Specific and sensitive detection of Nosema bombi (Microsporidia: Nosematidae) in bumble bees (Bombus spp.; Hymenoptera: Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 10(7) down to 10 spores diluted in 100 microl of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of approximately 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to be more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy.     

Interspecific geographic distribution and variation of the pathogens Nosema bombi and Crithidia species in United States bumble bee populations

Several bumble bee (Bombus) species in North America have undergone range reductions and rapid declines in relative abundance. Pathogens have been suggested as causal factors, however, baseline data on pathogen distributions in a large number of bumble bee species have not been available to test this hypothesis. In a nationwide survey of the US, nearly 10,000 specimens of 36 bumble bee species collected at 284 sites were evaluated for the presence and prevalence of two known Bombus pathogens, the microsporidium Nosema bombi and trypanosomes in the genus Crithidia. Prevalence of Crithidia was ≤10% for all host species examined but was recorded from 21% of surveyed sites. Crithidia was isolated from 15 of the 36 Bombus species screened, and were most commonly recovered from Bombus bifarius, Bombus bimaculatus, Bombus impatiens and Bombus mixtus. Nosema bombi was isolated from 22 of the 36 US Bombus species collected. Only one species with more than 50 sampled bees, Bombus appositus, was free of the pathogen; whereas, prevalence was highest in Bombus occidentalis and Bombus pensylvanicus, two species that are reportedly undergoing population declines in North America. A variant of a tetranucleotide repeat in the internal transcribed spacer (ITS) of the N. bombi rRNA gene, thus far not reported from European isolates, was isolated from ten US Bombus hosts, appearing in varying ratios in different host species. Given the genetic similarity of the rRNA gene of N. bombi sampled in Europe and North America to date, the presence of a unique isolate in US bumble could reveal one or more native North American strains and indicate that N. bombi is enzootic across the Holarctic Region, exhibiting some genetic isolation.     

Differential virulence in a multiple‐host parasite of bumble bees: resolving the paradox of parasite survival?

Parasite virulence determines both the impact that parasites have on their hosts and parasite fitness. While most studies of virulence have involved single-species host–parasite interactions, the majority of parasites are likely to use multiple concurrent host species. Our understanding of how this impacts on parasite epidemiology and virulence is limited. Using the bumble bee Bombus lucorum , which exists in sympatry with B. terrestris in multi-species assemblages, and their generalist micosporidian parasite Nosema bombi , we tested whether the apparent paradox of parasite maintenance due to parasite virulence in a single host, B. terrestris , could be resolved through understanding the parasite's virulence in this sympatric host species. Nosema bombi significantly impacted colony growth, individual longevity, and individual development in B. lucorum . However, these effects were different both qualitatively and quantitatively to the parasite's impact in B. terrestris . Infected colonies of B. lucorum successfully produced both male and female reproductives, and infected female reproductives were capable of successful mating. Variation in life-history across host species may explain differences in the virulence, or impact of the parasite in B. terrestris and B. lucorum , with species with shorter life-cycles being more likely to transfer the parasite from one annual generation to the next. These results suggest that to understand the virulence and epidemiology of multi-host parasites we need to examine their ecological interactions across their various host species.     

不同株的熊蜂短膜虫与宿主生存的关系

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Factors influencing Nosema bombi infections in natural populations of Bombus terrestris (Hymenoptera: Apidae)

Bumblebees are of profound ecological importance because of the pollination services they provide in natural and agricultural ecosystems. Any decline of these pollinators is therefore of great concern for ecosystem functioning. Increased parasite pressures have been discussed as a major factor for the loss of pollinators. One of the main parasites of bumblebees is Nosema bombi, an intracellular microsporidian parasite with considerable impact on the vitality of the host. Here we study the effect of host colony density and host genetic variability on N. bombi infections in natural populations of the bumblebee Bombus terrestris. We sampled males and workers from six B. terrestris populations located in an agricultural landscape in Middle Sweden to determine the prevalence and degree of N. bombi infections. All individuals were genotyped with five microsatellite markers to infer the colony densities in the sampled populations and the genetic variability of the host population. We confirmed that genetic variability and sex significantly correlate with the degree of infection with N. bombi. Males and workers with lower genetic variability had significantly higher infection levels than average. Also colony density had a significant impact on the degree of infection, with high density populations having higher infected individuals.     

An interspecific comparison of foraging range and nest density of four bumblebee (Bombus) species

Bumblebees are major pollinators of crops and wildflowers in northern temperate regions. Knowledge of their ecology is vital for the design of effective management and conservation strategies but key aspects remain poorly understood. Here we employed microsatellite markers to estimate and compare foraging range and nest density among four UK species: Bombus terrestris, Bombus pascuorum, Bombus lapidarius, and Bombus pratorum. Workers were sampled along a 1.5-km linear transect across arable farmland. Eight or nine polymorphic microsatellite markers were then used to identify putative sisters. In accordance with previous studies, minimum estimated maximum foraging range was greatest for B. terrestris (758 m) and least for B. pascuorum (449 m). The estimate for B. lapidarius was similar to B. pascuorum (450 m), while that of B. pratorum was intermediate (674 m). Since the area of forage available to bees increases as the square of foraging range, these differences correspond to a threefold variation in the area used by bumblebee nests of different species. Possible explanations for these differences are discussed. Estimates for nest density at the times of sampling were 29, 68, 117, and 26/km2 for B. terrestris, B. pascuorum, B. lapidarius and B. pratorum, respectively. These data suggest that even among the most common British bumblebee species, significant differences in fundamental aspects of their ecology exist, a finding that should be reflected in management and conservation strategies.     

Effects of Nosema bombi and its treatment fumagillin on bumble bee (Bombus occidentalis) colonies

We examined the effects of Nosema bombi (Microsporidia: Nosematidae) on colonies of bumble bees, Bombus occidentalis Greene (Hymenoptera: Apidae), used to pollinate tomatoes in commercial greenhouses. We assessed methods of detecting N. bombi and tested the effectiveness of fumagillin to control this parasite. N. bombi did not affect adult population size or amount of brood in B. occidentalis colonies. Fumagillin was not effective against N. bombi at the doses we tested, and frass samples did not provide accurate estimates of the intensity of N. bombi infections. The number of N. bombi spores per bee was highly variable among bumble bees within colonies, and accurate estimates could only be obtained by sampling a large proportion of bees in each colony. Therefore, whole bee and frass sampling is useful for determining if N. bombi is present or absent, but not for obtaining accurate estimates of the intensity of N. bombi infections.     

A field experiment on the effect of Nosema bombi in colonies of the bumblebee Bombus terrestris

Abstract 1. Nosema bombi is a microsporidian parasite of bumblebees that is of potentially great ecological and economic importance. Nosema bombi has been shown to have detrimental fitness effects on the bumblebee Bombus terrestris under laboratory conditions. The present study investigates whether N. bombi also has negative fitness effects under natural conditions. 2. A field experiment was conducted with B. terrestris spring queens caught from the wild. In the laboratory these queens were assigned to either infected or control treatment groups. On the emergence of workers, colonies were placed in the field and a number of fitness and life‐history traits were measured. 3. The size of colonies produced by infected queens was significantly smaller than those of control queens. None of the 14 infected colonies produced sexual offspring, whereas four of the 14 control colonies reached the male production stage. Results of the field experiment are in agreement with prior laboratory‐based results, and even imply that such effects are amplified under natural conditions. 4. The evidence suggests that, on entering the colony early, N. bombi has a highly detrimental effect on the fitness of its host. It would seem that this parasite reduces its opportunities to transmit into the next host generation.     

Nosema bombi: A pollinator parasite with detrimental fitness effects

Nosema bombi is an obligate intracellular parasite that infects different bumblebee species at a substantial, though variable, rate. To date its pathology and impact on host fitness are not well understood. We performed a laboratory experiment investigating the pathology and fitness effects of this parasite on the bumblebee Bombus terrestris. We experimentally infected one group of colonies with N. bombi spores at the start of the worker production, while a second uninfected group of colonies served as controls. During colony development we collected live workers for dissections to measure infection intensities. In parallel, we measured several life history traits, to investigate costs to the host. We succeeded in infecting 11 of 16 experimental colonies. When infection occurred at an early stage of colony development, virtually all individuals were infected, with spores being found in a number of tissues, and the functional fitness of males and young queens was reduced to zero. Further, the survival of workers from infected colonies and infected males were reduced. With such severe effects, N. bombi appears to decrease its opportunities for transmission to the next host generation.     

Invasive Bombus terrestris (Hymenoptera: Apidae) parasitized by a flagellate (Euglenozoa: Kinetoplastea) and a neogregarine (Apicomplexa: Neogregarinorida)

The flagellate Crithidia bombi and the neogregarine Apicystis bombi have been found in individuals of Bombus terrestris, a Palaearctic species of bumble bee commercially reared and shipped worldwide for pollination services. B. terrestris has recently entered into the northwestern Patagonia region of Argentina from Chile, where it was introduced in 1998. Prevalence was 21.6% for C. bombi and 3.6% for A. bombi (n = 111). The pathogens were not detected in 441 bumble bees belonging to five of the eight known Argentine native species (Bombus atratus, Bombus morio, Bombus bellicosus, Bombus opifex, Bombus tucumanus) collected elsewhere in the country. Although the absence of natural occurrence of C. bombi and A. bombi in Argentine native bumble bees cannot be ascertained at present due to the limited surveys performed, it is important to report their detection in invasive B. terrestris. The invasion event is relatively recent and the accompanying pathogens are not species specific within the genus Bombus.     

Absolute configuration of chiral terpenes in marking pheromones of bumblebees and cuckoo bumblebees

The absolute configurations of citronellol, 2,3-dihydrofarnesol, and 2,3-dihydrofarnesal in male marking pheromones of seven species of bumblebees and cuckoo bumblebees were determined by enantioselective gas chromatography on a capillary column coated with 60% heptakis(2,3-di-O-acetyl-6-O-TBDMS)-beta-cyclodextrin in polysiloxane PS 268. Pure (-)-S-enantiomers of all three terpenes were found in the labial glands of all investigated specimens of the following species: Bombus (Bombus) terrestris, B. (Bombus) lucorum, B. (Pyrobombus) pratorum, B. (Pyrobombus) pyrenaeus, B. (Pyrobombus) jonellus, B. (Pyrobombus) impatiens, and the cuckoo bumblebee B. (Ashtonipsithyrus) bohemicus. Within species, specimens were collected at different localities and in different years. Except for 2,3-dihydrofarnesol in B. terrestris, this is the first report on the absolute configuration of terpenes in marking pheromones of bumblebees.     

Does Pathogen Spillover from Commercially Reared Bumble Bees Threaten Wild Pollinators?

The conservation of insect pollinators is drawing attention because of reported declines in bee species and the 'ecosystem services' they provide. This issue has been brought to a head by recent devastating losses of honey bees throughout North America (so called, 'Colony Collapse Disorder'); yet, we still have little understanding of the cause(s) of bee declines. Wild bumble bees (Bombus spp.) have also suffered serious declines and circumstantial evidence suggests that pathogen 'spillover' from commercially reared bumble bees, which are used extensively to pollinate greenhouse crops, is a possible cause. We constructed a spatially explicit model of pathogen spillover in bumble bees and, using laboratory experiments and the literature, estimated parameter values for the spillover of Crithidia bombi, a destructive pathogen commonly found in commercial Bombus. We also monitored wild bumble bee populations near greenhouses for evidence of pathogen spillover, and compared the fit of our model to patterns of C. bombi infection observed in the field. Our model predicts that, during the first three months of spillover, transmission from commercial hives would infect up to 20% of wild bumble bees within 2 km of the greenhouse. However, a travelling wave of disease is predicted to form suddenly, infecting up to 35-100% of wild Bombus, and spread away from the greenhouse at a rate of 2 km/wk. In the field, although we did not observe a large epizootic wave of infection, the prevalences of C. bombi near greenhouses were consistent with our model. Indeed, we found that spillover has allowed C. bombi to invade several wild bumble bee species near greenhouses. Given the available evidence, it is likely that pathogen spillover from commercial bees is contributing to the ongoing decline of wild Bombus in North America. Improved management of domestic bees, for example by reducing their parasite loads and their overlap with wild congeners, could diminish or even eliminate pathogen spillover.     

Cophylogeny of Nosema (Microsporidia: Nosematidae) and bees (Hymenoptera: Apidae) suggests both cospeciation and a host-switch

Some microsporidian parasites belonging to the genus Nosema infect bees. Previous phylogenies of these parasites have produced alternative, conflicting relationships. We analyzed separately, and in combination, large and small subunit ribosomal DNA sequences of Nosema species infecting bees under neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian frameworks. We observed a sister relationship between Nosema ceranae and Nosema bombi, with Nosema apis as a basal member to this group. When compared to their respective hosts (Apis cerana, Bombus spp., and A. mellifera), 2 plausible evolutionary scenarios emerged. The first hypothesis involves a common ancestor of N. bombi host-switching from a historical Bombus lineage to A. cerana. The second suggests an ancestral N. ceranae host-switching to a species of Bombus. The reported events offer insight into the evolutionary history of these organisms and may explain host specificity and virulence of Nosema in these economically important insects.     

The Abundance and Pollen Foraging Behaviour of Bumble Bees in Relation to Population Size of Whortleberry (Vaccinium uliginosum)

Habitat fragmentation can have severe effects on plant pollinator interactions, for example changing the foraging behaviour of pollinators. To date, the impact of plant population size on pollen collection by pollinators has not yet been investigated. From 2008 to 2010, we monitored nine bumble bee species (Bombus campestris, Bombus hortorum s.l., Bombus hypnorum, Bombus lapidarius, Bombus pascuorum, Bombus pratorum, Bombus soroensis, Bombus terrestris s.l., Bombus vestalis s.l.) on Vaccinium uliginosum (Ericaceae) in up to nine populations in Belgium ranging in size from 80 m² to over 3.1 ha. Bumble bee abundance declined with decreasing plant population size, and especially the proportion of individuals of large bumble bee species diminished in smaller populations. The most remarkable and novel observation was that bumble bees seemed to switch foraging behaviour according to population size: while they collected both pollen and nectar in large populations, they largely neglected pollen collection in small populations. This pattern was due to large bumble bee species, which seem thus to be more likely to suffer from pollen shortages in smaller habitat fragments. Comparing pollen loads of bumble bees we found that fidelity to V. uliginosum pollen did not depend on plant population size but rather on the extent shrub cover and/or openness of the site. Bumble bees collected pollen only from three plant species (V. uliginosum, Sorbus aucuparia and Cytisus scoparius). We also did not discover any pollination limitation of V. uliginosum in small populations. We conclude that habitat fragmentation might not immediately threaten the pollination of V. uliginosum, nevertheless, it provides important nectar and pollen resources for bumble bees and declining populations of this plant could have negative effects for its pollinators. The finding that large bumble bee species abandon pollen collection when plant populations become small is of interest when considering plant and bumble bee conservation.     

Multiple rRNA variants in a single spore of the microsporidian Nosema bombi

To understand the source of the multiple DNA sequence variants of Nosema bombi ribosomal RNA (rRNA) found in a single bumble bee host, we PCR amplified, cloned, and sequenced the partial rRNA gene from 125 clones, which were derived from four out of 46 spores individually isolated from a single host by laser microdissection. At least two rRNA variants, characterized by either (GTTT)(2) or (GTTT)(3) repeat units within the internal transcribed spacer (ITS) region, were found per spore in approximately equal proportions, variants which were also found in approximately equal proportions in 55 clones of the two DNA extracts of multiple spores from the same host. Firstly, we demonstrate for the first time that DNA sequences can be obtained from single-binucleate microsporidia. Secondly, it appears that concerted evolution has not homogenized the sequences of all rRNA copies within a single N. bombi spore or even within a single nucleus. We thereby demonstrate unequivocally that two or more rRNA sequence variants exist per N. bombi spore, and urge caution in the use of multicopy rRNA genes for population genetic and phylogenetic analysis of this and other Microsporidia unless homologous copies can be reliably typed.     

Natural variation in the genetic architecture of a host-parasite interaction in the bumblebee Bombus terrestris

The genetic architecture of fitness-relevant traits in natural populations is a topic that has remained almost untouched by quantitative genetics. Given the importance of parasitism for the host's fitness, we used QTL mapping to study the genetic architecture of traits relevant for host-parasite interactions in the trypanosome parasite, Crithidia bombi and its host, Bombus terrestris. The three traits analysed were the parasite's infection intensity, the strength of the general immune response (measured as the encapsulation of a novel antigen) and body size. The genetic architecture of these traits was examined in three natural, unmanipulated mapping populations of B. terrestris. Our results indicate that the intracolonial phenotypic variation of all three traits is based on a network of QTLs and epistatic interactions. While these networks are similar between mapping populations in complexity and number of QTLs, as well as in their epistatic interactions, the variability in the position of QTL and the interacting loci was high. Only one QTL for body size was plausibly found in at least two populations. QTLs for encapsulation and Crithidia infection intensity were located on the same linkage groups.     

Serial passage of the parasite Crithidia bombi within a colony of its host, Bombus terrestris, reduces success in unrelated hosts

In the wild, Bombus spp. bees may contract infections of the trypanosome parasite Crithidia bombi from their nestmates or from others while foraging on contaminated flowers. We expected that as C. bombi is transmitted repeatedly among related workers within a colony, the parasite population would become more successful in this relatively homogeneous host population and less successful in individuals from unrelated colonies of the same or different species. To test our prediction, we serially passaged cocktails of C. bombi strains through workers from the same colony, taking the intensity of infection in related versus unrelated workers as a measure of parasite success at each step in the serial transfer. Using a repeated measures ANOVA, we found the ability of C. bombi to exploit Bombus spp. hosts did not increase within a colony, but did decrease for infections in workers from unrelated colonies. This reduction in success is most likely due to a gradual loss of appropriate C. bombi strains from the infecting the population as the cocktail is 'filtered' during the serial passage within a given colony, without a corresponding increase in overall intensity of the surviving strains.     

Detection of Deformed wing virus, a honey bee viral pathogen, in bumble bees (Bombus terrestris and Bombus pascuorum) with wing deformities

Honey bees (Apis mellifera) productively infected with Deformed wing virus (DWV) through Varroa destructor (V. destructor) during pupal stages develop into adults showing wing and other morphological deformities. Here, we report for the first time the occurrence of bumble bees (Bombus terrestris, Bombus pascuorum) exhibiting wing deformities resembling those seen in clinically DWV-infected honey bees. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. Since such deformed bumble bees are not viable DWV infection may pose a serious threat to bumble bee populations.     

Genetic analysis of spatial foraging patterns and resource sharing in bumble bee pollinators

Conservation biologists, evolutionary ecologists and agricultural biologists require an improved understanding of how pollinators utilize space and share resources. Using microsatellite markers, we conducted a genetic analysis of space use and resource sharing at several spatial scales among workers of two ecologically dissimilar bumble bee species (Bombus terrestris and B. pascuorum) foraging in an urban landscape (London, UK). At fine scales, the relatedness of workers visiting small patches of flowers did not differ significantly from zero. Therefore, colonies shared flower patches randomly with other colonies, suggesting that worker scent-marks deterring visits to unrewarding flowers have not evolved as signals benefiting nestmates. To investigate space use at intermediate scales, we developed a program based on Thomas & Hill's maximum likelihood sibship reconstruction method to estimate the number of colonies utilizing single sites. The average number of colonies (95% confidence limits) sending workers to forage at sites of approximately 1 ha in area was 96 colonies (84-118) in B. terrestris and 66 colonies (61-76) in B. pascuorum. These values are surprisingly high and suggested that workers travelled far from their colonies to visit the sites. At the landscape scale, there was little or no genetic differentiation between sites. We conclude that urban habitats support large bumble bee populations and are potentially valuable in terms of bumble bee conservation. In addition, bumble bee-mediated gene flow in plants is likely to occur over large distances and plant-bumble bee conservation requires landscape-scale action.     

Identification of Nosema bombi Fantham and Porter 1914 (Microsporidia) in Bombus impatiens and Bombus sandersoni from Great Smoky Mountains National Park (USA)

Ninety three bumble bees belonging to the genus Bombus, subgenus Pyrobombus (three Bombus vagans, seven Bombus bimaculatus, 17 B. sandersoni and 68 B. impatiens) from Great Smoky Mountains National Park were examined for microsporidia. Light microscopy of calcoflour and trichrome-stained smears, and PCR revealed infection with N. bombi in one specimen each of B. sandersoni and B. impatiens. Sizes and shapes of spores in both N. bombi isolates were similar to those described for European isolates of the microsporidium. A region of the rRNA gene from the B. impatiens isolate (1689 bp, accession GQ254295) aligned with homologous sequences from eight European isolates, with only three variable sites. Sequence variability of this region between novel isolates and the European ones was the same as among European isolates.