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 will be collected from traps with tunnels lined with paper straws. Paper straws containing nests will be shipped immediately to the Pollinating Insects Research Unit (PIRU) in Logan, UT. The straws will be sliced longitudinally so each cell can be visually inspected to select only cells with unhatched eggs or second instars. These nest cells will be divided into two treatment groups from each of the three sources until up to 200 cells are available for each region and treatment (total cells = 1,200). For the “unmanaged” treatment, one group of cells will be held in growth chambers programmed to mimic the natural daily temperature cycles of the climates of their origin starting at the time of collection (see Pitts-Singer 2014). Temperatures will be 5-year averages obtained from local weather stations. For the “managed” treatment, the other group will be contained in a growth chamber set at a constant temperature of 26°C (Bosch & Kemp 2000) where larvae will continue to develop without changes in diurnal thermal cycles. Each cell will be visually inspected daily to determine mortality and developmental stages until larvae (5th instars) complete the spinning of their cocoons (marking the start of the prepupal stage) (similar to Pitts-Singer et al. 2014). The bees will then be x-rayed every two days to record when the bees reach pupal and adult stages. Thirty days after reaching the adult stage, the managed bees will be subjected to two intermediate temperatures over a two-week period until the winter storage temperature of 4°C is reached (Bosch & Kemp 2001). The unmanaged group will remain at fluctuating temperatures of their climates of origin. In March/April 2018, as if needed for pollination of 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. Generalized linear mixed models will be performed using PROC GLIMMIX to look for the effects of native region (California, Washington, and Utah) 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, adult bees from California and Utah will be allowed to mate with each other in controlled experiments, having reciprocal (UT ♀ × CA♂; CA♂ × UT♀) and controlled (same source) crosses. Preliminary work suggests that mixing populations from different geographic locations results 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 will monitor mating behavior and reproductive output. Receptivity of males and females to mating attempts will be scored. Mated females will be allowed to nest in a greenhouse, and then the offspring will be raised at constant temperatures. Developmental time and survival will be scored, and the number of cells and sex ratio will be recorded.
For Objective 3, in 2017 for molecular analysis at PIRU, we will obtain BOB females through trap nesting and net collection from where populations have previously been located in California, Washington, Idaho and Utah. In addition, we will use previously collected bees from Maryland, Virginia and Michigan as outgroups in analyses. A minimum of 15 female (diploid) adults will be obtained for each population that we will analyze. We will extract DNA from a single leg of each bee using a Chelex® extraction protocol (modified from Walsh et al. 1991). Extracted DNA will be stored at -20 °C. We will use PCR to amplify microsatellites. The 10-μl multiplex reactions will 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 will be followed by a final extension period of 15 min at 72 °C. We will use four previously developed loci (OruA8, OruC4, OruS4, and OruS8) in addition to six unnamed loci currently being developed by J. Strange. We will separate the DNA amplifications on an Applied Biosystems 3730xl automatic sequencer (Life Technologies), and we will score allele sizes using GeneMapper™ v4.0 Software (Applied Biosystems). During 2017-2018, we will determine population genetics and perform spatial analysis. To account for differences in sample sizes across regions, we will use 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) 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 will use the Bayesian methodology implemented in the program STRUCTURE (Pritchard et al. 2000) to infer population structure. We will estimate 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 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 will be 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.
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 Utah and California will be introduced to spring-blooming commercial cherry orchards in both source and reciprocal states. 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). 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. At least 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. 16 other nesting sites will be placed 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), at which time up to 10 females per nesting site will be collected, stored at -20°C, and later evaluated for the presence and type of protein marker/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.
We have recruited a graduate student, Morgan Dunn, to perform Objectives 1, 2, 5 and 6. She began her Master’s Degree program in January 2018. 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 have 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.
A subaward to transfer funds to Foothill Bee Ranch (Steve Peterson) is being dispensed in an ARS-Foothill Bee Ranch cooperative agreement.
For Objective 1, we have coordinated with cooperating bee managers 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 has found a collaborator orchardist in Orland, California, and together they have prepared ground and seeded a plot of spring-blooming flowers known to be appropriate for blue orchard bee nesting. In late Spring 2018, Morgan (with ARS technical support) will transport chilled cocooned bees and field cages to Orland so that the experiment can be set up before the flowers bloom.
For Objective 3, a technician has been hired and already begun to develop and use primers for examining the genetic differences in bee populations from the three different regions. Eight DNA microsatellite primers were designed, ordered and tested for utility in studying population differentiation. These primers were evaluated for variability in blue orchard bees, and of the eight primers, four were found to have utility. A second iteration of primer design and development is underway to increase the number of molecular markers available for this study.
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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 will be collected from orchards and production facilities with a high quality video camera.
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 finings 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
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