Maïté Marquine

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.

Evolution of insecticide resistance in Culex pipiens populations: The Corsican paradox

Culex pipiens mosquitoes from Corsica have been subject to insecticide treatments since 1971, using temephos (an organophosphate). After 17 years, resistance has not developed beyond a 14‐fold level. This relatively low resistance is due to the presence of several identified resistance genes, including the insensitive target ( AceR ) and overproduced esterases (A1, A4 and B4). The fact that only a low resistance has developed after 17 years of treatment and that this low resistance level is the result of a relatively large number of resistance genes constitute a paradox. To understand this situation and explain why a higher temephos resistance level has not evolved in Corsica as in other parts of the world, it is proposed that the occurrence (through mutation or migration) of efficient resistance genes was a limiting step, and that the only resistance genes available at that time through migration from the surrounding Mediterranean countries had a low cross‐resistance to temephos. The local situation of Corsica is discussed in the light of recent data on the world distribution of the known organophosphate resistance genes in this species, and the relative role of mutation and migration in the evolution of insecticide resistance in natural populations.

POPULATION STRUCTURE AND DYNAMICS OF SELECTED GENES IN THE MOSQUITO CULEX PIPIENS

To determine the outcome of the combined effects of gene flow, genetic drift, and selection on the evolution of insecticide‐resistance genes in the mosquito Culex pipiens, samples were collected along three transects crossing treated and nontreated areas in northern Spain and southern France. Electrophoretic polymorphisms of five presumably neutral genes disclosed that differentiation among samples was low, and that both Wright F‐statistics and Slatkin private‐alleles methods provided a high estimate for Nm. In contrast, there was a strong differentiation in the distribution of resistance genes closely associated with insecticide treatments. These divergent situations are explained in relation to both the very recent origin of some resistance genes that are still localized geographically (A2‐B2 and C1), and the high fitness cost of the older and ubiquitous ones in nontreated areas (A1, A4‐B4, and AceR).

Dissecting the cost of insecticide resistance genes during the overwintering period of the mosquito Culex pipiens.

In several insects, there appears to be a high fitness cost associated with insecticide resistance genes during the overwintering period. In order to understand when and how this cost operates, all mosquitoes entering a natural cave for overwintering were regularly sampled, and their resistance genes at two loci (Ester and Ace.1) were individually identified. During the main period of entry (October and November), susceptible mosquitoes were first observed, followed by resistant ones, this trend being similar for both loci. This observation is best explained by a migration phenomenon, northern and more susceptible populations starting to overwinter first, followed by southern and more resistant ones. During the remaining part of the winter (December–March), mosquitoes entering the cave were still caught and they probably corresponded to individuals leaving a former overwintering site in search of a more suitable one. A lower overall frequency of resistant phenotypes was found in the second part of the winter at Ester, suggesting that a large fitness cost (42%) had operated. A decreasing frequency of resistant phenotypes was also found at Ace.1, indicating a large survival cost of resistant mosquitoes (7% for the homozygote Ace.1R) in their former overwintering site. These results are discussed in the light of the local evolution of these resistance genes in southern France.

Resistance to Bacillus sphaericus in Culex pipiens (Diptera: Culicidae): Interaction Between Recessive Mutants and Evolution in Southern France

Abstract In southern France, failure to control Culex pipiens L. with Bacillus sphaericus Neide toxin (Bs) was first detected in 1994, at the extreme east of the Languedoc-Roussillon coast. This failure was due to a single recessive mutant, sp-1R. Two complementary strategies were used to test whether sp-1R had invaded the Bs-controlled area by 1998. First, a strain (BP) was selected from resistant larvae sampled in the western part of the Bs-controlled area. In BP strain, resistance involved a single recessive gene, sp-2R, distinct from sp-1R, that conferred a similarly high resistance in the homozygous state (≈6,000-fold). Combining one copy of sp-1R and one of sp-2R conferred a > 100-fold resistance. Second, Bs-resistance was monitored among the offspring of field females crossed to sp-1RR homozygous males. Females were sampled in 20 localities of southern France and three localities of the Llobregat delta (Barcelona, Spain) where C. pipiens control is also intensive. The 537 females in the study produced enough larvae to infer their genotype: 462 progenies were susceptible and the survival rate of 51 others was explained by the presence of sp-1R and/or sp-2R. The remaining 24 cases indicated that other factors could confer resistance when combined with sp-1R. The current data showed that, even when recessive, resistant mutants can rapidly increase in frequency, providing some interactions that protect them from disappearance. We discuss the consequences of this finding on the current strategies aimed to avoid or delay resistance in the pests controlled with B. sphaericus or B. thuringiensis Berliner toxins.

Insecticide resistance in Chinese populations of the Culex pipiens complex through esterase overproduction

In most parts of China, mosquitoes have been subjected to organophosphate (OP) insecticide treatments since the mid‐1960s, and resistance gene monitoring in the Culex pipiens complex (Diptera: Culicidae) started in only a few locations from the end of the 1980s. Many resistant alleles at the Ester locus have been found in field populations, including those commonly found around the world (EsterB1 and Ester2), and those endemic to China (EsterB6, EsterB7, Ester8, and Ester9). This situation is atypical, and may represent a complex situation for the evolution of insecticide resistance genes in China. To increase our understanding of the Chinese situation and our ability to manage resistance in the C. pipiens complex, a large study was performed. Twenty field populations were sampled from Beijing to Guangzhou. Bioassays with five insecticides (dichlorvos, parathion, chlorpyrifos, 2‐sec‐butylphenyl methyl carbamate, and propoxur) disclosed resistance levels variable according to the geographic origin, and up to 85‐fold for dichlorvos. Six overproduced esterases were identified, including two that have not been previously described. Most of them were found in all samples, although at variable frequencies, suggesting variable selection or a transient situation, e.g., each one was recently restricted to a particular geographic area. The results are discussed in the context of recent alterations to insecticide campaigns, and of the evolution of resistance genes in Chinese C. pipiens populations.

Evolution of resistance under insecticide selection pressure in Culex pipiens quinquefasciatus (Diptera, Culicidae) from Martinique.

Abstract Population surveys of Culex pipiens quinquefasciatus carried out in 1991 and 1999 were compared with data collected in 1990 before the beginning of the control program against this mosquito. Larval samples collected in 1999 displayed resistance to the four tested insecticides: permethrin, propoxur, temephos, and chlorpyrifos. Temephos resistance ratio at LC50 (RR50) ranged between 8.1- and 42-fold compared with 2.9- and 4.6-fold in 1990, and chlorpyrifos RR50 ranged between 8.6- and 123-fold compared with 6.4- and 19-fold in 1990. This increased resistance to organophosphorus insecticides was associated with a sharp decrease of susceptible genotypes at two loci (Ester and ace-1), as well as to an allele replacement at the Ester locus.

The role of passive migration in the dispersal of resistance genes in Culex pipiens quinquefasciatus within French Polynesia

We have investigated the distribution of resistance genes in relation to genetic structure and gene flow between various islands of French Polynesia in Culex pipiens quinquefasciatus . We show that (1) resistance to organophosphorous insecticides, mediated by A2–B2 esterases, is present in all islands, (2) A2–B2 frequency decreases as distance from Tahiti increases, and (3) genetic differentiation (measured as estimates of the F st , parameter from allozyme polymorphism) between islands is significantly correlated to geographic distances which is also correlated to air or sea commercial traffic. These data are discussed in relation to A2–B2 invasion of French Polynesian islands.

A New Esterase Gene Amplification Involved in OP Resistance in Culex pipiens Mosquitoes from China

Two overproduced esterases (A8 and B8) notpreviously described were found in southern China. Theyprovide a low resistance level to organophosphate (OP)insecticides, and correspond to a coamplification of both esterase loci (Est-2 and Est-3)classically involved in OP resistance for this mosquitospecies. This coamplification is distinct from all othersimilar events thus far reported. The peculiar situation in southern China, where numerous OP resistancealleles at these two loci were found, is discussed incomparison with the Mediterranean situation, the onlyone with a similar diversity of overproducedesterases.

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.