Bombi Infection Research Articles

Parasites of the genus Nosema, Crithidia and Lotmaria in the honeybee and bumblebee populations: a case study in India

The populations of honeybees and bumblebees have been decreasing around the world in the recent decades. A variety of pathogens and parasites, including bacteria, fungi, protozoa, nematodes, mites and insects play signi ficant role in honeybee and bumblebee colonies loss. Pa rasites of the genus Nosema (Microsporidia: Nosematidae) and the genera Crithidia and Lotmaria (Kinetoplastida: Trypanosomatidae) have a significant negative impact on honeybee and bumblebee colonies. Recent studies of nuclear DNA markers of these parasites allowed to describe new species and genetic variants. The aim of this study was to investigate the Microsporidia (Nosema spp.) and Trypanosomatidae (Crithidia spp. and Lotmaria passim) prevalence and genetic diversity in honeybee and bumble bee populations of Indian territories that haven’t been studied before. In total 119 specimens of 4 honeybee and 5 bumblebee species were analyzed in this study. The prevalence of parasites in honeybee and bumblebee po pulations of the two Indian states (Jammu and Kashmir, Karnataka) were identified using PCR with primers specific for the ribosomal RNA genes cluster of Nosema, Crithidia and Lotmaria species. Co­infection by microsporidian and trypanosomatid parasites was detected in several honeybee and bumblebee specimens from Jammu and Kash mir state. Comparative analysis of ribosomal RNA genes sequences showed that honeybee samples from India studied were infected by N. bombi, N. ceranae and L. passim. Bumblebee populations were infected by Nosema D, Crithidia bombi and Crithidia expoeki. No honeybee’s specimen with trypanosomatid infection was found in Karnataka state. For the first time N. bombi infection was detected in the honeybee population. The studies of distribution of microsporidia and trypanosomatid parasites among the honeybee and bumblebee populations all over the World were summarized and supplemented.

A scientific note on Nosema bombi infection intensity among different castes within a Bombus auricomus nest

Bumble bees (Bombus) provide vital services to agricultural and natural plant systems (Kremen et al. 2002; Fontaine et al. 2006). A survey across diverse North American Bombus species detected higher prevalence of infection by the microsporidian parasite Nosema bombi in declining relative to non-declining species, indicating a correlation between Nosema and Bombus population declines (Cameron et al. 2011). Although it has been shown in the laboratory that N. bombi infection is detrimental to the fitness of Bombus terrestris and Bombus lucorum (Otti and Schmid-Hempel 2007; Rutrecht and Brown 2009), much remains unclear regarding N. bombi’s ecology and how it interacts with Bombus in the field.

Differential gene expression and alternative splicing in insect immune specificity.

BackgroundEcological studies routinely show genotype-genotype interactions between insects and their parasites. The mechanisms behind these interactions are not clearly understood. Using the bumblebee Bombus terrestris/trypanosome Crithidia bombi model system (two bumblebee colonies by two Crithidia strains), we have carried out a transcriptome-wide analysis of gene expression and alternative splicing in bees during C. bombi infection. We have performed four analyses, 1) comparing gene expression in infected and non-infected bees 24 hours after infection by Crithidia bombi, 2) comparing expression at 24 and 48 hours after C. bombi infection, 3) determining the differential gene expression associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours after infection and 4) determining the alternative splicing associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours post infection.ResultsWe found a large number of genes differentially regulated related to numerous canonical immune pathways. These genes include receptors, signaling pathways and effectors. We discovered a possible interaction between the peritrophic membrane and the insect immune system in defense against Crithidia. Most interestingly, we found differential expression and alternative splicing of immunoglobulin related genes (Dscam and Twitchin) are associated with the genotype-genotype interactions of the given bumblebee colony and Crithidia strain.ConclusionsIn this paper we have shown that the expression and alternative splicing of immune genes is associated with specific interactions between different host and parasite genotypes in this bumblebee/trypanosome model.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1031) contains supplementary material, which is available to authorized users.

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.

Ecological effects on gut bacterial communities in wild bumblebee colonies

1. Animal hosts harbour diverse and often specific bacterial communities (microbiota) in their gut. These microbiota can provide crucial services to the host such as aiding in digestion of food and immune defence. However, the ecological factors correlating with and eventually shaping these microbiota under natural conditions are poorly understood. 2. Bumblebees have recently been shown to possess simple and highly specific microbiota. We here examine the dynamics of these microbiota in field colonies of the bumblebee Bombus terrestris over one season. The gut bacteria were assessed with culture-independent methods, that is, with terminal restriction fragment length profiles of the 16S rRNA gene. 3. To further understand the factors that affect the microbiota, we experimentally manipulated field-placed colonies in a fully factorial experiment by providing additional food or by priming the workers' immune system by injecting heat-killed bacteria. We furthermore looked at possible correlates of diversity and composition of the microbiota for (i) natural infections with the microbial parasites Crithidia bombi and Nosema bombi, (ii) bumblebee worker size, (iii) colony identity, and (iv) colony age. 4. We found an increase in diversity of the microbiota in individuals naturally infected with either C. bombi or N. bombi. Crithidia bombi infections, however, appear to be only indirectly linked with higher microbial diversity when comparing colonies. The treatments of priming the immune system with heat-killed bacteria and additional food supply, as well as host body size, had no effect on the diversity or composition of the microbiota. Host colony identity had only a weak effect on the composition of the microbiota at the level of resolution of our method. We found both significant increases and decreases in the relative abundance of selected bacterial taxa over the season. 5. We present the first study on the ecological dynamics of gut microbiota in bumblebees and identify parasite infections, colony identity and colony age as important factors influencing the diversity and composition of the bacterial communities. The absence of an effect of our otherwise effective experimental treatments suggests a remarkable ability of the host to maintain a homoeostasis in this community under widely different environments.

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.

Pathways to immunity: temporal dynamics of the bumblebee (Bombus terrestris) immune response against a trypanosomal gut parasite

Immune response dynamics in insects from natural host-parasite associations are poorly understood, despite accumulating evidence of ecological immune phenomena in these systems. Using a gene discovery approach, we have identified genes relating to signalling, enzymatic processes and respiration that were up-regulated in the bumblebee, Bombus terrestris, during infection with the trypanosomatid parasite, Crithidia bombi. In addition, we have mapped dynamic changes in the temporal expression of these genes and three candidate antimicrobial peptide (AMP) immune genes, Abaecin, Defensin and Hymenoptaecin, from 1 to 24 h after C. bombi infection. We show that dynamic changes in expression occur for individual genes at distinct phases of the immune response to C. bombi that correspond to early, intermediate and late stages of infection.

Factors affecting parasite prevalence among wild bumblebees

1. Bumblebees are important pollinators in North America and are attacked by a range of parasites that impact their fitness; however, few studies have investigated the extent or causes of parasitism in North America.2. This study used a 2‐year multi‐site survey of bumblebee parasitism to ask: (i) how common are parasitoid conopid flies and the internal parasites Crithidia bombi and Nosema bombi in Massachusetts; and (ii) what factors are correlated with parasitism?3. Infection rates by all three parasites were higher in this study than previously documented in North America. Overall, conopids infected 0–73% of bees in each sample, C. bombi infected 0–82% of bees, and N. bombi infected 0–32%.4. Conopid flies infected female bees more than males and intermediate‐sized bees more than large or small bees. Crithidia bombi infection rates were higher in certain bee species and sites, and exhibited a unimodal pattern of prevalence over time. Nosema bombi parasitism was higher in male than female bees.5. Infection by N. bombi in two rare bumblebee species was higher than expected based on parasitism rates of common bee species but C. bombi infection was lower.If high prevalence of N. bombi in these bumblebee species is common, parasitism may be a potential cause of their decline.6. Given the documented effects of these parasites, the high levels of infection may affect bee populations in Massachusetts and threaten the stability of their valuable ecosystem services.

Infection with the trypanosome Crithidia bombi and expression of immune-related genes in the bumblebee Bombus terrestris

Social bees and other insects are frequently parasitized by a large range of different microorganisms. Among these is Crithidia bombi (Kinetoplastida: Trypanosomatidae), a common gut parasite of bumblebees, Bombus spp. (Insecta: Apidae). Bumblebees are important pollinators in commercial and natural environments. There are clear detrimental effects of C. bombi infections on the fitness of bumblebees. However, little has been known about how the bee's immune system responds to infections with trypanosome parasites. Here, we study the immune response of Bombus terrestris on infection by C. bombi. We measured the expression of four immune-related genes ( Hemomucin, MyD88, Relish, and TEP7) using RT-qPCR in adult B. terrestris workers that were either healthy or infected with the trypanosome parasite C. bombi. The potential recognition gene Hemomucin was significantly upregulated in the infected bees. Further, there was substantial and significant variation in all four genes among different bumblebee colonies irrespective of infection status.

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.

Infection and transmission ofNosema bombiinBombus terrestriscolonies and its effect on hibernation, mating and colony founding

The impact of the microsporidium Nosema bombi on Bombus terrestris was studied by recording mating, hibernation success, protein titre in haemolymph, weight change during hibernation, and colony founding of queens that were inoculated with N. bombi in the larval phase. Infection with N. bombi was diagnosed in 36% of B. terrestris queens exposed to N. bombi. Mating and hibernation of queens was not significantly affected by N. bombi infection but colony founding was reduced significantly. Haemolymph protein titre of N. bombi diseased queens was reduced, possibly indicating a disturbance of the metabolism. It was demonstrated that N. bombi infection was transmitted to the successive age cohorts in a colony and to the adults that were already in the colony prior to the introduction of the infection. The study showed a significant negative impact of N. bombi on B. terrestris colony development and indoor rearing.

Effects of natal and novel Crithidia bombi (Trypanosomatidae) infections on Bombus terrestris hosts

Bombus terrestris queens may contract infections of the trypanosome parasite Crithidia bombi from their natal nests; alternatively, the queens may also become infected after leaving their natal nests while foraging on contaminated flowers. We expected that, because C. bombi adapts to the natal colony during the previous generation, C. bombi infections from the natal colony will be more damaging to queens than a novel infection acquired from an unrelated colony. To test our prediction, we used queens exposed to three treatment groups: natal infection, novel infection, and control (no infection). We found that the infected queens produced fewer males and had a lower overall fitness, but we did not find any differences based on the source of the infections. We noted a strong matriline effect on the likelihood of a queen surviving hibernation and successfully founding a colony. Taken together, our results suggest that while C. bombi affects the fitness of B. terrestris, one vertical transmission event is no more damaging than randomly encountered infections. Furthermore, we found that, at least under laboratory conditions, matriline effects on fitness could override the effect of infection status.

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 (323 bp 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 μl 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 ∼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.

Does parasitic infection impair the ability of bumblebees to learn flower-handling techniques?

Although the capacity to learn how to manipulate flowers plays an integral role in the foraging of bumblebees, little is known about the effects of parasitic infection on the motor learning and memory of host bees. In the laboratory experiment reported here, we examined whether infection by the intestinal protozoan Crithidia bombi affected the ability of bumblebees, Bombus impatiens, to learn the specialized motor pattern required to handle a novel flower type. Using videotaped records of foraging behaviour, we related the motor performance of bees to the intensity of C. bombi infection. Low intensities of infection had no effect on the ability of bees to learn a novel flower-handling method; however, a high intensity of infection significantly reduced both motor-learning rate and maximum handling proficiency. In addition, highly infected bees showed a 200% increase in the amount of time and the number of visits required to learn how to manipulate flowers. These results indicate that C. bombi can influence the foraging behaviour of host bumblebees in subtle but ecologically significant ways.

The role of male disease susceptibility in the evolution of haplodiploid insect societies.

Heterozygosity at loci affecting resistance against parasites can benefit host fitness. We predict that, in haplodiploid species, haploid males will suffer decreased parasite resistance relative to diploid females. We suggest that elevated susceptibility in haploid males has shaped the evolution of social behaviour in haplodiploid species. Male susceptibility will select for behavioural adaptations that limit males' exposure to pathogens and that limit male transmission of pathogens within and between colonies. The relatedness-asymmetry hypothesis that has been advanced to explain female-only workers does not make these predictions. We review the relevant evidence for genetic effects on parasite resistance in insects and summarize empirical evidence that relates to the haploid-susceptibility hypothesis.

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.

PREVALENCE OF SPHAERULARIA BOMBI (NEMATODA: TYLENCHIDA: SPHAERULARIIDAE) IN BUMBLE BEE QUEENS (HYMENOPTERA: APIDAE) ON CAPE BRETON ISLAND, NOVA SCOTIA, CANADA

AbstractSphaerularia bombi Dufour is an internal nematode parasite of bumble bee queens in North America and Europe. Infection functionally castrates the bee. Here we document the prevalence and intensity of S. bombi infections in seven species of Bombus and three species of Psithyrus on Cape Breton Island, Nova Scotia, Canada. We found dramatic variation in prevalences among Bombus spp., and some evidence that prevalence increased as the nesting season progressed. Also, we report S. bombi in the bumble bee nest parasite Psithyrus insularis (Smith), the first record for the genus in North America.