Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens

Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI), resulting in abortive embryonic development. One Wolbachia host, Culex pipiens complex mosquitoes, displays high levels of variability in both CI crossing types (cytotypes) and DNA markers. We report here an analysis of 14 mosquito strains, containing 13 Wolbachia variants, and with 13 different cytotypes. Cytotypes were Wolbachia-dependent, as antibiotic treatment rendered all strains tested compatible. Cytotype distributions were independent of geographical distance between sampling sites and host subspecies, suggesting that Wolbachia does not promote a reproductive isolation depending on these parameters. Backcross analysis demonstrated a mild restoring effect of the nuclear genome, indicating that CI is mostly cytoplasmically determined for some crosses. No correlation was found between the phenotypic and genotypic variability of 16 WO prophage and transposon markers, except for the WO prophage Gp15 gene, which encodes a protein similar to a bacterial virulence factor. However, Gp15 is partially correlated with CI expression, suggesting that it could be just linked to a CI gene.     

Forty years of erratic insecticide resistance evolution in the mosquito Culex pipiens

One view of adaptation is that it proceeds by the slow and steady accumulation of beneficial mutations with small effects. It is difficult to test this model, since in most cases the genetic basis of adaptation can only be studied a posteriori with traits that have evolved for a long period of time through an unknown sequence of steps. In this paper, we show how ace-1, a gene involved in resistance to organophosphorous insecticide in the mosquito Culex pipiens, has evolved during 40 years of an insecticide control program. Initially, a major resistance allele with strong deleterious side effects spread through the population. Later, a duplication combining a susceptible and a resistance ace-1 allele began to spread but did not replace the original resistance allele, as it is sublethal when homozygous. Last, a second duplication, (also sublethal when homozygous) began to spread because heterozygotes for the two duplications do not exhibit deleterious pleiotropic effects. Double overdominance now maintains these four alleles across treated and nontreated areas. Thus, ace-1 evolution does not proceed via the steady accumulation of beneficial mutations. Instead, resistance evolution has been an erratic combination of mutation, positive selection, and the rearrangement of existing variation leading to complex genetic architecture.     

Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens

Gene duplication is thought to be the main potential source of material for the evolution of new gene functions. Several models have been proposed for the evolution of new functions through duplication, most based on ancient events (Myr). We provide molecular evidence for the occurrence of several (at least 3) independent duplications of the ace-1 locus in the mosquito Culex pipiens, selected in response to insecticide pressure that probably occurred very recently (<40 years ago). This locus encodes the main target of several insecticides, the acetylcholinesterase. The duplications described consist of 2 alleles of ace-1, 1 susceptible and 1 resistant to insecticide, located on the same chromosome. These events were detected in different parts of the world and probably resulted from distinct mechanisms. We propose that duplications were selected because they reduce the fitness cost associated with the resistant ace-1 allele through the generation of persistent, advantageous heterozygosis. The rate of duplication of ace-1 in C. pipiens is probably underestimated, but seems to be rather high.     

Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.     

Characterization of a novel high-activity esterase in Tunisian populations of the mosquito Culex pipiens

AIn the mosquito Culex pipiens (L.) (Diptera: Culicidae) esterases contribute to insecticide resistance by their increased activity. These esterases display a heterogeneous geographical distribution, particularly in Tunisia, where they are very diverse. In this study, we extended the characterization of a highly active esterase first detected in 1996: B12. Esterase B12 displayed the fastest electrophoretic mobility of all the previously described highly active esterases. We showed that it was encoded by the Ester(B12) allele at the Ester locus, and we isolated a strain, TunB12, homozygous for this allele. TunB12 displayed a low (approximately two- to three-fold) but significant resistance to the organophosphates temephos and chlorpyrifos, and to the pyrethroid permethrin. Only temephos resistance was synergized by S,S,S-tributyl-phosphorotrithioate. Real-time quantitative polymerase chain reaction revealed that the Ester(B12) allele was not amplified in TunB12 strain, indicating that B12 high activity could be due to a gene up-regulation mechanism. Ester(B12) allele frequencies also were estimated in 20 Tunisian populations collected in 2005. Analyses revealed a large distribution of this allele all over the country. Finally, sequences of Ester(B12) were acquired and genetic distance trees were constructed with the resistance Ester alleles already published, providing indications about allele's origins. The diverse array of highly active esterases in C. pipiens from Tunisia and the possible scenario of the origin of their coding alleles are discussed in the context of their possible evolution.     

Insecticide resistance in the mosquito Culex pipiens: what have we learned about adaptation?

Resistance to organophosphate (OP) insecticide in the mosquito Culex pipiens has been studied for ca. 30 years. This example of micro-evolution has been thoroughly investigated as an opportunity to assess precisely both the new adapted phenotypes and the associated genetic changes. A notable feature is that OP resistance is achieved with few genes, and these genes have generally large effects. The molecular events generating such resistance genes are complex (e.g., gene amplification, gene regulation) potentially explaining their low frequency of de novo occurrence. In contrast, migration is a frequent event, including passive transportation between distant populations. This generates a complex interaction between mutations and migration, and promotes competition among resistance alleles. When the precise physiological action of each gene product is rather well known, it is possible to understand the dominance level or the type of epistasis observed. It is however difficult to predict a priori how resistance genes will interact, and it is too early to state whether or not this will be ever possible. These resistance genes are costly, and the cost is variable among them. It is usually believed that the initial fitness cost would gradually decrease due to subsequent mutations with a modifier effect. In the present example, a particular modifier occurred (a gene duplication) at one resistance locus, whereas at the other one reduction of cost is driven by allele replacement and apparently not by selection of modifiers.     

Recombination between two amplified esterase alleles in Culex pipiens

Esterase gene amplification at the Ester superlocus provides organophosphate resistance in the mosquito Culex pipiens (L.). In this study we explored the possibility of recombination between two amplified esterase alleles, thus generating a composite amplified allele. To do that, females heterozygous for two distinct amplified alleles (Ester(2) and Ester(4)) were crossed with males homozygous for a third resistance allele (Ester(8)). Among analyzed offspring, one recombinant composite allele (Ester(2-4)) was detected, providing a rate of recombination of approximately 0.2%. This is the first report of a recombination between two distinct amplified esterase alleles. This phenomenon renders the predictability of allele evolution considerably more complex than was previously thought.     

Parasitism increases and decreases the costs of insecticide resistance in mosquitoes

Adaptations conferring resistance to xenobiotics (antibiotics, insecticides, herbicides, etc.) are often costly to the organism's fitness in the absence of the selecting agent. In such conditions, and unless other mutations compensate for the costs of resistance, sensitive individuals are expected to out-reproduce resistant individuals and drive resistance alleles to a low frequency, with the rate and magnitude of this decline being proportional to the costs of resistance. However, this evolutionary dynamic is open to modification by other sources of selection acting on the relative fitness of susceptible and resistant individuals. Here we show parasitism not only as a source of selection capable of modifying the costs of organophosphate insecticide resistance in mosquitoes, but also that qualitatively different interactions (increasing or decreasing the relative fitness of resistant individuals) occurred depending on the particular form of resistance involved. As estimates of the parasite's fitness also varied according to its host's form of resistance, our data illustrate the potential for epidemiological feedbacks to influence the strength and direction of selection acting on resistance mutations in untreated environments.     

Insecticide resistance genes confer a predation cost on mosquitoes, Culex pipiens

Newly occurring adaptive genes, such as those providing insecticide resistance, display a fitness cost which is poorly understood. In order to detect subtle behavioural changes induced by the presence of resistance genes, we used natural predators and compared their differential predation on susceptible and resistant Culex pipiens mosquitoes, using strains with a similar genetic background. Resistance genes were either coding an overproduced detoxifying esterase (locus Ester), or an insensitive target (locus ace-1). Differential predation was measured between susceptible and resistant individuals, as well as among resistant mosquitoes. A backswimmer, a water measurer, a water boatman and a predaceous diving beetle were used as larval predators, and a pholcid spider as adult predator. Overall, the presence of a resistance gene increased the probability of predation: all resistance genes displayed predation costs relative to susceptible ones, at either the larval or adult stage, or both. Interestingly, predation preferences among the susceptible and the resistance genes were not ranked uniformly. Possible explanations for these results are given, and we suggest that predators, which are designed by natural selection to detect specific behavioural phenotypes, are useful tools to explore non-obvious differences between two classes of individuals, for example when they differ by the presence or absence of one recent gene, such as insecticide resistance genes.     

Insecticide resistance genes induce a mating competition cost in Culex pipiens mosquitoes

Resistance to organophosphorus insecticides (OP) in Culex pipiens mosquitoes represents a convenient model for investigating the fitness cost of resistance genes and its origin, since both the environmental changes in nature and the adaptive genes are clearly identified. Two loci are involved in this resistance--the super-locus Ester and the locus Ace.1--each displaying several resistance alleles. Population surveys have shown differences in fitness cost between these resistance genes and even between resistance alleles of the same locus. In order to better understand this fitness cost and its variability, the effects of these resistance genes on several fitness-related traits are being studied. Here, through competition experiments between two males for the access to one female, we analysed the effect on paternity success associated with three resistance alleles--Ester4, Ester1 and Ace.1R--relative to susceptible males and relative to one another. The eventual effect of female genotype on male mating success was also studied by using susceptible and resistant females. The strains used in this experiment had the same genetic background. Susceptible males had a mating advantage when competing with any of the resistant males, suggesting a substantial cost of resistance genes to this trait. When competing against susceptible males, the paternity success did not vary among resistant males, whatever the genotype of the female. When competing against other resistant males, no difference in paternity success was apparent, except when the female was Ester1.