Pierrick Labbé

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

On the worldwide spread of an insecticide resistance gene: a role for local selection

Adaptation occurs by gene replacement (or transient balanced polymorphism). Replacement may be caused by selection (local or global) and/or genetic drift among alleles. In addition, historical events may blur the respective effects of selection and drift during the course of replacement. We address the relative importance of these processes in the evolution of insecticide resistance genes in the mosquito Culex pipiens. The resistance allele, Ester2, has a broad geographic distribution compared to the other resistance alleles. To distinguish between the different processes explaining this distribution, we reviewed the literature and analysed updated data from the Montpellier area of southern France. Overall, our data indicate that Ester2 prevails over other Ester resistance alleles in moderately treated areas. Such conditions are common and favour the hypothesis of selection acting at a local level. This places an emphasis on the importance of ecological conditions during the evolution of resistance. Finally, we highlight that historical events have contributed to its spread in some areas.

Insecticide resistance in Culex pipiens quinquefasciatus and Aedes albopictus mosquitoes from La Réunion Island.

Resistance to insecticides was monitored on Culex pipiens quinquefasciatus mosquitoes collected in twelve localities of La Réunion, a geographically isolated island of the Indian Ocean. This mosquito is of medical concern in the region as a known vector for filariasis and a potential vector for West Nile and Rift Valley Fever viruses. Our bioassays indicated the presence of resistance to all tested insecticides, i.e. organochlorides, organophosphates and pyrethroids. A molecular investigation revealed a higher frequency of resistance genes in the coastal areas compared to elevated rural sites, probably reflecting the different nature of insecticide pressures together with the genetic cost of resistance alleles. A simple molecular test was developed to detect Rdl allele, encoding a gamma-aminobutyric acid (GABA) receptor resistant to dieldrin. Unexpectedly high Rdl frequencies were recorded over the whole island, despite this insecticide having been banned for over 15 years. This resistant allele was also detected for the first time in two samples of Aedes albopictus, a species recently involved in severe Chikungunya epidemics on the island. Rdl selection in these two mosquito species discloses current insecticide pressures in urban areas, from unknown origins, that should be taken into account to develop vector control strategies.

Resistance Gene Replacement in the Mosquito Culex pipiens : Fitness Estimation From Long-Term Cline Series

How adaptation appears and is later refined by natural selection has been the object of intense theoretical work. However, the testing of these theories is limited by our ability to estimate the strength of natural selection in nature. Using a long-term cline series, we estimate the selection coefficients acting on different alleles at the same locus to analyze the allele replacement observed in the insecticide resistance gene Ester in the mosquito Culex pipiens in the Montpellier area, southern France. Our method allows us to accurately account for the resistance allele replacement observed in this area since 1986. A first resistance allele appeared early, which was replaced by a second resistance allele providing the same advantage but at a lower cost, itself being replaced by a third resistance allele with both higher advantage and cost. It shows that amelioration of the adaptation (here resistance to insecticide) through allele replacement was successively achieved by selection of first a generalist allele (i.e., with a low fitness variance across environments) and later a specialist allele (i.e., with a large fitness variance across environments). More generally, we discuss how precise estimates of the strength of selection obtained from field data help us understand the process of amelioration of adaptation.

Wolbachia Age-Sex-Specific Density in Aedes albopictus : A Host Evolutionary Response to Cytoplasmic Incompatibility?

Background Wolbachia bacteria have invaded many arthropod species by inducing Cytoplasmic Incompatibility (CI). These symbionts represent fascinating objects of study for evolutionary biologists, but also powerful potential biocontrol agents. Here, we assess the density dynamics of Wolbachia infections in males and females of the mosquito Aedes albopitcus, an important vector of human pathogens, and interpret the results within an evolutionary framework. Methodology/Principal Findings Wolbachia densities were measured in natural populations and in age controlled mosquitoes using quantitative PCR. We show that the density dynamics of the wAlbA Wolbachia strain infecting Aedes albopictus drastically differ between males and females, with a very rapid decay of infection in males only. Conclusions/Significance Theory predicts that Wolbachia and its hosts should cooperate to improve the transmission of infection to offspring, because only infected eggs are protected from the effects of CI. However, incompatible matings effectively lower the fertility of infected males, so that selection acting on the host genome should tend to reduce the expression of CI in males, for example, by reducing infection density in males before sexual maturation. The rapid decay of one Wolbachia infection in Aedes albopictus males, but not in females, is consistent with this prediction. We suggest that the commonly observed reduction in CI intensity with male age reflects a similar evolutionary process. Our results also highlight the importance of monitoring infection density dynamics in both males and females to assess the efficiency of Wolbachia-based control strategies.

Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations.

Two amino acid substitutions in acetylcholinesterase 1 (AChE1), G119S and F290V, are responsible for resistance to organophosphate and carbamate insecticides in Culex pipiens mosquitoes. These mutations generate very different levels of insensitivity to insecticide inhibitors. We described here a biochemical method that rapidly identifies AChE1 variants (susceptible, G119S and F290V, named S, R and V, respectively) present in individual mosquitoes. We investigated the frequency of AChE1 phenotypes in 41 field samples collected around the Mediterranean Sea. F290V substitution was found only in 15 samples and at low frequency, whereas G119S was highly spread in all samples. However, seven V distinct alleles were identified whereas only one R allele was present. The [V] enzymatic phenotype was never observed alone, and the V allele was always found associated with the susceptible and/or G119S AChE1 ([VS], [VR] or [VRS] phenotypes). Furthermore, we showed the presence of duplicated alleles, associating a susceptible and a V copy of the ace-1 gene, in most individuals analyzed for its presence. Evolutionary forces driving the large number of F290V ace-1 alleles and their low frequency in Mediterranean countries are discussed.

Multiple Insecticide Resistances in the Disease Vector Culex p. Quinquefasciatus from Western Indian Ocean

Several mosquito-borne diseases affect the Western Indian Ocean islands. Culex pipiens quinquefasciatus is one of these vectors and transmits filariasis, Rift Valley and West Nile viruses and the Japanese encephalitis. To limit the impact of these diseases on public health, considerable vector control efforts have been implemented since the 50s, mainly through the use of neurotoxic insecticides belonging to Organochlorines (OC), Organophosphates (OP) and pyrethroids (PYR) families. However, mosquito control failures have been reported on site, and they were probably due to the selection of resistant individuals in response to insecticide exposure. In this study, we used different approaches to establish a first regional assessment of the levels and mechanisms of resistance to various insecticides. Bioassays were used to evaluate resistance to various insecticides, enzyme activity was measured to assess the presence of metabolic resistances through elevated detoxification, and molecular identification of known resistance alleles was investigated to determine the frequency of target-site mutations. These complementary approaches showed that resistance to the most used insecticides families (OC, OP and PYR) is widespread at a regional scale. However, the distribution of the different resistance genes is quite heterogeneous among the islands, some being found at high frequencies everywhere, others being frequent in some islands and absent in others. Moreover, two resistance alleles displayed clinal distributions in Mayotte and La Réunion, probably as a result of a heterogeneous selection due to local treatment practices. These widespread and diverse resistance mechanisms reduce the capacity of resistance management through classical strategies (e.g. insecticide rotation). In case of a disease outbreak, it could undermine the efforts of the vector control services, as only few compounds could be used. It thus becomes urgent to find alternatives to control populations of Cx. p. quinquefasciatus in the Indian Ocean.

High incidence of ace-1 duplicated haplotypes in resistant Culex pipiens mosquitoes from Algeria

The status of genes conferring resistance to organophosphate and carbamate insecticides has been examined in Culex pipiens pipiens mosquitoes sampled in Algeria. Presence of overproduced esterases was sporadic, but acetylcholinesterase-1 resistant alleles were observed in almost all samples. We focused our study on the AChE1 G119S substitution characterized in almost all samples, mostly at the heterozygous state. A genetic test revealed the presence of ace-1 duplication associating a susceptible and a resistant ace-1 copy. Molecular characterization showed a high occurrence of ace-1 duplication with six distinct duplicated alleles out of four samples. The inferred frequency of duplicated allele suggests that it is replacing the single resistant G119S allele. Finally, we discuss the mechanism at the origin of these duplicated haplotypes and their consequences on the management of insecticide resistance.

Wolbachia divergence and the evolution of cytoplasmic incompatibility in Culex pipiens.

Many insect species harbor Wolbachia bacteria that induce cytoplasmic incompatibility (CI), i.e. embryonic lethality in crosses between infected males and uninfected females, or between males and females carrying incompatible Wolbachia strains. The molecular mechanism of CI remains unknown, but the available data are best interpreted under a modification–rescue model, where a mod function disables the reproductive success of infected males’ sperm, unless the eggs are infected and express a compatible resc function. Here we examine the evolution of CI in the mosquito Culex pipiens, harbouring a large number of closely related Wolbachia strains structured in five distinct phylogenetic groups. Specifically, we used a worldwide sample of mosquito lines to assess the hypothesis that genetic divergence should correlate with the divergence of CI properties on a low evolutionary scale. We observed a significant association of Wolbachia genetic divergence with CI patterns. Most Wolbachia strains from the same group were compatible whereas those from different groups were often incompatible. Consistently, we found a strong association between Wolbachia groups and their mod-resc properties. Finally, lines from the same geographical area were rarely incompatible, confirming the conjecture that the spatial distribution of Wolbachia compatibility types should be constrained by selection. This study indicates a clear correlation between Wolbachia genotypes and CI properties, paving the way toward the identification of the molecular basis of CI through comparative genomics.