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The Learning Curve - Part 3: The Natural Miticides
The Learning Curve: Part 3 Randy Oliver ScientificBeekeeping.com First Published in ABJ in July 2009 I added a number of updates on May 2015, marking 15 years of successful commercial beekeeping in my operation without the use of synthetic miticides. "It is not the strongest of the species that survives, nor the most intelligent [...]
Read More | Varroa Management Archives - Page 8 of 8 - Scientific Beekeeping | https://scientificbeekeeping.com/varroa-management/page/8/ |
Pesticide exposure pathways
Sorry for the low resolution of this snip of this Powerpoint slide that I created for a presentation. I've color coded the ellipses and arrows. Red is the pesticide active ingredient. Blue is the initial mode of exposure. Orange are the ages/temporal tasks of the bees involved. Green are the contaminated foods or combs.
Note how colony organization is set up to avoid exposing the queen and brood to toxins (whether natural or manmade). Note the special case (the pollen hogs at the lower right) in which newly emerged workers and drones get killed by planting dust.
Category: Topics | Pesticide exposure pathways - Scientific Beekeeping | https://scientificbeekeeping.com/pesticide-exposure-pathways/ |
The Short Version
The Major Problems Facing Bees and Beekeepers
Number one is varroa. Varroa changed the virus dynamics within the colony. If either the bees or the beekeeper don't keep the varroa infestation rate down to fewer than 5 mites per 100 bees, colonies start to suffer. At about 15 mites per 100, viruses go epidemic, and the colony will generally die. Lack of varroa management is the number one reason that colonies die, other than from sheer neglect (see Feeding).
Many recreational beekeepers want to go "natural" and "treatment free." I commend you on those sentiments, and hold that as a goal to reach (it is much harder to do so if you are making your living from your bees). But keep this in mind: you wouldn't expect a broiler-type chicken bred for living under controlled conditions to survive in the wild. And you can't expect commercial bee stock bred for living under intense beekeeper management to survive in the wild just because you want to be Mr. or Ms. Natural. I strongly suggest that beginning beekeepers make life easier on themselves and their bees by first following standard practices until you get the hang of beekeeping; then you can try going treatment free (see Varroa Management or Treatment Free).
Feeding
It is not necessary to feed a colony. I ran hundreds of healthy colonies for years without ever feeding a drop of syrup or a single pollen patty. But that was only possible because I moved my hives to good forage throughout the year.
It became far more difficult to keep bees healthy after the arrival of varroa. In my traditional yards in the dry Sierra foothills during summer, colonies went downhill without supplemental protein. In order to cut out my summer migration to better forage, I learned to feed pollen supplement, with fantastic results!
And then I learned how sugar syrup, fed at critical times, could also greatly improve colony buildup, health, and wintering. The short version is to feed light syrup for stimulation, heavy syrup for winter stores. Feeding nucs, packages, or even overwintered colonies during spring buildup really helps them. In our dry late summers, light syrup along with protein patties encourages broodrearing and greatly improves colony health.
Caution: if you are growing a new colony, it is possible to overfeed syrup to the extent that you plug out the broodnest, leaving the queen no place to lay eggs! You can kill a colony with kindness. Don't keep feeding unless you are inspecting the broodnest from time to time.
I normally leave enough natural honey on the hives for them to winter on (in my moderately cold area, I want a strong double-deep hive to weigh about 120-130 lbs total. However, if the bees put on dark honeydew as winter stores, it may help to replace the combs of honeydew directly above the cluster with dark drawn combs, and then feed heavy syrup for the bees to refill those combs with.
The type of sugar does not appear to be critical. Cane or beet sugar work fine. Commercial sucrose/HFCS blends may even be better. Some beekeepers feel that inverting the sugars also helps, but that is beyond the scope of this short version.
The one clear responsibility that a beekeeper has is to make sure that his colonies don't starve! There is no excuse for allowing animals in your care to starve. Starvation usually happens in late winter/early spring. Heft your hives regularly to make sure that the bees always have a reserve of honey or syrup honey. In a pinch you can even feed dry sugar poured over a piece of newspaper laid on the top bars.
If you live in an area with pollen dearths, feeding high-quality pollen supplements can make all the difference in the world to your bees. I know, it's not natural, but neither is the food that you feed your dog or cat, or even your children. Get over it!
Varroa Management
Treatment Free
Nosema
AFB
Small Cell
Although the use of small cell foundation to help the bees control varroa has an impassioned following, there is little empirical evidence that it actually helps (see http://www.elgon.es/diary/?p=37 for a review). However, in the single trial in which I tested it, the results appeared to be positive, so I keep an open mind. I can say this-many have tried it, sometimes at large scale, and were disappointed in the results. Others find that mite resistant stocks do not necessarily require small cell to be successful at controlling the mite.
If I wanted to practice truly "natural" beekeeping, I'd allow the bees to build their own combs in foundationless frames, rather than trying to force them into any specific cell size. In my own operation, we keep a single foundationless drone frame in each hive (in the upper brood chamber, to the outside of the broodnest), as I feel that it is completely unreasonable to expect the bees to maintain 20 brood combs without any drone brood! See "Drone Trap Frames."
Drone Trap Frames
Summer Nucs
See Mike Palmer's video https://www.youtube.com/watch?v=nznzpiWEI8A
Category: Topics | The Short Version - Scientific Beekeeping | https://scientificbeekeeping.com/the-short-version/ |
IPM 6 Fighting Varroa The Arsenal: Our Choice of Chemical Weapons
IPM 6 Fighting Varroa The Arsenal: Our Choice of Chemical Weapons Randy Oliver ScientificBeekeeping.com First Published in ABJ in June 2007 I'm clearly in the "minimal chemical" camp, yet all my commercial buddies, without exception, depend upon "off label" use of agricultural miticides to keep their colonies alive. These are top-notch beekeepers, and I [...]
Read More | Varroa Management Archives - Page 7 of 8 - Scientific Beekeeping | https://scientificbeekeeping.com/varroa-management/page/7/ |
Pesticide exposure pathways
Sorry for the low resolution of this snip of this Powerpoint slide that I created for a presentation. I've color coded the ellipses and arrows. Red is the pesticide active ingredient. Blue is the initial mode of exposure. Orange are the ages/temporal tasks of the bees involved. Green are the contaminated foods or combs. Note [...]
Read More | Topics Archives - Page 4 of 4 - Scientific Beekeeping | https://scientificbeekeeping.com/topics/page/4/ |
Mite Washer; Still Improving
First published in: American Bee Journal, August 2015
Mite Washer; Still Improving Randy Oliver ScientificBeekeeping.com First Published in ABJ in August 2015 The quickest and most accurate way to monitor varroa levels is by the alcohol wash. After the publication of my "improved" washer design, I've gotten some great suggestions from readers. Unless you monitor for varroa, you have no idea as to [...]
Read More | Varroa Management Archives - Page 6 of 8 - Scientific Beekeeping | https://scientificbeekeeping.com/varroa-management/page/6/ |
General public presentation
Here is a slide show for general public presentation. Contains some old 35mm photos, which I hope to soon replace.
Public general presentationPublic general presentation
Category: Topics | General public presentation - Scientific Beekeeping | https://scientificbeekeeping.com/general-public-presentation/ |
DWV sampling instructions
Dear U.S. beekeeper,
Recent studies by Dr. Stephen Martin and associates have found that there is apparently a benign form of DWV that can out compete the virulent form, thus allowing colonies to survive despite varroa infestation.
If this is true, it raises the possibility that we may be able to minimize the effect of varroa by inoculating our colonies with the benign form of DWV.
We obtained funding from Project Apism to survey bee colonies across the U.S. to determine the distribution of the strains of DWV. We're especially interested in adult bee samples from feral and survivor stock that have survived for some time without treatment. We also need reference samples from "normal" managed apiaries.
If you are interested in contributing samples, please write to Randy at randy@randyoliver.com, with the word "kit" in the subject line, the sort(s) of hive(s) that you're able to sample, and the state in which the hives are located. Please also include your mailing address.
I will reply, and send a postpaid sampling kit. It should take less than an hour of your time to contribute to this research.
Please print out these sampling instructions
Sampling Instructions PDF
Category: Topics | DWV sampling instructions - Scientific Beekeeping | https://scientificbeekeeping.com/dwv-sampling-instructions/ |
Building a Better Mite Washer
Building a Better Mite Washer - Larry Clamp
Notes from Randy:
Tinkerer Larry Clamp put together a very nice set up illustrated instructions for building mite washer cups, and is generously sharing them. Thanks, Larry!
The thin black screen from package bee cages (or some older veils) is easier to work with than hardware cloth, and
Depending upon the type of plastic cup, some expensive glues intended for plastics may work (I found that the alcohol may eventually work under the silicone). Brion Dunbar tells me that he's had good luck with 3M ScotchWeld High Performance Industrial Plastic Adhesive 4693H.
Category: Topics | Building a Better Mite Washer - Scientific Beekeeping | https://scientificbeekeeping.com/building-a-better-mite-washer/ |
What's Happening with the Bees 2015
2015 What's Happening Auburn
Category: Topics | What's Happening with the Bees 2015 - Scientific Beekeeping | https://scientificbeekeeping.com/whats-happening-with-the-bees-2015/ |
International Websites of Interest
I'm open to suggestions for interesting websites on beekeeping in countries other than the U.S. to link to-please email me suggestions.
Ukraine:A commercial honey sales website, but with a nice summary of the history of beekeeping in that country http://www.honey-export.com/
Category: Topics | International Websites of Interest - Scientific Beekeeping | https://scientificbeekeeping.com/international-websites-of-interest/ |
Donze 1998 A look under the cap
donze-1998-a-look-under-the-cap
Category: Topics | Donze 1998 A look under the cap - Scientific Beekeeping | https://scientificbeekeeping.com/donze-1998-a-look-under-the-cap/ |
Extended-Release Oxalic Acid Progress Report - Part 1
First published in: American Bee Journal, July 2017
Extended-Release Oxalic Acid Progress Report Part 1 Randy Oliver ScientificBeekeeping.com First published in ABJ July 2017 In January I wrote about an exciting extended-release application method for oxalic acid [[1]]. I'm currently collaborating with the USDA Agricultural Research Service and the EPA to get this application method added to the current label for oxalic acid. [...]
Read More | Varroa Management Archives - Page 5 of 8 - Scientific Beekeeping | https://scientificbeekeeping.com/varroa-management/page/5/ |
K.I.S.S. Breeding for varroa resistance
Open the link below to view the annotated pictorial presentation. 2017 KISS Breeding and if you want to see us doing smokin' hot mite washin' in real time, Rachel surprised me by figuring out how to prop up her cell phone to take a video of us washing a yard-to see the 36-second video, click [...]
Read More | Topics Archives - Page 3 of 4 - Scientific Beekeeping | https://scientificbeekeeping.com/topics/page/3/ |
2019 EcoFarm
Beekeeping is more difficult today than it used to be.
Our changing agricultural landscape provides less forage, and growers still apply pesticides to freely (although the pesticide situation for bees today is far better than it used to be in the '60s and '70s).
The main problem for honey bees worldwide is the recent invasion of the varroa mite, which acts as a vector for Deformed Wing Virus.
The long-term solution is to breed bees naturally resistant to the mite. In this presentation I offer a brief version of how to go about doing it. There is more information at my website.
Until such bee stock is more widely available, good bee husbandry requires occasional treatments to control the mite. My sons and I run a successful commercial beekeeping operation, and have used only organically-approved treatments since 2001.
I also give a progress report on our registration of extended-release oxalic acid for mite control. This organic treatment will help us to keep healthy, thriving bees.
You can find instructions for keeping bees healthy at https://scientificbeekeeping.com/first-year-care-for-your-nuc/
The slides for my presentation can be viewed at 2019 EcoFarm short (this is a large file with many photos, so may take a while to download).
Happy beekeeping!
Randy
Category: Topics | 2019 EcoFarm - Scientific Beekeeping | https://scientificbeekeeping.com/2019-ecofarm-2/ |
Being Part of The Solution
Click on the link below to view a ppt presentation.
Being Part of the Solution
Category: Topics | Being Part of The Solution - Scientific Beekeeping | https://scientificbeekeeping.com/being-part-of-the-solution/ |
Guessing our future with varroa
Category: Topics | Guessing our future with varroa - Scientific Beekeeping | https://scientificbeekeeping.com/guessing-our-future-with-varroa/ |
Glyphosate fact checking
This is a very contentious subject, but how much of the media alarm over glyphosate is based upon actual risk assessment?
Having run my garden and orchard organically for many years, I was faced with invasive Vinca major and Himalaya blackberries that were taking an excessive amount of my time to control. Weed whacking, horticultural vinegar, or ammoniated soaps would kill the aboveground foliage, but the vines would sprout right back up and laugh at me. I'm anything but a corporate shill for spraying unnecessary or dangerous chemicals, so I researched, for my own health concern, about the safety of using a glyphosate herbicide (which also meant that I could no longer consider my property as "organic").
I found that my own independent review of the literature did not support glyphosate as being of risk to me. Keep in mind that the label instructs the applicator to wear normal protective gear anyway! So I don't use it in my garden beds, but have now used it in other areas on my property, spot-treating only those two specific invasives (with impressive results). That said, I am not a proponent of widespread use of glyphosate on landscapes, as eliminating all native vegetation and "weeds" eliminates the food plants necessary for pollinators and wildlife, and good soil structure is dependent upon vegetation coverage that provides plenty of plant roots. Any pesticide should be used only sparingly and in a sustainable manner.
I also must point out that we citizens hire the scientists at EPA to perform risk assessments for us. The EPA risk assessors also have families that they wish to protect. I have the honor of knowing the head of risk assessment personally from informal meetings and meals at conferences, as well as many phone and email conversations about protecting bees from pesticides. EPA knows how to do risk assessment, and can better analyze the data from any published or unpublished study better than any can layman or activist group. Bottom line: I have no reason to question EPA's risk assessments, summarized at https://www.epa.gov/ingredients-used-pesticide-products/glyphosate
For an excellent and objective scientific review, read (of interest, search the word "honey"): Residues of glyphosate in food and dietary exposure https://ift.onlinelibrary.wiley.com/doi/10.1111/1541-4337.12822
For a very good review of glyphosate residues in off-the-shelf foods, the CFIA (Canadian Food Inspection Agency) analyzed 7955 samples of foods to determine the level of compliance of foods in the Canadian marketplace against established MRLs. Read the full report here: https://pubs.acs.org/doi/10.1021/acs.jafc.9b07819
That said, the best layman's review of glyphosate was recently published at https://geneticliteracyproject.org/gmo-faq/is-glyphosate-roundup-dangerous/?mc_cid=527d29e2df&mc_eid=dc9006049b
I've snipped three informative graphics from the above article, since in my own prior research, they were three data sets that I had already graphed out myself:
Money-hungry, fear-provoking "activist groups" such as the Environmental Working Group, hire skilled marketers and lawyers to generate and post scary correlations about glyphosate and cancer. But correlation does not imply causality, as evidenced by the graph below:
For more humorous spurious correlations, see https://www.tylervigen.com/spurious-correlations
I had previously checked the databases of countries that had kept track of incidence of Non-Hodgkin's Lymphoma and glyphosate use, and had found that although glyphosate use has skyrocketed over the years, the incidence of NHL has remained flat, which does not support a link between the two. Take a look at the two graphs below (from the GLP article):
If there was really any evidence that glyphosate caused NHL, we'd see it in the farmers who get it splashed all over their jeans all day long when applying it.
The maps below would clearly indicate if there was any connection between glyphosate application and NHL. There doesn't appear to be one.
Bottom line: I'm far more likely to harm myself from picking, digging, and pulling out blackberries than I am from spraying them (once is usually enough) with glyphosate. I you have hard data that indicates that I should revisit my current assessment, please forward it to me!
Category: Topics | Glyphosate fact checking - Scientific Beekeeping | https://scientificbeekeeping.com/glyphosate-fact-checking/ |
About Randy
About me
To start with, among other things, I'm a honey bee researcher, so if you're not comfortable with honey bees being around, read no more
I'm a healthy, youthful, happy, high-energy, and loving guy, finally over the heartache of losing my beloved wife. So I'm now looking for a new life partner and soul mate with whom to share my exciting, productive, and joyful days.
I am a biologist, nature lover, family man, teacher, inventor, writer, gardener, and builder. My friends would describe me as the busiest guy they know, and the guy who gets things done. The answer man to any question. Playful and funny. My joy is sharing with others - I've always been a teacher and giver. Helpful, loving, sharing. Widely loved by many.
I maintain amicable relationships with those with whom I've been in previous relationships. They, and the women that I work with, will all vouch for my loving good nature, honesty, and trustworthiness. And if you're familiar with the five love languages, mine is clearly touch -- hugs and intimacy warm my soul.
Disclaimer: My eharmony photo is from 2018 , but it best captures my essence. Here are some more from this year.
Possible Incompatibilities
Since I'm not in any way trying to seduce you (and have no desire to waste our time), please let me share some things about me and my lifestyle that might suggest incompatibility:
I'm a honey bee researcher (writer and international invited speaker). You'd need to be OK with being around honey bees.
If you're looking for a quiet life in front of the TV, read no more. I'm a high-energy, high IQ, vibrant, scientifically-minded guy, biologically about 10 years younger than my chronological age, lean and very active, and I work nonstop -- so am not the right partner for a low self-esteem or low-energy lady (although I'm an intense and focused hard worker, I'm also very happy and humorous, always smiling, laughing, and joking).
I've got many skills. And just like the cobbler who can't find time to shoe his children, my houses are in various stages of remodeling and disarray.
Although I'm financially set up, I live a simple, rustic, eco-friendly lifestyle, and wear my jeans for more than one day at a time.
I am looking for someone who wants to move to my place. I've got a rural property in the California foothills (Grass Valley), where we get a bit of snow in the winter. I grow a large garden and orchard, and am looking for a partner who would enjoy being involved and engaged in the garden and the property (woodsy with a lovely view).
I'm more of a cat person than a dog person. I like the wildlife around my home, and don't want a dog scaring everything off.
I'm not the right person for someone who is intimidated by intelligence, or put off by me being so engaged in my research, writing, shop work, and property maintenance. I can also be kinda messy, since I always have so many projects in the works.
As far as a partner, I'd prefer an energetic, physically active, preferably lean, and affectionate woman who enjoys a simple rustic, eco-friendly lifestyle. I follow the Golden Rule, but have no interest in organized religion.
More about me
I'm likeable, happy, and appreciated by others. I've got no issues or traumas, and am responsible and caring. I exemplify the Scout Laws (I'm the inverse of our ex-president). You will never hear an untruth cross my lips.
I'm an ex hippy child of the '60s, now a responsible, environmental, fiscally-conservative, socially-liberal adult. I was raised to honor and respect women, and have no desire to dominate a relationship.
I've got many skills, and am highly competent, capable, and accomplished. I'm always happy, free of stress, loving, generous, and looking for a soul mate and partner to share the everyday joys of life with -- like a new flower blooming, or the taste of the first fruits from my orchard, or a beautiful sunset.
I live on a dream property with garden, orchard, and a view, am in good health, and financially stable. I've got two houses next to each other (one vacant since my mother passed away), and I'd be happy to remodel either one to my new partner's likings.
I've handed my sons our beekeeping business, so am now free to follow my passion of performing honey bee research and providing scientific information to beekeepers worldwide. My sons run the business from my property (one lives next door), so we often have visitors and things going on at the property.
I lead a simple, frugal, rustic life, in an older house - nothing fancy (other than traveling to speak). I'm early to bed, early to rise. In my office before sunrise, cranking data, writing, email correspondence, and reading scientific papers. After breakfast, in and out for the rest of the day. Other than the news, I watch very little TV. Back in for dinner, which I enjoy cooking. I love spending evenings watching a movie together with the one I love.
I'm home or nearby most days, and don't go to town much, but do travel to speaking engagements, which I try to limit to no more than once a month.
I respect and honor women, and have worked with ladies most of my life. I'm well liked and loved - any of my ex's or female coworkers will give me a thumbs up.
The partner I'm looking for
My previous relationships have been based upon love, attractiveness, and intimacy, but after signing up for eharmony, I realize that I'd be wise to find someone whose energy and personality traits match mine. I'm a likeable guy, so I'm not desperate or trying to sell myself, seduce or mislead you, so I will be honest and straightforward. I do not want to waste your time or mine.
As far as relationship, since I'm a brainy, strong guy, I want an equally strong, self-assured partner who can stand up for herself, and communicate and articulate her feelings and wants. I don't argue or criticize, and have zero desire to dominate or always get my way.
Although I very much love doing things together, I need a partner who also has her own interests and hobbies to happily entertain herself while I'm working on my own projects. I'm far more interested in an active tomboy "country girl" in dirty jeans than a beauty queen in high heels. I want a gal who doesn't mind getting dirt on her hands, and will enjoy working in the garden and orchard with me, and be my third hand when I'm working on projects (and ditto for me helping with yours).
I ain't perfect, and don't expect you to be either. I want my partner to accept me in full as I am, and I will do the same in return. I don't want to merely love my partner, I want to be in love with her every moment of my life!
Category: Topics | About Randy - Scientific Beekeeping | https://scientificbeekeeping.com/about-randy/ |
Fair demo cage
Category: Topics | Fair demo cage - Scientific Beekeeping | https://scientificbeekeeping.com/fair-demo-cage/ |
The Varroa Problem: Part 16b - Bee Drift and Mite Dispersal (cont.)
First published in: American Bee Journal, May 2018
Contents Bee Drift and Mite Dispersal (continued) 1 So why do colonies allow bees to drift in?. 1 The sheer numbers involved. 4 The amount of mite drift into other hives. 5 Collapse and Robbing. 7 What happens to all the mite-infested bees when a colony collapses?. 8 Swarms coming back to bite you in [...]
Read More | Varroa Management Archives - Page 4 of 8 - Scientific Beekeeping | https://scientificbeekeeping.com/varroa-management/page/4/ |
Screenshots of hive weights
Supplementary material for A Study on Bee and Mite Drift, Part 5
Due to unfamiliarity with data preservation for the scale hives, we lost all our raw data when we removed the batteries from the scales at the end of the trial. Luckily, I had been following the weight data during the course of the trial, and had taken a few screenshots. I fully expected to see evidence of robbing correlating with mite immigration, but did not.
A screenshot of scale data for the Donors.The only sudden weight losses that we observed were for Donors D6 and D8 (not shown in this chart) -- the rest of the Donors showed no indication of getting robbed during their collapse. But the bulk of those weight reductions may well have simply represented the body mass of the workers that abandoned the hive (the bees covering 8 combs weigh roughly 4 pounds).
A screenshot of typical scale data for the Receivers, with weight gains by R4 and R7.
Category: Topics | Screenshots of hive weights - Scientific Beekeeping | https://scientificbeekeeping.com/screenshots-of-hive-weights/ |
Sick Bees - Part 14: An Update on the "Nosema Cousins"
Contents
Worldwide Status and Distribution
Ceranae vs. apis
Coinfection
Seasonality
Sample Interpretation
What if You're Dealing with N. apis?
Seasonality
Recommendations
Acknowledgements
References
Sick Bees 14:
An Update On The "Nosema Cousins"
First published in ABJ December 2011
Randy Oliver
ScientificBeekeeping.com
In my last article, I described how to quickly sample for nosema. So what do the spore counts actually mean as far as colony health is concerned? I wrote an article a little over two years ago with the tongue in cheek title "Nosema ceranae: Kiss of Death, or Much Ado about Nothing." Well, N. ceranae is still an enigma, but it appears that the answer lies somewhere in between.
Dr. Mariano Higes (2005, 2006) was the first to raise the flag to alert beekeepers worldwide that a new species of nosema had invaded Europe, and appeared to be the cause of the unusual colony collapses that plagued Spain (a major beekeeping country) in 2003 and 2004. Then in 2007, just as Colony Collapse Disorder was rampaging through our own bee operations, we found out that Nosema ceranae had somehow spread throughout the U.S. right under our eyes!
Drs. Diana Cox-Foster and Ian Lipkin (2007) then published a paper suggesting that a newly-described virus was involved in CCD, but later research indicated that IAPV wasn't the only culprit, leaving N. ceranae as a leading suspect.
Shortly afterward, Higes (2008) described in great detail the progression of N. ceranae infection (in his Spanish apiaries) through four stages: Asymptomatic, Replacement, False Recovery, and finally the dreaded Depopulation. The logic, the numbers, and the devastating final result were all clear and compelling. The specter of N. ceranae ravaging our hives resulted in unnerved beekeepers boosting the sales of fumagillin to the point that supplies ran short.
I had never previously worried about nosema, but I pulled out a microscope and found out that N. ceranae was indeed widespread in my operation. I ran trials, and found out that the danged parasite could flourish despite being drowned in fumagillin (Oliver 2008a), but more surprisingly, that colonies here at Comedy of Errors Apiaries thrived despite exhibiting spore counts in the millions. To try to reconcile the differences between the very different outcomes of N. ceranae infection in my operation with those reported for Spain, I began an ongoing correspondence with Dr. Higes, which continues to this day.
To be frank, some other Spanish researchers dispute Higes' conclusions (debate leads to better science), so I have often questioned and challenged him on details of methodology and interpretation, which he and his team of collaborators have generally clarified with additional research. In this series of articles I will be citing a number of the Higes team's papers, since they have clearly led the pack in N. ceranae research, meticulously investigating nearly every aspect of this pathogen's effects upon bees.
I've previously written at length about N. ceranae in my "Nosema Twins" series (all available at ScientificBeekeeping.com), but feel that there has been so much recent research completed that it would benefit the reader for me to write a digest of our current state of knowledge. I've scoured the literature for every relevant research paper (including a number still in press), and have discussed as well current findings with many of the world's nosema researchers. I wish that at this time I could say that I have the answers to all your questions about Nosema ceranae, but unfortunately, in many aspects this parasite still remains an enigma.
Worldwide Status And Distribution
Nosema ceranae has now spread into the European honey bee populations of most areas of the world, roughly concurrent with the spread of varroa (and its altering of virus dynamics), which greatly confuses analysis of the effect of these two novel parasites upon bee health. It is difficult to tell in which countries N. ceranae has already reached equilibrium, and in which it is still invading.
Since the first invasive wave of a novel parasite into naive hosts is generally that most damaging, it would be helpful to know when ceranae actually arrived in various countries. For example, we know from analysis of archived bee samples that N. ceranae has been present on the East Coast for at least two decades (Chen 2008). Unfortunately, any initial effects of its invasion may have been masked by our focus upon the massive impact of the arrival of varroa at about the same time.
Since no one was looking for N. ceranae in the U.S. until 2007, we obviously didn't start studying it until long after it was well established and likely homogenized throughout the bee population via migratory beekeeping practices. And it is also likely that by the time we started studying the impact of N. ceranae upon the health of colonies, natural selection may have already weeded out the bees least tolerant of the emergent pathogen.
In Europe, however, N. ceranae only recently invaded bee populations already suffering from varroa and viruses, miticide failure and comb contamination, extreme weather events, plus changes in agricultural practices and pesticide use--the combination of which likely factor into colony losses in that region.
In a fresh study (Botias 2011), the Higes team analyzed archived Spanish honey samples (frozen) and adult bee samples (in alcohol) dating back to 1998. They found that N. ceranae first appeared beginning in 2000 and increased in prevalence through 2009 (the latest samples analyzed), concurrent with a decrease in the prevalence of N. apis. It is noteworthy that Spain concurrently suffered from devastating drought during much of that period, which led to serious colony stress.
N. ceranae is still in the process of extending its range worldwide, and appears to be most successful in warmer climates. It is of interest that in varroa-free Australia, its invasion does not appear to be causing significant colony losses. Interestingly, although it is well-established in Canada, it is not yet common in some northern European countries, but this may be due to restrictions upon bee imports (Fries 2010).
N. ceranae is widely distributed throughout the U.S., but surprisingly, there were great differences in the percent of colonies infected in a recent state-by-state survey (Fig. 1).
Figure 1. Prevalence (percent of samples infected) of N. ceranae in various states as determined by PCR analysis (more sensitive than spore counts) of aggregate samples collected from 8 randomly selected colonies per apiary, 4 apiaries per state. Note that in some states over 70% of samples were infected! From Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
Ceranae Vs. Apis
In a widely cited paper by Martin-Hernandez (2007), her arresting graph of nosema positive samples over time clearly shows a definite shift over the period from 1999 to 2005--there initially were only spikes in spring and fall (ostensibly from apis), transitioning to nearly 100% of samples being positive every month of the year (due to ceranae). Of note is that her data has an inherent bias, in that the samples were voluntarily sent to the lab by beekeepers for diagnosis of problems, suggesting that the data may reflect the change in nosema loads in sick hives. Also of note, is that despite this graph being widely cited, it is often misunderstood--it did not plot spore levels, but rather only the yes/no detection of nosema spores.
What the graph did strongly indicate was that N. ceranae rapidly and thoroughly invaded Spain over a period of only a few years! This initial finding has now been confirmed by Botias (2011). Likely, a similar phenomenon occurred in the U.S., since Chen (2008) found N. ceranae to already be widespread in archived U.S. bee samples dating back to 1995.
The general trend appears to be that N. ceranae now predominates in warmer countries, whereas N. apis is better adapted to colder areas. It has been often stated that N. ceranae has displaced N. apis, but more careful analysis suggests that that may not actually be the case!
When Dr. Robb Cramer asked me in 2007 to send him infected bees so that he could culture pure N. ceranae, he found that the samples often contained some N. apis as a "contaminant." In Dr. Diana Cox-Foster's (2007) analysis of CCD colonies, they also found both species of nosema. Later studies by Bourgeois (2010) and Runckel (2011) of commercial operations in the U.S. also found N. apis, but in far fewer hives than its cousin, only in spring and/or fall, and notably, at much lower spore levels than N. ceranae.
The differences between the detectability of the two nosema species (N. apis typically produces much lower spore counts and is generally only seen in spring and fall) may lead "to an increased chance of detecting N. ceranae over N. apis, which could have biased the impression that N. apis has been displaced" (Higes 2010).
So, has ceranae actually displaced apis, or have we merely been overlooking its cousin? In order to answer that question, Dr. Raquel Martin-Hernandez (2011) carefully analyzed over 2000 bee samples from all across Spain. She found ceranae and apis coexisting throughout country, with ceranae clearly predominant (in roughly 40% of hives), apis hanging in there (in up to 15%), and occasional mixed infections (below 7%). She also found that infection by ceranae was favored in hotter areas of the country, whereas apis succeeded better where winters are colder.
I'm seeing similar indications from other countries (e.g., Gisder 2010), which are appearing to confirm that apis is the more cold-adapted species. As far as seasonality, Martin-Hernandez found apis only in the spring and fall, whereas ceranae could be found all year, and notably, once ceranae infects a colony, it almost always persists (detectable with PCR, even if not obvious via spore counts).
Practical note: these studies indicate that N. ceranae remains present as an infection in a colony throughout the year, even if it is not detectable by microscopy. But we don't know whether these inapparent infections affect colony health.
I found one last study to be of special interest: Dr. Judy Chen (2009) looked at nosema invasion from the other direction--in a turn of the tables, N. apis appears to have been introduced from the Western honey bee (Apis mellifera) into the Eastern honey bee (Apis cerana) in Asia, and is now an emergent parasite in that species, which had historically been infected only by N. ceranae! She analyzed bee samples from China, Taiwan, and Japan. Her findings:
"N. apis was detected in 31% of examined bees and N. ceranae was detected in 71% of examined bees and that the copy number of N. ceranae was 100-fold higher than that of N. apis in co-infected bees, showing that N. ceranae is the more abundant of two Nosema species in the Eastern honey bees."
This study suggests that N. apis can not only hold its own against N. ceranae, but can actually invade into ceranae's turf! Interestingly, in the Eastern honey bee, despite its long coevolution with N. ceranae, ceranae still produces higher spore counts than its invading cousin.
Coinfection
This brings up the question of what happens when bees are infected simultaneously by both species of nosema? Dr. Zachary Huang (pers comm) found that in both cage trials and field observations that longevity was substantially shorter for coinfected bees as opposed to those infected by either species of nosema alone (unpublished data).
Note that in Cox-Foster's (2007) CCD study that they found "a trend for increased CCD risk in samples positive for N. apis" (100% of CCD colonies tested positive for ceranae and 90% for apis, but remember that apis is easy to miss when samples consist of house bees). As Jim Fischer noted in a post to Bee-L, "What was striking was that every hive showing CCD symptoms tested positive for BOTH Nosema apis and Nosema ceranae, and this correlation was better than the correlation between CCD and IAPV that was the focus of the paper."
These findings leave me very curious about the impact of coinfection by two nosema species upon colony health!
Seasonality
Spore counts of N. ceranae generally reach a peak in May, then drop spontaneously during summer, and may spike sporadically in fall and winter. But there is more to the picture than this. Dr. Ingemar Fries (2010), who has studied nosema for decades, explains thusly:
"The typical pattern for N. apis infections in temperate climates is low prevalence or hardly detectable levels during the summer with a small peak in the fall. During the winter there is a slight increased prevalence with a large peak in the spring before the winter bees are replaced by young bees... The pattern is similar both in the southern and northern hemisphere... Unfortunately, very few data exist for N. apis on the seasonal prevalence from tropical or subtropical conditions. The only published year round sampling under conditions where bees could fly all year round, revealed detectable levels of N. apis with no seasonal pattern of prevalence."
Along that line, Dr. Denis Anderson in Australia (pers comm) tells me that, "there are also many unseasonal occurrences of N. apis -- I get many samples sent in in the mid summer here that are loaded with N. apis." This could well be happening in the U.S., where, as far as I can tell, there have been few studies on N. apis in warmer areas, other than the fact that it was commonly found in package bees produced in the southern states.
Practical application: we need to learn more about the prevalence and seasonality of N. apis in the warmer parts of our country!
I've now seen data and presentations on N. ceranae seasonal prevalence from researchers from all over the world. Since a picture is worth a thousand words, I've summarized them in a crude graph below (Fig. 2).
Figure 2. A generic graph of typical N. ceranae spore counts over the course of the year in my operation. Important note: Counts of house bees would follow the same trend, but at much lower levels. The late-season spikes are often sporadic flare ups that spontaneously "go away."
Practical application: It is not unusual to see high nosema spore counts in April and May. Counts will typically drop in summer whether you treat or not. I'll cover treatments in a subsequent article.
But new technology is showing something surprising about nosema sampling--that spore counts do not necessarily reflect degree of actual nosema infection (Meana 2010)! Look at the following graph (Fig. 3), from a recent nationwide study of pathogens in U.S. bees--instead of measuring spore counts, the blue bars indicate the percentage of colonies infected by N. ceranae as determined by DNA analysis (PCR).
Figure 3. The blue bars indicate the percentage prevalence of N. ceranae in sampled colonies (e.g., 0.7 = present in 70% of hives). Note that even though spore counts suggest that N. ceranae disappears for much of the year (previous graph), a substantial proportion of colonies actually remain infected to some degree by the parasite. Also note how closely the coinfection with another intestinal parasite (the presumably opportunistic trypanosomes) tracks nosema infection. No one is sure whether there is a causal relationship, or whether the simple explanation is that both parasites flourish in stressed bees. Graph from Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
As opposed to the above graph, Runckel (2011) also measured the amount of nosema DNA in samples, which presumably correlates with the intensity of the infection. They found high levels of N. ceranae transcripts in midsummer, at a time when spore counts are generally quite low (Fig. 5)! Their data indicated that N. apis was only present in spring and fall (which does correspond to spore counts). Go figure!
So what's up with high levels of N. ceranae DNA transcripts without correspondingly high spore counts? No one to my knowledge has answered that important question. What we do know is that N. ceranae can exist in the vegetative stage for a while before it produces spores (Martin-Hernandez 2009). But we're not clear on to what extent N. ceranae produces "autoinfective spores," as opposed to the "environmental" spores that are discharged into the gut contents (Cali 1999), and whether such autoinfective spores show up under microscopy. What is clear, however, is that N. ceranae appears to be able to reproduce within a bee without producing spores that are observable by microscopy.
Practical note: although N. ceranae spore counts may disappear in summer, DNA analysis indicates that the bees may still be infected. This is something of a mystery, as the bee population turns over rapidly during the summer, suggesting that N. ceranae is somehow infecting new bees without spores being evident!
So the next question is, is an infection by N. ceranae more pathogenic than one by N. apis? Although some initial cage trials indicated extreme virulence for the new nosema, trials in which bees were allowed to feed upon natural pollen generally found that both species affect bee longevity about the same (Forsgren 2010, Porrini 2011, Huang pers comm) despite the fact that spore levels get much higher with N. ceranae.
Take home: We clearly still have lots to learn about N. ceranae! It does not appear to cause rapid death of well-fed bees. The inapparent summer infections are puzzling.
So what's the cause of the seasonality of nosema spore counts? With N. apis it is presumed to be due to the requisites of transmission via dysentery by infected bees in the hive during the winter and colony nutritional stress, and limited by its sensitivity to high temperature. Martin-Hernandez (2009, 2010) demonstrated that N. apis can only grow in a narrow range of temperature (about 33degC). N. ceranae, on the other hand, grows readily over a range from 25degC to 37degC. However, N. ceranae spores are surprisingly susceptible to chilling (Fries 2010), which may limit their infectivity at lower temperatures.
Studies from a number of countries coinfected with both of the nosema cousins suggest that N. apis will continue to be the historical problem during winter and spring, with typical fall and spring spikes, whereas ceranae will be more prevalent in warmer climes, present throughout much of the year, spiking in late spring (perhaps tracking pollen flows), and then again sporadically in fall through winter.
Take home: if Nosema apis was a problem in your area prior to the invasion of N. ceranae, it may still contribute to colony health issues during the fall and spring!
Sample Interpretation
It would sure be easier if there were a simple sampling protocol that everyone could follow, and if there were clear treatment (or worry) thresholds based upon nosema spore counts, as there are for varroa (Fig. 4), but alas, I'm sorry to say that there aren't.
Figure 4. Average varroa infestation rates from 2700 colonies in 13 states (many of which received mite treatments). Sampling for varroa infestation level is relatively straightforward and simple to interpret. Typical treatment thresholds are below 5 mites per 100 bees. Graph from Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
Unlike sampling for varroa, which are easily seen with the naked eye, sampling for nosema requires either a microscope or laboratory apparatus that can perform PCR. However, a number of researchers (Meana 2010, Bourgeouis 2010, Traver 2010) have demonstrated that spore counts alone do not give an accurate picture of the actual degree of infection. Unfortunately, as far as assessment methods available to Joe Beekeeper, spore counts will have to suffice as a surrogate measure of the actual degree of infection (Fig 5).
Figure 5. Average nosema spore counts from the same 2700 hives. Note the typical huge spike in spore counts (predominantly from N. ceranae) in spring, and then again lesser spikes in fall and winter. Important note: these spore counts were from samples of bees from brood frames--counts from entrance bees would likely be several times higher (compare to Figure 2). Graph from Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
That said, let's return to sampling for a bit. If you want to find spores, then sample older bees, such as foragers at the entrance (Meana 2010)--spore counts will typically be about 10 times higher in older bees, since it takes a while for the infection to build up in a bee (Smart 2011). He found that in infected colonies with a background spore count of 0.5-1M in bees from under the inner cover, almost no bees younger than 12 days old contained spores (at least detectable by microscopy).
This is not at all surprising, since El-Shemy (1989) found the same to be true for N. apis--spore counts were an order of magnitude higher in bees from the entrance. Indeed, he suggested that it was best to sample exiting bees at the entrance, since returning bees have likely defecated. The magnitude of the spore counts from an infected colony generally increases in samples (in order from lowest to highest), of bees from the broodnest, outer areas of the cluster, entrance bees, exiting foragers, returning foragers.
Both El-Shemy and Higes (2008) found that the best indicator of degree of infection was to squash bees from an entrance sample one at a time in order to determine the percentage of bees infected. My own sampling of sick colonies supports this recommendation. But in reality, few of us have time to squash dozens of bees one at a time for each sample--so I won't even suggest that you go there!
The next best method may be to do a spore count for a pooled sample of 50 bees from the entrance (but don't forget that even one or two highly-infected bees can greatly skew the count). In practice, however, it is often danged difficult and time consuming to collect 50 entrance bees, even if you use a special vacuum (Oliver 2008b), especially in cool weather or from sick colonies with few foragers.
For this reason, many researchers simply take standardized samples of bees from under the cover, or from an outside comb. There is support for this, as Gajda (2009) found that although spore counts were much higher in entrance bees, the relative proportion of infected bees was similar in samples taken from an outside comb.
Practical application: If you want to find out whether N. ceranae is present to any significant extent in your operation, sample bees from the entrance. If you want to know if the infection is serious, sample house bees from under the cover.
If you are curious as to whether you have gotten old or young bees in your sample, here is an easy general observation that I've made: since only nurse bees normally eat pollen, they are the only ones that will have it in their guts (duh). But my point is, that this is really easy to use that pollen as an indicator of bee age if you use the ziplock bag method for processing samples (see my previous article, and Fig. 6).
Figure 6. How to tell if your sample contains young or old bees. (Left photo) when you crush samples of nurse bees in a ziplock bag, and then mush them in water, the fluid will typically turn opaque yellow (since the guts of nurse bees are full of pollen). (Right photo) on the other hand, the fluid from the guts of entrance bees will typically be a tan/gray color (since foragers and guards don't eat pollen).
What If You're Dealing With N. Apis?
Oh, that it were only so simple as dealing with only one nosema, but the previously cited studies suggest that many of us actually may still have N. apis popping up in fall and early spring. To make things even harder, spore counts of N. apis, on a per bee, or per pooled sample basis, are generally only a fraction (about 1/10th, as best I can tell from previous studies) of what we see with N. ceranae. But it also appears that an infection by N. apis at that low level can be as serious as an infection by N. ceranae at a much higher spore count!
Important note: Martin-Hernandez (2011) easily found N. ceranae in samples of either foragers or house bees, whereas she only found N. apis in foragers and drones. So if N. apis is your concern, then you should take entrance samples! N. apis infection may be serious at a much lower spore level!
Seasonality
The other consideration is that you must put any spore count into the context of time of year, the climate that your bees are in, the nutritional status of the colonies, and especially the load of other pathogens. I will discuss these points in the next article.
In cold climates, nosema management may have other considerations. Hedtke (2011) performed a detailed 6-year study of 220 hives in Germany, and (surprisingly) found that "No statistical relation between N. ceranae detection in autumn and the following spring could be demonstrated, meaning that colonies found to be infected in autumn did not necessarily still carry a detectable infection in spring, and colonies which developed a detectable infection over winter had not been detectably infected in autumn." So much for careful sampling!
Recommendations
Heck, I'd be crazy to stick my neck out and give any recommendations! So let's look at what sort of nosema levels are involved in crashing colonies. The CCD colonies analyzed by Cox-Foster (2007) had mean spore counts in the range of tens to hundreds of millions from broodnest samples! Is it really any surprise that those colonies collapsed? The house bees in Higes' (2008) winter-collapsing colonies hit 20M before they went down (field bees hit 50M), but those that collapsed in summer only hit 3M.
But note that in the U.S. survey graph above, that 2M was the average spore count across the U.S. in April and May of this year, yet I'm not hearing of massive colony collapses, despite very poor conditions in many states.
In my own California foothill operation (we get snow during the winter, and move to almonds in February), it is not unusual to see entrance spore counts in May in the millions or tens of millions, but they generally drop during summer, provided that colonies are not stressed by other factors. Entrance counts during summer and fall are typically in the zero to 5M range (25 spores per field of view if you follow the protocol in my previous article--I'll call these FOV counts (Oliver 2008c)). I have not looked at near as many samples of house bees, but counts are generally zero to a fraction of a million, even in colonies running at 10M at the entrance.
I am by no means suggesting that you follow my lead, but I simply no longer worry about high spore counts in spring, as they generally spontaneously drop later in the season, and I haven't experienced winter losses associated with N. ceranae (unless I've intentionally inoculated the hives with viruses). However, I do keep my mite levels down, and feed pollen supplement to maintain good nutrition if necessary. And I monitor nosema levels throughout the year so that I don't get blindsided!
I've never treated for nosema (except in experiments), yet have not experienced colony collapses since 2006. But I'm not saying that you have no reason for concern--I will be writing about a trial in which I did compare survival of treated vs. untreated colonies that had virus issues, and fumagillin appeared to help.
I'd be concerned if counts for house bees got above 5 per FOV at any time, although I know several large commercial beekeepers who routinely ignore such counts with no dire consequences so far. I just checked a number of samples of house bees today (late October), and they ran from zero to 2 spores per FOV, despite there often being counts of 100-200 per FOV of entrance samples this spring.
In some operations where N. ceranae apparently got out of hand, treatment and comb sterilization seemed to help. However, in other operations with sky-high spore counts in spring, lack of treatment did not result in any noticeable problems. Due to these huge discrepancies, it is confoundingly difficult to come up with recommendations. However, the more beekeepers who start tracking spore counts, the more we will learn about appropriate treatment decisions.
If you are in an area with a long, cold winter which keeps the bees confined, you may be dealing with Nosema apis, for which the economic threshold of 1M (5 per FOV) for house bees has been well established.
Practical application: since spore counts for N. apis generally only reach levels about 1/10th of those for N. ceranae, you'd be wise to ask your local university determine which nosema species you're dealing with, since it follows that the economic threshold for treatment for N. apis may be far less than that for N. ceranae.
I will continue this review of N. ceranae in the next issue, including treatments, and its relationship to colony mortality and honey production.
Acknowledgements
Thanks to you, my readers! It just occurred to me that I've recently passed the 5 year mark in writing for ABJ, and it's been one wild ride! If I had any idea what I was getting into, I would probably have chickened out. But your feedback and appreciation keep me going--my motivation is simply the gratification that I get from sharing what I've learned with other beekeepers. Your donations also allow me to perform the sort of quick and dirty research necessary to answer burning questions. I am constantly on the learning curve, and greatly appreciate hearing information that is relevant to better bee management--feel free to contact me (no beginners questions please) randy@randyoliver.com.
As always, Peter Loring Borst has helped me greatly with research. I thank Dr. Mariano Higes for his patience in discussing his research. Dr. Steve Pernal and Ingemar Fries have been gracious with their time. I also thank all the other nosema researchers who have patiently answered my questions.
References
Botias, C, et al (2011) The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade. Research in Veterinary Science (in press).
Bourgeois, AL (2010) Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee. Journal of Invertebrate Pathology 103: 53-58.
Cali, A and PM Takvorian (1999) Developmental morphology and life cycles of the microsporidia. P. 121. in Wittner, M and LM Weiss, eds. The Microsporidia and Microsporidiosis.,American Society for Microbiology.
Chen, Y.P., et al (2008). Nosema ceranae is a long-present and widespread microsporidian infection of the European honeybee (Apis mellifera) in the United States. J Invertebr Pathol 582 97: 186-188.
Chen, YP, et al (2009) Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees. Journal of Invertebrate Pathology 101 (2009) 204-209.
Cox-Foster, DL, et al. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318(5848): 283-287.
El-Shemy, A.A.M. and RS Pickard (1989) Nosema apis Zander infection levels in honeybees of known age. J. Apic. Res. 28 (2), 101-106.
Forsgren, E, and I Fries (2010) Comparative virulence of Nosema ceranae and Nosema apis in individual European honey bees. Veterinary Parasitology 170: 212-217.
Fries, I (2010) Nosema ceranae in European honey bees (Apis mellifera). Journal of Invertebrate Pathology 103: S73-S79. https://bienenkunde.uni-hohenheim.de/uploads/media/Nosema_ceranae_in_European_honey_bees__Fries.PDF
Gajda, A (2009) The size of bee sample for investigation of Nosema sp. infection level in honey bee colony. http://www.coloss.org/publications/Nosema-Workshop-Proceedings.pdf
Gisder S, et al. (2010) Five-year cohort study of Nosema spp. in Germany: does climate shape virulence and assertiveness of Nosema ceranae? Appl Environ Microbiol 76: 3032-3038.
Hedtke, K, et al (2011) Evidence for emerging parasites and pathogens influencing outbreaks of stress-related diseases like chalkbrood. Journal of Invertebrate Pathology 108:167-173.
Higes, M (2010) Nosema ceranae in Europe: an emergent type C nosemosis. Apidologie 14(3): 375 - 392.
Higes, M., et al (2005) El sindrome de despoblamiento de las colmenas en Espana. Consideraciones sobre su origen. Vida Apicola 133: 15-21.
Higes M, et al (2006) Nosema ceranae, a new microsporidian parasite in honeybees in Europe, Invertebr Pathol. 92(2):93-5.
Higes, M, et al (2007) Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia). J Invertebr Pathol. 94(3):211-7
Category: Nosema Summaries and Updates
Tags: N. ceranae, Nosema cereanae, Nosema Cousins, part 14, sick bees | Nosema Cousins Archives - Scientific Beekeeping | https://scientificbeekeeping.com/tag/nosema-cousins/ |
Old Bees/ Cold bees/ No bees? Part 2
Old Bees/ Cold Bees/ No Bees? Part 2
©️ Randy Oliver
ScientificBeekeeping.com
First Published in ABJ in July 2008
One day during his tenure as a professor, Albert Einstein was visited by a student. "The questions on this year's exam are the same as last year's!" the young man exclaimed.
"Yes," Einstein answered, "but this year all the answers are different."
Beekeepers today may feel that they are in a similar situation. Colony management that was previously successful may no long work! Here at Next Year We're Going to Make the Big Bucks Apiaries, unexpected losses of colonies have been eating up our profit margin. We've been making increase like crazy each spring, only see some fail to build, or crash during fall or winter.
However, being the optimist that I am, I think that I'm beginning to get a handle on the situation!
Last month, I was describing how a forager has limited longevity at best. Even that brief lifespan can be shortened further by disease or poor nutrition.
Protein levels
Protein intake of newly-emerged bees has recently been correlated with their subsequent behavior (Nelson, et al 2007). The authors found that bees with low vitellogenin levels begin foraging earlier in life, and tend to forage more for nectar than pollen. This is an insidious effect of lack of pollen in a colony--that the bees will begin to forage, and thus begin aging, sooner in life. (Research on this topic is ongoing, and sometimes contradictory--see Matilla and Otis 2006).
The "salary" that the hard-working foragers get from the colony is a daily allotment of protein-rich jelly. Schmickl & Crailsheim (2004) point out that although the older bees acting as foragers collect the pollen, most of it is eaten by the nurse bees. The nurse bees then process it into protein-rich jelly, which is then fed back to returning foragers to fulfill their protein requirements. Since the forager immune system and antioxidant system is dependent upon vitellogenin, their lifespan as seniors is partially limited by how well the younger bees feed them.
Predictably, the nurse bees prioritize first the hungry older larvae (since the colony has invested considerable resources in them). Foragers will feel the pinch during a protein shortage. Indeed, this appears to be part of a feedback loop that stimulates foragers to collect more pollen.
So imagine the situation during a drought--foragers work extra hard to locate pollen, yet there is not enough to spare to keep their own body protein levels up. Ditto when inclement weather during broodrearing keeps the foragers inside. The colony eats up all available pollen in a few days. The nurse bees cut back on the jelly fed to brood, may cannibalize eggs and younger larvae, and are ultimately forced to steal protein from their own bodies to continue feeding the oldest brood to maturity. No jelly will be left for the foragers. The bottom line is that when pollen resources get scarce, the foragers may suffer from lack of protein. This could again shorten their lifespan.
From a practical standpoint, it is important to remember that a low protein level in a colony will not only initiate premature aging of the bees, but will depress the immune system of the colony overall, thus making the colony as a whole more susceptible to any diseases.
As an aside, we beekeepers must be careful when feeding our bees protein supplements or sugar syrup. Poor quality ingredients-such as aged soy flour, or "off spec" HFCS can be quite toxic to bees. In addition, foragers can be poisoned by mycotoxins (fungal toxins) in fermented nectar or sugar syrup, leading to large losses (Manning 2008).
General infections
Sick bees are short-lived bees. Bees that had to fight infection (bacterial or viral) or parasitism (varroa) when they were in the larval or pupal stage may never reach their full potential as adults. Adult bees then have their own set of problems. A forager carrying a varroa mite, or infected with nosema has less chance of returning from each foray than an unparasitized bee. Or perhaps they don't return on purpose-- "This behavior can be interpreted as suicidal pathogen removal, serving as a disease defense mechanism which reduces the colony's load of parasites or pathogens" (Kralj, et al 2006). Nature has likely selected for a behavior in which dying foragers remove themselves from the hive, rather than forcing undertaker bees to carry them out.
When a bee is "challenged" by a pathogen or parasite, it activates its immune system. This process is metabolically expensive (Moret & Schmid-Hempel 2000). A bee (or colony) fighting a disease requires more food, and is not as productive. The extra metabolic effort required for cold-weather flight may prove to be more than it can summon. For an older bee with a low vitellogenin titer, similar to an elderly malnourished human, a normally minor disease may be fatal.
Parasites
As I mentioned earlier, colony collapse events have a recurrent history--which suggests that weather and/or the subtle effects of well-established pathogens are involved. However, in the last 25 years three new serious players have entered the picture: tracheal mites in 1984, varroa mites in 1988, and Nosema ceranae (apparently) sometime in the past decade. These parasites did not enter the scene quietly, but rather have each wreaked havoc. In the aftermath, they have added continual new stresses to our beleaguered bees, by weakening them, suppressing their immune systems, creating points of entry for pathogens, and adding entirely new vectors for viruses. Therefore, the current widespread collapses could be caused by the action of historical environmental stresses and pathogens, exacerbated by the additional parasite stresses.
The internal parasite, nosema, has long been called the "invisible killer" --since by shortening forager lifespan, it can devastate a colony without visible symptoms. Dr. Mariano Higes has detailed the pathology of N. ceranae that can lead to colony collapse. Nosema alone can be bad for a colony, but in concert with poor nutrition and/or viruses, it can be devastating. Indeed, some bee viruses are only found in conjunction with nosema infection.
Nosema can have an additional effect upon colony population. Some older research (Fisher 1964) suggested that nosema infection increases the level of juvenile hormone (JH) in the insect. In the case of honey bees, JH is antagonistic to vitellogenin, and higher levels of JH would cause premature aging.
I've corresponded with JH expert Dr. Zachary Huang, and vitellogenin expert Dr. Gro Amdam about this. Dr. Huang has unpublished data showing that nosema does not produce JH directly, but that infected bees can indeed have elevated JH titers, which cause bees to begin foraging earlier. (In some colonies, bees do not respond to the infection by showing earlier foraging, but he did not examine these colonies to see if the infected bees also had higher JH titers). The actual mechanism has not been determined, but likely involves vitellogenin, just as in healthy bees.
The colony-to-colony difference in response to nosema infection is notable, since it may explain why nosema is harder on some colonies than others. Anything that induces bees in a colony to begin foraging at an earlier age, will accelerate the aging of the workers, and restrain population growth.
Parasitic mites have also clearly demonstrated their impact upon bee longevity. Both varroa and tracheal mites can cause colony depopulation. The tracheal mite is especially rough on wintering bees, and the varroa mite on fall and winter bees. However, it is possible that neither mite kills the colony directly, but rather initiates a viral epidemic that polishes the bees off.
From a practical standpoint, it is moot whether the parasite actually kills a colony, or rather just sets it up for a fatal blow from one or more viruses. Control the parasite, and the bees can generally keep viral infections to a low level on their own.
Viruses
Bees are ubiquitously afflicted by continuously morphing RNA viruses, much as beekeepers are afflicted by the continuously morphing cold and flu RNA viruses. However, bee viruses tend to lie latent in the bees--only occasionally causing observable illness. In this manner they act more analogously to herpes viruses--virtually all humans carry them, but they only flare up when we are stressed, infected by another disease, or immune compromised.
Bee viruses were a relatively unimportant issue to beekeepers until the arrival of varroa. Then we quickly discovered that the combination of varroa and nearly any virus can be lethal. Dr. Norman Carreck (2008) writes:
"Infestation by Varroa and subsequent infection by ABPV and KBV can lead to many of the symptoms associated with CCD, namely the spectacular and rapid loss of strong colonies, leaving empty hives with just the queen and a few workers remaining."
So the obvious question is, can a normally latent virus, or one of its mutants, flare up periodically to cause an epidemic (or more properly, epizootic) in the bee population, perhaps due to an earlier nutritional stress event, and especially with the assistance of nosema? Such a virus could be newly identified, such as Israeli Acute Paralysis Virus (IAPV--still a suspect in my book), or an old timer like Sacbrood Virus (SBV).
Sacbrood is a good example of how the effect of a virus can be overlooked. We generally think of sacbrood as an uncommon virus of bee larvae in spring and summer, generally associated with poor weather or nutritional stress. In actuality, it has always been an extremely common virus, and can be found in adult bees on all continents at high frequency throughout the year (Tentcheva 2004, Koglberger 2005, Berenyi 2006, Nielson 2008). It can exist as an inapparent infection in pupae, which then emerge as infected adults (Dall 1985).
Not surprisingly, sacbrood is commonly found in collapsing colonies (Kulincevic 1984, Cox-Foster 2007, Bromenshenk 2008). However, one would expect such a common virus to be found in any survey--that doesn't necessarily mean that it is creating the problem.
But get this: in my own operation, I have historically rarely seen sacbrood. Yet the past two years, I've seen it commonly. Notably, I'm seeing it with regularity in colonies that are collapsing, or recovering from collapse. These observations certainly make me a bit suspicious!
SBV is only noticed when it kills last-instar larvae (which die stretched out on their backs, with their heads upturned--see Goodwin (2003) to download photos). It is normally spread as workers remove infected larvae, and get exposed to the highly-infected ecdysial fluid under the skin. Only young workers are easily infected by ingesting the virus--but these are the very workers that typically clean cells. Once infected, the nurse bees or foragers can spread the virus through their saliva, jelly, and stored pollen. Shen, et al (2005), additionally found that SBV can be transmitted by the queen to her eggs, and likely by the varroa mite. I will go into more detail about bee viruses later, but first let me quote from the legendary bee pathologist, Dr. Lesley Bailey (1972):
"sacbrood virus accumulates in the brains of infected bees...without causing symptoms [emphasis mine]. However, infected individuals fly earlier in life than healthy bees and infected foragers fail to collect pollen....The few infected bees that gather pollen contaminate their loads with much sacbrood virus. Infection...much shortens the...lives of workers that have eaten pollen."
Bailey found that the median number of days for foragers to failure to return to the colony went from 14 days for healthy bees, down to 5 days for SBV-infected foragers! This fact could have major implications on colony population (recall Figure 3), and there are no symptoms for infected adults other than that they just disappear. Now I am not suggesting that Sacbrood Virus is the cause of CCD, but merely pointing out how easy it is to overlook the potential contribution of an inapparent infection by a virus.
Yet another tidbit from the paper was especially striking to me: "Infected workers...are unable to maintain the usual metabolic rates of bees at temperatures below 35oC [brood nest temperature], or to resist chilling." Remember how colonies often dwindle during cold spells? This brings us to the subject of...
Hot-Blooded Ladies
The honey bee is a tropical insect that has adapted to temperate climates, much as humans have done--by living in heated shelters. Harvard zoologist Bernd Heinrich describes bee thermal strategies in two fascinating books: The Thermal Warriors and Bumblebee Economics. Unlike most insects, honey bees normally maintain body temperatures above ambient temperature, both individually, and as a colony. They do so in a clever way--they can "uncouple" their wings from the massive flight muscles in the thorax, and shiver--much as we shiver to warm up. However, bees have refined their shivering to such an extent, that they do it without visible shaking.
And shiver they do. They shiver to keep the brood nest at 94oF. Individual bees shiver to maintain a flight muscle temperature of at least 85oF, below which they are unable to fly. A bee readying itself to take off shivers to warm its flight motor up to about 100oF, and typically maintains it at about 95oF. Bees at the outside of a cluster hold their temperature to a minimum of 41oF, since below that temperature they are too cold to initiate shivering, and will die.
Although the bee's thorax is covered with an insulating pile, it will still chill quickly in cool air if it is not constantly generating heat. Due to the laws of thermodynamics, for every 20oF that the ambient temperature drops, the bee needs to work about twice as hard to stay warm. As I mentioned before, foraging in cool weather is very wearing on the bees!
When bees are returning from foraging during a cool day, one can see them occasionally stop to "rest." They are hardly resting! Rather, they have lost too much heat from their flight motor (thoracic muscles) due to the 15 mph "wind" passing over their bodies as they fly. They need to stop to warm back up. Indeed, an apparently resting bee may be working its flight muscles harder than it would while flying!
A bee stores about 15 minutes worth of fuel in its flight muscles, and about another 15 minutes worth in its blood (Southwick 1992). Once these sources are depleted, it is dependent upon whatever nectar it has in the honey sac--and can fly much faster if the sac contains high-sugar nectar. Should a forager run out of sugar fuel while it is flying or shivering, it will die in the field.
So how can you tell if a bee is really resting, or whether it is working hard to warm itself up? Simple: look at its abdomen. From the drawing in Figure 5, you can see that much of the bee's abdomen is taken up with air sacs. These sacs function as pumps to move fresh air efficiently through the bee (more efficiently than our own lungs). Insects obtain oxygen, and dump carbon dioxide by using branched tubes called trachea that open to the outside air at holes called spiracles on the sides of their bodies--three pairs on the thorax, and six on the abdomen.
In order to ventilate, the bee "pumps" its abdomen like an accordion, and opens and closes its spiracles so that it sucks air into the tracheal sacs in the thorax, and expels it from the abdominal spiracles (Stoffolano nd). The largest intake spiracle is the first thoracic, and it is screened by "hairs" to prevent the entry of dust and parasites (although the tracheal mite can enter this spiracle in newly-emerged bees of susceptible stocks).
The illustration above may mainly apply to a bee that has high respiratory needs, such as when flying or producing heat. Otherwise, the the breathing may occur mainly in the thorax (Bailey 1954), with air entering through the first thoracic spiracle, and exiting through the third thoracic spiracle (rather than out the abdominal spiracles).
So if an apparently "resting" bee is pumping its abdomen, you know that it is in actuality working as hard as it can to warm up--pumping oxygen to its flight muscles, and carbon dioxide out. This one-way flow-through system of ventilation is extremely efficient. Indeed, when not flying or shivering, the bee stops the pumping action in order to minimize its tissue exposure to harmful oxygen.
The bee has a clever countercurrent heat exchange system at its waist (the petiole) which prevents thoracic heat from being lost to the abdomen. The abdomen remains unheated. However, the bee instead uses its haemolymph (blood) to pump heat to the head! By placing its head or thorax against a cell wall or capping, the bee can transfer considerable heat to the brood (Figure 6).
Figure 6. At left is a top-view thermograph of three bees inside empty cells adjacent to brood. The upper bee is generating the most heat. Note how the heat transfers to the head. At right is a side view of two bees in cells. The upper is resting, the lower generating heat. The asterisks mark the walls of adjacent pupae. The white line is the comb midrib. From Marco Kleinhenz, Brigitte Bujok, Stefan Fuchs and Jurgen Tautz (2003) Hot bees in empty broodnest cells: heating from within ©️ 2003 The Company of Biologists Ltd, by permission.
The ability to transfer heat to the head allows honey bees to perform another neat trick--they can fly at temperatures that would kill most insects (up to 113oF). They do so by using their hot head as a radiator, and if necessary, exuding a droplet of nectar from their mouth to cool by evaporation! I am struck by what an amazing insect the bee is--it can maintain a constant body temperature similar to ours, cool itself when necessary, transfer heat to its offspring, and regulate the amount of oxygen that its tissues are exposed to!
OK, I've digressed, so let me return again to the question, What factor(s) could prevent the return of a bee that was initially healthy enough to fly away from the hive? Obviously, a pesticide kill, but those instances are generally pretty clear, and there are often piles of twitching bees in front of the hive. Instead, perhaps we should focus on the ability to fly. Sudden depopulation of a colony with no dead bees present, means that the bees must have flown away, and not flown back.
The non-returning bees were healthy enough rev up their flight motor and fly out, so were unlikely to have suddenly succumbed to mortality. More plausibly, they were simply unable to get their wing muscles back up to takeoff temperature once they cooled after leaving the warm colony. A bee can raise its thoracic temperature roughly 30oF above the ambient temperature while it is flying. That means that if it is flying in 55o weather, that once it leaves the warmth of the hive, it will barely be able to keep its wing muscles up to their minimum operating temperature (85oF)--hence bees don't fly much at temperatures below 55 degrees. And if they do, they often don't return.
So it appears that we should be looking for factors that affect ability of a bee to warm up its thoracic flight muscles. The most likely are age, poor nutrition, and/or disease--especially any disease that affects the flight musculature, the nerves that control it, or its energy conversion. The preliminary CCD report (van Englesdorp, et al 2007) describes some apparent pathologies of the flight muscles, including "white nodules" and "crystalline arrays." And remember Bailey's findings detailed earlier, that bees infected with sacbrood virus chilled more quickly, and were unable to maintain normal metabolic rates once cooled below broodnest temperature. I'm eager to hear of any research as to how any particular pathogen might accelerate the "aging" of the flight muscles.
Whatever the specific mechanism, the most likely reason that bees fly out, but don't return, is simply that once out of the hive, they couldn't generate flight muscle temperatures necessary for the return trip.
This brings up the additional question as to whether some factor is causing such flight-impaired bees to leave the colony at unfavorable temperatures. An intriguing hypothesis is based upon the observations that prior to some recent collapses, the colonies appeared "restless," bees may move away from the brood area toward the entrance(s), or that the combs may become repellent to bees. A pathogen or condition that changes the behavior of the bees to exit the hive at an early age, to initiate forays at inappropriate temperatures, or to abandon their hive and drift into others could certainly bring about a depopulation.
The Bottom Line
The dwindling of healthy-appearing colonies appears to be largely a function of the combined effects of the age of onset of foraging (or exit from the hive), and the number of days that the foragers then survive. Should the average age for forager "failure to return" drop to only a few days, the colony population dynamics go seriously into the red, and we observe that the colony "dwindles" as younger and younger bees are forced to shift from nursing responsibilities to foraging.
Several factors can promote early foraging, or accelerate the aging of foragers--especially poor nutrition or nosema, which also increase their vulnerability to disease and stress. In some collapses, something appears to cause a restlessness of the bees, or a repellency of the combs.
Some diseases, especially nosema, mites, and viruses, decrease the survivability of foragers without any apparent symptoms. The effect of any of these factors is greatest when foragers leave the warmth of the hive in cool weather, lose body heat, and are later unable to warm up for the return flight. Hence, spring and fall dwindling are often observed at the onset of cool weather events.
This is unfortunate for beekeepers preparing for almond pollination. A cold snap in fall or close to beginning of bloom can turn of profitable-looking yard of bees into a bunch of dinks seemingly overnight! We can only hope that further understanding of the causes leading up to such collapses, can help us to avert them in the future.
I have spoken with a number of beekeepers who were successful at taking strong colonies to almonds this season. There doesn't appear to be any single formula or magical potion for success, but rather, common sense husbandry may be the best approach:
* Be diligent with varroa! Don't let levels ever get high. Any number of methods will work to control the mite. But definitely get mite levels way down mid August at the latest. This will help keep viruses in check.
* Monitor nosema infestation, and treat in a timely manner if appropriate. Especially check colonies that fail to build normally.
* Don't baby colonies that aren't thriving, or have spotty brood. Kill or requeen them! (Some successful beekeepers requeen more than once a year!) Get sick colonies off to a hospital yard.
* Maintain good colony nutrition with regard to pollen, especially in late summer and fall. Move to better pasture, or feed your bees if necessary.
* It may be wise to maintain genetic diversity in your operation, since colonies vary in their resistance to different pathogens. Naturally resistant stocks go a long way toward success.
References
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Category: Aging and Thermoregulation | Old Bees/ Cold bees/ No bees? Part 2 - Scientific Beekeeping | https://scientificbeekeeping.com/old-bees-cold-bees-no-bees-part-2/ |
Pollen Supplement Formula
Update February 2023: Be sure to check out an updated Homebrew formula from my research in 2020 at https://scientificbeekeeping.com/a-comparative-trial-of-the-pollen-subs-part-5-revisiting-de-groot/
Update December 28, 2013
I am currently running a beekeeper-supported controlled trial of the major pollen supplements on the market, with a positive control of natural pollen, and a negative control of no protein feeding. 162 colonies total in the trial, 18 in each test group. I've recently completed the first grading for strength. The results were surprising, with two products outperforming natural pollen! As expected, the negative control is doing the worst. Both yeast-based formulations (including a variation of my homebrew formula below) are being poorly consumed. Final results will be in in February.
Interestingly, the Ontario Tech Team found that a yeast/egg homebrew formula similar to my original "homebrew" formula outperformed two off-the-shelf patties. I've copied it below. I am not promoting or recommending any formula. When my field trial is complete in February, I will post the results for you to interpret yourselves.
Update April 2012
There are new protein supplements on the market, most of which are very good. I am experimenting with several.
To most, I still add a corn/canola oil blend, food grade dried egg yolk, ground up multivitamin/minerals, some citric acid or lemon juice, and a splash of HBH or ProHealth as a feeding stimulant.
I see that the Kushlan mixer that I use is no longer on the market. Normal concrete mixers will not work-you need a mortar mixer or sausage mixer with rotating paddles. For mixing one sack at a time, it is surprising how quickly you can simply mix it with a garden hoe in a wheelbarrow!
UPDATES NOTE: If you don't see the changes, hit reload or clear your cashe.
added calc on Hackenberg formula
added Keith Jarret
Updated Feb 25, 2009 added comparison paragraph and table, corrected recipe % dry wt
Update 8/2/09 Jarret analysis
Update 8/25/09 Global patty link
Update 7/3/10 Protein sources
I've been asked again and again for the formula that I use. Well, I play with the formula often, and do not swear by any yet. The formula below was a composite of homebrews from various beekeepers, plus my own experimentation. It tested well against others, and I found it to be definitely better than brewers yeast/sugar alone. I will not comment on other proprietary products, since none have enough repeated data to support comparisons.
Testing by the USDA-ARS found that their product, licensed as MegaBee®️, performed better than the other products that they tested (www.megabeediet.com/). Commercial beekeepers report success with all the brands offered by the supply houses, Global, Norm Carey, Keith Jarret etc. See also http://www.honeybeeworld.com/misc/pollen/default.htm and http://globalpatties.com/pages/why.htm
Pollen supplement should be fed during pollen dearths. Appropriate times are prior to honey flows, in fall while the last rounds of brood are being raised, if bees are set in almonds prior to actual bloom, and immediately after almond bloom.
The most bang for the feeding buck is in late summer. California beekeepers should start feeding when natural pollen drops off in August, and by September at the latest. Continue as long as bees will take it, generally until about November 1st. Do not let colonies go long between feedings if they are actively rearing brood.
Field trials and practical experience have shown that colonies do well with about a pound a week of supplement, depending on protein content. Successful commercial migratory operations are feeding 10-20 lbs per colony per year, depending upon available natural pollen.
Please let me know of your successes and failures, so that we can figure out better formulas. Please do not post this formula to your web pages. Instead, send the link, or link back to this page, so that I can keep the formula updated.
Update: OK guys, there is a lot of confusion out there in comparing formulas. In general, when comparing livestock feeds, the number one thing that you pay for is protein. So if you want to see if you are getting the most bang for your buck, calculate out how much you are paying per pound of protein.
However, be aware of a few caveats!
1. This assumes that the proteins that you are comparing are equivalent nutritionally. For example, a protein that is either indigestible, or lacking in a single amino acid can't be compared to a digestible, complete protein.
2. Mann Lake's use of deGroot's amino acid ratio is not quite accurate. deGroot did not specify percentages of amino acids-he specified a "normalized" ratio of amino acid weights relative to the amount of tryptophan. deGroot never implied that a formula should be 1.00% trypotophan-rather, divide all other amino acid weights (or percentages) by the weight/percentage of tryptophan, to obtain their relative value. For example, brewers yeast runs about 0.62% tryptophan and 1.7% threonine. So you'd divide 1.7 by 0.62 to obtain a ratio of 2.74:1 threonine:tryptophan, which is close to deGroot's suggested ratio of 3:1.
The way you use these ratios is to see if any of the amino acids fall below the suggested ratio relative to tryptophan. Any that fall below would be called "limiting amino acids" since they would limit the amount of the total protein that can be utilized by the bees. To overcome that limitation, you would add a second protein source that was high in the limiting amino acid.
3. We are not sure what the optimum protein level is! Kleinschmidt found that the best pollens ran about 25% protein. The other 75% was fat, carbohydrates (some indigestible), minerals, sterols, and fiber. We don't really know how this works out in a pollen supplement that contains lots of sugar. We will probably find out by trial and error. Mann Lake, Dr. Gordon Wardell, and others selling supplements have done a great deal of research, but have clearly come to different conclusions as to what percent protein is best, or at least, most cost efficient (it's smart business practice to sell sugar at a dollar a pound).
4. When you are comparing formulas, make sure that you are comparing apples to apples! That is, you can't compare dry weight to wet weight, or with or without sugar. The only way to really compare them is in the finished, ready-to-feed patty.
There are two ways to determine the actual composition of a patty-by calculation, based upon the guaranteed analysis of the ingredients, or (most accurate) by actual lab analysis. Here are some actual comparisons of ready-to-feed patties. Those that were lab analyzed were guaranteed "fresh" from the distributors, and analyzed by an impartial agricultural lab in California.
Name Type analysis % Protein % Fat pH
MegaBee Lab 17.5 1.1 4.2
Bee-Pro Lab 10.3 5.3 4.8
Global 15% pollen Lab 14.8 2.4 5.5
Keith Jarrett Lab 17.6 7.5 4.9
"Kitchen Sink" Calc'd 19 6 low
Hackenberg Calc'd Can't compare, since amt water is not spec'd, but dry weight protein is ~15; I'm guessing it would take about 5 gal, which would put protein at 13%
The formula in the table below is experimental, but works well. It is likely that some of the ingredients can be deleted. I added the soy isolate in order to boost the protein level, and to balance the amino acids a bit, but need to test further to see if there is an added benefit. Compare the formula to David Hackenberg's formula following it.
Update 2022: The best formula that I've now tested is shown near the end of A Comparative Trial of the Pollen Subs: Part 5- Revisiting de Groot - Scientific Beekeeping
I mix all ingredients except the sugar and yeast into the water in a bucket with a motorized paint stirrer. I put the dry sugar into the mixer, then pour in the water solution from the bucket, and stir until mixed (put a bucket under the gate to catch any drip). Then I add the yeast on top, and stir until uniform (about 5 minutes). This procedure gives the best mixing in a Kushlan mixer. Water content is critical-too little gives a soupy mix, to much sets up too hard.
We pour the mix into oiled plastic storage tubs, and allow it to set up overnight. It will get firmer, so the poured mix must be soft. I sprinkle sugar over the top of it, so that you can pack it into the tub with your hands without it sticking.
In the field, we dump the tub out onto a sugared board, and cut it up with a spade or floor scraper into 2-3 lb slices, depending upon how much we want to feed. We place the slices between the brood boxes, after smoking the bees off the frames. If you make the formula soft, it will squeeze between the frames.
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Here is a formula developed, and successfully used, by Dave Hackenberg (by permission):
HACK'S SACK PROTEIN PATTIES
Protein Patty Recipe
1. 125 lbs. Sugar (Add water and keep wet. Should be a little thicker than pancake batter.)
2. Add either 3 cups citric acid or 4 quarts of lemon juice, (this is to put the ph at 4 1/2 to 5) 3. Add 1 cup Honey Bee Healthy (optional , but we prefer)
4. Add 1/2 bag Vitamins & Electrolytes (we use Russell's) (2 oz. worth)
5. Add 10 lbs. pollen (optional)
(keep the mix wet)
6. Mix in 25 lbs. of Inedible Dried eggs (available from Hackenberg Apiaries)
7. Add 3 1/2 cups Canola Oil
8. Mix in 24 lbs. (2 gallons) Honey
9. Finish by adding 50 lbs. Brewtech Brewers Yeast. Water until it has the consistency you desire. (available from Hackenberg Apiaries, Pat Heitkam or David Mendes)
David says that this formula tests out from 16-20% protei; however, by my math it would be about 15% before adding water.
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Note that his formula has much more sugar (plus honey). I tried inedible whole spray dried egg, but didn't like the consistency, and found that bees didn't much care for it at high percentage. The brand that I used also smelled like rotten eggs! I found that the dried egg yolks from Honeyville (human food grade) cost a bit more, but are of the highest quality (and smell good).
Sources of mixed supplement:
Keith Jarret ( cnhoney@att.netThis e-mail address is being protected from spam bots, you need JavaScript enabled to view it ) Ingredients not disclosed. Keith says that his product tests at "fat 9% & protein 16%, that is finished product just as the bees would eat. It comes in 200 lb tubs @ $1.05 lb."
Protein Sources
it is by far easiest in the U.S. to buy off-the-shelf pollen supplements, already tested and proven. There are several excellent ones on the market.
If you choose to mix your own, the most important item in any supplement is the protein source. A great review is in "Fat Bees, Skinny Bees"-a free download https://rirdc.infoservices.com.au/downloads/05-054.pdf Here is a list of some tried and true sources:
1. Natural pollen: the best, but problems with price, parasite spores, nutritional degradation, and pesticides.
2. Brewers Yeast: Brewtech brand works very well. Roller dried yeast may be better than spray dried. Debittered may be more palatable, but I'm not sure. Some brands (such as from corn ethanol production) dry rock hard if not eaten.
3. Torula yeast: good reviews
4. Soy flour: Expeller pressed best, not defatted. This process destroys the anti nutritional factors of soy. Problem with low B vitamins, and some complex sugars.
5. Soy protein isolate: expensive, but might have use as a protein booster and amino acid balancer.
6. Pea protein
7. Corn gluten
8. Barley flour
9. Quinoa flour
10. Dried egg or egg yolk
11. Milk products without lactose. Casein excellent, but too expensive.
Sources for ingredients:
(Broken Link!) http://store.honeyvillegrain.com/powdereddriedwholeeggsandeggwhites.aspx egg yolk, soy
Pat Heitkam, Orland, Calif. 530-865 -9562 Brewtech yeast, commercial orders
tackabery@aol.comThis e-mail address is being protected from spam bots, you need JavaScript enabled to view it commercial orders. soy isolate (tested to be free of melamine), yeast
This photo shows the desired consistency of the fresh mixture. I pour it into oiled tubs to set up. In the field, we dump the tub over onto a board sprinkled with sugar, and slice it up with a floor scraper.
Feedback:
Q: I have a question on the protein receipe. I see you have vitamins added to boost micro nutrients. Can I use kelp meal also? is this an exact receipe or can I add to it?
A: The recipe is a work in progress. It tested very well in a recent controlled trial by myself. I have no idea as to whether additions (or deletions) would help. Might I suggest making a batch in portions, tweaking one ingredient at a time. Then test side by side in colonies. Please let me know your results!
Category: Bee Nutrition | Pollen Supplement Formula - Scientific Beekeeping | https://scientificbeekeeping.com/pollen-supplement-formula/ |
Sick Bees - Part 14: An Update on the "Nosema Cousins"
Contents
Worldwide Status and Distribution
Ceranae vs. apis
Coinfection
Seasonality
Sample Interpretation
What if You're Dealing with N. apis?
Seasonality
Recommendations
Acknowledgements
References
Sick Bees 14:
An Update On The "Nosema Cousins"
First published in ABJ December 2011
Randy Oliver
ScientificBeekeeping.com
In my last article, I described how to quickly sample for nosema. So what do the spore counts actually mean as far as colony health is concerned? I wrote an article a little over two years ago with the tongue in cheek title "Nosema ceranae: Kiss of Death, or Much Ado about Nothing." Well, N. ceranae is still an enigma, but it appears that the answer lies somewhere in between.
Dr. Mariano Higes (2005, 2006) was the first to raise the flag to alert beekeepers worldwide that a new species of nosema had invaded Europe, and appeared to be the cause of the unusual colony collapses that plagued Spain (a major beekeeping country) in 2003 and 2004. Then in 2007, just as Colony Collapse Disorder was rampaging through our own bee operations, we found out that Nosema ceranae had somehow spread throughout the U.S. right under our eyes!
Drs. Diana Cox-Foster and Ian Lipkin (2007) then published a paper suggesting that a newly-described virus was involved in CCD, but later research indicated that IAPV wasn't the only culprit, leaving N. ceranae as a leading suspect.
Shortly afterward, Higes (2008) described in great detail the progression of N. ceranae infection (in his Spanish apiaries) through four stages: Asymptomatic, Replacement, False Recovery, and finally the dreaded Depopulation. The logic, the numbers, and the devastating final result were all clear and compelling. The specter of N. ceranae ravaging our hives resulted in unnerved beekeepers boosting the sales of fumagillin to the point that supplies ran short.
I had never previously worried about nosema, but I pulled out a microscope and found out that N. ceranae was indeed widespread in my operation. I ran trials, and found out that the danged parasite could flourish despite being drowned in fumagillin (Oliver 2008a), but more surprisingly, that colonies here at Comedy of Errors Apiaries thrived despite exhibiting spore counts in the millions. To try to reconcile the differences between the very different outcomes of N. ceranae infection in my operation with those reported for Spain, I began an ongoing correspondence with Dr. Higes, which continues to this day.
To be frank, some other Spanish researchers dispute Higes' conclusions (debate leads to better science), so I have often questioned and challenged him on details of methodology and interpretation, which he and his team of collaborators have generally clarified with additional research. In this series of articles I will be citing a number of the Higes team's papers, since they have clearly led the pack in N. ceranae research, meticulously investigating nearly every aspect of this pathogen's effects upon bees.
I've previously written at length about N. ceranae in my "Nosema Twins" series (all available at ScientificBeekeeping.com), but feel that there has been so much recent research completed that it would benefit the reader for me to write a digest of our current state of knowledge. I've scoured the literature for every relevant research paper (including a number still in press), and have discussed as well current findings with many of the world's nosema researchers. I wish that at this time I could say that I have the answers to all your questions about Nosema ceranae, but unfortunately, in many aspects this parasite still remains an enigma.
Worldwide Status And Distribution
Nosema ceranae has now spread into the European honey bee populations of most areas of the world, roughly concurrent with the spread of varroa (and its altering of virus dynamics), which greatly confuses analysis of the effect of these two novel parasites upon bee health. It is difficult to tell in which countries N. ceranae has already reached equilibrium, and in which it is still invading.
Since the first invasive wave of a novel parasite into naive hosts is generally that most damaging, it would be helpful to know when ceranae actually arrived in various countries. For example, we know from analysis of archived bee samples that N. ceranae has been present on the East Coast for at least two decades (Chen 2008). Unfortunately, any initial effects of its invasion may have been masked by our focus upon the massive impact of the arrival of varroa at about the same time.
Since no one was looking for N. ceranae in the U.S. until 2007, we obviously didn't start studying it until long after it was well established and likely homogenized throughout the bee population via migratory beekeeping practices. And it is also likely that by the time we started studying the impact of N. ceranae upon the health of colonies, natural selection may have already weeded out the bees least tolerant of the emergent pathogen.
In Europe, however, N. ceranae only recently invaded bee populations already suffering from varroa and viruses, miticide failure and comb contamination, extreme weather events, plus changes in agricultural practices and pesticide use--the combination of which likely factor into colony losses in that region.
In a fresh study (Botias 2011), the Higes team analyzed archived Spanish honey samples (frozen) and adult bee samples (in alcohol) dating back to 1998. They found that N. ceranae first appeared beginning in 2000 and increased in prevalence through 2009 (the latest samples analyzed), concurrent with a decrease in the prevalence of N. apis. It is noteworthy that Spain concurrently suffered from devastating drought during much of that period, which led to serious colony stress.
N. ceranae is still in the process of extending its range worldwide, and appears to be most successful in warmer climates. It is of interest that in varroa-free Australia, its invasion does not appear to be causing significant colony losses. Interestingly, although it is well-established in Canada, it is not yet common in some northern European countries, but this may be due to restrictions upon bee imports (Fries 2010).
N. ceranae is widely distributed throughout the U.S., but surprisingly, there were great differences in the percent of colonies infected in a recent state-by-state survey (Fig. 1).
Figure 1. Prevalence (percent of samples infected) of N. ceranae in various states as determined by PCR analysis (more sensitive than spore counts) of aggregate samples collected from 8 randomly selected colonies per apiary, 4 apiaries per state. Note that in some states over 70% of samples were infected! From Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
Ceranae Vs. Apis
In a widely cited paper by Martin-Hernandez (2007), her arresting graph of nosema positive samples over time clearly shows a definite shift over the period from 1999 to 2005--there initially were only spikes in spring and fall (ostensibly from apis), transitioning to nearly 100% of samples being positive every month of the year (due to ceranae). Of note is that her data has an inherent bias, in that the samples were voluntarily sent to the lab by beekeepers for diagnosis of problems, suggesting that the data may reflect the change in nosema loads in sick hives. Also of note, is that despite this graph being widely cited, it is often misunderstood--it did not plot spore levels, but rather only the yes/no detection of nosema spores.
What the graph did strongly indicate was that N. ceranae rapidly and thoroughly invaded Spain over a period of only a few years! This initial finding has now been confirmed by Botias (2011). Likely, a similar phenomenon occurred in the U.S., since Chen (2008) found N. ceranae to already be widespread in archived U.S. bee samples dating back to 1995.
The general trend appears to be that N. ceranae now predominates in warmer countries, whereas N. apis is better adapted to colder areas. It has been often stated that N. ceranae has displaced N. apis, but more careful analysis suggests that that may not actually be the case!
When Dr. Robb Cramer asked me in 2007 to send him infected bees so that he could culture pure N. ceranae, he found that the samples often contained some N. apis as a "contaminant." In Dr. Diana Cox-Foster's (2007) analysis of CCD colonies, they also found both species of nosema. Later studies by Bourgeois (2010) and Runckel (2011) of commercial operations in the U.S. also found N. apis, but in far fewer hives than its cousin, only in spring and/or fall, and notably, at much lower spore levels than N. ceranae.
The differences between the detectability of the two nosema species (N. apis typically produces much lower spore counts and is generally only seen in spring and fall) may lead "to an increased chance of detecting N. ceranae over N. apis, which could have biased the impression that N. apis has been displaced" (Higes 2010).
So, has ceranae actually displaced apis, or have we merely been overlooking its cousin? In order to answer that question, Dr. Raquel Martin-Hernandez (2011) carefully analyzed over 2000 bee samples from all across Spain. She found ceranae and apis coexisting throughout country, with ceranae clearly predominant (in roughly 40% of hives), apis hanging in there (in up to 15%), and occasional mixed infections (below 7%). She also found that infection by ceranae was favored in hotter areas of the country, whereas apis succeeded better where winters are colder.
I'm seeing similar indications from other countries (e.g., Gisder 2010), which are appearing to confirm that apis is the more cold-adapted species. As far as seasonality, Martin-Hernandez found apis only in the spring and fall, whereas ceranae could be found all year, and notably, once ceranae infects a colony, it almost always persists (detectable with PCR, even if not obvious via spore counts).
Practical note: these studies indicate that N. ceranae remains present as an infection in a colony throughout the year, even if it is not detectable by microscopy. But we don't know whether these inapparent infections affect colony health.
I found one last study to be of special interest: Dr. Judy Chen (2009) looked at nosema invasion from the other direction--in a turn of the tables, N. apis appears to have been introduced from the Western honey bee (Apis mellifera) into the Eastern honey bee (Apis cerana) in Asia, and is now an emergent parasite in that species, which had historically been infected only by N. ceranae! She analyzed bee samples from China, Taiwan, and Japan. Her findings:
"N. apis was detected in 31% of examined bees and N. ceranae was detected in 71% of examined bees and that the copy number of N. ceranae was 100-fold higher than that of N. apis in co-infected bees, showing that N. ceranae is the more abundant of two Nosema species in the Eastern honey bees."
This study suggests that N. apis can not only hold its own against N. ceranae, but can actually invade into ceranae's turf! Interestingly, in the Eastern honey bee, despite its long coevolution with N. ceranae, ceranae still produces higher spore counts than its invading cousin.
Coinfection
This brings up the question of what happens when bees are infected simultaneously by both species of nosema? Dr. Zachary Huang (pers comm) found that in both cage trials and field observations that longevity was substantially shorter for coinfected bees as opposed to those infected by either species of nosema alone (unpublished data).
Note that in Cox-Foster's (2007) CCD study that they found "a trend for increased CCD risk in samples positive for N. apis" (100% of CCD colonies tested positive for ceranae and 90% for apis, but remember that apis is easy to miss when samples consist of house bees). As Jim Fischer noted in a post to Bee-L, "What was striking was that every hive showing CCD symptoms tested positive for BOTH Nosema apis and Nosema ceranae, and this correlation was better than the correlation between CCD and IAPV that was the focus of the paper."
These findings leave me very curious about the impact of coinfection by two nosema species upon colony health!
Seasonality
Spore counts of N. ceranae generally reach a peak in May, then drop spontaneously during summer, and may spike sporadically in fall and winter. But there is more to the picture than this. Dr. Ingemar Fries (2010), who has studied nosema for decades, explains thusly:
"The typical pattern for N. apis infections in temperate climates is low prevalence or hardly detectable levels during the summer with a small peak in the fall. During the winter there is a slight increased prevalence with a large peak in the spring before the winter bees are replaced by young bees... The pattern is similar both in the southern and northern hemisphere... Unfortunately, very few data exist for N. apis on the seasonal prevalence from tropical or subtropical conditions. The only published year round sampling under conditions where bees could fly all year round, revealed detectable levels of N. apis with no seasonal pattern of prevalence."
Along that line, Dr. Denis Anderson in Australia (pers comm) tells me that, "there are also many unseasonal occurrences of N. apis -- I get many samples sent in in the mid summer here that are loaded with N. apis." This could well be happening in the U.S., where, as far as I can tell, there have been few studies on N. apis in warmer areas, other than the fact that it was commonly found in package bees produced in the southern states.
Practical application: we need to learn more about the prevalence and seasonality of N. apis in the warmer parts of our country!
I've now seen data and presentations on N. ceranae seasonal prevalence from researchers from all over the world. Since a picture is worth a thousand words, I've summarized them in a crude graph below (Fig. 2).
Figure 2. A generic graph of typical N. ceranae spore counts over the course of the year in my operation. Important note: Counts of house bees would follow the same trend, but at much lower levels. The late-season spikes are often sporadic flare ups that spontaneously "go away."
Practical application: It is not unusual to see high nosema spore counts in April and May. Counts will typically drop in summer whether you treat or not. I'll cover treatments in a subsequent article.
But new technology is showing something surprising about nosema sampling--that spore counts do not necessarily reflect degree of actual nosema infection (Meana 2010)! Look at the following graph (Fig. 3), from a recent nationwide study of pathogens in U.S. bees--instead of measuring spore counts, the blue bars indicate the percentage of colonies infected by N. ceranae as determined by DNA analysis (PCR).
Figure 3. The blue bars indicate the percentage prevalence of N. ceranae in sampled colonies (e.g., 0.7 = present in 70% of hives). Note that even though spore counts suggest that N. ceranae disappears for much of the year (previous graph), a substantial proportion of colonies actually remain infected to some degree by the parasite. Also note how closely the coinfection with another intestinal parasite (the presumably opportunistic trypanosomes) tracks nosema infection. No one is sure whether there is a causal relationship, or whether the simple explanation is that both parasites flourish in stressed bees. Graph from Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
As opposed to the above graph, Runckel (2011) also measured the amount of nosema DNA in samples, which presumably correlates with the intensity of the infection. They found high levels of N. ceranae transcripts in midsummer, at a time when spore counts are generally quite low (Fig. 5)! Their data indicated that N. apis was only present in spring and fall (which does correspond to spore counts). Go figure!
So what's up with high levels of N. ceranae DNA transcripts without correspondingly high spore counts? No one to my knowledge has answered that important question. What we do know is that N. ceranae can exist in the vegetative stage for a while before it produces spores (Martin-Hernandez 2009). But we're not clear on to what extent N. ceranae produces "autoinfective spores," as opposed to the "environmental" spores that are discharged into the gut contents (Cali 1999), and whether such autoinfective spores show up under microscopy. What is clear, however, is that N. ceranae appears to be able to reproduce within a bee without producing spores that are observable by microscopy.
Practical note: although N. ceranae spore counts may disappear in summer, DNA analysis indicates that the bees may still be infected. This is something of a mystery, as the bee population turns over rapidly during the summer, suggesting that N. ceranae is somehow infecting new bees without spores being evident!
So the next question is, is an infection by N. ceranae more pathogenic than one by N. apis? Although some initial cage trials indicated extreme virulence for the new nosema, trials in which bees were allowed to feed upon natural pollen generally found that both species affect bee longevity about the same (Forsgren 2010, Porrini 2011, Huang pers comm) despite the fact that spore levels get much higher with N. ceranae.
Take home: We clearly still have lots to learn about N. ceranae! It does not appear to cause rapid death of well-fed bees. The inapparent summer infections are puzzling.
So what's the cause of the seasonality of nosema spore counts? With N. apis it is presumed to be due to the requisites of transmission via dysentery by infected bees in the hive during the winter and colony nutritional stress, and limited by its sensitivity to high temperature. Martin-Hernandez (2009, 2010) demonstrated that N. apis can only grow in a narrow range of temperature (about 33degC). N. ceranae, on the other hand, grows readily over a range from 25degC to 37degC. However, N. ceranae spores are surprisingly susceptible to chilling (Fries 2010), which may limit their infectivity at lower temperatures.
Studies from a number of countries coinfected with both of the nosema cousins suggest that N. apis will continue to be the historical problem during winter and spring, with typical fall and spring spikes, whereas ceranae will be more prevalent in warmer climes, present throughout much of the year, spiking in late spring (perhaps tracking pollen flows), and then again sporadically in fall through winter.
Take home: if Nosema apis was a problem in your area prior to the invasion of N. ceranae, it may still contribute to colony health issues during the fall and spring!
Sample Interpretation
It would sure be easier if there were a simple sampling protocol that everyone could follow, and if there were clear treatment (or worry) thresholds based upon nosema spore counts, as there are for varroa (Fig. 4), but alas, I'm sorry to say that there aren't.
Figure 4. Average varroa infestation rates from 2700 colonies in 13 states (many of which received mite treatments). Sampling for varroa infestation level is relatively straightforward and simple to interpret. Typical treatment thresholds are below 5 mites per 100 bees. Graph from Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
Unlike sampling for varroa, which are easily seen with the naked eye, sampling for nosema requires either a microscope or laboratory apparatus that can perform PCR. However, a number of researchers (Meana 2010, Bourgeouis 2010, Traver 2010) have demonstrated that spore counts alone do not give an accurate picture of the actual degree of infection. Unfortunately, as far as assessment methods available to Joe Beekeeper, spore counts will have to suffice as a surrogate measure of the actual degree of infection (Fig 5).
Figure 5. Average nosema spore counts from the same 2700 hives. Note the typical huge spike in spore counts (predominantly from N. ceranae) in spring, and then again lesser spikes in fall and winter. Important note: these spore counts were from samples of bees from brood frames--counts from entrance bees would likely be several times higher (compare to Figure 2). Graph from Rennich, K, et al (2011) 2010-2011 National Honey Bee Pests and Diseases Survey Report.
That said, let's return to sampling for a bit. If you want to find spores, then sample older bees, such as foragers at the entrance (Meana 2010)--spore counts will typically be about 10 times higher in older bees, since it takes a while for the infection to build up in a bee (Smart 2011). He found that in infected colonies with a background spore count of 0.5-1M in bees from under the inner cover, almost no bees younger than 12 days old contained spores (at least detectable by microscopy).
This is not at all surprising, since El-Shemy (1989) found the same to be true for N. apis--spore counts were an order of magnitude higher in bees from the entrance. Indeed, he suggested that it was best to sample exiting bees at the entrance, since returning bees have likely defecated. The magnitude of the spore counts from an infected colony generally increases in samples (in order from lowest to highest), of bees from the broodnest, outer areas of the cluster, entrance bees, exiting foragers, returning foragers.
Both El-Shemy and Higes (2008) found that the best indicator of degree of infection was to squash bees from an entrance sample one at a time in order to determine the percentage of bees infected. My own sampling of sick colonies supports this recommendation. But in reality, few of us have time to squash dozens of bees one at a time for each sample--so I won't even suggest that you go there!
The next best method may be to do a spore count for a pooled sample of 50 bees from the entrance (but don't forget that even one or two highly-infected bees can greatly skew the count). In practice, however, it is often danged difficult and time consuming to collect 50 entrance bees, even if you use a special vacuum (Oliver 2008b), especially in cool weather or from sick colonies with few foragers.
For this reason, many researchers simply take standardized samples of bees from under the cover, or from an outside comb. There is support for this, as Gajda (2009) found that although spore counts were much higher in entrance bees, the relative proportion of infected bees was similar in samples taken from an outside comb.
Practical application: If you want to find out whether N. ceranae is present to any significant extent in your operation, sample bees from the entrance. If you want to know if the infection is serious, sample house bees from under the cover.
If you are curious as to whether you have gotten old or young bees in your sample, here is an easy general observation that I've made: since only nurse bees normally eat pollen, they are the only ones that will have it in their guts (duh). But my point is, that this is really easy to use that pollen as an indicator of bee age if you use the ziplock bag method for processing samples (see my previous article, and Fig. 6).
Figure 6. How to tell if your sample contains young or old bees. (Left photo) when you crush samples of nurse bees in a ziplock bag, and then mush them in water, the fluid will typically turn opaque yellow (since the guts of nurse bees are full of pollen). (Right photo) on the other hand, the fluid from the guts of entrance bees will typically be a tan/gray color (since foragers and guards don't eat pollen).
What If You're Dealing With N. Apis?
Oh, that it were only so simple as dealing with only one nosema, but the previously cited studies suggest that many of us actually may still have N. apis popping up in fall and early spring. To make things even harder, spore counts of N. apis, on a per bee, or per pooled sample basis, are generally only a fraction (about 1/10th, as best I can tell from previous studies) of what we see with N. ceranae. But it also appears that an infection by N. apis at that low level can be as serious as an infection by N. ceranae at a much higher spore count!
Important note: Martin-Hernandez (2011) easily found N. ceranae in samples of either foragers or house bees, whereas she only found N. apis in foragers and drones. So if N. apis is your concern, then you should take entrance samples! N. apis infection may be serious at a much lower spore level!
Seasonality
The other consideration is that you must put any spore count into the context of time of year, the climate that your bees are in, the nutritional status of the colonies, and especially the load of other pathogens. I will discuss these points in the next article.
In cold climates, nosema management may have other considerations. Hedtke (2011) performed a detailed 6-year study of 220 hives in Germany, and (surprisingly) found that "No statistical relation between N. ceranae detection in autumn and the following spring could be demonstrated, meaning that colonies found to be infected in autumn did not necessarily still carry a detectable infection in spring, and colonies which developed a detectable infection over winter had not been detectably infected in autumn." So much for careful sampling!
Recommendations
Heck, I'd be crazy to stick my neck out and give any recommendations! So let's look at what sort of nosema levels are involved in crashing colonies. The CCD colonies analyzed by Cox-Foster (2007) had mean spore counts in the range of tens to hundreds of millions from broodnest samples! Is it really any surprise that those colonies collapsed? The house bees in Higes' (2008) winter-collapsing colonies hit 20M before they went down (field bees hit 50M), but those that collapsed in summer only hit 3M.
But note that in the U.S. survey graph above, that 2M was the average spore count across the U.S. in April and May of this year, yet I'm not hearing of massive colony collapses, despite very poor conditions in many states.
In my own California foothill operation (we get snow during the winter, and move to almonds in February), it is not unusual to see entrance spore counts in May in the millions or tens of millions, but they generally drop during summer, provided that colonies are not stressed by other factors. Entrance counts during summer and fall are typically in the zero to 5M range (25 spores per field of view if you follow the protocol in my previous article--I'll call these FOV counts (Oliver 2008c)). I have not looked at near as many samples of house bees, but counts are generally zero to a fraction of a million, even in colonies running at 10M at the entrance.
I am by no means suggesting that you follow my lead, but I simply no longer worry about high spore counts in spring, as they generally spontaneously drop later in the season, and I haven't experienced winter losses associated with N. ceranae (unless I've intentionally inoculated the hives with viruses). However, I do keep my mite levels down, and feed pollen supplement to maintain good nutrition if necessary. And I monitor nosema levels throughout the year so that I don't get blindsided!
I've never treated for nosema (except in experiments), yet have not experienced colony collapses since 2006. But I'm not saying that you have no reason for concern--I will be writing about a trial in which I did compare survival of treated vs. untreated colonies that had virus issues, and fumagillin appeared to help.
I'd be concerned if counts for house bees got above 5 per FOV at any time, although I know several large commercial beekeepers who routinely ignore such counts with no dire consequences so far. I just checked a number of samples of house bees today (late October), and they ran from zero to 2 spores per FOV, despite there often being counts of 100-200 per FOV of entrance samples this spring.
In some operations where N. ceranae apparently got out of hand, treatment and comb sterilization seemed to help. However, in other operations with sky-high spore counts in spring, lack of treatment did not result in any noticeable problems. Due to these huge discrepancies, it is confoundingly difficult to come up with recommendations. However, the more beekeepers who start tracking spore counts, the more we will learn about appropriate treatment decisions.
If you are in an area with a long, cold winter which keeps the bees confined, you may be dealing with Nosema apis, for which the economic threshold of 1M (5 per FOV) for house bees has been well established.
Practical application: since spore counts for N. apis generally only reach levels about 1/10th of those for N. ceranae, you'd be wise to ask your local university determine which nosema species you're dealing with, since it follows that the economic threshold for treatment for N. apis may be far less than that for N. ceranae.
I will continue this review of N. ceranae in the next issue, including treatments, and its relationship to colony mortality and honey production.
Acknowledgements
Thanks to you, my readers! It just occurred to me that I've recently passed the 5 year mark in writing for ABJ, and it's been one wild ride! If I had any idea what I was getting into, I would probably have chickened out. But your feedback and appreciation keep me going--my motivation is simply the gratification that I get from sharing what I've learned with other beekeepers. Your donations also allow me to perform the sort of quick and dirty research necessary to answer burning questions. I am constantly on the learning curve, and greatly appreciate hearing information that is relevant to better bee management--feel free to contact me (no beginners questions please) randy@randyoliver.com.
As always, Peter Loring Borst has helped me greatly with research. I thank Dr. Mariano Higes for his patience in discussing his research. Dr. Steve Pernal and Ingemar Fries have been gracious with their time. I also thank all the other nosema researchers who have patiently answered my questions.
References
Botias, C, et al (2011) The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade. Research in Veterinary Science (in press).
Bourgeois, AL (2010) Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee. Journal of Invertebrate Pathology 103: 53-58.
Cali, A and PM Takvorian (1999) Developmental morphology and life cycles of the microsporidia. P. 121. in Wittner, M and LM Weiss, eds. The Microsporidia and Microsporidiosis.,American Society for Microbiology.
Chen, Y.P., et al (2008). Nosema ceranae is a long-present and widespread microsporidian infection of the European honeybee (Apis mellifera) in the United States. J Invertebr Pathol 582 97: 186-188.
Chen, YP, et al (2009) Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees. Journal of Invertebrate Pathology 101 (2009) 204-209.
Cox-Foster, DL, et al. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318(5848): 283-287.
El-Shemy, A.A.M. and RS Pickard (1989) Nosema apis Zander infection levels in honeybees of known age. J. Apic. Res. 28 (2), 101-106.
Forsgren, E, and I Fries (2010) Comparative virulence of Nosema ceranae and Nosema apis in individual European honey bees. Veterinary Parasitology 170: 212-217.
Fries, I (2010) Nosema ceranae in European honey bees (Apis mellifera). Journal of Invertebrate Pathology 103: S73-S79. https://bienenkunde.uni-hohenheim.de/uploads/media/Nosema_ceranae_in_European_honey_bees__Fries.PDF
Gajda, A (2009) The size of bee sample for investigation of Nosema sp. infection level in honey bee colony. http://www.coloss.org/publications/Nosema-Workshop-Proceedings.pdf
Gisder S, et al. (2010) Five-year cohort study of Nosema spp. in Germany: does climate shape virulence and assertiveness of Nosema ceranae? Appl Environ Microbiol 76: 3032-3038.
Hedtke, K, et al (2011) Evidence for emerging parasites and pathogens influencing outbreaks of stress-related diseases like chalkbrood. Journal of Invertebrate Pathology 108:167-173.
Higes, M (2010) Nosema ceranae in Europe: an emergent type C nosemosis. Apidologie 14(3): 375 - 392.
Higes, M., et al (2005) El sindrome de despoblamiento de las colmenas en Espana. Consideraciones sobre su origen. Vida Apicola 133: 15-21.
Higes M, et al (2006) Nosema ceranae, a new microsporidian parasite in honeybees in Europe, Invertebr Pathol. 92(2):93-5.
Higes, M, et al (2007) Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia). J Invertebr Pathol. 94(3):211-7
Category: Nosema Summaries and Updates
Tags: N. ceranae, Nosema cereanae, Nosema Cousins, part 14, sick bees | part 14 Archives - Scientific Beekeeping | https://scientificbeekeeping.com/tag/part-14/ |
Sick Bees - Part 15: An Improved Method for Nosema Sampling
Author's Note
Samples from Within the Hive
Soundbite Science
Infection Rate
So How Did We Get on the Wrong Track?
How to Determine the Colony Infection Rate
So What if I Count the Number of Infected Bees out of 10?
An Assessment of Our Situation
One HUGE Assumption
Validation of the Method
Update
Sequential Sampling
A Neat Little Shortcut
Which Hives to Sample?
What Time of Year?
From Where Should You Take Samples?
Practical Application: Completely Subject to Revision
Adds
Follow Up on the Quick Squash Method
Infection Prevalence
From Where Should We Take Samples?
Next Month
Acknowledgements
References
Sick Bees 15:
An Improved Method For Nosema Sampling
Randy Oliver
ScientificBeekeeping.com
In the previous articles in this series I showed how to use a microscope to view nosema spores and discussed from what part of the hive to take bee samples, and how researchers are interpreting spore counts. But spore counts don't tell us what we really need to know!
Author's Note
I'm hoping that the reader is benefitting from my digestion and summarization of current (and past) nosema research. There is a tremendous amount of information out there (much of it conflicting or confusing), but I'm trying my best to condense and simplify it into terms meaningful to Joe Beekeeper. The frustrating thing, though, is that it is clearly apparent that we still have a great deal more to learn about these parasites before anyone can make definitive recommendations as far as best management practices!
So my apologies in advance for the length and depth of this article. But I'm going someplace different, and feel that it would benefit the reader to follow the history and thought process that led to the conclusions that I reach at the end.
Samples From Within The Hive
Most researchers take bee samples for nosema testing from under the lid, or from an outside comb, since such samples are generally easier to take and presumably more consistent as far as bee age structure. The expectation also is that such a sample would be most representative of the colony infection as a whole, as opposed to samples from the entrance, in which spore counts are often sky high, or samples of nurse bees, in which counts are generally minimal. The assumption, of course, is that a "peripheral" sample would contain mostly mid-aged and older bees.
In early November, I had the pleasure of being visited by Dr. Dewey Caron, so I used the opportunity to put the above hypothesis to the test. We had experienced rainy weather and cold nights the two previous days, so the bees had not foraged nor broken cluster during that time. We went out to the bee yard and took samples of bees from the outermost portion of the cluster, from honey frames in the upper hive body (the bees were in fairly tight cluster, so I doubt that there had been much bee movement in the past two days).
As I illustrated in Figure 6 of my previous article, it is a normally a relatively simple matter to determine the "age" of the bees in a sample by seeing how much pollen is in their guts--it is generally assumed that only young bees (nurses) consume pollen. So we froze the bees and then spread them on a grid and crushed them to squash out their gut contents. As you can see in Figure 1, to our great surprise, the vast majority of bees in the samples from the three hives that we tested had guts full of pollen!
Figure 1. A sample of crushed bees from the periphery of the cluster (on an upper honey comb) after two days of confinement by cold weather in early November. Note that every single bee's gut was full of pollen (the orange stains), indicating that they were likely nurse bees, rather than older bees, and thus would be less likely to be infected by nosema.
We confirmed by microscopy that the orange coloration of the gut contents was indeed due to pollen grains. I have done similar squashes during summer, and again found substantial proportions of bees throughout the hive to have pollen in their guts, as opposed to bees in entrance samples, which rarely contain appreciable pollen. In the sample illustrated above, the extremely high proportion (100%) of bees containing pollen could possibly be due to the population turnover in November, when the forager bees fly off to die, leaving only young bees in the hive to winter (Mattila and Otis 2007). I have not yet confirmed this.
Practical application: So I'm not clear at this point whether these pollen-filled bees are chronologically old bees or not!
Another interesting finding that we made was that in a 50-bee subsample of one of the above samples, the spore count (which appeared to be N. ceranae), was about 100 spores in a field of view, indicating an infection level of about 20M. This high count in ostensibly young bees caught my attention, so I squashed individual bees one at a time--in the first six, only one of them contained an appreciable number of spores. Apparently, in the 50-bee sample, a few highly-infected bees skewed the spore count to that alarming level (Fig. 2). My point is that you should not allow any individual spore count to scare you!
Figure 2. One highly-infected bee can really skew a spore count! This photo, taken at 400x, shows thousands of nosema spores packed into the Malpighian tubules (bee "kidneys"), which are running at an angle crossways. The treelike structure is a tracheal (breathing) tube. A bee infected to this degree could contain 500 million nosema spores!
Practical application: I would normally have been alarmed by a mean spore count of 20M in a sample of fifty young bees, but upon closer inspection, most of the bees in the subsample of 6 bees were not infected to any degree.
I have written extensively about varroa. Varroa is easy to monitor, and if one makes an effort to understand its well-documented biology and population dynamics, then it is a relatively straightforward matter to make wise and effective management decisions for controlling its degree of damage to bee colonies. Unfortunately, we are nowhere near that state of confidence with N. ceranae. Worldwide data from actual field studies are so conflicting that no one can really make meaningful recommendations as to what level of infection, based upon simple spore counts, is economically tolerable. Add to that, there are potential down sides to treatment--first, fumagillin's expensive, it may have negative side effects upon bee health, may contaminate honey (and is not approved in many countries), and many beekeepers simply are adverse to adding one more danged treatment to their hives (Figure 3). I will discuss the above concerns, as well as the potential consequences (or lack thereof) of untreated nosema infection in later articles.
Practical application: I seriously question whether spore counts can be translated into meaningful treatment thresholds!
Figure 3. Let me warn you, that if you actually start sampling for nosema, it will give you much more to worry about! When we find high spore counts here at WishWeKnewWhatWeWereDoing Apiaries, my sons (Eric, on left, and Ian) and I worry about filling our almond pollination contracts.
Until recently, I pretty much blew off Nosema ceranae as not being much of an issue in my operation, despite finding spore counts in the millions or tens of millions, especially in spring. Our colonies have generally fared well, and I haven't noticed any strong correlation between colony strength and nosema counts. However, I'm a bit uneasy since spore counts have seemed to climb each year, and are higher this season than ever.
What most troubles me, though, is recent evidence that nosema is more of a problem in colony health and mortality than I have previously suspected--I will be covering this in subsequent articles in this series (I often am forced to choose which subject to cover first). So I'm in the same boat with the rest of you who are wondering how best to diagnose the degree of nosema infection in your operations, and whether treatment would be worthwhile.
Soundbite Science
We currently live in an age of information overload, largely due to the internet, with snippets of knowledge thrown at us faster then we can put them all together. This is a mixed blessing with scientific research, as sometimes quality is sacrificed in the race to be first to report some finding. Due to competition for limited funding, we are seeing a lot of "soundbite science" being published by grad students and post docs fighting to make a name for themselves, or faculty needing to "publish or perish."
But back in the day, the government subsidized the kind of painstaking, grinding, and detailed agricultural research seldom seen today. To see an example, download Dr. G.F. White's 1919 exhaustive 9-year study on nosema (Google Books "bulletin 780 nosema") undertaken after he discovered its presence in the U.S. --these guys with a government mandate were thorough! In this age of budget cutting and taxpayer support for giant agribusiness (less than 2% of USDA spending goes toward research these days), there is a strong case to be made for we beekeepers to encourage government funding of bee research.
Dr. White concluded:
"As a rule, colonies which in the spring of the year show less than 10 per cent of Nosema-infected bees gain in strength and the losses are not detected. This is often true also in cases where the infection is somewhat greater than 10 per cent. When the number of infected bees approaches 50 per cent the colonies become noticeably weakened and in many instances death takes place. When more than 50 per cent are infected they become weakened and usually die as a result of the infection. Generally speaking, therefore, it may be said that when a colony contains less than 10 per cent of Nosema-infected bees the prognosis is excellent; that when it contains more than 10 and less than 50 per cent the prognosis is unfavorable; and that when the number of Nosema-infected bees present approaches 100 per cent the prognosis is especially grave."
Pay attention: This prognosis is remarkably similar to that of Higes (2008)--that the tip point for colony health appears to occur when more than about 40% of the bees in the hive become infected (a 40% infection rate). The practical application is that spore counts may not be the best way to assess the impact of nosema infection upon colony health--it may be more important to determine the relative proportion of infected bees to healthy bees.
I will continue to return to Dr. White's findings in this series, as well as those by Dr. Mariano Higes and his collaborators in Spain, in which, by the way, there are about the same number of hives as in the entire contiguous U.S., in approximately 1/15th of the land area! What strikes me is the similarity in their conclusions, nearly a century apart, when they discovered, and then thoroughly investigated, nosema epidemiology and pathology in their respective countries!
Infection Rate
The proportion of infected bees in a hive is called the infection rate, and expressed as a percentage--if a quarter of the bees are infected, that would be a 25% infection rate. Remember, a colony of bees is a superorganism, with each individual bee somewhat akin to a single cell of your own body (of which millions die every day). A colony can easily handle the loss of a certain percentage of sick bees every day, especially if those bees are aged, and nosema infection is generally worse in the oldest bees (since aging allows more cycles of parasite reproduction within the bee).
Dr. White suggested that an average of about 10-15% infected bees in a hive is "normal." The rate in sick hives could go up to 100%! He found that the infection rate would often go to 70% in experimentally-inoculated hives.
So let's digest this. If only, say, 5 percent of the bees in a hive were actually infected (and only seriously infected during their last days of foraging life), the overall nosema infection would have little impact upon the colony, as they would be quickly replaced by the 1500-2000 new bees emerging each day. However, if 50 percent of the bees were infected, then that is entirely another matter! During the spring and summer, their shortened lifespan could seriously affect the population dynamics of the hive, reining back its normal population growth and ability to forage. And during the winter, when bees must live to a ripe old age in order for the cluster to survive until spring, a high nosema infection rate could lead to colony collapse. I will return to the details of this subject in an upcoming article.
Practical application: Nosema can be a serious problem during either winter or spring, should a high proportion of bees in the hive become infected. The infection rate is a more accurate measure of the seriousness of the infection than is a mean spore count, since a high spore count may merely reflect that one or a few highly-infected bees happened to be in the sample.
So why have we been focusing on spore counts, rather than infection rate? I just got off the phone with a large commercial queen producer who has closely tracked N. ceranae levels (and spent a large amount of money on treatments). He has nearly given up on looking at spore counts, since they simply did not appear to correlate to any significant degree with colony health and production. Ditto for my operation, and for much of the worldwide research. I feel that it is time for us to move beyond spore counts!
I'm not the only one who feels this way. Dr. Higes team's recently entreated: "There is an urgent need ... to decide on the reliability of standard methods to establish the levels of infection, a measure that will be necessary to standardize procedures to accurately, reliably and meaningfully quantify the degree of Nosema infection in honey bees" (Meana 2010). It's time for a paradigm shift of moving away from sample means, and to go back to looking at the actual percentage of infected individual bees.
So How Did We Get On The Wrong Track?
Good question! I've always wondered how the 10-bee sample size figure ever got engraved in stone. It appears that it evolved from a statement by Dr. White himself, who wrote that "Ten bees from a colony constitute a satisfactory sample as a rule."
So 10 bees became the typical sample size from the early 1900's until we found out that we had N. ceranae. At that point, I was misled by "discovery sampling" statistics (Oliver 2008), since I thought that I needed to "discover" whether I had nosema in my operation, and thus recommended taking samples of at least 50 bees. This number (or even 100) is commonly used by researchers these days, since it also helps to minimize the influence of any single highly-infected bee upon the mean spore count.
Unfortunately, many of us became seduced by the attractiveness of thinking that the number of spores counted in a hemacytometer actually reflected the seriousness of an N. ceranae infection in a hive. Fifty-bee samples are good for discovery, but in truth, once you've discovered that you have N. ceranae in your operation, they actually can be misleading. Here's the funny thing: Dr. White's 10-bee samples are actually a better assessment of the seriousness of a nosema infection! But when he recommended 10-bee samples, he wasn't talking about counting spores! What he actually recommended was:
"When a diagnosis of the disease is being made in practical apiculture, therefore, considerable caution should be observed. A colony showing only a small percentage of Nosema-infected bees and not other evidence of the disease is practically healthy. In reporting the presence of infection it would seem well to indicate in some way the amount of infection present. The percentage of infected bees among those examined might be given."
This is a major point! Nosema infection at the colony level is not about spore counts--rather, it is about the percentage of the bees that are infected!
So why the heck did most everyone go from determining the infection rate to counting spores? Well, several researchers found that, at least with Nosema apis, spore counts of a 10-bee sample roughly correlated with infection rate. Then some Canadian scientists (Fingler 1982) found that a 25-bee sample was an even more "reliable method of assessing the degree to which colonies are infected by nosema." But again, those researchers clearly understood that spore counts were merely crude proxies for the actual rate of infection. I doubt that beekeepers (or even many subsequent researchers) ever fully grasped that message.
So is a 10-bee sample enough? Look at it this way: since a single nosema-infected bee typically contains more than 10M spores (Forsgren 2010; Smart 2011), then having even one single infected bee in a 10-bee sample (indicating > than a 10% infection rate) would put the mean spore count above 1M--the typical rule of thumb for treatment. So what's the chance of hitting at least one infected bee in a 10-bee sample.
In order to answer this question, we need to use probability theory, which was ironically, initially developed to help gamblers make better decisions in games of chance. As an aside, doesn't it seem funny that the ABF national convention is going to be in Las Vegas? I mean, commercial beekeepers already live their lives gambling their life savings on the weather, the price of honey, varroa treatments, and honey flows, and are going to be in Las Vegas just before the big roulette wheel stops spinning and tells them whether they'll hit the jackpot in the almonds the next month!
But I digress. Probability theory can be used to predict, for example, the chance of being dealt two aces in a hand of five cards (4/52 x 3/51 = 1/221, or less than half of one percent probability). Bee samples can be looked in a similar manner, since when you are squashing bee guts, nosema infections generally show as either positive (tons of spores) or negative (zero to a very few spores)--sort of a sick/not sick litmus test. So I did some homework with probability tables, and was able to answer my question about hitting 1 infected bee out of 10 (Figure 4).
Figure 4. In this graph the bottom scale is the actual infection rate of the colony. The blue line plots the chance of hitting at least one infected bee in a sample of 10 bees. The red line indicates "negatives," in which you would not find a single infected bee. You can see that it's almost impossible to miss getting at least one infected bee in a 10-bee sample if the colony infection rate is over 30%.
So the 1M spore rule of thumb is very conservative, meaning that you certainly wouldn't miss a nosema infection, but also means that you'd often wind up feeding fumagillin to apiaries that in actuality were dealing just fine with relatively "safe" infection rates. In the case of N. ceranae, in which individual bee spore counts may exceed 100M, having even a single infected bee would result in a mean spore count of 10M, which might scare the pants off you, despite the substantial likelihood that the colony was only infected at a minimal rate!
A recent study by Traver and Fell (2011a) supports the above interpretation--they found that colonies that tested low for nosema DNA exhibited zero spores in 10-bee samples about a third of the time, whereas samples from colonies with "high-level" infections seldom were free of spores. So it appears to me that the good old 10-bee spore count works fairly well as a crude but conservative proxy for the actual infection rate, with spore counts stepping up sharply with each additional infected bee in the sample. However, it should not be interpreted as any sort of linear measure of the degree of infection. It worked, but it likely led to too many unnecessary treatments.
The problem with spore counts: spore counts from a pooled homogenate of many bees are more or less a measure of the reproductive success of nosema in a relatively few bees. The infection rate (percentage of bees actually infected) is a much better measure of the actual impact of nosema upon colony health.
How To Determine The Colony Infection Rate
OK, I hope that I've convinced you now that it's time to move away from counting spores--but that certainly doesn't mean that you should throw away that shiny new microscope that I earlier convinced you to buy!
You may have wondered why, when I was squashing bees in my kitchen with Dr. Caron, that I stopped after crushing only six bees. Well, in truth, squashing individual bees is time consuming, and my gut feeling was that I would have hit more than one infected bee in the sample should the actual infection rate have been high.
Of course, my readers should know that I'm not about publishing "gut feelings." So, being the curious sort of guy, I bit the bullet and plowed into an investigation to see whether I could come up with some sort of shortcut for determining a colony's infection rate without having to individually squash a whole bunch of bees. I spent some serious time working out the math (much to my long-suffering wife's dismay, such as when she groggily walked into the kitchen first thing in the morning, and was immediately barraged by me excitedly showing her the results of some probability calculations that I've been working on since before dawn).
My personal issues aside, what I found was that the problem with extrapolating from samples is that you want to avoid false negatives (missing a serious infection; easy to do with samples of only a few bees), while at the same time not misdiagnosing false positives (erroneously concluding that a healthy hive is seriously infected--which is a problem with the mean (average) spore count from of a pooled bee sample).
Scientists just love hard, accurate figures out to the third decimal place, with 99% confidence levels. But in reality, there is rarely that kind of certainty when you're dealing with any data derived from bee samples! And there's a lot of elbow room when making management decisions. So first, let's perform a reality check. Suppose that you have a colony that is infected at the 40% rate, and that the infected bees are evenly distributed in the hive. And then suppose that you take a sample of 100 bees from that colony.
You'd expect that the sample would contain 40 infected bees (40 per 100 = 40%). And the average sample would indeed contain 40 bees. But no single sample is an average! Any single sample has only an 8% chance of containing exactly 40 infected bees!
That's fine, you say--all that I really care about is whether that sample contains at least 40 bees. The chance of that happening with a single sample of 100 bees is still only 54%! You would still get 46% false negatives. A 46% chance at losing a bet isn't bad if you're betting five bucks in Las Vegas. But it's pretty poor odds if you're risking your bee operation on it!
Motivational message: For the arithmetically-challenged among you whose eyes are starting to glaze over because I'm using three-syllable words and talking about math, please hang in there!
So What If I Count The Number Of Infected Bees Out Of 10?
You'd sure think that this would make sense! After all, Dr. White recommended this method. But surprisingly, it's not that accurate. Let's look at the probabilities. Suppose that a colony is actually infected at the 40% rate, and that you took a perfectly representative sample of 10 bees. You'd still have only a 25% chance of finding exactly 4 infected bees (but a 67% chance of hitting between 3 and 5 bees). So counting the number of infected bees in a 10-bee sample will give you only a very rough assessment of the actual infection rate.
But here's a big surprise-counter intuitively, as the sample size goes down, your chances of missing that infection actually go down too! For that same 40% infected colony, here are the probabilities of underestimating the infection rate (Table 1):
Sample Size Probability of getting fewer than 40% infected bees in the sample
100 46% (46 times out of 100)
10 38%
5 34%
Table 1. Probabilities of underestimating the infection rate of a colony in which 40 out of 100 bees were actually infected, by sample size (number of bees in the sample), assuming a perfectly representative sample.
I'm hoping that you're catching my drift here--that we may be able to streamline the process of estimating the degree to which a colony is infected, by utilizing 5-bee samples.
An Assessment Of Our Situation
So let's review where we stand with regard to nosema sampling methods and interpretation:
We want to avoid dangerous false negatives, since they might lead you to not treat a truly sick apiary.
However, you (and your bees) could live with false positives, since the worst that you'd do is to give unnecessary treatments.
But you're still a penny pinching beekeeper who doesn't want to waste money (or you don't want to use treatments for other reasons).
Sending a sample of 10 bees to the lab for a spore count has an unacceptably high rate of false positives--at least two-thirds of the time.
Spore counts of even 25-bee samples of either house or forager bees are still unreliable predictors of colony health (Meana 2010).
And even individually squashing 10 bees one at a time will underestimate a serious 40% infection rate over a third of the time!
So what to do? I'm not telling you all this merely to frustrate you--I and my sons live off the income from our bees, so I've got a vested interest in finding a way out of this quandary! Researchers worldwide are coming to the conclusion that simple spore counts generally have little correlation with observed colony health status. What you really need to know is one of two things--are your bees in the "safe" zone (under 20% infected) or in the danger zone (over 40% infected). And what you don't want to do is to spend all day squashing bees one at a time and viewing their guts under a scope. Are we all agreed on the above?
So I got out the probability tables, a pocket calculator, found a handy online binomial distribution calculator (http://stattrek.com/Tables/Binomial.aspx), and started playing with the numbers. I found that for our purposes of differentiating between a benign nosema prevalence and serious infection,that the sweet spot for sample sizes lies in the range of 5-10 bees (sort of a Goldilocks "not too many, but not too few"). See for yourself (Fig. 5):
Figure 5. This graph is for 5-bee samples. Compare identical colored markers between the left column and the right column--the greater the vertical spread, the better the discrimination between infection rates. Note that at "benign" infection rates (green and blue dots) you'd nearly always hit either zero or 1 infected bee, but rarely 2 or more. At dangerous infection rates (red markers) the reverse holds true--you'd rarely hit zero infected bees (not shown), and seldom even 1, but nearly always at least 2 positive bees if the colony infection rate exceeded 60%. I will post this article to ScientificBeekeeping.com for handy reference.
One HUGE Assumption
All of these probabilities are contingent upon your taking a representative sample that reflects the overall infection rate of the hive. Would this be the case in real life? Would 5-bee samples give consistent results? I didn't know, so l decided to put it to the test the day before I sent this article off to press!
Validation Of The Method
The "boys" and I were treating colonies with an oxalic acid dribble in November (bees were still flying most days), so I took samples of bees from the weakest hives in each yard, and later processed subsamples of 5 bees at a time. Here are the results (Table 2):
Colony number
Number of nosema-positive bees per 5-bee sample, and (below) per 10-bee sample (by subsequent pairs)
Overall infection rate of sampled bees
Notes
1
0/5, 0/5
0/10
0/10 =
0%
Appeared to be free of nosema.
2
0/5, 0/5
0/10
0/10 =
0%
Appeared to be free of nosema.
3
2/5, 1/5, 2/5, 1/5, 2/5, 4/5, 0/5
3/10, 3/10, 3/10, 3/10, 6/10, 4/10
12/35 =
34%
Only 1 zero in the 5-bee samples. Note the consistency of the 10-bee samples.
4
0/5, 0/5
0/10
0/10 =
0%
Appeared to be free of nosema.
5
3/5, 2/5, 1/5, 3/5, 1/5, 1/5
5/10, 3/10, 4/10, 4/10, 2/10
11/30 =
37%
No zeroes. Only the last pair of 1/5's would have missed the infection.
6
4/5, 0/5, 3/5, 5/5, 2/5
4/10, 3/10,8/10,7/10
14/25 =
56%
The 10-bee samples certainly picked up the infection! This colony had the most intensely infected bees, plus a serious amoeba infection.
7
0/5, 0/5
0/10
0/10 =
0%
Appeared to be free of nosema.
8
0/5, 1/5, 0/5, 0/5, 1/5
1/10, 1/10/ 0/10, 1/10
2/25 =
<1%
Very consistent results
9
3/5, 0/5, 3/5, 1/5, 0/5
3/10, 3/10, 4/10, 1/10
7/25 =
28%
2 bees were only slightly infected. One 10-bee sample underestimated.
10
2/5, 2/5, 3/5, 0/5, 2/5
4/10, 5/10, 3/10, 2/10
9/25 =
36%
2 bees were only slightly infected. The last 2/10 missed, but the 2/5 would have flagged the infection.
Table 2. Results of bee samples from 10 weak colonies in the fall. I sub sampled each sample, 5 bees at a time, with each bee being individually squashed (total of 205 bees), and rated each bee as to whether it was positive for nosema spores or not. I stopped counting after two groups of 5 if I hadn't yet detected any nosema. Out of 31 pairs of 5-bee samples (the lower figures in column 2), in only 2 cases out of 31 would I have underestimated the actual colony infection rate (by not hitting either 2 positive bees in 5, or 3 in 10). Note how consistently the paired 10-bee samples reflected the overall infection rate!
Practical application: I found the above reality check instructive, to say the least! In fact, I could say that I learned more about the degree of nosema infection in my operation in three hours of bee squashing than I'd learned in the last four years of counting spores! I doubt that I will ever do another spore count.
I love this method! For one, I learned that nosema was only associated with half of my weakest hives, so I can now sleep a bit better. On the other hand, half of those weak hives did have high nosema levels, so I need to address this (spot treatment?). I'm now eager to go sample some strong colonies. What is also apparent is that the method worked remarkably well! It's not perfect, but it appears that I'd rarely miss an infection if I processed two samples of 5 bees for each tested hive. And the method readily picked out the really sick hive! Clearly, this is only a preliminary test of the procedure, and needs to be repeated with a lot more hives, but the apparent accuracy of the method is very encouraging to me.
The only remaining problem is that most beekeepers will choke at the thought of how much time it would take them to squash and microscopically view 10 bees out of each sampled hive. And that leads us to:
Sequential Sampling
Think of this Quick Squash method as similar to doing an alcohol wash of 300 bees. If I only see 1 mite, no worries for a while, as mite populations take about a month to double. If I see more than 6 mites, I treat. In between, I make a note to check back soon. It's a similar case for nosema sampling (although it may take less time for the infection rate to double).
I'm immensely grateful to Dr. Jose Villa of the Baton Rouge Bee Lab for bringing to my attention that I was reinventing the wheel--this sort of decision making process based upon small sample sizes already has a fancy name: it's called "sequential sampling," and was develped for quality control inspections during World War II. Furthermore, Dr. Villa dug into the library and forwarded me existing "Decision Tables" for tracheal mite sampling produced by Tomasko (1993) and Frazier (2000). They exactly fit the bill for what I was crudely trying to work out!
Sequential sampling is all about the tradeoff between tedium (the number of bees that you need to squash and view) and confidence (the error rate which you are willing to accept). And it appears that for our purposes I estimated the minimum number of bees to sample right on the nose!
So here's the gist (backed by some complex math) for the following parameters. Given that you want to decide whether about 10% or fewer of the bees in the population are infected (the "tolerable" level), or if the rate is above the 30-40% range ("intolerable"), and are willing to allow an error rate of 20% for overestimating ("false positives"), but only a 10% limit for underestimating a serious infection (I'm intentionally avoiding most of the associated mathematical jargon). The cutoffs are:
Practical application: it appears that in order to make a decision whether to treat or not, that a couple of 5-bee samples should be adequate, interpreted as follows:
0 positive bees out of 5, or no more than 1 positive out of 10 indicates < 10% infection
3 positive bees out of 5, or at least 4 positives out of 10 indicates > 30% infection
Any number of positive bees lying between these cutoffs (e.g., 2 bees out of 5, or 3 out of 10) suggest an infection level that lies in the gray zone, but I doubt that going beyond a 10-bee sample is worth the effort--I'd just move on to the next sample.
So I've got us down to 10-bee samples. But even so, I must advise you that nosema infection appears to exist in "pockets" of bees in the hive, so any single small sample is inadequate for making an apiary-level decision (Botias 2011). It's obvious that what is needed is a quick method for processing samples of 10 bees at a time!
Acknowledgements
I wish to thank my wife Stephanie for her patience, and helpful comments on my manuscripts. As always Peter Borst helped with the research for this article. Thanks to Dr. Jerry Bromenshenk for his helpful suggestions. And a big thanks to Drs. Mariano Higes, Aranzazu Meana, and Raquel Martin-Hernandez for their diligent work on nosema! For financial support toward this research, I've very appreciative of Joe Traynor, Heitkam's Honey Bees, Jester Bee Company, the Virginia State Beekeepers Assoc, and individual beekeepers Paul Limbach, Chris Moore, and Keith Jarret.
References
Botias, C, et al (2011) Critical aspects of the Nosema spp. diagnostic sampling in honey bee (Apis mellifera L.) colonies. Parasitology Research (in press).
Fingler BG, WT Nash, and TI Szabo (1982) A comparison of two techniques for the measurement of nosema disease in honey bee colonies wintered in Alberta, Canada. ABJ 122(5):369-371.
Forsgren, E, and I Fries (2010) Comparative virulence of Nosema ceranae and Nosema apis in individual European honey bees. Veterinary Parasitology 170: 212-217.
Frazier, MT, et al (2000) A sequential sampling scheme for detecting infestation levels of tracheal mites (Heterostigmata: Tarsonemidae) in honey bee (Hymenoptera: Apidae) colonies. Journal of Economic Entomology 93(3):551-558.
Higes, M, et al (2008) How natural infection by Nosema ceranae causes honeybee colony collapse. Environ Microbiol 10: 2659-2669.
Mattila HR, and GW Otis (2007) Dwindling pollen resources trigger the transition to broodless populations of long-lived honeybees each autumn. Ecol Entomol 32:496-505.
Meana, A, et al (2010) The reliability of spore counts to diagnose Nosema ceranae infections in honey bees. Journal of Apicultural Research and Bee World 49(2): 212-214.
Oliver, R (2008) The Nosema Twins Part 3: Sampling. ABJ 148(2): 149-154. https://scientificbeekeeping.com/the-nosema-twins-part-3-sampling/
Porrini, MP, et al (2011) Nosema ceranae development in Apis mellifera: influence of diet and infective inoculum. Journal of Apicultural Research 50(1): 35-41
Smart, MD and WS Sheppard (2011, in press) Nosema ceranae in age cohorts of the western honey bee (Apis mellifera). J. Invertebr. Pathol. doi:10.1016/j.jip.2011.09.009
Tomasko, M. Finley, J. Harkness, W. Rajotte, E. 1993. A sequential sampling scheme for detecting the presence of tracheal mite (Acarapis woodi) infestations in honey bee (Apis mellifera L.) colonies. Penn. State College of Agricultural Sciences, Agricultural Experiment Station Bulletin 871.
Traver, B., and RD Fell (2011a) Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia. J Invertebr Pathol 107 (1):43-49.
Traver, BE MR Williams, and RD Fell (2011b; in press) Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies. Journal of Invertebrate Pathology.
White, GF (1919) Nosema-Disease. USDA Bulletin No. 780.
Category: Nosema Summaries and Updates, Sampling
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