Background Coevolution between pairs of antagonistic species is generally considered an endless "arms race" between attack and defense traits to counteract the adaptive responses of the other species.
Background
Coevolution between pairs of antagonistic species is generally considered an endless "arms race" between attack and defense traits to counteract the adaptive responses of the other species.Presentation of the hypothesis When more than two species are involved, diffuse coevolution of hosts and parasitoids could be asymmetric because consumers can choose their prey whereas preys do not choose their predator. This asymmetry may lead to differences in the rate of evolution of the antagonistic species in response to selection. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of the hypothesis is that the consumer trophic level is more likely to win the coevolution race.
Presentation of the hypothesis
When more than two species are involved, diffuse coevolution of hosts and parasitoids could be asymmetric because consumers can choose their prey whereas preys do not choose their predator. This asymmetry may lead to differences in the rate of evolution of the antagonistic species in response to selection. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of the hypothesis is that the consumer trophic level is more likely to win the coevolution race.Testing the hypothesis We propose testing the asymmetry hypothesis by focusing on the tritrophic system plant/aphid/aphid parasitoid. The analysis of the genetic variability in the virulence of several parasitoid populations and in the defenses of several aphid species or several clones of the same aphid species could be compared. Moreover, the analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation may complement the detection of differences between host and parasitoid trophic specialization.
Testing the hypothesis
We propose testing the asymmetry hypothesis by focusing on the tritrophic system plant/aphid/aphid parasitoid. The analysis of the genetic variability in the virulence of several parasitoid populations and in the defenses of several aphid species or several clones of the same aphid species could be compared. Moreover, the analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation may complement the detection of differences between host and parasitoid trophic specialization.Implications of the hypothesis Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots. Thus this hypothesis profoundly changes our understanding of coevolution and may have important implications in terms of pest management.
Implications of the hypothesis
Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots. Thus this hypothesis profoundly changes our understanding of coevolution and may have important implications in terms of pest management.

Background Coevolution is the result of reciprocal selective pressures exerted by interacting species. Many studies have been devoted to the hypothesis of an endless "arms race" between antagonistic species, in which each species develops escalating attack and defense traits to counteract the adaptive responses of the other species. In reference to Lewis Carroll's book "Through the Looking Glass", Van Valen [ 1 ] named this model of coevolution "the Red Queen Hypothesis" (RQH) because, even in a constant physical environment, interacting species must evolve continuously to maintain their position. The RQH can be seen as an arms race between "resistance" and "virulence" where, following recent reviews on coevolution (e.g. [ 2 ]), "resistance" is the target's ability to survive attacks by the consumer, and "virulence" is the consumer's ability to defeat the target's defenses. The RQH was initially developed in the context of multiple species interactions, to account for the constant probability of species extinction. However, as modeling the coevolution of many species involves a number of difficulties, later studies based on the RQH have mostly been limited to interactions between pairs of species. From this restricted situation, two main predictions can be made: first, arms races induce an advantage of rare genotypes of which resistance or virulence is more efficient and thus, frequency-dependent fluctuations of resistance and virulence may be predicted. Second, a geographic view of the coevolutionary process suggests a dynamic mosaic structure, with local and temporary "hot spots" of antagonistic species coevolution [ 3 ]. However, for plant-pathogen interactions [ 4 ] and for animal host-parasite interactions [ 5 - 7 ], empirical observations and experimental tests of RQH have not given entirely convincing results. For instance, there is considerable evidence of genetic variation for resistance, but parasite-driven genetic change in resistance has never been observed directly [ 8 ]. Conversely, in certain systems of interacting species, matching genetic diversity, which is expected under the RQH, is lacking [ 9 ]. Another study [ 2 ] has suggested that, for a given host-parasitoid association, the rank order of survival of different host strains exposed to different parasitoid strains remains constant. This lack of frequency dependence has been interpreted as evidence against the RQH. Nevertheless, the RQH continues to lie at the heart of the debate concerning the coevolution of antagonistic species, probably because of the lack of plausible alternative hypotheses. Although metapopulation structures or time lags between the responses of antagonistic species to selective pressures have been evoked as reasons for the lack of clear experimental evidence in favor of the RQH [ 10 ], the additional hypothesis that is considered the most satisfactory relates to the cost of resistance [ 11 ]. Defense is thought to carry costs when it is not needed, i.e. the fitness of resistant individuals in the absence of enemies is reduced compared to susceptible individuals. Following this hypothesis, the arms race is constrained by the resistance cost and thus resistance and virulence of antagonistic species reach fixed levels that can be considered optimal. This hypothesis may result in a low level of genetic polymorphism of resistance and virulence in spatially restricted areas. In contrast, resistance and virulence are expected to be polymorphic on a larger spatial scale if, because of ecological factors, the densities of antagonistic species are spatially heterogeneous. However, although resistance costs have often been demonstrated, expected geographical patterns of resistance and virulence are not observed: antagonistic species densities are not clearly spatially associated with resistance and virulence variability levels [ 2 ]. Thus, although taking account the resistance cost hypothesis is a useful addition to Red Queen arms race models, this theoretical view of coevolutionary process does not provide predictions that fit well with most situations observed in the field [ 10 , 11 ].
Background
Coevolution is the result of reciprocal selective pressures exerted by interacting species. Many studies have been devoted to the hypothesis of an endless "arms race" between antagonistic species, in which each species develops escalating attack and defense traits to counteract the adaptive responses of the other species. In reference to Lewis Carroll's book "Through the Looking Glass", Van Valen [ 1 ] named this model of coevolution "the Red Queen Hypothesis" (RQH) because, even in a constant physical environment, interacting species must evolve continuously to maintain their position. The RQH can be seen as an arms race between "resistance" and "virulence" where, following recent reviews on coevolution (e.g. [ 2 ]), "resistance" is the target's ability to survive attacks by the consumer, and "virulence" is the consumer's ability to defeat the target's defenses.The RQH was initially developed in the context of multiple species interactions, to account for the constant probability of species extinction. However, as modeling the coevolution of many species involves a number of difficulties, later studies based on the RQH have mostly been limited to interactions between pairs of species. From this restricted situation, two main predictions can be made: first, arms races induce an advantage of rare genotypes of which resistance or virulence is more efficient and thus, frequency-dependent fluctuations of resistance and virulence may be predicted. Second, a geographic view of the coevolutionary process suggests a dynamic mosaic structure, with local and temporary "hot spots" of antagonistic species coevolution [ 3 ].However, for plant-pathogen interactions [ 4 ] and for animal host-parasite interactions [ 5 - 7 ], empirical observations and experimental tests of RQH have not given entirely convincing results. For instance, there is considerable evidence of genetic variation for resistance, but parasite-driven genetic change in resistance has never been observed directly [ 8 ]. Conversely, in certain systems of interacting species, matching genetic diversity, which is expected under the RQH, is lacking [ 9 ]. Another study [ 2 ] has suggested that, for a given host-parasitoid association, the rank order of survival of different host strains exposed to different parasitoid strains remains constant. This lack of frequency dependence has been interpreted as evidence against the RQH.Nevertheless, the RQH continues to lie at the heart of the debate concerning the coevolution of antagonistic species, probably because of the lack of plausible alternative hypotheses. Although metapopulation structures or time lags between the responses of antagonistic species to selective pressures have been evoked as reasons for the lack of clear experimental evidence in favor of the RQH [ 10 ], the additional hypothesis that is considered the most satisfactory relates to the cost of resistance [ 11 ]. Defense is thought to carry costs when it is not needed, i.e. the fitness of resistant individuals in the absence of enemies is reduced compared to susceptible individuals. Following this hypothesis, the arms race is constrained by the resistance cost and thus resistance and virulence of antagonistic species reach fixed levels that can be considered optimal. This hypothesis may result in a low level of genetic polymorphism of resistance and virulence in spatially restricted areas. In contrast, resistance and virulence are expected to be polymorphic on a larger spatial scale if, because of ecological factors, the densities of antagonistic species are spatially heterogeneous. However, although resistance costs have often been demonstrated, expected geographical patterns of resistance and virulence are not observed: antagonistic species densities are not clearly spatially associated with resistance and virulence variability levels [ 2 ]. Thus, although taking account the resistance cost hypothesis is a useful addition to Red Queen arms race models, this theoretical view of coevolutionary process does not provide predictions that fit well with most situations observed in the field [ 10 , 11 ].Presentation of the hypothesis Adaptive responses to selective pressures exerted by antagonistic organisms are not necessarily the same for the target species and the consumer species. There is a first level of potential evolutionary asymmetry between them because the failure of virulence for a consumer is a delay in fitness acquisition whereas the failure of defense for a prey is the loss of its entire fitness. Such a difference was underlined by Dawkins [ 12 ] noting Aesop's fable of the hare outrunning the fox, because the former runs for life whereas the latter runs for a meal. The opposite asymmetry has been suggested in the case of host-parasitoid relationships [ 2 , 13 ]: all consumers are obliged to overcome the defenses of their target whereas not all targets suffer attacks from consumers. Extending the field of interest to interactions and coevolution between more than two species (i.e. diffuse coevolution) may offer new perspectives. Considering the reciprocal selective pressures exerted by many species leads us to take into account the specificity of virulence and resistance. Until now, few works have been devoted to this subject (but see [ 14 ]). However, when considering diffuse coevolution, there is a second level of potential asymmetry because a consumer may choose its targets, whereas targets cannot be certain which of its enemies will attack it. A recent model described the evolution of resistance of one host subjected to the attacks of two types of parasitoids differing in their virulence and specificity (different genotypes of a species or different species) and the evolution of virulence of one parasitoid attacking two types of hosts differing in their resistance and specificity [ 15 ]. The results suggested that the level of specialization of resistance traits was not affected by the total probability of being attacked. They also suggested that, if the probabilities of encounters fluctuate and differ between trophic levels, generalist traits of resistance and partially specialist traits of virulence are favored. Finally, this model showed that fluctuating host encounter probabilities across or within generations will promote a partial specialization of the parasitoid virulence rather than a total one. The asymmetry hypothesis (AH) may lead to differences in the rate of evolution of the antagonistic species in response to selection. For a specialist consumer capable of target choice, chosen targets constitute a more or less "constant environment". On the other hand, for a generalist defender, the diversified, facultative and fluctuating attacks by a set of enemies constitute a "variable environment". The constancy of the environment may lead to the faster adaptation of specialists (mostly consumers under AH) than of generalists (mostly targets under AH) [ 16 ]. Thus, under the asymmetry hypothesis, the evolution of host defense traits is likely to be slower than the evolution of parasitoid virulence traits. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of AH is that the consumer trophic level is more likely to win the coevolution race [ 17 ]. This may account for the paradox described by Holt & Hochberg [ 18 ] – the lack of clear published examples of an increase in host resistance after biological control using parasitoids, despite the potentially strong selective pressure associated with parasitism. The asymmetry hypothesis seems to fit well with numerous data concerning geographic structures published in the literature (reviewed in [ 15 ] but see also [ 19 ]). In most of these studies, the interaction trait under consideration is the capability of  Drosophila  species to encapsulate parasitoid eggs. In this case, it can be also noted that the resistance trait is not specific (artificial eggs are encapsulated as well as parasitoid ones) when virulence traits (reviewed in [ 20 ]) are diversified and specialized (i.e., escaping the host's immune response, just swamping the host with virus-like particles, or mimicking host tissue on the egg surface).
Presentation of the hypothesis
Adaptive responses to selective pressures exerted by antagonistic organisms are not necessarily the same for the target species and the consumer species. There is a first level of potential evolutionary asymmetry between them because the failure of virulence for a consumer is a delay in fitness acquisition whereas the failure of defense for a prey is the loss of its entire fitness. Such a difference was underlined by Dawkins [ 12 ] noting Aesop's fable of the hare outrunning the fox, because the former runs for life whereas the latter runs for a meal. The opposite asymmetry has been suggested in the case of host-parasitoid relationships [ 2 , 13 ]: all consumers are obliged to overcome the defenses of their target whereas not all targets suffer attacks from consumers.Extending the field of interest to interactions and coevolution between more than two species (i.e. diffuse coevolution) may offer new perspectives. Considering the reciprocal selective pressures exerted by many species leads us to take into account the specificity of virulence and resistance. Until now, few works have been devoted to this subject (but see [ 14 ]). However, when considering diffuse coevolution, there is a second level of potential asymmetry because a consumer may choose its targets, whereas targets cannot be certain which of its enemies will attack it.A recent model described the evolution of resistance of one host subjected to the attacks of two types of parasitoids differing in their virulence and specificity (different genotypes of a species or different species) and the evolution of virulence of one parasitoid attacking two types of hosts differing in their resistance and specificity [ 15 ]. The results suggested that the level of specialization of resistance traits was not affected by the total probability of being attacked. They also suggested that, if the probabilities of encounters fluctuate and differ between trophic levels, generalist traits of resistance and partially specialist traits of virulence are favored. Finally, this model showed that fluctuating host encounter probabilities across or within generations will promote a partial specialization of the parasitoid virulence rather than a total one.The asymmetry hypothesis (AH) may lead to differences in the rate of evolution of the antagonistic species in response to selection. For a specialist consumer capable of target choice, chosen targets constitute a more or less "constant environment". On the other hand, for a generalist defender, the diversified, facultative and fluctuating attacks by a set of enemies constitute a "variable environment". The constancy of the environment may lead to the faster adaptation of specialists (mostly consumers under AH) than of generalists (mostly targets under AH) [ 16 ]. Thus, under the asymmetry hypothesis, the evolution of host defense traits is likely to be slower than the evolution of parasitoid virulence traits. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of AH is that the consumer trophic level is more likely to win the coevolution race [ 17 ]. This may account for the paradox described by Holt & Hochberg [ 18 ] – the lack of clear published examples of an increase in host resistance after biological control using parasitoids, despite the potentially strong selective pressure associated with parasitism.The asymmetry hypothesis seems to fit well with numerous data concerning geographic structures published in the literature (reviewed in [ 15 ] but see also [ 19 ]). In most of these studies, the interaction trait under consideration is the capability of  Drosophila  species to encapsulate parasitoid eggs. In this case, it can be also noted that the resistance trait is not specific (artificial eggs are encapsulated as well as parasitoid ones) when virulence traits (reviewed in [ 20 ]) are diversified and specialized (i.e., escaping the host's immune response, just swamping the host with virus-like particles, or mimicking host tissue on the egg surface).DrosophilaTesting the hypothesis We propose a dedicated test of the hypothesis of the specialization of the parasitoid virulence and the absence of specialization in the defense of the aphids, against the RQH. The study will focus on the tritrophic system plant/aphid/aphid parasitoid. Two complementary experimental approaches could be considered: A- The analysis of the genetic variability of virulence and defense of the parasitoid  Lysiphlebus testaceipes  and the aphid  Aphis gossypii . To measure this variability, several populations of the parasitoid collected on different clones of the aphid will be confronted to different clones of this aphid. The different clones of the aphid will be confronted to the different parasitoid populations. This system that considers intra specific genetic variability for each trophic level will be used to evaluate the fitness of the consumers and of the targets in the different parasitoid /aphid combinations. An important point is that virulence will be estimated from the success rate of parasitism (i.e. the production of offspring) whereas defense will be estimated through the measure of the aphid fitness, i.e. the number of offspring produced by the aphid whatever the outcome of the parasitism (success or failure). Two key factors of the host fitness will be considered: 1) the host survival rate in case of parasitism failure (an aphid may die or not, even if the parasitism does not result in parasitoid offspring production); and 2) the number of offspring produced by the host after parasitism: after a parasitoid's sting, the host may produce offspring until mummification in case of parasitoid embryo development or until a variable date in the case of parasitism failure. B- The analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation. The specialization of  L. testaceipes  on different aphid clones can lead to a neutral genetic differentiation of the parasitoid as a function of the host and the plant because the reproduction of the parasitoid occurs soon after the emergence of the adults from their host. The genetic differentiation between populations of the parasitoid sampled from different aphid clones and from different plant species could be evaluated and compared to a putative effect of isolation by distance. This test may allow local verification or rejection of the predictions of the AH (specialization of the parasitoid and generalism of the host). When performed several times on animals from diverse geographic origins, this should eventually allow rejection of the classical interpretation of RQH and the host spots theory of coevolution [ 3 ].
Testing the hypothesis
We propose a dedicated test of the hypothesis of the specialization of the parasitoid virulence and the absence of specialization in the defense of the aphids, against the RQH. The study will focus on the tritrophic system plant/aphid/aphid parasitoid. Two complementary experimental approaches could be considered: A- The analysis of the genetic variability of virulence and defense of the parasitoid  Lysiphlebus testaceipes  and the aphid  Aphis gossypii . To measure this variability, several populations of the parasitoid collected on different clones of the aphid will be confronted to different clones of this aphid. The different clones of the aphid will be confronted to the different parasitoid populations. This system that considers intra specific genetic variability for each trophic level will be used to evaluate the fitness of the consumers and of the targets in the different parasitoid /aphid combinations. An important point is that virulence will be estimated from the success rate of parasitism (i.e. the production of offspring) whereas defense will be estimated through the measure of the aphid fitness, i.e. the number of offspring produced by the aphid whatever the outcome of the parasitism (success or failure). Two key factors of the host fitness will be considered: 1) the host survival rate in case of parasitism failure (an aphid may die or not, even if the parasitism does not result in parasitoid offspring production); and 2) the number of offspring produced by the host after parasitism: after a parasitoid's sting, the host may produce offspring until mummification in case of parasitoid embryo development or until a variable date in the case of parasitism failure. B- The analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation. The specialization of  L. testaceipes  on different aphid clones can lead to a neutral genetic differentiation of the parasitoid as a function of the host and the plant because the reproduction of the parasitoid occurs soon after the emergence of the adults from their host. The genetic differentiation between populations of the parasitoid sampled from different aphid clones and from different plant species could be evaluated and compared to a putative effect of isolation by distance.Lysiphlebus testaceipesAphis gossypiiL. testaceipesThis test may allow local verification or rejection of the predictions of the AH (specialization of the parasitoid and generalism of the host). When performed several times on animals from diverse geographic origins, this should eventually allow rejection of the classical interpretation of RQH and the host spots theory of coevolution [ 3 ].Implications of the hypothesis The consequences of the asymmetry hypothesis are important. Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots [ 17 ]. However some consequences of the AH are compatible with the resistance cost or the Red Queen hypotheses. Under the AH, a low variability in generalist resistance level, as expected under the hypothesis of regulation by the cost of resistance, would be selected for locally by the global pressure exerted by all the species of the upper trophic level. Also, higher levels of local variability in consumer specialist virulence would be selected, as in situations for which the classical RQH holds true. The AH cannot be applied to every situation. In the case of host-parasitoid associations, the high level of genetic variability in resistance observed within some local host populations [ 20 , 21 ] is better explained by RQH if one dominant parasitoid species is present rather than a complex of species. Moreover, as generalism can be seen as a bet-hedging response to unpredictable environmental variations [ 15 ], the advantages of specificity or generalism depend on the level of such fluctuations. One can predict that host defenses will be less generalist when facing more stable enemy communities. Another key point deals with the supposed difference of speed in evolutionary responses between generalist and specialist traits. It may depend on other important biological factors like the discrepancy between population sizes, generation times and the reproductive mode (e.g. a sexual parasitoid versus an asexual aphid) or the genetic make up (e.g. an haplo-diploid parasitoid versus a diploid aphid) of the considered species. The relative speed of evolutionary responses of antagonistic species has thus to be evaluated in each biological model studied. Asymmetry may be suspected in cases involving successive trophic levels other than host-parasitoid associations, such as plant-insect or parasitoid-hyperparasitoid combinations, as soon as individuals of the upper trophic level can choose their target. However, random attacks, such as those due to plant pathogens, may favor more generalist traits of virulence, but the specificity of consumers, and therefore asymmetry, may be restored through indirect choices: pathogens transmitted by vectors may use or manipulate the specificity traits of the vector. Large sets of species interactions may thus lead to asymmetric coevolution. The AH may have consequences in terms of pest management. For instance, it is generally thought that variations in parasitism outcome result from a variability of host resistance due to the selection for higher resistance [ 2 , 22 ]. However, under the AH, virulence of the parasitoid is expected to evolve faster than does the resistance of the host. Therefore, the asymmetry hypothesis implies that variations in parasitism outcome more probably result from variability in parasitoid virulence. Asymmetry could also help to understand some surprising resistance-virulence patterns issuing from biological control. For instance, in the U.S.A., the pea aphid  Acyrthosiphon pisum  is parasitized by the hymenopteran  Aphidius ervi  and is specialized on different plant species. Hufbauer & Via [ 23 ] observed that pea aphids that are specialized on alfalfa are successfully parasitized less often than are pea aphids specialized on clover. Hufbauer [ 24 ] also observed that parasitoids collected from alfalfa and clover fields do not differ in their ability to overcome pea aphid resistance. They concluded that alfalfa host population is more resistant to  A. ervi  than is the clover host population. If the association they studied dealt with two aphid and one parasitoid populations, under the AH, virulence specialization rather than resistance level variations can explain the observations: the parasitoid population is specialized on the clover host population, but keeps a partial virulence on the alfalfa aphid biotype. This suggests that short-term parasitoid specialization may be a key factor in biological control efficiency. For instance, consumers introduced to control a pest could rapidly specialize against non-target hosts. The asymmetry hypothesis thus provides food for thought concerning diffuse coevolution and could be applied to domains beyond host-parasitoid coevolution. Similar thoughts may be applicable to the durability and efficiency of plant resistance or immunological responses to diseases transmitted by vectors. Its theoretical implications and its consequences in terms of population management are potentially important and remain unexplored.
Implications of the hypothesis
The consequences of the asymmetry hypothesis are important. Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots [ 17 ]. However some consequences of the AH are compatible with the resistance cost or the Red Queen hypotheses. Under the AH, a low variability in generalist resistance level, as expected under the hypothesis of regulation by the cost of resistance, would be selected for locally by the global pressure exerted by all the species of the upper trophic level. Also, higher levels of local variability in consumer specialist virulence would be selected, as in situations for which the classical RQH holds true.The AH cannot be applied to every situation. In the case of host-parasitoid associations, the high level of genetic variability in resistance observed within some local host populations [ 20 , 21 ] is better explained by RQH if one dominant parasitoid species is present rather than a complex of species. Moreover, as generalism can be seen as a bet-hedging response to unpredictable environmental variations [ 15 ], the advantages of specificity or generalism depend on the level of such fluctuations. One can predict that host defenses will be less generalist when facing more stable enemy communities. Another key point deals with the supposed difference of speed in evolutionary responses between generalist and specialist traits. It may depend on other important biological factors like the discrepancy between population sizes, generation times and the reproductive mode (e.g. a sexual parasitoid versus an asexual aphid) or the genetic make up (e.g. an haplo-diploid parasitoid versus a diploid aphid) of the considered species. The relative speed of evolutionary responses of antagonistic species has thus to be evaluated in each biological model studied.Asymmetry may be suspected in cases involving successive trophic levels other than host-parasitoid associations, such as plant-insect or parasitoid-hyperparasitoid combinations, as soon as individuals of the upper trophic level can choose their target. However, random attacks, such as those due to plant pathogens, may favor more generalist traits of virulence, but the specificity of consumers, and therefore asymmetry, may be restored through indirect choices: pathogens transmitted by vectors may use or manipulate the specificity traits of the vector. Large sets of species interactions may thus lead to asymmetric coevolution.The AH may have consequences in terms of pest management. For instance, it is generally thought that variations in parasitism outcome result from a variability of host resistance due to the selection for higher resistance [ 2 , 22 ]. However, under the AH, virulence of the parasitoid is expected to evolve faster than does the resistance of the host. Therefore, the asymmetry hypothesis implies that variations in parasitism outcome more probably result from variability in parasitoid virulence. Asymmetry could also help to understand some surprising resistance-virulence patterns issuing from biological control. For instance, in the U.S.A., the pea aphid  Acyrthosiphon pisum  is parasitized by the hymenopteran  Aphidius ervi  and is specialized on different plant species. Hufbauer & Via [ 23 ] observed that pea aphids that are specialized on alfalfa are successfully parasitized less often than are pea aphids specialized on clover. Hufbauer [ 24 ] also observed that parasitoids collected from alfalfa and clover fields do not differ in their ability to overcome pea aphid resistance. They concluded that alfalfa host population is more resistant to  A. ervi  than is the clover host population. If the association they studied dealt with two aphid and one parasitoid populations, under the AH, virulence specialization rather than resistance level variations can explain the observations: the parasitoid population is specialized on the clover host population, but keeps a partial virulence on the alfalfa aphid biotype. This suggests that short-term parasitoid specialization may be a key factor in biological control efficiency. For instance, consumers introduced to control a pest could rapidly specialize against non-target hosts.Acyrthosiphon pisumAphidius erviA. erviThe asymmetry hypothesis thus provides food for thought concerning diffuse coevolution and could be applied to domains beyond host-parasitoid coevolution. Similar thoughts may be applicable to the durability and efficiency of plant resistance or immunological responses to diseases transmitted by vectors. Its theoretical implications and its consequences in terms of population management are potentially important and remain unexplored.List of abbreviations RQH: Red Queen Hypothesis AH: Asymmetry Hypothesis
List of abbreviations
RQH: Red Queen HypothesisAH: Asymmetry HypothesisAuthors' contributions Both authors have been involved in the elaboration of the hypothesis and the evaluation of its consequences, and in the drafting of the paper.
Authors' contributions
Both authors have been involved in the elaboration of the hypothesis and the evaluation of its consequences, and in the drafting of the paper.


Background Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset.
Background
Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset.Methods To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector  Anopheles gambiae  were challenged with several isolates of the human malaria parasite  Plasmodium falciparum  (obtained from naturally infected children in Kenya).
Methods
To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector  Anopheles gambiae  were challenged with several isolates of the human malaria parasite  Plasmodium falciparum  (obtained from naturally infected children in Kenya).Anopheles gambiaePlasmodium falciparumResults Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes.
Results
Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes.Conclusions These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria.
Conclusions
These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria.

Background In the last few years, exciting advances in the biology and molecular genetics of the development of  Plasmodium  parasites in their mosquito vectors [ 1 , 2 ] have led to the creation of transgenic mosquitoes that are partially resistant to malaria infection [ 3 ], bringing the efforts to control malaria with the techniques of transgenesis a major step forward [ 4 , 5 ]. A crucial aspect of these advances is, of course, the fact that the mosquito's genetic make-up determines, at least partly, its resistance to malaria infection [ 6 , 7 ], giving hope for the possibility that key genes controlling resistance may be identified. This hope has been reinforced by the recent identification, in a rodent model of malaria, of several mosquito immune genes that affect parasite development [ 8 , 9 ]. Unfortunately, several aspects of the current knowledge make it difficult to estimate the relevance of such laboratory-based studies to control malaria in natural conditions [ 10 ]. One of the crucial aspects is that most studies on the genetics of resistance have considered the response of a mosquito line or colony to a single malaria genotype, while any malaria control programme based on the release of resistant mosquitoes in highly endemic areas can be effective only if mosquitoes are resistant to all the genotypes of the parasite [ 11 ]. Because of the limited genetic variation in laboratory colonies compared to natural populations of mosquitoes [ 12 ] and the large diversity of natural populations of malaria parasites [ 13 ], it is currently far from clear whether this will be possible. One potential problem of the genetic diversity in natural populations could be that, as in many other host-parasite systems (e.g. plant-fungus [ 14 ], snail-schistosome [ 15 ], bumble-bee-trypanosome [ 16 ],  Daphnia -bacterium [ 17 ]), the outcome of the interaction is determined by an interaction between host and parasite genotypes. In systems governed by such genotype by genotype interactions, individual hosts are resistant to only a portion of the parasite genotypes and, reciprocally, individual parasites can infect only particular host genotypes [ 18 ]. In other words, no parasite is best at infecting all hosts, and no host is best at resisting all parasites, so that the success of infection depends on the specific combination of the two opponents. Despite its potentially important role for malaria epidemiology and control, such a genetic specificity of host-parasite compatibility between malaria parasites and their insect vectors have never been investigated. This study examines the potential for genotype by genotype interactions in the combination that is most important for the epidemiology of malaria –  Plasmodium falciparum  and  Anopheles gambiae . Malaria genotype by mosquito genotype interactions were tested with a standard procedure of quantitative genetics from measurements of the resistance of different genetic backgrounds of the mosquito  A. gambiae , a major malaria vector in sub-Saharan Africa, to different isolates of the human malaria parasite  P. falciparum . The parasite isolates were obtained from naturally infected children in western Kenya that harboured gametocytes, the infective stage of the parasite. The genetic backgrounds of mosquitoes were 'mosquito families' generated as the F1 offspring of single egg-laying females. Each mosquito family was challenged with each parasite isolate, and all mosquitoes were simultaneously fed on the blood of the gametocyte carriers via membrane-feeding. This basic design was repeated three times throughout three successive experimental blocks that involved different families and isolates, giving a total of 18 mosquito families, 11 parasite isolates, and 62 specific interactions. As in previous studies [ 7 ], the resistance of mosquitoes was quantified with the proportion of blood-fed females that developed oocysts and with the number of oocysts. The genotype by genotype interaction on mosquito resistance was estimated according to standard quantitative genetic methods as the interaction effect in a statistical analysis between the parasite isolate and the mosquito family [ 17 , 19 ]. These methods are based on the idea that sibs are genetically more similar that non-sibs. Therefore, partitioning the variance of any trait (e.g. number of oocysts) among families (individuals sharing a mother) and within groups of sibs give an indication of the extent to which the trait has a genetic basis [ 20 ].
Background
In the last few years, exciting advances in the biology and molecular genetics of the development of  Plasmodium  parasites in their mosquito vectors [ 1 , 2 ] have led to the creation of transgenic mosquitoes that are partially resistant to malaria infection [ 3 ], bringing the efforts to control malaria with the techniques of transgenesis a major step forward [ 4 , 5 ]. A crucial aspect of these advances is, of course, the fact that the mosquito's genetic make-up determines, at least partly, its resistance to malaria infection [ 6 , 7 ], giving hope for the possibility that key genes controlling resistance may be identified. This hope has been reinforced by the recent identification, in a rodent model of malaria, of several mosquito immune genes that affect parasite development [ 8 , 9 ]. Unfortunately, several aspects of the current knowledge make it difficult to estimate the relevance of such laboratory-based studies to control malaria in natural conditions [ 10 ]. One of the crucial aspects is that most studies on the genetics of resistance have considered the response of a mosquito line or colony to a single malaria genotype, while any malaria control programme based on the release of resistant mosquitoes in highly endemic areas can be effective only if mosquitoes are resistant to all the genotypes of the parasite [ 11 ]. Because of the limited genetic variation in laboratory colonies compared to natural populations of mosquitoes [ 12 ] and the large diversity of natural populations of malaria parasites [ 13 ], it is currently far from clear whether this will be possible. One potential problem of the genetic diversity in natural populations could be that, as in many other host-parasite systems (e.g. plant-fungus [ 14 ], snail-schistosome [ 15 ], bumble-bee-trypanosome [ 16 ],  Daphnia -bacterium [ 17 ]), the outcome of the interaction is determined by an interaction between host and parasite genotypes. In systems governed by such genotype by genotype interactions, individual hosts are resistant to only a portion of the parasite genotypes and, reciprocally, individual parasites can infect only particular host genotypes [ 18 ]. In other words, no parasite is best at infecting all hosts, and no host is best at resisting all parasites, so that the success of infection depends on the specific combination of the two opponents.PlasmodiumDaphniaDespite its potentially important role for malaria epidemiology and control, such a genetic specificity of host-parasite compatibility between malaria parasites and their insect vectors have never been investigated. This study examines the potential for genotype by genotype interactions in the combination that is most important for the epidemiology of malaria –  Plasmodium falciparum  and  Anopheles gambiae . Malaria genotype by mosquito genotype interactions were tested with a standard procedure of quantitative genetics from measurements of the resistance of different genetic backgrounds of the mosquito  A. gambiae , a major malaria vector in sub-Saharan Africa, to different isolates of the human malaria parasite  P. falciparum . The parasite isolates were obtained from naturally infected children in western Kenya that harboured gametocytes, the infective stage of the parasite. The genetic backgrounds of mosquitoes were 'mosquito families' generated as the F1 offspring of single egg-laying females. Each mosquito family was challenged with each parasite isolate, and all mosquitoes were simultaneously fed on the blood of the gametocyte carriers via membrane-feeding. This basic design was repeated three times throughout three successive experimental blocks that involved different families and isolates, giving a total of 18 mosquito families, 11 parasite isolates, and 62 specific interactions. As in previous studies [ 7 ], the resistance of mosquitoes was quantified with the proportion of blood-fed females that developed oocysts and with the number of oocysts. The genotype by genotype interaction on mosquito resistance was estimated according to standard quantitative genetic methods as the interaction effect in a statistical analysis between the parasite isolate and the mosquito family [ 17 , 19 ]. These methods are based on the idea that sibs are genetically more similar that non-sibs. Therefore, partitioning the variance of any trait (e.g. number of oocysts) among families (individuals sharing a mother) and within groups of sibs give an indication of the extent to which the trait has a genetic basis [ 20 ].Plasmodium falciparumAnopheles gambiaeA. gambiaeP. falciparumMethods Mosquitoes The mosquitoes used in this study came from a colony that had been established in 2001 from  A. gambiae s.s . caught in the area surrounding Mbita, a small village on the shore of Lake Victoria in Suba District (western Kenya). These mosquitoes had been initially adapted to feed on a Parafilm ®  membrane, and then maintained in standard insectary conditions using a rabbit as a blood source for routine maintenance. Females of the colony were blood-fed on a rabbit and allowed to lay eggs in individual vials. Immediately after hatching, each larva was individually placed in one well of a 12-well plate with three mL of filtered lake water. They were fed daily on a standard diet of Tetramin ®  fish food (0.06 mg per larva on day 0; 0.12 mg on day 1; 0.24 mg on day 2; 0.36 mg on day 3; 0.48 mg on day 4; 0.6 mg on the following days). Adults were kept in an insectary and supplied with a 6% glucose solution and cotton soaked with distilled water. The temperature and humidity in the insectary followed the daily environmental fluctuations. So that mosquito age at pupation did not affect the success of infection, only females that pupated seven days after hatching were used. The wing length of the mosquitoes, measured from the tip (excluding the fringe) to the distal end of the allula with a precision of 0.02 mm, was used as an indication of body size [ 21 ]. Where both wings could be measured, the mean of the two lengths was used. Gametocyte carriers P. falciparum  carriers were recruited from the two- to 10-year old children in the rural area around Mbita, from December 2003 to January 2004. Finger-prick blood samples were collected and thick blood smears were air-dried, stained with 8% Giemsa during 15 minutes, and examined microscopically for the presence of  P. falciparum . Children with asexual parasitemia (>1,000 parasites/μL) were immediately treated with sulfadoxine-pyrimethamine according to national guidelines. Asymptomatic gametocyte-positive children were recruited for the study after their parents or guardians had signed an informed consent form. The Kenyan and the United States National Institute of Health ethical review committees approved this recruitment procedure. Experimental infections For logistic reasons, the experiment was repeated three times, and within each experimental block infections were done simultaneously. For each of the three blocks,  P. falciparum  isolates were collected from gametocyte carriers that had been identified one or two days before, and used to feed the mosquitoes on the same single day (block 1: December 14, 2003; block 2: January 23, 2004; block 3: January 28, 2004). The gametocyte densities were assessed just before blood withdrawal on a blood smear (as described above) by counting against 500 leukocytes, and converted to numbers of parasites per μL by assuming a standard leukocyte count of 8,000/μL. Although gametocyte densities in the venous blood and the peripheral finger-prick blood might differ, potential differences were assumed to be proportional among the isolates. A sample of five mL of venous blood was collected from each gametocyte carrier in a heparinized tube, 400 μL of which were stored at -20°C for further parasite genotyping. So that the importance of human factors such as transmission-blocking immunity [ 22 ] was reduced, the blood was centrifuged at 37°C for three minutes at 2,000  g  and the autologous serum was replaced with the same volume of a pool of AB serum from two French blood donors without any malaria exposure (the same pool of AB serum was used for all experimental blocks). The mixture was used to feed mosquitoes, which had been starved for 12–16 h before blood feeding, with a standard membrane-feeding system [ 23 ]. For each mosquito family, i.e. each group of mosquitoes that was derived from the eggs of a single female, equal groups of three-day old females were randomly chosen and fed separately with each isolate. Depending on the size of the family, each feeding cage contained between four and 15 females. Mosquitoes were allowed to take a blood meal for 40 minutes, after which unfed and partially fed mosquitoes were discarded. Seven or eight days after the infective blood meal, mosquitoes were dissected and their midguts were stained with 2% mercurochrome in distilled water in order to detect the presence and number of oocysts by light microscopy. Microsatellite genotyping P. falciparum  DNA was extracted from the blood samples using the QIAamp DNA blood kit following the manufacturer's instructions (Qiagen, CA). The isolates were typed using the semi-nested PCR method slightly modified from a previous study [ 24 ] (details are available upon request to PD) and the markers used [ 25 ] and their GenBank accession number in parenthesis are as follows: PJ2 (G37826), UIDG (G37823), Polyα (L18785), TA60 (AF010556), ARA2 (G37848), Pfg377 (L04161), PfPK2 (X63648), TA87 (AF010571), TA109 (AF010508). The microsatellite PCR products were size-genotyped using a standard size Genescan 500 LIZ on an ABI Prism 310 Genetic Analyser (PE Applied Biosystems, CA). Data analysis Only those mosquito families in which at least four individuals had been fully fed and had survived infection with each isolate were included in the analyses. The likelihood that a mosquito had been infected was analysed with a nominal logistic analysis. The intensity of infection was analysed with an analysis of variance (ANOVA). In this analysis, the square root of the number of oocysts was used, so that the assumptions of the statistical tests (in particular, normality of the residuals) were satisified. As the study was run in three successive experimental blocks, both analyses included the effect of block, and the effects of family, isolate and their interaction. The effect of wing length was also included as a potential confounder [ 26 ]. As different families and isolates were used in each experimental block, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors.
Methods
Mosquitoes The mosquitoes used in this study came from a colony that had been established in 2001 from  A. gambiae s.s . caught in the area surrounding Mbita, a small village on the shore of Lake Victoria in Suba District (western Kenya). These mosquitoes had been initially adapted to feed on a Parafilm ®  membrane, and then maintained in standard insectary conditions using a rabbit as a blood source for routine maintenance. Females of the colony were blood-fed on a rabbit and allowed to lay eggs in individual vials. Immediately after hatching, each larva was individually placed in one well of a 12-well plate with three mL of filtered lake water. They were fed daily on a standard diet of Tetramin ®  fish food (0.06 mg per larva on day 0; 0.12 mg on day 1; 0.24 mg on day 2; 0.36 mg on day 3; 0.48 mg on day 4; 0.6 mg on the following days). Adults were kept in an insectary and supplied with a 6% glucose solution and cotton soaked with distilled water. The temperature and humidity in the insectary followed the daily environmental fluctuations. So that mosquito age at pupation did not affect the success of infection, only females that pupated seven days after hatching were used. The wing length of the mosquitoes, measured from the tip (excluding the fringe) to the distal end of the allula with a precision of 0.02 mm, was used as an indication of body size [ 21 ]. Where both wings could be measured, the mean of the two lengths was used.
Mosquitoes
The mosquitoes used in this study came from a colony that had been established in 2001 from  A. gambiae s.s . caught in the area surrounding Mbita, a small village on the shore of Lake Victoria in Suba District (western Kenya). These mosquitoes had been initially adapted to feed on a Parafilm ®  membrane, and then maintained in standard insectary conditions using a rabbit as a blood source for routine maintenance. Females of the colony were blood-fed on a rabbit and allowed to lay eggs in individual vials. Immediately after hatching, each larva was individually placed in one well of a 12-well plate with three mL of filtered lake water. They were fed daily on a standard diet of Tetramin ®  fish food (0.06 mg per larva on day 0; 0.12 mg on day 1; 0.24 mg on day 2; 0.36 mg on day 3; 0.48 mg on day 4; 0.6 mg on the following days). Adults were kept in an insectary and supplied with a 6% glucose solution and cotton soaked with distilled water. The temperature and humidity in the insectary followed the daily environmental fluctuations. So that mosquito age at pupation did not affect the success of infection, only females that pupated seven days after hatching were used. The wing length of the mosquitoes, measured from the tip (excluding the fringe) to the distal end of the allula with a precision of 0.02 mm, was used as an indication of body size [ 21 ]. Where both wings could be measured, the mean of the two lengths was used.A. gambiae s.sGametocyte carriers P. falciparum  carriers were recruited from the two- to 10-year old children in the rural area around Mbita, from December 2003 to January 2004. Finger-prick blood samples were collected and thick blood smears were air-dried, stained with 8% Giemsa during 15 minutes, and examined microscopically for the presence of  P. falciparum . Children with asexual parasitemia (>1,000 parasites/μL) were immediately treated with sulfadoxine-pyrimethamine according to national guidelines. Asymptomatic gametocyte-positive children were recruited for the study after their parents or guardians had signed an informed consent form. The Kenyan and the United States National Institute of Health ethical review committees approved this recruitment procedure.
Gametocyte carriers
P. falciparum  carriers were recruited from the two- to 10-year old children in the rural area around Mbita, from December 2003 to January 2004. Finger-prick blood samples were collected and thick blood smears were air-dried, stained with 8% Giemsa during 15 minutes, and examined microscopically for the presence of  P. falciparum . Children with asexual parasitemia (>1,000 parasites/μL) were immediately treated with sulfadoxine-pyrimethamine according to national guidelines. Asymptomatic gametocyte-positive children were recruited for the study after their parents or guardians had signed an informed consent form. The Kenyan and the United States National Institute of Health ethical review committees approved this recruitment procedure.P. falciparumP. falciparumExperimental infections For logistic reasons, the experiment was repeated three times, and within each experimental block infections were done simultaneously. For each of the three blocks,  P. falciparum  isolates were collected from gametocyte carriers that had been identified one or two days before, and used to feed the mosquitoes on the same single day (block 1: December 14, 2003; block 2: January 23, 2004; block 3: January 28, 2004). The gametocyte densities were assessed just before blood withdrawal on a blood smear (as described above) by counting against 500 leukocytes, and converted to numbers of parasites per μL by assuming a standard leukocyte count of 8,000/μL. Although gametocyte densities in the venous blood and the peripheral finger-prick blood might differ, potential differences were assumed to be proportional among the isolates. A sample of five mL of venous blood was collected from each gametocyte carrier in a heparinized tube, 400 μL of which were stored at -20°C for further parasite genotyping. So that the importance of human factors such as transmission-blocking immunity [ 22 ] was reduced, the blood was centrifuged at 37°C for three minutes at 2,000  g  and the autologous serum was replaced with the same volume of a pool of AB serum from two French blood donors without any malaria exposure (the same pool of AB serum was used for all experimental blocks). The mixture was used to feed mosquitoes, which had been starved for 12–16 h before blood feeding, with a standard membrane-feeding system [ 23 ]. For each mosquito family, i.e. each group of mosquitoes that was derived from the eggs of a single female, equal groups of three-day old females were randomly chosen and fed separately with each isolate. Depending on the size of the family, each feeding cage contained between four and 15 females. Mosquitoes were allowed to take a blood meal for 40 minutes, after which unfed and partially fed mosquitoes were discarded. Seven or eight days after the infective blood meal, mosquitoes were dissected and their midguts were stained with 2% mercurochrome in distilled water in order to detect the presence and number of oocysts by light microscopy.
Experimental infections
For logistic reasons, the experiment was repeated three times, and within each experimental block infections were done simultaneously. For each of the three blocks,  P. falciparum  isolates were collected from gametocyte carriers that had been identified one or two days before, and used to feed the mosquitoes on the same single day (block 1: December 14, 2003; block 2: January 23, 2004; block 3: January 28, 2004). The gametocyte densities were assessed just before blood withdrawal on a blood smear (as described above) by counting against 500 leukocytes, and converted to numbers of parasites per μL by assuming a standard leukocyte count of 8,000/μL. Although gametocyte densities in the venous blood and the peripheral finger-prick blood might differ, potential differences were assumed to be proportional among the isolates. A sample of five mL of venous blood was collected from each gametocyte carrier in a heparinized tube, 400 μL of which were stored at -20°C for further parasite genotyping. So that the importance of human factors such as transmission-blocking immunity [ 22 ] was reduced, the blood was centrifuged at 37°C for three minutes at 2,000  g  and the autologous serum was replaced with the same volume of a pool of AB serum from two French blood donors without any malaria exposure (the same pool of AB serum was used for all experimental blocks). The mixture was used to feed mosquitoes, which had been starved for 12–16 h before blood feeding, with a standard membrane-feeding system [ 23 ]. For each mosquito family, i.e. each group of mosquitoes that was derived from the eggs of a single female, equal groups of three-day old females were randomly chosen and fed separately with each isolate. Depending on the size of the family, each feeding cage contained between four and 15 females. Mosquitoes were allowed to take a blood meal for 40 minutes, after which unfed and partially fed mosquitoes were discarded. Seven or eight days after the infective blood meal, mosquitoes were dissected and their midguts were stained with 2% mercurochrome in distilled water in order to detect the presence and number of oocysts by light microscopy.P. falciparumgMicrosatellite genotyping P. falciparum  DNA was extracted from the blood samples using the QIAamp DNA blood kit following the manufacturer's instructions (Qiagen, CA). The isolates were typed using the semi-nested PCR method slightly modified from a previous study [ 24 ] (details are available upon request to PD) and the markers used [ 25 ] and their GenBank accession number in parenthesis are as follows: PJ2 (G37826), UIDG (G37823), Polyα (L18785), TA60 (AF010556), ARA2 (G37848), Pfg377 (L04161), PfPK2 (X63648), TA87 (AF010571), TA109 (AF010508). The microsatellite PCR products were size-genotyped using a standard size Genescan 500 LIZ on an ABI Prism 310 Genetic Analyser (PE Applied Biosystems, CA).
Microsatellite genotyping
P. falciparum  DNA was extracted from the blood samples using the QIAamp DNA blood kit following the manufacturer's instructions (Qiagen, CA). The isolates were typed using the semi-nested PCR method slightly modified from a previous study [ 24 ] (details are available upon request to PD) and the markers used [ 25 ] and their GenBank accession number in parenthesis are as follows: PJ2 (G37826), UIDG (G37823), Polyα (L18785), TA60 (AF010556), ARA2 (G37848), Pfg377 (L04161), PfPK2 (X63648), TA87 (AF010571), TA109 (AF010508). The microsatellite PCR products were size-genotyped using a standard size Genescan 500 LIZ on an ABI Prism 310 Genetic Analyser (PE Applied Biosystems, CA).P. falciparumData analysis Only those mosquito families in which at least four individuals had been fully fed and had survived infection with each isolate were included in the analyses. The likelihood that a mosquito had been infected was analysed with a nominal logistic analysis. The intensity of infection was analysed with an analysis of variance (ANOVA). In this analysis, the square root of the number of oocysts was used, so that the assumptions of the statistical tests (in particular, normality of the residuals) were satisified. As the study was run in three successive experimental blocks, both analyses included the effect of block, and the effects of family, isolate and their interaction. The effect of wing length was also included as a potential confounder [ 26 ]. As different families and isolates were used in each experimental block, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors.
Data analysis
Only those mosquito families in which at least four individuals had been fully fed and had survived infection with each isolate were included in the analyses. The likelihood that a mosquito had been infected was analysed with a nominal logistic analysis. The intensity of infection was analysed with an analysis of variance (ANOVA). In this analysis, the square root of the number of oocysts was used, so that the assumptions of the statistical tests (in particular, normality of the residuals) were satisified. As the study was run in three successive experimental blocks, both analyses included the effect of block, and the effects of family, isolate and their interaction. The effect of wing length was also included as a potential confounder [ 26 ]. As different families and isolates were used in each experimental block, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors.Results The three successive experimental blocks involved three, five and three parasite isolates, and nine, four and five mosquito families, respectively. A third (151) of the 455 mosquitoes of the study were infected by  P. falciparum  oocysts, and the number of oocysts in infected mosquitoes ranged from one to 97 (mean 11.0, median 3). The prevalence and the number of oocysts differed among blocks (block effect, Table  1 ), and were lower in larger mosquitoes (wing length effect, Table  1 ). Table 1 Statistical analysis of the effects of mosquito family and parasite isolate on the success of infection. The proportion of infected mosquitoes (a, nominal logistic analysis) and the square root of the number of oocysts (b, ANOVA) were analysed as a function of the mosquito family, the parasite isolate, and their interaction. In both analyses, the mosquito's wing length was included as a confounder. As the study was run in three experimental blocks using different families and isolates, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors. (a) Proportion infected (b) Intensity of infection Source d.f. χ 2 P Sum of Squares F P Experimental Block 2 85.5 <0.001 265.2 2.40 0.155 Wing Length 1 3.1 0.029 6.4 7.14 0.008 Family (within Block) 14 3.8 0.927 29.5 0.66 0.800 Isolate (within Block) 8 15.5 0.482 462.5 19.40 <0.001 Family*Isolate (within Block) 34 127.2 <0.001 110.2 3.59 <0.001 Error (for analysis b) 395 356.8 Family by isolate interaction While the crude variation among families (averaged across all parasite isolates) was substantial (mean infection rate ranging from four to 83%; mean number of oocysts ranging from 0.1 to 16.4), most of this variation was due to differences among blocks (Fig.  1 ), so that there was no evidence that families differed in the overall proportion of infected individuals or in the intensity of infection (family effect, Table  1 ). Similarly, most of the crude differences among isolates (averaged across mosquito families) were due to differences among blocks. Thus, isolates did not vary in the proportion of mosquitoes they infected (ranging from four to 94%), although they did vary in the number of oocysts they produced (median ranging from zero to 35) (isolate effect, Table  1 ). More importantly in the context of our study, while neither the families of mosquitoes nor the parasites differed in their average responses to all of their partners, the interaction between mosquito family and parasite isolate (an estimation of the genotype by genotype interaction) had a highly significant effect on the likelihood and the intensity of infection (family by isolate effect, Table  1 ). Thus, no parasite isolate was most infectious to every host genotype. Rather, isolates that were most infectious on one host tended to be less infectious than the other isolates on other hosts (Fig.  1 ). Similarly no host genotype was most resistant to every parasite isolate (Fig.  1 ). Figure 1 Graphic representation of the mosquito family by parasite isolate interactions underlying (a) the probability and (b) the intensity of infection . Each point represents the proportion of infected mosquitoes (in a) or the mean of the square root of the number of oocysts (in b) for a given combination of family and isolate. The families are indicated on the x-axes, and are separated into the three experimental blocks of the study with vertical lines. Different colours represent different isolates (squares: isolates containing two clones; circles: isolates containing three clones), and the lines connect points representing the same isolate. Crossing lines give an indication of family by isolate interactions. Genetic characterization of  P. falciparum  isolates While the quantitative genetic analysis of the data gives an adequate representation of the genetic basis of the mosquito's resistance [ 20 ], the use of natural isolates may complicate the interpretation, as (i) they do not necessarily consist of different malaria clones and (ii) isolates often contain several clones in areas where transmission is high [ 27 , 28 ]. However, genotyping the blood samples at nine microsatellite markers showed that the isolates differed. The overall genetic diversity was high, ranging from four to 15 allelic variants per locus. Each isolate had an allelic pattern that differed from all other isolates at, at least, one locus (data not shown), showing that the isolates were genetically distinct. Using the maximum number of alleles at a single locus as a conservative estimate of the number of clones, each isolate was found to contain either two or three distinct clones of  P. falciparum  (Table  2 ). Table 2 Description of  P. falciparum  isolates. The number of gametocytes per 500 leukocytes, converted to numbers of parasites per μL (assuming a standard leukocyte count of 8,000/μL) and the maximum number of alleles at a single locus found for 9 microsatellite markers (a conservative estimate of the number of clones) are given for each isolate. Experimental block Isolate Gametocyte density (parasites/μL) Number of clones 1 A 176 3 1 B 32 3 1 C 32 2 2 D 32 3 2 E 16 2 2 F 16 3 2 G 32 3 2 H 32 2 3 I 48 2 3 J 16 2 3 K 16 3 Potential confounding effects Separate analyses of the data for the two numbers of clones (two or three) ensured that the number of clones contained in each isolate did not confound the interpretation. The effect of the mosquito family by parasite isolate interaction on the likelihood of infection was significant in both cases (two clones:  P  = 0.050; three clones:  P  = 0.003) and the effect of the interaction on the number of oocysts was significant in one of the cases and showed a tendency in the other case (two clones:  P  = 0.219; three clones:  P  = 0.008). In addition, differences in gametocyte density between isolates (Table  2 ) may be expected to bias infection success [ 23 ]. There were sufficient data to analyse the effects of the mosquito family by parasite isolate interaction separately for the isolates with 16 or 32 gametocytes/μL (i.e. one or two gametocytes per 500 leukocytes). At both gametocyte densities, the interaction significantly influenced the probability of infection (16 gametocytes/μL:  P  < 0.001; 32 gametocytes/μL:  P  < 0.001) and the number of oocysts (16 gametocytes/μL:  P  < 0.001; 32 gametocytes/μL:  P  < 0.001). In conclusion, the two potential confounders – number of clones per isolate and gametocyte density – had no qualitative influence on the results of the analysis.
Results
The three successive experimental blocks involved three, five and three parasite isolates, and nine, four and five mosquito families, respectively. A third (151) of the 455 mosquitoes of the study were infected by  P. falciparum  oocysts, and the number of oocysts in infected mosquitoes ranged from one to 97 (mean 11.0, median 3). The prevalence and the number of oocysts differed among blocks (block effect, Table  1 ), and were lower in larger mosquitoes (wing length effect, Table  1 ).P. falciparumStatistical analysis of the effects of mosquito family and parasite isolate on the success of infection. The proportion of infected mosquitoes (a, nominal logistic analysis) and the square root of the number of oocysts (b, ANOVA) were analysed as a function of the mosquito family, the parasite isolate, and their interaction. In both analyses, the mosquito's wing length was included as a confounder. As the study was run in three experimental blocks using different families and isolates, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors.Family by isolate interaction While the crude variation among families (averaged across all parasite isolates) was substantial (mean infection rate ranging from four to 83%; mean number of oocysts ranging from 0.1 to 16.4), most of this variation was due to differences among blocks (Fig.  1 ), so that there was no evidence that families differed in the overall proportion of infected individuals or in the intensity of infection (family effect, Table  1 ). Similarly, most of the crude differences among isolates (averaged across mosquito families) were due to differences among blocks. Thus, isolates did not vary in the proportion of mosquitoes they infected (ranging from four to 94%), although they did vary in the number of oocysts they produced (median ranging from zero to 35) (isolate effect, Table  1 ). More importantly in the context of our study, while neither the families of mosquitoes nor the parasites differed in their average responses to all of their partners, the interaction between mosquito family and parasite isolate (an estimation of the genotype by genotype interaction) had a highly significant effect on the likelihood and the intensity of infection (family by isolate effect, Table  1 ). Thus, no parasite isolate was most infectious to every host genotype. Rather, isolates that were most infectious on one host tended to be less infectious than the other isolates on other hosts (Fig.  1 ). Similarly no host genotype was most resistant to every parasite isolate (Fig.  1 ). Figure 1 Graphic representation of the mosquito family by parasite isolate interactions underlying (a) the probability and (b) the intensity of infection . Each point represents the proportion of infected mosquitoes (in a) or the mean of the square root of the number of oocysts (in b) for a given combination of family and isolate. The families are indicated on the x-axes, and are separated into the three experimental blocks of the study with vertical lines. Different colours represent different isolates (squares: isolates containing two clones; circles: isolates containing three clones), and the lines connect points representing the same isolate. Crossing lines give an indication of family by isolate interactions.
Family by isolate interaction
While the crude variation among families (averaged across all parasite isolates) was substantial (mean infection rate ranging from four to 83%; mean number of oocysts ranging from 0.1 to 16.4), most of this variation was due to differences among blocks (Fig.  1 ), so that there was no evidence that families differed in the overall proportion of infected individuals or in the intensity of infection (family effect, Table  1 ). Similarly, most of the crude differences among isolates (averaged across mosquito families) were due to differences among blocks. Thus, isolates did not vary in the proportion of mosquitoes they infected (ranging from four to 94%), although they did vary in the number of oocysts they produced (median ranging from zero to 35) (isolate effect, Table  1 ). More importantly in the context of our study, while neither the families of mosquitoes nor the parasites differed in their average responses to all of their partners, the interaction between mosquito family and parasite isolate (an estimation of the genotype by genotype interaction) had a highly significant effect on the likelihood and the intensity of infection (family by isolate effect, Table  1 ). Thus, no parasite isolate was most infectious to every host genotype. Rather, isolates that were most infectious on one host tended to be less infectious than the other isolates on other hosts (Fig.  1 ). Similarly no host genotype was most resistant to every parasite isolate (Fig.  1 ).Genetic characterization of  P. falciparum  isolates While the quantitative genetic analysis of the data gives an adequate representation of the genetic basis of the mosquito's resistance [ 20 ], the use of natural isolates may complicate the interpretation, as (i) they do not necessarily consist of different malaria clones and (ii) isolates often contain several clones in areas where transmission is high [ 27 , 28 ]. However, genotyping the blood samples at nine microsatellite markers showed that the isolates differed. The overall genetic diversity was high, ranging from four to 15 allelic variants per locus. Each isolate had an allelic pattern that differed from all other isolates at, at least, one locus (data not shown), showing that the isolates were genetically distinct. Using the maximum number of alleles at a single locus as a conservative estimate of the number of clones, each isolate was found to contain either two or three distinct clones of  P. falciparum  (Table  2 ). Table 2 Description of  P. falciparum  isolates. The number of gametocytes per 500 leukocytes, converted to numbers of parasites per μL (assuming a standard leukocyte count of 8,000/μL) and the maximum number of alleles at a single locus found for 9 microsatellite markers (a conservative estimate of the number of clones) are given for each isolate. Experimental block Isolate Gametocyte density (parasites/μL) Number of clones 1 A 176 3 1 B 32 3 1 C 32 2 2 D 32 3 2 E 16 2 2 F 16 3 2 G 32 3 2 H 32 2 3 I 48 2 3 J 16 2 3 K 16 3
Genetic characterization of  P. falciparum  isolates
P. falciparumWhile the quantitative genetic analysis of the data gives an adequate representation of the genetic basis of the mosquito's resistance [ 20 ], the use of natural isolates may complicate the interpretation, as (i) they do not necessarily consist of different malaria clones and (ii) isolates often contain several clones in areas where transmission is high [ 27 , 28 ]. However, genotyping the blood samples at nine microsatellite markers showed that the isolates differed. The overall genetic diversity was high, ranging from four to 15 allelic variants per locus. Each isolate had an allelic pattern that differed from all other isolates at, at least, one locus (data not shown), showing that the isolates were genetically distinct. Using the maximum number of alleles at a single locus as a conservative estimate of the number of clones, each isolate was found to contain either two or three distinct clones of  P. falciparum  (Table  2 ).P. falciparumDescription of  P. falciparum  isolates. The number of gametocytes per 500 leukocytes, converted to numbers of parasites per μL (assuming a standard leukocyte count of 8,000/μL) and the maximum number of alleles at a single locus found for 9 microsatellite markers (a conservative estimate of the number of clones) are given for each isolate.P. falciparumPotential confounding effects Separate analyses of the data for the two numbers of clones (two or three) ensured that the number of clones contained in each isolate did not confound the interpretation. The effect of the mosquito family by parasite isolate interaction on the likelihood of infection was significant in both cases (two clones:  P  = 0.050; three clones:  P  = 0.003) and the effect of the interaction on the number of oocysts was significant in one of the cases and showed a tendency in the other case (two clones:  P  = 0.219; three clones:  P  = 0.008). In addition, differences in gametocyte density between isolates (Table  2 ) may be expected to bias infection success [ 23 ]. There were sufficient data to analyse the effects of the mosquito family by parasite isolate interaction separately for the isolates with 16 or 32 gametocytes/μL (i.e. one or two gametocytes per 500 leukocytes). At both gametocyte densities, the interaction significantly influenced the probability of infection (16 gametocytes/μL:  P  < 0.001; 32 gametocytes/μL:  P  < 0.001) and the number of oocysts (16 gametocytes/μL:  P  < 0.001; 32 gametocytes/μL:  P  < 0.001). In conclusion, the two potential confounders – number of clones per isolate and gametocyte density – had no qualitative influence on the results of the analysis.
Potential confounding effects
Separate analyses of the data for the two numbers of clones (two or three) ensured that the number of clones contained in each isolate did not confound the interpretation. The effect of the mosquito family by parasite isolate interaction on the likelihood of infection was significant in both cases (two clones:  P  = 0.050; three clones:  P  = 0.003) and the effect of the interaction on the number of oocysts was significant in one of the cases and showed a tendency in the other case (two clones:  P  = 0.219; three clones:  P  = 0.008).PPPPIn addition, differences in gametocyte density between isolates (Table  2 ) may be expected to bias infection success [ 23 ]. There were sufficient data to analyse the effects of the mosquito family by parasite isolate interaction separately for the isolates with 16 or 32 gametocytes/μL (i.e. one or two gametocytes per 500 leukocytes). At both gametocyte densities, the interaction significantly influenced the probability of infection (16 gametocytes/μL:  P  < 0.001; 32 gametocytes/μL:  P  < 0.001) and the number of oocysts (16 gametocytes/μL:  P  < 0.001; 32 gametocytes/μL:  P  < 0.001). In conclusion, the two potential confounders – number of clones per isolate and gametocyte density – had no qualitative influence on the results of the analysis.PPPPDiscussion While the specificity of mosquito-malaria interactions at the species level is well documented [ 29 ], the present results are the first experimental evidence of the genetic specificity of mosquito infection by malaria parasites at the intraspecific level. This finding corroborates an earlier study, where a mosquito line selected for resistance to malaria infection varied considerably in its response against different  Plasmodium  species and strains [ 6 ]. The present study goes one step further by suggesting that mosquito resistance to malaria is at least partly determined by the specific interaction between its own and the parasite's genotype. This idea is supported by two other studies showing that, in a strain of mosquitoes selected to resist infection by a wide variety of malaria species, different genetic loci are involved in the responses against different  Plasmodium  parasites [ 30 , 31 ]. The present study suggests, moreover, that the genes conferring resistance to a particular parasite depend on the genetic background of the mosquito. The specificity of host-parasite interactions is often postulated to occur at the level of parasite recognition. While the molecular mechanisms of  Plasmodium  recognition by mosquitoes are still largely unknown, a group of thioester-containing proteins (TEPs) represents a promising family of candidate recognition molecules. One of them, the complement-like protein TEP1, has recently been shown to bind to and mediate the killing of the rodent malaria parasite  P. berghei  by the mosquito  A. gambiae  [ 9 ]. Moreover, two allelic variants of the  TEP1  gene are associated to susceptible and refractory strains of  A. gambiae  [ 9 ]. It is therefore tempting to speculate that this protein may be involved in the specific recognition of particular malaria genotypes by the insect's immune system. The mosquito genotype by parasite genotype interactions shown in this paper may help to understand some puzzling aspects of the epidemiology of malaria. Thus, even in areas with intense transmission, the probability that a mosquito becomes infected is generally low [ 26 , 32 ]. Furthermore, the probability of infection is generally low even when, as in our study, mosquitoes fed on a blood-meal known to contain infectious gametocytes [ 33 , 34 ]. This could have several explanations: mosquitoes fail to pick up infective gametocytes, transmission-blocking immunity in the human hosts prevents the parasite's development within the mosquito [ 22 , 35 ], or parasites are cleared by mosquitoes that mount a sufficiently effective immune response [ 2 ]. The present results indicate an additional reason: that many parasites are incompatible with many of the mosquitoes in a natural population. The epidemiological consequences of mosquito genotype by malaria genotype interactions are perhaps most obvious in the context of malaria control with mosquitoes transformed to be resistant against malaria. The strong genetic specificity of compatibility between parasite isolates and individual insect vectors suggests that most studies on the mechanisms underlying the resistance of mosquitoes against  Plasmodium  might be misleading for the development of malaria control strategies. Indeed, most of these laboratory-based studies focus on the response of one mosquito line or colony against a single parasite strain and thus do not represent the genetic diversity of mosquitoes and parasites in natural populations [ 12 , 13 ]. However, any malaria control programme based on the release of mosquitoes harbouring 'resistance genes' is unlikely to be effective if resistance is expressed against only a subset of the parasite genotypes of the local population. Indeed, as parasites facing resistant mosquitoes will be under strong selective pressure to avoid mosquito defence mechanisms, genotypes that are eliminated by the resistance genes might be replaced rapidly by genotypes that cannot be controlled. Thus, before a possible release of transgenic mosquitoes, it will be crucial to ensure that the transformed mosquitoes are resistant to all of the parasite genotypes in the local population. This reinforces the idea that any release of genetically modified mosquitoes for reducing transmission of mosquito-borne diseases must be preceded by studies that have moved from the laboratory to the field [ 10 ].
Discussion
While the specificity of mosquito-malaria interactions at the species level is well documented [ 29 ], the present results are the first experimental evidence of the genetic specificity of mosquito infection by malaria parasites at the intraspecific level. This finding corroborates an earlier study, where a mosquito line selected for resistance to malaria infection varied considerably in its response against different  Plasmodium  species and strains [ 6 ]. The present study goes one step further by suggesting that mosquito resistance to malaria is at least partly determined by the specific interaction between its own and the parasite's genotype. This idea is supported by two other studies showing that, in a strain of mosquitoes selected to resist infection by a wide variety of malaria species, different genetic loci are involved in the responses against different  Plasmodium  parasites [ 30 , 31 ]. The present study suggests, moreover, that the genes conferring resistance to a particular parasite depend on the genetic background of the mosquito.PlasmodiumPlasmodiumThe specificity of host-parasite interactions is often postulated to occur at the level of parasite recognition. While the molecular mechanisms of  Plasmodium  recognition by mosquitoes are still largely unknown, a group of thioester-containing proteins (TEPs) represents a promising family of candidate recognition molecules. One of them, the complement-like protein TEP1, has recently been shown to bind to and mediate the killing of the rodent malaria parasite  P. berghei  by the mosquito  A. gambiae  [ 9 ]. Moreover, two allelic variants of the  TEP1  gene are associated to susceptible and refractory strains of  A. gambiae  [ 9 ]. It is therefore tempting to speculate that this protein may be involved in the specific recognition of particular malaria genotypes by the insect's immune system.PlasmodiumP. bergheiA. gambiaeTEP1A. gambiaeThe mosquito genotype by parasite genotype interactions shown in this paper may help to understand some puzzling aspects of the epidemiology of malaria. Thus, even in areas with intense transmission, the probability that a mosquito becomes infected is generally low [ 26 , 32 ]. Furthermore, the probability of infection is generally low even when, as in our study, mosquitoes fed on a blood-meal known to contain infectious gametocytes [ 33 , 34 ]. This could have several explanations: mosquitoes fail to pick up infective gametocytes, transmission-blocking immunity in the human hosts prevents the parasite's development within the mosquito [ 22 , 35 ], or parasites are cleared by mosquitoes that mount a sufficiently effective immune response [ 2 ]. The present results indicate an additional reason: that many parasites are incompatible with many of the mosquitoes in a natural population.The epidemiological consequences of mosquito genotype by malaria genotype interactions are perhaps most obvious in the context of malaria control with mosquitoes transformed to be resistant against malaria. The strong genetic specificity of compatibility between parasite isolates and individual insect vectors suggests that most studies on the mechanisms underlying the resistance of mosquitoes against  Plasmodium  might be misleading for the development of malaria control strategies. Indeed, most of these laboratory-based studies focus on the response of one mosquito line or colony against a single parasite strain and thus do not represent the genetic diversity of mosquitoes and parasites in natural populations [ 12 , 13 ]. However, any malaria control programme based on the release of mosquitoes harbouring 'resistance genes' is unlikely to be effective if resistance is expressed against only a subset of the parasite genotypes of the local population. Indeed, as parasites facing resistant mosquitoes will be under strong selective pressure to avoid mosquito defence mechanisms, genotypes that are eliminated by the resistance genes might be replaced rapidly by genotypes that cannot be controlled. Thus, before a possible release of transgenic mosquitoes, it will be crucial to ensure that the transformed mosquitoes are resistant to all of the parasite genotypes in the local population. This reinforces the idea that any release of genetically modified mosquitoes for reducing transmission of mosquito-borne diseases must be preceded by studies that have moved from the laboratory to the field [ 10 ].PlasmodiumConclusions This study demonstrated that the resistance of an anopheline mosquito to  P. falciparum  development, a major component of its vector competence, varies considerably between different combinations of parasite isolates and individual, genetically variable, vectors. Optimal transmission may thus require some specific compatibility between the insect's and the parasite's genotypes. This result has important consequences for the epidemiology of malaria. Overall, it suggests that conclusions from a particular subset of mosquito and malaria genotypes will not necessarily hold for other combinations of genotypes. Therefore, field studies taking into account the full diversity of mosquito and parasite populations are necessary to reach valid conclusions concerning the technologies developed in laboratories for the control of malaria.
Conclusions
This study demonstrated that the resistance of an anopheline mosquito to  P. falciparum  development, a major component of its vector competence, varies considerably between different combinations of parasite isolates and individual, genetically variable, vectors. Optimal transmission may thus require some specific compatibility between the insect's and the parasite's genotypes. This result has important consequences for the epidemiology of malaria. Overall, it suggests that conclusions from a particular subset of mosquito and malaria genotypes will not necessarily hold for other combinations of genotypes. Therefore, field studies taking into account the full diversity of mosquito and parasite populations are necessary to reach valid conclusions concerning the technologies developed in laboratories for the control of malaria.P. falciparumAuthors' contributions LL participated in the design and the coordination of the study, carried out the fieldwork, participated in the molecular analysis, performed the statistical analysis, and wrote the manuscript. JH participated in the fieldwork. PD carried out the molecular analyses and helped to draft the manuscript. LCG supervised and coordinated the fieldwork. JCK conceived and designed the study, performed the statistical analysis, and wrote the manuscript.
Authors' contributions
LL participated in the design and the coordination of the study, carried out the fieldwork, participated in the molecular analysis, performed the statistical analysis, and wrote the manuscript. JH participated in the fieldwork. PD carried out the molecular analyses and helped to draft the manuscript. LCG supervised and coordinated the fieldwork. JCK conceived and designed the study, performed the statistical analysis, and wrote the manuscript.