POLLINATOR PROTECTION VIA THE STUDY OF SYMBIOSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006491
• Project Start Date: May 1, 2015
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
Entomology, Riverside

Non Technical Summary
There is strong evidence that both wild and managed pollinators are declining in parts of the world, including North America. As these pollinators contribute to about a third of global food production, protecting pollinator populations is of utmost importance. In the McFrederick Lab, we study the microscopic bacteria, fungi, and viruses (microbes) that assiociate with wild and managed bees in order to understand how we can increase bee health.We use a combination of modern DNA sequencing, field surveys, and experiments to understand how microbes affect bee health. For example, we have used deep DNA sequencing to find bacteria that may aid in the digestion of pollen. Pollen is an extremely hard food to digest due to the presence of a tough pollen cell walls. The bacteria we study make chemicals that can break down pollen cell walls, and we can use sterilized pollen to manipulate the presence of these bacteria then determine how well bees digest pollen in the bacteria's presence or absence. In this way, we can determine if pollen is more digestable when these bacteria are present.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
315	3099	1130	100%

Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3099 - Bees, honey, and other pollinators, general;

Field Of Science
1130 - Entomology and acarology;

Keywords

pollinator

microbe

bacteria

fungus

virus

plant

pollinator decline

Goals / Objectives
Research in the McFrederick Laboratory centers around the ulttimate goal of protecting both wild and managed pollinator populations by studying their microbes. Bees are affected by sundry microbial pathogens, but also associate with commensal and putatively beneficial microbes. we aim to understand the interactions between microbes and their bee hosts and what that ultimately means for the bee host.The goals and objectives of this project are as follows, divided into four seperate categories:(A) The role of Lactobacillus kunkeei in alfalfa leafcutter bee nutrition(1)Test for bacterial-mediated effects on Megachile rotundata. We will test whether L. kunkeei and other bacteria aid in pollen wall breakdown, and if this in turn benefits M. rotundata nutrition.(B)The influence of cover crop forage on honey bee nutrition and gut microbes, and on colony growth and activity(1) Develop methods to exploit data from continuous monitoring of bee hives in the field.(2) Determine the effect of strategically planted oilseed cover crops that bloom prior to, and shortly after the almond pollination, on honey bee nutrition, health, and queen quality.(3) Determine if cover crops affect the honey-bee gut microbiota when compared to bees fed high fructose corn syrup.(4) Understand the interplay between cover crops, honey bee nutrition, health, queen quality, and microbes by synthesizing the results from objectives 1-3.(C)The probiotic potential of Lactobacillus kunkeei for honey production(1) To test the hypothesis that L. kunkeei acts as a probiotic that can boost honey production if fed to bees in winter supplemental feed.

Project Methods
(A) The role ofLactobacillus kunkeeiin alfalfa leafcutter bee nutritionWe will add microbes that we have isolated from M. rotundata to the sterilized provisions according to the treatments in Table 1. For each treatment, we will randomly select 30 eggs, which we will first surface sterilize using a wash in EquateTM Multipurpose Solution, followed by three washes in sterile saline solution. For each provision, we will measure initial provision size, place the sterilized egg on the provision, and allow the larvae to consume the provision. Once the larvae defecates, we will measure protein, carbohydrate, and lipid digestion levels in the frass as in Dobson and Peng (1997). As a control, we will also measure these nutrient levels in fresh, uningested pollen. We will divide samples into 3 equal parts, and use separate stains for proteins, carbohydrates, and lipids (see budget for details on stains). Using a phase contrastTable 1. Experimental treatments.TreatmentOrganisms1L. kunkeei strain 1 (M. rotundata)2L. kunkeei strain 2 (H. ligatus)3Mixture of 30 bacterial strains4Sterile pollen only5Unsterilized no treatment controlmicroscope, we will measure the percent of the pollen's cytoplasm showing stain uptake in pollen from frass as compared to the control. We will take digital pictures of the stained pollen, and use imageJ to measure the intensity of stain uptake. We will additionally measure offspring size, weight, and record sex and survival. The bee measurements will allow us to directly determine the effect of L. kunkeei on larval growth, while the protein, carbohydrate, and lipid measurements will allow us to determine if bacteria facilitate pollen digestion. We will analyze these data with standard analysis of variance or, if deemed necessary, linear mixed models (if multiple trials are necessary) or Aster models (if fitness components exhibit different distributions) (Shaw et al. 2008) . To verify that bacteria are present in large enough numbers to effect bee nutrition, we will quantify the amount of bacteria in the guts of 10 larvae per treatment using qPCR. We have developed qPCR assays to quantify all bacteria using the universal 16S gene and to quantify L. kunkeei only using the single-copy protein-coding gene TefG. We will rear ten larvae per treatment for qPCR assays in addition to and simultaneously with the 30 larvae per treatment for the nutrition assay outlined above.(B)The influence of cover crop forage on honey bee nutrition and gut microbes, and on colony growth and activityObjective 3: Effects of cover crops on gut microbiota We propose to study the effects of the different cover crop treatments on the honey-bee gut microbiota, to test the hypothesis that feeding of high fructose corn syrup will favor the growth of some gut microbes compared to gut microbiota from bees that were given cover crops to forage on. We will collect 3 worker bees and the queen (requeening in the process) from each hive. We will make our collections directly before the almond bloom, so that the bees have only corn syrup or cover crop forage before collection. We will surface sterilize each sample by rinsing each sample in a 10% bleach solution, followed by three rinses in sterilized, deionized water. We will then dissect the gut from each sample, using sterilized dissection trays and tools. To quantify bacterial communities found in the workers and the queens, we will use a combination of next-generation sequencing and quantitative PCR. We have extensive experience with these methods (McFrederick et al. 2012; 2013; 2014), and recently co-authored a paper creating standard methods for analyses of the honey bee gut microbiota as part of the COLOSS BEEBOOK (Engel et al. 2013b). To identify gut bacteria and determine their relative abundances, we will sequence a gene found in all bacteria, using modern sequencing technology that allows us to generate thousands of DNA sequences from a single sample (Illumina DNA sequencing). We will then compare the sequences that we obtain to sequences from known bacteria and thereby determine what species of bacteria are present in our samples. To determine the absolute number of bacteria in each gut sample, we will additionally use quantitative PCR, which is a technique that allows us to determine the starting number of copies of a gene in a sample. Combined with the DNA sequence data, this will allow us to determine what microbes are present, their abundance relative to each other, and the overall size of the gut microbiota. We will analyze the next generation sequencing data using standardized methods (Engel et al. 2013). We will use the QIIME pipeline (Kuczynski et al. 2011)and UniFrac analyses (Hamady and Lozupone 2009)to determine if the gut microbiota differ by treatment. To determine if the absolute abundance of bacteria differ by treatment, we will use standard Analysis of Variance.(C)The probiotic potential ofLactobacillus kunkeeifor honey productionWe propose to conduct an experiment over the winter, spring, and summer of 2014-2015.Experimental treatmentsWe will use 32 colonies that have been exposed to similar conditions from the past year and that are at the same strength going into the experiment, which we will begin in late November/early December 2014, upon notification of funding. For treatments, we will randomly select 15 of these colonies for the probiotic treatment group, while the other fifteen will be the control group. To control for similar diet between the probiotic treatment and the control treatment, we will sterilize high fructose corn syrup (HFCS) and pollen substitute patties by autoclaving (HFCS) or gamma irradiation (pollen substitute patties). We will then inoculate half of the sterilized pollen substitute and HFCS with a strain of L. kunkeei that we isolated from honey and currently have cryopreserved, at a concentration of 104 bacteria per ml/mg. We will provide syrup and pollen substitute patties to hives in both treatment groups by checking and adding supplemental food every week until sufficient natural forage is available. For geographical replication, we will keep half of the colonies (8 control and 8 probiotic treatment hives) at the Carl Hayden Bee Research Center (CHBRC), and half of the colonies in an apiary on Agricultural Operations land on the campus of UC Riverside (Ag Ops). For statistical analysis, site (CHBRC or AgOps) will be treated as a block, in order to account for variation in hive metrics due to site. This geographic replication will increase the ability to generalize our findings.Hive monitoring We will monitor hives at monthly intervals throughout the experiment, beginning in December 2014 and ending June 2015, after the spring bloom has ceased. To assess colony growth and honey production, we will take monthly measurements. At each inspection point we will examine each colony to check for signs of diseases or parasites. We will weigh each frame separately on a portable balance, and take digital photographs (using a frame/camera mount to standardize the distance of the camera to the frame) of each side of each frame. To determine how much honey, pollen, and brood each colony is producing, we will estimate the area of each of these per frame with the image analysis software ImageJ. We will estimate the weight of honey in the hive, along with the weight of adult bees, brood, pollen, and wax by subtracting the weight of the hive parts from the total hive (as in Meikle et al. 2008).Statistical analyses Honey production will be tested for significant differences between the treatment and control groups using a repeated measures ANOVA, with month as a within subjects factor and treatment as a between subjects factor. We will include block (CHBRC and Ag Ops) as a random factor.

Progress 05/01/15 to 09/30/17

Outputs
Target Audience: The research in the McFrederick laboratory targets both agriculutral and general public stakeholders. On the agricultural side, I conducted research for the Almond Board of California, the National Honey Board, Project ApisM,and the Western Alfalfa Seed Grower's Association. I also presentedat the Almond Board of California'sand the Western Alfalfa Seed Grower's Association's annual meetings. I gave talks at CAPCA meetings (3) and various other public outlets such as beekeeper's associations and Botanic Gardens. Finally, we conducted outreach for elementary schools and the general public. For example, we staffeda booth at the annual Riversie Insect Fair (3 yaers), which drew about 14,000 visitors in 2017. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Nine undergraduate students from diverse backgrounds were trained in microbiology, experimentation, molecular biology, and bee biology. One of these undergrads won a research award, presented her results at a campus-wide research symposium, and was accepted to a Ph.D. program. Four graduate students were trained in microbiology, experimentation, molecular biology, bioinformatics, and bee biology. One student received research fellowships from NASA and the USDA, as well as published the first graduate student-led paper from the lab. One postdoc was trained in microbiology, experimentation, molecular biology, bioinformatics, and bee biology. The postdoc published one paper and recently received a prestigious fellowship in the UK. How have the results been disseminated to communities of interest?I presented at the Almond Board of California's and the Western Alfalfa Seed Grower's Association's annual meetings anually for all three years of the project. I gave talks at CAPCA meetings (3) and various other public outlets such as beekeeper's associations and Botanic Gardens. Finally, we conducted outreach for elementary schools and the general public. For example, we staffed a booth at the annual Riversie Insect Fair (3 yaers), which drew about 14,000 visitors in 2017. I also gave 2 interviews for the local newspaper (the Press Enterprise), maintained my lab's website, and fielded numerous phone calls and questions. Finally, we have been maintaining our productive publication record. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (A) The role of Lactobacillus kunkeei in alfalfa leafcutter bee nutrition In 2016 we conducted two experiments with ALCBs. First, we used 16S rRNA gene sequencing to investigate the microbiome of parasites and parasitoids of ALCBs. Next, we measured protein content and fat content of ALCB larvae, while also sequencing microbes in the guts of these same larvae. We looked for correlations between the nutritional parameters and specific microbes, but were surprised to find none. In 2017, we tested the inhibitory ability of lactobacilli on fungal bee pathogen and saprohytic fungi found in bee pollen provisions. We used inhibition assays with 30 strains of Lactobacillus and 13 fungal isolates. We discovered that all fungal strains could be inhibited by one or more bacterial strains, but no one bacteria could inhibit all fungi. Fungal cocktails are therefore important for protecting bee pollen provisions from saprophytic fungi, and bee larvae from fungal pathogens. (B) The influence of cover crop forage on honey bee nutrition and gut microbes, and on colony growth and activity In 2016, we conducted a large scale experiment with Mark Carrol and William Meikle of the Carl Hayden Bee Research center. We found that honey bee colonies that were given forage survived much better than those denied forage fopr several weeks in the winter. Interestingly, the forage treatment had little effect on the gut microbiome, but larrge effects on bee health. This work has now been published by my graduate student Jason Rothman. In 2017, we continued this work in collaboration with Elina Nino and Neal Williams of UC Davis and Kirk Anderson of the Carl Hayden Bee Research Center. We are investigating the effect of supplemental forage on pathogen load and honey bee immune function. We are still processing samples in the lab for this project. (C) The probiotic potential of Lactobacillus kunkeei for honey production We finished this experiment in 2016. We found that one strain did not affect honey bee survival while a seperate strain negatively affected survival. When developing probiotics, strain identity is therefore extremely important. We have recently sequenced the genomes of these two strains, and are hoping to identify differences that may explain the different phenotypes.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Marcus J Pennington, Jason A Rothman, Michael B Jones, Quinn S McFrederick, Jay Gan, John T Trumble. Effects of contaminants of emerging concern on Megaselia scalaris (Lowe, Diptera: Phoridae) and its microbial community. Scientific Reports 7(1) 8165
• Type: Journal Articles Status: Published Year Published: 2017 Citation: MJ Pennington, JA Rothman, SL Dudley, MB Jones, QS McFrederick, Jay Gan, John T Trumble. Contaminants of emerging concern affect Trichoplusia ni growth and development on artificial diets and a key host plant Proceedings of the National Academy of Sciences 114 (46), E9923-E9931
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Longitudinal Effects of Supplemental Forage on the Honey Bee (Apis mellifera) Microbiota and Inter-and Intra-Colony Variability JA Rothman, MJ Carroll, WG Meikle, KE Anderson, QS McFrederick Microbial ecology, 1-11
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Effects of contaminants of emerging concern on Myzus persicae (Sulzer, Hemiptera: Aphididae) biology and on their host plant, Capsicum annuum MJ Pennington, JA Rothman, MB Jones, QS McFrederick, J Gan, John T. Trumble Environmental monitoring and assessment 190 (3), 125
• Type: Journal Articles Status: Published Year Published: 2017 Citation: P Graystock, SM Rehan, QS McFrederick. 2017. Hunting for healthy microbiomes: determining the core microbiomes of Ceratina, Megalopta, and Apis bees and how they associate with microbes in bee collected pollen. Conservation Genetics 18 (3), 701-711

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The research in the McFrederick laboratory targets both agriculutural and general public stakeholders. On the agricultural side, I condiuct research for the Almond Board of California and the Western Alfalfa Seed Grower's Association. I also present at both opf these association's annual meetings. We also had resewarch projects sponsored by the Honey Board and Project ApisM. I give talks at CAPCA meetings and various other public outlets. Finally, we conduct outreachforelementary schools and the general public. For example, we man a booth at the annual Riversie Insect Fair, which drew about 10,000 visitors in 2016. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students, seven undergrads, and one postdoc were trained on experimentation, field work, molecular biology, microbiology, and bioinformatics. How have the results been disseminated to communities of interest?We presented goal A to the Alfalfa Seed Grower's Association annual winter seed meeting. We presented Goal B at the Almond Conference, and we submitted a reprt of goal C to the Honey Board. What do you plan to do during the next reporting period to accomplish the goals?We will continue with the bioinformatic postions of Goal C, an extension of Goal B looking at immune system function, and an extension of goal A using meta-transcriptromics and microbiol;ogical assays.

Impacts
What was accomplished under these goals? (A) The role ofLactobacillus kunkeeiin alfalfa leafcutter bee nutrition In 2016 we conducted two experiments with ALCBs. First, we used 16S rRNA gene sequencing to investigate the microbiome of parasites and parasitoids of ALCBs. Next, we measured protein content and fat content of ALCB larvae, while also sequencing microbes in the guts of these same larvae. We looked for correlations between the nutritional parameters and specific microbes, but were surprised to find none. (B)The influence of cover crop forage on honey bee nutrition and gut microbes, and on colony growth and activity In 2016, we conducted a large scale experiment with Mark Carrol and William Meikle of the Carl Hayden Bee Research center. We found that honey bee colonies that were given forage survived much better than those denied forage fopr several weeks in the winter. Interestingly, the forage treatment had little effect on the gut microbiome, but larrge effects on bee health. (C)The probiotic potential ofLactobacillus kunkeeifor honey production We finished this experiment in 2016. We found that one strain did not affect honey bee survival while a seperate strain negatively affected survival. When developing probiotics, strain identity is therefore extremely important. We have recent;y sequenced the genomes of these two strains, and are hoping to identify differences that may explain the different phenotypes.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Meirelles, L., McFrederick, Q.S., Rodrigues, A., Mantovani, J.D., de Melo Rodovalho, C., Ferreira, H., Bacci, M., Mueller, U.G. Bacterial microbiomes from vertically?transmitted fungal inocula of the leaf?cutting ant Atta texana. Environmental Microbiology Reports. (Accepted 03/26/2016. 11 galley pages.) (Refereed, Electronic) doi: 10.1111/1758-2229.12415.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: McFrederick, Q.S., Thomas, J.M., Neff, J.L., Russell, K.A., Vuong, H.Q., Hale, A.R., Mueller, U.G. Flowers and wild megachilid bees share microbes. Microbial Ecology [IF 3.232]. (Accepted 09/15/2016. 13 galley pages.) (Refereed, Electronic) doi: 10.1007/s00248-016-0838-1.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Gordon, E.RL., McFrederick, Q.S., Weirauch, C. Phylogenetic evidence for ancient and persistent environmental symbiont reacquisition in Largidae (Hemiptera: Heteroptera). Applied and Environmental Microbiology [IF 3.668]. (Accepted 09/27/2016. 34 manuscript pages. 0 galley pages.)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Graystock, P., Blaine, E.J., McFrederick, Q.S., Goulson , D., Hughes, W.O. 2016. Do managed bees drive parasite spread and emergence in wild bees? . International Journal for Parasitology: Parasites and Wildlife. Vol. 5: p.64-75. (Refereed, Electronic) doi: 10.1016/j.ijppaw.2015.10.001.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Engel, P., Kwong, W.K., McFrederick, Q.S., Anderson, K.E., Barribeau, S., Chandler, J.A., Cornman, R.S., Dainat, J., de Miranda, J., Doublet, V., Emery, O., Evans, J.D., Farinelli, L., Flenniken, M., Granberg, F., Grasis, J., Gauthier, L., Hayer, J., Koch, H., Kocher, S., Martinson, V., Moran, N., Munoz-Torres, M., Newton, I., Paxton, R., Powell, E., Sadd, B., Schmid-Hempel, P., Schmid-Hempel, R., Song, S., Schwarz, R., vanEngelsdorp, D., Dainat, B. 2016. The Bee Microbiome: Impact on Bee Health and Model for Evolution and Ecology of Host-Microbe Interactions. MBio [IF 6.650]. Vol. 7: 2 p.e02164-15
• Type: Journal Articles Status: Published Year Published: 2016 Citation: McFrederick, Q.S., Rehan, S. 2016. Characterization of pollen and bacterial community composition in brood provisions of a small carpenter bee . Molecular Ecology [IF 5.947]. Vol. 25: p.2302-2311. (Refereed, Electronic) doi: 10.1111/mec.13608.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: De Palma, A., Abrahamczyk, S., Aizen, M., Albrecth, M., Basset, Y., Bates, A., Blake, R., Boutin, C., Bugter, R., Connop, S., Cruz-Lopez, L., Cunningham, S., Darvill, B., Diekotter, T., Dorn, S., Downing, N., Entling, M., Farwing, N., Felicioli, A., Fonte, S., Fowler, R., Franzen, M., Goulson , D., Grass, I., Hanley, M., Hendrix, S., Herrmann, F., Herzog, F., Holzschuh, A., Jauker, B., Kessler, M., Knight, M.E., Kruess, A., Lavelle, P., Le Feon, V., Lentini, P., Malone, L., Marshall, J., Martinez Pachon, E., McFrederick, Q.S., Morales, C., Murdi-Stojnic, S., Nates-Parra, G., Nilsson, S., Ockinger, E., Osgathorpe, L., Parra-H, A., Peres, C., Persson, A., Petanidou, T., Poveda, K., Power, E., Quaranta, M., Quintero, C., Rader, R., Richards, M., Roulston, T.H., Rousseau, L., Sadler, J., Samnegard, U., Schellhorn, N., Schuepp, C., Schweiger, O., Smith-Pardo, A., Steffan-Dewenter, I., Stout, J., Tonietto, R., Tscharntke, T., Tylianakis, J., Verboven, H., Vergara, C., Verhulst, J., Westphal, C., JooYoon, H., Purvis, A. 2016. Predicting bee community responses to land-use changes: Effects of geographic and taxonomic biases . Scientific Reports [IF 5.228]. Vol. 6: p.31153. (Refereed, Electronic) doi: 10.1038/srep31153.

Progress 05/01/15 to 09/30/15

Outputs
Target Audience:Our target audience includes alfalfa seed producers, almond growers, alfalfa leafcutting bee keepers, honey bee keepers, scientists (in symbiosis research and Entomology), and the general public. We reach this broad audience by presenting and publishing in the outlets mentioned in the products section. In addition, we actively talk to the media in an effort to publicize our science. Finally, we maintain a blog for the general public. As bee declines are big news, in our view outreach is extremelyimportant. Changes/Problems:We have refined some of our research questions and posed additional questions based on the results obtained to date. We detail these tweaks above. None of the tweaks constiute major changes or problems. What opportunities for training and professional development has the project provided?Three graduate students, one postdoctoral researcher, and 4 undergraduate research assistants helped on these projects. They were trained in sterile technique, microbiology, beekeeping, experimental design, statistics, and molecular techniques. How have the results been disseminated to communities of interest?McFrederick presented the results of project A to the Alfalfa Seed Growers Association on January 25 2016. McFrederick also presented the results of project B on December 9th, 2015to the Almond Board of California annual Almond Meeting attendees. Publications from both of these projects are in preparation. What do you plan to do during the next reporting period to accomplish the goals?A) The role ofLactobacillus kunkeeiin alfalfa leafcutter bee nutrition We are planningwork this summer thatwill be aimed at identifyingmicrobes that negatively affect ALCB nutrition. We have also proposed work to investigate microbes associated with ALCB parasites, in response to requests from alfalfa seed producers for research to help control these pernicious parasites. (B)The influence of cover crop forage on honey bee nutrition and gut microbes, and on colony growth and activity We are currently repeating this experiment in field season 2. We have also proposed research to extend this work to look at honey bee immunity. The objectives of this research are: To verify and extend previous data, we will determine whether the availability of supplemental forage in or near almond orchards affects colony growth. Determine if supplemental forage affects honey bee immunity by measuring pathogen load. Determine if the availability of supplemental forage affects honey bee immunity by measuring production of immune compounds. Determine if rapini cover crops and native plant cover crops provide the same quality of forage in regards to honey bee immunity. (C)The probiotic potential ofLactobacillus kunkeeifor honey production We are in the process of conducting individual bee experiments to determine if L. kunkeeican increase the health of individual bees. We are also exploring whether this bacterium cam inhibit the growth of molds in stored food in honey bee colonies..

Impacts
What was accomplished under these goals? A) The role ofLactobacillus kunkeeiin alfalfa leafcutter bee nutrition In the summer of 2015 we conducted an experiment to test for the role thatLactobacillus kunkeei (which we have in the meantime discovered is a new species that we are currently describing as Lactobacillus micheneri)plays in alfalfa leafcutting bee (ALCB) nutrition. Briefly, we used gamma irridiation to sterilize pollen provisions. We then treated the pollens in 4 ways (1) sterile control (2)L. micheneri only (3)L. micheneriand microbes from unsterilized pollen provisions and (3) all microbes from pollen proviosins. We then sterilized ALCB eggs, added these eggs to the treated provisions, and allowed the larvae to develop. Once the larvae had defecated and entered the overwintering state, we collected them and prepared them for protein analysis. We then conducted the bicinchoninic acid assay on the larvae to measure the amount of protein that each larvae had assimilated. We found that the sterile andL. michenerionly treatments had the highest levels of protein. This tells us that some components of the microbiome are lowering the amount of protein that a larvae can assimilate. Future work will be aimed at identifying these detrimental microbes. (B)The influence of cover crop forage on honey bee nutrition and gut microbes, and on colony growth and activity As this research is conducted during February-March, we doi not have any work to report in the period of05/01/2015- 09/30/2015. (C)The probiotic potential ofLactobacillus kunkeeifor honey production In April 2015, we set up 15 honey bee colonies frompackages. We treated 8 of these colonies withL. kunkeeiand left the other 15 sterile. We then fed them sugar solution withL. kunkeeimonthly, and weighed each colony monthly through July 2015. We additionally did whole colony evaluations on June 12th, 2015. We did not find differences in colony performance across the experiment. Probiotic effects may be hard todetect on the colony level. We are therefore in the process of conducting individual bee experiments to answer these questions.

Publications