BACKGROUND
parasitic species' strategies of host exploitation fall into a very wide range, from specialists, which can infect and develop in a restricted variety of hosts, to generalists, which can use a wide range of hosts.
specialization on a given host is a selective process that increases the fitness of the parasite on this host.
if the host population is homogeneous and remains constant through time, evolution is expected to favor parasites that are best adapted to this host, and therefore select for specialist strategies.
on the other hand, the selective pressures operating in variable or heterogeneous host populations can mitigate this specialization process  <cit> .
if evolution of the parasite is rapid with respect to the time scale of variation, temporal variation in a host population might lead to repeated evolution of parasites specialized on the different hosts, as has been observed for example in bacteriophages adapting to alternate hosts  <cit> .
in a more rapidly changing or spatially heterogeneous host population, a parasite might find itself exposed to a variety of possible hosts.
in this context, theory predicts that evolution favors parasitic strategies with the highest geometric mean fitness across different hosts  <cit> .
because this geometric mean increases as the variance of fitness over the hosts decreases  <cit> , strategies associated with similar fitnesses across different hosts are positively selected.
the range of possible strategies is shaped by the assumption that specialization to one host is associated with a lower fitness on other hosts.
this cost of adaptation is generally considered to be a necessary condition for the maintenance of diversity in heterogeneous environments  <cit> .
in the case of parasitic species, an illustration of such a trade-off is the decreased ability of parasites to infect their original host after being maintained on a novel species for many generations  <cit> .
while most studies of specialization of parasites deal with differences among host species , the ideas should also be valid for genetic differences within a single host species.
trade-offs in genotype-specific specialization have been demonstrated in microbial  <cit>  as well as eukaryotic  <cit>  systems.
we provide direct experimental confirmation of these ideas and quantification of this trade-off with the microsporidian brachiola algerae , a common insect parasite  <cit> , and one of its hosts, the yellow fever mosquito aedes aegypti.
b. algerae spores infect host larvae when they are ingested, and the parasite then proliferates within host tissues as the larva grows.
mortality in a. aegypti due to b. algerae microsporidiosis is usually quite low, while the parasite's virulence is rather reflected by a delay of the host's pupation.
we let parasites evolve in three types of regimes differing in the combinations of the mosquito's genotypes .
first, in four single line regimes, the parasites were maintained on only one of the four isofemale lines.
second, in a mixture regime the parasites were maintained on an equitable mixture of the four lines, providing a regime where host population is heterogeneous.
third, in an alternating regime, the parasites were maintained on a single isofemale line in any given generation, but the line was alternated in a regular sequence among generations.
after thirteen generations, we tested the parasites of each of the evolved parasite lines on each of isofemale host line by measuring the parasites' infectivity  and the number of spores produced in infected individuals as two components of the parasite's transmission success.
the first  regime is expected to select for specialist strategies.
if specialization is costly, the performance of specialized parasites on other host lines is expected to be significantly lower, while parasites evolved in the second and third regimes should exhibit intermediate success across different host lines.
RESULTS
after  <dig> generations of evolution, the parasites had developed clear differences in infectivity .
thus, adapted parasites  infected  <dig> %  of the larvae, parasites that had evolved in a mixture of isofemale lines infected  <dig> % , parasites that had evolved on an alternating sequence of isofemale lines infected  <dig> % , and mismatched parasites  infected  <dig> % .
the two regimes that were expected to lead to generalists  did not differ significantly in their infectivity, but both differed from adapted and mismatched parasites.
in contrast, when we considered only the mosquitoes that were infected, we detected no significant differences in the log-transformed number of spores among the four types of parasites .
the analysis compares the four types of tests: specialized parasites on their own host, specialized parasites on mismatched hosts, parasites evolved on mixtures of hosts and parasites evolved on alternating hosts.
'evolution regime' refers to the isofemale line  the parasites evolved on.
to test more formally whether the parasites had indeed specialized on their hosts, i.e.
whether they adapted to the hosts they had evolved on, we compared the adapted parasites with the mismatched ones.
the analysis was done in two steps .
first, we performed a logistic analysis  or anova  that included the interaction between selection line and test line.
in a second step, we split the interaction term into two components, the first describing the difference between adapted and mismatched combinations and the second accounting for the rest of the interaction .
the analysis showed that the difference between adapted and mismatched parasites explained a significant component of the variation of the parasites' infectivity , confirming that the parasites had indeed evolved to be more infectious on the isofemale line they had evolved on.
however, as in the first analysis we did not observe any specialization concerning the number of spores within infected hosts.
the analysis compares only specialized parasites tested on their own host and specialized parasites tested on mismatched hosts.
'isofemale line' refers the isofemale line the parasites evolved on; 'test line' is the isofemale line the evolved parasites were tested on.
in , the factors written in italics are the components of the previous interaction  note that the second component of the interaction testline * replicate is pooled into the error term.
the component 'adapted'  gives the importance of specialization onto a given host line.
in  we did not decompose the interaction terms, as they were far from statistically significant.
the cost of specialization is illustrated in fig.
<dig>  where we plot, for each parasite, its mean index values on the three mismatched hosts  against its value on a matched host.
our results show a negative correlation between the two types of index values , confirming the cost of specialization in this system.
discussion
our results confirm the idea that homogeneous and constant host populations let parasites evolve to be specialists on certain genotypes of the host, while diverse or temporally variable populations lead to the evolution of generalist strategies.
it is striking that this specialization is reflected in the probability that the parasites infect their host, but not in the number of spores  once the host is infected.
this might correspond to the lack of a known effective immune response against microsporidian parasites in insects .
alternatively, a rapid innate response could be responsible for the difference in results for infectivity and for the number of spores in successful infections.
in addition, our results confirm that specialization to a given host is associated with being less well adapted to alternative hosts.
this negative correlation is expected under the hypothesis of an evolutionary cost of adaptation, i.e.
that increased success on one type of hosts is associated with decreased success on other  hosts.
previous studies  have observed such a cost, and indeed it seems likely that in the long term there must be a cost of adaptation  <cit> , although this is not always observed  <cit> .
it should be noted that our experimental design does not allow us to demonstrate what evolutionary processes led to the observed differences in infectivity.
in particular, our interpretations are limited by the impossibility to preserve the ancestral parasite strain throughout the time of the experiment.
because b. algerae spores cannot be viably stored or frozen for a long period of time, they have to be maintained on their hosts, which renders the true ancestral state unavailable.
we are also limited in our knowledge of the existing diversity within the microsporidian population, which hinders our understanding of the evolutionary processes at work in this study.
therefore, we interpret the observed trade-off as a cost only in the general sense that infectivity on non-passaged lines is lower than that on passaged lines, but cannot attribute these differences specifically to an increased infectivity of specialists on the line in which they were passaged, a decreased infectivity on the other lines, or a combination of both.
these conclusions rely on the critical assumption that parasites that had evolved on a mixture of hosts behave as one generalist strain, as opposed to a mix of specialized strains.
without a direct assessment of the genotypic diversity within that strain, it is impossible to disprove this alternative hypothesis.
we believe however that we are indeed in presence of a generalist strain, based on two main observations.
first, the results remain qualitatively similar when we use the temporally variable alternating regime as a source of generalists, a regime intuitively less likely to maintain a mix of specialists from generation to generation.
second, if this strain was in fact a mix of specialist strains, it should be able to infect each isofemale line as effectively as the best adapted specialist.
therefore, as long as infectivity is not limited by the concentration of spores, such a mix of specialists would not explain the observed lower infectivity.
we chose our spore concentration  to be fairly high to avoid as much as possible these potential dose effects, so that our preliminary tests as well as other studies  <cit>  confirm that lower concentrations lead to similar levels of infectivity.
one important caveat of this interpretation comes from the limited number of generations that we used in our selection treatments.
an alternative explanation for the reduced infectivity of parasites evolved on a mixture of hosts is that they have had less time to adapt to each of their hosts  <cit> .
under this hypothesis, even without any cost to adaptation, it is expected that parasites selected on a mixture of hosts would initially exhibit a lower success on a single host line because of the lower time spent within this host, but ultimately reach similar infectivity if selection is carried out for a long enough time.
therefore, the observed pattern could be due to the difference in true selection time rather than to a cost of adaptation if the duration of our selection phase  falls in the range during which such transient differences would be observed.
our choice of  <dig> generations was limited by practical considerations, and the relatively long generation time of our mosquito host for experimental evolution studies.
to discriminate between these hypotheses, similar experiments could be carried out with an extended selection period.
if there is a cost of adaptation, differences in infectivity will hold no matter how long selection acts, while under the alternative hypothesis of differences in time on each host, longer selection would eventually lead to similar infectivities.
if the observed differences are indeed due to a cost of specialization, the underlying genetic mechanism could be antagonistic pleiotropy, where the alleles responsible for the adaptation to one environment are detrimental in other environments  <cit> , or accumulation of mutations that have no or an only slightly deleterious effect in the evolved environment, but are strongly deleterious and thus become an important load in other environments  <cit> .
both mechanisms have received empirical support from other studies of ecological specialization  <cit> .
although our experiment does not enable us to discriminate between the two possibilities, the accumulation of deleterious mutations seems less probable, as the number of mutations is limited by a small number of generations and by a bottleneck of the size of the parasite's population when hosts ingest the parasite's spores.
therefore, it is more likely that the trade-off revealed in our experiment is based on antagonistic pleiotropy of the alleles involved in the adaptation of b. algerae to its host.
because of the cost of specialization, generalist strategies should be expected to have higher average success in population with diverse hosts.
indeed, if we pool the data from the four isofemale lines, the parasites that had evolved in the two generalist regimes  tended to have higher infectivity on average  than the specialist parasites , although this difference was not quite statistically significant .
however, the long-term fitness of our parasites in heterogeneous environments should be assessed by the geometric mean fitness  rather than the above average infectivities.
although this gmf cannot be calculated here without measuring fitness across several generations, one might observe that the specialist strategies are associated with higher variance of infectivity across the genotypes of the host .
given that gmf decreases with the variance of the fitness across generations  <cit> , we can speculate that the above difference in average infectivity is an underestimation of the actual difference in gmf between specialists and generalists.
thus, specialization on a specific genotype does not pay in a heterogeneous or varying environment, enabling generalist strategies to dominate populations.
CONCLUSIONS
overall, our experiment provided experimental support for the two main ideas about the evolution of specialization by giving two clear results.
first, the microsporidian parasite had a high potential for specialization on individual genotypes of its host.
indeed, parasites that had evolved for thirteen generations on a specific isofemale line of their host were about 10% more likely to infect this isofemale line than if they had evolved on a mixture of isofemale lines.
second, the adaptation to a given host genotype was costly in that it led to a lower infectivity on other isofemale lines.
because of this cost of specialization, generalist strategies were, on average, more successful over the four mosquito lines used in the experiment.
this cost, preventing the evolution of a generalist that would be superior on all genotypes, could explain the high level of specificity generally observed in invertebrate host-parasite interactions  <cit> .

 parasitic species' strategies of host exploitation fall into a very wide range, from specialists, which can infect and develop in a restricted variety of hosts, to generalists, which can use a wide range of hosts. specialization on a given host is a selective process that increases the fitness of the parasite on this host. if the host population is homogeneous and remains constant through time, evolution is expected to favor parasites that are best adapted to this host, and therefore select for specialist strategies. on the other hand, the selective pressures operating in variable or heterogeneous host populations can mitigate this specialization process. if evolution of the parasite is rapid with respect to the time scale of variation, temporal variation in a host population might lead to repeated evolution of parasites specialized on the different hosts, as has been observed for example in bacteriophages adapting to alternate hosts. in a more rapidly changing or spatially heterogeneous host population, a parasite might find itself exposed to a variety of possible hosts. in this context, theory predicts that evolution favors parasitic strategies with the highest geometric mean fitness across different hosts. because this geometric mean increases as the variance of fitness over the hosts decreases, strategies associated with similar fitnesses across different hosts are positively selected. the range of possible strategies is shaped by the assumption that specialization to one host is associated with a lower fitness on other hosts. this cost of adaptation is generally considered to be a necessary condition for the maintenance of diversity in heterogeneous environments. in the case of parasitic species, an illustration of such a trade-off is the decreased ability of parasites to infect their original host after being maintained on a novel species for many generations. while most studies of specialization of parasites deal with differences among host species, the ideas should also be valid for genetic differences within a single host species. trade-offs in genotype-specific specialization have been demonstrated in microbial as well as eukaryotic systems. we provide direct experimental confirmation of these ideas and quantification of this trade-off with the microsporidian brachiola algerae, a common insect parasite, and one of its hosts, the yellow fever mosquito aedes aegypti. b. algerae spores infect host larvae when they are ingested, and the parasite then proliferates within host tissues as the larva grows. mortality in a. aegypti due to b. algerae microsporidiosis is usually quite low, while the parasite's virulence is rather reflected by a delay of the host's pupation. we let parasites evolve in three types of regimes differing in the combinations of the mosquito's genotypes. first, in four single line regimes, the parasites were maintained on only one of the four isofemale lines. second, in a mixture regime the parasites were maintained on an equitable mixture of the four lines, providing a regime where host population is heterogeneous. third, in an alternating regime, the parasites were maintained on a single isofemale line in any given generation, but the line was alternated in a regular sequence among generations. after thirteen generations, we tested the parasites of each of the evolved parasite lines on each of isofemale host line by measuring the parasites' infectivity and the number of spores produced in infected individuals as two components of the parasite's transmission success. the first regime is expected to select for specialist strategies. if specialization is costly, the performance of specialized parasites on other host lines is expected to be significantly lower, while parasites evolved in the second and third regimes should exhibit intermediate success across different host lines.  after generations of evolution, the parasites had developed clear differences in infectivity. thus, adapted parasites infected % of the larvae, parasites that had evolved in a mixture of isofemale lines infected %, parasites that had evolved on an alternating sequence of isofemale lines infected %, and mismatched parasites infected %. the two regimes that were expected to lead to generalists did not differ significantly in their infectivity, but both differed from adapted and mismatched parasites. in contrast, when we considered only the mosquitoes that were infected, we detected no significant differences in the log-transformed number of spores among the four types of parasites. the analysis compares the four types of tests: specialized parasites on their own host, specialized parasites on mismatched hosts, parasites evolved on mixtures of hosts and parasites evolved on alternating hosts.'evolution regime' refers to the isofemale line the parasites evolved on. to test more formally whether the parasites had indeed specialized on their hosts, i.e. whether they adapted to the hosts they had evolved on, we compared the adapted parasites with the mismatched ones. the analysis was done in two steps. first, we performed a logistic analysis or anova that included the interaction between selection line and test line. in a second step, we split the interaction term into two components, the first describing the difference between adapted and mismatched combinations and the second accounting for the rest of the interaction. the analysis showed that the difference between adapted and mismatched parasites explained a significant component of the variation of the parasites' infectivity, confirming that the parasites had indeed evolved to be more infectious on the isofemale line they had evolved on. however, as in the first analysis we did not observe any specialization concerning the number of spores within infected hosts. the analysis compares only specialized parasites tested on their own host and specialized parasites tested on mismatched hosts.'isofemale line' refers the isofemale line the parasites evolved on;'test line' is the isofemale line the evolved parasites were tested on. in, the factors written in italics are the components of the previous interaction note that the second component of the interaction testline * replicate is pooled into the error term. the component'adapted' gives the importance of specialization onto a given host line. in we did not decompose the interaction terms, as they were far from statistically significant. the cost of specialization is illustrated in fig. where we plot, for each parasite, its mean index values on the three mismatched hosts against its value on a matched host. our results show a negative correlation between the two types of index values, confirming the cost of specialization in this system. discussion our results confirm the idea that homogeneous and constant host populations let parasites evolve to be specialists on certain genotypes of the host, while diverse or temporally variable populations lead to the evolution of generalist strategies. it is striking that this specialization is reflected in the probability that the parasites infect their host, but not in the number of spores once the host is infected. this might correspond to the lack of a known effective immune response against microsporidian parasites in insects. alternatively, a rapid innate response could be responsible for the difference in results for infectivity and for the number of spores in successful infections. in addition, our results confirm that specialization to a given host is associated with being less well adapted to alternative hosts. this negative correlation is expected under the hypothesis of an evolutionary cost of adaptation, i.e. that increased success on one type of hosts is associated with decreased success on other hosts. previous studies have observed such a cost, and indeed it seems likely that in the long term there must be a cost of adaptation, although this is not always observed. it should be noted that our experimental design does not allow us to demonstrate what evolutionary processes led to the observed differences in infectivity. in particular, our interpretations are limited by the impossibility to preserve the ancestral parasite strain throughout the time of the experiment. because b. algerae spores cannot be viably stored or frozen for a long period of time, they have to be maintained on their hosts, which renders the true ancestral state unavailable. we are also limited in our knowledge of the existing diversity within the microsporidian population, which hinders our understanding of the evolutionary processes at work in this study. therefore, we interpret the observed trade-off as a cost only in the general sense that infectivity on non-passaged lines is lower than that on passaged lines, but cannot attribute these differences specifically to an increased infectivity of specialists on the line in which they were passaged, a decreased infectivity on the other lines, or a combination of both. these conclusions rely on the critical assumption that parasites that had evolved on a mixture of hosts behave as one generalist strain, as opposed to a mix of specialized strains. without a direct assessment of the genotypic diversity within that strain, it is impossible to disprove this alternative hypothesis. we believe however that we are indeed in presence of a generalist strain, based on two main observations. first, the results remain qualitatively similar when we use the temporally variable alternating regime as a source of generalists, a regime intuitively less likely to maintain a mix of specialists from generation to generation. second, if this strain was in fact a mix of specialist strains, it should be able to infect each isofemale line as effectively as the best adapted specialist. therefore, as long as infectivity is not limited by the concentration of spores, such a mix of specialists would not explain the observed lower infectivity. we chose our spore concentration to be fairly high to avoid as much as possible these potential dose effects, so that our preliminary tests as well as other studies confirm that lower concentrations lead to similar levels of infectivity. one important caveat of this interpretation comes from the limited number of generations that we used in our selection treatments. an alternative explanation for the reduced infectivity of parasites evolved on a mixture of hosts is that they have had less time to adapt to each of their hosts. under this hypothesis, even without any cost to adaptation, it is expected that parasites selected on a mixture of hosts would initially exhibit a lower success on a single host line because of the lower time spent within this host, but ultimately reach similar infectivity if selection is carried out for a long enough time. therefore, the observed pattern could be due to the difference in true selection time rather than to a cost of adaptation if the duration of our selection phase falls in the range during which such transient differences would be observed. our choice of generations was limited by practical considerations, and the relatively long generation time of our mosquito host for experimental evolution studies. to discriminate between these hypotheses, similar experiments could be carried out with an extended selection period. if there is a cost of adaptation, differences in infectivity will hold no matter how long selection acts, while under the alternative hypothesis of differences in time on each host, longer selection would eventually lead to similar infectivities. if the observed differences are indeed due to a cost of specialization, the underlying genetic mechanism could be antagonistic pleiotropy, where the alleles responsible for the adaptation to one environment are detrimental in other environments, or accumulation of mutations that have no or an only slightly deleterious effect in the evolved environment, but are strongly deleterious and thus become an important load in other environments. both mechanisms have received empirical support from other studies of ecological specialization. although our experiment does not enable us to discriminate between the two possibilities, the accumulation of deleterious mutations seems less probable, as the number of mutations is limited by a small number of generations and by a bottleneck of the size of the parasite's population when hosts ingest the parasite's spores. therefore, it is more likely that the trade-off revealed in our experiment is based on antagonistic pleiotropy of the alleles involved in the adaptation of b. algerae to its host. because of the cost of specialization, generalist strategies should be expected to have higher average success in population with diverse hosts. indeed, if we pool the data from the four isofemale lines, the parasites that had evolved in the two generalist regimes tended to have higher infectivity on average than the specialist parasites, although this difference was not quite statistically significant. however, the long-term fitness of our parasites in heterogeneous environments should be assessed by the geometric mean fitness rather than the above average infectivities. although this gmf cannot be calculated here without measuring fitness across several generations, one might observe that the specialist strategies are associated with higher variance of infectivity across the genotypes of the host. given that gmf decreases with the variance of the fitness across generations, we can speculate that the above difference in average infectivity is an underestimation of the actual difference in gmf between specialists and generalists. thus, specialization on a specific genotype does not pay in a heterogeneous or varying environment, enabling generalist strategies to dominate populations.  overall, our experiment provided experimental support for the two main ideas about the evolution of specialization by giving two clear results. first, the microsporidian parasite had a high potential for specialization on individual genotypes of its host. indeed, parasites that had evolved for thirteen generations on a specific isofemale line of their host were about 10% more likely to infect this isofemale line than if they had evolved on a mixture of isofemale lines. second, the adaptation to a given host genotype was costly in that it led to a lower infectivity on other isofemale lines. because of this cost of specialization, generalist strategies were, on average, more successful over the four mosquito lines used in the experiment. this cost, preventing the evolution of a generalist that would be superior on all genotypes, could explain the high level of specificity generally observed in invertebrate host-parasite interactions. 
evolutionary theory predicts that the pressure for parasites to specialize on one host or to become generalists on a wide range of hosts is driven by the diversity or temporal variability of the host's population and by genetic trade-offs in the adaptation to different hosts. the success of a specialist on its matched mosquito line was negatively correlated with its success on other lines, suggesting an evolutionary cost to specialization. the specialized parasites were most infective on their own isofemale line and least infective on other isofemale lines, while the generalist parasites had intermediate infection success on all lines. this trade-off was corroborated by the observation that the generalists had higher average mean infectivity than the specialists over all isofemale lines. overall, our experiment reveals the potential for specialization of a parasite to individual genotypes of its host and provides experimental evidence of the cost associated with the evolution of specialization, an important feature for understanding the coevolutionary dynamics between hosts and parasites.
 evolutionary theory predicts that the pressure for parasites to specialize on one host or to become generalists on a wide range of hosts is driven by the diversity or temporal variability of the host's population and by genetic trade-offs in the adaptation to different hosts. we give experimental evidence for this idea by letting the parasite brachiola algerae evolve on one of four genetically homogeneous lines of the mosquito aedes aegypti, on a mixture of the four lines or on an alternating sequence of the four lines. the first regime was expected to lead to specialists, the other two to generalists. after generations, we tested the evolved parasites on each of the four lines of the mosquito.  the specialized parasites were most infective on their own isofemale line and least infective on other isofemale lines, while the generalist parasites had intermediate infection success on all lines. the success of a specialist on its matched mosquito line was negatively correlated with its success on other lines, suggesting an evolutionary cost to specialization. this trade-off was corroborated by the observation that the generalists had higher average mean infectivity than the specialists over all isofemale lines.  overall, our experiment reveals the potential for specialization of a parasite to individual genotypes of its host and provides experimental evidence of the cost associated with the evolution of specialization, an important feature for understanding the coevolutionary dynamics between hosts and parasites. 