Claire Berticat

Comparative genomics: Insecticide resistance in mosquito vectors

Resistance to insecticides among mosquitoes that act as vectors for malaria (Anopheles gambiae) and West Nile virus (Culex pipiens) emerged more than 25 years ago in Africa, America and Europe; this resistance is frequently due to a loss of sensitivity of the insects acetylcholinesterase enzyme to organophosphates and carbamates. Here we show that this insensitivity results from a single amino-acid substitution in the enzyme, which we found in ten highly resistant strains of C. pipiens from tropical (Africa and Caribbean) and temperate (Europe) areas, as well as in one resistant African strain of A. gambiae. Our identification of this mutation may pave the way for designing new insecticides.

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.

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.

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.

HIGH WOLBACHIA DENSITY CORRELATES WITH COST OF INFECTION FOR INSECTICIDE RESISTANT CULEX PIPIENS MOSQUITOES

Abstract In the mosquito Culex pipiens, insecticide resistance genes alter many life‐history traits and incur a fitness cost. Resistance to organophosphate insecticides involves two loci, with each locus coding for a different mechanism of resistance (degradation vs. insensitivity to insecticides). The density of intracellular Wolbachia bacteria has been found to be higher in resistant mosquitoes, regardless of the mechanism involved. To discriminate between costs of resistance due to resistance genes from those associated with elevated Wolbachia densities, we compared strains of mosquito sharing the same genetic background but differing in their resistance alleles and Wolbachia infection status. Life‐history traits measured included strength of insecticide resistance, larval mortality, adult female size, fecundity, predation avoidance, mating competition, and strength of cytoplasmic incompatibility (CI). We found that: (1) when Wolbachia are removed, insecticide resistance genes still affect some life‐history traits; (2) Wolbachia are capable of modifying the cost of resistance; (3) the cost of Wolbachia infections increases with their density; (4) different interactions occurred depending on the resistance alleles involved; and (5) high densities of Wolbachia do not increase the strength of CI or maternal transmission efficiency relative to low Wolbachia densities. Insecticide resistance genes generated variation in the costs of Wolbachia infections and provided an interesting opportunity to study how these costs evolve, a process generally operating when Wolbachia colonizes a new host

Costs and benefits of multiple resistance to insecticides for Culex quinquefasciatus mosquitoes

BackgroundThe evolutionary dynamics of xenobiotic resistance depends on how resistance mutations influence the fitness of their bearers, both in the presence and absence of xenobiotic selection pressure. In cases of multiple resistance, these dynamics will also depend on how individual resistance mutations interact with one another, and on the xenobiotics applied against them. We compared Culex quinquefasciatus mosquitoes harbouring two resistance alleles ace-1Rand KdrR(conferring resistance to carbamate and pyrethroid insecticides, respectively) to mosquitoes bearing only one of the alleles, or neither allele. Comparisons were made in environments where both, only one, or neither type of insecticide was present.ResultsEach resistance allele was associated with fitness costs (survival to adulthood) in an insecticide-free environment, with the costs of ace-1Rbeing greater than for KdrR. However, there was a notable interaction in that the costs of harbouring both alleles were significantly less than for harbouring ace-1Ralone. The two insecticides combined in an additive, synergistic and antagonistic manner depending on a mosquitos resistance status, but were not predictable based on the presence/absence of either, or both mutations.ConclusionInsecticide resistance mutations interacted to positively or negatively influence a mosquitos fitness, both in the presence or absence of insecticides. In particular, the presence of the KdrRmutation compensated for the costs of the ace-1Rmutation in an insecticide-free environment, suggesting the strength of selection in untreated areas would be less against mosquitoes resistant to both insecticides than for those resistant to carbamates alone. Additional interactions suggest the dynamics of resistance will be difficult to predict in populations where multiple resistance mutations are present or that are subject to treatment by different xenobiotics.

High Wolbachia density in insecticide–resistant mosquitoes

Wolbachia symbionts are responsible for various alterations in host reproduction. The effects of the host genome on endosymbiont levels have often been suggested, but rarely described. Here, we show that Wolbachia density is strongly modified by the presence of insecticide–resistant genes in the common house mosquito, Culex pipiens. The Wolbachia density was estimated using a real–time quantitative PCR assay. Strains harbouring different genes conferring resistance were more infected than a susceptible strain with the same genetic background. We show that this interaction also operates in natural populations. We propose that mosquitoes may control Wolbachia density less efficiently when they carry an insecticide–resistant gene, i.e. when they suffer from a physiological resistance cost.

Acetylcholinesterase genes within the Diptera: takeover and loss in true flies

It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by the ace-1 gene, distinct and divergent from the ace-2 gene, which performs this function in Drosophila. This is an unprecedented situation within the Diptera order because both ace genes derive from an old duplication and are present in most insects and arthropods. Nevertheless, Drosophila possesses only the ace-2 gene. Thus, a secondary loss occurred during the evolution of Diptera, implying a vital function switch from one gene (ace-1) to the other (ace-2). We sampled 78 species, representing 50 families (27% of the Dipteran families) spread over all major subdivisions of the Diptera, and looked for ace-1 and ace-2 by systematic PCR screening to determine which taxonomic groups within the Diptera have this gene change. We show that this loss probably extends to all true flies (or Cyclorrhapha), a large monophyletic group of the Diptera. We also show that ace-2 plays a non-detectable role in the synaptic AChE in a lower Diptera species, suggesting that it has non-synaptic functions. A relative molecular evolution rate test showed that the intensity of purifying selection on ace-2 sequences is constant across the Diptera, irrespective of the presence or absence of ace-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover of ace-2 and the loss of ace-1, taking into account our limited knowledge of non-synaptic functions of ace genes and some specific adaptations of true flies.

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.

PARASITISM INCREASES AND DECREASES THE COSTS OF INSECTICIDE RESISTANCE IN MOSQUITOES

Abstract Adaptations conferring resistance to xenobiotics (antibiotics, insecticides, herbicides, etc.) are often costly to the organisms 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 parasites fitness also varied according to its hosts 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.