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.     

Chalkbrood transmission in the alfalfa leafcutting bee: the impact of disinfecting bee cocoons in loose cell management systems

Understanding pathogen transmission could illuminate new methods for disease prevention. A case in point is chalkbrood in the alfalfa leafcutting bee [Megachile rotundata (F.)]. Propagation of this solitary bee is severely hampered by chalkbrood, a larval disease caused by Ascosphaera aggregata (Ascomycota). Alfalfa leafcutting bees nest in existing cavities in wood or hollow reeds and overwinter as larvae. In the early summer, emerging adults frequently must chew through dead, diseased siblings that block their exit, becoming contaminated with chalkbrood spores in the process. When alfalfa leafcutting bees are used as a commercial pollinator, the cocoons are removed from nesting boards to reduce chalkbrood transmission, but the disease is still common. To determine if these removed cocoons (called loose cells) are an important source of disease transmission, they were disinfected with a fungicide before bees were incubated, and released in the field. Chalkbrood prevalence among the progeny of the treated bees was reduced up to 50% in one field trial, but not significantly when tested in an on-farm trial. Thus, substantial disease transmission still occurred when the loose cells were disinfected, and even when clean nesting materials were used. In conclusion, pathogen transmission must still be occurring from another source that has yet to be identified. Another possible source of transmission could arise from bees that emerge midsummer in populations with a high percent of multivoltinism, but dirty nesting boards and feral bees also may be minor sources of transmission.     

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.     

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.     

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.     

Solitary and group nesting in the orchid bee <Emphasis Type="Italic">Euglossa hyacinthina</Emphasis> (Hymenoptera,Apidae)

Summary Orchid bees (Euglossini) provide a potentially informative contrast for examining origins of advanced social behaviour in bees because they are the only tribe in the apine clade that do not form large colonies or have queens and workers. We investigated natural nests of Euglossa hyacinthina Dressler, an orchid bee that nests singly or in groups. By comparing the two types of nests, we examined if individuals in a group merely share the nest (are communal) or exhibit a level of social organization where there is reproductive division of labour among the females. Observations are consistent with communal nesting, indicating that all females in group nests are reproductively similar to the solitary nesting females because the provisioning of young, as well as the ovary development and mating status of females sharing nests were not different than that of solitary-nesting females. Also, multiple female nests did not produce a female-biased brood as predicted for nests with reproductive division of labour. We also investigated potential advantages of group nesting vs. individual nesting. We demonstrate that per capita offspring production is lower in nests with more than one female. However, we found that nests with single females were left unattended for longer periods of time during foraging, and that there was a high incidence of natural enemy attack in nests when females were absent. Group and solitary nesting may be advantageous under different conditions.Received 3 December 2002; revised 7 March 2003; accepted 2 April 2003.     

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.     

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.     

Spatial Nest‐Settlement Decisions in Digger Wasps: Conspecifics Matter more than Heterospecifics and Previous Experience

Research into the driving forces behind spatial arrangement of wasp nests has considered abiotic environmental factors, but seldom investigated attraction or repulsion towards conspecifics or heterospecifics. Solitary female digger wasps (Hymenoptera) often nest in dense aggregations, making these insects good models to study this topic. Here, we analysed the nesting patterns in an area shared by three species of the genus Bembix, in a novel study to discover whether female wasps are attracted to or repulsed by conspecific nests, heterospecific nests or their own previously established nests when choosing nest‐digging locations. Early in the season, each species showed a clumping pattern of nests, but later in the season, a random distribution of nests was more common, suggesting an early conspecific attraction. Such behaviour was confirmed by the fact that females started building their nests more frequently where other females of their species were simultaneously digging. The distances between subsequent nests dug by individual females were shorter than those obtained by random simulations. However, this pattern seemed to depend on the tendency to dig close to conspecifics rather than remain in the vicinity of previous nests, suggesting that females' experience matters to future decisions only on a large scale. Nesting patches within nest aggregations largely overlapped between species, but the nests of each species were generally not closer to heterospecific nests than expected by chance, suggesting that females are neither repulsed by, nor attracted to, congenerics within nest aggregations. A role of the spatial distribution of natural enemies on the observed nesting patterns seemed unlikely. Bembix digger wasp nest aggregations seem thus to be primarily the result of female–female attraction during nest‐settlement decisions, in accordance with the ‘copying’ mechanisms suggested for nesting vertebrates.     

Group nesting and individual variation in behavior and physiology in the orchid bee <Emphasis Type="Italic">Euglossa nigropilosa</Emphasis> Moure (Hymenoptera,Apidae)

The Euglossini are a key group for studying the traits that promote or hinder highly social behavior in bees because it is the only tribe in the Apine clade without large colonies or females with distinct life histories, e.g. queens and workers. There have been few studies on behavior of orchid bee females in nests because these nests are not found easily. Taking advantage of the relatively high abundance of Eg. nigropilosa nests at Reserva Natural La Planada, Colombia, we examined social behavior of Eg. nigropilosa individuals in five nests (3 original and 2 reused) for nine months. We report this species to have the largest colonies known for Euglossa, with nests reaching up to 22 individuals, and all nests containing more than one female bee from the same generation. These nests presented many traits that correspond to communal insect colonies. No generational overlap and no cooperative brood care were detected. We examined natural enemies and resource limitation as important factors for group nesting. We examined parasitoid attacks to cells in a nest with females and one without females. We also searched for nesting locations and examined nest re-use as indicators of nest site limitation. Lastly, we examined behavioral and physiological differences among females in the same nest. Such differences could be the bases for evolution of alternative life histories among group living females. We examined extent of ovary development and oviposition rates in similarly aged females in the same nest. We found large variation in reproductive effort of young females. We also examined differences in resin foraging and cell usurpation behaviors. Behavioral specialization was observed, with some individuals bringing only resin to the nest. Inside the nests, bees had territories in which they constructed and defended cells. This territoriality may be a defense against usurpation of provisioned cells by nest mates. Received 10 December 2007; revised 2 May 2008; accepted 7 May 2008.     

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.     

Colony Social Organisation and Alternative Social Strategies in the Eastern Carpenter Bee, <Emphasis Type="Italic">Xylocopa virginica</Emphasis>

The eastern carpenter bee, Xylocopa virginica, can nest either solitarily or in groups. In southern Ontario, Canada, near the northern edge of the range, most nests are social, containing groups of two to five adult females. Although social nests were much more frequent than solitary ones, they produced no more brood, so per capita brood productivity was actually lower for social females. Social females exhibited several reproductive strategies inferred from wing wear patterns, which reflect flight activity, and mandibular wear patterns, which reflect nest construction activity. Primary females accumulated a large degree of wing and mandibular wear and were presumed to be the primary reproductives in social nests. Secondary females accumulated less wing and mandibular wear and were probably subordinates awaiting opportunities to supersede primaries as dominant foragers and egg-layers. Tertiary females remained inactive, apparently deferring reproduction to the subsequent year. Social nesting, serial replacement of dominant females in social nests, and deferred reproduction are probably responses to severe competition for nests and nesting substrate.     

Honey‐Making Bee Colony Abundance and Predation by Apes and Humans in a Uganda Forest Reserve1

Honey‐making bee colonies in Bwindi Impenetrable National Park were investigated with Batwa Pygmies locating 228 nests of Apis and five stingless bees (Meliponini). The relative importance of predation, food supply, nesting site, and elevation affecting abundance were studied for meliponines in particular. Nest predation and overall nest abundance had no correlation with elevation along a 1400 m gradient, nor did flowering phenology or pollen collection. Many suitable, large trees were unoccupied by bee nests. In 174 ha of forest plots, 2 Meliponula lendliana, 13 M. nebulata, 16 M. ferruginea, 16 M. bocandei, and 20 Apis mellifera adansonii nests occurred, suggesting a habitat‐wide density of 39 nests/km². Compared to other studies, Ugandan Meliponini were uncommon (0.27 colonies/ha, tropical mean = 1.9/ha), while Apis mellifera was numerous (0.12 nests/ha, tropical mean = 0.06/ha), despite park policy allowing humans to exploit Apis. Meliponine colony mortality from predators averaged 12 percent/yr and those near ground were most affected. Tool‐using humans and chimpanzees caused 82 percent of stingless bee nest predation. Selective factors affecting nest heights and habit may include auditory hunting by predators for buzzing bees, and indirect mutualists such as termites that leave potential nesting cavities. Mobility and free‐nesting by honey bee colonies should enable rapid community recovery after mortality, especially in parks where human honey hunting is frequent, compared to sedentary and nest‐site‐bound Meliponini.     

A description of nesting behaviors,including factors impacting nest site selection,in black‐and‐white ruffed lemurs (Varecia variegata)

Nest site selection is at once fundamental to reproduction and a poorly understood component of many organisms’ reproductive investment. This study investigates the nesting behaviors of black‐and‐white ruffed lemurs, Varecia variegata, a litter‐bearing primate from the southeastern rainforests of Madagascar. Using a combination of behavioral, geospatial, and demographic data, I test the hypotheses that environmental and social cues influence nest site selection and that these decisions ultimately impact maternal reproductive success. Gestating females built multiple large nests throughout their territories. Of these, females used only a fraction of the originally constructed nests, as well as several parking locations as infants aged. Nest construction was best predicted by environmental cues, including the size of the nesting tree and density of feeding trees within a 75 m radius of the nest, whereas nest use depended largely on the size and average distance to feeding trees within that same area. Microhabitat characteristics were unrelated to whether females built or used nests. Although unrelated to nest site selection, social cues, specifically the average distance to conspecifics’ nest and park sites, were related to maternal reproductive success; mothers whose litters were parked in closer proximity to others’ nests experienced higher infant survival than those whose nests were more isolated. This is likely because nesting proximity facilitated communal crèche use by neighboring females. Together, these results suggest a complex pattern of nesting behaviors that involves females strategically building nests in areas with high potential resource abundance, using nests in areas according to their realized productivity, and communally rearing infants within a network of nests distributed throughout the larger communal territory.     

Colony disturbance and solitary nest initiation by workers in the obligately eusocial sweat bee, Halictus ligatus

Sweat bees are one of the most socially polymorphic lineages on the planet. In obligately eusocial species, newly enclosed females may become either queens or workers, depending on the environmental and social circumstances of the nest into which they emerge. In socially polymorphic species, females also have the option of nesting solitarily, founding a nest and raising future reproductives alone, without the help of other adult females. Halictus ligatus is a widespread Nearctic, ground-nesting sweat bee. It has been particularly well studied in Ontario, where detailed studies have described it as obligately eusocial. Here we report evidence that the flexibility of female H. ligatus actually extends to expressing behaviour more typical of socially polymorphic species, those in which some individuals reproduce solitarily. In a population in southern Ontario, black wasps (Astata sp.) emerged from the soil beneath the nesting aggregation and proceeded to excavate their own nesting tunnels, dislocating many H. ligatus nest entrances. Young workers whose natal nests were destroyed by the wasp activity constructed new nests, so under very specific circumstances, it is possible for potential altruists to nest solitarily.     

Nest site preference and success in a gregarious, ground-nesting bee Dieunomia triangulifera

1. The characteristics of preferred nest sites of the solitary bee Dieunomia triangulifera, which nests gregariously in alluvial soil, were investigated. Nest presence was used as an indication of preference. In those cases where nests were more than a few days old, therefore, this study also investigated nest success. 2. Independent variables tested were soil surface moisture, presence of nesting conspecifics, smooth vs. irregular soil surface, distance to visual landmarks, percentage of vegetation cover, soil compaction, vehicular traffic, soil surface temperature, and light intensity. 3. Bees preferred to nest in moist, compact soil with an irregular surface. More bees nested near visual landmarks and in places with little or no vegetation. They preferred nesting in areas with warmer soil surface temperatures and brighter illumination. The presence of nesting conspecifics did not influence nesting decisions. Vehicular traffic also seemed to have no impact on nest initiation but did seem to have a negative impact on nest success. 4. Although it might seem that ground-nesting bees should not be limited by nesting sites, relatively little of the study area met all their apparent criteria. This might account, in part, for gregarious nesting in this species.     

An overview of proximate factors affecting the nesting behavior of solitary wasps and bees (Hymenoptera: Aculeata) in preexisting cavities in wood

Guilds of Aculeate solitary wasps and bees that nest in preexisting cavities in wood are important components of terrestrial ecosystems because they engage in several ecological interactions (e.g. predation and pollination) with other species of plants and animals. Spatial and temporal variations in richness and abundance of solitary wasps and bees can be related to changes in environmental structure and in the diversity of other groups of organisms. The nesting period of these Aculeata is their most critical life cycle stage. Females of solitary wasp and bee species invest relatively more time constructing and provisioning their nests than do females of social species. Differently from species that nest in the soil or construct exposed nests, the main factors affecting the reproductive success of solitary species nesting in preexisting wood holes are still unknown. Our objective is to provide an overview of the role of proximate causes of nesting failure or success among solitary wasps and bees (Aculeata), for designing effective conservation and management strategies for these Hymenoptera.     

Mate Choice and Mating Pattern in a Stream Goby of the Genus Rhinogobius

The mate choice and mating pattern of a benthic goby Rhinogobius sp. CB (cross band type) were investigated in the Kamo River, Shikoku, Japan. During the breeding season, gravid females assumed a nuptial color and either males or females initiated a courtship display. Males preferentially courted a female of similar size to lead her to his nest, whereas females courted more frequently when they encountered a large male. Eggs in any one nest were always at the same developmental stage. Sampling data of nesting males and females indicated that, in more than half the nests, males gathered more than one female before spawning. In some nests with eggs, two or three females had spent ovaries, indicating that the eggs were laid by multiple females within a short span of time. However, a comparison between the total number of eggs which females would spawn in one nest and the number of eggs actually deposited suggested that eggs were contributed by one female in most nests. This low level of polygyny in spite of multiple female availability is attributed to a limited available spawning area of the nest.     

Buildings promote higher incubation temperatures and reduce nest attentiveness in a Neotropical thrush

Incubation is an energetically costly parental task of breeding birds. Incubating parents respond to environmental variation and nest‐site features to adjust the balance between the time spent incubating (i.e. nest attentiveness) and foraging to supply their own needs. Non‐natural nesting substrates such as human buildings impose new environmental contexts that may affect time allocation of incubating birds but this topic remains little studied. Here, we tested whether nesting substrate type (buildings vs. trees) affects the temperature inside the incubation chamber (hereafter ‘nest temperature’) in the Pale‐breasted Thrush Turdus leucomelas, either during ‘day’ (with incubation recesses) or ‘night’ periods (representing uninterrupted female presence at the nest). We also tested whether nesting substrate type affects the incubation time budget using air temperature and the day of the incubation cycle as covariates. Nest temperature, when controlled for microhabitat temperature, was higher at night and in nests in buildings but did not differ between daytime and night for nests in buildings, indicating that buildings partially compensate for incubation recesses by females with regard to nest temperature stability. Females from nests placed in buildings exhibited lower nest attentiveness (the overall percentage of time spent incubating) and had longer bouts off the nest. Higher air temperatures were significantly correlated with shorter bouts on the nest and longer bouts off the nest, but without affecting nest attentiveness. We suggest that the longer bouts off the nest taken by females of nests in buildings is a consequence of higher nest temperatures promoted by man‐made structures around these nests. Use of buildings as nesting substrate may therefore increase parental fitness due to a relaxed incubation budget, and potentially drive the evolution of incubation behaviour in certain urban bird populations.