Integrative and Comparative Biology Advance Access originally published online on May 5, 2006
Integrative and Comparative Biology 2006 46(4):373-380; doi:10.1093/icb/icj044
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Fitness consequences of selfing and outcrossing in the cestode Schistocephalus solidus
Department of Evolutionary Ecology, Max-Planck-Institute of Limnology August-Thienemann-Strasse 2, 24306 Plön, Germany
Correspondence: 1E-mail: milinski{at}mpil-ploen.mpg.de
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Mixed-mating, that is reproduction by both self-fertilization and cross-fertilization is common in hermaphroditic parasites. Its maintenance poses, however, a problem for evolutionary biology. The tapeworm Schistocephalus solidus Müller 1776, served as a model to study experimentally the consequences of selfing and outcrossing in its 2 consecutive intermediate hosts, a copepod (Macrocyclops albidus Jurine) and the three-spined stickleback fish (Gasterosteus aculeatus). Size-matched tapeworms were allowed to reproduce either alone or in pairs in an in vitro system that replaced the definitive bird host's gut. Selfed eggs from singletons had a 4 times lower hatching success than outcrossed eggs from pairs. Outcrossed offspring achieved both a higher infection success and a higher weight in the copepod, and a higher number of parasites per host in both intermediate hosts, but only under competition. Outcrossed offspring were generally more successful. If a S. solidus plerocercoid has a partner in the bird's gut, they should outcross unless they differ in size and thus cannot solve the Hermaphrodite's Dilemma cooperatively. Using microsatellite markers, the proportion of selfed offspring and the total reproductive output of each worm within pairs varying in mean weight and in weight difference was measured. Worms produced more selfed offspring not only with increasing weight difference as expected but also with decreasing total weight of the pair. If small worms were selfed, they have already purged deleterious mutations and would thus be better selfers in a year with low parasite density when worms cannot find partners. To maintain this advantage they should self a higher proportion of their eggs even with a partner. Here I review recent exprimental evidence.
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Introduction |
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Many hermaphroditic parasites (that is cestodes, trematodes, monogeneans) reproduce by both self-fertilization and cross-fertilization (Williams and McVicar1968
; Nollen1983). To understand the maintenance of such mixed mating systems it is necessary to compare the fitness consequences of the 2 reproductive modes. Mixed mating systems are a challenge for evolutionary biology. Although self-fertilizers have a reduced cost of sex (Fisher1941; Maynard 1978), the low fitness of inbred progeny selects directly against self-fertilization. Most models of mating system evolution predict that a mixture of selfed and outcrossed progeny should not exist. Lande and Schemske(1985) and Charlesworth and others (1990) showed in quantitative genetic models that the balance between the cost of outcrossing and inbreeding depression should result in either complete selfing or complete outcrossing. However, Cheptou and Dieckmann (2002) using an adaptive dynamics modeling approach predicted that both deterministic and stochastic environmental effects leading to fluctuating populations can produce evolutionarily stable mixed mating systems. Endoparasitic simultaneous hermaphrodites may be suitable to detect mixed mating systems that are predicted by this model, because stochastic density fluctuations are influencing the probability for outcrossing in the definitive host. Hermaphroditic parasites are at risk to end up without a mating partner in the final host, and selfing may thus be maintained in these species mainly because it offers reproductive assurance (Lloyd 1980). To test whether self-fertilization is only such a best-of-bad-job-strategy Christen and others (2002) compared experimentally the fitness consequences of selfing and outcrossing in the hermaphroditic tapeworm Schistocephalus solidus.
The cestode S. solidus is a simultaneous hermaphrodite that reproduces in its definitive bird (heron Ardea cinera, cormorant Phalacrocorax carbo, kingfisher Alcedo atthis, and other fish-eating birds) host's gut either by outcrossing with a partner or by selfing when alone (Fig. 1). The parasite has a complex life cycle involving a cyclopoid copepod (first intermediate host) and the three-spined stickleback, Gasterosteus aculeatus (second intermediate host). After the parasite's eggs have passed into the water with the bird's feces, coracidia, the first larval stage, hatch from the eggs. They have to be eaten by the first intermediate host. In the copepod's body cavity the larva grows and develops into the procercoid. Only when the infected copepod is eaten by the second intermediate host, the third larval stage, the plerocercoid, can develop and grow to become infective to the definitive host after several months (Hopkins and Smyth1951; Clarke1954). The evolution of this complex life cycle can be understood as the parasite's strategy to maximize its reproductive output (Parker, Chubb, Roberts, and others2003; Parker, Chubb, Roberts, Michaud, and others2003). All the participants of this system can be bred and handled in the lab, except for the bird, which has been replaced by an in vitro system. This method has been pioneered by Smyth(1954) and was modified by Wedekind(1997) and Schärer and Wedekind (1999).
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Hatching rate, infecting success, and growth in the first intermediate host
Christen and others (2002)
started with plerocercoids that had been removed from wild-caught sticklebacks from a brackish water population near Neustadt, Northern Germany, in autumn. They selected 10 triplets of three size-matched worms each. One of the worms was randomly assigned to become a selfer in the in vitro bird, the remaining two a pair that was allowed to outcross in another in vitro bird. All eggs that these worms produced were collected during 8 days, which is the total reproductive period. As first fitness measures of selfing and outcrossing the total number of eggs per worm, egg size, and the hatching rate of the eggs was determined (Fig. 2).
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Then single lab-bread copepods (Macrocyclops albidus), the parents of which had also been caught near Neustadt, were exposed to 6 coracidia each, produced either by the selfing worm or by the outcrossing pair of each triplet. After 6 days each copepod was screened for procercoids under the microscope. There was no competition between selfed and outcrossed procercoids in one host in these treatments (Fig. 2). In a third treatment, single copepods were exposed to a mixture of 3 selfed and 3 outcrossed coracidia. This treatment allowed for competition between selfed and outcrossed procercoids in one host. The 2 types were distinguishable under the microscope by a fluorescent labeling technique. A previous study (Kurtz and others2002
) had shown that this technique does not influence infection success or growth of the parasites, and there was no statistical interaction between labeling and reproductive mode with regard to tapeworm size.
Figure 3 shows the infection success, the number of parasites per copepod, and parasite volume of selfed and outcrossed parasites both without and with competition. Altogether 444 copepods were exposed in this study. Selfing and outcrossing worms produced almost the same number of eggs, and these eggs had a very similar size. These results were confirmed with S. solidus from the same population in a later study (Christen and Milinski2003). However, selfing S. solidus from a population near Bochum, central Germany, produced smaller eggs than did pairs from this area (Wedekind and others1998; Schärer and Wedekind1999).
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There was, however, a dramatic difference in hatching rate of selfed and outcrossed eggs in most triplets (Fig. 4). On average, selfed eggs had a 4 times lower hatching rate than outcrossed eggs (Fig. 3). This is an enormous disadvantage of selfing. We found even lower hatching rates of selfed eggs in later studies.
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Next, infection success, intensity (that is number of parasites per copepod) and growth (that is volume on day six postinfection) in the copepod was determined. Figure 5 shows the difference between outcrossed and selfed parasites per triplet for each of the 3 measures. When the value is positive, outcrossed parasites are more successful, when it is negative, selfed ones are better. There was no significant difference in the success of selfed and outcrossed parasites in any measure (Fig. 5).
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Under competition, however, outcrossed procercoids were significantly better in all 3 measures. Especially, they grew much better than selfed parasites, this is depicted by the volume that they had reached until day 6 (Fig. 5).
Infecting success and growth in the second intermediate host
Christen and Milinski (2003) determined experimentally the infection rate and the success of selfed and outcrossed parasites under competition in single three-sticklebacks, the second intermediate host. We started with 9 triplets of size-matched plerocercoids that had been removed from wild-caught sticklebacks as in the previous study (Christen and others2002). Single copepods were exposed either to 6 selfed or to 6 outcrossed coracidia (Fig. 6). Twelve days later we exposed single sticklebacks that had been bred parasite-free in the lab to 2 copepods each. Each of the two copepods carried the same number of procercoids, one carried only selfed the other only outcrossed procercoids. After 60 days the fish were dissected and the plerocercoids were counted and measured. Their origin, selfed or outcrossed, was determined by using microsatellite markers (see Binz and others2000; Christen and Milinski2003, for details).
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The infection success, the number of parasites per fish, and the weight of parasites were measured on day 60, postinfection. The results are shown again as the difference between outcrossed and selfed parasites in Figure 6. All differences were positive. So outcrossed worms were more successful. But only the number of plerocercoids per fish was significantly higher for outcrossed parasites. This effect was, however, rather strong.
In conclusion: we found (1) a four times higher hatching rate of outcrossed eggs, (2) that outcrossed parasites achieved a higher infection success, (3) a higher number of parasites per host, and (3) higher weight in copepods, the first intermediate host, but only under competitive conditions. (4) Furthermore, outcrossed parasites achieved a higher number of plerocercoids per host in the stickleback, the second intermediate host, again under competitive conditions. If anything, outcrossed parasites were more successful.
An experimental test of predictions of mixed-mating and Hermaphrodite's Dilemma theory
If a plerocercoid of S. solidus finds a partner in the bird's gut, they should outcross. This is what both the majority of mixed-mating models and our previous results (Christen and others2002; Christen and Milinski2003) suggest. However, there is a further inevitable complex conflict, which is called the Hermaphrodite's Dilemma (Leonard1990; Michiels1998). It arises when 2 simultaneous hermaphrodites meet for reproduction and describes the conflict between the mating partners over which individual of the pair will play the preferred role, that is sperm donor or sperm acceptor, in any encounter. The cooperative solution of the dilemma will be a mating system based on reciprocity with potential cheating in the preferred sexual role. Hermaphrodite's Dilemma theory predicts that especially asymmetric pairs might have conflicts of interest, which might lead to some selfing as a best of bad job strategy. Bigger worms of S. solidus are of higher female quality than small worms, because female fecundity is strongly correlated with body size (Wedekind and others1998; Schärer and Wedekind1999). The higher female quality and attractiveness of big worms was confirmed in a choice experiment where small worms preferred big worms (Lüscher and Wedekind2002). We therefore expect that mating partners that differ in size solve their conflict noncooperatively more often than partners of similar size. So if pairs of S. solidus produce any selfed eggs, this should occur only in asymmetric pairs; symmetric pairs are expected to completely outcross.
Lüscher and Milinski(2003) staged this situation in in vitro birds with twenty-nine pairs of plerocercoids that had been removed from wild-caught sticklebacks as before (Fig. 7). The pairs had either no asymmetry in size (all the dots directly above 1) or were asymmetric up to a twofold difference in weight. At the same time pairs differed in mean weight. The 2 arrows at the left in Figure 7 point to 2 symmetric pairs that differ strongly in their weight. The 2 right arrows point to 2 pairs that were highly asymmetric and again differed in weight. There were various combinations of weight and asymmetry. To be able to analyze asymmetry and mean weight independently of each other, it was essential that there was no correlation between the 2 variables. About 90 000 eggs of each worm pair were transferred to Petri dishes for hatching. Ninety coracidia of each pair were used for microsatellite analysis to determine if each larva resulted from outcrossing, selfing by the bigger worm, or selfing by the smaller worm of the pair. Only seventeen worm pairs fulfilled the requirement not to share identical alleles on at least 1 of the 4 microsatellite loci.
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Figure 8 shows the percentage of selfed eggs that these seventeen pairs produced. Eight pairs selfed about 5% of their eggs, others 10, 18, 40, 50, up to 100%. We found this result surprising, given the disadvantages of selfing that we had documented. Did the high selfing rates predominantly occur in asymmetric pairs? We performed a multiple regression analysis of outcrossing rate on asymmetry and mean weights of pairs.
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The lower part (b) of Figure 9 shows that indeed outcrossing rate decreased significantly with increasing asymmetry of pairs, when the effect of weight was removed. So more asymmetric pairs selfed more eggs, as expected from Hermaphrodite's Dilemma theory. However, the upper part (a) of Figure 9 shows that outcrossing rate increased with weight of the pairs, when the effect of asymmetry was removed. This result is not easily explained.
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Discussion |
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Outcrossing is the preferred strategy of most pairs of S. solidus (Lüscher and Milinski2003
). Singleton worms even delay the start of their reproduction probably to wait for a potential partner to arrive (Schjoerring 2004). Outcrossed eggs have both a higher hatching rate and competitive advantages in both intermediate hosts (Christen and others 2002, Christen and Milinski 2003). However, all worms in pairs engaged in some self-fertilization, although they had the opportunity to completely outcross (Lüscher and Milinski 2003). In asymmetric pairs this is expected from Hermaphrodite's Dilemma theory (Leonard 1990). Symmetric pairs, however, also produced selfed eggs especially when the partners were small. A functional explanation for this finding may be the following. Inbreeding depression is expected to be strongest for the first selfed generation. Thereafter it is expected to decrease during consecutive selfing events, because of purging of deleterious mutations with large homozygous effects on fitness (Charlesworth and Charlesworth 1987, 1998). Hence, in a fully outcrossing population, offspring of a worm that invests both in outcrossing and in selfing may have a higher fitness when almost all individuals have to reproduce by selfing because of low density. The evolutionary stability of intermediate selfing rates for such a scenario has been confirmed in a model by Cheptou and Dieckmann (2002). But why do especially small worms self at relatively high rates?
If small worms were small because they had been produced by selfing, we found a trend for this expectation in a previous study (Fig. 6), then small worms had already purged some of their deleterious mutations and their offspring should suffer less from selfing than those of outcrossed worms. By being selfed they should have become better selfers. So, were those pairs that selfed a higher proportion of their eggs better selfers? We have an indirect hint. In the first study (Christen and others 2002) we found that outcrossed eggs hatch at a 4 times higher rate than selfed eggs. Therefore we expect a strong positive correlation between outcrossing rate and hatching rate for worm pairs. The higher the proportion of selfed eggs is, the lower should be the overall hatching rate. Surprisingly, when pairs decided to self a certain proportion of their eggs, there was no significant correlation between outcrossing rate and hatching rate (Lüscher and Milinski 2003). Does this mean that better selfers "knew" that they were better selfers and thus selfed a higher proportion of their eggs as expected? But why should they self at all? Because there may be years with low prevalence when each worm has a very low probability to meet a partner (see above). Then almost every worm has to self. Selfed parasites might then be very successful because they have already survived the low hatching rate and have purged deleterious mutations. "When you are a selfed worm, go on selfing, the next year might be a low prevalence year." With each generation with uninterrupted selfing, these simultaneous hermaphrodites should become better selfers, that is an advantage that would be lost by only one episode of outcrossing. It would be interesting to test whether the proportion of eggs that these worm decide to self increases with the number of consecutive selfed generations. Such a finding would imply the existence of a specific memory, a kind of genomic imprinting, of whether one's parent (and grandparent) had selfed.
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Acknowledgements |
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I thank Mira Christen, Joachim Kurtz, and Annelis Lüscher for collaboration on the studies discussed in this paper, and Janet Leonard for organizing a most stimulating symposium.
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From the symposium "Sexual Selection and Mating Systems in Hermaphrodites" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2005, at San Diego, California.
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References |
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