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.

Variation in Expression of Reference Genes across Life Stages of a Bee, Megachile rotundata.

The alfalfa leafcutting bee, Megachile rotundata is widely used in the western United States as a pollinator for alfalfa seed production. Unfortunately, immatures experience high mortality in agriculturally managed populations. Quantified gene expression could be used to identify how this bee responds during different life stages to pathogens, environmental toxins, and other stresses, but stably expressed reference genes are needed to normalize transcription data. We evaluated twelve candidate genes for their transcription stability across different life stages, including during and after diapause. RPS18 and RPL8 were the two most stably expressed genes, followed by RPS5 and RPL27A. These genes were also very stable even during and after diapause, while the most variable genes being APN, PMIIM, NPC2, and Cr-PII had increased expression levels during larval growth and were also variable during and after diapause. The four reference genes we identified in M. rotundata may prove useful for transcriptomic studies in other bees as well, such as honey bees.

Mixed infections reveal virulence differences between host-specific bee pathogens.

Dynamics of host-pathogen interactions are complex, often influencing the ecology, evolution and behavior of both the host and pathogen. In the natural world, infections with multiple pathogens are common, yet due to their complexity, interactions can be difficult to predict and study. Mathematical models help facilitate our understanding of these evolutionary processes, but empirical data are needed to test model assumptions and predictions. We used two common theoretical models regarding mixed infections (superinfection and co-infection) to determine which model assumptions best described a group of fungal pathogens closely associated with bees. We tested three fungal species, Ascosphaera apis, Ascosphaera aggregata and Ascosphaera larvis, in two bee hosts (Apis mellifera and Megachile rotundata). Bee survival was not significantly different in mixed infections vs. solo infections with the most virulent pathogen for either host, but fungal growth within the host was significantly altered by mixed infections. In the host A. mellifera, only the most virulent pathogen was present in the host post-infection (indicating superinfective properties). In M. rotundata, the most virulent pathogen co-existed with the lesser-virulent one (indicating co-infective properties). We demonstrated that the competitive outcomes of mixed infections were host-specific, indicating strong host specificity among these fungal bee pathogens.

PCR reveals high prevalence of non/low sporulating Nosema bombi (microsporidia) infections in bumble bees (Bombus) in Northern Arizona.

About 20% of bumble bee species are in decline in North America, and the microsporidian pathogen, Nosema bombi, has been correlated with these declines. We conducted a comprehensive survey of N. bombi infections in the bumble bee communities throughout the flight season along an elevation gradient in Northern Arizona. Focusing on two species, Bombus (Pyrobombus) huntii and Bombus (Pyrobombus) centralis, we used a combination of PCR and microscopy to distinguish between sporulating and non/low, sporulating N. bombi infections. Surprisingly high levels of PCR-positive infections with no detectable spore loads were found in B. huntii (31-63%) and B. centralis (56.5-66.5%), while the prevalence of sporulating infections was low (3.0-11.8% and 0-12.9% respectively). We determined the prevalence of sporulating N. bombi infection in six other co-occurring, but rarer, bumble bee species (0-62.5%,), but did not test them using PCR. The prevalence of sporulating N. bombi infections in B. (Bombias) nevadensis was significantly higher than in either B. huntii or B. centralis (29%). The declining bumble bee, Bombus sensu strico occidentalis, had the highest prevalence of sporulating N. bombi infections (62.5%), but we purposely captured very few B. occidentalis because of its declining status. PCR was a more sensitive measure of N. bombi prevalence and revealed that wild bumble bees have a much higher prevalence of N. bombi than has previously been recognized. Microscopy and PCR together provide complementary, not redundant, information that deepens our understanding of the dynamic interactions between N. bombi and their bumble bee hosts.

Interactions between fungi and bacteria influence microbial community structure in the Megachile rotundata larval gut.

Recent declines in bee populations coupled with advances in DNA-sequencing technology have sparked a renaissance in studies of bee-associated microbes. Megachile rotundata is an important field crop pollinator, but is stricken by chalkbrood, a disease caused by the fungus Ascosphaera aggregata. To test the hypothesis that some gut microbes directly or indirectly affect the growth of others, we applied four treatments to the pollen provisions of M. rotundata eggs and young larvae: antibacterials, antifungals, A. aggregata spores and a no-treatment control. We allowed the larvae to develop, and then used 454 pyrosequencing and quantitative PCR (for A. aggregata) to investigate fungal and bacterial communities in the larval gut. Antifungals lowered A. aggregata abundance but increased the diversity of surviving fungi. This suggests that A. aggregata inhibits the growth of other fungi in the gut through chemical or competitive interaction. Bacterial richness decreased under the antifungal treatment, suggesting that changes in the fungal community caused changes in the bacterial community. We found no evidence that bacteria affect fungal communities. Lactobacillus kunkeei clade bacteria were common members of the larval gut microbiota and exhibited antibiotic resistance. Further research is needed to determine the effect of gut microbes on M. rotundata health.

Detoxification and stress response genes expressed in a western North American bumble bee, Bombus huntii (Hymenoptera: Apidae).

BACKGROUND: The Hunt bumble bee (Bombus huntii Greene, Hymenoptera: Apidae) is a holometabolous, social insect important as a pollinator in natural and agricultural ecosystems in western North America. Bumble bees spend a significant amount of time foraging on a wide variety of flowering plants, and this activity exposes them to both plant toxins and pesticides, posing a threat to individual and colony survival. Little is known about what detoxification pathways are active in bumble bees, how the expression of detoxification genes changes across life stages, or how the number of detoxification genes expressed in B. huntii compares to other insects. RESULTS: We found B. huntii expressed at least 584 genes associated with detoxification and stress responses. The expression levels of some of these genes, such as those encoding the cytochrome P450s, glutathione S-transferases (GSTs) and glycosidases, vary among different life stages to a greater extent than do other genes. We also found that the number of P450s, GSTs and esterase genes expressed by B. huntii is similar to the number of these genes found in the genomes of other bees, namely Bombus terrestris, Bombus impatiens, Apis mellifera and Megachile rotundata, but many fewer than are found in the fly Drosophila melanogaster. CONCLUSIONS: Bombus huntii has transcripts for a large number of detoxification and stress related proteins, including oxidation and reduction enzymes, conjugation enzymes, hydrolytic enzymes, ABC transporters, cadherins, and heat shock proteins. The diversity of genes expressed within some detoxification pathways varies among the life stages and castes, and we typically identified more genes in the adult females than in larvae, pupae, or adult males, for most pathways. Meanwhile, we found the numbers of detoxification and stress genes expressed by B. huntii to be more similar to other bees than to the fruit fly. The low number of detoxification genes, first noted in the honey bee, appears to be a common phenomenon among bees, and perhaps results from their symbiotic relationship with plants. Many flowering plants benefit from pollinators, and thus offer these insects rewards (such as nectar) rather than defensive plant toxins.

Temperature stress affects the expression of immune response genes in the alfalfa leafcutting bee, Megachile rotundata.

Environmental stresses are thought to be associated with increases in disease suceptibility, attributable to evolutionary trade-offs between the energy demands required to deal with stress vs pathogens. We compared the effects of temperature stress and pathogen exposure on the immune response of a solitary bee, Megachile rotundata. Using an oligonucleotide microarray with 125 genes (375 probes), we determined that both high and low temperatures increased the expression of immune response genes in M. rotundata and reduced levels of a disease called chalkbrood. In the absence of the pathogen, trypsin-like serine and pathogen recognition proteases were most highly expressed at the lowest rearing temperature (20°C), while immune response signalling pathways and melanization were highly expressed at the warmest temperature tested (35°C). In pathogen-exposed bees, immune response genes tended to be most highly expressed at moderate temperatures, where we also saw the greatest infection levels. Temperature stress appears to have activated immunity before the pathogen elicited a response from the host, and this early activity prevented infection under stressful conditions. In this insect, the trade-off in energetic costs associated with stress and infection may be partially avoided by the use of conserved responses that reduce the effects of both.

Polymorphic DNA sequences of the fungal honey bee pathogen Ascosphaera apis.

The pathogenic fungus Ascosphaera apis is ubiquitous in honey bee populations. We used the draft genome assembly of this pathogen to search for polymorphic intergenic loci that could be used to differentiate haplotypes. Primers were developed for five such loci, and the species specificities were verified using DNA from nine closely related species. The sequence variation was compared among 12 A. apis isolates at each of these loci, and two additional loci, the internal transcribed spacer of the ribosomal RNA (ITS) and a variable part of the elongation factor 1α (Ef1α). The degree of variation was then compared among the different loci, and three were found to have the greatest detection power for identifying A. apis haplotypes. The described loci can help to resolve strain differences and population genetic structures, to elucidate host-pathogen interaction and to test evolutionary hypotheses for the world's most important pollinator: the honey bee and one of its most common pathogens.

Prewinter management affects Megachile rotundata (Hymenoptera: Megachilidae) prepupal physiology and adult emergence and survival.

The alfalfa leafcutting bee, Megachile rotundata F. (Hymenoptera: Megachilidae), is widely used as a pollinator for production of alfalfa, Medicago sativa L., seed, and populations of these bees can be maintained by alfalfa seed growers or can be purchased from mostly Canadian bee providers. M. rotundata raised in Canada have higher survival rates during the incubation that occurs after winter storage than do bees produced in the northwestern United States, but no reason has been found for this difference. We investigated whether storing immature M. rotundata for various time periods at a warm temperature (16 degrees C) before winter or allowing them to remain unmanaged at ambient temperatures affects physiological aspects of prepupae during the winter as well as the survival and longevity of adult bees after spring or summer incubation. Our results show that the timing of the onset of winter storage and incubation does affect prepupal weights, prepupal lipid and water contents, adult emergence, and adult female longevity. Winter storage of prepupae in November or December with a late June incubation resulted in heavier adults that emerged more readily than bees incubated in late May. However, adult females incubated in May thrived longer than June-incubated bees if fed a honey-water diet. Thus, some prewinter management regimes for M. rotundata commercial stocks may be more effective than others for achieving optimal adult emergence synchrony, as well as adult survival and longevity for pollination of a summer crop.

Do weather conditions correlate with findings in failed, provision-filled nest cells of Megachile rotundata (Hymenoptera: Megachilidae) in western North America?

Cavity-nesting alfalfa leafcutting bees, Megachile rotundata (F.) (Hymenoptera: Megachilidae), are excellent pollinators of alfalfa, Medicago savita L., for seed production. In commercial settings, artificial cavities are placed in field domiciles for nesting and, thereby, bee populations are sustained for future use. For this study, cells from leafcutting bee nests were collected in late summer from commercial seed fields. Over 3 yr (2003-2005), 39 samples in total of approximately equal to 1,000 cells each were taken from several northwestern U.S. states and from Manitoba, Canada. X-radiography of 500 cells from each sample was used to identify "pollen balls" (i.e., cells in which the pollen-nectar provision remained, but the egg or larva, if present, was not detectable on an x-radiograph). Most U.S. samples seemed to have higher proportions of pollen ball cells than Manitoba samples. Pollen ball cells were dissected to determine the moisture condition of the mass provision and true contents of each cell. Most pollen ball cells from Manitoba samples contained fungus, the frequency of which was positively correlated with cool, wet weather. In the United States, most pollen ball cells had moist provisions, and many of them lacked young brood. Correlation analysis revealed that pollen ball cells occurred in greater proportions in fields with more hot days (above 38 degrees C). Broodless pollen ball cells occurred in greater proportions under cool conditions, but dead small larvae (second-third instars) seemed to occur in greater proportions under hot conditions. Pollen ball cells with unhatched eggs and first instars (in the chorion) occurred in lesser proportions under hot conditions.

Effects of three-dimensional and color patterns on nest location and progeny mortality in alfalfa leafcutting bee (Hymenoptera: Megachilidae).

ABSTRACT In alfalfa, Medicago sativa L., seed production where high bee densities are released, alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae), females may enter several nesting holes before locating their nests. Such levels of "wrong hole" visits lead to an increase in the time spent by females locating their own nests, thereby decreasing alfalfa pollination efficiency and possibly healthy brood production. The objectives of this study were to determine the effect of different nesting board configurations in commercial alfalfa leafcutting bee shelters (separating nesting boards, applying a three-dimensional pattern to the boards, applying a color contrast pattern, or applying a combination of three-dimensional and color contrast patterns) on nest location performance, on the incidence of chalkbrood disease, and on the incidence of broodless provisions. Separating the nesting boards inside shelters improved the ability of females to locate their nests. An increase in nest location performance also occurred in boards with the three-dimensional pattern and the combined three-dimensional and color contrast pattern, compared with the uniform board (a standard configuration currently used commercially). The percentage of provisioned cells that were broodless was not statistically different between treatments, but the percentage of larvae infected with chalkbrood decreased by half in the three-dimensional board design, compared with the uniform board.

Emergence success and sex ratio of commercial alfalfa leafcutting bees from the United States and Canada.

Samples of overwintering alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae), cells were sent to the laboratory as loose cells or in nesting boards from bee managers in the United States and in Canada. X-radiographs of cells were used for determining cell contents. Cells containing live prepupae were incubated, and the sex of emerging adults was recorded daily. Cells from which no adult emerged were dissected to determine the developmental stage of dead bees and sex of dead pupae or adults. Bee cells incubated in commercial settings and placed in alfalfa fields by the same bee managers described above also were evaluated to determine adult emergence success. The proportion of live bees in wood nesting boards from the United States was much lower than the live proportion in polystyrene nesting boards from Canada and loose cells overwintered in the United States. For laboratory-incubated loose cells, survival and sex ratios of bees from Canadian sources were statistically higher than those of U.S. bees, but the onset and duration of emergence times were similar. Fewer bees survived in the commercial setting than in the laboratory. Prepupal mortality was significantly higher than pupal or adult mortality, but there was no significant difference between the sexes in the likelihood of survival during incubation. This study supports the commonly held belief that alfalfa leafcutting bees raised in Canada and then sold to the United States represent a more viable source of bees than most bees produced in the United States.

Field trials using the fungal pathogen, Metarhizium anisopliae (Deuteromycetes: Hyphomycetes) to control the ectoparasitic mite, Varroa destructor (Acari: Varroidae) in honey bee, Apis mellifera (Hymenoptera: Apidae) colonies.

The potential for Metarhizium anisopliae (Metschinkoff) to control the parasitic mite, Varroa destructor (Anderson and Trueman) in honey bee colonies was evaluated in field trials against the miticide, tau-fluvalinate (Apistan). Peak mortality of V. destructor occurred 3-4 d after the conidia were applied; however, the mites were still infected 42 d posttreatments. Two application methods were tested: dusts and strips coated with the fungal conidia, and both methods resulted in successful control of mite populations. The fungal treatments were as effective as the Apistan, at the end of the 42-d period of the experiment. The data suggested that optimum mite control could be achieved when no brood is being produced, or when brood production is low, such as in the early spring or late fall. M. anisopliae was harmless to the honey bees (adult bees, or brood) and colony development was not affected. Mite mortality was highly correlated with mycosis in dead mites collected from sticky traps, indicating that the fungus was infecting and killing the mites. Because workers and drones drift between hives, the adult bees were able to spread the fungus between honey bee colonies in the apiary, a situation that could be beneficial to beekeepers.