A contribution to the knowledge of infections in bumble bees, Bombus spp Paul Schmid-Hempel, Roland Loosli

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Paul Schmid-Hempel, Roland Loosli. A contribution to the knowledge of Nosema infections in bumble bees, Bombus spp. Apidologie, Springer Verlag, 1998, 29 (6), pp.525-535. ￿hal-00891554￿

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A contribution to the knowledge of Nosema infections in bumble bees, Bombus spp.

Paul Schmid-Hempel Roland Loosli

ETH Zürich, Experimental Ecology, ETH-Zentrum, NW, CH-8092 Zürich, Switzerland

(Received 1 December 1997; accepted 22 June 1998)

Abstract - Experimental infections of adult and larval workers and adult males of with the microsporidium Nosema bombi showed all stages and both sexes to be susceptible. On average 19-29 % of infections were successful and no significant differences among these host cat- egories were found. Different sources of Nosema spores differed in their success in infecting differ- ent host colonies, suggesting genotype-genotype interactions at the level of colonies and parasite sources. In a second experiment, N. bombi obtained from B. terrestris were found to be infective for workers of B. lapidarius and B. hypnorum, although less so in these foreign hosts. On the other hand, case mortality was significantly higher in foreign hosts than in B. terrestris. Infection and high spore loads correlated with early death of the host. In addition, a factorial analysis showed that variation among-colonies-within-species explained more of the variation in infection success than the factor species per se. © Inra/DIB/AGIB/Elsevier, Paris parasite / brood / host specificity / Bombus / Nosema bombi

1. INTRODUCTION have been reported from all major groups, i.e. the termites, wasps, ants and bees [33]. In particular, Nosema apis Z. are an of Microsporidia important group the honey bee, Apis mellifera L., of insects, infecting protozoan parasites, especially has been studied in some detail because of where often infect cells or they epithelial the economic importance of its host [3]. the fat body [6, 40]. More recently, infec- tions by microsporidia in humans have Bumble bees, Bombus spp., are eusocial become more important, owing to the bees with an annual life cycle and are of increase in the number of patients with increasing economic interest as pollinators immunodeficiency [7, 8]. In social insects, of crop. In addition, the importance of their

* Correspondence and reprints E-mail: psh @ eco.umnw.ethz.ch diseases, for example caused by Nosema 1 275 non-infected ones was found by Mac- bombi Fantham & Porter, has been recog- Farlane et al. [26]. In fact, not all colonies nized since early in this century [16]. In fact, and individuals are affected in the same way Nosema has been reported for a number of and the effects are not universally detri- Bombus species [26, 33]. So far, all infec- mental. Several authors have found few or tions in bumble bees have been attributed no externally visible effects of experimental to N. bombi Fantham & Porter, although it is infections [17, 27] or even found increased unclear whether all infections are in fact by production of sexuals in naturally infected the same parasite. Typically, identification colonies [22]. Similarly, overwintered is by light microscopy and inspection of queens that have become infected in their spores, a procedure that is not always reli- maternal colony often manage to found a able [25]. Some authors have even consid- colony, to raise brood and to complete an ered N. bombi to be the same species as apparently normal life cycle (pers. obs.). N. apis [38, 40], and suggested that these Given the biology of N. bombi, infection two are cross-infective for the two parasites of hibernating queens should be the most hosts But later and more recent [15, 23, 35]. or even route of vertical work has demonstrated that these important only clearly transmission between the annual genera- two parasites are different and that reports of tions. Nosema spores are susceptible to UV- successful cross-infections are ambiguous light and desiccation [5], conditions likely to we the [14, 27, 41]. Here, follow currently be encountered outside the host. of nomenclature of N. bombi Spores accepted using N. apis can persist for 1-2 years or more in for all of our sources. honey stores of honey bees [29], but such In the field, prevalences of infection a long-lasting reservoir is not available in bumble bees. Transovarial trans- range widely. For example, up to 55 % of (vertical) workers and 8-50 % of males in European mission is known to occur in other Nosema but so far has not been populations of various species were found to species [28] reported be infected by N. bombi [13, 36]. According from N. bombi and seems unlikely from detailed of struc- to Betts [4], all hibernated spring queens of investigations reproductive tures Within the N. bombi B. lapidarius collected in England were [27]. colony, found to be infected. Severe infestations of spreads by ingestion of spores shed by infected workers colonies autumn queens of B. terrestris collected in [14]. However, also contract novel infections from Denmark were found by Skou et al. [37]. outside, In New Zealand, where bumble bees have as previously uninfected colonies in the field been imported in the last century, 76 % of all may suddenly contain infected workers [22]. such horizontal transmission nests (n = 76) and 10 % of queens (n = 275) Perhaps, occurs of B. terrestris were found to be infected between colonies via spores [26]. deposited on flowers as has been shown for the trypanosome C. bombi in the same hosts The effects of N. bombi on its host are [12], or by increased drifting rates of quite variable. According to our own obser- infected workers as is known for honey bees vations, infected workers often become slug- infected by the Varroa mite [32]. But apart gish, die early and the affected colonies may from a few studies on the phenology of the eventually perish. MacFarlane et al. [26] parasite within its host [27,41], not much is suggest that Nosema infection paralyses the known of the general biology and ecology of abdomen of the queen and completely pre- N. bombi, despite the potential importance of vents mating. A similar observation was microsporidia in general and of this para- also made by DeJonghe [11]. On the other site in an economically interesting host in hand, no difference in body mass of 12 particular. Here, we report the results of two severely infected queens as compared to infection experiments with N. bombi and three species of bumble bees. The aim of pension of 12 000 spores·μL-1. The same vol- our study is to broaden our knowledge of ume of sugar water (Apinvert®) was then added. a standard inoculum of 10 60 000 an important microsporidian parasite and, Finally, μL (i.e. spores; similar to ref. [27]) was prepared. We at the same time, to elucidate its ecology by considered these two solutions as representing whether this can cross- investigating parasite different sources of Nosema, probably containing infect to other Bombus species and to what a different set of ’strains’. This assumption was extent the effect of infection varies among later justified by the results. Workers from three colonies within and between host species. non-infected colonies of B. terrestris (K162, K166, K226), raised from the same stock of queens, were chosen at random to receive the inocula. Each was offered the standard of 10 2. MATERIALS AND METHODS μL after starvation for 3 h; in each case, the entire dose was quickly imbibed. For the infection of of N. bombi were identified Spores by light larvae, a similarly sized piece of brood was cut microscopy [27]. Results from pilot experiments out from the same colonies and the number of based on 95 workers and males suggested an larvae in it estimated. With a standard of overall modest infection success when spore sus- 10 μL·larva-1, a dose for the entire piece of brood are fed to workers. pensions Furthermore, spores was calculated. This amount of spore suspen- often in the host’s faeces 2-3 appeared only sion was then applied with a fine brush to the weeks Guided these post-infection. by previous entire clump. All larvae were 1-5 days old when observations, we evaluated infection success infected. We also added three non-infected nurse (’infected’ versus ’not infected’) 21 days post- workers to the brood clump for the daily routine infection. In the second experiment, we also of brood care. These were removed as soon as scored spore loads in the freshly dead workers the larvae pupated and before the first worker if they had died before these 21 days. We used an hatched. To evaluate infection success, we arbitrary scale for infection intensity, i.e. spore checked the workers (that had hatched from these concentration in the as follows: 0 = no host, larvae) on day 21 post-emergence. spores found (not infected), 1 = low infection (up to 100 spores·μL-1), 2 = moderate infection (up to 1 000·μL-1), 3 = heavy infection (up to 2.2. Experiment 2: 10 000·μL-1), 4 = very heavy infection (more than 10 000·μL-1); grades 1-3 were considered to infection of different hosts species be infected. To measure spore concentration, the abdomen of the test worker was homogenized In this experiment, the abdomen of one in 0.5 mL water. were then taken Subsamples worker of an infected colony of B. terrestris, lab- and counted in a haemocytometer (Neubauer oratory-raised from a queen caught around chamber) under the microscope. All experimen- Zürich, Switzerland (no. 198), in spring 1996, infected workers were in tally kept individually was homogenized in 0.5 mL of water and spores small wooden boxes with sugar water (diluted standardized to 12 000·μL-1. An equal volume ad libitum and under controlled con- Apinvert®) of sugar water (Apinvert®) was then added. All % ditions (28 °C, 60-80 R.H.). If not indicated bees were infected with the standard inoculum of values are as means and S.E. otherwise, given 10 μL (i.e. 60 000 spores) gained from this source All analyses were performed with SPSS6.1 and and offered after 3 h of starvation. Workers of two-tailed probabilities are reported. healthy colonies of B. terrestris, B. lapidarius L. and B. hypnorum L., laboratory-raised from queens caught in the same area around Zürich 2.1. 1: infection with two Experiment at the same time, served as experimental hosts. sources (A, B) of spores All workers were infected at age 1-4 days. Whether or not an infection had occurred, Spores of Nosema were prepared as follows. together with the assessment of infection inten- One worker each was taken from two infected sity (grades 0-4), was evaluated either 21 days colonies of B. terrestris L. (labelled as sources A, post-infection or in freshly dead workers for B). These colonies were laboratory-raised from those that had died earlier. In addition, the num- queens caught around Zürich, Switzerland, in ber of surviving or dead animals up to 21 days spring 1995. The abdomen was homogenized in was used to calculate case mortality rates (per- water, spore load counted and diluted to a sus- centage dead before 21 days). 3. RESULTS were used (difference between sources A versus B, U-test: z = 2.66, P = 0.008; dif- 3.1. Experiment 1: ference among colonies: Kruskal-Wallis H infection with two sources (A, B) = 10.79, df = 2, P = 0.005). For two of the of spores colonies (K162, K166) we had enough data to test for a host versus parasite interaction A total of 87 individual hosts could be effect with a factorial analysis. Both the analysed (table I). Only 25 (28.7 %) of those analysis with infection intensities (0-4) and infection success showed a actually had spores of Nosema 21 days post- (yes/no) clearly infection. Thus, the average infection suc- visible, but only marginally significant, cess was rather low. Infection success interaction effect (figure 1). (’infected’ versus ’not infected’) in worker larvae as compared to adult workers showed no difference (χ2 = 0.036, P = 0.84). 3.2. Experiment 2: Although male larvae were more frequently infection of different hosts species infected (38.9 %, N = 18) than worker larvae (27.0 %, N = 37), the difference was also In this second experiment, spores of not significant = 0.798, P = 0.37). Many (χ2 N. bombi were extracted from the host infected larvae died or during pupation B. terrestris. A total of 327 workers from afterwards and showed defor- shortly strong 28 colonies of three mations. species (B. terrestris, B. lapidarius, B. hypnorum) could be inoc- On the other hand, there was a clear dif- ulated with this source, of which 305 could ference between colonies (colony K 162: be reliably scored for infection. A total of 40.5 % infected, N = 37; K166: 34.6 %, 60 workers ( 19.7 %) contained spores N = 26; K226: 4.2 %, N = 24) (χ2 = 10.03, 21 days post-infection or at the time of their P = 0.007) in infection success of workers, death if before this census day, while 245 irrespective of adult or larval stage. Simi- remained free of infection (table II). When larly, a strong effect for source of infection the data were analysed per individual worker was found (average success with source A: irrespective of colony, a clear difference 17.6 % of workers, N = 51; with source B: among species was found for both, the suc- 44.4 %, N = 36) (χ2 = 7.40, P = 0.007). The cess of infection (infected/non-infected) same pattern emerged when, instead of using (c2 = 8.89, df = 2, P = 0.012) and the inten- the dichotomous infected/non-infected clas- sity of infection per worker (scale 0-4: sification, infection intensities (scale 0-4) H = 8.59, df = 2, P = 0.014). A species dif- ference was also found for the average mor- nificantly higher (37.1 ± 5.4 %, N = 16 tality rate per colony (table II; figure 2). colonies) than in colonies of the original However, the species differences in infection host (B. terrestris: 16.4 ± 7.1 %, N = 12) success and intensity disappeared when the (U-test: z = 2.727, P = 0.006, N = 28 average colony values instead of individual colonies). In contrast, the average preva- values were considered (figure 2). lence and infection intensity seemed higher on the original host, although not signifi- Across all colonies and of irrespective cantly so (figure 2; P > 0.4 in both cases). species, the average infection intensity per worker was highly correlated with the preva- The average infection intensity in bees lence of infection (percentage infected work- that were infected at all and that had died ers) in its colony (Spearman’s rS = 0.99, before the census day 21 (mean = 1.344 ± P < 0.0001, N = 28 colonies). Similarly, 0.12, N = 32) was higher than in those that prevalence (arcsin-transformed: r = 0.399, had survived to this day (mean = 1.107 ± P = 0.036) and infection intensity (sqrt-trans- 0.08, N = 28) although this difference was formed: r = 0.424, P = 0.024) were posi- not significant (U-test: z = 1.588, N = 60, tively correlated with case mortality rate in P = 0.112). On the other hand, we found the colony. Interestingly, the average case that the survival of infected animals (sur- mortality rate in colonies of the two foreign vival 46.7 %, N = 60 bees) was significantly hosts (B. lapidarius, B. hypnorum) was sig- lower than that of non-infected animals, i.e. where the inoculation did not lead to infec- 4. DISCUSSION tion (survival 77.5 %, N = 240) (χ2 = 22.32, P < 0.001). Both findings suggest that heavy The results of this study demonstrate that Nosema infections lead to premature host different sources of Nosema bombi vary death. considerably in their potential to infect a given colony (figure 1). This makes a geno- Figure 2 shows that no species differ- type-genotype interaction between parasite ences for infection success and intensity ’strain’ and host colony very likely. On the exist when the average values per colony other hand, the results of cross-species infec- N. bombi from B. terrestris to are used instead of average values per tions with a certain worker. In fact, by looking at the data (table II), different species showed that since work- it is evident that variation at the level of the degree of host specificity exists, ers of B. lapidarius or B. hypnorum were colony is more pronounced than variation less likely to become infected than those of at the species level. Consequently, we also the original host B. terrestris (figure 2, table II). infection intensities with a hier- compared Remarkably, the infection caused higher archical factorial (colony nested analysis case mortality in the foreign than in the orig- within a total of 28 colonies in three species; inal host, while infection success showed When species; table III). colony identity the opposite tendency (figure 2). This sug- was explicitly taken into account in this way, gests that populations of N. bombi in dif- a highly significant effect was found for ferent host species may be differentiated variation among colonies while the species from each other but are probably not dif- effect disappeared (table III). ferent parasite species altogether. The result also supports an earlier observation of Both bee races can otherwise be hybridized DeJonghe [11] that the subspecies B. ter- with each other. Hybrids also show higher restris xanthopus from Sardinia, Italy is susceptibility. more susceptible to a given Nosema infec- However, a strong result is that the intrin- tion than B. t. terrestris from Central Europe. sic variation among colonies can over- shadow differences among species. This is dependent [18]. In careful investigations of suggested by the results of table III, as well infected tissue, Mclvor and Malone [27] as by the fact that colony-wide averages for found that mature spores of N. bombi - infection intensity or prevalence did not dif- which are typically recognized in the fae- fer sufficiently to generate significant ces - appear 5 days (120 h) post-infection in species effects (figure 2). To be sure, our its host B. terrestris at the earliest. These results do suggest that Nosema from B. ter- time spans must be compared to the aver- restris does not perform as well on other age life span of bumble bee workers which species (figure 2). However, strong colony is normally around 20-30 days [20], or to a effects have come to light in both experi- mean weekly mortality rate in the order of ments. In fact, similar differences in the sus- 20-35 % [34]. Assuming a maximum pre- ceptibility of different honey bee colonies patent phase of approximately 20 days and to infection N. have been in by apis reported immediate infection after emergence from a number of for in studies, example Furgala the pupa, the chances of the host to reach also Such versus [19] (see [3, 30]). colony this age are around 50 % and half of the strain effects are for parasite important infections would be lost before they can be understanding the ecological and evolu- transmitted further. Hence, the temporal of interac- tionary dynamics host-parasite dynamics of N. bombi infections warrants tions. In the current case, it suggests that further studies. colony-level variation may be important in determining the dynamics of Nosema infec- Similarly, the overall level of infection tions in field populations and perhaps entire success (28.7 % in the first experiment; 19.7 % communities of co-existing Bombus species. in the second), despite forced inoculations, is low. Of course, temperature In the honey bee, N. apis seems unable to remarkably and condition of the host are to affect infect the larvae, as freshly emerged bees likely the of Nosema, as is known for N. are normally free of the disease [1, 3, 21]. cycle apis in bees. In bees, Spores detected in other organs, e.g. fat body honey honey sporonts pro- duced in summer differ from those in win- and hypopharyngeal glands, are probably ter bees concluded not N. apis [2]. Nevertheless, other species [39, 40, 42]. Weiser [40] of microsporidia in honey bees have been that diapause in the host also arrests Recent studies found to kill the pupae by infection [9]. We microsporidian development. now find that N. bombi is able to infect both also found that the observation of emptied brood and adults of bumble bees, contrary to spores of N. apis is similar to observations of the study of Eijnde and Van den Vette [14] dimorphic spores in other Nosema spp., who only found larvae to be susceptible. although it is doubtful whether true dimor- However, these authors checked their adults phism occurs [10, 18, 33]. Perhaps, some 10-12 days post-infection. This may some- spores of N. apis germinate inside the host times be too early, since we found that new cytoplasm, whereas others spores remain spores are often shed by an infected host ungerminated and spread to other hosts or only after a considerable length of time (up reinfect the gut epithelium (I. Fries, pers. to 2-3 weeks). Unfortunately, the data did comm.). Such characteristics could be rele- not allow a firm statistical inference for this vant for microsporidia in hosts with annual issue. Similarly long periods have also been life cycles as in the bumble bees. More stud- reported by L’Arrivée [24] for N. apis in ies are clearly indicated to further elucidate honey bees, but other studies report a period the intricate relationship of these of around 5 days [3] or even shorter (2 days: microsporidia with their hosts. According Kellner 1980, cited in Fries [18], who found to our results, such studies should be highly 3 days). These differences appear to be dose rewarding. ACKNOWLEDGEMENTS En outre l’intensité de l’infection (nombre de spores) était plus élevée chez les ouvrières We thank Regula Schmid-Hempel for help mortes jeunes, alors que la probabilité de in raising colonies, collecting parasites and com- survie des insectes infectés était inférieure à ments. Helpful comments were also provided by celle des insectes non infectés. Une attaque Fries and two Ingemar anonymous reviewers. intense N. bombi conduit donc à la mort the Swiss National Sci- par Financially supported by de l’hôte. factorielle ence Foundation (no. 3100-049040.95 to PSH) prématurée L’analyse de la résussite de l’infection fait ressortir and a grant towards Training and Mobility of Researchers (TMR, provided by the Federal que la variation entre colonies au sein d’une Office of Science and Education, BBW no. même espèce hôte est plus importante que 95.0575). les composantes de la variation entre les espèces hôtes. Des différences entre les colonies au sein d’une même espèce hôte peuvent donc cacher les différences entre Résumé - Contribution à la connaissance espèces hôtes. © Inra/DIB/AGIB/Elsevier, des infections dues à Nosema chez les Paris bourdons, Bombus spp. Le but de ce travail est d’étudier quels stades de l’hôte peuvent Bombus / Nosema bombi / parasite / être parasités par la microsporidie Nosema spécificité hôte / couvain bombi et s’il existe des résistances entre les colonies et entre les diverses espèces de bourdons. Le parasite a été prélevé sur des ouvrières de Bombus terrestris et a servi à Zusammenfassung - Ein Beitrag zur infecter expérimentalement des larves, des Kenntnis der Nosema-Infektionen bei ouvrières et des mâles de B. terrestris. Les Hummeln. Das Ziel dieser Studie war, zu deux stades et les deux sexes ont pu être untersuchen, welche Stadien des Wirts von infectés avec succès. En moyenne 19 à 29 % Nosema bombi befallen werden können und des infections ont réussi. Aucune différence ob es Unterschiede in der Resistenz zwi- n’a été mise en évidence entre les diverses schen Kolonien und verschiedenen Arten catégories d’hôtes. Dans une première expé- von Bombus gibt.Das Mikrosporidium rience on a utilisé deux sources de spores Nosema bombi wurde aus Arbeiterinnen von de N. bombi (provenant de deux colonies B. terrestris gewonnen und experimentell de B. terrestris). Là, on a observé des dif- Larven und adulte Arbeiterinnen, sowie férences dans la réussite de l’infection selon adulte Männchen von B. terrestris infiziert. la colonie hôte (n = 3 ; de 4,2 à 40,5 %) et Beide Stadien und Geschlechter konnten selon la source de spores (de 17,6 à 44,4 %) infiziert werden. Durchschnittlich waren (figure 1). En outre il existe une interaction 19-29 % der Infektionen erfolgreich. Es génotype-génotype entre l’origine du para- wurde kein Unterschied zwischen diesen site et la colonie hôte. Dans une seconde Wirtskategorien festgestellt. Im Versuch expérience on a pu transmettre N. bombi wurden zwei verschiedene Quellen von provenant de l’hôte B. terrestris à des Nosema-Sporen verwendet (ursprünglich ouvrières de 28 colonies de B. Lapidarius, isoliert aus zwei verschiedenen Kolonien B. hypnorum et B. terrestris. Le succès et von B. terrestris). Dabei zeigten sich Unter- l’intensité de l’infection sur les hôtes étran- schiede im Infektionserfolg von 4.2 % bis gers a été légèrement plus faible que sur 40.5 % zwischen drei getesteten Wirtsko- l’hôte propre (figure 2 ; tableau II). En lonien, und ebenfalls zwischen den beiden revanche, la mortalité finale chez les hôtes verwendeten Sporen-Quellen (17.6 %, 44.4 %; étrangers a été plus élevée que chez B. ter- Abb. 1). Ausserdem wurde eine Interaktion restris (37,1 % contre 16,4 % ; figure 2). deutlich, was auf Genotyp-Genotyp Inter- aktion in bezug auf Parasiten-Herkunft und [7] Canning E.U., Hollister W., Human infections Wirtskolonie schliessen lässt. In einem zwei- with microsporidia, Rev. Med. Microbiol. 3 (1992) 35-42. ten konnte N. bombi aus der Experiment [8] Canning E.U., Hollister W.S., The importance of Wirtsart B. terrestris auf die Arbeiterinnen microsporidia as opportunistic infections in aus 28 Kolonien von B. lapidarius, B. hyp- patients with acquired immune deficience syn- sowie auf B. terrestris selber über- drome, Eur. J. Gastroent. Hepatol. 4 (1992) norum , 422-427. werden. Der und die Inten- tragen Erfolg [9] Clark T.B., A second microsporidian in the sität der Infektion von N. bombi in den honey bee, J. Invertebr. Pathol. 35 (1980) fremden Wirtsarten war leicht geringer als 290-294. auf der eigenen Wirtsart (Abb. 2, Tabelle I). [10] De Graaf D.C., Raes H., Jacobs F.J., Spore in Nosema apis Andererseits war die Fallmortalität in den dimorphism (Microsporida, ) developmental cycle, J. Inver- fremden Wirtsarten höher als in B. terre- tebr. Pathol. 63 (1994) 92-94. stris (37.1 % gegenüber 16.4 %; Abb. 2). [11] DeJonghe R., Crossing experiments with Bom- Ausserdem war die Infektionsintensität bus terrestris terrestris (Linnaeus, 1758) and bei früh Arbeite- Bombus terrestris xanthopus (Kriechbaumer, (Sporenzahl) gestorbenen 1870) and some notes on diapause and nose- rinnen höher, während die Überlebens- mose (Nosema) (Hymenoptera, Apoidea), wahrscheinlichkeit infizierter Tiere kleiner Phegea 14 (1986) 19-23. war als diejenige nicht infizierter Tiere. [12] Durrer S., Schmid-Hempel P., Shared use of Intensiver Befall durch Nosema führt daher flowers leads to horizontal pathogen transmis- sion, Proc. R. Soc. Lond. B 258 (1994) 299-302. zum Tod des Wirts. Eine fak- vorzeitigen [13] Durrer S., Schmid-Hempel P., Parasites and the torielle Analyse des Infektionserfolgs ergab regional distribution of bumble bee species. ausserdem, dass die Variation zwischen Ecography 18 (1995) 114-122. 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