Ascosphaera aggregata contamination on alfalfa leafcutting bees in a loose cell incubation system

The alfalfa leafcutting bee, a solitary bee used to pollinate alfalfa seed crops, is seriously affected by chalkbrood, a larval disease caused by the fungus Ascosphaera aggregata. One attempt to control the disease includes removing nests from the nesting boards (the “loose cell” system). We report here that adults emerging from the loose cells are heavily contaminated with A. aggregata spores. The contamination levels are not as high as previously reported for bees emerging directly from the boards, but they are still a likely focus for disease spread and may need to be targeted in chalkbrood control strategies.     

Impact of disinfecting nesting boards on chalkbrood control in the alfalfa leafcutting bee

The alfalfa leafcutting bee, Megachile rotundata (F.), is a solitary, cavity-nesting bee that has been managed in large numbers to pollinate alfalfa, Medicago spp., seed crops since the 1960s. Propagation of these bees from 1 yr to the next has been seriously hampered by chalkbrood, a larval disease caused by the fungus Ascosphaera aggregata Skou. In the United States, attempts to control the disease have been fairly unsuccessful, but include removing nests from the nesting boards and then disinfecting the boards with heat treatments or a fumigant. The problem is that many boards are made of polystyrene (so heat cannot be used), and very few fumigants are registered for this use. In this study, ozone was tested as a fumigant and compared with heat treatments and methyl bromide fumigation. Ozone was found to be inadequate for killing A. aggregata spores and for reducing chalkbrood levels in the field. Methyl bromide and heat treatments did greatly reduce spore viability in the boards, but did not reduce chalkbrood levels in the field. Surprisingly, larvae in new nesting boards (boards free of contamination) and chalkbrood infection levels were similar to those from nests in contaminated, used boards. Disinfecting nesting boards may be necessary for controlling chalkbrood, but the results reported here indicate that it is not sufficient in and of itself. Some other source of spores was present in the field that was greater than the effect of contamination from the boards, but the source still needs to be determined.     

Laboratory bioassays to evaluate fungicides for chalkbrood control in larvae of the alfalfa leafcutting bee (Hymenoptera: Megachilidae)

Chalkbrood, a fungal disease in bees, is caused by several species of Ascosphaera. A. aggregata is a major mortality factor in populations of the alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae) used in commercial alfalfa seed production. Four formulated fungicides, Benlate 50 WP, Captan, Orbit, and Rovral 50 WP were tested in the laboratory for efficacy against hyphal growth of A. aggregata cultures. The same fungicides, with the addition of Rovral 4 F, were tested for their effects on incidence of chalkbrood disease, and toxicity to M. rotundata larvae. Benlate, Rovral 50 WP, and Rovral 4 F reduced incidence of chalkbrood with minimal mortality on larval bees. Benlate and Rovral 50 WP also reduced hyphal growth. Orbit was effective in reducing hyphal growth, but it did not reduce incidence of chalkbrood and was toxic to bee larvae. Captan was not effective in reducing hyphal growth or chalkbrood incidence, and it was toxic to bee larvae. Fungicides that reduce incidence of chalkbrood and larval mortality in this laboratory study are candidates for further study for chalkbrood control.     

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.     

Fungicide tests on adult alfalfa leafcutting bees (Hymenoptera: Megachilidae)

Chalkbrood is a serious disease of alfalfa leafcutting bee Megachile rotundata (F.) (Hymenoptera: Megachilidae) larvae, causing upward of 20% infection in the field. The causative agent is the fungus Ascosphaera aggregata. This bee is used extensively for alfalfa seed pollination in the United States. Using laboratory bioassays, we previously demonstrated that fungicides can reduce chalkbrood levels in the larvae. Here, we evaluate the toxicity of four fungicides, Benlate, Captan, Orbit, and Rovral, to adult bees by using three different bioassays. In the first test, fungicides were applied to bees' thoraces. In the second test, mimicking foliage residue, a piece of filter paper soaked in fungicide was placed on the bottom of a container of bees. The third test evaluated oral toxicity by incorporating fungicides into a sugar-water solution that was fed to the bees. The filter paper test did not discriminate among the fungicides well, and the oral test resulted in the greatest mortality. Toxicity to males was greater than to females. The use of fungicides for chalkbrood control is a logical choice, but caution should be used in how they are applied in the presence of bees.     

Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundata

The spore load of Ascosphaera species spores on larval chalkbrood cadavers and newly emergent adults of the alfalfa leafcutting bee, Megachile rotundata, was determined. The spore content of chalkbrood cadavers ranged from 3 × 10⁶ to 5 × 10⁸. Adults emerging through zero to nine cadavers carried spores on all body parts examined by scanning electron microscopy. Estimates of the total number of spores obtained from a series of adult washes ranged from 9 × 10⁴ to 8 × 10⁷. Some adult males which emerged through no cadavers carried 10⁴ to 10⁵ spores, indicating that nesting materials might also have been contaminated. However, the control of chalkbrood in commercial bee populations may not be accomplished simply by providing clean nesting materials as adults may still emerge through diseased larvae.     

Etiology and symptomatology of chalkbrood in the alfalfa leafcutting bee,Megachile rotundata

Aseptically reared larvae of the alfalfa leafcutting bee, Megachile rotundata, are susceptible to infection by spores but not mycelial cultures of Ascosphaera aggregata when introduced per os. The symptoms and signs of chalkbrood vary, depending upon host age at inoculation. Larvae inoculated early in life did not undergo the internal color changes after death that characterized larvae inoculated later. A longer time to death was also evident among larvae inoculated at an early age. Changes in the aerobic state of the host gut at the molt to the fourth instar may account for the difference in average time to death.     

Lipids stimulate spore germination in the entomopathogenic ascomycete <Emphasis Type="Italic">Ascosphaera aggregata</Emphasis>

The alfalfa leafcutting bee (Megachile rotundata) is solitary and managed on a large scale for pollination of alfalfa seed crops. The bees nest in holes drilled in wood or polystyrene blocks, and their larvae are highly prone to a fungal disease called chalkbrood. The most prevalent form of chalkbrood is caused by Ascosphaera aggregata, but this ascomycete is difficult to culture. Hyphae will grow on standard fungal media, but spore germination is difficult to achieve and highly variable. We found that germination can be enhanced with oils. Lipids derived from plants and bee larvae increased germination from 50% (without oil) to 75–85% (with oil). Percent germination was significantly greater in the presence of lipids but germination was not significantly different when different oils, including mineral oil, were used. A. aggregata spores oriented along the oil--aqueous interface in the broth in a polar fashion, with swelling and germ tube formation always occurring into the aqueous portion of the broth. The other half of the spore tended to attach to a lipid droplet, where it remained, without swelling, during germ tube formation. The physical attachment of spores to the oil--aqueous interface is what most probably stimulates spore germination, as opposed to some nutritional stimulation. However, further research is needed to determine if and where the spores encounter such an interface when germinating in the host gut, where germination normally occurs.     

Effects of the insecticide phosmet on solitary bee foraging and nesting in orchards of Capitol Reef National Park, Utah

Capitol Reef National Park, in southcentral Utah, contains 22 small orchards planted with antique fruit varieties by Mormon pioneers beginning over a century ago. The orchards continue to be managed in a pick-and-pay program, which includes spraying with phosmet to suppress codling moth (Cydia pomonella L.). The park is also home to a rich diversity of flowering plants, many of which are rare, bee-pollinated, and have populations within 1 km of the orchards. Over 3 yr, we studied the short-term effects of phosmet spraying on bee populations: (1) foraging on plants within the orchard understory and adjacent to it; and (2) nesting in, and at several distances from, the orchards. We recorded a rich bee fauna (47 taxa) in the orchards and on plants nearby. In 2 yr (2002 and 2004), we found no difference in the number of native bee visits to several species of plants flowering in and near to orchards immediately before and 1 d after spraying. Conversely, our nesting studies using the semidomesticated alfalfa leafcutting bee, Megachile rotundata (F.), showed strong significant declines in the number of adult males, nesting females, and progeny production subsequent to spraying at distances up to 160 m from sprayed orchards where the bees were presumably foraging. We showed that M. rotundata is negatively affected by phosmet spraying and suggest that caution should be exercised in its use in areas where bees are apt to forage.     

PCR diagnostic methods for Ascosphaera infections in bees

Fungi in the genus Ascosphaera are the causative agents of chalkbrood, a major disease affecting bee larval viability. Identification of individual Ascosphaera species based on morphological features has been difficult due to a lack of distinguishing characteristics. Most identifications are based on the size and shape of the ascomata, spore balls and conidia. Unfortunately, much overlap occurs in the size of these structures, and some Ascosphaera species will not produce sexual structures in vitro. We report a quick and reliable diagnostic method for identifying Ascosphaera infections in Megachile bees (leafcutting bees) using PCR markers that employ genus-specific primers for Ascosphaera, and species-specific primers for species known to be associated with Megachile spp. Using these methods, species identifications can be performed directly on bees, including asymptomatic individuals. Furthermore, the PCR markers can detect co-infections of multiple Ascosphaera species in a single host. We also identified a marker for Ascosphaera apis, the predominant cause of chalkbrood in Apis mellifera, the honey bee. Our diagnostic methods eliminate the need for culturing samples, and could be used to process a large number of field collected bee larvae.     

A fluctuating thermal regime improves survival of cold-mediated delayed emergence in developing Megachile rotundata (Hymenoptera: Megachilidae)

A significant concern in the commercial application of the alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae), for pollination is synchronizing bee emergence and activity with peak crop bloom. Previous studies have demonstrated that the commercial spring incubation of this species can be successfully interrupted by low temperature incubation, thereby slowing development and giving producers flexibility in timing emergence to weather conditions or crop bloom. In this study, we demonstrate that the implementation of a fluctuating thermal regime, during which bees are given a daily one hour pulse of high temperature, markedly increases the "shelf-life" of individuals of this species. Although constant temperatures can be used to store bees for up to 1 wk with no decrease in survival, properly staged bees can be stored for up to 6 wk without a significant decrease in percentage of emergence. Hence, we expect fluctuating thermal regime protocols to become a valuable tool for M. rotundata managers, especially when timing nesting activity with peak bloom to maximize effectiveness.     

Predation Cues in Solitary bee Nests

Predation at the nesting site can significantly affect solitary bees’ reproductive success. We tested female red mason bees’ (Osmia bicornis L.) acceptance of potential nesting sites, some of which were marked with cues coming from predated conspecifics (crushed bees) or from a predator itself (rodent excreta). In our experiment, females did not avoid nests marked with either of the two predator cues. We suggest that bee females do not recognize these two cues as risky. Alternatively, costs of abandoning natal aggregation might be too high compared with any perceived predation risk of staying. Moreover, the presence of crushed bees can provide positive information about the presence of conspecifics and, possibly, information about a nesting aggregation that may be preferred by bees when choosing a nesting site.     

In vivo development and pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the alfalfa leafcutting bee, Megachile rotundata

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 × 10³ to 3 × 10⁶) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.     

Pollinating bees (Hymenoptera: Apiformes) of U.S. alfalfa compared for rates of pod and seed set

Alfalfa (=lucerne) flowers require visiting bees to trip the sexual column, thereby providing pollination and subsequent pod and seed set. Previous studies have compared the pollination values of different bee species solely by the speed with which they handle flowers and the proportion of visited flowers tripped. In this greenhouse study, five species of bees, including the three commercially managed U.S alfalfa pollinators, are likewise compared for their floral tripping frequencies. These bee species are also compared for the pod set and mature seed that results from their single visits to virgin flowers. Regardless of the identity of the pollinating bee, tripped flowers had the same probabilities of pod set and seed set. Thus, differences in the single-visit pollination efficiencies of the various bee species are entirely attributable to the proportion of visited flowers that they trip. Females of the alkali bee, Nomia melanderi Cockerell, and the alfalfa leafcutting bee, Megachile rotundata F, tripped 81 and 78% of visited flowers, respectively. Males of these species are significantly less effective (61 and 51%, respectively), but still significantly superior to the honey bee, Apis mellifera L. (22% of visited flowers tripped). These relationships are supported by field data for tripping frequencies. One candidate pollinator, Osmia sanrafaelae Parker, shows promise (44% tripped), but not the congeneric O. aglaia Sandhouse (13% tripped).     

Detection of deformed wing virus in the greenhouse for possible horizontal transmission of virus in honey bee colony

The honey bee Apis mellifera L. is a crucial insect in the agricultural industry and natural ecosystem by being a major pollinator. Nevertheless, honey bee population has been recently facing a decline. Among the several factors responsible for this decline, deformed wing virus (DWV) is considered a primary cause that negatively affects honey bee health. DWV is a cosmopolitan honey bee pathogen and causes morphological disadvantages in individual honey bees and colony collapse. Regarding the horizontal transmission of DWV, in addition to Varroa destructor, a well-known major vector of DWV, flowers have recently been implied as a transmission route. Therefore, in this study, we detected DWV from various substances, including flowers, honey bee feces, pupa, larva, nurse bee, surface of nurse bee, pollen collected by forager bee, and forager bee samples in four strawberry greenhouses, which could suggest the potential for the horizontal transmission of DWV in the semi-field condition. We also detected DWV in pollen collected by DWV-negative forager bees, implying that flowers can serve as a potential source of virus infection. These findings suggest that the surrounding environment such as shared floral sources affects the spread of DWV.     

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.     

采用竹管蜂巢繁育苜蓿切叶蜂

对5种繁育苜蓿切叶蜂的蜂巢材料进行了筛选测定,结果表明,竹管的繁蜂效果较好,子代蜂的雌雄性比为1：1,而且竹管内的蜂茧均未被寄生蜂寄生。     

Olfactory cues and nest recognition in the solitary bee Osmia lignaria

Abstract.  The use of olfactory cues for nest recognition by the solitary bee Osmia lignaria is studied in a greenhouse environment. Glass tubes are provided as nesting cavities to allow the in-nest behaviour of bees to be observed. In addition, each glass tube is cut into three sections for experimental manipulation and for subsequent chemical analysis. Nesting females drag their abdomen along the tube before exiting, spiral inside the tube, and sometimes deposit fluid droplets from the tip of the abdomen. For the manipulation, the outer section, the middle section, or both sections are removed and replaced with similar clean glass tube sections, and the behaviour exhibited by test females is recorded upon arrival in front of the nesting site and inside the nesting tubes. The resulting hesitation behaviour displayed by females after treatments appears to indicate the loss of some olfactory cues used for nest recognition inside the entire nest. Chemical analysis of the depositions inside the nesting tube, as well as analysis of the cuticular lipids of the nesting bees, reveals the presence of free fatty acids, hydrocarbons and wax esters.