Progress 09/01/09 to 08/31/13
Outputs
Target Audience: The following individuals were trained under the grant during the last year: Michelle Duennes, graduate student in Cameron lab, Charles Dean, graduate student in Cameron lab, and Haw Chuan Lim, postdoc in the Cameron lab. The entomological/pollinator scientific community was served through multiple talks/posters at scientific meetings, including the Evolution meeting 2012, Pollinator Conference at Penn State 2013 and ESA meeting 2013. In addition, academic communities were served through formal colloquia presentations at universities. Outreach efforts targeted conservation groups, including Xerces Society as well as lectures at Fisher High School, a rural high school within 50 miles of the U of I, which targeted a student population with a fraction of socially, economically and educationally disadvantaged individuals-- this included formal and informal classroom instruction. Additional community groups targeted include Master Naturalists, Master Gardeners and NRCS/FSA employees, Extension educators and growers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Dr. Jeffrey Lozier, a postdoc in the PI's lab, was a co-PI on the project, and he completed his tenure in the Cameron lab and accepted a faculty position at the University of Alabama, beginning Fall 2011. Dr. Lozier's training in pollinator population genetic research was an important factor in obtaining his faculty position. A second postdoc, Dr. Haw Chuan Lim, also completed a 3-yr postdoctoral tenure on this project, and he recently accepted a new postdoc at the Smithsonian Institution. The experience he gained in next generation sequencing and analysis was an important factor in obtaining his new postdoc. A graduate student, Michelle Duennes, ran and tested the Nosema PCR reactions from museum material and thus learned how to work with historical specimens for DNA extraction and PCR. We have also trained several undergraduates and a technical assistant to help extract DNA from the museum samples and run PCR reactions. We also enhanced the career opportunities of a high school teacher (Isaac Stewart) working in rural Illinois (Fisher, IL), who has incorporated his training as a student in the lab into his teaching and research on pollinator decline in the U.S. Several of Mr. Stewart's high school students have participated in the project. How have the results been disseminated to communities of interest? We have participated in outreach throughout the project, giving presentations on the importance of wild pollinators at National Pollinator Week and at venues comprising Master Naturalists and Master Gardeners, and USDA NRCS and FSA supported outreach lectures to farmers and extension. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have accomplished all of the listed goals plus an additional component comprising a metagenomic analysis ofthe gut microbiota of multiple bumble bee species. Our findings are based on sampling museum specimens of declining U.S. bumble bee populations for Nosema bombi. We sampled from two time periods: 1985-1990 (prior to importation of colonies from Europe for commercial pollination) and 1994-2007 (after commercial colonies had been brought in from Europe and the pollination services industry was well developed). Our findings indicate that N. bombi prevalence in the U.S. increased significantly during the mid to late 1990s, after commercial pollination began in the U.S. Before this time period, prevalence was very low. Prevalence in European bumble bee populations were constant across the time period 1985-2007. Additional analysis of 16S rRNA and amplicon sequences from the Nosema genome suggest that Nosema bombi was present in U.S. populations prior to importation of colonies from Europe, but that before 1990 there were several different strains. After importation, a single strain—identical to the “European” strain— is dominant in U. S. populations and throughout Europe. This situation contrasts strikingly with the remarkably high amount of genetic diversity found in N. bombi from Asian bumble bees. These data suggest there may have been a sweep in the U.S. of a dominant European strain sometime during he mid-1990s, and are consistent with the hypothesis that development of commercial pollination in the U.S. influenced the spread of Nosema into populations of U.S. bumble bees. These results should have an impact on the manner in which colonies are both screened for pathogens and are allowed to be transported. We are recommending that colonies not be moved outside of their native ranges and we strongly encourage additional research on the population structure and genetic diversity of bumble bees in the U.S. to assist in decisions regarding colony movements and commercial development. We also encourage critical research to determine the direct impact of Nosema on the health of different bumble bee populations in the U.S. Due to the sensitive nature of the research findings we have waited to submit any manuscripts for publication until assessing multiple lines of evidence. We have two manuscripts in the works at this time.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2013
Citation:
Lim, H. C., Lozier, J. D., Duennes, M. A., Thorp, R. W., and Cameron, S. A. A test of the invasive pathogen hypothesis of bumble bee decline in North America. PLoS Pathogens.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Lim, H. C., Chu, C.-C., Seufferheld, M., and Cameron. S. A. 2013. Metagenomic analysis of bumble bee microbiota. ISME-Nature (Multidisciplinary Journal of Microbial Ecology).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2012
Citation:
Lim, H. C., Lozier, J. D., Thorp, R. W., and Cameron, S. A. Testing the Nosema invasion hypothesis. Evolution 2012 international meeting, Ottawa (proceedings abstract).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Cameron, S. A., Lim, H. C., Lozier, J. D., and Thorp, R. W. 2013. Causal factors in bumble bee decline: Testing the role of Nosema bombi. Entomological Society of America Annual Meeting (proceedings abstract).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Lim, H. C., Lozier, J. D., Thorp, R. W. and Cameron, S. A. Causal factors in bumble bee decline: Testing the role of Nosema bombi. International Conference on Pollinator Biology, Health and Policy (Penn State, Aug. 2013).
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: We have nearly completed this research on testing the hypothesis that Nosema bombi, introduced from Europe, is the cause of decline in U.S. bumble bees. This research has resulted in data collected from museum specimens from across the U.S., enabling us to collect pathogen prevalence from historical records. We developed a protocol that allowed us to obtain the Nosema DNA without destructive sampling of the specimens, a new approach to looking back in time at pathogen loads in dead animals. This can be used in other similar studies to look back in time. During the last year we gave a presentation of the results to date at the Evolution meetings in Ottawa, July 2012. We also nearly completed the development of the next generation genotyping of N. bombi for analysis of strain variation in Europe and the U.S. Multiple undergraduates, a graduate student and a postdoc have received training on this project over the last 12 months. In particular, the postdoc has developed the bioinformatic techniques required for analysis of the next generation RAD sequences. We also participated in outreach throughout, including presentations about the importance of wild pollinators at National Pollinator Week and to our local Master Naturalists group. We are nearing completion of this project and expect to submit our first manuscripts on the results in April and May 2013. PARTICIPANTS: Dr. Jeffrey Lozier, a postdoc in the PI's lab, is a co-PI on the project, and he completed his tenure in the Cameron lab and accepted a faculty position at the University of Alabama, beginning Fall 2011. Dr. Lozier is continuing to contribute to the research in terms of data analysis. A second postdoc, Dr. Haw Chuan Lim, was recruited to work specifically on this project and he has developed the next generation amplicon sequencing. We have also trained several undergraduates to help extract DNA from the museum samples and run PCR reactions. Dr. Robbin Thorp, a second co-PI, has completed his museum contacts and assistance with obtaining the museum specimens for the research. TARGET AUDIENCES: The primary target audience is the international community of scientists and conservation groups interested in global decline of wild pollinators, in particular the possible role of pathogens. There is interest in the role of the commercial bumble bee pollination services industry in aiding in the spread of potentially harmful pathogens. We are hoping to elucidate this potential by careful scientific analysis, which should be useful to conservationists as well as the commercial industry. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our findings from sampling museum specimens of declining U.S. bumble bee species for Nosema bombi both before and after bumble bee importation from Europe was allowed (1992-1994) indicate that N. bombi prevalence in the U.S. increased after the time period when commercial colonies were imported into the U.S. (1992-1994). Before this time period, prevalence was very low. After 1994, the prevalences in declining species increased significantly through the mid-late 1990s. Prevalences in European bumble bee populations were constant across the time period 1985-2000. Additional analyses from 16S rRNA and amplicon sequences from the whole genome suggest that Nosema bombi was present in U.S. populations prior to importation of colonies from Europe, but that before 1990 there were several different strains. After importation, only a single strain can be found in U.S. populations. These data in sum are consistent with the hypothesis that importation of bumble bees from Europe influenced the spread of Nosema bombi into populations of U.S. bumble bees.
Publications
- No publications reported this period
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: To test the Nosema invasion hypothesis, we have obtained museum specimens from both western and Mid-western U.S. institutions for analysis of N. bombi from pre- and post-1992 Bombus host specimens. We have also completed N. bombi screening of western European B. terrestris hosts from museum collections, and collected B. terrestris workers from their native range (focusing on the original populations from which the captive rearing industry selected its queens for commercial colony production) in southern France and Belgium; these native populations have been screened for N. bombi. Our proposed non-destructive DNA extraction techniques work well with highly sensitive PCR primers for the internal transcribed spacer (ITS) region of the N. bombi genome. To find sufficient variation among potentially different strains from different Nosema populations obtained from museum and field collections in the U.S. and Europe (goal to elucidate the geographic origin of Nosema bombi found in North American Bombus), the major output of the second year was development of the Next Generation genome reduction approach. We first had to develop an efficient method to purify N. bombi spores from single individuals, including triple confirmation methods to insure against contamination (a major issue when working with fungal spore DNA). Next Gen sequence data requires use of an appropriate computational method (i.e., pipeline) to handle the 454 data, which we modified from one under development (PRGmatic). We also collected the material from European field sites It is too early to disseminate any of the information we have collected thus far. PARTICIPANTS: PD Cameron oversees the entire research effort. She has also organized and assisted with field collections. J. D. Lozier, co-PD, assisted with European field collections, extracts DNA from museum specimens and PCR screens for presence of N. bombi in the specimens. Postdoc H. C. Lim developed the N. bombi spore purification procedure and the Next Gen sequencing effort, including learning and working with the computational pipeline for data handling. Co-PD R. Thorp assisted with European field collections of B. terrestris hosts and obtained host specimens from western museum collections. Contacts at Louisiana State University (Sara Hird) assisted with application of the pipeline (PRGmatic, in press in Molecular Ecology Resources) for Next Gen data handling. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
From the screening of B. terrestris from western European collections we have determined that N. bombi is present in the representative museum specimens from pre-1992 (before putative introduction to U.S.) and post-1992. We detected positive infections in 4 individuals (10%*) collected from 1988-1991. We have conducted spore isolation and purification, reduced genome amplification and next-generation sequencing for seven samples from Europe and North America (pools of N. bombi from seven infected bees). Sequences generated were clustered and aligned with a program pipeline comprising CAP3 and BWA. Genetic variation at individual loci was detected using SAMtools and VarScan. To ensure that the loci detected can be attributed to the N. bombi genome (rather than to contaminating genomes such as bacteria), we compared them to loci present in N. ceranae and N. apis using BLASTn and BLASTx. We will carry out genetic characterization on eight additional samples to ensure that a large number of polymorphic markers are recovered.
Publications
- No publications reported this period
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: MULTI-LOCUS-SEQUENCE-TYPING (MLST) UPDATE: During the first year of this project we have used the Nosema ceranae genome to develop primers for PCR amplification of N. bombi genes from spores found within declining populations of North American Bombus species. For each of these genes, amino acid and nucleotide sequences from N. ceranae were aligned with other available reference sequences in GenBank and primers were designed to conserved regions surrounding areas of high sequence polymorphism. We currently have four genes that perform well, although no sequence variation between North America and European isolates have been found. DNA sequences obtained from our samples were blasted against those in GenBank to ensure that they are derived from species of Nosema. DNA FROM HISTORICAL SPECIMENS UPDATE: A researcher recently inquired whether our proposed whole-specimen DNA extraction procedure would cause the specimen to become unsuitable for any later DNA work. We felt that others might have similar concerns and we tested whether we could successfully extract DNA (using a Chelex protocol) from forelimbs of specimens that had previously undergone whole-specimen extraction. Using appropriate sterile conditions, we were able to successfully extract DNA and amplify a Bombus-specific locus of ~165bp. These results demonstrate that a brief (~1 hour) incubation of a whole bumble bee specimen in a Chelex/Proteinase-K solution does not prohibit future use of that individual for additional DNA work. We have obtained specimens of B. terrestris from Belgium (1980s-1990s), from which we have successfully identified N. bombi infections. We have obtained specimens of various North American species currently affected by N. bombi from institutions in MI, MN, KS, and OH. We plan to screen these specimens for N. bombi once we have completed analyzing the contemporary samples to avoid possible PCR contamination. COLLECTION OF BOMBUS TERRESTRIS POPULATIONS: We (Cameron, Lozier and Thorp) completed fieldwork in southern France, collecting individuals from original sites used for supplying queens for commercial rearing in the early days of the industry (1980s). We collected from 23 populations across the range of southern Provence, the Catalan region, near the border with Spain, and near Paris. In total we collected nearly 500 individuals, from which we are currently screening for presence of N. bombi and sequencing for genetic variation for comparison with North American N. bombi from closely related (declining) species. Thus far, we have examined ~200 B. terrestris samples, in addition to ~20 specimens of B. affinis+B. terricola collected in the US this summer, and identified ~28 N. bombi infections that are currently being sequenced. This rate of infection is typical of values found in the literature. The PCR-based screening of N. bombi is supplemented by inspection of the bumble bee midgut under a phase-contrast microscope. PARTICIPANTS: PI Cameron devoted about 10% time to this project during the first year, including organizing and participating in fieldwork to collect Bombus terrestris in southern France to obtain specimens for screening Nosema bombi. Co-PIs J. D. Lozier and R. Thorp participated in this collection, with the assistance of our host and European collaborator, Pierre Rasmont (Univ. Mons Hinaut, Belgium). Dr. Haw Chuan Lim was hired as a postdoc in August to engage in research on the genetic variation of N. bombi, and is assisting co-PI Lozier with genetic screening of spores. He has worked full time on this project since his August appointment. Dr. Lim will attend a genomics workshop at Cold Spring Harbor Laboratories on Long Island to advance his knowledge of next-generation genomic approaches that will add new technical expertise to our Nosema research. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Thus far, all available sequence data has proven to be identical between North American specimens and European specimens, although the sample size of sequenced individuals is still very small. We will continue to sequence various genes of N. bombi as we detect them in European and North American samples.
Publications
- No publications reported this period
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