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5E-mail: [email protected]. Abstract. Host-parasite coevolution is often described as a process of reciprocal adaptation and counter adaptation, driven by ...
Evolution, 60(6), 2006, pp. 1177–1186

EXPERIMENTAL EVOLUTION OF RESISTANCE IN PARAMECIUM CAUDATUM AGAINST THE BACTERIAL PARASITE HOLOSPORA UNDULATA KONRAD LOHSE,1,2 ARNAUD GUTIERREZ,3,4

AND

OLIVER KALTZ3,5

1 Environmental

and Evolutionary Biology, School of Biology, University of St. Andrews, St. Andrews, Fife, KY16 9TH, United Kingdom 2 E-mail: [email protected] 3 Laboratoire de Parasitologie Evolutive, CNRS-UMR 7103, Universite ´ Pierre et Marie Curie, 75252 Paris Cedex 05, France 4 E-mail: [email protected] 5 E-mail: [email protected] Abstract. Host-parasite coevolution is often described as a process of reciprocal adaptation and counter adaptation, driven by frequency-dependent selection. This requires that different parasite genotypes perform differently on different host genotypes. Such genotype-by-genotype interactions arise if adaptation to one host (or parasite) genotype reduces performance on others. These direct costs of adaptation can maintain genetic polymorphism and generate geographic patterns of local host or parasite adaptation. Fixation of all-resistant (or all-infective) genotypes is further prevented if adaptation trades off with other host (or parasite) life-history traits. For the host, such indirect costs of resistance refer to reduced fitness of resistant genotypes in the absence of parasites. We studied (co)evolution in experimental microcosms of several clones of the freshwater protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. After two and a half years of culture, inoculation of evolved and naive (never exposed to the parasite) hosts with evolved and founder parasites revealed an increase in host resistance, but not in parasite infectivity. A cross-infection experiment showed significant host clone-by-parasite isolate interactions, and evolved hosts tended to be more resistant to their own (local) parasites than to parasites from other hosts. Compared to naive clones, evolved host clones had lower division rates in the absence of the parasite. Thus, our study indicates de novo evolution of host resistance, associated with both direct and indirect costs. This illustrates how interactions with parasites can lead to the genetic divergence of initially identical populations. Key words.

Coevolution, costs of resistance, cross-infection, infectivity, local adaptation. Received November 28, 2005.

Host-parasite coevolution is often described as an ongoing process of adaptation and counteradaptation, driven by frequency-dependent selection (Thompson 1994; Woolhouse et al. 2002). That is, adaptation of the parasite to the most common genotype in the population selects for rare resistant genotypes, which then increase in frequency until they are again tracked by the parasite, and so on (Bell and Maynard Smith 1987; Nee 1989). Because of the constant need for novelty, antagonistic coevolution may be a powerful engine creating and maintaining genetic diversity (Haldane 1949; Frank 1991; Agrawal and Lively 2002). It may thus promote the maintenance of sexual reproduction (Hamilton 1980; Bell and Maynard Smith 1987), and shape the geographical distribution of genetic variation (Thompson 1994, 1999). However, frequency-dependent cycling stops if one of the two players evolves genotypes of universal resistance or infectivity. Their spread to fixation can be prevented in two ways. First, there may be direct costs of adaptation. Evolving resistance against one parasite genotype reduces resistance against other parasite genotypes, due to antagonistic pleiotropy or conditionally deleterious mutations. The resulting host genotype 3 parasite genotype interactions facilitate frequency-dependent selection and the maintenance of genetic diversity (Agrawal and Lively 2002). By the same logic, adaptation to locally common genotypes reduces performance on genotypes from other populations, thus generating geographic patterns of host or parasite local adaptation (Morand et al. 1996; Gandon 2002). Second, fixation of omnipotent genotypes may be prevented by indirect costs of adaptation (Antonovics and Thrall 1994; Parker 1994). For the host, indirect costs of resistance can be defined as reduced

Accepted March 25, 2006.

fitness in the absence of the parasite. Thus, genes conferring higher resistance may have negative pleiotropic effects on other life-history traits, such as fecundity or growth, which are unrelated to the direct interaction with the parasite. For the parasite, indirect costs may arise from trade-offs between infectivity, propagule production or virulence (Frank 1991; Bergelson et al. 2001; Thrall and Burdon 2003). These indirect costs of adaptation can maintain genetic variation (Antonovics and Thrall 1994; Parker 1994; Bergelson et al. 2001; Agrawal and Lively 2002), but, because of their nonspecificity, they do not necessarily create patterns of local adaptation. Naturally occurring host and parasite genotypes often vary in resistance, infectivity, or virulence or display genotype 3 genotype interactions for these traits (Burdon 1987; Sorci et al. 1997; Carius et al. 2001; Kaltz and Shykoff 2002; Little 2002; Webster et al. 2004; Lambrechts et al. 2005). Tradeoffs between resistance and other host fitness components have been demonstrated in several studies, although they are far from being ubiquitous (Rausher 1996; Webster and Woolhouse 1999; Bergelson et al. 2001; Rigby et al. 2002; Strauss et al. 2002; Brown 2003). Furthermore, cross-infection studies have revealed patterns of parasite (or host) local adaptation in natural populations of various host-parasite systems (Kaltz and Shykoff 1998; Van Zandt and Mopper 1998; Bergelson et al. 2001), indicating population-specific adaptation of parasites to their hosts, and vice versa. However, direct evidence for the coevolutionary process in natural populations is still scarce (Little 2002; Woolhouse et al. 2002; Webster et al. 2004). Alternatively, mechanisms of antagonistic coevolution can

1177 q 2006 The Society for the Study of Evolution. All rights reserved.

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be studied through artificial selection or experimental (co)evolution in the laboratory (Chao et al. 1977; Lenski and Levin 1985; Boule´treau 1986; Boots and Begon 1993; Ebert and Mangin 1997; Kraaijeveld and Godfray 1997; Ebert 1998; Mackinnon and Read 1999; Messenger et al. 1999; Turner and Chao 1999; Elena 2001; Buckling and Rainey 2002a; Woolhouse et al. 2002; Capaul and Ebert 2003; Haag and Ebert 2004; Webster et al. 2004; Fischer and SchmidHempel 2005; Morgan et al. 2005; Stewart et al. 2005). Studies on parasite-mediated selection for resistance are quite rare. For example, the presence of microsporidian parasites altered the genetic composition of initially diverse experimental Daphnia magna populations, sometimes resulting in fixation of a single genotype (Capaul and Ebert 2003; Haag and Ebert 2004). Experiments with Escherichia coli and the bacteriophage T7 showed how parasite adaptation was rapidly countered by the evolution of universal host resistance against both ancestral and evolved phage (Chao et al. 1977). However, indirect costs of resistance (slower growth in the absence of the parasite) facilitated the maintenance of both susceptible and resistant genotypes, as well as that of the phage, in the population (Chao et al. 1977). Such trade-offs between resistance and fecundity can lead to the genetic divergence of selection lines exposed or unexposed to parasites, as shown for insect hosts (Boots and Begon 1993; Kraaijeveld and Godfray 1997). So far, only a few studies have investigated how direct costs of adaptation shape patterns of genetic variation within and across coevolving experimental populations. Ongoing cycles of antagonistic coevolution between Pseudomonas fluorescens and a bacteriophage increased genetic divergence among experimental populations (Buckling and Rainey 2002b). In this system, hosts seem to evolve more rapidly than their parasites, resulting in average patterns of local host adaptation (Buckling and Rainey 2002a; Morgan et al. 2005), whereas in the above E. coli–phage system, average patterns of local parasite adaptation emerged (Forde et al. 2004). To our knowledge, these studies represent the only long-term coevolutionary experiments testing for reciprocal selection and local adaptation. We used the freshwater protozoan Paramecium caudatum and its bacterial parasite Holospora undulata to investigate host-parasite coevolution experimentally. This micronucleusspecific parasite sterilizes its host, reduces asexual (mitotic) division rates and increases host mortality (Fokin 2004; Restif and Kaltz 2006). Genetic variation in resistance and infectivity (Fokin and Skovorodkin 1997; Kaltz and Nidelet 2004; Fels and Kaltz 2005) indicates the potential for (co)evolution. We set up experimental microcosms, each initially composed of a single host clone infected with parasites from a single stock culture, and the corresponding uninfected control lines. We regularly replaced a fraction of the population with fresh culture medium, but otherwise let epidemiological or evolutionary processes act freely. After two and a half years of culture, we carried out three experiments. First, we tested for reciprocal selection. If the parasite selected for increased resistance, evolved hosts should be more resistant parasites than naive (control) hosts. Conversely, selection for increased infectivity should result in evolved parasites that are more infectious than founder

parasites, at least when compared on the naive hosts. Second, a cross-infection experiment tested for the evolution of host genotype 3 parasite genotype interactions and whether hosts were more resistant against parasites from their own microcosm than against parasites from other microcosms. Such a pattern of local adaptation would indicate direct costs of adaptation and the evolution of specialists. Third, to test for an indirect fitness cost of evolving resistance against H. undulata, we compared the growth rates of evolved and naive hosts in the absence of the parasite. MATERIALS

AND

METHODS

Study Organisms The ciliate P. caudatum lives in ponds and lakes of the Northern Hemisphere (Wichtermann 1986). It feeds on bacteria and detritus, ingested from the water column. For the most part of their life cycle, paramecia reproduce asexually by mitotic division. Like all ciliates, P. caudatum has two nuclei. The polyploid macronucleus serves a vegetative function; the smaller micronucleus is an ‘‘ordinary’’ nucleus capable of mitosis and meiosis, and active mainly during sexual reproduction (Wichtermann 1986; Go¨rtz 1988). The gram-negative H. undulata is a micronucleus-specific endoparasite of P. caudatum and belongs to the a-group of Proteobacteria, together with other endosymbionts like Rickettsia or Wolbachia (Amann et al. 1991). Species of Paramecium are known to be infected by different Holospora species, specific to either micro- or macronucleus, but all with similar life cycles (Fokin et al. 1996; Go¨rtz and Brigge 1998; Fokin 2004). Generally, there are two distinct morphs of the parasite. The long (15–20 mm) infectious forms of the parasite are ingested from the water column. Once inside a food vacuole, they direct their transport to the nucleus. Within 24 h post infection, infectious forms differentiate into the short (5–10 mm), round reproductive forms, which multiply and begin to fill out the nucleus. After 7–10 days, reproductive forms can differentiate into infectious forms, which are released during cell division, or upon host death, to infect novel hosts horizontally (Go¨rtz and Wiemann 1989). Reproductive forms are unable to infect new hosts horizontally, but are transmitted vertically along with the mitotically dividing micronucleus. Culturing Techniques and Design of the Long-Term Experiment Of the five Paramecium clones used in this experiment, four (K3, K6, K8, K9) were established from a mating between two uninfected stocks of opposite mating type (KNZ 5, mating type O3 and KNZ 2, mating type E3, provided by T. Watanabe, Tohoku University, Japan). Each clone was derived from one of the exconjugant cells obtained from one conjugating pair of paramecia. Thus, these clones are full siblings, derived from different matings between the same parents. Another clone (O3) was derived from a single individual isolated from a P. caudatum stock, collected in Germany and provided by H.-D. Go¨rtz (University of Stuttgart, Germany).

EXPERIMENTAL EVOLUTION OF RESISTANCE

In October 2002, a mass culture of each clone was split: one half remained uninfected, and the other half was inoculated with infectious forms from a strain collected in Germany (provided by H.-D. Go¨rtz) and kept as a mass stock culture in our lab since 2001. Three days after inoculation, the cultures were split into three to seven replicate lines (50ml Falcon tubes; Fisher Labosi, Elancourt, France), each consisting of several thousands of paramecia in 50 ml of growth medium. This medium was initially made of Protozoan Pellets (Carolina Biological Supply Company, Burlington, NC), suspended at a concentration of 0.7 g/L in Volvic mineral water (Danone, Puy de Dome, France), autoclaved, and then inoculated with the bacterium Serratia marcenscens (Institut Pasteur, Paris, France) as a food resource for the paramecia. For economic reasons, the pellets were replaced after several months with organically grown lettuce (dried for two days at 808C, then ground up with mortar and pestle and autoclaved at a concentration of 0.7 g/L prior to adding the food bacteria). The lines were kept at 238C. To ensure long-term survival of the cultures, once a week 10 ml of each line were removed and replaced with fresh medium inoculated with food bacteria. Thus, weekly, a 20% mortality regime was imposed on both host and parasite. A protocol of cryopreservation was not available at the beginning of the experiment. In an attempt to preserve the founder parasite culture in its ancestral state, replicate tubes were maintained at 58C and supplied with fresh medium once or twice a month. These conditions nearly arrest development of host and parasite (Fokin 2004) and thus reduce the potential for evolutionary change. Adaptation Assay Preparation of host subclones. In February 2005, one infected and one uninfected replicate line from each of the five clones were randomly chosen for the adaptation assay. For infected lines, prevalence was determined by fixation of 40– 50 randomly picked paramecia with lacto-aceto orcein (Go¨rtz and Dieckmann 1980) and inspection for presence or absence of infection at 1000x magnification (phase contrast). From each replicate line, 10 randomly chosen subclones were established from single, uninfected individuals. To this end, we picked 30–50 individuals from each replicate line, washed them three times in 200 ml food solution and placed them individually into 2-ml Eppendorf tubes (Eppendorf International, Hamburg, Germany), containing 100 ml of growth medium. By adding 50 ml of medium every second day, clonal cultures (subclones) were grown from these individuals. After four to six days, 10 uninfected subclones were chosen and kept for another 10 days in optimal growth, with 100 ml of medium added every three to four days. Two days prior to the experiment, 200 ml of Volvic water were added to ensure that the clones were at stationary phase for the adaptation assay. Preparation of inocula. Ten days prior to the experiment, the 50 ml of each of the five infected replicate lines were divided in two tubes and gradually regrown until they reached again 50 ml to ensure sufficient quantities of parasites for the inoculations. Similarly, the cultures infected with the founder parasite were brought to 238C and kept under optimal

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growth conditions for 10 days prior to the experiment. Inocula were prepared 24 h prior to the first experiment. We filtered 80 ml of an infected culture through one layer of medical gauze to remove larger particles. The filtrate was centrifuged for 25 min at 2500 g and 48C, the supernatant discarded and the concentrated infected paramecia ground with a tissue homogenizer (Thomas, Fisher Labosi). Then the infectious forms were concentrated by density-gradient centrifugation (25 min at 2500 g, with 90% Percoll (Fisher Labosi) in the dense layer of the gradient). Inocula were washed twice with Volvic by centrifugation for 25 min at 2500 g. All inocula were diluted to a density of 90 infectious forms/ml and kept at 58C prior to inoculation. Experiment 1: Evolved and naive hosts versus evolved and founder parasites. If coevolution is reciprocal and timelagged, we predict that contemporary parasites perform better on past hosts than do past parasites. Conversely, because of the lag phase in the cycling dynamics, contemporary hosts should be more resistant to past parasites than past hosts (Buckling and Rainey 2002a). Here, we made the simplified assumption that past parasites are adequately represented by the founder parasite, and past hosts by the naive (control) hosts. Thus we tested the evolved parasites from the five replicate lines (K3, K6, K8, K9, O3) against the 10 host subclones with which they (potentially) coevolved, and against the 10 subclones originating from the same host clone, but never exposed to the parasite. All subclones were tested against the founder parasite. These tests were carried out as follows. On 9 March 2005, two groups of 25 individuals from each subclone were washed in 200 ml autoclaved food solution and then transferred to 500 ml tubes containing 50 ml of the inoculum with either evolved or founder parasites. Only seven subclones were available for each of the two selection lines from clone O3. In total, we prepared 188 assay tubes. Three days post inoculation all individuals were fixed and prevalence determined. Experiment 2: Direct costs of adaptation (cross-infection test). The aim of this experiment was to test for the evolution of host clone 3 parasite isolate interactions and for a pattern of (local) host-parasite adaptation. To this end, evolved hosts were confronted with evolved parasites in all possible combinations. Because there was not enough inoculum available from the O3 parasite, only hosts and parasites from the other four clones (K3, K6, K8, K9) were used. On 10 March 2005, each combination of parasite and host was replicated over six host subclones (five host subclones in combinations with the K9 parasite). Each of the 92 assay tubes consisted of 25 paramecia, inoculated as described above. After 72 h, individuals were fixed and prevalence determined. Experiment 3: Indirect costs of resistance. The first experiment showed that evolved host clones were more resistant than their naive, unselected counterparts (see Results). Therefore, we tested for an indirect cost of evolving resistance, that is, whether evolved hosts were less fit than naive hosts in the absence of the parasite. The subclones used for the above tests had been fed with 200 ml of growth medium and then stored at 108C prior to this experiment. On 26 March 2005, the tubes were brought back to room temperature. Ten individuals from each of 89 evolved and naive subclones (5–

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TABLE 1. Mean (6 SE) proportion of infected paramecia, shown for the different combinations of host type and parasite type, tested for the five host clones in experiment 1. Means taken over subclones (mean n 5 9.5; range: 7–10). Parasite type Clone

Host type

K3

naive evolved naive evolved naive evolved naive evolved naive evolved

K6 K8 K9 O3

Founder

0.070 0.101 0.075 0.032 0.257 0.109 0.131 0.032 0.094

6 6 6 6 6 6 6 6 6 0

0.024 0.064 0.028 0.022 0.047 0.030 0.017 0.012 0.026

Evolved

0.033 0.037 0.132 0.043 0.069 0.047 0.093 0.031 0.038

6 6 6 6 6 6 6 6 6 0

0.017 0.017 0.026 0.017 0.020 0.020 0.030 0.013 0.011

10 subclones per clone and host type, mean 8.3 6 0.7 SE) were washed once in 200 ml of diluted medium and then grown for four days in a 2-ml tube containing 200 ml of growth medium. After this conditioning period, three individuals from each tube were randomly chosen and placed individually into a 500 ml assay tube containing 50 ml of growth medium. They were allowed to divide for five days, with 100 ml of new medium added on the second and fourth day. The content of each tube was then transferred to a multiwell glass slide and the paramecia were counted under a dissecting microscope. Data Analysis Evolved and naive hosts versus evolved and founder parasites. A fully factorial, nested analysis of deviance (ANODEV) investigated variation in infection success (proportion of infected paramecia). The model consisted of host clone, host type (evolved vs. naive), parasite type (evolved vs. founder), and host subclone. Based on a logistic regression approach, ANODEV uses mean deviances (Pearson x2/ degrees of freedom) to calculate pseudo-F values for hypothesis testing (Schmid and Dolt 1994). Model terms were fitted sequentially, analogous to a SAS type 2 procedure for analysis of variance (ANOVA; SAS 1996). Cross-infection experiment. Variation in infection success in the cross-infection experiment was analyzed by means of ANODEV, in fully factorial models with host clone, host subclone, and parasite strain. To test for an overall pattern

of local host-parasite adaptation, the diagonal of the combination matrix (sympatric host-parasite combinations) was tested against the off-diagonal (allopatric host-parasite combinations). An alternative analysis was carried out on the differences in resistance of subclones against sympatric and allopatric parasites. Costs of resistance. Variation in (log-transformed) densities of paramecia was analyzed in an ANOVA, with host type, host clone, and subclone as explanatory variables. In all analyses, host (sub)clone and parasite strain were considered as random factors; backward elimination of nonsignificant model terms was performed to obtain minimal adequate models (Crawley 1993). ANODEVs were carried out with the SAS statistical package using the Proc Genmod procedure and the logit link function (SAS 1996). Other analyses were carried out with JMP statistical package (SAS 2003). RESULTS Experiment 1: Evolved and Naive Hosts versus Evolved and Founder Parasites Of the 188 assay tubes, nine were lost during fixation and another two were excluded from analysis because all individuals died after inoculation. Mean prevalence across all tubes was 7.42%, ranging from 0% to 60%. Seventy-two of the 177 tubes were uninfected. Infection success of evolved and founder parasites on evolved and naive hosts is summarized in Table 1. Analysis of deviance (Table 2) revealed a significant overall difference in resistance between evolved and naive hosts. Evolved hosts were more resistant in four of the five clones (evolved O3 subclones even were entirely resistant), whereas there was no consistent difference for clone K3, resulting in a marginally significant host type 3 host clone interaction (Fig. 1). A relatively stronger resistance of evolved hosts against founder parasites would have indicated a time-lagged response to selection. However, there was no significant host type 3 parasite type interaction: the difference in resistance between evolved and naive hosts did not significantly change with parasite type (evolved or founder; Table 1, 2). We did not find a consistent overall difference in infection success between evolved and founder parasites. Rather, the pattern varied with the origin of the evolved parasites (significant parasite type 3 host clone interaction; Table 2).

TABLE 2. Analysis of deviance testing effects of host clone, parasite type (evolved vs. ancestral), host type (evolved vs. naive), and host subclone (nested within clone and host) on the proportion of infected paramecia. The parasite 3 clone 3 host interaction (F4,72 5 0.46, not significant) and the parasite 3 host type interaction (F1,76 5 2.04, P 5 0.1570) were eliminated from the full model. The denominator column shows the factors used for hypothesis testing. The scale parameter was fixed at the value of one in the estimation procedure to correct for overdispersion. MD, mean deviance. Source

df

MD

Denominator

F

P

Clone Parasite type Host type Clone 3 parasite type (4) Clone 3 host type (5) Subclone [clone 3 host type] (6) (Scaled) error (7)

4 1 1 4 4 85 77

11.78 19.79 58.06 6.19 4.33 1.84 1.00

(6) (4) (5) (7) (6) (7)

6.40 4.57 13.41 6.19 2.35 1.84

0.0002 0.0993 0.0215 0.0002 0.0606 0.0035

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FIG. 1. Difference (6 SE of the difference) in mean resistance between evolved and naive hosts in experiment 1, shown for the five host clones. Averages taken over host subclones (mean n 5 9.5; range: 7–10), after combining infection success over evolved and founder parasites. Negative values indicate higher resistance of evolved hosts.

Evolved parasites from host clone K6 were more infectious than founder parasites, whereas the reverse was the case for at least two host genetic backgrounds (Fig. 2). Prior to the experiment, the prevalences in the five infected replicate lines were approximately 0.5 in K3, K6, and K8, but much lower in K9 and O3 (0.086 and 0.04, respectively). There was a significant positive correlation between these prevalences and the mean prevalences measured in the evolved subclones derived from these cultures (Spearman rank test, r 5 0.90, P 5 0.0374). Thus, subclones derived from populations of low ‘‘natural’’ levels of infection were also more resistant in the adaptation assay. Experiment 2: Cross-Infection of Evolved Hosts and Parasites Mean prevalence across all 16 host-parasite combinations was 0.025 (6 0.005), ranging from 0% to 25%. Of the 92 assay tubes, 64 remained uninfected. Because of the low overall infection success, ANODEVs were performed not only on the proportion of infected individuals within assay tubes, but also on the presence or absence of infection within assay tubes (Table 3). Both approaches revealed significant differences in resistance among subclones, as well as significant host 3 parasite interactions. These interactions are illustrated by the crossing reaction norms of parasite infection success across the different host clones, that is, parasites of different origin performed differently on different hosts (Fig. 3, Table 4). For instance, parasites from host clone K3 had the highest infection success on clone K8, but ranked last on K3, their own host clone. Although repartitioning of the deviance of the interaction terms (Table 3) for a comparison between sympatric and allopatric combinations did not reveal a significant pattern of local adaptation (for both response variables: F1,8 # 1.80, not significant), inspection of the means in Table 4 showed that resident (local) parasites were less infectious than nonresident (foreign) parasites on three (K3, K6, K8) of the four host clones. On clone K9, resident parasites were more in-

FIG. 2. Difference (6 SE of the difference) in mean infection success of evolved and founder parasites in experiment 1, measured on the five host clones. Averages taken over replicate assay tubes (mean n 5 17.7; range: 12–20), pooled over evolved and naive hosts. Positive values indicate higher infectivity of evolved parasites.

fectious than nonresidents; however, they can be considered as locally maladapted in the sense that they were less infectious on their own than on other hosts (Table 4). In an alternative test for local adaptation, we calculated the difference between the resistance of each host subclone against its sympatric parasite and against allopatric parasites (combined over parasite origin; n 5 23 differences). The mean values were negative for three of the four clones (Fig. 4), indicating higher resistance against sympatric parasites than against allopatric parasites. In a one-way ANOVA, using this new response variable, the variation among host clones was not significant (F3,19 5 1.35, P 5 0.2873), but, overall, differences were significantly different from zero (t22 5 2.08, P 5 0.0497). That is, when considering subclones as units of replication, an overall trend of locally adapted hosts emerged. Experiment 3: Indirect Costs of Resistance Divisions occurred in 96% of the 254 assay tubes. On average, paramecia accomplished approximately five mitotic divisions (mean number per tube: 37.4 6 1.6 SE). For all TABLE 3. Analyses of deviance testing effects of host clone, parasite strain, and host subclone (nested within subclone) on infection success (proportion of infected paramecia or presence/absence of infection in the assay tubes) in the cross-infection experiment. The denominator (Den.) column shows the factors used for hypothesis testing. Scale parameter fixed at one in the estimation procedure to correct for overdispersion. MD, mean deviance.

Source

Host clone Parasite strain Host clone 3 parasite (3) Subclone [host] (4) (Scaled) error (5)

df

3 3 9 20 56

Proportion of Proportion of infected infected Den. paramecia MD tubes MD

3 3 5 5

7.80 2.47 2.69*** 2.87** 1.00

* P 5 0.0005; ** P , 0.0025; *** P 5 0.0113.

3.54 3.12 3.39** 3.04* 1.00

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FIG. 3. Mean infection success in the different combinations of evolved hosts and parasites in the cross-infection experiment. Values represent the proportion of the five to six replicate assay tubes, where paramecia became infected.

five host clones tested, evolved subclones (derived from infected replicate lines) divided less often (Wilcoxon signedrank test on overall means: T 5 7.5, P 5 0.062) than did naive subclones (derived from uninfected replicate lines). However, this difference varied among host clones (significant host type 3 host clone interaction; Table 5): it was most pronounced for the clones O3 and K8, but only marginal for the other three clones (Fig. 5). DISCUSSION Reciprocal Selection? Experiment 1 showed that, overall, the presence of the parasite led to higher levels of resistance. The difference in resistance between evolved and naive hosts varied among the five replicate lines, each representing a different clonal background. We note that evolved hosts from the O3 clone, which were 100% resistant, originated from the same geographical region as the founder parasite. However, to establish whether different genotypes evolve resistance more easily than others will require replication of lines of the same host clone. One explanation for the difference between naive and evolved hosts is the de novo evolution of resistance due to parasitemediated selection. Infection with H. undulata reduces division rates and survival (Restif and Kaltz 2006), so that such a response to selection within more than 100 generations (see below) seems plausible. However, strictly speaking, a response to selection should be measured against the ancestral genotype. Here, control replicate lines were cultured just like infected lines to account for evolutionary changes of resis-

FIG. 4. Mean differences (6 SE) in infection success between resident and nonresident parasites on each host clone in the crossinfection experiment, averaged over host subclones. Negative values indicate higher resistance against resident parasites and thus local adaptation of the host.

tance due to culture conditions alone. Thus, a second explanation could be that resistance is costly in the absence of the parasite and therefore selected against. Hence, from this first experiment alone we cannot decide which of the two mechanisms, gain or loss of resistance, explains our data. In the sections below, we argue that both processes play a role. Although the presence of the parasite resulted in increased resistance in the host, there was little evidence that exposure to the five (novel) host clones caused an increase in infectivity in the parasite. However, there are several caveats. We tested for reciprocal selection at one point in time, with naive hosts and founder parasites as reference points of the ‘‘past.’’ Both reference points may have their limitations. First, keeping the founder parasite at 58C may not have preserved its ancestral state. We cannot entirely rule out this possibility, although the extremely slow development of both host and parasite (Fokin 2004) and the stably maintained prevalences (near 100%) at this temperature suggest that the potential for genetic change is small. Second, selection for increased infectivity may indeed have occurred in the evolved lines, but remained undetected. If the system coevolves rapidly, the present evolved parasites may have adapted to novel host genotypes evolving within their populations. In this case, comparing evolved and founder parasites on the original (naive) hosts does not necessarily reveal this adaptation. It may therefore be more appropriate to test host and parasite at multiple points in time to account for the coevolutionary

TABLE 4. Mean (6 SE) proportion of infected paramecia for the different combinations of evolved hosts and parasites in the crossinfection experiment. Each mean represents the average across five to six replicate assay tubes. Sympatric combinations between parasite and host isolated from the same selection line are shown in bold. Parasite origin Host origin

K3

K6

K3 K6 K8 K9

0 0.0227 6 0.0227 0.0663 6 0.0380 0.0221 6 0.0150

0 0.0088 6 0.0088 0.0643 6 0.0268 0

K8

0.0174 0.0093 0.0263 0.0076

6 6 6 6

K9

0.0111 0.0093 0.0169 0.0076

0.0517 0.0352 0.0515 0.0224

6 6 6 6

0.0163 0.0254 0.0337 0.0141

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TABLE 5. Analyses of variance testing effects of host clone, host type (evolved, naive), and host subclone (nested within host clone and type) on division frequency (log-transformed densities). The denominator (Den.) column shows the factors used for hypothesis testing. MS, mean squares. Source

df

MS

Den.

F

P

Host clone Host type Host clone 3 type (3) Subclone [host clone, type] (4) Error (5)

3 3 9 20 56

5.96 44.84 9.58 1.39 0.48

3 3 5 5

4.31 4.68 6.94 2.90

0.0034 0.0965 ,0.0001 ,0.0001

dynamics (Buckling and Rainey 2002a; Brockhurst et al. 2004; Forde et al. 2004). In addition, selection in the parasite may operate on traits other than infectivity. In this long-term experiment, populations grow back to carrying capacity after weekly removal of 20% of the culture. This may select for more efficient vertical rather than horizontal transmission. Indeed, in a previous adaptation assay, using a different set of replicate lines, evolved parasites showed consistently higher prevalences than did the founder parasite after a period of intense vertical transmission (Kaltz and Nidelet 2004). On the other hand, considerable loads of infectious forms in infected individuals in our long-term cultures, together with a constant level of new infections (5–10%, O. Kaltz and O. Restif, unpubl. data), indicate a nonnegligible role of horizontal transmission. Local Host Adaptation In the cross-infection experiment, hosts tended to be more resistant to parasites with which they coevolved than to parasites from other clones. This supports the idea of specific, de novo evolution of resistance in these lines, and it is consistent with the apparent asymmetry in experiment 1, which suggested adaptation on the host’s rather than the parasite’s side. Our long-term replicate lines can be considered isolated subpopulations within a metapopulation. Thus, the specificity of resistance has created a geographic pattern of locally adapted hosts, at least in three of the four lines. Sign and

FIG. 5. Difference (6 SE of the difference) in mean density between evolved and naive host clones, after five days of growth in the absence of the parasite. Averages taken over subclone means (mean n 5 8.3; range: 5–11).

magnitude of local adaptation are expected to fluctuate in both time and space (Ebert and Hamilton 1996; Kaltz and Shykoff 1998; Buckling and Rainey 2002a; Forde et al. 2004), and, therefore, testing a larger number of lines may reveal a clearer picture of the generality of this pattern. Furthermore, one may argue that our finding of local adaptation results from exclusive sampling of healthy hosts in the population. However, we think that this should not alter the conclusions. In fact, it merely shifts the coevolutionary time frame to consider, from (infected) hosts of the recent past to (uninfected) hosts the parasite faces at present or in the near future (Buckling and Rainey 2002b). Either way, the positive correlation between ‘‘natural’’ prevalences in the replicate lines and those obtained in the experiment indicates that we captured at least partly the coevolutionary status quo in these tubes. Experiments 1 and 2 contradict conventional wisdom of more rapidly evolving and thus locally adapted parasites (Ebert and Hamilton 1996; Morand et al. 1996; Gandon 2002). Other such counterexamples have been documented in natural populations of host-parasite systems (Kaltz and Shykoff 1998; Bergelson et al. 2001), and in experimental coevolution studies involving bacteria and phage (Buckling and Rainey 2002b; Morgan et al. 2005). In these cases, hosts may have increased their evolutionary potential through sexual recombination, migration, larger population sizes, or larger and more versatile genomes. In our case, hosts were kept strictly asexual (i.e., clonal) in unconnected replicate lines. Furthermore, H. undulata should benefit from the typical generation time and population size advantage of parasites. In our long-term cultures, paramecia divide perhaps once or twice a week, on average, which adds up to 130–260 generations, at population sizes of 2500–5000 individuals. In contrast, within only 7–10 days, a singly infected host can produce several hundreds of bacterial cells. Thus, even if only a small fraction of the population is infected, the population size of the parasite exceeds that of the host easily by a factor of 10. What, then, accelerates host evolution? As mentioned above, selection may also act on other life-history traits of the parasite, such as the efficiency of vertical transmission or virulence. Genetic trade-offs between these traits and infectivity (Ebert and Mangin 1997; Thrall and Burdon 2003) may then obstruct the evolution of more infectious parasites. Another explanation may be the high ploidy level (probably up to several thousands of copies; Freiburg 1988) in the macronucleus, where most gene expression occurs during the asexual phase of the host life cycle. Thus, despite smaller population size in terms of number of individuals, the total

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number of gene copies may exceed that of the parasite. Moreover, multiple copies of genes within the same macronucleus may facilitate the accumulation of different resistance mutations within a single clonal lineage and thus speed up rates of adaptation in the host (Lively et al. 1998). Direct and Indirect Costs of Resistance Selection for increased resistance may incur two types of costs. First, differential resistance of hosts against different parasites and the pattern of local adaptation in experiment 2 are likely to reflect direct costs of adaptation. They arise if increased resistance against local parasites trades off with resistance against foreign parasites; in principle, these tradeoffs can be generated by negative pleiotropy of the genes conferring resistance and/or by the accumulation of conditionally neutral mutations reducing resistance against foreign parasites. Secondly, indirect costs arise if resistance trades off with other fitness traits. Our data support the existence of this type of cost: evolved hosts were more resistant than naive hosts (experiment 1) but divided less rapidly in the absence of the parasite (experiment 3). Pleiotropic effects of resistance genes can have various causes (Simms and Triplett 1994; Coustau et al. 2000; Rigby et al. 2002). In our system, the infection process involves interactions between the membranes of the digestive vacuole of the host and the parasite (Fokin et al. 2003). Similar to other systems (Frank 1994; De Wit 1997; Buckling and Rainey 2002a), this may set the stage for the evolution of genotype-specific receptors, which could explain pleiotropic effects and direct costs of resistance. Trade-offs with other fitness traits may arise if resistance evolves via changes in feeding behavior or in the functioning of digestive vacuoles, thus providing a direct physiological link with resource acquisition and division rates. Direct and indirect costs of resistance can counteract the spread to fixation of omnipotent genotypes and provide the basis for frequency-dependent selection (Bell and Maynard Smith 1987; Nee 1989; Antonovics and Thrall 1994; Parker 1994; Agrawal and Lively 2002). The two types of cost may act in a complementary fashion. For example, had we been able to include the evolved O3 host clones (100% resistant in experiment 1, Table 1) in the cross-infection experiment, we might have found a case of universal resistance and no direct costs of resistance. However, of all five clones, evolved O3 hosts showed the strongest reduction in division rates relative to the naive hosts (Fig. 5). Thus, insufficient direct costs of resistance may be compensated by indirect costs, as proposed for classical gene-for-gene models (Agrawal and Lively 2002) and shown experimentally for the E. coli–phage system (Chao et al. 1977). All three experiments suggest that the response to selection for resistance and its associated costs varies with host genetic background (Strauss et al. 2002). Such variation in trade-off structure may provide one explanation why parasite-mediated selection on existing genetic variation in resistance is sometimes unpredictable (Little 2002). With experimental (co)evolution approaches, we can explicitly test for the effect of genetic background, by setting up replicate lines of host (and/or parasite) genotypes (e.g., Kaltz and Nidelet 2004).

Conclusions Our experiments have demonstrated genetic differentiation in freely coevolving microcosms of host and parasite, likely to be driven by antagonistic selection. The interplay between the costs and benefits of increased resistance resulted in patterns of local adaptation and in the genetic divergence of an important life-history trait (growth rate) in parasite-exposed versus parasite-free populations. As in other experimental systems (Chao et al. 1977; Buckling and Rainey 2002b), our parasite appears to lag behind in the coevolutionary race, which challenges conventional wisdom of host-parasite coevolution. Whether this race consists of repeated coevolutionary cycles can only be answered by sampling at multiple time points. Further experiments will investigate the repeatability of coevolutionary outcomes and include more realistic scenarios of metapopulation dynamics, namely patterns of migration or extinction/recolonization. ACKNOWLEDGMENTS This project was financed by a research grant ACI Jeunes Chercheurs (Ministe`re de Recherche, France) to OK and an ERASMUS program student mobility grant to KL. We thank L. Millot for maintaining the long-term cultures, J. Brusini and T. Nidelet for help with establishing the subclones, and an anonymous referee for useful comments. LITERATURE CITED Agrawal, A. F., and C. M. Lively. 2002. Infection genetics: genefor-gene versus matching-alleles models and all points in between. Evol. Ecol. Res. 4:79–90. Amann, R., N. Springer, W. Ludwig, H.-D. Go¨rtz, and K.-H. Schlaifer. 1991. Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature 351:161–164. Antonovics, J., and P. H. Thrall. 1994. The cost of resistance and the maintenance of genetic polymorphism in host-pathogen systems. Proc. R. Soc. Lond. B 257:105–110. Bell, G., and J. Maynard Smith. 1987. Short-term selection for recombination among mutually antagonistic species. Nature 328: 66–68. Bergelson, J., G. Dwyer, and J. Emerson. 2001. Models and data on plant-enemy coevolution. Annu. Rev. Genet. 35:469–499. Boots, M., and M. Begon. 1993. Trade-offs with resistance to a granulovirus in the Indian meal moth examined by a laboratory evolution experiment. Funct. Ecol. 7:528–534. Boule´treau, M. 1986. The genetic and coevolutionary interactions between parasitoids and their hosts. Pp. 160–200 in J. Waage and D. Greathead, eds. Insect parasitoids. Academic Press, London. Brockhurst, M. A., A. D. Morgan, P. B. Rainey, and A. Buckling. 2004. Population mixing accelerates coevolution. Ecol. Lett. 6: 975–979. Brown, J. K. 2003. A cost of disease resistance: paradigm or peculiarity? Trends Genet. 19:667–671. Buckling, A., and P. B. Rainey. 2002a. Antagonistic coevolution between a bacterium and a bacteriophage. Proc. R. Soc. Lond. B 269:931–936. ———. 2002b. The role of parasites in sympatric and allopatric host diversification. Nature 420:496–499. Burdon, J. J. 1987. Diseases and plant population biology. Cambridge Univ. Press, Cambridge, U.K. Capaul, M., and D. Ebert. 2003. Parasite-mediated selection in experimental Daphnia magna populations. Evolution 57:249–260. Carius, H. J., T. J. Little, and D. Ebert. 2001. Genetic variation in a host-parasite association: potential for coevolution and frequency-dependent selection. Evolution 55:1136–1145.

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