Honey bees have become increasingly unavailable and expensive for crop pollination since Colony Collapse Disorder was described and the world was alerted to CCD and the many factors that negatively impact honey bee health. Although other managed pollinators, such as bumble bees, alfalfa leafcutting bees, and blue orchard bees (BOBs), are available to pollinate certain crops, a ready BOB supply and well-developed management system for their use are especially lacking. BOB suppliers and managers need more information to supply customers with disease- and pest-free bees, to manage bees for optimal performance for pollination, and to maintain populations in numbers large enough for profitable business and to accommodate large commercial operations. We seek to meet six objectives that tackle primary stakeholder concerns: 1) understanding variation in developmental phenology of regionally-distinct BOB populations; 2) finding the genetic basis for regional differences of BOB sources through cross-breeding/mating experiments; 3) studies of population genetics; 4) examining pest and pathogen communities of BOBs from distinct sources; 5) describing patterns and seeking causes of BOB dispersal/loss in commercial settings; and 6) disseminating user-friendly information to the general public. Field collections, orchard and laboratory behavioral studies, molecular bioassays, and visual, x-radiographic, and microscopic diagnoses all will be employed to meet the research objectives. Dissemination of information and development of public education materials and events will be developed under consultation with Extension personnel in Washington, Oregon, California, and Utah. Findings will reduce the need to trap BOBs from the wild where the impact of trapping is yet unknown and, thus, will preserve local diversity of native populations. Better bee management would provide a more reliable source of alternative bees for pollination of suitable, pollinator-dependent crops. Understanding incidence of pests and disease will reduce the chance of epidemic outbreaks in managed populations and possible spillover to native bees. We will continuously educate the public and primary stakeholders, while seeking valuable input from stakeholders as results unfold and new research approaches are developed.
1. Determine the variation in developmental phenology of regional populations of BOBs by maintaining regionally-specific bees under managed or unmanaged conditions. Year 2 January-December; Year 3 January-June.
2. Determine the heritability of regional phenology traits for BOBs from California and Utah by examining population crosses in controlled experiments. Year 2 January-December; Year 3 January-June.
3. Using population genetic tools, assess the extent of population genetic differentiation among regions where BOBs are sourced and where they are deployed, detect the current structure of populations, and understand the potential for future admixture. Year 1 September – Year 3 May.
4. Using visual or molecular examinations, identify parasites and pathogens obtained from collections of bees from wild-trapped and managed populations, and use findings to infer how BOB stocking density, co-pollination with honey bees, and other management strategies may effect disease transmission within the pollination or mass propagation systems. Year 1 September – Year 2 August.
5. Determine the difference in the retention of females between California and Utah BOBs used as pollinators in regions outside of their geographic origin by examining the dispersal and flight range of these populations in cherry orchards in regionally distinct environments. Year 3 March-November.
6. Deliver high quality educational products and training on BOBs through extension and outreach to maximize information sharing and adoption of new technologies. Year 2 October – Year 3 December.
Hypothesis Objective 1: Developmental phenotypes of blue orchard bees are regionally adapted to match their local climatic conditions.
Hypothesis Objective 2: Allowing reproductive crossing of blue orchard bee populations from different geographic locations results in development timing in progeny according to sex that causes female bees to emerge as adults before male bees.
Hypothesis Objective 3: Blue orchard bee populations can be distinguished genetically according to regions where they naturally occur.
Hypothesis Objective 4: Some parasites and pathogens found in blue orchard bee populations are regionally-distinct, while others are common to most populations.
Hypothesis Objective 5: The propensity to disperse is increased when blue orchard bee populations are released for crop pollination in non-native regions and out of synchrony with natural adult emergence timing.
For Objective 1, in 2018, recently-provisioned (≤ one-week old) BOB nests traps in California, Washington, and Utah were collected from traps with tunnels lined with paper straws. Paper straws containing nests were shipped to the Pollinating Insects Research Unit (PIRU) in Logan, UT. The straws were sliced longitudinally so each cell could be visually inspected to select only cells with unhatched eggs or second instars. Unfortunately, most of the nests shipped from WA and CA contained cells with developing larvae that were too old for our experiment, and numbers of appropriate cells were too low for use in the experiment. The Utah nest cells, however, were collected by the PIRU research team and contained ample numbers of eggs and early instars. These nest cells were divided into two treatment groups for developing in either Washington temperatures (which is similar to Utah temperatures) or California temperatures (= 2 treatments, 300 cells each). For the “unmanaged” treatment, one group of cells were held in growth chambers programmed to mimic the natural daily temperature cycles of the climates of their origin (Wenatchee, WA) starting at the time of collection (see Pitts-Singer 2014). Programmed temperatures were 5-year averages obtained from local weather stations. Each cell was visually inspected daily to determine mortality and developmental stages until larvae (5th instars) completed the spinning of their cocoons (marking the start of the prepupal stage) (similar to Pitts-Singer et al. 2014). The bees were then x-rayed every two days to record when the bees reached pupal and adult stages. Thirty days after reaching the adult stage, a winter-managed subsample of bees were subjected to two intermediate temperatures over a two-week period until the winter storage temperature of 4°C was reached (Bosch & Kemp 2001). An unmanaged subsample of bees remained at fluctuating Logan temperatures. In March/April 2019, as if needed for pollination of WA and CA cherries, half of the cocooned adults from each treatment will be incubated at 22ºC (Bosch & Kemp 2000), and the other half will remain unmanaged until they emerge naturally. The length of each developmental stage, the time to emergence, duration of emergence period, and weight of adults for each group of bees will be measured. Mortality (and any known cause of it) also will be recorded for all developmental stages.
For this same objective, the collection of just-created bee cells from traps in CA, WA, and UT will again occur in spring 2019, and the experiment repeated with all three bee sources represented. Once all data are collected in 2019 and 2020, as was done in 2018-2019, generalized linear mixed models will be performed using PROC GLIMMIX to look for the effects of native region (CA, WA, and UT) and treatment (managed or unmanaged) on 1) developmental parameters for immature and adult bees, and 2) parameters concerning adult survival and duration of emergence period. Results will determine the value of trapping and/or propagating regional populations for local crop pollination, and whether efforts could be made to breed populations to be more locally synchronized with crop bloom.
For Objective 2, in spring 2018 in Winters, CA, adult bees from California and Utah were allowed to mate in screened field cages placed over blooming Phacelia tanacetifolia, and using supplied nesting tunnels and floral resources, the females could build nests.
Preliminary work suggested that mixing populations from different geographic locations may result in male progeny emerging after female progeny, which is the reverse of the natural protandrous system for this species (Trostle et al., unpublished). To determine if there are reproductive consequences of pairing bees from regionally-distinct trapping sites, we monitored cell production by each female and the progeny sex ratio. Offspring were allowed to develop to late larval or pupal stages in CA and then were transported to PIRU to be raised at constant temperatures using standard management procedures. Developmental time and survival were scored, and in spring 2019, these progeny will be used in cage studies for Year 2 to perform back crosses.
Two cages each contained mixed sources of adult bees (UT ♀ × CA♂; CA♂ × UT♀) and two cages each contained sames sources of adults as control crosses. Preliminary work suggested that mixing populations from different geographic locations may result in male progeny emerging after female progeny, which is the reverse of the natural protandrous system for this species (Trostle et al., unpublished). To determine if there are reproductive consequences of pairing bees from regionally-distinct trapping sites, we monitored cell production by each female and the progeny sex ratio. Offspring were allowed to develop to late larval or pupal stages in CA and then were transported to PIRU to be raised at constant temperatures using standard management procedures. Developmental time and survival were scored, and in spring 2019, these progeny will be used in CA cage studies for Year 2 to perform back crosses. The adults and progeny in the 2019 cage study will be monitored and reared as in 2018.
For Objective 3, in 2017 for molecular analysis at PIRU, we obtained BOB females through trap-nesting and net collection from where populations have previously been located in California, Washington, Idaho and Utah. In addition, we used previously collected bees from Maryland, Virginia and Michigan as outgroups in analyses. A minimum of 15 female (diploid) adults were obtained for each population for analysis. We extracted DNA from a single leg of each bee using a Chelex® extraction protocol (modified from Walsh et al. 1991). Extracted DNA was stored at -20 °C. We used PCR to amplify microsatellites. The 10-μl multiplex reactions contain: 1 μl extracted DNA, 1x Promega (Madison, WI) reaction buffer, 0.6 mM dNTP mixture, 0.1-0.4 μM primer, 0.001 mg BSA, 0.4 units Taq polymerase (Promega, Madison WI), and the MgCl2 concentration will be adjusted to 1.4 mM. The PCR conditions for multiplex reactions are: one 4 min cycle at 95 °C, 30 cycles of 95 °C for 30 sec, and annealing at 54 °C for 75 sec, then 72 °C for 45 sec. The cycles were followed by a final extension period of 15 min at 72 °C. We used four previously developed loci (OruA8, OruC4, OruS4, and OruS8) in addition to six unnamed loci currently being developed by J. Strange. We separated the DNA amplifications on an Applied Biosystems 3730xl automatic sequencer (Life Technologies), and we scored allele sizes using GeneMapper™ v4.0 Software (Applied Biosystems). During 2017-2018, we determined population genetics and performed spatial analysis. To account for differences in sample sizes across regions, we used the program HP-Rare (Kalinowski 2005) to calculate allelic richness and private allelic richness. Arlequin v.3.5.x (Excoffier and Lischer 2010) or Genepop v.4.2 (Raymond and Rousset 1995; Rousset 2008) were used to determine the fixation index FST for each pair of groups heterozygoseties, population pairwise comparisons, analyses of molecular variance (AMOVA), and isolation by distance (IBD). In 2018, we used the Bayesian methodology implemented in the program STRUCTURE (Pritchard et al. 2000) to infer population structure. We estimated the optimal number of populations by employing the methods of Evanno et al. (2005) in STRUCTURE HARVESTER (Earl and vonHoldt 2012). Pairing this method with our IBD analysis verified that farm-by-farm and region-by-region structure in populations would not be overlooked.
For FY18 Objective 3 molecular analysis, we obtained BOB females through trap-nesting and net collection from where populations have previously been located in California, Washington, and Utah. We extracted DNA from a single leg of each bee. We sampled DNA from 20 individuals at multiple sites in each state. We used PCR to amplify microsatellites at four previously developed loci (OruA8, OruC4, OruS4, and OruS8) in addition to six unnamed loci currently recently developed by J. Strange. DNA amplifications have been completed, but analyses have not been done on the raw data files. During FY19, we will determine population genetics and perform spatial analysis. We have developed and used these primers for examining the genetic differences in bee populations from the three different regions. Eight DNA microsatellite primers designed in 2017 were ordered and tested for utility in studying population differentiation. These primers were evaluated for variability in BOBs, and of the eight primers, four were found to have utility. We are continuing a second iteration of primer design and development to increase the number of molecular markers available for this study.
For Objective 4, to examine the health of the pollinator species, adult and immature bees plus cells with pests will be collected from trap nests from all sites where they are collected in 2017 and 2018 to evaluate pest and pathogen levels. In the nest cells, parasites (cleptoparasitic bees and pollen mites) and parasitoids will be removed, preserved in ethanol, counted, and identified (using DNA barcoding if necessary). The adult bees and developing bees will be frozen immediately in liquid nitrogen. While frozen, adults will be identified and photographed using high resolution Z-stacks, and then they will be bisected. One half were homogenized in TRIzol® reagent (Invitrogen) for analysis as individual bees, and the other half used for analysis of spores of the protozoan parasites and Nosema. Both sets of samples will be kept at -80°C until full analysis. In 2018, O. lignaria (BOBs) and other Osmia species were trap-nested from areas in CA and UT and sampled from the CA mating cage experiment (Objective 2). Significant numbers from the stock for the cage experiment died in the pre-adult stage and, thus, were collected and later frozen them at -80°C; they are awaiting analysis. A limited number of bee samples have been extracted with TRIzol® reagent (Invitrogen) for molecular analyses, and the DNA and RNA have been isolated. Because the PCR work resulted in the detection of chalkbrood (Ascosphaera torchioi) in several of the collected BOBs, samples of the fungus were sent for DNA sequencing to gain additional genetic sequences for the pathogen and to allow for comparison of samples from the different regions. Sequences have been received and are being analyzed. PCR probes for the protozoan diseases are continuing to be tested and resolved for specificity. In addition, in a separate set of samples of O. lignaria and O. ribifloris, the microbiome was sequenced and characterized from pollen provisions. Further data will be collected in the months to come, to characterize the pathogens, parasites, and parasitoids.
For Objective 5 in 2019, to measure dispersal and BOB establishment with respect to source population and management history (Sgolastra et al. 2016), wild-trapped bees collected from California and Utah will be introduced to spring-blooming commercial cherry orchards in both California and Utah. Nests collected in the previous year will be maintained in cold storage as cocooned adults in containers until ready to be incubated for emergence in spring 2018. Prior to incubation, loose cocoons from each population will be dusted with a combination of egg white protein powder and a unique powdered fluorescent marker, and emerging females will contaminate themselves with these markers as they crawl about in the container (Hagler et al. 2011, Boyle et al. unpublished). Since the use of protein markers has not yet been field-tested, we will also add a thoracic paint mark to denote whether a female BOB is sourced from California or Utah. Bees will be released into the orchards at a rate of 300 females and 750 males per acre into commercial cherry orchards at onset of bloom, with equal numbers of Utah- and California-sourced bees. Three orchards (isolated by at least 1 km) in California and Utah will be selected for this experiment. Within each orchard, a three-acre section (110 m × 110 m) will serve as the study site, and 16 corrugated plastic nest boxes containing 100 cardboard nesting tubes each (with paper straw inserts) will be distributed evenly throughout each three-acre section. Sixteen other nesting sites will be placed both far (up to 500 m) and very far (1 km) from release points (up to 48 nest boxes per orchard) so that bees collected at all sites can be evaluated for their dispersal distance from release sites. Bees will be permitted to forage and nest in orchards until bloom is at peak (100% bloom), during which time up to 10 females per nesting site will be collected twice during active nesting at each of the distances from release sites. While contained by researchers, the color of the bee will be recorded and also the bee will be rinse in buffer that will be later analyzed for the distinctive protein mark/fluorescent dye. This evaluation will provide a thorough estimate of overall BOB dispersal in an orchard environment and whether dispersal (and conversely local retention) varies due to a mismatch in orchard setting and source population/life history. In Utah, wild blue orchard bee populations have been observed foraging and nesting in managed cherry orchards (Pitts-Singer, unpub. data), which makes the use of the markers imperative for this study. Thus, in addition to evaluating emigration of managed bees from the orchard, we can also detect the immigration of (unmarked) wild bees into the orchard. By analyzing each nesting bee from all orchards in all both regions, we can obtain a thorough estimate of BOB retention over the course of the pollination season and statistically evaluate, how dispersal may vary due to source population. Statistical comparisons will be conducted using PROC GLIMMIX in SAS.
Graduate student, Morgan Dunn, is performing Objectives 1, 2, 5 and 6. She began her Master’s Degree program in January 2018. Ms. Dunn has had an MS committee meeting to establish a degree curriculum, has completed several hours of her coursework towards meeting degree requirements, has completed her project proposal narrative required by USU Graduate School, and has been preparing for projects for 2019 bee season.
Bee nests containing cocooned adults, natural enemies and dead cells were collected in summer 2017 and (by winter) shipped to PIRU. From these nests, we designated live bees for Objectives 1, 3, and 4. Cells with natural enemies and dead bees (larvae and adults) or unused provision are preserved for Objective 4. These nests have been x-rayed for determining the number and category of each cell type.
Subawards to transfer funds to Diane Alston of Utah State University for support of the graduate student and to Stephen Peterson of Foothill Bee Ranch have been dispensed as cooperative agreements with ARS PIRU.
For Objective 1, we continue to coordinate with cooperating bee managers and ARS staff to obtain newly sealed bee nests from Washington, California and Utah where they trap bees. Those nests will be sent to the Pollinating Insects Research Unit (PIRU) in Logan, UT for rearing at the various temperatures of the different regions.
For Objective 2, Steve Peterson identified Hedgerow Farms as a collaborator to provide floral resources for CA cage experiments. In late Spring 2018, Morgan Dunn and PI Pitts-Singer transport chilled cocooned bees and field cages to Winters, CA and set up the project equipment, painted and measured adult females, and released bees in cages. Steve Peterson assisted Morgan in obtaining 5 weeks of data in CA and he also monitored larval development. From the data collected thus far, there were no significant differences between BOB crosses in time to initiate nests and in duration of the nesting period, nor in mean cell production per female. Utah adult females were found to be significantly larger than the California females. Utah females were twice as likely to nest as were California females. The Utah females also produced significantly more female offspring than did the California females, regardless of which of the source of males with whom they mated.
Hedgerow Farms has been contracted (paid) to plant a field of P. tanacetifolia for the cage experiment work in 2019. The hiring of technical support is underway for helping in CA projects (Objectives 2&5). Cocooned adults collected from the cage project in 2018 are being managed for early spring emergence.
For Objective 3, a technician was hired to develop and use primers for examining the genetic differences in bee populations. Eight DNA microsatellite primers were designed, ordered and tested for utility in studying population differentiation. These primers were evaluated for variability in BOBs and of the eight primers, four were found to have utility.
For Objective 4, chalkbrood (Ascosphaera torchioi) was detected in several of the collected BOB samples using PCR. Additionally, the sequencing and characterization of the microbiome of pollen provisions of O. lignaria and O. ribifloris revealed some species-specific differences among the two species.
For Objective 5, Stephen Peterson has identified cherry producers that will allow 2019 research work to occur. Dr. Peterson will also provide CA-sourced bees, and UT-sourced bees have already been purchased for use. All are currently in cold storage. Nest boxes are being assembled and outfitted with cardboard tubes that provide at least two tunnels for each female released. The hiring of technical support is underway for helping in CA projects (Objectives 2&5).
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Objective 6 Producer and Ag Professional Education Activities: Several activities will be pursued to develop and deliver high quality and convenient educational products and training for BOB producers and vendors, growers interested in BOBs as orchard pollinators, extension agents, and researchers. First, a series of three one-hour webinar lectures will be developed and delivered, one in each of the three project years. The webinars will be offered in a “Learn at Lunch” format, scheduled mid-day during a winter month when bee producers, growers, and other stakeholders are most likely to be available to view their computers. Selected researchers, collaborators, and cooperators with new and relevant knowledge will share best management practices for BOB production as part of each lecture. The webinars will be delivered as live-streaming interactive audio/video via Zoom, a video conferencing software program, and will be hosted by eXtension. Utah State University (USU) Extension has a membership with eXtension that includes a site license for Zoom events. To better engage the audience, each presenter will join the webinar via webcam, so that the audience can see their face alongside their slideshow. Additionally, participants can use their computer mics to ask questions, or use chat windows to submit written questions and comments. Webinars will be advertised well ahead of time, and the number and location of participants tracked to assist with impact assessment. Second, a series of 5-6 YouTube videos will be developed to address management “how to” topics, e.g., how to: prepare bees for winter storage; assess and clean dead, diseased, and parasitized cells; situate nesting sites in or around orchards for maximizing pollination; best safeguard bees from hazards; track BOB nesting activity; use alternative floral resources during the nesting season. Video recordings were made with a high quality video camera by Extension collaborator and media specialist Dennis Hinkamp to portray bee management in orchards and how bees are processed in the laboratory. Using a microscope camera, photographs were taken by graduate student Morgan Dunn to show stages of bees development.
BOB videos will provide current and novel information on BOB management practices that can reach a wide swath of stakeholders. The YouTube video format will reach and engage stakeholders who rely on digital media for mobile and convenient information. Third, two public outreach workshops will be offered as a final day of the annual Orchard Bee Association meeting. Attended by registered members, including BOB producers and vendors of bees and accessories/equipment, orchardists interested in BOBs as alternative or complimentary pollinators, extension agents, researchers and students, this annual meeting is hosted at different locations across the western states. Registered attendees and all interested public stakeholders will be invited to attend the workshop. The workshops will be advertised through pollinator and fruit and nut tree crop producers, extension offices, the Natural Resource Conservation Service, and other relevant mailing lists and channels. The goal of the public workshops is to engage traditional and non-traditional stakeholders in obtaining the latest information about management and application of alternative pollinators.
Scholarly Publication and Educational Materials: A second volume to a popular and essential guide book on blue orchard bee management practices will be written and published. The first volume was authored by two former USDA ARS Pollinating Insects Research Unit scientists, J. Bosch and W. Kemp, entitled “How to manage the blue orchard bee as an orchard pollinator” and was published by the Sustainable Agriculture Network. New and expanded knowledge on BOB management and implementation will be included in volume 2, and will include various topics such as the benefits of alternative floral resources on BOB propagation, optimal nest box spacing and distribution within orchards, the risks and impacts of pesticide exposure to nesting BOBs, and BOB preferences for nesting materials/substrates.
All of the research objectives will be achieved with methodologies that will result in publication in peer-reviewed publications written by PIs (and student). PIs and student also will present findings at professional and industry meetings (e.g., Entomological Society of America national and branch meetings). Fact sheets will be created under consultation with extension collaborators as education materials that can be uploaded onto university-maintained websites.
Educational & Outreach Activities
List of presentations:
Dunn, M., D. Alston, T. Pitts-Singer, and S. Peterson. Mixing regionally-distinct populations of blue orchard bees, Osmia lignaria, to understand development, emergence, and reproductive success. 2018 Joint meeting of Entomological Society of America, Entomological Society of Canada, and Entomological Society of British Columbia, 10 min student competition talk, Vancouver, BC, November 11-14.
(Ms. Dunn was awarded the President’s Prize (1st place) for her presentation.)
Dunn, M., D. Alston, T. Pitts-Singer, and S. Peterson. Mixing regionally-distinct populations of blue orchard bees, Osmia lignaria, to understand development, emergence, and reproductive success. 2018 Orchard Bee Association, 15 min contributed research talk, University of California, Davis, December 7-9.
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