Mylène Weill

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.

The unique mutation in ace-1 giving high insecticide resistance is easily detectable in mosquito vectors.

High insecticide resistance resulting from insensitive acetylcholinesterase (AChE) has emerged in mosquitoes. A single mutation (G119S of the ace‐1 gene) explains this high resistance in Culex pipiens and in Anopheles gambiae. In order to provide better documentation of the ace‐1 gene and the effect of the G119S mutation, we present a three‐dimension structure model of AChE, showing that this unique substitution is localized in the oxyanion hole, explaining the insecticide insensitivity and its interference with the enzyme catalytic functions. As the G119S creates a restriction site, a simple PCR test was devised to detect its presence in both A. gambiae and C. pipiens, two mosquito species belonging to different subfamilies (Culicinae and Anophelinae). It is possibile that this mutation also explains the high resistance found in other mosquitoes, and the present results indicate that the PCR test detects the G119S mutation in the malaria vector A. albimanus. The G119S has thus occurred independently at least four times in mosquitoes and this PCR test is probably of broad applicability within the Culicidae family.

A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace–1) being a new locus and the other (ace–2) being homologous to the gene previously described in Drosophila. The gene ace–1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace–1 and ace–2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace–1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace–1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace–2 in at least the Drosophilidae and Muscidae and ace–1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.

Insecticide Control of Vector-Borne Diseases: When Is Insecticide Resistance a Problem?

Many of the most dangerous human diseases are transmitted by insect vectors. After decades of repeated insecticide use, all of these vector species have demonstrated the capacity to evolve resistance to insecticides. Insecticide resistance is generally considered to undermine control of vector-transmitted diseases because it increases the number of vectors that survive the insecticide treatment. Disease control failure, however, need not follow from vector control failure. Here, we review evidence that insecticide resistance may have an impact on the quality of vectors and, specifically, on three key determinants of parasite transmission: vector longevity, competence, and behaviour. We argue that, in some instances, insecticide resistance is likely to result in a decrease in vector longevity, a decrease in infectiousness, or in a change in behaviour, all of which will reduce the vectorial capacity of the insect. If this effect is sufficiently large, the impact of insecticide resistance on disease management may not be as detrimental as previously thought. In other instances, however, insecticide resistance may have the opposite effect, increasing the insects vectorial capacity, which may lead to a dramatic increase in the transmission of the disease and even to a higher prevalence than in the absence of insecticides. Either way—and there may be no simple generality—the consequence of the evolution of insecticide resistance for disease ecology deserves additional attention.

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.

The kdr mutation occurs in the Mopti form of Anopheles gambiae s.s. through introgression.

Anopheles gambiae s.s. is a complex of sibling taxa characterized by various paracentric inversions. In west and central Africa, where several taxa are sympatric, a kdr mutation responsible for pyrethroid resistance has been described in only one (the S taxon), suggesting an absence of gene flow between them. Following a thorough sampling, we have found a kdr mutation in another taxon (M). To establish whether this mutation is the same event or not, the large intron upstream of the kdr mutation was sequenced to find polymorphic sites in susceptible/resistant and M/S mosquitoes. The low genetic diversity found in this DNA region indicates that a local genetic sweep has recently occurred. However, some polymorphic sites were found, and it is therefore concluded that the kdr mutation in the M taxon is not an independent mutation event, and is best explained by an introgression from the S taxon. These results are discussed within the context of possible gene flow between members of An. gambiae s.s. taxa, and with the possible spread of the kdr mutation in other closely related malaria vectors of the An. gambiae complex.

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

Evidence of Introgression of the ace-1R Mutation and of the ace-1 Duplication in West African Anopheles gambiae s. s

Background The role of inter-specific hybridisation is of particular importance in mosquito disease vectors for predicting the evolution of insecticide resistance. Two molecular forms of Anopheles gambiae s.s., currently recognized as S and M taxa, are considered to be incipient sibling species. Hybrid scarcity in the field was suggested that differentiation of M and S taxa is maintained by limited or absent gene flow. However, recent studies have revealed shared polymorphisms within the M and S forms, and a better understanding of the occurrence of gene flow is needed. One such shared polymorphism is the G119S mutation in the ace-1 gene (which is responsible for insecticide resistance); this mutation has been described in both the M and S forms of A. gambiae s.s. Methods and Results To establish whether the G119S mutation has arisen independently in each form or by genetic introgression, we analysed coding and non-coding sequences of ace-1 alleles in M and S mosquitoes from representative field populations. Our data revealed many polymorphic sites shared by S and M forms, but no diversity was associated with the G119S mutation. These results indicate that the G119S mutation was a unique event and that genetic introgression explains the observed distribution of the G119S mutation within the two forms. However, it was impossible to determine from our data whether the mutation occurred first in the S form or in the M form. Unexpectedly, sequence analysis of some resistant individuals revealed a duplication of the ace-1 gene that was observed in both A. gambiae s.s. M and S forms. Again, the distribution of this duplication in the two forms most likely occurred through introgression. Conclusions These results highlight the need for more research to understand the forces driving the evolution of insecticide resistance in malaria vectors and to regularly monitor resistance in mosquito populations of Africa.

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.