Arnaud Berthomieu

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.

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.

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.

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.

Trifling variation in truffles

Of the ten species of European truffle (fungi of the genus Tuber, phylum Ascomycota), some have economic value because of their organoleptic properties (taste and perfume), in particular the black truffle (Tuber melanosporum Vitt.) and the summer and burgundy truffles,. The black truffle is mainly found in Spain, France and Italy (Fig. 1a), and it shows variation in several traits, including in its famous organoleptic properties, across this geographical range. Here we show that this variation probably results from environmental, rather than genetic, influences.

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.

Fossil Rhabdoviral Sequences Integrated into Arthropod Genomes: Ontogeny, Evolution, and Potential Functionality

Retroelements represent a considerable fraction of many eukaryotic genomes and are considered major drives for adaptive genetic innovations. Recent discoveries showed that despite not normally using DNA intermediates like retroviruses do, Mononegaviruses (i.e., viruses with nonsegmented, negative-sense RNA genomes) can integrate gene fragments into the genomes of their hosts. This was shown for Bornaviridae and Filoviridae, the sequences of which have been found integrated into the germ line cells of many vertebrate hosts. Here, we show that Rhabdoviridae sequences, the major Mononegavirales family, have integrated only into the genomes of arthropod species. We identified 185 integrated rhabdoviral elements (IREs) coding for nucleoproteins, glycoproteins, or RNA-dependent RNA polymerases; they were mostly found in the genomes of the mosquito Aedes aegypti and the blacklegged tick Ixodes scapularis. Phylogenetic analyses showed that most IREs in A. aegypti derived from multiple independent integration events. Since RNA viruses are submitted to much higher substitution rates as compared with their hosts, IREs thus represent fossil traces of the diversity of extinct Rhabdoviruses. Furthermore, analyses of orthologous IREs in A. aegypti field mosquitoes sampled worldwide identified an integrated polymerase IRE fragment that appeared under purifying selection within several million years, which supports a functional role in the hosts biology. These results show that A. aegypti was subjected to repeated Rhabdovirus infectious episodes during its evolution history, which led to the accumulation of many integrated sequences. They also suggest that like retroviruses, integrated rhabdoviral sequences may participate actively in the evolution of their hosts.

Insecticide resistance: a silent base prediction

Response to a challenging environment proceeds through adaptation, the result of stochastic processes (chance) and of the influence of history (constraint) [1]. Adaptations, such as pesticide resistance, provide an opportunity to study historical constraints. Insecticides, widely used since the mid 1950s, have elicited numerous cases of resistance. Specific amino acid changes at unique or few critical positions of the target protein explain resistance to the major classes of insecticides, such as cyclodienes, organochlorines, pyrethroids, carbamates and organophosphates (OPs) [2–6] and sometimes lead to extremely high resistance levels (>1000 fold). In mosquitoes, a single glycine (Gly) to serine (Ser) substitution at position 119 (Torpedo nomenclature) in the acetylcholinesterase (AChE1) gene confers high levels of resistance to carbamates and OPs [3]. This G119S substitution was selected at least twice independently in Culex pipiens, once in Anopheles albimanus and once in Anopheles gambiae, suggesting that there are only few possibilities to generate high AChE1 insensitivity [7]. Although heavily controlled with carbamates and OPs, the mosquito vector of dengue and yellow fever, Aedes aegypti, never developed high levels of resistance. We first checked whether the G119S mutation in AChE1 is ineffective in this species. We cloned the complete AChE1 cDNA, encoding a protein 96.4% similar to C. pipiens AChE1, (supplemental data) and produced wild-type and G119S mutant recombinant proteins. Ae. aegypti AChE1 behaved exactly like C. pipiens AChE1: wild-type proteins were inhibited at identical doses of the carbamate insecticide propoxur (IC50 = 5 x 10–7 M), while G119S proteins remained insensitive up to 10–2 M propoxur (Figure 1). It is, therefore, unlikely that the low resistance in Ae. aegypti resulted from particular biochemical properties of its AChE1. Alternatively, Ae. aegypti AChE1 may not be able to evolve to the G119S substitution. Notably, in Ae. aegypti glycine 119 of AChE1 is encoded by a GGA codon, whereas GGC was found in all the other species analysed so far [3,7]. This silent third base change represents an extraordinarily heavy constraint. It decreases the probability of a spontaneous G119S substitution by several orders of magnitude . As Ser can be encoded by AGY or TCN, substitution to Ser requires only one mutation when Gly is encoded by GGY, whereas two are required when Gly is encoded by GGR. We thus hypothesized that high levels of resistance cannot emerge if the G119S substitution requires more than a single step mutation, a situation that could be described as a ‘codon constraint’. Accordingly, knowing the sequence of codon 119 should allow the prediction of the ability of a given mosquito species to develop high OP-resistance. We first checked all known acetylcholinesterase amino acid sequences (79 animal species), and found that a glycine is present at position 119 in all species, except in ascidians and Schistosoma, which show a serine. This suggests that presence of a glycine is critical, and that no other amino acids are allowed in this position, except serine. We next analysed the sequence of codon 119 in 26 natural populations of Ae. aegypti collected in 12 countries. In all samples, the glycine was encoded by GGA (serineimmutable), which fits with the