Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Local Farmers (especially pumpkin and squash growers) NY State residents The scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate and undergraduate training: Our project involved significant student training. Kristen Brochu, a graduate student in the department of Entomology, took a lead role in all projects reported above. Kristen worked closely with a lab technician (Maria van Dyke) to conduct the field experiments. Additional grad students involved in the project included Mary Centrella and Katherine Urban-Mead. Undergrad Erin Krichilsky provided additional support for bumblebee microcolony experiments. Post-doc Elizabeth Murray was involved in screening of viral pathogens in solitary and social bees. The graduate students, undergraduate students, and post-docs associated with the project gained considerable expertise in bee biology, taxonomy, molecular biology, computational biology, microbial ecology, and pollination biology. How have the results been disseminated to communities of interest?We presented our results at the Entomological Society of America Annual Meeting and several symposia this past year. We also interacted significantly with growers over the course of the last year, through both in-person meetings and formal letters. What do you plan to do during the next reporting period to accomplish the goals?We plan to: 1. analyze pesticide data from pollen provisions 2. publish research reports 3. write up extension publications
Impacts
What was accomplished under these goals?
a) Major activities completed: 1. Microbial metagenomics - In order to fully characterize the microbial community of squash bee pollen provisions, we sampled pollen provisions collected from squash bee nests over three years (2014-2016) and 16 nesting aggregations in upstate NY. Nests were excavated and pollen provisions were collected using ethanol-sterilized tools and stored in sterile screw-cap vials which were immediately preserved in liquid nitrogen. Samples were stored at -80°C until DNA extraction. We collected 5-10 pollen provisions from each of the 16 nesting aggregations for a total of 136 samples. In order to compare pollen provisions to the local environmental microbial community, we also collected soil samples at the same depth as nest brood cells at each site as well as pollen and nectar samples from a random sampling of Cucurbita flowers in fields adjacent to nest sites. Adult bees were collected at each site both on flowers and in nests in order to assess the microbial community of adult digestive systems. To characterize the microbial community associated with pollen provisions of the squash bee (Peponapis pruinosa), we used standard amplicon sequencing protocols. For DNA extraction, we mechanically ground samples in liquid nitrogen and then extracted large molecular weight DNA using a modified phenol-chloroform protocol (Brochu 2018). We assessed DNA concentration and quality using a NanoDrop 2000. For bacterial sequencing we used primers 27F and 519 to amplify the V4 variable region of the 16S gene (Brochu 2018). For fungal sequencing we used primers ITS1F and ITS4R to amplify the internal transcribed spacer 1 gene (ITS1) (Brochu 2018). Bacterial and fungal taxa were amplified using the HotStarTaq Plus Master Mix Kit (Qiagen, USA). PCR products were run on a 2% agarose gel to verify amplification success and relative band intensity. Samples were pooled (proportionally according to their molecular weight and DNA concentration) and purified using calibrated Ampure XP beads. The resulting product will be used to construct an Illumina library and then sequenced according to the manufacturer's guidelines on an Illumina MiSeq instrument available from the Cornell Biotechnology Resource Center (http://www.biotech.cornell.edu/biotechnology-resource-center-brc). Sequence data were processed using QIIME software. Sequences were joined, demultiplexed and filtered for quality (removing sequences <150bp or with ambiguous base calls). Chimaeras were removed and Operational Taxonomic Units (OTUs) were generated by clustering at 97% sequence similarity. Reference sequences were picked from each cluster and taxonomy was assigned by comparison of reference OTUs to the Greengenes database for 16S and the UNITE repository for ITS1 genes. 2. Bumblebee microcolony experiment - Previous studies have suggested that generalist bees may not be capable of digesting certain pollens that are used by closely related specialists. Cucurbit pollen is large, spiny, and rich in toxic cucurbitacins and is therefore potentially either chemically or mechanically indigestible to generalist bees. Field studies conducted by Brian Nault in Geneva, NY had suggested that free foraging bumblebees, even when placed in cucurbit fields, avoided collecting cucurbit pollen (although they will readily collect cucurbit nectar). Are bumblebees avoiding cucurbit pollen because it is indigestible to their offspring or simply because there are better alternative sources of pollen in the area? We designed an experiment to test the hypothesis that cucurbit pollen is toxic to generalist bumblebees. Our experiment consisted of "microcolonies" (small groups of workers obtained from commercially sourced bumble bee colonies) fed different diets in order to assess the impact of cucurbit pollen on bee development. Our "microcolonies" consisted of 5 workers obtained from one of three source colonies purchased from Biobest Canada (Leamington, ON, Canada). Each source colony provided three microcolony replicates for each of five treatments (thus a total of 45 individual microcolonies). The five treatments were as follows: (1) CONTROL: standard, commercially available bumblebee diet, (2) SOLVENT: DMSO added to standard bumblebee diet, (3) CHEMISTRY: cucurbitacins (dissolved in DMSO) added to standard bumblebee diet, (4) CRUSHED: crushed cucurbit pollen, and (4) NATURAL: natural (intact) cucurbit pollen. Each treatment consisted of the unique pollen diet mixed with 30% sucrose solution. We monitored nectar and pollen consumption daily in order assess how much bees were consuming. For each microcolony, we recorded the following measures of fitness daily: (1) number of dead workers, (2) number of larval cells produced, and (3) number of ejected larvae (a measure of stress). Microcolonies were terminated once the first adult offspring eclosed or at 50 days from inception, whichever came first. b) Significant results achieved (major findings, developments, conclusions): 1. Microbial metagenomics - Our final data set consisted of 136 pollen provisions collected from 16 nesting aggregations across three years. We documented a diverse but stable microbial community in Peponapis pruinosa (Apidae), based on 16S and ITS1 amplicon sequencing (Brochu 2018). The brood cell microbiome is diverse, consisting of 162 bacterial operational taxonomic units (OTUs) and 243 fungal OTUs. We found four bacteria and 14 fungi that comprised the "core" microbiota of the pollen provisions. Among the bacteria, Lactobacillus micheneri was the dominant species (representing 79% of bacterial reads). The core fungal community consisted of Fusarium oxysporum (18%), Cladosporium cladosporioides (14%), Alternaria alternata (11%), and Starmerella spp. (6%). The microbial community of pollen provisions contained elements of adult digestive tract microbes, soil microbes, and floral microbes (obtained from analyses of pollen and nectar separately) but was distinct from those communities in terms of taxonomic composition. The core bacterial community of pollen provisions was shared with pollen and nectar of the cucurbit host-plants. The core fungal community of pollen provisions was shared with soil, nectar, and pollen from the cucurbit host-plants. While the brood cell microbiome is acquired from a variety of sources (soil, pollen, nectar, and adult digestive tract), the composition of this community is unique in the brood cell. Such a stable but unique microbial community may provide a pathway for a coevolved mutualism between bees and microbes. 2. Bumblebee microcolony experiment - At the end of our microcolony experiment it was clear that cucurbit pollen has a negative impact on bumblebee development. Pollen consumption per microcolony increased over time in all treatments except CRUSHED and NATURAL.In terms of mortality, the CRUSHED diet treatments showed significantly higher adult mortality than the other four treatments. Adult offspring production differed dramatically among treatments. While all microcolonies produced eggs, we found that treatment significantly affected the probability of a microcolony rearing offspring to adulthood. Microcolonies fed CONTROL, SOLVENT, and CHEMISTRY treatments were significantly more likely to produce adult offspring than microcolonies fed CRUSHED and NATURAL cucurbit treatments, which never produced adults. When larvae are sick or adult bees are stressed, they will eject larvae from their brood cells and discard them in the trash (where they defecate) or smear the larvae along the wall of the microcolony chamber. We found a significant effect of diet treatment on the number of ejected larvae per day, with post-hoc Tukey analyses showing that microcolonies fed the NATURAL cucurbit treatment ejected significantly more larvae than microcolonies in all other treatments (Brochu 2018).
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Brochu, K. 2018. Differential impacts of pollen quality and microbial communities on generalist and specialist bees visiting a shared food resource. PhD thesis, Cornell University.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Murray, E.A., J. Burand, N. Trikoz, J. Schnabel, H. Grab, B.N. Danforth (2018). Viral transmission in honey bees and native bees, supported by a global black queen cell virus phylogeny. Environmental Microbiology [published online 10 December 2018; https://doi-org.proxy.library.cornell.edu/10.1111/1462-2920.14501]
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Brochu, K. and Danforth, B.N. (2019) Microbes in the nests of a solitary specialist ground-nesting bee (Peponapis pruinosa) are diverse, stable, and distinct. Microbial Ecology (under review)
- Type:
Books
Status:
Awaiting Publication
Year Published:
2019
Citation:
Danforth, B.N., R.L. Minckley, J.L. Neff (2019). The Solitary Bees: Biology, Evolution, Conservation. Princeton, NJ: Princeton University Press (due August 2019).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Brochu, K. and B.N. Danforth (2018). Love it or leave it: Digestive adaptations of specialist and generalist bees on cucurbits. Joint Entomological Society of America and Entomological Society of Canada meeting, Vancouver, BC, Canada (November 10-14, 2018).
|
Progress 10/01/15 to 09/30/18
Outputs
Target Audience:Local Farmers (especially pumpkin and squash growers) NY State residents The scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate and undergraduate training: Our project involved significant student training. Kristen Brochu, a graduate student in the department of Entomology, took a lead role in all projects reported above. Kristen worked closely with a lab technician (Maria van Dyke) to conduct the field experiments. Additional grad students involved in the project included Mary Centrella and Katherine Urban-Mead. Undergrad Erin Krichilsky provided additional support for bumblebee microcolony experiments. Post-doc Elizabeth Murray was involved in screening of viral pathogens in solitary and social bees. The graduate students, undergraduate students, and post-docs associated with the project gained considerable expertise in bee biology, taxonomy, molecular biology, computational biology, microbial ecology, and pollination biology. How have the results been disseminated to communities of interest?We presented our results at the Entomological Society of America Annual Meeting in all three years of the grant. We also interacted significantly with squash and pumpkin growers over the course of the grant year, through both in-person meetings and formal letters. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
There is increasing evidence that bees host a diverse and biologically important microbial community in the pollen provisions that they gather, store, and feed to their developing larvae. This microbial community consists of diverse bacteria and fungi that likely play a key role in bee larval nutrition.The heavy use of fungicides in agroecosystems has the potential to disrupt the beneficial fungal communities present in the pollen provisions of solitary bees and thus to impact the diversity and abundance of these pollinator communities in agroecosystems. Our project sought to examine the interacting effects of agrochemicals (herbicides, fungicides, and insecticides) on the microbial community of one economically important wild pollinator of squash and pumpkin (the squash bee: Peponapis pruinosa). The squash bee is a native, ground-nesting, solitary bee and the single most important pollinator of squash and pumpkin across the United States. We found that the pollen provisions of the squash bee host a diverse community of microbes. Based on an analysis of 136 pollen provisions collected from 16 nesting aggregations across three years, we documented a microbial community of 162 bacterial and 243 fungal species. These bacteria and fungi are likely obtained from the environment, including flowers and soil. The microbial community of the pollen provisions of the squash bee are, however, unique from flowers and soil and are remarkably stable across space and time. Our studies provide new insights into ways to preserve and maintain native pollinators in agricultural habitats in the United States. Objective I. Screen pollen provisions of P. pruinosa collected from squash farms for pesticide residues. 1) Major activities: we surveyed agrochemical use and pesticide exposure in 16 squash and pumpkin farms in central NY. 2) Data collected: we obtained data on fungicides, pesticides and herbicides applied in squash and pumpkin fields in central NY over a three year period. 3) Results: We detected 20 fungicides (Chlorothalonil, Copper Ammonium complex, Copper Octanoate, Copper sulfate, Cyazofamid, Cyflufenamid, Famoxadone, Cymoxanil, Fluopicolide, Mefenoxam, Propamocarb, Propamocarb hydrochloride, Pyraclostrobin, Boscalid, Quinoxyfen, Rhamnolipid Biosurfactant, Triflumizole, Azoxystrobin, and Fludioxonil), 6 herbicides (Clomazone, Ethalfluralin, Glyphosate-isopropylammonium, Glyphosate-potassium, Halosulfuron-methyl, and S-metolachlor), and 4 insecticides (Acetamiprid, Lambda-Cyhalothrin, Spinosad, and Thiamethoxam) in the pollen provisions of the squash bee. 4) Key outcomes: we provided growers with information on the exposure of squash bees to pesticides in nesting aggregations in squash field in central NY via grower meetings. Objective II. Use metagenomic screening to identify the microbial community present in P. pruinosa pollen provisions. 1) Major activities: In order to fully characterize the microbial community of squash bee pollen provisions, we sampled pollen provisions collected from squash bee nests over three years (2015-2017) and 16 nesting aggregations in upstate NY. Nests were excavated and pollen provisions were collected using sterilized tools and stored in sterile screw-cap vials which were immediately preserved in liquid nitrogen. Samples were stored at -80°C until DNA extraction. We collected 5-10 pollen provisions from each of the 16 nesting aggregations for a total of 136 samples. In order to compare pollen provisions to the local environmental microbial community, we also collected soil samples at the same depth as nest brood cells at each site as well as pollen and nectar samples from a random sampling of Cucurbita flowers in fields adjacent to nest sites. Adult bees were collected at each site both on flowers and in nests in order to assess the microbial community of adult digestive systems. 2) Data collected: To characterize the microbial community associated with the squash bee, we used standard amplicon sequencing protocols. For DNA extraction, we mechanically ground samples in liquid nitrogen and then extracted large molecular weight DNA using a modified phenol-chloroform protocol (Brochu 2018). We assessed DNA concentration and quality using a NanoDrop 2000. For bacterial sequencing we used primers 27F and 519 to amplify the V4 variable region of the 16S gene (Brochu 2018). For fungal sequencing we used primers ITS1F and ITS4R to amplify the internal transcribed spacer 1 gene (ITS1) (Brochu 2018). Samples were pooled (proportionally according to their molecular weight and DNA concentration) and purified using calibrated Ampure XP beads. The resulting product was used to construct an Illumina library and then sequenced according to the manufacturer's guidelines on an Illumina MiSeq instrument available from the Cornell Biotechnology Resource Center (http://www.biotech.cornell.edu/biotechnology-resource-center-brc). Sequence data were processed using QIIME software. We excluded all mitochondrial, chloroplast, and Viridiplantae sequences. 3) Results: Our final data set consisted of 136 pollen provisions collected from 16 nesting aggregations across three years. We documented a diverse, but stable, microbial community in the squash bee based on 16S and ITS1 amplicon sequencing (Brochu 2018). The brood cell microbiome consisted of 162 bacterial and 243 fungal species. We found four bacteria and 14 fungi that comprised the "core" microbiota of the pollen provisions. Among the bacteria, Lactobacillus micheneri was the dominant species (representing 79% of bacterial reads). The core fungal community consisted of Fusarium oxysporum (18%), Cladosporium cladosporioides (14%), Alternaria alternata (11%), and Starmerella spp. (6%). The microbial community of pollen provisions contained elements of adult digestive tract microbes, soil microbes, and floral microbes (obtained from analyses of pollen and nectar separately) but was distinct from those communities in terms of taxonomic composition. The core bacterial community of pollen provisions was shared with pollen and nectar of the cucurbit host-plants. The core fungal community of pollen provisions was shared with soil, nectar, and pollen from the cucurbit host-plants. Unlike polylectic bees, in which the microbial community changes dramatically from site to site, the microbial community of the pollen provisions in P. pruinosa was stable across sites and the three years of the study. 4) Key outcomes: While the brood cell microbiome is acquired from a variety of sources (soil, pollen, nectar, and adult digestive tract), the composition of this community is unique in the brood cell. Such a stable but unique microbial community may provide a pathway for a coevolved mutualism between bees and microbes. Objective III. Evaluate the effect of pesticides on pathogens and mutualists of P. pruinosa by correlating our data on pesticides in pollen provisions with our data on microbial communities in pollen provisions. 1) Major activities: We combined data from Obj. 1 (pesticide exposure) with data from Obj. 2 (characterization of the brood cell microbiome) to examine how pesticide exposure impacts the brood cell microbiome. 2) Data collected: We combined data on the variation in microbial associates of the pollen provisions (Obj. 2) with data on agrochemicals present in the pollen provisions at the same sites (Obj. 1) and examined whether the microbial community varied with agrochemical use. 3) Results: Our studies are still underway but we have initial evidence that the microbial community of the pollen provisions is impacted by agrochemical use. 4) Key outcomes: Demonstrating a correlation between agrochemical use and microbial community composition is a first step toward understanding how agrochemicals may be impacting bee-microbe associations and ultimately bee health.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Brochu, K. 2018. Differential impacts of pollen quality and microbial communities on generalist and specialist bees visiting a shared food resource. PhD thesis, Cornell University.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Murray, E.A., J. Burand, N. Trikoz, J. Schnabel, H. Grab, B.N. Danforth (2018). Viral transmission in honey bees and native bees, supported by a global black queen cell virus phylogeny. Environmental Microbiology [published online 10 December 2018; https://doi-org.proxy.library.cornell.edu/10.1111/1462-2920.14501]
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Brochu, K. and Danforth, B.N. (2019) Microbes in the nests of a solitary specialist ground-nesting bee (Peponapis pruinosa) are diverse, stable, and distinct. Microbial Ecology (in review)
- Type:
Books
Status:
Awaiting Publication
Year Published:
2019
Citation:
Danforth, B.N., R.L. Minckley, J.L. Neff (2019). The Solitary Bees: Biology, Evolution, Conservation. Princeton, NJ: Princeton University Press (due August 2019).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Brochu, K.; and Danforth, B.N. (2016) Microbial Ecology of the Bee Brood Cell. Oral Presentation. 2016 International Congress of Entomology. Orlando, FL. (September 28, 2016)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Brochu, K.; and Danforth, B.N. (2017). Microbial Communities in Ground-Nesting Bee Brood Cells: Diverse, Stable, and Unique. Oral presentation. 2017 Annual Meeting of the Entomological Society of America. Denver, CO (November 4-8).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Brochu, K.; van Dyke, M.; Milano, N.; McArt, S.; Petersen, J.; Nault, B.; Kessler, A.; and Danforth, B.N. (2017) Effects of Pollen Defenses on a Generalist Pollinator. Oral Presentation. Symposium on the Physiology and Toxicology of Insect Pollinators. Cornell University, NY.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Brochu, K.; and Danforth, B.N. (2017) Microbial Ecology of the Squash Bee Brood Cell. Oral Presentation. 6th Annual Entomology Department Symposium. Cornell University, NY.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Brochu, K. and B.N. Danforth (2018). Love it or leave it: Digestive adaptations of specialist and generalist bees on cucurbits. Joint Entomological Society of America and Entomological Society of Canada meeting, Vancouver, BC, Canada (November 10-14, 2018).
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Local Farmers (especially pumpkin and squash growers) NY State residents The scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate and undergraduate training: Our project involved significant student training. Kristen Brochu, a graduate student in the department of Entomology, took a lead role in all projects. Kristen worked closely with a lab technician (Maria van Dyke) to conduct the field experiments. Additional grad students involved in the project included Mary Centrella and Katherine Urban-Mead. Undergrad Erin Krichilsky provided additional support for bumblebee microcolony experiments. These graduate and undergraduate students gained considerable expertise in bee biology, taxonomy, computational biology, microbial ecology, and pollination biology. How have the results been disseminated to communities of interest?We presented our results at the Entomological Society of America Annual Meeting and several symposia this past year. We also interacted significantly with growers over the course of the last year, through both in-person meetings and formal letters. What do you plan to do during the next reporting period to accomplish the goals?We plan to: 1. analyze pesticide data from pollen provisions 2. publish research reports 3. write up extension publications
Impacts
What was accomplished under these goals?
a) Major activities completed: Microbial Screening - We continued to use high-throughput microbial amplicon sequencing to detect both bacteria and fungi in pollen provisions that were collected during nest excavations. We completed the screening process for our third field season of data, adding 10 samples from each of five nest sites found in 2016, as well as nectar and bee gut samples from each site across all three years of the study to complement the pollen and soil samples that we have already sequenced. In addition, we sequenced several pollen provisions where the larvae had succumbed to fungal infection. Sequenced reads were then compared to existing databases to determine their identity. Species accumulation curves indicate that we have adequately sampled both the communities of bacteria and fungi that are associated with bee brood cells. Our sampling is the most extensive ever conducted for a ground-nesting bee species. Differential Expression Analysis - While in the field conducting fieldwork, we observed bumblebees and honey bees (Bombus spp. and Apis mellifera respectively) grooming cucurbit pollen off their bodies, but not collecting it. We conducted a microcolony experiment to assess the fitness costs of consuming cucurbit pollen for generalist bees and distinguished between the mechanisms of defense, either mechanical or chemical. The results of this experiment suggest strong physiological costs for generalist bees in consuming cucurbit pollen. We then prepared gut samples from A. mellifera (feeding on a standard pollen diet), B. impatiens (feeding on a standard pollen diet or cucurbit pollen) and P. pruinosa (feeding on cucurbit pollen) for transcriptome analysis in order to compare the genetic basis of the physiological costs of feeding on the cucurbit diet. Our microbial analysis of the guts of P. pruinosa will also enable us to compare their microbiome to that of honey bees and bumble bees to assess the relative contribution of the gut microbiome in protecting bees against a defended pollen diet. Chemical Comparison of Cucurbit varieties- Previous chemical analyses on different varieties of cucurbit pollen found strong chemical differences between varieties suggesting that chemistry may play a role in attracting bees to these plants. We conducted a follow-up bee choice experiment utilizing six varieties of squash and four varieties of pumpkin that were transplanted from the greenhouse to the field to assess differences in preference between specialist and generalist bees. It appears that specialist bees (P. pruinosa) prefer particular varieties of squash, but the preferences vary by bee gender, while generalist bees (honey bees and bumblebees) exhibit no preferences. We also know from previous work that generalists suffer physiological costs when fed on a cucurbit diet. These results together suggest that pollen chemistry may be interacting in interesting ways with the microbial community of P. pruinosa and could have important impacts on bee health. We thus conducted chemical analyses on the pollen provisions for which we had microbial community data and are now comparing the chemical variation in our pollen provisions to the microbial community. Soil Condition Modeling - We also conducted analyses for soil texture, organic matter, and aggregate stability from two field seasons (2015 and 2016) at the Cornell Nutrient Analysis Lab. Preliminary results show that P. pruinosa has specific requirements for nest site soils with a high sand content and aggregate stability as well as a narrow range of organic matter and moisture content. These results will be combined with GIS data to create a predictive model for squash bee nesting preferences. We will also assess whether or not different soil features might influence the microbial community or larval mortality at local (nest) and landscape scales. b) Significant results achieved (major findings, developments, conclusions): Microbial screening - We found surprising stability across all three years of the study. There were slightly greater differences in samples from 2016; however, there was a major drought in the region during the summer of 2016, so it is almost surprising that the microbial community is not more different. The microbial community was very similar across all sites of the study and across individual seasons (i.e. from August to September each year and from egg to larval developmental stages). The microbial community of the pollen provisions are also distinct from all other types of samples, suggesting a unique and consistent microbial community that may even have an adaptive function. Bee guts share microbes largely with pollen, nectar, and pollen provisions, having few community members in common with soil. Soil overlapped most with pollen provisions, suggesting that bees were moving a significant number of microbes into the brood cell from the surrounding soil. Nectar had a limited microbial community that largely overlaps with pollen and to some degree pollen provisions. Microbes seem to be ubiquitous in the environment with bees acquiring microbes from all available sources, although in different ratios. We also found that the microbial community is different in brood cells that have succumbed to fungal infection. There is a remarkable loss of diversity, notably a reduction in Lactobacillus, the single most common bacteria found in healthy provisions. Differential Expression Analysis - The results of our microcolony study suggest that mechanical defenses are important in reducing the pollen consumed by generalists and consuming cucurbit pollen renders generalist bees unable to rear offspring to adulthood. These severe physiological costs were previously unknown and could have an impact on the success of these generalist pollinators in agricultural environments. Our transcriptome results will shed light on the genetic basis for P. pruinosa's adaptation to a cucurbit pollen diet as well as the possible role that microbes might play in buffering bees from the physiological costs of feeding on a specialist diet. Chemical Comparison of Cucurbit varieties- Our previous chemical analyses suggest that variation in pollen chemistry may affect the floral preferences of specialist (P. pruinosa), but not generalist bees, while generalists suffer physiological costs when fed on a cucurbit diet. Taken together, these results suggest that pollen chemistry may be interacting with the microbial community to impact the health of P. pruinosa. We are preparing to test this hypothesis by comparing our chemical analyses to our data on the microbial community of pollen provisions. This analysis will allow us to assess the effect of pollen chemistry on the microbial community or how influential the microbial community is in changing the chemistry of pollen provisions. Soil Condition Modeling - Our results show that P. pruinosa has specific requirements for nesting site soil which may be related to acquiring beneficial microbes or avoiding harmful ones. Soil features may also affect which microbes are able to colonize the brood cell. In addition, we are using GIS data to build a predictive framework for preferred nest site conditions. Our current work is exploring how the microbial community is affected by local soil conditions as well as nest site features at the landscape level.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Brochu, K.; and Danforth, B.N. (2017). Microbial Communities in Ground-Nesting Bee Brood Cells: Diverse, Stable, and Unique. Oral presentation. 2017 Annual Meeting of the Entomological Society of America. Denver, CO (November 4-8).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Brochu, K.; van Dyke, M.; Milano, N.; McArt, S.; Petersen, J.; Nault, B.; Kessler, A.; and Danforth, B.N. (2017) Effects of Pollen Defenses on a Generalist Pollinator. Oral Presentation. Symposium on the Physiology and Toxicology of Insect Pollinators. Cornell University, NY.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Brochu, K.; and Danforth, B.N. (2017) Microbial Ecology of the Squash Bee Brood Cell. Oral Presentation. 6th Annual Entomology Department Symposium. Cornell University, NY.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Local Farmers (especially apple farmers) NY State residents The scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Post-doctoral, Graduate and undergraduate training: Our project involved significant post-doctoral and student training. Kristen Brochu, a graduate student in the Department of Entomology, took a lead role in the microbial screening and pesticide detection projects. Kristen worked closely with a lab technician (Maria van Dyke) to conduct the field experiments. These students gained considerable expertise in bee biology, taxonomy, computational biology, microbial ecology, and pollination biology. Elizabeth Murray, a post-doc on the lab participated in the analysis of pathogen and pesticide data. Elizabeth has developed expertise in the analysis of bee pathogen data as well as microbial metagenomics. How have the results been disseminated to communities of interest?We presented our scientific results during the International Congress of Entomology in Miami Florida in October, 2016as well as to stakeholders and state officials at meetings for the NYS Pollinator Protection Planin Syracuse and Albany. We also interacted significantly with growers over the course of the last year, through both in-person meetings, email, and written correspondence. What do you plan to do during the next reporting period to accomplish the goals?We plan to: 1. continue microbial analyses 2. analyze pesticide data from pollen provisions 3. write up research reports and extension publications
Impacts
What was accomplished under these goals?
a) Major activities completed: 1. Larval mortality assessment - We surveyed 24 farms in the Finger Lakes region of New York in the summer of 2014, 2015, and 2016 for squash bee (Peponapis pruinosa) nesting aggregations. We found six aggregations in 2014, five in 2015, and five in 2016, spanning a range of agricultural practices (i.e. both conventional and organic fields). Nest sites were carefully excavated multiple times over the course of the season, in order to collect pollen provisions and assess larval mortality, due to both fungal infection and cleptoparasite infection. We found high levels of fungal infection with increasing prevalence towards the end of the season (<10% of nest cells were infected with fungi in August, as opposed to just over 31% in September). Incidence of fungal infection was also found to be highly variable across sites ranging from 8% to 38% of nest cells infected. Rates of cleptoparasitism also varied, but were generally quite low (on average, about 2% of nest cells). We also found at least three different species of nest parasites affecting larval mortality. 2. Microbial screening - We used high-throughput microbial amplicon sequencing to detect both bacteria and fungi in pollen provisions that were collected during nest excavations. We screened 10 pollen provisions from each site, as well as soil and pollen from each site. Sequenced reads were then compared to existing databases to determine their identity. Thus far, 91 unique bacterial species were found in significant abundance in P. pruinosa pollen provisions. Lactobacillus spp., a bacteria also found in social bee nests that has exhibited anti-microbial properties, was the dominant bacteria, accounting for 84% of sequences. Four other common species account for an additional 10% of sequences. Three of these species (Saccharibacter floricola, Fructobacillus tropaeoli, and Fructobacillus pseudoficulneus) are associated with flowers and fruits. The other, Acinetobacter rhizosphaerae (1%), is associated with the rhizosphere and has been found to increase growth in several plant species. Fungal communities were more diverse, with 151 unique fungal species found overall, and with 11 species comprising 72% of the sequences. Fusarium oxysporum, a common soil fungus and occasional plant pathogen, was the most common species (accounting for 22% of sequences). We have identified this species as the primary agent of fungal infection detected in our pollen provisions. Plant pathogens comprise the majority of the other common species (Reniforma strues, Alternaria alternate, Bionectria ochroleuca, Cylindrocarpon olidum, Cylindrocarpon sp. Podosphaera fusca, and Epicoccum nigrum), together accounting for 28% of sequences. Cladosporium cladosporioides (12%) and Trichoderma hamatum (3%) are both species that are used in control of other fungi, either through parasitism, production of anti-fungal compounds, or competition. Starmerella sp. (7%) is a species of yeast in a genus that is commonly associated with flowers and bees. 3. Pesticide detection - We consulted with squash growers at 24 farms about the various chemicals used on their farms. Among these farmers, fungicides with 19 unique active ingredients are commonly used, with only six herbicides, and four insecticides. Based on these results, we will examine pollen provisions of squash bees for a diverse array of pesticides, with a special focus on fungicides. We are currently processing samples in order to begin the pesticide screening process. b) Significant results achieved (major findings, developments, conclusions): 1. Larval mortality assessment - Cleptoparasitism of Peponapis pruinosa pollen provisions occurs at very low rates in this system, with about 2% of nest cells affected. By far the most common cleptoparasite is Triepeolus, a generalist cuckoo bee species, but we also found low incidence of two as yet unidentified species, one fly and one beetle. We found that fungal infection represents the most important source of larval mortality in this system, representing about 30% of nest cells. Due to the high number of nest cells affected by this fungal infection, provisionally identified as Fusarium oxysporum, microbes likely play a major role in bee health and are likely a strong factor selecting for anti-microbial defenses in bees. We will continue to explore this idea through our microbial screening and pesticide analyses. We have also discovered a species of mite in P. pruinosa nest cells that may play a 'cleaning' role by feeding on the fungal invader. We will continue to explore the potential role of this mite, first identifying the species. 2. Microbial screening - We detected a diverse community of both bacteria and fungi in pollen provisions of P. pruinosa. This community included species of bacteria and fungi that are known to have anti-microbial effects and could potentially play a beneficial role in bee health. We also identified plant pathogens that could potentially play a role in pollen provision spoilage. We also found that the bacterial and fungal communities in the pollen provisions change exceptionally little from site to site and between 2014 and 2015. We have yet to analyze data from the 2016 field season. We observed 41 bacterial species in common between pollen provisions and pollen, including all but one of the most common bacterial species found in pollen provisions (Fructobacillus tropaeoli). We also observed 52 bacterial species in common between pollen provisions and soil, but of the most common bacterial species found in pollen provisions, only Lactobacillus spp. was found in soil. Lactobacillus spp. was the only bacterial species commonly found in pollen provisions that was found in both pollen and soil. These results suggest that pollen is a more important source of bacterial species than soil. We observed 29 fungal species in common between pollen provisions and pollen, including 7 of the most common fungi found in pollen provisions. We observed 61 fungal species in common between pollen provisions and soil, including 7 of the most common fungi found in pollen provisions. Of the most common fungal species in pollen provisions that were found in both soil and pollen, four are plant pathogens (Fusarium oxysporum, Alternaria alternate, Podosphaera fusca, and Epicoccum nigrum), and one is a biocontrol agent (Cladosporium cladosporioides). We also found that both the bacterial and fungal community in the pollen provisions was significantly different than the microbial communities found in pollen and soil, with respect to the species present and their abundances. These results suggest that nest cells have a unique microbial community that is resilient to environmental changes. We are further exploring how this microbial community impacts bee health. 3. Pesticide detection - Based on the feedback from growers in the Finger Lakes region, it is clear that fungicides are the most common type of pesticides sprayed by squash and pumpkin growers. Fungicides are thus more likely to appear in the pollen provisions of squash bees, impacting their health and development. Our analyses will focus on the impacts of fungicides and their interacting effects with the fungal community in squash bee nests. Particularly in light of the fungal infection, that is a significant source of mortality in this system, and the diverse fungal community, which includes known fungal biocontrol agents, we expect that fungicides will have both direct and indirect effects on bee health.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Brochu, K.; And Danforth, B.N. (2016) Microbial Ecology of the Bee Brood Cell. Oral Presentation. 2016 International Congress of Entomology. Orlando, FL. (September 28, 2016)
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