduction
51 84
However, these recent works focused mainly on the relevance of serine de novo synthesis via
85
phosphoglycerate dehydrogenase (PHGDH) itself instead of addressing the subsequent catabolism of serine
86
via SOC for metastatic progression. Mechanistic studies on the role of SOC in context of metastasis were
87
mainly focused on the proliferative capacities essential for metastatic outgrowth [11, 12, 14] rather than
88
analyzing metabolic requirements that support the growth-independent metastatic processes of dissemination. 89
In the absence of such growth demands and under certain metabolic stress conditions, we have previously
90 However, changes in cancer cell metabolism during stages of reduced biomass production, for example upon
61
chemotherapy or when cancer cells transition along the metastatic cascade, are far less described [3]. 62
Targeting metabolic cues to not only reduce cancer growth but also to metabolically interfere with invasive and
63
migratory properties of cancer cells represents a desirable therapeutic approach to ultimately prevent
64
metastatic progression of the initial disease. We have previously shown that a reduction of biomass demand
65
can result in increased formate release from cancer cells to their surrounding microenvironment [4]. The
66
mechanistic basis for this formate overflow phenomenon particularly in growth inhibited cancer cells, is the
67
rate of serine catabolism through serine one-carbon cycle (SOC). 68 Within this metabolic pathway, which spans between cytosol and mitochondrion [5], extracellular and de novo
69
synthesized serine is catabolized to support all three essential pillars of metabolism: biomass production, redox
70
equivalent and bioenergy generation [6]. Especially metastasizing cancer cells need to selectively and
71
dynamically adopt their cellular metabolism to fulfil these three major metabolic constraints along every step
72
of the metastatic cascade [3, 7]. Previous studies have shown that metabolic rewiring during the growth-
73
independent process of tumor cell escape and dissemination is dominated by bioenergetic and redox demands
74
rather than biosynthetic needs [3, 7, 8]. Importantly, serine plasticity allows to readjust the metabolic outputs
75
of serine catabolism through the various routes of SOC towards either of the three metabolic hallmarks [6]. 76
For example, upon high growth rates, serine, glycine and formate production from SOC is required to support
77
the synthesis of anabolic building blocks such as proteins, lipids, heme, glutathione, and nucleotides. Abstract
29 We found that cytosolic inhibition
36
of DHFR by MTX only abrogates cytosolic folate cycle, while mitochondrial one-carbon cycle remains highly
37
active. Despite a decreased cellular demand for biomass production, de novo serine synthesis and formate
38
overflow are increased, suggesting that mitochondria provide a protective environment that allows serine
39
catabolism to support cellular motility during nucleotide synthesis inhibition. 40 Enhanced motility of growth-arrested cells was reduced by inhibition of PHGDH-dependent de novo serine
41
synthesis and genetic silencing of mitochondrial one-carbon cycle. In vivo targeting of mitochondrial one-
42
carbon cycle and formate overflow strongly and significantly reduced lung metastasis formation in an orthotopic
43
breast cancer model. In summary, we identified mitochondrial serine catabolism as a targetable, growth-
44
independent metabolic vulnerability to limit metastatic progression. 45 50 Introduction
51 Many primary tumors can be targeted by specific treatment schemes. Thus survival rates of patients harboring
52
primary tumors steadily increases. However, in case of tumor relapse and metastatic disease, effective
53
therapies are mostly lacking with the consequence that secondary tumors account for the majority of cancer
54
deaths [1]. 55
Classical chemotherapeutic approaches to counteract cancer growth aim to target biomass production by
56
interfering with the synthesis of proteins, lipids, and nucleotides and thereby directly interfere with central
57
pathways of cancer cell metabolism. Consequently, it is well studied that beneficial metabolic reprogramming
58
is fundamental for cancer cells to maximize the synthesis of essential lipids, proteins and nucleotides in order
59
to drive the excessive growth rates observed within the primary tumor and secondary metastasis [2, 3]. 60
However, changes in cancer cell metabolism during stages of reduced biomass production, for example upon
61
chemotherapy or when cancer cells transition along the metastatic cascade, are far less described [3]. 62
Targeting metabolic cues to not only reduce cancer growth but also to metabolically interfere with invasive and
63
migratory properties of cancer cells represents a desirable therapeutic approach to ultimately prevent
64
metastatic progression of the initial disease. We have previously shown that a reduction of biomass demand
65
can result in increased formate release from cancer cells to their surrounding microenvironment [4]. The
66
mechanistic basis for this formate overflow phenomenon particularly in growth inhibited cancer cells, is the
67
rate of serine catabolism through serine one-carbon cycle (SOC). 68 Many primary tumors can be targeted by specific treatment schemes. Thus survival rates of patients harboring
52
primary tumors steadily increases. However, in case of tumor relapse and metastatic disease, effective
53
therapies are mostly lacking with the consequence that secondary tumors account for the majority of cancer
54
deaths [1]. 55
Classical chemotherapeutic approaches to counteract cancer growth aim to target biomass production by
56
interfering with the synthesis of proteins, lipids, and nucleotides and thereby directly interfere with central
57
pathways of cancer cell metabolism. Consequently, it is well studied that beneficial metabolic reprogramming
58
is fundamental for cancer cells to maximize the synthesis of essential lipids, proteins and nucleotides in order
59
to drive the excessive growth rates observed within the primary tumor and secondary metastasis [2, 3]. Introduction
51 60
However, changes in cancer cell metabolism during stages of reduced biomass production, for example upon
61
chemotherapy or when cancer cells transition along the metastatic cascade, are far less described [3]. 62
Targeting metabolic cues to not only reduce cancer growth but also to metabolically interfere with invasive and
63
migratory properties of cancer cells represents a desirable therapeutic approach to ultimately prevent
64
metastatic progression of the initial disease. We have previously shown that a reduction of biomass demand
65
can result in increased formate release from cancer cells to their surrounding microenvironment [4]. The
66
mechanistic basis for this formate overflow phenomenon particularly in growth inhibited cancer cells, is the
67
rate of serine catabolism through serine one-carbon cycle (SOC). 68
Within this metabolic pathway, which spans between cytosol and mitochondrion [5], extracellular and de novo
69
synthesized serine is catabolized to support all three essential pillars of metabolism: biomass production, redox
70
equivalent and bioenergy generation [6]. Especially metastasizing cancer cells need to selectively and
71
dynamically adopt their cellular metabolism to fulfil these three major metabolic constraints along every step
72
of the metastatic cascade [3, 7]. Previous studies have shown that metabolic rewiring during the growth-
73
independent process of tumor cell escape and dissemination is dominated by bioenergetic and redox demands
74
rather than biosynthetic needs [3, 7, 8]. Importantly, serine plasticity allows to readjust the metabolic outputs
75
of serine catabolism through the various routes of SOC towards either of the three metabolic hallmarks [6]. 76
For example, upon high growth rates, serine, glycine and formate production from SOC is required to support
77
the synthesis of anabolic building blocks such as proteins, lipids, heme, glutathione, and nucleotides. In
78
contrast, upon energy limitation, most of serine derived carbon is excreted in the form of formate or CO2 and
79
SOC is maximized towards energy production in form of NADH and ATP [4]. Alternatively, upon oxidative
80
stress, the demand for glycine to support GSH synthesis is prioritized [9]. We have previously shown that the
81
rate of serine catabolism to formate is significantly increased in adenomas of the small intestine and breast
82
tumors compared to normal tissue and, that increased formate levels can increase the invasiveness of cancer
83
cells [10]. In addition, other labs also reported a role of serine metabolism in the context of metastasis [11-13]. Introduction
51 Given this pro-migratory effect of nucleotide synthesis inhibition
129
in growth-arrested cancer cells, we generated MTX-resistant MDA-MB468 cells by long-term cultivation in
130 demonstrated that cells increase their rate of formate overflow via SOC [4, 10], which further manifests that
91
regulation of SOC is a key metabolic switch especially in, but not limited to, the growth-independent stages of
92
metastatic outgrowth. 93 Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells
106
To assess the relative importance of different metabolic pathways for cancer cell migration, we employed a
107
panel of metabolic perturbations targeting glycolysis, mitochondrial oxidative phosphorylation (OxPhos) as well
108
as lipid, protein, and nucleotide synthesis (Figure 1A). All metabolic interventions effectively inhibited the
109
growth and cell cycle progression of MDA-MB468 breast cancer cells and the chosen drug concentrations had
110
none to mild toxic effects after 48 h treatment (Figure 1B-C, S1A). Inhibition of glycolysis, OxPhos, lipid, and
111
protein synthesis significantly reduced migration of MDA-MB468 cells (Figure 1D). Surprisingly and in contrast
112
to all other growth-inhibiting conditions, inhibition of nucleotide synthesis with various drugs did not diminish
113
migration of MDA-MB468 cells (Figure 1E). Calculation of the area under curve (AUC) allowed us to
114
quantitatively compare wound closure upon the diverse metabolic perturbations over time (Figure 1D, E) and
115
confirmed that wound closure was significantly reduced upon rotenone (Rot), galactose (Gal), simvastatin
116
(SIM) and sirolimus (rapamycin, SIR) treatment. However, treatment with the antifolates methotrexate (MTX)
117
and pemetrexed (PEM), as well as with hydroxyurea (HU) and clofarabine (CLO) did not affect or did even
118
significantly increase wound closure in scratch assays in comparison to fully proliferative, untreated control
119
cells (Figure 1D, E). This divergent effect of nucleotide synthesis inhibition from all other metabolic
120
perturbations was further realized, when directly correlating our data on MDA-MB468 cell migration with cell
121
growth in response to treatment (Figure 1F, G). While cell migration correlates with reduced cell growth for all
122
other metabolic perturbations, nucleotide synthesis inhibition does not reduce cell migration in correlation to
123
growth inhibition (Figure 1F, G). This finding was verified in LN229 glioblastoma and 4T1 breast cancer cells
124
(Figure S1B-D). To further confirm this finding, we assessed migration of MDA-MB468 cells in a trans-well
125
assay. Introduction
51 In
78
contrast, upon energy limitation, most of serine derived carbon is excreted in the form of formate or CO2 and
79
SOC is maximized towards energy production in form of NADH and ATP [4]. Alternatively, upon oxidative
80
stress, the demand for glycine to support GSH synthesis is prioritized [9]. We have previously shown that the
81
rate of serine catabolism to formate is significantly increased in adenomas of the small intestine and breast
82
tumors compared to normal tissue and, that increased formate levels can increase the invasiveness of cancer
83
cells [10]. In addition, other labs also reported a role of serine metabolism in the context of metastasis [11-13]. 84
However, these recent works focused mainly on the relevance of serine de novo synthesis via
85
phosphoglycerate dehydrogenase (PHGDH) itself instead of addressing the subsequent catabolism of serine
86
via SOC for metastatic progression. Mechanistic studies on the role of SOC in context of metastasis were
87
mainly focused on the proliferative capacities essential for metastatic outgrowth [11, 12, 14] rather than
88
analyzing metabolic requirements that support the growth-independent metastatic processes of dissemination. 89
In the absence of such growth demands and under certain metabolic stress conditions, we have previously
90 demonstrated that cells increase their rate of formate overflow via SOC [4, 10], which further manifests that
91
regulation of SOC is a key metabolic switch especially in, but not limited to, the growth-independent stages of
92
metastatic outgrowth. 93
In the present study, our goal was to identify metabolic processes that are specifically important to support
94
cancer cell motility in the growth independent process of metastatic dissemination. We have discovered, that
95
antifolates, while having strong growth arresting properties do not decrease the cellular motility potential. Using
96
MTX as a tool compound to separate metabolic processes of growth from motility processes, we have identified
97
SOC as a central metabolic pathway that supports cellular motility potential. Specifically, the mitochondrial
98
SOC is required to support the observed motility, while the cytosolic part, which is essential for proliferation, is
99
dispensable for the cellular motility potential. Finally, we provide evidence that formate overflow via
100
mitochondrial SOC is a targetable growth-independent vulnerability to limit breast cancer metastasis. This
101
identified metabolic vulnerability might have clinical implications for metastatic progression in context of
102
growth-inhibiting chemotherapies. Introduction
51 103
104
Results
105
Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells
106
To assess the relative importance of different metabolic pathways for cancer cell migration, we employed a
107
panel of metabolic perturbations targeting glycolysis, mitochondrial oxidative phosphorylation (OxPhos) as well
108
as lipid, protein, and nucleotide synthesis (Figure 1A). All metabolic interventions effectively inhibited the
109
growth and cell cycle progression of MDA-MB468 breast cancer cells and the chosen drug concentrations had
110
none to mild toxic effects after 48 h treatment (Figure 1B-C, S1A). Inhibition of glycolysis, OxPhos, lipid, and
111
protein synthesis significantly reduced migration of MDA-MB468 cells (Figure 1D). Surprisingly and in contrast
112
to all other growth-inhibiting conditions, inhibition of nucleotide synthesis with various drugs did not diminish
113
migration of MDA-MB468 cells (Figure 1E). Calculation of the area under curve (AUC) allowed us to
114
quantitatively compare wound closure upon the diverse metabolic perturbations over time (Figure 1D, E) and
115
confirmed that wound closure was significantly reduced upon rotenone (Rot), galactose (Gal), simvastatin
116
(SIM) and sirolimus (rapamycin, SIR) treatment. However, treatment with the antifolates methotrexate (MTX)
117
and pemetrexed (PEM), as well as with hydroxyurea (HU) and clofarabine (CLO) did not affect or did even
118
significantly increase wound closure in scratch assays in comparison to fully proliferative, untreated control
119
cells (Figure 1D, E). This divergent effect of nucleotide synthesis inhibition from all other metabolic
120
perturbations was further realized, when directly correlating our data on MDA-MB468 cell migration with cell
121
growth in response to treatment (Figure 1F, G). While cell migration correlates with reduced cell growth for all
122
other metabolic perturbations, nucleotide synthesis inhibition does not reduce cell migration in correlation to
123
growth inhibition (Figure 1F, G). This finding was verified in LN229 glioblastoma and 4T1 breast cancer cells
124
(Figure S1B-D). To further confirm this finding, we assessed migration of MDA-MB468 cells in a trans-well
125
assay. Similar to our previous findings, there was no observable reduction of cell migration in response to MTX
126
or PEM (Figure 1H). Furthermore, using ECM-collagen coating in trans-well assay, we found that nucleotide
127
synthesis inhibition had no impact on cancer cell invasion (Figure 1H). These findings were also confirmed
128
using LN229 and 4T1 cells (Figure S1E, F). MTX treatment triggers a ROS-driven EMT phenotype MTX treatment triggers a ROS driven EMT phenotype
142
One of the most established and extensively described nucleotide synthesis-inhibiting clinical drugs is the
143
dihydrofolate reductase inhibitor MTX. MTX impedes the regeneration of tetrahydrofolate (THF), which is a
144
central enzymatic cofactor during purine and pyrimidine synthesis [15]. As MTX induced the strongest growth
145
repression without impacting cell death (Figure 1B, C) and had no effect on cell migration (Figure 1E), we
146
selected it as a tool compound to identify metabolic processes that are essential to support cell migration and
147
metastasis independent of cell proliferation. We tested multiple MTX concentrations and chose a concentration
148
of 50 nM, which is not only significantly growth- and cell cycle-arresting in MDA-MB468 cells (Figure 1B, S1A,
149
S2A), but has also been shown to be within the therapeutic window [16-19]. We observed that 48 h MTX
150
treatment of MDA-MB468 cells resulted in altered cellular morphology with individual cells visibly increasing in
151
cell size (Figure 2A). Following treatment, single cells exhibited an outstretched cell shape with protruding
152
edges, indicative of a remodeling of the cellular cytoskeleton (Figure 2A). MTX-resistant MDA-MB468 cells
153
showed comparable morphologic alterations (Figure S2B). We found that the observed cell cycle and
154
proliferation arrest upon MTX treatment was reversible, as MDA-MB468 cells promptly regained growth
155
properties (Figure S2C) and re-established proliferation by resuming cell cycle progression (Figure S2D) upon
156
MTX removal. Using [U-13C]serine tracer and metabolic flux analysis, we confirmed that 50 nM MTX was
157
sufficient to significantly diminish the flux of labelled serine carbon units via methylenetetrahydrofolate to ATP
158
(Figure 2B). Of note, nucleotide synthesis was partially restored in MTX-resistant MDA-MB468 cells (Figure
159
S2E). Using stable isotope labeling by amino acids in cell culture (SILAC) and subsequent quantitative
160
proteomics analysis, we confirmed the efficiency of the selected MTX dose by determining expression changes
161
of mechanistic targets of MTX such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and
162
thymidine kinase (TK) which were indeed regulated by treatment as expected (Figure 2C, S2F). Interestingly,
163
we also found superoxide dismutase (SOD) to be one of the most significantly upregulated enzymes in
164
response to treatment (Figure 2C, S2F), which corresponds to prior reports that depict MTX as a potent
165
inducer of oxidative stress [20-22]. Introduction
51 Similar to our previous findings, there was no observable reduction of cell migration in response to MTX
126
or PEM (Figure 1H). Furthermore, using ECM-collagen coating in trans-well assay, we found that nucleotide
127
synthesis inhibition had no impact on cancer cell invasion (Figure 1H). These findings were also confirmed
128
using LN229 and 4T1 cells (Figure S1E, F). Given this pro-migratory effect of nucleotide synthesis inhibition
129
in growth-arrested cancer cells, we generated MTX-resistant MDA-MB468 cells by long-term cultivation in
130 50 nM MTX for 2 months. The resulting MTX-resistant MDA-MB468 cells proliferate in the presence of
131
increasing concentrations of MTX and show enhanced migratory capacity when compared to the parental
132
MDA-MB468 cell line (Figure 1I, J). 133
In conclusion, the perturbation of any major anabolic route results in growth repression, its impact on cell
134
migration however highly depends on the targeted metabolic pathway, with nucleotide synthesis being an
135
ineffective target to abrogate cell motility. Considering that cell proliferation contributes to the experimental
136
outcome in scratch and trans-well assays, cell migration must in fact be increased upon nucleotide synthesis
137
inhibition to compensate for the reduced growth rate. Different means of metabolic growth repression thus
138
differentially impact cell motility and do not necessarily result in decreased cell migration. Furthermore,
139
prolonged nucleotide synthesis inhibition in drug-resistant cells acts as a selection pressure for more motile
140
cancer subpopulations. 141 50 nM MTX for 2 months. The resulting MTX-resistant MDA-MB468 cells proliferate in the presence of
131
increasing concentrations of MTX and show enhanced migratory capacity when compared to the parental
132
MDA-MB468 cell line (Figure 1I, J). 133 In conclusion, the perturbation of any major anabolic route results in growth repression, its impact on cell
134
migration however highly depends on the targeted metabolic pathway, with nucleotide synthesis being an
135
ineffective target to abrogate cell motility. Considering that cell proliferation contributes to the experimental
136
outcome in scratch and trans-well assays, cell migration must in fact be increased upon nucleotide synthesis
137
inhibition to compensate for the reduced growth rate. Different means of metabolic growth repression thus
138
differentially impact cell motility and do not necessarily result in decreased cell migration. Furthermore,
139
prolonged nucleotide synthesis inhibition in drug-resistant cells acts as a selection pressure for more motile
140
cancer subpopulations. 141 MTX treatment triggers a ROS-driven EMT phenotype We validated these prior findings by measuring a moderate but significant
166
increase of mitochondrial and cytosolic reactive oxidant species (ROS) in MDA-MB468 cells in response to
167
MTX (Figure 2D, E). Moreover, the mesenchymal marker protein vimentin (VIM) and transforming growth
168
factor-beta2 (TGFB2) were found to be highly upregulated in the global proteomics approach following MTX
169
treatment (Figure 2C, S2F). This finding corresponds to earlier reports, which highlight the potential of
170 oxidative stress as a driver of cancer cell migration and the transformation of cancer cells via epithelial-
171
mesenchymal transition (EMT) [6, 23-28]. We thus analyzed expression levels of the major regulators of EMT
172
- the transcription factors ZEB1, ZEB2, SNAIL, and SLUG - in MDA-MB468 cells and found that ZEB1 and
173
ZEB2 expression as well as VIM expression was upregulated in a time-dependent manner in response to MTX
174
treatment (Figure 2F). Similar upregulation of ZEB1, ZEB2, and VIM expression was observed in the MTX-
175
resistant MDA-MB468 cell line compared to the parental MDA-MB468 cell line (Figure 2G). Similarly to mRNA
176
expression, there was an upregulation of VIM at the protein level following MTX-treatment as well as in the
177
MTX-resistant MDA-MB468 cell line (Figure 2H, I). MTX-treatment also induced the expression of the
178
collagenase MMP9 (Figure 2J), an enzyme which is involved in the degradation of the extracellular matrix
179
during cancer cell migration [29]. To investigate the relevance of ROS as a potential trigger for the observed
180
EMT phenotype in response to MTX treatment, we treated cell with the antioxidant N-acetylcysteine (NAC)
181
which reduced ROS levels in MTX-treated MDA-MB468 cells (Figure 2K). While NAC alone already reduced
182
the cell migration, combined treatment with NAC and MTX resulted in a significant reduction of MTX-induced
183
cell migration (Figure 2L). Correspondingly, NAC treatment also prevented the EMT phenotype induction in
184
MTX-treated MDA-MB468 cells with the exception of ZEB1 (Figure 2M). These findings indicate that ROS
185
induction upon MTX promotes EMT and sustains migration. In conclusion, the moderate increase in cellular
186
ROS levels after MTX acts as a pro-migratory stimulus that subsequently results in the establishment and
187
long-term selection of phenotypically altered cancer cell subpopulations. MTX treatment triggers a ROS-driven EMT phenotype However, research in the past has
188
raised awareness, that antioxidant treatment is not a promising cancer treatment strategy with mixed, and not
189
always beneficial, outcomes in clinical trials [30, 31]. Therefore, our aim was to further investigate the metabolic
190
mechanisms that support enhanced cell migration in the presence of the nucleotide synthesis and growth-
191
inhibiting tool compound MTX. 192 MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis Having established that growth arrested MDA-MB468 cells maintain full migratory and invasive properties
194
under MTX treatment, we wanted to define metabolic mechanisms that maintain this pro-invasive EMT
195
phenotype. To this end, we focused on central carbon metabolism using [U-13C]glutamine and [U-13C]glucose
196
to monitor glycolytic activity and glucose- or glutamine-derived carbon oxidation through the TCA cycle (Figure
197
3A, D). Absolute uptake rates of glutamine and glutamate were sustained in response to MTX treatment
198
(Figure 3B), while the relative flux of [U-13C]glutamine throughout the TCA-cycle was significantly increased
199
in response to MTX (Figure 3C). Absolute quantification of uptake and release rates of glucose and lactate
200
also revealed sustained high glycolytic rates upon MTX treatment (Figure 3E). This came as a surprise, as
201
lactate release rates were previously shown to generally correlate with cell growth rates [32]. Here however,
202
MTX-treated and growth-arrested cells maintained their glycolytic rate and associated lactate release rates at
203
constant high levels. This indicates a constant generation of glycolysis-derived ATP even in the presence of
204
reduced energetic demand for anabolic reactions under growth-arresting conditions. Consequently, [U-
205
13C]glucose distribution within the TCA-cycle was comparable in untreated and MTX-treated MDA-MB468 cells
206
(Figure 3F) as well as in 4T1 and LN229 cells (Figure S3A, B). It has previously been shown that MTX inhibits
207
OCR in HCT116 cells [33]. We could replicate this finding and observed a ~ 30 % reduction in OCR in HCT116
208
cells upon MTX (Figure S3C). However, OCR in MDA-MB468, LN229, and 4T1 cells was sustained at control
209
level in response to MTX (Figure 3G), which indicates cell line specific effects of MTX on OCR. Overall, these
210 findings indicate that, despite decreased growth rates, central carbon metabolism and mitochondrial
211
respiration are sustained in response to MTX treatment, with an additional increase in oxidative glutamine
212
metabolism. Furthermore, we observed that MTX treatment significantly increased the relative flux of glucose
213
to serine via the serine de novo synthesis pathway despite decreased anabolic demands for nucleotide
214
synthesis (Figure 3H, S3D). The serine de novo synthesis pathway is catalyzed by three enzymes - PHGDH,
215
PSPH, and PSAT1, whose protein levels remained unchanged in response to MTX treatment in MDA-MB468,
216
MDA-MB231, 4T1 and LN229 cells (Figure S3E-H). In agreement with Diehl et al. Mitochondria protect SOC-dependent serine catabolism in the presence of MTX Mitochondria protect SOC-dependent serine catabolism in the presence of MTX
226
Given our finding of increased [U-13C]glucose-derived formate (Figure 3J), we hypothesized that the influence
227
of MTX-mediated DHFR inhibition on mitochondrial SOC is, in contrast to cytosolic SOC, limited (Figure 4A). 228
To test this hypothesis, we employed [U-13C]serine tracing to further characterize serine flux through SOC. We
229
observed that despite inducing growth-arrest, MTX did not alter the rates of serine consumption and glycine
230
release in MDA-MB468 or LN229 cells (Figure 4B, S4A). This is a remarkable observation for two reasons:
231
(i) since the major route of serine catabolism is via SOC [4], one would have expected that MTX-mediated
232
inhibition of DHFR would also cause a decrease in cellular serine consumption; (ii) since cell dry mass
233
composition is constituted by around 60 % of proteins [35], growth arresting conditions upon MTX should result
234
in decreased consumption rates of proteinogenic amino acids. In fact, a general trend for decreased
235
consumption of other essential amino acids was observed (Figure S4B) in response to MTX. Most strikingly,
236
the total release rate of formate as well as of labeled M+1 formate derived from [U13C]serine into the culture
237
medium was significantly increased (Figure 4C) in MDA-MB468 cells and sustained in LN229 cells (Figure
238
S4C). We could confirm this observation of sustained serine and glycine consumption and release (CORE)
239
rates and increased formate overflow via SOC in the presence of MTX in Plasmax medium [36], which is a
240
culture medium closer to the physiological state of human physiology (Figure S4D). SOC follows a cycle in
241
which serine can be resynthesized in the cytoplasm and mitochondrion from glycine via serine
242
hydroxymethyltransferase 1 and 2 (SHMT1, SHMT2) (Figure 4A). As the mitochondrial SHMT2 reaction is
243
highly reversible, the serine labeling pattern from [U13C]serine is composed of a mix of fully labeled M+3
244
isotopologues as well as M+1 and M+2 isotopologues, which represent the different recombination events with
245
labeled and unlabeled glycine [4, 5, 37]. Consequently, M+1 and M+2 serine isotopologues are expected to
246
be absent upon complete SOC inhibition. Intriguingly, DHFR inhibition with MTX neither completely abrogated
247
serine M+1 and M+2 isotopologues upon [U-13C]serine nor did it affect the serine to glycine ratio in MDA-
248
MB468 or LN229 cells (Figure 4D, E, S4E, F). MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis 228
To test this hypothesis, we employed [U-13C]serine tracing to further characterize serine flux through SOC. We
229
observed that despite inducing growth-arrest, MTX did not alter the rates of serine consumption and glycine
230
release in MDA-MB468 or LN229 cells (Figure 4B, S4A). This is a remarkable observation for two reasons:
231
(i) since the major route of serine catabolism is via SOC [4], one would have expected that MTX-mediated
232
inhibition of DHFR would also cause a decrease in cellular serine consumption; (ii) since cell dry mass
233
composition is constituted by around 60 % of proteins [35], growth arresting conditions upon MTX should result
234
in decreased consumption rates of proteinogenic amino acids. In fact, a general trend for decreased
235
consumption of other essential amino acids was observed (Figure S4B) in response to MTX. Most strikingly,
236
the total release rate of formate as well as of labeled M+1 formate derived from [U13C]serine into the culture
237
medium was significantly increased (Figure 4C) in MDA-MB468 cells and sustained in LN229 cells (Figure
238
S4C). We could confirm this observation of sustained serine and glycine consumption and release (CORE)
239
rates and increased formate overflow via SOC in the presence of MTX in Plasmax medium [36], which is a
240
culture medium closer to the physiological state of human physiology (Figure S4D). SOC follows a cycle in
241
which serine can be resynthesized in the cytoplasm and mitochondrion from glycine via serine
242
hydroxymethyltransferase 1 and 2 (SHMT1, SHMT2) (Figure 4A). As the mitochondrial SHMT2 reaction is
243
highly reversible, the serine labeling pattern from [U13C]serine is composed of a mix of fully labeled M+3
244
isotopologues as well as M+1 and M+2 isotopologues, which represent the different recombination events with
245
labeled and unlabeled glycine [4, 5, 37]. Consequently, M+1 and M+2 serine isotopologues are expected to
246
be absent upon complete SOC inhibition. Intriguingly, DHFR inhibition with MTX neither completely abrogated
247
serine M+1 and M+2 isotopologues upon [U-13C]serine nor did it affect the serine to glycine ratio in MDA-
248
MB468 or LN229 cells (Figure 4D, E, S4E, F). This observation of diminished, but not abolished serine M+1
249
and M+2 isotopologues indicates a sole inhibition of cytosolic but not mitochondrial SOC under MTX treatment. 250 MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis [34] who showed that
217
changes in NAD+/NADH ratio can increase PHGDH activity and in consequence serine de novo synthesis
218
rates, we observed an increased NAD+/NADH ratio upon MTX (Figure 3I). Congruently, yet somewhat
219
surprisingly, MTX treatment did also increase the proportion of labeled, extracellular formate derived from [U-
220
13C]glucose through serine and the mitochondrial SOC (Figure 3J). This may indicate that the mitochondrial
221
part of the SOC is not as inhibited by MTX treatment as its cytosolic part. In summary, we demonstrate that,
222
despite decreased metabolic demand for biomass production, MTX-treated cells sustain high metabolic rates
223
comparable to fully proliferating cells. Additionally, treated cells exhibit increased rates of serine synthesis and
224
formate excretion. 225 findings indicate that, despite decreased growth rates, central carbon metabolism and mitochondrial
211
respiration are sustained in response to MTX treatment, with an additional increase in oxidative glutamine
212
metabolism. Furthermore, we observed that MTX treatment significantly increased the relative flux of glucose
213
to serine via the serine de novo synthesis pathway despite decreased anabolic demands for nucleotide
214
synthesis (Figure 3H, S3D). The serine de novo synthesis pathway is catalyzed by three enzymes - PHGDH,
215
PSPH, and PSAT1, whose protein levels remained unchanged in response to MTX treatment in MDA-MB468,
216
MDA-MB231, 4T1 and LN229 cells (Figure S3E-H). In agreement with Diehl et al. [34] who showed that
217
changes in NAD+/NADH ratio can increase PHGDH activity and in consequence serine de novo synthesis
218
rates, we observed an increased NAD+/NADH ratio upon MTX (Figure 3I). Congruently, yet somewhat
219
surprisingly, MTX treatment did also increase the proportion of labeled, extracellular formate derived from [U-
220
13C]glucose through serine and the mitochondrial SOC (Figure 3J). This may indicate that the mitochondrial
221
part of the SOC is not as inhibited by MTX treatment as its cytosolic part. In summary, we demonstrate that,
222
despite decreased metabolic demand for biomass production, MTX-treated cells sustain high metabolic rates
223
comparable to fully proliferating cells. Additionally, treated cells exhibit increased rates of serine synthesis and
224
formate excretion. 225
Mitochondria protect SOC-dependent serine catabolism in the presence of MTX
226
Given our finding of increased [U-13C]glucose-derived formate (Figure 3J), we hypothesized that the influence
227
of MTX-mediated DHFR inhibition on mitochondrial SOC is, in contrast to cytosolic SOC, limited (Figure 4A). argeting of serine de novo synthesis decreases MTX-dependent cancer cell migration Targeting of serine de novo synthesis decreases MTX-dependent cancer cell migration
264
Having confirmed increased serine synthesis from glucose and subsequent formate overflow through
265
mitochondrial SOC in response to MTX treatment, we sought to determine whether interfering with this
266
metabolic pathway is a means to counteract enhanced cell migration upon MTX. To identify a suitable inhibitor
267
of serine de novo synthesis, we tested multiple available allosteric and one competitive inhibitor of PHGDH. 268
We found that the competitive inhibitor BI-4916 (BI) had superior efficiency when compared to the allosteric
269
inhibitors WQ-2101 (WQ), NCT-502 (NCT), and CBR-5884 (CBR) as can be observed in the isotopic labelling
270
pattern of serine from [U-13C]glucose tracer (Figure 5A). Isotopic labelling of released formate from [U-
271
13C]glucose tracer was also profoundly diminished after BI treatment alone or in combination with MTX (Figure
272
5B). Additionally, routine screened for metabolic side effects revealed that treatment with the allosteric
273
inhibitors WQ, NCT, and CBR alone did result in a significant reduction of mitochondrial OCR (Figure 5C),
274
which was not observed with the competitive inhibitor BI (Figure 5C). WQ and NCT also negatively affected
275
TCA cycle activity as characterized by a reduction of M+2 isotopologue abundance of prominent TCA
276
metabolites following [U-13C]glucose tracing (Figure 5D). In contrast, BI had no adverse effect on TCA cycle
277
activity or proliferation rate and thus emerged as the preferred PHGDH inhibitor in all subsequent experiments
278
(Figure 5D, S5A-D). Combined treatment with the specific PHGDH inhibitor BI significantly reduced MTX-
279
mediated cell migration (Figure 5E), which further indicated that mitochondrial SOC provides a metabolic
280
foundation to support cancer cell motility upon cytosolic DHFR inhibition. This inhibitory effect on MTX-
281
mediated cell motility was further enhanced upon combined PHGDH inhibition and serine and glycine (S/G)
282
starvation (Figure 5F). While serine and glycine starvation alone resulted only in moderate inhibitory effects
283
on migration of MTX-treated cells, combined treatment with PHGDH inhibition fully blunted cell migration,
284
indicating that serine is not only essential to support biomass production but also to support the metabolic
285
program required for migration (Figure 5F). Mitochondria protect SOC-dependent serine catabolism in the presence of MTX Overall, these results indicate that mitochondria provide a protected cellular
261
environment that permits serine catabolism with formate overflow via SOC even in the presence of the
262
antifolate MTX. 263 Mitochondria protect SOC-dependent serine catabolism in the presence of MTX This observation of diminished, but not abolished serine M+1
249
and M+2 isotopologues indicates a sole inhibition of cytosolic but not mitochondrial SOC under MTX treatment. 250 To comparatively demonstrate how sole or dual inhibition of cytosolic and mitochondrial SOC affects serine
251
MID, we analyzed a panel of CRISPR generated knockouts in HAP1 cells in which either the cytosolic
252
(SHMT1), the mitochondrial (MFT, SHMT2), or both compartments (FPGS, MFT+SHMT1) of SOC were
253
abrogated [38]. In concordance with our previous findings and resulting conclusion that MTX solely abolishes
254
cytosolic SOC, cytosolic SHMT1 KO did not eliminate intermediary M+1 and M+2 serine isotoplogues from [U-
255
13C]serine, whereas mitochondrial or combined cytosolic and mitochondrial inhibition of SOC upon MFT KO,
256
SHMT2 KO, FPGS KO and MFT+SHMT1 KO resulted in a complete loss of M+1 and M+2 serine isotopologues
257
(Figure 4F). SHMT1 KO reduced serine M+1 and M+2 isotopologue abundance comparable to MTX treatment
258
(Figure 4F). In mutants harboring a full inhibition of SOC or mitochondrial SOC, we additionally observed an
259
increase of the serine to glycine ratio, whereas MTX treatment and SHMT1 KO slightly reduced serine to
260
glycine ratio (Figure 4G). Overall, these results indicate that mitochondria provide a protected cellular
261
environment that permits serine catabolism with formate overflow via SOC even in the presence of the
262
antifolate MTX. 263 To comparatively demonstrate how sole or dual inhibition of cytosolic and mitochondrial SOC affects serine
251
MID, we analyzed a panel of CRISPR generated knockouts in HAP1 cells in which either the cytosolic
252
(SHMT1), the mitochondrial (MFT, SHMT2), or both compartments (FPGS, MFT+SHMT1) of SOC were
253
abrogated [38]. In concordance with our previous findings and resulting conclusion that MTX solely abolishes
254
cytosolic SOC, cytosolic SHMT1 KO did not eliminate intermediary M+1 and M+2 serine isotoplogues from [U-
255
13C]serine, whereas mitochondrial or combined cytosolic and mitochondrial inhibition of SOC upon MFT KO,
256
SHMT2 KO, FPGS KO and MFT+SHMT1 KO resulted in a complete loss of M+1 and M+2 serine isotopologues
257
(Figure 4F). SHMT1 KO reduced serine M+1 and M+2 isotopologue abundance comparable to MTX treatment
258
(Figure 4F). In mutants harboring a full inhibition of SOC or mitochondrial SOC, we additionally observed an
259
increase of the serine to glycine ratio, whereas MTX treatment and SHMT1 KO slightly reduced serine to
260
glycine ratio (Figure 4G). Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility
302 Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility
302 Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility
302
To further reinforce the role of mitochondrial SOC in sustaining cell migration under nucleotide synthesis
303
inhibition, we employed MDA-MB468 cells with a shRNA mediated knockdown (KD) of MTHFD2, a key enzyme
304
of the mitochondrial SOC [39]. This KD cell line exhibited significantly reduced MTHFD2 expression (Figure
305
S6A) and displayed a significant reduction of formate overflow even in the presence of MTX (Figure 6A),
306
indicating effective abrogation of MTHFD2 activity. MTHFD2 KD did not significantly alter cell proliferation and
307
both cell lines were comparatively sensitive to the growth-arresting effects of MTX (Figure 6B). We confirmed
308
that MTX treatment itself did not upregulate protein levels of MTHFD2 or any of the other SOC enzymes
309
SHMT1, SHMT2, or MTHFD1L (Figure S6B-E). In line with our prediction, MTHFD2 KD markedly reduced
310
MTX-mediated cell motility (Figure 6C), confirming that mitochondrial SOC activity is a major metabolic
311
determinant of cell migration in growth-arrested cells under MTX treatment. Of note, cell death rates upon MTX
312
were not affected by MTHFD2 KD, which further confirmed that the observed reduction upon MTX and
313
MTHFD2 KD was not related to enhanced cell death or further reduced growth rates, but solely based on an
314
inherent reduction of migratory capacity upon loss of mitochondrial SOC activity (Figure 6B, 6D). In summary,
315
we uncovered that mitochondrial SOC acts as a metabolic facilitator of cell migration under stress conditions
316
such as chemotherapeutic nucleotide-synthesis inhibition. Based on these observations, we speculate that
317
different growth inhibiting and stress inducing conditions, such as antifolate therapy or energy limitation [4],
318
can trigger increased rates of formate overflow and concomitant accumulation of formate within the tumor
319
microenvironment. Such increased concentrations of formate might act in an autocrine and/or paracrine
320
manner to trigger a pro-invasive phenotype, which represents the first stage to enter the metastasis process. 321
Conclusively, targeting formate overflow emerges as a conceivable intervention to limit metastasis formation. 322
G
ti
t
ti
f
it
h
d i l SOC
d
t
t
i
f
ti
i
i
323 argeting of serine de novo synthesis decreases MTX-dependent cancer cell migration Furthermore, while neither BI treatment alone nor serine and
286
glycine starvation alone were sufficient to prevent the induction of the previously observed EMT-phenotype in
287
response to MTX (Figure 2G, 5G), combined inhibition and starvation did minimize ZEB1, ZEB2, and VIM
288
upregulation (Figure 5G). This indicated that the efficient restriction of both, extracellular and de novo-
289
synthesized serine to inhibit mitochondrial SOC, is effective to prevent the pro-migratory, phenotypic change
290 upon MTX. Notably, serine and glycine starvation did not further increase cell death after combined MTX and
291
BI treatment (Figure 5H). Since MTX-treated cells were already in full growth arrest, the additional perturbation
292
with S/G starvation and/or PHGDH inhibition reveals that serine catabolism fulfills an important function to
293
support cell motility. This function is independent of serine’s anabolic function to support biosynthesis
294
processes. (Figure S5E). Using the MTX-resistant MDA-MB468 cell line, we found that BI treatment was also
295
sufficient to reduce the migratory capacity of such selected, pro-migratory, and potentially metastatic cancer
296
subpopulations (Figure 5I). In summary, depletion of de novo-synthesized serine using a specific PHGDH
297
inhibitor uncovered that the mitochondrial SOC is essential to sustain maximal cancer cell migration under
298
growth-arresting stimuli such as nucleotide synthesis inhibition by MTX. This implies that the mitochondrial
299
SOC represents a metabolic vulnerability that can be targeted to limit cell migration during certain
300
chemotherapeutic interventions (Figure 5J). 301 Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Having previously reported that formate overflow via SOC is a characteristic of
360
invasive cancer cells [10], and knowing that MTX has a direct inhibiting effect on SOC and proliferation,
361
studying this metabolic pathway emerged as a conceivable starting point for such further mechanistic
362
investigations. Within SOC, the non-essential amino acid serine is catabolized in the mitochondrion to glycine
363
and formyl-tetrahydrofolate (formyl-THF), which are required building blocks for cytosolic purine and thymidine
364
(a pyrimidine) synthesis. During thymidine synthesis, the co-factor THF is oxidized to dihydrofolate (DHF),
365
which subsequently requires regeneration to keep the folate in its bioactive form. This reduction of DHF to THF
366
is dependent on the enzyme DHF reductase (DHFR), which is a target of MTX. Hence, upon MTX treatment
367 mammary fat pads of immunocompetent BALB/c mice and monitored primary tumor growth over 6 weeks
331
(Figure 7A). In concordance with in vitro data, primary tumor growth in vivo and tumor weight at the endpoint
332
were not affected by Mthfd1l knockdown (Figure 7B, C). After 6 weeks, mice were sacrificed and lung tissue
333
was stained with H&E staining. Staining shows that micrometastases formation was visibly reduced in
334
response to MTHFD1L KD (Figure 7D). In addition, the number of macroscopic lung metastases were counted
335
and we found primary tumors derived from Mthfd1l-depleted 4T1 cells had significantly less pulmonary
336
metastases compared to 4T1 cells expressing MTHFD1L (Figure 7E). While all mice that were injected with
337
MTHFD1L-expressing 4T1 cells, exhibited lung metastases over 50% of mice injected with Mthfd1l knockdown
338
cells did not show metastatic lesions (Figure 7E). These in vivo findings further strengthen the key relevance
339
of mitochondrial SOC and specifically mitochondrial formate generation as a cornerstone that supports cancer
340
cell migration independent of primary tumor growth and cancer cell proliferation rates. 341
342 In this study, we uncovered a growth-independent function for serine and its mitochondrial catabolism that
344
drives cancer cell dissemination. Importantly, by using MTX as a tool compound to study migration
345
independent of proliferation, we could show that mitochondria sustain an autarkic SOC that is sufficient to
346
support the cells migratory capacity. This finding implies the dismal possibility that chemotherapeutic
347
approaches with nucleotide synthesis inhibiting drugs, which are undertaken to reduce primary cancer growth,
348
might not sufficiently suppress the motility capacities of remaining cancer cells. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo The fact, that in our hands,
349
breast cancer cells even showed a transitioning phenotype and were selected over time to be drug resistant,
350
while at the same time showing enhanced migratory potential, could warrant further work to comparatively
351
investigate metastatic disease progression after such therapies. Of note, aside from its use in cancer therapy,
352
the antifolate MTX represents the “anchor drug” for chronic treatment of autoimmune diseases such as
353
rheumatoid arthritis. Here, intracellular erythrocyte concentration of MTX and mean MTX plasma concentration
354
after chronic, low-dose MTX were previously reported to be in a comparable, even slightly higher, nM range
355
to our chosen drug concentration of 50 nM [40, 41]. This implies an additional, potential significance of our
356
findings in cancer patients that undergo chronic MTX therapy for an arthritic comorbidity and might warrant
357
further clinical retrospective and prospective investigations beyond the scope of this study. Our aim here was
358
however to comprehensively analyze the metabolic pathways that foster such sustained cell migration under
359
growth-arresting conditions. Having previously reported that formate overflow via SOC is a characteristic of
360
invasive cancer cells [10], and knowing that MTX has a direct inhibiting effect on SOC and proliferation,
361
studying this metabolic pathway emerged as a conceivable starting point for such further mechanistic
362
investigations. Within SOC, the non-essential amino acid serine is catabolized in the mitochondrion to glycine
363
and formyl-tetrahydrofolate (formyl-THF), which are required building blocks for cytosolic purine and thymidine
364
(a pyrimidine) synthesis. During thymidine synthesis, the co-factor THF is oxidized to dihydrofolate (DHF),
365
which subsequently requires regeneration to keep the folate in its bioactive form. This reduction of DHF to THF
366
is dependent on the enzyme DHF reductase (DHFR), which is a target of MTX. Hence, upon MTX treatment
367 mammary fat pads of immunocompetent BALB/c mice and monitored primary tumor growth over 6 weeks
331
(Figure 7A). In concordance with in vitro data, primary tumor growth in vivo and tumor weight at the endpoint
332
were not affected by Mthfd1l knockdown (Figure 7B, C). After 6 weeks, mice were sacrificed and lung tissue
333
was stained with H&E staining. Staining shows that micrometastases formation was visibly reduced in
334
response to MTHFD1L KD (Figure 7D). Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo In addition, the number of macroscopic lung metastases were counted
335
and we found primary tumors derived from Mthfd1l-depleted 4T1 cells had significantly less pulmonary
336
metastases compared to 4T1 cells expressing MTHFD1L (Figure 7E). While all mice that were injected with
337
MTHFD1L-expressing 4T1 cells, exhibited lung metastases over 50% of mice injected with Mthfd1l knockdown
338
cells did not show metastatic lesions (Figure 7E). These in vivo findings further strengthen the key relevance
339
of mitochondrial SOC and specifically mitochondrial formate generation as a cornerstone that supports cancer
340
cell migration independent of primary tumor growth and cancer cell proliferation rates. 341
342
Discussion
343
In this study, we uncovered a growth-independent function for serine and its mitochondrial catabolism that
344
drives cancer cell dissemination. Importantly, by using MTX as a tool compound to study migration
345
independent of proliferation, we could show that mitochondria sustain an autarkic SOC that is sufficient to
346
support the cells migratory capacity. This finding implies the dismal possibility that chemotherapeutic
347
approaches with nucleotide synthesis inhibiting drugs, which are undertaken to reduce primary cancer growth,
348
might not sufficiently suppress the motility capacities of remaining cancer cells. The fact, that in our hands,
349
breast cancer cells even showed a transitioning phenotype and were selected over time to be drug resistant,
350
while at the same time showing enhanced migratory potential, could warrant further work to comparatively
351
investigate metastatic disease progression after such therapies. Of note, aside from its use in cancer therapy,
352
the antifolate MTX represents the “anchor drug” for chronic treatment of autoimmune diseases such as
353
rheumatoid arthritis. Here, intracellular erythrocyte concentration of MTX and mean MTX plasma concentration
354
after chronic, low-dose MTX were previously reported to be in a comparable, even slightly higher, nM range
355
to our chosen drug concentration of 50 nM [40, 41]. This implies an additional, potential significance of our
356
findings in cancer patients that undergo chronic MTX therapy for an arthritic comorbidity and might warrant
357
further clinical retrospective and prospective investigations beyond the scope of this study. Our aim here was
358
however to comprehensively analyze the metabolic pathways that foster such sustained cell migration under
359
growth-arresting conditions. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo We sought to confirm our hypothesis on the central role of mitochondrial SOC as a facilitator of cancer cell
324
migration and ultimately metastasis formation in vivo. To specifically inhibit formate overflow, we generated a
325
stable knockdown of Mthfd1l in 4T1 breast cancer cells. The engineered cells exhibited a functional knockdown
326
of the enzyme as seen from the prominent reduction in formate release rates when compared to non-targeting
327
shRNA transfected 4T1 cells (Figure S7A). In line with our previous reports [4, 10], knockdown of Mthfd1l had
328
no effect on cell proliferation in vitro (Figure S7B) and reduced invasion (Figure S7C). To test if formate
329
overflow suppression can reduce metastasis formation in vivo, we injected both murine cell lines into the
330 mammary fat pads of immunocompetent BALB/c mice and monitored primary tumor growth over 6 weeks
331
(Figure 7A). In concordance with in vitro data, primary tumor growth in vivo and tumor weight at the endpoint
332
were not affected by Mthfd1l knockdown (Figure 7B, C). After 6 weeks, mice were sacrificed and lung tissue
333
was stained with H&E staining. Staining shows that micrometastases formation was visibly reduced in
334
response to MTHFD1L KD (Figure 7D). In addition, the number of macroscopic lung metastases were counted
335
and we found primary tumors derived from Mthfd1l-depleted 4T1 cells had significantly less pulmonary
336
metastases compared to 4T1 cells expressing MTHFD1L (Figure 7E). While all mice that were injected with
337
MTHFD1L-expressing 4T1 cells, exhibited lung metastases over 50% of mice injected with Mthfd1l knockdown
338
cells did not show metastatic lesions (Figure 7E). These in vivo findings further strengthen the key relevance
339
of mitochondrial SOC and specifically mitochondrial formate generation as a cornerstone that supports cancer
340
cell migration independent of primary tumor growth and cancer cell proliferation rates. 341
342
Discussion
343
In this study, we uncovered a growth-independent function for serine and its mitochondrial catabolism that
344
drives cancer cell dissemination. Importantly, by using MTX as a tool compound to study migration
345
independent of proliferation, we could show that mitochondria sustain an autarkic SOC that is sufficient to
346
support the cells migratory capacity. This finding implies the dismal possibility that chemotherapeutic
347
approaches with nucleotide synthesis inhibiting drugs, which are undertaken to reduce primary cancer growth,
348
might not sufficiently suppress the motility capacities of remaining cancer cells. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo The fact, that in our hands,
349
breast cancer cells even showed a transitioning phenotype and were selected over time to be drug resistant,
350
while at the same time showing enhanced migratory potential, could warrant further work to comparatively
351
investigate metastatic disease progression after such therapies. Of note, aside from its use in cancer therapy,
352
the antifolate MTX represents the “anchor drug” for chronic treatment of autoimmune diseases such as
353
rheumatoid arthritis. Here, intracellular erythrocyte concentration of MTX and mean MTX plasma concentration
354
after chronic, low-dose MTX were previously reported to be in a comparable, even slightly higher, nM range
355
to our chosen drug concentration of 50 nM [40, 41]. This implies an additional, potential significance of our
356
findings in cancer patients that undergo chronic MTX therapy for an arthritic comorbidity and might warrant
357
further clinical retrospective and prospective investigations beyond the scope of this study. Our aim here was
358
however to comprehensively analyze the metabolic pathways that foster such sustained cell migration under
359
growth-arresting conditions. Having previously reported that formate overflow via SOC is a characteristic of
360
invasive cancer cells [10], and knowing that MTX has a direct inhibiting effect on SOC and proliferation,
361
studying this metabolic pathway emerged as a conceivable starting point for such further mechanistic
362
investigations. Within SOC, the non-essential amino acid serine is catabolized in the mitochondrion to glycine
363
and formyl-tetrahydrofolate (formyl-THF), which are required building blocks for cytosolic purine and thymidine
364
(a pyrimidine) synthesis. During thymidine synthesis, the co-factor THF is oxidized to dihydrofolate (DHF),
365
which subsequently requires regeneration to keep the folate in its bioactive form. This reduction of DHF to THF
366
is dependent on the enzyme DHF reductase (DHFR), which is a target of MTX. Hence, upon MTX treatment
367
the folate co-factor accumulates in its inactive form DHF, which prevents nucleotide synthesis and cell growth
368
through obstruction of SOC activity [5] [42]. In agreement, our tool compound MTX effectively abolishes SOC-
369
dependent nucleotide synthesis in the cytosol. However, using [U13C]serine- and [U13C]glucose-assisted
370 metabolic flux analyses, we found that mitochondrial one-carbon cycle was preserved under antifolate-induced
371
nucleotide synthesis inhibition. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Such compartmentalization of SOC is on the one hand enabled by the lack of
372
oxidation of THF to DHF in the mitochondrial compartment, but is also based on the chemical modification of
373
folate species and their transport activities across the mitochondrial membrane. For maximal biologic activity,
374
folate species need to be polyglutamated by folylpolyglutamate synthase (FPGS) and the resulting
375
polyglutamate species were shown to be poorly transported across the mitochondrial membrane [43, 44]. 376
Consequently, polyglutamated folate species can be chemically trapped within the mitochondria to sustain an
377
autarkic mitochondrial SOC especially upon growth arrest and subsequent unaltered mitochondrial content. 378
Furthermore, there have been implications that the mitochondrial folate transporter is specific for reduced
379
folates with the possibility of limiting the transport of MTX and folic acid itself across the mitochondrial
380
membrane [45].Taken together, such compartmentalization of SOC and autarkic function of mitochondrial
381
SOC emerges as a selective advantage upon perturbation of cytosolic SOC and indicates an additional
382
metabolic functionality of the mitochondrial SOC pathway during growth-arresting conditions. Specifically,
383
plasticity of serine catabolism through this sustained mitochondrial pathway could offer a metabolic reserve in
384
the form of ATP and redox equivalent generation that might allow growth-arrested cancer cells to undergo
385
epithelial-mesenchymal transition and to enhance their migratory capacity in order to escape from sites of
386
nutrient deprivation or chemotherapeutic targeting. In concert with this notion, we identified mitochondrial SOC
387
as a targetable metabolic vulnerability by using genetic and pharmacologic inhibition of mitochondrial one-
388
carbon cycle activity to reduce migration in vitro and metastasis formation in vivo. These findings are in line
389
with previous reports indicating that serine de novo synthesis rates and serine uptake rates are associated
390
with disease progression and even metastatic dissemination [11-13, 46]. However to our knowledge, this is
391
the first report to show that it is indeed the enhanced activity of mitochondrial serine catabolism to formate via
392
SOC which translates these enhanced serine synthesis rates or serine uptake rates to a pro-migratory signal
393
downstream. Based on these findings, we hypothesize that serine plasticity and the alternative usage of serine
394
catabolism in the mitochondria can selectively promote formate overflow in response to extrinsic or intrinsic
395
stress stimuli such as growth inhibition or nutrient deprivation. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Consequently, the resulting increase in local
396
formate concentrations in the TME could subsequently serve as a local stress signal in an auto- or paracrine
397
manner to promote the formation of invasive cancer cells. These cells are, as a result, more prone and capable
398
in escaping a non-beneficial primary TME, which eventually advances dissemination and metastasis formation. 399
Thus, restricting mitochondrial SOC, which is at the root of this stress signaling cascade, emerges as a
400
promising, preventive strategy to hinder metastatic tumor progression early on. 401
402 We are grateful to Saverio Tardito (Cancer Research UK Beatson Institute, Glasgow, UK) for providing
404
Plasmax medium to us. We are also thankful to Clément Thomas (LIH, Luxembourg) for providing 4T1 cells,
405
Lewis Cantley (Weill Cornell Medical College) for providing MDA-MB468 cells and to Patel’s laboratory
406
(Cambridge, UK) for providing HAP1 cells [38]. 407 Plasmax medium to us. We are also thankful to Clément Thomas (LIH, Luxembourg) for providing 4T1 cells,
405
Lewis Cantley (Weill Cornell Medical College) for providing MDA-MB468 cells and to Patel’s laboratory
406
(Cambridge, UK) for providing HAP1 cells [38]. 407 We would like to thank: the LCSB Metabolomics Platform, especially Xiangyi Dong and Floriane Vanhalle, for
408
providing technical and analytical support; the National Cytometry Platform (Quantitative Biology Unit, LIH)
409
and especially Thomas Cerutti for support with flow cytometric analyses; Magretta Adiamah for her diligent
410 We would like to thank: the LCSB Metabolomics Platform, especially Xiangyi Dong and Floriane Vanhalle, for
408
providing technical and analytical support; the National Cytometry Platform (Quantitative Biology Unit, LIH)
409
and especially Thomas Cerutti for support with flow cytometric analyses; Magretta Adiamah for her diligent
410 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411
All graphical figures were produced with BioRender.com. 412
J.M. is supported by the FNR-ATTRACT program (A18/BM/11809970), INTER-BMBF grant (18/13399110)
413
and i2Tron (PRIDE19/14254520). N.K. is supported by the LIH Career Launchpad program (Legs Baertz). 414
D.B. is supported by the FNR-ATTRACT program (A14/BM/7632103), the FNR-CORE (C18/BM/12691266),
415
i2Tron (PRIDE19/14254520) and the FNR-RIKEN (TregBar/11228353) grant. D.B. and L.S.B. are funded by
416
the FNR-PRIDE (PRIDE/11012546/NEXTIMMUNE) scheme. E.L. is supported by the FNR-CORE program
417
(C16/BM/11282028 and C20/BM/14591557), by a Proof of Concept FNR grant (PoC/18/12554295), a
418
PRIDE17/11823097 and by i2Tron (PRIDE19/14254520). G.D. and F.X. are supported by FNR-CORE
419
(C17/BM/11642138). B.J. and M.Z.N. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo are supported by Luxembourg National Research Fund
420
(C18/BM/12670304/COMBATIC) and FNRS Televie grant (7.4579.20). 421
422
Author Contributions
423
Conceptualization, J.M., N.K.; Methodology, J.M., N.K., C.J., M.Z.N., B.J., E.L., D.B., G.D., V.I.P.; Software,
424
A.V., F.X.; Validation, N.K., J.M., L.N., C.D.; Formal Analysis, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S.,
425
C.J.; Investigation, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S., E.L.; Resources, C.J., A.V., J.M., E.L.;
426
Data Curation, N.K., J.M., F.X., A.V.; Writing – Original Draft, N.K., J.M.; Writing – Review & Editing, All
427
Authors; Visualization, N.K., J.M., L.S.B., F.X., V.I.P.; Supervision, J.M., D.B., A.V., E.L., G.D.; Project
428
Administration, J.M., Funding Acquisition, J.M., D.B., E.L., A.V., G.D.. 429
430
Materials & Methods
431
Chemicals
432
Methotrexate, rotenone, PKUMDL-WQ-2101, galactose, hydroxyurea, NAC, and fluorouracil were purchased
433
from Sigma Aldrich. Clofarabine, pemetrexed disodium hydrate, sirolismus were purchased from Bio Connect. 434
Simvastatin was purchased from Sanbio. CBR-5884 and BI-4916 were purchased from MedChemExpress. 435
NCT-502 was purchased from ApeXBio. 436
Cell Culture
437
All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) without phenol red, glucose, and
438
glutamine (Thermo Fisher Scientific) and supplemented with 2 mM glutamine, 17 mM glucose, and 10 % fetal
439
bovine serum (FBS) at 37°C and 5 % CO2. For galactose treatment, supplemented glucose was replaced by
440
17 mM galactose at the beginning of the experiment. MDA-MB468 and LN229 were obtained from ATCC. 441
HCT-116 was obtained from the group of E. Letellier (LSRU, University of Luxembourg), and 4T1 from C. 442
Thomas (LIH, Luxembourg). HAP1 cells were obtained from Patel’s laboratory [38] and cultured in IMDM
443
medium supplemented with 10 % FBS. MTX-resistant MDA-MB468 cells were generated through prolonged
444
culturing of MDA-MB468 cells with 50 nM MTX for 2 months until cells started to regrowth under MTX. All cell
445
lines were routinely tested for mycoplasma contamination. 446
Metabolic Characterization
447
For metabolic characterization of cell lines, previously established protocols for absolute quantification of
448
exchange fluxes and intracellular fluxes of one-carbon metabolism were applied [4]. 449
Stable Isotope Tracing and Metabolite Extraction
450 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411
All graphical figures were produced with BioRender.com. 412
J.M. is supported by the FNR-ATTRACT program (A18/BM/11809970), INTER-BMBF grant (18/13399110)
413
and i2Tron (PRIDE19/14254520). N.K. is supported by the LIH Career Launchpad program (Legs Baertz). Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 414
D.B. is supported by the FNR-ATTRACT program (A14/BM/7632103), the FNR-CORE (C18/BM/12691266),
415
i2Tron (PRIDE19/14254520) and the FNR-RIKEN (TregBar/11228353) grant. D.B. and L.S.B. are funded by
416
the FNR-PRIDE (PRIDE/11012546/NEXTIMMUNE) scheme. E.L. is supported by the FNR-CORE program
417
(C16/BM/11282028 and C20/BM/14591557), by a Proof of Concept FNR grant (PoC/18/12554295), a
418
PRIDE17/11823097 and by i2Tron (PRIDE19/14254520). G.D. and F.X. are supported by FNR-CORE
419
(C17/BM/11642138). B.J. and M.Z.N. are supported by Luxembourg National Research Fund
420
(C18/BM/12670304/COMBATIC) and FNRS Televie grant (7.4579.20). 421
422
Author Contributions
423
Conceptualization, J.M., N.K.; Methodology, J.M., N.K., C.J., M.Z.N., B.J., E.L., D.B., G.D., V.I.P.; Software,
424
A.V., F.X.; Validation, N.K., J.M., L.N., C.D.; Formal Analysis, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S.,
425
C.J.; Investigation, N.K., C.D., L.N., M.B., L.S.B., F.X., V.I.P., M.S., E.L.; Resources, C.J., A.V., J.M., E.L.;
426
Data Curation, N.K., J.M., F.X., A.V.; Writing – Original Draft, N.K., J.M.; Writing – Review & Editing, All
427
Authors; Visualization, N.K., J.M., L.S.B., F.X., V.I.P.; Supervision, J.M., D.B., A.V., E.L., G.D.; Project
428
Administration, J.M., Funding Acquisition, J.M., D.B., E.L., A.V., G.D.. 429
430
Materials & Methods
431
Chemicals
432
Methotrexate, rotenone, PKUMDL-WQ-2101, galactose, hydroxyurea, NAC, and fluorouracil were purchased
433
from Sigma Aldrich. Clofarabine, pemetrexed disodium hydrate, sirolismus were purchased from Bio Connect. 434
Simvastatin was purchased from Sanbio. CBR-5884 and BI-4916 were purchased from MedChemExpress. 435
NCT-502 was purchased from ApeXBio. 436
Cell Culture
437 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411
All graphical figures were produced with BioRender.com. 412 proofreading of the manuscript; all our collaboration partners for fruitful discussions and constructive feedback. 411
All graphical figures were produced with BioRender.com. 412 Methotrexate, rotenone, PKUMDL-WQ-2101, galactose, hydroxyurea, NAC, and fluorouracil were purchased
433
from Sigma Aldrich. Clofarabine, pemetrexed disodium hydrate, sirolismus were purchased from Bio Connect. 434
Simvastatin was purchased from Sanbio. CBR-5884 and BI-4916 were purchased from MedChemExpress. 435
NCT-502 was purchased from ApeXBio. 436 Stable isotope tracing experiments with [U-13C]-glucose tracer or [U-13C]-glutamine tracer (Cambridge Isotope
451
Laboratories, CLM-1396) were performed in DMEM supplemented with 2 mM glutamine, 17 mM glucose
452
tracer, and 10 % FBS. Stable isotope tracing experiments with [U-13C]-serine tracer (Cambridge Isotope
453
Laboratories) were performed in MEM (Thermo Fisher Scientific) supplemented with 400 μM glycine, 2 mM
454
glutamine, 17 mM glucose, 400 μM serine tracer, and 10 % FBS. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo GC-
476
MS analysis was performed using an Agilent 7890A GC coupled to an Agilent 5975C inert XL Mass Selective
477
Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in
478
split mode (20:1) at 270 °C. The gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm)
479
DB-5MS capillary column (Agilent J&W GC Column, 122-5532G). Helium was used as carrier gas with a
480
constant flow rate of 1.4 ml/min. GC oven temperature was held at 80 °C for 1 min and increased to 130 °C at
481
10 °C/min followed by a post run time of 4 min at 280 °C. Total run time was 15 min. Transfer line temperature
482
was set to 280 °C. Mass selective detector (MSD) was operating under electron ionization at 70 eV. MS source
483
was held at 230 °C and the quadrupole at 150 °C. For precise quantification, measurements were performed
484
in selected ion monitoring mode. Target ions (m/z) and dwell times are shown in Table S2. GC-MS
485
chromatograms were processed using Agilent MassHunter Quantitative Analysis for GC-MS, Version B.08.00. 486
Final determination of release rates was performed as described in [4]. 487
Determination of MIDs of Intracellular TCA Cycle Metabolites following Stable Isotope Tracing:
488
Polar metabolites were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c = 20 mg/ml) in pyridine
489
under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MTBSTFA w/ 1% TBDMCS. GC-
490 y
Formate derivatization in the culture medium was performed using MCF derivatization as described in [4]. GC-
476
MS analysis was performed using an Agilent 7890A GC coupled to an Agilent 5975C inert XL Mass Selective
477
Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in
478
split mode (20:1) at 270 °C. The gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm)
479
DB-5MS capillary column (Agilent J&W GC Column, 122-5532G). Helium was used as carrier gas with a
480
constant flow rate of 1.4 ml/min. GC oven temperature was held at 80 °C for 1 min and increased to 130 °C at
481
10 °C/min followed by a post run time of 4 min at 280 °C. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo To allow for adaptation, cells were cultivated
455
in MEM for at least one passage prior to [U-13C]-serine tracer experiments. 150,000 to 200,000 cells were
456
seeded in 12-well plates in triplicates for each experimental condition. Identical triplicate wells were seeded to
457
allow for cell count and cell volume determination (to calculate the packed cell volume (PCV)) at the start and
458
end of each tracing experiment. The day after seeding, growth medium was replaced by tracer medium and
459
cells were cultured for 24 h. In parallel, 3 wells per condition were counted to assess starting PCV. After 24 h,
460
triplicate wells were counted to assess PCV at the end of the experiment and one set of triplicates was used
461
for subsequent metabolite extraction. Medium of these triplicates were collected and analyzed for exchange
462
rates. To determine the basal medium composition for the subsequent calculation of exchange rates, identical
463
medium was incubated in triplicates in empty 12 wells throughout the experiment and analyzed in parallel to
464
the medium samples. Collected medium samples were centrifuged at 300 g for 5 min. Supernatant was
465
collected and stored at -20°C until extraction of metabolites. Formate extraction, derivatization, and
466
quantification as well as metabolite extraction for LC-MS analysis was performed as described in [4]. For
467
metabolite extraction for GC-MS analysis after [U-13C]-glucose tracing and [U-13C]-glutamine tracing, cells
468
were washed with cold 0.9 % NaCl solution. 400 μl ice-cold MeOH/H2OMQ [(ratio, 1:1) containing the internal
469
standards pentanedioc-d6 acid and [U-13C]-ribitol at a final concentration of 1 μg/ml and Tridecanoid-d25 acid
470
at a final concentration of 5 μg/ml] was added to each well. Plates were incubated for 5 min at 4°C on a rocking
471
shaker. Supernatant was collected, mixed with 200 μl CHCl3, and centrifuged for 5 min at 13,000 g at 4°C. 472
Upper polar phase was collected and stored at -20°C for subsequent MS analysis of polar metabolites. 473
GC-MS Measurements
474 Stable isotope tracing experiments with [U-13C]-glucose tracer or [U-13C]-glutamine tracer (Cambridge Isotope
451
Laboratories, CLM-1396) were performed in DMEM supplemented with 2 mM glutamine, 17 mM glucose
452
tracer, and 10 % FBS. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Stable isotope tracing experiments with [U-13C]-serine tracer (Cambridge Isotope
453
Laboratories) were performed in MEM (Thermo Fisher Scientific) supplemented with 400 μM glycine, 2 mM
454
glutamine, 17 mM glucose, 400 μM serine tracer, and 10 % FBS. To allow for adaptation, cells were cultivated
455
in MEM for at least one passage prior to [U-13C]-serine tracer experiments. 150,000 to 200,000 cells were
456
seeded in 12-well plates in triplicates for each experimental condition. Identical triplicate wells were seeded to
457
allow for cell count and cell volume determination (to calculate the packed cell volume (PCV)) at the start and
458
end of each tracing experiment. The day after seeding, growth medium was replaced by tracer medium and
459
cells were cultured for 24 h. In parallel, 3 wells per condition were counted to assess starting PCV. After 24 h,
460
triplicate wells were counted to assess PCV at the end of the experiment and one set of triplicates was used
461
for subsequent metabolite extraction. Medium of these triplicates were collected and analyzed for exchange
462
rates. To determine the basal medium composition for the subsequent calculation of exchange rates, identical
463
medium was incubated in triplicates in empty 12 wells throughout the experiment and analyzed in parallel to
464
the medium samples. Collected medium samples were centrifuged at 300 g for 5 min. Supernatant was
465
collected and stored at -20°C until extraction of metabolites. Formate extraction, derivatization, and
466
quantification as well as metabolite extraction for LC-MS analysis was performed as described in [4]. For
467
metabolite extraction for GC-MS analysis after [U-13C]-glucose tracing and [U-13C]-glutamine tracing, cells
468
were washed with cold 0.9 % NaCl solution. 400 μl ice-cold MeOH/H2OMQ [(ratio, 1:1) containing the internal
469
standards pentanedioc-d6 acid and [U-13C]-ribitol at a final concentration of 1 μg/ml and Tridecanoid-d25 acid
470
at a final concentration of 5 μg/ml] was added to each well. Plates were incubated for 5 min at 4°C on a rocking
471
shaker. Supernatant was collected, mixed with 200 μl CHCl3, and centrifuged for 5 min at 13,000 g at 4°C. 472
Upper polar phase was collected and stored at -20°C for subsequent MS analysis of polar metabolites. 473
GC-MS Measurements
474
Analysis of Formate Release Rates:
475
Formate derivatization in the culture medium was performed using MCF derivatization as described in [4]. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Total run time was 15 min. Transfer line temperature
482
was set to 280 °C. Mass selective detector (MSD) was operating under electron ionization at 70 eV. MS source
483
was held at 230 °C and the quadrupole at 150 °C. For precise quantification, measurements were performed
484
in selected ion monitoring mode. Target ions (m/z) and dwell times are shown in Table S2. GC-MS
485
chromatograms were processed using Agilent MassHunter Quantitative Analysis for GC-MS, Version B.08.00. 486
Final determination of release rates was performed as described in [4]. 487
Determination of MIDs of Intracellular TCA Cycle Metabolites following Stable Isotope Tracing:
488 Polar metabolites were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c = 20 mg/ml) in pyridine
489
under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MTBSTFA w/ 1% TBDMCS. GC-
490 MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977A Mass Selective Detector
491
(Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in splitless
492
mode at 270 °C. Gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) ZB-35MS capillary
493
column with 5 m guard column (Phenomenex). Helium was used as carrier gas with a constant flow rate of 1.2
494
ml/min. GC oven temperature was held at 100 °C for 2 min and increased to 300 °C at 10 °C/min and held for
495
4 min. Total run time was 26 min. Transfer line temperature was set to 280 °C. Mass selective detector (MSD)
496
was operating under electron ionization at 70 eV. MS source was held at 230 °C and the quadrupole at 150
497
°C. For precise quantification of the MID, measurements were performed in selected ion monitoring mode. 498
Target ions (m/z) and dwell times are shown in Table S3. 499
The MetaboliteDetector software package (Version 3.220180913) was used for mass spectrometric data post
500
processing, quantification, MID calculations, correction of natural isotope abundance, and determinations of
501
fractional carbon contributions [47]. 502
Analysis of Medium Exchange Rates:
503
Polar metabolites of the culture medium were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c =
504
20 mg/ml) in pyridine under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MSTFA. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 505
GC-MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977A Mass Selective
506
Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in
507
splitless mode at 270 °C. Gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) ZB-35MS
508
capillary column with 5 m guard column (Phenomenex). Helium was used as carrier gas with a constant flow
509
rate of 1.2 ml/min. GC oven temperature program: 90° C for 1 min, 9° C/min to 270° C, 25° C/min to 320° C
510
and held for 7 min. Total run time was 30 min. Transfer line temperature was set to 280° C. MSD was operated
511
under electron ionization at 70 eV. MS source was held at 230° C and the quadrupole at 150° C. Full scan
512
mass spectra were acquired between m/z 70 and 700. 513
The MetaboliteDetector software package (Version 3.220180913) was used for quantification. Briefly, peak
514
areas of all isotopologues of defined quantification ions were summed up and divided by the peak area of the
515
internal standard for normalization. In addition, a calibration curve was prepared to calculate absolute
516
concentrations. Absolute uptake and release rates were calculated as described in [4]
517
LC-MS Measurements
518
Untargeted LC-MS analysis was carried out as previously described in [4]. Targeted analysis of GSH/GSSG
519
was performed as described in [48]. 520
Nucleotide and NAD/NADH Analysis
521
The following analytical conditions are based on a protocol from [4]. Metabolite analyses were performed using
522
a Thermo Vanquish Flex Quaternary LC coupled to a Thermo Q Exactive HF mass spectrometer. 523
Chromatography was carried out with a SeQuant ZIC-pHILIC 5µm polymer (150 x 2.1 mm) column connected
524
to the corresponding SeQuant ZIC-pHILIC Guard (20 x 2.1 mm) pre-column. Column temperature was
525
maintained at 45 °C. The flow rate was set to 0.2 mL/min and the mobile phases consisted of 20 mmol/L
526
ammonium carbonate in water, pH 9.2 (Eluent A) and Acetonitrile (Eluent B). The gradient was: 0 min, 80% B;
527 The MetaboliteDetector software package (Version 3.220180913) was used for mass spectrometric data post
500
processing, quantification, MID calculations, correction of natural isotope abundance, and determinations of
501
fractional carbon contributions [47]. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 536 follows: sheath gas flow rate, 25; aux gas flow rate, 15; sweep gas flow rate, 0; spray voltage, 4.5 kV (+) / 3.5
531
kV (–); capillary temperature, 325 °C; S-lense RF level, 50; aux gas heater temperature, 50 °C. The Orbitrap
532
mass analyzer was operated at a resolving power of 30,000 in full-scan mode (scan range: m/z 75…1000;
533
automatic gain control target: 1e6; maximum injection time: 250 ms). Data were acquired with Thermo Xcalibur
534
software (Version 4.3.73.11) and analyzed with TraceFinder (Version 4.1). Subsequent data analysis for
535
normalization and natural isotope subtraction were performed using in house scripts as in [4]. 536
Western Blot
537
Total cell lysates were prepared by 30 min incubation of cell pellets on ice in cell lysis buffer (150 mM NaCl,
538
1 mM EDTA, 50 mM Tris-HCl, 1% NP-40). Lysis efficiency was maximized by sonification. Lysis solution was
539
centrifuged at 13,000 g for 15 min at 4°C and supernatant was collected and stored at -80°C. Protein
540
concentration was determined by Bradford assay. 15 – 25 μg of total protein were loaded on NuPAGE 4-12%
541
Bis-Tris gels (Life Technologies) or RunBlue 4-12% Bis-Tris gels (Westburg) using 4x NuPage LDS Sample
542
buffer (Thermo Fisher Scientific) supplemented with 10 mM DTT (Sigma Aldrich) and blotted on nitrocellulose
543
membrane according to standard protocols. Membranes were stained with REVERT staining solution (LI-COR)
544
and analyzed for total protein abundance. Subsequently, membranes were blocked with Odyssey TBS
545
blocking buffer (LI-COR) or 5% milk-powder in TBST and incubated with the indicated primary antibodies over
546
night at 4°C. Incubation with secondary antibody occurred for 2 h at RT. Detection was performed with the
547
Odyssey CLx Infrared Imaging System (LI-COR). ImageStudioLite Software Vers.5.2 (LI-COR) was used for
548
image analysis. Antibodies used for Western blot analysis in this study: MTHFD1L (16113-1-AP) from
549
Proteintech; vimentin (3390), β-actin (3700), and MTHFD2 (41377) from Cell Signaling Technology; PHGDH
550
(HPA021241), PSAT1 (HPA042924), PSPH (HPA020376), SHMT1 (HPA023314), and SHMT2 (HPA020549)
551
from Sigma Aldrich; IRDye 680RD Goat Anti-Mouse IgG (H+L) and IRDye 800CW Donkey Anti-Rabbit IgG
552
(H+L) from LI-COR. 553 Total cell lysates were prepared by 30 min incubation of cell pellets on ice in cell lysis buffer (150 mM NaCl,
538
1 mM EDTA, 50 mM Tris-HCl, 1% NP-40). Lysis efficiency was maximized by sonification. Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo 502 Polar metabolites of the culture medium were derivatized for 90 min at 45 °C with 20 μl of methoxyamine (c =
504
20 mg/ml) in pyridine under continuous shaking and subsequently for 90 min at 45 °C with 20 μl of MSTFA. 505
GC-MS analysis was performed using an Agilent 7890B GC coupled to an Agilent 5977A Mass Selective
506
Detector (Agilent Technologies). A sample volume of 1 μl was injected into a Split/Splitless inlet, operating in
507
splitless mode at 270 °C. Gas chromatograph was equipped with a 30 m (I.D. 250 μm, film 0.25 μm) ZB-35MS
508
capillary column with 5 m guard column (Phenomenex). Helium was used as carrier gas with a constant flow
509
rate of 1.2 ml/min. GC oven temperature program: 90° C for 1 min, 9° C/min to 270° C, 25° C/min to 320° C
510
and held for 7 min. Total run time was 30 min. Transfer line temperature was set to 280° C. MSD was operated
511
under electron ionization at 70 eV. MS source was held at 230° C and the quadrupole at 150° C. Full scan
512
mass spectra were acquired between m/z 70 and 700. 513 The MetaboliteDetector software package (Version 3.220180913) was used for quantification. Briefly, peak
514
areas of all isotopologues of defined quantification ions were summed up and divided by the peak area of the
515
internal standard for normalization. In addition, a calibration curve was prepared to calculate absolute
516
concentrations. Absolute uptake and release rates were calculated as described in [4]
517 Untargeted LC-MS analysis was carried out as previously described in [4]. Targeted analysis of GSH/GSSG
was performed as described in [48]. follows: sheath gas flow rate, 25; aux gas flow rate, 15; sweep gas flow rate, 0; spray voltage, 4.5 kV (+) / 3.5
531
kV (–); capillary temperature, 325 °C; S-lense RF level, 50; aux gas heater temperature, 50 °C. The Orbitrap
532
mass analyzer was operated at a resolving power of 30,000 in full-scan mode (scan range: m/z 75…1000;
533
automatic gain control target: 1e6; maximum injection time: 250 ms). Data were acquired with Thermo Xcalibur
534
software (Version 4.3.73.11) and analyzed with TraceFinder (Version 4.1). Subsequent data analysis for
535
normalization and natural isotope subtraction were performed using in house scripts as in [4]. Seahorse Measurements
560 The day prior to measuring OCR, 40,000 cells were seeded on poly-L-lysine coated plates. Cells were treated
561
as indicated. XF96 Extracellular Flux Analyzer (Seahorse Bioscience) was used to measure basal OCR
562
following manufacturer’s instructions. Basal OCR was normalized to the protein concentration in the wells
563
following the protocol described in [49] using Bradford assay. 564 Poly-L-lysine Coating
554 Poly-L-lysine (P1274) was purchased from Sigma-Aldrich and reconstituted at 500 μg/ml in H2OMQ. Wells were
555
coated with PLL prior to Seahorse measurement and migration analysis by Scratch assay. To this end, PLL
556
solution was diluted 1:20 in H2OMQ and added to the plates at least 1 h prior to seeding. Following incubation
557
with PLL and before cells were seeded, plates were washed twice with H2OMQ and allowed to dry under the
558
hood. 559 Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo Lysis solution was
539
centrifuged at 13,000 g for 15 min at 4°C and supernatant was collected and stored at -80°C. Protein
540
concentration was determined by Bradford assay. 15 – 25 μg of total protein were loaded on NuPAGE 4-12%
541
Bis-Tris gels (Life Technologies) or RunBlue 4-12% Bis-Tris gels (Westburg) using 4x NuPage LDS Sample
542
buffer (Thermo Fisher Scientific) supplemented with 10 mM DTT (Sigma Aldrich) and blotted on nitrocellulose
543
membrane according to standard protocols. Membranes were stained with REVERT staining solution (LI-COR)
544
and analyzed for total protein abundance. Subsequently, membranes were blocked with Odyssey TBS
545
blocking buffer (LI-COR) or 5% milk-powder in TBST and incubated with the indicated primary antibodies over
546
night at 4°C. Incubation with secondary antibody occurred for 2 h at RT. Detection was performed with the
547
Odyssey CLx Infrared Imaging System (LI-COR). ImageStudioLite Software Vers.5.2 (LI-COR) was used for
548
image analysis. Antibodies used for Western blot analysis in this study: MTHFD1L (16113-1-AP) from
549
Proteintech; vimentin (3390), β-actin (3700), and MTHFD2 (41377) from Cell Signaling Technology; PHGDH
550
(HPA021241), PSAT1 (HPA042924), PSPH (HPA020376), SHMT1 (HPA023314), and SHMT2 (HPA020549)
551
from Sigma Aldrich; IRDye 680RD Goat Anti-Mouse IgG (H+L) and IRDye 800CW Donkey Anti-Rabbit IgG
552
(H+L) from LI-COR. 553 Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death Cell
591
proliferation was either determined as measurement of cell density (confluence) or and through quantification
592
of red objects by using the IncuCyte ® NucLight Rapid Red Reagent (Essen Bioscience). Staining was
593
performed according to manufacturer’s instructions. Viable cell number was determined by trypan blue staining
594
and automatic counting using a Countess™ Cell Counting Chamber Slide (Thermo Fisher). 595
Migration Assay (Wound Closure)
596
Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen
597
Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells
598
per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker Tool
599
(Essen Bioscience) and indicated individual treatment conditions were applied simultaneously. Measurement
600
for individual treatment conditions was performed in biological replicates (n = 4 – 8) and repeated as
601
independent experiments as stated for the respective experiment (see figure legends). 602
Invasion and Migration Assay (Transwell Assay)
603
Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018
604
[10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. 605
Treatment was carried out for 18 h to 24 h. 606
RNA Extraction cDNA Synthesis and qPCR Analysis
607 Subsequent centrifugation yielded a pellet that was resuspended in 100 μl PBS and fixed with ice-cold 80%
571
EtOH. Fixed cells were stored at -20°C for at least 1 h and maximum 5 days prior to measurement. Cells were
572
centrifuged, pellet was incubated for 1 h in 200 μl RNAse A in PBS (30 μg/ml) at RT. Immediately prior to
573
measurement, 98 μl propidium iodide (PI) in PBS (50 μg/ml) was added. Flow cytometric analysis was
574
performed using BD FACSCanto and BD FACSDiva software. Analysis was performed in FlowJo. 575
C ll D
h A
l
i
6 A fresh pellet following centrifugation was resuspended in 50 μl AnnexinV-FITC staining solution (5%
577
AnnexinV-FITC in AnnexinV binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA
578
in ddH2O)) and incubated for 15 min on ice in the dark. Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death 200,000 cells were seeded in 2 ml DMEM and treated the subsequent day as indicated. After incubation,
566
medium was collected and cells were washed with PBS. PBS fraction was collected and cells were detached
567
with trypsin. Trypsination was stopped by DMEM addition and this fraction as well as a subsequent PBS
568
washing fraction were collected. The combined solutions were centrifuged and washed with PBS. 569
Cell Cycle Distribution
570 Subsequent centrifugation yielded a pellet that was resuspended in 100 μl PBS and fixed with ice-cold 80%
571
EtOH. Fixed cells were stored at -20°C for at least 1 h and maximum 5 days prior to measurement. Cells were
572
centrifuged, pellet was incubated for 1 h in 200 μl RNAse A in PBS (30 μg/ml) at RT. Immediately prior to
573
measurement, 98 μl propidium iodide (PI) in PBS (50 μg/ml) was added. Flow cytometric analysis was
574
performed using BD FACSCanto and BD FACSDiva software. Analysis was performed in FlowJo. 575
Cell Death Analysis
576
A fresh pellet following centrifugation was resuspended in 50 μl AnnexinV-FITC staining solution (5%
577
AnnexinV-FITC in AnnexinV binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA
578
in ddH2O)) and incubated for 15 min on ice in the dark. 450 μl PI-staining solution (1.1 μg/ml PI in AnnexinV
579
binding buffer) was added immediately prior to measurement using BD FACSCanto and BD FACSDiva
580
software (for wild-type cells) or NovoCyte Quanteon (for GFP-positive transfected cells). Analysis was
581
performed in FlowJo. 582
Flow Cytometric Analysis of ROS Levels
583
250,000 MDA-MB468 cells were seeded in 2 ml medium in 6 well plates and treated as indicated. Following
584
incubation, adherent cells were detached with trypsin, centrifuged at 350 g for 5 min and washed with warm
585
DMEM. Live cells were stained in 100 μl DMEM supplemented with 1:2,000 DAPI and 1:500 DCFDA for 30 min
586
at 37°C. Following incubation, samples were centrifuged at 350 g for 5 min and washed with PBS. Following
587
centrifugation at 350 g for 5 min, cells were resuspended in 100 μl PBS and measured using the BD
588
LSRFortessaTM system and BD FACSDiva software. Data analysis was performed using FlowJo software. 589
Cell Proliferation
590
Cell proliferation was determined using the IncuCyte® Live-Cell Analysis system (Essen Bioscience). Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death 605
Treatment was carried out for 18 h to 24 h. 606
RNA Extraction, cDNA Synthesis, and qPCR Analysis
607
Total RNA was extracted directly from the cell culture dishes using RNeasy Mini Kit (Qiagen, 74104). For
608
qPCR, RNA was converted to cDNA using High-capacity cDNA Reverse Transcription Kit (ThermoFisher,
609
4368814). qPCR was performed from 20 ng cDNA per samples using Fast SYBR™ Green Master Mix
610 Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen
597
Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells
598
per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker Tool
599
(Essen Bioscience) and indicated individual treatment conditions were applied simultaneously. Measurement
600
for individual treatment conditions was performed in biological replicates (n = 4 – 8) and repeated as
601
independent experiments as stated for the respective experiment (see figure legends). 602 Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018
604
[10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. 605
Treatment was carried out for 18 h to 24 h. 606 Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018
604
[10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. 605
Treatment was carried out for 18 h to 24 h. 606 Flow Cytometric Analysis of Cell Cycle Distribution and Cell Death 450 μl PI-staining solution (1.1 μg/ml PI in AnnexinV
579
binding buffer) was added immediately prior to measurement using BD FACSCanto and BD FACSDiva
580
software (for wild-type cells) or NovoCyte Quanteon (for GFP-positive transfected cells). Analysis was
581
performed in FlowJo. 582 software (for wild type cells) or NovoCyte Quanteon (for GFP positive transfected cells). Analysis was
581
performed in FlowJo. 582
Flow Cytometric Analysis of ROS Levels
583
250,000 MDA-MB468 cells were seeded in 2 ml medium in 6 well plates and treated as indicated. Following
584
incubation, adherent cells were detached with trypsin, centrifuged at 350 g for 5 min and washed with warm
585
DMEM. Live cells were stained in 100 μl DMEM supplemented with 1:2,000 DAPI and 1:500 DCFDA for 30 min
586
at 37°C. Following incubation, samples were centrifuged at 350 g for 5 min and washed with PBS. Following
587
centrifugation at 350 g for 5 min, cells were resuspended in 100 μl PBS and measured using the BD
588
LSRFortessaTM system and BD FACSDiva software. Data analysis was performed using FlowJo software. 589
Cell Proliferation
590
Cell proliferation was determined using the IncuCyte® Live-Cell Analysis system (Essen Bioscience). Cell
591
proliferation was either determined as measurement of cell density (confluence) or and through quantification
592
of red objects by using the IncuCyte ® NucLight Rapid Red Reagent (Essen Bioscience). Staining was
593
performed according to manufacturer’s instructions. Viable cell number was determined by trypan blue staining
594
and automatic counting using a Countess™ Cell Counting Chamber Slide (Thermo Fisher). 595
Migration Assay (Wound Closure)
596
Cell migration was determined using the IncuCyte® Scratch Wound Assay system for 96 well plates (Essen
597
Bioscience). 96 well image lock plates (Essen Bioscience) were PLL coated prior to seeding of 40,000 cells
598
per well. Wound application was performed 24 h post seeding using the 96-pin IncuCyte WoundMaker Tool
599
(Essen Bioscience) and indicated individual treatment conditions were applied simultaneously. Measurement
600
for individual treatment conditions was performed in biological replicates (n = 4 – 8) and repeated as
601
independent experiments as stated for the respective experiment (see figure legends). 602
Invasion and Migration Assay (Transwell Assay)
603
Cellular invasion and migration was analyzed by Boyden chamber assay as described in Meiser et al., 2018
604
[10]. Transwells were coated with ECM-Collagen for invasion assays and un-coated for migration assay. RNA Extraction, cDNA Synthesis, and qPCR Analysis Total RNA was extracted directly from the cell culture dishes using RNeasy Mini Kit (Qiagen, 74104). For
608
qPCR, RNA was converted to cDNA using High-capacity cDNA Reverse Transcription Kit (ThermoFisher,
609
4368814). qPCR was performed from 20 ng cDNA per samples using Fast SYBR™ Green Master Mix
610 Total RNA was extracted directly from the cell culture dishes using RNeasy Mini Kit (Qiagen, 74104). For
608
qPCR, RNA was converted to cDNA using High-capacity cDNA Reverse Transcription Kit (ThermoFisher,
609
4368814). qPCR was performed from 20 ng cDNA per samples using Fast SYBR™ Green Master Mix
610 (ThermoFisher, 4385612). All samples were analyzed in technical duplicates or triplicates. qPCR using Fast
611
SYBR Green was conducted at 95°C for 20 s and then 40 cycles of 95°C for 1 s and 60°C for 20 s. Specificity
612
of the reaction was verified by melt curve analysis. The relative quantification of each mRNA was performed
613
using the comparative Ct method. Experiments were performed using the QuantStudio 5 Real-Time PCR
614
System (Applied Biosciences, ThermoFisher Scientific) and data processing was performed using
615
QuantStudio Design&Analysis v1.5.1 software (Applied Biosciences, ThermoFisher Scientific). All expression
616
data was normalized to two housekeeping controls (GAPDH and CycloA). All human primers used in this study
617
are listed in the table below:
618 Gene Name
Sequence Fwd (5’ – 3’)
Sequence Rev (5’ – 3’)
CDH1
AGTGCCAACTGGACCATTCA
TCTTTGACCACCGCTCTCCT
CycloA
CAGACAAGGTCCCAAAGACA
CCATTATGGCGTGTGAAGTC
GAPDH
CATGAGAAGTATGACAACAGCCT
AGTCCTTCCACGATACCAAAGT
MMP9
CGCGCTGGGCTTAGATCATT
GGTTCAGGGCGAGGACCATA
SLUG
CTACAGCGAACTGGACACACA
TGGAATGGAGCAGCGGTAGT
SNAIL
CGAAAGGCCTTCAACTGCAAA
TGACATCTGAGTGGGTCTGGA
VIM
CCTTGAACGCAAAGTGGAATC
GACATGCTGTTCCTGAATCTGAG
ZEB1
AAGAACTGCTGGGAGGATGACA
CCTCTTCAGGTGCCTCAGGAAAA
ZEB2
GCAAGAGGCGCAAACAAGC
GGGTTGGCAATACCGTCATCC
619 shRNA mediated Gene Silencing
620
Gene knockdown with shRNA was performed in MDA-MB468 and 4T1 cells using lentiviral particles
621
expressing two different shRNAs targeting the gene of interest and one non-silencing control vector (pGIPZ-
622
shSCR: TTACTCTCGCCCAAGCGAG (Code: RHS4346). For silencing of the MTHFD2 gene in MDA-MB468
623
cells, pGIPZ shMTHFD2-2: ATTGCATTTCTATTGGCCT (V2LHS_90968) purchased from Horizon Discovery
624
was used. For silencing of the Mthfd1l gene in 4T1 cells, pGIPZ shMTHFD1L-2: TAGATTTCAATTTCATCTG
625
(Code: V2LHS_96542) purchased from Horizon Discovery was used. All plasmids used for generation of
626
lentivirus were first amplified from glycerol stocks, isolated (Machery&Nagel plasmid isolation kit), and used
627
for transfection of HEK293T cells. RNA Extraction, cDNA Synthesis, and qPCR Analysis Survey scans of peptide precursors from 375
682 For H&E staining, snap-frozen lungs were cut in 15 μm sections using Leica Cryostat (Leica Biosystems) and
654
embedded in Tissue-Tek® O.C.T (Sakura). 5 sections in a distance of 150 μm to each other were stained per
655
lung. Staining was performed according to established protocols. Briefly, selected sections were dehydrated
656
with MeOH and stained with Gill 2 hematoxylin. Sections were neutralized with successive washes of tap
657
water, hard water (10g MgSO4 and 0.7g NaHCO3 per L), and distilled water. Subsequently, sections were
658
stained with Eosin-solution and dehydrated through successive washes with 80%, 95%, 100% EtOH, and Xylol
659
prior to mounting. 660 SILAC (Stable Isotope Labeling with Amino acids in Cell culture) strategy was used for the proteomic analysis. 662
In general, MDA-MB468 cells were cultivated in DMEM-F12 SILAC medium supplemented either with Lys0
663
and Arg0 (light channel) or Lys8 and Arg10 (heavy channel). After 6 passages, labeling efficiency of heavy
664
channel was checked using LC-MS/MS. Cells in light channel were treated with 50 nM MTX for 24h and 72h,
665
while heavy channel was used as control. Cell pellets of biological duplicates were collected after the
666
treatment. Proteins were extracted in lysis buffer (50mM ammonium bicarbonate, 6M Urea, 2M Thio-urea, pH
667
8) following a 30 min incubation at 4 °C in the presence of protease inhibitors (cOmplete™ EDTA-free Protease
668
Inhibitor Cocktail, Roche). Following centrifugation at 16,000 g for 10 min, supernatants were taken for protein
669
quantification. Samples from light channel (50 μg protein) were mixed with control heavy channel (50 μg
670
protein) for protein reduction (5 mM DTT, 1 h incubation at 37°C) and alkylation (10 mM IAA, 45min in dark at
671
room temperature). Protein digestion was performed with Lys-C (FUJIFILM Wako, 125-05061) at 1:30 ratio
672
(enzyme/protein substances) for 4 h at 37 °C, then samples were diluted 4 times with 50 mM ammonium
673
bicarbonate and digested overnight with 1 μg of trypsin at 37 °C. Digestion was terminated through addition of
674
formic acid (1% final concentration). Digested peptides were cleaned up with reverse phase Sep-Pak C18 1
675
cc Vac Cartridge (Waters, WAT054955) and eluted with 1 mL 50% ACN. Eluted peptides were dried by
676
Speedvac (Thermo Fisher Scientific) and re-suspended in 0.1% formic acid. Peptide concentration was
677
measured with Nanodrop. RNA Extraction, cDNA Synthesis, and qPCR Analysis 662
In general, MDA-MB468 cells were cultivated in DMEM-F12 SILAC medium supplemented either with Lys0
663
and Arg0 (light channel) or Lys8 and Arg10 (heavy channel). After 6 passages, labeling efficiency of heavy
664
channel was checked using LC-MS/MS. Cells in light channel were treated with 50 nM MTX for 24h and 72h,
665
while heavy channel was used as control. Cell pellets of biological duplicates were collected after the
666
treatment. Proteins were extracted in lysis buffer (50mM ammonium bicarbonate, 6M Urea, 2M Thio-urea, pH
667
8) following a 30 min incubation at 4 °C in the presence of protease inhibitors (cOmplete™ EDTA-free Protease
668
Inhibitor Cocktail, Roche). Following centrifugation at 16,000 g for 10 min, supernatants were taken for protein
669 housed in a specific pathogen free (SPF) facility at a relative humidity of 40–70%, at 22 °C, and in 12 h
643
dark/light cycles. 644 housed in a specific pathogen free (SPF) facility at a relative humidity of 40–70%, at 22 °C, and in 12 h
643
dark/light cycles. 644
Syngeneic 4T1 mammary carcinoma cells were orthotopically implanted into the left mammary fat pads (1
645
injection/mouse) of immune competent female Balb/c mice according to established protocols [50]. Briefly,
646
each injection contained 2000 cells in a mixture of 25 μL PBS and 25 μL matrigel. 9 mice per group were
647
injected with either 4T1 SCR or 4T1 MTHFD1L KD cells. Primary tumor growth was monitored between day 7
648
and day 35 of the experiment. Weight was monitored over time and no weight loss was observed. Experiment
649
was terminated after 6 weeks and lung and liver were prepared for examination of metastatic outgrowth. No
650
metastases were found in the liver. Macroscopic lung metastases were blindly counted under a microscope
651
and microscopic lung metastases were visualized by H&E staining. 652
H&E staining
653
For H&E staining, snap-frozen lungs were cut in 15 μm sections using Leica Cryostat (Leica Biosystems) and
654
embedded in Tissue-Tek® O.C.T (Sakura). 5 sections in a distance of 150 μm to each other were stained per
655
lung. Staining was performed according to established protocols. Briefly, selected sections were dehydrated
656
with MeOH and stained with Gill 2 hematoxylin. Sections were neutralized with successive washes of tap
657
water, hard water (10g MgSO4 and 0.7g NaHCO3 per L), and distilled water. RNA Extraction, cDNA Synthesis, and qPCR Analysis Subsequently, sections were
658
stained with Eosin-solution and dehydrated through successive washes with 80%, 95%, 100% EtOH, and Xylol
659
prior to mounting. 660
Proteomics
661
SILAC (Stable Isotope Labeling with Amino acids in Cell culture) strategy was used for the proteomic analysis. 662
In general, MDA-MB468 cells were cultivated in DMEM-F12 SILAC medium supplemented either with Lys0
663
and Arg0 (light channel) or Lys8 and Arg10 (heavy channel). After 6 passages, labeling efficiency of heavy
664
channel was checked using LC-MS/MS. Cells in light channel were treated with 50 nM MTX for 24h and 72h,
665
while heavy channel was used as control. Cell pellets of biological duplicates were collected after the
666
treatment. Proteins were extracted in lysis buffer (50mM ammonium bicarbonate, 6M Urea, 2M Thio-urea, pH
667
8) following a 30 min incubation at 4 °C in the presence of protease inhibitors (cOmplete™ EDTA-free Protease
668
Inhibitor Cocktail, Roche). Following centrifugation at 16,000 g for 10 min, supernatants were taken for protein
669
quantification. Samples from light channel (50 μg protein) were mixed with control heavy channel (50 μg
670
protein) for protein reduction (5 mM DTT, 1 h incubation at 37°C) and alkylation (10 mM IAA, 45min in dark at
671
room temperature). Protein digestion was performed with Lys-C (FUJIFILM Wako, 125-05061) at 1:30 ratio
672
(enzyme/protein substances) for 4 h at 37 °C, then samples were diluted 4 times with 50 mM ammonium
673
bicarbonate and digested overnight with 1 μg of trypsin at 37 °C. Digestion was terminated through addition of
674
formic acid (1% final concentration). Digested peptides were cleaned up with reverse phase Sep-Pak C18 1
675
cc Vac Cartridge (Waters, WAT054955) and eluted with 1 mL 50% ACN. Eluted peptides were dried by
676
Speedvac (Thermo Fisher Scientific) and re-suspended in 0.1% formic acid. Peptide concentration was
677
measured with Nanodrop. Peptides were measured by LC-MS/MS on Q-Exactive HF mass spectrometer
678
(Thermo Fisher) connected to a Dionex Ultimate 3000 (Thermo Fisher). 500 ng of peptides were loaded onto
679
a trap column (Acclaim PepMap 75 μm x 2 cm, C18, 3 μm) and separated on a 25 cm Acclaim pepmap RSLC
680
column (75 μm x 25 cm, C18, 2 μm) using a 150 min gradient (2% to 90% acetonitrile) with a flow rate of 0.3
681
μL/min. MS data were acquired in data dependent mode (DDA). RNA Extraction, cDNA Synthesis, and qPCR Analysis Lentivirus was produced for 24 h in HEK293T cells co-transfected with the
628
viral core packaging construct pCMVR8.74, the VSV-G envelope protein vector pMD2.G and the respective
629
pGIPZ-shRNA-target plasmid using lipofectamine 3000 (Invitrogen #L3000008) as a transfection reagent. 630
Filtered virus supernatant was used to transduce MDA-MB468 and 4T1 cells in DMEM supplemented with 2%
631
FBS. After incubation at 37°C for 24 h, the virus was removed and cells were cultured in DMEM supplemented
632
with 10 % FBS for 48 h. 96 h post transduction cells were cultured in selection media containing 2 µg/mL of
633
puromycin (Sigma #P8833) for five passages to obtain stably transduced GFP-positive cells. 634 housed in a specific pathogen free (SPF) facility at a relative humidity of 40–70%, at 22 °C, and in 12 h
643
dark/light cycles. 644
Syngeneic 4T1 mammary carcinoma cells were orthotopically implanted into the left mammary fat pads (1
645
injection/mouse) of immune competent female Balb/c mice according to established protocols [50]. Briefly,
646
each injection contained 2000 cells in a mixture of 25 μL PBS and 25 μL matrigel. 9 mice per group were
647
injected with either 4T1 SCR or 4T1 MTHFD1L KD cells. Primary tumor growth was monitored between day 7
648
and day 35 of the experiment. Weight was monitored over time and no weight loss was observed. Experiment
649
was terminated after 6 weeks and lung and liver were prepared for examination of metastatic outgrowth. No
650
metastases were found in the liver. Macroscopic lung metastases were blindly counted under a microscope
651
and microscopic lung metastases were visualized by H&E staining. 652
H&E staining
653
For H&E staining, snap-frozen lungs were cut in 15 μm sections using Leica Cryostat (Leica Biosystems) and
654
embedded in Tissue-Tek® O.C.T (Sakura). 5 sections in a distance of 150 μm to each other were stained per
655
lung. Staining was performed according to established protocols. Briefly, selected sections were dehydrated
656
with MeOH and stained with Gill 2 hematoxylin. Sections were neutralized with successive washes of tap
657
water, hard water (10g MgSO4 and 0.7g NaHCO3 per L), and distilled water. Subsequently, sections were
658
stained with Eosin-solution and dehydrated through successive washes with 80%, 95%, 100% EtOH, and Xylol
659
prior to mounting. 660
Proteomics
661
SILAC (Stable Isotope Labeling with Amino acids in Cell culture) strategy was used for the proteomic analysis. RNA Extraction, cDNA Synthesis, and qPCR Analysis 716
6. Benzarti, M., et al., Metabolic Potential of Cancer Cells in Context of the Metastatic Cascade. Cells,
717
2020. 9(9). 718
7. Elia, I., G. Doglioni, and S.M. Fendt, Metabolic Hallmarks of Metastasis Formation. Trends Cell Biol,
719
2018. 28(8): p. 673-684. 720
8. Faubert, B., A. Solmonson, and R.J. DeBerardinis, Metabolic reprogramming and cancer
721
progression. Science, 2020. 368(6487). 722
9. LeBoeuf, S.E., et al., Activation of Oxidative Stress Response in Cancer Generates a Druggable
723
Dependency on Exogenous Non-essential Amino Acids. Cell Metab, 2020. 31(2): p. 339-350 e4. 724
10. Meiser, J., et al., Increased formate overflow is a hallmark of oxidative cancer. Nat Commun, 2018. 725
9(1): p. 1368. 726
11. Pollari, S., et al., Enhanced serine production by bone metastatic breast cancer cells stimulates
727
osteoclastogenesis. Breast Cancer Res Treat, 2011. 125(2): p. 421-30. 728
12. Rossi, M., et al., Heterogeneity in PHGDH protein expression potentiates cancer cell dissemination
729
and metastasis. bioRxiv, 2021: p. 2021.01.24.427949. 730 Unpaired t-test with Welch’s correction was applied for pairwise comparison (two-sided) using GraphPad
699
Software Vers.8. For normalization, data points of one experiment were either normalized to the untreated
700
control or divided by the global mean of this individual experiment. We define one n as one independent
701
biological experiment (consisting of several wells, e.g. triplicate wells for all stable isotope tracing experiments). 702
The mean of one experiment was considered as one n. The mean values of several independent experiments
703
(as indicated in figure legends) were plotted and used for statistical analysis as indicated. 704 2. DeBerardinis, R.J. and N.S. Chandel, Fundamentals of cancer metabolism. Sci Adv, 2
e1600200. Bergers, G. and S.M. Fendt, The metabolism of cancer cells during metastasis. Nat Rev Cancer
2021. 4. Meiser, J., et al., Serine one-carbon catabolism with formate overflow. Sci Adv, 2016. 2(10): p. 713
e1601273. 714 4. Meiser, J., et al., Serine one-carbon catabolism with formate overflow. Sci Adv, 2016. 2(10): p. 713
e1601273. 714 5. Tibbetts, A.S. and D.R. Appling, Compartmentalization of Mammalian folate-mediated one-carbon
715
metabolism. Annu Rev Nutr, 2010. 30: p. 57-81. 716 5. Tibbetts, A.S. and D.R. Appling, Compartmentalization of Mammalian folate-mediated one-carbon
715
metabolism. Annu Rev Nutr, 2010. 30: p. 57-81. 716 6. Benzarti, M., et al., Metabolic Potential of Cancer Cells in Context of the Metastatic Cascade. Cells,
717
2020. 9(9). 718 6. RNA Extraction, cDNA Synthesis, and qPCR Analysis Peptides were measured by LC-MS/MS on Q-Exactive HF mass spectrometer
678
(Thermo Fisher) connected to a Dionex Ultimate 3000 (Thermo Fisher). 500 ng of peptides were loaded onto
679
a trap column (Acclaim PepMap 75 μm x 2 cm, C18, 3 μm) and separated on a 25 cm Acclaim pepmap RSLC
680
column (75 μm x 25 cm, C18, 2 μm) using a 150 min gradient (2% to 90% acetonitrile) with a flow rate of 0.3
681
μL/min. MS data were acquired in data dependent mode (DDA). Survey scans of peptide precursors from 375
682 to 1500 m/z were performed at 70,000 resolution with a 3×106 ion count target and the top 12 abundant peaks
683
from survey scan were selected for fragmentation. Tandem MS was performed by isolation at 1.4 m/z with the
684
quadrupole, HCD fragmentation with a normalized collision energy of 28. The MS2 ion count target was set to
685
1×105 and the max injection time was 45 ms. Only precursors with a charge state of 2–7 were sampled for
686
MS2. The dynamic exclusion duration was set to 20 s with a 10 ppm mass tolerance around the selected
687
precursor and its isotopes. Each sample was analyzed twice as technical replicates. All raw data was analyzed
688
with MaxQuant (version 1.6.7.0) and searched with Andromeda against the Homo sapiens database from
689
Uniprot. The minimal peptide length was set to 7 amino acids and the maximum of 3 missed cleavages were
690
allowed. The search included variable modifications of methionine oxidation and N-terminal acetylation,
691
deamidation (N and Q) and fixed modification of carbamidomethyl cysteine. The “Match between run” was
692
checked within 1 min retention time window. Mass tolerance for peptide precursor and fragments were set as
693
10 ppm and 20 ppm, respectively. The FDR was set to 0.01 for peptide and protein identifications. SILAC
694
based protein quantification (MaxQuant built-in) was used for quantitative evaluation of identified protein. 695
ProTIGY (https://github.com/broadinstitute/protigy), an R based tool, was used for differential analysis of
696
MaxQuant output. 697 to 1500 m/z were performed at 70,000 resolution with a 3×106 ion count target and the top 12 abundant peaks
683
from survey scan were selected for fragmentation. Tandem MS was performed by isolation at 1.4 m/z with the
684
quadrupole, HCD fragmentation with a normalized collision energy of 28. RNA Extraction, cDNA Synthesis, and qPCR Analysis The MS2 ion count target was set to
685
1×105 and the max injection time was 45 ms. Only precursors with a charge state of 2–7 were sampled for
686
MS2. The dynamic exclusion duration was set to 20 s with a 10 ppm mass tolerance around the selected
687
precursor and its isotopes. Each sample was analyzed twice as technical replicates. All raw data was analyzed
688
with MaxQuant (version 1.6.7.0) and searched with Andromeda against the Homo sapiens database from
689
Uniprot. The minimal peptide length was set to 7 amino acids and the maximum of 3 missed cleavages were
690
allowed. The search included variable modifications of methionine oxidation and N-terminal acetylation,
691
deamidation (N and Q) and fixed modification of carbamidomethyl cysteine. The “Match between run” was
692
checked within 1 min retention time window. Mass tolerance for peptide precursor and fragments were set as
693
10 ppm and 20 ppm, respectively. The FDR was set to 0.01 for peptide and protein identifications. SILAC
694
based protein quantification (MaxQuant built-in) was used for quantitative evaluation of identified protein. 695
ProTIGY (https://github.com/broadinstitute/protigy), an R based tool, was used for differential analysis of
696
MaxQuant output. 697
Statistics
698
Unpaired t-test with Welch’s correction was applied for pairwise comparison (two-sided) using GraphPad
699
Software Vers.8. For normalization, data points of one experiment were either normalized to the untreated
700
control or divided by the global mean of this individual experiment. We define one n as one independent
701
biological experiment (consisting of several wells, e.g. triplicate wells for all stable isotope tracing experiments). 702
The mean of one experiment was considered as one n. The mean values of several independent experiments
703
(as indicated in figure legends) were plotted and used for statistical analysis as indicated. 704
705
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706
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739
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741
Concentration of MTX and Toxicities in 63 Pediatric Mature B Cell Lymphoma in Chines
742
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744
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746
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749
critically dependent upon the production of reactive oxygen species. Br J Pharmacol, 20
750
p. 501-11. 751
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752
16(4): p. 283-93. 753
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759
373-TXNIP-HIF1alpha-TWIST signaling axis in breast cancer. Oncotarget, 2015. 6(32):
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26. Reczek, C.R. and N.S. Chandel, ROS-dependent signal transduction. Curr Opin Cell Bi
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p. 8-13. 762
27. Hunter, M.V., et al., Oxidative Stress Orchestrates Cell Polarity to Promote Embryonic W
763
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789
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Methotrexate in Patients with Rheumatoid Arthritis. Clin Drug Investig, 2020. 40(9): p. 827-838. 792
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793
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795
maintain the subcellular compartmentalization of folates. J Biol Chem, 2014. 289(42): p. 29386-96. 796
44. Lowe, K.E., et al., Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of
797
folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and
798
folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism. J Biol Chem,
799
1993. 268(29): p. 21665-73. 800
45. Horne, D.W., R.S. g
p
50.
Kocaturk, B. and H.H. Versteeg, Orthotopic injection of breast cancer cells into the mammary fat pad
811
of mice to study tumor growth. J Vis Exp, 2015(96).
812
813 RNA Extraction, cDNA Synthesis, and qPCR Analysis J Nutr, 1992. 122(11): p. 2204-9. 45. Horne, D.W., R.S. Holloway, and H.M. Said, Uptake of 5-formyltetrahydrofolate in isolated rat liver
801
mitochondria is carrier-mediated. J Nutr, 1992. 122(11): p. 2204-9. 802
46
T j
M
t
l
S
i
th
i
th
i hibiti
t
ith di t
i
d
l
i
803 46. Tajan, M., et al., Serine synthesis pathway inhibition cooperates with dietary serine an
limitation for cancer therapy. Nat Commun, 2021. 12(1): p. 366. py
( ) p
Hiller, K., et al., MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted
GC/MS based metabolome analysis. Anal Chem, 2009. 81(9): p. 3429-39. 47. Hiller, K., et al., MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted
805
GC/MS based metabolome analysis. Anal Chem, 2009. 81(9): p. 3429-39. 806
48. Mak, T.W., et al., Glutathione Primes T Cell Metabolism for Inflammation. Immunity, 2017. 46(6): p. 807
1089-1090. 808 49. Dranka, B.P., et al., Assessing bioenergetic function in response to oxidative stress by metabolic
809
profiling. Free Radic Biol Med, 2011. 51(9): p. 1621-35. 810 49. Dranka, B.P., et al., Assessing bioenergetic function in response to oxidative stress by metabolic
809
profiling. Free Radic Biol Med, 2011. 51(9): p. 1621-35. 810 p
g
( ) p
50. Kocaturk, B. and H.H. Versteeg, Orthotopic injection of breast cancer cells into the mammary fat pad
811
of mice to study tumor growth. J Vis Exp, 2015(96). 812
813 50. Kocaturk, B. and H.H. Versteeg, Orthotopic injection of breast cancer cells into the mammary fat pad
811
of mice to study tumor growth. J Vis Exp, 2015(96). 812 Figure 1: Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells. (A) Schematic
depicting synthesis routes of central metabolic building blocks (lipids, nucleotides, and proteins) and pharmacologic intervention points of
the selected drug panel. (B) MDA-MB468 cells were treated with 100 nM Sirolimus (SIR), 1 μM Simvastatin (SIM), 50 nM Rotenone (Rot),
galactose (Gal) supplementation, 50 nM Methotrexate (MTX), 1 μM Pemetrexed (PEM), 0.5 mM hydroxyurea (HU), and 100 nM
Clofarabine (CLO) for 48 h. Viable cell number was determined by trypan blue staining. Each dot represents an independent experiment;
mean ± SD; ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Comparisons to control apart from the labeled are all
significant with p<0.0001. (C) MDA-MB468 cells were treated for 48 h as in (B). RNA Extraction, cDNA Synthesis, and qPCR Analysis Cell death induction was assessed by flow cytometric
analysis of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3 - 8). 2way ANOVA with Dunnett’s multiple comparisons test. (D) Migration
of MDA-MB468 cells in response to 50 nM Rot, Gal, 1 μM SIM, and 100 nM SIR and respective area under curve (AUC); mean ± SEM (n
= 3 - 9); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (E) Migration of MDA-MB468 cells in
response to 50 nM MTX, 1 μM PEM, 0.5 mM HU, and 100 nM CLO and respective AUC; mean ± SEM (n = 3 - 12); Brown-Forsythe and
Welch one-way ANOVA with Dunnett’s multiple comparisons test. (F) Correlation of cell migration assessed as AUC over 40h and
proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468 cells were treated as in (B) and (D). (G) Correlation
of cell migration assessed as AUC over 40h and proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468
cells were treated as in (B) and (E). (H) Migration and invasion of MDA-MB468 cells treated for 24 h with 50 nM MTX was assessed using
non-coated (migration) or ECM-Collagen-coated (invasion) Boyden chambers. Each dot represents an independent experiment; mean ±
SEM; Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (I) Proliferation of MTX-resistant MDA-
MB468 cells upon the indicated concentrations of MTX [nM] measured as cell density; mean ± SEM (n = 4). (J) Migration of parental and
MTX-resistant MDA-MB468 cells and respective AUC; mean ± SEM (n = 6 - 15); unpaired t-test with Welch’s correction. For comparison, 815
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Figure 1: Inhibition of major anabolic synthesis routes differentially impacts the motility potential of cancer cells. (A) Schematic
816
depicting synthesis routes of central metabolic building blocks (lipids, nucleotides, and proteins) and pharmacologic intervention points of
817
the selected drug panel. (B) MDA-MB468 cells were treated with 100 nM Sirolimus (SIR), 1 μM Simvastatin (SIM), 50 nM Rotenone (Rot),
818
galactose (Gal) supplementation, 50 nM Methotrexate (MTX), 1 μM Pemetrexed (PEM), 0.5 mM hydroxyurea (HU), and 100 nM
819
Clofarabine (CLO) for 48 h. Viable cell number was determined by trypan blue staining. RNA Extraction, cDNA Synthesis, and qPCR Analysis Graph shows mean ± SEM of five
independent experiments each measured in triplicate wells. (C) Quantitative proteomics analysis of SILAC-labeled MDA-MB468 cells
treated with and without 50 nM MTX for 72h. The right side of the volcano plot shows the upregulated proteins after MTX treatment (red),
while downregulated ones on the left side (blue). Differential expressions were defined as absolute log2 Fold change ≥ 1 with adjusted p-
value ≤ 0.05. (D) Mitochondrial ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of MitoSOX mean
fluorescence intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (E)
Intracellular ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of DCF-DA mean fluorescence
intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (F) mRNA expression
from the indicated target genes in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time
RT-qPCR. Each dot represents an independent experiment; mean ± SEM. No data shown for SLUG expression at 72 h. (G) mRNA
expression from the indicated target genes in MTX-resistant MDA-MB468 cells relative to parental MDA-MB468 cells as measured using
real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. (H) Expression of vimentin in MDA-MB468 cells after
240 h 50 nM MTX treatment; β-actin serves as loading control. Quantification of vimentin signal intensity relative to total protein stain. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (I) Expression of vimentin in parental
d MTX
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Figure 2: MTX treatment triggers a ROS-driven EMT phenotype. (A) Morphological changes of MDA-MB468 cells in response to 48 h
839
50 nM MTX. Bright-field images are representative of independent experiments. Scale bars correspond to 60 and 300 μm. (B) MID of
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intracellular ATP upon [U-13C]serine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Graph shows mean ± SEM of five
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independent experiments each measured in triplicate wells. (C) Quantitative proteomics analysis of SILAC-labeled MDA-MB468 cells
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treated with and without 50 nM MTX for 72h. RNA Extraction, cDNA Synthesis, and qPCR Analysis Each dot represents an independent experiment;
820
mean ± SD; ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Comparisons to control apart from the labeled are all
821
significant with p<0.0001. (C) MDA-MB468 cells were treated for 48 h as in (B). Cell death induction was assessed by flow cytometric
822
analysis of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3 - 8). 2way ANOVA with Dunnett’s multiple comparisons test. (D) Migration
823
of MDA-MB468 cells in response to 50 nM Rot, Gal, 1 μM SIM, and 100 nM SIR and respective area under curve (AUC); mean ± SEM (n
824
= 3 - 9); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (E) Migration of MDA-MB468 cells in
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response to 50 nM MTX, 1 μM PEM, 0.5 mM HU, and 100 nM CLO and respective AUC; mean ± SEM (n = 3 - 12); Brown-Forsythe and
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Welch one-way ANOVA with Dunnett’s multiple comparisons test. (F) Correlation of cell migration assessed as AUC over 40h and
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proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468 cells were treated as in (B) and (D). (G) Correlation
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of cell migration assessed as AUC over 40h and proliferation assessed as fold cell growth after 48 h compared to control. MDA-MB468
829
cells were treated as in (B) and (E). (H) Migration and invasion of MDA-MB468 cells treated for 24 h with 50 nM MTX was assessed using
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non-coated (migration) or ECM-Collagen-coated (invasion) Boyden chambers. Each dot represents an independent experiment; mean ±
831
SEM; Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (I) Proliferation of MTX-resistant MDA-
832
MB468 cells upon the indicated concentrations of MTX [nM] measured as cell density; mean ± SEM (n = 4). (J) Migration of parental and
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MTX-resistant MDA-MB468 cells and respective AUC; mean ± SEM (n = 6 - 15); unpaired t-test with Welch’s correction. For comparison,
834
the time point of 50% wound closure is indicated for both cell lines. 835
836 Figure 2: MTX treatment triggers a ROS-driven EMT phenotype. (A) Morphological changes of MDA-MB468 cells in response to 48 h
50 nM MTX. Bright-field images are representative of independent experiments. Scale bars correspond to 60 and 300 μm. (B) MID of
intracellular ATP upon [U-13C]serine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. RNA Extraction, cDNA Synthesis, and qPCR Analysis Graph shows mean ± SEM (n = 3); Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple
862
comparisons test. (M) mRNA expression from the indicated target genes in MDA-MB468 cells in response to 10 mM NAC and 50 nM
863
MTX at 72 h as measured using real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. 864
865
866 MMP9 in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time RT-qPCR. Each dot
857
represents an independent experiment; mean ± SEM. (K) Intracellular ROS levels in response to 24 h 50 nM MTX and 10 mM NAC as
858
measured by flow cytometric quantification of DCF-DA mean fluorescence intensity. Each dot represents an independent experiment. 859
Graph shows mean ± SD; Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple comparisons test. (L) Migratory potential
860
of MDA-MB468 cells in response to 10 mM NAC and 50 nM MTX treatment as time-dependent quantification of relative wound density in
861
IncuCyte and respective AUC. Graph shows mean ± SEM (n = 3); Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple
862
comparisons test. (M) mRNA expression from the indicated target genes in MDA-MB468 cells in response to 10 mM NAC and 50 nM
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MTX at 72 h as measured using real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. 864
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866 857
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866 Figure 3: MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis. (A) Schematic depicting
interdependence of glycolysis and tricarboxylic acid (TCA) cycle as well as metabolic 13C label pattern from [U-13C]glutamine tracer. (B)
Absolute consumption and release (CORE) rates of glutamine and glutamate in the culture medium from MDA-MB468 cells in response
to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (C) Enrichment of
representative isotopomers of glutamine (Gln), glutamate (Glut), α-ketoglutarate (αKG), fumarate (Fum), malate (Mal), aspartate (Asp),
and citrate (Cit) upon [U-13C]glutamine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent
experiment in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (D) Schematic depicting interdependence of glycolysis
and TCA cycle as well as expected metabolic 13C label pattern from [U-13C]glucose tracer. RNA Extraction, cDNA Synthesis, and qPCR Analysis (E) Absolute CORE rates of lactate and glucose
in the culture medium of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of
triplicate wells; mean ± SEM. (F) Enrichment of representative isotopologues of pyruvate (Pyr), Cit, αKG, Fum, Mal, and Asp upon [U-
13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment with triplicate
wells; mean ± SEM; unpaired t-test with Welch’s correction. (G) Basal cellular respiration in response to 24 h 50 nM MTX treatment was
determined in MDA-MB468, LN229, and 4T1 cells as quantification of mitochondrial oxygen consumption rate (OCR). Each dot represents
an individual experiment composed of six technical replicates; mean ± SEM. (H) Enrichment of M+3 isotopologue of serine upon [U-
13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate
wells; mean ± SEM; unpaired t-test with Welch’s correction. (I) Ratio of absolute intracellular NAD+ to NADH in MDA-MB468 cells in
response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (J) M+1
isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon 24 h treatment with 50 nM MTX. Each dot
indicates an independent experiment measured in triplicate wells; mean ± SEM. 867
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887 Figure 3: MTX-treated cells sustain high metabolic rates and enhance de novo serine synthesis. (A) Schematic depicting
interdependence of glycolysis and tricarboxylic acid (TCA) cycle as well as metabolic 13C label pattern from [U-13C]glutamine tracer. (B)
Absolute consumption and release (CORE) rates of glutamine and glutamate in the culture medium from MDA-MB468 cells in response
to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (C) Enrichment of
representative isotopomers of glutamine (Gln), glutamate (Glut), α-ketoglutarate (αKG), fumarate (Fum), malate (Mal), aspartate (Asp),
and citrate (Cit) upon [U-13C]glutamine tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent
experiment in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (D) Schematic depicting interdependence of glycolysis
and TCA cycle as well as expected metabolic 13C label pattern from [U-13C]glucose tracer. RNA Extraction, cDNA Synthesis, and qPCR Analysis The right side of the volcano plot shows the upregulated proteins after MTX treatment (red),
843
while downregulated ones on the left side (blue). Differential expressions were defined as absolute log2 Fold change ≥ 1 with adjusted p-
844
value ≤ 0.05. (D) Mitochondrial ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of MitoSOX mean
845
fluorescence intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (E)
846
Intracellular ROS levels in response to 24 h 50 nM MTX measured by flow cytometric quantification of DCF-DA mean fluorescence
847
intensity. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (F) mRNA expression
848
from the indicated target genes in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time
849
RT-qPCR. Each dot represents an independent experiment; mean ± SEM. No data shown for SLUG expression at 72 h. (G) mRNA
850
expression from the indicated target genes in MTX-resistant MDA-MB468 cells relative to parental MDA-MB468 cells as measured using
851
real-time RT-qPCR. Each dot represents an independent experiment; mean ± SEM. (H) Expression of vimentin in MDA-MB468 cells after
852
240 h 50 nM MTX treatment; β-actin serves as loading control. Quantification of vimentin signal intensity relative to total protein stain. 853
Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (I) Expression of vimentin in parental
854
and MTX-resistant MDA-MB468 cells; β-actin serves as loading control. Quantification of vimentin signal intensity relative to total protein
855
stain. Each dot represents an independent experiment; mean ± SD; unpaired t-test with Welch’s correction. (J) mRNA expression from
856 MMP9 in MDA-MB468 cells in response to 50 nM MTX at the indicated time points as measured using real-time RT-qPCR. Each dot
857
represents an independent experiment; mean ± SEM. (K) Intracellular ROS levels in response to 24 h 50 nM MTX and 10 mM NAC as
858
measured by flow cytometric quantification of DCF-DA mean fluorescence intensity. Each dot represents an independent experiment. 859
Graph shows mean ± SD; Brown-Forsythe and Welch ANOVA test with Games-Howell’s multiple comparisons test. (L) Migratory potential
860
of MDA-MB468 cells in response to 10 mM NAC and 50 nM MTX treatment as time-dependent quantification of relative wound density in
861
IncuCyte and respective AUC. RNA Extraction, cDNA Synthesis, and qPCR Analysis (E) Absolute CORE rates of lactate and glucose
in the culture medium of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of
triplicate wells; mean ± SEM. (F) Enrichment of representative isotopologues of pyruvate (Pyr), Cit, αKG, Fum, Mal, and Asp upon [U-
13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment with triplicate
wells; mean ± SEM; unpaired t-test with Welch’s correction. (G) Basal cellular respiration in response to 24 h 50 nM MTX treatment was
determined in MDA-MB468, LN229, and 4T1 cells as quantification of mitochondrial oxygen consumption rate (OCR). Each dot represents
an individual experiment composed of six technical replicates; mean ± SEM. (H) Enrichment of M+3 isotopologue of serine upon [U-
13C]glucose tracer in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate
wells; mean ± SEM; unpaired t-test with Welch’s correction. (I) Ratio of absolute intracellular NAD+ to NADH in MDA-MB468 cells in
response to 24 h 50 nM MTX. Each dot represents an individual experiment composed of triplicate wells; mean ± SEM. (J) M+1
isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon 24 h treatment with 50 nM MTX. Each dot
indicates an independent experiment measured in triplicate wells; mean ± SEM. 88
Figure 4: Mitochondria protect SOC-dependent serine catabolism in the presence of MTX. (A) Schematic of the cytosolic and
89
mitochondrial compartments of SOC and the pharmacologic intervention points of MTX. (B) Absolute CORE rates of serine and glycine
90
of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents the mean of an individual experiment each measured in
91
triplicate wells; mean ± SEM. (C) MID of extracellular formate upon [U-13C]serine tracer in MDA-MB468 cells in response to 24 h 50 nM
92
MTX. Graph shows mean ± SEM of three independent experiments each measured in triplicate wells; unpaired t-test with Welch’s
93
correction for M+1 isotopologue enrichment. (D) MID of intracellular serine upon [U-13C]serine tracer in MDA-MB468 cells in response to
94
24 h 50 nM MTX; mean ± SEM of four independent experiments each measured in triplicate wells. (E) Ratio of absolute intracellular serine
95
and glycine in response to 24 h 50 nM MTX in MDA-MB468 cells. RNA Extraction, cDNA Synthesis, and qPCR Analysis Each dot represents an independent experiment in triplicate wells; mean
96
± SEM. (F) MID of intracellular serine upon [U-13C]serine tracer in SHMT1, MFT, SHMT2, and FPGS-depleted HAP1 cells; mean ± SEM
97
of independent experiments each measured in triplicate wells (n = 2 – 8). (G) Ratio of absolute intracellular serine and glycine levels in
98
SHMT1, MFT, SHMT2, and FPGS depleted HAP1 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM. 99
00 8
Figure 4: Mitochondria protect SOC-dependent serine catabolism in the presence of MTX. (A) Schematic of the cytosolic and
9
mitochondrial compartments of SOC and the pharmacologic intervention points of MTX. (B) Absolute CORE rates of serine and glycine
0
of MDA-MB468 cells in response to 24 h 50 nM MTX. Each dot represents the mean of an individual experiment each measured in
triplicate wells; mean ± SEM. (C) MID of extracellular formate upon [U-13C]serine tracer in MDA-MB468 cells in response to 24 h 50 nM
2
MTX. Graph shows mean ± SEM of three independent experiments each measured in triplicate wells; unpaired t-test with Welch’s
3
correction for M+1 isotopologue enrichment. (D) MID of intracellular serine upon [U-13C]serine tracer in MDA-MB468 cells in response to
4
24 h 50 nM MTX; mean ± SEM of four independent experiments each measured in triplicate wells. (E) Ratio of absolute intracellular serine
5
and glycine in response to 24 h 50 nM MTX in MDA-MB468 cells. Each dot represents an independent experiment in triplicate wells; mean
6
± SEM. (F) MID of intracellular serine upon [U-13C]serine tracer in SHMT1, MFT, SHMT2, and FPGS-depleted HAP1 cells; mean ± SEM
7
of independent experiments each measured in triplicate wells (n = 2 – 8). (G) Ratio of absolute intracellular serine and glycine levels in
8
SHMT1, MFT, SHMT2, and FPGS depleted HAP1 cells. Each dot represents an independent experiment in triplicate wells; mean ± SEM. 9
0 Figure 5: Targeting de novo serine synthesis decreases MTX-dependent cancer cell migration. (A) MID of intracellular serine upon
[U-13C]glucose tracer in MDA-MB468 cells in response to 24 h treatment with 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, 15
μM BI-4916, and 50 nM MTX as indicated; mean ± SEM of independent experiments each measured in triplicate wells (n = 1 – 9). RNA Extraction, cDNA Synthesis, and qPCR Analysis (B)
M+1 isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon treatment as in (A). Each dot indicates
an independent experiment measured in triplicate wells; mean ± SEM. (C) Basal cellular respiration in response to 24 h 10 μM WQ-2101
10 μM NCT-502, 30 μM CBR-5884, and 15 μM BI-4916 treatment was determined in MDA-MB468 cells as quantification of mitochondria
OCR. Graph shows fold OCR compared to untreated control. Each dot represents an individual experiment composed of six technic a
replicates; mean ± SEM. (D) M+2 isotopologues of indicated TCA-metabolites in MDA-MB468 cells using [U-13C]glucose tracer upon
treatment as in (B). Each dot indicates an independent experiment measured in triplicate wells; mean ± SEM. (E) Migration of MDA-
MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective AUC; mean ± SEM (n = 5); Brown-Forsythe and Welch one-
way ANOVA with Dunnett’s multiple comparisons test. (F) Migration of MDA-MB468 cells in response to 50 nM MTX, 15 μM BI-4916, and
serine- and glycine starvation and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s
multiple comparisons test. (G) mRNA expression of the indicated target genes in MDA-MB468 cells in response to 72 h 50 nM MTX, 15
μM BI-4916, and serine- and glycine starvation as measured using real-time RT-qPCR. Each dot represents an independent experiment;
mean ± SEM. (H) MDA-MB468 cells were treated for 48 h with 50 nM MTX and 15 μM BI-4916 under serine- and glycine starvation. Cel
death induction was assessed by flow cytometry of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3). 2way ANOVA with Dunnett’s
multiple comparisons test. (I) Migration of MTX-resistant MDA-MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective
AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (J) Schematic depiction
of the cytosolic and mitochondrial compartments of SOC and the pharmacologic intervention points of MTX and inhibitors of PHGDH. 901
Figure 5: Targeting de novo serine synthesis decreases MTX-dependent cancer cell migration. (A) MID of intracellular serine upon
902
[U-13C]glucose tracer in MDA-MB468 cells in response to 24 h treatment with 10 μM WQ-2101, 10 μM NCT-502, 30 μM CBR-5884, 15
903
μM BI-4916, and 50 nM MTX as indicated; mean ± SEM of independent experiments each measured in triplicate wells (n = 1 – 9). RNA Extraction, cDNA Synthesis, and qPCR Analysis (B)
904
M+1 isotopologue of extracellular formate in MDA-MB468 cells using [U-13C]glucose tracer upon treatment as in (A). Each dot indicates
905
an independent experiment measured in triplicate wells; mean ± SEM. (C) Basal cellular respiration in response to 24 h 10 μM WQ-2101,
906
10 μM NCT-502, 30 μM CBR-5884, and 15 μM BI-4916 treatment was determined in MDA-MB468 cells as quantification of mitochondrial
907
OCR. Graph shows fold OCR compared to untreated control. Each dot represents an individual experiment composed of six technic al
908
replicates; mean ± SEM. (D) M+2 isotopologues of indicated TCA-metabolites in MDA-MB468 cells using [U-13C]glucose tracer upon
909
treatment as in (B). Each dot indicates an independent experiment measured in triplicate wells; mean ± SEM. (E) Migration of MDA-
910
MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective AUC; mean ± SEM (n = 5); Brown-Forsythe and Welch one-
911
way ANOVA with Dunnett’s multiple comparisons test. (F) Migration of MDA-MB468 cells in response to 50 nM MTX, 15 μM BI-4916, and
912
serine- and glycine starvation and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s
913
multiple comparisons test. (G) mRNA expression of the indicated target genes in MDA-MB468 cells in response to 72 h 50 nM MTX, 15
914
μM BI-4916, and serine- and glycine starvation as measured using real-time RT-qPCR. Each dot represents an independent experiment;
915
mean ± SEM. (H) MDA-MB468 cells were treated for 48 h with 50 nM MTX and 15 μM BI-4916 under serine- and glycine starvation. Cell
916
death induction was assessed by flow cytometry of AnnexinV-FITC/PI-stained cells; mean ± SD (n = 3). 2way ANOVA with Dunnett’s
917
multiple comparisons test. (I) Migration of MTX-resistant MDA-MB468 cells in response to 50 nM MTX and 15 μM BI-4916 and respective
918
AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch one-way ANOVA with Dunnett’s multiple comparisons test. (J) Schematic depiction
919
of the cytosolic and mitochondrial compartments of SOC and the pharmacologic intervention points of MTX and inhibitors of PHGDH. 920 21
Figure 6: Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility. (A) Absolute formate CORE rates of
22
MDA-MB468 cells in response to MTHFD2 knockdown and treatment for 24 h with 50 nM MTX. RNA Extraction, cDNA Synthesis, and qPCR Analysis Each dot indicates an independent
23
experiment measured in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (B) Proliferation of NucLight Rapid Red
24
stained MDA-MB468 cells harboring a MTHFD2 knockdown in response to 50 nM MTX; mean ± SEM (n = 6). (C) Migration of MDA-
25
MB468 cells in response to MTHFD2 knockdown and 50 nM MTX and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch
26
one-way ANOVA with Dunnett’s multiple comparisons test. (D) MDA-MB468 cells carrying a MTHFD2 knockdown were treated for 48 h
27
with 50 nM MTX. Cell death induction was assessed by flow cytometry of AnnexinV-APC/PI-stained cells; mean ± SD (n = 4). 2way
28
ANOVA with Dunnett’s multiple comparisons test. 29 921
922
923
924
925
926
927
928
929 921
Figure 6: Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility. (A) Absolute formate CORE rates of
922
MDA-MB468 cells in response to MTHFD2 knockdown and treatment for 24 h with 50 nM MTX. Each dot indicates an independent
923
experiment measured in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (B) Proliferation of NucLight Rapid Red
924
stained MDA-MB468 cells harboring a MTHFD2 knockdown in response to 50 nM MTX; mean ± SEM (n = 6). (C) Migration of MDA-
925
MB468 cells in response to MTHFD2 knockdown and 50 nM MTX and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch
926
one-way ANOVA with Dunnett’s multiple comparisons test. (D) MDA-MB468 cells carrying a MTHFD2 knockdown were treated for 48 h
927
with 50 nM MTX. Cell death induction was assessed by flow cytometry of AnnexinV-APC/PI-stained cells; mean ± SD (n = 4). 2way
928
ANOVA with Dunnett’s multiple comparisons test. 929 921
Figure 6: Targeting mitochondrial serine catabolism reduces MTX-dependent cell motility. (A) Absolute formate CORE rates of
922
MDA-MB468 cells in response to MTHFD2 knockdown and treatment for 24 h with 50 nM MTX. Each dot indicates an independent
923
experiment measured in triplicate wells; mean ± SEM; unpaired t-test with Welch’s correction. (B) Proliferation of NucLight Rapid Red
924
stained MDA-MB468 cells harboring a MTHFD2 knockdown in response to 50 nM MTX; mean ± SEM (n = 6). RNA Extraction, cDNA Synthesis, and qPCR Analysis (C) Migration of MDA-
925
MB468 cells in response to MTHFD2 knockdown and 50 nM MTX and respective AUC; mean ± SEM (n = 4); Brown-Forsythe and Welch
926
one-way ANOVA with Dunnett’s multiple comparisons test. (D) MDA-MB468 cells carrying a MTHFD2 knockdown were treated for 48 h
927
with 50 nM MTX. Cell death induction was assessed by flow cytometry of AnnexinV-APC/PI-stained cells; mean ± SD (n = 4). 2way
928
ANOVA with Dunnett’s multiple comparisons test. 929 0
Figure 7: Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo. (A) Experimental layout: 4T1 breast cancer
1
cells transfected with non-targeting control (SCR, n=9) and 4T1 cells with knockdown of Mthfd1l (n=9) were injected into the mammary
2
fat pads of immunocompetent female Balb/c mice (1 injection/mouse in the left mammary fat pad). Primary tumor growth was monitored
3
over time and lung metastases formation was evaluated at the end-point. (B) Primary tumor size was measured at the indicated time
4
points and mean tumor volume was calculated. Data are presented as tumor volume ± SEM at each time point following injection with
5
4T1 SCR or Mthfd1l KD cells. (C) Primary tumor weight was measured at the end-point. Each dot indicates an independent animal; mean
6
± SEM; unpaired t-test with Welch’s correction. (D) Lung tissue was stained with H&E stain. Representative pictures show microscopic
7
lung metastasis in mice after orthotopic injection of 4T1 SCR and Mthfd1l KD cells. Arrows indicate metastatic nodules. Scale bar
8
corresponds to 100 μm. (E) Macroscopic lung metastases were counted and number of metastases per lung is depicted. Each dot
9
indicates an independent animal; mean ± SEM; unpaired t-test with Welch’s correction. 0
1 Figure 7: Genetic targeting of mitochondrial SOC reduces metastasis formation in vivo. (A) Experimental layout: 4T1 breast cancer
cells transfected with non-targeting control (SCR, n=9) and 4T1 cells with knockdown of Mthfd1l (n=9) were injected into the mammary
fat pads of immunocompetent female Balb/c mice (1 injection/mouse in the left mammary fat pad). Primary tumor growth was monitored
over time and lung metastases formation was evaluated at the end-point. (B) Primary tumor size was measured at the indicated time
points and mean tumor volume was calculated. Data are presented as tumor volume ± SEM at each time point following injection with
4T1 SCR or Mthfd1l KD cells. (C) Primary tumor weight was measured at the end-point. Each dot indicates an independent animal; mean
± SEM; unpaired t-test with Welch’s correction. (D) Lung tissue was stained with H&E stain. Representative pictures show microscopic
lung metastasis in mice after orthotopic injection of 4T1 SCR and Mthfd1l KD cells. Arrows indicate metastatic nodules. Scale bar
corresponds to 100 μm. (E) Macroscopic lung metastases were counted and number of metastases per lung is depicted. Each dot
indicates an independent animal; mean ± SEM; unpaired t-test with Welch’s correction. Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementalFiguresKiweleretal.pdf
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https://openalex.org/W2918520968
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https://journal.umy.ac.id/index.php/jiwp/article/download/5832/4062
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Indonesian
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GERAKAN ISLAM INDONESIA: MENGURAI BELENGGU, MEMBANGUN PERADABAN
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Journal of Islamic World and Politics
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cc-by-sa
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GERAKAN ISLAM INDONESIA: MENGURAI
BELENGGU, MEMBANGUN PERADABAN* Mohammad Syifa A. Widigdo
Magister Studi Islam
Universitas Muhammadiyah Yogyakarta
Email: syifamin@gmail.com Abstrak “Gerakan Islam Indonesia (GII) pada umumnya tidak mempunyai pengaruh,
kontribusi, dan peran global yang berarti meskipun kiprah dan kontribusinya
di tingkat nasional tidak perlu disangsikan lagi. Kalaupun ada bagian dari
GII yang mempunyai jaringan internasional, mereka berada dalam posisi
inferior karena hanya memosisikan dirinya sebagai cabang, proksi, atau
simpatisan dari organisasi Islam internasional. Akibatnya, GII tidak saja
hanya menjadi gerakan lokal atau gerakan proksi organisasi transnasional,
tapi juga tidak sanggup menghadapi tantangan global yang semakin
kompleks. Dalam artikel ini, saya berusaha memetakan kelemahan dan
keterbatasan GII tersebut dan menyajikan beberapa tawaran jalan keluarnya. Salah tawaran solusinya adalah bahwa Gerakan Islam se-Asia Tengara
penting untuk berkolaborasi dan bergerak secara aktif sehingga Islam di Asia
Tenggara tidak lagi dianggap sebagai pinggiran. Sebab, Gerakan Islam di
Asia Tenggara mempunyai cukup potensi dan modal untuk menjadi salah
satu pusat peradaban Islam di dunia.” Kata Kunci: Gerakan Islam, Jaringan Internasional, Asia Tenggara, dan
Tantangan Global Tantangan Global Abstract Indonesian Islamic Movementsgenerally do not show a global influence and
contribution although in a national level its crucial role and contribution are
undeniable. Some of these movements may have an international network
Sebagian (diskusi tentang tantangan dan belenggu Gerakan Islam Indonesia) dari
artikel ini pernah dipublikasikan di Harian Umum Republika dengan judul
“Peran Global Gerakan Islam Indonesia” pada tanggal 10 Februari 2016. Artikel
ini merupakan versi yang lengkap dan mendalam, termasuk diskusi tentang
solusi untuk menaklukkan tantangan dan keluar dari belenggu, yang penulis olah
dari dari hasil penelitian terhadap Gerakan Islam Indonesia. Vol.2. No.2 July-December 2018 ISSN: 2614-0535 E-ISSN: 2655-1330 Mohammad Syifa A. Widigdo 387 y
g
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban but their power and authority are limited since their position is merely as a
branch, proxy, or sympathizer of a certain transnational Islamic organization. As a result, these local and proxy movements are unable to cope with global
challenges, which are increasingly complex. In this article, I attempt to map
weaknesses and limitations of Indonesian Islamic Movements and offer some
altenative solutions to cope with such weaknesses and limitations. One of the
altenative solutions is for Indonesian Islamic Movements to operate and
collaborate more actively with other Islamic movements in Southeast Asia. In
this way, Islamic movements in Indonesia in particular, and in Southeast Asia in
general, are not deemed peripheral. They have sufficient potentials and
capabilities to be one of the centers of Islamic civilization. Key words: Islamic Movement, International networking, Southeast Asia,
and Global Challenge September 2013; Chaq, Mei 2013). Tidak mengherankan jika kemudian
gerakan Islam diidentikkan dengan
gerakan
politik
yang
bertujuan
merubah tatanan rezim di tingkat
nasional
maupun
global(Lihat
Basyir, November 2016, pp. 339-
362) .Sementara
kegiatan
dan
perjuangan organisasi Islam yang
menggunakan cara-cara transformatif
dan
damai
seringkali
tidak
mendapatkan label sebagai “Gerakan
Islam.”Istilah yang dipakai untuk
menggambarkan
gerakan
ini,
misalnya, adalah “organisasi September 2013; Chaq, Mei 2013). Tidak mengherankan jika kemudian
gerakan Islam diidentikkan dengan
gerakan
politik
yang
bertujuan
merubah tatanan rezim di tingkat
nasional
maupun
global(Lihat
Basyir, November 2016, pp. 339-
362) .Sementara
kegiatan
dan
perjuangan organisasi Islam yang
menggunakan cara-cara transformatif
dan
damai
seringkali
tidak
mendapatkan label sebagai “Gerakan
Islam.”Istilah yang dipakai untuk
menggambarkan
gerakan
ini,
misalnya, adalah “organisasi 3
Untuk
mempelajari
lebih
lanjut
perbedaan antara tajdīd, iṣlāḥ, dan iḥyā’,
silahkan baca Abū Khalil, As‟ad and
Mahmoud
Haddad.
“Revival
and
Renewal.” In The Oxford Encyclopedia
of the Islamic World. Oxford Islamic
Studies
Online,
http://www.
oxfordislamicstudies.com/article/opr/
t236/e0682 (accessed Apr 30, 2013). PENDAHULUAN Istilah “Gerakan Islam” belakangan
cenderung
mengalami
peyorasi
karena
seringkali
diasosiasikan
dengan
gerakan-gerakan
yang
berorientasi politik, radikal, at au
meng ha l a l kanc ara - c ara
kekerasan untuk mencapai tujuan
politik.Peyorasi ini tampak dalam
beberapa
kajian
dan
penelitian
membahas gerakan Islam, biasanya
dikaitkan dengan varian radikal dari
gerakan
tersebut
(Lihat
tulisan
Ahyar, Mei 2015 pp. 1-26; Ar, 8 Islamic World and Politics
Vol.2. No.2 July-December 2018 88 Islamic World and Politics
Vol.2. No.2 July-December 2018 388 Islam”(Lihat misalnya Collins, Oct.,
2004). Padahalistilah
“gerakan”
dalam
konteks
sejarah
Islam
mengacu pada dua konsep, yakni
pembaharuan (tajdīd) dan reformasi
(iṣlāḥ), yang meniscayakan makna
perubahandan transformasi secara
damai
dan
beradab.3Misalnya,
gerakan
pembaharuan
(tajdīd)
Muhammad Abduh (w. 1905) di
Mesir di bidang pemikiran melalui
jurnal “Al-Urwat al- Wuthqā” dan
reformasi
(iṣlāḥ)
di
bidang
pendidikan dengan memperkenalkan
bidang studi sains di kurikulum
Universitas al-Azhar(Hourani, 1983,
pp. 130-192).Oleh sebab itu, dalam
artikel ini, saya menggunakan istilah
“Gerakan Islam” dalam pengertian
yang luas, tidak hanya untuk gerakan
yang berorientasi politik dan radikal,
tapi
juga
(bahkan
utamanya)
merujuk pada organisasi dan gerakan
keagamaan
berorientasi
sosial,
intelektual, atau kultural, seperti NU
(Nahdlatul Ulama), Muhammadiyah,
Persis
(Persatuan
Islam),
Hidayatullah,
HMI
(Himpunan
Mahasiswa Islam), Islam”(Lihat misalnya Collins, Oct.,
2004). Padahalistilah
“gerakan”
dalam
konteks
sejarah
Islam
mengacu pada dua konsep, yakni
pembaharuan (tajdīd) dan reformasi
(iṣlāḥ), yang meniscayakan makna
perubahandan transformasi secara
damai
dan
beradab.3Misalnya,
gerakan
pembaharuan
(tajdīd)
Muhammad Abduh (w. 1905) di
Mesir di bidang pemikiran melalui
jurnal “Al-Urwat al- Wuthqā” dan
reformasi
(iṣlāḥ)
di
bidang
pendidikan dengan memperkenalkan
bidang studi sains di kurikulum
Universitas al-Azhar(Hourani, 1983,
pp. 130-192).Oleh sebab itu, dalam
artikel ini, saya menggunakan istilah
“Gerakan Islam” dalam pengertian
yang luas, tidak hanya untuk gerakan
yang berorientasi politik dan radikal,
tapi
juga
(bahkan
utamanya)
merujuk pada organisasi dan gerakan
keagamaan
berorientasi
sosial,
intelektual, atau kultural, seperti NU
(Nahdlatul Ulama), Muhammadiyah,
Persis
(Persatuan
Islam),
Hidayatullah,
HMI
(Himpunan
Mahasiswa Islam), dan PII (Pelajar Islam Indonesia),
bahkan perguruan tinggi-perguruan
tinggi Islam negeri maupun swasta. Saya
melihat
adanya
beberapa
kelamahan
dalam
“Gerakan
IslamIndonesia
(GII)”
dalam
konteks pembaharuan (tajdīd) dan
reformasi (iṣlāḥ) tersebut. Di satu
sisi, GII yang direpresentasikan oleh
organisasi-organisasi
Islam
yang
sudah
mapan
tidak
mempunyai
kredensial dan jaringan di tingkat
internasional
yang
membuatnya
hanya
berpengaruh
di
tingkat
nasional saja. Organisasi-organisasi
Islam seperti NU, Muhammadiyah,
Persis,
Hidayatullah,
HMI,
dan
PIImeskipun mempunyai pengaruh
kuat
di
tingkat
nasional,
tapi
pengaruh
visi
dan
gerakan
keislamannya tidak merambah ke
tingkat yang lebih luas. PENDAHULUAN Di
sisi
yang
lain,
GII
yang
mempunyai
jaringan
di
tingkat
internasional
kebanyakan
hanya
merupakan cabang, proksi, atau
simpatisan
dari
gerakan-gerakan
Islam
transnasional. Daripada
menyebarkan visi berislam yang
bercorak keindonesiaan ke tingkat
internasional,
kelompok-kelompok
Islam ini lebih bersemangat untuk
menerjemahkan
doktrin-
doktrin
induk
organisasinya
di
tanah
Indonesia. Hal ini terjadi, misalnya,
dalam kasus gerakan salafi, HTI Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
u, Membangun Peradaban 389 bidang merambah ke dunia muslim,
kaum muslim mempunyai respon
yang beragam. Sebagian dari mereka
menyambut
modernisasi
dengan
antusias. Nilai-nilai yang lahir dari
rahim modernitas seperti demokrasi,
kebebasan individu, kemerdekaan
berfikir, persamaan hak, hak asasi
manusia, ide kemajuan (the idea of
progress), dan sejenisnya diadopsi
dan diinjeksikan ke dunia muslim
tanpa melalui proses penyaringan
yang ketat. Mereka kemudian sering
disebut sebagai muslim liberal. (Hizbut Tahrir Indonesia), berbagai
kelompok simpatisan Negara Islam
Iraq dan Suriah (NIIS). Dua mainstream Gerakan Islam
Indonesia sebagaimana disebutkan
di atas, selain bermasalah dalam
konteks skala pengaruh dan corak
pemikiran,
juga
mempunyai
kelemahan dalam cara pandang
dunianya. Walhasil, Gerakan Islam
Indonesia tidak saja hanya menjadi
gerakan lokal atau gerakan proksi
organisasi transnasional, tapi juga
tidak
sanggup
menghadapi
tantangan global yang semakin
kompleks. Melalui refleksi pendek
atas
fenomena
Gerakan
Islam
Indonesiakontemporer dan literatur
yang saya baca, saya berusaha
memetakan
kelemahan
dan
keterbatasan
GII
tersebut
dan
menyajikan beberapa tawaran jalan
keluarnya. Salah
satu
jalan
keluarnya
adalah
penggalakan
internasionalisasi Gerakan Islam
Indonesia, minimal dimulai dari
lever Asia Tenggara. Sebagian yang lain melihat
modernitas
dengan
nilai-nilai
dibawanya sebagai sebuah ancaman
dan karenanya harus ditolak dan
dilawan. Modernitas diidentikkan
dengan nilai, budaya, dan politik
negara Barat yang dinilai tidak cocok
dengan Islam. Mereka kemudian
melihat Islam tidak hanya sebagai
agama tapi juga sebagai ideologi
politik, ekonomi, sosial, dan budaya. Islam, bagi mereka, adalah alternatif
sistem nilai yang orisinal dan solusi
bagi segala permasalahan (al-Islām
huwa
al-ḥall).Beberapa
kalangan
bahkan berpendapat bahwa tawaran
sistem dan peradaban Islam lebih
superior dan bisa menggantikan
peradaban Barat modern. Kalim
Siddiqui(1983) bahkan menganggap
bahwa kohesi peradaban Islam yang
dianggap superior itu dapat TANTANGAN Dunia modern yang semakin
terbuka
dan
saling
terkoneksi
memberikan
tantangan
yang
kompleks baik di sektor ekonomi,
politik,
sosial,
budaya,
maupun
dalam kehidupan keagamaan. Ketika
proses modernisasi di hampir segala 390 Islamic World and Politics
Vol.2. No.2 July-December 2018 390 direduksi
dalam
bentuk
negara
Islam. Dia
mengakatan,
“The
Islamic movement, as demonstrated
in
earlier
discussion,
has
the
network
of
mosques,
political
culture,
common
memory
and
shared expectations necessary to
hold together until a higher state of
cohesion is reached. The highest
stage of this cohesion is of course
the Islamic state”(Siddiqui, 1983). Meskipun mereka juga menikmati
dan menggunakan hasil modernitas
seperti teknologi dengan berbagai
produknya, namun secara umum
mereka
tetap
resisten
terhadap
modernitas dengan pelbagai turunan
nilai
dan
sistemnya. Untuk
mencapai tujuan-tujuan perlawanan
tersebut, sebagian memakai cara-
cara yang persuasif dan akademik,
namun sayangnya sebagian yang
lain menghalalkan dan memakai
cara-cara kekerasan. Karena klaim
keislamannya,
mereka
sering
disebut sebagai kelompok Islamis. direduksi
dalam
bentuk
negara
Islam. Dia
mengakatan,
“The
Islamic movement, as demonstrated
in
earlier
discussion,
has
the
network
of
mosques,
political
culture,
common
memory
and
shared expectations necessary to
hold together until a higher state of
cohesion is reached. The highest
stage of this cohesion is of course
the Islamic state”(Siddiqui, 1983). Meskipun mereka juga menikmati
dan menggunakan hasil modernitas
seperti teknologi dengan berbagai
produknya, namun secara umum
mereka
tetap
resisten
terhadap
modernitas dengan pelbagai turunan
nilai
dan
sistemnya. Untuk
mencapai tujuan-tujuan perlawanan
tersebut, sebagian memakai cara-
cara yang persuasif dan akademik,
namun sayangnya sebagian yang
lain menghalalkan dan memakai
cara-cara kekerasan. Karena klaim
keislamannya,
mereka
sering
disebut sebagai kelompok Islamis. dengan nilain-nilai modern tapi juga
berakar dalam tradisi khazanah
intelektual Islam. Di sisi yang lain,
ketika nilai-nilai tersebut dipisahkan
dari akar keagamaan, dijadikan
jargon ideologis, dan kemudian
dipaksakan
melalui
dominasi
politik,
represi
ekonomi,
dan
kekuatan
militer,
mereka
me-
nolaknya. Penolakan
tersebut,
misalnya, terefleksi dalam resistensi
mereka terhadap sekularisme se-
bagai sebagai faham ideologis, ter-
hadap struktur kapitalisme atau neo
-liberalisme yang eksploitatif, dan
terhadap
imperialisme
politik-
militer
negara-negara
Barat
di
negeri-negeri muslim. Persepsi Gerakan Islam ter-
hadap tantangan global tergantung
pada model penyikapan mereka
terhadap
fenomena
modernitas
tersebut. Bagi
kaum
muslim
liberal, tantangan utama yang ada
dalam benak mereka dalam sikap
konservatif,
apologetik,
dan
resisten dari kalangan muslim
sendiri. Bagi mereka, sikap seperti
itu malah menarik Islam ke dalam
kemunduran
dan
menghambat
potensi partisipasi dan kontribusi
Islam bagi peradaban modern. TANTANGAN Watt,
hubungan antara Barat dan Islam itu
tidak melulu konfliktual, tapi juga
saling memberi dan menerima,” The
history of Islam and Europe is not
only a history of mutual conquests
(jihad and crusade), but is also a
history of cultural encounters in the
course of which decisive process of
cultural borrowing took place in
both sides”(Tibi, 2012, p. 17; Watt,
1991). Gerakan Islam Indonesia yang
memiliki masalah berupa belenggu
ideologis dan peimikiran adalah
kalangan muslimliberal dan Islamis. Kalangan
liberal
mengalami
kesulitan
untuk
membayangkan
masa
depan
peradaban
tanpa
menjadikan Barat sebagai referensi
utama.Imajinasi mereka terjerat dan
terbatas pada apa yang telah dicapai
dan dimiliki oleh Barat. Barangkali
mereka memang realitis karena
kemajuan peradaban Barat TANTANGAN Sementara bagi kalangan Islamis
yang resisten terhadap modernitas,
tantangan utama yang menghantui
pikiran mereka adalah peradaban Namun demikian, sebagian
kaum muslimin yang lain meng-
hadapi modernitas dengan sikap
kritis dan selektif. Di satu sisi,
mereka
menghargai
nilai-nilai
modern tersebut karena dianggap
paralel
dan
ekuivalen
dengan
semangat ajaran Islam. Mereka
bahkan mampu merumuskan nilai-
nilai yang tidak saja bersesuaian Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 391 Barat
yang
menginisiasi
dan
mengendalikan modernisme. Islam
sebagai
sebuah
agama
dan
peradaban
dianggap
berlawanan
secara diametral dengan peradaban
Barat modern tersebut. Sedangkan
bagi kalangan muslim kritis, tan-
tangan utamanya adalah memilah
bagian-bagian yang positif dari
peradaban
Barat
modern
dan
khazanah Islam untuk kemudian
dijembatani dan disinergikan, dan
pada
yang
bersamaan,
menanggalkan bagian-bagian yang
negatifdari keduanya. Bagi mereka,
masa depan dunia bukanlah konflik
antar dua peradaban, Barat dan
Islam, melainkan dialog dan sinergi
antar berbagai tradisi peradaban di
dunia.Dalam bahasa Bassam Tibbi
dengan mengutip William M. Watt,
hubungan antara Barat dan Islam itu
tidak melulu konfliktual, tapi juga
saling memberi dan menerima,” The
history of Islam and Europe is not
only a history of mutual conquests
(jihad and crusade), but is also a
history of cultural encounters in the
course of which decisive process of
cultural borrowing took place in
both sides”(Tibi, 2012, p. 17; Watt,
1991). respon kaum muslimin terhadap
tantangan modernitas sebagaimana
dijelaskan di atas. Hanya saja,
Gerakan
Islam
Indonesia
(GII)
mempunyai- beberapa keter- batasan
dan kelemahan yang mem- buatnya
tidak maksimal dalam berpartisipasi
dan berkontribusi bagi konstruksi
peradaban
dunia,
khususnya
peradaban Islam. Keter- batasan dan
kelemahan tersebut muncul bukan
semata dipicu oleh sebab eksternal
seperti
proses
modernisasi(Tibi,
2001),tapi lebih karena sebab-sebab
internal yang saya sebut sebagai
belenggu-belenggu
yang
menghambat
dan
membatasi
kemampuan eksplorasi, kreatifitas,
dan kebebasan mereka. Belenggu-
belenggu tersebut adalah, pertama,
belenggu
ideologis
danepistemologis (pemikiran), dan
kedua, belenggu pergerakan. Barat
yang
menginisiasi
dan
mengendalikan modernisme. Islam
sebagai
sebuah
agama
dan
peradaban
dianggap
berlawanan
secara diametral dengan peradaban
Barat modern tersebut. Sedangkan
bagi kalangan muslim kritis, tan-
tangan utamanya adalah memilah
bagian-bagian yang positif dari
peradaban
Barat
modern
dan
khazanah Islam untuk kemudian
dijembatani dan disinergikan, dan
pada
yang
bersamaan,
menanggalkan bagian-bagian yang
negatifdari keduanya. Bagi mereka,
masa depan dunia bukanlah konflik
antar dua peradaban, Barat dan
Islam, melainkan dialog dan sinergi
antar berbagai tradisi peradaban di
dunia.Dalam bahasa Bassam Tibbi
dengan mengutip William M. BELENGGU Tipologi
Gerakan
Islam
di
Indonesia tidak jauh dari tiga bentuk 392 Islamic World and Politics
Vol.2. No.2 July-December 2018 392 bisa dilihat secara empirik di abad
ke-21 ini. Namun demikian, sikap
ideologisdan
pemikiran
yang
membebek kepada Barat dan hanya
berorientasi untuk menerjemahkan
pencapaian Barat dalam konteks
Indonesia
seperti
itu
membuat
mereka tidak kreatif untuk mencari
referensi
lain
dalam
konteks
pembangunan peradaban di masa
depan. Hal yang serupa terjadi pada
kalangan Islamis. Mereka secara
ideologis
dan
epistemologis
menjadikan
pencapaian
masya-
rakat Islam di masa lalu sebagai
rujukan utama. Bahkan, mereka
tidak bisa melepaskan imajinasi dan
pemikiranya dari bayangan kejayaan
masa lalu untuk dibang- kitkan
kembali di masa kini atau masa
depan. Idealisasi
tentang
kekhalifahan, generasi masa awal
Islam yang saleh (salaf al-ṣāliḥ),
atau para imam yang terjaga dari
dosa
(maʿṣūm)
mendominasi
imajinasi dan aspirasi mereka. Sikap
seperti ini tidak saja utopis tapi juga
membuat mereka tidak kreatif dalam
konteks menerjemahkan nilai-nilai
luhur
yang
terpendam
dalam
khazanah teks dansejarah Islam
dalam konteks kekinian. Mereka
lebih
terobsesi
pada
bentuk,
institusi, dan struktur dari sejarah
masa lalu Islam daripada isi dan
nila-nilai luhur yang diwariskannya. Kedua sikap dan pemikiran tersebut
di atas, baik dari kalangan liberal
maupun Islamis, mempunyai dua
konsekuensi yang kontraproduktif
bagi pembangunan peradaban masa
depan:
satu,
membelenggu
kemerdekaan
dan
kemampuan
Gerakan Islam Indonesia untuk
secara kreatif dan orisinal merumus-
kan sendiri model per-adabannya,
dua,
membuat
Gerakan
Islam
Indonesia kedap rasa dan empati
sehingga tidak bisa mendengar dan
menyelami
suasana
batin
masyarakat Indonesia yang bergelut
dengan berbagai pemasalahannya. Oleh sebab itu, hemat saya, satu-
satunya yang bisa diharapkan untuk
mempimpin agenda pembangunan
peradaban
masa
depan
adalah
Gerakan Islam Indonesia yang kritis
dan transformatif. Gerakan ini tidak
saja terbuka terhadapkebijaksanaan
(wisdom) dan keutamaan (virtue)
baik yang datang dari Barat maupun
dari khazanah teks dan sejarah masa
lalu Islam, tapi juga, mempunyai
kemampuan
untuk
menyelami
suasana batin masyarakat Indonesia
karena mereka memang bergelut
dengan permasalahan masyarakat
dalam aktivitas kesehariannya. Hanya saja, Gerakan Islam bisa dilihat secara empirik di abad
ke-21 ini. Namun demikian, sikap
ideologisdan
pemikiran
yang
membebek kepada Barat dan hanya
berorientasi untuk menerjemahkan
pencapaian Barat dalam konteks
Indonesia
seperti
itu
membuat
mereka tidak kreatif untuk mencari
referensi
lain
dalam
konteks
pembangunan peradaban di masa
depan. Hal yang serupa terjadi pada
kalangan Islamis. Mereka secara
ideologis
dan
epistemologis
menjadikan
pencapaian
masya-
rakat Islam di masa lalu sebagai
rujukan utama. BELENGGU Bahkan, mereka
tidak bisa melepaskan imajinasi dan
pemikiranya dari bayangan kejayaan
masa lalu untuk dibang- kitkan
kembali di masa kini atau masa
depan. Idealisasi
tentang
kekhalifahan, generasi masa awal
Islam yang saleh (salaf al-ṣāliḥ),
atau para imam yang terjaga dari
dosa
(maʿṣūm)
mendominasi
imajinasi dan aspirasi mereka. Sikap
seperti ini tidak saja utopis tapi juga
membuat mereka tidak kreatif dalam
konteks menerjemahkan nilai-nilai
luhur
yang
terpendam
dalam
khazanah teks dansejarah Islam
dalam konteks kekinian. Mereka
lebih
terobsesi
pada
bentuk,
institusi, dan struktur dari sejarah
masa lalu Islam daripada isi dan
nila-nilai luhur yang diwariskannya. Hanya saja, Gerakan Islam
Indonesia
yang
kritis
tersebut
mempunyai belenggunya tersendiri. Tidak seperti kalangan liberal dan Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
u Membangun Peradaban 393 Timur Tengah, khususnya Jazirah
Arab bagi kalangan Sunnī dan Iran
bagi
kalangan
Syi‟ah,
sebagai
episentrum gerakan. Kiprah mereka
di Indonesia tidak lebih dari sekedar
menjadi simpatisan, cabang, atau
proksi bagi pusat pergerakan mereka
di Timur Tengah. Dalam istilah
Bassam
Tibi,
mereka
adalah
tumpahan (spill-over) dari pro-
blematika dan pergerakan di Timur
Tengah yang dianggap sebagai pusat
(the core)(Tibi, 2012, p. 22).Oleh
sebab itu, tidak mengherankan jika
pergerakan
mereka
umumnya
mengandung orientasi politik dan
hanya
ditujukan
untuk
men-
dapatkan legitimasi, afirmasi, dan
pengaminan
dari
kelompok-
kelompok induk mereka di Timur
Tengah. Islamis yang mempunyai jaringan
internasional kuat karena afinitas
ideologis dan epistemologisnya, GII
yang kritis dan transformatifbelum
ditunjang oleh kredensial, integritas,
dan
jaringan
untuk
dapat
berpartisipasi
dan
berkontribusi
secara global. Itulah yang menjadi
belenggu pergerakannya. Berbeda dengan jenis beleng-
gu GII yang bersifat kritis-trans-
formatif,
GII
yang
berkarakter
liberal
dan
Islamis
memiliki
belenggu pergerakannya tersendiri. Mereka
tidak
terlalu
memiliki
masalah dengan jaringan. Masalah
mereka terletak pada integritas dan
kredensial sebagai Gerakan Islam
yang mempunyai jiwa keislaman
dan
keindonesiaan. Gerakan
kalangan liberal menempatkan Barat
sebagai pusat orientasi per- gerakan
dan Indonesia sebagai cabangnya. Pergerakan
mereka
umumnya
berada di level pemikiran dan
ditujukan hanya untuk mendapatkan
legitimasi, afirmasi, atau anggukan
dari Barat bahwa mereka telah
menjadi
pegerakan
Islam
yang
modern dan liberal di tanah air. Orientasi
keislaman
dan
keindonesiaan seringkali absen dari
agenda-agenda
pemikiran
dalam
pergerakannya. Gerakan
kaum
Islamis memiliki belenggu yang
serupa. Mereka menjadikan GII yang berkarakter kritis-
transformatif
tidak
memiliki
problem atau belenggu sebagaimana
yang dimiliki oleh gerakan liberal
dan Islamis dalam konteks kehausan
untuk mendapat legitimasi, afirmasi,
dan persetujuan dari pihak luar. BELENGGU Mereka telah memiliki legitimasi,
afirmasi,
dan
dukungan
sosialmaupun kultural dari masya-
rakat Indonesia. Mereka juga telah
bergelut dengan teks, sejarah, dan
menjadi bagian tak terpisahkan dari
kehidupan masyarakat Islam di
dunia. Hanya saja, sebagaimana 394 Islamic World and Politics
Vol.2. No.2 July-December 2018 394 yang tadi dikemukakan, masalah
utama
bagi
mereka
adalah
minim-nya tingkat partisipasi dan
kontribusi mereka di tingkat global. Masalah ini diperparah dengan
belenggu-belenggu yang membatasi
pergerakan dan kiprah mereka,
seperti krisis kredensial, integritas,
dan jaringandi dalam pergerakan
mereka. Namun, dalam hal krisis
kredensial dan integritas, mereka
berbeda dengan kalangan liberal dan
Islamis. Jika
kalangan
liberal
mengalamai krisis kredensial dan
integritas dalam konteks komitmen
keislaman dan kalangan Islamis
dalam
konteks
komitmen
keindonesiaan,maka
krisis
kredensial
dan
integritas
yang
dimiliki oleh gerakan Islam kritis
berada dalam konteks pergaulan
global. Mereka masih dianggap
sebagai pemain figuran. Keislaman
mereka masih dianggap Islam yang
pinggiran dan inferior(Tibi, 2012,
p. 22).Bagaimana cara mengatasi
belenggu-belenggu gerakan Islam
Indonesia yang seperti ini? di Madinah. Kota -kota lain diang-
gap sebagai kota pinggiran yang
tidak
mempunyai
otoritas
ke-
agamaan
maupun
kekuasaan. Setelah berhasil merebut kekuasaan
dari tangan Alī b. Abī Tālib pada
tahun 661, Mu‟āwīyah mendirikan
Daulah Umayyah yang berpusat di
Damaskus, Syria, hingga tahun 750. Pusat kekuasaan dan keagamaan
pun berpindah ke sana. Kota-kota
lain dianggap sebagai pinggiran dan
tidak punya signifikansi kekuasaan
dan keagamaan yang setara dengan
pusat. Termasuk
Mekah
dan
Madinah,
Kufah,
dan
Basrah. Pesaing-pesaing utama politik yang
tinggal
di
kota-kota
pinggiran
tersebut dihabisi, seperti Husayn b. „Alī di Kufah pada tahun 680 dan
„Abdullāh b. Zubayr di Mekah pada
tahun 692. Namun demikian pusat-
pusat kajian kagamaan di luar
Damaskus mulai merebak. Hasan al-
Basri (642-728) mempunyai pusat
kajian yang ramai dan berpengaruh
di Basrah. „Abdullāh b. „Abbās (w. 687), „Abdullāh b. „Umar (w. 693),
dan Mālik b. Anas (711-795)
mengembangkan tradisi kesarjanaan
tersendiri di Mekkah dan Madinah. Abū Hanīfah (699-
767) merumuskan dan menularkan
t d l
i h k
t
di i di p. 22).Bagaimana cara mengatasi
belenggu-belenggu gerakan Islam
Indonesia yang seperti ini? p. 22).Bagaimana cara mengatasi
belenggu-belenggu gerakan Islam
Indonesia yang seperti ini? PINGGIRAN
MEMBANGUN
KREDENSIAL, INTEGRITAS,
DAN JARINGAN 767) merumuskan dan menularkan
metodologi hukum tersendiri di
Kufah. Tradisi keilmuan yang
berkembang baik di kota-kota Pada awal Islam, dari zaman
Nabi
hingga
al-Khulafā‟
al-
Rāshidūn
(622-661),
pusat
keagamaan dan kekuasaan berada Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 395 perdagangan, kota-kota tersebut juga
menjadi pusat-pusat keilmuan yang
menjadi tiang penyangga zaman
keemasan Islam. „Ulamā‟ di bidang
ilmu al-Qur‟an, Hadīth, fiqih, kalām,
ta‟awwuf, filsafat, dan kesusastraan
kebanyakan berasal dari kota-kota
pinggiran tersebut. Demikian pula
dengan para ilmuwan di bidang
matematika, optik, kimia, astronomi,
kedokteran dan ilmu-ilmu rasional
lainnya. Mereka
banyak
yang
berasal dan berkarya di wilayah luar
pusat
kekuasaanitu. Dinamika
kesarjana-
an
dan
perdagangan
semacam inilah yang membuat kota-
kota
tersebut
tetap
memiliki
kredensial di bidang kesarjanaan
Islam, integritas di bidang ekonomi,
dan jaringan yang kuat baik di
bidang keilmuan maupun ekonomi. Dengan demikian, daerah-daerah
pinggiran tersebut mempunyai daya
tawar,
tidak
dipandang
sebelah
mata, dan mampu berkontribusi
secara signifikan dalam konteks
kejayaan peradaban Islam di abad
pertengahan(Hodgson,
1974a,
1974b) pinggiran
kekuasaan
tersebut
membuatnya
tidak
kehilangan
signifikansi keagamaan. Kota-kota
tersebut
tetap
mempunyai
legitimasi keislaman yang setara,
bahkan
beberapa
lebih
kuat,
dibandingkan dengan Damaskus. Kecenderungan seperti ini terus
terjadi
ketika
pusat
kekuasaan
beralih ke Daulah Abbasiyah di
tahun 750 hingga 1258. Pusat ke-
kuasaan dunia Islam boleh saja
berada di Baghdad dan sebagian
dibagi dengan Dinasti Fatimiyyah
yang berpusat di Kairo (dengan
kekuasaan meliputi Afrika Utara dan
Mesir) dan Dinasti Umayyah di
Andalusia
yang
berpusat
di
Kordoba. Namun demikian, kota-
kota baru di luar lingkaran pusat
kekuasaan juga berkembang. Di
wilayah
Khurāsān,
terdapat
Nīshāpūr, Herat, Marw, dan Balkh. Di Asia Tengah, terdapat kota-kota
seperti Samarqand, Bukhara, dan
Khawarizm. Di Andalusia, selain
Cordoba juga ada Granada. Kota-
kota di pinggir kekuasaan tersebut
tidaklah inferior terhadap pusat-
pusat kekuasaan. Sebagian menjadi
kota perdagangan yang strategis dan
ramai,
seperti
Samarqand
dan
Nishapur, salah satunya karena
memang
berada
di
jalur
perdagangan Silk Road. Namun
demikian, selain menjadi kota Narasi sekilas tentang kotadan
wilayah pinggiran dala sejarah Islam
tersebut memberi gambaran tentang
peran dan sumbangan kota-kota itu
terhadap pembangunan peradaban. Kota-kota tersebut 396 Islamic World and Politics
Vol.2. No.2 July-December 2018 396 tidak hanya menjadi salah satu tiang
kuat
penyangga
peradaban. Beberapa kotabahkan menjadi pusat
kesarjanaan, ekonomi, dan politik
yang
baru,
termasuk
Baghdad,
Kairo, Cordoba, dan Samarqand. PINGGIRAN
MEMBANGUN
KREDENSIAL, INTEGRITAS,
DAN JARINGAN Catatan sejarah ini dapat dijadikan
inspirasi dan pijakan bagi negeri-
negeri di luar pusat dan sumber
diskursus keislaman di masa kini,
termasuk Indonesia. Gerakan Islam
di Indonesia tidak perlu meminta
dan
mencari
legitimasi
keberagamaan
dan
kesarjanaan
Islam dari negeri -negeri di Timur
Tengah. Mereka bisa mengakses
sendiri sumber-sumber keislaman
dari yang primer seperti al-Qur‟an
dan Sunnah hingga yang sifatnya
literatur kesarjanaan seperti yang
ditulis
oleh
para
sarjana
dan
„ulamā‟. Oleh sebab itu, salah satu
hal yang penting dilakukan adalah
pembangunan
kredensial
kesarjanaan Islam di Indonesia,
terutama melalui tradisi riset dan
publikasi yang kuat. Hal ini penting
dilakukan agar para sarjana Islam di
Indonesia dapat berdiksusi secara
setara dengan koleganya di negara-
negara Timur Tengah maupun Barat
dan memberikan kontribusi yang
penting bagi kesarjanaan Islam di
tingkat dunia. Hanya saja, tradisi kesarjanaan
Islam saja tidak c k p Pem bangunan kredensial dan integritas
keislaman di Indonesia juga perlu
ditopang
oleh
kredensial
dan
integritas di bidang-bidang yang
lain, terutama di bidang ekonomi,
ilmu
pengetahuan,
sosial,
serta
politik. Negara-negara Islam di
kawasan Timur Tengah cenderung
menganggap negeri-negeri Islam di
luar kawasan mereka sebagai kurang
Islam
(less Islamic)
dan
oleh
karenanya mereka perlu diislamkan
lagi. Anggapan semacam ini akan
luntur dengan sendirinya apabila
negeri-negeri Muslim di luar Timur
Tengah itu mempunyai kredensial
dan integritas di bidang ekonomi,
ilmu
pengetahuan,
sosial,
serta
politik. Pengalaman
hubungan
antara negara-negara Timur Tengah
dengan negara-negara Barat meng-
ajarkan tentang pola hubungan yang
penuh penghormatan itu. Timur
Tengah lebih respek (untuk tidak
mengatakan,
inferior)
terhadap
negara-negara
Barat
daripada
negara-negara
Muslim
di
luar
kawasan
Timur
Tengah
bukan
karena
bertambahnya
jumlah
populasi Muslim atau majunya-
kesarjanaan
Islam
di
Barat,
melainkan, karena sumber daya
ekonomi,
keunggulan
ilmu
pengetahuan, tatanan sosialnya yang Hanya saja, tradisi kesarjanaan
Islam saja tidak cukup. Pem- Mohammad Syifa A. Widigdo
39
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 397 kekuatan politiknya.4Oleh sebab itu,
agar peradaban Islam di Indonesia
mempunyai
daya
tawar,
posisi
sejajar, dan tidak lagi dianggap
remeh, figuran, inferior, atau lemah
oleh koleganya di Timur Tengah dan
di belahan dunia yang lain, maka
Gerakan
Islam
Indonesia
dituntutuntuk menjadi peolopor bagi
pembangunan bukan saja iklim dan
tradisi kesarjanaan yang kuat, tapi
juga kekuatan ekonomi, keunggulan institusi pendidikan,
keteraturan sosial, dan stabilitas
politik. PINGGIRAN
MEMBANGUN
KREDENSIAL, INTEGRITAS,
DAN JARINGAN Dalam konteks Indonesia kini,
stabilitas politik Indonesia dan
negara-negara yang berpenduduk
Muslim di kawasan Asia Tenggara
jauh lebih baik daripada kondisi
politik di negara-negara Timur
Tengah. Gerakan Islam Indonesia
tidak perlu untuk terobsesi di
sektor politik, apalagi dengan
hanya glorifikasi struktur politik
masa lalu (baca: khilāfah atau
imāmah). Pada
dasarnya,yang
diperlukan di sektor inihanyalah
terusmerawat demokrasi sehingga
proses pergantian politik dapat
berlangsung secara damai dan
problem-problem kerakyatan dapat
diselesaikan. Pekerjaan rumahGII
kemudian tinggal mengerucutpada
tigabidang,
yaitu
membangun
kekuatan ekonomi, mewujudkan
tatanan sosial yang adil dan damai,
dan keunggulan ilmu pengetahuan. 4
Hal ini tercermin dalam kenyataan
bahwa negera-negara Timur Tengah
mengirimkan pelajar-pelajar terbaiknya
untuk belajar di Barat. Para sarjana dan
professional terbaik mereka juga banyak
yang berkarir di negera-negara Barat. Bahkan, para pembesar politik maupun
tokoh
intelektual
mereka
pun
kebanyakan
pernah
menempuh
pendidikan di Barat. Ini menunjukkan
betapa mereka mempunyai respek yang
besar terhadap kemajuan peradaban dan
perkembangan ilmu pengetahuan di
Barat. Bukan itu saja, beberapa negara di
Timur Tengah, seperti Qatar, UEA, dan
Arab Saudi bahkan membuka cabang-
cabang perguruan tinggi Barat di negara
mereka atau merekrut secara besar-
besaran para pengajar lulusan Barat
untuk mengajar di perguruan tinggi
mereka. Hal yang sama terjadi dalam
konteks
politik
dan
ekonomi. Ketergantungan mereka terhadap Barat,
terutama Amerika dan sekutunya, sangat
besar baik dalam konteks menyelesaikan
konflik di wilayahnya, mengkonsumsi
minyak yang diproduksinya, atau untuk
membangun gedung, hotel, dan toko-
toko di kawasannya. Gerakan Islam di Indonesia
sebenarnya mempunyai kapasitas
yang cukup untuk dapat menger-
jakan dan menyelesaikan pekerjaaan
rumah tersebut. Untuk hal yang
pertama, yakni membangun ke-
kuatan ekonomi, gerakan Islam di
Indonesia
mempunyai
lembaga
perbankan syariah, keuangan mikro,
pengelola zakat atau wakaf, 398 Islamic World and Politics
Vol.2. No.2 July-December 2018 398 dan bahkan asosiasi pengusaha
yang dapat menjadi aktor utama
dalampemberdayaan
ekonomi
umat dan bangsa. dalam
konteks
mengembangkan
pemahaman keislaman yang progresif
dan transormatif di kalangan generasi
muda Islam Indonesia. Sementara
organisasi non-pemerintah (ornop)
atau yayasan, seperti The Wahid
Institute, Maarif Institute, Fahmina
Institute, dan LSAF (Lembaga Agama
dan Filsafat) berperan penting dalam
konteks
mengadvokasi
kelompok-
kelompok yang terpinggirkan, hak-
hak
perempuan,
dan
pluralisme
kepercayaan
berdasarkan
pema-
haman- Islam yang progresif dan
transformatif. Sementara untuk hal yang kedua,
yaitu mewujudkantatanan sosial yang
adil dan damai, gerakan Islam di
Indonesia juga memiliki organisasi-
organisasi Islam yang mapan untuk
dijadikan sebagaiujuk tombak dalam
mewujudkan- cita- cita sosial tersebut. 5
Berikut
kepanjangan
dari
beberapa
singkatan yang disebut di atas: UIN
(Universitas
Islam
Negeri),
IAIN
(Institut Agama Islam Negeri), STAIN
(Sekolah Tinggi Agama Islam), UII
(Universitas
Islam
Indonesia)
di
Yogyakarta, Unisma (Universitas Islam
Malang), UMI (Universitas Muslim
Indonesia) di Makassar, PPIM (Pusat
Pengkajian Islam dan Masyarakat). PINGGIRAN
MEMBANGUN
KREDENSIAL, INTEGRITAS,
DAN JARINGAN Dengan
demikian,
diharapkan,
kapasitas dan kompetensi mereka
dapat meningkat dan karya-karya
mereka dapat memberi sumbangan
yang berarti bagi kesarjanaan dan
kemanusiaan dalam skala global. pembangunan
peradaban. Tidak
perlu di tingkat dunia tampaknya
masih terlalu muluk dan utopis. Partisipasi dan kontribusi Gerakan
Islam di Indonesia dapat dimulai
dengan membangut jaringan yang
kuat di lingkup negara-negara Asia
Tenggara. Mungkin
selama
ini
asosiasi jaringan gerakan Islam di
Asia Tenggara dilekatkan dengan
kelompok-kelompok radikal yang
menghalalkan kekerasan. Jaringan
kelompok-kelompok
radikal
(Islamis) tersebut akan kalah dan
terhenti
dengan
sendirinya
jika
gerakan Islam yang kritis dan
transformatif di Asia Tenggara dapat
berkolaborasi dan bersinergi secara
struktural maupun kultural di sektor
ekonomi,
sosial,
serta
ilmu
pengetahuan.Jaringan formal secara
ekonomi
telah
terbuka
melalui
pemberlakuan Masyarakat Ekonomi
Asean (MEA) pada akhir tahun
2015. Masyarakat Asia Tenggara ke
depan akan semakin terkoneksi dan
terintegrasi antara yang satu dengan
yang
lain. Kenyataan
ini
memberikan
kesempatan
bagi
gerakan Islam di Indonesia. Mereka
dapat mempelopori kolaborasi dan
sinergi elemen-elemen masyarakat
dan
perguruan
tinggi
untuk
meningkatkan
kompetensi
diri,
menciptakan karya unggul di bidang
ilmu pengetahuan dan teknologi, PINGGIRAN
MEMBANGUN
KREDENSIAL, INTEGRITAS,
DAN JARINGAN Organisasi
masyarakat
(ormas)
keagamaan
seperti
NU,
Muhammadiyah,
Persis,
dan
Hidayatullah menjadi aktor penting
tidak
saja
dalam
menyebarkan
pemahaman-
keagamaan
yang
moderat dan damai, tapi juga menjadi
pemain utama dalam membangun
hubungan yang saling menghargai
dan menghormati antar agama serta
membela hak-hak kelompok yang
dianggap
sesat
dan
minoritas. Organisasi pelajar dan mahasiswa
seperti
HMI,
PMII
(Pelajar
Mahasiswa Islam Indonesia), dan
IMM
(Ikatan
Mahasiswa
Muhammadiyah)telah
menjadi
organisasi perkaderan dan perjuangan
yang teruji. Mereka tidak saja telah
berhasil mendidik generasi muda
Indonesia untuk menjadi pemimpin di
berbagai sektor ke- hidupan. Lebih
dari itu, mereka juga merupakan
pemain utama Untuk hal yang ketiga, yakni
membangun
keunggulan
ilmu
pengetahuan,-
gerakan
Islam
di
Indonesia juga mempunyai banyak
institusi pendidikan dan lembaga
penilitian yang dapat didorong untuk
menjadi pusat-pusat keunggulan ilmu
pengetahuan. Selain
pesantren,
madrasah,
dan
sekolah
Islam,
Indonesia juga mempunyai perguruan
tinggi Islam yang negeri (i.e. UIN,
IAIN, STAIN) maupun swasta (e.g. Perguruan Tinggi Muhammadiyah,
UII, Unisma, UMI) yang tersebar di
seluruh pelosok negeri. Lembaga-
lembaga penelitian yang terkoneksi
dengan perguruan tinggi (e.g. PPIM
UIN Syarif Hidayatullah) maupun di
luar perguruan tinggi (e.g. The
Habibie Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 399 pembangunan
peradaban. Tidak
perlu di tingkat dunia tampaknya
masih terlalu muluk dan utopis. Partisipasi dan kontribusi Gerakan
Islam di Indonesia dapat dimulai
dengan membangut jaringan yang
kuat di lingkup negara-negara Asia
Tenggara. Mungkin
selama
ini
asosiasi jaringan gerakan Islam di
Asia Tenggara dilekatkan dengan
kelompok-kelompok radikal yang
menghalalkan kekerasan. Jaringan
kelompok-kelompok
radikal
(Islamis) tersebut akan kalah dan
terhenti
dengan
sendirinya
jika
gerakan Islam yang kritis dan
transformatif di Asia Tenggara dapat
berkolaborasi dan bersinergi secara
struktural maupun kultural di sektor
ekonomi,
sosial,
serta
ilmu
pengetahuan.Jaringan formal secara
ekonomi
telah
terbuka
melalui
pemberlakuan Masyarakat Ekonomi
Asean (MEA) pada akhir tahun
2015. Masyarakat Asia Tenggara ke
depan akan semakin terkoneksi dan
terintegrasi antara yang satu dengan
yang
lain. Kenyataan
ini
memberikan
kesempatan
bagi
gerakan Islam di Indonesia. Mereka
dapat mempelopori kolaborasi dan
sinergi elemen-elemen masyarakat
dan
perguruan
tinggi
untuk
meningkatkan
kompetensi
diri,
menciptakan karya unggul di bidang
ilmu pengetahuan dan teknologi, Center) juga ada.5 Yang diperlukan
selanjutnya adalah pembangunan
kapasitas dan kompetensi mereka
agar dapatmendidik, meneliti, dan
berkarya di level yang setara, atau
bahkan, mengungguli, pusat-pusat
keunggulan dunia. Di samping
perlu
keberpihakan
dan
keterlibatan pemerintah, mereka
juga
perlu
untuk
membuka
jaringan dan berkolaborasi dengan
pusat-pusat
keunggulan
dunia. 6 Saya memilih untuk memakai istilah
“dereligiusisasi” daripada “sekularisasi”
dengan pemikiran bahwa yang paling
dikhawatirkan dari proses sekularisasi
adalah
tersingkirnya
aspek-aspek
religius dari nafas kehidupan masya-
rakat. Penghargaan terhadap etika dan
norma
agama
dihilangkan
dan
digantikan dengan etika dan norma yang
bersifat positivis dan materialistik. SINERGI UNTUK PERADABAN Chicago:
The University of Chicago Press. Hodgson, M. G. S. (1974b). The
Venture of Islam 2. Chicago: The
University of Chicago Press. and History in a World of
Civilization
(Vol. 1:
The
Classical Age of Islam). Chicago:
The University of Chicago Press. Hodgson, M. G. S. (1974b). The
Venture of Islam 2. Chicago: The
University of Chicago Press. Hourani,
A. (1983). Arabic
Thought in the Liberal Age,
1798-1939. Cambridge:
Cambridge University Press. SINERGI UNTUK PERADABAN Dengan sumber daya gerakan
Islam di Indonesia seperti yang
dikemukakan di atas, seharusnya
umat Islam Indonesia melalui
gerakan -gerakan tersebut di atas
mampu mengambil peran aktif dan
strategis dalam konteks 400 Islamic World and Politics
Vol.2. No.2 July-December 2018 400 dan sekaligus memberi sumbangan
penting bagi peradaban dunia. (wasa’iyyah ) dalam beragama,
pluralisme
keagamaan
dan
kepercayaan di Asia Tenggara, hak-
hak kaum minoritas, pemberdayaan
dan pembelaan perempuan, serta
ekonomi
kerakyatan
dan
pemberdayaan, maka mereka akan
mendapatkan dua manfaat sekaligus. Pertama,
mereka
akan
dapat
mencegah dan mengobati penyakit
radikalisme dalam gerakan Islam
melalui
pemahaman
keagamaan
yang
moderat
dan,
kedua,
menangkal penyingkiran nilai, etika,
dan
norma
keagamaan
dari
masyarakat melalui penyajian Islam
di
Asia Tenggara
yang
dapat
berdialog dengan modernitas dan
realita yang terus berkembang. Di samping itu, kolaborasi dan
sinergi pusat-pusat keunggulan yang
dipelopori oleh gerakan Islam di Asia
Tenggara
juga
diharapkan
dapat
mengatasi beberapa tan- tangan yang
dihadapi oleh umat Islam selama ini. Tantangan internal masyarakat Asia
Tenggara,
seperti
kemiskinan,
menjadi masalah yang diharapkan
dapat diatasi dengan pembukaan
MEA. Namun tantangan-tantangan
lain, terutama ideologi kekerasan
yang dibawa oleh sebagian gerakan
Islamis
dari
Timur
Tengah
dandereligiusasi6pemikiran
dan
budaya yang diimpor kalangan liberal
dari dunia Barat, bukan menjadi
domain dan konsentrasi MEA untuk
menyelesaikannnya. Dalam hal ini,
gerakan Islam di Asia juga dapat
mengambil
peran
penting. Jika
organisasi Islam dan pusat-pusat
keunggulan
Asia
Tenggara
berkomitmen
danterus
mengeksplorasi
hubungan
antara
Islam denganpemahaman moderat Pada
akhirnya,
diharapkan,
masyarakat Islam di Asia Tenggara
mampu
menampilkan
peradaban
tersendiri. Yaitu, peradaban yang
menunjukkan bahwa Islam di Asia
Tenggara dapat berjalan beriringan
dengan
demokrasi
politik
dan
ekonomi, mendorong keunggulan
ilmu pengetahuan dan teknologi,
menghargai keragaman agama dan
kepercayaan,
melindungi
kaum
minoritas, membela hak-hak kaum
perempuan,
serta
dapat
hidup
harmonis dalam perbedaan sekte,
etnis, dan agama. Dengan demikian,
jika peradaban Islam di belahan
dunia lain, misalnya Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban Mohammad Syifa A. Widigdo
Gerakan Islam Indonesia: Mengurai Belenggu, Membangun Peradaban 401 Timur Tengah, ingin belajar tentang
hal-hal tersebut di atas, mereka akan
pergi
belajar
ke
pengalaman
paradaban Islam di Asia Tenggara. Saat itulah, peradaban Islam yang
awalnya dianggap pinggiran, akan
sejajar atau bahkan mengungguli
peradaban yang menganggap dirinya
sebagai pusat Islam. and History in a World of
Civilization
(Vol. 1:
The
Classical Age of Islam). Daftar Pustaka Ahyar, M. (Mei 2015 ). Membaca
Gerakan Islam radikal dan
Deradikalisasi Gerakan Islam. Walisongo, 23(1), 1-26. Siddiqui, K. (1983). Struggle for
the supremacy of Islam—
some critical dimensions. In
K. Siddiqui (Ed.), Issues in the
Islamic Movement 1981-82. London:
The
Open
Press
Limited. Ar, E. H. (September 2013). Pola
Gerakan Islam Garis Keras di
Indonesia. Jurnal Khatulistiwa
– Journal Of Islamic Studies,
3(2). Tibi, B. (2001). Islam Between
Culture and Politics. New
York: Palgrave. Basyir,
K. (November
2016). Ideologi Gerakan Politik Islam
di Indonesia. Al-Tahrir, 16(2),
339 - 362. Tibi, B. (2012). Islam in Global
Politics: Conflict and cross-
civilizational bridging. New
York: Routledge. Chaq, M. D. u. (Mei 2013). Pemikiran Hukum Gerakan
Islam Radikal. Tafaqquh, 1(1),
16-42. Watt, W. M. (1991). Muslim-
Christian Encounters. New
York: Routledge. Collins, E. F. (Oct., 2004). Islam
and the Habits of Democracy:
Islamic Organizations in Post-
New Order South Sumatra. Indonesia(78), 93-120. Hodgson, M. G. S. (1974a). The
Venture of Islam 1: Conscience
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Cognitive sub-Nyquist hardware prototype of a collocated MIMO radar
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Cognitive Sub-Nyquist Hardware Prototype of a Collocated
MIMO Radar Kumar Vijay Mishra, Eli Shoshan, Moshe Namer, Maxim Meltsin, David Cohen,
Ron Madmoni, Shahar Dror, Robert Ifraimov, and Yonina C. Eldar the Xampling framework where Fourier coefficients of the received
signal are acquired from their low-rate samples (or Xamples) [8, p. 387-388] [10]. Application of Xampling in space and time enables
sub-Nyquist sampling without loss of any of the aforementioned radar
resolutions. The Xamples are expressed as a matrix of unknown target
parameters and the reconstruction algorithm is derived by extending
the orthogonal matching pursuit (OMP) [8] to simultaneously solve
a system of CS matrix equations. In sub-Nyquist MIMO, the radar
antenna elements are randomly placed within the aperture (see [11]
for introduction and [3] for recent updates on random arrays), and
signal orthogonality is achieved by frequency division multiplexing
(FDM). In a conventional MIMO radar, the use of non-overlapping
FDM waveforms results in a strong range-azimuth coupling [12–14]
in the receiver processing, and therefore, it is common to use
orthogonal code signals (i.e. code division multiplexing or CDM). The coupling due to FDM can be reduced by randomizing the carrier
frequencies across transmitters [13]. The FDM-based sub-Nyquist
MIMO mitigates the range-azimuth coupling by randomizing the
element locations in the aperture. the Xampling framework where Fourier coefficients of the received
signal are acquired from their low-rate samples (or Xamples) [8, p. 387-388] [10]. Application of Xampling in space and time enables
sub-Nyquist sampling without loss of any of the aforementioned radar
resolutions. The Xamples are expressed as a matrix of unknown target
parameters and the reconstruction algorithm is derived by extending
the orthogonal matching pursuit (OMP) [8] to simultaneously solve
a system of CS matrix equations. In sub-Nyquist MIMO, the radar
antenna elements are randomly placed within the aperture (see [11]
for introduction and [3] for recent updates on random arrays), and
signal orthogonality is achieved by frequency division multiplexing
(FDM). In a conventional MIMO radar, the use of non-overlapping
FDM waveforms results in a strong range-azimuth coupling [12–14]
in the receiver processing, and therefore, it is common to use
orthogonal code signals (i.e. code division multiplexing or CDM). The coupling due to FDM can be reduced by randomizing the carrier
frequencies across transmitters [13]. The FDM-based sub-Nyquist
MIMO mitigates the range-azimuth coupling by randomizing the
element locations in the aperture. I.
Introduction Multiple input multiple output (MIMO) radar is a novel radar
paradigm [1] that uses an array of several transmit and receive antenna
elements, with each transmitter radiating a different waveform. In a
collocated MIMO radar [2], the antenna elements are placed close to
each other so that the radar cross-section of a target appears identical
to all the elements. The waveform diversity in a collocated MIMO
is based on the mutual orthogonality - usually in time, frequency
or code - of different transmitted signals. The receiver separates
and coherently processes the target echoes corresponding to each
transmitter. The angular resolution of MIMO is same as a virtual
phased array with the same antenna aperture but many more antenna
elements than MIMO. In this work, we present the first hardware prototype of a
sub-Nyquist MIMO that can demonstrate reduction in both spatial and
spectral sampling using real-time analog signals. This implementation
follows the recommendations of [9] for signal orthogonality, array
structure and reconstruction algorithms. The prototype can be
configured either as a filled or thinned array, thereby allowing
comparison of Nyquist and sub-Nyquist spatial sampling using the
same hardware. Our previous work in [15] presented the hardware
realization of spectral sub-Nyquist sampling in radar. In [15], a few
randomly chosen, narrow subbands of the received signal spectrum
are pre-filtered before being sampled by low-rate analog-to-digital
converters (ADCs). Since this implementation uses a bandpass filter
and an ADC for each subband, a similar implementation of spectral
sub-Nyquist sampling in each channel of a MIMO receiver would
require enormous hardware resources. To circumvent such a simplistic
and excessively large design, we instead transmit only in those
subbands that are sampled by the receiver. This eliminates all
pre-filtering band-pass stages and each receiver channel requires only
one low-rate ADC to sample all subbands. While a radar achieves high angular resolution by using a
large virtual aperture, its range-time resolution can be improved
by transmitting signals with large bandwidth. In other words, the
conventional processing resolution is limited by the number of
elements and the receiver sampling rate. Several methods have been
proposed to address the problem of preserving the MIMO radar
resolution when either the number of antennas [3] or the number
of received samples [4–6] is reduced. Most exploit the fact that the
target scene is sparse facilitating the use of compressed sensing (CS)
methods [7, 8]. 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Cognitive Sub-Nyquist Hardware Prototype of a Collocated
MIMO Radar Abstract—We present the design and hardware implementation of
a radar prototype that demonstrates the principle of a sub-Nyquist
collocated multiple-input multiple-output (MIMO) radar. The setup
allows sampling in both spatial and spectral domains at rates much lower
than dictated by the Nyquist sampling theorem. Our prototype realizes
an X-band MIMO radar that can be configured to have a maximum of
8 transmit and 10 receive antenna elements. We use frequency division
multiplexing (FDM) to achieve the orthogonality of MIMO waveforms
and apply the Xampling framework for signal recovery. The prototype
also implements a cognitive transmission scheme where each transmit
waveform is restricted to those pre-determined subbands of the full signal
bandwidth that the receiver samples and processes. Real-time experiments
show reasonable recovery performance while operating as a 4×5 thinned
random array wherein the combined spatial and spectral sampling factor
reduction is 87.5% of that of a filled 8 × 10 array. Keywords—MIMO radar, sub-Nyquist, compressed sensing, collocated,
cognitive radar The authors are with the Andrew and Erna Viterbi Faculty of Electrical and
Computer Engineering, Technion - Israel Institute of Technology, Haifa, Israel,
e-mail: {mishra@ee, elis@ee, namer@ee, maxim.meltsin@ee, davidco@tx,
mron@t2, shahard@t2, rifraimov@ef, yonina@ee}.technion.ac.il
This project has received funding from the European Union’s Horizon
2020 research and innovation programme under grant agreement No.
646804-ERC-COG-BNYQ. K.V.M. acknowledges partial support via Andrew
and Erna Finci Viterbi Fellowship. The authors are with the Andrew and Erna Viterbi Faculty of Electrical and
Computer Engineering, Technion - Israel Institute of Technology, Haifa, Israel,
e-mail: {mishra@ee, elis@ee, namer@ee, maxim.meltsin@ee, davidco@tx,
mron@t2, shahard@t2, rifraimov@ef, yonina@ee}.technion.ac.il This project has received funding from the European Union’s Horizon
2020 research and innovation programme under grant agreement No.
646804-ERC-COG-BNYQ. K.V.M. acknowledges partial support via Andrew
and Erna Finci Viterbi Fellowship. C. Cognitive Transmission Let Bh be the set of all frequencies in the transmit signal spectrum
of effective bandwidth Bh. In the cognitive radar transmission, the
spectrum ˜Hm(ω) of each of the transmitted waveforms ˜hm(t) is limited
to a total of Nb non-overlapping frequency bands Bi, 1 ≤i ≤Nb
(Figure 1): xq (t) =
M−1
X
m=0
L
X
l=1
αlhm (t −τl) e j2πβmqθl,
(3) (3) where αl denotes the complex-valued reflectivity of the lth target and
βmq is governed by the array structure. Our goal is to estimate the
range-time τl and azimuths θl of each target. ˜Hm(ω) =
γ(ω)Hm(ω),
ω ∈
Nb
[
i=1
Bi ⊂Bh
0,
otherwise
(7) A. MIMO Radar Model Let the operating wavelength of the radar be λ and the total
number of transmit and receive elements be T and R respectively. The classic approach to collocated MIMO adopts a virtual uniform
linear array (ULA) structure [17], where the receive antennas spaced
by λ
2 and transmit antennas spaced by R λ
2 form two ULAs (or vice
versa, see e.g. [18]). Here, the coherent processing of a total of TR
channels in the receiver creates a virtual equivalent of a phased array
with TR λ
2-spaced receivers and normalized aperture Z = TR
2 . per channel. Xampling obtains a set κ of arbitrarily chosen Fourier
coefficients from low rate samples of the received channel signal such
that |κ| = K < N. Let us now consider the mth transmission. Suppose Y m is the
K × Q matrix with qth column given by ym,q[k], k ∈κ. The matrix
Y m can be expressed as Let us now consider a collocated MIMO radar system that has
M < T transmit and Q < R receive antennas. The locations of these
antennas are chosen uniformly at random within the aperture of the
virtual array mentioned above. The mth transmitting antenna sends a
unique pulse sm (t) given by Y m = AmX (Bm)T ,
(5) (5) where X is a sparse matrix in which the location and values of the
non-zero elements correspond to the locations and reflectivity of the
targets respectively. The matrices Am and Bm are known functions of
radar parameters (T, R, fm, κ, τ, and transmit bandwidth), and each of
their columns correspond to a range and an azimuth cell, respectively. The sparse matrix X can be recovered from the set of equations (5)
for all 0 ≤m ≤M −1, by solving the optimization problem sm (t) = hm (t)e j2π fct,
0 ≤t ≤τ,
(1) (1) where τ denotes the pulse repetition interval (PRI), fc is the common
carrier frequency at the RF (radio-frequency) stage, and {hm (t)}M−1
m=0
is a set of narrowband, orthogonal FDM pulses each with the
continuous-time Fourier transform (CTFT) minimize
X
||X||0
subject to Y m = AmX (Bm)T ,
0 ≤m ≤M −1. (6) Hm(ω) =
∞
Z
−∞
hm(t)e−jωtdt. (2) (2) (6) An approximate solution to this problem can be obtained through an
extension of the matrix OMP method [20]. II.
Sub-Nyquist Collocated MIMO Radar With the exception of cognitive transmission, the array and signal
models of sub-Nyquist MIMO realized by our prototype closely
follow that detailed by [9] and, hence, we only summarize them here. Fig. 1. A conventional radar with signal bandwidth Bh transmits in the band
Bh. A cognitive radar transmits only in subbands {Bi}Nb
i=1, but with more
in-band power than the conventional radar. I.
Introduction Limiting the signal transmission to selective subbands allows
for more in-band power resulting in an increase in signal-to-noise
ratio (SNR). This approach has recently been proposed in the
context of sub-Nyquist cognitive radars [16]. Our prototype, therefore,
additionally demonstrates application of cognitive transmission in
sub-Nyquist MIMO radar. Additionally, a radar that transmits in
several disjoint narrow bands has advantages of disguising the
transmit frequencies as an effective electronic counter measure
(ECM), and also escape radio-frequency (RF) interference from other
licensed radiators in the vacant non-transmit subbands. Recently, [9] proposed a sub-Nyquist collocated MIMO radar
(sub-Nyquist MIMO hereafter) that can recover the target range and
azimuth by simultaneously thinning an antenna array and sampling
received signals at sub-Nyquist rates. The recovery algorithm uses In the following section, we briefly review the signal and system
model of sub-Nyquist MIMO. We then describe the design philosophy 56 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Fig. 1. A conventional radar with signal bandwidth Bh transmits in the band
Bh. A cognitive radar transmits only in subbands {Bi}Nb
i=1, but with more
in-band power than the conventional radar. of our prototype and its major sub-modules in Section III. Finally, we
present results obtained by the prototype in real-time experiments. A. MIMO Radar Model We refer the reader to [9]
for full details of this recovery algorithm. Suppose the target scene consists of L non-fluctuating point targets
(Swerling-0 model) [19] whose location is given by their range-time τl
and direction-of-arrival (DoA) θl, 1 ≤l ≤L. The pulses transmitted by
the radar are reflected back by the targets and collected at the receive
antennas. When the received waveform is downconverted from RF to
baseband, we obtain the following signal at the qth antenna, Suppose the target scene consists of L non-fluctuating point targets
(Swerling-0 model) [19] whose location is given by their range-time τl
and direction-of-arrival (DoA) θl, 1 ≤l ≤L. The pulses transmitted by
the radar are reflected back by the targets and collected at the receive
antennas. When the received waveform is downconverted from RF to
baseband, we obtain the following signal at the qth antenna, B. Sub-Nyquist Range-Azimuth Recovery (7) The application of Xampling in both space and time enables
recovery of range and direction at sub-Nyquist rates. The performance
guarantees of this procedure are provided in [9]. The received
signal xq(t) is separated into M channels, aligned and normalized. The Fourier coefficients of the received signal corresponding to the
channel that processes the mth transmitter echo are given by where γ(ω) = Bh/|Bi| for ω ∈Bi. The total transmit power Pt remains
the same such that the power relation between the conventional and
cognitive waveforms is )
Z Bh/2
−Bh/2
|Hm(ω)|2 dω =
Nb
X
i=1
Z
Bi
| ˜Hm(ω)|2 dω = Pt
(8) (8) ym,q[k] =
L
X
l=1
αle j2πβmqθle−j 2π
τ kτle−j2πfmτl,
(4) (4) In a cognitive radar, the sub-Nyquist receiver obtains the set κ of the
Fourier coefficients only from the subbands Bi. where −N
2 ≤k ≤−N
2 −1, fm is the (baseband) carrier frequency
of the mth transmitter and N is the number of Fourier coefficients 57 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) TABLE I. Technical characteristics of the prototype
Parameters
Mode 1
Mode 2
Mode 3
Mode 4
#Tx, #Rx
8,10
8,10
4,5
8,10
Element placement
Uniform
Random
Random
Random
Equivalent aperture
8x10
8x10
8x10
20x20
Angular resolution (sine of DoA)
0.025
0.025
0.025
0.005
Range resolution
1.25 m
Signal bandwidth per Tx
12 MHz (15 MHz including guard-bands)
Pulse width
4.2 µs
Carrier frequency
10 GHz
Unambiguous range
15 km
Unambiguous DoA
180◦(from -90◦to 90◦) TABLE I. Technical characteristics of the prototype
Parameters
Mode 1
Mode 2
Mode 3
Mode 4
#Tx, #Rx
8,10
8,10
4,5
8,10
Element placement
Uniform
Random
Random
Random
Equivalent aperture
8x10
8x10
8x10
20x20
Angular resolution (sine of DoA)
0.025
0.025
0.025
0.005
Range resolution
1.25 m
Signal bandwidth per Tx
12 MHz (15 MHz including guard-bands)
Pulse width
4.2 µs
Carrier frequency
10 GHz
Unambiguous range
15 km
Unambiguous DoA
180◦(from -90◦to 90◦)
Fig. 2. Tx and Rx element locations for the hardware prototype modes over a
6 m antenna aperture. Mode 4’s virtual array equivalent is the 20 × 20 ULA. chosen Fourier coefficients. Therefore, sub-Nyquist radar prototype
in [15] opted for sampling random disjoint subsets of κ, with each
subset containing consecutive Fourier coefficients. B. System Description Figure 3 shows the sub-Nyquist MIMO prototype, user interface
and radar display. The inset graph depicts the signal flow through
a simplified block diagram. The user selects the prototype mode
from the control interface and passes the control triggers to the
transmit waveform generator card, where an FPGA device reads out
the pre-stored received waveform from an on-board memory. Two
separate digital-to-analog converters (DACs) - one each for I and Q
samples - convert the resulting signal to baseband analog domain. The
transmit waveform generator is off-the-shelf Xilinx VC707 evaluation
board that is custom fit with a 4DSP FMC204 16-bit DAC card. Each
of the I and Q analog signals are then passed on to their respective
analog pre-processor cards. B. Sub-Nyquist Range-Azimuth Recovery The prototype in
[15] used four random Fourier coefficient groups, pre-filtered the
baseband signal to corresponding four subbands (or Xampling slices),
and sampled each subband via separate low-rate ADC. If we use the same pre-filtering approach as in [15] for each
of the eight channels of our sub-Nyquist MIMO prototype, then
the hardware design would need a total of 4 × 8 = 32 bandpass
filters (BPFs) and ADCs excluding the analog filters to separate
transmit channels. We sidestep this requirement by adopting cognitive
transmission wherein the analog signal of each channel lives only in
certain pre-determined subbands and consequently, a BPF stage is
not required. More importantly, for each channel, a single low-rate
ADC can subsample this narrow-band signal as long as the subbands
are coset bands so that they don’t aliase after sampling [21]. This
implementation needs only eight low-rate ADCs, one per channel. Another advantage of this approach is flexibility of the prototype in
selecting the Xampling slices. Unlike [15], the number and spectral
locations of slices are not permanently fixed, and they can be changed
(within the constraints of aliasing due to subsampling). Fig. 2. Tx and Rx element locations for the hardware prototype modes over a
6 m antenna aperture. Mode 4’s virtual array equivalent is the 20 × 20 ULA. The prototype can be configured to operate in various array
configurations or modes. When operating at its maximum strength
of 8 Tx and 10 Rx elements, it can be programmed as either a
ULA (Mode 1) or a random array (Mode 2), each with the equivalent
aperture of an 8 × 10 virtual array, i.e., 1.2 m. For the same aperture,
the system can be operated as a thinned 4 × 5 array (Mode 3). In
this configuration fewer receivers are serialized and the channels
corresponding to the removed transmit elements are not processed
by the digital receiver. Mode 3, hence, demonstrates the spatial
sub-Nyquist sampling. Finally, the prototype can also function as
a 8x10 thinned array (Mode 4) which can be viewed as a spatial
sub-Nyquist version of a 20x20 virtual array with aperture of 6 m. III.
Hardware Design We consider the implementation of a MIMO radar architecture
that has a maximum of 8 transmit (Tx) and 10 receive (Rx)
antenna elements. As explained in subsequent sections, the spatial
and spectral sampling aspects of sub-Nyquist MIMO that we intend to
demonstrate manifest only in the receiver processing. Therefore, our
radar prototype does not physically radiate the transmit waveforms
from an antenna and receive data from actual targets in real-time. Instead, multiple transmit waveforms are pre-computed externally at
baseband, their echoes from simulated targets are recorded and the
complex samples (in-phase I and quadrature-phase Q pairs) are stored
in an on-board memory of a custom-designed waveform generator
board. The prototype then processes these pre-recorded signals in
real-time. Similarly, we omit the implementation of the up-conversion
to RF carrier frequency in the transmitter and the corresponding
down-conversion in the receiver from this prototype. We would
assume that the physical array aperture and simulated target response
correspond to an X-band ( fc = 10 GHz) radar. The choice of radar
frequency band also affects the clutter response that we intend to
consider in a future extension of this prototype. Figure 2 shows exact details of element locations for all four
modes. Table I summarizes the technical parameters of the prototype
for all four array configurations. As mentioned before, this system
employs FDM-based signal orthogonality with each transmit signal
hm(t) chosen to be approximately flat in spectrum, over the extent
of 12 MHz (one-sided band). Each of the transmit waveforms is
separated from its nearest neighbor by a 3 MHz guard-band. A. Design Philosophy A conventional 8x10 MIMO radar receiver would require
simultaneous hardware processing of 80 (or 160 I/Q) data streams. Since a separate sub-Nyquist receiver for each of these 80 channels is
expensive, we implement the eight channel analog processing chain
for only one receive antenna element and serialize the received signals
of all 10 elements through this chain. This approach allows the
prototype to implement a number of receivers greater than 10 as the
eight-channel hardware only limits the number of transmitters. Given a particular receive element, we intend to extract Fourier
coefficient set κ for each of its transmit channels using low-rate ADCs. It has been shown [7, pp. 210-268] that high recovery performance is
promised when these coefficients are drawn uniformly at random. An
ADC can not, however, individually acquire each of the randomly A custom-built analog pre-processor (APP) splits the 120 MHz
baseband analog signal from the waveform generator in 8 channels. The 9 dB attenuation due to 8-channel splitter is compensated with
the use of 10 dB amplifier for each channel. The signal corresponding 58 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Fig. 3. Sub-Nyquist MIMO prototype and user interface. The analog pre-processor module consists of two APP cards mounted on opposite sides of a common
chassis. The inset shows the simplified block diagram of the system. The subscript r represents received signal samples for rth receiver. Wherever applicable,
the second subscript corresponds to a particular transmitter. The square brackets (parentheses) are used for digital (analog) signals. Fig. 3. Sub-Nyquist MIMO prototype and user interface. The analog pre-processor module consists of two APP cards mounted on opposite sides of a common
chassis. The inset shows the simplified block diagram of the system. The subscript r represents received signal samples for rth receiver. Wherever applicable,
the second subscript corresponds to a particular transmitter. The square brackets (parentheses) are used for digital (analog) signals. Fig. 3. Sub-Nyquist MIMO prototype and user interface. The analog pre-processor module consists of two APP cards m
chassis. The inset shows the simplified block diagram of the system. The subscript r represents received signal samples
the second subscript corresponds to a particular transmitter. The square brackets (parentheses) are used for digital (analo Fig. 5. Plan Position Indicator (PPI) display of results for Mode 1 and
3. The origin is the location of the radar. A. Design Philosophy Only the first transmitter channel uses a low-pass filter as it is
difficult to practically realize a bandpass filter with a passband close
to zero. The first five channels use Chebyshev filter design and the
rest are elliptic filters, all with a passband ripple of 0.1 dB. Since
subsampling raises the out-of-band noise, all of these front-end filters
are designed to provide approximately 30 dB stopband attenuation. The imbalance in gain and spectral distortion are corrected by placing
tunable equalizers at the end of APP chain. The channelized I/Q
analog signals are then digitized using low-rate 16-bit ADCs in a
digital receiver card. A digital receiver is realized using a single Xilinx
VC707 evaluation board with two eight-channel 4DSP FMC168
digitizer daughter cards, one each for I and Q signals. The digital
receiver output is transferred over LAN to a radar display. As
shown in Figure 4a, the cognitive radar signal occupies only certain
subbands in a 15 MHz band. Here, the sliced transmit signal has eight
subbands each of width 375 kHz with the frequency range of 1.63-2,
2.16-2.53, 3.05-3.42, 3.88-4.25, 5.66-6.03, 6.51-6.88, 8.64-9.01 and
12.32-12.69 MHz before subsampling. The resulting coherence [22] Fig. 5. Plan Position Indicator (PPI) display of results for Mode 1 and
3. The origin is the location of the radar. The red dot indicates the north
direction relative to the radar. Positive (negative) distances along the horizontal
axis correspond to the east (west) of the radar. Similarly, positive (negative)
distances along the vertical axis correspond to the north (south) of the radar. The estimated targets are plotted over the ground truth. for this selection of Fourier coefficients is 0.42. The total signal
bandwidth is 0.375 × 8 = 3 MHz. This signal is subsampled at 7.5
MHz and, as shown in Figure 4b, there is no aliasing between different
subbands. A non-cognitive signal would have occupied entire 15 MHz
spectrum requiring a Nyquist sampling rate of 30 MHz. Therefore,
use of cognitive transmission enables spectral sampling reduction by
a factor of 4 (= 30 MHz/7.5 MHz) for each channel. Depending on
whether the guard-bands of non-cognitive transmission are included
in the computation or not, the effective signal bandwidth is reduced
by a factor of 5 (= 15 MHz/3 MHz) or 4 (= 12 MHz/3 MHz)
respectively for each channel. Mode 3 has 50% spatial sampling
reduction when compared with Mode 1 or 2. A. Design Philosophy The red dot indicates the north
direction relative to the radar. Positive (negative) distances along the horizontal
axis correspond to the east (west) of the radar. Similarly, positive (negative)
distances along the vertical axis correspond to the north (south) of the radar. The estimated targets are plotted over the ground truth. (a) Before subsampling
(b) After subsampling
Fig. 4. The normalized one-sided spectrum of one channel of a given receiver
(a) before and (b) after subsampling with a 7.5 MHz ADC. Each of the
subbands span 375 kHz and is marked with a numeric label. In a non-cognitive
processing, the signal occupies the entire 15 MHz spectrum before sampling. (a) Before subsampling
(b) After subsampling Fig. 4. The normalized one-sided spectrum of one channel of a given receiver
(a) before and (b) after subsampling with a 7.5 MHz ADC. Each of the
subbands span 375 kHz and is marked with a numeric label. In a non-cognitive
processing, the signal occupies the entire 15 MHz spectrum before sampling. to each transmitter is then filtered using BPFs with 12 MHz passband. Only the first transmitter channel uses a low-pass filter as it is
difficult to practically realize a bandpass filter with a passband close
to zero. The first five channels use Chebyshev filter design and the
rest are elliptic filters, all with a passband ripple of 0.1 dB. Since
subsampling raises the out-of-band noise, all of these front-end filters
are designed to provide approximately 30 dB stopband attenuation. The imbalance in gain and spectral distortion are corrected by placing
tunable equalizers at the end of APP chain. The channelized I/Q
analog signals are then digitized using low-rate 16-bit ADCs in a
digital receiver card. A digital receiver is realized using a single Xilinx
VC707 evaluation board with two eight-channel 4DSP FMC168
digitizer daughter cards, one each for I and Q signals. The digital
receiver output is transferred over LAN to a radar display. As
shown in Figure 4a, the cognitive radar signal occupies only certain
subbands in a 15 MHz band. Here, the sliced transmit signal has eight
subbands each of width 375 kHz with the frequency range of 1.63-2,
2.16-2.53, 3.05-3.42, 3.88-4.25, 5.66-6.03, 6.51-6.88, 8.64-9.01 and
12.32-12.69 MHz before subsampling. The resulting coherence [22] to each transmitter is then filtered using BPFs with 12 MHz passband. C. Experimental Results [8]
Y. C. Eldar,
Sampling
Theory:
Beyond
Bandlimited
Systems. Cambridge University Press, 2015. We evaluated the performance of all the modes through hardware
simulations. Only one pulse was transmitted in all experiments
and all modes were evaluated against the identical target scenarios. We injected the received signal corresponding to the echoes from
10 targets, placed at arbitrary range and azimuths, in the transmit
waveform generator. In the first experiment, when the minimum
angular spacing (in terms of the sine of DoA) between any two targets
was approximately 0.025, the recovery performance of the thinned
4×5 array in Mode 3 was not worse than Modes 1 and 2 until the noise
was dramatically increased. Figure 5 shows the detection performance
of all the modes for this experiment when the signal SNR = −5
dB; the injected noise is complex white Gaussian. Here, a successful
detection (green circle) occurs when the estimated target is within
two range cells and one DoA bin of the ground truth (blue circle);
otherwise, the estimated target is labeled as a false alarm (magenta
circle). In case of high SNR or absence of noise, our criterion for
successful detection is sensu stricto, i.e. the estimated target must lie
at the exact location of the ground truth for a successful detection. [9]
D. Cohen, D. Cohen, Y. C. Eldar, and A. M. Haimovich, “Sub-Nyquist
collocated MIMO radar in time and space,” in IEEE Radar Conf., May
2016. [10]
O. Bar-Ilan and Y. C. Eldar, “Sub-Nyquist radar via Doppler focusing,”
IEEE Trans. on Signal Proc., vol. 62, no. 7, pp. 1796–1811, 2014. [11]
Y. Lo, “A mathematical theory of antenna arrays with randomly spaced
elements,” IEEE Trans. Antennas Propag, vol. 12, no. 3, pp. 257–268,
1964. [12]
O. Rabaste, L. Savy, M. Cattenoz, and J.-P. Guyvarch, “Signal
waveforms and range/angle coupling in coherent colocated MIMO
radar,” in Int. Conf. on Radar, 2013, pp. 157–162. [13]
H. Sun, F. Brigui, and M. Lesturgie, “Analysis and comparison of mimo
radar waveforms,” in IEEE Radar Conf., 2014, pp. 1–6. [14]
J. Xu, G. Liao, S. Zhu, L. Huang, and H. C. So, “Joint range and
angle estimation using MIMO radar with frequency diverse array,” IEEE
Trans. Signal Process., vol. 63, no. 13, pp. 3396–3410, 2015. [15]
E. Baransky, G. Itzhak, I. Shmuel, N. Wagner, E. Shoshan, and Y. C. Eldar, “A sub-Nyquist radar prototype: Hardware and applications,”
IEEE Trans. Aerosp. Electron. References [1]
E. Fishler, A. Haimovich, R. Blum, D. Chizhik, L. Cimini, and
R. Valenzuela, “MIMO radar: An idea whose time has come,” in IEEE
Radar Conf., 2004, pp. 71–78. [2]
J. Li and P. Stoica, “MIMO radar with colocated antennas,” IEEE Signal
Process. Mag., vol. 24, no. 5, pp. 106–114, 2007. g ,
,
, pp
,
[3]
M. Rossi, A. M. Haimovich, and Y. C. Eldar, “Spatial compressive
i
f
MIMO
d
”
Si
l
l 62
2 g ,
,
, pp
,
[3]
M. Rossi, A. M. Haimovich, and Y. C. Eldar, “Spatial compressive
sensing for MIMO radar,” IEEE Trans. Signal Process., vol. 62, no. 2,
pp. 419–430, 2014. Fig. 6. As in Fig. 5, but for a closely-spaced target scenario. The inset plots
show the selected region in each PPI display at a magnified scale. sensing for MIMO radar,” IEEE Trans. Signal Process., vol. 62, no. 2,
pp. 419–430, 2014. pp
[4]
Y. Yu, A. P. Petropulu, and H. V. Poor, “MIMO radar using compressive
sampling,” IEEE J. Sel. Topics in Signal Process., vol. 4, no. 1, pp. 146–163, 2010. spatial and spectral sampling reduction in Mode 3, then we use a
total one-eighth of the Nyquist sampling rate and one-tenth of the
Nyquist signal bandwidth (guard-bands included). The sampling rate
reduction is, therefore, seven-eighth or 87.5% in Mode 3. The receiver
processes 80 and 20 channels in 8x10 and 4x5 arrays, respectively. So, the hardware resources are reduced by 75% in Mode 3. [5]
D. S. Kalogerias and A. P. Petropulu, “Matrix completion in colocated
MIMO radar: Recoverability, bounds & theoretical guarantees,” IEEE
Trans. Signal Process., vol. 62, no. 2, pp. 309–321, 2014. [6]
S. Sun, W. U. Bajwa, and A. P. Petropulu, “MIMO-MC radar: A MIMO
radar approach based on matrix completion,” IEEE Trans. Aerosp. Electron. Syst., vol. 51, no. 3, pp. 1839–1852, 2015. [7]
Y. C. Eldar and G. Kutyniok, Compressed Sensing: Theory and
Applications. Cambridge University Press, 2012. IV.
Summary We presented the first hardware prototype of sub-Nyquist
MIMO that demonstrates real-time operation of both spatial and
spectral reduction in sampling. The thinned 4x5 array achieves the
detection performance of its filled array counterparts even though
the combined reduction of spatial and spectral sampling is 87.5%. While we did not analyze the performance for Doppler recovery and
clutter contamination, the prototype design does not restrict such
an evaluation. Future theoretical insights on the selection of best
subbands and improved recovery algorithms can further enhance the
performance. A. Design Philosophy If we account for both 59 4th Int. Workshop on Compressed Sensing on Radar, Sonar, and Remote Sensing (CoSeRa 2016) Fig. 6. As in Fig. 5, but for a closely-spaced target scenario. The inset plots
show the selected region in each PPI display at a magnified scale. C. Experimental Results Syst., vol. 50, no. 2, pp. 809–822, 2014. [16]
D. Cohen, A. Dikopoltsev, R. Ifraimov, and Y. C. Eldar, “Towards
sub-Nyquist cognitive radar,” in IEEE Radar Conf., May 2016. In the second experiment, the minimum angular spacing between
the two targets was reduced to 0.02, and the SNR of the injected
signal remained at −5 dB. Since the angular resolution of Mode 4
is better than the other three modes (see Table I), all the targets
are detected successfully in Mode 4. Mode 1 and 3 showed a false
alarm as seen in the inset plots of Figure 6. The Mode 2 also shows
successful recovery in the broad sense of our detection criterion. The
strict sense location error in Mode 2 is clearly larger than that in Mode
4. However, relatively better performance of Mode 2 over Modes 1
and 3 is not entirely fortuitous here. Figure 2 shows that both Tx and
Rx array elements in Mode 2 are distributed such that its virtual array
is wider than Modes 1 and 3. Thus, the effective angular resolution
for Mode 2 could be better than 1 and 3, but still worse than 4. yq
g
y
[17]
C.-Y. Chen, “Signal processing algorithms for MIMO radar,” Ph.D. dissertation, California Institute of Technology, 2009. [18]
E. Brookner, “MIMO radars and their conventional equivalents
An
update,” in IET Int. Radar Conf. IET, 2015, pp. 1–8. [19]
M. Skolnik, Radar handbook. McGraw-Hill, 1970. [20]
T. Wimalajeewa, Y. C. Eldar, and P. K. Varshney, “Recovery of sparse
matrices via matrix sketching,” arXiv preprint arXiv:1311.2448, 2013. [21]
K. M. Cohen, C. Attias, B. Farbman, I. Tselniker, and Y. C. Eldar,
“Channel estimation in UWB channels using compressed sensing,” in
IEEE Int. Conf. on Acoustics, Speech and Signal Processing, 2014, pp. 1966–1970. [22]
P. Xia, S. Zhou, and G. B. Giannakis, “Achieving the Welch bound with
difference sets,” IEEE Trans. Inf. Theory, vol. 51, no. 5, pp. 1900–1907,
2005. 60
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https://openalex.org/W2181412895
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https://etj.uotechnology.edu.iq/article_38169_6c0fb55dc2c4f16290019a37514f204e.pdf
|
English
| null |
A Simplified Method For Estimating The Peak Load In The Large Cold Stores
|
Maǧallaẗ al-handasaẗ wa-al-tiknūlūǧiyā
| 2,009
|
cc-by
| 5,505
|
Nomenclature Nomenclature
A : surface area that heats transfer
through it (m
2 )
if ,fo : Heat transfer coefficient for the
external and internal air layers (W/
2
m
C
o Nomenclature
A : surface area that heats transfer
through it (m
2 ) if ,fo : Heat transfer coefficient for the
external and internal air layers (W/
2
m
C
o Cp : Specific heat of the product
(J/kg. C
o
) * Mechanical Engineering Department, University of Technology/Baghdad * Mechanical Engineering Department, University of Technology/Baghdad ** College of Engineering, University of Anbar / Anbar
*** Mechanical Engineering Department, Technology Institute /Baghdad ** College of Engineering, University of Anbar / Anbar
*** Mechanical Engineering Department, Technology Institute /Baghdad A Simplified Method For Estimating The Peak Load
In The Large Cold Stores Dr. Ahmed A. M. Saleh* , Dr. Obaid T. Fadhil**
& Aziz. J. Aziz***
Received on:28/9/2008
Accepted on:2/4/2009 2009
,
10
.
No
,
27
.l
No
,
Journal
.
Tech
&
.
Eng 2009
,
10
. No
,
27
.l
No
,
Journal
. Tech
&
. Eng Abstract In this paper a simplified method for estimating the peak load in the large cold
stores is found and its occurrence day by utilizing two new variables A & B. The
first variable represents the number of days that is necessary for cooling the
product (storage) up to the needed temperature according to the daily temperature
differences only. The second variable represents the number of days required for
cooling the total product (storage) depending upon the daily storage mass only
without returning back to the details of distribution of the daily thermal load that is
followed in calculating thermal load for cold stores. g
By the demonstrative and analysis of the thermal load in cold stores with detailing
study for each effective factors in estimating the thermal load characteristic is
observed that the factors daily temperature differences (∆T) and storage mass
(m) per day affect significantly in estimating the peak load values and day of
occurrence. Keywords: Cold store, Peak Load Calculation, Daily Temperature Difference,
Daily Storage Mass Daily Storage Mass
ﻁﺭﻴﻘﺔ ﻤﺒﺴﻁﺔ ﻟﺘﺨﻤﻴﻥ ﺤﻤل ﺍﻟﺫﺭﻭﺓ ﻓﻲ ﺍﻟﻤﺨﺎﺯﻥ ﺍﻟﻤﺒﺭﺩﺓ ﺍﻟﻜﺒﻴﺭﺓ
ﺍﻟﺨﻼﺼﺔ
ﺘﻡ ﻓﻲ ﻫﺫﺍ ﺍﻟﺒﺤﺙ
ﺇﻴﺠﺎﺩ ﻤﻌﺎﺩﻟﺔ ﻴﻤﻜﻨﻨﺎ ﺒﻭﺍﺴﻁﺘﻬﺎ ﺘﺨﻤﻴﻥ ﺤﻤل ﺍﻟﺫﺭﻭﺓ ﻭ ﻴﻭﻡ ﺤﺩﻭﺜـﻪ ﻓـﻲ
ﻤﺨﺎﺯﻥ ﺍﻟﺘﺒﺭﻴﺩ ﺍﻟﻜﺒﻴﺭﺓ ﻭﺫﻟﻙ ﺒﺎﻻﺴﺘﻔﺎﺩﺓ ﻤﻥ ﺍﻟﻤﺘﻐﻴﺭﺍﺕ ﺩﻭﻥ ﺍﻟﺭﺠﻭﻉ ﺍﻟﻰ ﺘﻔﺎﺼﻴل ﺘﻭﺯﻴـﻊ ﺍﻟﺤﻤـل
ﺍﻟﺤﺭﺍﺭﻱ
. ﻴﻤﺜل ﺍﻟﻤﺘﻐﻴﺭ
ﺍﻻﻭل ﻋﺩﺩﺍﻷﻴﺎﻡ ﺍﻟﻤﻁﻠﻭﺒﺔ ﻟﺘﺒﺭﻴﺩﺍﻟﻤﻨﺘﺞ
)
ﺍﻟﺨﺯﻴﻥ
(
ﺍﻟـﻰ ﺩﺭﺠـﺔ ﺍﻟﺤـﺭﺍﺭﺓ
ﺍﻟﻤﻁﻠﻭﺒﺔ ﻁﺒﻘﺎﹰ
ﻟﻔﺭﻕ ﺩﺭﺠﺎﺕ ﺍﻟﺤﺭﺍﺭﺓ ﺍﻟﻴﻭﻤﻴﺔ ﻓﻘﻁ،
ﺃﻤﺎ ﺍﻟﻤﺘﻐﻴﺭ ﺍﻟﺜﺎﻨﻲ ﻓﻴﻤﺜل ﻋﺩﺩ ﺍﻷﻴﺎﻡ ﺍﻟﻤﻁﻠﻭﺒـﺔ
ﺇﻋﺘﻤﺎﺩﺍﻋﻠﻰ ﻤﻘﺩﺍﺭﺍﻟﺨﺯﻥ ﺍﻟﻴﻭﻤﻲ
. ﺘﺒﻴﻥ ﻤﻥ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻋﻼﻩ ﺒﺄﻥ ﺍﻟﻌﺎﻤﻠﻴﻥ ﻓﺭﻕ ﺩﺭﺠﺎﺕ ﺍﻟﺤﺭﺍﺭﺓ ﺍﻟﻴﻭﻤﻲ ﻭﻜﻤﻴﺔ ﺍﻟﻜﺘ
ﻠﺔ
ﺍﻟﻤﺨﺯﻭﻨـﺔ ﻟﻜـل
ﻴﻭﻡ
)∆
T
،
m
(؛
ﻟﻬﻤﺎ ﺘﺄﺜﻴﺭ ﻤﻠﻤﻭﺱ ﻓﻲ ﺘﺤﺩﻴﺩ ﻤﻘﺩﺍﺭ ﺤﻤل ﺍﻟﺫﺭﻭﺓ ﻭﺯﻤﻥ ﺤﺩﻭﺜﻪ ﻭﺫﻟـﻙ
ﺒﻌﺩ
ﺘﺤﻠﻴل ﻭﺩﺭﺍﺴﺔ ﻜل ﺍﻟﻌﻭﺍﻤل ﺍﻟﻤﺅﺜﺭﺓ
ﺍﻟﺘﻲ ﺘﺤﺩﺩ
ﻤﻭﺍﺼﻔﺎﺕ ﺍﻟﺤﻤل ﺍﻟﺤـﺭﺍﺭﻱ ﻓـﻲ ﺍﻟﻤﺨـﺎﺯﻥ
ﺍﻟﻤﺒﺭﺩﺓ
ﺍﻟﻜﺒﻴﺭﺓ
. air
ρ
: Air density (kg/
3
m )
1. Introduction air
ρ
: Air density (kg/
3
m )
1. Introduction K : Thermal conduction coefficient
for materials forming the wall (W/m. C
o ) Cold stores are vessels or rooms
properly thermal insulation supplied by
electrical and mechanical machines that
decrease the temperature of the space
and its contents to a proper temperature
thus; this lowered proper temperature
reduces or prevents the factors which
help to corrupt the storage products. The
suitable temperature for storing depends
on two main factors; type of the storage
and the duration of store [1]. m : Daily storing mass in aday (kg)
mTot: Total capacity mass storage( kg) L
N : Number of lighting L
N : Number of lighting P
N : Number of persons P
N : Number of persons m
N : Number of equipment m
N : Number of equipment NOC: Numbers of the air change per
day m
OH
: operating of hours of the m
OH
: operating of hours of the equipment per a day These stores are frequently supplied
with cooling equipment, which its
capacity reaches thousands of tons of
cooling,
and
other
completion
equipment that support in works of
separate, transfer, pouch and so on. Also
the initial cost of the cold stores is very
expensive. The cooling system forms a
very important part from the total cost,
thus the equipment selection is very
important and it is a real effective factor
upon the total coast of the project. L
OH : Operating hours of lighting per
day (hr/day) P
OH : Hours of work in the day m
P : Input Power of equipment (W) LP : Power of lighting (W) 1
Q
: Heat transfer through wall, roof,
and ground (W) 2
Q : The amount of heat transfer due
to the air change (W) 3
Q : Thermal load from lighting (W) Jing Xie et al. [2] they used CFD
technique to study a two-dimensional
mathematical model for the inside a
mini type construction cold store (4.5 m
× 3.3 m × 2.5 m). The simulation results
reflected the characteristics of airflow
and temperature distribution, and a
several design parameters (corner baffle,
the stack mode of food stuffs, etc.) were
analyzed. The results of calculation
indicated
that
all
these
designed
parameters influenced the flow and
temperature fields. ∆T : Daily temperature reduction C
o
∆TTot : Total temperature differences
required ( C
o
) ∆T : Daily temperature reduction C
o
∆TTot : Total temperature differences
required ( C
o
) y
p
∆TTot : Total temperature differences
required ( C
o
) in
h : Enthalpy of the internal air (inside
cold store condition) (J/kg) air
ρ
: Air density (kg/
3
m )
1. Introduction 4
Q : Thermal load from equipment
(W) 5
Q : Amount of heat from persons
(W) (
)
Q6 : Sensible heat (W) Q6 : Sensible heat (W) Q7 : Amount of heat results from
respiration (W) P
Q : Amount of heat generates from
each person (W/person) qres : Heat respiration (J/kg) qres : Heat respiration (J/kg) V : daily air change (m3/day) s
V
: Volume of cold store (
3
m ) S. Akdemir [3] studies the cooling
load and choosing of cooling system
elements for designing of the cold store. He calculated transmissions, infiltration,
production, and other source of heat. In U : over all heat transfer coefficients
for wall, roofs and ground (W/m2. C
o
)
g,r,
w
T
∆
: Temperature difference between
two sides of wall, roof and ground ( C
o
) https://doi.org/10.30684/etj.27.10.17 2412-0758/University of Technology-Iraq, Baghdad, Iraq This is an open access article under the CC BY 4.0 license http://creativecommons.org/licenses/by/4.0 A Simplified Method For Estimating The Peak Load
In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 ∆T : Daily temperature reduction C
o
∆TTot : Total temperature differences
required ( C
o
)
air
ρ
: Air density (kg/
3
m )
1. Introduction out
h
: Enthalpy of the external air
(outside condition) (J/kg)
in
h : Enthalpy of the internal air (inside
cold store condition) (J/kg)
K : Thermal conduction coefficient
for materials forming the wall (W/m. C
o )
m : Daily storing mass in aday (kg)
mTot: Total capacity mass storage( kg)
L
N : Number of lighting
P
N : Number of persons
m
N : Number of equipment
NOC: Numbers of the air change per
day
m
OH
: operating of hours of the
equipment per a day
L
OH : Operating hours of lighting per
day (hr/day)
P
OH : Hours of work in the day
m
P : Input Power of equipment (W)
LP : Power of lighting (W)
1
Q
: Heat transfer through wall, roof,
and ground (W)
2
Q : The amount of heat transfer due
to the air change (W)
3
Q : Thermal load from lighting (W)
4
Q : Thermal load from equipment
(W)
5
Q : Amount of heat from persons
(W)
Q6 : Sensible heat (W)
Q7 : Amount of heat results from
respiration (W)
P
Q : Amount of heat generates from
each person (W/person)
qres : Heat respiration (J/kg)
V : daily air change (m3/day)
s
V : Volume of cold store (
3
m )
U : over all heat transfer coefficients
for wall, roofs and ground (W/m2. C
o
)
g,r,
w
T
∆
: Temperature difference between ∆T : Daily temperature reduction C
o out
h
: Enthalpy of the external air
(outside condition) (J/kg) 2.2.2
Equipment Load The equipment load can be
calculated
using
the
following
equation: m
m
m
4
OH
. P. N
Q =
/24 ……. (5) m
m
m
4
OH
. P. N
Q =
/24 ……. (5) 2.2.5
Respiration load It is the amount of heat generates
from product due to the vitality
chemical reactions in any plan and it
can calculate using the following
equation [5]: g,r,
w
1
T
A
U
Q
∆
⋅
⋅
=
……(1-a) o
n
n
2
2
1
1
i
f/1
K
/
X
...... K
/
X
K
/
X
f/1
U
/1
+
+
+
+
=
……(1-b) 3600
24
q. m
Q
res
7
×
=
……….. (8) (8) 2.1.1 Heat transfer through wall, roof,
and ground 3600
24
T
. C
. m
Q
p
6
×
∆
= Can
be
calculated
using
the
following equation (Searle engineers
hand book 1988) [5]: PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load
In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 addition the amount of the cooling fluid
was also calculated. addition the amount of the cooling fluid
was also calculated. 2.2.3
Persons
(workers) The thermal load per day in the
cold stores can be divided into two main
parts: it is calculated using the following
equation: 24
/
OH
. Q
. N
Q
P
p
P
5 =
……..(6) 24
/
OH
. Q
. N
Q
P
p
P
5 =
……..(6) 2.2 Internal thermal load This thermal load generated inside
the cold store and it contains the
following: Erol,
N. [4]
stated
that
the
determination of total input cooling load
would not be possible, for this reason
there may be some deviation in the
cooling load and the focus point must be
minimizing the deviation. He explained
that the ambient temperature of cold
store, the situation of store product
before entering the cold store, the daily
working hours and the determination of
which product will be stored are
important for calculation of the cooling
load. 2.2.1 Lighting load It is the thermal energy generated
from lighting used inside a store. It can
be
calculated
using
the
following
equation [5]: 24
/
OH
. P. N
Q
L
L
L
3 =
…… (4) 2. Elements of thermal load in the
cold stores 2.2.3
Persons
thermal
load
(workers) 2.2.3
Persons
thermal
load
(workers) load it is calculated using the following
equation: The external thermal load is due to
transfer of the heat from external to
internal space of the store. It consists of:
2.1.1 Heat transfer through wall, roof,
and ground The external thermal load is due to
transfer of the heat from external to
internal space of the store. It consists of:
2.1.1 Heat transfer through wall, roof,
and ground 2.1.2 Air change heat gain 3. Factor affects thermal load 5. Case Study-Sample of Calculation
Input data 5. Case Study-Sample of Calculation
Input data p
- Dimension: (25 × 15 × 5) m - Store Room Temperature: 4 C
o - Store Room Temperature: 4 C
o - Product : Potato - Capacity : 550 ton - Product Initial Temperature :
35 C
o - Outside Temperature : 50 C
o
& RH= 20%
2 - Wall U-value : 0.34 W/m2. C
o - Wall U-value : 0.34 W/m2. C
o (for 10 mm Polystyrene board) (for 10 mm Polystyrene board)
Peak load calculation (traditional (for 10 mm Polystyrene board)
Peak load calculation (traditional method) Due to the probability of miss-match
of all components loads in one day so
it can not be able to find the peak
load directly although the correct
choice of the daily storing mass and
the daily temperature difference was
used. Due to the probability of miss-match
of all components loads in one day so
it can not be able to find the peak
load directly although the correct
choice of the daily storing mass and
the daily temperature difference was
used. )
- Q1 : Wall & Roof , use
equation -1 )
- Q1 : Wall & Roof , use
equation -1 q
Q1)wall = 0.34 × 400 × (50 – 5)
= 6256 W/day
Q1)Roof = 0.34 × 375 × (50 – 5)
= 5865 W/day
Q1 = 6256 + 5865
= 12121 W/day q
Q1)wall = 0.34 × 400 × (50 – 5)
= 6256 W/day
Q1)Roof = 0.34 × 375 × (50 – 5)
= 5865 W/day
Q1 = 6256 + 5865
= 12121 W/day 3. Factor affects thermal load It can be calculated using the
following equation [5]: In the revision of the previous
thermal loads which are used for design
any cold store; it can be noticed that: ….. (2)
(
)
24
3600
h
h
. V
. Q
in
out
air
2
×
−
ρ
= •
T
he internal thermal load forms the
larger part of the overall load
and
the heat from product has a larger
part of the internal load (specific heat
and respiration heat of potato are
3.56 kJ/kg. C
o
, 270 kJ/kg and for (V) Is calculated from the knowledge of
store's volume and the number of air
change in a day as shown below (Air-
Conditioning & Refrigeration Institute
1987) [6]. NOC
. V
V
s
=
…….(3) NOC
. V
V
s
= NOC
. V
V
s
= PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load
In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 meat 3.2 kJ/kg. C
o
, 231 kJ/kg )
returning to ASHRAE Handbook
1998 [7]. The other loads take
different ratio from overall load. meat 3.2 kJ/kg. C
o
, 231 kJ/kg )
returning to ASHRAE Handbook
1998 [7]. The other loads take
different ratio from overall load. T
T
A
Tot
∆
∆
=
(day) …….(9) (day) * Factor -B: It represents number of
days required to fill the store by
products depending on the daily storing
mass (m) only, i.e. : It
is clear that there are two important
factors in calculation of the product
load, the daily storing mass (m) and
the daily temperature difference ∆T
which affect directly in the value of
the load, so the wrong selection of
those two factors may give a very far
result from the actual and then
choose incompatible equipment to
the
real
load
(expensive
unnecessarily cost). The time is taken
by the product to reach the optimum
storage
temperature
(sometimes
called the pull- down time) will be
limited by the overall refrigeration
capacity
of
the
equipment
(M.Eltawil, D.Samuel and O.Singhal
2006 [8]. m
m
B
Tot
=
(day) …..(10) m
m
B
Tot
=
(day) …..(10) 5. Case Study-Sample of Calculation 4. Determination of the peak load and
starting day To simplifying the analysis of the
calculation, it’s important to redefine
some variables: * External load = Q1 + Q2 (W)
* Base load=Q3+Q4+Q5 (W)
* Internal load = Q6+ Q7 (W)
And two new factors: * External load = Q1 + Q2 (W)
* Base load=Q3+Q4+Q5 (W)
* Internal load = Q6+ Q7 (W)
And two new factors: = 3287.7 W/day
-
Q3 : Lighting, use equation - 4
( operating hour = 10) And two new factors: * Factor -A : It represents number of
days necessary for cooling the product
(storage) up to the required temperature
depending upon the daily temperature
differences (∆T) only i.e. : y
- Q4 : Equipment, use equation- 5
( fan power 4×3 kW & heater
of 8 kW)
Q4 = 20 × 1000 × 18 / 24
= 1500 W/day PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com 6. Daily load distribution Curves A collection of different cases of
storing were taken depending upon
storing masses and the daily temperature
difference which covered most of the
expected states in the loads of cold
stores. Table -2 shows those chosen
cases and the two factors A and B. y
- Q6 : Product(sensible), use
equation - 7 ( Cp of
product= 3.56 kJ/kg. C
o
) 3600
24
)
4
35
(
10
56
.3
10
550
Q
3
3
6
×
−
×
×
×
×
= Figures (1) to (5) represent the
distribution of the daily load during the
first period of storing for cases in the
Table-2. = 702523 W/day
- Q7 : Respiration(latent), use
equation - 8
3600
24
10
270
10
550
Q
3
3
7
×
×
×
×
=
= 1718750
W/day = 702523 W/day
- Q7 : Respiration(latent), use
equation - 8
10
270
10
550
3
3
×
×
× 7. Results and Discussion The graphical analysis and study of
the load component distribution of
previous figures could build Table-3
which represents a summary of the
obtained results and offers simple
equations to calculate peak load for the
most cases without returning back to the
details of loads distribution or graphical
analysis or backup figures that is
required to calculate throughout the first
days of storing reaching to last days with
algebraic sum to determine the peak
load and day of it. y
Table -1 shown the summary of
calculation y
Table -1 shown the summary of
calculation y
Table -1 shown the summary of
calculation PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load
In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 - Q5 : Person, use equation - 6
( operating hour = 10 )
Q3 = 10 × 240 × 8 / 24
= 800 W/day
- Q6 : Product(sensible), use
equation - 7 ( Cp of
product= 3.56 kJ/kg. C
o
)
3600
24
)
4
35
(
10
56
.3
10
550
Q
3
3
6
×
−
×
×
×
×
= = 343750 W
6. Daily load distribution Curves Load calculation with ∆T and ∆m
( new method) Load calculation with ∆T and ∆m
( new method) - Let ∆T per day = 2 C
o
Therefore number of days
required (Factor- A) = (35-4)/2
= 15.5 day y
- Mass charging per day (m) =
110 ton
Therefore number of days
required (Factor- B) = 550/110
= 5 day In general, these are the obtained
simplified equations Peak load = Base Load + External oad y
- Load calculation
Q1=0.34×2×
(400+375)
(where ∆T=2 ○C )
= 527 W
24
3600
1000
87
.4
5. 2962
27
.1
Q2
×
×
×
×
= - Load calculation
Q1=0.34×2×
(400+375)
(where ∆T=2 ○C )
= 527 W + B×Q7 + minimum of (A or B) ×Q6 + B Q7 + minimum of (A or B) Q6
Peak load Day = maximum of (A or B)
In order to verify the validity of the
above equation, it can be applied on the
case study example 24
3600
1000
87
.4
5.
2962
27
.1
Q2
×
×
×
×
= •
Base load= Q3 + Q4 + Q5 = 17.36
kW/day •
Base load= Q3 + Q4 + Q5 = 17.36
kW/day = 212 W
Q3, Q4, Q5 same as before
Q6
using
equation-7
and
equation-8, with ∆T=2 ○C and
m=110 ton •
External load = Q1 + Q2 = 15.41
kW/day •
External load = Q1 + Q2 = 15.41
kW/day •
B × Q7 = 5 × 343.7 = 1718.5
kW/day •
B × Q7 = 5 × 343.7 = 1718.5
kW/day y
•
Min(A, B) × Q6 = 5 × 9.05 =
45.3 kW/day •
Min(A, B) × Q6 = 5 × 9.05 =
45.3 kW/day 3600
24
2
10
56
.3
10
110
Q
3
3
6
×
×
×
×
×
=
= 9064.8 W
3600
24
10
270
10
110
Q
3
3
7
×
×
×
×
= 3600
24
2
10
56
.3
10
110
Q
3
3
6
×
×
×
×
×
=
= 9064.8 W
3600
24
10
270
10
110
Q
3
3
7
×
×
×
×
= y
Total load = 17.36 + 15.41 + 1718.5 Total load = 17.36 + 15.41 + 1718.5 +45.3 =1796.57( kW/day) and the peak
load day is 20. +45.3 =1796.57( kW/day) and the peak
load day is 20. +45.3 =1796.57( kW/day) and the peak
load day is 20. PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load
In The Large Cold Stores A Simplified Method For Estimating The Peak Load
In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 Eng. & Tech. Journal ,Nol.27, No. 10, 2009 The result of the total peak load above is
about 1796 kW/day, whenever, the peak
load calculated with traditional method
listed in table-1 is 2454 kW/day. i.e. the
actual peak load (suggested) is about
26% less than the other (traditional). calculation and the probability of wrong
estimation of the load in large cold
stores which causes a loss of money by
choosing over size machine to overcome
the load due to the overestimate of the
cooling load. References MarAjans, Istanbul, Turkey, 1993 MarAjans, Istanbul, Turkey, 1993
[5] Searle engineers hand book, cold
room calculation, A division of Hall –
Thermo
tank
product,
London
office,1988 [6]Air-Conditioning
&
Refrirration
Institute,
Refrigeration
and
Air-
conditioning, Second Edition, Prentice-
Hall, Inc., Englewood Cliffs, 1987 [7] ASHRAE Handbook, Refrigeration,
American
Society
of
Heating,
Refrigeration
and
Air-Conditioning
Engineers, Inc. SI Edition. 1998
[8] M.Eltawil, D.Samuel and O.Singhal,
Potato storage Technology and store
Design Aspect, Agricultural Engineering
International, the CIGRE journal Invited
Overview No. 11, Vol. VIII, April,
2006. [7] ASHRAE Handbook, Refrigeration,
American
Society
of
Heating,
Refrigeration
and
Air-Conditioning
Engineers, Inc. SI Edition. 1998 In general, the peak load will decrease
(practically is preferable) when the value
of A and B is large; if there are no
technical limitations. References [1] Design of Room cooling Facilities
structural and energy Requirements,
North
Carolina
State
University,
Department
of
Biological
and
Agricultural Engineering, Post harvest
commodity series, A6-414-2,1988 [1] Design of Room cooling Facilities
structural and energy Requirements,
North
Carolina
State
University,
Department
of
Biological
and
Agricultural Engineering, Post harvest
commodity series, A6-414-2,1988
[2] Jing Xie, Xiao-Hua Qu, Jun-yesh
and Da-wen sun, Effect Of Design
Parameter on Flow and Temperature
Field of a Cold store by CFD simulation,
Journal of Food Engineering, Volume
77, Issue 2, November 2005
[3] S. Akdemir, Designing of Cold
Store and Choosing of Cooling system
Elements, Journal of Applied Sciences
8(5):788-794, 2008 [1] Design of Room cooling Facilities
structural and energy Requirements,
North
Carolina
State
University,
Department
of
Biological
and
Agricultural Engineering, Post harvest
commodity series, A6-414-2,1988
[2] Jing Xie, Xiao-Hua Qu, Jun-yesh
and Da-wen sun, Effect Of Design
Parameter on Flow and Temperature
Field of a Cold store by CFD simulation,
Journal of Food Engineering, Volume
77, Issue 2, November 2005
[3] S. Akdemir, Designing of Cold
Store and Choosing of Cooling system
Elements Journal of Applied Sciences [2] Jing Xie, Xiao-Hua Qu, Jun-yesh
and Da-wen sun, Effect Of Design
Parameter on Flow and Temperature
Field of a Cold store by CFD simulation, Journal of Food Engineering, Volume
77, Issue 2, November 2005 The final peak load equation shows
that the last term is the major factor
affected on the result of the peak load
calculation
therefore
the
relation
between 1/A or ∆T/ ∆T Tot and 1/B or
m/m Tot can
be
plotted
against
the
increasing in the load as a percentage of
product load (Q6), as shown in figure
(6). [3] S. Akdemir, Designing of Cold
Store and Choosing of Cooling system
Elements, Journal of Applied Sciences
8(5):788-794, 2008 [4] Erol N., Fundamental method for
refrigeration
and
air-conditioning,
Istanbul Technical university, Alummi
Association of vocational school of
Maritime, [4] Erol N., Fundamental method for
refrigeration
and
air-conditioning,
Istanbul Technical university, Alummi
Association of vocational school of
Maritime, Figure (6) shows the increasing of 1/A
(increases the ratio of daily temperature
difference to the total of temperature
difference(∆T/∆T Tot )causes
increasing
the peak load while the value of 1/B is
constant until it reaches its maximum
value when A=B. As mentioned above
the load ratio increases when 1/B is
significantly increased with constant
value of 1/A. 24
3600
1000
87
.4
5.
2962
27
.1
Q2
×
×
×
×
= From Table-3 and figures (1) to (5),
in some cases it appears that peak load
duration might not be exceeded more
than
one
day
and
then
reduced
gradually, that means the possibility to
pass (avoid) this period of highly load in
this day by changing the storing method. Table-3 enables the store manager or
staff to determine previously the value
and time of peak load to take the
necessary technical arrangements. 7. Conclusions The result of applying the present
prediction
method
of
peak
load
estimation
reveals
the
significant
difference between the two method of PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com A Simplified Method For Estimating The Peak Load
In The Large Cold Stores Eng. & Tech. Journal ,Nol.27, No. 10, 2009 Table (1) Summary of load calculation
Q
Load type
kW/day
1
2
External Load
12.121
3.29
3
4
5
Base Load
1.56
15.0
0.8
6
7
Internal (Product) Load
702.5
1718.7
TOTAL
2454 Table (1) Summary of load calculation Table (2) The chosen cases for percentage of the daily storing
mass and the temperature difference Table (2) The chosen cases for percentage of the daily storing
mass and the temperature difference
Case
∆T/∆T Tot
%
Factor-A
(day)
m/m Tot
%
Factor-B
(day)
1
25
4
10
10
2
10
10
25
4
3
50
2
50
2
4
33
3
10
10
5
20
5
50
2 Table (3) A summary of the result
case
Peak
load day
Nominal
load starting
day
Peak load
(W)
Nominal Load
(W)
A=B
A or B
A+B
Base load + External
Load + B*Q7+A*Q6
Base load + External
Load + B*Q7
A>B
A
A+B
Base load + External
Load + B*Q7+B*Q6
Base load + External
Load + B*Q7
B>A
B
A+B
Base load + External
Load + B*Q7+A*Q6
Base load + External
Load + B*Q7 Table (3) A summary of the result PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Loa
In The Large Cold Stores
0
20
40
60
80
100
120
140
160
180
200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale )
Product Load
Respiration Load
External Load
Base Load
Figure (1) Load distribution case No. 1 (A= 4 , B= 10)
0
20
40
60
80
100
120
140
160
180
200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale )
Product Load
Respiration Load
External Load
Base Load
Figure (2) Load distribution case No .2 (A=10, B=4) Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Load
In The Large Cold Stores 0
20
40
60
80
100
120
140
160
180
200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale )
Product Load
Respiration Load
External Load
Base Load
Figure (1) Load distribution case No. 1 (A= 4 , B= 10) Figure (1) Load distribution case No. 1 (A= 4 , B= 10) 0
20
40
60
80
100
120
140
160
180
200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale )
Product Load
Respiration Load
External Load
Base Load
Figure (2) Load distribution case No .2 (A=10, B=4) 0
20
40
60
80
100
120
140
160
180
200
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale )
Product Load
Respiration Load
External Load
Base Load 0
20
40
60
80
100
120
140
Load ( not to scale ) Figure (2) Load distribution case No .2 (A=10, B=4) PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Load
In The Large Cold Stores
0
10
20
30
40
50
60
70
80
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale)
Product Load
Resipration Load
External Load
Base Load
Figure (3) Load distribution case No. 3 (A=2, B=2)
Figure (4) Load distribution case No. 4 (A= 3 , B=10)
0
20
40
60
80
100
120
140
160
180
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale )
Product Load
Respiration Load
External Load
Base Load Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Load
In The Large Cold Stores 0
10
20
30
40
50
60
70
80
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale)
Product Load
Resipration Load
External Load
Base Load
Figure (3) Load distribution case No. 3 (A=2, B=2) Figure (3) Load distribution case No. 3 (A=2, B=2) Figure (4) Load distribution case No. 4 (A= 3 , B=10)
0
20
40
60
80
100
120
140
160
180
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Day
Load ( not to scale )
Product Load
Respiration Load
External Load
Base Load Figure (4) Load distribution case No. 4 (A= 3 , B=10) PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com Eng. & Tech. Journal ,Nol.27, No. 10, 2009 A Simplified Method For Estimating The Peak Loa
In The Large Cold Stores
0.0
20.0
40.0
60.0
80.0
100.0
1/A %
Product Load Increse %
1/B=100 %
1/B=75 %
1/B=50 %
1/B=25 %
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Rendering near-field speckle statistics in scattering media
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Rendering Near-Field Speckle Statistics in Scattering Media Rendering Near-Field Speckle Statistics in Scattering Media CHEN BAR, Department of Electrical Engineering, Technion, Israel
IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA
ANAT LEVIN, Department of Electrical Engineering, Technion, Israel
OD = 1
OD = 5
OD = 10
Tissue
Sensor
Image of illum i1
Image of illum i2
Fig. 1. Rendering near-field speckle patterns. We propose an efficient and physically-accurate algorithm that can simulate speckle patterns produced by
coherent illumination sources located or focused very close to the material. One of the important properties of speckle is the memory effect: small translations of
the illuminator produce shifted, highly-correlated speckle patterns. In the figure, we used our algorithm to simulate light scattering in a medium with realistic
tissue parameters, at thicknesses of 50 µm, 250 µm, 500 µm, and a mean free path of 50 µm, equivalent to optical depths (OD) of 1, 5 and 10, respectively. In
each case the figure presents two speckle images obtained with a small shift of the input illumination, leading to correlated shifted speckle patterns (e.g.,
the shift is visible in the insets of the first two columns). As expected, when the thickness increases, more scattering is present, and thus the memory-effect
correlation becomes weaker (the correlation is less visible in the inset of the third column). CHEN BAR, Department of Electrical Engineering, Technion, Israel
IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA
ANAT LEVIN, Department of Electrical Engineering, Technion, Israel
OD = 1 CHEN BAR, Department of Electrical Engineering, Technion, Israel
IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA
ANAT LEVIN, Department of Electrical Engineering, Technion, Israel
OD
1 CHEN BAR, Department of Electrical Engineering, Technion, Israel
IOANNIS GKIOULEKAS, Robotics Institute, Carnegie Mellon University, USA
ANAT LEVIN, Department of Electrical Engineering, Technion, Israel
OD = 1 Image of illum i1 Image of illum i2 Fig. 1. Rendering near-field speckle patterns. We propose an efficient and physically-accurate algorithm that can simulate speckle patterns produced by
coherent illumination sources located or focused very close to the material. One of the important properties of speckle is the memory effect: small translations of
the illuminator produce shifted, highly-correlated speckle patterns. In the figure, we used our algorithm to simulate light scattering in a medium with realistic
tissue parameters, at thicknesses of 50 µm, 250 µm, 500 µm, and a mean free path of 50 µm, equivalent to optical depths (OD) of 1, 5 and 10, respectively. Rendering Near-Field Speckle Statistics in Scattering Media In
each case the figure presents two speckle images obtained with a small shift of the input illumination, leading to correlated shifted speckle patterns (e.g.,
the shift is visible in the insets of the first two columns). As expected, when the thickness increases, more scattering is present, and thus the memory-effect
correlation becomes weaker (the correlation is less visible in the inset of the third column). We introduce rendering algorithms for the simulation of speckle statistics
observed in scattering media under coherent near-field imaging conditions. Our work is motivated by the recent proliferation of techniques that use
speckle correlations for tissue imaging applications: The ability to simulate
the image measurements used by these speckle imaging techniques in a
physically-accurate and computationally-efficient way can facilitate the
widespread adoption and improvement of these techniques. To this end,
we draw inspiration from recently-introduced Monte Carlo algorithms for
rendering speckle statistics under far-field conditions (collimated sensor
and illumination). We derive variants of these algorithms that are better
suited to the near-field conditions (focused sensor and illumination) required
by tissue imaging applications. Our approach is based on using Gaussian
apodization to approximate the sensor and illumination aperture, as well
as von Mises-Fisher functions to approximate the phase function of the
scattering material. We show that these approximations allow us to derive
closed-form expressions for the focusing operations involved in simulating
near-field speckle patterns. As we demonstrate in our experiments, these approximations accelerate speckle rendering simulations by a few orders of
magnitude compared to previous techniques, at the cost of negligible bias. We validate the accuracy of our algorithms by reproducing ground truth
speckle statistics simulated using wave-optics solvers, and real-material
measurements available in the literature. Finally, we use our algorithms to
simulate biomedical imaging techniques for focusing through tissue. Authors’ addresses: Chen Bar, Department of Electrical Engineering, Technion, Haifa,
Israel; Ioannis Gkioulekas, Robotics Institute, Carnegie Mellon University, Pittsburgh,
PA, USA; Anat Levin, Department of Electrical Engineering, Technion, Haifa, Israel. CCS Concepts: • Computing methodologies →Computational
photography; Rendering. Additional Key Words and Phrases: Monte Carlo rendering, speckle,
memory effect ACM Reference Format:
Chen Bar, Ioannis Gkioulekas, and Anat Levin. 2020. Rendering Near-Field
Speckle Statistics in Scattering Media. ACM Trans. Graph. 39, 6, Article 187
(December 2020), 26 pages. https://doi.org/10.1145/3414685.3417813 Permission to make digital or hard copies of all or part of this work for personal or
classroom use is granted without fee provided that copies are not made or distributed
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on the first page. Copyrights for components of this work owned by others than ACM
must be honored. Abstracting with credit is permitted. To copy otherwise, or republish,
to post on servers or to redistribute to lists, requires prior specific permission and/or a
fee. Request permissions from permissions@acm.org.
© 2020 Association for Computing Machinery.
0730-0301/2020/12-ART187 $15.00
https://doi.org/10.1145/3414685.3417813 ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 1
INTRODUCTION A core challenge in medicine is the development of technologies
for imaging deep inside biological tissues at high spatial resolutions. What makes this type of imaging possible is the fact that, when a
light source illuminates tissue, a significant amount of light enters,
travels inside, and re-emerges out of the tissue. What makes this
type of imaging difficult is the fact that, when inside the tissue, light
scatters multiple times. Thus, the fundamental challenge that needs
to be solved to enable imaging inside tissue is inverting the multiple
scattering process, in order to extract the information that light
carries about the tissue it interacted with. 187:2
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin In recent years, several imaging techniques have emerged that
address this challenge by taking advantage of the fact that images
of tissue under coherent (e.g., laser) illumination contain significant
speckles: These are pseudo-random, high-frequency spatial varia-
tions in the intensity of the captured images. The statistical proper-
ties of these speckle patterns is a classical research area within optics,
having been the subject of several textbooks [Erf 1978; Goodman
2007; Jacquot and Fournier 2000; Kaufmann 2011]. This research
has revealed that, despite its random appearance, a speckle has
strong statistical properties that provide rich information about the
underlying scattering material (e.g., tissue). Perhaps best known
among them is the memory effect (ME) property, illustrated in Fig. 1,
which describes how speckle fields remain correlated under small
changes in imaging conditions. The memory effect is at the core of
speckle-based techniques for tissue imaging applications such as
fluorescence imaging and adaptive optics focusing inside tissue. importance sampling scheme for starting the volume path trac-
ing process that takes into account the focused beams inside the
medium. In our experiments, we validate the physical accuracy of
our algorithms by showing that they can reproduce speckle corre-
lation statistics simulated using wave-equation solvers, as well as
speckle correlation measurements of real materials that are publicly
available in the literature [Osnabrugge et al. 2017]. Our algorithms
match this synthetic and measured groundtruth more accurately
than existing simulation techniques (e.g., multi-slice layer based
algorithms [Schott et al. 2015]) and analytical models (e.g., tilt-shift
memory effect [Osnabrugge et al. 2017]) from optics. Additionally,
we show that our algorithms allow us to simulate focusing-inside-
tissue applications, which require near-field conditions. 1.1
Why render near-field speckle statistics? Speckle statistics have strong potential for applications in the con-
text of tissue imaging, where scattering by cells and other variations
of the local index of refraction in the tissue drastically degrade
image contrast. For example, several papers have suggested using
speckle correlations to detect incoherent fluorescence sources inside
the tissue. As Katz et al. [2014] have observed, due to the memory
effect, the auto-correlation of random speckle images, together with
a phase retrieval algorithm, can help remove the effect of scattering
and reveal the location of the sources under the skin. Unfortunately,
this idea has been successfully demonstrated mostly in the far-
field setting, with the sources located at a large distance outside
the scattering medium, rather than inside it. The only successful
demonstration of this idea in the near-field we are aware of is by
Chang et al. [2018], whose experiments were able to recover fluo-
resent particles spanning a small spatial range of 10 µm. We argue
that this state of affairs is due to the limited exploration of near-field
speckle statistics, an issue we hope our paper will help address. For this, we draw inspiration from Bar et al. [2019], who intro-
duced a Monte Carlo framework for simulating speckle correlations
in a way that combines physical accuracy and computational ef-
ficiency. Unfortunately, despite offering orders-of-magnitude ac-
celeration compared to previous physically-accurate simulation
techniques (e.g., wave equation solvers), the algorithms of Bar et
al. [2019] are primarily intended for simulating far-field speckle sta-
tistics, and remain impractical for the near-field case. For example,
as we show in our experiments, using their framework to simulate
current techniques for focusing inside scattering [Judkewitz et al. 2014] can take several days on a large cluster. These performance
characteristics significantly constrain the scope of investigations
that can be performed using these algorithms (e.g., evaluation of
effectiveness of existing techniques under different imaging param-
eters or for tissue samples of different optical parameters). Another important application of speckle techniques in tissue
imaging is the use of adaptive optics [Mertz et al. 2015] to focus
light at points deep inside tissue. Achieving this type of focusing
requires using a coherent wavefront of a shape specific to the tissue
sample being imaged. Determining the exact wavefront is challeng-
ing, and typically involves using external information or a guiding
star [Horstmeyer et al. 2015]. 1
INTRODUCTION For the
small volumes we simulate, our algorithm is already 3000× faster
than an adaptation of the far-field algorithm [Bar et al. 2019]; and
this performance difference will only increase for real-sized volumes. We believe that the ability to simulate near-field speckle statistics
outside the lab will accelerate ongoing research on speckle-based
techniques for tissue imaging applications. To fortify this effort, we
have made our implementation publicly available [Bar et al. 2020]. l
g
g
p
p
g
Unfortunately, most previous studies of speckle statistical proper-
ties and of the memory effect, have been performed under imaging
conditions that are not suitable for tissue imaging. Typically, most
studies assume that both the light sources and the sensors are out-
side and at a large distance from the scattering volume, a set of
conditions referred to as far-field imaging. By contrast, tissue imag-
ing applications require both sources and sensors to be focused
very close to the tissue (e.g., confocal microscopy), or even located
inside it (e.g., fluorescent particles), a set of conditions known as
near-field imaging. Far-field imaging simplifies analysis, simulation,
and experiments relating to speckles. However, inferences drawn
for the far-field case do not necessarily generalize to the near-field
one. Our goal is to develop physically-accurate and efficient render-
ing algorithms that can help improve our understanding of speckle
statistical properties under near-field conditions. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 1.1
Why render near-field speckle statistics? Once this wavefront is found, the
memory effect can be used to scan an area inside tissue, e.g., by
shifting and tilting the wavefront to focus at neighboring points [Os-
nabrugge et al. 2017]. In our experiments, we simulate this approach,
and show preliminary investigations on the effectiveness of using
the tilt-shift memory effect. By enabling researchers to perform such
investigations in simulation, without the need for lab experiments,
we hope that our paper can help expand the scanning range and
operational capabilities of techniques for focusing inside tissue.fi With these considerations in mind, we develop a computationally-
efficient algorithm for simulating near-field (focused) speckle sta-
tistics inside scattering media. To this end, we extend the Monte
Carlo rendering algorithm of Bar et al. [2019] in several ways that
make it better-suited for near-field simulations. Our innovations
are three-fold: First, we derive a path-integral expression for near-
field speckle statistics. Second, we approximate optical apertures
and material phase functions using von Mises-Fisher functions, to
obtain analytical expressions for connecting paths traced inside
a volume to near-field sources and sensors. Third, we develop an p
p
q
g
Furthermore, the ability to efficiently render speckle patterns
can facilitate the widespread adoption of data-driven approaches in Rendering Near-Field Speckle Statistics in Scattering Media
•
187:3 limitation is due to the fact that our algorithms use volume path trac-
ing to sample light paths, and is thus shared with standard Monte
Carlo volume rendering algorithms for simulating incoherent inten-
sity [Novak et al. 2018]. As in the intensity case, techniques based
on the diffusion approximation [Jensen et al. 2001] would be better-
suited for simulating speckle statistics at volumes of larger optical
depths. However, we note that near-field speckle correlations decay
as a function of optical depth much faster than intensity does; and
are essentially non-existent for volumes of optical depths signif-
icantly larger than what we simulate. Consequently, biomedical
imaging techniques based on speckle correlations are typically only
applicable for optical depths below the diffusive regime, coincid-
ing with the range of optical depths we emphasize in our experi-
ments. Considering that neither single-scattering [Narasimhan et al. 2006] nor diffusion approximations [Feng et al. 1988] are effective
in this range, our algorithms provide a suitable simulation tool for
researchers investigating these biomedical imaging techniques. tissue imaging. 1.2
Limitations Our algorithms are subject to a few limitations that suggest im-
portant directions for future research. First, they assume that the
simulated scattering medium is homogeneous, meaning that its
optical scattering parameters are the same at all spatial locations
inside the volume. Our algorithms additionally assume that the
medium is exponential, meaning that it comprises uncorrelated scat-
terers [Bitterli et al. 2018; d’Eon 2018; Jarabo et al. 2018]; and scatters
light isotropically, meaning that its phase function is rotationally-
invariant [Jakob et al. 2010]. These assumptions are commonly used
in biomedical imaging research and applications to approximate
how biological tissues scatter light at optical and infrared wave-
lengths. Therefore, our algorithms can be used to simulate imaging
applications involving such materials. Second, our algorithms are primarily geared towards transmission
mode imaging configurations, where illumination and sensing hap-
pen at opposite sides of a scattering volume. This is a consequence
of our use of von Mises-Fisher functions to approximate the imaging
aperture and material phase function. When using these approxi-
mations to simulate reflection mode imaging configurations, where
illumination and sensing are on the same side of a scattering volume,
accuracy will depend on the exact material parameters. In particu-
lar, when simulating materials with phase functions that have very
little back-scattering, accuracy suffers because of numerical issues. We provide a detailed analysis in App. A.4. In practical terms, this
means that our algorithms can be used to simulate tissue imaging
applications such as fluorescence imaging and focusing through
tissue, both of which correspond to transmissive configurations;
but should be used with caution for applications such as confocal
microscopy, which corresponds to a reflective configuration.fi Analytical models for speckle statistics. Quantifying differences in
speckle characteristics between the near-field and far-field cases can
be done using analytical tools that approximate speckle statistics
with closed-form mathematical expressions. Most available such
tools are for the far-field case [Akkermans and Montambaux 2007;
Baydoun et al. 2016; Berkovits and Feng 1994; Dougherty et al. 1994;
Feng et al. 1988; Freund and Eliyahu 1992; Fried 1982], though re-
cently tools have been introduced for the near-field case [Judkewitz
et al. 2014; Osnabrugge et al. 2017]. For example, Osnabrugge et
al. [2017] derived a tilt-shift model for the memory effect, assuming
that the underlying scattering material is optically thin and very
forward-scattering. 2
RELATED WORK Speckle in computational imaging. Speckle statistics have found
wide applicability in computational imaging. Example applications
include motion tracking [Jacquot and Rastogi 1979; Jakobsen et al. 2012; Smith et al. 2017], looking around the corner [Batarseh et al. 2018; Freund 1990; Katz et al. 2012], and seeing through [Abooka-
sis and Rosen 2004; Bertolotti et al. 2012; Katz et al. 2014; Rosen
and Abookasis 2003; Takasaki and Fleischer 2014] or focusing
through [Choi et al. 2011; Edrei and Scarcelli 2016; Katz et al. 2010,
2012; Lai et al. 2015; Mosk et al. 2012; Nixon et al. 2013; Rueckel et al. 2006; van Putten et al. 2011; Vellekoop and Aegerter 2010; Vellekoop
et al. 2012, 2010; Vellekoop and Mosk 2007; Yaqoob et al. 2008] tissue
and other scattering layers. Most of these imaging techniques rely
on the memory effect of speckles, and therefore are based on spatial
correlations between speckle images. Alternatively, imaging tech-
niques such as diffusing wave spectroscopy [Pine et al. 1988], laser
speckle contrast imaging [Boas and Yodh 1997], and dynamic light
scattering [Goldburg 1999] use temporal speckle correlations [Berne
and Pecora 2000; Dougherty et al. 1994] to estimate flow (e.g., blood
flow [Durduran et al. 2010]) and liquid composition parameters. 1.1
Why render near-field speckle statistics? The use of such approaches is in part motivated
by analogous successes in the far-field case, where it has been
demonstrated that machine learning algorithms can improve the
performance of memory-effect-based imaging around the corner
and through scattering [Li et al. 2018; Metzler et al. 2020, 2018]. The successful deployment of machine learning algorithms requires
large, physically-accurate datasets. Previously, the lack of physically-
accurate simulation tools meant that datasets had to be collected
using lab measurements, an approach that is not scalable. We hope
that our rendering tools can help reduce the data collection over-
head, making machine learning approaches tractable. Last but not least, accurate speckle rendering algorithms can
be useful for inverse rendering problems involving speckle mea-
surements. In particular, prior work has shown that differentiable
rendering techniques can be used to recover accurate scattering pa-
rameters of real-world materials from incoherent intensity measure-
ments [Gkioulekas et al. 2013]. We expect that our algorithms can be
combined with modern differentiable rendering techniques [Nimier-
David et al. 2020; Zhang et al. 2020, 2019]. In turn, this has the
potential to enable recovering accurate high-resolution models of
important material classes, such as biological tissue, from measure-
ments of speckle fields and speckle correlations. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 1.2
Limitations Deriving closed-form expressions requires re-
strictive assumptions (single scattering, diffusion, or Fokker-Planck
limits), which typically do not apply to the predominantly-turbid Third, our algorithms become inefficient for very optically-deep
(e.g., more than 10 times the mean free path) volumes, where light
is expected to perform a large number of scattering events. This 187:4
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin 𝐨𝐨
𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐
ෞ
𝒗𝒗𝟏𝟏
ෞ
𝒗𝒗𝟐𝟐
𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐
𝒗𝒗𝟐𝟐
𝒗𝒗𝟏𝟏
(a) Directional
(b) Focused
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐
ෞ
𝒗𝒗𝟏𝟏
ෞ
𝒗𝒗𝟐𝟐
𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐
𝒗𝒗𝟐𝟐
𝒗𝒗𝟏𝟏
(c) Directional paths
(d) Focused paths
Fig. 2. Transmissive imaging configurations. Previous work [Bar et al. 2019] simulated speckle statistics by directional sources and sensors, as in
(a). In contrast this work considers speckles by focused camera and sources,
as in (b). (c-d) demonstrate what is implied when evaluating such speckle
correlations using a Monte Carlo algorithm. In the directional formulation,
one draws 4 directional connections from each path toward the illumination
and viewing directions. In contrast, to simulate speckles through a focused
lens, one needs to trace all paths via the aperture. 𝐨𝐨
𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐
ෞ
𝒗𝒗𝟏𝟏
ෞ
𝒗𝒗𝟐𝟐
(a) Directional
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐
𝒗𝒗𝟐𝟐
𝒗𝒗𝟏𝟏
(b) Focused
𝒊𝒊𝟏𝟏
𝒊𝒊𝟐𝟐 tissue scattering. Developing efficient and physically-accurate ren-
dering tools for near-field speckle statistics can help assess the accu-
racy of these approximations, and facilitate their broader adoption
in application regimes where they are accurate. Rendering wave-optics effects. Monte Carlo rendering techniques
within computer graphics have typically focused on simulating inco-
herent light transport. More recently, there have been a few works
on simulating wave optics effects, such as diffraction and speckle
due to rough surface geometry [Bergmann et al. 2016; Cuypers et al. 2012; Sadeghi et al. 2012; Stam 1999; Sur et al. 2018; Werner et al. 2017; Yan et al. 2018; Yeh et al. 2013]. Variants of volumetric path
tracing for simulating speckle in scattering have appeared in op-
tics [Lu et al. 2004; Mout et al. 2016; Pan et al. 1995; Sawicki et al. 2008; Schmitt and Knüttel 1997; Xu 2004], though these typically can-
not estimate second-order statistics (e.g., the memory effect). Bar et
al. [2019] addressed this shortcoming by introducing a Monte Carlo
algorithm that uses simultaneous path connections to multiple light
sources and sensors, to correctly model speckle correlations between
them. 3
BACKGROUND ON SPECKLE STATISTICS We begin by providing background on speckle statistics and on their
Monte Carlo modeling for the far-field case. We use this background
in Sec. 4 to contrast the far-field and near-field cases, which helps
highlight the challenges involved in rendering the latter case, and
positions our work relative to the prior art of Bar et al. [2019]. relative to MFP. For example, a volume has optical depth OD = 2
if its thickness is equal to 2 · MFP, meaning that light travelling
through the volume undergoes on average two scattering events.i Notation and setting. We use bold letters for vectors (e.g., points
o, i, v), with a circumflex for unit vectors (e.g., directions ˆω,ˆi, ˆv). We consider scattering volumes V ∈R3 that satisfy the assump-
tions underlying classical radiative transfer for isotropic [Jakob et al. 2010] exponential media [Bitterli et al. 2018; d’Eon 2018; Jarabo et al. 2018]: Each volume comprises a set of scatterers, whose locations
in the volume are statistically independent. These scatterers are
assumed to be small enough relative to the wavelength of light
to be considered infinitesimal points. They are also assumed to
be spherically symmetric, and thus scatter incident light waves in
a rotationally-invariant way. We model speckle fields arising in
such volumes due to incident illumination that we assume to be
monochromatic, fully-coherent and unpolarized. These fields are a
function of the volumes’ bulk properties, which we describe next. The scattering amplitude function s(cosθ) describes how a field
interacts with a scatterer: if a scatterer is illuminated from direction
ˆi, the complex scattered field u at direction ˆv is uˆi
ˆv = s(ˆi · ˆv). The
phase function is defined as ρ(cosθ) ≡|s(cosθ)|2. It is commonly
characterized by an anisotropy parameter −1 ≤д ≤1, equal to the
average cosθ: д = 0 corresponds to scattering equally in all direc-
tions, and д = 1 to fully forward scattering. Tissue is characterized
by very forward scattering (д > 0.9) [Cheong et al. 1990]. These parameters are a function of wavelength, and the scatterers’
shape, size, and refractive index. For spherical scatterers, they can
be computed using Mie theory [Bohren and Huffman 1983; Frisvad
et al. 2007]. The three coefficients also depend on the density ς, equal
to the expected number of scatterers in a unit volume. 1.2
Limitations Unfortunately, their algorithm is primarily tailored to far-field
imaging and becomes inefficient for the near-field case, as discussed
in Sec. 3. The difference between the two cases is reminiscent of
the challenges in rendering depth-of-field effects in incoherent light
transport, where special rendering algorithms are required due to
the need to sample many rays on the aperture plane [Barsky and
Kosloff 2008; Kolb et al. 1995; Soler et al. 2009]. (a) Directional𝒊𝟏𝒊𝟐 ෞ
𝒗𝒗𝟏𝟏
ෞ
𝒗𝒗𝟐𝟐
(c) Directional paths 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺
𝒗𝒗𝟐𝟐
𝒗𝒗𝟏𝟏
(d) Focused paths 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝒗𝟏 (c) Directional paths (d) Focused paths Fig. 2. Transmissive imaging configurations. Previous work [Bar et al. 2019] simulated speckle statistics by directional sources and sensors, as in
(a). In contrast this work considers speckles by focused camera and sources,
as in (b). (c-d) demonstrate what is implied when evaluating such speckle
correlations using a Monte Carlo algorithm. In the directional formulation,
one draws 4 directional connections from each path toward the illumination
and viewing directions. In contrast, to simulate speckles through a focused
lens, one needs to trace all paths via the aperture. Fig. 2. Transmissive imaging configurations. Previous work [Bar et al. 2019] simulated speckle statistics by directional sources and sensors, as in
(a). In contrast this work considers speckles by focused camera and sources,
as in (b). (c-d) demonstrate what is implied when evaluating such speckle
correlations using a Monte Carlo algorithm. In the directional formulation,
one draws 4 directional connections from each path toward the illumination
and viewing directions. In contrast, to simulate speckles through a focused
lens, one needs to trace all paths via the aperture. 3
BACKGROUND ON SPECKLE STATISTICS We assume
that scattering volumes are spatially homogeneous, meaning that
scatterers are uniformly distributed, or equivalently, that the bulk
parameters are the same everywhere inside a volume. Bulk material properties. We use a statistical description of the
optical properties of scattering volumes. In particular, the scattering
and absorption coefficients σs and σa model, respectively, the portion
of energy that is scattered and absorbed upon interaction with a
scatterer. Their sum is the extinction coefficient σt ≡σa + σs, and
its inverse is the mean free path, MFP = 1/σt , which is the average
distance in the volume light travels between two scattering events. Given a volume V, it is common to express its geometric dimensions ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 3.1
Modeling and rendering far-field speckle statistics Transmissive far-field imaging. We focus on the geometry illus-
trated in Fig. 2(a): Scatterers are placed at a configuration of locations Rendering Near-Field Speckle Statistics in Scattering Media
•
187:5 than wave-equation solvers, and scalable to much larger volumes. We proceed to review this Monte Carlo rendering approach. O = {o1, o2, . . .} inside the volume V, each sampled independently
from the others, using the volume density ς. This configuration is
imaged using light sources and sensors that are on opposite sides
of the volume, a setting we refer to as transmission mode imaging. Additionally, in this background section, we assume that the volume
is illuminated by a directional plane wave ˆi, and imaged with a direc-
tional sensor ˆv. We refer to these conditions together as the far-field
imaging conditions. If we know the exact scatterer locations, and
incoming and outgoing directions, we can solve the wave equation
to obtain the complex-valued scattered field uˆi,O
ˆv
, arising from the
interaction of the incident illumination with the scattering volume. Covariance rendering. Bar et al. [2019] derive their algorithm
from a path integral expression for speckle covariance, obtained by
considering the correlation of fields that travel along all possible
pairs of paths from ˆi1 to ˆv1, and from ˆi2 to ˆv2. They showed that
this expression can be simplified to use only pairs of paths that
coincide everywhere, except for their connections to ˆi1, ˆv1,ˆi2, ˆv2. We review this simplified formulation. Consider the space P of sub-
paths ®xs = o1 →· · · →oB, B ≥1, where each vertex ob ∈V;
we denote by ˆωb ≡
ob, ob+1 the direction of the b-th edge of the
sub-path. These vertices correspond to the shared part of two full
paths ®x1 = ˆi1→o1→. . .→oB →ˆv1, ®x2 = ˆi2→o1→. . .→oB →ˆv2,
formed by connecting the sub-path to ˆi1, ˆv1 and ˆi2, ˆv2. Then, the
speckle covariance of Eq. (2) can be expressed as: Defining speckle statistics. For any volume with a given scatterer
configuration O, the scattered field typically contains large fluctu-
ations with a semi-random noise structure known as speckle (see,
e.g., Fig. 1). We can characterize speckle using the first and second-
order statistics of fields due to different volumes with the same bulk
material properties. 3.1
Modeling and rendering far-field speckle statistics In particular, we can define the speckle mean, Cˆi1,ˆi2
ˆv1,ˆv2 =
∫
P
cˆi1,ˆi2
ˆv1,ˆv2(®xs) d®xs,
(3) (3) mˆi
ˆv ≡EO
h
uˆi,O
ˆv
i
,
(1) (1) where the far-field path contribution function cˆi1,ˆi2
ˆv1,ˆv2 equals the cor-
relation of the fields that travel along ®x1, ®x2. For B ≥2, this equals: where the far-field path contribution function cˆi1,ˆi2
ˆv1,ˆv2 equals the cor-
relation of the fields that travel along ®x1, ®x2. For B ≥2, this equals: and the speckle covariance, Cˆi1,ˆi2
ˆv1,ˆv2 ≡EO
h
uˆi1,O
ˆv1
· uˆi2,O
ˆv2
∗i
−mˆi1
ˆv1 · mˆi2
ˆv2
∗
,
(2) cˆi1,ˆi2
ˆv1,ˆv2(®xs) = f (®xs) · υ(ˆi1→o1)s(ˆi1 · ˆω1)
· υ(oB→ˆv1)s( ˆωB−1 · ˆv1)
· υ(ˆi2→o1)∗s(ˆi2 · ˆω1)∗
· υ(oB→ˆv2)∗s( ˆωB−1 · ˆv2)∗,
(4) cˆi1,ˆi2
ˆv1,ˆv2(®xs) = f (®xs) · υ(ˆi1→o1)s(ˆi1 · ˆω1) (2) · υ(oB→ˆv1)s( ˆωB−1 · ˆv1)
· υ(ˆi2→o1)∗s(ˆi2 · ˆω1)∗ · υ(oB→ˆv1)s( ˆωB−1 · ˆv1) where (·)∗is complex conjugation.uˆi1,O
ˆv1
,uˆi2,O
ˆv2
are two speckle fields
generated by the same scatterer configuration O, when illuminated
by two monochromatic, mutually-coherent incident waves from ˆi1,ˆi2,
and measured at two sensors ˆv1, ˆv2. The expectation EO is taken
with respect to all scatterer configurations O sampled from the
same density ς. As we detail in App. A.1, the speckle mean mˆi
ˆv can
be computed using a closed-form expression and is typically zero. Th
f
f
d li
th
kl
i
Cˆi1 ˆi2 · υ(oB→ˆv2)∗s( ˆωB−1 · ˆv2)∗, (4) and for B = 1: and for B = 1: cˆi1,ˆi2
ˆv1,ˆv2(®xs) = σs · υ(ˆi1→o1)υ(o1→ˆv1)s(ˆi1 · ˆv1)
· υ(ˆi2→o1)∗υ(o1→ˆv2)∗s(ˆi2 · ˆv2)∗. (5) · υ(ˆi2→o1)∗υ(o1→ˆv2)∗s(ˆi2 · ˆv2)∗. (5) (5) Therefore, we focus on modeling the speckle covariance Ci1,i2
ˆv1 ˆv2.i v ,v
The definition of Eq. (2) suggests a straightforward approach for
computing this covariance: randomly sample many scatterer con-
figurations O from the material bulk parameters, solve the wave
equation numerically to compute uˆi1,O
ˆv1
,uˆi2,O
ˆv2
, and use averaging to
approximate the expectation in Eq. (2). Unfortunately, while exact
wave-equation solvers exist [Thierry et al. 2015; Treeby and Cox. 2010; Yee 1966], their computational complexity is prohibitive, typi-
cally making them intractable for volumes of width larger than a few
dozen wavelengths. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 3.1
Modeling and rendering far-field speckle statistics This computational cost is further exacerbated
by the need to use the solvers multiple times for averaging.i In the above, f (®xs) is the standard radiometric throughput of ®xs,
augmented by scattering coefficients at the first and last vertex, f (®xs) = (σs)B
B−1
Ö
b=1
e−σt ∥ob+1−ob ∥
∥ob+1 −ob ∥2 ρ( ˆωb−1 · ˆωb). (6) (6) Finally, υ(·) is the complex volumetric throughput, defined as: υ( ˆω→o) = e−1
2 σtd( ˆω→o)eik( ˆω ·o),
(7)
υ(o→ˆω) = e−1
2 σtd(o→ˆω)e−ik( ˆω ·o),
(8) (7) (7)
(8) (8) (8) For an alternative, note first that, when ˆi1 = ˆi2 = ˆi, ˆv1 = ˆv2 = ˆv
and mˆi
ˆv = 0, Cˆi,ˆi
ˆv,ˆv reduces to the intensity Iˆi
ˆv. This intensity is typ-
ically modeled in computer graphics using the radiative transfer
equation, or its integral form, the volume rendering equation. The
latter gives rise to Monte Carlo volume rendering algorithms, which
compute intensity using as input only the volume’s bulk mate-
rial properties [Novak et al. 2018]. Bar et al. [2019] derived analo-
gous Monte Carlo volume rendering algorithms for computing the
speckle covariance Cˆi1,ˆi2
ˆv1,ˆv2 for any directions ˆi1,ˆi2, ˆv1, ˆv2. Like their
intensity counterparts, these algorithms take as input bulk material
parameters, and not particle positions. Bar et al. [2019] showed that
their approach is physically accurate, orders-of-magnitude faster where k ≡2π/λ is the wavenumber and λ the wavelength of the
illumination; and d( ˆω →o),d(o →ˆω) denote the distance a ray
entering or leaving, respectively, o at direction ˆω, travels inside the
scattering volume V. Fig. 2(c) visualizes these terms. The covariance rendering algorithm of Bar et al. [2019] uses
a Monte Carlo path sampling approach to evaluate the speckle
covariance integral of Eq. (3). This algorithm takes advantage of
the presence of the radiometric throughput term in Eq. (4), and
samples sub-paths ®xs using standard volumetric path tracing. Then,
for each sampled sub-path, the endpoints o1, oB are connected to
the far-field illuminations ˆi1, ˆi2 and sensors ˆv1, ˆv2, to compute the
complex volumetric throughput terms in Eqs. (4) and (5). ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 4
NEAR-FIELD SPECKLE STATISTICS As mentioned in the introduction, many important applications
require imaging speckle fields using a sensor that is focused at some
point, rather than being at infinity; and using illumination that is
better modeled as a point than as a directional source. Both of these
points can be near or even inside the scattering volume. To distin-
guish them from the far-field case of the previous section, we refer
to these conditions together as the near-field imaging conditions. Scattered fields formed under far-field and near-field conditions
often have very different characteristics, as we discuss in the rest
of the section. As in Sec. 3.1, we focus on transmission mode imag-
ing configurations, where illumination and sensing are on opposite
sides of the volume. The imaging configuration is shown in Fig. 2(b). N = 2W sin(Θmax)
λ
. (12) (12) For some representative numbers, we consider simulating a rel-
atively thin volume of thickness 400 µm. We assume that we im-
age a spatial area of size 100 µm × 100 µm using a numerical aper-
ture sin(Θmax) = 0.5 and illumination wavelength λ = 0.5 µm. We need to simulate a volume that is at least as wide as the de-
focused beam, as light contributing to the scattered field can be
due to scattering anywhere inside the beam (see Fig. 3). For the
dimensions mentioned, the defocus blur has size 400 µm at the far
edge of the target, suggesting that we need to simulate a volume of
widthW = 100 µm+400 µm = 500 µm. Then Eq. (12) suggests using
N = 1000 samples on each coordinate axis of the four directions
we integrate in Eq. (11). Therefore, to simulate the near-field covari-
ance, we need (1000)8 samples, which is prohibitively large. We note
that real near-field scenes used in, e.g., Osnabrugge et al. [2017] are
bigger and would require an even more far-field samples. Modeling near-field speckle statistics. We use the notation uˆi
ˆv,uiv
to indicate scattered fields due to far-field and near-field imaging
conditions, respectively, with the circumflex distinguishing between
directional to point sources/sensors. If we know the scattered field
uˆi
ˆv for all illumination and sensing directions ˆi, ˆv, we can compute
the scattered fielduiv by integrating over the sensor and illumination
apertures (Fig. 2(d)) [Goodman 1968; Mertz 2019]. 3.1
Modeling and rendering far-field speckle statistics d Anat Levin 187:6
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin We note that when ˆi1 = ˆi2 = ˆi, ˆv1 = ˆv2 = ˆv, the above algorithm
becomes equivalent to the standard volumetric path tracing algo-
rithm used in computer graphics to render intensity Iˆi
ˆv [Novak et al. 2018]. A key observation by Bar et al. [2019] is that, in the case of
covariance Cˆi1,ˆi2
ˆv1,ˆv2, each sampled sub-path ®xs needs to contribute to
two pairs of input-output directions (blue and green connections
in Fig. 2(c)). Using the same sub-paths for both (ˆi1, ˆv1), (ˆi2, ˆv2) pairs
is necessary to account for the correlation of fields from different
illuminator-viewpoint combinations, and this correlation cannot be
modeled using paths sampled independently for each pair. transform with respect to the variables ˆi and ˆv, and thus uˆi
ˆv is often
referred to as the Fourier field [Goodman 1968; Mertz 2019].i
1
2i The near-field speckle covariance Ci1,i2
v1,v2 can be defined analo-
gously to the far-field covariance in Eq. (2). Combining this defini-
tion with Eq. (9), we can relate near-field and far-field covariances: Ci1,i2
v1,v2 =
∫∫∫∫
av1(ˆv1)ai1(ˆi1)av2(ˆv2)∗ai2(ˆi2)∗Cˆi1,ˆi2
ˆv1,ˆv2dˆi1dˆi2dˆv1dˆv2. (11) (11) Why is rendering near-field covariance difficult? Eq. (11) provides a
conceptually simple way to compute near-field covariance statistics:
We sample, or discretize, the domains of ˆv1, ˆv2,ˆi2,ˆi1, estimate the
corresponding far-field covariance values as in Sec. 3.1, and form
their weighted average using the weights in Eq. (11). Before concluding this section, we mention that Bar et al. [2019]
present a variant of the above-described algorithm that simulates
scattered fields uˆi1
ˆv1, uˆi2
ˆv2 in a way that accurately reproduces their
correlations. In the rest of the paper, we show how to adapt both of
these rendering algorithms to the near-field case. We present most
of our theory in Secs. 4-6 in the context of covariance rendering,
then adapt it in Sec. 7 for field rendering. In practice, this approach is computationally impractical, because
of the very large number of samples of the far-field covarianceCˆi1,ˆi2
ˆv1,ˆv2
we need to compute. This number is determined by the width W
of the volume V and the numerical aperture Θmax, and not by the
size of the sensor. As we show in App. A.2, Nyquist sampling rate
implies the number of samples per axis of integration should be: ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 4
NEAR-FIELD SPECKLE STATISTICS Concretely: gg
qi
p
Our discussion so far has been about covariance rendering, but
similar arguments apply for field rendering, where using Eq. (9)
would require N 4 samples, or (1000)4 for the above example. We dis-
cuss the field rendering case in detail in Sec. 7, but for demonstration,
we compare in Fig. 3 speckle images rendered by our proposed field
rendering algorithm and using Eq. (9). Due to memory constraints,
the far-field directions were sampled at 10% of the Nyquist sampling
rate; this aliased sampling results in clear replica artifacts in the cor-
responding images. Even under these aliased sampling conditions,
rendering using Eq. (9) was 100× slower than our algorithm, while
requiring 30 GB GPU memory. These challenges cannot be allevi-
ated by using a large number of samples for single scattering and
fewer samples for multiple scattering, as is often done in intensity
rendering [Belcour et al. 2014]: even though intensity images from
multiple scattering have low spatial frequency, coherent multiple
scattering still creates high-frequency speckle patterns. ui
v =
∫
ˆi∈S2
∫
ˆv∈S2 ai(ˆi)av(ˆv)uˆi
ˆv dˆv dˆi,
(9) (9) where S2 is the unit sphere and, assuming an ideal lens, av(ˆv) ≡m(ˆv)eik(ˆv·v),
ai(ˆi) ≡m(ˆi)e−ik ˆi·i
. (10) (10) The functions m(ˆi) and m(ˆv) denote illumination and viewing
aperture amplitudes. Typically, these are binary functions indicating
which directions pass through an aperture of a finite extent. We
indicate the width of these masks using the angle Θmax between
the optical axis and the propagating direction that most deviates
from this axis, and we refer to sin Θmax as the numerical aperture
(NA) (Fig. 3(a)). The term exp(ik(ˆv · v)) is the phase accumulated in
direction ˆv when focusing at the point v; and analogously for the
term exp(−ik(ˆi · i)). Using paraxial optics approximations, it is also
possible to express the two integrals of Eq. (9) as a double Fourier ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. Rendering Near-Field Speckle Statistics in Scattering Media
•
187:7 F.F. image with i1
F.F. image with i2
N.F. image with i1
N.F. image with i2
𝜽
𝟒𝟎𝟎𝝁𝒎
𝟓𝟎𝟎𝝁𝒎
𝟒𝟎𝟎𝝁𝒎
image area
𝟏𝟎𝟎𝝁𝒎
NA:
𝐬𝐢𝐧𝜽= 𝟎. 𝟓
viewing objective
(a)
(b)
(c)
Fig. 3. Near-field challenges. Complex volumetric throuput : Complex volumetric throuput :
Aperture function focused at ˆi :
Aperture function × throughput :
Scattering amplitude function:
Convolved aperture function:
(throughput up to first scatterer) 𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐨𝐨𝟏𝟏) ∫
i∈S𝐢
(a) ˜ai(ˆi, o1)
(b) ϒ( ˆω1, o
s
ϒ( ˆω|o1, i) = ˜ai( ˆω|o1) ∗s
˜av( ˆω|o1)
˜av( ˆω|o1) · ϒ( ˆω|o1, i)
(c) (a) ˜ai(ˆi, o1) (b) ϒ( ˆω1, o1, i) ˜av( ˆω|o1) · ϒ( ˆω|o1, i) ˜av( ˆω|o1) ˜av( ˆω|o1) ˜ai( ˆω|o1) Fig. 4. Notation summary and visualization. (a) The aperture function ˜ai(ˆi, o1) includes an apodization mask m(ˆi), a complex wave focusing at i and a
conjugate wave focusing at o1, the first point on the sampled path (attenuation exp(−1/2σtd( ˆω →o)) is not visualized). (b) The aperture function convolved
angulary with the scattering amplitude function leads to ϒ( ˆω1, o1, i), the throughput up to the first point and direction. That is, to compute the field
propagating from o1 at direction ˆω1 we integrate over all directions ˆi in the aperture. For each direction we consider the value of the aperture function in this
direction, times the amount of energy scattered from ˆi to ˆω1. (c) Visualizing the pipeline of the single-scattering covariance in Eq. (18). Illumination aperture ˜ai
is convolved with scattering function s to generate ϒ( ˆω, o1, i), which is then multiplied with the viewing aperture ˜av. For visualization the real component of
the involved spherical functions is projected onto the 2D ˆωx , ˆωy plane (north hemisphere only). where now the contribution of each sub-path is determined by the
near-field path contribution function ci1,i2
v1,v2. For B ≥2, this equals: 4
NEAR-FIELD SPECKLE STATISTICS Note the ME shift demonstrated in the insets.𝒎𝐢 𝒎𝒎( ̂𝐢𝐢)
𝐨𝐨𝟏𝟏
𝐢𝐢
𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐢𝐢)
𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐨𝐨𝟏𝟏)
(a) ˜ai(ˆi, o1) Complex volumetric throuput :
υ(ˆi→o) = e−1
2 σtd( ˆω→o)eik( ˆω ·o),
Aperture function focused at ˆi :
ai(ˆi)=m(ˆi)e−ik ˆi·i
Aperture function × throughput :
˜ai(ˆi, o1) = ai(ˆi)υ(ˆi→o)
Scattering amplitude function:
s( ˆω1 · ˆω2)
Convolved aperture function:
ϒ( ˆω, o1, i) =
∫
ˆi∈S2 ˜ai(ˆi, o1)s(ˆi · ˆω)
(throughput up to first scatterer)
𝒎𝒎( ̂𝐢𝐢)
𝐨𝐨𝟏𝟏
𝐢𝐢
𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐢𝐢)
𝒆𝒆𝑖𝑖𝑖𝑖( ̂𝐢𝐢ȉ𝐨𝐨𝟏𝟏)
𝐨𝐨𝟏𝟏
𝐢𝐢𝟏𝟏
𝒔𝒔( ̂𝐢𝐢ȉ ෝ𝝎𝝎)
𝐢𝐢𝟐𝟐
𝐢𝐢𝟑𝟑
ෞ
𝛚𝛚𝟏𝟏
(a) ˜ai(ˆi, o1)
(b) ϒ( ˆω1, o1, i)
˜ai( ˆω|o1)
s
ϒ( ˆω|o1, i) = ˜ai( ˆω|o1) ∗s
˜av( ˆω|o1)
˜av( ˆω|o1) · ϒ( ˆω|o1, i)
(c)
Fig. 4. Notation summary and visualization. (a) The aperture function ˜ai(ˆi, o1) includes an apodization mask m(ˆi), a complex wave focusing at i and a
conjugate wave focusing at o1, the first point on the sampled path (attenuation exp(−1/2σtd( ˆω →o)) is not visualized). (b) The aperture function convolved
angulary with the scattering amplitude function leads to ϒ( ˆω1, o1, i), the throughput up to the first point and direction. That is, to compute the field
propagating from o1 at direction ˆω1 we integrate over all directions ˆi in the aperture. For each direction we consider the value of the aperture function in this
direction, times the amount of energy scattered from ˆi to ˆω1. (c) Visualizing the pipeline of the single-scattering covariance in Eq. (18). Illumination aperture ˜ai
is convolved with scattering function s to generate ϒ( ˆω, o1, i), which is then multiplied with the viewing aperture ˜av. For visualization the real component of
the involved spherical functions is projected onto the 2D ˆωx , ˆωy plane (north hemisphere only). 𝐨𝐨𝟏𝟏
𝐢𝐢𝟏𝟏
𝒔𝒔( ̂𝐢𝐢ȉ ෝ𝝎𝝎)
𝐢𝐢𝟐𝟐
𝐢𝐢𝟑𝟑
ෞ
𝛚𝛚𝟏𝟏
(b) ϒ( ˆω1, o1, i) 4
NEAR-FIELD SPECKLE STATISTICS Consider an imaging setup as in (a) where one wants to image a 100 µm × 100 µm area through a 400 µm thick tissue with a
0.5N A objective. The resulting imaging cone is 400 µm wide. As scattering can arise from anywhere inside the imaging cone, for realistic simulation the
medium should be at least as wide as the defocus cone, requiring us to simulate a slab of wider than 500 µm. Assuming e.g. λ = 0.5 µm, this results in
N = 1000 samples in each axis. (b) Two speckle images generated by two nearby illuminators, rendered using far-field covariances, sampled at 10% of the
Nyquist limit revealing aliasing. The simulation still runs 100× slower than our near-field approach, and occupies as much as 30 GB GPU global memory. (c)
Aliasing-free speckle images by our suggested near field approach. Note the ME shift demonstrated in the insets.𝒎𝐢 N.F. image with i1
N.F. image with i2
(c) N.F. image with i2
c) 𝜽
𝟒𝟎𝟎𝝁𝒎
𝟓𝟎𝟎𝝁𝒎
𝟒𝟎𝟎𝝁𝒎
image area
𝟏𝟎𝟎𝝁𝒎
NA:
𝐬𝐢𝐧𝜽= 𝟎. 𝟓
viewing objective
(a)i F.F. image with i1
(b F.F. image with i2
b) N.F. image with i
(c (b) (c) (a)i Fig. 3. Near-field challenges. Consider an imaging setup as in (a) where one wants to image a 100 µm × 100 µm area through a 400 µm thick tissue with a
0.5N A objective. The resulting imaging cone is 400 µm wide. As scattering can arise from anywhere inside the imaging cone, for realistic simulation the
medium should be at least as wide as the defocus cone, requiring us to simulate a slab of wider than 500 µm. Assuming e.g. λ = 0.5 µm, this results in
N = 1000 samples in each axis. (b) Two speckle images generated by two nearby illuminators, rendered using far-field covariances, sampled at 10% of the
Nyquist limit revealing aliasing. The simulation still runs 100× slower than our near-field approach, and occupies as much as 30 GB GPU global memory. (c)
Aliasing-free speckle images by our suggested near field approach. 4.1
Near-field covariance path integral where now the contribution of each sub-path is determined by the
near-field path contribution function ci1,i2
v1,v2. For B ≥2, this equals: To overcome the computational challenges of evaluating near-field
speckle covariance, we first derive for it a path-integral expression,
which will absorb the directional integrations of Eq. (11) into the
path contribution function. Then, in Sec. 5, we introduce an approx-
imation that allows us to compute this path contribution function
analytically. This completely removes the need for directional inte-
gration, drastically reducing computational complexity.i ci1,i2
v1,v2(®xs) = f (®xs) · ϒ( ˆω1, o1, i1)ϒ( ˆωB−1, oB, v1)
· ϒ( ˆω1, o1, i2)∗ϒ( ˆωB−1, oB, v2)∗,
( (14) where ϒ denotes integration over the aperture of terms in Eq. (4): where ϒ denotes integration over the aperture of terms in Eq. (4): denotes integration over the aperture of terms in Eq. ( ϒ( ˆω1, o1, i)
≡
∫
ˆi∈S2 ˜ai(ˆi, o1)s(ˆi · ˆω1) dˆi,
(15)
ϒ( ˆωB−1, oB, v)
≡
∫
ˆv∈S2 ˜av(ˆv, oB)s( ˆωB−1 · ˆv) dˆv,
(16) (15) By combining Eq. (3) and Eq. (11), we can express the near-field
covariance as a path integral on the same space of sub-paths P: and ˜a denotes weighed aperture functions a: and ˜a denotes weighed aperture functions a: Ci1,i2
v1,v2 =
∫
P
ci1,i2
v1,v2(®xs) d®xs,
(13) (13) ˜ai(ˆi, o1) ≡ai(ˆi)υ(i→o1),
˜av(ˆv, oB) ≡av(ˆv)υ(oB→ˆv). (17) (17) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 187:8
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin Similarly, for B = 1, the contribution function equals: in a narrow function that is sharply peaked around µr, whereas
γr = 0 results in a uniform function over the sphere. Von Mises-
Fisher functions have found use in various applications in computer
graphics [Han et al. 2007], though we emphasize that, compared to
this prior work, we use complex functions (γi , 0). ci1,i2
v1,v2(®xs) = σs(o1) ·
∫
ˆv1∈S2 ˜av1(ˆv1, o1)ϒ(ˆv1, o1, i1) dˆv1
·
∫
ˆv2∈S2 ˜av2(ˆv2, o1)ϒ(ˆv2, o1, i2) dˆv2
∗
. (18) (18) Von Mises-Fisher functions can be thought of as a generalization
of the Gaussian distribution to the unit sphere, with variance σ2 =
1
γ [Mardia and Jupp 2000]. 4.1
Near-field covariance path integral Intuitively, we can see that this is the
case by considering that, when we constrain ∥ˆµ∥= ∥ˆω∥= 1, We use ˜ai(ˆi|o1), ϒ( ˆω1|o1, i) to denote versions of these functions
with respect to only their first argument, conditioned on fixed val-
ues for their other arguments. These are complex functions on the
unit sphere S2. In particular, ϒ( ˆω1|o1, i) can be thought of as a convo-
lution of the aperture function ˜ai(ˆi|o1) with the scattering amplitude
function s(ˆi · ˆω). We summarize and visualize these terms in Fig. 4. e−γ
2 ∥ˆω−ˆµ ∥2 ∝e−γ ( ˆµ· ˆω),
(22) e−γ
2 ∥ˆω−ˆµ ∥2 ∝e−γ ( ˆµ· ˆω), (22) where ∝denotes equality up to a multiplicative scale. where ∝denotes equality up to a multiplicative scale. With Eqs. (14) and (18) at hand, conceptually we can compute
the near-field covariance Ci1,i2
v1,v2 using a Monte Carlo rendering al-
gorithm exactly analogous to the one proposed by Bar et al. [2019]
for the far-field case: First, we sample sub-paths ®xs using standard
volumetric path tracing. Second, we compute the path contribution
function for each sampled path, and accumulate the results. We
note however that, unlike the far-field case, in the near-field case
the path contribution function cannot be computed analytically,
as it requires spherical integration for evaluating ϒ. Theoretically,
this could be done using a second-stage Monte Carlo integration
procedure, by importance sampling one or more directions in the
aperture plane. However, as we show experimentally in Sec. 8, be-
cause these integrals have complex integrands with highly-varying
phases, Monte Carlo estimates have very high variance. Considering
that this second-stage Monte Carlo integration procedure needs to
be performed separately for each sampled sub-path ®xs, it quickly
results in an overwhelming computational overhead, making this
overall rendering procedure intractable. In the next section, we side-
step this overhead by deriving closed-form approximations to these
integration and convolution operations, which can be computed
analytically without the need for Monte Carlo integration. 5.1
Working with von Mises-Fisher functions Fitting with von Mises-Fisher functions. We aim to approximate the
spherical functions that appear in the near-field path contribution
function ci1,i2
v1,v2(®xs) using von Mises-Fisher functions.ii We first consider the aperture function ˜ai(ˆi), defined in Eqs. (10)
and (17). We reproduce the definition here for convenience:
˜ai(ˆi)=m(ˆi)e−ik(ˆi·i)υ(i→o1)=m(ˆi)e−ik(ˆi·i)−1
2 σtd( ˆω→o)+ik( ˆω ·o). (23) (23) We want to express ˜ai(ˆi) as a von Mises-Fisher function. To achieve
this, we first choose to approximate the aperture mask m(·) as a real
von Mises-Fisher function. Denoting the optical axis of the system
by ˆµa (usually this is the z-axis ˆµa = ˆz = [0, 0, 1]), we have, m( ˆω) ≈e−γaeγa( ˆµa · ˆω). (24) (24) This approximation is a form of apodization: a binary aperture,
which completely blocks or transmits fields propagating in different
directions, is replaced by a non-binary mask, which attenuates the
amplitude of transmitted fields by an increasing amount at larger
propagation angles. Such non-binary apertures are typical of sys-
tems that use short-focal-length lenses with strong aberrations. On
the illumination side, these non-binary apertures are also represen-
tative of the Gaussian profiles of laser beams. As we show in Sec. 8,
even when the underlying aperture is binary, using the apodiza-
tion of Eq. (24) produces accurate speckle statistics for transmissive
imaging configurations. We note that the mean width of the non-
binary aperture equals σa = 1/√γa, and should be set to match the
width of the true binary aperture. Additionally, we can use γa = 0
to model isotropic point sources (e.g., fluorescent particles). ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 5
NEAR-FIELD USING VON MISES-FISHER FUNCTIONS g=0.97
Liquid Clay
Coffee
Whole Milk
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2 mixtures
3 mixtures
4 mixtures
6 mixtures
G.T. Fig. 5. Phase function fitting. Visualizing the von Mises-Fisher mixture fit of some Henyey Greenstein phase functions as well as real world phase functions
measured by [Gkioulekas et al. 2013]. A small number of mixture components provides a good fit even when the phase function includes a back lobe (e.g. liquid
clay). The top row shows a full [−π , π] range, and the lower row zooms around the central forward/backward lobe of the function for better visualization. H.G. g=0.2
H.G. g=0.9
H.G. g=0.97
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4 mixtures
6 mixtures
G.T. ome Henyey Greenstein phase functions as well as real world phase functions H.G. g=0.97 4 mixtures 6 mixtures
G.T. Fig. 5. Phase function fitting. Visualizing the von Mises-Fisher mixture fit of some Henyey Greenstein phase functions as well as real world phase functions
measured by [Gkioulekas et al. 2013]. A small number of mixture components provides a good fit even when the phase function includes a back lobe (e.g. liquid
clay). The top row shows a full [−π , π] range, and the lower row zooms around the central forward/backward lobe of the function for better visualization. of Banerjee et al. [2005] to approximate s as a mixture of von Mises-
Fisher functions centered at ˆi: of Banerjee et al. [2005] to approximate s as a mixture of von Mises-
Fisher functions centered at ˆi: where βo =
q
µ + γs ˆωo,
co = βo −γs
βo
(µ · ˆωo). (32) (32) s(ˆi, ˆv) ≈
Õ
m
πmeγs,m(ˆi·ˆv). (28) (28) We discuss the selection of ˆωo in App. A.3, but the simplest strategy
is to select it as the direction at the center of the viewing aperture. We compare approximated and exact convolutions in App. A.4. 5
NEAR-FIELD USING VON MISES-FISHER FUNCTIONS In this section, we present two main technical results. First, we show
how to use mixtures of von Mises-Fisher functions to approximate
the various spherical functions that appear in the near-field path con-
tribution function ci1,i2
v1,v2(®xs) of Eqs. (14) and (18). Second, we show
how this approximation allows us to derive closed-form expressions
for the convolution and other integral terms in ci1,i2
v1,v2(®xs). Overall,
this allows us to compute covariance contribution analytically for
each sampled sub-path ®xs, avoiding Monte Carlo integration. The phase terms in Eq. (23) already form a complex von Mises-
Fisher function. Thus, to complete our treatment of ˜ai(ˆi), we need to
add the attenuation term, for which we assume that the attenuation
is approximately constant over the aperture: Von Mises-Fisher functions. We begin with background on complex
von Mises-Fisher functions, defined as [Mardia and Jupp 2000]: e−0.5σtd( ˆω→o) ≈e−0.5σtd( ˆµa→o). (25) (25) h( ˆω) = η · e(µ· ˆω),
(19) (19) Putting things together, we approximate the aperture function as: Putting things together, we approximate the aperture function as: s together, we approximate the aperture function as: ˜ai( ˆω, o) ≈η · eµ· ˆω,
(26) ˜ai( ˆω, o) ≈η · eµ· ˆω,
(26) (26) where ˆω ∈S2, and µ = µr + iµi is a complex three-dimensional
vector parameterizing the von Mises-Fisher function. We define: where ˆω ∈S2, and µ = µr + iµi is a complex three-dimensional
vector parameterizing the von Mises-Fisher function. We define: with γr = ∥µr∥,
γi = ∥µi ∥,
γ = ∥µ∥= γr −γi + 2i(µr · µi),
(20)
ˆµ = µ/γ,
ˆµr = µr/γr,
ˆµi = µi/γi. (21) η = e−γa−1
2 σtd( ˆµa→o1),
µ = γa ˆµa + ik(o1 −i). (27) (27) (21) We approximate the viewing aperture function similarly. We approximate the viewing aperture function similarly. The value |h( ˆω)| is maximized when ˆω = ˆµr. The scaleγr is inversely
proportional to the support of the function: Large γr values result The value |h( ˆω)| is maximized when ˆω = ˆµr. The scaleγr is inversely
proportional to the support of the function: Large γr values result We now turn our attention to the scattering amplitude function s
in Eqs. (14) and (18). We use the expectation-maximization algorithm Rendering Near-Field Speckle Statistics in Scattering Media
•
187:9 H.G. g=0.2
H.G. g=0.9
H.G. 5
NEAR-FIELD USING VON MISES-FISHER FUNCTIONS Note
that, since the scattering amplitude function is approximated by
a mixture of von Mises-Fisher functions, we need to compute the
convolution with each mixture element separately. In Fig. 5, we show fits of this kind for Henyey-Greenstein phase func-
tions, as well as real-world phase functions measured by Gkioulekas
et al. [2013]; in all cases, the phase function is accurately approxi-
mated using a small number of mixture components. Integration and convolution of von Mises-Fisher functions. The
approximations we derived facilitate computing the covariance in-
tegrals Eqs. (14) and (18). In particular, these evaluation can now
be done analytically, without the need for Monte Carlo integration,
using the properties of von Mises-Fisher functions.i 5.2
Visualizing convolutioni The function ϒ( ˆω, o1, i), defined in Eq. (15) as the convolution of the
aperture function with the scattering function, equals the through-
put of a path up until the first scattering event. This function encodes
the contribution of a path starting at node o1 and emerging from it
at direction ˆω. This function is a fundamental building block of the
near-field correlation, and as such we study and visualize its struc-
ture. In the next section, we use this to devise importance sampling
schemes for accelerating our Monte Carlo rendering algorithms. We consider first the spherical integration in Eq. (18). By approx-
imating the integrand as a von Mises-Fisher function, as described
above, we can compute this integral analytically using: ∫
ˆω ∈S2 ηeµ· ˆω = η · 4π sinh(√µ)
õ
= η · 2π e
√µ −e−√µ
õ
. (29) (29) Spatial structure. The aperture function ˜ai(ˆi; o1) is complex, and
its phase depends on the distance between the focus point i and
location o1 of the first scatterer in the path (Fig. 4(a)). When the
phase variation is rapid, blurring this complex function will reduce
the magnitude to zero. We want to understand for which o1 positions
the throughput contribution ϒ( ˆω, o1, i) is not zero. This will be
valuable for defining an importance sampling strategy that avoids
sampling o1 in areas receiving no energy. To this end we define In this equation, √µ equals: √µ ≡
q
µ2x + µ2y + µ2z. (30) (30) where each term in the summation involves complex square power
rather than squared amplitude, and thus õ is a complex number. We now consider the spherical convolution in Eq. (14). We use
the same approach as for integration, and replace the two functions
that are being convolved with their approximation in terms of von
Mises-Fisher functions we derived above. The resulting convolution
of two von Mises-Fisher functions is also available through a simple
analytical form. We note that, even though the exact convolution
result is not itself a von Mises-Fisher function, it can be accurately
approximated as one, as is necessary to facilitate subsequent com-
putation steps in our rendering algorithm. In particular, in App. A.3,
we show that the convolution can be approximated as: e(o|i) ≡
∫
ˆω ∈S2 |ϒ( ˆω, o, i)| . (33) (33) Fig. 6 visualizes the shape of e(o|i) for an x −z volume slice. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 5.2
Visualizing convolutioni This
is similar to the optics concept of a Gaussian beam [Yariv 1997],
focused at i. Considering that our illumination is a beam focused at
i, it is expected that scattering points o1 that are not located in the
area of the illumination beam will not receive light. However, while
the shape of a focused beam depends only on the aperture width
γa, e(o|i) also depends on the width of the scattering function γs. In
particular, assuming for ease of notation that the aperture axis is Fig. 6 visualizes the shape of e(o|i) for an x −z volume slice. This
is similar to the optics concept of a Gaussian beam [Yariv 1997],
focused at i. Considering that our illumination is a beam focused at
i, it is expected that scattering points o1 that are not located in the
area of the illumination beam will not receive light. However, while
the shape of a focused beam depends only on the aperture width
γa, e(o|i) also depends on the width of the scattering function γs. In
particular, assuming for ease of notation that the aperture axis is ∫
ˆψ ∈S2 e(µ· ˆψ) · eγs( ˆω · ˆψ) ≈2π
βo
e
γs
βo (µ· ˆω)+co, (31) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 187:10
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin North
South
North
South
North
South
North
South
North
South
γa = 16; γs = 2
˜a
-0.5
0
0.5
-0.5
0
0.5
-0.5
0
0.5
-0.5
0
0.5
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0
0.5
s
-0.2
0
0.2
-0.2
0
0.2
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0.2
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0
0.2
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0
0.2
˜a ∗s
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0
0.1
-0.01
0
0.01
-0.005 0 0.005
-0.01
0
0.01
-0.001
0
0.001
γa = 16; γs = 2
˜a
-0.5
0
0.5
-0.5
0
0.5
-0.5
0
0.5
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0
0.5
-0.5
0
0.5
s
-10
0
10
-10
0
10
-10
0
10
-10
0
10
-10
0
10
˜a ∗s
-0.5
0
0.5
-0.5
0
0.5
-0.5
0
0.5
-0.2
0
0.2
-0.1
0
0.1
Fig. 6. Visualizing spatial and angular throughput components. Left: e(o|i), the energy of path starting points, as an x −z slice through the volume. This resembles the shape of a Gaussian beam, which is narrow at the focus plane and wide at out-of-focus depths. 5.2
Visualizing convolutioni Right: the angular part of
the throughput, ϒ( ˆω |o, i) = ˜a ∗s as a function of direction ˆω, for a subset of o positions marked with corresponding colors on the Gaussian beams. These are
functions on the 3D sphere, and we show the north (forward) and south (backward) hemispheres projected on the ˆωx −ˆωy plane. The directions with high
throughput shift for points o at the periphery of the beam (e.g., orange and pink points). We display only the real part of these complex spherical functions aligned with the north pole ˆµa = ˆz, we show in App. A.5 that: aligned with the north pole ˆµa = ˆz, we show in App. A.5 that: Mises-Fisher functions, showing good agreement. The directions
with highest power after convolution can shift, and may not be
located at the center of the sphere (e.g., the orange and pink points
in lower panel). This happens at the periphery of the beam. Below
we use the directional density ϒ( ˆω|i, o) for importance sampling. e(o|i) = G(oxy |oz, i) = β(z)e
−∥oxy −ix,y ∥2
2w(z)2
, (34) with with w(z;γs,γa) =
s
γa + γs
k2
+ z2
γa
,
(35)
β(z) = e−1
2 σtd( ˆz→oz)π k2γ 2a(γs + γa)
2w(z;γs,γa)2 eγa+γs . (36) (35) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 5.2
Visualizing convolutioni The panels visualize two scattering functions
defined by γs = 2 (wide) and γs = 100 (narrow), showing that the beam waist is wider for narrow forward-scattering phase functions. Right: the angular part of
the throughput, ϒ( ˆω |o, i) = ˜a ∗s as a function of direction ˆω, for a subset of o positions marked with corresponding colors on the Gaussian beams. These are
functions on the 3D sphere, and we show the north (forward) and south (backward) hemispheres projected on the ˆωx −ˆωy plane. The directions with high
throughput shift for points o at the periphery of the beam (e.g., orange and pink points). We display only the real part of these complex spherical functions. ˜a ∗s γa = 16; γs = 2
˜a
-0.5
0
0.5
s
-10
0
10
˜a ∗s
-0.5
0
0.5 -0.5
0
0.5
-10
0
10
-0.1
0
0.1 -0.5
0
0.5
-10
0
10
-0.5
0
0.5 Fig. 6. Visualizing spatial and angular throughput components. Left: e(o|i), the energy of path starting points, as an x −z slice through the volume. This resembles the shape of a Gaussian beam, which is narrow at the focus plane and wide at out-of-focus depths. The panels visualize two scattering functions
defined by γs = 2 (wide) and γs = 100 (narrow), showing that the beam waist is wider for narrow forward-scattering phase functions. Right: the angular part of
the throughput, ϒ( ˆω |o, i) = ˜a ∗s as a function of direction ˆω, for a subset of o positions marked with corresponding colors on the Gaussian beams. These are
functions on the 3D sphere, and we show the north (forward) and south (backward) hemispheres projected on the ˆωx −ˆωy plane. The directions with high
throughput shift for points o at the periphery of the beam (e.g., orange and pink points). We display only the real part of these complex spherical functions. Fig. 6. Visualizing spatial and angular throughput components. Left: e(o|i), the energy of path starting points, as an x −z slice through the volume
This resembles the shape of a Gaussian beam, which is narrow at the focus plane and wide at out-of-focus depths. The panels visualize two scattering functions
defined by γs = 2 (wide) and γs = 100 (narrow), showing that the beam waist is wider for narrow forward-scattering phase functions. 7
FIELD RENDERING So far we focused on evaluating speckle covariance. A complemen-
tary question is how to directly render speckle images. Suppose, for
example, that we want to generate Ni images from Ni different illu-
mination points, where each image includes Nv sensor points. This
requires sampling Ni,v = Ni · Nv complex numbers. One approach
for this is to evaluate the corresponding covariance matrix of size
Ni,v × Ni,v, and then use it to sample values. However, for large
Ni,v, the covariance matrix can be impractically large. Instead, we
seek an algorithm with complexity O(Ni,v) rather than O(N 2
i,v). g
p
y
,
i,v
For this, we follow Bar et al. [2019], who note that the covariance
matrix as given by Eq. (3), (14) and (18) is essentially an infinite
summation of rank-1 matrices, decomposed over the path space Cik ,im
vk ,vm =
∫
P
f (®xs)b(ik, vk)b(im, vm)∗d®xs,
(43)
for all (k,m) pairs k,m ∈{1, . . . , Ni,v}, with
b(ik, vk) =
( ∫
ˆv∈S2 ˜avk (ˆv, o1)ϒ(ˆv, o1, ik),
B = 1,
ϒ( ˆω1, o1, ik)ϒ( ˆωB, oB, vk),
B ≥2. (44) Cik ,im
vk ,vm =
∫
P
f (®xs)b(ik, vk)b(im, vm)∗d®xs,
(43) Cik ,im
vk ,vm =
∫
P
f (®xs)b(ik, vk)b(im, vm)∗d®xs,
(43)
for all (k,m) pairs k,m ∈{1, . . . , Ni,v}, with
b(ik, vk) =
( ∫
ˆv∈S2 ˜avk (ˆv, o1)ϒ(ˆv, o1, ik),
B = 1,
ϒ( ˆω1, o1, ik)ϒ( ˆωB, oB, vk),
B ≥2. (44) (43) b(ik, vk) =
( ∫
ˆv∈S2 ˜avk (ˆv, o1)ϒ(ˆv, o1, ik),
B = 1,
ϒ( ˆω1, o1, ik)ϒ( ˆωB, oB, vk),
B ≥2. (44) (44) Despite the fact that this rank-one decomposition is over-
complete, we can use it to sample from the covariance matrix. We
sample sub-paths ®xs ∼p(®xs), using the same importance function
as in Sec. 6. The field is updated using b(ik, vk), resulting in Bar et al. [2019] sample sub-paths from a distribution p(®xs) ∝f (®xs),
where the first node o1 and direction ˆω1 are sampled uniformly. This uniform sampling strategy can be problematic, because the
term c (Eq. (37)) includes the throughput ϒ of the start and end
segments. In particular, as discussed in Sec. 5.2, the spatial part
of ϒ has a shape similar to a Gaussian beam (Fig. 6). The first scattering point is sampled from We can importance sample the last segment when
iluminators are more than camera pixels, or importance sample both
the first and last segment in bidirectional algorithms. as in standard volumetric path tracing. The sampling scheme is
summarized in Alg. 1, and we provides details in App. A.6. We note
that we choose to importance sample the first segment of the path
rather than the last one because, for most imaging configurations in
this paper, we had a small number of illuminators and a large number
of camera pixels. We can importance sample the last segment when
iluminators are more than camera pixels, or importance sample both
the first and last segment in bidirectional algorithms. The first scattering point is sampled from ALGORITHM 1: Monte Carlo rendering of covariance Ci1,i2
v1,v2. ◃Initialize covariance estimate. Set C = 0. for iteration = 1 : N do
◃Sample a subpath:
◃Sample first vertex from the sum of Gaussian beams. Sample beam index j ∈{1, 2}
Sample point o1 ∼e(o1 |ij) . ◃Update covariance with single scattering path. Update C +=
1
p(o1)
∫
ˆv1∈S2 ˜av1(ˆv1, o1)ϒ(ˆv1, o1, i1)
·
∫
ˆv2∈S2 ˜av2(ˆv2, o1)ϒ(ˆv2, o1, i2)
∗
. ◃Sample first direction from the angular throughput. Sample direction ˆω1 ∼|ϒ( ˆω1 |o1, ij)|2. ◃Continue tracing the subpath:
◃Sample second vertex of subpath. Sample distance d ∼σt e−σt d. Set point o2 = o1 + d · ˆω1. Set b = 2. while ob inside medium do
◃Update covariance with next-event estimation. Update C +=
1
p(o1)p( ˆω1|o1)ϒ( ˆω1, o1, i1)ϒ( ˆωb−1, ob, v1)
ϒ( ˆω1, o1, i2)∗ϒ( ˆωb−1, ob, v2)∗
◃Sample next vertex of subpath:
◃Sample direction from phase function. Sample direction ˆωb ∼|s( ˆωb−1 · ˆωb)|2. ◃Sample free path. Sample distance d ∼σt e−σt d. ◃Create next vertex of subpath. Set point ob+1 = ob + d · ˆωb. ◃Account for absorption. Sample scalar a ∼Unif[0, 1]. if a > σs/σt then
◃Terminate subpath at absorption event. break
end
Set b = b + 1. end
end
◃Produce final covariance estimate. Update C =
1
N C. return C. ALGORITHM 1: Monte Carlo rendering of covariance Ci1,i2
v1,v2. p(o1) ∝1
2
|e(o1|i1)|2 + |e(o1|i2)|2
. (40)i (40) We implement this sampling by first uniformly sampling one of
he two beams in the summand, and then sampling o1 from the
Gaussian beam G(oxy |oz, ij) of Eq. (34), where j ∈1, 2 denotes the
index of the sampled beam. The first direction is sampled from p( ˆω1|o1) ∝|ϒ( ˆω1|o1, ij)|2. (41) p( ˆω1|o1) ∝|ϒ( ˆω1|o1, ij)|2. (41)
The rest of the path is sampled from (41) The rest of the path is sampled from p(o2 →, . . . , →oB |o1, ˆω1) ∝f (o1 →, . . . , →oB),
(42) (42) as in standard volumetric path tracing. The sampling scheme is
summarized in Alg. 1, and we provides details in App. A.6. We note
that we choose to importance sample the first segment of the path
rather than the last one because, for most imaging configurations in
this paper, we had a small number of illuminators and a large number
of camera pixels. 6
IMPORTANCE SAMPLING In Sec. 4.1 the covariance is expressed as an integral over path space,
where each path contributes a termc(®xs). For convenience we repeat
here the definition for paths of length B ≥2: (36) and z ≡oz −iz. For every z plane, e(o|i) is a planar Gaussian with
standard deviation equal to w(oz −iz). The Gaussian is narrowest
when oz −iz = 0, that is, when o1 is at the same depth as i. The
beam expands at depths away from the focus depth. c(®xs) = f (®xs) · ϒ( ˆω1, o1, i1)ϒ( ˆωB−1, oB, v1)
· ϒ( ˆω1, o1, i2)∗ϒ( ˆωB−1, oB, v2)∗. (37) (37) We use Monte Carlo approximation of this integral, by sampling N
sub-paths ®xs,n from a distribution p(®xs,n) and computing We use Monte Carlo approximation of this integral, by sampling N
sub-paths ®xs,n from a distribution p(®xs,n) and computing Angular structure. Fig. 6 also provides a visualization of the an-
gular part of the throughput, namely the variation of ϒ( ˆω|o, i) as
a function of ˆω, in a few positions of the first point o. For that, we
display the aperture function ˜ai(ˆi|o) as a spherical function (i.e., a
function of direction), before and after convolution with s. We com-
pare the exact convolution against the approximation based on von C = 1
N
Õ
n
c(®xs,n)
p(®xs,n). (38) (38) The quality of this estimator depends on the sampling distribution
p, and estimation variance reduces when p closely approximates c. The quality of this estimator depends on the sampling distribution
p, and estimation variance reduces when p closely approximates c. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. Rendering Near-Field Speckle Statistics in Scattering Media
•
187:11 The first scattering point is sampled from 7
FIELD RENDERING As the beam
assigns zero weight to most points in space, uniform sampling of the
starting node o1 will produce many paths with zero contribution. When the phase function is narrow, a similar argument holds for
the first direction ˆω1. We address this issue by deriving importance
sampling strategies for the start node and direction. u(ik, vk) =
1
√
N
Õ
n
b(ik, vk |®xs,n)
s
f (®xs,n)
p(®xs,n) e2πiζn,
(45) (45) where ζn ∈[0, 1] is a random phase ensuring
E[b(ik, vk |®xs,n)e2πiζn ] = 0. (46) where ζn ∈[0, 1] is a random phase ensuring ζn
[ , ]
p
g
E[b(ik, vk |®xs,n)e2πiζn ] = 0. (46) E[b(ik, vk |®xs,n)e2πiζn ] = 0.f E[b(ik, vk |®xs,n)e2πiζn ] = 0. (46)f (46) As different paths are sampled independently, and given the zero-
mean property, contributions from different sub-paths are uncorre-
lated. Namely, for n1 , n2: To this end, we sample the path from a distribution E
h
b(ik, vk |®xs,n1)e2πiζn1 · b(im, vm|®xs,n2)∗e−2πiζn2
i
= 0,
(47) (47) (39) p(o1 →. . .→oB) = p(o2 →. . .→oB | ˆω1, o1)p( ˆω1|o1)p(o1). (39) ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 187:12
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin д = 0.9 , σa = 0.6
д = 0.9 , σa = 0.25
д = 0.2 , σa = 0.25
𝒊𝟐
𝒊𝟏
𝒗𝟐
ation objective
wing objective
N.F. F.F. Binary
N.F. F.F. Binary
N.F. F.F. Binary
∆= 0λ
∆= λ
∆= 2λ
0
0.01
0.02
0.03
0.04
0.05
0
1
2
3
10-3
0
0.5
1
10-4
(a)
(b)
(c)
(d)i д = 0.9 , σa = 0.6
д = 0.9 , σa = 0.25
д = 0.2 , σa = 0.25
𝒊𝟐
𝒊𝟏
𝒗𝟏
𝒗𝟐
illumination objective
viewing objective
N.F. F.F. Binary
N.F. F.F. Binary
N.F. F.F. Binary
∆= 0λ
∆= λ
∆= 2λ
0
0.01
0.02
0.03
0.04
0.05
0
1
2
3
10-3
0
0.5
1
10-4
(a)
(b)
(c)
(d)
Fig. 7. Comparisons with far field rendering. (a) Imaging setup. We use illumination and viewing pairs defined by ∆= i2x,y −i1x,y = v2x,y −v1x,y and
τ = v1x,y −i1x,y = v2x,y −i2x,y. (This setup is also used in Fig. 8-11.) (b-d) We compare covariances rendered using our near-field (N.F.) algorithm against those
obtained by rendering far-field (F.F.) correlations and applying focusing. 7
FIELD RENDERING 1 shows speckle images rendered with this algorithm. We
note that in Eq. (45), the same set of paths is used to update all
illuminators and sensors. As a result, the speckle images due to
different illuminators in Fig. 1 are correlated, shifted versions of each
other, corresponding to the memory effect property. Rendering each
of these images independently, using a standard Monte Carlo volume
rendering approach, would fail to reproduce these correlations. We denote by i1x,y, i2x,y, v1x,y, v2x,y the x −y coordinates of the
corresponding 3D points on the focus plane. We denote by ∆≡
i2x,y −i1x,y the 2D displacement between the illumination direc-
tions, and by τ ≡v1x,y −i1x,y the 2D displacement between the
illumination and viewing directions (Fig. 7(a)). Classical memory
effect theory [Feng et al. 1988] states that strong correlations ex-
ist between fields for illumination and viewing pairs satisfying
i2x,y −i1x,y = v2x,y −v1x,y = ∆, for small ∆values. With this in
mind, in our experiments, we evaluate and visualize correlations of
the form: 7
FIELD RENDERING We also compare with covariances rendered with a binary aperture of equivalent
width to our apodized aperture. As binary apertures cannot be well-approximated using von Mises-Fisher functions, we only render them using the far-field
algorithm. The volume has size 50λ × 50λ × 20λ. We evaluate two aperture widths in (b,c) and two phase functions in (c,d). Each square visualizes correlation
as a function of τ , and different rows correspond to different x-axis illuminator shifts ∆= i2x −i1x . We see close agreement in all cases. h
k
k
i
1
2 д = 0.9 , σa = 0.25
д = 0.2 , σa = 0.25
N.F. F.F. Binary
N.F. F.F. Binary
0
1
2
3
10-3
0
0.5
1
10-4
(c)
(d) д = 0.9 , σa = 0.6
N.F. F.F. Binary
∆= 0λ
∆= λ
∆= 2λ
0
0.01
0.02
0.03
0.04
0.05
0
(b)i 𝒊𝟐
𝒊𝟏
𝒗𝟏
𝒗𝟐
illumination objective
viewing objective
∆= 0λ
∆= λ
∆= 2λ
(a) (a) (b) (c) Fig. 7. Comparisons with far field rendering. (a) Imaging setup. We use illumination and viewing pairs defined by ∆= i2x,y −i1x,y = v2x,y −v1x,y and
τ = v1x,y −i1x,y = v2x,y −i2x,y. (This setup is also used in Fig. 8-11.) (b-d) We compare covariances rendered using our near-field (N.F.) algorithm against those
obtained by rendering far-field (F.F.) correlations and applying focusing. We also compare with covariances rendered with a binary aperture of equivalent
width to our apodized aperture. As binary apertures cannot be well-approximated using von Mises-Fisher functions, we only render them using the far-field
algorithm. The volume has size 50λ × 50λ × 20λ. We evaluate two aperture widths in (b,c) and two phase functions in (c,d). Each square visualizes correlation
as a function of τ , and different rows correspond to different x-axis illuminator shifts ∆= i2x −i1x . We see close agreement in all cases. sensing focus points v1, v2 are all located at the same depth plane,
at the back plane of the volume. This configuration corresponds, for
example, to the fluorescent imaging setting, where a fluorescent par-
ticle is deep inside a medium such as tissue, and a camera attempts
to observe it by focusing through the scattering at the illuminator’s
plane. for all (k,m) pairs. Therefore, we see that E
h
u(ik, vk) · u(im, vm)∗i
equals the desired covariance of Eq. (43). Fig. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 8
EVALUATIONfi 7,
our near-field approach is 400× faster than the tabulated far-field
approach, and 3000× faster than the Monte Carlo approach. The
performance advantage will become even larger for volumes with
sizes corresponding to realistic lab experiments. All algorithms were
implemented and run on a V100 NVIDIA GPU. Table 1. Runtimes of different algorithms for achieving RMSE < 0.01. 10λ × 10λ × 4λ
25λ × 25λ × 10λ
50λ × 50λ × 20λ
N.F. 0.46 s
2.12 s
6 s
F.F. Tab. 31 s
221 s
2.4 × 103 s
F.F. M.C. 199 s
1.47 × 103 s
2.04 × 104 s Table 1. Runtimes of different algorithms for achieving RMSE < 0.01. 10λ × 10λ × 4λ
25λ × 25λ × 10λ
50λ × 50λ × 20λ
N.F. 0.46 s
2.12 s
6 s
F.F. Tab. 31 s
221 s
2.4 × 103 s
F.F. M.C. 199 s
1.47 × 103 s
2.04 × 104 s note that, in Sec. 4, we presented the formal approach for estimat-
ing near-field covariance using far-field rendering, based on the
far-field covariance Eq. (11) rather than fields. In these experiments,
we opt for the field-based approach, because the number of far-field
samples it requires scales more favorably with the width W of the
simulated volume (W 4 scaling for the field-based approach, W 8
scaling for the covariance-based approach).i Table 2 reports RMSE from equal-time comparisons of the three
approaches using volumes of different sizes. Error was measured
against a reference rendering produced by running the far-field
approach till convergence. For each volume size, we report RMSE
for both a small and a long rendering time. The latter allows us to
quantify the bias of the near-field approach. While any such bias is
too small to be noticeable in the visual comparisons of Fig. 7, the nu-
merical difference between the near-field and far-field covariances
does not fully converge to zero. We anticipate that most of the bias
is due to the assumption that volumetric attenuation is constant
for all directions through the aperture (see Eq. (25)). In the second
row of Table 2 we compare our near-field approach against a refer-
ence evaluated using the far-field approach but with an equivalent
constant directional attenuation, showing smaller error. 8
EVALUATIONfi We consider the case where the illumination focus points i1, i2 and Rendering Near-Field Speckle Statistics in Scattering Media
•
187:13 Table 2. Equal-time comparisons of different algorithms. 10λ × 10λ × 4λ 25λ × 25λ × 10λ 50λ × 50λ × 20λ
1 s
60 s
5 s
500 s
15 s
6000 s
N.F. 0.0089 0.0076 0.0096 0.0093 0.0135 0.0110
N.F. biased ref. 0.0053 0.0021 0.0050 0.0018 0.0080 0.0045
F.F. Tab. 0.0396 0.0078 0.0672 0.0056 0.0915 0.0051
F.F. M.C. 0.1140 0.0198 0.2024 0.0207 0.3317 0.0632 Table 2. Equal-time comparisons of different algorithms. N.F. F.F. Aliasing F.F. M.C. eq. time F.F. M.C. ×10
∆= 0λ
∆= λ
∆= 2λ
(a)
(b)
(c)
(d)
(e)
Fig. 8. Acceleration of far-field rendering. (a) N.F (1× runtime). (b) F.F. with dense discretization of aperture integral (400× runtime). (c) F.F. with
sparser discretization of aperture integral (100× runtime) (d, e) F.F. with
Monte Carlo sampling of aperture integral (1× and 10× runtime). N.F. F.F. Aliasing F.F. M.C. eq. time F.F. M.C. ×10
∆= 0λ
∆= λ
∆= 2λ
(a)
(b)
(c)
(d)
(e)i covariance is computed when evaluating the integral of Eq. (11). The number of random samples is selected to achieve the lowest
possible error for equal runtime. As the integrand is complex, using
Monte Carlo approximation results in very high variance. At the
cost of increased runtime, Fig. 8(e) demonstrates a better estimate. (a) (e) Fig. 8. Acceleration of far-field rendering. (a) N.F (1× runtime). (b) F.F. with dense discretization of aperture integral (400× runtime). (c) F.F. with
sparser discretization of aperture integral (100× runtime) (d, e) F.F. with
Monte Carlo sampling of aperture integral (1× and 10× runtime). Runtime and bias evaluation. We compare the runtime of our
near-field approach against two versions of the far-field approach: a
tabulated version, computing all far field directions before applying
near field transformation, as in Fig. 8(b); and a Monte Carlo version,
randomly sampling directions, as in Fig. 8(d-e). Table 1 shows the
runtime each approach requires to converge to a root-mean-square-
error (RMSE) relative to a reference rendering below 0.01. In all
cases, our near-field approach has the best performance, and the
performance improvement increases as the widthW of the simulated
volume increases. For the volume size we use for the results in Fig. 8
EVALUATIONfi We now evaluate the efficiency and accuracy of our proposed ren-
dering algorithms. We compare our algorithms against three alterna-
tives: First, we compare with an approach that uses the Monte Carlo
rendering algorithm of Bar et al. [2019] to produce far-field esti-
mates, and then converts them to near-field estimates using Eqs. (2)
and (9). We also consider a few variants of this approach that benefit
from various acceleration techniques. Second, we compare with
groundtruth estimates produced by a wave-equation solver. Third,
we compare with estimates produced using a layered propagation
approach popular in the optics literature. Additionally, we show
experiments evaluating performance improvements due to the im-
portance sampling scheme of Sec. 6. In Sec. 9, we show experiments
relating to the tilt-shift memory effect [Osnabrugge et al. 2017], in-
cluding experiments validating our algorithms against groundtruth
measurements of real materials. C(∆,τ) = C
i1
x,y,i1
x,y+∆
i1x,y+τ,i1x,y+τ+∆,
(48) (48) for different displacements ∆and τ. For sufficiently wide volumes,
C(∆,τ) is approximately invariant to i1. Comparison with far-field approach. We compare first with the
simulation approach based on the far-field rendering algorithms of
Bar et al. [2019]. To reduce the computational burden, we compare
with an approach based on a combination of Eqs. (2) and (9), rather
than Eq. (11): We first discretize the illumination and viewing aper-
tures into a set of directions ˆi and ˆv. For each set of (ˆi, ˆv) values,
we use the implementation provided by Bar et al. [2019] to sample
far-field scattered waves uˆi
ˆv, which we subsequently convert to the
near-field scattered waves using Eq. (9). By running the field ren-
dering algorithm of Bar et al. [2019] multiple times, we end up with
multiple samples of near-field scattered waves, corresponding to
different scatterer configurations. Finally, we use these near-field
scattered waves to approximate the covariance as in Eq. (2). We Experimental configurations. Our experiments focus on illumina-
tion and imaging configurations that are known from literature to
produce strongly-correlated fields. We describe these configurations
using Fig. 7(a) as reference: As we focus on transmission mode imag-
ing, the focused illumination is placed at the back of the sample, and
the focused sensor is placed at the front. We refer to App. A.4 for
an evaluation of our technique under reflective imaging conditions. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 8
EVALUATIONfi V = 500λ × 500λ × 100λ
OD = 4 , д = 0.97
∆= 0λ
∆= 3λ
∆= 6λ The left part of Fig. 11 shows correlations valuesC(∆,τ) (Eq. (48)),
as a function of τ for a few ∆and д values, simulated using µ-diff
and our technique. Note that, as these simulations are in 2D, τ is
a scalar, and thus C(∆,τ) is a 1D curve. We observe that the plots
computed with our technique closely match those produced by the
wave-equation solver, demonstrating the accuracy of our technique. At the same time, for this small example our technique is three
orders of magnitude faster, and can scale to much larger volumes. Fig. 9. The benefit of importance sampling. The figure compares uni-
form sampling vs. importance sampling (I.S.) of the first scatterer on the
path o1, vs. importance sampling of both the first scatterer and the first
direction o1, ˆω1. Top panel: a small target whose size is only 20λ ×20λ ×20λ. Fig. 9. The benefit of importance sampling. The figure compares uni-
form sampling vs. importance sampling (I.S.) of the first scatterer on the
path o1, vs. importance sampling of both the first scatterer and the first
direction o1, ˆω1. Top panel: a small target whose size is only 20λ ×20λ ×20λ. For such small targets uniform sampling converges as well, although re-
quiring a larger number of path samples (compare results with 103 samples
to 105 samples), demonstrating that our importance sampling strategy is
consistent. Middle panel: When expanding the size of the target volume,
convergence of uniform sampling is very slow. As the phase function is
rather wide, importance sampling of the first direction does not improve
much. Lower panel: If we also use a very forward scattering phase function,
convergence is much accelerated by importance sampling the first direction. Comparison with multi-slice layered propagation. We compare
additionally with the multi-slice beam propagation method [Schott
et al. 2015], which is a popular numerical approach in optics for
simulating wave propagation. This approach accounts for multiple-
scattering effects by approximating the simulated volume as a se-
quence of planar slices orthogonal to the optical axis; layers are
modeled as infinitesimally-thin 2D phase masks, separated by free
space. The phase mask of each layer is selected to scatter light with
an angular spread matching the phase function of the simulated
volume. 8
EVALUATIONfi In our experiments, to keep the number of far-field samples man-
ageable, we simulate a relatively small volume of size 50λ×50λ×20λ
with mean free path MFP = 10λ, leading to an optical depth OD = 2. We consider two choices of aperture width, σa = 0.6 and σa = 0.25,
and both wide and narrow forward-scattering Henyey-Greenstein
phase functions (low and high д values, respectively). When using
the far-field approach, we simulate both von Mises-Fisher-apodized
and binary aperture masks m(·) of the same width, to quantify the ef-
fect of our apodization approximation on accuracy (binary apertures
can only be evaluated at the far-field, at increased computational
complexity, as the von Mises-Fisher approximation does not ap-
ply). We show the results in Fig. 7. We observe that, in all cases,
our near-field rendering algorithm produces very similar results to
the far-field approaches. We also note that the far-field approach
produces very close results when using apodization and binary
masks, indicating that our apodization approximation does not in-
troduce significant bias. At the same time, our near-field approach
is orders-of-magnitude faster, as we quantify in detail below. The benefit of importance sampling. To evaluate the effect of the
importance sampling scheme we introduced in Sec. 6, we compare
in Fig. 9 covariance estimates produced by our Monte Carlo al-
gorithm, using three different sampling strategies: first, uniform
sampling of both the first path vertex and direction; second, impor-
tance sampling of the first vertex and uniform sampling of the first
direction; and third, importance sampling of both the first vertex
and direction. We observe first that, when run till convergence (e.g.,
in the top panel where the sample is small and the phase function
has significant side-scattering), all three combinations converge
to the same result. This confirms that importance sampling does
not introduce any addiional bias. We additionally observe that the
estimate using importance sampling of both the first vertex and In Fig. 8, we additionally compare against two accelerated variants
of the far-field approach. We reuse the configuration of Fig. 7(d), this
time with a wider τ grid. First, we render far-field covariance only
for a (fixed) regular subset of directions. As the number of samples is
significantly lower than that required by the Nyquist sampling rate
in Eq. (12), aliased replicas appear. 8
EVALUATIONfi Second, we use a Monte Carlo
strategy that randomly samples the directions at which far-field 187:14
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin V = 20λ × 20λ × 20λ
V = 500λ × 500λ × 100λ
OD = 2 , д = 0.5
OD = 4 , д = 0.97
log10(RMSE)
103
104
105
106
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
103
104
105
106
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
Sample number
Sample number
Fig. 10. Convergence plots. log(RMSE) of different sampling strategies as
a function of sample number, for the first and third volumes in Fig. 9. 103 Samples
105 Samples
Uniform
I.S. o1
I.S. o1, ˆω1
Uniform
I.S. o1
I.S. o1, ˆω1
V = 20λ × 20λ × 20λ
OD = 2 , д = 0.5
∆= 0λ
∆= 3λ
∆= 6λ
V = 500λ × 500λ × 100λ
OD = 4 , д = 0.5
∆= 0λ
∆= 3λ
∆= 6λ
V = 500λ × 500λ × 100λ
OD = 4 , д = 0.97
∆= 0λ
∆= 3λ
∆= 6λ
Fig. 9. The benefit of importance sampling. The figure compares uni-
form sampling vs. importance sampling (I.S.) of the first scatterer on the
path o1, vs. importance sampling of both the first scatterer and the first
direction o1, ˆω1. Top panel: a small target whose size is only 20λ ×20λ ×20λ. For such small targets uniform sampling converges as well, although re-
quiring a larger number of path samples (compare results with 103 samples
to 105 samples), demonstrating that our importance sampling strategy is
consistent. Middle panel: When expanding the size of the target volume,
convergence of uniform sampling is very slow. As the phase function is
rather wide, importance sampling of the first direction does not improve
much. Lower panel: If we also use a very forward scattering phase function,
convergence is much accelerated by importance sampling the first direction. V = 20λ × 20λ × 20λ
OD = 2 , д = 0.5
log10(RMSE)
103
104
105
106
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
Sample number V = 500λ × 500λ × 100λ
OD = 4 , д = 0.97
103
104
105
106
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
Sample number Sample number Sample number Sample number Sample number p
p
Fig. 10. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 8
EVALUATIONfi Convergence plots. log(RMSE) of different sampling strategies as
a function of sample number, for the first and third volumes in Fig. 9. p
p
Fig. 10. Convergence plots. log(RMSE) of different sampling strategies as
a function of sample number, for the first and third volumes in Fig. 9. Comparison with a wave-equation solver. Bar et al. [2019] demon-
strated the accuracy of their far-field covariance rendering algo-
rithm by comparing against numerical wave-equation solvers. For
additional validation, we also compare directly with the µ-diff
solver [Thierry et al. 2015] they use. The solver takes as input a
configuration of scatterer locations, and uses numerical techniques
to solve the wave equation and output an estimate of the complex
scattered field. To compute covariance, we run the solver for multi-
ple scatterer instantiations sampled from the same distribution, then
use the results to estimate the speckle covariance of Eq. (2). We note
that this simulator only works in 2D; thus, for these comparisons
only, we restrict our Monte Carlo rendering algorithm to 2D as well. The left part of Fig. 11 shows correlations valuesC(∆,τ) (Eq. (48)),
as a function of τ for a few ∆and д values, simulated using µ-diff
and our technique. Note that, as these simulations are in 2D, τ is
a scalar, and thus C(∆,τ) is a 1D curve. We observe that the plots
computed with our technique closely match those produced by the
wave-equation solver, demonstrating the accuracy of our technique. At the same time, for this small example our technique is three
orders of magnitude faster, and can scale to much larger volumes. Comparison with a wave-equation solver. Bar et al. [2019] demon-
strated the accuracy of their far-field covariance rendering algo-
rithm by comparing against numerical wave-equation solvers. For
additional validation, we also compare directly with the µ-diff
solver [Thierry et al. 2015] they use. The solver takes as input a
configuration of scatterer locations, and uses numerical techniques
to solve the wave equation and output an estimate of the complex
scattered field. To compute covariance, we run the solver for multi-
ple scatterer instantiations sampled from the same distribution, then
use the results to estimate the speckle covariance of Eq. (2). We note
that this simulator only works in 2D; thus, for these comparisons
only, we restrict our Monte Carlo rendering algorithm to 2D as well. 8
EVALUATIONfi (a) Setup: two beams with spatial
displacement ∆a and angular displacement θ enter the volume. We compute
the correlation between the speckle patterns measured at the b plane as a
function of both tilt and shift, assuming that the input displacement ∆a
is selected to maximize Eq. (50). The bottom images visualize the results. (b,c) Analytic and measured correlation, reproduced from Osnabrugge et
al. [2017]. (d-e) Correlation computed by our algorithm for two different
phase functions with the same average cosine д = 0.98. Fig. 12. The tilt-shift memory effect. (a) Setup: two beams with spatial
displacement ∆a and angular displacement θ enter the volume. We compute
the correlation between the speckle patterns measured at the b plane as a
function of both tilt and shift, assuming that the input displacement ∆a
is selected to maximize Eq. (50). The bottom images visualize the results. (b,c) Analytic and measured correlation, reproduced from Osnabrugge et
al. [2017]. (d-e) Correlation computed by our algorithm for two different
phase functions with the same average cosine д = 0.98. C(∆a, ∆b,θ) ≈δ(∆b −∆a −Lθ)e
−L3k2
2ℓtr
θ 2
12 +
θ
2 +
∆b
L
2
,
(50) (50) where δ(·) is the Dirac delta function, and ℓtr is the transport mean
free path ℓtr ≡MFP/(1 −д). Their derivation is based on three
simplifying assumptions: it uses a layered representation similar
to that of the multi-slice layered propagation technique in Sec. 8;
at each layer, it assumes forward-only propagation; and it uses a
differential equation to integrate over multiple scattering planes. propagation technique, as the latter requires for each layer a high-
resolution discretization of the wave, resulting in large dense arrays
that need to be convolved to model propagation between layers. Additionally, Osnabrugge et al. [2017] show measurements of the
function C(∆a, ∆b,θ) for a tissue phantom of thickness L = 258 µm,
made of silica microspheres immersed in agarose gel. By combining
Mie theory [Frisvad et al. 2007] with the dispersion and sizing
properties of the materials used for fabrication, the authors estimate
for the phantom an anistropy parameterд = 0.98 and mean free path
MFP = 296 µm. In Fig. 12(b,c), we replicate from their paper (using
data provided by the authors) the measured correlationC(∆a, ∆b,θ),
as well as the analytical prediction using the model of Eq. (50). 8
EVALUATIONfi Our simulator is physically accurate, producing results in close agreement with those of the solver, while being orders of magnitude faster.t Our approach
Multi-slice simulator
д = 0.98
д = 0.3
д = 0.98
д = 0.3
C(∆,τ)
-20
0
20
0
0.02
0.04
0.06
0.08
0.1
-20
0
20
0
0.002
0.004
0.006
0.008
0.01
-20
0
20
0
0.02
0.04
0.06
0.08
0.1
-20
0
20
0
0.002
0.004
0.006
0.008
0.01
= 0
=
= 2
Wave solver
= 0
=
= 2
Monte-carlo /
Multi-slice
τx
τx
τx
τx
Fig. 11. Wave optics alternatives. We compare covariances obtained with our approach, against those by a wave-equation solver, and a multi-slice simulator
from optics. Our simulator is physically accurate, producing results in close agreement with those of the solver, while being orders of magnitude faster. The multi-slice approach is valid for a very forward-scattering phase function (д = 0.98) and small optical depth. For a wider phase function (д = 0.3), the
multi-slice approach produces inaccurate results, due to incorrect modeling of back and side scattering angles. The comparison is performed in 2D due to the
limitations of the solver. The simulated volume is 200λ × 70λ wide with OD = 3.i Multi-slice simulator
д = 0.98
д = 0.3
20
0
20
-20
0
20
0
0.002
0.004
0.006
0.008
0.01
= 0
=
= 2
Wave solver
= 0
=
= 2
Monte-carlo /
Multi-slice
τx
τx
h
h
b
l
d
l
l
l 20 20 20 Fig. 11. Wave optics alternatives. We compare covariances obtained with our approach, against those by a wave-equation solver, and a multi-slice simulator
from optics. Our simulator is physically accurate, producing results in close agreement with those of the solver, while being orders of magnitude faster. The multi-slice approach is valid for a very forward-scattering phase function (д = 0.98) and small optical depth. For a wider phase function (д = 0.3), the
multi-slice approach produces inaccurate results, due to incorrect modeling of back and side scattering angles. The comparison is performed in 2D due to the
limitations of the solver. 8
EVALUATIONfi This technique has high computational efficiency compared
to exact wave-equation solvers, but cannot model back-scattering,
and has worse accuracy for wide scattering angles. direction results in reduced noise in all cases. The improvement
becomes more pronounced as the width of the volume increases
(second panel in Fig. 9); this is because, as the size of the Gaussian
beam relative to the volume decreases, uniform vertex sampling
will result in more paths starting in points of the volume that do
not receive any light. Finally, the improvement achieved by using
importance sampling increases even further as the phase function
becomes more forward-scattering (third panel in Fig. 9); in this case,
it is necessary to importance sample the first direction as well, oth-
erwise the majority of path-starting directions will have near-zero
contribution. Fig. 10 additionaly shows convergence plots for the
first and third volumes in Fig. 9. The right part of Fig. 11 shows correlation estimates from this
technique, using the same experimental settings as in our com-
parisons with the wave-equation solver. We observe that, for very
forward-scattering phase functions, both our Monte Carlo algo-
rithm and the layered propagation technique closely match the
groundtruth produced by the solver. However, for phase functions
with significant side-scattering, the accuracy of the layered propaga-
tion technique is significantly worse than that of ours. Additionally,
our Monte Carlo algorithm is significantly faster than the layered Rendering Near-Field Speckle Statistics in Scattering Media
•
187:15 Our approach
Multi-slice simulator
д = 0.98
д = 0.3
д = 0.98
д = 0.3
C(∆,τ)
-20
0
20
0
0.02
0.04
0.06
0.08
0.1
-20
0
20
0
0.002
0.004
0.006
0.008
0.01
-20
0
20
0
0.02
0.04
0.06
0.08
0.1
-20
0
20
0
0.002
0.004
0.006
0.008
0.01
= 0
=
= 2
Wave solver
= 0
=
= 2
Monte-carlo /
Multi-slice
τx
τx
τx
τx
Fig. 11. Wave optics alternatives. We compare covariances obtained with our approach, against those by a wave-equation solver, and a multi-slice simulator
from optics. 8
EVALUATIONfi The
correlation is displayed as a function of (∆b,θ) alone, with ∆a
selected according to the Dirac delta relationship in Eq. (50).i 8
EVALUATIONfi The simulated volume is 200λ × 70λ wide with OD = 3.i
2
2
a plane
b plane
(a) Setup
2
2
a plane
b plane
(a) Setup
θ[rad]
-5
0
5
-0.0319
-0.0158
0
0.0158
0.0319
-5
0
5
-0.0319
-0.0160
0
0.0160
0.0319
-5
0
5
-0.0319
-0.0158
0
0.0158
0.0319
-5
0
5
-0.0319
-0.0158
0
0.0158
0.0319
∆[µm]
∆[µm]
∆[µm]
∆[µm]
(b) Analytic
(c) Measured
(d) VMF
(e) Mie
[Osnabrugge]
Our simulation
Fig. 12. The tilt-shift memory effect. (a) Setup: two beams with spatial
displacement ∆a and angular displacement θ enter the volume. We compute
the correlation between the speckle patterns measured at the b plane as a
function of both tilt and shift, assuming that the input displacement ∆a
is selected to maximize Eq. (50). The bottom images visualize the results. (b,c) Analytic and measured correlation, reproduced from Osnabrugge et
al. [2017]. (d-e) Correlation computed by our algorithm for two different
phase functions with the same average cosine д = 0.98. of the speckle fields due to the two beams if we additionally tilt
the beams at angles −θ/2, θ/2 respectively. Intuitively, appropriately
selecting the tilting angle as a function of the displacement ∆a
helps increase the overlap between the defocused beams inside the
medium, and thus increases the correlation of the resulting speckle
fields.i This observation motivates evaluating how speckle field correla-
tion varies as a function of tilt angle and shift displacements at the
input and output planes. Concretely, we can write this as a function: (a) Setup
θ[rad]
-5
0
5
-0.0319
-0.0158
0
0.0158
0.0319
-5
0
5
-0.0319
-0.0160
0
0.0160
0.0319
-5
0
5
-0.0319
-0.0158
0
0.0158
0.0319
-5
0
5
-0.0319
-0.0158
0
0.0158
0.0319
∆[µm]
∆[µm]
∆[µm]
∆[µm]
(b) Analytic
(c) Measured
(d) VMF
(e) Mie
[Osnabrugge]
Our simulationf θ[rad] C(∆a, ∆b,θ) ≡E
"Õ
τ
u1(τ −∆b/2) · u2(τ + ∆b/2)∗eikτθ
#
,
(49)i (49) where the expectation is taken over all fields with the same material
parameters (e.g., fields generated by different scatterer instantiations
O sampled from the same density). Osnabrugge et al. [2017] derived
an analytic approximation for this function that takes the form: ∆[µm]
(e) Mie ∆[µm]
(b) Analytic
[Osn ∆[µm]
(c) Measured [Osnabrugge] Fig. 12. The tilt-shift memory effect. (a) Setup: two beams with spatial Fig. 12. The tilt-shift memory effect. ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 9
THE TILT-SHIFT MEMORY EFFECTi In this section, we use our near-field rendering technique to study
the tilt-shift memory effect property introduced by Osnabrugge et
al. [2017]. We first briefly review this property, using Fig. 12(a) as a
reference: A scattering volume of thickness L is illuminated by two
input beams from its top surface, denoted in Fig. 12(a) as the a plane. The scattered fieldsu1,u2 due to the two beams are imaged by a cam-
era focused at the lower plane of the volume, denoted in Fig. 12(a)
as the b plane. The two illumination beams have a displacement
relative to each other equal to i2 −i1 = ∆a. The key observation
of Osnabrugge et al. [2017] is that we can increase the correlation To evaluate the accuracy of our near-field covariance rendering
algorithm, we use it to estimate the function C(∆a, ∆b,θ) for the 187:16
•
Chen Bar, Ioannis Gkioulekas, and Anat Levin Shift
Tilt-Shift
In
Out
In
Out
∆= 0λ
∆= 1.5λ
∆= 3λ
Analytic S
Analytic S+T
MC S
MC S+T
-20
-10
0
10
20
0
0.2
0.4
0.6
0.8
1
-20
-10
0
10
20
0
0.2
0.4
0.6
0.8
1
-20
-10
0
10
20
0
0.2
0.4
0.6
0.8
1
∆[µm]
∆[µm]
∆[µm]
(a) OD = 1, g = 0.98 (b) OD = 3, g = 0.98
(c) OD = 6, g=0.99
Fig. 13. Adaptive optics focusing. Illuminating a scattering slab with the
complex wave visualized at the first and third columns of the top row leads
to a sharp focused point at the other edge of the random media slab. Due
to the ME, a small shift of the same pattern can focus at a nearby point,
but focusing power degrades with displacement (note the weak power at
the third row). Applying both shift and tilt to the input pattern (rightmost
columns) leads to a stronger power at the same displacement (compare the
power for the non-zero displacements at rows 2 and 3). Lower panel: Using
our approach to evaluate the expected power one can achieve with the shit
only and shift+tilt approaches, as a function of displacement (that is, the
average power of the yellow dot at the three top rows). The scan range
predicted by our accurate simulator is wider than the analytical prediction. 9.1
Focusing through turbid media Illuminating a scattering slab with the
complex wave visualized at the first and third columns of the top row leads
to a sharp focused point at the other edge of the random media slab. Due
to the ME, a small shift of the same pattern can focus at a nearby point,
but focusing power degrades with displacement (note the weak power at
the third row). Applying both shift and tilt to the input pattern (rightmost
columns) leads to a stronger power at the same displacement (compare the
power for the non-zero displacements at rows 2 and 3). Lower panel: Using
our approach to evaluate the expected power one can achieve with the shit
only and shift+tilt approaches, as a function of displacement (that is, the
average power of the yellow dot at the three top rows). The scan range
predicted by our accurate simulator is wider than the analytical prediction. Note that configurations (b) and (c) have the same transport mean free
path and should be equivalent according to the simplified analytic model
of Eq. (50), yet they are very different according to an accurate MC simulator. The memory effect property of scattered fields provides a way
to achieve this refocusing task. The way this works is that we first
use a guiding star to measure the scattered field that is created
due to emission from a single point ix,y at the a plane (Fig. 12(a)). Measuring this fieldu(vx,y) at all points vx,y at theb plane provides
us with exactly the wavefront shape we need to focus at point ix,y. Then, the memory effect property suggests that a shifted wave refocusing simulations using only shifting (Eq. (51)) versus using
both shifting and tilting (Eq. (52)). We observe that using the tilted-
shifted wave improves refocusing, making the yellow focused spots
of the second column in Fig. 13 stronger than the fourth one, and
thus validating the observation of Osnabrugge et al. [2017]. We refer
to App. A.8 for more details regarding this simulation. u(vx,y + ∆),
(51) (51) can be used to roughly focus at ix,y + ∆. Osnabrugge et al. [2017]
improve upon this idea by recommending to refocus using a wave
that is both shifted and tilted; that is, a wave of the form We can additionally use our rendering algorithms to more ac-
curately evaluate the scanning range over which this refocusing
technique remains effective. 9
THE TILT-SHIFT MEMORY EFFECTi Note that configurations (b) and (c) have the same transport mean free
path and should be equivalent according to the simplified analytic model
of Eq. (50), yet they are very different according to an accurate MC simulator. Shift
Tilt-Shift
In
Out
In
Out
∆= 0λ
∆= 1.5λ
∆= 3λ same tissue phantom. We refer to App. A.7 for details on how to sim-
ulate this with our framework. We perform simulations using both
the exact Mie-theory phase function describing the phantom, as
well as a von Mises-Fisher phase function with the same д = 0.98 av-
erage cosine. Comparing with the measured data and the analytical
approximation in Fig. 12(b,c), we make the following observations:
Both our renderings and the analytical approximation produce a
correlation function with a dominant lobe that has the same orien-
tation as the one in the measured data. However, our simulations
match the dimensions of this lobe more closely than the analytical
model. Overall, our simulations reproduce the important qualitative
features of the measuremenets, confirming the accuracy of our algo-
rithms. Differences between our renderings and the measurements
are likely due to inaccurate modeling of the true material properties
of the phantom—as seen in Fig. 12(d-e), replacing the exact reported
phase function with an approximation results in a better match to
the measurements—and due to aberrations in the imaging optics. We also note that Osnabrugge et al. [2017] measured correlation
with an interferometric setup, which typically produces very noisy
estimates of small signals such as weak speckle correlations. In Analytic S
Analytic S+T
MC S
MC S+T
-20
-10
0
10
20
0
0.2
0.4
0.6
0.8
1
-20
-10
0
10
20
0
0.2
0.4
0.6
0.8
1
-20
-10
0
10
20
0
0.2
0.4
0.6
0.8
1
∆[µm]
∆[µm]
∆[µm] ACM Trans. Graph., Vol. 39, No. 6, Article 187. Publication date: December 2020. 9.1
Focusing through turbid media Having shown that our rendering algorithms can accurately repro-
duce the tilt-shift memory effect, we now use them to analyze this
effect in the context of a specific biomedical imaging application: we
simulate focusing through turbid media with adaptive optics. This
involves using, e.g., a spatial light modulator to produce a coherent
wavefront whose shape is specific to the tissue sample being imaged. Finding the exact shape of this wavefront is challenging and usually
requires having external information or a guiding star [Horstmeyer
et al. 2015]. Once we know the wavefront needed to focus at a spe-
cific point inside the volume, an important practical consideration
is whether we can use this information to refocus at other points in
a neighborhood around the original point [Judkewitz et al. 2014].fi 0
∆[µm] 0
∆[µm] ∆[µm]
∆[µm]
∆[µm]
(a) OD = 1, g = 0.98 (b) OD = 3, g = 0.98
(c) OD = 6, g=0.99 (a) OD
1, g
0.98 (b) OD
3, g
0.98
(c) OD
6, g 0.99
Fig. 13. Adaptive optics focusing. Illuminating a scattering slab with the
complex wave visualized at the first and third columns of the top row leads
to a sharp focused point at the other edge of the random media slab. Due
to the ME, a small shift of the same pattern can focus at a nearby point,
but focusing power degrades with displacement (note the weak power at
the third row). Applying both shift and tilt to the input pattern (rightmost
columns) leads to a stronger power at the same displacement (compare the
power for the non-zero displacements at rows 2 and 3). Lower panel: Using
our approach to evaluate the expected power one can achieve with the shit
only and shift+tilt approaches, as a function of displacement (that is, the
average power of the yellow dot at the three top rows). The scan range
predicted by our accurate simulator is wider than the analytical prediction. Note that configurations (b) and (c) have the same transport mean free
path and should be equivalent according to the simplified analytic model
of Eq. (50), yet they are very different according to an accurate MC simulator. ( )
, g
( )
, g
( )
, g
Fig. 13. Adaptive optics focusing. 10
CONCLUSION Brian A. Barsky and Todd J. Kosloff. 2008. Algorithms for Rendering Depth of Field
Effects in Computer Graphics. ICCOMP (2008). We presented computationally-efficient algorithms for simulating
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of closed-form expression for transforming far-field correlations
to the near-field ones, for individual Monte Carlo paths. We also
derive efficient importance sampling strategies for path generation. f
p
p
(
)
Mahed Batarseh, Sergey Sukhov, Zhean Shen, H. Gemar, Roxana Rezvani, and Aristide f
p
p
(
)
Mahed Batarseh, Sergey Sukhov, Zhean Shen, H. Gemar, Roxana Rezvani, and Aristide
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p
p
(
)
Mahed Batarseh, Sergey Sukhov, Zhean Shen, H. Gemar, Roxana Rezvani, and Aristide
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scattering and absorption. ACM TOG (2014). The closed-form expressions are made possible through the use
of von Mises-Fisher functions to approximate all spherical functions
appearing in the path integral expression of near-field speckle co-
variance. We make four such approximations: (i) We use apodization
in the aperture plane to convert the aperture mask into a spheri-
cal von Mises-Fisher function. (ii) We assume the exponential at-
tenuation is constant through all aperture directions. (iii) We ap-
proximate the phase function as a mixture of von Mises-Fisher
functions. (iv) We approximate the analytical convolution of von
Mises-Fisher functions, which is not in general a von Mises-Fisher
function, as such a function. We have shown through simulations
that: (i) Even though apodization cannot express an exactly binary
aperture, if one matches its variance to the desired NA, a similar
depth of field and similar speckle statistics are produced. (ii) The
assumption of constant directional attenuation introduces negligible
bias. (iii) Real-word and common parametric phase functions can
be well-approximated using a small number of von Mises-Fisher
functions. (iv) The convolution of von Mises-Fisher functions can
be closely approximated as a von Mises-Fisher function. 10
CONCLUSION For narrow
phase functions emitting little energy in the backward direction, nu-
merical problems arise in back directions. As a result, our approach
mostly applies for transmission mode imaging, and further research
is required for reflection mode imaging.f Stephan Bergmann, Mahsa Mohammadikaji, Stephan Irgenfried, Heinz Worn, Jürgen
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Tom Cuypers, Tom Haber, Philippe Bekaert, Se Baek Oh, and Ramesh Raskar. 2012. Reflectance Model for Diffraction. ACM TOG (2012). lf
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Sketch and motivation. Journal of Computational and Theoretical Transport (2018). As an application of our algorithms, we evaluated different per-
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what predicted by previous approximate analytical models. Expeditions award 1730147, DARPA REVEAL grant HR0011-16-C-
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ical model depends only on the transport mean free path ℓtr, our
simulations suggest that the scanning range varies significantly
for materials with the same transport mean free path, but different
phase function and actual mean free path (compare Fig. 13(b,c)). 10
CONCLUSION We hope
that our algorithms will open the door for better understanding of
speckle statistics under near-field imaging conditions, in turn al-
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(
) Arindam Banerjee, Inderjit S. Dhillon, Joydeep Ghosh, and Suvrit Sra. 2005. Clustering
on the unit hypersphere using von Mises-Fisher distributions. JMLR (2005). j
,
j
, J y
p
,
g
on the unit hypersphere using von Mises-Fisher distributions. JMLR (2005). Ch
B
M
i
Alt
I
i Gki
l k
d A
t L
i
2019 A M
t C
l on the unit hypersphere using von Mises-Fisher distributions. JMLR Chen Bar, Marina Alterman, Ioannis Gkioulekas, and Anat Levin. 2019. A Monte Carlo Chen Bar, Marina Alterman, Ioannis Gkioulekas, and Anat Levin. 2019 Framework for Rendering Speckle Statistics in Scattering Media. ACM TOG (2019). h
k
l k
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b
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h b Framework for Rendering Speckle Statistics in Scattering Media. ACM TOG (2019). Chen Bar, Ioannis Gkioulekas, and Anat Levin. 2020. Project Website. https://github. / h b
/
i
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fi ld Framework for Rendering Speckle Statistics in Scattering Media. ACM TOG (2019). Chen Bar, Ioannis Gkioulekas, and Anat Levin. 2020. Project Website. https://github. com/chabner/gaussianBeam-field.f 9.1
Focusing through turbid media In the lower panel of Fig. 13 we plot
the expected power we can measure at focus points for different
displacements ∆, comparing the prediction by our model with the
analytical prediction of Osnabrugge et al. [2017]. We do simulations
for a material with a Henyey-Greenstein phase function of д = 0.98
and mean free path of MFP = 75 µm at wavelength λ = 0.5 µm. We test two material thicknesses L = 75 µm and L = 225 µm, cor-
responding to optical depths of OD = 1 and OD = 3. We observe
that the Monte Carlo simulations predict that the effective scanning u(vx,y + ∆)eikθ(∆)vx,y,
(52) (52) where θ(∆) is selected according to Eq. (50). For example, the
strength of the yellow spot at the fourth column of Fig. 13 is slightly
higher than at the second column. Using Eq. (50), Osnabrugge et
al. [2017] also predict the range of shifts for which this refocusing
is effective; that is, they estimate the scanning range for which
sufficient memory effect correlations exists. We use our rendering algorithms to evaluate these refocusing
techniques, as shown in Fig. 13. The top part of the figure compares Rendering Near-Field Speckle Statistics in Scattering Media
•
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METODOLOGIAS ATIVAS NAS AULAS DE LÍNGUA PORTUGUESA DO ENSINO FUNDAMENTAL I: UM RELATO DE EXPERIÊNCIA SOBRE O USO DO SMARTPHONE NO PROCESSO DE ENSINO E APRENDIZAGEM
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Resumo Este estudo tem como objetivo relatar uma experiência, com a utilização de metodologias ativas,
vivenciada no transcorrer das aulas de Língua Portuguesa, do ensino fundamental I, a partir do uso
do smartphone. Na contemporaneidade, observamos o quão as tecnologias digitais de informação e
comunicação (TDIC) articulam-se com a realidade que, há tempos, transforma-se em virtude dos
avanços tecnológicos. Sendo assim, a escola como agência de fomento cognitivo não pode estar à
parte de todo esse processo social, envolvendo, sobretudo, os smartphones que já fazem parte da
rotina da população, principalmente, crianças e adolescentes. Dessa forma, amparamo-nos na
pesquisa-ação como metodologia, partindo de uma breve revisão de literatura para, na sequência,
descrevermos sucintamente o desenvolvimento do trabalho, envolvendo as metodologias ativas e o
uso do smartphone. Partimos da hipótese de que esse recurso atrelado à escolha teórico-metodológica
pode elevar o nível de engajamento dos estudantes, bem como os resultados de aproveitamento. Palavras-chave: Metodologias Ativas. Smartphone. Ensino fundamental. Língua portuguesa. CÉLIA APARECIDA REGINATO1
PAULO ALEXANDRE FILHO2 DOI: 10.29327/23860.14.25-8 2 Possui graduação em Letras - Alemão pela Universidade Estadual Paulista Júlio de Mesquita Filho - UNESP - Câmpus
de Assis. Mestrado em Letras, na área de concentração de Linguagens e Letramentos, pela UNESP - Câmpus de Assis.
Atualmente, professor efetivo do Quadro de Magistério, na Secretaria Estadual de Educação - SP, para o componente
curricular de Língua Portuguesa. Doutorando em Educação pela Faculdade de Filosofia e Ciências, UNESP - Câmpus
de Marília. Desenvolve seu doutorado na linha de pesquisa Teoria e Práticas Pedagógicas em que a tese focaliza os
estudos de natureza teórico - metodológica, relacionando-se com as diversas áreas do currículo da Educação Básica
e Superior, em especial, com a Didática, a Metodologia, a Psicologia, a Linguística e as políticas educacionais no
Brasil. Participa do grupo de pesquisa TECLIN - Tecnologias, Culturas e Linguagens, coordenado pelo professor Dr.
Fábio Marques de Souza (UEPE). Tem experiência na área de Linguagens e Letramentos. Keywords: Active Methodologies. Smartphone. Elementary School. Portuguese language. Keywords: Active Methodologies. Smartphone. Elementary School. Portuguese language. Keywords: Active Methodologies. Smartphone. Elementary School. Portuguese language. ACTIVE METHODOLOGIES IN PORTUGUESE LANGUAGE
CLASSES OF ELEMENTARY EDUCATION I: AN EXPERIENCE
REPORT ON THE USE OF SMARTPHONE IN THE TEACHING AND
LEARNING PROCESS 1 Mestranda em Educação pela Faculdade de Filosofia e Ciências - Campus de Marília/SP. Possui Licenciatura Plena
em Pedagogia pela Faculdade de Filosofia e Ciências - Campus de Marília/SP (2001). Possui Curso de Pós-
Graduação Lato Sensu - Especialização em Coordenação Pedagógica pela Universidade Federal de São Carlos em
parceria com o Ministério da Educação - Programa Escola de Gestores da Educação Básica (2016). Especialização
Pós-Graduação Lato Sensu em Gestão e Docência na Educação Básica pela Universidade Santa Cecília em parceria
com o IFGE - Instituto de Formação e Gestão Educacional (2013); Habilitação Específica de 2º Grau para o Magistério
pelo Centro Específico de Formação e Aperfeiçoamento para o Magistério - CEFAM (1994). 2 Possui graduação em Letras - Alemão pela Universidade Estadual Paulista Júlio de Mesquita Filho - UNESP - Câmpus
de Assis. Mestrado em Letras, na área de concentração de Linguagens e Letramentos, pela UNESP - Câmpus de Assis. Atualmente, professor efetivo do Quadro de Magistério, na Secretaria Estadual de Educação - SP, para o componente
curricular de Língua Portuguesa. Doutorando em Educação pela Faculdade de Filosofia e Ciências, UNESP - Câmpus
de Marília. Desenvolve seu doutorado na linha de pesquisa Teoria e Práticas Pedagógicas em que a tese focaliza os
estudos de natureza teórico - metodológica, relacionando-se com as diversas áreas do currículo da Educação Básica
e Superior, em especial, com a Didática, a Metodologia, a Psicologia, a Linguística e as políticas educacionais no
Brasil. Participa do grupo de pesquisa TECLIN - Tecnologias, Culturas e Linguagens, coordenado pelo professor Dr. Fábio Marques de Souza (UEPE). Tem experiência na área de Linguagens e Letramentos. 137 Abstract This study aims to report an experience, with the use of active methodologies, experienced in the
course of Portuguese language classes of elementary school I from the use of smartphones. Nowadays, we observe how digital information and communication technologies (TDIC) are
articulated with the reality that, for some time, has been transformed by virtue of technological
advances. Thus, the school as a cognitive development agency cannot be apart from this entire social
process, involving, above all, smartphones that are already part of the population's routine, especially
children and adolescents. Thus, we rely on action research as a methodology, starting from a brief
literature review and then briefly describing the development of the work, involving active
methodologies and the use of smartphones. We start from the hypothesis that this resource linked to
the theoretical-methodological choice can raise the level of engagement of students, as well as the
results. 1 INTRODUÇÃO Este relato é fruto de uma experiência vivenciada durante as aulas de Língua Portuguesa, no
Ensino Fundamental I, tendo as metodologias ativas como suporte teórico-metodológico e o
smartphone como recurso pedagógico, no ano de 2019, portanto, anterior ao contexto de pandemia. De início, faz-se necessário descrever brevemente a escola onde se deu o desenvolvimento deste
trabalho, uma vez que acreditamos que cada realidade traz consigo suas peculiaridades que devem
ser tomadas como base para reflexão de cada sujeito quando confrontado com realidades específicas
de diferentes lugares. A unidade escolar localiza-se no interior de São Paulo e faz parte da rede municipal de
educação. Os alunos são do 5º ano do ensino fundamental I, e grande parte deles apresenta
dificuldade de leitura e escrita, demonstrando pouco engajamento com as atividades propostas. Quanto à infraestrutura, a escola não possuía número suficiente de computadores e internet com sinal
estável a ponto de promovermos atividades mais longas e com toda a classe que era composta por 28
alunos. No transcorrer do primeiro bimestre, pudemos comprovar as dificuldades dos alunos por
meio de avaliação diagnóstica, estudo das fichas de acompanhamento individual de cada um e
compartilhamento de experiências com a professora deles no 4º ano. Durante essas reuniões de
formação, a professora do ano anterior sempre me relatava que apesar das defasagens apresentadas
pela turma, o que mais lhe preocupava era a apatia desses estudantes que, na maioria das vezes, não
se mostravam interessados ou desafiados durante a realização das atividades propostas. Apesar de uma comunidade carente e com grandes vulnerabilidades socioemocionais,
observei no transcorrer do bimestre que a maioria dos alunos possuía smartphone e, não raro, era
preciso chamar a atenção deles para que não se dispersassem mexendo no aparelho. Dessa forma,
notei que grande parte deles se interessava pelo uso desse recurso, porém apenas para fins de 138 comunicação simultânea por meio do aplicativo WhatsApp e acesso às redes sociais. Assim, vi nesse
aparelho uma possibilidade de implementar seu uso efetivo para construção do conhecimento e
promover o engajamento dos estudantes a partir das metodologias ativas. comunicação simultânea por meio do aplicativo WhatsApp e acesso às redes sociais. 1 INTRODUÇÃO Assim, vi nesse
aparelho uma possibilidade de implementar seu uso efetivo para construção do conhecimento e Ao levar a proposta para a coordenadora da escola, fui muito bem acolhida e, após detalhar
os acontecimentos e observações de sala de aula, ela me solicitou que compartilhasse com os demais
colegas minhas ideias para que todos pudessem opinar. Para minha surpresa, grande parte dos
professores, até os mais tradicionais, receberam muito bem a ideia, mas não deixaram de colocar os
“contras” como, por exemplo, a possibilidade dos alunos filmarem/gravarem as aulas sem
consentimento do professor, uso de internet móvel, uma vez que a escola não possuía wi-fi de
qualidade a ponto de fornecer acesso para todos os alunos, entre outras questões. Eu e a coordenadora tomamos nota de todas as observações das demais colegas. Na
sequência, marcamos uma reunião para que juntas pensássemos em ações que pudessem solucionar
ou minimizar esses entraves, pois o uso de metodologias ativas articulado às TDIC poderia oferecer
bons resultados. Chegamos à conclusão de que deveríamos mandar comunicado para os pais ou
responsável, esclarecendo nossos objetivos, bem como suas necessidades para a formação dos alunos
e, por fim, colocando-nos à disposição para quaisquer dúvidas. Amparamo-nos na metodologia qualitativa em que a pesquisa-ação ofereceu o suporte
necessário para o desenvolvimento da prática conforme supracitado. Nesse sentido, Severo (2020, p. 2) ressalta que “dessa forma, não há mais espaço para o modelo de ensino centrado em práticas
tradicionais, baseadas fortemente na pura transmissão de conteúdos sem alguma relação com a
realidade e necessidades do sujeito aprendente.” A seguir, faremos uma breve revisão de literatura em que buscamos na teoria dos
multiletramentos e das metodologias ativas o aporte teórico indispensável para a elaboração de
estratégias pedagógicas, bem como promover a reflexão necessária sobre a experiência aqui descrita
na forma de relato de experiência. Assim, Rojo e Moura (2019) e Moran (2018) nos ofertaram os
estudos acerca dos temas tratados, neste trabalho, que em nenhum momento pretendeu ser recebido
de maneira a-crítica. Reiteramos que cada local tem suas especificidades e, assim, este relato além
do objetivo, busca também estender as possibilidades de aplicação desse recurso e dessa
metodologia. 2 FUNDAMENTAÇÃO TEÓRICA Para esta seção, procuramos subsídios teóricos nas metodologias ativas e na Pedagogia dos
multiletramentos para fins de respaldar nossa prática, envolvendo o uso do smartphone, bem como
as intencionalidades das ações e estratégias implementadas no transcorrer do processo de ensino e
aprendizagem. . 139 Partimos da premissa de que os estudos aqui apresentados nos serviram de base para a
compreensão da nossa realidade como forma de estabelecer entre teoria e prática as pontes
necessárias para ressignificar nosso lugar de fala enquanto educadores e, ao mesmo tempo, promover
alternativas que pudessem viabilizar nosso trabalho, à medida que os desafios iam surgindo. Partimos da premissa de que os estudos aqui apresentados nos serviram de base para a
compreensão da nossa realidade como forma de estabelecer entre teoria e prática as pontes
necessárias para ressignificar nosso lugar de fala enquanto educadores e, ao mesmo tempo, promover
alternativas que pudessem viabilizar nosso trabalho, à medida que os desafios iam surgindo. 2.1 A PEDAGOGIA DOS MULTILETRAMENTOS NAS PRÁTICAS DE
ENSINO E APRENDIZAGEM Concebemos a realidade atual como um cenário repleto de tecnologias em que as pessoas
passaram a depender sistematicamente de algum desses recursos para realizar suas ações do
cotidiano. Se pensarmos na vivência de nossos alunos, comparando-as com aquilo que ofertamos em
nossas aulas, constatamos que ainda estamos longe de atingir uma aprendizagem mais significativa
e mais próxima daquilo que nos circunda. Nesse sentido, concebemos a Pedagogia dos multiletramentos como possibilidade teórico-
metodológica viável no contexto atual de educação, em que as demandas passaram a exigir das
pessoas competências, não apenas instrumentais, mas também interpessoais quando se trata das
TDIC. Para Rojo e Moura (2019, p. 20), “multiletramentos [...] aponta, a um só tempo, para a
diversidade cultural das populações em êxodo e para a diversidade de linguagens dos textos
contemporâneos, o que vai implicar, é claro, uma explosão multiplicativa dos letramentos, que se
tornam multiletramentos.” Dessa forma, notamos a necessidade da escola, enquanto agenciadora de letramento,
incorporar em suas práticas a perspectiva dos multiletramentos, sobretudo, no intuito de oferecer uma
educação voltada para as transformações de mundo assistidas nos últimos tempos. Trata-se, portanto,
de legitimar as diferentes formas de construir sentido e valorizar as práticas sociais que agora passam
a ocorrer em sintonia com a digitalidade. Não podemos deixar de considerar todas essas transformações e, ao mesmo tempo, buscar
em nossos alunos a parceria necessária para a construção do conhecimento, uma vez que eles têm
muito a nos oferecer de suas experiências com os recursos digitais. Logo, o professor se torna
mediador, orientando sobre as potencialidades desses artefatos e também esclarecendo o quão
importante é a tecnologia para a aprendizagem quando utilizada adequadamente. Rojo e Moura
(2019, p. 24) observam que “[...] o conceito de multiletramentos abre cada vez mais espaço aos
conceitos de mídia e de modalidade de linguagem [...].” Trazer para o espaço escolar uma teoria que visa à valorização das linguagens
multissemióticas significa, neste momento, reformular o conceito de aprendizagem de que o
conhecimento só se constrói a partir de textos canônicos ou expositivos presentes nos livros didáticos
e apostilas. Ao articularmos nossas práticas levando em consideração a teoria dos multiletramentos, 140 estamos tornando a educação menos verticalizada e ofertando aos alunos a possibilidade de construir, tamos tornando a educação menos verticalizada e ofertando aos alunos a possibilidade de construi por meio do cooperativismo, seu próprio conhecimento. por meio do cooperativismo, seu próprio conhecimento. 2.2 AS METODOLOGIAS ATIVAS COMO ESTRATÉGIAS TEÓRICO-
METODOLÓGICAS NA EDUCAÇÃO BÁSICA 141 Dessa maneira, concebemos a ideia de que, em um primeiro momento, precisamos realocar Dessa maneira, concebemos a ideia de que, em um primeiro momento, precisamos realoc nossa posição de educadores e estarmos sempre abertos a novas experimentações, sem perder de
vista que as crianças e jovens têm muito a nos oferecer e, portanto, colaborar com nossas visões sobre
nossa profissão. Não se trata de descaracterizar o papel do professor e do aluno, mas sim promover
uma pedagogia em que todos possam ser valorizados e protagonistas de suas próprias ações. nossa posição de educadores e estarmos sempre abertos a novas experimentações, sem perder de
vista que as crianças e jovens têm muito a nos oferecer e, portanto, colaborar com nossas visões sobre
nossa profissão. Não se trata de descaracterizar o papel do professor e do aluno, mas sim promover
uma pedagogia em que todos possam ser valorizados e protagonistas de suas próprias ações. 2.2 AS METODOLOGIAS ATIVAS COMO ESTRATÉGIAS TEÓRICO-
METODOLÓGICAS NA EDUCAÇÃO BÁSICA A concepção de aprendizagem ativa extrapola a ideia de uma educação pautada em aulas
expositivas. Tornar o processo de ensino e aprendizagem ativo requer do educador novas posturas e
maneiras de conceber a construção do conhecimento. Sabemos das dificuldades em nos
desvencilharmos de modelos pedagógicos tradicionais em que cumprimos com a função de ensinar
e o aluno de aprender como se os papéis de um e de outro fossem bem definidos e obedecessem a
um roteiro com começo, meio e fim. Para Moran (2018, p. 04), “metodologias ativas são estratégias de ensino centradas na
participação efetiva dos estudantes na construção do processo de aprendizagem, de forma flexível,
interligada e híbrida.” Assim, podemos compreender que a educação por esse modelo de
aprendizagem exige uma concepção mais reflexiva daquilo que objetivamos ao final de cada aula,
bimestre ou ano letivo, uma vez que precisamos estar atentos ao percurso formativo de nossos alunos,
bem como às estratégias que empregamos. Ou seja, trazer as metodologias ativas para o nosso campo
de atuação exige novas atitudes frente aos inúmeros desafios que nossa profissão de educadores nos
lançam no cotidiano. As ações que implementamos em sala de aula nem sempre oportunizam aos alunos espaço
para o diálogo como forma de escuta reflexiva em que eles podem inclusive externar seus pontos de
vista sobre aquilo que acham relevante para sua formação. Na maioria das vezes, cumprimos com o
currículo determinado pelas autoridades competentes, deixando de lado a valorização do diálogo,
tornando esses estudantes pessoas silenciadas pelo sistema que busca massificar as inteligências, as
competências e as relações afetivas. Moran (2018, p. 2) observa que “as metodologias predominantes
no ensino são dedutivas: o professor transmite primeiro a teoria e depois o aluno deve aplicá-la a
situações mais específicas.” Não estamos aqui colocando em xeque a aula expositiva, tampouco desvalorizando o papel
do professor. Pelo contrário, neste momento, buscamos estender as reflexões de modo que as crianças
e jovens da atualidade possam aprender de forma mais significativa, engajando-se mais em seu
percurso formativo. Desse modo, torna-se imprescindível mobilizar outras competências, tão
importantes quanto aquelas de tempos em que a tecnologia ainda não havia tomado proporções como
na contemporaneidade, uma vez que “os processos de aprendizagem são múltiplos, contínuos,
híbridos, formais e informais, organizados e abertos, intencionais e não intencionais (MORAN, 2018,
p. 3). 3 O USO DO SMARTPHONE NO PROCESSO DE ENSINO E
APRENDIZAGEM: RELATANDO A EXPERIÊNCIA Para tal, também recorremos à estratégia do agrupamento produtivo, para 142 que o nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido que o nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido que o nível cognitivo de cada integrante não destoasse a ponto de um ou outro se sentir desmerecido
ou pouco importante. Inicialmente, trabalhamos a ferramenta de busca de maior acesso na internet,
ou seja, como pesquisar o sentido das palavras no Google. A priori, pode parecer uma atividade
muito simples para o 5º ano, contudo pudemos constatar o contrário, uma vez que nem todos sabiam
explorar essa plataforma e suas facilidades. Então, solicitamos que aqueles alunos que já sabiam
sobre esse recurso, compartilhassem com os outros colegas, inclusive, relatando em quais momentos
faziam uso. Assim, durante as aulas os alunos passaram a contar com o Google como ferramenta
indispensável para pesquisa. Com o passar do tempo, um deles me disse que em vez de pesquisar no
Google, poderíamos baixar aplicativos de dicionário. Foi então que lhes apresentei o aplicativo de
busca Play Store3 e por meio dele pudemos selecionar alguns dicionários que poderiam nos servir de
material de apoio. Logo, notamos que muito da capacidade em operar com as tecnologias quando se
trata de crianças e jovens não condiz com a realidade, pois sem orientação e os devidos
encaminhamentos, nem todos conseguiriam chegar até aquele ponto da aprendizagem. Ao retornarmos das férias de julho para o terceiro bimestre, fui chamada pela coordenadora
para descrever como foi o uso do smartphone em sala de aula, expondo se a prática adotada havia se
refletido na aprendizagem e se valia a pena continuar. Após relatar a experiência, a coordenadora
disse que seria muito positivo continuar, porém promovendo outros desafios como forma de
demonstrar às crianças o potencial dessa ferramenta quando utilizada adequadamente. Desse modo, avançamos nos objetivos de uso do smartphone, ampliando as possibilidades
de utilização desse artefato. Em um determinado momento, um dos alunos perguntou se eu conhecia
um determinado filme ao qual havia assistido e gostaria de me recomendar, mas não se lembrava do
título. 3 A Google Play Store — ou simplesmente Play Store, como é popularmente conhecida — é a loja de
aplicativos oficial do Google. É por ela que os usuários Android podem fazer download de apps de forma
gratuita ou paga — ou, ainda, baixar um programa gratuitamente, mas usá-lo mediante pagamento de
assinatura. Disponível em: https://www.techtudo.com.br/listas/2020/10/o-que-e-google-play-loja-de-apps-do-
android-oferece-filmes-e-livros.ghtml. Acesso em: 22 set. 2021. 3 O USO DO SMARTPHONE NO PROCESSO DE ENSINO E
APRENDIZAGEM: RELATANDO A EXPERIÊNCIA Conforme relatamos, a classe era composta por 28 alunos do ensino fundamental I e grande
parte deles apresentava defasagens de leitura e escrita sem contar o pouco envolvimento com a
resolução das atividades propostas. Muitos deles perdiam o foco durante a realização das tarefas e
isso quando não pegavam o aparelho celular para se comunicarem com os colegas ou até mesmo com
os próprios familiares. Após afinar as ideias coletadas durante a reunião com os demais colegas, eu e a coordenadora
realinhamos e redefinimos nossas ações para que o trabalho acontecesse a partir do segundo bimestre. Assim, todos os pais e/ou responsável foram comunicados e, nesse sentido, ficamos muito surpresos,
pois ninguém se opôs à proposta de utilização do smartphone como recurso pedagógico. Pelo
contrário, houve um responsável que, inclusive, elogiou para a coordenadora, em conversa telefônica,
a estratégia de usar essa ferramenta com a finalidade de promover conhecimento. Assim, definimos que, logo de início, os estudantes precisariam conhecer os objetivos do uso
do smartphone e, ao mesmo tempo, estabelecer com eles algumas regras de conduta para que nossos
objetivos fossem alcançados ao final do bimestre. Logo, a coordenadora foi convidada para
apresentar junto comigo a proposta de trabalho que seria desenvolvido nesta classe. Por fim,
reiteramos as regras de comportamento e a responsabilidade durante a utilização do smartphone. Ao
final, fomos questionadas por um dos alunos acerca de como participaria das aulas, sendo que ele
não tinha esse recurso. Então, respondemos que todo o trabalho seria desenvolvido em grupo. Reiteramos que se trata de uma prática ocorrida em 2019, momento anterior à pandemia, portanto
sem necessidade de distanciamento. Quanto à metodologia empregada, optamos pelas metodologias ativas, pois “[essas] são
caminhos para avançar mais no conhecimento profundo nas competências socioemocionais e em
novas práticas” (MORAN, 2015, p. 01). Acreditamos que promover uma aprendizagem baseada nos
princípios de autonomia, pesquisa e cooperativismo seja o propósito de todo educador e, nesse
sentido, reconhecemos a potencialidade dos artefatos tecnológicos, no caso o smartphone. Dessa forma, estabelecemos que aqueles que dispunham de internet iriam se sentar com
aqueles que não possuíam. 3 O USO DO SMARTPHONE NO PROCESSO DE ENSINO E
APRENDIZAGEM: RELATANDO A EXPERIÊNCIA Nesse momento, aproveitei para trabalhar com eles a pesquisa por meio de palavras-chave
com o objetivo de evidenciar que o Google trata-se de um compêndio virtual onde praticamente
podemos encontrar tudo. Na sequência, pedi que todos se reunissem em grupos e solicitei para que o estudante nos
falasse um pouco sobre a história/enredo do filme. Em seguida, perguntei à classe quais trechos
descritos pelo aluno seriam os mais importantes para a compreensão da obra. Desse modo, elenquei
na lousa três excertos que poderiam ser fundamentais para o processo de busca na plataforma Google. 143 Logo depois, solicitei que aqueles que estivessem com internet entrassem no Google e no campo de
busca digitassem um dos trechos Logo depois, solicitei que aqueles que estivessem com internet entrassem no Google e no campo de
busca digitassem um dos trechos. Eles se surpreenderam com as inúmeras possibilidades de filmes que apareceram. O aluno
responsável por desencadear toda a atividade nos disse que não era nenhum daqueles filmes que
apareceu. Então, falei que precisaríamos consultar cada uma das páginas mostradas, mas logo as
crianças perceberam que seria um trabalho muito penoso. Nessa etapa, falei da necessidade de
refinarmos a pesquisa, ou seja, deveríamos ser mais objetivos com as palavras no campo de busca. Novamente, voltamo-nos para a lousa e escolhemos apenas palavras que pudessem nos ajudar a
encontrar o título do filme. Por fim, após a discussão, retomamos a busca e, nesse momento, o aluno achou o filme o
qual havia assistido e que gostaria de me recomendar. Pedi para ele que lesse as informações sobre
a obra de modo a compartilhar conosco o enredo da trama. Nesse dia, a aula foi extremamente
produtiva, todos participaram e tiveram seu espaço de fala, inclusive, sugerindo novos filmes para
os demais colegas. até aquele momento. A nosso ver, a prática foi extremamente exitosa, tendo em vista que esses alunos tiveram a
oportunidade de se tornarem sujeitos ativos e protagonistas de sua aprendizagem, desvencilhando-se
do papel passivo e receptor de informação que, infelizmente, ainda persiste na educação. Não
descartamos ao longo deste relato de experiência os obstáculos, principalmente, aqueles de
infraestrutura, que mesmo em meio a uma cultura altamente tecnológica e digital ainda, ainda
dificultam muito a implementação de uma aprendizagem mais coerente com as transformações de
mundo vigentes. 4 REFLEXÕES ACERCA DO USO DO SMARTPHONE A PARTIR DO
USO DE METODOLOGIAS ATIVAS Utilizar as tecnologias digitais de informação e comunicação (TDIC) é de suma importância
na atualidade. Todavia, sabemos dos inúmeros desafios acerca da infraestrutura das escolas de nível
básico, sobretudo, as públicas. Muito se fala a respeito dos recursos tecnodigitais no processo de
ensino e aprendizagem, mas nem sempre encontramos as instituições devidamente paramentadas e
prontas para a efetivação de ações mais arrojadas com a realidade de nossos alunos. Durante a implementação da prática de uso do smartphone, alguns colegas me questionavam
sobre a monitoração dos aparelhos e se eu não me sentia insegura quanto ao acesso dos alunos a
outros sites ou possíveis dispersões. Sempre respondia amparada em estudos e pesquisadores como
Rojo e Moura (2019) e Moran (2018) que falam da necessidade dos educadores tornarem as
ferramentas tecnológicas suas aliadas diante das demandas educacionais do século XXI. Sabemos
que ainda se trata de inovações e, portanto, muitas incertezas ainda nos assolam, tornando o processo
de ensino e aprendizagem totalmente desafiador. Por outro lado, houve professores que sentiram a necessidade de relatar durante nossas
reuniões o avanço da classe que agora parecia mais engajada, mais responsável e ciente da função
do smartphone durante as aulas. A coordenadora também descreveu algumas falas de alguns pais,
dizendo que os filhos passaram a utilizar o aparelho não apenas para diversão, mas também para
estudar e aprofundar os conhecimentos. As avaliações internas puderam comprovar esse avanço e os 144 próprios estudantes comentavam sobre seus êxitos, reconhecendo a sua evolução do início do ano próprios estudantes comentavam sobre seus êxitos, reconhecendo a sua evolução do início CONSIDERAÇÕES FINAIS Promover uma educação de qualidade requer do educador, não apenas competências teórico-
metodológicas, mas também um olhar crítico sobre o contexto em que atua. As crianças de hoje
vivem em um mundo onde a virtualidade é a principal característica e as relações passam pela
mediação da tela. Desse modo, não podemos concentrar nossos esforços em manter práticas
pedagógicas que pouco podem auxiliar os estudantes de hoje em suas vivências e inserção em
práticas sociais. As transformações sociais, dos últimos tempos, trouxeram à tona muitos aspectos que
precisam ser (re)pensados pelas instituições de ensino de modo que os muros da escola não se
mostrem impermeáveis aos avanços de uma sociedade totalmente digital e dependente de recursos
móveis como, por exemplo, o smartphone. Concebemos, neste relato, a utilização de metodologias
ativas aliada aos recursos digitais como uma das muitas possibilidades de se construir conhecimento. Não tivemos a intenção, em nenhum momento, de transmitir uma receita ou passo a passo efetivo
para o processo de aprendizagem, pois acreditamos que por se tratar de um tema muito complexo
cada local exige dos profissionais novas estratégias e ações, respeitando sempre a realidade de cada
escola. Não podemos deixar de citar, neste trabalho, a atual conjuntura da educação em que grande
parte das instituições vivencia a implementação do ensino emergencial remoto em virtude do
distanciamento sanitário imposto pelas autoridades públicas. Diante disso, temos observado
inúmeros debates e formações acerca das possibilidades ofertadas pelas metodologias ativas e,
principalmente, no vasto potencial das TDIC. Nesse sentido, podemos dizer que muitos profissionais
têm buscado ressignificar suas práticas, buscando nos recursos tecnológicos instrumentos capazes de
minimizar as perdas, tendo em vista o distanciamento sociossanitário. Por fim, vislumbramos, por
meio deste breve relato, suscitar novos diálogos, novas reflexões e novos rumos para a educação em
nosso país. 145 , p
,
p
https://www2.ifrn.edu.br/ojs/index.php/RBEPT/article/view/6717. Acesso em: 22 set. 2021. REFERÊNCIAS MORAN, J. M. Educação Híbrida: um conceito-chave para a educação, hoje. In: BACICH, L.;
TANZI NETO, A.; TREVISANI, F. M. (Orgs.). Ensino híbrido: personalização e tecnologia na
educação. Porto Alegre: Penso, 2015. MORAN, J. M. Metodologias ativas para uma aprendizagem mais profunda. In: BACICH, L.;
MORAN, J. (Orgs.). Metodologias ativas para uma educação inovadora. 1. ed. Porto Alegre:
Penso, 2018. p. 02-25. CÉLIA APARECIDA REGINATO Mestranda em Educação pela Faculdade de Filosofia e Ciências - Campus de Marília/SP. Possui Licenciatura Plena em Pedagogia pela Faculdade de Filosofia e Ciências - Campus
de Marília/SP (2001). Possui Curso de Pós-Graduação Lato Sensu - Especialização em
Coordenação Pedagógica pela Universidade Federal de São Carlos em parceria com o
Ministério da Educação - Programa Escola de Gestores da Educação Básica (2016). Especialização Pós-Graduação Lato Sensu em Gestão e Docência na Educação Básica pela
Universidade Santa Cecília em parceria com o IFGE - Instituto de Formação e Gestão
Educacional (2013); Habilitação Específica de 2º Grau para o Magistério pelo Centro
Específico de Formação e Aperfeiçoamento para o Magistério - CEFAM (1994). ROJO, R.; MOURA, E. Letramentos, mídias e linguagens. 1. ed. São Paulo: Parábola, 2019. ROJO, R.; MOURA, E. Letramentos, mídias e linguagens. 1. ed. São Paulo: Parábola, 2019. SEVERO, C. E. P. Aprendizagem baseada em projetos: uma experiência educativa na educação
profissional e tecnológica. Revista Brasileira da Educação Profissional e Tecnológica, v. 2, n. 19, p. 1-13, 2020. Disponível em: , p
,
p
https://www2.ifrn.edu.br/ojs/index.php/RBEPT/article/view/6717. Acesso em: 22 set. 2021. 146 PAULO ALEXANDRE FILHO Graduação em Letras - Alemão pela Universidade Estadual Paulista Júlio de Mesquita Filho
- UNESP - Câmpus de Assis. Mestrado em Letras, na área de concentração de Linguagens e
Letramentos, pela UNESP - Câmpus de Assis. Atualmente, professor efetivo do Quadro de
Magistério, na Secretaria Estadual de Educação - SP, para o componente curricular de
Língua Portuguesa. Doutorando em Educação pela Faculdade de Filosofia e Ciências,
UNESP - Câmpus de Marília. Desenvolve seu doutorado na linha de pesquisa Teoria e
Práticas Pedagógicas em que a tese focaliza os estudos de natureza teórico - metodológica,
relacionando-se com as diversas áreas do currículo da Educação Básica e Superior, em
especial, com a Didática, a Metodologia, a Psicologia, a Linguística e as políticas
educacionais no Brasil. Participa do grupo de pesquisa TECLIN - Tecnologias, Culturas e
Linguagens, coordenado pelo professor Dr. Fábio Marques de Souza (UEPE). Tem
experiência na área de Linguagens e Letramentos. Artigo Recebido em 25/09/2021
Aceito para Publicação em 30/01/2022 Artigo Recebido em 25/09/2021
Aceito para Publicação em 30/01/2022 REGINATO, Célia Aparecida; FILHO, Paulo Alexandre. METODOLOGIAS
ATIVAS NAS AULAS DE LÍNGUA PORTUGUESA DO ENSINO
FUNDAMENTAL I: UM RELATO DE EXPERIÊNCIA SOBRE O USO
DO SMARTPHONE NO PROCESSO DE ENSINO E APRENDIZAGEM
Revista Paidéi@, UNIMES VIRTUAL, Volume 14, Número 25, Jan.2022. Disponível
em: https://periodicos.unimesvirtual.com.br/index.php/paideia/index 147
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A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting
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Review
A Review on the Adaption of Alginate-Gelatin Hydrogels for
3D Cultures and Bioprinting wska 1
, Karolina Cierluk 2, Agnieszka M. Jankowska 1
, Julita Kulbacka 3
, Jerzy Detyna
ak 4 Magdalena B. Łabowska 1
, Karolina Cierluk 2, Agnieszka M. Jankowska 1
, Julita Kulbacka 3
, Jerzy Detyna 1,*
and Izabela Michalak 4 Magdalena B. Łabowska 1
and Izabela Michalak 4 1
Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering,
Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland; 1
Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering,
Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland;
magdalena.labowska@pwr.edu.pl (M.B.Ł); agnieszka.jankowska@pwr.edu.pl (A.M.J.)
2 1
Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering,
Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland;
magdalena.labowska@pwr.edu.pl (M.B.Ł); agnieszka.jankowska@pwr.edu.pl (A.M.J.) y
gy,
g
,
,
;
magdalena.labowska@pwr.edu.pl (M.B.Ł); agnieszka.jankowska@pwr.edu.pl (A.M.J.) 2
Faculty of Chemistry, Wroclaw University of Science and Technology, Norwida 4/6, 50-373 Wroclaw, Pol
karolinacierluk@gmail.com 3
Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University,
Borowska 211A, 50-556 Wroclaw, Poland; julita.kulbacka@umed.wroc.pl
4
Department of Advanced Material Technologies, Faculty of Chemistry, Wroclaw University of Science and
Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland; izabela.michalak@pwr.edu.pl
*
Correspondence: jerzy.detyna@pwr.edu.pl Abstract: Sustaining the vital functions of cells outside the organism requires strictly defined pa-
rameters. In order to ensure their optimal growth and development, it is necessary to provide a
range of nutrients and regulators. Hydrogels are excellent materials for 3D in vitro cell cultures. Their ability to retain large amounts of liquid, as well as their biocompatibility, soft structures, and
mechanical properties similar to these of living tissues, provide appropriate microenvironments that
mimic extracellular matrix functions. The wide range of natural and synthetic polymeric materials,
as well as the simplicity of their physico-chemical modification, allow the mechanical properties to be
adjusted for different requirements. Sodium alginate-based hydrogel is a frequently used material for
cell culture. The lack of cell-interactive properties makes this polysaccharide the most often applied
in combination with other materials, including gelatin. The combination of both materials increases
their biological activity and improves their material properties, making this combination a frequently
used material in 3D printing technology. The use of hydrogels as inks in 3D printing allows the
accurate manufacturing of scaffolds with complex shapes and geometries. materials materials
Keywords: alginate; gelatin; hydrogels; cell cultures; bioprinting Academic Editor: Maurice Collins
Received: 8 December 2020
Accepted: 2 February 2021
Published: 10 February 2021 Citation: Łabowska, M.B.; Cierluk,
K.; Jankowska, A.M.; Kulbacka, J.;
Detyna, J.; Michalak, I. A Review on
the Adaption of Alginate-Gelatin
Hydrogels for 3D Cultures and
Bioprinting. Materials 2021, 14, 858.
https://doi.org/10.3390/ma14040858 Review
A Review on the Adaption of Alginate-Gelatin Hydrogels for
3D Cultures and Bioprinting The aim of this paper is to
provide an overview of the materials used for 3D cell cultures, which are mainly alginate–gelatin
hydrogels, including their properties and potential applications. 1. Introduction Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Hydrogels are three-dimensionally cross-linked networks of hydrophilic polymeric
materials capable of absorbing large amounts of liquid between their polymeric chains. These materials are created through the reaction of one or more monomers or by association
bonds (hydrogen bonds and strong van der Waals forces between chains). The ability
of these materials to attract water molecules and their tendency to dissolve in water
result from the presence of hydrophilic functional groups connected with the polymeric
backbone (−OH, −COOH, −COO, > C = O, −CHNH2, −CONH, −CONH2, −NH2, and
−SO3H) [1–6]. The amount of water that polymer networks of hydrogels are capable
of absorbing is between 10% and 1000 times greater than dry weight [7]. However, the
content—and thus the volume—of a hydrogel can change over time, depending on the
external parameters (e.g., the nature of the solvent, temperature, pH), as well as on the
drying process [5]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). There are numerous literature reports on the classification of hydrogels. They can
be grouped according to their source, configuration (amorphous, semi-crystalline, crys- https://www.mdpi.com/journal/materials Materials 2021, 14, 858. https://doi.org/10.3390/ma14040858 Materials 2021, 14, 858 2 of 24 talline) [2,3,8,9], type of cross-linking (chemically cross-linked, physically cross-linked, en-
zymatically cross-linked) [2,10], and polymeric composition (homopolymeric, copolymeric,
multipolymer interpenetrating polymeric—which consists of at least two independent
cross-linked polymers (synthetic or natural), however they are not covalently bonded to
each other) [2,9,11]. Hydrogels can also be divided according to their durability. Despite
their high endurance and stability in swelling environments, durable and degradable
hydrogels can be distinguished. In comparison to natural sources of hydrogels, synthetic
sources have better mechanical strength but a slower degradation rate. However, they
are not biocompatible in vivo, unlike natural hydrogels [9,12,13]. Hydrogels can also be
divided depending on the presence or absence of electrical charge in the polymeric chain. Hydrogels that have no charge in their backbone are non-ionic or neutral. Conversely,
ionic hydrogels are cationic, which are positive-charge-bearing, or anionic, which have
a negative charge in their chains. 1. Introduction These states
depend on various factors, such as the chemical compositions of the materials, the amounts
and types of solvents, the nature of the bonds, the polarity of the polymers, the charges,
and the temperature. However, through physico-chemical modification of the structure
and cross-linking, it is possible to obtain hydrogels with desired viscous, rheological, and
mechanical properties [4,5,18]. p
p
The universality of hydrogels enables their application as biosensors in the food in-
dustry, agriculture, water purification, diagnostics, cosmetics, dentistry, and biomedical
fields (e.g., in pharmaceuticals, drug delivery, and wound dressings) [2,10,19]. In bio-
engineering, they are mainly utilized due to their biocompatibility, biodegradability, and
low immunogenicity. The water content in hydrogels supports their integrity, solubility,
and diffusion of their substances, which are useful factors in delivering a therapeutic
agent. The properties of hydrogels, such as their soft structures, elasticity, and similarity
to living tissues, make them excellent for tissue engineering and regenerative medicine
applications, as well as in stem cell cultures and cell implantation. Tissues and organs in
the human body are characterized by their unique structures, differing in water content, as
well as their mineral and protein contents, therefore it is necessary to use a biopolymer to
manufacture a hydrogel with common building blocks [2,5,6,12]. Hydrogels are excellent
materials for the production of substrates for cell cultures, for example three-dimensional
porous scaffolds that can imitate the extracellular matrix (ECM), ensuring cell proliferation
and differentiation in the in vitro microenvironment. Furthermore, their mechanical and
structural properties allow imitation of native tissues and provide cell protection against
possible damage caused by external factors [6,17,20]. p
g
y
In contrast to 2D cell cultures, 3D structures provide more space for cell proliferation
and more closely mimic the natural environment [17]. The use of hydrogel materials for 3D
cell cultures involves significant challenges, such as the in vitro behavior of the cells and
the influence of external conditions on their appropriate development. Although each cell
line has different requirements and the preparation of a universal in vivo environment is
not possible, the simplicity with which hydrogel properties can be modified enables them
to be customized according to the specific requirements. Furthermore, the utilization in
the biomedical field of 3D printing technology makes it possible to achieve more accurate
geometries and shapes for the manufactured structures [21,22]. 1. Introduction The presence of a charge in the backbones affects their
dissociation in different media environments, i.e., cationic hydrogels swell in media at
lower pH (< 7) and anionic hydrogels at higher pH (> 7) [14]. There is also a group of
ampholyte hydrogels that contain both cationic and anionic functional groups. These are
characterized by their hydrophilicity and good biocompatibility [8,9,15]. The classification
of hydrogels is summarized in Figure 1. Figure 1. Classification of hydrogels. The materials used in the hydrogel manufacturing process include both natural and
synthetic polymers, as well as hybrid hydrogels, which involve combinations of natural and
synthetic polymers [2,12]. These natural polymers can be obtained from natural sources
such as plants, bacterial cultures, or animals, including polysaccharides (e.g., agarose, algi-
nate, carrageenan, chitosan), glycosaminoglycans (e.g., heparin, hyaluronic acid, keratan
sulfate), and polypeptides or proteins (e.g., collagen, fibrin, gelatin, silk). Examples of
synthetic polymers used to manufacture hydrogels include poly(ethylene glycol) (PEG),
poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(vinyl alcohol) (PVA) [1,16,17]. Hy- Figure 1. Classification of hydrogels. Figure 1. Classification of hydrogels. Figure 1. Classification of hydrogels. The materials used in the hydrogel manufacturing process include both natural and
synthetic polymers, as well as hybrid hydrogels, which involve combinations of natural and
synthetic polymers [2,12]. These natural polymers can be obtained from natural sources
such as plants, bacterial cultures, or animals, including polysaccharides (e.g., agarose, algi-
nate, carrageenan, chitosan), glycosaminoglycans (e.g., heparin, hyaluronic acid, keratan
sulfate), and polypeptides or proteins (e.g., collagen, fibrin, gelatin, silk). Examples of
synthetic polymers used to manufacture hydrogels include poly(ethylene glycol) (PEG),
poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(vinyl alcohol) (PVA) [1,16,17]. Hy- The materials used in the hydrogel manufacturing process include both natural and
synthetic polymers, as well as hybrid hydrogels, which involve combinations of natural and
synthetic polymers [2,12]. These natural polymers can be obtained from natural sources
such as plants, bacterial cultures, or animals, including polysaccharides (e.g., agarose, algi-
nate, carrageenan, chitosan), glycosaminoglycans (e.g., heparin, hyaluronic acid, keratan
sulfate), and polypeptides or proteins (e.g., collagen, fibrin, gelatin, silk). Examples of
synthetic polymers used to manufacture hydrogels include poly(ethylene glycol) (PEG),
poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(vinyl alcohol) (PVA) [1,16,17]. Hy- Materials 2021, 14, 858 3 of 24 drogels can occur in different forms, such as solid, semi-solid, and liquid states. 1. Introduction Three-dimensional printing
is a versatile technique for creating complex biomimetic tissue structures with extraordinary
spatial precision [23]. The aim of this review is to present trends in the utilization of alginate–gelatin hydro-
gels as substrates in 3D cell cultures, as well as an overview of their material characteristics. Due to the lack of biological activity, sodium alginate cannot be employed alone for the
production of hydrogel matrices. It is most often used in combination with other polymers
to allow the adhesion of cells and their healthy development. The combination of sodium
alginate and gelatin provides an excellent hydrogel for use as a substrate for 3D cell cultures
due to its unique biological properties, such as its biocompatibility, biodegradability, and
non-toxicity. Some of the recent additions to this field are cellulose nanomaterials, which
improve the chemical, physical, and biological properties of the created hydrogels. The eas-
ily modifiable mechanical properties of these materials can be adapted to the living tissue,
making them ideal environments for cell culture development. Moreover, the rheological
properties of alginate-gelatin hydrogels make them excellent candidates for utilization in
additive technologies at room temperature. Thus, it is possible to use them as hydrogel
inks within the cell content, without risking damage. 2. Materials and Methods for the Preparation and Characterization of Hydrogel Substrates The most important characteristics of a hydrogel are its permeability and water
retention capacity. In contact with water, the polar hydrophilic groups are hydrated, which
leads to the creation of primary bound water and swelling of the network. This results in the
exposure of hydrophobic groups capable of interacting with water molecules and forming Materials 2021, 14, 858 4 of 24 secondary bound water. Often total bound water is also formed by combining primary
and secondary bound water. The hydrogel reaches a balanced swelling level by creating an
elastic network retracting force by opposing covalent or physical cross-linking [24,25]. g
y pp
g
p y
g
The biocompatibility of a hydrogel depends on the interaction of the material and
its degradation products with living tissue or body fluids—it should not induce toxic or
immunological responses. The low interfacial free energy of the hydrophilic surface in
contact with body fluids results in a low tendency of proteins and cells to adhere to these
surfaces, which makes the hydrogel satisfactorily biocompatible [26,27]. Hydrogels are
characterized by their flexibility and soft structures. Therefore, these hydrated polymeric
biomaterials are often utilized in biomedical fields as ECM substitutes. The biological,
physical, and mechanical properties, as well as the permeability control of hydrogels,
provide a biomimetic microenvironment similar to that of living tissue. The physico-
chemical viscosity modification of a hydrogel allows control of its mechanical properties,
as well as enabling various forms of the material to be achieved for 3D cell cultures, such
as beads, injectable gels, moldable gels, and macroporous structures [28,29]. A hydrogel’s
porosity is a very important aspect, because large surface areas foster attachment and
ingrowth of cells. Moreover, pores connected by channels enable transport of gases,
nutrients, and waste [20]. Hydrogel cell culture substrates can be prepared as mono- or multi-component mix-
tures of different polymers ratios. Composite substrates are preferred due to their ability
to combine the functions of the individual components, as opposed to single-component
substrates [30]. Among the natural polymers used for hydrogel fabrication for 3D cell
cultures are alginate, collagen, gelatin, and fibrin. One of the most popular materials is
based on sodium alginate [29,31]. Alginate hydrogel is an excellent material for imitating
the extracellular matrix, which is the outer environment of the cell. 2. Materials and Methods for the Preparation and Characterization of Hydrogel Substrates However, this natu-
ral polymer does not provide mammalian cell-adhesive ligands, i.e., short polypeptide
sequences in ECM proteins, which mimic ligands used in integrin-mediating cell adhesion
formation and facilitate cell attachment [32,33]. In order to form three-dimensional struc-
tures in hydrogels for cells, components that are part of the in vivo matrix are required. On the surfaces of cells are receptors, which allow the cells to bind with matrix ligands. Typically, cells have receptors for many different ECM factors, which allow them to simul-
taneously bind to various ligands and to form networks. The cells themselves also produce
components of the extracellular matrix; nevertheless, supplementation of hydrogels with
ligands is required [34]. Thus, to mimic the ECM, this material is utilized in combination
with other materials, such as gelatin, chitosan [32], elastin, agarose, or hyaluronic acid [20]. Moreover, additives such as nanocellulose act as reinforcing agents in hydrogel materials,
increasing the mechanical properties and improving the printability [35]. In this review,
we would like to focus mainly on alginate–gelatin hydrogels for cell cultures. 2.1. Alginate Alginate is commonly used to manufacture hydrogels due to its ability to retain water. This linear polysaccharide is obtained from brown seaweed or bacteria. The quantity and
quality of alginate extracted from brown seaweed may depend on the species, as well as the
harvest season. Typically, the amount of extracted polysaccharide is up to 40% of the dry
matter of brown seaweed and also strongly depends on the extraction method [17,36,37]. Alginate consists of two hexuronic acids—α-L-guluronic (G) and β-D-mannuronic (M),
linked by 1–4 glycosidic bonds, as shown in Figure 2 [20,38]. Ionic gel formation depends
on the chemical composition and block distribution in the alginate molecule [17,28]. The
physical properties are determined by the numbers of MM, GG, and MG blocks. The high
viscosity is dependent on the high content of M blocks, while the high content of G gives
higher gelling properties [39]. Alginate-based gels can also be evaluated using the M/G
ratio. Flexible gels are obtained using a high M/G ratio, while brittle gels are obtained
from sodium alginate with a low M/G ratio [40]. Although alginate is biocompatible,
biodegradable, and non-toxic, it has several disadvantages, such as low its bioadhesivity Materials 2021, 14, 858 5 of 24 and biological inertness, which limit its applications, particularly in tissue engineering. The
main drawback of alginate is the difficulty of obtaining pure alginate from contaminated
marine algae. Removal of impurities is possible through further purification steps, which
generate additional costs and are time-consuming [41,42]. and biological inertness, which limit its applications, particularly in tissue engineering. The
main drawback of alginate is the difficulty of obtaining pure alginate from contaminated
marine algae. Removal of impurities is possible through further purification steps, which
generate additional costs and are time-consuming [41,42]. Figure 2. Chemical structure of alginate (M and G blocks). Reproduced with permission from [43]. Copyright 2020 IOSR Journal of Pharmacy. Figure 2. Chemical structure of alginate (M and G blocks). Reproduced with permission from [43]. Copyright 2020 IOSR Journal of Pharmacy. In order to increase the mechanical properties of alginate hydrogels, a variety of
covalent cross-linking methods have been used. However, the mechanical properties of
ionically cross-linked alginate hydrogels decrease over time on account of external streams
of cross-linking ions into the surrounding environment [44]. The mechanical properties of
alginate-based hydrogels may also depend on the gel homogeneity, which can be controlled
by the gelling rate. 2.1. Alginate An important parameter that affects the gelling rate is the temperature
of gelation [45]. The combination of alginate hydrogels with other materials can lead to the
improvement or changes of their physical and mechanical properties [46]. Alginates are available in various forms, including hydrogels, microcapsules, micro-
spheres, fibers, foams, and sponges. However, the most commonly used form, especially
in the medical field, is the hydrogel form [46]. The rheological properties of the alginate
hydrogel and its ease of gelation, biodegradability, and lack of toxicity make it suitable for a
wide range of industrial applications (e.g., in the food industry, agriculture, cosmetics, pack-
aging, and textiles). Its biomedical applications include dentistry, pharmaceuticals, wound
healing, drug delivery, cell therapy, and as a bone graft substitute for spine fusion [4,47]. Fabrication of this hydrogel in the medical industry requires the use of high-purity sodium
alginate to prevent immunogenicity [28,29]. 2.2. Gelatin Gelatin is a type of protein manufactured through hydrolysis of collagen of animal
origin (i.e., bovine, porcine, or fish collagen), connective tissues, and bones [32,48]. Its
biocompatibility, lack of immune responses in the body, degradability, and absence of
toxicity resulted in its approval by the U.S. FDA. Thus, it is used in the food industry,
as well as in the biomedical sector (e.g., pharmacy, wound healing, drug delivery, gene
therapy, tissue engineering, regenerative medicine). It is one of the most often utilized
materials for cell cultures due to its retention of collagen’s bioactive sequences. This enables
the creation of a suitable microenvironment for cell adhesion, migration, proliferation, and
differentiation [17,32]. Despite its numerous advantages, gelatin also has some drawbacks. One of them is its solubility, because at around 30–40 ◦C it passes from a gel state to
a solution, which limits its long-term use in transplantation. In order to prolong the
degradation time and increase the water resistance of gelatin, it is necessary to apply
cross-linking [49]. In Figure 3, the basic chemical composition of gelatin is shown. Materials 2021, 14, 858 6 of 24 Figure 3. Chemical structure of gelatin. Reproduced with permission from [50]. Copyright 2020 John Wiley & Sons. Figure 3. Chemical structure of gelatin. Reproduced with permission from [50]. Copyright 2020 John Wiley & Sons. Gelatin is exposed to factors such as temperature, pH changes, and the presence of
solvents, which modify its flexibility, meaning it takes various forms. Below 40 ◦C it can
occur in a gel state (the reaction is thermally reversible), while above this temperature it
can become an aqueous solution. Chemical modifications and cross-linking change the
structure of gelatin hydrogel, as well as its properties and the forms it acquires [32,51]. Depending on the animal species, gelatin may have different properties. Wang
et al. [52] provided a comparison of methacrylate gelatin from cold water fish and pigs
and cold-soluble gelatin. Cold-soluble gelatin has a balance between thermal stability
and physical and biological properties compared to the other types of gelatin. Cold water
fish gelatin also has thermal stability when compared to pork [52]. A comparison of the
properties of gelatin from cold water fish and pigs was also made by Yoon’s [53] research
team. The research showed the same results as in Wang’s study [53]. 2.3. Alginate Dialdehyde-Gelatin Hydrogels Lately, there has been a lot of interest in hydrogels, which are a combination of
natural polysaccharides (e.g., alginate) and proteins (e.g., gelatin). This is due to the
adverse properties of alginate itself, such as its inefficient cell attachment, poor alginate–
cell interactions, and slow degradability with uncontrolled kinetics [54]. The solution to
these limitations may be the use of alginate that has been previously oxidized to form
alginate dialdehyde (ADA) and which is subsequently covalently cross-linked with gelatin
(GEL). Between aldehyde groups of ADA and free amino groups of gelatin, Schiff’s base is
formed [51,55,56]. The chemical structure of ADA-GEL is shown in Figure 4. The obtained alginate-gelatin cross-linked hydrogel (ADA-GEL) can be used to pro-
duce microcapsules (for encapsulation of bioactive compounds or cells and for drug
delivery) [56–58] and can be applied as a non-cytotoxic biomaterial with good mechanical
strength and biocompatibility in regenerative medicine (e.g., bone tissue regeneration) [55],
or as a soft tissue adhesive in wound healing [59]. The obtained alginate-gelatin cross-linked hydrogel can differ in its microstructure
and physico-chemical properties, depending on the oxidation degree of the ADA and
the cross-linking degree and gelation time of the ADA-GEL [51,55,59]. Microcapsules
produced from ADA-GEL are characterized by their higher degradability when compared
to alginate microcapsules [51], and exhibit good cell adhesion, proliferation, and migration
properties [51,57,60]. An interesting application of alginate dialdehyde cross-linked gelatin
hydrogel was suggested by Manju et al. [61]. This hydrogel was used as a coating material
for polyethylene terephthalate (PET) vascular graft prostheses, which are intended to
replace compromised arteries in the body. The ADA-GEL was non-toxic and was charac-
terized by its biocompatibility, biodegradability, and reduced water permeability, while
it had no effect on the mechanical properties of the graft and improved the adhesion and
proliferation of fibroblast cells. Materials 2021, 14, 858 7 of 24 Figure 4. Chemical structure of alginate dialdehyde-gelatin (ADA-GEL). Reproduced with permis-
sion from [51]. Copyright 2020 Royal Society of Chemistry. Figure 4. Chemical structure of alginate dialdehyde-gelatin (ADA-GEL). Reproduced with permis-
sion from [51]. Copyright 2020 Royal Society of Chemistry. ADA-GEL can be also used as an ink, but this application may be limited due to its
viscosity [58]. Nevertheless, there are some studies using ADA-GEL as an ink in 3D printing
technology. In the work by Dranseikiene et al. [58], ring-shaped structures containing
alginate dialdehyde-gelatin cell-laden hydrogel were fabricated. 2.3. Alginate Dialdehyde-Gelatin Hydrogels The cell viability (MG-63
osteosarcoma cells) in samples depended on the cross-linking agent (CaCl2 or BaCl2),
with better results in terms of increased cell viability and cell network formation being
observed after cross-linking with Ba2+. Ruther et al. [60] investigated the printing of three-
dimensional, cell-containing, vessel-like structures using ADA-GEL ink. This artificial
vessel supported the viability, proliferation, and migration of immobilized cells (human
dermal fibroblast and human umbilical vein endothelial cells). 3. Material Additives Used in Gel Systems Apart from the basic materials forming the gel systems, additives are also used to
increase their efficiency. Material additives are inactive ingredients designed to structurally
improve and enhance the hydrogel’s characteristics, as well as influence the degradation
profile and interactions between cells and the matrix. Among the additives, materials
from natural (e.g., collagen, pectin), synthetic (e.g., polyvinyl alcohol, poloxamers), and
semi-synthetic sources (e.g., cellulose derivatives) can be distinguished. The used mate-
rials include peptides that increase cell adhesion, nanomaterials that adjust rheological
properties to specific applications, as well as growth factors that enable direct stem cell
differentiation [62]. Additives added to alginate hydrogel culture media are used to influ-
ence cell growth and attachment. Furthermore, they improve resistance to degradation and
facilitate the production of the hydrogel, which is a benefit during additive manufacturing
and processing in terms of bioprinting [63]. To enhance the strength and increase the mechanical properties of hydrogels when
using additive manufacturing technologies in tissue engineering, additives in the form
of microfibers, nanofibers, microparticles, and nanoparticles, as well as woven and non-
woven scaffolds, are also used. Reinforcement of hydrogels in this manner eliminates
the need for dense cross-linking, allowing them to obtain similar mechanical properties,
thereby improving cell migration and promoting the formation of neo-tissues [64]. The
nanomaterials included in hydrogels, such as cellulose and chitin, can affect the structures
of the hydrogels, thus improving their mechanical properties and drug release profiles [65]. Materials 2021, 14, 858 8 of 24 Zhang et al. [66] created hydrogel composites with alginate microcapsules cross-linked with
Ca2+ ions and cellulose whiskers. As a result, the mechanical properties of the hydrogels
were improved and the release profile of the drug was more stable, without causing
cytotoxicity. In Kinneberg’s work [67], the application of a microporous, fibrous collagen
scaffold increased the tangent modulus and toughness of the hydrogel. Research carried
out by Park et. Al. [68] showed that the addition of nanocellulose to alginate hydrogel
enhanced its mechanical strength, but also improved the cells’ ability to proliferate and
prolonged their viability. Similar research was carried out by Siquiera et al. [69], whereby
the viability of L929 fibroblast cells was examined in alginate hydrogels and compared to
nanocellulose–alginate hydrogels containing nanofibers and nanocrystals. The addition
of nanocellulose supported the cell bioadhesion and cell growth on the created medium. 3. Material Additives Used in Gel Systems Nanocellulose is one of the materials that improves not only the mechanical properties of
the created substrates, but also has a direct effect on cells and their development. 3.1. Hydrogel Fabrication from Materials Useful for 3D Cell Culture Various materials can be used to create different types of hydrogel matrices. The
compositions of these media depend on the type of culture and the results expected by the
researchers. There is no doubt that 3D cultures and systems are more reliable setups that
faithfully recreate cellular responses. The available data show that 3D cells do not have
receptors that recognize alginates. Having high purity is of special importance, because
some residual endotoxins can interact with receptor CD14 (a receptor for complexes of
lipopolysaccharide and lipopolysaccharide binding protein) [28]. Thus, highly purified
alginates are crucial in future applications. Natural substrates such as sodium alginate
have been used by Zimmermann et al. [70], Dvir-Ginzberg et al. [71], and Bauer et al. [72]. It was also noted that alginates can be functionalized to obtain specific properties. For
instance, it was found that arginine–glycine–aspartic acid conjugated to sodium alginate
(RGD-alginate) can stimulate cell proliferation and can affect cell adhesion and differentia-
tion [73]. De Melo et al. [74] investigated PEG poly(ethylene glycol) and sodium alginate
concentrations to optimize a polymer blend for usage as a mechanically resistant support
hydrogel. It was also shown that a combination of alginate with gelatin can decrease
gelatin leakage over prolonged culture while simultaneously enhancing cell adhesion
and the secretion of vascular endothelial growth factor (VEGF) [57]. Chung et al. [75]
and Li et al. [76] prepared a sodium alginate-gelatin hydrogel in order to examine cell
development. The gelatin strengthened the cell-filled sodium alginate scaffolds. Demirta¸s
et al. [77] fabricated sodium alginate and alginate-hydroxyapatite (HA) hydrogels and
used them for printing. Schwarz et al. [78] manufactured 3D-printed, grid-like structures
for cartilage tissue engineering by using a system composed of an oxidized alginate-gelatin
hydrogel. The examples of the applied materials for the 3D cell culture substrates and their
concentrations are shown in Table 1. 9 of 24 Materials 2021, 14, 858 Table 1. The examples of hydrogel materials based on alginate and gelatin used for the production of 3D cell culture substrates. 3.1. Hydrogel Fabrication from Materials Useful for 3D Cell Culture Hydrogel
Type of Cells
Sodium Alginate
Concentrations (w/v%)
Gelatin/Other Additive
Concentrations (w/v%)
The Aim of Research
Reference
sodium alginate
C2C12 mouse myoblasts
2.5
testing the influence of substrate stress–relaxation on the
regulation of muscle cell (myoblast) proliferation and spreading
in vitro
[72]
sodium alginate-gelatin
BL6 primary mouse myoblasts
1, 2, 4
10
optimization of the bioink consistency and investigation of
printability with live cells
[75]
sodium alginate-gelatin
rat Schwann cell line RSC96s
4
20
examination of the cell behavior in the created microenvironment
[76]
sodium alginate-gelatin
L929, mouse fibroblast cell line
1
4
evaluation of the cell viability and possibility to develop in the
created microenvironment
[44]
2–2.5
4–8
analysis of the printability with the cell-filled bioink and
optimization of the parameters to obtain structures for
cell cultures
[79]
2.5
8
sodium alginate-gelatin
Non-Small Cell Lung Cancer (NSCLC)
PDX (EGFR T790M) cell line
1–6
3–8
research on the cells growth on the manufactured hydrogel;
modification of the alginate-gelatin concentration to achieve
good printability
[80]
sodium alginate-gelatin
mesenchymal stem cells (MSCs) from
adult sheep adipose tissue
1, 3, 5, 7, 9
6
examination of the printing possibilities according to the
individual component concentrations
[81]
5
2, 4, 6, 8, 10
sodium alginate-gelatin
human adipose-derived mesenchymal
stem cells (MSCs)
3
10
evaluation of the cell development on produced substrates
[82]
sodium alginate-gelatin with mouse
plantar dermis (PD)
mouse mesenchymal stem cell (sMSCs)
1
3
investigation of the chemical and physical properties of the
Alg-Gel-PD bioink, and its effect on embedded mouse
mesenchymal stem cells (MSCs)
[83]
sodium alginate-gelatin mixed with
hECM
HepaRG human bipotent hepatic
progenitor cells; A549 human epithelial
lung carcinoma cells
2
3
the optimization of a bioink composed of hydrogel and human
extracellular matrix (hECM) to print human HepaRG liver cells
and testing of its suitability for the study of transduction using an
adeno-associated virus (AAV) vector and infection with human
adenovirus 5 (hAdV5)
[84]
hECM
0; 0.25; 0.5; 1; 2
alginate dialdehyde-gelatin (ADA-GEL)
osteosarcoma cells MG-63
5
5
evaluation of the cell encapsulation possibility in prepared
hydrogel and monitoring of cell activity
[58]
alginate dialdehyde–gelatin (ADA-GEL)
HUVECs human umbilical vein
endothelial cells; rat Schwann cells
2; 3; 6
2
characterization of the printability and cell viability of various
concentrations of alginate dialdehyde (ADA)–gelatin (Gel)
hydrogels for bioprinting
[85]
2
3; 6
sodium alginate with d-gluconic acid
rat liver cell
1
0.2
analysis of the cell growth on the created substrate
[71]
sodium alginate/PEO-bisamine
(polyoxyethylene bis(amine))
L929, mouse fibroblast cell line
2
5 and 20
evaluation of the cell viability and possibility of developing in
created microenvironment
[44]
sodium alginate/PEO with gelatin
mesenchymal stem cells (MSCs)
2, 4, 5
PEO 1:1 with alginate; gelatin: 10%
examination of the cell viability and possibility of proliferating in
created microenvironment
[86] of hydrogel materials based on alginate and gelatin used for the production of 3D cell culture substrates. 3.1. Hydrogel Fabrication from Materials Useful for 3D Cell Culture PEO 1:1 with alginate; gelatin: 10% Materials 2021, 14, 858 10 of 24 10 of 24 According to the type of cell, different conditions are needed for growth, proliferation,
or differentiation, and therefore a surface is required for culture, which will provide the
cells with a specific environment. Alginate-gelatin-based hydrogels are mainly found
as substrates in laboratory studies, however there are already commercially available
cell culture substrates made from other materials, which are intended for specific cell
types. One of them is Matrigel, a gelatinous protein mixture obtained from Englebreth–
Holm–Swarm tumor cells from mice. It is used as a basement membrane matrix for stem
cells and maintains them in an undifferentiated state. However, this substrate is not
precisely defined, thus it may not reflect the accuracy of the research results [87]. Similar
to Matrigel is Cultrex® BME, which is used as its substitute. Cultrex® BME is also a
homogenate of mouse tumor tissue, therefore both substrates differ in composition from
human transmembrane proteins [88]. HyStem®-C is a biodegradable and biocompatible
material consisting of three blocks: Glycosil® (thiolated hyaluronic acid), Gelin® (tioltated
gelatin), and Extralink® (polyethylene glycol diacrylate). It is capable of mimicking ECM,
and therefore is used as a tissue model in developing new drugs and cell cultures. It is a
suitable material for bioprinting and for designing organ models [89]. Another hydrogel
suitable for cell culture is Biogelx™, obtained from synthetic peptide powder functionalized
with collagen, fibronectin, and laminin, which was created to mimic the ECM. PuraMatrix™
is a peptide hydrogel consisting of amino acids that is suitable for 3D cell culture [6]. Most
of these products are used for research purposes. However, new materials for 3D cell
cultivation and bioprinting, such as bacterial cellulose–gelatin hydrogels [90]; synthetic,
thermo-responsive polyisocyanopeptide hydrogel [91]; chitosan–fibrin hydrogel for tissue
engineering [92]; collagen-based hydrogels [93]; and others are currently being fabricated
and tested. 3.2. Hydrogel Cross-Linking Methods Cross-linking is a process that leads to the formation of a three-dimensional polymer
network structure by binding one polymer to another. It is a stabilization process—the
attached polymers lose their ability to move as a single chain. The consequence of cross-
linking is a change in the physical and chemical properties of a substance. Cross-linked
polymers are mechanically stronger but less flexible and less heat- or solvent-resistant in
comparison to liquid polymers [94]. p
q
p
y
Cross-linking of hydrosols (liquid polymers) has positive effects on the physico-
chemical properties of hydrogels—including their water holding capacity, hardness, and
structural integrity, which affect their cytocompatibility—as well as on their biochemical
properties [94,95]. In bioprinting, the cells are mixed in hydrosol and the cross-linking of
the hydrogel takes place after extrusion from the bioprinter. Thus, the cross-linker should
not be toxic to cells and should have fast cross-linking capability to maintain the highest
cell viability [96]. Sodium alginate is most often physically cross-linked through the application of ionic
interactions [45,94,95]. Cross-linking with divalent or trivalent ions occurs under mild
conditions, at room temperature and neutral pH [94,96]. Such cross-linking conditions
are suitable when using cells. Cross-linking of sodium alginate occurs through a bond
between G blocks (guluronate blocks) and divalent ions. This is an “egg box” cross-linking
model [45,95]. Ca2+, Ba2+, and Zn2+ are divalent ions, which are usually used in such ap-
plications [45,95,96]. An example of ionic cross-linking is presented in Figure 5. The choice
and concentration of ionic cross-linkers is a critical factor in order to obtain enhanced cell
viability, better printability, and mechanical stability of a hydrogel during bioprinting [96]. Other studies have shown that the subsequent addition of cross-linkers in appropriate
ratios can improve the cell viability and mechanical stability of hydrogels [96–98]. The sta-
bility of hydrogel cross-linking in cell cultures is an important issue. Under physiological
conditions, divalent ions can be exchanged with monovalent cations, which contribute to
the depletion of the network [45]. 11 of 24 11 of 24 Materials 2021, 14, 858 Figure 5. Graphical illustration of ionic cross-linking of polymer chains (own source). Figure 5. Graphical illustration of ionic cross-linking of polymer chains (own source). Photo cross-linking is another covalent cross-linking approach. Modified sodium
alginate with the addition of methacrylate can be cross-linked via exposure to an argon ion
laser (514 nm) for 30 s in the presence of eosin and triethanolamine. 3.3. 3D Cell Cultures Cell cultures growing in the three-dimensional form behave quite differently than
in a monolayer. Due to the use of 3D culture, the impact of cell interaction and the
influence of the external environment on cell development have been investigated. This is
why the research on three-dimensional cell cultures is so important. In vitro experiments
aim to reflect the in vivo conditions to the greatest extent. Each cell line has individual
requirements and conditions that must be met to achieve the most reliable test results. It
is not realistic to work out a uniform template of optimized culture conditions for all cell
lines. However, for faster optimization of three-dimensional cell culture conditions, it is
worth paying attention to certain aspects described below. This review focuses on the use
of 3D-printed alginate-gelatin hydrogel scaffolds in three-dimensional cell cultures. Hydrogels obtained from sodium alginate and gelatin imitate the extracellular matrix. Both components of the gel, which are cross-linked under mild conditions, are transparent;
decomposition of the network can take place under controlled conditions; and the porosity
of the material provides the cells with access to nutrients and metabolite exchange [28,105]. It is worth noting that gelatin as an animal-derived material could be an immunogenic
agent. However, the use of sodium alginate alone does not provide optimal conditions for
the development of culture [28,106]. Depending on the application of the hydrogel, the selection of the type and con-
centration of sodium alginate and gelatin affects the properties of the hydrogel and the
cell growth [28,107]. The number and length of G blocks that are involved in ion cross-
linking depend on the porosity, stability, and elasticity of the gel [28,108]. The mechanical
properties can affect cell proliferation and conditions. Alginate has different affinities for divalent ions, which is why their selection can
affect such properties as swelling, stability, and cell growth [28]. Sarker et al. [96] studied
the effects of three ionic cross-linkers of alginate—CaCl2, ZnCl2, and BaCl2—in terms
of the viability of Schwann cells (the main glial cells of the peripheral nervous system)
and the mechanical properties of hydrogels. Research showed that the best of these three
cross-linking ions in terms of the impact on mechanical properties was Ba2+, while Ca2+
had the best effect on cell viability. The research team suggested using both cross-linking
compounds, namely CaCl2 and BaCl2 [96]. 3.2. Hydrogel Cross-Linking Methods Materials 2021, 14, 858 12 of 24 12 of 24 Gelatin, as a product of collagen hydrolysis, can be cross-linked with transglutaminase,
which catalyzes the acyl transfer reaction between the γ-carboxamide group of peptides
or protein-bound glutamine residues (acyl donors) and primary amines (acyl acceptor)
containing ε-amino groups of lysine. A network of isopeptide bonds is formed [102]. This approach was used in research conducted by Schwarz et al. [78] and Chen et al. [95]. The properties of interpenetrating alginate-gelatin hydrogel have been studied. Sodium
alginate has been cross-linked with calcium ions, while gelatin has been cross-linked with
transglutaminase [95]. Another gelatin cross-linking agent could be water-soluble 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide (EDC), which specifically forms amide or peptide
bonds between the carboxylic acid and protein amino groups [103]. One way to cross-link gelatin is to modify it with tyramine (TA). TA–gelatin is obtained
by carbodiimide coupling of tyramine groups on gelatin. Then, TA–gelatin is covalently
cross-linked in the presence of horseradish peroxidase (HRP) and hydrogen peroxide
(H2O2). Tyrosine–tyramine, tyrosine–tyrosine, and tyramine–tyramine bonds are formed. The addition of TA–gelatin increases the mechanical stability and gelation rate and slows
the enzymatic degradation of silk hydrogels cross-linked in physiological buffers. Ma-
nipulation of the amount of TA–gelatin added allows modulation of the silk hydrogel’s
bioactivity, in turn affects the cell’s microenviroment [104]. 3.2. Hydrogel Cross-Linking Methods Cross-linking takes
place in mild conditions with the proper selection of chemical initiators. This method can
be used directly in contact with drugs and cells. To avoid harmful cross-linking conditions
(where a light sensitizer or acid release is required as a by-product), polyallylamine partially
modified with acetylphenoxyinninamyldiene chloride is used as an alternative, which
converts to a dimer under the influence of light at about 330 nm and does not release toxic
by-products during the cross-linking reaction [45,99]. Additionally, using light radiation,
the resulting hydrogel from sodium alginate and polyallylamine has improved mechanical
properties [45]. Thermal gelation is the next cross-linking approach. The use of this method for sodium
alginate is not common due to the poor sensitivity of sodium alginate to temperature. How-
ever, semi-interpenetrating polymer networks (semi-IPN) can be used. An example is a
semi-IPN prepared from copolymerization of N-isopropylacrylamide (NIPAAm) with a
poly(ethylene glycol)-co-poly(-caprolactone) (PEG-co-PCL) macromer in the presence of
sodium alginate using UV irradiation. The use of sodium alginate in semi-IPNs improves
the mechanical strength. The increase in hydrogel swelling with the increase in the con-
centration of sodium alginate at a constant temperature and decrease in swelling with the
increase in temperature can be used for drug delivery [45,100]. Covalent cross-linking is another method. Covalent networks are formed by poly-
merization of end-functionalized monomers or macromers (Figure 6) [94]. In this type of
network, cross-linking dissociation and bond transformation do not occur. Water migration
causing stress relaxation is also preserved. Chemically modified sodium alginate is used
for covalent cross-linking [28]. One of the covalent cross-linkers used in the cross-linking
of sodium alginate is poly(ethylene glycol) (PEG), which is used for PEG-diamines. It
has been shown that the mechanical properties and swelling can be controlled by using
covalent cross-linking agents. Using hydrophilic cross-linkers can compensate for the lack
of the hydrophilic nature of the gel due to the cross-linking reaction [45]. Figure 6. Graphical illustration of covalent cross-linking of polymer chains (own source). Figure 6. Graphical illustration of covalent cross-linking of polymer chains (own source). An interesting approach to create cross-linked hydrogels is to use the cells themselves. For example, cells added to adhesive ligand-modified sodium alginate (arginine-glycine-
aspartic acid peptide sequence-modified (RGD-modified)) can form networks by binding
multiple polymer chains based on the receptor–ligand binding. This method of cross-
linking occurs without the addition of cross-linkers [45,101]. 3.3. 3D Cell Cultures When forming hydrogels for cell cultures, an important aspect is sodium alginate
modification in order to adapt and control the type of and interaction between cells. Examples of such modifications are the covalent attachment of heparin-binding peptides
(HBP) [28,109] or peptides found in extracellular matrix proteins [28,109,110]. In a ligand– Materials 2021, 14, 858 13 of 24 13 of 24 receptor approach, cells can interact with the hydrogel network. For example, attachment
of the RGD (arginine–glycine–aspartic acid) peptide sequence has also been shown to
affect the initiation of interactions with cells via recognition of an adhesive molecule (RGD
sequence) by cell receptors. Some cell lines may show accelerated proliferation when
sodium alginate is modified by the RGD sequence [28,111]. As it turns out, the sequence
fragments flanking the RGD and the conformation (linear or cyclic) and length of the RGD
sequence all impact the integrin receptor affinity and selectivity, depending on the cell type. Additionally, depending on the cell type, the used RGD sequence density may affect the
development of the culture [28,112]. An important issue when establishing a 3D cell culture is the printing process. Atten-
tion should be paid to parameters such as the rheological properties, printing process, and
needle diameter relative to time; the dimensions of the printing thread; and the impact
on cell survival. Currently, computational simulation can be used to implement process
design strategies, as well as comparisons with other inks [113]. g
g
p
Table 2 shows a comparison of studies on alginate and gelatin-based hydrogels in three-
dimensional cell cultures. Depending on the composition of the hydrogel, the modification
strategy, and the cross-linking methods used, different concentrations of alginate and
gelatin were used. Based on these few examples, it can be seen that studies on three-
dimensional cell cultures have been carried out on different cell lines. Table 2. Comparison of the hydrogel compositions depending on the cross-linking methods and modifications used. Cell Line
Concentration (w/v%)
Cross-Linking Method
Modification
Reference
Sodium Alginate
Gelatin
Sodium Alginate
Gelatin
NB SH-SY5Y *
0.50–0.75
2.00–5.00
Ionic (CaCl2)
Covalent
(transglutaminase)
n.a. [95]
1) Hbmsc *;
2) D1 stem cells *;
3) MC3T3-E1 *
2.00
n.a. n.a. n.a. 1) linear RGD
2) cyclic RGD
[114]
RSC96 *
1.00–2.50
n.a. Ionic (CaCl2)
n.a. 1) added poly-L-lysine
2) added fibronectin
3) added RGD
4) covalent binding RGD
[115]
ACPCs *
n.a. 8.00
n.a. 3.3. 3D Cell Cultures 1) Free radical
polymerization
2) Free radical
polymerization and
covalent
1) methacrylic anhydride
(GelMA)
2) methacrylic anhydride
and tyramine (GelMA-Tyr)
[116]
1) HepaRG *
2) A549 *
2.00
3.00
1) Ionic
(CaSO4)—before
printing
2) Ionic (CaCl2)—after
printing
3) Ionic
(CaCl2)—during
incubation
n.a. human extracellular matrix
(hECM)
[84]
1) Saos-2 *
2) hBMSCs *
4.00
5.00
Ionic (CaCl2)
n.a. 1) cellulose nanofiber (CNF)
2) bioactive glass (BaG)
[117]
1) HUVECs *
2) MSCs *
1.00; 2.00; 3.00
GelMA:5.00;
7.00
Ionic (CaCl2)
Free-radical
polymerization
1) 4-arm poly(-ethylene
glycol)-tetra-acrylate
(PEGTA)
2) methacrylic anhydride
[118]
Note: NB SH-SY5Y—human neuroblastoma cell line; hBMSC—human bone marrow stem cells; D1—mesenchymal stem cells; MC3T3-E1—
osteoblastic cell line; RSC96—neuronal Schwann cell; ACPCs—articular cartilage progenitor cells. Table 2. Comparison of the hydrogel compositions depending on the cross-linking methods and modifi he hydrogel compositions depending on the cross-linking methods and modifications used. Y—human neuroblastoma cell line; hBMSC—human bone marrow stem cells; D1—mesenchymal stem cells; MC3T3-E1—
ne; RSC96—neuronal Schwann cell; ACPCs—articular cartilage progenitor cells. Note: NB SH-SY5Y—human neuroblastoma cell line; hBMSC—human bone marrow stem cells; D1—mesenchymal stem cells; MC3T3-E1—
osteoblastic cell line; RSC96—neuronal Schwann cell; ACPCs—articular cartilage progenitor cells. Alginate-hydrogel-based cell constructs are good candidates for tissue engineering
and regenerative medicine applications (Figure 7). The available studies indicate that stem
cells can be combined with biomaterial scaffolds for in vivo and in vitro research. There
are various biomaterial formulations for tissue engineering, e.g., hydrogels, electrospun
nanofibers, and microspheres. Alginate material is recommended for hydrogels and
electrospun nanofibers [119]. This material was efficiently used with adipose-derived adult
stem cells (ASCs) and bone marrow-derived MSCs (mesenchymal stem cells) to generate
replacement cartilage [120] and was also used with neural tissue [121]. In another study,
the authors blended calcium alginate gels for internal and external gelation. The gels
were injectable and could be applied as immobilization matrices for human MSCs [122]. Materials 2021, 14, 858 14 of 24 14 of 24 Pandolfiet al. [123] verified that alginate-based microencapsulations can be efficiently
applied for transplants in patients that are not eligible for immunosuppressive therapies. Consequently, alginate-based materials seem to be good candidates for tissue engineering
and regeneration. Figure 7. Comparison of 2D culture and 3D model based on alginate hydrogel (own source). Figure 7. Comparison of 2D culture and 3D model based on alginate hydrogel (own source). Clinical applications for the materials will be possible in the relatively near future. 3.3. 3D Cell Cultures g
y
y
p
y
There are reports of clinical trials concerning the utility of gelatin with selected renal
cells in patients with type 2 diabetes [131] and chronic kidney disease, and of gelatin with
renal autologous cells in patients with chronic kidney disease from congenital anomalies of
the kidneys and urinary tract [132]. 3.3. 3D Cell Cultures The application of 3D hydrogels in clinical trials is still in progress, however there are some
available data that show that they could be alternatives in regeneration or transplantation
treatments. Forte et al. [124] developed a composite hydrogel based on gelatin to form brain
tissue phantom . The authors suggested that this newly developed model will be useful
in traumatic brain injury (TBI) studies. Gelatin-based hydrogels are used as extracellular
matrices (ECMs) in 3D bioprinting technologies [125]. p
g
g
Alginate has been used in wound-healing processes in the form of electrospun hy-
drogels and sponges [126,127]. The authors of a previous study observed a significant
improvement of the healing process and monocyte stimulation, as well as higher cytokine
levels, such as tumor necrosis factor-α and interleukin-6 [128]. It was also proven that
alginate might be useful in reconstructive surgery and bone regeneration, e.g., alginate
scaffolds helped in bone tissue formation [129]. p
[
]
Alginate was also used in cardiac muscle regeneration in the form of a hydrogel
and porous 3D scaffold [130]. The most advanced clinical trials involving alginate-based
systems for cardiac regeneration have included Algisyl-LVR™sponsored by LoneStar
Heart, Inc (Laguna Hills, CA, USA), and PRESERVATION 1 sponsored by Bellerophon
BCM LLC (Hampton, NJ, USA). It is also noted that alginate implants are under clinical
investigation for intramyocardial delivery in patients with acute myocardial infarct (MI). There are reports of clinical trials concerning the utility of gelatin with selected renal
cells in patients with type 2 diabetes [131] and chronic kidney disease, and of gelatin with
renal autologous cells in patients with chronic kidney disease from congenital anomalies of
the kidneys and urinary tract [132]. Alginate was also used in cardiac muscle regeneration in the form of a hydrogel
and porous 3D scaffold [130]. The most advanced clinical trials involving alginate-based
systems for cardiac regeneration have included Algisyl-LVR™sponsored by LoneStar
Heart, Inc (Laguna Hills, CA, USA), and PRESERVATION 1 sponsored by Bellerophon
BCM LLC (Hampton, NJ, USA). It is also noted that alginate implants are under clinical
investigation for intramyocardial delivery in patients with acute myocardial infarct (MI). 4. Mechanical Properties of Alginate-Based Hydrogels The mechanical properties of sodium alginate hydrogels depend on the number and
sequence of monomer units (M and G), as well as their molecular weight, chain stiffness,
and cross-linking density [133,134]. Similarly, the mechanical stiffness of ion-cross-linked
sodium alginate hydrogels depends on the molecular weight of the sodium alginate and
the concentration of binding cations [135]. There is a relationship between the rheological
properties of the pre-gel solution and the mechanical stiffness of the post-hydrogel with
the particle concentration and molecular weight distribution of the polymer used to form
them [133]. The mechanical properties of biomaterials are an important parameter in regulating
the behavior of cell adhesion. Indeed, cells can remodel the mechanical, structural, and
chemical composition of the surrounding ECM. It is also known that the mechanical
properties of the extracellular microenvironment change the behavior of cells, including Materials 2021, 14, 858 15 of 24 15 of 24 their spreading, proliferation, or differentiation [136]. Most studies have focused on the
influence of matrix stiffness on cells using hydrogel substrates, which show purely elastic
behavior. Examples of mechanical properties for the most commonly used hydrogels are
given in Table 3. Importantly, the matrix stiffness influences the ability of stem cells to
differentiate towards specific lineages. Mesenchymal stem cells differentiate towards a
neurogenic lineage when cultured on soft 0.1–1 kPa substrates, towards a myogenic lineage
when cultured on 8–17 kPa substrates, and towards an osteogenic lineage when cultured
on stiffer 25–40 kPa substrates [137]. Besides the stiffness of the matrix, the viscoelasticity
and stress relaxation are also important [72]. Commercially available sodium alginate reaches molecular weights in the range of
32,000–400,000 g/mol. A decrease in the pH of the sodium alginate solution results in
increased viscosity (reaching a maximum around pH 3–3.5). The shear modulus of the
hydrogel depends on the sodium alginate concentration [133]. The physical properties of
the obtained gel can be improved by increasing the molecular weight of the sodium alginate. Solutions made from high molecular weight polymers of sodium alginate become more
viscous, which is often unsatisfactory during processing [138]. The mechanical strength also
depends on the sources from which the alginates are obtained, e.g., Azotobacter alginates
have comparatively high stiffness [139]. The viscosity of the pre-gel solution and its stiffness
after gelation can be successfully controlled through manipulation of the molecular weight
and distribution [133]. Table 3. Mechanical properties of hydrogels. 4. Mechanical Properties of Alginate-Based Hydrogels Material
Example
Tensile Strength
Tensile Modulus
Compressive Strength
Compressive Modulus
Reference
Traditional hydrogel
(PVA), PEG
1 ~ 100 kPa
<100 kPa
10 ~ 100 kPa
1 ~ 100 kPa
[140,141]
Alginate hydrogel
n.a. ~20 kPa
~78 kPa
n.a. n.a. [142]
Alginate-gelatin
hydrogel
n.a. ~0.5 MPa
~1 MPa
2 ~ 12 MPa
30 ~ 50 kPa
[81,142]
Alginate-gelatin-
nanocellulose
hydrogel
n.a. n.a. ~220 kPa
~320 kPa
60 ~ 110 kPa
[143,144]
Nanocomposite
hydrogel
PEG/clay
255 kPa
16 kPa
3.7 MPa
38 kPa
[145–147]
Cartilage
n.a. ~3 MPa
~9 MPa
~35 MPa
~15 MPa
[148,149]
Collagen fiber
n.a. ~75 MPa
~1000 MPa
n.a. n.a. [140,150]
Gelatin hydrogel
n.a. n.a. 3–25 kPa
n.a. n.a. [151] Table 3. Mechanical properties of hydrogels. Table 3. Mechanical properties of hydrogels. 5. Alginate-Gelatin Hydrogel as an Ink in 3D Bioprinting 5. Alginate-Gelatin Hydrogel as an Ink in 3D Bioprinting 5. Alginate-Gelatin Hydrogel as an Ink in 3D Bioprinting Additive manufacturing is a technology that could be widely applied in biomedicine. Materials and technologies used in medical and biological fields have to fulfill specific
requirements. Bioprinting technology is a branch of additive technology, commonly
known as 3D printing, which is utilized as an innovative tool in manufacturing living
functional tissues from cells and supporting biomaterials as bioinks in computed printing
systems. This technology enables a wide range of applications in tissue engineering or
regenerative medicine, for example replacing injured or damaged tissue manufactured in an
automated way with high repeatability [152]. Moreover, 3D printing, including bioprinting
technology, consists of sequential printing with precursor bioink (which consists of a
polymer or composite) in a predefined trajectory and within cells. Multilayer ink deposition
enables controlled spatial positioning of cells, thus facilitating the precise creation of
scalable structures, which cannot be provided by two- and standard three-dimensional cell
cultures [153]. Bioink is a specially prepared ink for biological and medical applications
that is non-toxic, enabling cell and tissue viability. Bioink is composed mainly of hydrogel
due to its biodegradability and ability to mimic the anatomy and mechanical features of the
body, however additionally it contains living cells, cell aggregates, or bioactive molecules,
creating entirely biologically functional products. The potential application and cell type
determine the choice of bioink and bioprinter to be adopted [28,152]. The widely used 3D printing methods for plastics, metals, and ceramics are not ad-
equate or appropriate for biomaterials applied in cell culture media. 4. Mechanical Properties of Alginate-Based Hydrogels Conventional 3D Materials 2021, 14, 858 16 of 24 16 of 24 printing strategies described by Bishop [154], such as inkjet printing, microextrusion, and
laser printing, have also been developed to generate 3D biological structures directly
surrounding cells and bioactive agents, including various biomimetic tissues, e.g., bone
tissue, cartilage and osteochondral tissue, heart tissue, blood vessels, liver tissue, and
tumors [155,156]. Over the last decade, there has been tremendous progress in the de-
velopment of three-dimensional bioprinting. The most commonly used 3D bioprinting
techniques include inkjet printing or droplet bioprinting, microextrusion, and laser-assisted
bioprinting (LaBP) [152,157] (Figure 8). Figure 8. Schematic representation of the most common methods of bioprinting. (A) Thermal inkjet
bioprinting. (B) Piezoelectric inkjet bioprinting. (C) Pneumatic extrusion bioprinting. (D) Mechanical
extrusion bioprinting. (E) Laser-assisted bioprinting. Copyright 2017, Chongqing Medical University. Production and hosting by Elsevier B.V [154] Figure 8. Schematic representation of the most common methods of bioprinting. (A) Thermal inkjet
bioprinting. (B) Piezoelectric inkjet bioprinting. (C) Pneumatic extrusion bioprinting. (D) Mechanical
extrusion bioprinting. (E) Laser-assisted bioprinting. Copyright 2017, Chongqing Medical University. Production and hosting by Elsevier B.V [154] Three-dimensional bioprinting is a versatile technique for creating biomimetic tissue
structures [23]. The production of alginate substrates using the additive method is an
excellent choice due to the uncomplicated processing procedure and reduction of waste,
which affect the material yield and programmable viscosity (ranges from 0.03 Pa/s to
6 × 104 Pa/s). However, in addition to its accuracy, the procedure is limited by its reso-
lution (200 µm) [158]. The printability of alginate bioinks is dependent on their viscosity,
however 3D biostructures exhibit constant rigidity in relation to certain conditions [153]. k h
b
bl f
f k
f
h Three-dimensional bioprinting is a versatile technique for creating biomimetic tissue
structures [23]. The production of alginate substrates using the additive method is an
excellent choice due to the uncomplicated processing procedure and reduction of waste,
which affect the material yield and programmable viscosity (ranges from 0.03 Pa/s to
6 × 104 Pa/s). However, in addition to its accuracy, the procedure is limited by its reso-
lution (200 µm) [158]. The printability of alginate bioinks is dependent on their viscosity,
however 3D biostructures exhibit constant rigidity in relation to certain conditions [153]. Bioinks have to be suitable for a range of key requirements for processing with a 3D
bioprinting machine. 4. Mechanical Properties of Alginate-Based Hydrogels It is possible to increase the viscosity by applying higher pressure on the
hydrogel extrusion, which eliminates the gel outflow from the nozzle and provides higher
accuracy to the printed structures [161]. The efficiency of bioprinting depends on both the bioink printability and the achieved
cell viability, as well as the process parameters (e.g., printing speed, resolution, pres-
sure) [165,166]. The accuracy of the obtained structure is also affected by the solidification
of the bioinks during the bioprinting process, which usually takes place through cross-
linking. The gentle solidification of the printout is the main prerequisite for the selected
bioinks, as they cannot cause damage or cell death [166]. Certain 3D printing applications
(e.g., when building more complex structures for tissue regeneration) do not provide the
smart response needed for such an artificially created structure to properly work in the
body, creating the opportunity to utilize 4D printing [167]. Sodium alginate hydrogel is widely used in the bioprinting process due to its biocom-
patibility, rapid ability for cross-linking, and simple regulation of its mechanical properties
through physico-chemical modifications [156,168]. Due to its hydrophilic nature, alginate
scaffolds allow the cell viability to be maintained during the culture, because media and
cells are pulled into the scaffold via capillary action. One of the benefits of possessing
dense cell cultures is the maximization of the contact interactions between cells [165]. Sodium alginate is not suitable for direct printing because the structure obtained is
not rigid enough and takes a liquid form after printing. Recently, multi-material bioinks
have been used more frequently, as these combinations can benefit from the advantages
of different materials [160]. However, it is possible to strengthen the structure of sodium
alginate by adding gelatin while maintaining its biocompatibility, because dissolved gelatin
forms a solution at body temperature (37 ◦C) and can form a gel after cooling (<29 ◦C). These thermal properties of gelatin guarantee good flow characteristics for bioinks and
allow quicker gelation during the bioprinting process compared to alginate combined with
Ca2+, which improves the initial stability of the manufactured structure [75,153,169]. One
well-known and accessible composite bioink is based on a combination of alginate and
gelatin. Alginate allows fast cross-linking, whereas gelatin provides thermoresponsive
properties [160]. Alginate-gelatin bioink combines the rapid ionic cross-linking of alginate
and the temperature sensitivity of gelatin for multilayer printing and is widely used as
bioink in cell bioprinting applications [159]. 4. Mechanical Properties of Alginate-Based Hydrogels Printability should be determined by rheological properties, bioink
consistency measurements, and sample dimensions [159–161]. The most important feature
of the rheological properties is the bioink viscosity, which can directly affect the printing
pressure required for the material dosage, and thus the cell viability in bioink and shape
fidelity of the printouts, as well as the elastic recovery, viscoelastic shear moduli, and shear
stress. Excessive pressure in the printing nozzle can damage the cells during printing,
thus it is important to control shear stress during extrusion because the cells suspended Bioinks have to be suitable for a range of key requirements for processing with a 3D
bioprinting machine. Printability should be determined by rheological properties, bioink
consistency measurements, and sample dimensions [159–161]. The most important feature
of the rheological properties is the bioink viscosity, which can directly affect the printing
pressure required for the material dosage, and thus the cell viability in bioink and shape
fidelity of the printouts, as well as the elastic recovery, viscoelastic shear moduli, and shear
stress. Excessive pressure in the printing nozzle can damage the cells during printing,
thus it is important to control shear stress during extrusion because the cells suspended Materials 2021, 14, 858 17 of 24 17 of 24 in the ink have differing sensitivity [162,163]. Gao et al. [162] examined the rheological
properties of an alginate-gelatin hydrogel and their influence on the bioink and cells placed
in it during the manufacturing process. In a study carried out by Diamantides et al. [164],
the effect of the density of cells contained in a hydrogel on its rheological properties was
examined. Another factor that may affect the rheological properties of some hydrogels
is temperature. The extrusion of bioinks during using 3D bioplotters does not require
increased temperature, and therefore does not affect cell destruction either. Such bioinks,
which are exist sol form or are gelated after being printed from the nozzle, are not suitable
for bioprinting because the mechanical strength of the structure is not sufficient to support
the cell culture. However, when bioink is too viscous, the filament is irregular and unstable
during printing. The manufactured structure requires the use of a semi-viscous hydrogel
to maintain the integrity of the structure consisting of regular fibers, while for the highest
cell viability, the viscosity of the bioink should be as low as possible to reduce the induced
shear stress [159]. 4. Mechanical Properties of Alginate-Based Hydrogels The substrate properties can also be modified
by using different solvents and gelatin concentrations. Their suitability for printing can
also be supported by adding an extruder heating system. The rheological properties of
alginate-gelatin hydrogels are influenced by temperature, in addition to the concentrations
of both substances. Temperature should not lead to cell apoptosis in the culture [161]. Other
materials used to support the biological activity of alginate-based bioinks include fibrin,
which improves interactions with cells, as well as polyvinyl alcohol and hydroxyapatite
(HAp), which enable multicomponent bioinks to be obtained. Cellulose can also be used,
which provides a bioink with appropriate shear-thinning action and can accurate reproduce
the shapes of the created structures [156]. Materials 2021, 14, 858 18 of 24 18 of 24 Alginate, which has a higher molecular weight and is highly concentrated, is a shear-
thinning physical gel. It is important to adjust its concentration in order to obtain the
appropriate properties when printing. There are ways to process unprintable alginate
compositions using different cross-linking strategies, such as printing on calcium chloride
solutions, spraying calcium chloride mist onto the solution coming out of the nozzle, or
pre-cross-linking with calcium chloride [160]. p
g
Traditional 3D structures manufactured using 3D bioprinting technology with static
mechanical properties might not be suitable for biomedical applications [170]. A new solu-
tion, which can more accurately mimic the dynamics of native tissues, has recently been
developed using four-dimensional bioprinting (4D) technology, based on three-dimensional
bioprinting with additional shape-shifting capability [171]. It is possible to design biologi-
cally active structures that respond to impulses, and after appropriate stimulation, may
change their shape to achieve the required functionality. Programmable bioinks capable of
being used in 4D bioprinting are being produced to obtain various stimulus-responsive
biomaterials, including thermal, humidity, electrical, magnetic, and photo-sensitive materi-
als [172]. 6. Conclusions Sodium alginate in the form of hydrogel is a low-cost biomaterial that demonstrates
good printability and excellent biocompatibility. This is why it is used in a wide range of
applications, such as in wound healing, regenerative medicine, and drug delivery, as well
as for cell culture. Alginate-gelatin hydrogel has very beneficial properties; the combination
of these two substances provides a substrate with mechanical properties and a structure
adapted to free cell growth. While sodium alginate is a less rigid structure, gelatin increases
the rigidity and provides a substrate with a precise shape. Bioprinting technology offers
promising opportunities for cell cultures. It allows the preparation of media with specific
structures and mechanical properties using a variety of substances. The production process providing accurate structures using alginate-gelatin bioink in
additive manufacturing technology enables the creation of hydrogel substrates of any shape
and high porosity, imitating the ECM. This facilitates the correct ingrowth, proliferation,
and differentiation of cells outside the body, which will significantly contribute to the
development of tissue engineering and regenerative medicine. In vitro 3D cell cultures will
enable the future preparation of tissues or parts of organs and could even be an opportunity
for drug testing systems. Author Contributions: Conceptualization, M.B.Ł., K.C., A.M.J., J.K., J.D. and I.M.; writing—original
draft preparation, M.B.Ł., K.C. and A.M.J.; writing—review and editing, J.K., J.D. and I.M.; visual-
ization, M.B.Ł. and A.M.J.; supervision, J.K., J.D. and I.M.; project administration, M.B.Ł.; funding
acquisition, J.D. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.B.Ł., K.C., A.M.J., J.K., J.D. and I.M.; writing—original
draft preparation, M.B.Ł., K.C. and A.M.J.; writing—review and editing, J.K., J.D. and I.M.; visual-
ization, M.B.Ł. and A.M.J.; supervision, J.K., J.D. and I.M.; project administration, M.B.Ł.; funding
acquisition, J.D. All authors have read and agreed to the published version of the manuscript. Funding: This research is a part of Statutory Subsidy No. 8201003902 at the Department of Mechanics,
Materials, and Biomedical Engineering (K58), Faculty of Mechanical Engineering, Wrocław University
of Science and Technology, Smoluchowskiego 25, 50-372 Wrocław, Poland. Data Availability Statement: No new data were created or analyzed in this study. Conflicts of Interest: Authors declare no conflict of interest. 1.
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Evaluation of the Agronomic Performance of Atrazine-Tolerant Transgenic japonica Rice Parental Lines for Utilization in Hybrid Seed Production
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Luhua Zhang1., Haiwei Chen1.¤, Yanlan Li1, Yanan Li1, Shengjun Wang2, Jinping Su2, Xuejun Liu2,
Defu Chen1*, Xiwen Chen1* ory of Molecular Genetics, College of Life Sciences, Nankai University, Tianjin, China, 2 Tianjin Crop Research Institute, Tianjin, China Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Key Project of Tianjin Science and Technology Support Program (11ZCGYNC01000), the Key Program of the Natural
Science Foundation of Tianjin (12YFJZJC01700, 14JCZDJC34100), the grant of Natural Science Foundation of China (No. 31070273, 31070717) and the 111 Project
(No. B08011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * Email: chendefu@nankai.edu.cn (DC); xiwenchen@nankai.edu.cn (XC) * Email: chendefu@nankai.edu.cn (DC); xiwenchen@nankai.edu.cn (XC) * Email: chendefu@nankai.edu.cn (DC); xiwenchen@nankai.edu.cn (XC) . These authors contributed equally to this work. . These authors contributed equally to this work. ¤ Current address: College of Life Sciences, Chifeng College, Chifeng, China ¤ Current address: College of Life Sciences, Chifeng College, Chifeng, China Evaluation of the Agronomic Performance of
Atrazine-Tolerant Transgenic japonica Rice Parental
Lines for Utilization in Hybrid Seed Production Luhua Zhang1., Haiwei Chen1.¤, Yanlan Li1, Yanan Li1, Shengjun Wang2, Jinping Su2, Xuejun Liu2,
Defu Chen1*, Xiwen Chen1* October 2014 | Volume 9 | Issue 10 | e108569 Abstract Currently, the purity of hybrid seed is a crucial limiting factor when developing hybrid japonica rice (Oryza sativa L.). To
chemically control hybrid seed purity, we transferred an improved atrazine chlorohydrolase gene (atzA) from Pseudomonas
ADP into hybrid japonica parental lines (two maintainers, one restorer), and Nipponbare, by using Agrobacterium-mediated
transformation. We subsequently selected several transgenic lines from each genotype by using PCR, RT-PCR, and
germination analysis. In the presence of the investigated atrazine concentrations, particularly 150 mM atrazine, almost all of
the transgenic lines produced significantly larger seedlings, with similar or higher germination percentages, than did the
respective controls. Although the seedlings of transgenic lines were taller and gained more root biomass compared to the
respective control plants, their growth was nevertheless inhibited by atrazine treatment compared to that without
treatment. When grown in soil containing 2 mg/kg or 5 mg/kg atrazine, the transgenic lines were taller, and had higher
total chlorophyll contents than did the respective controls; moreover, three of the strongest transgenic lines completely
recovered after 45 days of growth. After treatment with 2 mg/kg or 5 mg/kg of atrazine, the atrazine residue remaining in
the soil was 2.9–7.0% or 0.8–8.7% respectively, for transgenic lines, and 44.0–59.2% or 28.1–30.8%, respectively, for control
plants. Spraying plants at the vegetative growth stage with 0.15% atrazine effectively killed control plants, but not
transgenic lines. Our results indicate that transgenic atzA rice plants show tolerance to atrazine, and may be used as
parental lines in future hybrid seed production. Citation: Zhang L, Chen H, Li Y, Li Y, Wang S, et al. (2014) Evaluation of the Agronomic Performance of Atrazine-Tolerant Transgenic japonica Rice Parental Lines
for Utilization in Hybrid Seed Production. PLoS ONE 9(10): e108569. doi:10.1371/journal.pone.0108569 Editor: Jin-Song Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China Editor: Jin-Song Zhang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China Received January 27, 2014; Accepted September 1, 2014; Published October 2, 2014 Received January 27, 2014; Accepted September 1, 2014; Published October 2, 2014 pyright: 2014 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L
restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ng et al. Atrazine Tolerant Transgenic japonica Rice Parental Lines supplemented with 2 g/L proline, 3 mg/L 2, 4-D and 300 mg/L
casein hydrolysate in dark at 28uC. After 2–3 weeks, the scutellum-
derived calli were excised and subcultured every four weeks on the
same medium but with 0.5 g/L proline, 2 mg/L 2, 4-D in dark at
28uC. The highly embryogenic compact calli (3–5 mm in
diameter) that subcultured for less than five generations, were
selected and co-cultivated with A. tumefaciens EHA105 harboring
p1301C-ubi-22-14 on the co-cultivation medium (subculture
medium but with 100 mM acetosyringone) for 3 days in dark at
28uC. Following that, the explants were then transferred into
selection medium (subculture medium but with 50 mg/L hygro-
mycin and 500 mg/L cefotaxime) in dark at 28uC for selection. After two cycles of selection, hygromycin-resistant calli were
transferred onto pre-regeneration medium (NB medium with
0.5 g/L proline, 2 mg/L 6-BA, 1 mg/L NAA, 5 mg/L ABA,
300 mg/L casein hydrolysate and 50 mg/L hygromycin) for 14 to
21 days in dark at 28uC, then to regeneration medium (pre-
regeneration medium but without 5 mg/L ABA) for 30 days under
54 mmol/m2/s light at 28uC and finally to the rooting medium
(MS medium with 1 mg/L IBA) for 15 days under light at 28uC. production of false hybrids. Furthermore, as the Chinese economy
develops, increasing numbers of young men are leaving their home
towns to seek work in the cities, leaving the elderly and women to
work on the farms. The transformation of heavy and complex
farming to light and simple farming is therefore becoming
increasingly important. Thus, ensuring hybrid seed purity and
reducing labor costs are two key issues in hybrid japonica rice seed
production. Genetic engineering, especially herbicide resistance engineering,
provides an efficient means of controlling purity in hybrid seed
production. Yan first proposed a strategy of utilizing herbicide
resistance genes to chemically control purity in hybrid seed
production [5]. Since then, two-line hybrid rice production has
been extensively investigated [6–12] and progress has recently
been reviewed [13]. Additionally, some transgenic hybrid rice
combinations have been used in field trials [7,8,10,12]. However,
the research has mainly focused on the bar gene isolated from
Streptomyces hygroscopicus [6,8,10,12]; other genes, such as the
EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene from
Agrobacterium strain CP4, and the protoporphyrinogen oxidase
gene from Bacillus subtilis, have rarely been investigated [11]. Molecular analysis of transgenic lines Genomic DNA was isolated from young leaves using a modified
CTAB method [19]. PCR was performed to preliminarily select
the transformed plants using primers atzA7/atzA8 [20] and SL-
Ubi-C-F/SL-Ubi-C-R (Table 1). RT-PCR was performed to
further confirm the expression of atzA in the PCR-positive
transformed plants. Total RNA was extracted from young leaves
using an RNAultra Extraction Kit (Qiagen). cDNA was synthe-
sized using atzA8 and oligo(dT) as primers and SuperScriptTM II
RNase H2 (Invitrogen) as reverse transcriptase. RT-PCR was
amplified using the cDNA as template and atzA7/atzA2 [20] as
primers. b-actin (AB047313) was also amplified as the internal
control using actin-R/actin-F as primers [21]. Atrazine
(6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-di-
amine) is a triazine herbicide, and is commonly used in maize,
sorghum, and sugarcane fields [14]. By inhibiting electron
transport to plastoquinone in the photosystem PSII, atrazine
terminates photosynthesis and kills weeds [15]. Atrazine was once
the most widely used herbicide worldwide, because of its low cost
and high effectiveness [14]. We previously isolated an atrazine
chlorohydrolase gene (atzA) from a soil bacterium Pseudomonas
ADP [16], and modified this gene by using directed evolution, to
improve the enzymatic activity [17]. In the present study, we
transferred the improved atzA gene into breeding hybrid japonica
parental lines. Our results indicate that the transgenic atzA rice
lines show tolerance to atrazine, and may be used as parental lines
to chemically improve seed purity in hybrid seed production. Germination and seedling growth in the presence of
atrazine Fifty seeds from each transgenic line and the respective control
plant were directly sown on the surface of filter paper in plates
containing 0, 75 or 150 mM atrazine. Seeds were placed in a
growth chamber at 28uC with 16 h of 54 mmol/m2/s light per
day. Germination (based on radicles .2 mm) was recorded daily
and the cumulative values at day 3 and day 7 were calculated to
represent as germination potential and germination percentage. The shoot length, root length and their biomass were measured
after 7 days. For seedling growth test, seven-day old seedlings
germinated in absence of atrazine were placed on filter paper in
pots, and incubated in Kimura B nutrition solution [22]
containing 0, 75 or 150 mM atrazine in growth chamber described
above. During the period, the nutrition solution containing the
respective concentration of atrazine was added to keep the filter
paper wet. The growth parameters as described above were
measured after 10 days. Construction of plant expression vector Construction of plant expression vector Ubiquitin promoter and an improved atrazine chlorohydrolase
gene atzA-22-4 [17] were respectively amplified from pSTAR-
LING, an RNAi intermediate vector for monocots (a kind gift
from the Commonwealth Scientific and Industrial Research
Organization, Australia), and AtzA-22-4 using primers SL-Ubi-
F/SL-Ubi-R and atzA-TJ-F/atzA-TJ-R (Table 1). After respec-
tively inserted into TaKaRa pMD19T-simple vector and con-
firmed by sequencing, the pAtzA-22-4-19T-simple was restricted
with EcoRV to collect the 1.4 kb fragment, and then lignated with
pUbi-19T-simple. The resulting plasmid was restricted with SacI/
SpeI to collect the 3.4 kb fragment, and then ligated with
pCAMBIA1301. The recombinant plasmid p1301-ubi-22-14 was
then introduced into Agrobacterium tumefaciens EHA105 by the
freeze-thaw method [18]. Soil-grown transgenic T2 lines in the presence of atrazine
Two-week old seedlings of similar size that germinated in
absence of atrazine were transplanted into pots containing 1.12 kg
of soil with 0, 2, or 5 mg/kg of atrazine. Nine plants were planted
in each pot, and irrigated with the same amount of water every
day and with Kimura B nutrition solution [22] twice a week. Plants were incubated in the greenhouse in a 14-h light/10-h dark
cycle (28/25uC) at 300 mmol m22s21 light and 75% relative
humidity. Plant growth and chlorophyll content were measured at Atrazine Tolerant Transgenic japonica Rice Parental Lines If
the strategy is proven to be effective, chemical control of hybrid
seed purity will be mainly dependent on an herbicide with a single
mode of action, and this will hinder sustainable weed manage-
ment. Introduction erably to meet consumer’s demand for high-quality food both in
China and globally. Rice (Oryza sativa) is one of the most important staple food
crops globally. According to the National Grain and Oil
Information Center, the area of China planted with rice in 2012
was 3.06107 hm2, including 9.06106 hm2 of japonica rice [1]. Japonica rice is mainly planted in the northern region of the
Qinling Mountains–Huai River, and its planted area has increased
in recent years because of its high quality and good taste. Japonica
rice production is currently dominated by conventional varieties,
with hybrid rice accounting for only 3% of the cultivated area. On
the other hand, indica hybrid rice represents 70–80% of the total
planted area of indica rice [2]. Therefore, there is considerable
potential for the development of japonica hybrid rice. An increase
in the annual planted area of japonica hybrid rice from 3% to
50%, i.e., to reach 4.06106 hm2, is estimated to lead to the
production of 3.56109 kg of high-quality grain (www.cngrain. com/Publish/qita/200503/207290), thereby contributing consid- The three-line system is a traditional and effective production
method for hybrid japonica rice seed [3]. The most widely used
male sterile line in the system is BT-type cytoplasmic male sterile
(CMS). However, the panicle of this line is loosely enclosed when
heading, and this appearance closely resembles that of the
maintainer. This makes it difficult for farmers to distinguish the
BT-CMS line when eliminating off-type plants [2]. Furthermore,
the BT CMS line has good restorability, and may therefore be
easily pollinated with exotic pollens that contaminated during
mechanical harvesting and storage of seeds, and also with exotic
pollens from other plants [4]. The use of contaminated CMS lines
in seed production results in decreased hybrid seed purity. Therefore, off-type contamination must be eliminated as early as
possible. This is largely a manual process and requires consider-
able labor input, particularly in Asia. On the one hand, the need
for increased labor will increase the price of hybrid seeds, while on
the other hand, the increase in manual procedures may lead to the October 2014 | Volume 9 | Issue 10 | e108569 October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 1 Atrazine Tolerant Transgenic japonica Rice Parental Lines Germination of transgenic atzA lines in the presence of
atrazine Atrazine and hydroxyatrazine in the rice leaves and soil were
also determined in the soil-grown experiment. One gram of leaves,
or five grams of soil samples that collected from the soil layer
mixture as thick as possible, were extracted with 5 mL dichloro-
methane. After 30 min of incubation at room temperature, the
mixture was centrifuged at 15,294 g for 10 min. The supernatant
was transferred to a new tube and filtered through a 0.2-mm filter
and then subjected to CoM 6000 HPLC system analysis on an
analytical C18 column (5 mm, 250 mm64.6 mm) at 30uC with
linear gradients phase as follows: 0 to 6 min, 10% to 25%
acetonitrile; 6 min to 21 min, 25% to 65% acetonitrile; 21 min to
23 min, 65% to 100% acetonitrile; and 23 min to 25 min, 100%
acetonitrile [24] at the wavelength of 228 nm. To investigate the atrazine tolerance of transgenic lines during
germination, we germinated seeds of three lines of each genotype
and the respective controls in the presence of atrazine (Fig. 1A,
Table 2). The germination potential of the wild types decreased as
the atrazine concentration increased (58.3–98%, 22.4–64.2%, and
0%, respectively, in 0 mM, 75 mM, and 150 mM atrazine); on the
other hand, the germination percentage significantly decreased
only in presence of 150 mM atrazine (95.2–100%, 72.1–96.7%,
and 0–42.2%, respectively, in 0 mM, 75 mM, and 150 mM
atrazine). In the presence of the investigated atrazine concentra-
tions, the highest germination potential was determined for
Nipponbare, followed by Jinhui1, Jindao8, and Jindao7. In the
presence of 0 mM or 75 mM atrazine, the germination potentials
and percentages of almost all of the transgenic lines did not differ
significantly from those of the respective controls. The exceptions
were the germination potential for Jindao8 in the presence of
75 mM atrazine and the germination percentage for Jinhui1 in the
presence of 75 mM atrazine. On the other hand, in the presence of
150 mM atrazine, all of the transgenic lines showed significantly
higher germination potentials and germination percentages than
did the respective controls. Moreover, two of the wild types
(Nipponbare and Jinhui1) failed to germinate or only rarely
germinated at day 3 or day 7. Interestingly, in the presence of
150 mM
atrazine,
the
highest
germination
percentage
was
determined for Jindao7, followed by Jindao8, Jinhui1, and
Nipponbare; on the other hand, Jindao7 showed the lowest
germination potential. Genetic transformation and plant regeneration Oryza sativa L. Nipponbare and japonica hybrid rice parental
lines in the three-line system, Jindao7 (maintainer), Jindao8
(maintainer) and Jinhui3 (restorer) were used for transformation. Mature seeds were dehulled, surface-sterilized and placed on NB
medium (N6 macro elements, B5 micro elements and vitamins) October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org 2 Atrazine Tolerant Transgenic japonica Rice Parental Lines Atrazine Tolerant Transgenic japonica Rice Parental Lines Table 1. Primers used in this study. Table 1. Primers used in this study. Primer
Sequence (59R39)
SL-Ubi-F
tgagctcctgcagtgcagcgtgacccggtcgt, with SacI site
SL-Ubi-R
tgatatcctgcagaagtaacaccaaacaacag, with EcoRV site
atzA-TJ-F
tgatatcatgcaaacgctcagcatccag, with EcoRV site
atzA-TJ-R
tgatatcactagtctagaggctgcgccaagctg, with EcoRV and SpeI site
SL-Ubi-C-F
cccgccgtaataaatagacac
SL-Ubi-C-R
accacaccacatcatcacaac
doi:10.1371/journal.pone.0108569.t001 Table 1. Primers used in this study. Primer
Sequence (59R39)
SL-Ubi-F
tgagctcctgcagtgcagcgtgacccggtcgt, with SacI site
SL-Ubi-R
tgatatcctgcagaagtaacaccaaacaacag, with EcoRV site
atzA-TJ-F
tgatatcatgcaaacgctcagcatccag, with EcoRV site
atzA-TJ-R
tgatatcactagtctagaggctgcgccaagctg, with EcoRV and SpeI site
SL-Ubi-C-F
cccgccgtaataaatagacac
SL-Ubi-C-R
accacaccacatcatcacaac
doi:10.1371/journal.pone.0108569.t001 doi:10.1371/journal.pone.0108569.t001 15 -day intervals. For chlorophyll content analysis, approximately
10 mg of leaves was extracted with 5 ml acetone and then
quantified
the
absorbance
at
663.6 nm and
646.6 nm,
as
described by Porra et al. [23]. Chlorophyll a = 12.256A663.6–
2.556A646.6, Chlorophyll b = 20.316A646.6–4.916A663.6. lines that showed a segregation pattern of 3:1 resistant/sensitive in
the germination test were selected to produce the T2 generations. Individual plants, whose herbicide tolerance did not segregate in
the germination test, were considered as homogenous lines and
selected. For simplicity, in further research, we used only two or
three independent transgenic T2 lines for each genotype. Germination of transgenic atzA lines in the presence of
atrazine When germinated in the presence of
atrazine, all of the transgenic lines produced larger seedlings (with
taller shoots and longer roots) than did the respective control
plants (Fig. 1A). However, the presence of atrazine significantly
inhibited the growth of transgenic lines and wild types (Fig. 1A). Spraying atrazine to transgenic T2 lines Two-week old seedlings from the germination experiment were
also transplanted into pots with soil. Each pot contained 16 plants. After 30 days’ growth, the last second leaves were taken and cut
into 2–3 cm section, soaked in 0, 75 and 150 mM atrazine, and
incubated at 25uC under light for 2 days. The color of the leaves
was observed every day. After 40 days’ growth, each pot of plants
was sprayed with 20 ml of 0.15% atrazine solution. Statistical analysis All the experiments were performed for three times. Significant
difference between the specific transgenic line and the respective
control was performed using independent-samples t-test at 95% or
99% confidence with IBM SPSS Statistics 11.0. Values indicated
by * or ** represented significantly difference at P,0.05 or P,
0.01. Tolerance of soil-grown transgenic atzA lines to atrazine To further investigate the tolerance of transgenic lines to
atrazine, we transplanted 15-day-old seedlings germinated in the
absence of atrazine, to pots of soil containing 0 mg/kg, 2 mg/kg,
or 5 mg/kg of atrazine. We measured the plant height (as a non-
destructive assay of plant growth) and chlorophyll content at 15-
day intervals. In the absence of atrazine, we determined no significant
differences in plant height between the transgenic lines and the
respective controls (Fig. 2A). In soil containing 2 mg/kg or 5 mg/
kg of atrazine, all the transgenic lines were significantly taller than
were the respective control plants. However, the presence of
atrazine significantly inhibited the growth of transgenic lines and
control plants (by 68.1–111.9% and 46.4–68.2%, respectively). In
contrast to the control plants, all of the transgenic lines (except for
the Jindao8) were slightly taller in soil containing 2 mg/kg of
atrazine than in soil containing 5 mg/kg of atrazine. The growth
of transgenic lines gradually recovered with an increase in the
growth time. Further, three of the strongest transgenic lines
(Jindao7–4, Jinhui1–5, and Jindao8–4) completely recovered after
45 days of growth (data not shown). Figure 1. Germination and seedling growth of transgenic atzA
rice lines in the presence of atrazine. (A) Representative images of
seeds from the transgenic lines and the respective controls sown on
plates containing 0, 75 or 150 mM atrazine for 7 days. (B) Representative
images from seven-day old seedlings germinated in absence of atrazine
were transplanted into pots containing 0, 75 or 150 mM atrazine for 10
days. For simplicity, only images of transgenic Jindao8 (JD8) and/or
Nipponbare (N) were shown here. doi:10.1371/journal.pone.0108569.g001 Atrazine may disrupt the electron flow of photosystem II and
destroy photosynthetic pigments in plant leaves [15]. In the
absence of atrazine, we observed no difference in chlorophyll
content between transgenic lines and control plants (Fig. 2B). However, in soil containing 2 mg/kg or 5 mg/kg of atrazine, the
transgenic lines retained more chlorophyll than did the respective
control plants. With a few exceptions, the chlorophyll content of
the transgenic lines decreased significantly in soil containing
5 mg/kg of atrazine, but not in soil containing 2 mg/kg of
atrazine; on the other hand, the chlorophyll content of the control
plants decreased significantly under both conditions. For each
genotype grown in soil containing the same atrazine concentra-
tion, we observed no difference in chlorophyll content at different
growth stages. Selection of transgenic atzA plants PCR analysis revealed that 18 (100%) Nipponbare transfor-
mants, 15 out of 18 (83%) Jindao7 transformants, 14 out of 16
(88%) Jinhui1 transformants, and 17 (100%) Jindao8 transfor-
mants were positive. RT-PCR analysis further confirmed the
expression of atzA in the PCR-positive lines (data not shown). Subsequently, the self-pollinated seeds (T1 progenies) from each
plant were germinated in the presence of hygromycin. Transgenic To investigate the tolerance of transgenic lines to atrazine, we
transplanted 7-day old seedlings germinated in the absence of
atrazine, to pots of soil containing different concentration of
atrazine (Fig. 1B and Table 3). In the absence of atrazine, we
determined no differences in shoot or root growth between any of
the transgenic lines and the respective controls. However, in the October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org 3 Atrazine Tolerant Transgenic japonica Rice Parental Lines Atrazine Tolerant Transgenic japonica Rice Parental Lines Figure 1. Germination and seedling growth of transgenic atzA
rice lines in the presence of atrazine. (A) Representative images of
seeds from the transgenic lines and the respective controls sown on
plates containing 0, 75 or 150 mM atrazine for 7 days. (B) Representative
images from seven-day old seedlings germinated in absence of atrazine
were transplanted into pots containing 0, 75 or 150 mM atrazine for 10
days. For simplicity, only images of transgenic Jindao8 (JD8) and/or
Nipponbare (N) were shown here. doi:10.1371/journal.pone.0108569.g001 Plants became yellowish, and subsequently rotted and died after
10 days (Fig. 1B). Note: In each independent treatment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates significant difference (P,0.05) or
highly significant difference (P,0.01) between the treatment and the control.
aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. For simplicity, only two transgenic lines for each genotype were
shown here.
doi:10.1371/journal.pone.0108569.t002 Note: In each independent treatment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates
highly significant difference (P,0.01) between the treatment and the control. g y
g
(
)
aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. For simplicity, only two transge
shown here. tment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates significant difference (P,0.05) o
,0.01) between the treatment and the control. Tolerance of soil-grown transgenic atzA lines to atrazine After 45 days, the chlorophyll contents of the three presence of 75 mM atrazine, all of the transgenic lines (except for
Jindao8) were significantly taller than were the respective controls;
further, all of the transgenic lines (except for Jinhui1) produced
significantly more root biomass than did the respective controls. On the other hand, in the presence of 150 mM atrazine, shoot and
root growth of almost all of the transgenic lines and the respective
controls were significantly inhibited. The exceptions were the
shoot growth of Nipponbare and the root growth of Jinhui1–5. Table 2. Germination of transgenic atzA rice lines in the presence of atrazine. Line
0 mM
75 mM
150 mM
3 d
7 d
3 d
7 d
3 d
7 d
Na-WT
98.062.0
10060.0
54.061.6
94.561.4
0.060.0
0.060.0
N-3
98.061.6
94.260.0
60.062.2
98.261.1
64.361.6**
96.261.8**
N-5
96.062.0
10060.0
64.061.3
92.161.5
62.362.6**
92.661.5**
JD7-WT
58.361.6
10060.0
24.362.1
90.262.3
0.060.0
42.261.1
JD7-2
56.061.1
98.161.4
22.361.7
88.262.1
2.360.1**
88.561.6**
JD7-4
62.362.2
98.661.6
26.361.3
90.260.8
2.460.2**
86.561.3**
JH3-WT
86.362.0
95.262.8
64.262.2
72.161.1
0.060.0
4.560.0
JH-3
82.361.2
94.261.8
58.761.6
90.261.2*
32.360.9**
78.661.2**
JH3-5
88.363.2
92.261.3
66.362.7
94.161.0*
38.761.1**
80.561.2**
JD8-WT
82.064.2
98.760.8
22.461.6
96.761.0
0.060.0
26.561.3
JD8-1
78.362.5
99.260.8
48.361.1**
97.260.2
34.360.7**
94.562.8**
JD8-4
78.062.6
10060.0
54.062.6**
98.361.0
35.261.6**
96.861.3**
Note: In each independent treatment 50 seeds were used for the experiment. Values are shown as mean 6SEM (n = 3). * or ** indicates significant difference (P,0.05) or
highly significant difference (P,0.01) between the treatment and the control. aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. For simplicity, only two transgenic lines for each genotype were
shown here. doi:10.1371/journal.pone.0108569.t002 Table 2. Germination of transgenic atzA rice lines in the presence of atrazine. October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 4 Atrazine Tolerant Transgenic japonica Rice Parental Lines Table 3. Seedling growth performance of transgenic atzA rice lines in the presence of atrazine. Table 3. Seedling growth performance of transgenic atzA rice lines in the presence of atrazine. showed tolerance to the atrazine concentration used in weed
control. showed tolerance to the atrazine concentration used in weed
control. strongest lines (Jindao7–4, Jinhui1–5, and Jindao8–4) were almost
identical when grown in soil containing 5 mg/kg of atrazine and
when grown in the absence of atrazine (data not shown). Ability of soil-grown T2 transgenic lines to degrade
atrazine Current research on the utilization of herbicide resistance genes
in hybrid rice is focused on the two-line hybrid production system. Sterile lines are not stable in the two-line system, and therefore
most japonica hybrid combinations are still produced by using
three-line system. In the present study, we have developed
atrazine-tolerant transgenic japonica rice parental lines with the
potential to be used in future hybrid seed production. To investigate the ability of the soil-grown transgenic plants to
degrade atrazine, we determined the atrazine residue in the soil at
15-day intervals, after growth of seedlings in pots of soil containing
0 mg/kg, 2 mg/kg, or 5 mg/kg of atrazine. We observed that the
atrazine residue in the soil decreased with an increase in growth
time, for transgenic and control plants (Fig. 2C). However, the
decrease was greater for transgenic lines than for control plants. After treatment with 2 mg/kg of atrazine, the atrazine residue
remaining in the soil was 2.9–7.0% for transgenic lines, and 44.0–
59.2% for control plants; after treatment with 5 mg/kg of atrazine,
the atrazine residue remaining in the soil was 0.8–8.7% for
transgenic lines and 28.1–30.8% for control plant. For each
genotype at the same growth stage, the atrazine residue in the soil
was higher after growth in 2 mg/kg atrazine than after growth in
5 mg/kg atrazine. We also observed that hydroxyatrazine, not
atrazine (only trace) appeared in the leaves of the transgenic plants
(Fig. S1), indicating that atrazine was metabolized rather than
accumulated in the transgenic plants. Herbicide tolerance during germination is very important when
utilizing transgenic atzA lines in hybrid seed production, because
off-type plants should be eliminated as early as possible. Atrazine is
a slightly water-soluble herbicide, with a saturated concentration
of 153 mM. Kawahigashi [25] previously showed that an atrazine
concentration of 100 mM did not affect the germination of rice
plants. Therefore, we used two atrazine concentrations (75 mM
and 150 mM) in the germination test, to evaluate the tolerance of
transgenic rice lines. In contrast to Kawahigashi [25], we observed
that the transgenic lines germinated well in the presence of
atrazine whereas the respective controls did not. However, as the
seedling growth of transgenic lines were also inhibited by atrazine,
chemical control of contamination during germination may not be
appropriate. Tolerance of soil-grown transgenic atzA lines to atrazine Line
0 mM
75 mM
150 mM
Shoot length (cm)
Root biomass (mg)
Shoot length (cm)
Root biomass (mg)
Shoot length (cm)
Root biomass (mg)
N a-WT
9.9061.37
32.0063.16
6.5460.86 (66.0)a
21.0062.55 (65.6)
5.0761.17 (51.2)
21.7563.77 (68.0)
N-3
10.5561.49
30.2563.50
9.5662.08* (90.7)
29.5063.14** (97.5)
7.5561.20* (71.6)
21.5066.40 (71.1)
N-5
8.7862.42
32.7564.50
8.9461.33* (101.8)
29.8561.83** (91.2)
9.3960.51** (106.9)
21.0062.71 (64.1)
JD7-WT
8.4760.37
28.5062.56
5.2860.57 (62.4)
20.0063.10 (70.2)
6.5460.61 (77.2)
17.7565.19 (62.3)
JD7-2
9.4062.61
31.5063.00
7.0860.78* (75.3)
32.5062.65** (103.2)
5.8160.56 (61.8)
17.0063.46 (54.0)
JD7-4
7.9961.65
27.5063.35
8.1661.27* (102.2)
27.5062.65** (100.0)
6.7860.53 (84.8)
17.7564.19 (64.5)
JH3-WT
9.8361.25
24.0063.07
5.8060.43 (59.0)
21.7563.36 (90.6)
4.6260.42 (47.0)
13.2566.24 (55.2)
JH-3
10.4461.47
26.7562.75
8.2760.59* (79.2)
24.2562.22* (90.7)
5.1260.61 (49.0)
15.2562.63 (57.0)
JH3-5
8.8961.58
25.5063.57
8.2560.78* (92.8)
23.2562.87* (83.8)
6.0460.19* (67.9)
22.0063.37** (86.3)
JD8-WT
7.8860.93
26.7563.55
7.2061.85 (91.4)
21.7562.87 (81.3)
6.4360.35 (81.5)
12.2563.46 (45.8)
JD8-1
8.1161.51
27.5062.15
7.1860.40 (88.6)
27.5061.52** (100.0)
5.6561.12 (69.6)
11.5061.91 (41.8)
JD8-4
7.9060.72
27.7562.53
7.6460.69 (96.7)
27.7561.79** (100.0)
5.6460.78 (71.3)
11.7562.99 (42.3)
Note: The parameters were recorded after 10 days. Values are shown as mean 6 SEM (n = 3). * or ** indicates significant difference (P,0.05) or highly significant
difference (P,0.01) between the treatment and the control. aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. bData in the bracket are the percentage of the treated values to the mock values. doi:10.1371/journal.pone.0108569.t003 Note: The parameters were recorded after 10 days. Values are shown as mean 6 SEM (n = 3). * or ** indicates significant difference (P,0.05) or highly significant
difference (P,0.01) between the treatment and the control. aN, JD7, JH3 and JD8 were the abbreviations of Nipponbare, Jindao7, Jindao8 and Jinhui3, respectively. bData in the bracket are the percentage of the treated values to the mock values. doi:10.1371/journal.pone.0108569.t003 October 2014 | Volume 9 | Issue 10 | e108569 Utilization of transgenic plants in hybrid seed production Utilization of transgenic plants in hybrid seed production
To investigate the tolerance of transgenic plants to the atrazine
concentration used in weed control, we first examined the
tolerance of leaf sections to atrazine solution. In the presence of
75 mM atrazine, the leaf sections of transgenic lines remained
green, whereas those of control plants became bleached (Fig. 3A). In the presence of 150 mM atrazine, the leaf sections of transgenic
lines became slightly bleached, but remained greener than those of
control plants. We subsequently sprayed plants with 0.15%
atrazine, and observed that wild-type plants became curled and
withered after 6 days, whereas transgenic lines continued to grow
well (Fig. 3B). Our results indicate that the transgenic rice plants To evaluate the tolerance of seedlings to atrazine, we used 7-
day-old seedlings germinated in the absence of atrazine as a
starting material, to avoid cumulative effects derived from
germination. We observed that almost all of the transgenic lines
grew well in the presence of 75 mM atrazine with no difference
from that in absence of atrazine. Therefore, 75 mM atrazine may
be used for effective control of off-type plants during the early
seedling growth stage. Our data also suggested spraying plants
with 0.15% atrazine (equivalent to the standard dosage used in the
field for weed control, i.e., 200–250 g 40% suspension concentrate
in 30,50 kg of water) at the subsequently vegetative growth stage
could serve as an alternative means of chemically controlling October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org October 2014 | Volume 9 | Issue 10 | e108569 5 Atrazine Tolerant Transgenic japonica Rice Parental Lines h and physiology of transgenic atzA rice lines in the presence of atrazine. Fifteen-day old seedlings germinated in absence
transplanted into pots with 1.12 kg soil containing 0, 2 or 5 mg/kg of atrazine. (A) plant height; (B) chlorophyll content; and (C)
were measured at 15-day intervals. Values are shown as mean 6SEM (n = 3). * or ** over the bar indicates significant difference (P,
gnificant difference (P,0.01) between the treatment and the control. For simplicity, only data of transgenic Nipponbare (N) and
re shown here. al.pone.0108569.g002 Figure 2. Growth and physiology of transgenic atzA rice lines in the presence of atrazine. Fifteen-day old seedlings germinated in absence
of atrazine were transplanted into pots with 1.12 kg soil containing 0, 2 or 5 mg/kg of atrazine. October 2014 | Volume 9 | Issue 10 | e108569 Utilization of transgenic plants in hybrid seed production (A) plant height; (B) chlorophyll content; and (C)
atrazine residue were measured at 15-day intervals. Values are shown as mean 6SEM (n = 3). * or ** over the bar indicates significant difference (P,
0.05) or highly significant difference (P,0.01) between the treatment and the control. For simplicity, only data of transgenic Nipponbare (N) and
Jindao8 (JD8) were shown here. doi:10.1371/journal.pone.0108569.g002 off-type plants. However, we did not test the tolerance of
transgenic atzA lines at the reproductive growth stage, which is
important for the utilization in mechanical harvesting of hybrid
seed production. Further studies are required to investigate
whether the lethal dosage for untransformed parental lines is
tolerated at the reproductive stage. growth of transgenic lines. Our data also suggested that almost all
of the atrazine was degraded after growth of the transgenic rice
plants for 45 days. This was also verified by the appearance of
hydroxyatrazine in the leaves of transgenic plants. Besides,
atrazine metabolite hydroxyatrazine could be directly determined
in transgenic plants. However, as the leave, stem and root of the
plants all possibly metabolize atrazine [28], it would be more
complicated if we want to quantitatively evaluate the atrazine
metabolized by plants. That is why we chose to determine the
atrazine residue in the soil after growth of the transgenic plants in
this study. Atrazine degrades relatively slowly, with an average half-life of
4–57 weeks [26]. Therefore, application of this herbicide will
inevitably lead to accumulation in the field, which may affect
current crop growth or damage sensitive succession crops [27]. The transgenic rice lines were taller, and had higher chlorophyll
contents than did the respective control plants when growing in
the soil of 2 mg/kg or 5 mg/kg atrazine. Three of the transgenic
lines (Jindao7–4, Jinhui1–5, and Jindao8–4) completely recovered
after 45 days of growth in the presence of atrazine, suggesting that
an atrazine concentration up to 5 mg/kg does not affect sustained Utilization
of
herbicide
resistance
genes
in
hybrid
seed
production may be achieved by introducing the genes into a
maintainer or restorer line in the three-line system. When an
herbicide-resistant maintainer is obtained, its corresponding CMS
line, which possesses herbicide resistance, may be created by using October 2014 | Volume 9 | Issue 10 | e108569 PLOS ONE | www.plosone.org 6 Atrazine Tolerant Transgenic japonica Rice Parental Lines Figure 3. Tolerance of mature transgenic atzA plants to atrazine. Supporting Information Figure S1
HPLC analysis for determination of atrazine
and its metabolite hydroxyatrazine in leaves of trans-
genic and WT rice plants after grown in 5 mg/kg
atrazine soil for 45 days. (A) Standard of hydroxyatrazine (I)
and atrazine (II). (B) Wild
type (WT). (C–E) Transgenic
Nipponbare (N-6), Jindao7 (JD7-4) or Jindao8 (JD8-4), respective-
ly. (TIF) Figure S1
HPLC analysis for determination of atrazine
and its metabolite hydroxyatrazine in leaves of trans-
genic and WT rice plants after grown in 5 mg/kg
atrazine soil for 45 days. (A) Standard of hydroxyatrazine (I)
and atrazine (II). (B) Wild
type (WT). (C–E) Transgenic
Nipponbare (N-6), Jindao7 (JD7-4) or Jindao8 (JD8-4), respective-
ly. (TIF) Author Contributions Field trials with genetically modified rice are strictly controlled
in China, and therefore we were unable to evaluate our herbicide-
resistant hybrid rice combinations on a field scale. Further studies
are required to verify the validity of utilizing atrazine-tolerant
transgenic lines in the field, and to determine whether herbicide- Conceived and designed the experiments: DFC XWC. Performed the
experiments: LHZ HWC YLL YNL SJW JPS. Analyzed the data: LHZ
DFC XWC. Contributed reagents/materials/analysis tools: SJW XJL
DFC XWC. Wrote the paper: DFC XWC. Utilization of transgenic plants in hybrid seed production (A) Representative images of leave sections in the presence of atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 30 days’ growth, the last second leaves were cut into 2–3 cm section, and
soaked in 0, 75 and 150 mM atrazine, and incubated at 25uC under light for 2 days. (B) Representative images of mature plants after sprayed with
atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 40 days’ growth, each pot of transgenic lines (9 plants) was
sprayed with 20 mL of 0.15% atrazine solution. For simplicity, only data of transgenic Jindao8 (JD8) was shown here. doi:10.1371/journal.pone.0108569.g003 Figure 3. Tolerance of mature transgenic atzA plants to atrazine. (A) Representative images of leave sections in the presence of atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 30 days’ growth, the last second leaves were cut into 2–3 cm section, and
soaked in 0, 75 and 150 mM atrazine, and incubated at 25uC under light for 2 days. (B) Representative images of mature plants after sprayed with
atrazine. Fifteen-day old seedlings were transplanted into pots containing soil. After 40 days’ growth, each pot of transgenic lines (9 plants) was
sprayed with 20 mL of 0.15% atrazine solution. For simplicity, only data of transgenic Jindao8 (JD8) was shown here. doi:10.1371/journal.pone.0108569.g003 a backcross. The resulting herbicide-resistant CMS breeder seeds
may be used to cross with the original maintainer (herbicide
sensitive), to multiply the herbicide-resistant foundation seeds. During this process, the selfed maintainer and off-type plants may
easily be eliminated by herbicide spraying, thus ensuring the purity
of CMS seeds. The herbicide-resistant CMS line may also be used
in mechanical harvesting, by spraying herbicide to kill the pollen
plants after pollination, and harvesting seeds from the surviving
CMS plants. A similar result may be achieved when crossing the
herbicide-resistant CMS line with the herbicide-sensitive restorer
line. Alternatively, the herbicide-resistant restorer line may be
crossed with the herbicide-sensitive sterile line during the hybrid
seed production process, by spraying herbicide to kill the off-type
plants. Further, the herbicide-resistant maintainer and restorer
lines may be useful for eliminating off-type plants, and for weed
management when growing hybrid rice. resistant hybrid rice combinations retain their original superiority
in terms of yield. Nevertheless, the development of atrazine-
tolerant transgenic japonica rice parental lines represents a
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s-triazines. Crit Rev Environ Control 19: 1–14. 26. Erickson LE, Lee KH, Sumner DD (1989) Degradation of atrazine and related
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Oral Fluorescein angiography in the diagnosis of paediatric optic nerve abnormalities.
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Oral Fluorescein angiography in the diagnosis of
paediatric optic nerve abnormalities.
Brinda Muthusamy
(
brinda.muthusmamy@nhs.net
)
Addenbrooke's Hospital
Robert Thomas Brady
Addenbrooke's Hospital, Cambridge, UK
https://orcid.org/0000-0001-5839-8566
Hrishikesh Kaza
Nikhil Jain
Article
Keywords:
Posted Date: November 14th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3417412/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
Licen
Read Full License
Additional Declarations: There is no conflict of interest Oral Fluorescein angiography in the diagnosis of
paediatric optic nerve abnormalities. Brinda Muthusamy
(
brinda.muthusmamy@nhs.net
)
Addenbrooke's Hospital
Robert Thomas Brady
Addenbrooke's Hospital, Cambridge, UK
https://orcid.org/0000-0001-5839-8566
Hrishikesh Kaza
Nikhil Jain
Article
Keywords:
Posted Date: November 14th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3417412/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
Licen
Read Full License
Additional Declarations: There is no conflict of interest Brinda Muthusamy
(
brinda.muthusmamy@nhs.net
) Brinda Muthusamy
(
brinda.muthusmamy@nhs.net
) OBJECTIVE FFA is a well-established investigation for the diagnosis of optic nerve abnormalities, requiring an
intravenous cannula and extended imaging acquisition time. Cannulation can present a challenge in
paediatric patients and whilst oral FFA has been used for decades, it has been limited by imaging
technology and unconfirmed image acquisition timings. For years, we have used a modern ultra-widefield
retinal camera, and established imaging time-points to demonstrate dynamic optic nerve head changes
upon ingestion of fluorescein and collected a database of oFFA images for various presentations. RESULTS Our study presents a series of images and descriptions for common optic nerve abnormalities in
paediatric populations. In the interpretation part of the study, overall sensitivity of 76.8% in the consultant
group vs 63.3% in the combined consultant + trainees and specificity of 87.5% vs 68.4% in the combined
group. CONCLUSIONS This is the first study that describes characteristic features of several common, and serious, optic nerve
abnormalities specifically for oFFA interpretation in a paediatric population. It also highlights the rapid
accumulation of oFFA interpretation skills in non-specialist consultant and trainee ophthalmologists such
as to obtain a high diagnostic accuracy with high sensitivity and specificity. METHODS Using an established protocol, optic nerve colour images were obtained, followed by oral administration
of fluorescein dye. The optic nerves are then imaged at established intervals. An interpretation of oFFA
tutorial was delivered to consultant ophthalmologists and trainees. Subsequently, these groups were
assessed using a series of fifteen cases with sensitivity and specificity of the test determined. Keywords: License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Additional Declarations: There is no conflict of interest Additional Declarations: There is no conflict of interest Version of Record: A version of this preprint was published at Eye on February 10th, 2024. See the
published version at https://doi.org/10.1038/s41433-024-02956-y. Page 1/11 Page 1/11 Page 1/11 INTRODUCTION Fundus Fluorescein angiography (FFA) is a well-established investigatory tool for the diagnosis of optic
nerve abnormalities. The test typically requires the intravenous administration of a fluorescein solution
followed by the sequential imaging of ocular structures as the dye is perfused through the vasculature
(1). Filling defects and leakage are two commonly observed features which can be observed and used to
determine a diagnosis. Characteristic findings of paediatric optic nerve abnormalities have been
described in paediatric patients to discern between optic nerve head drusen and papilloedema (2). A Page 2/11 study by Chang et al described fluorescein angiography as the single most important imaging modality to
distinguish between these presentations (3). Intravenous FFA requires the placement of an intravenous cannula and an extended imaging acquisition
time to capture the different sequences of dye perfusion through the vasculature. Whilst this is not
typically difficult to complete in adults, it can be a challenge in paediatric patients. In particular, those
who are very young, are needle phobic, and those with learning difficulties (4). Therefore, an alternative
oral administration technique could overcome these barriers. Oral FFA (oFFA) has been used for decades
but was limited by the quality of imaging technology and unconfirmed optimised timings of photograph
acquisition (5). In our unit we utilise a retinal camera with a rapid, high-quality, image capture time with
established timing protocol to obtain images at different phases of fluorescein dye perfusion. This
technique has the benefit of avoiding cannulation and obtaining quality images to aid diagnosis. Having utilised this protocol for several years, we have collected a database of oFFA images for various
optic nerve presentations including optic nerve head drusen, papilloedema, inflammatory papillitis and
normal optic nerves. A selection of images were used to construct an oFFA interpretation tutorial for
consultant ophthalmologists and trainee ophthalmologists. This study outlines the clinical findings of
oFFA in optic nerve abnormalities and the results of an assessment upon interpretation which found it to
have a high diagnostic accuracy with high sensitivity and specificity. METHODS Completing an oFFA investigation. Completing an oFFA investigation. Protocol outlined in Fig. 1. Pre-procedure, informed written consent is obtained. We advise parents that
appointment will take approximately two hours and there are risks due to exposure to fluorescein. This
includes pruritus, nausea, vomiting and allergy. Children will typically have discolouration of their urine. A
patient information leaflet is provided which details post-procedure care. Staffing required: medical photographer, a paediatric nurse, and a doctor to cross-check prescription and
supervise the child during the one-hour period post-ingestion of the fluorescein. The oral dose of
fluorescein is calculated and prescribed based on the child’s current weight. Pupils are dilated on arrival using gutt cyclopentolate 1% & gutt phenylephrine 2.5%. Initial optic nerve
colour images are captured using a Silverstone SS OCT - Optos ultra-widefield (UWF™) retinal imaging
device (Nikon Co. LTD. Japan), followed by oral administration of fluorescein dye diluted in 20ml fruit
juice with a straw to prevent staining of teeth and mouth. Post-ingestion the optic nerves are imaged at
15-, 30-, 45-, and 60-minute intervals or as requested. The paediatric nurse records vital signs at 15-minute
intervals during imaging. The nurse will also monitor the child for one-hour post-procedure prior to
discharge. Impact of interpretation tutorial. Page 3/11 An interpretation of oFFA tutorial describing the features of normal and 3 optic nerve pathologies was
prepared and delivered over an electronic meeting platform (Zoom video communications, Qumu
corporation, USA). This format facilitated the inclusion of consultant and trainee ophthalmologists from
across a wide geographical area to participate. Following the tutorial, participants were invited to complete an assessment of magnified coloured optic
disc photos and oFFA images of 15 optic nerves. Each nerve had five associated questions. Importantly,
participants were given no clinical context to the patient’s presenting signs, symptoms, or other
investigations. The diagnosis around the optic nerve images were confirmed by a consultant paediatric neuro-
ophthalmologist who had access to all the clinical data for these patients. Images were used from
patients who had a full neuro-ophthalmic examination, investigations, and duration of follow-up in order
to be certain of the accuracy of the diagnosis. Answers were collated and analysed using a Chi-squared statistical test to compare results between
consultant and trainee ophthalmologist groups. Test sensitivity and specificity was determined. The final
analysis was done with the use of Statistical Package for Social Sciences (SPSS) software,
IBM(manufacturer), Chicago, USA, version 25.0. Completing an oFFA investigation. For statistical significance, p value of less than 0.05 was
considered significant. RESULTS Interpreting images from an oFFA investigation. Normal optic nerve: Figure 2 illustrates the common oFFA features observed in the normal optic nerve. There is a gradual
increase in hyperfluorescence through the three phases. There should not be any expansion of the dye
beyond the optic nerve margin. The vasculature on and surrounding the optic nerve should be clearly
visible throughout all phases. Papilloedema: In the case of papilloedema, Fig. 3, hyperfluorescence observed at the optic nerve increases over time as
the series progresses. There is leakage beyond the margins of the optic nerve. There is obscuration of the
vessels on the optic nerve head. Maximal hyperfluorescence occurs in the late phase. Optic nerve head drusen (ONHD): Impact of interpretation tutorial. There were a total of 37 participants in the study – who were divided into two groups – consultants
(group A, n = 8) and trainees who ranged from year 2 to year 7 + of their ophthalmology training (group B,
n = 29). Total number of optic discs evaluated by each participant was 15, with 5 photograph-based
questions linked with each optic disc and FFA. These questions included comments on all aspects of the
optic disc and oFFA as described above. The total responses evaluated were 2769 (group A + group B). The total number of correct and incorrect responses received were 2020 (72.95%) and 749 (27.94%)
respectively. The diagnosis given matched the actual diagnosis, in 81.7% of the responses amongst the
consultant group and 58.8% in the trainee group. The responses to the first question, to comment on whether the optic discs were normal versus abnormal,
revealed the following – nearly 4/5ths of the trainees gave the correct response. This compares to more
than 90% with the correct response in the consultant group, and this was statistically significant (p =
0.004). The proportion of correct responses on disc vessels (clear at the disc margins or not clear) were
significantly higher in consultant group as compared to the trainees. (85% vs 71.59% respectively (p
value = 0.003). The correct responses received on the presence or absence of optic disc swelling was
again higher in consultant as compared to ophthalmology trainees, and this was statistically significant. (76.67% vs 66.74% respectively, p value = 0.038). The two groups were also asked to comment on disc leak and a possible diagnosis on the basis of disc
and oFFA photos. Correct responses to both these questions were higher in the consultant group than
trainees and this was statistically significant (p = 0.002 and p < 0.0001 respectively). Sensitivity and
specificity for detection of disc leak and correct diagnosis were calculated from the data and both were
higher in the consultant group. Sensitivity of 76.8% in the consultant group vs 63.3% in the combined
consultant + trainees and Specificity of 87.5% vs 68.4% in the combined group. Subsequently, diagnostic
accuracy was calculated in the consultant’s group (82.5%) and combined consultants + trainees
(65.95%). The positive and negative predictive value of both groups are shown in Table:7 (supplementary
information). Inflammatory papillitis: With inflammatory optic neuropathy, there is early phase hyperfluorescence of the optic nerve head. There
is leakage of fluorescein beyond the margins of the optic nerve. The hyperfluorescence increases
moderately as the series progresses. Significantly, there is obscuration of the blood vessels overlying the
optic nerve head. Optic nerve head drusen (ONHD): There is uptake of fluorescein by the optic nerve drusen in the early phase of the series. This forms a well
demarcated area of hyperfluorescent drusen that gradually increases in hyperfluorescence as the series Page 4/11 Page 4/11 progresses, Fig. 4. The overlying vasculature should be clearly visible and not obscured even in the late
images. progresses, Fig. 4. The overlying vasculature should be clearly visible and not obscured even in the late
images. Impact of interpretation tutorial. The area under curve (AUC) was calculated from Sensitivity with True negatives and
presented as a probability for the consultant and combined groups, which was 0.82 and 0.66 respectively. DISCUSSION Page 5/11 This study discusses the clinical findings of a range of optic nerve pathologies as revealed by oral fundus
fluorescein angiography (oFFA). We describe a protocol for administering oral fluorescein and acquiring
angiographic images in a population where intravenous cannulation may be too difficult or too
distressing. In addition to this, we found that the images we generated can be interpreted as for images
obtained by intravenous (IV) FFA. Specifically, these clinical observations support those previously
published by Pineles and Arnold (2) who described that early and late leakage on IV FFA can be used to
reliably distinguish papilloedema from pseudo-papilloedema (mainly optic disc drusen). The same
authors produced a subsequent study years later that compared various imaging modalities to
differentiate papilloedema from pseudo-papilloedema and found FFA to be the most accurate in the
paediatric population (3), highlighting the benefit and need of accurate interpretation of these images in
day-to-day clinical work. Moreover, this manuscript highlights the rapid accumulation of oFFA interpretation skills in non-specialist
consultant and trainee ophthalmologists. We can highlight a significant difference in accuracy, sensitivity
and specificity between consultant interpretation and trainee interpretation. This inter-observer variability
could have ramifications in clinical practice as patients present to a variety of clinics and, should an
ophthalmologist not feel comfortable in interpreting oFFA images, they may not be able to benefit from
their high sensitivity and specificity of oFFA. There have been multiple studies since the 1960s in which FFA has been used to identify papilloedema in
an adult population (6) and how to differentiate the findings from those in pseudo-papilloedema (7). The
clear consensus between these studies is that leakage around the disc is suggestive for papilloedema
and directs away from pseudo-papilloedema. Our study corroborates these findings and expands on
them to represent the paediatric population. It is clear from our observations, and others, that timing plays
a critical role is discerning signs such as leakage which are fundamental to an accurate diagnosis. Our
findings support the dynamic nature of changes occurring over time, that further aid in diagnosis when
compared to investigations using a single image at a single time point. This is particularly relevant when
comparing studies that quote a lower diagnostic accuracy of this investigation but do not capture earlier
time points (8). In addition, our study presents a series of images and description of pertinent features for inflammatory
papillitis. DISCUSSION The presence of disc leakage in inflammatory optic nerve head pathology has been well
described in patients with an underlying diagnosis of sarcoidosis (9) and Vogt-Koyangai-Harada
Syndrome (10). Our findings in paediatric patients with inflammatory papillitis secondary to panuveitis
are very similar in description to the adult patients with the above conditions. It is therefore apparent that
paediatric inflammatory optic nerve pathology looks very similar on oFFA to adult IV FFA. Differentiating
between underlying conditions may be very limited using FFA but occult granulomas that may be not
visible on clinical examination can be become apparent on FFA (9). In addition, our study presents a series of images and description of pertinent features for inflammatory
papillitis. The presence of disc leakage in inflammatory optic nerve head pathology has been well
described in patients with an underlying diagnosis of sarcoidosis (9) and Vogt-Koyangai-Harada
Syndrome (10). Our findings in paediatric patients with inflammatory papillitis secondary to panuveitis
are very similar in description to the adult patients with the above conditions. It is therefore apparent that
paediatric inflammatory optic nerve pathology looks very similar on oFFA to adult IV FFA. Differentiating
between underlying conditions may be very limited using FFA but occult granulomas that may be not
visible on clinical examination can be become apparent on FFA (9). Inter observer variability has been well demonstrated before and in addition to their other findings Chang
et al (3) demonstrated a range of accuracy between neuro-ophthalmologists of 83%-97% in interpreting Page 6/11 FFA images of paediatric optic nerve pathology. Our study further extends the finding of variability to the
interpretation of ophthalmologists fully trained in other subspecialties as well as a wide range of training
grades. This suggests a need for a training model for ophthalmologists of all grades to standardise
interpretation acumen. A limitation of this study was the presentation of clinical images without any clinical context. The signs
and symptoms upon presentation, complete with investigations and additional imaging modalities is
fundamental to reaching a correct diagnosis. We would expect the diagnostic accuracy to be higher if the
participants were provided with this information. An additional limitation was the imposition of an
arbitrary time limit of one minute to answer all questions on each image. Finally, the presentation of
images did not allow the interpreter to magnify optic nerve features which may have limited their ability
to observe key findings such as vessel obscuration. DISCUSSION In reality, such time limits do not exist and clinicians
can magnify areas of interest to further evaluate image features. Future work will focus on validating the investigation by assessing additional images presented to the
same body of interpreters. An additional goal would be to optimise a teaching strategy and delivery of an
oFFA tutorial to maximise participants engagement, understanding, and application of new skills. FUNDING The authors did not receive any additional financial resources for completion of this work. CONCLUSION This is the first study that describes the characteristic features of several common, and serious, optic
nerve abnormalities specifically for oFFA interpretation. We found that our methodology achieved
comparable clinical features to those previously published. Moreover, we found that interpretation skills
could be rapidly developed such as to obtain a high diagnostic accuracy with high sensitivity and
specificity. CONFLICT OF INTEREST The authors declare no confilicts of interests . References Int Ophthalmol. 2007;27(2–3):155–61. ACKNOWLEDGMENTS The authors would like to acknowledge the paediatric nursing team and imaging technician team at
Addenbrooke’s hospital for their support in completing the oFFA investigations described above. We Page 7/11 would also like to acknowledge the participation of consultant and trainees colleagues who participated
in the study. would also like to acknowledge the participation of consultant and trainees colleagues who participated
in the study. References 1. Cavallerano AA. Ophthalmic fluorescein angiography. Optom Clin. 1996;5(1):1–23. 1. Cavallerano AA. Ophthalmic fluorescein angiography. Optom Clin. 1996; 2. Pineles SL, Arnold AC. Fluorescein angiographic identification of optic disc drusen with and without
optic disc edema. J Neuroophthalmol. 2012;32(1):17–22. 3. Chang MY, Velez FG, Demer JL, Bonelli L, Quiros PA, Arnold AC, et al. Accuracy of Diagnostic Imaging
Modalities for Classifying Pediatric Eyes as Papilledema Versus Pseudopapilledema. Ophthalmology. 2017;124(12):1839–48. 3. Chang MY, Velez FG, Demer JL, Bonelli L, Quiros PA, Arnold AC, et al. Accuracy of Diagnostic Imaging
Modalities for Classifying Pediatric Eyes as Papilledema Versus Pseudopapilledema. Ophthalmology. 2017;124(12):1839–48. 4. Mohan S, Raman R. Recommended protocol and technique for doing oral fundus fluorescein
angiography for adults and children. Indian J Ophthalmol. 2022;70(12):4430–3. 4. Mohan S, Raman R. Recommended protocol and technique for doing oral fundus fluorescein
angiography for adults and children. Indian J Ophthalmol. 2022;70(12):4430–3. 5. Kelley JS, Kincaid M. Retinal Fluorography Using Oral Fluorescein. Archives of Ophthalmology. 1979;97(12):2331–2. 5. Kelley JS, Kincaid M. Retinal Fluorography Using Oral Fluorescein. Archives of Ophthalmology. 1979;97(12):2331–2. 6. Hayreh SS. Pathogenesis of optic disc edema in raised intracranial pressure. Progress in retinal and
eye research. 2016;50:108–44. 6. Hayreh SS. Pathogenesis of optic disc edema in raised intracranial pressure. Progress in retinal and
eye research. 2016;50:108–44. 7. Miller SJ, Sanders MD, Ffytche T. Fluorescein fundus photography in the detection of early
papilloedema and its differentiation from pseudo-papilloedema. Lancet. 1965;2(7414):651–4. 7. Miller SJ, Sanders MD, Ffytche T. Fluorescein fundus photography in the detection of early
papilloedema and its differentiation from pseudo-papilloedema. Lancet. 1965;2(7414):651–4. 8. Elhusseiny AM, Fong JW, Hsu C, Grigorian F, Grigorian AP, Soliman MK, et al. Oral Fluorescein
Angiography for the Diagnosis of Papilledema Versus Pseudopapilledema in Children. American
Journal of Ophthalmology. 2023;245:8–13. 8. Elhusseiny AM, Fong JW, Hsu C, Grigorian F, Grigorian AP, Soliman MK, et al. Oral Fluorescein
Angiography for the Diagnosis of Papilledema Versus Pseudopapilledema in Children. American
Journal of Ophthalmology. 2023;245:8–13. 9. Moschos MM, Guex-Crosier Y. Anterior segment granuloma and optic nerve involvement as the
presenting signs of systemic sarcoidosis. Clin Ophthalmol. 2008;2(4):951–3. 9. Moschos MM, Guex-Crosier Y. Anterior segment granuloma and optic nerve involvement as the
presenting signs of systemic sarcoidosis. Clin Ophthalmol. 2008;2(4):951–3. 10. Arellanes-García L, Hernández-Barrios M, Fromow-Guerra J, Cervantes-Fanning P. Fluorescein fundus
angiographic findings in Vogt-Koyanagi-Harada syndrome. Int Ophthalmol. 2007;27(2–3):155–61. 10. Arellanes-García L, Hernández-Barrios M, Fromow-Guerra J, Cervantes-Fanning P. Fluorescein fundus
angiographic findings in Vogt-Koyanagi-Harada syndrome. Figures Page 8/11 gure 1 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating papilloedema. Figure 1 Pathway to completing an oFFA investigation. This begins with the decision to undertake the
investigation, the informed consent of the paediatric patient and guardians. The fluorescein is then
prescribed, and the dose is adjusted by weight of the patient. The dosing phase involves baseline
observations of blood pressure, heart rate, temperature, and baseline colour photographs. This is
followed by fluorescein ingestion, dilated in apple juice, using a straw. Finally, the patient is imaged at Page 9/11 time points of 15-, 30-, 45-, and 60-minutes post ingestion. The patient is monitored for any adverse
effects and the sequence of images can be reviewed by the ordering physician once completed. Figure 2
Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating the normal optic nerve. Figure 5 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating inflammatory papillitis. Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating inflammatory papillitis. Figure 4 Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating optic nerve head drusen. Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating optic nerve head drusen. Figure 5
Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating inflammatory papillitis. Figure 2 Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating the normal optic nerve. Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating the normal optic nerve. Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating the normal optic nerve. Figure 3
Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating papilloedema. Figure 3 Figure 3 Right eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating papilloedema. Page 10/11 Figure 4
Left eye colour image of optic nerve with subsequent oFFA series taken at 15-, 30-, and 45 minutes
illustrating optic nerve head drusen. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SUPPLEMENTARYINFORMATION.docx Page 11/11
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Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap Kinerja Guru SMK Swasta Se-Kecamatan Bangil
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Kelola/Kelola: Jurnal Manajemen Pendidikan
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cc-by
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Magister Manajemen Pendidikan
FKIP Universitas Kristen Satya Wacana
jurnalkelola@gmail.com Magister Manajemen Pendidikan
FKIP Universitas Kristen Satya Wacana
jurnalkelola@gmail.com e-ISSN 2549-9661
Volume: 6, No. 2, Juli-Desember 2019
Halaman: 188-192 Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap
Kinerja Guru SMK Swasta Se-Kecamatan Bangil Windasari
Manajemen Pendidikan Universitas Negeri Surabaya
windasari@unesa.ac.id Muchammad Zulham Yahya
SMK Darut Tauhid Bangil
zulwamyahya27@gmail.com ABSTRACT This research objective to find out the impact of motivation and compensation through
teachers performance Vocational High School in Bangil district. Collecting data technique
used questionnaire and distributed to respondent. Sampling technique is cluster random
sampling. Sample for every school are 3 teachers from each major. Totally sample 42
respondents from 5 schools. Validity test used pearson product moment. Analisis technique to
answer the research questions are inferential statistic multiple linier regression. Research
results showed motivation and compensation have an impact through teachers performance
Vocational High School in Bangil District. Keywords: Motivation, Compensation, Performance, Teachers
Article Info
Received date: 3 Oktober 2019
Revised date: 28 November 2019
Accepted date: 20 Desember 2019 Keywords: Motivation, Compensation, Performance, Teachers Article Info
Revised date: 28 November 2019
Accepted date: 20 Desember 2019 Received date: 3 Oktober 2019 Received date: 3 Oktober 2019 PENDAHULUAN besar dalam mengembangkan potensi peserta
didik di sekolah. Pendidikan memiliki peranan penting
dalam mencerdaskan manusia. Berdasarkan
Undang-Undang Nomor 20 tahun 2003 tentang
Sistem Pendidikan Nasional, Pasal 3 dijelaskan
bahwa tujuan pendidikan nasional adalah
mengembangkan potensi peserta didik agar
menjadi manusia yang beriman dan bertakwa
kepada Tuhan Yang Maha Esa, berakhlak
mulia, sehat, berilmu, cakap, kreatif, mandiri,
dan menjadi warga negara yang demokratis
serta bertanggung jawab. Untuk mencapai
tujuan pendidikan tersebut, kualitas guru
menjadi salah satu faktor penentu keberhasilan
peserta didik dalam proses belajar mengajar. Kinerja guru memiliki kontribusi yang cukup Motivasi
guru
memiliki
dampak/
implikasi terhadap pemimpin dan pengelola
sekolah karena motivasi guru juga berpengaruh
terhadap motivasi peserta didik. Guru dengan
tingkat motivasi yang tinggi akan mampu
menciptakan lingkungan sosial dan psikologi
yang lebih baik untuk peserta didik. Oleh
karena itu, motivasi guru juga merupakan kunci
keberhasilan kinerja organisasi atau sekolah. Bukti empiris menunjukkan terdapat
hubungan yang kuat diantara kompensasi dan
kualitas
guru. Penelitian
yang
ada
mengindikasikan bahwa kompensasi berpotensi
untuk meningkatkan kinerja guru. Kompensasi 188 Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap Kinerja Guru … | Windasari & Muchammad Z. Yahya masih memiliki kinerja yang kurang. Penelitian
sebelumnya yang sudah pernah dilakukan
terkait motivasi merupakan penelitian yang
dilakukan untuk mengetahui kinerja guru di
sekolah negeri. Kompensasi yang diberikan
kepada guru negeri memiliki system yang
berbeda dengan sistem kompensasi yang
diberikan kepada guru swasta. Oleh karena itu
peneliti ingin mengetahui pengaruh motivasi
dan kompensasi terhadap kinerja guru SMK
swasta di kecamatan bangil. Adapun tujuan dari
penelitian ini adalah untuk mengukur seberapa
besar pengaruh motivasi kerja dan kompensasi
terhadap kinerja guru SMK Swasta se-
Kecamatan Bangil? yang diterima oleh guru baik berupa financial
ataupun non finanasial juga akan berdampak
kepada kepuasan kerja. Guru yang memiliki
kepuasan kerja bagus maka akan terus
menunjukkan kinerja yang maksimal. Motivasi dan kompensasi merupakan
faktor yang berpengaruh terhadap kinerja guru. Motivasi menjadi poin yang penting mengingat
tanggung jawab guru adalah menyampaikan
ilmu pengetahuan dan keterampilan kepada
peserta
didik. Sedangkan
tujuan
dari
kompensasi yang baik adalah untuk menarik,
memotivasi dan mempertahankan orang-orang
yang berkinerja baik untuk pencapaian tujuan
organisasi. Kompensasi yang diberikan kepada
individu merupakan suatu bentuk apresiasi jasa
dan upaya dalam menyelesaikan tugas dan
pekerjaan mereka. Saat ini negara maju dan
berkembang mengalami kekurangan guru
berkualitas di bidang pendidikan. Sebagai
factor penting dalam keberhasilan dalam
pembelajaran, guru seharunya memiliki kinerja
yang bagus. PENDAHULUAN Sekolah Menengah Kejuruan
(SMK) adalah salah satu lembaga pendidikan di
Indonesia yang berperan dalam menghasilkan
lulusan yang memiliki kompetensi siap kerja. Sehingga, kualitas SMK sangat menentukan
mutu Sumber Daya Manusia (SDM) bangsa
Indonesia. Data (Dikbud, 2016) menunjukkan
bahwa kinerja guru SMK masih dalam kategori
kurang yaitu dnegan nilai masih kurang yaitu
77.60%. Kelola: Jurnal Manajemen Pendidikan, Vol. 6, No. 2, Juli-Desember 2019 Desian penelitian ini akan mengukur variable
motivasi dan kompensasi secara bersama-sama
terhadap kinerja. Keterangan:
X1
: Motivasi Kerja
X2
: Kompensasi Guru
Y
: Kinerja Guru erhadap kinerja. Gambar 1.1 Pengaruh Variabel X1 dan X2
terhadap Y
X1
X2
Y Hasil dari analisis data menggunakan
spps diperoleh hasil regresi ganda seperti tabel
1.1 dibawah ini. Gambar 1.1 Pengaruh Variabel X1 dan X2
terhadap Y Gambar 1.1 Pengaruh Variabel X1 dan X2 Tabel 1.1 Analisis Persamaan Linier Regresi Ganda
Coefficientsa
Model
Unstandardized Coefficients Standardized Coefficients
t
Sig. B
Std. Error
Beta
1
Constant
3.684
2.826
1.304 .200
Tot Mot
.359
.079
.367 4.530 .000
Tot Komp
1.096
.138
.645 7.972 .000 Karena untuk motivasi kerja t hit = 4,530
>1.68 = t 0,05;39, maka motivasi berpengaruh
signifikan terhadap kinerja guru. Begitu juga
untuk kompensasi guru, t hit = 7,972 >1.68= t
0,05;39, maka kompensasi guru berpengaruh
signifikan terhadap kinerja guru. Dari tabel diatas, persamaan linier
regresi ganda Kinerja Guru (Y) dalam Motivasi
Kerja (X1) dan Kompensasi Guru (X2) adalah
sebagai berikut : Y = 3,684 + 0,0356 X1 + 1,096 X2 ANOVAa
Model
Sum of Squares df Mean Square F
Sig. 1
Regression
621.882
2
310.941 102.253 .000b
Residual
118.595 39
3.041
Total
740.476 41 Dari tabel diatas terlihat F hit = 102,253
> 3,23 = F 0,05(2;39), maka regresi linier ganda
signifikan. mengacu kepada penguasaan konsep dan
keterampilan
mengajar
di
dalam
kelas. Semakin tinggi motivasi guru untuk mengajar,
guru akan menunjukkan kemampuan optimal di
dalam kelas sehingga kinerjanya akan terus
meningkat. METODE PENELITIAN Penelitian ini merupakan penelitian
kuantitatif deskriptif dengan metode survey. Penelitian ini dilaksanakan di 5 SMK di Bangil
dengan subyek penelitian ini adalah 3 orang
guru dari tiap jurusan pada setiap sekolah. Setiap guru akan mengisi dan menjawab
kuesioner yang telah disebarkan. Metode
penarikan sampel yang digunakan adalah
menggunakan Central Limit Teorema (sebuah
teorema yang menyatakan bahwa kurva
distribusi sampling untuk ukuran sampel 30
atau lebih akan berpusat pada nilai parameter
populasi dan akan memiliki semua sifat-sifat
distribusi normal). Agar tidak berpusat pada
satu titik maka digunakan metode cluster
random sampling dengan mengambil 3 guru
dari tiap jurusan yang ada di sekolah masing-
masing sehingga jumlah sample yang diambil
dari populasi yang ada sebanyak 42 guru dari 5
sekolah yang terbagi 14 jurusan. Berdasarkan
penelitian
sebelumnya
yang
dilakukan
oleh
(Nowsu,
2016)
kompensasi
memiliki
pengaruh
terhadap
motivasi guru di sekolah negeri. Penelitian ini
juga
menyatakan
bahwa
motivasi
juga
berpengaruh terhadap kinerja guru di sekolah
negeri di Kenya.Selain itu, berdasarkan hasil
penelitian (Sukrapi, 2014) menunjukkan bahwa
motivasi kerja guru berpengaruh positif dan
signifikan terhadap kinerja guru. Dalam penelitian ini akan digunakan uji
validitas product moment dan uji reliabilitas
croanbach’s alpha dengan bantuan software
SPSS for windows versi 21. Teknik analisis
data yang digunakan untuk menguji dalam
penelitian ini yaitu Regresi Linier Berganda
untuk menguji apakah ada pengaruh variabel
motivasi dan kompensasi terhadap kinerja guru. Dari data rendahnya kinerja guru SMK
yang dikeluarkan oleh Kemendikbud dapat
dilihat bahwa secara umum kualitas guru SMK 189 Kelola: Jurnal Manajemen Pendidikan, Vol. 6, No. 2, Juli-Desember 2019 Simpulan Motivasi dan kompensasi merupakan
dua variabel penting dalam mempengaruhi
tinggi rendahnya kinerja guru. Keinginan guru
untuk mengajar dengan baik di kelas serta
didukung pemberian kompensasi pelatihan
peningkatan kompetensi dari sekolah akan
memberikan dampak yang sangat positif
terhadap kinerja guru di sekolah. Hasil penelitian ini menunjukkan hasil
yang signifikan korelasi antara motivasi dan
kompensasi terhadap kinerja guru. Pengaruh
motivasi kerja dan kompensasi terhadap kinerja
guru SMK Swasta se-Kecamatan Bangil positif
dan signifikan sebesar 83,98%. Jika semakin
tinggi motivasi kerja yang dimiliki dan
kompensasi yang diterima oleh guru maka
kinerja guru SMK swasta se-Kecamatan Bangil
semakin tinggi. Selain
itu,
penelitian
ini
juga
membuktikan
bahwa
kompensasi
guru
berpengaruh positif juga terhadap kinerja guru
SMK Swasta se-Kecamatan Bangil. Semakin
besar atau tinggi kompensasi yang diberikan
oleh sekolah terhadap guru maka akan semakin
tinggi dan baik pula kualitas kinerja guru. Hal
ini
sejalan
dengan
pendapat
Handoko
(2008:157) kompensasi hendaknya mendorong
perilaku-perilaku yang diinginkan. Prestasi
kerja yang baik, pengalaman, kesetiaan,
tanggung jawab baru dan perilaku-perilaku lain
dapat dihargai melalui rencana kompensasi
yang efektif. Pembahasan Berdasarkan data analisis, diperoleh
hasil bahwa motivasi kerja dan kompensasi
guru
secara
bersama-sama
berpengaruh
terhadap kinerja guru SMK Swasta se-
kecamatan Bangil. Motivasi guru sangat
penting
untuk
menumbuhkan
dan
mengembangkan proses pembelajaran yang
baik di dalam kelas. Motivasi guru di sekolah Kompensasi dan insentif finansial yang
diberikan kepada guru merupakan factor
penting dalam persaingan yang kompetitif di
antara SMK swasta yang ada, bentuk lain
pemberian kompensasi berupa kesempatan
guru
untuk
mengikuti
pelatihan
dan 190 Pengaruh Motivasi Kerja Dan Kompensasi Guru Terhadap Kinerja Guru … | Windasari & Muchammad Z. Yahya swasta di Bangil merupakan sekolah SMK
dalam pengelolaan SDM nya sudah terstruktur. Beberapa guru yang direkrut merupakan guru-
guru yang memiliki kompetensi sesuai dengan
bidang yang diampunya dan juga merupakan
lulusan dari universitas yang bereputasi. pengembangan diri juga sangat berpengaruh
terhadap kinerja guru. Guru yang memiliki
kinerja
bagus,
melaksanakan
tugas
pembelajaran di kelas dengan baik didukung
dengan teknikal skills atau kompetensi yang
bagus. Artinya pemberian kompensasi baik
dalam bentuk finasial ataupun pelatihan
keduanya sangat berpengaruh terhadap kinerja
guru di sekolah. Hasil tersebut sesuai dengan
pendapat
(Hasibuan,2008)
yang
mendefinisikan
motivasi
sebagai
suatu
perangsang
(want)
dan
daya
penggerak
kemauan bekerja seseorang. Selain input SDM yang cukup memiliki
kualifikasi dan motivasi yang bagus, sistem
kompensasi di beberapa sekolah SMK memang
sudah baik. Saran Adapun
saran
untuk
peneliti
kedepannya adalah bahwa ruang lingkup studi
ini difokuskan pada guru SMK Swasta se-
Kecamatan Bangil yang berdampak pada
generalisasi studi yang terbatas. Keterbatasan
ini mengisyaratkan perlunya studi studi
lanjutan untuk menggeneralisasi hasil-hasil
yang diperoleh pada konteks yang berbeda dan
lebih luas, sehingga konsep-konsep yang diuji
dalam model dapat ditingkatkan validitas
eksternalnya. Seorang guru yang memiliki tingkat
motivasi bagus akang sangat membantu dan
memudahkan organisasi dalam meraih tujuan
organisasi. Guru dengan tingkat motivasi yang
tinggi akan melaksanakan tugas pengajaran
dengan sebaik mungkin kepada peserta didik. Begitu pula sebaliknya, guru dengan tingkat
motivasi yang rendah akan berdampak kepada
kualitas pengajaran yang diberikan kepada
peserta didik. Hampir sebagian besar SMK DAFTAR PUSTAKA DAFTAR PUSTAKA
Afifah, Marnesya. (2011). Pengaruh Motivasi,
Kompensasi, dan Pengembangan Karir
terhadap Kinerja Guru (Studi Kasus
Pada
SMK
Nusantara
Jl. Tarumanegaran dalam No.1 Ciputat). Jakarta : UIN Syarif Hidayatullah. 191 Kelola: Jurnal Manajemen Pendidikan, Vol. 6, No. 2, Juli-Desember 2019 Mulyasa. (2013). Uji Kompetensi dan Penilaian
Kerja Guru. Bandung : PT. Remaja
Rosdakarya. Arikunto,
Suharsimi. (1999). Prosedur
Penelitian: Suatu Pendekatan Praktek. Yogyakarta: Rineka Cipta. Fauzie, Ahmad. (2009). Pengaruh Motivasi dan
Kompensasi Terhadap Kinerja Pegawai
Perpustakaan UIN Syarif Hidayatullah
Jakarta (Tesis). Jakarta : UIN Syarif
Hidayatullah. Nowsu, J (2016). Motivation And Teachers’
Performance
In
Selected
Public
Secondary Schools In Ikenne Local
Government Area Of Ogun State.. British Journal of Psychology Research
Vol.5, No.3, pp 40-50. Ghozali, Imam. (2005). Aplikasi Analisis
Multivariate dengan Program SPSS. Semarang: Universitas Diponegoro. Robbins, Stephen P. (2002). Prinsip-prinsip
Perilaku Organisasi. Jakarta : Erlangga. Handoko,
T. Hani. (2008). Manajemen
Personalia & Sumberdaya Manusia. Yogyakarta: BPFE. Robbins, Stephen P dan Timothy A. Judge. (2009). Perilaku
Organisasi
Organizational Behavior Edisi 12. Jakarta : Salemba Empat. Hasibuan, Malayu S.P. (2008). Manajemen
Sumber Daya Manusia. Jakarta :
Penerbit Bumi Aksara. Sarimaya, Farida. (2008). Sertifikasi Guru. Jakarta : Yrama Widya. Kadarisman,
M
(2012). Manajemen
Kompensasi. Jakarta : Rajagrafindo
Persada. Wekesa, Jane N, dan Silas Nyaroo. (2013). “Effect
of
Compensation
on
Performance of Public Secondary
School Teachers in Eldort Municipality
Kenya”. International
Journal
of
Scientific and Research Publications
(ISSN 2250-3153) Vol. 03 - Issue 6,
June 2013. Keran, Kristina N. (2012). Pengaruh Motivasi
Kerja, Kompetensi, Kompensas dan
Kinerja Karyawan di Yayasan Bintang
Timur Tangerang (Tesis). Jakarta :
Universitas Esa Unggul. 192
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Presence of a Haloarchaeal Halorhodopsin-Like Cl<sup>−</sup> Pump in Marine Bacteria
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Microbes and environments
| 2,018
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cc-by
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Presence of a Haloarchaeal Halorhodopsin-Like Cl– Pump in Marine Bacteria Yu Nakajima1,2*, Takashi Tsukamoto3†, Yohei Kumagai1,2, Yoshitoshi Ogura4, Tetsuya Hayashi4, Jaeho Song5,
Takashi Kikukawa6,7, Makoto Demura6,7, Kazuhiro Kogure1,2, Yuki Sudo3, and Susumu Yoshizawa1,2 Yu Nakajima1,2*, Takashi Tsukamoto3†, Yohei Kumagai1,2, Yoshitoshi Ogura4, Tetsuya Hayashi4, Jaeho Song5,
Takashi Kikukawa6,7, Makoto Demura6,7, Kazuhiro Kogure1,2, Yuki Sudo3, and Susumu Yoshizawa1,2 1Atmosphere and Ocean research Institute (AORI), The University of Tokyo, 5–1–5, Kashiwanoha, Kashiwa, Chiba 277–8564,
Japan; 2Department of Natural Environmental Studies, Graduate School of Frontier Sciences, the University of Tokyo, Chiba
277–8563, Japan; 3Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama
700–8530, Japan; 4Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka-shi, Fukuoka 812–8582,
Japan; 5Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea; 6Faculty of Advanced Life Science,
Hokkaido University, Sapporo 060–0810, Japan; and 7Global Station for Soft Matter, Global Institution for Collaborative Research
and Education, Hokkaido University, Sapporo 060–0810, Japan Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone
of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl–-pumping
rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one
of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus
halorhodopsin (RmHR) was identified as a light-driven inward Cl– pump. Spectroscopic assays showed that the estimated
dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl–-transporting
photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial
ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial
HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and
crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related
genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl–-pumping rhodopsin is
present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR. Key words: family Rhodothermaceae, rhodopsin, genome analysis, chloride ion pump Rhodopsins are seven-transmembrane proteins composed
of a protein moiety (opsin) and light-absorbing chromophore
(retinal). * Corresponding author. E-mail: yunakajima@aori.u-tokyo.ac.jp;
Tel: +81–4–7136–6168; Fax: +81–4–7136–6169.
† Present address: Faculty of Advanced Life Science, and Global
Station for Soft Matter, Global Institution for Collaborative
Research and Education, Hokkaido University, Sapporo 060–0810,
Japan Microbes Environ. Vol. 33, No. 1, 89-97, 2018 Microbes Environ. Vol. 33, No. 1, 89-97, 2018 https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME17197 Key words: family Rhodothermaceae, rhodopsin, genome analysis, chloride ion pump Materials and Methods Strain information and genomic sequencing of R. marinus SG-29T,
R. marina SAORIC-28T, and R. profundi SAORIC-476T. R. marinus SG-29T was isolated from surface seawater (30° 40' N,
138° 00' E; depth 50 m), and additional information has been
reported previously (27). R. marina SAORIC-28T was isolated from
a deep seawater sample obtained from the western North Pacific
Ocean (32° 00' N, 138° 13' E; depth 3000 m) by the R/V Tansei
maru (Atmosphere and Ocean Research Institute, The University of
Tokyo and Japan Agency for Marine-Earth Science and Technology
[JAMSTEC]) on 3 July 2010 (KT-10-12 cruise). R. profundi SAORIC-
476T was isolated from a deep seawater sample obtained from the
western North Pacific Ocean (32° 00' N, 140° 00' E; depth 3000 m)
during the research cruise (MR-11-05) of R/V Mirai (JAMSTEC) in
May 2011. Details of the culture conditions and phenotypic traits of
these strains have been described previously (28, 30). The genomic
DNA of strains SAORIC-28T and SAORIC-476T was extracted
using phenol-chloroform and ethanol precipitation (21). Library
preparation was performed for strain SAORIC-28T, as previously
reported for strain SG-29T (24). A KAPA HyperPlus Kit (Kapa
Biosystems, Boston, MA, USA) was used for library preparation for
strain SAORIC-476T. Each end of the libraries (300 bp) of SAORIC-
28T and SAORIC-476T were sequenced on a MiSeq instrument with
MiSeq Reagent kit version 3 (Illumina, San Diego, CA, USA). ( pp
μ
)
Retinal isomer compositions were analyzed using normal-phase
high-performance liquid chromatography (HPLC). The procedures
for the retinal oxime extraction and HPLC analysis were the same as
those described in our previous study (35). Regarding measurements
under dark conditions, the sample was maintained in the dark for
more than 1 week. In measurements under light conditions, the
sample was illuminated with 540±10 nm light for 5 min before retinal
oxime extraction. The retinal oxime extracted from the purple
membrane (PM) containing Halobacterium salinarum bacteriorho-
dopsin (HsBR) was used as a reference. The eluting retinal oxime
was monitored by its absorption at 360 nm. The flow rate was 1 mL/
min. All measurements were performed at 25°C. p
All UV-Vis spectra were recorded at 25°C using a UV-1800
spectrophotometer (Shimadzu, Kyoto, Japan). Presence of a Haloarchaeal Halorhodopsin-Like Cl– Pump in Marine Bacteria pH was measured using a pH meter
(LAQUA F-72; HORIBA, Kyoto, Japan). Measurements were repeated
under the same conditions after the addition of the protonophore
carbonyl cyanide m-chlorophenylhydrazone (CCCP, final concen-
tration, 30 μM). All measurements were performed at 4°C. Different
solutes (100 mM NaCl, KCl, MgCl2, NaBr, NaI, NaNO3, and
Na2SO4) were used in the ion selectivity analysis of RmHR. (XeR) cluster, and the other is a unique cluster consisting of
rhodopsin containing a TSA motif (amino acid residues 85,
89, and 96 in BR numbering). A phylogenetic analysis
showed that this unique rhodopsin cluster is closely related to
the HR of halophilic archaea. Therefore, we named these
HR-like rhodopsins RmHR (R. marinus SG-29T halorhodopsin),
R28HR1, and R28HR2 (R. marina SAORIC-28T halorhodopsin
1 and 2). A functional analysis using heterologously expressed
RmHR was subsequently conducted to identify its ion specificity. We further performed a spectroscopic analysis using purified
RmHR to clarify its spectroscopic characteristics because
even rhodopsins with the same ion transport specificity have
intermediates with different ion affinities and structural
changes, and the properties are important factors that define
rhodopsins. In the present study, we performed genomic and
phylogenetic analyses, and also characterized RmHR spec-
troscopically, and these properties were compared with those
of known microbial rhodopsins. Spectroscopic analysis of RmHR. E. coli BL21 (DE3) cells were used for the functional expression
of RmHR as a recombinant protein. The procedures for protein
expression and purification were the same as those described in our
previous study (35). In spectroscopic measurements, the buffer was
sufficiently exchanged by centrifugation (AmiconUltra centrifuge
unit; 30,000 molecular weight cut-off, Merck Millipore, Bedford,
MA, USA) and the PD-10 column (GE Healthcare, Waukesha, WI,
USA) with 10 mM 3-(N-morpholino)-propanesulfonic acid (MOPS)
buffer (Dojindo Laboratories, Kumamoto, Japan) containing 0.1%
(w/v) n-dodecyl-β-D-maltoside (DDM, Dojindo Laboratories) and
NaCl at the desired concentrations (1 mM to 4 M). In order to
maintain ionic strength at 4 M, Na2SO4 was added to the samples at
the desired concentrations (0–1.333 M). Neither Na+ nor SO42– was
a substrate ion for RmHR (see the Results and Discussion section). The optical density of DDM-solubilized RmHR was adjusted to
approximately 0.5 optical density (OD) at its absorption maximum
(approx. 10 μM). Presence of a Haloarchaeal Halorhodopsin-Like Cl– Pump in Marine Bacteria They are widely distributed among animals and
prokaryotes and have been classified into two groups: type-1
(microbial origin) and type-2 (eukaryotic origin). Microbial
type-1 rhodopsins were initially discovered in extremely
halophilic archaea: a light-driven outward H+-pumping rhodopsin
(bacteriorhodopsin [BR] [26]), inward Cl–-pumping rhodopsin
(halorhodopsin [HR] [22]), and light-sensing rhodopsins
(sensory rhodopsin I and II [SRI and SRII]) (31). In 2000, an
H+-pumping rhodopsin was also found in a marine bacterial
genomic fragment using a metagenomic approach and was
named proteorhodopsin (PR) (4). Recent studies have shown
that PR genes are widely present among marine bacteria and
archaea inhabiting the euphotic layer of the ocean (7). Furthermore, outward Na+-pumping rhodopsin (NaR) (15) and
inward Cl–-pumping rhodopsin (ClR) (43) were discovered in
marine bacteria. Of note, BR and HR of haloarchaea and PR
and ClR of marine bacteria belong to distinct phylogenetic
lineages, suggesting that these ion-pumping rhodopsins
evolved independently in their respective habitats. The family Rhodothermaceae belongs to the class Cytophagia
in the phylum Bacteroidetes. This family includes 7 genera
(Longimonas [40], Longibacter [41], Rhodothermus [1],
Rubricoccus [27], Rubrivirga [28], Salinibacter [3], and Salisaeta
[36]) and has been found in diverse environments, e.g.,
hydrothermal vents, hypersaline environments, and oceans. The genera Longimonas, Longibacter, Salisaeta, and Salinibacter
include halophilic bacteria, and members of the genus
Rhodothermus are thermophiles. In contrast, members of the
genera Rubricoccus and Rubrivirga have been isolated from
surface or deep seawater and are not extremophiles. Salinibacter,
a halophilic bacterium, dominates with halophilic archaea in
hypersaline environments (5–25% according to fluorescence
in situ hybridization [2] and 7–9% according to a metagenomic
analysis [8]). Previous studies reported that Salinibacter ruber
M31T carries multiple rhodopsin genes: xanthorhodopsin (XR),
HR, and SRI (23). In addition, a genomic analysis suggested
that S. ruber acquired not only the HR and SRI genes, but also
many other genes that are necessary for salt adaptation from
halophilic archaea by lateral gene transfer (LGT) because
both of these halophilic prokaryotes inhabit and dominate the
same environment (23). Following the genome information of Rubricoccus marinus
SG-29T (24), we herein report two genome sequences of
Rubrivirga marina SAORIC-28T and R. profundi SAORIC-
476T. A genomic analysis revealed that R. marinus SG-29T and
R. marina SAORIC-28T have five rhodopsin genes belonging
to two different clusters: one is included in the xenorhodopsin 90 Nakajima et al. then irradiated through a 520±10 nm bandpass filter (MX0520;
Asahi Spectra) for 3 min. Gene preparation and ion transport measurements of RmHR. The DNA fragments encoding RmHR, R28HR1, and R28HR2
were chemically synthesized by Eurofins Genomics (Eurofins Genomics,
Tokyo, Japan) with codon optimization for Escherichia coli. These
gene fragments were inserted into the NdeI and XhoI sites of the
pET21a vectors (Novagen, Darmstadt, Germany); consequently, the
plasmids encoded RmHR, R28HR1, and R28HR2 with hexahistidines
at the C terminus. The vectors were transformed into E. coli strain
C41 (DE3) (Lucigen, Middleton, WI, USA). E. coli cells with the
plasmid were incubated at 37°C on an LB medium agar plate supple-
mented with ampicillin (final concentration, 100 μg mL–1). Cells
carrying the plasmid were then grown at 37°C in 200 mL of 2×YT
medium supplemented with ampicillin (final concentration, 100 μg
mL–1), and protein expression was induced at an OD 660 nm (OD660)
of 0.4–0.6 with 0.2 mM isopropyl β-D-1-thiogalactopyranoside
(IPTG) and 10 μM all-trans-retinal (Sigma-Aldrich, St Louis, MO,
USA). Rhodopsin-expressing cells were collected by centrifugation
(8,000×g for 3 min), washed three times with 100 mM NaCl, and
then re-suspended in the solvent for measurement. The light source
was a 300 W xenon lamp (MAX-303; Asahi Spectra, Tokyo, Japan),
and 6 mL of the cell suspension was initially placed in the dark and ΔA=1/(1+10(pKa-pH)) Materials and Methods In the Cl– titration
experiment, the absorption maximum wavelength (λmax) of RmHR
was plotted against the logarithm of the Cl– concentration ([Cl–]) and
analyzed by fitting the data to the following Hill equation: λmax=a+b·[Cl–]n/([Cl–]n+[Kd]n) λmax=a+b·[Cl–]n/([Cl–]n+[Kd]n) where a, b, Kd, and n represent the offset, the amplitude of the λmax
change, the dissociation constant for Cl–, and the Hill coefficient,
respectively. In the pH titration experiment, the sample was suspended
in a 6-mix buffer (0.89 mM citrate, 0.89 mM MES, 1.1 mM TES,
0.78 mM TAPS, 1.1 mM CHES, 0.33 mM CAPS; MES, TES, TAPS,
CHES, and CAPS are Good’s buffers and were purchased from
Dojindo Laboratories) (34) containing 0.05% DDM, 0–4 M NaCl,
and 0–1.333 M Na2SO4. The initial pH was approximately 3.8 and
was then adjusted to the desired value by adding a very small
amount of 2 M NaOH, followed by measurements of the UV-Vis
absorption spectrum. The concentration change in the RmHR sample
was almost negligible. The acid dissociation constant, pKa, of the
protonated Schiff base of the retinal chromophore was estimated by
fitting the data to the Henderson-Hasselbalch equation: Gene preparation and ion transport measurements of RmHR. ΔA=1/(1+10(pKa-pH)) where P0 and P1–P4 represent the unphotolyzed original pigment and
the 1st–4th kinetically defined states, respectively. All data on
time-dependent changes in absorbance were simultaneously fit with
a sum of 4 exponential decay functions. The appropriate number of
exponents was selected based on reductions in the standard deviation
of the residuals. The P1–P4 states were allowed to contain a few
physically defined photointermediates such as L, N, and O with
individual absorption maxima when a quasi-equilibrium state
existed between them. The time constants τ1–τ4 and the absorbance
differences Δε1–Δε4 between the P1–P4 states and the original P0
state were assessed according to the fitting results. Independently,
the pure retinal spectrum of the P0 state was extracted from the
UV-Vis absorption spectrum of the initial state (unphotolyzed)
RmHR by spectral decomposition using the skewed Gaussian function
as described previously (10). Finally, the absolute spectra of each
P1–P4 state were obtained by adding the P0 spectrum to each absor-
bance difference Δε1–Δε4. Results and Discussion Genomic and phylogenetic analysis of R. marinus SG-29T,
R. marina SAORIC-28T, and R. profundi SAORIC-476T. A previous study showed that R. marinus SG-29T has a
4.43-Mbp chromosome encoding 3847 CDSs (24). The present
genome sequence analysis of R. marina SAORIC-28T and R. profundi SAORIC-476T revealed that these strains have 4.98 Mbp
and 4.48 Mbp chromosomes encoding 4267 and 3842 CDSs,
respectively (Table S1). The G+C contents of both strains
from the genus Rubrivirga were two of the highest (72.5%
and 71.3%) among the available genomes of class Cytophagia
in the NCBI database. The genera Rubrivirga and Rubricoccus
belong to the family Rhodothermaceae, and the 16S rRNA
phylogenetic tree is shown in Fig. 1. This family contains
members of thermophilic (genus Rhodothermus), halophilic
(genera Salinibacter, Salisaeta, Longimonas, and Longibacter),
and slightly halophilic (genera Rubricoccus and Rubrivirga,
isolated from ocean) strains. Growth ranges for the NaCl
concentration of thermophilic strains were similar to those of
the genera Rubricoccus and Rubrivirga (Fig. 1). In contrast,
halophilic bacteria in the family Rhodothermaceae grew
under high-salinity conditions (20–30%). In addition, the
lower limit of the growth range for NaCl of the genera
Longimonas, Longibacter, and Salisaeta (2–4%), was lower
than that of the genus Salinibacter (12–15%). The tempera-
ture growth range for the genera Rubricoccus and Rubrivirga
was approximately 4–42°C (Fig. 1). In contrast, halophilic
Rhodothermaceae strains grew at 20–50°C, and this range
was between the temperature growth range for thermophiles
(50–85°C) and marine isolates (4–42°C). These physiologi-
cal traits clearly showed that the family Rhodothermaceae
contained two different types of extremophiles, indicating
that the common ancestor of this family was a halophilic or
thermophilic bacterium. Therefore, the genera Rubricoccus
and Rubrivirga with members that inhabit the ocean may
have a higher G+C content in class Cytophagia as a remnant
of their ancestors. As described in the Results and Discussion section, the P3 state
was obtained as a quasi-equilibrium mixture of N-like and O-like
intermediates with relative fractions that were dependent on the Cl–
concentration. P3 state spectra contained not only the main absorption
bands of the N-like and O-like intermediates, but also the respective
β-bands. The procedures for the spectral decomposition of the P3
state were the same as those described in previous studies (10, 29). Assuming both β-bands are identical to that of the P0 state, the
spectrum of the P3 state is described as: ΔA=1/(1+10(pKa-pH)) salinarum R1, genes with an
e-value less than 1e-10 were counted, and a threshold with a
sequence identity >50% was established. We used the genome
family Rhodothermaceae, Cytophaga hutchinsonii ATCC 33406T,
which is a type species of the class Cytophagia, Indibacter alkaliphilus
LW1T, Spirosoma linguale DSM 74T, N. marinus S1-08T, and
Flavobacterium aquatile LMG 4008T. Since I. alkaliphilus and S. linguale, belonging to the class Cytophagia, have a rhodopsin gene,
these genome data were used for this assay. Furthermore, because N. marinus is a marine flavobacterium with the ClR gene and F. aquatile is a type species of the class Flavobacteriia, these genome
data were also used. with a Nd:YAG laser (5 mJ pulse–1, 532 nm, 7 ns) as an actinic light
source as described previously (18, 35). Due to the setting for the
response time (0.5 μs) of a homemade I-V converter, a large scattering
artifact from the laser flash appeared before 10 μs. Therefore, plots
were started from 10 μs. Time-dependent changes in absorbance
were measured from 400 to 710 nm at 10-nm intervals. Data before
the laser pulse were adopted as a baseline. In order to improve the
signal-to-noise ratio, the results of 30 flashes were averaged at each
wavelength. No further data processing was applied. The temperature
was kept at 20°C using a thermostat. We analyzed the data using a sequential model based on our
previous studies (10, 18) as follows: P0→P1→P2→P3→P4→P0 where P0 and P1–P4 represent the unphotolyzed original pigment and
the 1st–4th kinetically defined states, respectively. All data on
time-dependent changes in absorbance were simultaneously fit with
a sum of 4 exponential decay functions. The appropriate number of
exponents was selected based on reductions in the standard deviation
of the residuals. The P1–P4 states were allowed to contain a few
physically defined photointermediates such as L, N, and O with
individual absorption maxima when a quasi-equilibrium state
existed between them. The time constants τ1–τ4 and the absorbance
differences Δε1–Δε4 between the P1–P4 states and the original P0
state were assessed according to the fitting results. Independently,
the pure retinal spectrum of the P0 state was extracted from the
UV-Vis absorption spectrum of the initial state (unphotolyzed)
RmHR by spectral decomposition using the skewed Gaussian function
as described previously (10). Finally, the absolute spectra of each
P1–P4 state were obtained by adding the P0 spectrum to each absor-
bance difference Δε1–Δε4. Spectrum of the P3 state=f×Abs(N,λ)+(1–f)×Abs(O,λ)+Abs(β,λ) where f, Abs (N, λ), Abs (O, λ), and Abs (β, λ) represent the fraction
of the N-like intermediate, the main absorption band of the N-like
intermediate, the main absorption band of the O-like intermediate,
and the β-band, respectively. We employed skewed Gaussian functions
to describe these three absorption bands and then decomposed the
spectrum of the P3 state to estimate f, which we assumed to be the
only parameter dependent on the Cl– concentration. The fractions of
the N-like and O-like intermediates were plotted against the Cl–
concentration, and data were simultaneously analyzed by the Hill
equation as follows: ΔA=1/(1+10(pKa-pH)) where pH and ΔA are the adjusted pH value and absorption change
at 380 nm, which corresponds to the deprotonated Schiff base,
respectively.l where pH and ΔA are the adjusted pH value and absorption change
at 380 nm, which corresponds to the deprotonated Schiff base,
respectively.l Time-dependent changes in absorbance were measured by flash-
photolysis using a homemade computer-controlled apparatus equipped 91 HR-Like Cl– Pump in Marine Bacteria order to compare gene synteny around the Cl–-pumping rhodopsin
gene and blh gene, we acquired the genomes of H. salinarum R1, S. ruber M31T, Synechocystis sp. PCC 7506, Mastigocladopsis repens
PCC 10914, Salisaeta longa DSM 21114T, and Rhodothermus
marinus DSM 4252T from the NCBI RefSeq database. The ortholog
clustering of the CDSs of each genome was performed using
eggNOG-mapper (13) and the bactNOG dataset in the eggNOG
database, version 4.5 (14). We visualized the operon structure and
colored each gene based on each eggNOG ortholog cluster using R
software. In order to evaluate the potential LGT, a BLAST analysis
was performed on the genomes of H. salinarum R1, genes with an
e-value less than 1e-10 were counted, and a threshold with a
sequence identity >50% was established. We used the genome
family Rhodothermaceae, Cytophaga hutchinsonii ATCC 33406T,
which is a type species of the class Cytophagia, Indibacter alkaliphilus
LW1T, Spirosoma linguale DSM 74T, N. marinus S1-08T, and
Flavobacterium aquatile LMG 4008T. Since I. alkaliphilus and S. linguale, belonging to the class Cytophagia, have a rhodopsin gene,
these genome data were used for this assay. Furthermore, because N. marinus is a marine flavobacterium with the ClR gene and F. aquatile is a type species of the class Flavobacteriia, these genome
data were also used. order to compare gene synteny around the Cl–-pumping rhodopsin
gene and blh gene, we acquired the genomes of H. salinarum R1, S. ruber M31T, Synechocystis sp. PCC 7506, Mastigocladopsis repens
PCC 10914, Salisaeta longa DSM 21114T, and Rhodothermus
marinus DSM 4252T from the NCBI RefSeq database. The ortholog
clustering of the CDSs of each genome was performed using
eggNOG-mapper (13) and the bactNOG dataset in the eggNOG
database, version 4.5 (14). We visualized the operon structure and
colored each gene based on each eggNOG ortholog cluster using R
software. In order to evaluate the potential LGT, a BLAST analysis
was performed on the genomes of H. Fraction of N-like (or O-like)=a+b·[Cl–]n/([Cl–]n+[Kd]n) where a, b, Kd, and n represent the offset, amplitude of the λmax change,
dissociation constant for Cl–, and Hill coefficient, respectively. Phylogenetic and comparative genome analyses Multiple alignments of amino acid sequences were performed
using the CLUSTALW option in MEGA 6.0 software (32). Phylogenetic trees were constructed using the maximum-likelihood
(ML) method (5) with 1,000 bootstrap replications (6). The nucleotide
sequences of 16S rRNA and the amino acid sequences of rhodopsins,
15,15'-β-carotene dioxygenase (blh), and lycopene cyclase (crtY and
crtYcd) genes were collected from the NCBI database and RAST
annotated sequences for the genera Rubricoccus and Rubrivirga. In 92 Nakajima et al. Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences. Phylogenetic tree of 16S rRNA showing the taxonomic
positions of strains SG-29T, SAORIC-28T, and SAORIC-476T among related species of the family Rhodothermaceae in the classes Cytophagia and
Sphingobacteria. The habitats of each strain are indicated by different-colored squares. The red and open circles indicate strains containing the rhodopsin
gene and no rhodopsin gene, respectively. The G+C content and growth range for temperature and NaCl concentrations are also shown on the right
side. Black closed circles indicate G+C content of each strain. ND: No genome data. +
content Fig. 1. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences. Phylogenetic tree of 16S rRNA showing the taxonomic
positions of strains SG-29T, SAORIC-28T, and SAORIC-476T among related species of the family Rhodothermaceae in the classes Cytophagia and
Sphingobacteria. The habitats of each strain are indicated by different-colored squares. The red and open circles indicate strains containing the rhodopsin
gene and no rhodopsin gene, respectively. The G+C content and growth range for temperature and NaCl concentrations are also shown on the right
side. Black closed circles indicate G+C content of each strain. ND: No genome data. The genome sequence analysis showed that the genomes of
R. marinus SG-29T and R. marina SAORIC-28T encoded two
different types of rhodopsin genes (RmXeR [WP_094549673]
and RmHR [WP_094550238]) and three rhodopsin genes
(R28XeR [WP_095509440], R28HR1 [WP_095509924],
and R28HR2 [WP_095512583]), respectively. The identities
of amino acid sequences among RmHR, R28HR1, and R28HR2
were 73% (RmHR-R28HR1), 62% (RmHR-R28HR2), and
57% (R28HR1-R28HR2) respectively. No rhodopsin gene
was found in the genome of R. profundi SAORIC-476T. Light-induced pH changes and ion selectivity of RmHR. In order to clarify the function of this unique rhodopsin
cluster, codon-optimized RmHR, R28HR1, and R28HR2 genes
were chemically synthesized and each gene was heterologously
overexpressed in E. coli. Light-induced pH changes were
observed in suspensions of E. coli cells expressing each gene
in 100 mM NaCl. However, light-induced pH changes in
R28HR1 were smaller than those in RmHR, and were not
observed in R28HR2. Therefore, RmHR was used in subse-
quent analyses. Light-induced alkalization was observed in
100 mM NaCl, and this pH change was not abolished by the
addition of CCCP (Fig. 3A). Since CCCP is a protonophore,
this alkalization may be explained by passive proton influx
due to the negative membrane potential, which was created
by outward Na+ or inward Cl– translocation. In order to eluci-
date the ion selectivity of RmHR, we performed similar
observations in different salt solutions (Fig. 3B). The signal
almost disappeared in 100 mM NaI and Na2SO4, suggesting
that RmHR cannot transport Na+. In contrast, strong signals
were observed in 100 mM KCl, NaBr, and MgCl2, and a slight
pH change was noted in 100 mM NaNO3. These anion-dependent
transport properties are very similar to those of archaeal HRs
in halophilic archaea, suggesting that RmHR functions as a
light-driven inward anion (Cl–, Br–, and NO3–) pump. Fraction of N-like (or O-like)=a+b·[Cl–]n/([Cl–]n+[Kd]n) Although R28HR1 and R28HR2 were found from the deep-
sea isolate, it currently remains unclear whether these are
deep-sea adapted rhodopsins because the habitat of the genus
Rubrivirga is not yet understood. In the BLASTP search
against the metagenome database (env_nr), no sequence with
>50% identity with RmHR was found. This result implies
that the RmHR gene is very rare in the marine environment. In the family Rhodothermaceae, four strains (S. ruber M31T,
S. longa DSM 21114T, R. marinus SG-29T, and R. marina
SAORIC-28T) have rhodopsin genes. In contrast, no rhodopsin
gene was found in the two thermophilic strains belonging to
the genus Rhodothermus (Fig. 1). A recent study showed that
RmXeR of SG-29T functions as a light-driven inward proton
pump (17). R28XeR also belongs to the same cluster of
RmXeR, suggesting that the function of R28XeR is an inward
H+ pump. RmHR, R28HR1, and R28HR2 formed a unique
cluster close to HR and SyHR (Fig. 2 and S1). Although HR
and SyHR were discovered from halophilic prokaryotes and a
freshwater cyanobacterium, RmHR, R28HR1, and R28HR2
formed a unique marine bacterial cluster. RmHR and
R28HR1 had the same TSA motif as HR, and R28HR2 has
TTD motif sequences (Table 1). In contrast, cyanobacterial
HRs had the TSD, TSV, or TSL motif sequence (9). Spectroscopic analysis of RmHR. Spectroscopic analysis of RmHR. In order to investigate the spectroscopic properties of
RmHR, we prepared a detergent DDM-solubilized sample. Under this condition, the results described below were able to
be precisely compared to those of all the other anion-pumping
rhodopsins investigated, such as Natronomonas pharaonis
halorhodopsin (NpHR), H. salinarum halorhodopsin (HsHR),
Nonlabens marinus S1-08T rhodopsin 3 (NM-R3), Synechocystis 93 HR-Like Cl– Pump in Marine Bacteria Fig. 2. Unrooted maximum-likelihood phylogenetic tree of microbial rhodopsins. (A) Amino acid sequences of microbial rhodopsins were aligned
using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based
on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. All rhodopsin amino acid sequence data used in this study were obtained
from the public database (http://www.ncbi.nlm.nih.gov/). Dark gray indicates the clade consisting of the freshwater cyanobacterial rhodopsin clade,
and light gray indicates halorhodopsin. An open circle and closed gray circle indicate the positions of strain SG-29T and SAORIC-28T, respectively. Sodium ion pump rhodopsin (NaR); chloride ion pump rhodopsin (ClR); xanthorhodopsin-like rhodopsin (XLR); proteorhodopsin (PR); xenorhodopsin
(XeR); sensory rhodopsin (SR); bacteriorhodopsin (BR); halorhodopsin (HR). The motif sequence (amino acid residues 85, 89, and 96 in BR numbering)
is shown under the rhodopsin name. (B) Detailed phylogenetic tree of the SR, BR, HR, cyanobacterial HR, and RmHR homologues. The habitats of
each strain are indicated by squares of different colors. Bootstrap values >80% are indicated as a percentage of the replicates tested. Asterisk represents
the strain that was isolated from seawater by using medium for freshwater cyanobacteria. Fig. 2. Unrooted maximum-likelihood phylogenetic tree of microbial rhodopsins. (A) Amino acid sequences of microbial rhodopsins were aligned
using CLUSTALW, and evolutionary distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based
on 1,000 replications; evolutionary analyses were conducted in MEGA 6.0. All rhodopsin amino acid sequence data used in this study were obtained
from the public database (http://www.ncbi.nlm.nih.gov/). Dark gray indicates the clade consisting of the freshwater cyanobacterial rhodopsin clade,
and light gray indicates halorhodopsin. An open circle and closed gray circle indicate the positions of strain SG-29T and SAORIC-28T, respectively. Sodium ion pump rhodopsin (NaR); chloride ion pump rhodopsin (ClR); xanthorhodopsin-like rhodopsin (XLR); proteorhodopsin (PR); xenorhodopsin
(XeR); sensory rhodopsin (SR); bacteriorhodopsin (BR); halorhodopsin (HR). Spectroscopic analysis of RmHR. The motif sequence (amino acid residues 85, 89, and 96 in BR numbering)
is shown under the rhodopsin name. (B) Detailed phylogenetic tree of the SR, BR, HR, cyanobacterial HR, and RmHR homologues. The habitats of
each strain are indicated by squares of different colors. Bootstrap values >80% are indicated as a percentage of the replicates tested. Asterisk represents
the strain that was isolated from seawater by using medium for freshwater cyanobacteria. Table 1. Comparison of important residues, particularly motifs, among ion pumps. Residue numbers in BR (above) and PR (below)
85
86
87
88
89
90
91
92
93
94
95
96
Rhodopsin type
Taxonomic group
Motif
97
98
99
100
101
102
103
104
105
106
107
108
BR
Halobacterium salinarum R1
DTD
D
W
L
F
T
L
P
L
L
L
L
D
PR
Ca. Pelagibacter ubique HTCC 1062
DTE
D
W
L
I
T
V
P
L
L
M
L
E
ClR
Nonlabens marinus S1-08T
NTQ
N
W
M
A
T
I
P
C
L
L
L
Q
SyHR
Synechocystis sp. PCC 7506
TSD
T
W
F
L
S
T
P
L
L
L
L
D
HR
Salinibacter ruber M31T
TSA
T
W
A
F
S
T
P
F
I
L
L
A
HR
Halobacterium salinarum R1
TSA
T
W
A
L
S
T
P
M
I
L
L
A
RmXeR
Rubricoccus marinus SG-29T
DTA
D
W
V
V
T
T
P
L
L
L
T
A
R28XeR
Rubrivirga marina SAORIC-28T
DTA
D
W
V
V
T
T
P
L
L
L
A
A
RmHR
Rubricoccus marinus SG-29T
TSA
T
W
F
T
S
T
P
L
L
L
L
A
R28HR1
Rubrivirga marina SAORIC-28T
TSA
T
W
F
L
S
T
P
L
L
L
L
A
R28HR2
Rubrivirga marina SAORIC-28T
TTD
T
W
F
L
T
T
P
L
L
L
L
D Table 1. Comparison of important residues, particularly motifs, among ion pumps. sp. PCC 7509 halorhodopsin (SyHR), and Mastigocladopsis
repens PCC 10914 halorhodopsin (MrHR) (11, 25, 33, 35,
42). Based on this background, we used DDM-solubilized
RmHR in the subsequent spectroscopic analysis. We also
focused on the photochemical properties of RmHR in the
presence of NaCl because Cl– is considered to be a candidate
substrate anion for RmHR in the native habitat of R. marinus
SG-29T (Cl– concentration of approximately 500 mM). sp. Spectroscopic analysis of RmHR. coli cell suspensions expressing RmHR in solutions containing (A) 100 mM
NaCl without (black solid line) and with (gray broken line) 30 μM CCCP and (B) 100 mM KCl, MgCl2, NaBr, NaI, NaNO3, and Na2SO4 without
CCCP. The cell suspension was illuminated with green light (520 nm). Temperature was maintained at 4°C. The word “div.” represents division. dependency, Cl–-binding affinity in the initial state (practically
the dissociation constant, Kd,int.) was estimated to be 7.6±1.7 mM
by the Hill equation (Fig. S2C). The Hill coefficient was
1.2±0.23, indicating positive cooperativity for Cl– binding. The estimated Kd,int. value of RmHR was in the same order as
those of NpHR, HsHR, and MrHR (2–6 mM) (10, 11, 18, 33,
42), but was approximately 100-fold lower than that of SyHR
(approximately 0.1 mM) (25) and approximately 3- and
5-fold larger than those of NM-R3 (24 mM) and Fulvimarina
pelagi HTCC 2506T rhodopsin (FR) (84 mM), respectively
(16, 35). In addition, the acid dissociation constant, pKa, of
the Schiff base was dependent on the Cl– concentration (Fig. S2D), which is similar to archaeal HsHR and NpHR (39). At
low concentrations of NaCl (0 - several tens of mM), the
absorption band of the deprotonated Schiff base appeared in
the shorter wavelength region at approximately 380 nm (Fig. S2B). Therefore, subsequent experiments were performed in
the presence of more than 100 mM NaCl. section). The P1 and P2 states each contained a blue-shifted
photointermediate with a similar absorption maximum at 510 nm. Analogous to NpHR, we tentatively assigned these interme-
diates as L1- and L2-like, respectively. The following P3 state
contained at least two photointermediates with absorption
maxima at 520 and 590 nm, which we tentatively assigned as
N- and O-like intermediates, respectively, by referring to the
P3 state of NpHR. In the case of NpHR, the molar ratio of N
and O changed in a Cl– concentration-dependent manner (10,
18). In order to evaluate this phenomenon, we performed
flash photolysis measurements on RmHR with varying Cl–
concentrations. It is important to note that the Cl– concentration-
dependent absorption change was significant in the P3 state
(Fig. S4). Fig. S3D shows the Cl–-dependent change in the
molar ratio of N-like and O-like intermediates between 100 mM
and 4 M NaCl. The fraction of the N-like intermediate increased
with elevations in the Cl– concentration, indicating that this
intermediate is the Cl–-binding form. Spectroscopic analysis of RmHR. PCC 7509 halorhodopsin (SyHR), and Mastigocladopsis
repens PCC 10914 halorhodopsin (MrHR) (11, 25, 33, 35,
42). Based on this background, we used DDM-solubilized
RmHR in the subsequent spectroscopic analysis. We also
focused on the photochemical properties of RmHR in the
presence of NaCl because Cl– is considered to be a candidate
substrate anion for RmHR in the native habitat of R. marinus
SG-29T (Cl– concentration of approximately 500 mM). rhodopsin encoded in the R. marinus SG-29T gene (RmXeR)
showed a light-dependent retinal composition change in the
initial state (75 and 45% of all-trans retinal under dark and
light conditions, respectively) (17). The biological significance
of RmXeR currently remains unclear. In order to evaluate this feature in RmHR, we measured the
UV-Vis absorption spectra under various Cl– concentrations
ranging between 1 mM and 4 M (Fig. S2B). Many anion-
pumping rhodopsins show a spectral blue-shift upon substrate
anion binding. In the experiment, ionic strength was maintained
at 4 M by adding Na2SO4 at appropriate concentrations because
in many Cl–-pumping rhodopsins, Cl– binding reaches a plateau
at 4 M and SO42– is not a substrate ion for RmHR, as shown
in Fig. 3B. As shown in Fig. S2B, RmHR exhibited a Cl–-
dependent spectral blue-shift from 550 nm in the presence of
1 mM Cl– to 542 nm in the presence of 4 M Cl–. Based on this We initially examined the retinal isomer composition of
RmHR under dark and light conditions in the presence of 1 M
NaCl. As shown in Fig. S2A, RmHR predominantly possesses
all-trans retinal as a chromophore, which is responsible for
the light-driven anion pump function, at 99.6 and 96.2%
under dark and light conditions, respectively. This property is
the same as those of the other anion-pumping rhodopsins
reported to date (11, 25, 33, 35). On the other hand, another Nakajima et al. 94 Nakajima et al. 94
Fig. 3. Anion transport activity of RmHR. Light-induced pH changes in E. coli cell suspensions expressing RmHR in solutions containing (A) 100 mM
NaCl without (black solid line) and with (gray broken line) 30 μM CCCP and (B) 100 mM KCl, MgCl2, NaBr, NaI, NaNO3, and Na2SO4 without
CCCP. The cell suspension was illuminated with green light (520 nm). Temperature was maintained at 4°C. The word “div.” represents division. Fig. 3. Anion transport activity of RmHR. Light-induced pH changes in E. Spectroscopic analysis of RmHR. In contrast, the fraction
of the O-like intermediate decreased with the increase in the
Cl– concentration, indicating that the O-like intermediate is
the Cl–-releasing form. Data were simultaneously fit by the
skewed Gaussian function in order to identify the fractions of
the N-like and O-like intermediates. As shown in Fig. S3E,
we plotted these fractions against NaCl concentrations and
then estimated the dissociation constant for Cl– release, Kd,rel,
as 308±45.6 mM using the Hill equation. The value was
approximately 40-fold larger than that of the initial state
(Kd,int.=7.6 [mM]), the behavior of which was the same as
NpHR, whereas the magnitude of the change was different
(Kd,int.=2 [mM] and Kd,rel=1.2 [M]) (33). In the P4 state (Fig. S3C), a photointermediate with the same absorption maximum
at 540 nm as the initial state (P0) was identified, and we named
it the RmHR’-intermediate by referring to other anion-pumping
rhodopsins, including NpHR (18, 35). Analogous to NpHR,
in the O-like-to-RmHR’ transition, Cl– was entered into the
initial binding site on the extracellular side of RmHR, which
was supported by the blue-shifted absorption from O-like to
RmHR’ and the same absorption maximum of RmHR’ as that
of the initial state. We summarized the Cl– transporting pho- The Cl–-transporting photoreaction, called a photocycle, of
RmHR was analyzed using a time-resolved flash-photolysis
technique. Fig. S3A shows the flash-induced light-minus-
dark difference absorption spectra in the 10-μs to 388-ms
time domain. After flash excitation, photointermediates with
absorption bands at approximately 460 nm and 610 nm,
respectively, were generated with the concomitant disappear-
ance of the initial state absorption band at approximately
550 nm. Time-dependent absorption changes in these three
absorption bands are shown in Fig. S3B. Data were simulta-
neously fit to the exponential decay function with 4 exponents,
which was the same number as NpHR (10, 18), indicating
that RmHR passed through 4 kinetically intermediate states,
P1–P4, in its photocycle. The time constants between each
transition, τ1–τ4, were 0.105, 0.483, 2.14, and 23.5 ms,
respectively. According to the sequential model (10, 18), we
calculated the absolute spectra of the P1–P4 states of RmHR
in the presence of 1,000 mM NaCl, as shown in Fig. S3C. The
spectrum of P0 corresponded to the initial state pure absorption
spectrum of RmHR, which was extracted from the experimentally
measured absorption spectrum (see the Materials and Methods HR-Like Cl– Pump in Marine Bacteria 95 tocycle model of RmHR in Fig. Phylogenetic analyses based on retinal biosynthesis pathway
genes. Functional and spectroscopic analyses revealed that RmHR
is a light-driven inward Cl–-pumping rhodopsin similar to the
HR of halophilic archaea rather than to ClR (e.g., NM-R3 and
FR) of marine flavobacteria. In order to further examine the
evolutionary history of RmHR, we performed phylogenetic
analyses on retinal biosynthesis pathway genes, such as crtY Table 2. Comparison of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. Rhodopsin (Type)
Retinal isomer
(dark/light)
(%)
Kd,int/Kd,rel
(mM)
Schiff base pKa
Photo-
intermediates
Existence of
N-like–O-like
equilibrium
Similarity of the
photocycle scheme
References
RmHR
(marine bacterial HR)
99.6/96.2
7.6/308
7.3 (0 M NaCl)–
10 (4 M NaCl)
(K), L1, L2, N, O,
RmHR’
Yes
(This study)
This study
HsHR
(archaeal HR)
50/86
2.6/N.D. Approx. 8
(10 mM NaCl)–
Approx. 11.5
(below 1 M NaCl)
K, L1, L2, N
No
Different from
RmHR
38, 39, 42
NpHR
(archaeal HR)
83/77
2/1200
Approx. 8
(10 mM NaCl)–
Approx. 10
(below 1 M NaCl)
(K), L1, L2, N, O,
NpHR’
Yes
Similar to RmHR
10, 18, 33,
37, 39
NM-R3
(marine bacterial ClR)
90.3/91.6
24/N.D. N.D. K, L, N, O1, O2,
NM-R3’
No
Different from
RmHR
35
FR
(marine bacterial ClR)
96.4/86.6
84/N.D. N.D. K, L, O, FR’
N.D. Different from
RmHR
16
MrHR
(cyanobacterial HR)
>98/>98
1.99/N.D. N.D. (K), L, N, O,
MrHR’
Yes
Different from
RmHR
9, 11
SyHR
(cyanobacterial HR)
N.D./97.5
0.1/N.D. 10.6 (1 M NaCl)
K, L, N, O, SyHR’ Yes
Similar to RmHR
25
N D indicates “Not determined” Table 2. Comparison of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. Rhodopsin (Type)
Retinal isomer
(dark/light)
(%)
Kd,int/Kd,rel
(mM)
Schiff base pKa
Photo-
intermediates
Existence of
N-like–O-like
equilibrium
Similarity of the
photocycle scheme
References
RmHR
(marine bacterial HR)
99.6/96.2
7.6/308
7.3 (0 M NaCl)–
10 (4 M NaCl)
(K), L1, L2, N, O,
RmHR’
Yes
(This study)
This study
HsHR
(archaeal HR)
50/86
2.6/N.D. Approx. 8
(10 mM NaCl)–
Approx. 11.5
(below 1 M NaCl)
K, L1, L2, N
No
Different from
RmHR
38, 39, 42
NpHR
(archaeal HR)
83/77
2/1200
Approx. 8
(10 mM NaCl)–
Approx. 10
(below 1 M NaCl)
(K), L1, L2, N, O,
NpHR’
Yes
Similar to RmHR
10, 18, 33,
37, 39
NM-R3
(marine bacterial ClR)
90.3/91.6
24/N.D. N.D. K, L, N, O1, O2,
NM-R3’
No
Different from
RmHR
35
FR
(marine bacterial ClR)
96.4/86.6
84/N.D. N.D. Spectroscopic analysis of RmHR. S3F. The initially captured
Cl– on the extracellular side of RmHR was transferred to the
intracellular side in the L2-like-to-N-like transition and was
then released into the intracellular bulk space in the N-like-
to-O-like transition. The next Cl– was entered into the initial
binding site through the intracellular side of the molecule in
the O-like to RmHR’ and RmHR’ to initial state transitions. The mechanism was similar to that of NpHR (18), but signifi-
cantly different from that of the bacterial anion pumps,
NM-R3 and FR, which are evolutionally distant from RmHR
(Table 2) (Fig. 2) (16, 35). (lycopene cyclase) and blh (15,15'-β-carotene dioxygenase). The enzymes encoded by the crtY and blh genes catalyze the
terminal cyclization reaction from all-trans-lycopene to all-
trans-β-carotene and cleave β-carotene to produce two mole-
cules of all-trans-retinal as a chromophore of rhodopsin. ML
phylogenetic trees based on the blh and crtY genes are shown
in Fig. 4 and S5. Regarding SAORIC-476T, since this strain
lacks rhodopsin and blh genes, only the crtY gene was used in
this analysis. In the phylogenetic tree of the blh gene,
although each cluster was mainly formed according to the
taxonomic group, the blh genes of SG-29T and SAORIC-28T
in the class Cytophagia belonged to the lineage containing
halophilic archaea and bacteria (Fig. 4). The phylogenetic
tree based on crtY showed that three clusters: Bacteroidetes
crtY and Proteobacteria crtY and crtYcd, had formed, and the
genes of strains SG-29T, SAORIC-28T, and SAORIC-476T
were included in the crtYcd cluster (Fig. S5). Carotenoid
cyclases such as crtY are considered to be diversified by LGT
or gene duplication, and some Actinobacteria (20), archaea
(12), and Bacteroidetes (19) contain crtYcd. Although the Phylogenetic analyses based on retinal biosynthesis pathway
genes. K, L, O, FR’
N.D. Different from
RmHR
16
MrHR
(cyanobacterial HR)
>98/>98
1.99/N.D. N.D. (K), L, N, O,
MrHR’
Yes
Different from
RmHR
9, 11
SyHR
(cyanobacterial HR)
N.D./97.5
0.1/N.D. 10.6 (1 M NaCl)
K, L, N, O, SyHR’ Yes
Similar to RmHR
25
N.D. indicates “Not determined”. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary
distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary
analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. (B)
Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap
values >80% are indicated as a percentage of the replicates tested. Table 2. Comparison of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. Rhodopsin (Type)
Retinal isomer
(dark/light)
(%)
Kd,int/Kd,rel
(mM)
Schiff base pKa
Photo-
intermediates
Existence of
N-like–O-like
equilibrium
Similarity of the
photocycle scheme
References
RmHR
(marine bacterial HR)
99.6/96.2
7.6/308
7.3 (0 M NaCl)–
10 (4 M NaCl)
(K), L1, L2, N, O,
RmHR’
Yes
(This study)
This study
HsHR
(archaeal HR)
50/86
2.6/N.D. Approx. 8
(10 mM NaCl)–
Approx. 11.5
(below 1 M NaCl)
K, L1, L2, N
No
Different from
RmHR
38, 39, 42
NpHR
(archaeal HR)
83/77
2/1200
Approx. 8
(10 mM NaCl)–
Approx. 10
(below 1 M NaCl)
(K), L1, L2, N, O,
NpHR’
Yes
Similar to RmHR
10, 18, 33,
37, 39
NM-R3
(marine bacterial ClR)
90.3/91.6
24/N.D. N.D. K, L, N, O1, O2,
NM-R3’
No
Different from
RmHR
35
FR
(marine bacterial ClR)
96.4/86.6
84/N.D. N.D. K, L, O, FR’
N.D. Different from
RmHR
16
MrHR
(cyanobacterial HR)
>98/>98
1.99/N.D. N.D. (K), L, N, O,
MrHR’
Yes
Different from
RmHR
9, 11
SyHR
(cyanobacterial HR)
N.D./97.5
0.1/N.D. 10.6 (1 M NaCl)
K, L, N, O, SyHR’ Yes
Similar to RmHR
25
N.D. indicates “Not determined”. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary
distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary
analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. Phylogenetic analyses based on retinal biosynthesis pathway
genes. similarity between the crtYcd and crtY genes is not high, the
crtYcd gene is in a carotenoid biosynthesis gene cluster and
has been shown to encode an enzyme that functions as a
lycopene cyclase-like crtY gene. Halophilic archaea, the
genera Natronomonas, Halobacterium, and Haloarcula, and
halophilic bacteria, S. ruber M31T and S. longa DSM 21114T,
possess the crtYcd, but not crtY gene. Similar to the phylogenetic
tree of the blh gene, the crtYcd genes of Rubricoccus and
Rubrivirga were not close to the genes of the Bacteroidetes
crtY clade, whereas these crtYcd genes were closely related
to the halophilic archaeal ones. Although the family
Rhodothermaceae belongs to the class Cytophagia in the
phylum Bacteroidetes, blh and lycopene cyclase genes formed
a phylogenetic lineage that is distinct from these genes in
Bacteroidetes. In conclusion, we herein report a novel light-driven inward
Cl–-pumping rhodopsin (RmHR), similar to the HR of halo-
philic archaea, in marine isolates of the family Rhodothermaceae
in the phylum Bacteroidetes. Spectroscopic assays also showed
that the properties of RmHR resembled those of HR from the
aspect of the intermediate states and mechanisms of Cl–
uptake and release. Phylogenetic analyses revealed that strains
of the genera Rubricoccus and Rubrivirga may have acquired
some genes by LGT, including the rhodopsin and blh genes
from halophilic archaea. These results suggest that these
genera and halophilic archaea had once lived in the same
environment. Collectively, the present results provide insights
into gene sharing between marine bacteria and halophilic
archaea and facilitate our understanding of the evolutionary
processes occurring in the ecologically diverse environments
of this family. Previous genomic and phylogenetic analyses showed that
S. ruber M31T acquired several genes from halophilic archaea
via LGT (23). Gene transfer beyond a domain may have
occurred because Salinibacter and halophilic archaea dominate
in the same niche, such as a hypersaline environment. The
best example of this gene transfer is the HR gene shared by
Salinibacter and halophilic archaea. In addition, not only this
case, but also other genes (e.g., blh, crtYcd, the cytochrome c
oxidase gene, and nitrous oxide reductase gene) were shared
among halophilic bacteria and archaea. Our results based on
the phylogenetic analysis suggest that the rhodopsin gene and
its related genes were shared not only between Salinibacter
and halophilic archaea, but also among those halophiles and
marine bacteria (R. marinus SG-29T and R. marina SAORIC-
28T). References 1. Alfredsson, G.A., J.K. Kristjansson, S. Hjörleifsdottir, and K.O. Stetter. 1988. Rhodothermus marinus, gen. nov., sp. nov., a thermophilic,
halophilic bacterium from submarine hot springs in Iceland. Microbiology
134:299–306. Visualizing genomic flanking regions of rhodopsin and blh genes. The results showing that several genes were shared among
the halophiles, Rubricoccus and Rubrivirga, indicated that a
common ancestor of strains SG-29T, SAORIC-28T, and
SAORIC-476T inhabited the same environment, such as salt
lakes, as halophiles. In order to clarify whether other genes
were acquired together with the rhodopsin and blh genes, we
compared the genomic flanking regions of these genes among
the genera Rubricoccus and Rubrivirga and the strains pos-
sessing genes closely related to this rhodopsin or the blh gene
(Fig. S6). The results of the analysis showed that there was
low similarity around rhodopsin genes (Fig. S6A). This
dissimilarity may suggest that only Cl–-pumping rhodopsin
genes were acquired by LGT or that a gene transfer event has
not recently occurred. The same analysis based on the blh
gene also showed no remarkable similarity to the flanking
regions of the rhodopsin gene. The flanking regions of R. profundi SAORIC-476T lacking the rhodopsin and blh genes
showed the presence of multiple conserved genes between
SAORIC-28T and SAORIC-476T (Fig. S6B). This result sug-
gested that only the blh gene was deleted in SAORIC-476T or
acquired in SAORIC-28T. The results of a BLAST analysis
revealed that although the numbers of potential LGT of
strains SG-29T, SAORIC-28T, and SAORIC-476T were lower
than that of S. ruber M31T, these numbers were approximately
2- to 4-fold higher than other phylum Bacteroidetes strains 2. Antón, J., R. Rosselló-Mora, F. Rodríguez-Valera, and R. Amann. 2000. Extremely halophilic bacteria in crystallizer ponds from solar
salterns. Appl. Environ. Microbiol. 66:3052–3057. pp
3. Antón, J., A. Oren, S. Benlloch, F. Rodríguez-Valera, R. Amann, and
R. Rosselló-Mora. 2002. Salinibacter ruber gen. nov., sp. nov., a
novel, extremely halophilic member of the Bacteria from saltern
crystallizer ponds. Int. J. Syst. Evol. Microbiol. 52:485–491. 4. Beja, O., L. Aravind, E.V. Koonin, et al. 2000. Bacterial rhodopsin:
evidence for a new type of phototrophy in the sea. Science 289:1902–
1906. 5. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a
maximum-likelihood approach. J. Mol. Evol. 17:368–376.i 6. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach
using the bootstrap. Evolution 39:783–791. g
7. Fuhrman, J.A., M.S. Schwalbach, and U. Stingl. 2008. Phylogenetic analyses based on retinal biosynthesis pathway
genes. Furthermore, these results indicate that the common
ancestor of the genera Rubricoccus and Rubrivirga acquired
rhodopsin and its related genes from halophilic archaea via
LGT in a hypersaline environment, as reported previously (23). Acknowledgements We are grateful to the captains and crews of R/V Mirai
(JAMSTEC) and R/V Tansei Maru (Atmosphere and Ocean Research
Institute, The University of Tokyo and JAMSTEC) for their assis-
tance and support with sample collection. This work was supported
by the Japan Society for the Promotion of Science (JSPS) to TT
(15K18519) and SY (15H02800) and the Interdisciplinary Collaborative
Research Program of the Atmosphere and Ocean Research Institute,
the University of Tokyo. This work was also supported by JST-
CREST and AMED to YS. Phylogenetic analyses based on retinal biosynthesis pathway
genes. (B)
Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap
values >80% are indicated as a percentage of the replicates tested. n of spectroscopic properties of RmHR with archaeal and cyanobacterial HRs and marine bacterial ClRs. N.D. indicates “Not determined”. N.D. indicates “Not determined”. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary
distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary
analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. (B)
Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap
values >80% are indicated as a percentage of the replicates tested. Fig. 4. Unrooted maximum-likelihood phylogenetic tree of the blh gene. Amino acid sequences were aligned using CLUSTALW, and evolutionary
distances were estimated using the LG with Freg model. The tree was constructed using bootstrap values based on 1,000 replications; evolutionary
analyses were conducted in MEGA 6.0. (A) Open and closed gray circles indicate the positions of strains SG-29T and SAORIC-28T, respectively. (B)
Detailed phylogenetic relationship of the blh gene among Flavobacteriia, Cytophagia, halophiles, strain SG-29T, and strain SAORIC-28T. Bootstrap
values >80% are indicated as a percentage of the replicates tested. 96 Nakajima et al. (Fig. S7). This result suggests that genes in strains SG-29T,
SAORIC-28T, and SAORIC-476T may be shared with H. salinarum, similar to S. ruber. similarity between the crtYcd and crtY genes is not high, the
crtYcd gene is in a carotenoid biosynthesis gene cluster and
has been shown to encode an enzyme that functions as a
lycopene cyclase-like crtY gene. Halophilic archaea, the
genera Natronomonas, Halobacterium, and Haloarcula, and
halophilic bacteria, S. ruber M31T and S. longa DSM 21114T,
possess the crtYcd, but not crtY gene. Similar to the phylogenetic
tree of the blh gene, the crtYcd genes of Rubricoccus and
Rubrivirga were not close to the genes of the Bacteroidetes
crtY clade, whereas these crtYcd genes were closely related
to the halophilic archaeal ones. Although the family
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Management of children's urinary tract infections in Dutch family practice: a cohort study
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BioMed Central BioMed Central Management of children s urinary tract infections in Dutch family
practice: a cohort study
Mirjam Harmsen*1, Michel Wensing1, Jozé CC Braspenning1,
René J Wolters1, Johannes C van der Wouden2 and Richard PTM Grol1 Address: 1Centre for Quality of Care Research (WOK), Radboud University Nijmegen Medical Centre, PO Box 9101 (114 kwazo), 6500 HB
Nijmegen, The Netherlands and 2Department of General Practice, Erasmus MC – University Medical Center Rotterdam, PO Box 1738, 3000 DR,
Rotterdam, The Netherlands Email: Mirjam Harmsen* - M.Harmsen@kwazo.umcn.nl; Michel Wensing - M.Wensing@kwazo.umcn.nl;
Jozé CC Braspenning - J.Braspenning@kwazo.umcn.nl; René J Wolters - R.Wolters@kwazo.umcn.nl; Johannes C van der
Wouden - J.vanderWouden@erasmusmc.nl; Richard PTM Grol - R.Grol@kwazo.umcn.nl * Corresponding author Received: 17 July 2006
Accepted: 13 March 2007 BMC Family Practice 2007, 8:9
doi:10.1186/1471-2296-8-9 This article is available from: http://www.biomedcentral.com/1471-2296/8/9 ;
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Family Practice Open Access Abstract Background: Optimal clinical management of childhood urinary tract infections (UTI) potentiates
long-term positive health effects. Insight into the quality of care in Dutch family practices for UTIs
was limited, particularly regarding observation periods of more than a year. Our aim was to
describe the clinical management of young children's UTIs in Dutch primary care and to compare
this to the national guideline recommendations. Methods: In this cohort study, all 0 to 6-year-old children with a diagnosed UTI in 2001 were
identified within the Netherlands Information Network of General Practitioners (LINH), which
comprises 120 practices. From the Dutch guideline on urinary tract infections, seven indicators
were derived, on prescription, follow-up, and referral. Results: Of the 284 children with UTI who could be followed for three years, 183 (64%) were
registered to have had one cystitis episode, 52 (18%) had two episodes, and 43 (15%) had three or
more episodes. Another six children were registered to have had one or two episodes of acute
pyelonephritis. Overall, antibiotics were prescribed for 66% of the children having had ≤ 3 cystitis
episodes, two-thirds of whom received the antibiotics of first choice. About 30% of all episodes
were followed up in general practice. Thirty-eight children were referred (14%), mostly to a
paediatrician (76%). Less than one-third of the children who should have been referred was actually
referred. Conclusion: Treatment of childhood UTIs in Dutch family practice should be improved with
respect to prescription, follow-up, and referral. Quality improvement should address the low
incidence of urinary tract infections in children in family practice. Page 1 of 7
(page number not for citation purposes) BMC Family Practice 2007, 8:9 http://www.biomedcentral.com/1471-2296/8/9 http://www.biomedcentral.com/1471-2296/8/9 Design and setting A prospective cohort study in Dutch family practice was
performed. Study population We identified all children 0–6 years old (born between
1994 and 2001) with UTI diagnosed in 2001 within the
Netherlands Information Network of General Practice
(LINH). The LINH network contains 120 practices and is
representative of the Dutch population of patients, family
practitioners, and types of practices [11]. Informed con-
sent was arranged within the network (general board of
the National Institute for Health Services Research
(NIVEL), general board of the Centre for Quality of Care
Research (WOK), general board of the Dutch College of
General Practitioners (NHG), and the general board of the
National Association of General Practitioners (LHV)). According tothe Dutch Central Committee on Research
Involving Human Subjects (CCMO) regulations only
research in which the study participant has to be physi-
cally present during the study is subject to the Medical
Research Involving Human Subjects Act (WMO) and
therefore ethical approval is not required for studies that
use patient databases. The guideline on UTIs of the Dutch College of General
Practitioners (DCGP) states that every childhood UTI
should be treated with antibiotics because of the risk of
renal scarring. Amoxicillin/clavulanic acid or co-trimoxa-
zole are the medications of choice in an attempt to reach
effective tissue levels and to maximize the chance of the
most effective medicine until test results are available. The
follow-up recommendation takes into account that young
children may not express their complaints clearly, while
they have a high risk of renal scarring. The referral recom-
mendations are based on the patients' age and sex, which
predict the probability of anatomical abnormalities of the
urinary tract system [9]. The International Classification of Primary Care (ICPC)
[12] defines UTI as acute pyelonephritis (ICPC code U70)
or cystitis (ICPC code U71). These definitions imply that
UTI was diagnosed by urine testing, not just suspected or
assumed. The one-year incidence of UTIs in 0 to 6-year-old children
in Dutch family practices averages for girls 25.7 and for
boys 4.5 per 1000 life-years [10]. Insight into the quality
of care for UTI was limited, particularly regarding observa-
tion periods of more than a year. We have acquired the
necessary prospective data to provide insight into the pri-
mary-care-based management of childhood UTIs in the
Netherlands. We aimed to describe the clinical manage-
ment of young children's UTIs in Dutch primary care and
compare this to the national guideline recommendations. Methods
Design and setting g
Awareness of the importance of timely diagnosis and
treatment of childhood urinary tract infections (UTIs) is
growing. Without timely treatment, renal scarring can
occur [1], which is likely to affect approximately 5–15% of
young children with a UTI [2-5]. Renal scarring is associ-
ated with serious health problems in later life, such as
hypertension, complications during pregnancy, and renal
failure [1]. Optimal clinical management of childhood
UTI potentiates long-term positive health effects. There-
fore, guidelines across different countries advocate an
active approach concerning prescription, follow-up, and
referral [6-9]. Follow-up Twenty-eight percent to 37% of all episodes was followed-
up (Table 3). The overall follow-up rate was 32%. About
60% of all episodes with follow-up contacts consisted of
two contacts. The second contact took place within 14
days of the first for 90% of the episodes. There were no
differences for age or sex groups. Student's t-test or chi-square tests, as appropriate, were
used to investigate whether more boys or girls were treated
as recommended by the guideline, and whether younger
children were treated more consistently with the guideline
than older children. We also investigated whether recur-
rent childhood UTIs were more often treated according to
the guideline than single episodes. We considered a prob-
ability level of P < 0.05 statistically significant. Prescriptions Table 2 shows that, overall, 66% of the children received
antibiotics, varying from 61% to 70%. First choice medi-
cation, Amoxicillin/clavulanic acid or co-trimoxazole, was
prescribed for 55% to 83% of the children with antibiotics
(overall 66%). Another 4% to 25% (overall 13%) of the
children with antibiotics received ceftibuten, ofloxacin, or
nitrofurantoin. Since childhood UTIs should be treated as
complicated UTIs according to the DCGP-guideline, these
last three antibiotics should not be prescribed according
to the guidelines. Furthermore, 10% to 25% (overall
19%) of the children with antibiotics received amoxicillin
without clavulanic acid, which is also not according to the
DCGP-guideline. In eight cases, a child who did not
receive medication was referred to a medical specialist
directly after seeing the GP. No differences in prescription
for age or sex were found in any group. Descriptive statistics were applied to patient characteris-
tics, and the numbers of children receiving medication,
follow-up, or referrals. For each indicator, the number of
children (or episodes in the case of follow-up) to whom
family practitioners (FPs) offered the appropriate care was
divided by the total number of children (or episodes in
the case of follow-up) needing the provision of such care
(Table 1). We calculated the percentage of children with
more than one contact during a UTI episode as an indica-
tor of follow-up. Age groups were based on age when hav-
ing the first UTI. Measurements Although there is no specific DCGP guideline on UTIs in
children, the Dutch UTI guideline does include specific
recommendations for children [13]. We derived seven
clinical indicators of appropriate performance. Two indi-
cators focused on medication, one on follow-up, and four
on referring (Table 1). Page 2 of 7
(page number not for citation purposes)
Table 1: Indicators urinary tract infections (UTIs) in children in general practice
Indicator
Measurement
Numerator
Denominator
First-choice antibiotics
1. Antibiotics given
All children receiving antibiotics
All children
2. Amoxicillin/clavulanic acid OR co-
trimoxazole
Children receiving first-choice antibiotics
All children receiving antibiotics
Follow-up
3. Episodes with at least one follow-up contact
Number of UTI episodes with >1 contact
All UTI episodes
Referral
For all groups:
Total referred within the group
Total within the group
4. Children <1 year old
5. Boys <12 years old
6. Girls 1–4 years old with second UTI
7. Girls 5–12 years old with > 1 recurrent UTI Table 1: Indicators urinary tract infections (UTIs) in children in general practice Table 1: Indicators urinary tract infections (UTIs) in children in general practice Numerator Denominator All children
All children receiving antibiotics
All UTI episodes
Total within the group All children
All children receiving antibiotics
All UTI episodes
Total within the group Number of UTI episodes with >1 contact
For all groups:
Total referred within the group Page 2 of 7
(page number not for citation purposes) BMC Family Practice 2007, 8:9 http://www.biomedcentral.com/1471-2296/8/9 ences between included children and children excluded in
step 2 of the flow chart. Analyses The contacts were expressed in units of episodes. Episodes
were considered new episodes if the preceding contact for
UTI occurred more than 28 days previously. Prescriptions
and referrals within 28 days after the last contact for an
episode were linked to that episode. The first UTI in 2001
was assumed to be the child's first episode ever. To be able
to compare groups, four groups of children were created,
based on ICPC code and number of UTI episodes: group
1 (1 episode cystitis), group 2 (2 episodes cystitis), group
3 (≥ 3 episodes cystitis) and group 4 (1 or 2 episodes acute
pyelonephritis). Referrals Table 3 shows that overall 38 of 278 (14%) children were
referred for specialist treatment (range: 8–16%). Seventy-
six percent was referred to a paediatrician; 8% to a urolo-
gist; 3% to radiography; and from 13% it is not clear to
what specialist they were referred. Data collection In the LINH network, the family practice staff routinely
records the encoded patient information in electronic
medical records (EMR). For the period 2001–2003 data
were extracted from the EMR, concerning contacts with
the family practice, prescriptions, referrals, and patient
characteristics (age, sex). Of the 284 children included, 278 (98%) were diagnosed
with cystitis. Of these children, 66% had one episode
(group 1), 19% had two (group 2), and 15% had 3 to 10
(group 3). Six children were diagnosed with acute
pyelonephritis (2%; group 4). Practices were excluded from the analyses if they had reg-
istered fewer than 46 weeks in 2002 or 2003. Patients who
were not on the practice list and patients who had left the
practice before 1 January 2004 were also excluded. During the first UTI episode, the mean age varied from 2.8
(SD 2.5) to 4.1 years (SD 1.5). About 80% of the children
in group 1, 90% in groups 2 and 3, and 100% in group 4
were girls (figure 1). Because of the small numbers, no fur-
ther data are presented for group 4 and for episodes 4–10
in group 3. Page 3 of 7
(page number not for citation purposes) Study population Less than one-third of the children who should have been
referred concerning the guideline on referring specific age
and/or sex categories, was actually referred. Three of four
children younger than one year were referred, and fewer
than 25% of the boys were referred during at least one epi-
sode. In group 2, 9% of the girls aged 1–4 years were Figure 1 shows the selection of children included in this
study. Of 38,408 children in the 120 practices in the year
2001, 461 from 92 practices had a diagnosed UTI (1.2%). Of these 461 children, 284 (62%) in 59 practices could be
followed for three years. There were no age or sex differ- Page 3 of 7
(page number not for citation purposes) Page 3 of 7
(page number not for citation purposes) http://www.biomedcentral.com/1471-2296/8/9 BMC Family Practice 2007, 8:9 of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standar
TI = urinary tract infection
of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standar
TI = urinary tract infection. Study population All children born
between 1994 and
2001
(n=38,408)
All children with UTI
in 2001
(n=461)
Exclusion of
children not
having had a
UTI
All children with UTI
completely registered
for the years 2001-
2003
(n=284)
Exclusion of practices
with incomplete
registration and
patients not belonging
to the practice or with
incomplete
registration
Group 1:
Children with 1
episode cystitis/
UTI
(n=183)
78% female
mean (sd) age:
3.8 (1.6) years
Group 2:
Children with 2
episodes
cystitis/UTI
(n=52)
92% female
mean (sd) age:
3.6 (1.4) years
Group 3:
Children with 3
or more
episodes
cystitis/UTI
(n=43)
93% female
mean (sd) age:
4.1 (1.5) years
Group 4:
Children with
acute
pyelonephritis
(n=6)
100% female
mean (sd) age:
2.8 (2.5) years All children born
between 1994 and
2001
(n=38,408) Exclusion of
children not
having had a
UTI Exclusion of practices
with incomplete
registration and
patients not belonging
to the practice or with
incomplete
registration All children with UTI
completely registered
for the years 2001-
2003
(n=284) Group 3:
Children with 3
or more
episodes
cystitis/UTI
(n=43)
93% female
mean (sd) age:
4.1 (1.5) years Group 3:
Children with 3
or more
episodes
cystitis/UTI
(n=43) Group 4:
Children with
acute
pyelonephritis
(n=6) Group 2:
Children with 2
episodes
cystitis/UTI
(n=52) Group 1:
Children with 1
episode cystitis/
UTI
(n=183)
78% female
mean (sd) age:
3.8 (1.6) years Group 1:
Children with 1
episode cystitis/
UTI Group 1:
Children with 1
episode cystitis/
UTI
(n=183) 78% female
mean (sd) age:
3.8 (1.6) years Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi-
ation; UTI = urinary tract infection
Figure 1
Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi-
ation; UTI = urinary tract infection. Page 4 of 7
(page number not for citation purposes)
Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi-
ation; UTI = urinary tract infection
Figure 1
Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi-
ation; UTI = urinary tract infection. Study population Page 4 of 7
(page number not for citation purposes)
Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi-
ation; UTI = urinary tract infection
Figure 1
Selection of children with urinary tract infections for the analyses mean (sd) age = age during first episode; sd = standard devi-
ation; UTI = urinary tract infection. http://www.biomedcentral.com/1471-2296/8/9 BMC Family Practice 2007, 8:9 Table 2: Prescription of antibiotics in children with urinary tract infections
Group 1
(183 children)
Group 2
(52 children)
Group 3
(43 children)
Total
(278 children)
Percentage (number) of children/antibiotic prescription
Overall children with antibiotics (indicator 1)
66 (121/183)
70 (73/104)
61 (79/129)
66 (273/416)
First episode
66 (121/183)
73 (38/52)
67 (29/43)
68 (188/278)
Second episode
-
67 (35/52)
63 (27/43)
65 (62/95)
Third episode
-
-
53 (23/43)
53 (23/43)
Percentage (number) of children with antibiotics/choice of antibiotic
Amoxicillin/clavulanic acid or co-trimoxazole
Overall first-choice antibiotics (indicator 2)
65 (79/121)
56 (41/73)
76 (60/79)
66 (180/273)
First episode
65 (79/121)
55 (21/38)
69 (20/29)
64 (120/188)
Second episode
-
57 (20/35)
78 (21/27)
66 (41/62)
Third episode
-
-
83 (19/23)
83 (19/23)
Ceftibuten, ofloxacin, or nitrofurantoin
Overall
7 (9/121)
21 (15/73)
14 (11/79)
13 (35/273)
First episode
7 (9/121)
18 (7/38)
14 (4/29)
11 (20/188)
Second episode
-
23 (8/35)
15 (4/27)
19 (12/62)
Third episode
-
-
13 (3/23)
13 (3/23)
Amoxicillin
Overall
25 (30/121)
18 (13/73)
10 (8/79)
19 (51/273)
First episode
25 (30/121)
18 (7/38)
17 (5/29)
22 (42/188)
Second episode
-
17 (6/35)
7 (2/27)
13 (8/62)
Third episode
-
-
4 (1/23)
4 (1/23) Table 2: Prescription of antibiotics in children with urinary tract infections increased risk of complications or underlying pathology
in boys and younger children. Such unawareness may
lead to health complications when the child is older. increased risk of complications or underlying pathology
in boys and younger children. Such unawareness may
lead to health complications when the child is older. referred during the second episode, and in group 3 33%. Two girls in the 5–9 year olds group (7%) were referred
during the third episode. No differences regarding age and
sex subgroups were found. Study population The LINH network provided a unique opportunity for col-
lecting prospective data regarding clinical management in
routine healthcare settings, but one can question FP regis-
tration behaviour and whether all childhood UTIs were
identified with the ICPC codes 'acute pyelonephritis' and
'cystitis'. However, the incidence we found for 0 to 6-year-
olds, which is 12.0 (461*1000/38408), is comparable to
those in other Dutch studies: 15.1 [10] and 13.2 [14]. Since direct observation and hand-searching medical
records are infeasible, using databases of consultation reg-
istrations seems to be the optimal method for collecting
information about FP clinical behaviour. Page 5 of 7
(page number not for citation purposes) Discussion This study showed that the management of childhood
UTIs in the Netherlands varied substantially across
patients. Only 66% of the children received antibiotics
and of these 66% was prescribed first choice antibiotics. There was no follow-up in the majority of the episodes. Referral of children younger than one year was generally
consistent with the guidelines, but the referral rates for
boys, girls 1–4 years old with a second UTI, and girls 5–12
years old with more than one recurrent UTI should have
been much higher, if we consider the guidelines. It is difficult to compare our results on FP management
with other studies because our data are prospectively col-
lected, had a follow-up period of three years, and focussed
on primary care and individual young children; this in
contrast to other studies. A study by Kwok et al. [15]
already gave some insight in the management of chil-
dren's UTIs in Dutch family practice. But compared to our
study, this study concentrated on a much wider age range,
although the children most vulnerable to renal scarring
are the younger ones. The study also had a follow-up
period of only one year and did not pay attention to fol-
low-up after the antiobiotic treatment. One British study
found that 37% of children with proven UTI were sent for
renal tract imaging [16], and another Dutch study We found that the proportion of children receiving amox-
icillin decreased proportionally to the number of epi-
sodes. Perhaps FPs prescribe amoxicillin routinely
because this medication is much older than the combined
form with clavulanic acid, and has less side effects than
amoxicillin/clavulanic acid. If amoxicillin alone does not
work, they prescribe the combination. Prescribing ceftib-
uten, ofloxacin, or nitrofurantoin suggests that not all FPs
are aware that, according to the guidelines, childhood UTI
should be treated as complicated UTI. This is confirmed
by the fact that, for 98% of all children, the FP had regis-
tered the ICPC for cystitis instead of the code for compli-
cated UTI (pyelonephritis). Conclusion In order to prevent negative health outcomes, treatment
of childhood urinary tract infections in Dutch family prac-
tice should be improved with respect to prescription, fol-
low-up, and referral. Quality improvement should
address the low incidence of urinary tract infections in
children in family practice. 2. Pylkkänen J, Vilska J, Koskimies O: The value of level diagnosis of
childhood urinary tract infection in predicting renal injury. Acta Paediatr Scand 1981, 70:879-883. 3. Vernon SJ, Coulthard MG, Lambert HJ, Keir MJ, Matthews JNS: New
renal scarring in children who at age 3 and 4 had had normal
scans with dimercaptosuccinic acid: Follow up study. Brit Med
J 1997, 315:905-908. J
,
4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln
K: Epidemiology of symptomatic urinary tract infection in
childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. J
4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln
K: Epidemiology of symptomatic urinary tract infection in
childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. J
4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln
K: Epidemiology of symptomatic urinary tract infection in
childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. 5. Coulthard MG, Lambert HJ, Keir MJ: Occurence of renal scars in
children after their first referral for urinary tract infection. Brit Med J 1997, 315:918-919. Authors' contributions Improvement of professional performance might sub-
stantially improve clinical outcomes. This is demon-
strated in Sweden, where a more aggressive approach led
to no new cases of uraemia caused by non-obstructive
pyelonephritis during the years 1986–1995 [20]. But,
development and distribution of guidelines do not neces-
sarily lead to better patient care [21]. Future research
could focus on developing interventions to improve pre-
scription, follow-up, and referrals, but should also con-
sider motives for not following the guidelines. Because
not many childhood UTIs appear in family practice in the
Netherlands – our study saw an average of five children
per practice in one year – interventions should not be too
time consuming for the FPs. MH had primary responsibility for protocol development,
analyses, and writing the manuscript. MW, JCCB, RJW,
and JCvdW participated in the development of the proto-
col and contributed to the writing of the manuscript. RG
supervised the design and execution of the study and con-
tributed to the writing of the manuscript. All authors have
read and approved the final manuscript. Competing interests Competing interests The author(s) declare that they have no competing inter-
ests. References 1. Jacobson SH, Eklöf O, Eriksson CG, Lins LE, Tidgren B, Winberg J:
Development of hypertension and ureaemia after pyelone-
phritis in childhood: 27 year follow up. Brit Med J 1989,
299:703-706. 1. Jacobson SH, Eklöf O, Eriksson CG, Lins LE, Tidgren B, Winberg J:
Development of hypertension and ureaemia after pyelone-
phritis in childhood: 27 year follow up. Brit Med J 1989,
299:703-706. 2. Pylkkänen J, Vilska J, Koskimies O: The value of level diagnosis of
childhood urinary tract infection in predicting renal injury. Acta Paediatr Scand 1981, 70:879-883. 3. Vernon SJ, Coulthard MG, Lambert HJ, Keir MJ, Matthews JNS: New
renal scarring in children who at age 3 and 4 had had normal
scans with dimercaptosuccinic acid: Follow up study. Brit Med
J 1997, 315:905-908. 4. Winberg J, Andersen HJ, Bergström T, Jacobsson B, Larson H, Lincoln
K: Epidemiology of symptomatic urinary tract infection in
childhood. Acta Paediatr Scand 1974, Suppl 252:1-20. 5. Coulthard MG, Lambert HJ, Keir MJ: Occurence of renal scars in
children after their first referral for urinary tract infection. Brit Med J 1997, 315:918-919. Acknowledgements The Dutch Kidney Foundation (Nederlandse Nierstichting) provided a
f
hi
d (
j
OV02) W
h
k J
M ld
d H
k
d The Dutch Kidney Foundation (Nederlandse Nierstichting) provided a
grant for this study (project OV02). We thank Jan Mulder and Henk van den
Hoogen for their help with the data analyses. Discussion No significant differences of
age or gender might imply that GPs are unaware of the Page 5 of 7
(page number not for citation purposes) Page 5 of 7
(page number not for citation purposes) http://www.biomedcentral.com/1471-2296/8/9 BMC Family Practice 2007, 8:9 Table 3: Follow-up and referrals in children with urinary tract infections
Group 1
(183 children)
Group 2
(52 children)
Group 3
(43 children)
Total
(278 children)
Pecentage (number) of episodes with more than one contact during the episode
Overall episodes with more than one contact (indicator 3)
28 (52/183)
34 (35/104)
36 (46/129)
32 (133/416)
First episode
28 (52/183)
35 (18/52)
33 (14/43)
30 (84/278)
Second episode
-
33 (17/52)
37 (16/43)
35 (33/95)
Third episode
-
-
37 (16/43)
37 (16/43)
Percentage (number) of children with a referral
Overall
8 (14/183)
19 (10/52)
33 (14/43)
14 (38/278)
First episode
8 (14/183)
12 (6/52)
9 (4/43)
9 (24/278)
Second episode
-
8 (4/52)
16 (7/43)
12 (11/95)
Third episode
-
-
7 (3/43)
7 (3/43)
Percentage (number) of children with a referral, special groups of children
Children <1 year of age (indicator 4)
67 (2/3)
100 (1/1)
0 (0/0)
75 (3/4)
Boys overall (indicator 5)
18 (7/40)
25 (1/4)
0 (0/3)
17 (8/47)
Girls overall
5 (7/143)
19 (9/48)
38 (15/40)
13 (31/231)
Girls 1–4 years, second episode (indicator 6)
-
9 (2/22)
33 (7/21)
21 (9/43)
Girls 5–9 years, third episode (indicator 7)
-
-
7 (2/27)
7 (2/27) Table 3: Follow-up and referrals in children with urinary tract infections Table 3: Follow-up and referrals in children with urinary tract infections = International Classification of Primary Care; LINH =
Netherlands Information Network of General Practice;
UTI = urinary tract infection = International Classification of Primary Care; LINH =
Netherlands Information Network of General Practice;
UTI = urinary tract infection reported 4% of the children being referred [14]. Two other
British studies found much higher rates of referral [17,18]. However, these last two studies used postal question-
naires to measure FP behaviour, whereas our study and
the first two studies used medical records. Reporting
behaviour retrospectively may lead to overestimation of
guideline adherence because of social desirability bias
[19]. reported 4% of the children being referred [14]. Two other
British studies found much higher rates of referral [17,18]. Discussion However, these last two studies used postal question-
naires to measure FP behaviour, whereas our study and
the first two studies used medical records. Reporting
behaviour retrospectively may lead to overestimation of
guideline adherence because of social desirability bias
[19]. Page 6 of 7
(page number not for citation purposes) Abbreviations ,
pp
5. Coulthard MG, Lambert HJ, Keir MJ: Occurence of renal scars in
children after their first referral for urinary tract infection. Brit Med J 1997, 315:918-919. DCGP = Dutch College of General Practitioners; EMR =
electronic medical record; FP = family practitioner; ICPC Page 6 of 7
(page number not for citation purposes) Page 6 of 7
(page number not for citation purposes) BMC Family Practice 2007, 8:9 http://www.biomedcentral.com/1471-2296/8/9 http://www.biomedcentral.com/1471-2296/8/9 http://www.biomedcentral.com/1471-2296/8/9 6. American Academy of Pediatrics: Practice Parameter: The diag-
nosis, treatment, and evaluation of the initial urinary tract
infection in febrile infants and young children. Pediatrics 1999,
103:843-852. 7. Royal College of Physicians: Guidelines for the management of
acute urinary tract infection in childhood. Report of a Work-
ing Group of the research unit, Royal College of Physicians. J R Coll Physicians London 1991, 25:36-42. J
y
8. Jodal U, Lindberg U: Guidelines for management of children
with urinary tract infection and vesico-ureteric reflux. Rec-
ommendations from a Swedish state-of-the-art conference. Swedish Medical Research Council . Acta Paediatr Suppl 1999,
88(431):87-9. 9. van Haaren KAM, Visser HS, van Vliet S, Timmermans AE, Yadava R,
Geerlings SE, ter Riet G, van Pinxteren B: NHG-Standaard
Urineweginfecties - Tweede herziening [Guideline of the
Dutch College of General Practitioners on urinary tract
infections: second revision]. Huisarts Wet 2005, 48:341-352. 10. van der Linden MW, van Suijlekom-Smit LWA, Schellevis FG, van der
Wouden JC: Tweede nationale studie naar ziekten en verrichtingen in de
huisartspraktijk. Het kind in de huisartspraktijk [Second Dutch national sur-
vey of general practice. The child in general practice] Rotterdam/Utrecht,
Erasmus MC, afdeling Huisartsgeneeskunde/NIVEL; 2005. Erasmus MC, afdeling Huisartsgeneeskunde/NIVEL; 2005 11. Tacken MAJB, Braspenning JCC, de Bakker DH, Jabaaij L, van den
Hoogen HJM, Verheij R, Groenewegen PP, Grol RPTM: The Dutch
National Information Network of General Practice (LINH)
and quality-of-care data. In Quality of preventive performance in gen-
eral practice: The use of routinely collected data [thesis] Edited by: Tacken
MAJB. Nijmegen, Radboud University Nijmegen; 2005:23-44. 12. Lamberts H, Wood M: ICPC: International classification of primary care
Oxford, Oxford University Press; 1987. 13. Timmermans AE, Baselier PJAM, Winkens RAG, Arets H, Wiersma T:
NHG-Standaard Urineweginfecties: Eerste herziening [Guideline of the
Dutch College of General Practitioners on urinary tract infections: first revi-
sion] Utrecht, Nederlands Huisartsen Genootschap (NHG); 1999. 14. van de Lisdonk EH, Verstraeten J: Kinderen met urineweginfec-
ties: Verwijzen of niet? Abbreviations Grol R: Beliefs and evidence in changing clinical practice. Brit
Med J 1997, 315:418-421. Abbreviations Een retrospectief onderzoek in de
vier CMR-praktijken [Children with urinary tract infections:
To refer or not? A retrospective study in four CMR-prac-
tices]. Huisarts Wet 2000, 43:343-346. 15. Kwok WY, de Kwaadsteniet MCE, Harmsen M, van Suijlekom-Smit
LWA, Schellevis FG, van der Wouden JC: Incidence rates and
management of urinary tract infections among children in
Dutch general practice: Results from a nation-wide registra-
tion study. BMC Pediatr 2006, 6:10. y
16. South Bedfordshire Practitioners' Group: How well do general
practitioners manage urinary problems in children? Br J Gen
Pract 1990, 40:146-149. 17. Vernon S, Foo CK, Coulthard MG: How general practitioners
manage children with urinary tract infection: An audit in the
former Northern Region. Br J Gen Pract 1997, 47:297-300. g
J
18. Dighe AM, Grace JF: General practice management of child-
hood urinary tract infection. J R Coll Physicians London 1984,
34:324-327. 19. Paulus DL: Measurement and control of response bias. In Meas-
ures of personality and social psychological attitudes Edited by: Robinson
JP, Shaver PR and Wrightsman LS. San Diego (CA), Academic Press;
1991:17-59. 20. Esbjörner E, Berg U, Hansson S: Epidemiology of chronic renal
failure in children: A report from Sweden 1986-1994. Pediatr
Nephrol 1997, 11:438-442. Publish with BioMed Central and every
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Your research papers will be:
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BioMedcentral Publish with BioMed Central and every
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21. Grol R: Beliefs and evidence in changing clinical practice. Brit
Med J 1997, 315:418-421. p
21. Pre-publication history The pre-publication history for this paper can be accessed
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Structural analyses of wooden chairs by finite element method (FEM) and assessment of the cyclic loading performance in comparison with allowable design loads
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Maderas. Ciencia y tecnología
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1Bingöl University. Department of Material and Material Process Techniques. Bingöl, Turkey.
2Muğla Sıtkı Koçman University. Faculty of Engineering. Department of Civil Engineering. Muğla, Turkey.
3Muğla Sıtkı Koçman University. Faculty of Technology. Department of Woodworking Industrial Engineering. Muğla, Turkey.
♠Corresponding author:alikasal@mu.edu.tr
Received: 26.04.2020 Accepted: 19.11.2020 ABSTRACT Tests were carried out to investigate the cyclic loading performance of chairs constructed of Scotch pine
(Pinus sylvestris) and Oriental beech (Fagus orientalis). Totally, 30 real size chairs were constructed and
assembled with polyvinyl acetate adhesive. Chairs were tested under 3 cyclic loading directions;
namely, front to back, back to front, and backrest according to American Library Association (ALA)
specifications, and obtained loading performances of chairs were crosschecked with allowable design load levels. Additionally, specimens were analyzed as structures by using the finite element method in order to obtain
axial, shear, and bending stresses acting on each member under loadings and compare these values with design
stresses. As a result, it was concluded that finite element method analyses achieve reasonable estimates for strength
performances and failure behaviors of chairs. Depending on allowable design loads by American Library
Association; chairs constructed of beech could meet the medium service for front to back and back to front
loading, while heavy service for backrest loading. In the case of pine chairs; they could meet the medium
service for front to back and back to front loading, while light service for backrest loading. Keywords: Acceptable design loads, chair strength, cyclic loading, finite element analysis, furniture engi
neering design, structural analysis, wooden. https://orcid.org/0000-0002-4632-0072 https://orcid.org/0000-0002-4632-0072 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 ISSN impresa
ISSN online STRUCTURAL ANALYSES OF WOODEN CHAIRS BY FINITE
ELEMENT METHOD (FEM) AND ASSESSMENT OF THE CYCLIC
LOADING PERFORMANCE IN COMPARISON WITH ALLOWABLE
DESIGN LOADS Erkan Ceylan1
https://orcid.org/0000-0002-0497-7789
Ersan Güray2
https://orcid.org/0000-0001-5349-6511
Ali Kasal3,♠
https://orcid org/0000 0002 4632 0072 Erkan Ceylan1
https://orcid.org/0000-0002-0497-7789
Ersan Güray2 Erkan Ceylan1
https://orcid.org/0000-0002-0497-7789
Ersan Güray2
https://orcid.org/0000-0001-5349-6511
Ali Kasal3,♠
h
//
id
/0000 0002 4632 0072 Ersan Güray2 https://orcid.org/0000-0001-5349-6511
Ali Kasal3,♠ https://orcid.org/0000-0001-5349-6511 material economics, among others. (Eckelman 2003). It is well possible to utilize structural analyses and solid modelling
software to accomplish the design of furniture. In addition, the finite element method (FEM) provides a
furniture designer with enormous possibilities for analysis and design. In this context, furniture members, joints, and the whole system could be modelled parametrically via
FEM. It is required to make simplifications while modelling the structure, for instance, the geometry can be
taken as rough as possible and the connections are modelled by semi-rigid springs. If the numerical results do
not well satisfy the experimental output, then some detailing modifications are done on the model to provide
a better approximation. Strength calculations of the designed elements and connection points require realistic
modeling and loading of the system. Then, required constraints are provided to optimize the member sizes by
interpretation of these internal forces (Kasal et al. 2006). Recent studies show that the FEM has become widespread in the structural analysis of furniture
systems. In (Tankut et al. 2014), a bibliographical review of the FEM, used in the analysis of wooden furniture
products is listed. In another work, it is shown that the design of a chair can be very different if some simple
calculations are performed. In this study, additionally, it was underlined that there had been a need for the wood
analysis not in “building size” but “furniture size” (Gustafsson 1995). Some analytical models were developed
for the furniture frame analysis using FEM by Kasal and Pullela (1995). The load-deformation curves were
gathered by experiments and the stiffness is determined by incorporating the resulting curves with the loading. In the end, it was stated that FEM models had been helpful to simulate the furniture under loading (Kasal and
Pullela 1995). A computer program was developed for the strength analysis of wooden frames. In this study,
the side frame of a chair was analyzed and it was shown that accurate and practical strength analysis of the
wooden members could be performed by this program (Smardzewski 1998). He developed a mathematical
model for bent-mortise joints, in his other work, and tried to discover influencing factors on the strength of
glued-tenon-joints. Analyses were performed with a computer assisted program and according to results, the
flexural strength of the glued-mortise-tenon-joints was depending on mostly the shear strength of the adhesive
and the compression strength of the wood material (Smardzewski 2016). In the study carried by Kasalet al. material economics, among others. (Eckelman 2003). The design of furniture almost rests on traditional experiences in handcraft manufacturing. As far
as known, no manufacturer or designer applies static analyses to determine the internal forces inside
the wooden members of a furniture body. However, some researchers have shown interest in this field
(Gustafsson 1997). The section size and geometries of members that integrate the furniture frames should be
designed rationally and they should carry the acceptable loads probably imposed upon them in service. In the
methodology of furniture engineering; once forces and moments these acting to the ends of each member have been
analyzed, the internal stresses in these members can be calculated and by comparing their magnitudes with the
allowable design stresses for the material used, it can be seen whether or not each of the members has been safely
designed. This process provides a methodical way of designing a piece of furniture to meet any specified
service condition. In many types of furniture, members may be deliberately oversized so that no attempt is made to determine
the optimum or most efficient size for each member. The principal task of the designer in such cases is to verify
that each member can safely carry the forces imposed upon it without exceeding the allowable stress design
values. When members are used solely for structural purposes such as in upholstered furniture, however, they
often may be engineered exactly to meet specified strength requirements. The objective here is to produce an
optimum overall design in which just enough material is used to produce a piece of furniture that is capable of
carrying specified design loads with a given factor of safety. Such designs, being the most economical, provide
savings for both the manufacturer and consumer together (Eckelman 2003). Today, technology has rapidly developed. Just like in other industrial areas, technological developments
have been effective in the production of furniture from design to manufacturing. With the help of recent
technological improvements and innovations, furniture could be produced at high-speed and robustness;
furthermore, the constructional details are able to be analyzed in details. The precise analysis of furniture
frames is a complicated process in terms of computation, since there exist a large number of internal forces to
be determined for an effective solution. It is very important to use technological facilities in the strength
design of furniture (Kasal et al. 2006). INTRODUCTION The design of the new furniture should satisfy three main criteria such that it should have an
aesthetic looking, functional, durable and also feasible. The first one requires esthetic design; that is, the artistic
+development of the furniture structure, the design of the second is necessary for the use/functionality of the
furniture such that the furniture structure is planned to have the capability of its intended functionality, and the
last can be obtained by an engineering design; that is, the furniture can safely resist the loads imposed upon
it in its service life and manufactured with minimum cost. Until recently, the engineering design received
little attention. However, it is becoming increasingly important because of various reasons such as; consumer
demands for more reliable furniture, government pressures for warranties of furniture, increasing needs for Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío material economics, among others. (Eckelman 2003). (2016a), mortise and tenon joints in various sizes were analyzed by FEM and some strength properties like the
bending capacity, stiffness of the joint were reported. Joints became stronger and stiffer by increasing the tenon
sizes. The maximum stress was concentrated on the edge and corners of the glued surface and it was declared
that the strength properties of the joints have a shape-adhesive nature (Kasal et al. 2016b). The research was
aimed at designing flexible chairs consisting of lamellae using FEM. In this research, the ultimate strength 2 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 capacity of the flexible chairs was tested both experimentally and numerically. In the case, of a weight until
to 110 kg chairs met the requirements of actual standards but failed for heavy loadings (Langova et al. 2019). capacity of the flexible chairs was tested both experimentally and numerically. In the case, of a weight until
to 110 kg chairs met the requirements of actual standards but failed for heavy loadings (Langova et al. 2019). In the literature, there are many studies related to the ultimate and allowable strength of solid wood
and wooden materials. And, safety factors were recommended for calculating the allowable design loads
(Eckelman 2003, Forest Products Laboratory - USDA 2010). There are some studies related to the allowable
design loads for whole furniture systems. Among these studies, the most important references are GSA (FNAE
80-214) and ALA. In these references, allowable cyclic design loads for upholstered sofas and chairs were
given by classifying the light (domestic), medium, and heavy duty service (Eckelman 1982, Eckelman 1995a,
Eckelman 1995b, Eckelman 1999, Eckelman and Erdil 2001). The strengths of beech chairs were investigated
under cyclic and static loading. The cyclic front to back load performance of chairs was compared with the
acceptable design loads such that, it was recommended that the cyclic strength could be assumed to be 56
% of the strength of static loading case (Kuskun et al. 2018). Kiliç et al. (2018) investigated the effects of
tenon size, in the case of the front to back loading of Scotch pine chairs, and the strength of chairs wich were
evaluated according to the acceptable design loads. Chairs with joints connected by 40 mm in width and
50 mm in length tenons were capable of meeting light service which corresponds to the domestic usage. material economics, among others. (Eckelman 2003). However, a minimum of 50 mm x50 mm size was required for the width and length of each tenon, respectively,
to meet the requirement of the medium service (Kiliç et al. 2018). In the other study, the relationship between the
loading capacity of beech chairs and strength of side frame joints was investigated where the chair was
subjected to the front to back single push. In addition to this, a methodology was developed for predicting the
strength of chairs from the strength of joints (Kasal et al. 2016a). As seen in the literature, generally, the performances of chairs were evaluated in the case of the front
to back loading. There are very limited studies that evaluated the cyclic strength of the wooden chairs in all
loading directions. The purpose of this study was to measure the cyclic loading performance of wooden chair
frames by comparing the allowable design load levels in three loading directions given in ALA specifications. Furthermore, chair frames were structurally analyzed by using FEM and collected data were compared to
the actual strength test results. As a result, it was intended to promise information about the cyclic loading
performance of wooden chairs and to show the usage of structural analyses via FEM in furniture engineering
design. Materials properties Chair members were constructed from beech (Fagus orientalis L.) and pine (Pinus sylvestris L.) woods. They are procured from local commercial suppliers and are used quite often in the furniture industry. The
average density values of these species are 0,63 g/cm3 and 0,48 g/cm3, respectively. Other physical and
mechanical properties are taken as shown in ASTM D143-94 (2000) and ASTM D4442-92 (2003) standards. Whole chair frames were constructed by utilizing grooved beech dowels. They were 10 mm in diameter and
35 mm in length. 65% solid contained PVAc (polyvinyl acetate) glue is used to assemble the chair members. This type of glue is highly preferred since it is easily spread and dried, cold applied, odorless and fire resistant. Necessary properties of it are provided by the company (Polisan, Turkey) as density of 1,1 g/cm3, viscosity of
0,16 - 0,2Pa/s, ph = 5,00, ash rate of 3%. Construction of the chairs and individual joint specimens A total of 30 (2 wood species, 3 loading direction, 5 replication) chairs were prepared in 1/1 scale. 15
chairs were constructed of beech, and the remaining 15 were constructed of pine. The general configuration of
the chairs is shown in Figure 1. 3 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Figure 1: (a) Dimensions in mm (b) the real picture of the chairs. Figure 1: (a) Dimensions in mm (b) the real picture of the chairs. Figure 1: (a) Dimensions in mm (b) the real picture of the chairs. First of all, surfaces of wooden beams were smoothed at the planing machine and pieces with a thickness
of 21 mm were prepared. Then, they were cut to have a 60 mm width for all members except for stretchers. The stretcher was 21 mm thick and 30 mm wide. Assembling starts with the construction of side frames. Mortise and tenon joint techniques were utilized for constructing the side frames of the chair. The length and
the width of tenons were set to 40 mm at side rails, they were 30 mm for the stretcher since it was only 30 mm
in width. Corresponding mortise holes were drilled on back and front legs with a 7 mm thickness (1/3 of the
member thickness). Mortises were drilled and tenon joints were cut with appropriate laboratory machines. A
tight fit was obtained where the average mortise–tenon clearance was 0,076 mm ± 0,025 mm. In the assembly,
walls of the mortises and tenons were first adequately coated with adhesive. The adhesive was spread over
approximately with 150 gr/cm2 ± 10 gr/cm2. The tenons were then inserted into the mortises, and the assembly
was squeezed by bar clamps. Then a complete chair was built by these preassembled pieces utilizing dowel
joints. The depth of the embedment of dowels in the edge and the face were 20 mm, 15 mm, respectively. The
distance between the two dowels was chosen to be 30 mm. The dowels and corresponding holes at the top and
back rails and the lower front and back rails were coated with adhesive. Bar clamps were again used to squeeze
the side frames to force the dowels for a tight fit. Cyclic performance testing of chairs and moment-rotation characteristics of joints The “cyclic stepped increasing loading” is a recommended method to satisfy the requirements of the
performance test system. To do this, the furniture is subjected to a specified initial load at a prescribed cyclic
rate for a specified number of cycles. When cycles are accomplished, the subjected load is raised incrementally. Cycling loading is repeated after each increment. The procedure is repeated until the desired load level has
been reached, or failure occurs on the furniture frame, or horizontal deflection exceeds 50 mm on the side rail
and back leg connection (Eckelman 1988, Eckelman 1999). In the scope of the study, a total of 30 chair frames were constructed and tested. Chairs were tested
regarding the principles of the American Library Association (ALA) specification by applying the cyclic
front to back, back to front, and backrest loads to which the chairs were likely to be exposed in service
(Eckelman 1999). The cyclic loading tests were performed on the furniture performance testing equipment (Mates
2011, Ankara, Turkey) at the mechanical testing laboratory in the Wood Science and Industrial Engineering
Department of Mugla Sitki Kocman University. The cyclic loading test procedure was that 1) a chair was
subjected to a given load for 25000 cycles at a rate of 20 cycles per minute, 2) when 25000 cycles were
completed at this load level, the load was increased a specified amount and testing continued for next 25000
cycles, and 3) this procedure was repeated until the tested chair suffers disabling damage or the desired
acceptance level were reached. When performing the cyclic stepped increasing loading procedure, “initial
load,” “load increment,” “loading rate,” and “load cycles at each load level” practiced in the study are given in
Table 1. 25000 cycles at each load level took almost 21 hours (about 1 day) in cyclic loading for chairs. In the
tests, the ultimate failure loads (in Newton) and completed total cycles were recorded. Table 1: Loading program of cyclic loading tests for chair frames (Eckelman 1999). ble 1: Loading program of cyclic loading tests for chair frames (Eckelman 1999). In the front to back and back to front loading tests; horizontal loads in specified directions were applied
from 420 mm away from the supports. Construction of the chairs and individual joint specimens Not only whole chairs were constructed but also representative T-type and L-type specimens were
prepared for obtaining the moment rotation characteristics (stiffness coefficients) of each joint in order to
introduce them as semi-rigid connections in the structural analyses of chairs. Each joint type was tested
20 times since there had been 2 wood species and tests were replicated 10 times. Therefore, 20 identical
T-type joints for the connections between the “back leg to side rail” and 20 identical T-type joints for the
connections between the “back/front leg to stretchers” were prepared. Separately, 20 identical L-type specimens for
modelling the connection between the “front leg to side rails” were arranged. T-type and L-type joint
specimens and load directions are shown in Figure 2a, Figure 2b, and Figure 2c. Figure 2: T-type joint (a, b) and L-type joint (c) specimens (measurements in mm). Figure 2: T-type joint (a, b) and L-type joint (c) specimens (measurements in mm). 4 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 As a precaution for a unique moisture content (MC), before examining, all chairs and joint specimens were
kept in an environmentally controlled conditioning room to cure at least one month for reaching an average 12
% moisture content. As a precaution for a unique moisture content (MC), before examining, all chairs and joint specimens were
kept in an environmentally controlled conditioning room to cure at least one month for reaching an average 12
% moisture content. Cyclic performance testing of chairs and moment-rotation characteristics of joints In these tests, reaction brackets were fixed behind each of the back legs
for front to back loading while they were placed in front of each of the front legs for back to front loading in
order to prevent the chair from slipping. Steel chains then passed over the seat and attached to the air cylinders
that were used to apply the horizontal loads to the chair. The other end of the steel chains dropping over the
front edge of the seat for front to back loading, while the back edge of the seat for back to front loading, was
attached to the floor vertically. Chains provided the reaction forces preventing the chair from overturning. (Figure 3a and Figure 3b). In the case of the backrest loading test, a reaction bracket was placed just behind the back legs with the
same height of side rails, and another bracket was placed in front of the front legs. Here, just similar to cases
of back to front or front to back loadings, reaction brackets prevented the chair from tipping over backward
when front to back loads applied to the backrest. A short steel chain was tied around the backrest and fixed to
the air cylinder (Figure 3c). 5 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Figure 3: Tests; (a) front to back loading, (b) back to front loading, (c) backrest loading. Figure 3: Tests; (a) front to back loading, (b) back to front loading, (c) backrest loading. The allowable cyclic loading performance levels for light (domestic), medium and heavy usages are
listed in Table 2 as given in the ALA specifications (Eckelman 1999). Tests were performed and the resulting
performance values are to be declared and compared with these specified design loads in the next step. The allowable cyclic loading performance levels for light (domestic), medium and heavy usages are
listed in Table 2 as given in the ALA specifications (Eckelman 1999). Tests were performed and the resulting
performance values are to be declared and compared with these specified design loads in the next step. Table 2: Allowable design loads according to the loading direction (Eckelman 1999). Table 2: Allowable design loads according to the loading direction (Eckelman 1999). The moment rotation characteristics of joints in the side frame of the chair were determined to treat them
as semi-rigid connections in the structural analyses. Cyclic performance testing of chairs and moment-rotation characteristics of joints The moment-rotation characteristics of the joint were
determined by the relationship between the distortion angle between neighboring orthogonal members and the
applied moment. It could be considered as the rotational, linear behaving, stiffness coefficient and evaluated
by Equation 1: M
K
ϕ
=
(1) M
K
ϕ
=
(1) M
K
ϕ
=
(1) where K is the stiffness coefficient (Nm/rad), M is the moment (Nm) and ø designates the distortion angle
at the joint (rad). T-type and L-type joint specimens were examined under bending loads to simulate the condition in
which they are exposed to these loads as in the side frame of a full chair (Figure 2). Static loading tests were
performed on the 50 kN capacity universal-testing machine (Mares, İstanbul, Turkey) with the 6 mm/min
loading rate. The rail member of each specimen at a point 300 mm from the front edge of the leg was subjected
to a concentrated load, such that, the moment arm was 300 mm according to the literature (Kasal et al. 2016a,
Kiliç et al. 2018). Dial gage was clamped to the edge of joint rails and the measurements were recorded at regular intervals
as specimens were loaded. 6 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Structural analysis of chairs Table 4: Physical and mechanical properties of pine and beech woods used in the study. Wood
Species
MOE*
(MPa)
Tension
Strength
(MPa)
Compression
Strength (MPa)
Shear
Strength
(MPa)
G*
(MPa)
MOR*
(MPa)
Density
(kg/m3)
MC*
(%)
Pine
11000
95
55
6
4230
90
480
10,8
Beech
12500
125
75
11
4807
120
630
11,2
*MOE: modulus of elasticity; G: shear modulus; MOR: modulus of rupture, MC: moisture content. Table 4: Physical and mechanical properties of pine and beech woods used in the study *MOE: modulus of elasticity; G: shear modulus; MOR: modulus of rupture, MC: moisture content. Then, sectional properties, i.e., the cross-sectional area, the moment of inertia values about local (Y) and
(Z) axes, form factor and, torsional constant values were calculated and implemented into the workspace
(Table 5). Then, sectional properties, i.e., the cross-sectional area, the moment of inertia values about local (Y) and
(Z) axes, form factor and, torsional constant values were calculated and implemented into the workspace
(Table 5). Table 5: Sectional properties of the chair members. Table 5: Sectional properties of the chair members. After all physical and mechanical definitions for the problem, the supports should have been assigned. For
the front to back loading; front legs connection to the floor were treated with roller supports, whereas the back
legs were supposed to have pin connections with the floor. In other words, it was considered that back legs
were constrained to translations in (X), (Y), and (Z) directions, while they were free to rotate in any direction;
but the front legs, were constrained to the translation only in (Y) direction. For the back to front loading; unlike
the front to back loading, back legs of the chair were supported as roller, whereas the front legs were supported
as pinned. And finally, when it was loaded at the backrest; the back legs of the chair were supported as a roller,
whereas the front legs were supported as pinned, in addition, the back leg to side rail joints were supported as
the roller. Other than the support joints, all internal joints (nodes) were defined with six degrees of freedom, in
other words, all internal joints can carry the axial forces, shear forces, and bending moments in any direction. The numerical model was subjected to static analysis by loading properly. Structural analysis of chairs A chair structure is a frame structure where the members are assumed to be linear beams. Here, in this
work, every member is a prismatic member having a width, length, and depth. The model was created with
joints and the interconnected beam elements between them (Figure 4). Each joint was labelled from N1 to N20
and members were numerated from M1 to M24. This FEM modelling was performed on RISA-3D (2000) and
all internal forces were determined. Figure 4: Joints/nodes (a) and members (b) of the modelled chair. b
(b) f th
d ll d h i Figure 4: Joints/nodes (a) and members (b) of the modelled chair. Rotational linear springs were modelled at the T-type and L-type side frame connections to treat them
as semi-rigid connections. Since the software was able to assign semi-rigid connections by simply assuming
springs at these joints, simply, predefined K (rad/mm), stiffness coefficients are entered into the interface of
the program (Table 3). The stiffness coefficients were determined based on the bending tests of representative
T-type and L-type joints, as was previously explained. Table 3: Stiffness coefficients, K, obtained for each joint type according to wood species. Table 3: Stiffness coefficients, K, obtained for each joint type according to wood species. Next, some required physical and mechanical properties (Table 4) of beech and pine obtained from the
tests were entered into the software to calculate the axial, shear forces and, moments at the end of members. Poisson’s ratio (µ) was taken as 0,3 for both species (Kasal et al. 2006, Kasal et al. 2016b, Kiliç et al. 2018). 7 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Table 4: Physical and mechanical properties of pine and beech woods used in the study. Wood
Species
MOE*
(MPa)
Tension
Strength
(MPa)
Compression
Strength (MPa)
Shear
Strength
(MPa)
G*
(MPa)
MOR*
(MPa)
Density
(kg/m3)
MC
(%)
Pine
11000
95
55
6
4230
90
480
10 8 Table 4: Physical and mechanical properties of pine and beech woods used in the study. Wood
Species
MOE*
(MPa)
Tension
Strength
(MPa)
Compression
Strength (MPa)
Shear
Strength
(MPa)
G*
(MPa)
MOR*
(MPa)
Density
(kg/m3)
MC*
(%)
Pine
11000
95
55
6
4230
90
480
10,8
Beech
12500
125
75
11
4807
120
630
11,2
*MOE: modulus of elasticity; G: shear modulus; MOR: modulus of rupture, MC: moisture content. Structural analysis of chairs The ultimate loads that were
obtained from the actual tests were cyclic performance, but it was performed the FEM analyses with
static loads. Therefore, it was considered the relation between the cyclic and static strength. According to the
literature; cyclic strength is approximately half of the static strength, so in the structural analyses, it was
applied twice the loads that were obtained from the actual tests (Kasal et al. 2016a, Kuskun et al. 2018, Likos et
al. 2013). Ultimate loads were symmetrically applied at corresponding nodes that were on the two side frames
(Figure 5). Axial, shear forces, moments, and corresponding stresses acting on each member was obtained. Furthermore, axial force, shear force, and, moment diagrams and deflected shape of the chair was also
provided as the output of the software. 8 8 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Structural analyses of wooden..: Ceylan et al. Figure 5: Loading and supports of the numerical model, loaded in direction of (a) front to back,
(b) back to front, and (c) backrest. Figure 5: Loading and supports of the numerical model, loaded in direction of (a) front to back,
(b) back to front, and (c) backrest. The axial forces (tension or compression), shear forces, and moments were obtained from the structural
analyses for all members of the chair under each loading direction. Corresponding axial, shear and bending
stresses were also available and ready to be compared with the allowable design stresses. To find the allowable
design stresses, values given in Table 4 were multiplied by the appropriate reduction (safety) factors (Eckelman
2003). Table 6 also shows the allowable design stresses for the materials used in the construction of the chairs. Table 6: Allowable design stresses for the wood materials used in the construction of cha
*Reduction factors are taken according to Eckelman (Eckelman 2003). Table 6: Allowable design stresses for the wood materials used in the construction of chair*. *Reduction factors are taken according to Eckelman (Eckelman 2003). *Reduction factors are taken according to Eckelman (Eckelman 2003). As a result; deformation characteristics observed in actual tests were tried to verify by structural analysis. In other words, the consistency of the joints where deformations occur in actual tests was checked with the
results of structural analysis. Structural analysis results of chairs The chairs failed completely without tilting in average 10 days for front to back loading, 9 days for back to
front loading, and 5 days for backrest loading. Interconnecting joints softened suddenly, but they kept holding
loads after a while. In the chairs tested under the front to back or back to front loading; joint failures occurred
because of the glue line fractures, i.e. tenons withdrew truly from the front and back leg members. Softening
failures proceeded at the edge of the tenon and then propagated towards to another edge as the cycles and
load levels increased. As the adhesive bonding failed, the tenons were lead to be loaded in bending and shear 9 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío stresses. In the case of frames tested under backrest loading; failures occurred owing to fracture of the tenons
at their point of entry into the walls of the back and front legs or fracture of the back leg member at the top of
the back leg to side rail joints. The exaggerated deflected shapes of a chair under the three lading directions
obtained from the FEM analyses and actual tests are given in Figure 6 and Figure 7, respectively. stresses. In the case of frames tested under backrest loading; failures occurred owing to fracture of the tenons
at their point of entry into the walls of the back and front legs or fracture of the back leg member at the top of
the back leg to side rail joints. The exaggerated deflected shapes of a chair under the three lading directions
obtained from the FEM analyses and actual tests are given in Figure 6 and Figure 7, respectively. Figure 6: Deflected shape of chair obtained from the structural analyses under, (a) front to back loading,
(b) back to front loading, and (c) backrest loading. Figure 6: Deflected shape of chair obtained from the structural analyses under, (a) front to back loading,
(b) back to front loading, and (c) backrest loading. Figure 7: Observed deformation characteristics of chairs under, (a) front to back loading,
(b) back to front loading, and (c) backrest loading. Figure 7: Observed deformation characteristics of chairs under, (a) front to back loading,
(b) back to front loading, and (c) backrest loading. Structural analysis results of chairs As seen in Figure 6 and Figure 7, deflected shape of the chairs obtained from the structural analyses under
each loading direction have given reasonable estimates in terms of the overall deformation characteristics and
possible failure points of the chairs. In the strength analyses of the chairs, the axial forces and corresponding axial stresses under the front to
back, back to front, and backrest loadings were considered initially. Axial force diagrams are given in Figure 8
with the most critical tension and compression members. Figure 8: Axial force diagrams obtained from the structural analyses results for (a) front to back loading,
(b) back to front loading, and (c) backrest loading. Figure 8: Axial force diagrams obtained from the structural analyses results for (a) front to back loading,
(b) back to front loading, and (c) backrest loading. 10 10 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Regarding loading, the bottom part of the back leg member is exposed to a high compression due to the
front to back loads while the bottom part of the front leg member is pulled up from the test set-up platform, and
exposed to tensioning because of the pin supports. On the contrary, for the back to front loading; the bottom
part of the front leg member is subjected to high compression while the bottom part of the back leg member
is sustaining tension. In both loading directions, stretcher members are exposed to a considerable amount of
compression. In the case of the backrest loading; the side rail member is exposed to a tension force while the
stretcher member is exposed to a compression force. The members pointed out in Figure 8, were compared to the allowable axial design stress values
determined for each wood material used. It was found that the maximum tension stresses (T) occurred in the
bottom part of the front leg member (M14, M20) under the front to back loading, in the bottom part of the back
leg member (M13, M19) under back to front loading, and in the side rail member (M3, M7) under the backrest
loading. In the case of maximum compression stresses (C), has occurred in the stretcher member (M2, M6) for
all three loading directions. These members are the most critical members for carrying the axial forces under
the loadings. Structural analysis results of chairs The comparison results for the consequential ultimate axial stresses and allowable axial design
stresses were given in Table 7. Table 7: Comparison of the maximum axial stresses values and axial design stresses (MPa). Table 7: Comparison of the maximum axial stresses values and axial design stresses (MPa). As seen in Table 7, chairs made of both wood species were found to be strong enough to carry the axial
forces safely for all loading cases. The shear forces in direction (Y) and corresponding shear stresses under the
front to back, back to front, and backrest loadings were provided as the second phase. Shear force diagrams
were presented in Figure 9 with the most critical shear members for all loading directions. Table 7: Comparison of the maximum axial stresses values and axial design stresses (MPa). As seen in Table 7, chairs made of both wood species were found to be strong enough to carry the axial
forces safely for all loading cases. The shear forces in direction (Y) and corresponding shear stresses under the
front to back, back to front, and backrest loadings were provided as the second phase. Shear force diagrams
were presented in Figure 9 with the most critical shear members for all loading directions. Figure 9: Shear force diagrams obtained from the structural analyses results for (a) front to back,
(b) back to front, and (c) backrest loading. Figure 9: Shear force diagrams obtained from the structural analyses results for (a) front to back,
(b) back to front, and (c) backrest loading. The maximum values in members, shown in Figure 9, were compared to the allowable shear design
stresses. Maximum shear stresses (S) occurred in the bottom part of back leg and front leg members (M13,
M19 and M14, M20) under the front to the back and back to front loading, respectively. In the case of backrest 11 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío loading, maximum shear stresses occurred in the middle part (between the side rail and stretcher) of the back
leg member (M15, M16 and M21, M22). These critical members’ results for shear stresses which act upon the
back leg-bottom members and allowable shear design stresses were given in Table 8. loading, maximum shear stresses occurred in the middle part (between the side rail and stretcher) of the back
leg member (M15, M16 and M21, M22). Structural analysis results of chairs 12 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Structural analyses of wooden..: Ceylan et al. Table 9: Comparison of the maximum bending stresses and bending design stresses. Table 9: Comparison of the maximum bending stresses and bending design stresses. According to comparison results, the maximum bending stresses occurred in the back leg member where
it was joined the side rail member among the three loading directions. Therefore, the most critical place of the
chair is the back leg to side rail joints. Determined bending stress values that act upon these joints have gone
beyond the allowable bending design stresses for both wood species. The overall performance of the chairs has been seriously affected by the bending capacity of these joints
and the section sizes of the related members. In the engineering design of a chair, if these results are taken into
consideration, the strength performance of the chair can be upgraded. Here, in this study, all possible forces and consequential stresses are investigated for both wood species. When the literature is examined, it is stated that the bending moment is the most critical internal force through
them. The axial strength of even a very small sized wood member is generally much greater than needed, and
shear strength is rarely of concern (Eckelman 2003). In this study, it has been understood that either beech or
pine used chair members, and sectional sizes of each member of the chair had enough capacity to carry the
axial (tension or compression) forces, safely, whereas the members of the back leg where it was joined
the side rail or stretcher member has been imposed to over bending and shear forces. This fact is well
compromised with the actual performance test results in terms of failure locations. In other words, actual test failures
occurred in these joints where the structural analyses showed that the highest stresses occurred. In the
backrest loading tests; firstly, softening failures occurred in the mentioned joints, and catastrophically, other
joints were also untightened. In some chairs, the back leg member of the chair or tenons of the back leg to side
rail joints were broken. These results helped to confirm that the FEM model of the chair was consistent with
the experiment chair. Structural analysis results of chairs These critical members’ results for shear stresses which act upon the
back leg-bottom members and allowable shear design stresses were given in Table 8. Table 8: Comparison of the maximum shear stresses and shear design stresses. Table 8: Comparison of the maximum shear stresses and shear design stresses. According to Table 8, it was found that, chairs, nevertheless they are made of pine or beech, had enough
capability of carrying design loads in the case of both fronts to back or back to front loadings. However, the
shear stresses that occurred in the middle part of the back leg under backrest loading were higher than the
allowable shear design stresses for both chairs constructed of pine and beech. This result was consistent with
observations in actual backrest loading tests. In the actual tests, failures occurred at these members and/or
related (back leg to side rail or back leg to the stretcher) joints. In the last phase, the moments about the (Z) axis and consequential bending stresses were obtained from
the results of structural analyses. Moment diagrams about the (Z) axis obtained from the structural analysis
results were presented in Figure 10 with the most critical members and their ends. Figure 10: Moment diagrams obtained from the structural analyses results for (a) front to back,
(b) back to front, and (c) backrest loading. Figure 10: Moment diagrams obtained from the structural analyses results for (a) front to bac
(b) back to front, and (c) backrest loading. The maximum bending stresses (B) occurred in the (I) end of the bottom part of the back leg member
(M13-I, M19-I) where it was joined the stretcher member (M2, M6) under the front to back loading. Therefore,
the most critical point of the chair is the joint where the back leg connected to the stretcher. On the contrary, the
maximum bending stresses occurred in the (I) end of the bottom part of the front leg member (M14-I, M20-I)
where it was joined the stretcher member (M2, M6) under back to front loading. So, for this loading direction,
the most critical point of the chair is the joint where the front leg connected to the stretcher. In the case of
backrest loading, the back leg member was subjected to a considerable amount of bending stresses at the point
(M18-J) where it was joined the side rail member (M3). The results are summarized in Table 9. The chairs constructed of beech had mean cyclic loading performance of 1868 N, 1801 N, and 3469 N
whereas the chairs constructed of pine had mean cyclic loading performance of 1601 N, 1579 N, 1690 N for
front to back, back to front, and backrest loadings, respectively. Thus, the chairs constructed of beech were 14 Assessment of the cyclic loading performance of chairs with allowable design loads The mean ultimate cyclic strength performance values of tested chairs under each loading direction with
their coefficients of variation are summarized in Table 10. Table 10: Cyclic strength performance values and total completed cycles of the chairs Table 10: Cyclic strength performance values and total completed cycles of the chairs. The chairs constructed of beech had mean cyclic loading performance of 1868 N, 1801 N, and 3469 N
whereas the chairs constructed of pine had mean cyclic loading performance of 1601 N, 1579 N, 1690 N for
front to back, back to front, and backrest loadings, respectively. Thus, the chairs constructed of beech were 14 13 Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 Universidad del Bío-Bío Universidad del Bío-Bío %, 12 %, and 51 % stronger than the chairs constructed of pine for front to back, back to front, and backrest
loading, respectively. Test results indicated that the ultimate cyclic loading performance of the chairs was
significantly affected by the wood species. Differences in ultimate cyclic loading performance of the chairs
could be explained by differences in physical and mechanical properties, especially joint strengths, shear and
bending strength of the wood material used constructing the chairs. %, 12 %, and 51 % stronger than the chairs constructed of pine for front to back, back to front, and backrest
loading, respectively. Test results indicated that the ultimate cyclic loading performance of the chairs was
significantly affected by the wood species. Differences in ultimate cyclic loading performance of the chairs
could be explained by differences in physical and mechanical properties, especially joint strengths, shear and
bending strength of the wood material used constructing the chairs. According to Table 10, it has been seen that the front to back and back to front loading performance values
of the chairs were close to each other for both wood species. The backrest loading performance values of the
pine chairs were also close to the other loading directions. However, backrest loading performance values of
the beech chairs were significantly higher than the front to back and back to front loading performances. The cyclic loading performances of chairs under the front to back, back to front, and backrest loadings
were compared to the allowable light, medium, and heavy design load levels that were given in the ALA
specifications (Eckelman 1999). The allowable light design load levels represent the domestic use in practice. Assessment of the cyclic loading performance of chairs with allowable design loads The assessment results of chairs are given in Table 11 for each loading direction. Table 11: Comparison of loading performance values of chairs with allowable design loads. Table 11: Comparison of loading performance values of chairs with allowable design loads. According to results, chairs constructed of pine could meet the allowable medium service load
requirements for front to back and back to front loading directions, whereas they could only resist
allowable light service (domestic usage) load requirements for backrest loading direction. In the case of chairs
constructed of beech; they could satisfy the allowable medium service load requirements for front to back and
back to front loading directions, and could meet the heavy service load requirement for backrest loading. These
results were graphically shown in Figure 11. Figure 11: Assessment of performances of the chairs under the cyclic (a) front to back, (b) back to front, and
(c) backrest loading. Figure 11: Assessment of performances of the chairs under the cyclic (a) front to back, (b) back to front, and
(c) backrest loading. CONCLUSIONS This study was aimed to acquire quantitative information related to the cyclic front to back, back to front,
and backrest loading performances of chairs, constructed of Scots pine and Oriental beech. In addition to that,
it is intended to compare the cyclic loading performance of chairs with the allowable design loads that were 14 Structural analyses of wooden..: Ceylan et al. Maderas. Ciencia y tecnología 2021 (23): 19, 1-16 specified in ALA specifications. Furthermore, chairs were structurally analyzed by using the finite element
method (FEM). specified in ALA specifications. Furthermore, chairs were structurally analyzed by using the finite element
method (FEM). Generally, it was concluded that the overall design and construction of the pine chairs evaluated in this
study would be satisfactory for light service, while beech chairs would be satisfactory for medium service. Thus, it can be said that the chairs constructed of pine and beech are extremely suitable for domestic usage. In fact, it is possible to reduce the cross-sectional dimensions of these chairs in order to obtain economic and
ergonomic benefits if they are used for domestic usage. As expected, chairs constructed of beech yielded higher
strength than those of pine. Although beech yielded higher strength, pine could be commonly utilized in the
furniture frames in an engineering design approach because of its economic advantages. Chairs constructed of both species were given close cyclic loading performance under the front to back
and back to front directions. The backrest loading performance of pine chairs was also close to other loading
directions. However, the backrest loading performance of beech chairs was significantly higher than the front
to back and back to front loading directions. At the end of the structural analysis results, the most critical loading was backrest loading. Under this
loading, members and especially back leg to the side rail and back leg to stretcher joints of the chair were
subjected to considerable amounts of shear and bending stresses, and general failures occurred at these joints. Eventually, it can be concluded that the overall strength of the chairs by considering initially the
bending stress capacity of the members, secondly, section sizes and, finally the strength of the joints. Results
indicated that structural analysis by Finite Element Analysis (FEA) methods provide reasonable estimates
of the overall strength of these structures consistent with actual performance testing results. ACKNOWLEDGMENTS This paper is a part of MSc. thesis of the first author. The study was supported by Scientific and
Technological Research Council of Turkey (TUBITAK) with the project number; 216O013, and Muğla Sıtkı
Koçman University Scientific Research Project Office with the project number; 17/112. Preliminary data of
this paper were verbally presented at the V. International Furniture Congress which was held in Eskişehir,
Turkey. Then, data sets were expanded before converting into a scientific manuscript. CONCLUSIONS In addition, the
results also showed that performance testing is an important part of furniture engineering methodology and
provides quantitative feedback to designers. Performance testing enables exploration and investigation of
unforeseeable failures and ensures fruitful conception to actual usage and failure conditions. In conclusion, this study confirmed that product engineering methodology, integrating structural analyses
with performance testing is applicable to the design of chairs. A similar methodology can be followed for
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