Examining historical rates of leafcutting bee cell pathogens to establish baseline infectivity rates for alfalfa seed growers

The alfalfa leafcutting bee (Megachile rotundata) is one of the primary pollinators for the alfalfa seed industry. The alfalfa leafcutting bee is a solitary cavity nesting bee. Female Megachile rotundata bees will construct and provision individual brood cells lined with cut leaves (cocoon) and will gather nectar and pollen to place within the constructed cocoon. The female bee will lay a single egg within the constructed cocoon and leave the egg to undergo larval stage development and pupation into the adult stage. During this time multiple pathogens and parasitoids can prey on the developing larvae, resulting in the loss of the future adult bee. A major concern for commercial alfalfa seed growers is the presence of invertebrate pests and fugal pathogens. In the present study, we used historical data from the Parma Cocoon Diagnostic Laboratory to determine baseline rates of pathogen and parasite infection of Megachile rotundata cells and used this analysis to determine historical infection rates and cutoffs for management practices. Additionally, using a Faxitron (X-ray) analysis for Megachile rotundata cell obtained in 2020, we compared the presence of chalkbrood, pathogens, and parasitoids in samples collected from both growers stocks and newly purchased Canada bees. The results of the investigation demonstrate historical averages of the presence of chalkbrood, pathogens, and parasitoids. We also show a significant increase in chalkbrood and predators in 2007-2011 and a significant difference in chalkbrood and predators between bee samples obtained from Canada and grower stocks.

Multiplex PCR reveals unique trends in pathogen and parasitoid infestations of alfalfa leafcutting brood cells.

The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. Megachile rotundata is a solitary bee that forms brood cocoons where its larvae can develop. During the developmental stages of growth, broods can be preyed upon by multiple different fungal and bacterial pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollinator services provided by Megachile rotundata and reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of the most common bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. Co-infection of single brood cells by multiple Ascosphaera species was confirmed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species including multiple new predators and pathogenetic species not previously documented in associated with M. rotundata brood cells.

Adult body size measurement redundancies in Osmia lignaria and Megachile rotundata (Hymenoptera: Megachilidae)

Metrics to assess relative adult bee body size have included both mass and morphometrics, but these metrics may not equally or reliably estimate body size for all bee species and in all situations, due to bee age, diet, and/or environment. Understanding the relationships between different metrics and possible redundancies in the information they afford is important but not always known. Body size measurements provide valuable data for interpreting research outcomes for managed solitary bees, including Osmia lignaria Say and Megachile rotundata F. (Hymenoptera: Megachilidae). Applied studies of these important and readily available U.S. crop pollinators focus on refining commercial management practices, and basic empirical studies in various scientific disciplines (from genomics to ecology) employ them as model systems to study solitary bees. To examine common metrics of body size, we measured head capsule width (HCW), intertegular distance (ITD), and fresh and dry weights of newly emerged adults of both species. Using linear and exponential models, we determined relationships between these body size metrics. For M. rotundata, linear models best described relationships between ITD and all other metrics, and between HCW and fresh and dry weights. For O. lignaria, linear models best fit relationships between all metrics except for fresh weight with both ITD and HCW, which were fitted better with exponential models. For both species, model fits were strongest when males and females were pooled. Depending on the study question, knowing that only one metric may reliably measure body size can simplify evaluations of O. lignaria and M. rotundata responses to artificial or environmental variables.

Environmental impacts on diapause and survival of the alfalfa leafcutting bee, Megachile rotundata

Megachile rotundata exhibits a facultative prepupal diapause but the cues regulating diapause initiation are not well understood. Possible cues include daylength and temperature. Megachile rotundata females experience changing daylengths over the nesting season that may influence diapause incidence in their offspring through a maternal effect. Juvenile M. rotundata spend their developmental period confined in a nesting cavity, potentially subjected to stressful temperatures that may affect diapause incidence and survival. To estimate the impact of daylength and nest cavity temperature on offspring diapause, we designed a 3D printed box with iButtons that measured nest cavity temperature. We observed nest building throughout the season, monitored nest cavity temperature, and followed offspring through development to measure diapause incidence and mortality. We found that daylength was a cue for diapause, and nest cavity temperature did not influence diapause incidence. Eggs laid during long days had a lower probability of diapause. Siblings tended to have the same diapause status, explaining a lot of the remaining variance in diapause incidence. Some females established nests that contained both diapausing and nondiapausing individuals, which were distributed throughout the nest. Nest cavities reached stressful temperatures, which decreased survival. Mortality was significantly higher in nondiapausing bees and the individuals that were laid first in the nest. In conclusion, we demonstrate a maternal effect for diapause that is mediated by daylength and is independent of nest box temperature.

Honeybees, Bumblebees and Leaf-cutter Bees Vary in the Effect of a Simulated Pathogen Challenge on Individual Thermoregulation

Bees regulate their individual body temperatures by non-flight thermogenesis (NFT). The effects of a pathogen challenge on thermoregulation in bees generally is unknown, although honeybees have displayed opposing responses between two studies. To establish whether bees in general experience disruption of thermoregulation under pathogen challenge, we investigated a representative species of each of three major bee social backgrounds (honeybees, Apis mellifera; bumblebees, Bombus impatiens; and solitary bees, Megachile rotundata) and measured the body surface temperatures of individual bees as they recovered from cold torpor by NFT after injection with lipopolysaccharide (LPS) solution, which simulated a pathogen challenge. We found that LPS injection affected rewarming in the annually eusocial B. impatiens, but not A. mellifera or the solitary M. rotundata. Specifically, the pathogen challenge increased post-recovery body temperatures by 2 oC in B. impatiens individuals. Our findings indicate that immune responses by individual bees can interfere with thermoregulation, but this effect is not consistent among major bee species.

Eristalis (Diptera: Syrphidae) Flower Flies are Potential Non-host Vectors of the Common Trypanosome Bee Parasite, Crithidia Bombi

Flowers can be transmission platforms for parasites that impact bee health, yet bees share floral resources with other pollinator taxa, such as flies, that could be hosts or non-host vectors (i.e., mechanical vectors) of parasites. Here, we assessed whether the fecal-orally transmitted gut parasite of bees, Crithidia bombi, can infect Eristalis tenax flower flies. We also investigated the potential for two confirmed solitary bee hosts of C. bombi, Osmia lignaria and Megachile rotundata, as well as two flower fly species, Eristalis arbustorum and E. tenax, to transmit the parasite at flowers. We found that C. bombi did not replicate (i.e., cause an active infection) in E. tenax flies. However, 93% of inoculated flies defecated live C. bombi in their first fecal event, and all contaminated fecal events contained C. bombi at concentrations sufficient to infect bumble bees. Flies and bees defecated inside the corolla (flower) more frequently than other plant locations, and flies defecated at volumes comparable to or greater than bees. Our results demonstrate that Eristalis flower flies are not hosts of C. bombi, but they may be mechanical vectors of this parasite at flowers. Thus, flower flies may amplify or dilute C. bombi in bee communities.

Presence of Pathogen-killed Larvae Influences Nesting Behavior of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae)

The alfalfa leafcutting bee (Megachile rotundata (Fabricius)), a commercial pollinator used for alfalfa seed production, is susceptible to chalkbrood disease via ingested fungal spores. Diseases of insects can elicit behavioral changes in their hosts, but there are no recorded behaviors of alfalfa leafcutting bees in response to this fungal exposure. We conducted field studies to determine whether bees in pathogen-dense environments altered their nesting patterns, specifically if bees exposed to fungal spores produced higher numbers of nest cells and whether the proportions of nest cells that failed as eggs or small larvae (a state known as ‘pollen ball’) were greater. We found that our control bees, nontreated bees which were not exposed to chalkbrood spores other than those in the natural environment, had the highest proportion of pollen ball cells. Bees experimentally exposed to infective spores created the lowest number of nests and the fewest cells. Bees experimentally exposed to heat killed noninfective spores produced the greatest number of nests and cells overall and the greatest number of healthy progeny. We conclude that there are underlying behaviors that are elicited in response to the presence of chalkbrood spores that reduce the proportion of failed nest cells (grooming) and increase retention of bees at nesting sites (delay of bee emergence). Through further study of these behaviors, bee managers can potentially increase the productivity of their bee populations.