Best Management Practices for Regionally-Distinct Populations of the Blue Orchard Bee

Student Mentored

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 were dispensed as cooperative agreements with ARS PIRU.

Graduate student Morgan Dunn performed experiments and analyses for Objectives 1, 2, and 5 and lead efforts to obtain materials for extension videos for Objective 6. She began her Master of Science Degree program in January 2018.  Ms. Dunn had timely MS committee meetings and completed all of her coursework to assure that degree requirements were met.  She passed her qualifying exam in Fall 2019.  She is working to finish her thesis and journal articles from her research so that she will defend her thesis in May 2021 or shortly thereafter.

Bees Acquired

Bee nests containing cocooned adults, natural enemies and dead cells were collected in summers of 2017 and 2018 and (by winter) were 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 were preserved for Objective 4.  These nests were x-rayed for determining the number and category of each cell type.  Bees for Objective 5 were purchased from a Utah suppler and provided for free from collaborator Steve Peterson in California.  Other bees were collected through trapping or netting as described in methods .

Studies Performed

For Objective 1, we coordinated with cooperating bee managers and ARS staff to obtain newly sealed bee nests from Washington, California, and Utah where wild bees could be trapped.  Those nests were sent to the Pollinating Insects Research Unit (PIRU) in Logan, UT for rearing at the various temperatures of the different regions, as described in the methods.  Due to the problems incurred for getting bees from all regions in 2018, only bees obtained in 2019 were summarized and analyzed for this objective. The cells received from California, Washington, and Utah were randomly sorted so that approximately half of each population were put into incubators and allowed to develop at managed temperatures (constant 26°C) and the other half were exposed to unmanaged temperatures that mimicked their natural climate (6-year average annual temperatures by the hour, updated each week) (Table 1).  During larval development, bees were checked three times per week until cocoons were completed ("e" in figure).  After which, bees were X-rayed one per week to observe transitions from prepupa to pupa and adult ("f-h", respectively, in figure). Once bees reached adulthood, then bees were X-rayed once per month.  One month after bees reached adulthood, the managed bees were gradually cooled over a two-week period until reaching an overwintering temperature of 4°C.  Unmanaged bees remained at their natural temperatures throughout winter.

Mortality was high for all CA-sourced bees in both treatments due to desiccation (low humidity levels) in the chamber containing bees in the managed treatment and mold in the rearing chamber containing the bees in the unmanaged treatment. Therefore, all California bees were excluded from statistical analyses. We found that bees sourced from Utah and Washington both survived at a significantly higher rate when exposed to managed compared to unmanaged treatments (Table 1).  UT-sourced bees that were unmanaged also had significantly lower mortality overall than unmanaged WA-sourced bees (P = 0.01).  For bees in the unmanaged treatments, mortality at the prepupal stage was significantly higher in UT-sourced bees than in WA-sourced bees.  When comparing mortality of life stages regardless of bee population source, we found that there was significantly less mortality at the egg stage ("a" in figure) than any other life stage.  Regardless of treatment, significantly more WA-sourced bees died as larvae ("b-d" in figure) than did UT-sourced bees. For bees in both population sources, the entire immature development was significantly shorter for bees in the managed treatment than bees in the unmanaged treatment.  Analysis is ongoing to further compare the duration of life stages between treatments and bee sources, as well as the timing and duration of spring emergence ("i" in figure). 

For Objective 2, Steve Peterson identified Hedgerow Farms as a collaborator to provide floral resources (their own planting of P. tanacetifolia for seed production) for California cage experiments.  In late Spring 2018, Morgan Dunn and PI Pitts-Singer transported chilled cocooned bees and field cages to Winters, CA.  They set up the project equipment (e.g., cages and nest boxes), painted and measured adult females, and released bees in cages.  Steve Peterson assisted Morgan Dunn in obtaining 5 weeks of data in California and he also monitored larval development throughout the summer.  From the data collected for year 1, 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.

For the cage experiment work in 2019, Hedgerow Farms was contracted (paid) to plant a field of P. tanacetifolia.  An ARS technician was hired specifically for helping with California projects (Objectives 2&5).  Because cocooned adults collected from the cage project in 2018 were managed for early spring emergence for the 2019 cage study, all progeny that successfully emerged were again deployed in the same field cages for the 2019 component of the project.  We had enough progeny to have one cage per cross, except the “pure Utah” cross, which had 2 cages.  The experiment was conducted according to the 2018 protocols, and resulting offspring from these second-year crosses were again reared in a similar manner as the progeny produced from year 1 and were again monitored for spring emergence.  Results currently are being analyzed, although there are only small samples and data sets for some crosses.

For Objective 3, an ARS technician was hired to develop and use primers for examining the genetic differences in bee populations.  To date, 46 primer pairs were designed and tested for amplification by gel electrophoresis.  Pairs that yielded a product sized between approximately 100 and 500 base pairs in length as visualized on gels were then ordered as dye-labeled primers for closer scrutiny of fragment sizes.  Seventeen pairs met this criterion.  They were tested in single and multiplex reactions using bees from different regions.  Three new potential microsatellite loci resulted, and the remainder either were homozygous or amplified poorly.   We optimized multiplex reactions to examine genetic differences in bee populations, and we continued designing and testing new primers in 2020.

In 2020, an ARS technician was hired to develop and use primers for examining the genetic differences in bee populations.  In total, in silico analysis of microsatellites availability on the BOB genome identified 170,011 imperfect, 33,662 perfect, and 859 compound microsatellites.  After filtering for specific criteria, we identified 24 perfect microsatellites and 26 imperfect microsatellites for further testing.  No compound microsatellites fit our filtering criteria.  After confirming product size of the 50 loci with PCR amplification and agarose gel visualization, 31 microsatellites were identified for continued testing in the current study (Table 1).  16 perfect and 15 imperfect microsatellite loci were retained for further analysis.  The figure below shows an example a gel depicting the confirmation of four loci developed in the current study (Oli14, Oli021, Oli053, Oli064), as well as a locus developed for O. rufa (Oru4).  Here we only report the results of the 16 perfect microsatellites as we are still testing the 15 imperfect microsatellite using PCR amplification and agarose gel visualization.

Figure Legend: Image of PCR-amplified DNA of O. lignaria on an agarose gel (1.2%) for four loci (Oli14, Oli021, Oli053, Oli064) developed in this study and one locus (OruS4) developed in a previous study for O. rufa. Ladder is 1 KB (far left side of image); bright band in the middle indicates 500 nucleotide (base) mark.

For the 16 perfect microsatellite loci that produced product sizes < 250 nucleotides, average allelic richness and H_exp across all populations was 0.37 (±0.02 SEM) and 0.66 (±0.03 SEM), respectively. The average number of successfully genotyped chromosome sets to estimate these genetic diversity metrics is 70.67 (±0.59) (Table 2).  For these loci, we tested for differences in genetic diversity and structure in four populations of BOBs: Franklin, ID (n = 8), Placerville, CA (n = 13), El Dorado, CA (n = 16) and Fresno, CA (n = 11).

Figure Legend: Median allelic richness (A) and Expected heterozygosity H_e (B) across four BOB populations (El Dorado, CA; Franklin, ID; Fresno, CA; Placerville, CA) in western North America.

We failed to reject the null hypothesis that there are significant differences in average allelic richness (Kruskal Wallis Test: X² = 2.03, df = 3, P = 0.57) (Figure A) and H_exp (Kruskal Wallis Test: X² = 3.53, df = 3, P = 0.32) (Figure B) across BOB populations).  Furthermore, AMOVA results found only 12.20% of the genetic variation among the four populations, 64.60% among individuals within the four populations, and 23.19% within individuals.  Because most of the variance occurs among individuals within the four populations (64.60%), there is evidence of significant population genetic structure (overall F_ST = 1.24, p = 0.0009) (Table 3).  Furthermore, there is no evidence for inbreeding within populations (overall F_IS = 0.71, p = 0.98), which is supported by high estimates of allelic richness and H_exp.

In conclusion for Objective 3, we have identified 16 novel microsatellite loci for BOBs that are immediately available for population genetic analysis. These loci exhibited high genetic diversity (allelic richness, H_exp) and have the capacity to estimate genetic structure (F_ST, F_IS).  These loci have the potential to inform management and conservation decisions.  Continued research at PIRU will be pursued to determine the utility of the 15 novel imperfect microsatellites identified in this study (Table 2).

For Objective 4, BOB nests collected in 2017 from wildlands in California, Washington, and Utah (three sites in each state) contained x-ray detectable pests and one pathogen (Table 4).  Of cells examined, the natural enemies included the cleptoparasites Stelis sp. (cuckoo bee; Megachilidae), Tricrania (blister beetle; Meloidae), and Sapyga sp. (wasp, Sapygidae), and the parasitoid Monodontomerus obscurus (wasp, Pteromalidae).  The presence of some cells containing chalkbrood indicates that the fungal pathogen Ascosphaera sp., presumably A. torchioi, contributed to the death of some larvae.  There were also other dead immatures to which no cause could be assigned.  From these collections and rates of parasitism, we can conclude that the Utah samples were the most affected by Stelis, and the California samples had the most diverse array of natural enemies.  By far for these populations, most cells contained healthy, live adult bees (Table 4).  It was brought to our attention that some bee trappers clean out natural enemies and release bees back into trapping sites.  This activity may artificially reduce the number and types of natural enemies found at these particular sites.  Future studies are needed over multiple years to better elucidate the types and rates of attacks by pests and pathogens in sites where trappers are not influencing the bee populations. 

Given limited stocks of bees from the three different states, it was challenging to get adequate healthy bee samples from the different regions, in particular from Washington and California.  In addition, the COVID-19 pandemic created issues with performing the molecular assays for the pathogens with samples being extracted right before the lab work was affected (samples are being stored at -80 °C for future work).  Additional work will be performed when possible to gain a more complete record of the pathogens in BOBs from the different regions using these samples.  From California, 179 dead BOB larvae have been extracted for RNA and DNA and await analysis.  For newly emerged adult bees, we have extracted DNA and RNA from 52 bees from California, 64 from Utah, and 27 from Washington.  We plan to analyze these bees for most of the pathogens described below in a complete discussion of our research findings on pathogens in Osmia  and how their findings may impact use of Osmia in pollination or mass propagation systems.  Many of the Osmia are being sourced from wild populations and bee managers may sell their stock of bees to people in other states.  The chance for movement of pathogens from one area to another exists, so it is important to determine what pathogens to looks for.

The need for greater characterization of pathogens infecting Osmia and other cavity nesting bees was emphasized by an early study undertaken for comparing bee health for cavity-nesting bees and for Osmia species specifically.  In replication of nest trapping studies done in the 1970’s, 1980’s, and early 2000’s, decreased numbers of Osmia and other bee species were found in 10 sites near Logan, UT in the summers of 2018 and 2019.  The nest cells were analyzed, and the emerged adult bees identified.  The patterns across the two years varied, with a greater number of failed cells in early development (pollen balls) found the second year, especially in the Mendon, UT site (M) where the majority of BOBs had been found the previous year.  Dead larvae and pupae were detected without any known pathology.  Some dead larvae with recognizable chalkbrood infections were found.  Among the cells for the variety of bee species, a low % of parasitized cells were detected.  However, in 2020, another study in the mountains outside of Logan, UT with Osmia bruneri, found a very high proportion (>60%) of the larvae parasitized with Melittobia.  The same parasitoid has been reported in Osmia agalia in Oregon.  Additional studies are needed to determine how common this parasitoid wasp is in natural BOB populations.

Figure Legend.  Characterization of bee mortality in cavity-nesting bee species in wild areas.  During 2018 and 2019, nest  block traps were placed 12 sites outside of Logan, UT in replication of previous studies conducted by Dr. Frank Parker.  Multiple hole sizes were used, to allow for sampling a wide variety of the cavity nesting bees.  At the end of fall, the nests were opened, and the status of the cells determined.  Pupating or diapausing bees were put into gel capsules and incubated at the appropriate seasonal temperatures.  Cells with live bees that did not emerge in the subsequent summer were incubated again at the appropriate over wintering temperatures.  In 2019, dramatic changes in bee development were observed at site M, potentially correlated with increased rainfall and cooler temperatures.

Insight into pathogens of concern come from a study determining the bacterial microbiome in pollen provisions for BOBs and O. ribifloris from samples taken in 2015 and analyses performed in 2019 (Rothman et al. 2020).  In this comparison, bees were reared at the same four sites, a screenhouse without prior honey bee or bumble bee flights, an urban garden, and two wild land areas in USFS lands.  The two bee species have separate floral host preferences.  The microbiome of the pollen provisions was found to significantly differ between the two species with majority of bacterial species not being in common.  The bacterial members of the microbiome in the pollen provision were determined by barcode regions of the genomes.  No common set of microbes were associated with the samples for either bee species.  The pollen provisions from the four locations also differed in their microbiome compositions.  In areas with known honey bee and bumble bee colonies, several species of bacteria (Sodalis, Wolbachia, Lactobacillus, Snodgrassella, and Gillamelia) were detected at low levels in the pollen provisions for both species.  Sodalis was found in a significantly higher percentage of samples in BOB nests.   Sodalis and Wolbachia are both symbiotic bacteria found in other insects and have been suggested to potentially be pathogenic under some conditions.  Some Lactobacillus are considered to be a beneficial gut bacterium involved in digesting pollens.  Snodgrassella and Gillamelia are symbiotic gut bacteria found in both honey bees and bumble bees.  Future research is needed to determine if these bacteria are capable of colonizing or infecting Osmia species.
Rothman JA, Cox-Foster DL, Andrikopoulos C, McFrederick QS. Diet Breadth Affects Bacterial Identity but Not Diversity in the Pollen Provisions of Closely Related Polylectic and Oligolectic Bees. Insects. 2020 Sep 20;11(9):645. doi: 10.3390/insects11090645. 

Analysis of extracts of newly emerged BOB adults via DNA polymerase chain reactions did detect Spiroplasma, Rikettsia, Wolbachia, and Arsenophorus in a limited number of bees from California and Utah.  In addition, sequences for Wolbachia were found in genome projects for BOBs from Utah.  The one genomic project used isolated legs from bees for the DNA extractions, after the legs were rinsed of contaminating DNA, indicating that the bacterium was likely to be intracellular.  Wolbachia is known to be an intracellular endosymbiont in other insect species.  The detection of these four species in BOBs from California and Utah populations is a significant finding; in other insect species, it is known that insects having different strains of these species may have disrupted reproduction with death occurring in the embryos of the bees.  Additional research is needed to determine if there is strain variation in BOB populations and if these species are linked to impacts on reproduction.

RNA viruses found in multiple bee species are known to be transmitted via fecal-oral routes on pollen.  In addition, these pathogens may be vertically transmitted from mothers to their eggs via transovariole routes.  For common RNA viruses, viral pathogens were detected using RT-PCR in eastern populations of BOBs and O. cornifrons (Pennsylvania) and in BOBs in California and Utah.  In the Pennsylvania specimens, adult bees taken from nest cells had detectable levels of Deformed Wing Virus (DWV), Lake Sinai Virus (LSV), Blackened Queen Cell Virus (BQCV), Kashmir Bee Virus (KBV), and Israeli Acute Paralysis Virus (IAPV).  Interestingly, BOBs and O. cornifrons from nests in the same apples orchards differed in viruses being detected, with BOBS having detectable KBV and O. cornifrons having DWV.  In Utah and California populations, low levels of DWV and SBV have been detected in a limited number of samples tested.  Further research is needed to determine the impacts of these viruses upon BOB health.  In a related study, BOB larvae fed pollen contaminated with a viral slurry had significantly higher mortality than larvae fed pollen alone.  Additional research is needed to determine how viral infections correlate with bee health in the field.

For protozoan pathogens, no Trypanosome pathogens were detected in samples from Utah wild lands.  Primer sets recognizing Lotmaria and Crithidia were reacted with DNA extracts of 87 BOB adults and larvae from four sites in Utah; and these samples included both healthy and dying individuals.  A known positive was included in the PCR assays.  None of the extracts from the BOB samples had a reaction.  Studies in other solitary bees have not reported detection of trypanosomes; although, these are commonly found in honey bees and bumble bees.  A newly published study did report that BOBs can be infected with Crithidia when fed pollen with this protozoan; so future studies on unhealthy BOBs need to consider this potential infection.

For fungal pathogens, the best-known pathogen is Ascosphaera.  This genus has many species, and there is a lack of understanding on the full extent of the number of species and their host associations.  For BOBs, the chalkbrood is known to be caused by Ascosphaera torchioi.  In collaboration with Kathryn LeCroy, we participated in characterizing and sequencing chalkbrood isolated from samples of BOBs from the state of Virginia.  Some of the samples were characterized by sequencing PCR products and found to be Ascosphaera fusiformis.  This species was previously identified in Japan as infecting O. cornifrons.  Kate LeCroy’s research is determining if this fungal pathogen is newly introduced into the United States.  This finding is important, since there have been some reports of BOBs being shipped from the east coast to the west coast.  In future years, the species of chalkbrood needs to be confirmed in bee samples from the western United States, since the newly introduced pathogen may have different impacts on the larvae.

For other fungal pathogens, we have new findings.  Microsporidia are fungal pathogens that have evolved to become host-specific pathogens.  These are known from honey bees and bumble bees, with distinct microsporidia species infecting these bees.  For the first time, we have detected microsporidia in BOBs.  In bees taken from nests in four wild-land locations in Utah, 59 out of 87 sampled bees tested positive for presence of microsporidia, using DNA PCR reactions.  In these positive reactions, multiple bands were detected in some samples, suggesting the potential for multiple microsporidia species.  For the 87 samples tested, both live and dead bees (died during development in nests) were included.  A greater proportion of dead bees were found to have microsporidia as compared to the live bees (N=87, d.f.=1, Pearson Chi Square=6.939, p= 0.0084).

Figure Legend.  Detection of microsporidia sequences is significantly greater in dead BOBs than in live BOBs.

In two of the locations in which the success rate in nesting was monitored, the presence of microsporidia correlated with a greater chance in nest cell failure.  We are following up with characterizing the identity of the microsporida and determining the role of the bee health.

Figure Legend.  Detection of microsporidia in adult BOB samples is correlated with the proportion of failed cells in nests, in comparison of two locations near Logan, Utah.  The Cub River site has a known history of extensive illicit nest trapping and is associated with an increase in road traffic, while Birch Creek site is isolated and has no known history of illicit nest trapping.  More research is needed to understand the role of stress in microsporidian infections in Osmia.

For Objective 5, Stephen Peterson identified cherry producers that allowed use of their property for the 2019 research work to occur.  Dr. Peterson also provided CA-sourced bees, and UT-sourced bees were purchased for use.  In California sweet cherry orchards, we had low female retention; a mean of 3.9% of the females released were later detected nesting at site centers.  Comparing female retention based on population source, we found that significantly less CA-sourced females were retained at site centers than UT-sourced females (p<0.0001).  We detected marked bees nesting far and very far away, showing that BOBs are capable of dispersing at least 1 km in dense agricultural lands.  Only 3 CA-sourced females and 5 UT-sourced females were detected nesting far or very far away across all three sites.  Nine unmarked females were detected across all three sites only at site centers, suggesting that these bees were probably sourced from our cocoons released as males and are not from local populations.

Figure Legend. Results from California cherry orchards found low retention of nesting BOB females.

In Utah tart cherry orchards, a mean of 13.2% of the females released were later detected nesting at site centers. Here, we found no significant difference in female retention based on population source (p=0.2098).  Marked bees were again found far and very far away, but in higher numbers than in California; a total of 19 CA-sourced females and 41 UT-sourced females were detected nesting far or very far away across all three sites.  A total of 236 unmarked females were found nesting in our nest boxes across all three sites; 119 unmarked bees were found at site centers and 117 were found far and very far away.

Figure Legend. Results from Utah cherry orchards found equal retention of nesting BOB females from California and Utah sources.

The large numbers and even distribution of unmarked females throughout the sites suggests that these bees were from local populations, either wild or managed.  From a mean of 13.2% percent female retention, we recovered a mean of 44.9% of the female population at site centers.  When recovering progeny from entire sites (site center plus far and very far away), a mean of 79.3% of the female population was recovered.  At site 3, 61.4% and 114.6% of the female population was recovered at site center and far and very far away, respectively.  This highlights a potentially novel method for propagating managed populations in orchards in which we can use BOB’s apparent propensity to disperse to our advantage.  BOB nesting substrates can be deployed throughout common orchard growing regions, even 1 km from where any given population is released, to maximize population return.