LETTER Bumblebee workers from different sire groups vary in susceptibility to parasite infection

Transcription

1 Ecology Letters, (2003) 6: LETTER Bumblebee workers from different sire groups vary in susceptibility to parasite infection Boris Baer* and Paul Schmid- Hempel ETH Zurich, Ecology & Evolution, ETH-Zentrum NW, 8092 Zurich, Switzerland *Correspondence and present address: Boris Baer, Zoological Institute, Department of Population Ecology, Universitetsparken 15, 2100 Copenhagen, Denmark Abstract Female multiple mating with different males (polyandry) can be advantageous because the resulting genetic heterogeneity among offspring reduces the effects of parasitism. However, the underlying assumption that offspring fathered by different males vary in their susceptibility to parasites is so far only supported indirectly. Here we tested this crucial assumption using data from a study on the bumblebee Bombus terrestris L. with queens inseminated with sperm of either one or several males that originated from different sire groups (i.e. groups of brothers). We found that, under field conditions, workers from different sire groups, forming a patriline within a given colony, indeed differ in their susceptibility to the common intestinal parasite, Crithidia bombi, and do so independently of queen mating frequency. Keywords Multiple mating, parasitism, patriline, sire group, susceptibility. Ecology Letters (2003) 6: INTRODUCTION Females might prefer to mate with males carrying efficient resistance genes but this requires that females are able to assess a male s quality (Hamilton & Zuk 1982; Sherman et al. 1988; Baer & Schmid-Hempel 1999; Jennions & Petrie 2000; Kokko 2001). Social insect females are thought to lack this ability due to the short mating episodes in anonymous encounters, the presumed absence of appropriate cues, and, as is likely in the case of bumblebees, the poor predictability of resistant male genotypes given a parasite-driven antagonistic coevolution between host and parasite genotypes (Peters & Lively 1999; Schmid Hempel 2001; Strassmann 2001). A number of experimental studies in social insects, and especially in the bumblebee Bombus terrestris, have shown that under these circumstances multiple mating with several males can reduce parasitism and increase colony performance (Liersch & Schmid-Hempel 1998; Baer & Schmid- Hempel 1999; 2001; Cole & Wiernasz 1999; Tarpy in press). In other studies, however, such a beneficial effect has not been found (Page et al. 1995; Kraus & Page 1998; Neumann & Moritz 2000). In addition, queens of B. terrestris are mostly singly mated in Central Europe (Schmid-Hempel & Schmid- Hempel 2000) leading to the question as to why these queens are unable to take advantage of the demonstrated benefits of multiple mating. However, as recent work indicates, selfish male interests (males transfer a mating plug; Duvoisin et al. 1999; Sauter et al. 2001) and possible social costs of multiple mating (Baer & Schmid Hempel 2001) provide plausible explanations for this apparent discrepancy. Nevertheless, the polyandry vs. parasitism hypothesis remains highly controversial and competes with alternative explanations suggesting, for example, a beneficial effect of polyandry on genetic load at the sex locus (Page 1980), fertilization assurance (Fjerdingstad & Boomsma 1998), or increased colony efficiency through a more complex organization of division of labour (Crozier & Page 1985). The beneficial effect of the polyandry vs. parasitism hypothesis is supposed to result from increased genotypic diversity among worker offspring, perhaps leading to a reduction in parasite transmission or in the number of affected workers (Schmid-Hempel & Crozier 1999). An important requirement for this scenario (as well as for any such mating decision in animals other than social insects) is that an offspring s genotype, especially the paternally derived one, affects its susceptibility to parasitism. Here we have re-analysed and also added newly available data to an earlier experiment (Baer & Schmid-Hempel 2001) using the bumblebee B. terrestris and its internal parasite Crithidia bombi as a model system to test this assumption. In nature, bumblebees are known to be host of several parasites (Schmid-Hempel 1998). Among those, C. bombi is an abundant intestinal trypanosome parasite that negatively

2 Male lineages and parasite susceptibility 107 affects worker survival, ovary development and colony performance (Schmid-Hempel 1998; Brown et al. 2000). In addition, cross-infection experiments have demonstrated strong genotype genotype interactions at the level of entire colonies, between different lines of C. bombi and its host, B. terrestris (Schmid Hempel 2001; Schmid-Hempel et al. 1999). The interaction strongly affects average prevalence and intensity of infections among colonies, which suggests that the corresponding genotypic variance may indeed be relevant for eventual colony performance. As an aside, note that for the polyandry vs. parasitism hypothesis to work it is not necessary that genetic variation in susceptibility pertains to different parasite species. Rather, variation (as in the case of C. bombi) in the interaction with different strains of the same but crucial parasite is sufficient (Schmid-Hempel 1994; Ebert & Hamilton 1996). Bumblebees are annual social insects with colonies headed by a single queen. For the present experiment, we used 10 different and unrelated colonies as a source for groups of brothers (here, called sire groups ) whose sperm we used to artificially inseminate virgin queens either mimicking a single mating (sperm from one male only) or multiple mating (sperm from several males representing different sire groups). Insemination eliminated possible effects of mate choice. In singly mated colonies, all workers were derived from a single father but fathers belonging to a given sire group were replicated across colonies. In multiply mated colonies, workers derived from the same father form a patriline and thus several patrilines were present in each colony. Patrilines belonging to different sire groups occurred only once in each colony (except for inseminations with sperm from four brothers) but the respective sire groups were replicated across colonies. Hence, each sire group had representatives in the form of worker patrilines in colonies belonging to different treatment groups (i.e. singly and multiply mated colonies). We therefore tested whether workers that were sired by males of different sire groups varied in their susceptibility to C. bombi and whether this depended on the kind of colony (singly and multiply mated) they were reared in. MATERIAL AND METHODS The field experiment analysed here was performed with B. terrestris in 1999 and is also described elsewhere (Baer & Schmid-Hempel 2001). Here, in order to reduce unwanted variation that might be introduced by differences among female lineages and thus to increase experimental precision for the factor of interest (sire group), we used the largest subset of the experiment represented by females originating from the same mother colony (i.e. full sisters). Thus, a total of 43 colonies, all unrelated to the colonies used for sire groups, were available for the analysis. These could be classified according to four treatments: 16 colonies originated from queens inseminated with sperm from a single male, 11 from colonies where queens were inseminated with a mixture of sperm from two unrelated males, four from colonies with queens inseminated by four brother males and 12 colonies from colonies headed by a queen inseminated with sperm from four unrelated males. Paternity for each worker was identified with diagnostic microsatellite markers (Estoup et al. 1995) thus allowing assignment of sire group for each patriline in a given colony (Schmid-Hempel & Schmid-Hempel 2000). Artificial inseminations of queens were performed according to our standard protocol (Baer & Schmid-Hempel 2000). The design was balanced so that the presence of a given sire group was the same across treatments. However, due to uncontrollable variation in the biology of the system, the actual representation of different sire groups led to sparse cells in the design matrix (see statistical comments below). After the insemination procedure males were stored at )80 C and later genotyped to assign paternities among worker offspring with microsatellites (Estoup et al. 1995). The inseminated queens were allowed to start a colony in the laboratory. When colonies contained 10 workers, they were transferred to a field site, a typical bumblebee habitat close to Zurich where they remained under natural conditions throughout the whole colony cycle. After field placement, the colonies were checked once per week and a random subsample of c. 10% of the total workers present was removed and immediately freeze-killed in liquid nitrogen. Later these bees were dissected in the laboratory and prevalence (percentage of infected workers per group) as well as intensity (density of cells per microlitre in gut of infected bees) of C. bombi was determined using a haemocytometer (Neubauer chamber). Dissections were carried out with the observer blind to the identity and the origin of the inspected workers. A total of 860 workers were available for analysis. Data were analysed with SPSS 10 (Norusis, M. J. 1994, SPSS Statistics. SPSS Inc., Chicago, IL, USA) for Macintosh and all tests are reported with two-tailed probabilities. We used a univariate ANOVA with the following specifications. Crithidia bombi infection intensity or prevalence was used as the dependent variable (with prevalences either as arcsintransformed values or directly entered in a logistic regression). Intensity and prevalence typically increase with season and independently of other factors; therefore, we used the Julian date when a worker was collected as a covariate. Sire groups were introduced as a random factor in keeping with the standard usage as, for example, adopted for clones in comparable analyses (Via 1991; Jokela et al. 1997). Treatment as defined by insemination with different numbers of males was set as a fixed factor. We included an intercept to allow for an (unknown) intrinsic level of susceptibility in all

3 108 B. Baer and P. Schmid-Hempel individuals. Colony is not an explicit factor in this design but is part of the error term. Finally, the main results reported here are from a Type III sum-of-squares analysis, which has lower power for designs with sparse cells (as is the case here for some sire groups) but generates conservative estimates. For comparison, we also report the ANOVA results from a type IV analysis, which can handle sparse cells better but suffers from the problem of adding or dropping terms in a particular but not necessarily biologically justified sequence. In particular, random effects are estimated based on residuals after adjusting for fixed effects in an order for which there is no particular justification in the present study. RESULTS As reported elsewhere (Baer & Schmid-Hempel 2001), we found that colonies varying in genetic composition (as a result of different inseminations) also varied with respect to the intensity and prevalence of C. bombi. In particular, genetically heterogeneous colonies (i.e. those inseminated with sperm from several males) had lower parasite loads and generally higher fitness as expected under the hypothesis of adaptive polyandry. When we analysed the performance of different worker patrilines within the colonies, we found a significant effect of the sire group to which the father belonged on, Crithidia intensity as well as Crithidia prevalence (Fig. 1; Table 1). For both intensity and prevalence, this effect was independent of treatment, that is, whether or not the female had mated multiply (see nonsignificant interaction terms, Table 1). The significance of the results did not change when sire group number 8 (without repeated sampling, see Fig. 1) was removed from the analysis (for intensity: P ¼ and P ¼ 0.019, for Table 1 Effects on Crithidia bombi infections within experimental colonies for (a) intensity and (b) prevalence. See text for further explanations Source SS d.f. F P P IV a (a) Crithidia intensity Intercept <0.001 <0.001 Collecting date <0.001 <0.001 Treatment Sire group Treatment sire group Error (b) Crithidia prevalence Intercept <0.001 <0.001 Collecting date <0.001 <0.001 Treatment Sire group Treatment sire group Error a Significance levels under type IV analysis (see text). type III and IV-analyses, respectively; for prevalence: P ¼ and P ¼ 0.058, respectively). Similar results were obtained for prevalence when using a logistic regression instead of an ANOVA. Also in this case, sire group (Wald v 2 ¼ , d.f. ¼ 9, P ¼ 0.008) and collecting date (v 2 ¼ , d.f. ¼ 1, P < 0.001) had a significant effect, again no interaction between treatment and sire group (v 2 ¼ 8.440, d.f. ¼ 6, P ¼ 0.21), while treatment per se was marginally non-significant (v 2 ¼ 7.666, d.f. ¼ 3, P ¼ 0.053). Logistic regression and Type IV analyses therefore only differed from our standard analysis in the effect of treatment that became marginally non-significant. Again, removal of group number 8 did not change the conclusions (Wald v 2 ¼ , d.f. ¼ 8, P ¼ 0.005). DISCUSSION Figure 1 Intensity (open bars) and prevalence (black bars) of Crithidia bombi infection between workers of 10 Bombus terrestris sire groups. Groups differed significantly from each other and independently from worker origin (for statistics, see Table 1). Numbers below bars refer to the sample size of workers available per sire group, values are mean ± 1 SEM. In social hymenoptera, males are short-lived and contribute to offspring only by donating sperm that is later used by the female to build her colony (Simmons 2001). Given this biology a father s contribution to offspring performance is very likely restricted to genotypic effects alone. As our results now indicate with respect to parasitism under field conditions, colony workers vary in their susceptibility towards an important parasite, C. bombi, by the genetic background of their father, that is, by sire group. Consequently, such variation is likely to be due to genotypic effects carried by males with which the female has mated. Similar patriline effects in social insects have been reported in other studies before, mostly for worker behaviour but not for disease resistance in the field (Robinson 1992; Degrandi Hoffman et al. 1998; Arathi & Spivak 2001). Furthermore,

4 Male lineages and parasite susceptibility 109 these effects on colony performance were typically found to be non-additive, i.e. they depend on the presence of other patrilines (Fuchs & Schade 1994). In contrast, we found that the effects are additive (Table 1), that is, the effect of a sire group appears regardless of whether the colony contains additional sire groups or not. Consequently, queens have an incentive to mate with additional males regardless of their current mating frequency or with whom they have mated before. Additive effects should also facilitate the process of rapid host parasite co-evolution by negative frequency-dependent selection (Peters & Lively 1999), which in turn would help to maintain the appropriate genetic diversity in host (and parasite) populations that is a prerequisite for differences among sire groups. Obviously, the polyandry vs. parasitism hypothesis might still be wrong although basic assumptions are met; the hypothesis is not contradicted by the results presented but instead gains credibility. ACKNOWLEDGEMENT We thank B. Baer, P. Korner, C. Bertschi, C. Reber, R. Schmid-Hempel, C. Gerloff, R. Loosli, W. Hughes, J. Jokela and E. Magrot for help and comments. This work was supported by grants to PSH from the Swiss National Science Foundation and the Swiss Office of Science and Technology (within a European TMR network) and by a Marie Curie fellowship by the Swiss Science foundation to BB. REFERENCES Arathi, H.S. & Spivak, M. (2001). Influence of colony genotypic composition on the performance of hygienic behaviour in the honeybee, Apis mellifera L. Anim. Behav., 62, Baer, B. & Schmid-Hempel, P. (1999). Experimental variation in polyandry affects parasite loads and fitness in a bumblebee. Nature, 397, Baer, B. & Schmid-Hempel, P. (2000). The artificial insemination of bumblebee queens. Insectes Soc., 47, Baer, B. & Schmid-Hempel, P. (2001). Unexpected consequences of polyandry for parasitism and fitness in the bumblebee Bombus terrestris. Evolution, 55, Brown, M.J.F., Loosli, R. & Schmid-Hempel, P. (2000). Conditiondependent expression of virulence in a trypanosome infecting bumblebees. Oikos, 91, Cole, B.J. & Wiernasz, C. (1999). The selective advantage of low relatedness. Science, 285, Crozier, R.H. & Page, R.E. (1985). On being the right size: male contributions and multiple mating in social hymenoptera. Behav. Ecol. Sociobiol., 18, Degrandi Hoffman, G., Collins, A., Martin, J.H., Schmidt, J.O. & Spangler, H.G. (1998). Nest defense behavior in colonies from crosses between Africanized and European honey bees (Apis mellifera L.) (Hymenoptera: Apidae). J. Insect Behav., 11, Duvoisin, N., Baer, B. & Schmid-Hempel, P. (1999). Sperm transfer and male competition in a bumblebee. Anim. Behav., 58, Ebert, D. & Hamilton, W.D. (1996). Sex against virulence: the evolution of parasitic diseases. Trends Ecol. Evol., 11, Estoup, A., Scholl, A., Pouvreau, A. & Solignac, M. (1995). Monoandry and polyandry in bumble bees (Hymenoptera: Bombinae) as evidenced by highly variable microsatellites. Mol. Ecol., 4, Fjerdingstad, E.J. & Boomsma, J.J. (1998). Multiple mating increases the sperm stores of Atta colombica leafcutter ant queens. Behav. Ecol. Sociobiol., 42, Fuchs, S. & Schade, V. (1994). Lower performance in honeybee colonies of uniform paternity. Apidologie, 25, Hamilton, W.D. & Zuk, M. (1982). Heritable true fitness and bright birds: a role for parasites? Science, 218, Jennions, M.D. & Petrie, M. (2000). Why do females mate multiply? A review of the genetic benefits. Biol. Rev., 75, Jokela, J., Lively, J., Fox, A. & Dybdahl, M.F. (1997). Flat reaction norms and frozen phenotypic variation in clonal snails (Potamopyrgus antipodarum). Evolution, 51, Kokko, H. (2001). Fisherian and good genes benefits of mate choice: how (not) to distinguish between them. Ecol. Lett., 4, Kraus, B. & Page, R.E. (1998). Parasites, pathogens and polyandry in social insects. Am. Nat., 151, Liersch, S. & Schmid Hempel, P. (1998). Genetic variation within social insect colonies reduces parasite load. Proc. R. Soc. Lond. B, 265, Neumann, P. & Moritz, R.F.A. (2000). Testing genetic variance hypotheses for the evolution of polyandry in the honeybee (Apis mellifera L.). Insectes Soc., 47, Page, R.E. (1980). The evolution of multiple mating behavior by honey bee queens (Apis mellifera L.). Genetics, 96, Page, R.E., Robinson, G.E., Fondrk, M.K. & Nasr, M.E. (1995). Effects of worker genotypic diversity on honey bee colony development and behavior (Apis mellifera L). Behav. Ecol. Sociobiol., 36, Peters, A.D. & Lively, C.M. (1999). The Red Queen and fluctuating epistasis: a population genetic analysis of antagonistic coevolution. Am. Nat., 154, Robinson, G.E. (1992). Regulation of Division of Labor in Insect Societies. Ann. Rev. Entomol., 37, Sauter, A.F.B.M.J., Baer, B. & Schmid-Hempel, P. (2001). Males of social insects can prevent queens from multiple mating. Proc. R. Soc. Lond. B, 268, Schmid Hempel, P. (2001). On the evolutionary ecology of host parasite interactions: addressing the question with regard to bumblebees and their parasites. Naturwissenschaften, 88, Schmid-Hempel, P. (1994). Infection and colony variability in social insects. Phil. Trans. R. Soc. Lond. B, 346, Schmid-Hempel, P. (1998). Parasites in Social Insects. Monographs in Behavior and Ecology, Princeton University Press, Princeton. Schmid-Hempel, P. & Crozier, R.H. (1999). Polyandry versus polygyny versus parasites. Phil. Trans. R. Soc. Lond. B, 354, Schmid-Hempel, P., Puhr, K., Kruger, N., Reber, C. & Schmid- Hempel, R. (1999). Dynamic and genetic consequences of variation in horizontal transmission for a microparasitic infection. Evolution, 53,

5 110 B. Baer and P. Schmid-Hempel Schmid-Hempel, R. & Schmid-Hempel, P. (2000). Female mating frequencies in Bombus spp. from Central Europe. Insectes Soc., 47, Sherman, P.W., Seeley, T.D. & Reeve, H.K. (1988). Parasites, pathogens and polyandry in social Hymenoptera. Am. Nat., 131, Simmons, L.W. (2001). Sperm Competition and its Evolutionary Consequences in the Insectes. Monographs in Behaviour and Ecology, Princeton University Press, Oxford. Strassmann, J. (2001). The rarity of multiple mating by females in the social Hymenoptera. Insectes Soc., 48, Tarpy, D.R. (in press). Genetic diversity within honeybee colonies prevents severe infections and promotes colony growth. Proc. R. Soc. Lond. B. Via, S. (1991). The genetic structure of host plant adaptation in a spatial patchwork demographic variability among recipocally transplanted pea aphid clones. Evolution, 45, Manuscript received 2 October 2002 First decision made 22 October 2002 Manuscript accepted 6 November 2002