If you are a member and have anything that you feel is important to chemical free beekeeping, please email it to me. I will post it in this section in a future issue. Thank you. Dennis

Where ever you live in the world you should apply the information on working your bees that is given below when the weather conditions in your area are right. So take notes and be ready.

Cletus Notes  

 Hello Everyone,

Here on Lone Star Farms in Bryan, Texas our bees are working the aster and goldenrod plants. When that activity comes to a close we will be thinking about checking our mite levels in the hive.

We like to use the Kelley screen bottom board that comes with the slide in monitoring board. It is easy to use and it doesn`t disrupt the activities inside the hive. We merely paint a thin layer of vegetable oil on the board and slide it in the groove on the bottom board. We pull the board out and count the number of mites on the board after a twenty-four hour period. In my book “Beekeeping: A Personal Journey” I cover the acceptable levels of mite load according to the time of year.

If the mite load is too high, we will treat the hive using powdered sugar. Most books you read will tell you to treat the hive once every week for three weeks. This is not good information. You should treat each for a period of “four” weeks in order to cover the “Drone” brood which hatches in twenty-four days. Drone brood contains 80% of the mite load. If you only treat for twenty-one days you have missed the larger portion of mite load.

To treat a hive you should sprinkle one-cup of powdered sugar into each box on the hive. You should separate each box to perform the treatment not just dump the powdered sugar on the top box and hope that it goes down to the lower box. When you treat each box, leave some of the powdered sugar on the top bar of each frame. Most books will tell you to scrape it off down between the frames. If you leave some on the top bars, it will act like a time release. The bees will over time knock it down between the frames as they move around inside the hive.

You should perform this treatment once a week for four weeks. After that time, perform another mite load test. If the mite level is still too high after that second four week treatment, you should re-queen with a hygienic queen. If it is too late in the season to purchase a new queen, you will need to perform more powdered sugar treatments until it gets too cold in order to help keep the hive alive until a queen becomes available.

Finally, you should splash water under each have after each powdered sugar treatment. Powdered sugar will pass through the hive and land on the ground below the hive. The water will dissolve the powdered sugar. The bees in the area will forge under the hive to pick up the sweet sugar if present and in doing so, the mites that have fallen to the ground will merely hitch a ride on a forging bee and return to that bees hive.

I hope that this information helps and next month I will go into why mite levels go up. Enjoy your bees.

Dennis Brown    www.lonestarfarms.net

Author of “Beekeeping: A Personal Journey” and “Beekeeping: Questions and Answers”

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 Cassi J. Lee, Bryan D. Merrill, William J. Burnett, Sandra H. Burnett

 Bright red honey has been found in beehives in Utah, U.S.A.  KSL News reported that “red honey” was found in Davis, Salt Lake, Utah, and Washington Counties.  No local flower sources are known to generate red honey.  To date, at least 20 large and small-scale beekeepers have reported red in their hives.  Some beekeepers in Utah county estimate that their individual losses may be as high as $50,000 due to their projected loss in honey sales and potential problems in their bees. 

For two months, local beekeepers have speculated about the source of so much red in hives.  Recently, a local commercial beekeeper revealed that their operation had been open-pit feeding their bees with crushed candy cane byproduct dissolved in water.  Periodically during the summer, open troughs or perhaps pits containing this food were used throughout the four affected counties near locations where red honey was reported.  Scientists at Brigham Young University in Provo, Utah are working with the Utah Department of Agriculture and Food (UDAF) to determine whether the red honey is safe for bee or human consumption.  Samples of the crushed candy cane byproduct have been provided and samples of red honey have been collected from various locations. Laboratory tests are being performed to confirm ingredients in the food source and identify the concentration of components in the red honey .

 The crushed candy cane byproduct fed to honey bees may have potential hazards to both humans and bees due to red 40 dye and inverted sugar ingredients.  The Food and Drug Administration (FDA) allows a small amount of lead to be present in red 40.  Lead levels in the red honey may still be below FDA limits, but lab test results will need to confirm this.  Inverted sugar is also a potential problem.  If the syrup is warmed, the sugar components can convert into hydroxymethylfurfural (HMF).  Recent studies have shown that HMF is toxic to bees above a certain concentration.  Lab results will reveal if HMF, lead, and red 40 are present in the red honey. By strict interpretation of honey purity laws the UDAF may conclude that the “red honey” may not be considered honey at all.  The UDAF’s decision will affect the extent of financial losses in the state.  ; Even if the red honey is found safe for human consumption, its taste and appearance may be unappealing to the public, which may prevent normal sales.

 This event in Utah is reminiscent of recent honey contaminations in Red Hook, New York near a maraschino cherry factory and in France near an M&M waste-processing factory.  Both cases showed that the bees were gathering the factory byproduct which changed the color and taste of what the bees produced. 

             The results of the lab tests on the Utah red honey will be published in the November issue of Bee Culture. The article will discuss the extent of bees’ exposure to harmful ingredients and explain any health implications. For now, the UDAF officially recommends that Utah beekeepers not mix “red honey” with normal honey.

 This event is an example of how one beekeeper’s decision can impact many beekeepers in an area.  As beekeepers, like a hive of bees, we are interdependent.  We should work together for the health and safety of our bees and bee products.

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                                   Model of Dangerous Bee Disease in Jersey Provides Tool in Fight Against Honey Bee Infections

            Scientists at the University of Warwick have modeled an outbreak of the bee infection American foulbrood in Jersey, using a technique which could be applied to other honeybee diseases such as European foulbrood and the Varroa parasite.

  As well as modeling how bee infections spread, the method also allows scientists to simulate various disease control interventions in order to measure their efficacy.

  The researchers used two sets of data gathered two months apart during an outbreak of American foulbrood in Jersey in the summer of 2010. This provided two 'snapshots' of the disease from which they attempted to reconstruct the entire epidemic.

  Reconstructions like this are common for livestock infections, but this is the first time the method has been applied to bee disease.

  The research is published in the Journal of the Royal Society Interface

  American foulbrood is caused by the bacterium Paenibacillus larvae, which affects the larval stage of honey bees. It can cause the death of an entire hive within a matter of months.

  The Jersey data covered 450 honey bee hives, their location and their owners, from which the researchers built a computer simulation which modeled the speed at which the infection grew as well as how it spread geographically.

  Dr Samik Datta of the WIDER group, based at the School of Life Sciences at the University of Warwick, said: "Honeybees are one of the most important bee species in the world in terms of their contribution to food production through pollination.

  "But in the past 20 years there has been a marked increase in the level of disease among bee populations.

  "American foulbrood is an unusually virulent disease which can wipe out a hive within a few months.

  "By understanding how it is spreads from hive to hive, we then have a good basis to formulate interventions.

  "This is the first rigorous statistical analysis carried out on a honeybee disease epidemic that we are aware of."

  The model suggests that just under half of the 2010 Jersey infection spread was attributed to transmission by owners between their own hives.

  The researchers suggest that distance between colonies was another important factor in the spread of the disease, with the disease mostly spreading between hives less than 2km apart.

  The model also simulated the impact of different control strategies on controlling the epidemic and found that the measures taken by authorities in Jersey at the time – to inspect and destroy infected colonies – were the most effective.

  However, their model suggested an earlier intervention would have made disease extinction more likely.

  The researchers hope now to expand their model to investigate the spread of European Foulbrood, a more common bee disease in the UK. They also believe the same technique can be applied to the Varroa parasite.

  Dr Datta said: "Using just two snapshots of data we have been able to reconstruct this epidemic, and we are confident that our technique can be applied to a wide range of other outbreak scenarios."

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 The following article was sent in by member Tracey La Forge.

 Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees.

 Alejandra Vásquez mail,

* E-mail: Alejandra.Vasquez@med.lu.se(AV); Tobias.Olofsson@med.lu.se(TO)

Affiliation: Department of Laboratory Medicine, Medical Microbiology, Lund University, Lund, Sweden

Eva Forsgren,

Affiliation: Department of Ecology, Swedish University of Agricultural Sciences, Uppsala, Sweden

Ingemar Fries,

Affiliation: Department of Ecology, Swedish University of Agricultural Sciences, Uppsala, Sweden

Robert J. Paxton,

Affiliations: School of Biological Sciences, Queen's University Belfast, Belfast, United Kingdom, Institute for Biology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany

Emilie Flaberg,

Affiliation: Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden

Laszlo Szekely,

Affiliation: Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden

Tobias C. Olofsson equal contributor mail

 Abstract

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.

Citation: Vásquez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, et al. (2012) Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria and Honeybees. PLoS ONE 7(3): e33188. doi:10.1371/journal.pone.0033188

Editor: Niyaz Ahmed, University of Hyderabad, India

Received:January 20, 2012; Accepted: February 9, 2012; Published: March 12, 2012

Copyright:© 2012 Vásquez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded by grants from the Gyllenstierna Krapperup's Foundation, Ekhaga Foundation, the Swedish Board of Agriculture, The Swedish Research Council Formas, the EU FP7 project BeeDoc (244956), the Biotechnology and Biological Sciences Research Council's Insect Pollinators Initiative (grant BB/I000100/1), and the European Science Foundation COST (European Cooperation in Science and Technology) network COLOSS (FA0803).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:The authors have declared that no competing interests exist.

Introduction

Symbiosis is common in nature, in which symbionts as commensals or mutualists evolved to benefit each other. Culture-independent studies of the human microbiota identified recently a complex symbiotic environment with more than 1,000 bacterial phylotypes representing more than 7,000 strains [1]. The composition of this microbiota has been suggested to be a result of a highly coevolved symbiosis and commensalism influenced by nutrition, physiology and immunological factors [2], [3].

The insect gut has been described as the greatest unexplored reservoir of microbiological diversity [4]. Ryu and colleagues [5]established the importance of the normal flora in the fruit fly gut in order to sustain health. This small microbiota was sufficient to suppress growth of pathogens. While insects harbour a smaller number of symbionts compared to humans they may be even more important [6]. Studies have shown that symbiosis between social insects and microbial species are often highly coevolved [7]and that these symbionts are evolutionary shaped distinctly from the forces acting upon symbionts of solitary organisms, which normally lack a homeostatic fortress environment [8].

Lactic acid bacteria (LAB) are found as commensals within humans, insects and animals [9]. They confer an important bacterial group for the food industry and the fermentation of dairy products. In addition, strains within LAB are also generally recognized as safe (GRAS) food grade microorganisms and employed as probiotics bestowing human health [10], [11]. Genera within LAB are functionally related by phenotypic characteristics [12]and considered as beneficial organisms commonly found as both exogenous and endogenous microbes in healthy individuals. LAB found within humans and animals as commensals are known to protect their hosts via antimicrobial metabolites and modulation of host immune response [13], [14]. One of the most important genus within LAB is Lactobacillus, which is continuously under taxonomic discussion and includes at present 175 listed species [15].

We have discovered a rather special symbiotic lactic acid bacterial (LAB) microbiota within the honey crop of the Western honeybee Apis mellifera [16]. The crop is a central organ in the honeybee's food production between the oesophagus and ventriculus, used for collection and transport of nectar to hive. The crop microbiota of A. mellifera is composed of 13 bacterial species within the genera Lactobacillus and Bifidobacterium [16], [17], [18]and it plays a key role in the production of honey [16]and bee-bread [19], long term stored food for both adult honeybees and larvae. Our recent studies on the subspecies of A. mellifera have also demonstrated that the LAB microbiota is consistent across its native and introduced range [17].

Metagenomics has been used to identify a rich diversity of microbes within honeybees afflicted by Colony Collapse Disorder (CCD) [20], including emergent pathogens (i.e. Nosema ceranae and viruses) [21], while recent studies have picked up novel bacterial genera within the intestinal tract of bees by culture independent methods [20], [22], [23]. Some of these may comprise important symbionts for the maintenance of bee health; however, these descriptive methods do not inform on the functional role or importance of the bee crop microbiota or of individual symbionts within this niche.

We have demonstrated by both in vitro and in vivo studies that the LAB microbiota in A. mellifera inhibit one important honeybee pathogen, the bacterial brood pathogen Paenibacillus larvae that is the cause of the brood disease American foulbrood (AFB) [24]. In the current study we investigate if the LAB microbiota is consistent in all nine recognized honeybee (Apini) species plus stingless bee species (Meliponini), a phylogenetically close taxon that, like honeybees, are eusocial, live in colonies comprising one queen and 100's to 10,000's of workers, store large quantities of honey and bee bread and are managed commercially or exploited by ethnic groups across the tropics.

Functional characterization of the endogenous crop microbiota is essential in providing insights for the understanding of its role for bee health and disease. Here we explore the diversity, maintenance and dynamics of LAB in the honey crop and the pivotal role that they play in bee health, with major implications for research on bee decline and sustainable pollinator management.

Discussion

Our results reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect. A detection of ca. 50 novel species within these bacterial genera will make a huge impact in their current taxonomy. The findings of L. kunkeei as common symbionts with Apis and stingless bees highlight the importance of this organism. We have previously shown the consistent presence and dominance of this lactobacilli, in our studies of A. mellifera (25% of 158 isolates in Sweden [16], [17], [19]; 40% of 42 isolates in USA [18]; 28% of 50 isolates in Africa [17], and now in all Apis spp. and in the stingless bees. The most recent common ancestor to honeybees and stingless bees has been dated to >80 million years ago [27], suggesting that the L. kunkeei-dominated LAB flora is an ancient apine bee association. But invariance in L. kunkeei 16S rRNA gene sequences across host species and geographic locality (Figure 1) suggest possible horizontal transfer of LAB members between host species. However, in Borneo the five sympatric Apis spp. forage together with Trigona spp. on the same flowers but L. kunkeei was not found in any Trigona spp. investigated, arguing against horizontal transfer.

Our investigation shows how this microbiota is acquired (Table 2) and maintained within bees (Figure 2and Figure 3; Video S1and Video S2). Interestingly, LAB builds up gradually by trophallactic exchange with nestmates and L. kunkeei Fhon2 was found to dominate the crop microbiota at all sampling occasions again reflecting its importance (Table 2). However, that only six honeybee crop LAB members were found during the trial may reflect either the disadvantage for bacterial counting of using viable counts that display the dominant bacteria or the numerical variation across seasons. We know from our previous studies that the LAB members in the crop vary numerically across seasons with the flowers visited by bees and with the health status of bees [16]. On the other hand, we know that the microbiota is also rather consistent across Apis species. At first sight, it is surprising how this microbiota is maintained within the honey crop, with the extensive ebbing and flowing of sugars, enzymes, water and the constant invasion of foreign microbes following ingestion of flower nectar during foraging. Visualization of the crop and attached LAB reveals how this microbiota is conserved (Figure 2and Figure 3; Video S1and Video S2) in networks and biofilms. The property of biofilm formation is known in LAB species that resides in the human gut and vagina [28], [29], [30]. In addition to the well-described characteristic of LAB to produce exopolysaccharides, other likely mechanisms in biofilm formation and adhesion include the production of proteins, carbohydrates, enzymes, nucleic acids, lipids or membrane bound receptors. Exopolysaccharides are the main component in extracellular polymeric substances (EPS) and when, secreted into the environment, provide protection to bacteria; they are also involved in host colonization and cellular recognition [31]. It has been suggested that exopolysaccharides produced by food grade organisms (GRAS), in particular LAB, may confer health benefits in humans [32], [33], [34]; the same may be true for honeybees. The complexity of attachment and biofilm formation by this symbiotic community comprise yet unknown mechanisms of action. These may include membrane properties of the symbionts to avoid rejection by their host, as well as the production of potent antimicrobial substances.

During foraging, foreign microbes are introduced into bees and to their colony through their honey crop, with collected nectar, and through pollen (Table S1). When a flower blooms for the first time, its nectar and pollen are sterile but eventually become invaded by airborne microorganisms and microbes from insects. The composition and numbers of this transient microbiota may vary with time but also with flower type, visiting insects, temperature and the nutritional composition of the pollen and nectar. LAB members in the crop vary numerically [16]but are consistent within the same Apis species [17]. LAB diversity could be explained by variation in nutrient content of different nectars and pollen and also by variation in the microbes to which flowers are exposed. Transient floral microbes may trigger the growth of resident LAB microbiota in bees and their production of antimicrobial substances, a mechanism known for LAB strains in other niches (e.g. Lactobacillus reuteri when producing reuterin [35], [36], [37].

We raised the hypothesis that honeybee LAB possess antimicrobial properties against microorganisms present in nectars and on pollen in order to defend their niche (the honey crop) and prevent spoilage of honey and bee bread during their production, which may take from days to weeks. Our results show a preliminary overall inhibition of transient environmental microbes found in flowers (Table S1). Once again, L. kunkeei, the most common species in all Apis bees (Figure 1), was highlighted as the most potent, inhibiting all tested microorganisms. This bacterium was originally described as a wine-spoiling organism since it inhibited yeast in wine production [38], [39]. Interestingly, the extent of inhibition by single LAB members varied considerably with test microbes. Yet the LAB microbiota seems to work in a synergistic matter [24](Table S1); they produce antimicrobial agents including common organic acids, proteins, peptides, enzymes, and bacteriocins that we are currently characterising.

The LAB microbiota of the A. mellifera honey crop is added by bees to their brood food and corbicular pollen and is important in the production of honey and bee-bread [16], [19]. It is well known that species within Lactobacillus and Bifidobacterium inhibit pathogens; they have been used for centuries in food preservation to prevent microbial spoilage [40]. Commercial probiotic products for human consumption with viable LAB contain about the same quantity (108) of mostly one single LAB g−1 product [41], [42]. We hypothesise that the resident strains of Lactobacillus and Bifidobacterium in honey could function in a similar way as LAB for food preservation or as a defence against microorganisms invading humans.

Honey collected from managed or wild colonies of Apis spp. or stingless bees has been independently regarded as a therapeutic agent by many cultures throughout history, from the Maya in Mexico to the Pharaohs in Egypt [43], possibly reflecting beneficial effects of the viable honeybee microbiota when consumed or applied on wounds. It is feasible to believe that fresh honey represents a naturally occurring probiotic product, one with a great diversity and concentration of LAB species (Table 1) that may reflect a myriad of beneficial properties of every specific LAB member in the honey crop.

We believe that LAB antimicrobial mechanisms have evolved in synergy with bees to defend themselves and their hosts from environmental threats such as microbes found in nectars and pollen, and possibly for defence against specific honeybee pathogens. This ancient symbiotic relationship between LAB and bees seems to be of great benefit for bees and may be referred to as colonization resistance. The same phenomenon described for the normal flora in the fruit fly gut [6]. Honeybee brood are fed bee-bread containing viable LAB and their antimicrobial substances. Thus, our results strongly suggest that LAB linked to the honeybee crop have important implications for honeybee pathology, particularly for bacterial brood diseases such as AFB and EFB. Honeybees are considered to have only about a third of the innate immune genes compared to other insects [44], [45]. In addition to social defences that accrue to social insects [46], individual honeybees may also benefit from their LAB symbionts, which are probably of great importance in pathogen defence, possibly further reducing dependency on the innate immune system.

In order to secure honeybee pollination services, A. mellifera beekeepers replace harvested honey by feeding sugar solutions, occasionally mixed with antibiotics for prophylactic control of honeybee-specific bacterial diseases of bee brood such as AFB and microsporidia [47]. It is known that LAB antibiotic susceptibility varies [48], [49]. In vitro culturing of the 13 Apis individual LAB members with two antibiotics used in apiculture (oxytetracycline and tylosin) demonstrated high sensitivity of all to Tylosin, the most recently employed antibiotic within apicultural practices in the USA [26]. Nevertheless, strains L. kunkeei Fhon2 and Lactobacillus Fhon13, Hma11, Hma8 and Hon2 showed resistance to oxytetracycline that may reflect the extended use of this antibiotic in apiculture or their long-term exposure to environmental microbes from the surrounding environment that produce similar substances. The negative effects on honeybee health from damaging the honey crop microbiota by the use of these antibiotics need to be investigated further.

Perspectives

The economic value of commercial honeybee pollination is estimated at over US $14 billion in the USA alone and over US $220 billion worldwide [50]. Yet ongoing colony losses in the USA and Europe defy causal explanation despite intensive research effort [51], [52]and identification of emergent and exotic pathogens [20]. Our discovery of a diverse and novel honey crop LAB microbiota common to all recognized honeybee species plus 3 stingless bee species may be the missing link in this worldwide problem. Since related microbiotas are found across bee species, it strongly suggests a close evolutionary relationship between bacteria and hosts, as well as underscoring the importance of LAB symbionts for bees. Not only are LAB symbionts involved in honeybee food production and preservation, they are also of importance in host defence against pathogen and transient microbes intercepted during foraging. The importance of this crop microbiota for honeybees is additionally strengthened by the fact that it is immediately transferred to the sterile crop of newly emerged bees by trophallactic exchange with nestmates.

Any beneficial effect from this microbiota may be undermined where prophylactic use of antibiotics is practiced (e.g. USA) or where their natural foodstuffs, honey and pollen, are supplemented by the beekeeper with synthetic sugars and pollen substitutes lacking LAB or their beneficial substances. The absence of LAB is especially problematic when the bees attempt to produce and preserve food for themselves and their brood, when feeding their brood with pollen lacking LAB or LAB derived antimicrobial substances, when nestmates establish a LAB microbiota in callows by trophallactic exchange, or when pathogens invade their hive. Emphasis now needs to be given to discovering the mechanisms of action of LAB against pathogens and food spoiling microbes, and how they can be used to resolve ongoing honeybee colony losses, in which LAB may be the important missing link. Altered beekeeping husbandry practices that enhance LAB are needed, or direct manipulation by supplementary feeding of individual or composite LAB members and their products could help alleviate CCD. Further functional analysis of LAB in bees will certainly enrich our understanding of insect-microbe symbioses and their evolutionary dynamics within complex eusocial insect societies.

Materials and Methods

Ethics

No specific permits were required for the described field studies. Local colleagues (described in the acknowledgments) collected samples where permission was not required i.e. not from nature reserves or privately-owned locations. The field studies did not involve endangered or protected species.

LAB diversity in bees

We sampled the honey crop LAB microbiota of all 9 well recognized honeybee (Apini) species plus 3 stingless bee species (Meliponini). Apis andreniformis (n = 3 colonies), Apis cerana (n = 2 colonies), Apis koschevnikovi (n = 3 colonies), Apis nuluensis (n = 1 colony) and Apis dorsata (n = 1 colony) were collected from Borneo (Malaysia), Apis laboriosa (n = 2 colonies) from Nepal, Apis florea (n = 1 colony) from Thailand, Apis nigrocincta (n = 1 colony) from Indonesia, and A. mellifera (n = 25 colonies) from Sweden and Kenya. Samples of the stingless bee genera Trigona were collected from Thailand (n = 1 colony) and Borneo (n = 1 colony) (Malaysia), Meliponula bocandeei (n = 2 colony) from Kenya, and Melipona beecheii (n = 2 colonies) from Mexico. For each bee species, the honey crop LAB content of 10–20 bees was analysed. For A. nuluensis and A. dorsata we only analysed the honey crop content and the corbicular pollen from field collected bees as we were unable to sample colonies of these free-living bees. Isolation of LAB from honey crops, fresh honey, corbicular bee pollen and bee bread was performed as previously described [16], [19]. PCR-amplification of isolates for 16S rRNA gene sequencing, identification and phylogenetic analysis were performed according to Olofsson and Vásquez [16]. Additionally, the 16S rRNA gene sequences were also checked against the software RDP (Ribosomal Database Project II), accessible from the homepage (http://rdp.cme.msu.edu/). A total of 750 lactic acid bacterial isolates were retrieved in this study.

Ontogeny of LAB

To determine how the crop microbiota is acquired, we marked Western honeybees (A. mellifera) at eclosion from their wax brood cells (n = 30), returned them to the hive, collected them at different ages, cultivated the contents of their crops and identified the LAB (cultivation and identification as described previously [16]).

Maintenance of the honey crop microbiota

We performed in vitro and in vivo investigations of the symbionts with SEM and fluorescence microscopy. The SEM samples were prepared by freeze drying [53]and pictures were taken by Photographer Lennart Nilsson (Sweden).

The preparation and confocal fluorescence microscopy of bacteria in the honey crop was achieved as follows. Honeybees were fed with a mixture of honey and water containing (Sytox®, Green dye and BacLight™, Red bacterial stain, Molecular Probes) to discriminate the cells of the bee from the living bacteria. Following an incubation of approx. 15 min, the honey crop was dissected at room temperature. The crop was opened with a single longitudinal cut. To prevent the contraction of the muscles of the crop wall, the crop was rinsed in phosphate buffered saline (PBS) supplemented with 1 mM EDTA and mounted on a glass slide. Slides were examined using a TCS SP5 laser confocal microscope equipped with continuous spectrum white laser (Leica, Mannheim Germany). The images were captured using a 63× oil immersion objective (NA 1.4 HCX PL APO CS) with filter setup adapted to FITC and Texas Red dual colour illumination. The raw images were processed in ImageJ (NIH, Bethesda, USA) using median filtering. The Z-stacks was visualized using the ImageJ plugin 3D-viewer. The 3D-Movies (Video S1and Video S2) show a projection of 90 confocal z-sections through a z-depth of 45.3 µm, covering an area of 246×246 µm in the xy-direction.

Melissococcus plutoniusbioassay

We investigated possible inhibitory effects on European foulbrood (EFB), a major bacterial pathogen of larval honeybees, from the LAB microbiota using both in vitro and in vivo tests, as previously described [24]. As adult honeybee workers feed larvae with crop contents, this represents a typical means by which larval food acquires LAB. Bacterial suspensions of Melissococcus plutonius (provided by Dr. Jean-Daniel Charrière, Agaroscope, Switzerland, Accession nr: JN689233) were prepared fresh for each experiment and diluted in larval food for final concentrations of 107,106 and 105 bacteria per ml. A mixture of the thirteen previously described honeybee LAB [16], [17], [18]in approximately equal proportions was diluted in larval food for a final, total concentration of 107 LAB per ml. A. mellifera worker larvae were grafted and reared in vitro [24]. Briefly, first instar worker larvae were transferred to the surface of the larval diet of the different treatments. i) control group provided with uninfected diet, ii) control group initially fed uninfected diet but LAB supplemented food after 48 hours onwards and iii) experimental groups provided larval diet spiked with defined amounts of M. plutonius (107,106 and 105 bacteria ml−1). Twenty-four hours post exposure; larvae were transferred to wells containing uninfected diet and LAB supplemented food 48 hours post-infection onwards. The experiment was finished 21 days post-infection and larval mortality was monitored daily. A total of 420 larvae were used in two replicate experiments. The PoloPlus Probit and Logit Analysis program (version 2.0, LeOra software) was used to compare mortality rates between the experimental groups in the exposure bioassay.

Flowers, nectar, pollen and microorganisms

We analysed the microbial composition of 15 flowers frequently visited by A. mellifera in Sweden (Table S1). Flowers were collected aseptically in Kullaberg, Sweden. The flowers were then shaken in sterile buffer (PBS) and immediately transported to the Laboratory at Lund University. Dilutions were made with sterile peptone water (0.2% w/v), spread on MRS (Oxoid), APT (Oxoid) and TSB (Oxoid) agar plates incubated anaerobically at 35°C (MRS and APT agar plates) and aerobically at 22°C (TSB agar plates) during 5 days. Identification of the microbial isolates was achieved by sequencing the 16S rRNA genes (for bacteria) [16]and the D1-D2 regions of the LSU 26S rRNA genes (for yeasts) [54].

Dual-culture overlay assay

We analysed the inhibition properties of all honey crop LAB grown individually and together against the pathogen M. plutonius and also against the 55 bacterial strains and 5 yeast strains isolated from flowers. The assays were performed as earlier described [55]with the following modifications. LAB were inoculated on MRS agar plates (Oxoid, supplemented with 0.1% L-cysteine and 2.0% fructose) during 12 hours. We used the medium previously described for cultivation of M. plutonius [56], [57]and the same media as for the isolation of microbes from flowers for the over layer of soft agar. After an incubation of 2–4 days depending on growth rates the zone of inhibition was measured.

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  The following article was sent in by member Costa Kouzounis.

  ATTENTION ON VARROA

 Dr. Eric Mussen, University of California Davis, March/April 2013

 Researchers throughout the world still consider

 Varroa destructor to be one of the most important stresses on honey bee colonies around the world. It continues to have its biology examined by researchers from many places. The following is information on Varroa from four different laboratories.

 The first study, by researchers in USDA ARS, dealt with the question of whether honey bee stocks that had undergone strong, human-induced selection pressure could still compete in crop pollination with commercial Italian bees (CT) that had been treated twice for mites or (CU) that had not been treated. The highly selected stocks were Russian bees (RB) and outcrossed (50 percent, genetically) stocks of Varroa Sensitive Hygiene (VSH) bees. Coming through the first winter, 57 percent of the VSH stock, 56 percent of the CT stock, 39 percent of the RBs and 34 percent of the untreated Italian colonies (CU) were eight frames of bees or larger, which is recommended for almond pollination. By apple pollination time, all the colonies had built up to acceptable size. Mite counts showed that the treated Italian colonies continued to have the lowest mite populations. Mite population levels in the Russian and VSH colonies were lower than in the Italian colonies that had not been treated at all. Review the study on the Internet at: DOI:

 Researchers in Canada have been studying indoor wintering of honey bee colonies for a long time. Two important considerations are temperature and buildup of carbon dioxide. The researchers then wondered if those parameters could be adjusted to the detriment of varroa mites, without harming the bees. Clusters of approximately 300 infested adult honey bees were placed in self-contained glass chambers and incubated at 25 and 10 degrees Centigrade (77 and 50 degrees Fahrenheit, respectively) and with low, medium, and high ventilation. The air in the chambers started at 1-2 percent CO2. With high ventilation, it remained the same. Ventilation rate did not affect bee mortality at either temperature. There did appear to be an effect on the mites.

 At the cooler temperature, mite mortality was greatest with the highest ventilation. Medium and low ventilation, losses were about equal. At the warmer temperature, mite mortality was greatest under low ventilation conditions. The authors concluded that holding groups of bees at 25 degrees C and letting CO2 build up, might clear them of mites. However, indoor wintering usually is done at 4 degrees C (40 degrees F), so the mites are not too apt to be removed by increased CO2 levels in winter storage. For further information refer to: DOI:

 Due to the development of resistance to Apistan® strips (10 percent fluvalinate), researchers in Iran wished to determine how well Apivar®, Bayvarol®, and CheckMite+® controlled

  Varroa destructor in their hives. Each of these products is formulated as a plastic strips with 500 mg amitraz, 0.06 percent flumethrin, and 10 percent coumaphos, respectively.

 Scientists conducted a 43-day field trial in the fall of 2009 on 20 colonies containing about 10 frames of bees. Groups of five colonies were treated as follows: 1) two strips of Apivar in brood nest for 6 weeks; 2) four strips of Bayvarol in brood nest for 6 weeks; two strips of CheckMite+ in brood nest for 6 weeks; and 4) untreated control. Pre- and post-treatment percentages of mite infestations were: Apivar – 8.43 and 0.28; Bayvarol – 8.48 and 0.29; CheckMite+ - 9.64 and 0.14; control – 8.98 and 14.61. It is interesting to see how effective chemicals can be for

 Varroa control, when the mites first encounter them. The paper is: The efficacy of Apivar® and Bayvarol® and CheckMite+® in the Control of Varroa destructor in Iran, by Reza Shahrouzi. 2009. It can be accessed at: http://www.apiservices.com/articles/us/efficacy_of_bayvarol.pdf .

 Finally, from Arabia, researchers studied the effects of Apistan® (fluvalinate), Bayvarol® (flumethrin), Perizin® (amitraz), and "Bee Strips" [CheckMite+®] (coumaphos) for controlling

 Varroa in their colonies in 2003, 2004 and 2005. In 2003 two strips of Apistan left in the hives for 60 days were compared with four strips of Bayvarol for 45 days, two strips of Bayvarol for 45 days, and two strips of Apivar for 42 days. Controls were untreated. In 2004, most treatments were the same, except that the milder Bayvarol treatment was replaced with a Perizin® 50 ml emulsion treatment. The third season, the Apistan and Bayvarol treatments remained the same. This time, two Bee Strips were applied instead of Perizin and left in place for 45 days. Sticky boards were left in the hives throughout the treatment periods. Mites were counted periodically and at the end of the experiments, using either Perizin or Apivar for knockdown.

 Bayou Bee Bulletin

 The first season, four strips of Bayvarol knocked down the highest percentage of mites (96). Apivar (95 percent), two strips of Bayvarol (89 percent) and Apistan (85 percent) followed the leader. Only 25 percent of the control mites fell. The second season Apivar did best (95 percent) with Perizin (94 percent), Bayvarol (80 percent) and Apistan (80 percent) way ahead of the controls (18 percent). In year three, the newcomer, Bee Strips (95 percent), was most effective while Apivar (92 percent), Bayvarol (70 percent), and Apistan (60 percent) followed. Natural mortality this time was 11 percent. Throughout the study, the efficacy of Apistan was dropping. Bayvarol was showing the same trend. Ambient temperatures outside the hives did not impact the results in this study.

 At the time of these studies Apivar was very consistent in its effects on varroa mites. Where Apivar has been used in Europe for a long period of time, it still seems highly effective. Let’s hope that holds true for Apivar in the United States. This article is titled "Evaluation of the relative efficacy of different acaricides against

 Varroa destructor in Apis mellifera carnica" by Ahmad A. Al-Ghamdi. A PDF of this publication may be reviewed at: http://faculty.ksu.edu.sa/alkhazim/Documents/papers/2t.pdf .

 NEW DISEASE FOUND, HELPS PREDICT COLONY DEATH

  Bee Culture’s, Catch the Buzz, March 4, 2013*

 A new long-term study of honey bee health has found that a little-understood disease study authors are calling "idiopathic brood disease syndrome" (IBDS), which kills off bee larvae, is the largest risk factor for predicting the death of a bee colony.

 "Historically, we’ve seen symptoms similar to IBDS associated with viruses spread by large-scale infestations of parasitic mites," says Dr. David Tarpy, associate professor of entomology at North Carolina State University and co-author of a paper describing the study. "But now we’re seeing these symptoms – a high percentage of larvae deaths – in colonies that have relatively few of these mites. That suggests that IBDS is present even in colonies with low mite loads, which is not what we expected." The study was conducted by researchers from NC State, University of Maryland, Pennsylvania State University, and the U.S. Dept. of Agriculture (USDA).

 The study evaluated the health of 80 commercial colonies of honey bees (

 Apis mellifera) in the eastern United States on an almost monthly basis over the course of 10 months – which is a full working "season" for commercial bee colonies. The goal of the study was to track changes in bee colony health and, for those colonies that died off, to determine what factors earlier in the year may have contributed to colony death. Fifty-six percent of the colonies died during the study.

 "We found that colonies affected by IBDS had a risk factor of 3.2," says Dr. Dennis vanEnglesdorp of the University of Maryland, who was lead author on the paper. That means that colonies with IBDS were 3.2 times more likely to die than the other colonies over the course of the study.

 While the study found that IBDS was the greatest risk factor, a close runner-up was the occurrence of a so-called "queen event."

 Honey bee colonies have only one queen. When a colony perceives something wrong with its queen, the workers eliminate that queen and try to replace her. This process is not always smooth or successful. The occurrence of a queen event had a risk factor of 3.1.

 This is the first time anyone has done an epidemiological study to repeatedly evaluate the health of the same commercial honey bee colonies over the course of a season," Tarpy says. "It shows that IBDS is a significant problem that we don’t understand very well. It also highlights that we need to learn more about what causes colonies to reject their queens. These are areas we are actively researching. Hopefully, this will give us insights into other health problems, including colony collapse disorder."

 The paper, "Idiopathic brood disease syndrome and queen events as precursors of colony mortality in migratory beekeeping operations in the eastern United States," is published in the February issue of Preventive Veterinary Medicine. Co-authors of the study include Dr. Eugene Lengerich of Penn State and Dr. Jeffery Pettis of USDA. The work was supported by USDA and the National Honey Board.

 The study abstract follows:

 "Idiopathic brood disease syndrome and queen events as precursors of colony mortality in migratory beekeeping operations in the eastern United States"

 Authors: Dennis vanEnglesdorp, University of Maryland; David R. Tarpy, North Carolina State University; Eugene J. Lengerich, Pennsylvania State University; and Jeffery S. Pettis, USDA-ARS Bee Research Laboratory

 Published: February 2013, Preventive Veterinary Medicine

 Abstract: Using standard epidemiological methods, this study set out to quantify the risk associated with exposure to easily diagnosed factors on colony mortality and morbidity in three migratory beekeeping operations. Fifty-six percent of all colonies monitored during the 10-month period died. The relative risk (RR) that a colony would die over the short term (?50 days) was appreciably increased in colonies diagnosed with Idiopathic Brood Disease Syndrome (IBDS), a condition where brood of different ages appear molten on the bottom of cells (RR = 3.2), or with a "queen event" (e.g., evidence of queen replacement or failure; RR = 3.1). We also found that several risk factors—including the incidence of a poor brood pattern, chalkbood (CB), deformed wing virus (DWV), sacbrood virus (SBV), and exceeding the threshold of 5 Varroa mites per 100 bees—were differentially expressed in different beekeeping operations. Further, we found that a diagnosis of several factors were significantly more or less likely to be associated with a simultaneous diagnosis of another risk factor. These findings support the growing consensus that the causes of colony mortality are multiple and interrelated.

 This article was written by Matt Shipman and was brought to you by, "Bee Culture, The Magazine of American Beekeeping," published by the A.I. Root Company.