Biodiversity, impact and control of microsporidia in bumble bee (bombus spp.) pollinators

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 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 -speci W c 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-speci W c primer pair ITS-f2/r2, which amplifies a short fragment of È120 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. The results and the tools described, reduce the detection level of Microsporidia in bumble bees. This will aid in keeping breeding facilities free from Microsporidia infections. The method has been described in detail in scientific media and a lab manual for using the tool kit made available on the project web site and in a public technical report from the project presented at the project End User meeting.

The Pollinator Prasites project has identified the current state of knowledge based on methods and data from the project combined with previous experiences and available published data, for developing the best strategy for Microsporidia detection and control in bumble bee breeding. Microscopy: The simplest method for detection of N. bombi is search for spores in the Malpighian tubes and the ventriculus of bumble bees, using a squash preparation of the organs in tap water. A magnitude of 400 times is sufficient to detect the spores if present to some quantity. Dissection of the Malpighian tubes and ventriculus facilitates the checks. Detailed description of the detection and staining techniques of N. bombi see chapter II of the Pollinator Parasites Technical Report. Molecular methods - PCR : Comparative studies of light microscopy detection and the molecular techniques have demonstrated that PCR is a more sensitive way of detecting infection. An advantage using the molecular detection tool is that both spores and vegetative stages of the parasite will be detected, whereas light microscopy in squash preparations will detect spores only, unless preparations are also stained. The advantage of light microscopy is othat it is simple and rapid, and depending on the objective, may be sufficiently sensitive ( see chapter IV of the Pollinator parasites Technical report). Control strategies Quarantine of queens from nature: A queen caught in the wild should not be introduced directly into the rearing facility without quarantine. When queens caught in the wild are used for pollination purposes, they should be separate from the rearing unit until at least 20 to 30 worker bees have emerged. This offspring must be checked for N. bombi by light microscopy (spores), or by molecular techniques for infection. When the queens and males are used to increase the gene pool, it is best to hibernate the new queens and then to introduce the next generation of queens in the actual rearing unit for production of new colonies. Males become infected as well as queens and workers. Therefore the castes as well as the rearing box should be considered potential carriers of a nosema infection. Contact with nosema-diseased bees and faeces in the mating boxes, is a source of infection that should be avoided. Mating of non-infected queens with infected drones or even if they do not mate but are combined in a mating box, may transmit infection. Nosema infected colonies must be removed from the rearing facility as the infection will persist and will be passed on by infected queens. Drifting: Drifting of bumble bees is observed in greenhouses. Introduction of nosema spores via drifted workers or males is a serious risk if colonies are brought back from pollination assignments and reintroduced into rearing units. Reintroducing queens and males from old colonies into the rearing facility should always be avoided. In case it is necessary to return colonies, virgin queens or males used from pollination units should be placed in quarantine and be treated as colonies from the wild. Food: Nosema infections can be introduced via the food and food devices. Consequently, hygiene is important. All bumble bees in rearing facilities are fed with pollen, collected by honeybees and this material may be contaminated by Nosema apis. Spores of N. apis and spores of N. bombi are difficult to discriminate. In the wild, drones leave the colony and feed on and rest in flowers. In this way flowers and pollen may be contaminated with N. bombi spores. As pollen to be used in bumble bee rearing are collected by honey bees, N. bombi spores may possibly be introduced into rearing facilities. Therefore it is advisable to check the pollen to be fed to colonies in rearing units for nosema spores and avoid feeding Microsporidia spore contaminated pollen unless sensitive molecular detection techniques can exclude the presence of N. bombi (see chapter IV of the Technical Report). Hygiene It has been demonstrated that a low-level nosema infection may not be detected by light microscopy. Thus, even if no infection has been demonstrated using microscopy, general hygienic measures must be applied. This means: - No food, neither pollen nor sugar is transferred from one colony to another, - No food devices are transferred from one box to another, - Tweezers, food devices, mating chambers etc are cleaned using hot water and a disinfectant, - Food is stored in refrigerators, - Escaped bumble bees are captured and killed, - Spoiled food is cleaned up, - The complete facility is kept neatly, - Materials that are a risk for spreading diseases and parasites like old colonies, colonies that are not developing normally, used boxes and dead bumble bees are handled and carried away in a way that free flying bumble bees can not reach it.

Cross infectivity of Nosema bombi, induced by a one time application of 5 µl nosema spores suspension from Bombus species other than B. terrestris to worker brood of B. terrestris was tested. Using spores from Bombus pascuorum (500 and 250 000 per larva), spores from Bombus lapidarius (3 000 spores per larva) and spores from Bombus hypnorum (500 000 per larvae) did not induce a nosema infection in Bombus terrestris. Comparable amounts of nosema spores from B. terrestris could induce a nosema infection in worker brood on B. terrestris in a one-time application. This indicates that a one time administration of nosema spores in sugar solution, from species other than B. terrestris directly to the larvae does not induce a nosema infection in B. terrestris workers, whereas comparable amounts of nosema spores from B. terrestris do induce a nosema infection in B. terrestris workers. The results suggest that although it is the same parasite species that infects several species of bumble bees, cross infectivity between host species may required prolonged exposure or different exposure systems. The difficulty in establishing cross infection may also reflect locally adapted parasite strains. Although further work is needed to finally resolve the issue of cross infectivity, the project results to date will be published in scientific media. The results from these experiments adds to our understanding of parasite adaption in the host complex, but are of no immediate benefit for the bumble bee breeding industry.

Before the Pollinator Parasites project, the only genetic marker available with which to study Nosema bombi was based on ribosomal RNA. The Pollinator Parasites project has demonstrated that the entire sequence of this gene from eleven microsporidian infections of eight species of bumble bee revealed subtle variation at this locus (Tay et al. 2005). This variation could not be partitioned among populations of N. bombi found in different host species, but indicated a single panmictic species exhibiting little genetic variability. Ribosomal RNA therefore lacks the resolution necessary to investigate the population genetic processes and phylogeographic patterns within this parasite species, though it does allow for species differentiation (Klee et al. 2006). In order to improve upon this dearth of genetic markers, a partial genomic library was created from N. bombi DNA to provide a number of new genomic regions, which could subsequently be tested for their usefulness as population genetic and phylogeographic markers. We generated a genomic library of N. bombi and sequenced 38 clones containing DNA from anonymous regions of its genome. PCR primers designed on only 14 of these clones could successfully amplify N. bombi infected hosts but not uninfected hosts. Three of these 14 loci revealed considerable intraspecific variability in DNA sequence that could allow some separation of the microsporidia based on host species. These are the first genetic markers of their kind for microsporidia providing sufficient resolution to differentiate among genetic variants that display host species specificity. European bumble bee (Bombus spp.) species has revealed the presence of only one microsporidian species, N. bombi as revealed by Pollinator Parasites project results (Tay, OÕMahony and Paxton 2005). Yet significant sequence polymorphism was revealed even within isolates from one host individual. To understand the source of the multiple DNA sequence variants of N. bombi rRNA found in a single host, we PCR amplified, cloned and sequenced from many clones the rRNA from two individual spores, isolated by laser microdissection from one host. At least two rRNA variants were found per spore, which were also found in a DNA extract of multiple spores from the same host. Two possible explanations for this degree of polymorphism are that single bees can be infected with multiple ÔstrainsÕ of N. bombi and that different copies of the rRNA gene co-exist in one N. bombi genome. The reults provide evidence from single spore analyses that an individual N. bombi spore harbours multiple rRNA copies that are not homologous in sequence. This is the first time that DNA sequences have been obtained from single binucleate microsporidia, and they demonstrate that concerted evolution has not homogenized the sequences of all rRNA copies within a single N. bombi spore. The results presented represent major progress in the understanding of Microsporidia molecular genetics in general and of N. bombi in particular. All major results are either published in scientific medie , submitted for publication in scientific media, or in manuscript to be published.

This result adds information on rRNA gene sequences beyond the ssu 16S rRNA gene by providing information for the gentire rRNA gen of Nosema bombi. Characterisation of microsporidian species and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. We characterised the entire rRNA gene of a microsporidium from 11 isolates rep-resenting eight different European bumblebee (Bombus) species. We demonstrate that the microsporidium Nosema bombi infected all hosts that originated from a wide geographic area. A total of 16 variable sites (all single nucleotid polymorphisms (SNPs)) were detected in the small subunit (SSU) rRNA gene and 42 (39 SNPs and 3 indels) in the large subunit (LSU) rRNA sequence. Direct sequencing of PCR-amplified DNA products of the internal transcribed spacer (ITS) region revealed identical sequences in all isolates. In contrast, ITS fragment length determined by PAGE and sequencing of cloned amplicons gave better resolution of sequences and revealed multiple SNPs across isolates and two fragment sizes in each isolate (six short and seven long amplicon variants). Genetic variants were not unique to individual host species. Moreover, two or more sequence variants were obtained from individual bumblebee hosts, suggesting the existence of multiple, variable copies of rRNA in the same microsporidium, and contrary to that expected for a class of multi-gene family under concerted evolution theory. Our data on within-genome rRNA variability call into question the usefulness of rRNA sequences to characterise intraspecific genetic variants in the Microsporidia and other groups of unicellular organisms. The results have been published in 2005 in Journal of Eukaryotic Microbiology 52, 505-513. The results will be important for future studies of Microsporidia genetics in general and population genetic studies of this important group of parasites in particular.

Ultrastructural morphology of terrestrial Microsporidia is often difficult to describe because fixations and sectioning for transmission electron microscopy does not always yield optimal results. To improve our descriptions of pathology, gross morphology and ultrastructure of Microsporidia, needed species descriptions in this group efforts will be made to improve the standard protocol used in the project: For transmission electron microscopy, infected tissue to be embedded for TEM will be fixed using 2.5 % glutaraldehyde (v/v) in 0.2M sodium cacodylate buffer, pH 7.4, for 12 - 48 h and kept refrigerated (+7 to 8oC) during prefixation. The specimens will be post-fixed for 2h in 2 % OsO4 (w/v) in 0.1 M S-colloidine buffer after washing in Na-cacodylate buffer. The tissue pieces are then dehydrated in a series of ethanol solutions of ascending concentration, followed by fixation/washing/soaking in a propylene oxide solution. After dehydration, the tissue pieces are then embedded in Epoxy resin (Agar 100) by routine procedures for TEM.

A model of N.bombi epidemiology has been developed using a classical susceptible-infected scheme. This approach is very powerful even though no explicit genetics is taken into account and individual traits neither vary in the host nor in the parasite population. The general structure of the model reflect parasite transmission events as well as host and parasite mortality rates. Groups of individuals within a colony are classified as healthy (susceptible) or infected. In addition, there are several distinct classes: larvae (L), pupae (P), workers (W), young queens (Q) and males (M). Furthermore, spores of the parasite (Nosema) are contained outside hosts in a reservoir inside the colony and a reservoir outside the colony (i.e. in the field). Colonies in a population are connected by horizontal transmission of the parasite and produce successive generations of hibernating queens. Transmission events are modeled in form of the mass action principle. At a certain time (year), an infection enters the population with an infected queen (that may have migrated from somewhere else: in the example below, this is in year 3). An infected queen has a certain chance to found a colony (for the initial queen it is assumed that colony is established so that the infection enters the population). Hence, in later years, all infected queens can give raise to a colony but not all of them will succeed as determined by a stochastic filter. Once the infected colony grows, the parasite will spread from the mother queen to her larvae and workers. In turn, infected workers will spread the parasite into the field reservoir, from where it can be picked up by a worker from another colony. In this way, the parasite spreads within and among colonies as the season progresses. At the end of the season, a certain number of the daughter queens that are produced in the population will be infected. They over-winter with the same probability as the non-infected ones (according to our previous unpublished results). Hence, the model describes, both, an epidemic within a typical colony and an epidemic among colonies over years.The model is still based on a number of estimated parameters because conclusive quantified data is lacking for parasite transmission within and between colonies. The ambition of the project was to integrate the mathematical model with the recommendations fror disease control in breeding facilities. As the model still needs further refinements this has not been possible. Nevertheless, the Pollinator Parasites project has laid the foundation for a mathematical model describing the relationships between the host and the parasite. This model will be published in scientific media and developed into a tool that will be further refind and exploited. Undoubtedly, a model that captures the host-parasite adaptions is of wide interest not only to bumble bee breeders, but to scientists working on pests and diseases in pollinating social insects. This model represents a major step forward for the implementation of epidemiological theory into host-parasite relations in social insects.

Characterisation of microsporidian species and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. We characterised the entire rRNA gene of a microsporidium from 11 isolates rep-resenting eight different European bumblebee (Bombus) species. We demonstrate that the microsporidium Nosema bombi infected allhosts that originated from a wide geographic area. A total of 16 variable sites (all single nucleotid polymorphisms (SNPs)) were detected in the small subunit (SSU) rRNA gene and 42 (39 SNPs and 3 indels) in the large subunit (LSU) rRNA sequence. Direct sequencing of PCR-amplified DNA products of the internal transcribed spacer (ITS) region revealed identical sequences in all isolates. In contrast, ITS fragment length determined by PAGE and sequencing of cloned amplicons gave better resolution of sequences and revealed multiple SNPs across isolates and two fragment sizes in each isolate (six short and seven long amplicon variants). Genetic variants were not unique to individual host species. Moreover, two or more sequence variants were obtained from individual bumblebee hosts, suggesting the existence of multiple, variable copies of rRNA in the same microsporidium, and contrary to that expected for a class of multi-gene family under concerted evolution theory. Our data on within-genome rRNA variability call into question the usefulness of rRNA sequences to characterise intraspecific genetic variants in the Microsporidia and other groups of unicellular organisms. The results have been published in Journal of Eukaryotic Microbiology 52(6), 505-513. The results will be important for future studies of Microsporidia genetics as well as for population genetics of this important group of parasites.

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 10 000 000 down to 10 spores diluted in 100 microliters of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 1 000 000 spores down to 1 spore per PCR). Though the N.bombi -speci W c 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 È120 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. The results have been published in scientific media. Development of sensitive and specific tools for Microsporidia detection allows for the further development of protocols that can be used in any laboratory equipped for routine PCR analysis. This should have interest for molecular biologists as well as for those interested in bumble bee pathology.

Bumble bees collected from 7 European countries across 2003, 2004 and 2005 were examined for the incidence of the microsporidium Nosema bombi. In 5358 mostly visually examined bumble bees, we detected an average infection rate of 9 % across years and countries. Of the 30 European bumble bee species examined, N. bombi was detected in 16 of them, for which 9 species represent new records. The incidence of infection varied both spatially (country to country) and temporally (2003 versus 2004 and 2005). Of the two most commonly collected taxa, Bombus pascuorum (N = 1566) and Bombus terrestris/lucorum (N = 1842), the infection rate differed significantly between host species (18.2% of B. terrestris/lucorum individuals infected, 3.4% of B. pascuorum individuals). Bombus pascuorum showed no differences in the rate of infection among castes and sexes (worker, queen, male) or across years. In contrast, B. terrestris/lucorum showed a clear, significantly higher incidence of infection in males compared to workers or queens. The incidence of microsporidia was much higher in the latter half of the bumble bee flight season (July to September), with 10.6 % prevalence across species, years and countries, than in the early part (end of March to June), with a prevalence of 2.9 % of individuals infected. The same annual trend was seen for each of the castes and sexes.

A number of experiments and observations have been undertaken by several partners of the Pollinator Parasites project that address, directly or tangentially, the issue of how N. bombi is transmitted between hosts. This information is central to a full understanding of the biology of the microsporidium and how to control it. The relevant data are summarised in bullet form, under two headings of the main routes by which parasites are transmitted, with additional details of scientist and how the data will be more widely disseminated. Evidence concerning vertical transmission (VT): - (P2, Sandra Mustafa) (Evidence for VT) Overwintered queens often have ovaries that are infected with microsporidia (MSc. Thesis, manuscript in preparation). - (P2, Julia Klee and P4, Oliver Otti) (Evidence for VT) Ovaries of infected queens commonly contain microsporidia, as visualised by in-situ hybridisation (no publication). - (P3, Sjef van der Steen) (Evidence for VT) In the laboratory, infected larvae develop into infected queens that can successfully overwinter and found a colony the next spring, whose workers are infected (publication in preparation). - (P3, Sjef van der Steen) (Evidence against VT) Infected adult workers do not have developed ovaries and do not lay male-destined eggs (no publication). Evidence concerning horizontal transmission (HT): - (P2, Ulrich Ernst) (Evidence for HT) Young workers and males can be infected by feeding them with spores (MSc. Thesis, manuscript in preparation). - (P3, Sjef van der Steen) (Evidence for HT) Mid-aged larvae develop an infection when fed spores (manuscript ). - (P4, Oliver Otti) (Evidence for HT) Very young larvae fed spores are usually, though not always, infected as adults. - (P3, Sjef van der Steen and P4 Oliver Otti, in separate experiments) (Evidence for HT) Colonies experimentally infected develop infection even of individuals that were adult when spores were added to the colony (P3 manuscript in preparation). - (P2, Julia Klee and P4, Oliver Otti, in separate experiments) (Evidence against HT) When colonies are allowed to forage upon flowers which were artificially contaminated with spores or from which infected bumble bees have been collecting nectar, they do not become infected. - (P3, Sjef van der Steen), Administration of spores from B. pascuorum, B. lapidarius and B. hypnorum to B. terrestris larvae did not result in an infection of B. terrestris (P3 publication in preparation). Summary: Nosema bombi is an enigmatic species in many regards, and especially with respect to its modes of transmission. Evidence we have assembled suggests that both routes of transmission, vertical and horizontal, are used by the organism. However, the relative contribution of each to infection at the level of the individual, colony and population requires further investigation. Many of the above bullet points will be written up in manuscript form and submitted for publication in peer reviewed journals within the first 6 months of 2006. Then we aim to review the transmission of N. bombi in a manuscript, combining all experiments, published or not, for submission to a peer-reviewed journal by the end of 2006.

The impact of Nosema bombi on colony founding and transmission was studied by inducing N. bombi infection via administration of N. bombi spores from Bombus terrestris adults to individual worker and queen larvae in open cells of B. terrestris colonies. Administration of 312 500 spores to queen larvae resulted in 35 infected queens out of 113 queens (31%). There was no effect from treatments on mating success and hibernation mortality. The founding rate of new colonies after hibernation of the control queens was 33% whereas the success of ÒNosemaÓ queens was 4% (2 of 48 queens). It is concluded that N. bombi infection has a negative impact on colony founding. It should be noted that several queens where the parasite could not be detected in the intestines, had parasite DNA in ther ovaries using molecular detection techniques developed in the project. This suggests transovarial parasite transmission, although this transmission route remains to be verified. Using laboratory infection experiments the pathology and fitness effects from this parasite was studied. If infection occurs at an early stage of colony development, virtually all individuals are infected and spores are found in different tissues. Although the classical fitness parameters were not influenced by the treatment, the functional fitness of males and young queens was reduced to zero from the infection. Further, the survival of workers from infected colonies and infected males is highly reduced. With such severe effects, Nosema bombi even seems to decrease its opportunities for transmission to the next host generation. The results demonstrate the negative impact from infection and suggest that the parasite need to infect colonies and/or queens that will go into hibernation late in the season. Otherwise the negative impact from infection will impair parasite transfer to the following season. The results will be published in scientific media and have been presented at scientific conferences. The results are also communicated to the bumble bee breeding industry through the project End User meeting and, the Technical Report from the End User meeting and the project website. The results produced are important for understanding the host-parasite adaption. Such knowledge that can be used to develop sound control strategies for the breeding industry.

Bumble bees host lots of parasites including one microsporidium: Nosema bombi Fantham and Porter, 1914. The Pollinator Parasites project has documented this species in a large number of different bumble bee hosts. Numerous publications have treated various aspects on the ecology of this microsporidium and the impact on the hosts, but few have dealt with the cytology and none has done so in depth. Nosema bombi infected bumble bees were collected in southern Sweden and Denmark from B. hortorum, B. hypnorum, B. lapidarius, B. lucorum, B. pascuorum, B. pratorum, B. ruderarius, B. subterraneus and B. terrestris. Specimens were either dissected directly, killed by decapitation or freezing, or stored frozen for some time. Light microscopy were conducted on unstaied and fixed preparations using standard techniques. Ultrastructure was studied using standard techniques for electron microscopy but including the improved fixation of spores developed in the Pollinator Parasites project. Nosema bombi was most regularly observed in tissues samples from the Malpighian vessels. Small foci of developing microsporidia were often found in all regions of the gut wall, from the ventriculus and backwards. It was not observed that gut cells completely filled with microsporidia were released into the gut lumen, like is the case of Nosema apis, but in the regions of the gut posteriorly to the Malpighian vessels microsporidian spores were regularly observed in the lumen. In heavily infected hosts practically all Malpigian vessels, in their entire length, were infected. When squashed entire spore-filled cells were released. Presporal stages were most commonly seen in epithelium of the Malpighian vessels. In cases of advanced infection also the walls of the tracheal system were invaded. In two specimens, one male of B. pascuorum and one worker of B. lapidarius, microsporidian spores were also found in the musculature. In several specimens of B. hypnorum, B. lucorum, B. pascuorum and B. terrestris infection was restricted to peripheral fat cells, which means that samples from the Malpighian vessels and other tissues were negative. The presporal development was of Nosema type. The cells measured up to 6.1 µm in sections, the greatest diameter of sectioned nuclei was 1.9 µm. The cells had a typical, about 7 nm thick, plasma membrane. Cytoplasm and nuclei were both granular, but the nuclei were slightly more dense. When the merozoites matured to sporonts vesicular areas appeared in the cytoplasm, interpreted as a primordial Golgi apparatus. The thick external layer of the sporont wall, the primordium of the exospore, was added over large areas simultaneously, not as the regularly spaced strands typical for the genus Nosema. The sporogony was disporoblastic with the two elongated sporoblasts arranged like a short chain. In the sporoblasts the polar filament was initiated and extended forwards from a system of Golgi vesicles at the posterior pole. The earliest signs of a polaroplast became visible late in the maturation process, but prior to the first signs of the endospore layer. The diplokaryon, with distinct nucleoli in the nuclei, was located to the anterior half of the immature spore until it was pushed backwards by the developing polaroplast. The recorded length of living spores ranged from 2.73 to 6.63µm long. In one specimen of B. terrestris where two distinct classes of morphs were seen, the oval spores measured 1.95-2.60 x 2.77-5.85µm, the elongated spores 1.69-2.47 x 4.42-6.24µm. Spores were either completely uncoupled, or, when dealing with the more elongated morph, appeared pair-wise or arranged to strands of spores. The spores further differed in the willingness to eject the polar filament. The mature spores exhibited cytology of the most common microsporidian type. With exception of the spores found in one specimen of B. pratorum, the lowest number of polarfilament coils observed was 12 (spore length 3.1 µm), the greatest 24 coils (spore length 3.98 µm). Two intervals of coils were dominant, 13-15 and 18-20, the greatest number in the elongated spore morph. The coils were normally arranged as a single layer of coils, but with more than 15 coils, the last 2-4 coils were normally displaced to form an internal layer (Fig. 6 A, inset). Occasionally two almost complete layers of coils were observed (20-21 coils). The filament was isofilar, measuring about 100 nm wide (96-105 nm). The results presented complete the previous incomplete descriptions of N. bombi and document an unusual morphological variability. This documentation is useful to avoid future confusions on Microsporidia infections in bumblebees.

Introduction of a nosema infection into bumble bee colonies and into rearing facilities can be accomplished primarily in four ways, - By introducing queens caught in the wild for breeding purposes - By returning colonies from pollination assignments to the rearing unit - By feeding colonies material (primarily pollen) contaminated by N. bombi spores and by working hygienically. A nosema infection has a negative impact on colony development and therefore on the pollination performance of infected colonies. Nosema infection also has a negative impact on the continued rearing of new queens if the rearing colonies are infected. No cure is known for nosema disease in infected colonies. Thus, the only solution to deal with infection if found within rearing facilities, is to destroy all infected colonies, to avoid new introductions of the parasite and to maintain high hygienic standards. Nosema diagnosis on a regular basis is advisable. Simultaneously, other diseases and parasites can be checked for within colonies inspected for N. bombi. It is recommended to use random sampling of the rearing material every 4 week and diagnose for diseases and parasites. This check provides information of the health situation of the bees and developing infections or parasitation are detected before a general outbreak may occur. As discussed elsewhere, both microscopical and molecular techniques are available for dection of N. bombi. The PCR method developed in the Pollinator Parasites project, to detect N. bombi infections in Bombus spp., by amplifying partial rRNA of N. bombi, provides considerable advantages over microscopic detection techniques, especially through an increased level of detection sensitivity. This is because there is a risk of overlooking a low concentration of spores by light microscopy. Moreover, molecular detection reduces the risk of misidentification of N. bombi with other parasites and with starch grains, and gives the ability to detect all developmental stages of the microsporidium. The PCR based detection method will be useful in providing a quick and reliable identification of N. bombi infections as well as representing a sensitive tool for epidemiological studies of this little known microsporidium. Detailed protocols for using the tool-kit for N. bombi detection are available in the Technical report from Pollinator Parasites Professional meeting of bumble bee breeders, Wageningen, The Netherlands, December 2, 2005 and on the project web site.

Characterisation of microsporidian species and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. We characterised the entire rRNA gene of a microsporidium from 11 isolates rep-resenting eight different European bumblebee (Bombus) species. We demonstrate that the microsporidium Nosema bombi infected all hosts that originated from a wide geographic area. A total of 16 variable sites (all single nucleotid polymorphisms (SNPs)) was detected in the small subunit (SSU) rRNA gene and 42 (39 SNPs and 3 indels) in the large subunit (LSU) rRNA sequence. Direct sequencing of PCR-amplified DNA products of the internal transcribed spacer (ITS) region revealed identical sequences in all isolates. In contrast, ITS fragment length determined by PAGE and sequencing of cloned amplicons gave better resolution of sequences and revealed multiple SNPs across isolates and two fragment sizes in each isolate (six short and seven long amplicon variants). Genetic variants were not unique to individual host species. Moreover, two or more sequence variants were obtained from individual bumblebee hosts, suggesting the existence of multiple, variable copies of rRNA in the same microsporidium, and contrary to that expected for a class of multi-gene family under concerted evolution theory. Our data on within-genome rRNA variability call into question the usefulness of rRNA sequences to characterise intraspecific genetic variants in the Microsporidia and other groups of unicellular organisms.

To develop molecular genetic markers so as to differentiate between strains of the bumble bee infecting microsporidian Nosema bombi, we generated three partial genomic libraries for the microsporidia N. bombi and Nosema apis using both a non-enriched library and two libraries enriched for microsatellite repeat motifs. Our initial attempts to generate a partial genomic library of N. bombi (non-enriched library) were largely unsuccessful because extracted DNA was contaminated with host DNA. This is an unfortunate consequence of the fact that microsporidia, including N. bombi, are obligate intracellular parasites that are therefore closely associated with host tissue. All attempts to clone microsporidia DNA from host tissue will be similarly compromised. Our subsequent attempts to rid microsporidian spore extracts from host DNA using a DNAse treatment seem to have been largely successful. This method can be recommended in future attempts to clone microsporidian DNA. Our attempts subsequently to enrich a partial genomic library of N. bombi and N. apis seem to have been successful in that we have identified DNA sequences containing repeat motifs that show little or no similarity to host or other contaminant DNA. We were able to generate one microsatellite locus for N. bombi. There was a low number of copies of the microsatellite repeat motif at this locus. Such loci with low repeat copy number usually do not display variation. Indeed, as anticipated, the locus proved to be monomorphic in the tested samples. The density of repeat motifs in the enriched libraries for both N. bombi and N. apis was very low, possibly due to the highly reduced state of microsporidian genomes. The density of microsatellite containing clones generated from the enriched libraries is nevertheless extremely low. To place the results in context, other genomic libraries have been developed concurrently in the same laboratory by colleagues using the same techniques. When using orchid bee and colletid bee DNA as templates for cloning, ca 30% of clones from an enriched library were positive and contained repeat motifs. The low number of microsatellite motifs in microsporidian DNA may be a consequence of their highly reduced genomes. Indeed, we have been unable to detect any microsatellite repeat motifs in the microsporidian genomes: Encephalitozoon cuniculi and Antonospora locustae that are deposited in GenBank, suggesting that they are at a very reduced density in microsporidian genomes. Thus, the common practice to use polymorphic microsatellite repeat motifs for population getentic studies, may not be suitable for this group of organisms. The results are being prepared for scientific publication and should be of interest to all pathologists working on Microsporidia molecular biology, epidemiology and population genetics.

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