# Microbial ecology - HOW?



## Michael Bush (Aug 2, 2002)

First, I think it's important not to disrupt the microbes in a healthy hive. 

Second, as you already mentioned, I think you can often set a hive on the right track by giving it a frame of brood and pollen from a healthy hive. Think of it as the "germ theory of health". You want to share germs from those that are healthy. 

Any other actions pretty much require more knowledge than we currently have. We don't know for sure what beneficial organisms to innoculate them with, but we can be reasonably sure that a healthy hive has at least some of them.


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## BernhardHeuvel (Mar 13, 2013)

Watched the scientific activities by the Swedish scientists for years. They came out with this product and I start using it from next Spring on. Will report if anyone is interested. To me it does make sense to inoculate the hive with bee microbes just to give them a start. Just like starting off a sourdough or honey mead. Inoculate it and it runs by itself. 

http://symbeeotic.apicellae.se/


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## Kofu (Jan 26, 2011)

At the symposium yesterday, Ramona told us something I didn't quite get, that antimicrobial treatments common in the industry have completely eradicated certain strains of bacteria in most parts of the world, but not in Switzerland and New Zealand. Ah, here's a link:

After Decades of Antibiotic Treatment of Honeybee Colonies, Tetracycline-resistant Bacteria Often Found in US Bees

And that even Dee Lusby's bees, which haven't been exposed to treatment for 25+ years, are no different. So it seems like one area to explore is intercontinental inoculations...


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## Ramona (Apr 26, 2008)

Sorry I wasn't more clear. What I was discussing was honeybee gut bacteria showing resistance to antibiotics, not eradication of the bacteria.

Dee Lusby's bees are dramatically different from bees in the US that have been routinely treated with antibiotics. Her bee's gut bacteria more closely resemble gut bacteria from bees in parts of the world that have never been treated with antibiotics.

Although Dee's bees have not been treated in over 25 years, their gut microbes still show 
more antibiotic resistance than bees from places that have never been treated.

Here is a link to a more detailed analysis from the study done by the Moran Lab.


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


Am at airport with my fussy tablet waiting for delayed flight back to Boston...we had a great time at the Philly meeting!

Ramona


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## Kofu (Jan 26, 2011)

Ramona said:


> What I was discussing was honeybee gut bacteria showing resistance to antibiotics, not eradication of the bacteria.


Bacteria were eradicated that had no resistance, which is evidence of extensive long-term impact of chemical treatments on the microbial environment.


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## Phoebee (Jan 29, 2014)

Thanks, that was worth sharing. As a guy who studied symbiosis of this sort several decades ago, I'm surprised that this has not been done more completely by now.


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## Michael Bush (Aug 2, 2002)

>Bacteria were eradicated that had no resistance, which is evidence of extensive long-term impact of chemical treatments on the microbial environment. 

That is what worries me. People like to discount impacts on the microbes and act like everything will get back to normal in a few days or weeks or months, but actually they may never get back to normal.


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## WLC (Feb 7, 2010)

I have inoculated syrup and milk with probiotic bacteria and fed the fermented 'Beegurt' to my bees.

I've also used honey to ferment syrup and milk. I didn't feed it to my bees though.

So, Kofu, there is a practical way to do this.

However, it's still highly experimental.


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## Kofu (Jan 26, 2011)

WLC said:


> I have inoculated syrup and milk with probiotic bacteria and fed the fermented 'Beegurt' to my bees.
> ...
> So, Kofu, there is a practical way to do this. However, it's still highly experimental.


What kind of probiotic bacteria? What's the target, to get it into the 'honey stomach' where it will help make better honey, or ... ?

Learning the keywords. A search for 'probiotics' turns up another thread:


thenance007 said:


> Whatever your means of supplementation, there have been several scientific studies done that have concluded that adding probiotics to pollen supplement or syrup increases fat body weight and improves brood health and longevity


I'm learning all sorts of things here. Didn't quite realize there are commercial products such as Biogen-N, Trilac and Enterolactis Plus. (I skip that part of the catalogs.) Some of what I've seen says bacteria and fungii normally come with the pollen and nectar, so is the main reason for these products that emerging bees fed on high-fructose corn syrup are going to need pro-biotic supplements to help them digest it? Are these products designed for use by bees that have been flooded with antibiotics earlier, so they may not have the bacteria they need to survive, for themselves and to pass along to the youngsters in the colony?

That aspect doesn't seem so highly experimental, with the three citations given. What are you doing that's different from that?

How does this fit into a larger picture of a microbial environment engrained into how a honeybee colony operates?


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## Kofu (Jan 26, 2011)

BernhardHeuvel said:


> Watched the scientific activities by the Swedish scientists for years. They came out with this product and I start using it from next Spring on. Will report if anyone is interested. To me it does make sense to inoculate the hive with bee microbes just to give them a start. Just like starting off a sourdough or honey mead. Inoculate it and it runs by itself.
> 
> http://symbeeotic.apicellae.se/


Okay, so how is this different from the probiotics fed to emerging bees? What part of the biome in the hive, or the microbiome in the bees, are these microbes for?

This is getting pretty interesting. Maybe "everybody" knows but me, and I appreciate the opportunity to learn.

For me, a backyard beekeeper in a city environment, I'm not sure of the applications. I guess my main interest is in helping the bees to survive mites and other infestations, without much if any treatment, and I'm wondering how microbes fit into that picture.


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## WLC (Feb 7, 2010)

Kofu:

You're asking a whole bunch of questions at once. Step back a minute and clear your mind.

We know that Honeybees carry symbiotic bacteria, like lactic acid bacteria (LAB), in their honey stomachs that not only help them to maintain their own health, but are also important in fermenting honey and fermenting pollen into beebread.

We also know that organic acids, like lactic acid, have been used to control Varroa.

As an example, I've been able to get syrup and milk to ferment down into the lower pH 3 range. That's well within the effective range for treating Varroa.

There are other reasons to use probiotics in syrup. I wanted to try to improve its nutritional content for instance.

There's a lot we don't know at this point about the benefits of probiotics in beekeeping.

However, the possibilities are worth exploring.

I haven't, as yet, tried to ferment syrup and milk using fresh nectar or beebread.

But, I do know that raw honey, from around the world, does work.

It's like a whole new world waiting to be explored.

What's truly remarkable is that you can do this in your own kitchen.


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## Keith Jarrett (Dec 10, 2006)

WLC said:


> There's a lot we don't know at this point about the benefits of probiotics in beekeeping.
> However, the possibilities are worth exploring.
> It's like a whole new world waiting to be explored.


WLC, very well said.


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## WLC (Feb 7, 2010)

Keith, what's interesting is that I've also used LAB to ferment soy flour.

I bought some soy flour that was marked down (it was warm to the touch), and I fermented a sample of it in water with some LAB.

There are a lot of possibilities in applying fermentation to issues in beekeeping.


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## deknow (Jul 17, 2006)

Let me see if I can explain things.

The alimentary canal (mouth through gut and rectum) is a long tube. The environment wrt suitability for each of the core bacterial species varies dynamically...sometimes gradually and sometimes suddenly...as one travels down the canal, or observes different sections of it.
The illium is particularly suited for some specific strains that are novel and highly integrated into the cells of the bee. Last I knew, they had been unable to do a proper genome map of this strain because their diversity varies so much within each bee that they were unable to isolate a monoclonal sample (required to map the genome). These bacteria were not culturable by any of the means they had attempted and remain somewhat a mystery.

Presumably the use of antibiotics introduces levels (and/or concentrations) of 'naturally occurring antibacterial compounds' is many orders of magnitude higher than those produced and used by the existing (and proper) microbiotia of the hive, or really anywhere on the planet...the metabolic cost for nature to produce toxins of the concentration of terramycen powder is demonstrably prohibitive....therefore the cost that a robust complex culture would have to bear in order to be resilient as a whole against such concentrations of 'natural substances like antibiotics' is (and has been for 50 million years) too high a cost to bear against such an impossible level of toxin.

The (even single) antibiotic 'incident' is traumatic to the microflora...you can see that in the data and slides from the Moran study. The percentage of antibiotic resistance levels in bees recently treated, long ago treated, and never treated show antibiotic resistance levels to go down between 'incidents'...this is because such resistance is expensive to maintain in the population, and without exposure, it is largely selected against.

_but_ the incident itself hasn't just magically removed genes, it has almost certainly removed whole lines variation within the strains. It has selected (probably several times in the history of any bees that are kept in the US recently or not), for only the individual strains that could resist the unprecedented levels of toxin survive.

The result, after 'recovery' is a drastic thinning of the variation, and most certainly the niche specific subtle variation in genetic diversity...the continuum of gross and subtle traits that follow the continuum of niche through the alimentary canal.

Ever go to a restaurant that was unexpectedly understaffed and the managers were trying to serve the tables and cook the food? 

This thinning of the diversity of the gene pool of gut microbes by selecting for antibiotic resistance is a trauma to the system. The data shows that even after 26+ years, the impact (and no doubt damage) to the heritable complex microbial ecosystem of the hive is still measurable.

It would be like selecting (eugenicly speaking) 'the next generation of humans' solely on the individuals ability to win weight lifting competitions.

deknow


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## deknow (Jul 17, 2006)

My wife's (Ramona) obsession with the microbial component of the hive led to, I believe the first modern public presentation to beekeepers (the Nebraska state conference at the invitation of Michael Bush, the program director at the time). This presentation was given in November of 2008, and she cites and/or quotes from most of the literature available at that time...the first time that I'm aware that anyone has done this.

I think I do have video of this, and someday might get that up, but you could download the mp3 from our site as a 'cell cast'. 1.5 hours.
deknow

http://beeuntoothers.com/index.php/beekeeping/audiovideo/98-2008-microbe-talk


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## deknow (Jul 17, 2006)

Somewhere around 99% of the gut microbiota is in eight distinct groups (three of them named after Gilliaiam, Von Frisch, and Snodgrass). The other 1% is likely also significant and important, but this is obviously where the bulk of the research will focus.
When we are talking about diversity, we are talking about within these 8 groups.

deknow


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## deknow (Jul 17, 2006)

...and anyone interested to see what the mainstream beekeeping world's reaction to these concepts could try looking at the discussions on Bee-L in November of 2008.

edit: In fact, it was so bad that Michael Palmer (who was also speaking and whom I'd never met before) came up to me, and the first words out of his mouth were, "I think it's terrible what they are doing to you over on Bee-L".

That was a great conference...Dee, Michaels' Bush and Palmer, Ramona, me....we had a 'microbe party' in our (deserted) hotel...everyone bring something made by microbes...beer, wine, cheese, bread, salami...

Ramona and I bought 2 bottles of wine to make sure there was something to start with, about 50 people showed up, we collected donations for pizzas and watched "Queen of Trees" on PBS (get ahold of this video from the Nature series). 

In the end, the pizza and the tip were covered exactly (to the dollar) by what people had just given with their own judgement. The wine we bought had been drank, but there were 2 unopened bottles remaining in the morning, and we didn't want to check bags. We brought them to the liquor store next door where we had purchased our bottles (and these had come from there as well, it was the next building over from the hotel), explained our situation, and they took them back with no hassle.

Even Steven....a perfect ecosystem 

deknow


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## deknow (Jul 17, 2006)

Kofu said:


> It seems like the next set of questions is, *"How?"* How do we put that sort of information to practical use?


I think the most important thing is to stop selecting (even occasionally) for microbes that can resist antibiotics, or those that can repopulate the fastest after an organic acid or thymol treatment. Even intermittent exposures are well beyond what we can expect a well balanced and robust ecosystem to withstand and bounce back from...it just isn't evolved to maintain genes after such a trauma.


> Yesterday, at our Natural Beekeeping Symposium, Laurie R. Herboldsheimer gave a great introduction to this topic — "The Importance of the Microbial Environment in the Beehive." The Philadelphia Beekeepers Guild should have the video up sometime soon. (I'll post the link.)
> 
> Here are some practical questions:
> 
> How does it work to share comb between hives? Within an apiary? from apiary to apiary? What criteria for sharing (or not sharing)?


I can't give you a prescription, but Dee Lusby (who's bees are in the Moran study) brings comb in to extract while replacing them with combs from the previous days extraction from a different yard. All comb is deep, and there is no distinction between brood and honey frames. No treatments, no artificial feeds, very clean environment, and very different results in antibiotic resistance than the bees about 30-40 miles away at the Tucson Bee Lab that haven't seen antibiotics in 2+ years.
That is one working model that I can cite that is no doubt spreading things between yards.



> What factors affect the biome in the hive? and the microbiome in the bees' guts, etc.? — especially the factors that beekeepers can affect?


There is no question. The easyist thing for the beekeeper to affect...the low hanging fruit is also the thing that is having the most negative impact on the microbitota (by negative, I mean hard selection for a metabolically expensive trait that otherwise is of no use). It is what the beekeeper is putting in the hive. Fluvalinate and Coumaphos are consistently found in the highest rates and concentrations of any synthetic pesticides...even on trapped pollen because the bees collecting them are contaminated....by the beekeeper.

Essential oils are devistating to microbial cultures...that what they designed for.

Organic acids are used for sterilization...and when formic acid is removed from the hive, the concentration of the acid in the air inside the hive _goes up_...from the bees themselves releasing the acid they had been sequestering in their tiny lungs and bodies.


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## squarepeg (Jul 9, 2010)

dean, to what degree has the level of understanding regarding microbial ecology in the hive been advanced since 2008, and is mainstream beekeeping paying more attention than it was then?


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## deknow (Jul 17, 2006)

The biggest change has been the use of non culture based techniques to see the relative abundance of specific microbial strains.


Deknow


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## squarepeg (Jul 9, 2010)

understood, thanks dean. for you or mb, where does the number 'over 8000' come from pertaining to the number of different microorganisms found in the beehive?


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## deknow (Jul 17, 2006)

....from Martha Gillian's work. Some to much of that she found is likely incidental stuff that grows well in a petri dish. The entire body of her work came be downloaded from our website.

Deknow


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## squarepeg (Jul 9, 2010)

thanks again deknow. have you come across anything in your review of the literature that suggests feeding syrup alters bee gut microbiota?


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## deknow (Jul 17, 2006)

squarepeg said:


> dean, to what degree has the level of understanding regarding microbial ecology in the hive been advanced since 2008, and is mainstream beekeeping paying more attention than it was then?


Gilliam's work suggests that feeding and/or caging bees affects yeasts, fungi, and bacteria in the bee.
No such 'modern' work is yet being done. The current need is to study the basic functions, and will likely occupy the researchers for years.
Sugar syrup is a near ideal pH for most microbial bee diseases.
Work by Samataro and Yoder shows HFCS alters the balance of various fungi in beebread.

deknow


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## heaflaw (Feb 26, 2007)

squarepeg said:


> thanks again deknow. have you come across anything in your review of the literature that suggests feeding syrup alters bee gut microbiota?


If it does alter the mirobiota, will changing Ph of sugar water to approximate that of honey prevent the alteration?


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## deknow (Jul 17, 2006)

BernhardHeuvel said:


> Watched the scientific activities by the Swedish scientists for years.


Us as well. Unfortunately they stopped sharing any useful data with other researchers or beekeepers when they decided to go into the probiotic business.
I don't see anything wrong with this, but they can no longer be considered 'researchers that are hoping to advance our knowledge', they are producers of a product whos insights and discoveries are proprietary and untested by critique from others.

deknow


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## deknow (Jul 17, 2006)

pH is only one factor...an acidic sugar syrup is still not honey, is it?



heaflaw said:


> If it does alter the mirobiota, will changing Ph of sugar water to approximate that of honey prevent the alteration?


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## WLC (Feb 7, 2010)

Here's a new look:

"Honey Bees Avoid Nectar Colonized by Three Bacterial Species, But Not by a Yeast Species, Isolated from the Bee Gut"

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086494#pone-0086494-g003

I found it interesting that bees preferred artificial nectar inoculated with a yeast but not L. kunkeei.


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## squarepeg (Jul 9, 2010)

did i read it wrong? looks like the bees were neutral to the yeast inoculated 'nectar'.

like most good studies, this one generates more questions (grant proposals) than answers.

my take away - bees are picky eaters.


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## WLC (Feb 7, 2010)

I would call it 'basic research'.

However, its value to those interested in using probiotics is that 'nectar' inoculated with a non LAB species (a yeast) was preferred over L. Kunkeei.

It could point the way to making fermented syrup that more closely matches Honeybee's nectar foraging preferences, for instance.

PS-did you notice the pH of the nectar inoculated with the yeast? It was in the 3-4 range.


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## deknow (Jul 17, 2006)

I would be surprised if yeasts and bacteria in nectar contributed anything to the core (99% of the population) species...the foraging bees encountering these have well established microbial communtites.

Tobias and Alejandra (the Swedes) did document shifts in the flora with nectar flow, but it isn't clear if that is because of microbes in the nectar, or a change in the hospitablity of the gut do to different characteristics of the food source.

The other 1% likely has some (perhaps significant) unrecognized role, but it will be a while before these get looked at closely.

I agree with Squarepeg that WLC misstates things when he claims, "preferred artificial nectar inoculated with a yeast".
Perhaps you want to read the study (or even the conclusions) and then comment?
...but my reading is that it seemed to be the metabolites of the microbial fermentation that was being measured, not the actual presence of the cultures.


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## WLC (Feb 7, 2010)

The 'preferred' yeast isolated from the Honeybee's gut is commonly found in nectar.

>I agree with Squarepeg that WLC misstates things when he claims, "preferred artificial nectar inoculated with a yeast".<

It's in the second figure of the paper.


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## deknow (Jul 17, 2006)

WLC said:


> It's in the second figure of the paper.


Really? You really are just going to look at the picture...at that picture, and not see that the 'preference' wasn't consistent, and not significantly a preference.
From the discussion:


> Specifically, our data indicate that honey bees prefer nectar free of colonies of the three aerobic bacterial species we isolated from the bee gut, whereas the nectar-inhabiting yeast M. reukaufii had no effect on bees’ feeding preference.


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## WLC (Feb 7, 2010)

That's why I wrote 'preferred'. It was preferred over L. kunkeei.

Here's a recent paper on that yeast (Metschnikowia reukaufii ):

http://onlinelibrary.wiley.com/doi/10.1111/1574-6941.12284/abstract

It's an interesting species.

"Jack of all nectars, master of most: DNA methylation and the epigenetic basis of niche width in a flower living yeast"

http://ebd06.ebd.csic.es/pdfs/Herrera.et.al.2011_2.Mol.Ecol.pdf


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## deknow (Jul 17, 2006)

WLC said:


> That's why I wrote 'preferred'. It was preferred over L. kunkeei.


BS (and I don't mean Beesource).

You stated:


> I found it interesting that bees preferred artificial nectar inoculated with a yeast but not L. kunkeei.


...the 'but not' indicates that you are comparing each to a control. The bees had no preference for the yeast, but were less attracted to the bacteria.

I don't know much about the tagging feature here, but I'm betting this one is headed to 'WLC follies'.

deknow


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## squarepeg (Jul 9, 2010)

heaflaw said:


> If it does alter the mirobiota, will changing Ph of sugar water to approximate that of honey prevent the alteration?





WLC said:


> PS-did you notice the pH of the nectar inoculated with the yeast? It was in the 3-4 range.


on those rare occasions when feeding syrup is necessary i use vitamin c to lower the ph. (thanks mike bush)


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## WLC (Feb 7, 2010)

squarepeg said:


> on those rare occasions when feeding syrup is necessary i use vitamin c to lower the ph. (thanks mike bush)


The yeast has an acid protease. So, I found that it lowered the pH of 'nectar' to be of interest.

By the way, since you do add lactic acid bacteria to your syrup, why would you need vitamin C?

We're still discussing microbial ecology, right?

That Metschnikowia reukaufii is pretty cool for a yeast in my opinion.


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## squarepeg (Jul 9, 2010)

WLC said:


> By the way, since you do add lactic acid bacteria to your syrup, why would you need vitamin C?


i try to stay with an all natural (from the field) diet, and have never added lab. fortunately my location is pretty rich in floral biodiversity, and i believe nature is providing all of the essentials. this may be one of the reasons why my bees are thriving off treatments.


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## BernhardHeuvel (Mar 13, 2013)

Yeast presence at higher levels in a beehive is considered an indicator for stress or illnesses.

If you feed probiotics you do not do this via the syrup, especially not lactic acid bacteria. Bees have their own lactic acid bacteria species they want to preserve. They add those bacterias to sugary solutions so the bacteria can ferment the solutions. Would you add wine yeasts to an already ready fermented wine? Would you add lactic bacterias to an already fermented yoghurt? Doesn't make much sense. Lactic bacterias of the bees suppress yeasts in the fermenting process, to me that makes sense to choose the yeasts rather than the other competitive lactic bacteria.

The stuff the Swedish Symbeeotics sells is applicated by dribbling, within a sugarwater mix. Just like oxalic acid is dribbled. It gets taken and distributed within the hive by the dribbling.


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## deknow (Jul 17, 2006)

Bernhard,
Gilliam observed (with the bees in Arizona in the 70s or 80s) that unfed, free flying bees didn't show any yeast in their guts. Feeding and/or caging was enough to foster yeasts.

Almost every other account I've seen includes yeasts as generally found in bee guts.

We know from talking with some of the researchers that few are looking at the whole picture. Bacteria, fungi, and yeasts are all looked at separately by separate researchers and teams. I haven't seen much of the modern yeast work on honeybees...the Moran Lab is taking a deep look at the gut bacteria (not bacteria in other parts of the hive), Jay Yoder and Diane Samantaro are doing good work with the fungi in the beebread. I'll look at WLC's reference tomorrow...now I'm going to bed...been on the road for a couple of weeks and am ready to crash.

deknow


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## WLC (Feb 7, 2010)

I've noticed that many of the more recent studies have focused on identifying bacteria in the hive. L.kunkeei is turning up as one common species.

I thought that it was interesting to read about a yeast common in nectars and also found in the bee gut.

There is a growing interest in feeding bees probiotics. However, the main focus is on lactic acid bacteria.

Exploring the role of at least one common yeast may open up some debate in probiotic circles. That would be a good thing.


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## WLC (Feb 7, 2010)

Let me stir the pot a bit...

"Making artificial honey using yeast cells from salivary glands of honeybees"

http://nopr.niscair.res.in/bitstream/123456789/23184/1/IJEB 43(7) 664-666.pdf

Although the Asian Honeybee was used, it does suggest that there's a greater role for yeasts, rather than lactic acid bacteria, in creating honey.


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## deknow (Jul 17, 2006)

WLC said:


> Although the Asian Honeybee was used, it does suggest that there's a greater role for yeasts, rather than lactic acid bacteria, in creating honey.


Does it? How can it suggest a 'greater role for yeasts, rather than lactic acid bacteria' if the study only looks at yeasts only, and doesn't compare the fermentation to that of lactic acid bacteria?

Regardless, the writing in this is fast and loose enough to pretty much dismiss their conclusions as wishful thinking. The PDF is un-copyable without some work, which I'm not inclined to do (or to retype)....if interested, read the end of the conclusion where they claim honey can be produced artificially using yeast cells.

deknow


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## Phoebee (Jan 29, 2014)

I saw something similar in abbreviated form in Science News last summer, that a single yeast found in the honey stomach can turn sucrose syrup into something that is almost honey. About the same time I read about the discovery that was evidently unexpected by the researchers: fungicides can kill bees, and might explain CCD. 

Since yeasts are in essence single-celled fungi, well, there you go.

The yeast are certainly only one element in a complex web of organisms that complete the bee, but I would expect them to be important. Nothing is so simple as to point to single factors or causes, but definitely if you have foregone conclusions that a whole area is not important, you may just get blindsided from that area.


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## WLC (Feb 7, 2010)

The paper itself only has one citation. I would call it a 'basic research' paper.

The findings in both this and the previous 'yeast' paper were interesting enough to me that I've inoculated one of my LAB fermented cracked corn ferments with some comb honey.

If there are any yeasts in the honey that can both survive the low pH and use the remaining cracked corn as a feedstock, I might be able to see some new fermentation activity.

I think that others interested in using probiotics in beekeeping might share my own interest in the yeasts found in honey, and how they might be applied along with LAB.


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## Kofu (Jan 26, 2011)

This thread has a lot about specific individual species of bacteria and yeasts. They're important, obviously, but there's also another level of interest — in "communities" of microbes.



WLC said:


> We're still discussing microbial ecology, right?





deknow said:


> We know from talking with some of the researchers that few are looking at the whole picture. Bacteria, fungi, and yeasts are all looked at separately by separate researchers and teams. ...


One thing that interests me is the idea that there might be different combinations, similar to what researchers find in the human gut. I can't find a reference just now, but I remember reading that for humans — in the skin or in the gut— microbes can settle into any of several different ecological set-ups (aka microbiomes) depending on diet, place in the world, etc.

"Most of the microbes associated with humans appear to be not harmful at all, but rather assist in maintaining processes necessary for a healthy body. A surprising finding was that at specific sites on the body, a different set of microbes may perform the same function for different people. For example, on the tongues of two people two entirely different sets of organisms will break down sugars in the same way. This suggests that medical science may be forced to abandon the one-microbe model of disease, and rather pay attention to the function of a group of microbes that has somehow gone awry."

From Wikipedia: Human microbiome​
To me that suggests that with exposure to fungi-, herbi-, and pesticides, and beekeepers' "treatments," the microflora in the bees won't get totally wiped out, but might get pushed into impoverished but relatively stable ecologies. Maybe the bees are immunocompromised, or they have more trouble dealing with pests, or queen-rearing is harder, but at least they're alive.

For discussion's sake (why I didn't put this thread in the treatment-free area), what if organic acids knock the microflora back for awhile, as a side-effect in treating for varroa, but then it's easier to recover the previous balance, with the same basic ecology, than with the antibiotic treatments? Or the other way round?

It seems there are lots of different ways to go when we consider the microbiomes and their variations. (I want to come back and catalog/outline some of the different _practical_ approaches in another post, because it seems like beekeepers are already in a place where we can begin to speculate and experiment with these ideas, in some ways moving out ahead of the scientists.)


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## deknow (Jul 17, 2006)

1. Yeasts that are found in the bee gut and also found in the food they are bringing in are interesting, and clearly have a role in processes such as the early stages of fermentation of pollen. By any measure, they are part of the 'ecosystem' in the hive.

However, there is a huge distinction between this always renewing resource and the gut bacteria of the bees. Many of these are 'novel' and only exist in the guts of bees...some are so specialized that they integrate into the wall of the illium.

This community _can't_ be refreshed with what is found on/in flowers or anywhere in nature except in the guts of bees and/or insects similar to bees.

This culture is vertically heritable from one generation of bee to the next...impacts of damage can be measured for 26+ years...suggesting that it doesn't easily 'recover' to some ideal mean.

This is a separate set of genetic information...separate from the bee's nuclear and mitochodrial DNA that is passed on from one generation of bee to the next....with thousands? millions? of generations of selection pressures acting upon them within each generation of bees.

2. E.O. Wilson did an interesting experiment (can be seen in 'Lord of the Ants' video). He 'tented' some mangrove islands, in effect killing everything. 

Eventually, the biomass of critters living on the island returned to be the same as it was before tenting...but the makeup of that biomass was drastically different.

Every time you apply one of these things (and organic acids and essential oils make a 'broad spectrum antibiotic' look like a laser beam), you knock things back to a level ripe to be repopulated...by whatever traits were able to survive and/or repopulate and 'hold space' the most quickly.

...when this happens with antibiotics, there are specific genes we know to look at that shows us clearly the thinning of diversity of population that happens...when this happens with organic acids and essential oils, we don't (currently) have things cataloged in this way. Most likely it is the strains that grow the fastest when 'leaked' in through drifting bees.

One really must consider the 'hit' the microbial community comes under when soft treatments are applied...and compare that to what, in nature, this microbiome that hasn't seen the outside of a bee's gut in many millions of years, has been expected to contend with.

I maintain that if one wants to do _anything_ to support the microbiome, that one must stop putting anything into the hive as a first step.

deknow


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## WLC (Feb 7, 2010)

" How do we put that sort of information to practical use?"

I guess you could say that not disrupting the microflora of the hive is a 'practical' application.

It's certainly very important.

My own views on practical applications for the microflora found in a healthy colony is that we need to start growing it out ourselves and seeing what it can do.

For example, I know that 'store bought' LAB can produce a lot of starch from cracked corn.

I don't know what the microflora found in honey can do to cracked corn.

We can do a lot to understand it ourselves. Unless, of course, you would prefer to just 'read up' on it.


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## squarepeg (Jul 9, 2010)

"However, there is a huge distinction between this always renewing resource and the gut bacteria of the bees. Many of these are 'novel' and only exist in the guts of bees...some are so specialized that they integrate into the wall of the illium.

This community _can't_ be refreshed with what is found on/in flowers or anywhere in nature except in the guts of bees and/or insects similar to bees.

This culture is vertically heritable from one generation of bee to the next...impacts of damage can be measured for 26+ years...suggesting that it doesn't easily 'recover' to some ideal mean."

were any other differences found other than the loss of non-terramycin resistant bacteria in those studies?


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## Kofu (Jan 26, 2011)

WLC said:


> My own views on practical applications for the microflora found in a healthy colony is that we need to start growing it out ourselves and seeing what it can do.


I like that spirit of experimentation. At present I'm just getting established, with three hives in my backyard and other things going on in my life, so right now I am at the 'reading up on it' stage. Plus going to the symposium in Philadelphia a week ago to hear Ramona explain the little-big picture (wait till you see the video!), with lots of details but for me not enough info on practical applications. Plus trying to stir up conversation here. 

It doesn't hurts anyone to try things out and speculate about what's going on when we do X, Y, or Z. Some people are doing it commercially, with the pro-biotics on the market already. There's good reason for thinking it might help, and some evidence that it does help at least in some circumstances.

On the other hand, we can't replicate in a test tube everything that goes on in a honeybee's guts. One thing Ramona mentioned (and maybe Dean in this thread, too), is the turn in research from 'taking a culture' and growing it in a lab, to DNA analyses that show species that can't be grown outside the bee-guts.

Here's a nice set of pictures with one of an actual digestive tract, including the Ileum where apparently a lot important microbial activity occurs. And an article by Nancy Moran's lab, with the basic findings.


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## WLC (Feb 7, 2010)

Kofu:

Just for conversation...

Diastase Number is a measure of honey quality. Diastase is an enzyme found in honey that can break down starch into less complex sugars. While it has been assumed that diastase is produced by the honeybees themselves, there is the possibility that it's being produced by microflora.

Dean has mentioned the issue of the impact of treatments on colony microflora, and I have read that U.S. honey consistently tests at lower diastase numbers than European honey.

Since I already had a LAB cracked corn ferment, with a lot of soluble starch in the media, I thought that it would be interesting to see what happens to that ferment if I added comb honey (with some of the comb included as well).

I didn't add a lot, but if I do see that the starch is being digested, it could mean one of two things: either the diastase in that honey is doing its thing, or the honey microflora are digesting the starch.

It's a simple experiment with some potentially important ramifications.

So, there's a lot to be said for running your own experiments.


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## Phoebee (Jan 29, 2014)

Kofu said:


> On the other hand, we can't replicate in a test tube everything that goes on in a honeybee's guts. One thing Ramona mentioned (and maybe Dean in this thread, too), is the turn in research from 'taking a culture' and growing it in a lab, to DNA analyses that show species that can't be grown outside the bee-guts.


Back around 1975 I signed up for a course in symbiosis. It was so novel that the class consisted of two graduate students and one undergrad ... me. I can't understand why it was not swamped ... it was and remains a fascinating subject. I uncovered then-recent work in which it was documented that some snails could eat algae and incorporate the algae's chloroplasts into their own cells. Chloroplasts are believed to be symbiotic bacteria, now totally dependent on and part of their hosts. Evidently, though, it is possible to grow them in another host, in this case allowing an animal to photosynthesize.

Mitochondria in our bodies are also believed to be symbiotic bacteria. They have their own DNA and reproduce independently of the nucleus.

One of our field trips was to the dairy science department, where a bovine researcher unscrewed a lid from the side of a steer, and obtained a sample of microbes (mostly protozoa, but some bacteria) from the rumen. This stuff is almost impossible to grow in the lab, so they use a live animal to produce culture. One of the grad students wanted to know if this jar lid was bad for the steer. The guy laughed and said, "This animal is 18 months old, and we still need him. If not for the lid, he'd have been hamburger months ago."

It is not uncommon for symbionts to be so dependent on the host that they are difficult or impossible to grow outside the host. They've usually lost some process essential for independent life, which they get from the host, in exchange for something vital to the host.


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## Kofu (Jan 26, 2011)

deknow said:


> This community _can't_ be refreshed with what is found on/in flowers or anywhere in nature except in the guts of bees and/or insects similar to bees. This culture is vertically heritable from one generation of bee to the next... ...
> 
> Every time you apply one of these things (and organic acids and essential oils make a 'broad spectrum antibiotic' look like a laser beam), you knock things back to a level ripe to be repopulated...by whatever traits were able to survive and/or repopulate and 'hold space' the most quickly. ...
> 
> I maintain that if one wants to do _anything_ to support the microbiome, that one must stop putting anything into the hive as a first step.


Dean, I basically agree with you, but I want to be skeptical about it and test these ideas. So: I had a dental operation last year and had to take antibiotics. I ate a lot of yogurt and leafy green veggies, etc., and it feels like I recovered most of the basic functions within a few weeks. I assume that, living in an environment with lots of other humans around, I get to pick up some of the gut bacteria that I lost. On the other hand, it's possible "I'll never be the same."

With a lot of the treatments, presumably the whole ecosystem in the hive takes a hit. But the bees do seem to survive. Perhaps they are weaker and more susceptible to disease, etc. The whole hive is affected, so they can't pick up microbes from within the same hive, but as you say, there's drifting. And perhaps bees have developed a close relationship with the microbial environment outside, to replenish what they've lose. So it seems (and I'm a little tongue-in-cheek about this), you can't argue with success. Just look at the catalogs and see what sells. It works, right?

The treatment-free approach is based on the idea of building up integrated microbiomes, with the general feeling that within a few years an apiary can be on a relatively stable foundation. Add in cut-outs and feral bees, when possible. Share comb from your more successful beekeepers in the area.

But there's a range of possibilities between that model, where you say "one must stop putting anything into the hive as a first step," and industry-supported full-time reliance on agricultural chemistry. Thanks for staking out your position, and I personally hope to get there one of these years... In the meantime, it's mix-and-match for me, trying to learn as I go.


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## Phoebee (Jan 29, 2014)

Kofu, 

I've been put on antibiotics that failed to cure what they were supposed to, but temporarily cured my acne, and I noticed a curious lack of odor when performing certain essential functions. I suspect that puts me in the majority of Americans. I expect we all have out-of-whack biota, and probably one day we'll find out the long term consequences.

Several years ago I noticed an article on a new treatment, a transplant of, uh, the intestinal contents of a healthy human to one suffering from an out-of-whack intestinal flora. A little dab will do ya (boy, that dates me). 

Cattle transmit rumen organisms orally (they're messy kissers). Some people are reputed to have a habit that might do this for intestinal flora, but I suspect it is mostly just vicious rumor. Most people are too hygienic to pass flora this way. We might even be healthier if we were not such germophobes.


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## Kofu (Jan 26, 2011)

Phoebee, we're on the same wavelength. I read that article, and before Ramona gave her talk it was something she mentioned too. As 'gross' as it may sound, germs get around a lot more than we might want to believe. We are more hygenic in the imaginary world than in reality. People wash their hands after going to the bathroom a lot _less_ than they say they do, etc. And that's not a bad thing, as it turns out.


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## Phoebee (Jan 29, 2014)

The question is, can the true symbionts be transmitted by unwashed hands? The pathogens can button up for the ride, but symbionts may be so dependent on some contribution of the host that they can't be transmitted easily.

The cattle rumen symbionts are usually absolute anaerobes. A short exposure to oxygen is deadly for them. They need to be transmitted in a very sloppy manner, surrounded by enough other organisms that can utilize oxygen that they don't get a lethal dose on the way. 

You can kill termites by exposing them to too much oxygen ... it kills their symbionts and they starve to death. Like in cattle, it is the symbionts that break down cellulose. 

This may or may not have any similarity to what goes on in bees, just an indication of how difficult they could be to culture_ in vitro_.


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## WLC (Feb 7, 2010)

If you mean microflora that can only live within Honeybees, I have yet to hear of any (barring pathogens).

There are microflora that forms a biofilm in the honeycrop, but different studies have come up with different microflora.

I think that while these studies have done a fairly good job at culturing and characterizing the various microbes present in Honeybees, they're limited by the methods currently available to them.

Also, there a point that was made here before, some microflora might only be able to function as a community. So, when someone tries to isolate them on some specific type of media, they'll be lost from the culture.


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## Saltybee (Feb 9, 2012)

Pro microflora argument; cowpox protected from smallpox, how many centuries for mankind to figure that out. We are lucky man did not learn how to cure cowpox first.
Con; microflora will not prevent cancer, polio, etc. Varroa and viruses with microflora? I know I do not know.

If you beleave cows have any trouble passing flora, you have never stood behind one.


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## Phoebee (Jan 29, 2014)

Microflora operating as a community, that can't do it alone (I think this was discovered in the 1980's): Ever see a big bumpy rust lump on steel in a marine environment? That's not just rust, it is a complex layered community of microbes. At the core are sulfur-eating organisms that can get sustenance by consuming sulfur in some grades of steel and particularly from welds. But they can't dissolve steel. So they team up with organisms that can corrode steel. But they need layers of protection so they can go about this. Up pops this sophisticated arrangement for eating our stuff. Left alone you get those rusticles you see dripping off the Titanic. They are why it is dissolving faster than anyone would have believed. Nobody used to look for microbes in rust ... it was so obviously not a microbe habitat.

Where in the heck did this come from? Steel ships are recent inventions. The steel chemistries on which they feed are modern.


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## Kofu (Jan 26, 2011)

Practical applications — what then must we do?


Emulate the ancient hive, aka "paleo-beekeeping" 
Treatment-free, all the way
Natural ways of propagating microbes
Sharing comb
Nurse bees (will be accepted, right?)
Building nucs from survivor colonies
Adding from cut-outs, bringing survivor bees into apiary
Allow for drifting, robbing, recolonization

Inoculate
The hive location - biodiversity of flora
Probiotics, aimed at digestion, honey-making

Better targeting for treatments, find out which have "least-worst" effects
Antibiotics
Organic acids and essential oils
Other treatments

Consider "microbe-friendly" ways to buffer negative effects of other treatments, feeding
Use concepts of 'microbial ecology' to consider
Other factors that beekeepers can affect
Ventilation, humidity, location
Treatments
Find ways to measure the "balance" (see Sammataro video, their project this year)

Develop this conversation — beekeepers, beek clubs, science, industry ...

So this is my first pass at cataloging the different sorts of "how." In doing it, the "what" and "why" keep getting mixed in, of course.

*Sources*

I started this project a week ago, when I saw the topic for our Bee Guild's symposium, "The Importance of the Microbial Environment in the Beehive," and I wondered for those who accept that it's important, what then? I haven't been immersed in the topic, as others have. Most of these ideas above come from a handful of sources:

Three threads (and many individual contributors) in BeeSource
Microbial ecology of the bee and hive
Rethinking bee nutrition
And this thread, thus far
Diane Sammataro's presentation, on video, is a good overview.
A podcast including Phil Chandler was suggested by another Philly-area beek, where they're "talking about mimicking the microbial environment of a wild hive."

And of course, Laurie Ramona Herboldsheimer's presentation, Sunday, Feb. 9, 2014. She has one really excellent slide showing arrows for the interactions between different treatments and different genre of microbes, and a lot of other material and references. The Philly Bee Guild should have that video up on the web pretty soon.


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## Michael Bush (Aug 2, 2002)

>We are lucky man did not learn how to cure cowpox first.

Yes.

>Con; microflora will not prevent cancer, polio, etc.

I do not believe that is true. Polio was common in places that hygiene was common and rare in places where hygiene was rare... harder to say about cancer, but current theory on colon cancer is that the reason that fiber helps prevent it is because it provides a place for healthy bacteria to live in your gut. I think microflora WILL help prevent cancer and polio etc.

http://www.swedish.org/swedish/media/sni/multiple sclerosis/ms about ms/hygiene-hypothesis.pdf
http://chriskresser.com/the-hygiene-hypothesis-is-modern-disease-associated-with-being-too-clean
http://www.academia.edu/4463404/Dis..._hygiene_hypothesis_in_the_early_20th_century
http://books.google.com/books?id=gL...#v=onepage&q=polio hygiene hypothesis&f=false
http://books.google.com/books?id=mD...#v=onepage&q=polio hygiene hypothesis&f=false
http://en.wikipedia.org/wiki/Hygiene_hypothesis


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## Kofu (Jan 26, 2011)

Not wanting to steal Ramona's thunder, I've held off on sharing her most original (and controversial) claim. Now I find she announced it a year ago, :applause:



Ramona said:


> I'm actually in the middle of working on a talk to be given next Saturday as part of the "festooning" festival at Boston University - "Family Values" in the context of the beehive. ... "Potty Mouths" ... and many other topics.
> 
> Ramona


A video of that talk is up :thumbsup: on the web. :thumbsup: Check it out! Especially the 'censored' slide (22:16 to 25:37). She says she tried to find anyone who has suggested this before, and couldn't find it.

She started her talk in Philly with the same slide show, "Heather has two Mom-Bees." The whole thing is full of microbial dimensions.



Phoebee said:


> Cattle transmit rumen organisms orally (they're messy kissers). Some people are reputed to have a habit that might do this for intestinal flora, but I suspect it is mostly just vicious rumor.


And she says she wants to do a children's book with this material...


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## JBJ (Jan 27, 2005)

"I maintain that if one wants to do _anything_ to support the microbiome, that one must stop putting anything into the hive as a first step." deknow 

There are dosage thresholds that need to bee considered. All bee plants contain essential oils and bees naturally come into contact with these substances when they forage. Even the most potent antibiotic can be rendered useless if not delivered in an appropriate therapeutic dose. The same is true for essential oils.

Are you proposing there is no safe essential oil dosage to native bee/hive microflora? I have not seen the data to support this. Paracelsus maxim probably applies here: The dose makes the poison. 

At what concentration are EO's useless, helpful, and/or harmful? One could argue that there is a concentration of EO's that would fit into each of these 3 categories.


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## WLC (Feb 7, 2010)

There's another paper from the folks studying yeast/pollinator mutualism in nectar:

"Nectar bacteria, but not yeast, weaken a plant–pollinator mutualism."

http://www.stanford.edu/~fukamit/vannette-et-al-2013.pdf

If you're interested in symbiosis, this might 'float your boat'.


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## JBJ (Jan 27, 2005)

Interesting...
This study tells me we still have a lot to learn about both endogenous & exogenous microbiology of our favorite pollinator. 

Perhaps also that bees prefer alcohol to vinegar?


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## WLC (Feb 7, 2010)

There have been some major studies on the bacterial communities in Honeybees. They usually use 16s rRNA to identify them.

This group out of Stanford have identified a known yeast using 18s rRNA, and they've even gone as far to show that bees seem to prefer it to bacteria in their nectar.

It looks like our views on these bacterial communities in Honeybees and probiotics may have been wrong.


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## squarepeg (Jul 9, 2010)

WLC said:


> It looks like our views on these bacterial communities in Honeybees and probiotics may have been wrong.


wrong how wlc?


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## WLC (Feb 7, 2010)

We may have misidentified the real 'good microbes' in the Honeybee.

Lactic acid bacteria have gotten most of the spotlight to date.

But, a yeast as a 'good microbe'? 

Metschnikowia reukaufii?

I can barely spell it.


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## Kofu (Jan 26, 2011)

Can someone make the connection(s) here with honeybees? I'm sure it's there but I'm not seeing it.



WLC said:


> There's another paper from the folks studying yeast/pollinator mutualism in nectar:
> 
> "Nectar bacteria, but not yeast, weaken a plant–pollinator mutualism."


The mutualism in this paper is between hummingbirds and the bushes. A yeast helps, and a bacterium seems to hinder, that relationship. There's a question about how these microbes get around, and a suggestion that other pollinators might be involved. As I read it, nectar ferments while it's in the flowers, and that makes it more or less appealing to hummingbirds.

How might this connect with successful beekeeping — in the honey stomach or the honeybee gut, or in the hive — in terms of applied science?



JBJ said:


> Interesting...
> This study tells me we still have a lot to learn about both endogenous & exogenous microbiology of our favorite pollinator.


If the findings hold up, we can guess that yeasts and bacteria are involved together, both in the flowers and in the digestive processes. And probably for millions of years, so there has to be some mutualism between the yeast and bacteria too, right?


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## Kofu (Jan 26, 2011)

JBJ said:


> "I maintain that if one wants to do _anything_ to support the microbiome, that one must stop putting anything into the hive as a first step." deknow
> 
> ... Are you proposing there is no safe essential oil dosage to native bee/hive microflora? I have not seen the data to support this. Paracelsus's maxim probably applies here: "The dose makes the poison."


[Minor edit: I thought "Paracelsus maxim" was somehow Latin for 'the dose makes the poison,' :s but Paracelsus, well... let's say he reminds me of some folks around these days.]

This is the issue for me, because I used Apiguard last year, and it seemed to suppress a boom in varroa population, so my bees are alive now when they probably wouldn't be otherwise.

I believe the idea of microbial ecology and its benefits to a better understanding of beekeeping. And Dean and Ramona are probably right when they say that thymol in doses much, much heavier than could be found in nature is very likely a serious poison to the microbial community in a hive (while it's also killing the varroa mites, but usually not most of the bees, in the hive), and that some of the narrow-cast pesticides targeted at varroa might be less damaging to the microbes.

It seems like the scientists could get to JBJ's question pretty easily. Are the bees ingesting the thymol in its concentrated doses? Supposedly it gets tracked through the hive when the bees try to get it out of the hive, and it vaporizes in the brood nest after it's been tracked through, in concentrations enough to reduce the varroa load. If the bees ingest it, how does it affect the microbial balance in the honey stomach? in the rest of the bee-gut? in the bee bread? How seriously are bee functions affected? How long do these effects last?

And drawing on some of the ideas (3b and 6) I cataloged above, would it help to port a frame of brood comb and nurse bees into the hive, 1, 2 or 3 weeks later, from a hive that was not treated with thymol, in order to boost the populations of beneficial microbes?

Emerging brood would possibly get an inoculation of digestive bacteria by taking on the cleaning and grooming of other bees as the first of their duties... (Check out Ramona's 'Family Values' talk for details.)


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## WLC (Feb 7, 2010)

"How might this connect with successful beekeeping — in the honey stomach or the honeybee gut, or in the hive — in terms of applied science?"

Kofu:

They're marketing Probiotic supplements for Honeybees. However, what if they're using the wrong microbes?

Is Metschnikowia reukaufii (the yeast) being included?


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## BernhardHeuvel (Mar 13, 2013)

WLC said:


> ...


You put yeasts into your hives (poor bees...) and report back.  We await your results.

(You should consume more yeasts yourself and see what they do to your digestion. inch


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## WLC (Feb 7, 2010)

BernhardHeuvel said:


> You put yeasts into your hives (poor bees...) and report back.  We await your results.
> (You should consume more yeasts yourself and see what they do to your digestion. inch


Bernhard! I've already fed my bees LAB fermented syrup/milk. No, they didn't die.

But you know what, Perhaps someone would like to make their feed more attractive for their bees.

By the way, it's not just any old yeast that you want to feed to bees.

It looks like M. reukaufii, a yeast commonly found in both nectar and bees, might be the one.


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## Kofu (Jan 26, 2011)

WLC said:


> By the way, it's not just any old yeast that you [might] want to feed to bees.
> 
> It looks like M. reukaufii, a yeast commonly found in both nectar and bees, might be the one.


I'm new to all this, and will have to watch the videos from the talks this past Sunday, but I think it was suggested that the bacteria might help by keeping the yeast from going too far. (In the honey-stomach, perhaps.) So maybe there's a balance that benefits everyone — bushes, bacteria, yeasts, hummingbirds, bees, and other pollinators (not to mention, beekeepers and the rest of the planet). Maybe it's not one or the other.


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## WLC (Feb 7, 2010)

There is some mutualism between all of the above. The investigators felt that the yeast species was important though.

Yeast have been found in association with Nosemosis, for example. 

There are likely 'good yeast' and 'bad yeast' involved. How they interact with bacteria remains to be seen.


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## BernhardHeuvel (Mar 13, 2013)

WLC said:


> Bernhard! I've already fed my bees LAB fermented syrup/milk.


Yeah, another attack on bee hives that they just survived... If you know nothing about microbes and microbial communities, you should do less experimenting with living things like bees.



WLC said:


> There are likely 'good yeast' and 'bad yeast' involved.


This shows clearly that your knowledge about microbes is less than basic. There is no good or bad in microbes. You and I as humans do carry microbes that keep us alive. We depend on them. Those are the most dangerous microbes one can think of and they easily can turn on you and kill you pretty quick. In fact those microbes eat you up in the end, but that is another story.

Just like bees it is all about community in microbes. And balances. Putting stuff into the hive rather destroying the harmony than helping. So keep your yoghurt for yourself. The bees have their own bacteria that evolved with bees for millions of years. You can supplement those, but not foreign ones. 

Side note: You can identify persons by their microbes genes, since every human has a pattern of microbes with unique genetics. You can identify family members by identifying the family's microbes.

Supplementing microbes is only good to change the hive's environment, so the bees' own microbes are activated. 

You cannot do this with yeasts, because yeasts are big destroyers and this is true for almost all organisms, that they do more harm than good inside an organism. Wreck your health by eating live yeasts all day long. What do you reckon bread is baken for? Why eat not the dough freshly? 

Yeasts are not the bad guys, as said there is no good or bad. But one should be careful with yeasts. From what I learned about yeasts, I would never introduce yeasts into bee hives. Too dangerous.


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## BernhardHeuvel (Mar 13, 2013)

One hint for practical application. To stay ontopic, what or how to do. 

One observation was made that clustering of the bees reduces microbes count and species. So consolidates and strengthens the microbial community. While clustering the bees exchange microbes with each other through trophallaxis and microbes begin to balance each other out.

This is useful to know. Bees cluster during winter months, which they do anyway. (Normally, this winter was different.) But: bees also cluster when swarming. So swarming is another method for microbial consolidation. 

You either work with natural swarms (swarm beekeeping) or you shake all the bees at the end of the season after the last honey harvest of the year. Making artificial swarms on new foundation. A lot of beekeepers do this in Germany, even professionals and what you can see is an enhanced health in those bees. (You get rid of the pesticide contaminants this way, too.) Brood is removed and emerges in a seperate apiary, is treated and put back into the swarmed hives.


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## BernhardHeuvel (Mar 13, 2013)

Some time ago I put up a short document on microbes and humans:
http://www.immenfreunde.de/docs/Mikrobenmutter_engl.pdf

One can ask those questions you find there for the honeybees, too.


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## WLC (Feb 7, 2010)

Bernhard:

I instructed microbiology lab in college. So, I understand both the risks and the benefits to Honeybees of feeding them live cultures.

Yes, feeding them live cultures can disrupt their microfloral equilibrium.

As for yeasts, while I don't plan on feeding bees with a live culture of M. reukaufii, I find it to be interesting.


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## Kofu (Jan 26, 2011)

BernhardHeuvel said:


> One observation was made that clustering of the bees reduces microbes count and species. So consolidates and strengthens the microbial community. While clustering the bees exchange microbes with each other through trophallaxis and microbes begin to balance each other out.


It's nice to have a proper Latin-Greek hybrid term for something we've been talking about. Thanks!

*Trophallaxis*, n. "the transfer of food or other fluids among members of a community through mouth-to-mouth (_stomodeal_) or anus-to-mouth (_proctodeal_) feeding." —Wikipedia​


BernhardHeuvel said:


> Bees cluster during winter months, which they do anyway. ... But: bees also cluster when swarming. So swarming is another method for microbial consolidation.
> 
> You either work with natural swarms (swarm beekeeping) or you shake all the bees at the end of the season after the last honey harvest of the year. Making artificial swarms on new foundation. A lot of beekeepers do this in Germany, even professionals and what you can see is an enhanced health in those bees. (You get rid of the pesticide contaminants this way, too.) Brood is removed and emerges in a separate apiary, is treated and put back into the swarmed hives.


That's a great addition to this thread, and a new category of response to the question in the title of the thread. I'll put it in the next version of the catalog. 

(But everything up to now is on topic as far as I'm concerned. DIY pro-biotics, and fermentation experiments outside the hive with yeasts and bacteria... Why not? Seriously, think about the "Why not?" question rather than reject the idea automatically. There's more that we don't know than what we already know for sure.

It sounds like the flip side of microbes is the need to clear out the extra ones that wend their way into the "community." Clustering. Swarming. Artificial swarms.

Questions:

How is the brood 'treated' in this maneuver? for what, with what?
They're daughters of the same Queen, and will be welcomed, right?
Similar question about adding nurse bees from a microbially healthy hive. They'll be accepted, right? and can contribute microbes they've been hosting?
What about the bee bread? Do some of the microbes for that come from within the bees? or mostly from outside the hive?

There's lots more that can be added in this discussion. Of course we're beyond pure science already, into applications already in use and possible new applications to try.


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## Kofu (Jan 26, 2011)

Here's a reference...

4.4.2 Social Feeding Behaviors. From the textbook, _Insect Behavior_. By Robert W. Matthews, Janice R. Matthews. 2009.

Nothing about _proctodeal trophallaxis_ in honeybees, but it's just a textbook. The whole section (starting on the previous page) is good background reading. Termites are the main subject of study for this activity.


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## Kofu (Jan 26, 2011)

BernhardHeuvel said:


> Some time ago I put up a short document on microbes and humans:
> http://www.immenfreunde.de/docs/Mikrobenmutter_engl.pdf
> 
> One can ask those questions you find there for the honeybees, too.


I think you'll enjoy the talk Ramona (Laurie R. Herboldsheimer) gave at the Philadelphia Beekeepers Guild symposium last Sunday, because she goes into the same topics, both for humans and for bees, and draws the analogies. Our local beekeeper processing the videos says they'll be posted on the web in a week or two, probably through his YouTube channel (which also has Michael Bush's talks from last year).


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## JBJ (Jan 27, 2005)

Not all yeast are bad, some are probably endemic and beneficial.

Here is another consideration; propolis is antibacterial and antiviral... and its all over everywhere in the hive. I would bet that often times essential oils are being added at concentrations less antibiotic that natural propolis. There is documentation of propolis increasing brood viability. Now that interesting and worth thinking about IMO.


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## Kofu (Jan 26, 2011)

JBJ said:


> Propolis is antibacterial and antiviral... and its all over everywhere in the hive. I would bet that often times essential oils are being added at concentrations less antibiotic than natural propolis. There is documentation of propolis increasing brood viability.


Apiguard (thymol) apparently works by disturbing the bees and making them want to get rid of it. So they carry it out of the hive. (How they "carry" it, I'm curious to know.) Somewhere I read that the main effect is in the brood nest, where the bees track it across the brood comb. It evaporates and gets into the comb with the pupae, killing the mites (or disturbing their reproduction, or ...?).

... It can take from 2 to 10 days to be removed from the tray/dosing tray. The gel will
reduce as vapor is given off and as the bees detect the 'foreign material' they try to remove it. At high
temperatures the vapors are stronger. The bees will find the gel and try to clean it up quickly. Strong
colonies generally work faster than smaller or weaker ones. At lower temperatures, the gel sublimes
more slowly. It is not detected as readily by the workers and they do not remove it as quickly.
Even if the gel seems to have disappeared after only a few days, there is no need to apply a second 
treatment until 2 weeks have passed. The thymol, although not in the tray, is active throughout
the colony during this time, having been carried around by the housecleaning bees.

... The second dose usually lasts longer in the trays because the bees have become more accustomed 
to the odor of thymol in the hive by this time. The cleaning behavior is not as pronounced as for
the initial introduction.

— Apiguard - Q&A (pdf)​
Apparently when the brood are capped, a bit of propolis is added to the caps. And propolis is used elsewhere in the hive for its antimicrobial functions. But is propolis ever used in the hive to concentrate its effects in the brood comb, to kill mites?

As a protein denaturant, [thymol, in concentratration] disrupts cell membranes and affects all cellular 
processes. It is a very general mode of action rather than being highly specific. It should be more 
difficult for the varroa mite to change all of its body functions to become resistant to thymol. — Q&A​
Amazingly, the bees (most of them, at least) survive the treatment. The question is, how does it affect the microbial community in the hive that supports the bee population?


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## Kofu (Jan 26, 2011)

JBJ said:


> I would bet that often times essential oils are being added at concentrations less antibiotic than natural propolis.


JBJ, now I'm wondering if you meant something other than Apiguard/Thymol. I've heard only a little about other essential oils. John Seaborn talked about it a little, in Philly a couple of years ago. He adds oils to sugar syrup, and maybe he has other ways too.

I'll ask here, if you can just summarize other ways essential oils are used (effectively?) to control mites and/or for other pest control. (I can look elsewhere for more info.) How might those uses affect the microbes? In positive ways? And/or negative, but can it be done in ways that limit the impact at the microbial level?


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## deknow (Jul 17, 2006)

WLC said:


> If you mean microflora that can only live within Honeybees, I have yet to hear of any (barring pathogens).


This has been well documented and well reported for a while now:
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036393


> The most consistently present proteobacterial phylotypes in honey bees, the Gilliamella, Gamma2, and Snodgrassella phylotypes, are highly distinctive and so far found only in honey bees and bumble bees [8], but the Glliamella and Gamma2 phylotypes are nested within a larger clade that has been recovered from guts of other insects [10], [41].


One would do well to read these studies out of the Moran lab carefully...they are extremely well done, and extremely well written.

deknow


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## WLC (Feb 7, 2010)

"We sampled 2 localities, consisting of the USDA Agricultural Research Service Bee Labs in Tucson, Arizona and in Beltsville, Maryland, on 4/28/2011 and 4/20/2011 respectively. In each location, 5 bees were sampled from each of 4 colonies, for a total of 40 samples representing individual bees."

O.K., it's not what I would call a sufficient sample size.

However, most species haven't as yet been identified by DNA barcoding. So, it's conceivable that you can score some new species by sampling tissues in insects. (Hey, that sounds like a great project.)


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## Phoebee (Jan 29, 2014)

WLC said:


> "We sampled 2 localities, consisting of the USDA Agricultural Research Service Bee Labs in Tucson, Arizona and in Beltsville, Maryland, on 4/28/2011 and 4/20/2011 respectively. In each location, 5 bees were sampled from each of 4 colonies, for a total of 40 samples representing individual bees."
> 
> O.K., it's not what I would call a sufficient sample size.
> 
> However, most species haven't as yet been identified by DNA barcoding. So, it's conceivable that you can score some new species by sampling tissues in insects. (Hey, that sounds like a great project.)


A recent issue of Science News had a sidebar on how fast modern DNA analyzers are. They quoted a data output rate something like 36,000 streaming movies, simply mind-numbing, from a little desktop unit about the size of my laser printer. I wondered what used units are going for, looked on e-bay, and there was one selling for $128, supposedly in good condition, but obviously undesirable compared to newer units. I didn't look closely at the capability of the old one, but that gives some idea of how fast things are moving.

I consulted on an idea for a DNA analyzer with a friend ... he cancelled the project because someone beat him to it.

Analyzing an entire human genome down to base pairs is approaching $1000. One day we'll toss an occasional bee into a pocket analyzer and get more data than we'll really want.


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## deknow (Jul 17, 2006)

WLC, do your own homework.


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## deknow (Jul 17, 2006)

For those interested in the level of complexity we are talking about:
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050096
A single mutation in a gut microbe of an aphid can make it (the aphid) sterile if exposed (even briefly as juviniles) to heat that leaves aphids with unmutated bacteria unaffected. ...the same mutation gives an advantage if the temperatures are consistently low.


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## WLC (Feb 7, 2010)

deknow said:


> WLC, do your own homework.


Dean, we've already found 'new' DNA barcodes.

It's not difficult to do.

I've done my homework, and I do like the Moran study.

Phoebee:

Are you talking about the Personal Genome Machine? Or, the Ion Proton perhaps?


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## JBJ (Jan 27, 2005)

Kofu said:


> JBJ, now I'm wondering if you meant something other than Apiguard/Thymol. I've heard only a little about other essential oils. John Seaborn talked about it a little, in Philly a couple of years ago. He adds oils to sugar syrup, and maybe he has other ways too.
> 
> I'll ask here, if you can just summarize other ways essential oils are used (effectively?) to control mites and/or for other pest control. (I can look elsewhere for more info.) How might those uses affect the microbes? In positive ways? And/or negative, but can it be done in ways that limit the impact at the microbial level?


Various oils can be used for many different things/reasons in beekeeping, from keeping syrup fresh, simply calming the bees, and actual therapeutic reasons. It would be difficult to make broad statements about oils in general as the pharmacological, medicinal, metabolic properties vary substantially between particular plant essences.

I have more to add and several posts to respond to but duty calls for a bit.


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## thenance007 (May 25, 2011)

In my first year of beekeeping 4 years ago, I ran across an article on Evans & Lopez research on probiotics which indicated that when they fed probiotics, they had much stronger hives going into Spring than the controls and less foulbrood. They used human probiotics, so I began adding a capsule of whatever probiotic I was taking to a gallon of their sugar syrup every few months, along with a bit of vinegar or ascorbic acid and a pinch of sea salt for minerals. In the past year, I've fed very small quantities, probably less than a gallon spread out, sometimes mixing up a quart and pouring a cup over the brood nest areas of each of my 4 hives. 

I just had an inspection done, and the bee inspector said that mine were the healthiest 8 frame medium hives she'd ever seen this time of year. The bottom 3 boxes of each hive are full of bees, plenty of stores left, the 2 in the sun each have at least 6 frames of capped brood, the 2 in the shade are a bit further behind--and none of them are even bringing in pollen yet.

I reason that the bees spread disease-causing bacteria as they walk/crawl through the hive, and that inoculating the comb with good bacteria should offset some of that. Plus the nurse bees feed it to the brood. While I believe Michael Bush that a healthy hive shouldn't need it, I doubt that any hive could truly be considered healthy these days--too many environmental challenges even in a treatment free hive. While they are not the exact strains found in the bees gut, they are closely related, and if nothing else, possibly take space and resources away from the "bad" guys.

So I've been willing to take a risk and I have no controls; all I can say is that I haven't had any winter losses in 4 years and my bees are VERY healthy. It makes sense to me that the probiotics are likely contributing to that since that is about all I do that is different from most beekeepers. So if you want to try something inexpensive and without waiting for the ultimate research, I can tell you that in my experience (4 hives over 4 years) occasional human probiotics won't kill your bees and probably will do them some good.


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## WLC (Feb 7, 2010)

thenance:

Good to hear that.

I've got my own hypothesis going on besides the logical crowding out the bad, and lowering the pH.

My own opinion is that some of these strains may be producing diastase, invertase, or isomerase.

They're important in producing honey, for example.


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## Michael Bush (Aug 2, 2002)

>It would be difficult to make broad statements about oils in general as the pharmacological, medicinal, metabolic properties vary substantially between particular plant essences.

They are more similar than you might expect. All of them are the defense system of the plant so all of them are antimicrobial. They may be more or less antimicrobial and there may be other properties to some of them, but this antimicrobial activity is pretty consistent.


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## deknow (Jul 17, 2006)

Remember the 2011 2012 usda pollen survey showed thymol in 27.3% of beebread samples...at an average of 2,000+ ppb... ranging up to almost 40,000 ppb.

I know of one treatment free beekeeper who loaned a natural beekeeper some honey supers....guess what they came back smelling like?

When we spoke to the beekeepers on Long Island last year, we stayed with a retired Navy ships captain (loading ammo and fuel on the fly)....everything was beyond "ship shape".
She had taken over her father's rather large pidgin breeding "hobby".
One would think that a Navy person would be cleaning the bird stuff constantly (there were hundreds of birds). She loved Ramona's message, as she brushes the roosts and nests clean, but leaves plenty of litter on the floor. Every few days she puts the young birds on the floor to get exposed to everything, and more adult birds feed the young when they are on the floor. My understanding is that she wins races and sells/auctions plenty of very well regarded birds.

Deknow


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## lazy shooter (Jun 3, 2011)

This whole thread is just too complex for my engineering education. So, somewhere along the way, would one of you life science folks give me a synopsis of what this thread is about and any conclusions drawn from this thread?


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## Kofu (Jan 26, 2011)

I've been asking about Thymol/Apiguard, mainly because that's what I used last year. But what about Oxalic Acid, the new up-and-coming, low cost, "semi-natural" treatment?

What's the impact of Oxalic Acid on the microbiome? Both in the hive, generally, and inside the bees?

I'm thinking that the general environment in the hive is fairly antiseptic, with all the propolis around — except in the bee bread which gets a lot of its microbes from the flowers, with a little dab of nectar from the honey stomach. How many beneficial microbes live on the outside of the bees? They don't have colonies of germs on their skin like humans do, right?



deknow said:


> I think the most important thing is to stop selecting (even occasionally) for microbes that can resist antibiotics, or those that can repopulate the fastest after an organic acid or thymol treatment. Even intermittent exposures are well beyond what we can expect a well balanced and robust ecosystem to withstand and bounce back from ... it just isn't evolved to maintain genes after such a trauma. ...
> Organic acids are used for sterilization ... and when formic acid is removed from the hive, the concentration of the acid in the air inside the hive _goes up_ ... from the bees themselves releasing the acid they had been sequestering in their tiny lungs and bodies.


FatBeeMan says oxalic acid is good for killing tracheal mites, as well as varroa, which means the bees do take it up into their tiny lungs and bodies just as with formic acid.





http://www.youtube.com/watch?v=sQp9pdAOjdo​
With Oxalic Acid, the mortality rate for the bees is low, in the short run at least, and effectiveness against varroa is high. But! How do we factor in the microbial perspective?

Bees keep reservoirs of microbes in their honey stomach and lower intestines. If they have ways to repopulate the general microbial community from those reserves, how heavily are the microbes affected by Oxalic Acid vapor treatments? The selection process Dean explains above is obviously important. What are the specific ways it may play out?

It seems like the model is that Treatment Free beekeepers foster bees and their associated microbial communities, in order to get to a place (perhaps we're there already) where the bees can survive with a certain level of varroa mite infestation. For example, hygenic bee behaviors keep down the mite levels, in combination with robust immune systems which allow the bees to hold off the viral infections and other diseases that are often what really kill the bees.

In that model, any treatment that selects for other conditions (e.g., for microbes that can survive exposure to lower pH than is usual in a hive) is taking us down another path, to a different place, where the "metabolic cost" of having adapted to low pH treatments may reduce the ability of microbes in the bee-gut to support the robust immune system.

What other effects can we trace — via microbial ecology — from Oxalic Acid treatment to handicaps for bees and their normal life sustaining processes and their fight against infestation and disease?


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## Kofu (Jan 26, 2011)

deknow said:


> Remember the 2011 2012 usda pollen survey showed thymol in 27.3% of beebread samples...at an average of 2,000+ ppb... ranging up to almost 40,000 ppb.
> 
> I know of one treatment free beekeeper who loaned a natural beekeeper some honey supers....guess what they came back smelling like?


Here's scientific evidence of the same 'smell' test: "Propolis volatiles characterisation from acaricide-treated and -untreated beehives maintained at Algarve (Portugal)."

"Cluster II was mainly characterised by the high thymol content, followed by viridiflorol, n-tricosane and n-nonadecane. The presence of higher thymol levels in propolis samples from cluster II may reflect the long use of an acaricide with thymol as main active ingredient."​


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## Kofu (Jan 26, 2011)

lazy shooter said:


> This whole thread is just too complex for my engineering education. So, somewhere along the way, would one of you life science folks give me a synopsis of what this thread is about and any conclusions drawn from this thread?


I think a lot of the discussion is about "natural" beekeeping and whether so-called "soft" treatments are basically handicapping the bees in the natural ways that they manage to survive and thrive.

There are other discussions, too, about effects of pesticides, fungicides, herbicides, and antimicrobial treatments on bees and the microbial communities that are part of their support team.

And there's a side-bar discussion about "pro-biotics" — about ways to feed microbes back into the system if they've been depleted or otherwise might be helpful.


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## Michael Bush (Aug 2, 2002)

>So, somewhere along the way, would one of you life science folks give me a synopsis of what this thread is about and any conclusions drawn from this thread? 

1) Wiping out the microbes and letting them rebuild often results in one microbe overgrowing and taking over.
2) Once the microbes have been wiped out (via antibiotics, essential oils, organic acids etc.) the evidence is that in 25 years they have still not recovered.
3) Microbes often live in complex relationships between species. They live in communities where one microbe generates waste that is the food for another and often they supress each other enough that one does not take over. Once this complex relationship is destroyed, it may or may not ever get reestablished.
4) Microbes help the bees defend themselves from pathogens.
5) Microbes are necessary for the fermentation of bee bread. Not just one microbe, but several and the fermentation process is in stages involving different microbes.


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## WLC (Feb 7, 2010)

When I added human probiotics to cracked corn (my feedstock), I could easily detect a butter milk smell within a week.
Recently, I added pollen pellets to that ferment, and it now smells like vinegar (acetic acid).
I will also add some raw, unpasteurized honey, and pollen to cracked corn directly to see how it ferments.

I think that's there's a lot to learn about how 'living' hive products can be applied, which is one reason why I do these simple fermentation investigations.


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## pascal (Oct 1, 2010)

*Re: Microbial ecology - HOW? about probiotics*

I wish I'm not hacking this thread but, trying to add my 2 cents, I found this on the net: 



> Although colonies fed with pre- and probiotically modified
> feeds showed enhanced development during the first part of the
> season, in the autumn the number of combs covered with bees
> and the reserves of food for winter were comparable with those
> found in the control group.


At this adress http://world-food.net/download/journals/2013-issue_3&4/2013-issue_3&4-environment/e191.pdf

My opinion is that treatments occurs during winter and early spring, cleaning the so called "good" bacteria as well as the "bad" one. But bees rebuild them after like humans after antibiotherapy. Maybee probiotics in spring feeding will help a quicker recovery? I'll give them a try this spring on a few hives if somebody could help me finding what to use (the swedish lab bacteria can't be found in north america but maybe one biogen product will do the job?)

__________________________
Pascal Fournier 88 h (last count), 8 years, T (FA, OA)


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## Kofu (Jan 26, 2011)

Michael Bush said:


> 1) Wiping out the microbes and letting them rebuild often results in one microbe overgrowing and taking over.


One or more microbes take over, selected by the fact that they survived the treatment that wiped out the others, or that they moved in ahead of the others — in any case, a different set of microbes that's 'out of balance' compared to what existed before, and less likely to support the bees.



Michael Bush said:


> 2) Once the microbes have been wiped out (via antibiotics, essential oils, organic acids etc.) the evidence is that in 25 years they have still not recovered.


That's definitely an overstatement. True in some respects, with evidence that effects can linger for a very long time. If the 'evidence' is that the evidence itself isn't erased, then yes, the microbes "have still not recovered." That's where we get into the "metabolic costs" of microbial adaptation to treatments.



Michael Bush said:


> 3) Microbes often live in complex relationships between species. They live in communities where one microbe generates waste that is the food for another and often they supress each other enough that one does not take over. Once this complex relationship is destroyed, it may or may not ever get reestablished.


Some sort of new complex relationship is certainly reestablished, probably involving a different set of microbes that won't support the bees in the best way.

Dean mentioned E.O. Wilson's experiment, where Wilson 'tented' and fumigated an island in the Caribbean, then watched to see what happened to the ecological set-up as different species of insects moved in over time. A movie that mentions this famous experiment, 'Lord of the Ants,' is on YouTube,and here's a page with a transcript.

NARRATOR: Back in 1965, Ed Wilson teamed up with a mathematician-turned-biologist, Daniel Simberloff, to carry out an experiment. They wanted to work out how the number of species an isolated island could support depended on the island's size and distance from the mainland. Using a mangrove island as a simple model, the plan was to make an inventory of every living species, and then wipe them all out.

ED WILSON: We set out to observe the re-colonization, the rebirth of life, animal life, we didn't want to kill the island; we didn't want to kill the plants; but we wanted to watch to see what happened.​
[ . . . ]​
NARRATOR: After several years of experimenting, they were able to answer some fundamental questions about life on islands.

ED WILSON: If you start out with a certain number of species and then you remove them, all of them, and then you let other species come back in, would they return to an equilibrium—that is, back to the same level, and stay there—that they were at before you removed the old fauna? The answer to that was they do. They come back at that level. The second question we asked was, "Would it be the same species?" For the most part, no, they were different species.​
So there's a case to be made, by analogy and supported by research, that a different set of microbes will take the place of the set that bees evolved with.

.


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## Kofu (Jan 26, 2011)

A key citation in this discussion is a study by Nancy A. Moran and her team.

Long-term exposure to antibiotics has caused accumulation of resistance determinants in the gut microbiota of honeybees. Tian B, Fadhil NH, Powell JE, Kwong WK, and Moran NA. 2012. mBio 3(6):e00377-12. doi:10.1128/mBio.00377-12.

Specifically, they looked for genetic markers of bacterial resistance to tetracycline, used to treat for foulbrood diseases.

Since the 1950s, the antibiotic oxytetracycline has been widely applied to colonies of bees in the United States to control larval foulbrood diseases caused by the bacteria _Melissococcus pluton_ and _Paenibacillus larvae_; oxytetracycline was the only antibiotic approved for use in beekeeping until 2005. Antibiotic treatment potentially impacts the microbiota typical of healthy hosts. Compared to the gut microbiota of humans and other mammals, the honeybee gut microbiota provides a distinctive and relatively simple bacterial community exposed to a known antibiotic, and the differences in treatment history between honeybees in different localities provide an opportunity to observe the impact of selection pressure by application of a single antibiotic over several decades.​
They were able to find the 'markers' for tetracycline resistance in bees all over the U.S. Fewer in bees from Dee Lusby's apiaries in Arizona, but even the bacteria in her bees showed traces of exposure to tetracycline, in lines of bees that came off the commercial treatment wagon more than twenty-five years ago.

The variation in tetracycline resistance determinants observed among American honeybee colonies may reflect different recent histories of oxytetracycline treatment for individual colonies. Most of our samples had unknown histories of antibiotic treatment, largely due to their origin from mixing other colonies or from commercial bee packages. To determine whether resistance loci decline when antibiotic exposure is terminated, we obtained samples from four managed colonies in southern Arizona that were unusual in having not been treated directly or mixed with outside bees for over 25 years and samples from long-established feral colonies in Utah, also expected to have no recent exposure. These samples showed markedly lower copy numbers of resistance loci compared to other American samples. The FL, MD, and AZ (USDA) colonies, which had no antibiotic treatment for at least 2 years prior to sampling, showed intermediate levels of resistance loci. The highest frequencies were observed for colonies in CT and WA established from package bees purchased from commercial bee suppliers 0 to 12 months before sampling.​
Consequences:

Prolonged exposure to a single broad-spectrum antibiotic imposes strong selective pressure on a microbial community that is expected to result in loss of strain diversity. It is possible that antibiotic perturbation may shift the gut microbiota to an alternative state that is broadly similar but different in critical aspects. These shifts could affect host health: in the case of the distinctive gut bacteria of honeybees and bumblebees, metagenomic and experimental studies suggest beneficial roles in neutralization of dietary toxins, nutrition, and in defense against pathogens.​
That's why the microbes are important, and why any sort of treatment needs to be weighed carefully — or avoided entirely, some would say.


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## Phoebee (Jan 29, 2014)

WLC said:


> Phoebee:
> 
> Are you talking about the Personal Genome Machine? Or, the Ion Proton perhaps?


The SN article sidebar was talking about the Life Technologies Ion Proton P2 sequencing machine.

I think I need one of those.


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## WLC (Feb 7, 2010)

Phoebee said:


> The SN article sidebar was talking about the Life Technologies Ion Proton P2 sequencing machine.
> 
> I think I need one of those.


Phoebee:

Either your own institution has ion torrent sequencers, or you send them out to a sequencing company that does.

However, it's still costly and technically challenging enough that even bacterial sequencing usually gets done by institutions.


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## deknow (Jul 17, 2006)

Collecting data is one thing, analyzing it is another.
The Mattila study claimed to find a lot of new bacterial species...the Moran lab, using the same data set showed that it was poor statistical treatment (and poor use of the data in general) that led them to such conclusions.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041250

I'll note that Ramona used the conclusions in the Mattila study (that multiple mated queens had more microbial diversity in the workers) in some of her presentations...I think I've cited them before as well. It is pretty clear that the conclusions of the Mattila study were way off base...but remember the Moran version did nothing to collect more data....they used about twice as much of the raw data as the Mattila group did, and they verified the identity of species to a 97% criteria, whereas to the best of my reading, the Mattila team used half as much data and verified the identity of species to a 60% criteria. 

It is hard to know what happened here...but I'm always suspicious of math/statistical software packages (or specialists) where statistics are run like a video game, and the criteria is massaged until it makes a pretty graph.

Deknow


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## WLC (Feb 7, 2010)

One of the issues in the parade of bacterial community studies that have been done on Honeybees is that they use different methodologies. The other is that they're done using colonies from all over the map so that they're really just surveys of bacterial communities.

I think the real payoff is in applications using hive related products.

For example, I've noticed a difference in cracked corn ferments using pollen vs honey as an inoculant.


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## deknow (Jul 17, 2006)

WLC said:


> One of the issues in the parade of bacterial community studies that have been done on Honeybees is that they use different methodologies.


Unless someone can propose a 'perfect methodology' that will reveal all that we want to know (whether we are looking for it or not), and that everyone can agree upon it, and over time it shows itself to 'reveal all' better than any other methodology....then we are better off with different methodologies being explored.
One of the problem with children is that many are raised 'wrongly'...although this is a terrible state of affairs, it is more than slightly better than making sure all kids are raised 'rightly'.



> The other is that they're done using colonies from all over the map so that they're really just surveys of bacterial communities.


Given what we know about such complex microbial communities (that their makeup generally depends greatly on the other microbial communities that live nearby), the fact that these surveys that are made from all over the map (literally) show 95-99% of the individual gut microbes are from the same 8 core species, and the fact that we can see clear vertical heritability of these microbes from generation to generation of bees tells us quite a bit about the ecology of these communities.

I think the real payoff is the understanding of how complex such communities are and realizing that such complexity is at the root of 'bigger life'.

deknow



> For example, I've noticed a difference in cracked corn ferments using pollen vs honey as an inoculant.


It would be hard for me to imagine that someone with the level of expertise you claim would even consider that they might _not_ see a difference (assuming your inoculation is doing anything). You already know the inoculants are different.


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## Phoebee (Jan 29, 2014)

WLC said:


> Phoebee:
> 
> Either your own institution has ion torrent sequencers, or you send them out to a sequencing company that does.
> 
> However, it's still costly and technically challenging enough that even bacterial sequencing usually gets done by institutions.


"I* need *one of those" is my in-house joke to make my wife turn pale. You should see her response when I say that while drooling over a business jet or a WWII fighter.

Another recent Science News Magazine "special report" was featured in the Jan 11, 2014 issue. On the cover they called it "I, Ecosystem," but the actual title is "Microscopic Menagerie." Several other pieces in the issue bear on this topic as well, especially "Mother Lode", which has to do with some complex oligosaccharides found in human breast milk, and the profound effect they have on establishing intestinal flora in infants. There is even one on the effect microbes in the gut have on an Autism-like disease in mice, with speculation that something like it could be happening in humans.

The main article underscores the great difficulty in producing lab cultures of symbiotic microbes ("an estimated 99% of microbial life can't be cultured"). It also notes that while only about 3 lbs of a typical human is made of microbes (mostly bacteria), the cell count of the bacteria are much greater than the cell count of the host (bacteria are tiny) and the number of genes they carry may exceed the human nuclear genome by a factor of 400. 

Some things to think about when you're discussing what microbes they've been able to isolate and test in the lab: the true symbionts have lost genes needed to live independently, and depend on their host, just as their host depends on them.

And providing milk products or bacteria associated with digesting milk, or other "pro-biotics" not specific to bees, is no doubt going to wind up being a hilariously futile exercise.


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## deknow (Jul 17, 2006)

The other thought that I'll offer (especially to you, Kirk), is that we are looking at this all wrong.

Consider the aphid study I posted earlier in this thread. The mutation of ONE basepair in One gene in One species of gut bacteria changes the heat tolerance profile for the host aphid itself.

Even if we try to take 50 strains of these core bacteria and map their relationships....this one produces a substance that is antagonistic to that one....this one eats the waste of that one....the other one over there blooms in the early spring when temps are low and it is damp...this one eats the other one in warm weather when there is a flow....

Try to imagine the 'map' where each of these 50 strains is labled and lines drawn between it and other microbes it lives with to indicate each kind of interaction. 

Draw a bigger map around that to indicate the social and biological behavior of bees and draw the lines to show how they interact with each of these species.

Now draw a bigger map around that to indicate all the environmental, seasonal, weather related, and forage related influence on each one of these strains (pollution, pesticides, herbicides, nectar sources, temperature, other microbial communities in the area, quality of forage, etc)....draw the lines to show how they interact with the microbial community and with the bees.

Now, change (mutate) one SINGLE base pair in one SINGLE bacterial strain and something as dramatic as heat tolerance profile for the host changes...how must the whole ball of string be restrung?

There is so much data, so many individual (and dynamic) relationships, so many ways such a system is able to balance itself (not 'balance' as in 'to be balanced", but more as 'to be in the process of balancing') that other ways of looking at this data are necessary. There are probably fractal tools (the kinds of tools one would use to analyze or draw clouds), probably some quantum computing technique that can eventually be applied...but I think we can learn that the simplicity of running such a system comes from its complexity. 

How does an organism _happen_ to acquire gut microbes that can help it assimilate what it _happens_ to eat? Where do those microbes (or even just their digestion genes) come from in the host? 
Q...what kinds of bacteria live on 'new food x"?
A: I don't know what kinds, but some of them are bound to be producing enzymes that can digest 'new food x'....the bacteria is thriving on 'new food x' because it can metabolize abundant food.

deknow


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## WLC (Feb 7, 2010)

"And providing milk products or bacteria associated with digesting milk, or other "pro-biotics" not specific to bees, is no doubt going to wind up being a hilariously futile exercise."

While it's O.K. to be skeptical, I've already put some in a pro feeder, and they took about a quart before the nectar flow started.

I've also inoculated a completed LAB ferment with pollen pellets, and it made acetic acid (vinegar).

While you may not think that using pollen pellets to produce acetic acid is such a big deal, I assure you that to anyone with cases of Ball jars sitting around, it means plenty.

'Pollen for Pickles'.  

I think I've got another first going with that.


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## sqkcrk (Dec 10, 2005)

What about Prebiotics?


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## WLC (Feb 7, 2010)

I don't have any acacia gum handy.


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## JBJ (Jan 27, 2005)

Michael Bush said:


> >It would be difficult to make broad statements about oils in general as the pharmacological, medicinal, metabolic properties vary substantially between particular plant essences.
> 
> They are more similar than you might expect. All of them are the defense system of the plant so all of them are antimicrobial. They may be more or less antimicrobial and there may be other properties to some of them, but this antimicrobial activity is pretty consistent.


Some essential oils are for attraction of pollinators, this is why flowers smell good to bees. Further, the densities/boiling points of particular plant essences vary quite a bit. Some will volatilize at very low temperatures while others have higher vapor pressures. Also the LD50 values can be substantially different depending on the inherent toxicity of the particular material in question.


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## Phoebee (Jan 29, 2014)

Prebiotic? Define that in a way that doesn't mean "proper nutrients". The complex oligosaccharides in human milk qualify ... they may be indigestible to babies but they strongly affect bacteria, encouraging the good ones and banishing the bad. But those in human milk are very different from those in cow milk, and bees have their own very different systems. What bees get in their diets affects them, same as humans. For me, well, there's this polysaccharide found in legumes that makes _Clostridium perfingens _want to party ....

Here's an interesting segment from "Microscopic Menagerie." 

Two species of Jewel Wasps, _Nasonia giraulti _and _N. vitripennis_, only diverged around a million years ago. Usually the offspring of two such closely-related species could mate successfully, but the second-generation male offspring of such a hybrid are doomed. Their larvae become dark and die ... looks a bit like European foulbrood.

They exposed the hybrids to antibiotics, killing off the intestinal flora, and the offspring survived (I'd bet with some issues due to lack of flora, but at least this shows that the flora are the problem). If normal flora are then re-introduced the larvae darken and die.

So it would appear that a strong barrier to these species hybridizing, about as effective as, say, an ocean between them, is incompatibility of the microbes in their guts. Makes one wonder about problems with some honeybee hybrids.


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## Michael Bush (Aug 2, 2002)

>Prebiotic? Define that in a way that doesn't mean "proper nutrients". 

There is a difference between something that is directly a nutrient used by the host and one used by the symbiotic microbes. Prebiotic is proper nutrients for the microbes, not necessarily for the host.


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## Phoebee (Jan 29, 2014)

Michael Bush said:


> >Prebiotic? Define that in a way that doesn't mean "proper nutrients".
> 
> There is a difference between something that is directly a nutrient used by the host and one used by the symbiotic microbes. Prebiotic is proper nutrients for the microbes, not necessarily for the host.


Still, the term is broadly included in nutrition. Cows and termites can't digest cellulose. Their microbes can. So that would make the major portion of the bovine diet prebiotic. The complex oligosaccharides in human milk are clearly there for the microbes. Bees would starve without their unique flora, but we would probably die of vitamin deficiencies without ours. In each case, the host must eat what the microbes need.

It is good to know what these nutrients are, but we and the bees have been getting this in our diets for millions of years. Mostly, prebiotic is a term invented to market bottles of stuff.

Not that I think you need to be told this.


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## Kofu (Jan 26, 2011)

The video from the talk by Laurie Herboldsheimer (Ramona) is now online. I was there for the talk, and it's great to be able to watch it again, to be able to stop it and take notes, and think about the implications.





https://www.youtube.com/watch?v=fQvzIH016tc​
I suppose at least a few people have been thinking a lot about this sort of thing. But for those of us who haven't, the video is a very useful introduction to the basic ideas, with enough details about how microbes fit into different functions in the hive that we can now begin to talk about better beekeeping.

Ramona sells herself short when she says she doesn't have a lot of answers, on the level of practical applications. In her talk, she suggests a few applications. Thinking of _possible_ applications is just a different way of thinking about the problems.

One application is to be able to think more clearly about the impact that specific sorts of treatment may have, to anticipate the “side effects” aka collateral damage, and to find specific ways to buffer those effects or to help the bees to recover. (See the slide in the 'cover shot' of the video above for ideas.)

Ditto for agricultural chemicals — fungicides, pesticides, etc. - that the bees may be exposed to. For example, when the bees are unable to successfully make an emergency queen and there’s a chance that farmers in the area are using fungicides, it’s a practical step to find out who, why, and what, and perhaps to take steps to avoid that situation in the future. Perhaps it would be useful to be able to bring in beebread from an apiary that was not exposed? Anyway, thinking about the microbes (yeasts, molds, and bacteria, each of them with their own dynamics) helps us to understand “what then must we do.”

Another application of this knowledge relates to a point that Dean makes (when he steps on stage toward the end of the talk).

The key for all of us (collectively and individually) is to keep the bees long enough to get those populations (the bees and the different sorts of microbes) to stabilize, understanding that the microbial cultures are always balancing, but are never really “balanced.”​
This gives us the idea of a timeline. How many generations of brood, how many years, removed are we from the package, from the prophylactic antibiotic treatments that are common for shipping bees across state lines? How far removed are we from other chemicals, or from "soft" treatments? And when we start thinking about it that way, in terms of the impacts of _specific_ sorts of treatments on _specific_ sorts of microbes, and how these tend to affect the bees in _specific_ ways, we'll begin to understand what sorts of problems shake out at different stages over time. The time scale, we already know, can be as long as 25 years and more, but we can begin to find the increments on the scale between 0 and 25.


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## deknow (Jul 17, 2006)

WLC said:


> I've also inoculated a completed LAB ferment with pollen pellets, and it made acetic acid (vinegar).


is it your contention that the acetic acid was produced from microbes in the beebread metabolizing the lactic acid? If the acetic acid was produced by leftover sugars and alcohols being metabolized (or from the contents of the inoculation), that isn't a very big deal....beekeepers make vinegar from Mead all the time without adding pollen to the mix.



> While you may not think that using pollen pellets to produce acetic acid is such a big deal, I assure you that to anyone with cases of Ball jars sitting around, it means plenty.
> 
> 'Pollen for Pickles'.
> 
> I think I've got another first going with that.


We called beebread a "pollen pickle" in our book and in many of our talks and writings since.

Deknow


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## WLC (Feb 7, 2010)

Dean:

I've used raw pollen as an inoculant. YS Eco Bee Farms fresh bee pollen. Not bee bread.

I'm starting to form the opinion that it's the pollen that's critical in turning nectar into honey.

While I'm not an expert in bee foraging behavior, I've read that Honeybees will always mix nectar with pollen before they return to the hive.

So far, I wouldn't call the odor from non pollen ferments to be 'attractive' to my own sense of smell (although I do find the 'buttermilk' odor telling.

There was something pleasing about the pollen ferment of the completed LAB/cracked corn ferment. It had a pleasing, strong, vinegar smell.

I've some other pollen, honey, LAB, cracked corn, and milk ferments going on.

By examining what various combinations of ferments do, I think that we can gain some useful insights into the role of the different microbial communities in pollen, honey, and LAB/probiotics.


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## Michael Bush (Aug 2, 2002)

>Still, the term is broadly included in nutrition. Cows and termites can't digest cellulose. Their microbes can. So that would make the major portion of the bovine diet prebiotic. 

I can't digest "fiber" (which usually means cellulose). No amount of microbes can fix that nor what I eat. Fiber is not a nutrient for me at all. But it is a place for the microbes to live.

In the case of the cow, that broken down cellulose is their nutrition.


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## Kofu (Jan 26, 2011)

deknow said:


> The other thought that I'll offer (especially to you, Kirk), is that we are looking at this all wrong. [...]
> 
> There is so much data, so many individual (and dynamic) relationships, so many ways such a system is able to balance itself (not 'balance' as in 'to be balanced", but more as 'to be in the process of balancing') that other ways of looking at this data are necessary. There are probably fractal tools (the kinds of tools one would use to analyze or draw clouds), probably some quantum computing technique that can eventually be applied...but I think we can learn that the simplicity of running such a system comes from its complexity.


This describes the nature of "emergent systems." So we can see a friend looking through a menu and guess what they're thinking without mapping the individual species of bacteria in their gut.

Similarly, the observation that fungicides affect beebread which affects queen rearing can be understood in a general way, even though we don't understand all the details of what's happening. 

"Beebread involves mold. Mold is a fungus. Fungicides kill mold. Beebread isn't what it ought to be." I saw a picture recently, perhaps in your website or in a publication linked from it, of the molds that were cultured from clean beebread versus from tainted beebread, that's worth a thousand words. But try to convince the company that sold the fungicides to a local farmer to stop, and all of a sudden it gets immensely complex.

Or take the "can't see the forest for the trees" idea. A trained eye can see the differences between different sorts of forests, and can tell at a glance how well a forest has recovered after a fire. It's not necessary, although it would be helpful, to understand all the different species of flora and fauna involved. So my point is that ordinary beekeepers _can_ actually begin to see things in new ways, and we can change the way we do beekeeping, before the scientists have built a quantum fractal computer model to track everything at every level.


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## WLC (Feb 7, 2010)

I think that you're missing a key point: what is the impact of agricultural fungicides on the species diversity of nectar and pollen sources?

For instance, how are Metschnikowia reukaufii and M. gruessii impacted? They key nectar species. So, if they're not being brought back into the hive in sufficient numbers, it could impact overall hive productivity.


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## deknow (Jul 17, 2006)

WLC said:


> I've used raw pollen as an inoculant. YS Eco Bee Farms fresh bee pollen. Not bee bread.


I assume we are talking about 'trapped pollen'...which is not beebread, but by no means is it 'raw', 'uninoculated' or 'just pollen'.

The fermentation begins _as the bees pack the pollen in their corbicular baskets_. They wet the pollen with nectar/honey from their honey stomach, which both inoculates with whatever is in the nectar and/or honeystomach, and it wets the pollen, which allows all kinds of microbes to germinate (not sure if 'germinate' is the proper term'...'bloom' might be more accurate).

This is a multi-stage process that begins with many, many varieties of yeasts and molds, transforming into progressively more acidic states that are conducive to a less diverse community of LAB and the like (everyone should taste 'beebread' and 'trapped pollen' next to one another...beebread _is_ a pollen pickle).

But the point is that some (especially fungal) transformations happen very early in the process...like while it is still on the bee being transported, and in the first 12 hours _after_ being collected. At least one of these enhances the amount of 24 methalene cholestoral (a sterol required for the bees to make more bees).

I'm sure that some of the 'first 12 hour fermentations' that happen to beebread are happening to trapped pollen while it is sitting in the pollen trap...and I'm sure that some of them only happen when packed in the cells by the next set of bees. But to say (or think) that trapped pollen is, by any reasonable definition 'raw' is to mislead or be mislead.


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## deknow (Jul 17, 2006)

Michael Bush said:


> >Still, the term is broadly included in nutrition. Cows and termites can't digest cellulose. Their microbes can. So that would make the major portion of the bovine diet prebiotic.
> 
> I can't digest "fiber" (which usually means cellulose). No amount of microbes can fix that nor what I eat. Fiber is not a nutrient for me at all. But it is a place for the microbes to live.


I haven't looked into this claim critically...I think I heard it detailed on NPR.

In general, Japanese people can digest raw nori seaweed...non-Japanese people cannot. The culture in Japan for so long included eating raw nori, that the microbes (or their nori digesting genes) that live on the seaweed became incorporated into the cultural microflora.

This isn't happening in the US, even with so much sushi being eaten....because we all eat cooked nori...the microbes are killed (and presumably the cooking makes it possible for us to digest it without the microbes).

I found the story (audio and transcript...it's short)
http://www.npr.org/templates/story/story.php?storyId=125675700


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## Phoebee (Jan 29, 2014)

Fungi and yeasts are interesting in the types of mutualism they create. Leafcutter ants harvest leaves in order to grow fungi. However, not all fungi are good. I'm not up on zombie bees, but zombie ants are under the influence of particular fungi that take over the behavior of the ants. Fatally.

http://www.sciencedaily.com/releases/2011/03/110302171309.htm

There is an argument going around that yeasts have done the same thing to humans. Our species of happy hunter-gatherers took up farming. Good evidence exists that the reason we started growing grain was not for food, but for brewing beer, which was done for major gatherings. In fact, this "invention" of beer seems to have happened more than once. These events took planning and social organization. It is looking like beer is at the core of civilization, and you can argue (after a few beers) that yeasts created our civilization so that we would grow their food. The zombie influence is evident at happy hour.


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## WLC (Feb 7, 2010)

Even actual 'raw' pollen has it's own microflora, so it's a matter of the difference between uncollected pollen vs pollen pellets.

'Fresh' pollen pellets will do the job of representing a sample of microflora from outside the hive.

My own point was that it can be used for ferments.

I would be satisfied if it's a source of Metchinikowia reukaufii.


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## deknow (Jul 17, 2006)

WLC said:


> Even actual 'raw' pollen has it's own microflora, so it's a matter of the difference between uncollected pollen vs pollen pellets.


I think most hand collected pollen is 'dry' and not in the process of fermenting (at least to a large extent).

To call trapped pollen 'raw' is to imply something that just isn't true.



> 'Fresh' pollen pellets will do the job of representing a sample of microflora from outside the hive.


I think the air does a good job of representing a sample of microflora from outside the hive. Fresh pollen pellets contain much more (and much different) critters and substances than anything that one could call 'raw pollen'.


> My own point was that it can be used for ferments.


The air can be used for ferments...and often is for making vinegar.



> I would be satisfied if it's a source of Metchinikowia reukaufii.


What would this satisfy?

deknow


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## deknow (Jul 17, 2006)

WLC said:


> I think that you're missing a key point: what is the impact of agricultural fungicides on the species diversity of nectar and pollen sources?


...what is the effect of beekeeper applied essential oils (remember the amount of thymol found in the USDA pollen survey?) acting as fungicides in the hive? Remember why people use honeyBhealthy.

deknow


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## WLC (Feb 7, 2010)

"I would be satisfied if it's a source of Metchinikowia reukaufii. "
>What would this satisfy?<

Remember the paper you objected to showing a 'preference' for M. reukaufi inoculated 'nectar' vs other?

If I'm in the mood to feed probiotics to my bees, I would at least want them to find it more palatable.

Since feeding probiotics to colonies is an issue of interest, it might be better to use a more natural source of 'probiotics' like pollen, etc. .

Besides, there's no harm in having a favorite nectar yeast.

By the way, I still think that agricultural fungicides may be knocking down nectar yeasts in the field. That would mean less forage.


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## Ramona (Apr 26, 2008)

Phoebee said:


> ...Leafcutter ants harvest leaves in order to grow fungi.


Here is a link to a portion of a very old book (~1912?) on the fungal eating ants of North America:

http://antbase.org/ants/publications/10543/10543.pdf

The descriptions of the ants growing the fungus and feeding it to the young will bring tears to your eyes....very poetic.




> you can argue (after a few beers) that yeasts created our civilization so that we would grow their food.


I sometimes joke that the microbes (bacteria especially) evolved humans so that we could develop advanced technologies to transport them more quickly and effectively around the planet. Bacteria don't have legs or wings and, on their own, move rather slowly. Hitchhiking on an airplane can take them around the world in a matter of hours. Some bacteria have even survived on the bodies of spacecraft! 

http://microbepost.org/2013/07/30/n...ly-facility-and-the-extremotolerant-bacteria/


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## WLC (Feb 7, 2010)

Thanks Ramona, I'll check it out in a bit. (Oops-broken link.)

Have you read, "The Leafcutter Ants: Civilization by Instinct"? It's by Bert Holldobler and E.O. Wilson.

I enjoy drawing parallels between formic acid in ants and lactic acid in Honeybees from time to time.

By the way, nice hat in the video. Where's Dean's hat? 

Ramona, just get some mason jars, pick a substrate, and start growing your own. I don't want to be the only one in the world 'sniffing' this stuff.


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## WLC (Feb 7, 2010)

It looks like M reukaufii and even Metchnikowia gruessii are adapted for transportation from flower to flower by bees.

"Yeasts are also adapted to dispersion and then survival. One example of this is the crossshaped
yeast Metchnikowia gruessii that is dispersed by bees visiting flowers during its
feeding periods in the day. This cross-formed yeast species is adapted to the glossa or
tongue of the bees and so use the insect as a means of dispersal from one flower to the next."

http://cdn.intechopen.com/pdfs/23569/InTech Yeasts_biodiversity_and_its_significance_case_studies_in_natural_and_human_related_environments_ex_situ_preservation_applications and_challenges.pdf

I think I'm on the right track after all.


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## Kofu (Jan 26, 2011)

The "worth-a-thousand-words" picture of two lab cultures from bee bread, one affected by fungicide and the other not, is in Diane Sammataro's presentation (@ ~13:00) at the American Bee Research Conference in Orlando, FL, Jan. 15th, 2010.





http://www.youtube.com/watch?&v=NI9Z73UAX-g​
The video went up on YouTube just last summer, and Bernard posted it to BeeSource a few months ago. (thanks!)

An article on the web, published last year, gives a lot more information along these lines (and more pictures). "Fungicide Contamination Reduces Beneficial Fungi in Bee Bread Based on an Area-Wide Field Study in Honey Bee, _Apis mellifera_, Colonies," Jay A. Yoder, Andrew J. Jajack, Andrew E. Rosselot, Terrance J. Smith, Mary Clare Yerke, and Diana Sammataro. _Journal of Toxicology and Environmental Health, Part A_, 76:587–600, 2013.
http://naldc.nal.usda.gov/download/57094/PDF​
A search of BeeSource for discussion pulls up a thread of discussion, Fungicides Thought to be Safe Harming Bees?, with a number of other citations.


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## Phoebee (Jan 29, 2014)

I've not seen anything definitive on it ... I haven't looked yet ... but as yeasts are single-celled fungi, I would not be surprised in the least if some fungicides also killed beneficial yeasts.

EDIT: You don't have to look far. There are papers out on the effects of fungicides on fruits such as apples and grapes. The abstracts show a pronounced effect on yeast species on those, so that should raise concerns about their effects on beneficial yeasts employed by bees.

Not proof it does happen, but reason for caution.

http://link.springer.com/article/10.1007/BF03175547#page-1


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## Kofu (Jan 26, 2011)

An editorial in _Nature_ about regulating human faecal transplants got me looking for the studies that distinguish the different patterns of microbial communities in the human gut. Apparently there are three types, called enterotypes. And it looks like the enterotypes are really different from one another.

The lead study was published about three years ago: Enterotypes of the human gut microbiome. _Nature_. 473, 174–180 (12 May 2011). Here's a _Wired_ article about it, Gut-Bacteria Mapping Finds Three Global Varieties.

Research on honeybee microbiota is said to be 10-15 years behind research on humans. On the other hand, the technology is much further developed now, and can be readily applied. 

A Google search for apis mellifera enterotypes pulls up one scholarly hit, Paratransgenesis: An Approach to Improve Colony Health and Molecular Insight in Honey Bees (Apis mellifera)? And it looks like some people are already thinking about applications and not just the pure science.

The gut microbiota is also of interest as a resource for paratransgenesis; a Trojan horse strategy based on genetically modified symbiotic microbes that express effector molecules antagonizing development or transmission of pathogens. Paratransgenesis was originally engineered to combat human diseases and agricultural pests that are vectored by insects. We suggest an alternative use, as a method to promote health of honey bees and to expand the molecular toolbox for research on this beneficial social insect.​
The word 'enterotype' comes up only incidentally, in a citation of the other article. But it seems a good review of the research we've been talking about here.


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## WLC (Feb 7, 2010)

Thanks for the links Kofu.

I found this quote from the Wired article to be a useful summation: "The search returned three distinctive “enterotypes,” or bacterial communities dominated by a distinct genus — Bacteroides, Prevotella or Ruminococcus — each of which is found with a particular community of bacteria".

It would be interesting to know if the same thing is occurring in Honeybees since I have noted that some surveys of Microflora in Honeybees have resulted in different microbial communities being found.

I have read the paratransgenesis article before, and found it to be an interesting approach, but problematic if applied to Honeybees.


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## Michael Bush (Aug 2, 2002)

> a Trojan horse strategy based on genetically modified symbiotic microbes...

Great. Now we don't just have to worry about Monsanto suing us for "stealing" their GMO bee genetics when they show up in our bees, but suing us over the microbes in the gut of the bee... and we all know their engineered microbes will be SO much better and have no ill side effects, if there are, it will be so easy to remove them and change back... right?


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## Kofu (Jan 26, 2011)

Michael Bush said:


> Great. Now we don't just have to worry about Monsanto suing us for "stealing" their GMO bee genetics when they show up in our bees, but suing us over the microbes in the gut of the bee... and we all know their engineered microbes will be SO much better and have no ill side effects, if there are, it will be so easy to remove them and change back... right?


I wondered who was still reading this thread and if anyone would notice... Brave New World, isn't it?


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## FlowerPlanter (Aug 3, 2011)

Hopefully there's no money in GMO probiotics.

Has there been any studies to compare the microbes in CCD bees?
I heard there are a very small number of hives that did not fail to CCD. Someone should compare the microbes in them.

Symbeeotic suggests their product will work against CCD.


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## FlowerPlanter (Aug 3, 2011)

I too have experimented with fermenting probiotics.
After reading studies several by Gilliam. LABs cultures do better in fructuous. Made my own version of symbeeotic. Took honey (crush and strained with a lot of pollen) from a feral cut out that was at least 5 year old, and has done very well after being hived. Mixed with HFCS, a little soy isolate for nutrition and water. It’s been fermenting for over two months. It has a pleasant smell a little like yeast. Taste to confirm it's not mead or vinegar. I plan on adding it to some pollen patties in the spring. 

My opinion on feeding livestock probiotics is it can't hurt, but the benefits could be short lived (just like yogurt in humans) unless you feed it to them all the time. I don't think the livestock LAB are going to take up residence in the bees gut, it's not an environment that they can survive for very long. If it was then there are already there. Bees often drink from water and make other contact with live stock/wild animals to pick up microbes. Everything poops in ponds. Bees also taste everything they land on. 

There already in many patties like jester bee bread, nutrabee, feedbee, bee pro... and I bet this is one reason why the bee do so well on some of these patties. And pretty sure they are livestock probiotics. Seeing Symbeeotic is new technology and guarded secrets.


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## WLC (Feb 7, 2010)

I have fermented some different combinations of pollen, honey, and LAB using cracked corn as a feedstock, and I have found that one ferment had a distinctive 'sweet' note to it.

While it's far too early to say that I can do much more than subjectively characterize the ferments, I think that they've been instructive enough to say that it is a promising avenue of investigation for those interested in this field.

There are more than a few ways to use not only different feedstocks and sources of inoculants, there are also ways to change the 'balance' of the types of microbial communities that grow by using methods common to both microbiology and fermentation science.

For instance, metabisulphite (campden tablets) could be added to suppress the growth of LABs while favoring the growth of other microfloral communities like the yeasts. Adding amylase could breakdown starch in certain feedstocks making sugars available.

So, as it stands today, I think that it's safe to say that the field of microbial ecology as it relates to beekeeping is wide open.


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## irwin harlton (Jan 7, 2005)

Why not use the bacteria that is already in the bees gut by removing bee gut contents and growing that .Don't know what kind of environment you would need


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## WLC (Feb 7, 2010)

That would be the way to obtain cultures.

However, relatively large amounts of pollen and honey, some of it 'raw', are easily available.

So, it's easier. Eventually though, someone is going to start dissecting bees to do their own probiotic experiments.


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## Kofu (Jan 26, 2011)

For anyone following these threads about microflora in bees, here's a new study:

"How sweet it is: Honey bee bacteria help break down carbohydrates. First metatranscriptome of bee gut finds 19 different bacterial phyla", published in _Environmental Microbiology_. Irene Newton at Indiana University is named as the principle investigator.

Genes matching 19 bacterial phyla were identified, but dominating the communities were bacterial groups representing bacilli, gamma-proteobacteria and actinobacteria. Looking at how the entire group of microbiota could metabolize carbon-rich food sources by taking up sugars and fermenting carbohydrates, the team then developed a model of metabolism for the various microbial members.

One prominent microbial class in the bee gut, bacilli, is not able to make amino acids, but the other prominent classes -- actinobacteria and gamma-proteobacteria -- are believed to be able to synthesize all essential amino acids and may provide them to other members of the microbial community and to the bees themselves.

Bacilli, on the other hand, were found to encode and express enzymes involved in the production of short-chain fatty acids, such as acetate and lactate, that may be used by the host bee and by other microbial community members. As an interesting side note, two potential products of metabolism by the bacilli include a carbon storage and anti-freeze compound called 2,3-butanediol, which is used by microbes to prevent intracellular acidification, and acetoin, a chemical that produces a butter flavor in fermented foods.​
The press release linked above doesn't say which part of the bee gut was sampled. Other research says it makes a difference. Maybe the honey gut?

Via Professor Newton's page, here's the cite: 

Lee, F. J., Rusch, D. B., Stewart, F. J., Mattila, H. R. and Newton, I. L. G. (2014), Saccharide breakdown and fermentation by the honey bee gut microbiome. _Environmental Microbiology_. doi: 10.1111/1462-2920.12526​


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## deknow (Jul 17, 2006)

http://microdiv.blogspot.com/2014/07/how-sweet-it-is-metatranscriptomics-of.html

...this is a little more description of what was done. Note that the research here appears to have been done by analyzing 3 individual bees.

This data set appears to have been augmented by existing data..."As there was already a metagenome published for the honey bee gut, we took advantage of that dataset and used it in combination with the metatranscriptome to put together a genomic + transcriptomic view of metabolism performed by the major bacterial classes."
...I'm not sure how they were combined.

Of more concern is that a previous work in this area (identifying gut bacterial communities) was published by some of the same team:
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032962
...claimed to have found all kinds of new and important members of the bacterial community.

But

Through (what my limited understanding points to) statistical errors (in analyzing the gene sequences), and by throwing out about half the data they had.

This paper was completely (and specifically) refuted by researchers with the highest of credentials and reputation in this area:
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041250


> Mattila et al. [15] claimed to find novel groups of bacteria in honey bees. For example, they state “Importantly, the two dominant genera identified by our study (Succinivibrio and Oenococcus) have never been identified in honey bee colonies, which suggests that the major microbial players that are associated with honey bees were overlooked by previous methodologies.” In fact, 95% of the sequences they obtain from guts correspond to bacteria previously identified from honey bee guts, and their representative sequences that they assign to taxa such as “Succinivibrio” and “Oenococcus” show 100% identity to sequences from previous studies in honey bees (Table 3). The basis for their conclusions is not clear. Possibly only cultured isolates were considered in their analyses. We note that they report using a 60% confidence cutoff for assigning taxonomy descriptions. This criterion is expected to lead to some incorrect assignments, especially in combination with elimination of sequences from non-culture based studies.


Without looking closely at the paper (which I don't have access to), it would be hard to tell if they used a better methodology. What does seem odd is that many of these strains have had their genome mapped, and looking up what enzymes are coded for is something that can be done with a computer search.

deknow


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## Keith Jarrett (Dec 10, 2006)

Kofu said:


> For anyone following these threads about microflora in bees,


Kofu, thanks for posting.


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## Kofu (Jan 26, 2011)

Here's a new citation, from Nancy Moran's lab in Connecticut.

"Routes of acquisition of the gut microbiota of _Apis mellifera_." _Applied and Environmental Microbiology_. Doi:10.1128/AEM.01861-14, published online ahead of print on 19 September 2014.

Two words: proctodeal trophallaxis. Meticulously demonstrated.

And in answer to the question that started this thread, swapping frames of brood comb including nurse bees from hives that are doing well to hives that are struggling, seems like a good practical application.


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## Ramona (Apr 26, 2008)

Kofu, remember my "potty mouths" slide from the Philly talks last February?


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## Kofu (Jan 26, 2011)

Yes, I do. A lot of us remember, probably. Another beekeeper with better access to scholarly publications passed the article to me, and we all recognize the significance.

So, do you think this has practical applications? How would we know (or when would we have reason to think) that a hive suffering a poor bacterial mix would benefit from an "innoculation" from a healthier hive? I didn't read the article super-closely (all that _science_!), but it seems like just a frame of brood comb with accumulated bacteria from passing bees wouldn't be as good as a dose of healthy nurse bees and their fresh poop.


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