# Microbial ecology of the bee and hive



## Michael Bush (Aug 2, 2002)

*microflora*

Bacteria found in healthy bee gut and hive:

Bifidobacterium animalis
Bifidobacterium asteroides
Bifidobacterium coryneforme
Bifidobacterium cuniculi
Bifidobacterium globosum
Lactobacillus plantarum
Lactobacillus sp. (probably all but there are both general references and specific ones for Lactobacillus bifidus and Lactobacillus acidophilus)

If you want references simply try a search on any of these plus honeybee or bee. There are many.


Here are a few of them:


http://www.sciencedirect.com/scienc...serid=10&md5=6802f9ec81dd2483e31faf4c34d5758d

Oxytetracycline as a predisposing condition for chalkbrood in honeybee

"Antibiotics, particularly oxytetracycline, have been discussed as a possible predisposing condition in the appearance of chalkbrood in the honeybee (Apis mellifera L.). Nevertheless, the scientific data to support this belief have been insufficient. We have developed a method to study the effects of this antibiotic as a predisposing factor under different circumstances. We conclude that oxytetracycline does not increase the risk of chalkbrood in susceptible worker brood in the short or mid-term."

" ... use of antibiotics in the
honeybee can upset the balance of intestinal microflora, favoring the ..."


http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=15539925&dopt=Citation


http://web.uniud.it/eurbee/Proceedings/Diseases.pdf

"During a study aimed to characterize the intestinal microflora of honeybee larvae
and adults, we found that some lactic acid bacteria inhibit in vitro the growth of these
pathogens. These bacteria, belong to the genus Lactobacillus, are normal inhabitants of
the gut of honeybees and are GRAS (Generally Regarded As Safe).
Strains of this genus have been shown to have important metabolic and protective
functions in the gastrointestinal tract, interfering with enteric pathogens and
maintaining a healthy intestinal microflora."


http://www.blackwell-synergy.com/doi/abs/10.1111/j.1574-6968.1997.tb12678.x

"Emerging adult bees acquire intestinal microflora by food exchange with other bees in the colony and through consumption of pollen. Biochemical contributions of microorganisms to honey bees; the role of microorganisms in the conversion, enhancement, and preservation of pollen stored as bee bread in comb cells; and the production of antimycotic substances by molds and Bacillus spp. from honey bee colonies that are resistant to the fungal disease, chalkbrood, are discussed. An association of Bacillus spp. with bees including honey bees, stingless bees, and solitary bees from tropical and temperate zones appears to have evolved in which female bees inoculate food sources with these bacteria whose chemical products contribute to the elaboration and/or protection from spoilage of food that is stored in the nest."


http://iussi.confex.com/iussi/2006/techprogram/P1982.HTM

Age-dependent changes in intestinal microflora of honeybee

"Remi Kasahara1, Jun Nakamura2, Yoshikazu Koizumi3, Ayako Mitsui3, and Masami Sasaki4. (1) Graduate School of Agriculture, Tamagawa University, Machida, Tokyo, 1948610, Japan, (2) Honeybee Science Research Center, Tamagawa University, Machida, Tokyo, Japan, (3) Environmental Engineering Center Co., Ltd., Machida, Tokyo, 1948610, Japan, (4) Laboratory of Entomology, Faculty of Agriculture, Tamagawa University, Machida, Tokyo, 1948610, Japan

The intestinal microflora of honeybee was investigated by means of the PCR-DGGE (denaturing gradient gel electrophoresis) method based on the sequence-specific separation of PCR-derived rRNA gene amplicon, which have proven useful in analyses of wide ranged studies in microbial ecology. Entire intestinal contents of adult honeybees were removed with gut wall under sterile conditions and the whole genomic DNA was isolated. PCR was used to amplify 16S rRNA genes from the DNA with a set of bacterial specific GC-338F and universal primers. The former one contains a 40 base GC-rich sequence at the 5'-end. The result of DGGE profiles and the DNA sequence analyses confirmed that the intestinal microflora had already existed in the newly emerged workers (day 0), however, it was very simple at day 0 and consisted of only one or two common bacteria. The DNA sequence of one of those showed the homology to Lactobacillus alvei strain 1G2 with 97% similarity. Then the microflora tended to be complex with age, and in the foragers, the composition of bacteria was varied besides the several common ones. The age dependent changes in higher diversity of the intestinal microflora in foragers are probably due to the higher accessibility to the sources of bacteria, namely foods, nestmates, combs, and outside food sources. We discuss the effects of nutritional status and trophallaxis with other nestmates on the individual intestinal microflora, as well as the effects of season, location and food sources on the colony level intestinal microflora."

http://aem.asm.org/cgi/content/full/70/10/6197

"The genus Bifidobacterium includes gram-positive, pleomorphic, and strictly anaerobic bacteria, which are major constituents of the intestinal microflora of humans, of other warm-blooded animals, and even of honeybees"

http://www.pubmedcentral.nih.gov/articlerender.fcgi?&artid=154539

"The other probe, BAN, was able to detect a group of Bifidobacterium species isolated exclusively from blood-warm animals and honeybees (B. animalis, B. asteroides, B. coryneforme, B. cuniculi, and B. globosum), as well as two species isolated from sewage of uncertain origin (B. minimum and B. subtile)"

http://cmr.asm.org/cgi/content/full/19/1/12

"It is the subject of some speculation and debate as to when organisms currently in the genus Hafnia were first isolated. In 1919, L. Bahr worked on a bacterium that he designated "Bacillus paratyphi-alvei," an organism reputedly pathogenic for bees but not mice or guinea pigs (121). One of Bahr's apparently authentic "Bacillus paratyphi-alvei" strains (referred to as "Paratyphus alvei") was subsequently characterized in 1954 as belonging to a new group of enteric bacteria for which Møller coined the name "Hafnia group" during a systematic investigation of amino acid decarboxylase patterns among members of the family Enterobacteriaceae (89). Some groups subsequently questioned the legitimacy of this name in light of the fact that Bahr's strains differed in some biochemical characteristics from those described by Møller. However, Møller considered that Bahr's strain should be regarded as the type species of Hafnia, and he suggested the name Hafnia alvei."

"The specific epithet in the name Hafnia alvei is derived from the Latin noun alveus, meaning beehive, with "alvei" meaning "of a beehive." Ewing (33) questioned the epithet "alvei," stating that the name implied that these bacteria had something to do with bees or beehives although they did not. However, H. alvei has been recovered on occasion from the intestines of honeybees (Apis mellifera) as well as from honey, and several of these strains are included in the BCCM (Brussels, Belgium) collection (125)."


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## JBJ (Jan 27, 2005)

Thanks MB! I will wade through links shortly. 

It would be great to be able to inoculate with the right strains and confer some disease resistance and better nutrient assimilation.


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## Michael Bush (Aug 2, 2002)

Here are a few more:

Bartonella sp.
Gluconacetobacter sp.
Simonsiella sp.


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## sierrabees (Jul 7, 2006)

<It would be great to be able to inoculate with the right strains and confer some disease resistance and better nutrient assimilation.>
__________________
When we build nucs using bees from survivor colonies we are doing just that, although in an unconcrolled manner. However I don't worry about the control factor since I am convinced that the more control we humans exert the more we mess things up. When we start selecting our starter bees for other factors than survival is when we start getting weakened but production oriented stock which is what we see a lot of today.


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## jt9610 (Apr 20, 2010)

Michael has provided a great resource here. Is there anyone who has published a book that summarizes the recent findings about microbes in the honeybee and the honeybee hive? I tried to find such a book but am coming up empty. My guess is it will be a chapter buried in a broader book. The Idiot's Guide has a page or two but no real information. At this point someone will have done a good review but I can't find it.


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## Michael Bush (Aug 2, 2002)

If you search on:
research microbes honeybees Gilliam

You'll find a lot of research by Martha Gilliam. Leave off the Gilliam you'll find a little more, but not much since she seems to have done most of the research.

Some examples:
http://www.ars.usda.gov/is/ar/archive/aug98/bees0898.htm
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.1997.tb12678.x/full
http://www.springerlink.com/content/68g10110r6g70467/

These are more are listed here:
http://www.beeuntoothers.com/index.php?option=com_content&view=article&id=16&Itemid=23


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## sqkcrk (Dec 10, 2005)

And what does one do w/ this information? And how? Knowledge is a wonderful thing, but how does onme apply it? Or should we just be aware of it?


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## Keith Jarrett (Dec 10, 2006)

jt9610 said:


> At this point someone will have done a good review but I can't find it.


Hang on to your skivvies, News will be come via ABJ or BC soon.


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## JBJ (Jan 27, 2005)

"Hang on to your skivvies, News will be come via ABJ or BC soon."
Today 12:28 PM KJ

Cant wait!! This area of research has really blossomed since this thread was started. It is almost like somebody out there is listening

"And what does one do w/ this information? And how? Knowledge is a wonderful thing, but how does onme apply it? Or should we just be aware of it? " sqkcrk

Beneficial microbes are having a huge positive impact in many areas of agriculture. They have a role to play in disease control/prevention, enhancement of growth rates, and nutrition. The right microbes could be used to inoculate the hive and the bee gut to aid colony health and diet.


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## KQ6AR (May 13, 2008)

Hi John,
This is a good topic. I think about it all the time, but not from such a technical point of view. Every chemical we put into the hives kills some of the things the bees might need.


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## Ramona (Apr 26, 2008)

I put together a list of references from material I've gathered over the past few years on microbes and bees as well as some on microbes and other insects and humans. Most have links for downloading.

http://www.beeuntoothers.com/index.php/resources/microbe-refs

One of my favorites is "The Fungus Growing Ants of North America" (1907)(listed under "other symbiotic relationships"). Over 100 years ago it was understood that some ants engage in microbial farming for their food supply! We've known since Martha Gilliam's work (1970's-1990's) that pollen has to ferment properly to be a viable food source for the bees and that bees also engage in microbial farming.

Enjoy the links!

Ramona


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## rhaldridge (Dec 17, 2012)

bump. I was astonished to see so few comments in this old thread, despite the rich vein of information it contains.

I'm really beginning to think that a significant factor in successful treatment free beekeeping is the preservation of variety and population density of co-evolved organisms in the hive.

Here's an interesting quote I cam across on the biobees site:



> I want to share with you the most fascinating thing on bees I have ever read outside biobees.com. In the latest issue of NordBi-Aktuellt, the Journal of the Swedish Association for Preserving Apis mellifera mellifera, Swedish researcher Tobias Olofsson at the University of Lund describes his work on lactic acid bacteria. On the subject of hive atmosphere he writes (in my own humble translation from Swedish):
> 
> Lactic acid bacteria form organic acids such as lactic, acetic and formic acid. These are acids used by beekeepers to combat mites and nosema. Lactic acid bacteria are numerous and resemble small factories in the hive where they prosper in the honey stomach, bee bread, bee pollen and honey. Perhaps they produce an arsenal of substances dispersed in the hive's atmosphere? Perhaps the atmosphere in the hive is important to preserve and this would be a reason to disturb the bees as little as possible. Samples from the lab shows that the bacteria produces large amounts of organic acids that seep into the atmosphere. In modern beehives there are bottom screens and entrances at the bottom; how does this affect a potential atmosphere that might prevent disease? The answer is quite logical, but I put the question to Martin Ferm at the Swedish Environmental Research Institute (IVL) in Gothenburg. The organic acids accumulates in a fairly closed room but with a bottom screen with the full thrust of the wind at the bottom and with entrances at the bottom that aired these acids out according to Martin.
> Wild bees prefer a hollow tree with only a small gap as opening and they are very careful to seal every crack or hole. We will be investigating this properly: what is the atmosphere like inside a hive if it can be left alone and what does such an atmosphere do to mites? Our pilot study that was conducted in the summer of 2009, in a hive during a typical summer day and while winter fodder was given, was just the beginning. Formic acid and acetic acid were found in the hive atmosphere in the visible amount during a typical summer day and in even larger amounts when the fodder was given.
> ...


There is all kinds of interesting research on hive biota.


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## Rusty Hills Farm (Mar 24, 2010)

I have bookmarked this thread. Fantastic links and info. Lots to digest. I never cease to be amazed by how much there is out there to learn about EVERYTHING!

Rusty


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## Michael Bush (Aug 2, 2002)

>I'm really beginning to think that a significant factor in successful treatment free beekeeping is the preservation of variety and population density of co-evolved organisms in the hive.

Bingo.

Here is a very good recent one:
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033188

And a lot of older ones by Martha Gilliam:
http://www.beeuntoothers.com/index.php/beekeeping/gilliam-archives


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## gmcharlie (May 9, 2009)

is there any info on how to see if you have this "balance"?? or determine if you don't???


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## rhaldridge (Dec 17, 2012)

gmcharlie said:


> is there any info on how to see if you have this "balance"?? or determine if you don't???


I don't know how you'd determine that you do have a diverse and healthy micro-biota in hive and gut, but there's at least one way to be sure you don't have it.


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## VeggieGardener (Oct 4, 2011)

rhaldridge said:


> I don't know how you'd determine that you do have a diverse and healthy micro-biota in hive and gut, but there's at least one way to be sure you don't have it.


Yep!

As far as determining the presence of healthy micro-biota, examining samples under a microscope is one way that it is done with soil, so why couldn't that be a method to provide some evidence with bees also?


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## rhaldridge (Dec 17, 2012)

VeggieGardener said:


> Yep!
> 
> As far as determining the presence of healthy micro-biota, examining samples under a microscope is one way that it is done with soil, so why couldn't that be a method to provide some evidence with bees also?


It's probably pretty tough to look at a slide and tell much, unless you're trained to do it and can identify the different kinds of microbes, and have some factual basis for separating good from bad. A dying hive will likely have a lot of microbes too, just not the right ones.

Interesting piece on treated vs. untreated gut bacteria explained here:

http://www.beeuntoothers.com/index....tibiotic-resistant-gut-microbes-in-honey-bees


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## Solomon Parker (Dec 21, 2002)

A further difficulty would be that not all bacteria are able to be cultivated in a dish. Sometimes you just can't get the conditions right.


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## gmcharlie (May 9, 2009)

rhaldridge said:


> I don't know how you'd determine that you do have a diverse and healthy micro-biota in hive and gut, but there's at least one way to be sure you don't have it.


And that would be???


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## WLC (Feb 7, 2010)

All I had to do was add honey to syrup with a little milk.

You can grow this stuff in your kitchen.


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## rhaldridge (Dec 17, 2012)

gmcharlie said:


> And that would be???


Well, if you put stuff in the hive that kills bacteria and yeasts and arthropods, you would expect that stuff to kill those things.

Human beings on antibiotics lose their gut microflora, and it's not fun. From the research I cited above, it's even harder for bees to recover their original baseline microbiota. They compared bees from countries where no antibiotics have been used, Dee Lusby's bees, bees that hadn't been treated for 2 years, and commercial bees. The variety of gut microflora was in descending order, but even Dee Lusby's bees were not as diverse as the never-treated bees, even though hers haven't been treated for 25 years. At least that was my understanding.

It makes me wonder if part of the benefit of adding feral bees to your apiary is that they bring in more than just better genetics. Maybe they bring in co-evolved organisms too.


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## Keith Jarrett (Dec 10, 2006)

rhaldridge said:


> bump. I was astonished to see so few comments in this old thread, despite the rich vein of information it contains.


Rhaldrige, I agree with you..... There was a thread about pollen sub a while back, But as soon as you asked the folks to do there own home work they mostly threw you under the bus. American's are the most (not all) lazy bunch.


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## sqkcrk (Dec 10, 2005)

Keith,
Can you tell me what happens in a hive when you put 15lbs of protein patty into one of your hives? Besides the bees eating it, what exactly happens w/ that stuff? How does it effect the colony?

I usually feed one or sometimes two one lb pattys. Is that effective for growth or just maintainance?

Sorry if this is the wrong Thread in which to ask.


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## Keith Jarrett (Dec 10, 2006)

sqkcrk said:


> Keith,
> I usually feed one or sometimes two one lb pattys. Is that effective for growth or just maintainance?.


Mark, take a look at this months Sept issue of ABJ page 963 (1).

One or two pounds isn't going to do much Mark, not even maintainance.


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## sqkcrk (Dec 10, 2005)

What is the proper protein patty/syrup ratio? Because, if it takes one frame of pollen and one frame of honey to produce one frame of bees, one has to feed syrup as well as protein patty, doesn't one?


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## BernhardHeuvel (Mar 13, 2013)




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## squarepeg (Jul 9, 2010)

all i see is a black screen, is there a link to bernhard's video?


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## Rader Sidetrack (Nov 30, 2011)

Try this ...
http://www.youtube.com/watch?feature=player_embedded&v=NI9Z73UAX-g

I am confident that link is correct, but after I posted, I noticed that Bernhard's post was showing an error message with a link. The error link says ...


> If you’re getting a player error message, most of the time, the video should start working again in about 30 minutes. This error can sometimes happen if the uploader is making changes to the video, there’s an issue with your Internet connection speed, or the video is being removed or having other issues.
> 
> https://support.google.com/youtube/answer/3037019?p=player_error1&rd=1


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## squarepeg (Jul 9, 2010)

thanks rader, but i get the same thing on youtube - just a black screen. maybe it's my browser settings.


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## squarepeg (Jul 9, 2010)

updated the browser, it's working now.


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## BernhardHeuvel (Mar 13, 2013)

*Identifying Bacterial Predictors Of Honey Bee Health*

http://www.sciencedirect.com/science/article/pii/S0022201116301768


Giles E. Budge, Ian Adams, Richard Thwaites, Stéphane Pietravalle, Georgia C. Drew, Gregory D.D. Hurst, Victoria Tomkies, Neil Boonham, Mike Brown, Identifying bacterial predictors of honey bee health, Journal of Invertebrate Pathology, Available online 3 November 2016, ISSN 0022-2011, http://dx.doi.org/10.1016/j.jip.2016.11.003.
(http://www.sciencedirect.com/science/article/pii/S0022201116301768)

Abstract: 
Non-targeted approaches are useful tools to identify new or emerging issues in bee health. Here, we utilise next generation sequencing to highlight bacteria associated with healthy and unhealthy honey bee colonies, and then use targeted methods to screen a wider pool of colonies with known health status. Our results provide the first evidence that bacteria from the genus Arsenophonus are associated with poor health in honey bee colonies. We also discovered Lactobacillus and Leuconostoc spp. were associated with healthier honey bee colonies. Our results highlight the importance of understanding how the wider microbial population relates to honey bee colony health.
Keywords: probiotic; symbiont; microbiome


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## squarepeg (Jul 9, 2010)

very interesting study bernhard, thanks for posting it. do you have access to the entire paper and if so were there any implications as to how conventional treatments for mites may be impacting those particular microbes?


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## BernhardHeuvel (Mar 13, 2013)

Sean Patrick Leonard, *Engineering the gut microbiome of honey bees*, Dissertation, University of Texas at Austin, https://repositories.lib.utexas.edu/bitstream/handle/2152/82022/LEONARD-DISSERTATION-2020.pdf

Superb idea to fiddle with the bees' microbiome..


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## BernhardHeuvel (Mar 13, 2013)

Impact of Nutritional Stress on Honeybee Gut Microbiota, Immunity, and Nosema ceranae Infection

Abstract

Honeybees are important pollinators, having an essential role in the ecology of natural and agricultural environments. Honeybee colony losses episodes reported worldwide and have been associated with different pests and pathogens, pesticide exposure, and nutritional stress. This nutritional stress is related to the increase in monoculture areas which leads to a reduction of pollen availability and diversity. In this study, we examined whether nutritional stress affects honeybee gut microbiota, bee immunity, and infection by Nosema ceranae, under laboratory conditions. Consumption of Eucalyptus grandis pollen was used as a nutritionally poor-quality diet to study nutritional stress, in contraposition to the consumption of polyfloral pollen. Honeybees feed with Eucalyptus grandis pollen showed a lower abundance of Lactobacillus mellifer and Lactobacillus apis (Firm-4 and Firm-5, respectively) and Bifidobacterium spp. and a higher abundance of Bartonella apis, than honeybees fed with polyfloral pollen. Besides the impact of nutritional stress on honeybee microbiota, it also decreased the expression levels of vitellogenin and genes associated to immunity (glucose oxidase, hymenoptaecin and lysozyme). Finally, Eucalyptus grandis pollen favored the multiplication of Nosema ceranae. These results show that nutritional stress impacts the honeybee gut microbiota, having consequences on honeybee immunity and pathogen development. Those results may be useful to understand the influence of modern agriculture on honeybee health.

Castelli, L., Branchiccela, B., Garrido, M. et al. Impact of Nutritional Stress on Honeybee Gut Microbiota, Immunity, and Nosema ceranae Infection. Microb Ecol (2020). https://doi.org/10.1007/s00248-020-01538-1

https://link.springer.com/article/10.1007/s00248-020-01538-1


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## BernhardHeuvel (Mar 13, 2013)

Missing Microbes in Bees: How Systematic Depletion of Key Symbionts Erodes Immunity

Highlights
Honey bees possess a distinct core microbiota that contributes greatly to host stress tolerance and disease resistance.

Mounting evidence suggests that loss or depletion of key symbionts can weaken immunity, exacerbate pesticide toxicity, and reduce host nutritional status.

Agrochemicals, many of which exhibit hidden antimicrobial properties, pose a major threat and have the potential to irreversibly damage the honey bee microbiota.

Genetically engineered symbionts as well as naturally immunostimulatory Lactobacillus strains offer promise of being able to 'reset' dysbiotic microbiota phenotypes via modulation of host defense systems.

Consideration of the insect microbiota should become standard practice during agrochemical risk assessments.


Pesticide exposure, infectious disease, and nutritional stress contribute to honey bee mortality and a high rate of colony loss. This realization has fueled a decades-long investigation into the single and combined effects of each stressor and their overall bearing on insect physiology. However, one element largely missing from this research effort has been the evaluation of underlying microbial communities in resisting environmental stressors and their influence on host immunity and disease tolerance. In humans, multigenerational bombardment by antibiotics is linked with many contemporary diseases. Here, we draw a parallel conclusion for the case in honey bees and suggest that chronic exposure to antimicrobial xenobiotics can systematically deplete honey bees of their microbes and hamper cross-generational preservation of host-adapted symbionts that are crucial to health.

Brendan A. Daisley, John A.Chmiel, Andrew P.Pitek, Graham J.Thompson, Gregor Reid; Missing Microbes in Bees: How Systematic Depletion of Key Symbionts Erodes Immunity; Trends in Microbiology, Available online 14 July 2020, https://doi.org/10.1016/j.tim.2020.06.006

https://www.sciencedirect.com/science/article/abs/pii/S0966842X20301852


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## BernhardHeuvel (Mar 13, 2013)

*Secondary Metabolites Produced by Honey Bee-Associated Bacteria for Apiary Health: Potential Activity of Platynecine*

Abstract

Secondary metabolites of bacteria associated with honey bees were evaluated as part of an investigation on their potentiality for apiary health. Low molecular weight compounds released into culture filtrates by the four bacterial isolates taken from surface of healthy honey bees were analyzed using time-of-flight mass spectrometry. Only one low molecular weight compound was found in the culture filtrate of each isolate. Bacillus thuringiensis, Bifidobacterium asteroides and Acetobacteraceae bacterium, released into culture filtrates platynecine, a pyrrolizidine alkaloid of plant origin, which, until now, had never been reported as produced by bacteria. Lactobacillus kunkeei produced a 3,5-dinitropyridine, of unknown biological action never associated so far to bacteria. The highest relative concentration of platynecine was detected in B. thuringiensis (100%), B. asteroides and A. bacterium showed a concentration above 50% and below 25% that concentration. An in vitro assay on the potential for controlling the parasitic mite Varroa destructor by the culture filtrates of the three platynecine-producing bacteria was performed. Varroa mite mortality was proportional to the platynecine relative concentration into culture filtrate. Although miticidal activity of B. thuringiensis might be associated to other toxic proteins produced by this species, B. asteroides toxicity toward V. destructor along with the other findings of this study support the hypothesis that platynecine plays a direct or an indirect role in controlling varroa. Findings of this study suggest that secondary metabolites released by honey bee-associated bacteria represent a source of natural compounds to be considered in the challenge for counteracting the colony decline.

Manici, L.M., Saccà, M.L. & Lodesani, M. Secondary Metabolites Produced by Honey Bee-Associated Bacteria for Apiary Health: Potential Activity of Platynecine. Curr Microbiol (2020). https://doi.org/10.1007/s00284-020-02153-6

https://link.springer.com/article/10.1007/s00284-020-02153-6


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## squarepeg (Jul 9, 2010)

good post bernhard, and many thanks for your valued contributions.


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## BernhardHeuvel (Mar 13, 2013)

*Differences in honey bee bacterial diversity and composition in agricultural and pristine environments – a field study*

Agrochemicals and biocides are suspected to cause a dysbiosis of honey bee microbiota, decreasing colonies ability to respond to the environment. As a first step to investigate agriculture and beekeeping impact, hives bacteriomes from an anthropized environment (Agri-env) were compared to that of pristine’s (Prist-env). 16S rRNA sequencing evidenced differences in richness and composition between sample types (Gut (G), Brood (B), Bee-bread (BB)) and environments. Higher opportunist loads and shifts toward taxa capable of metabolizing insecticides were observed in G and B at Agri-env, while beneficial bacteria were enriched in Prist-env. Bacteria in BB did not differ, the acidity of the niche outweighing the influence of external factors. Results showed the environment plays a major role in shaping honey bee microbiota, the agricultural realm inducing a bacterial disruption that would let to colonies vulnerability. In contrast, a less susceptible bee will be promoted in less anthropized locations.

Muñoz-Colmenero, M., Baroja-Careaga, I., Kovačić, M. et al. Differences in honey bee bacterial diversity and composition in agricultural and pristine environments – a field study . Apidologie (2020). https://doi.org/10.1007/s13592-020-00779-w

https://link.springer.com/article/10.1007/s13592-020-00779-w


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## BernhardHeuvel (Mar 13, 2013)

*BIOLOGICAL PROPERTIES OF MICROORGANISMS ISOLATED FROM DRONE MILK OF HONEYBEES*

CONCLUSION
The results of the experiments showed that the probion strains isolated from drone milk of the honey bee (Bifidobacterium asteroides strain DFM5, Fructobacillus fructosus strain DF74 and Lactobacillus kunkeei strain DFM21) reacted differently to aggressive media with the best biological conditions. The studied microorganism strains showed high resistance to many antibiotic drugs, but as a result of repeated reseeding, antibiotic resistance in each culture was increased. The probion strains showed antagonistic properties against pathogenic test cultures, which were expressed by the growth inhibition zone of Escherichia coli and Staphylococcus aureus when grown with Lactobacillus kunkeei strain DFM21 at 5.4 and 4.1 mm when grown with Bifidobacterium asteroides strain 5 DM, 2 and 3.9 mm, when grown with Fructobacillus fructosus strain DF74 - 3.2 and 3.4 mm. When analyzing the production of organic acids, it was found that of the assortment present, the highest value was accounted for by lactic and acetic acids, the level of which was 1856.47 and 577.85 mg / l (Lactobacillus kunkeei strain DFM21), 1127.95 and 354.53 mg / l (Bifidobacterium asteroides strain DFM5), 895.21 and 276.49 mg/l (Fructobacillus fructosus strain DF74). The isolated cultures of microorganisms are not toxic to laboratory animals, both in acute and in chronic experience, since long-term use of microbial compositions is not affected negatively on the general condition of mice, their clinical status, morphologist and biochemical parameters of blood, and there are no negative effects on the organs and tissues of the experimental animals. According to the complex of the studies, the probiotic strain - Lactobacillus kunkeei strain DFM21 should be noted, since in several experiments it showed the best results.


Yury Lysenko, Andrei Koshchayev, Alexander Lysenko et al., Biological properties of microorganisms isolated from drone milk of honeybees, 2020 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. Volume 11 No.12 ISSN2228-9860 eISSN1906-9642 CODEN: ITJEA8 Paper ID:11A12K http://TUENGR.COM/V11A/11A12K.pdf DOI: 10.14456/ITJEMAST.2020.236 

https://tuengr.com/V11A/11A12K.pdf


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## BernhardHeuvel (Mar 13, 2013)

Dynamics of the Honeybee (Apis mellifera) Gut Microbiota Throughout the Overwintering Period in Canada

Microbial symbionts inhabiting the honeybee gut (i.e., gut microbiota) are essential for food digestion, immunity, and gut protection of their host. The taxonomic composition of the gut microbiota is dynamic throughout the honeybee life cycle and the foraging season. However, it remains unclear how drastic changes occurring in winter, such as food shortage and cold weather, impact gut microbiota dynamics. The objective of this study was to characterize the gut microbiota of the honeybee during the overwintering period in a northern temperate climate in Canada. The microbiota of nine honeybee colonies was characterized by metataxonomy of 16S rDNA between September 2017 and June 2018. Overall, the results showed that microbiota taxonomic composition experienced major compositional shifts in fall and spring. From September to November, Enterobacteriaceae decreased, while Neisseriaceae increased. From April to June, Orbaceae increased, whereas Rhizobiaceae nearly disappeared. Bacterial diversity of the gut microbiota decreased drastically before and after overwintering, but it remained stable during winter. We conclude that the honeybee gut microbiota is likely to be impacted by the important meteorological and dietary changes that take place before and after the overwintering period. Laboratory trials are needed to determine how the observed variations affect the honeybee health.


Naomie Bleau, Sidki Bouslama, Pierre Giovenazzo, Nicolas Derome, Dynamics of the Honeybee (Apis mellifera) Gut Microbiota Throughout the Overwintering Period in Canada, Microorganisms 2020, 8, 1146; doi:10.3390/microorganisms8081146

https://www.mdpi.com/2076-2607/8/8/1146


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## BernhardHeuvel (Mar 13, 2013)

*Effects of probiotics on the intestinal microbiota of the honey bee (Apis mellifera)*

*Effects of probiotics on the intestinal microbiota of the honey bee (Apis mellifera) and on the performance of colonies in spring in a northern climate*

Summary
The intestinal microbiota is made up of all the microorganisms that colonize the intestine of an animal. In bees, it participates in digestion and immune functions. Several environmental factors disrupt the microbiota of this insect, thus affecting its health. To prevent this from happening, adding probiotics to bee food is an interesting solution. Certain probiotics have been shown to promote colony growth and decrease the incidence of several diseases and parasites. However, no information is available on their effects in a northern climate, where winter survival and spring development of colonies are major issues for beekeepers.

The main objective of the project is to evaluate the effect of probiotic formulas on the taxonomic composition of the bee microbiota and the performance of colonies. Two commercial probiotic strains (Bactocell® and Levucell®SB), an endogenous bacterium (Parasaccharibacter apium) and an antibiotic were administered to a total of 45 bee colonies in the fall of 2017 and in the spring of 2018. Profiling of the bacterial microbiota was carried out at four times during the project: in September, November, April and June. Winter survival and colony performance were evaluated by measuring the number of larvae, number of bees in the colony, winter food consumption and hive weight.

The time of year influenced the composition of the bee's gut microbiota, suggesting adaptation to environmental conditions. In addition, taking probiotics only slightly modulated the bee microbiota. As for the performance of the colonies, the probiotic formulas Bactocell® and Levucell®SB improved the spring development of the colonies.

These results indicate that probiotics can improve the performance of bee colonies without disturbing their microbiota. This information will be useful in developing a probiotic formula for the Canadian beekeeping industry.

https://corpus.ulaval.ca/jspui/bitstream/20.500.11794/40141/1/36044.pdf


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## BernhardHeuvel (Mar 13, 2013)

*A prophylactic probiotic to fight paenibacillus larvae infection in honey bees*

Abstract
Honey bees are the most critical crop pollinators worldwide and for the last decades mostly a single antibiotic has been used to protect them from American foulbrood (AFB), a lethal larval disease caused by the bacterium Paenibacillus larvae. Oxytetracycline (OTC) is the only approved prophylactic antibiotic for P. larvae infection in Canada, which has triggered resistant strains. While only the first larvae stages are vulnerable to AFB, consequences are lethal.

Early microbiology, genomics and proteomics studies have identified molecular differences between the AFB resistant honey bee stages and the vulnerable larvae. Profiles of the immune factors, such as antimicrobial peptides (AMPs), and a characteristic commensal gut bacteria (CGB) community are key differences. 

Through this work we want to develop a new prophylactic for AFB, based on the molecular differences between the adult honey bee and the larvae. Specifically, a commensal bacterium strain that expresses and secretes naturally occurring AMPs targeting P. larvae vegetative cells. To identify AMPs candidates to be used as potential prophylactics, we pursued to uncover host-microbe specific responses of the adult honey bee. Through susceptibility assays we tested the inhibitory activity of characteristic honey bee and fruit fly naturally occurring AMPs against P. larvae, CGB (Bartonella apis, S. alvi and Bifidobacterium asteroides), and non- commensal bacteria (Bacillus subtills and Escherichia coli). 

To look for potential dysbiosis caused on the gut microbiota caused by the candidate AMPs we conducted an in vivo feeding experiment and proceeded with quantitative PCR (qPCR) and 16S rRNA deep amplicon sequencing, determining changes in size and composition. Jelleine and melittin (AMPs) were chosen as final candidates to attempt to be expressed and secreted by S. alvi.

Then, we predicted the Sec pathway-dependent extracellular proteins and with a mass spectrometry based-proteomics experiment explored the signal peptides secretion dynamics. 

Finally, we suggested the signal peptides to be used for the expression and secretion of jelleine and melittin in S alvi, offering a pioneering approach in the understanding and application of honey bee gut commensals.

Romero Guzmán, Atenas Sofia. 2020. “A Prophylactic Probiotic to Fight Paenibacillus Larvae Infection in Honey Bees.” Electronic Theses and Dissertations (ETDs) 2008+. T, University of British Columbia. doi:http://dx.doi.org/10.14288/1.0394247
https://open.library.ubc.ca/cIRcle/collections/ubctheses/24/items/1.0394247


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## Gray Goose (Sep 4, 2018)

Thanks Bernhard for the posting of info.

could be AFB is the Symptom of lack of the proper strains of Bacteria, in the hive.

Was there anything in the study referring to the build up of chems in the comb over time , thwarting some of these strains of bacteria?

was a time where 12 year old comb was find to use, Now I see some advocating 3-5 yr rotate them out, seems the comb would be a good place for the Bacteria to be.
Also considering making my own "patties" from bee bread, Pollen (trapped) uncapped honey (nectar) with the Microbes in mind.

In a related idea, Seems eating bee hive products helps humans as well, could be some symbiotic stuff in that thread as well. I recall in the book "hive and the honey bee" some +100yr old folks were interviewed, and they ate products from the hive, often the dark brood combs, containing larvae excreations, and microbes. As the light honey was what the "market" demanded. 

One could harvest from the healthy bees some of these microbes, propagate it, and add it to feed.

GG


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## BernhardHeuvel (Mar 13, 2013)

A Swedish scientist group extracted lactic acid bacteria from the bees, a special strain that only live in honeybees, and cultivated them in the laboratory. Those microbes were fed back to the hive – with very good results! Healthier bees, more honey. They made a product out of it: Symbeeotics. I tested it and it was brilliant. I don't know why they went off the market, but the idea was good.


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