Molecular data reveal a cryptic species within the Culex pipiens mosquito complex.

The Culex pipiens mosquito complex is a group of evolutionarily closely related species including C. pipiens and Culex quinquefasciatus, both infected by the cytoplasmically inherited Wolbachia symbiont. A Wolbachia-uninfected population of C. pipiens was however described in South Africa and was recently proposed to represent a cryptic species. In this study, we reconsidered the existence of this species by undertaking an extensive screening for the presence of Wolbachia-uninfected C. pipiens specimens and by characterizing their genetic relatedness with known members of the complex. We first report on the presence of Wolbachia-uninfected specimens in several breeding sites. We next confirm that these uninfected specimens unambiguously belong to the C. pipiens complex. Remarkably, all uninfected specimens harbour mitochondrial haplotypes that are either novel or identical to those previously found in South Africa. In all cases, these mitochondrial haplotypes are closely related, but different, to those found in other C. pipiens complex members known to be infected by Wolbachia. Altogether, these results corroborate the presence of a widespread cryptic species within the C. pipiens species complex. The potential role of this cryptic C. pipiens species in the transmission of pathogens remains however to be determined. The designation 'Culex juppi nov. sp.' is proposed for this mosquito species.

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes.

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l(-1) and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1(R) allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1(R) and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1(V) or the duplicated ace-1(D) allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.

Insecticide resistance in the Anopheles gambiae complex in Benin: a nationwide survey.

Benin has embraced World Health Organization-recommended preventive strategies to control malaria. Its National Malaria Control Programme is implementing and/or coordinating various actions and conducting evaluation trials of mosquito control strategies. Mosquito control is based on the use of insecticide-treated nets and indoor residual spraying, but the efficacy of these strategies to control malaria vectors is endangered by insecticide resistance. Here, we present the results of a nationwide survey on the status of insecticide susceptibility and resistance in Anopheles gambiae s.l. (Diptera: Culicidae) carried out in Benin in 2006-2007 (i.e. before extensive vector control was undertaken). Overall, our study showed that the S molecular form of An. gambiae s.s. predominates and is widely distributed across the country, whereas the frequency of the M form shows a strong decline with increasing latitude. Susceptibility to DDT, permethrin, carbosulfan and chlorpyrifos-methyl was assessed; individual mosquitoes were identified for species and molecular forms, and genotyped for the kdr and ace-1 loci. Full susceptibility to chlorpyrifos-methyl was recorded and very few samples displayed resistance to carbosulfan. High resistance levels to permethrin were detected in most samples and almost all samples displayed resistance to DDT. The kdr-Leu-Phe mutation was present in all localities and in both molecular forms of An. gambiae s.s. Furthermore, the ace-1(R) mutation was predominant in the S form, but absent from the M form. By contrast, no target modification was observed in Anopheles arabiensis. Resistance in the An. gambiae S molecular form in this study seemed to be associated with agricultural practices. Our study showed important geographic variations which must be taken into account in the vector control strategies that will be applied in different regions of Benin. It also emphasizes the need to regularly monitor insecticide resistance across the country and to adapt measures to manage resistance.

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito.

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens.

Reproductive incompatibilities called cytoplasmic incompatibilities are known to affect a large number of arthropod species and are mediated by Wolbachia, a maternally transmitted microorganism. The crossing relationships between strains of potential hosts define their incompatibility types and it is generally assumed that differences between strains of Wolbachia induce different crossing types. Among all the described host species, the mosquito, Culex pipiens, displays the greatest variability of cytoplasmic incompatibility crossing types. We analysed mitochondrial and bacterial DNA variability in Culex pipiens in order to investigate some possible causes of incompatibility crossing type variability. We sequenced fragments of the ftsZ gene, and the A + T-rich control region of the mtDNA. We also sequenced the second subunit of the mitochondrial cytochrome oxidase (COII) gene, in Culex pipiens and a closely related species, C. torrentium, in order to verify the usefulness of the A + T-rich region for the present purposes. No variability was found in the Wolbachia ftsZ gene fragment, and very limited variation of the mitochondrial marker whatever the compatibility type or the origin of the host. A low variability was found in the A + T-rich region and comparison of divergence of the A + T-rich region and COII gene between C. pipiens and C. torrentium did not reveal any special constraints affecting this region. In contrast to observations in other host species, variability of incompatibility crossing types is not due to multiple infections by distantly related Wolbachia strains.

Gene flow in the European corn borer Ostrinia nubilalis: implications for the sustainability of transgenic insecticidal maize.

Strategies proposed for delaying resistance to Bacillus thuringiensis toxins expressed by transgenic maize require intense gene flow between individuals that grew on transgenic and on normal (referred to as refuges) plants. To investigate gene flow in the European corn borer, Ostrinia nubilalis (Hübner), the genetic variability at 29 sampled sites from France was studied by comparing allozyme frequencies at six polymorphic loci. Almost no deviations from Hardy-Weinberg expectations occurred, and a high stability of allelic distribution was found among samples collected in the same site over two or three different generations, indicating a high stability of the genetic structure over time. The overall genetic differentiation was low at the region and whole country level, suggesting a high and homogeneous gene flow. These results are discussed in relation to the sustainability of transgenic insecticidal maize.

Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece.

We investigated the mechanisms conferring resistance to methyl-parathion (44-fold) and to methomyl (8-fold) in Tetranychus urticae from Greece by studying the effect of synergists on the resistance and the kinetic characteristics of various enzymes in a resistant strain (RLAB) and a susceptible reference strain (SAMB). It is shown that S,S,S-tributyl phosphorotrithioate, a synergist that inhibits esterases and glutathione S-transferases, and piperonyl butoxide, a synergist that inhibits cytochrome P450 mediated monooxygenases, did not affect the level of methyl-parathion or methomyl resistance in RLAB and that resistance ratios to both insecticides did not change significantly in the presence of either synergist. Isoelectric focusing of esterase allozymes on single mites revealed no differences in staining intensity and glutathione S-transferase activity was not significantly different in the two strains. The activity of two cytochrome P450 monooxygenase groups was compared. No significant difference of 7-ethoxyresorufin-O-diethylase activity was observed between strains that were two-fold higher in RLAB than in SAMB. The kinetic characteristics of acetylcholinesterase, the target enzyme of organophosphates and carbamates, revealed that acetylcholinesterase in RLAB was less sensitive to inhibition by paraoxon and methomyl in comparison with SAMB. I(50), the inhibitor concentration inducing 50% decrease of acetylcholinesterase activity was greater (119- and 50-fold with paraoxon and methomyl, respectively) and the bimolecular constant k(i) was lower (39- and 47-fold with paraoxon and methomyl, respectively) in RLAB compared to SAMB.

Mechanisms of insecticide resistance in the aphid Nasonovia ribisnigri (Mosley) (Homoptera: Aphididae) from France.

Nasonovia ribisnigri, a main pest of salad crops, has developed resistance to various insecticides in southern France, including the carbamate pirimicarb and the cyclodiene endosulfan, two insecticides widely used to control this aphid. Here we have investigated the mechanisms of resistance to these two insecticides by studying cross-resistance, synergism, activity of detoxifying enzymes, and possible modifications of the target proteins. Resistance to pirimicarb was shown to be mainly due to a decreased sensitivity of the target acetylcholinesterase; this modification conferred also, resistance to propoxur but not to methomyl and the two tested organophosphates (acephate and paraoxon). Endosulfan resistance was associated with a moderate level of resistance to dieldrin, and resistance to both insecticides was due, in part, to increased detoxification by glutathione S-transferases (GST). The endosulfan resistant strain displayed the same amino acid at position 302 of the Rdl gene (GABA receptor) as susceptible aphids (e.g. Ala), indicating that the Ala to Ser (or to Gly) mutation observed among dieldrin resistant strains of other insect species was not present.

Tissue localization of overproduced esterases in the mosquito Culex pipiens (Diptera: Culicidae).

We have investigated the tissue distribution of overproduced esterases A (A1 and A2) and B (B1 and B2) in strains of Culex pipiens L. by immunocytochemistry. S-LAB mosquitoes, lacking overproduced esterases, were used as reference. Tissues showing a strong specific reaction (fluorescence) were observed with anti-esterase A1 antiserum in S54 (with A1) and BOUAKE (with A2) strains, and with anti-esterase B1 antiserum in TEM-R and EDIT (with B1) and BOUAKE (with B2) strains. Overproduction of esterases A and B was tissue-specific. The most constant pattern for the two types of esterases was their overproduction in the alimentary canal and Malpighian tubes, although fluorescence varied in intensity depending on strains and developmental stages. There was no difference in the tissue distribution of esterases Al and A2. In contrast, esterases B pattern was highly variable among strains. Differences between TEM-R and EDIT were explained by the different overall overproduction and number of copies of the amplified gene (10-fold higher in TEM-R). The most striking difference in esterase B1 and B2 tissue localization concerned the nervous system where neurons were intenisely fluorescent in TEM-R and EDIT (B1), but not in BOUAKE (B2). All esterase B positive tissues in TEM-R contained large quantities of esterase B1 mRNA (in situ hybridization), indicating that at least part of the protein revealed by immunochemistry was produced in the tissues where it was observed. Our results are discussed in terms of the protection that the different esterases can confer during exposition to organophosphorous insecticides.

Resistance to organophosphorous insecticides in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Martinique.

Before beginning a widespread control program against Culex pipiens quinquefasciatus in Martinique, resistance to temephos, chlorpyrifos, and two organophosphorous insecticides, was investigated at seven breeding sites. At LC95, populations exhibited resistance ratios between 6.9 and 11.6 for temephos and between 6.4 and 51.4 for chlorpyrifos. Overproduced esterases A2-B2 and B1, known to be involved in organophosphorous-resistance, were present at all breeding sites; esterases A2-B2 frequency was > 50% at all sites but one; and esterase B1 frequency was < 7%. Experimental treatment of three breeding sites with temephos induced no significant increase in resistance, but our esterase studies indicated a significant increase in the frequencies of esterase B1 and of a new highly active esterase C2. These results indicate that a large-scale C. p. quinquefasciatus control program with organophosphorous insecticides will induce a rapid increase of these resistance genes throughout Martinique. However, this may not necessarily result in high levels of resistance, because, at present, the level of gene amplification of esterase B still appears to be low.

Resistance to Bacillus sphaericus in Culex pipiens (Diptera: Culicidae): interaction between recessive mutants and evolution in southern France.

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 (approximately 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 susceptibility in mosquitoes (Diptera: Culicidae) from French Polynesia.

Susceptibility to six organophosphate (OP), two pyrethroid (PY), and one carbamate (C) insecticides was investigated in Culex pipiens quinquefasciatus Say, Aedes aegypti (L.), and Aedes polynesiensis Marks larvae from the island of Tahiti. Cx. p. quinquefasciatus and Ae. aegypti were compared with susceptible reference strains treated simultaneously. A low, but significant, resistance to bromophos (4.6x), chlorpyrifos (5.7x), fenthion (2.4x), fenitrothion (5.0x), temephos (4.3x) and permethrin (2.1x) was found in Cx. p. quinquefasciatus, and to malathion (1.5x), temephos (2.3x), permethrin (1.8x) and propoxur (1.7x) in Ae. aegypti. Cx. p. quinquefasciatus was shown to possess over-produced esterases A2 and B2, which are known to be involved in resistance to OPs in other countries. Ae. polynesiensis was less resistant than the Ae. aegypti reference strain to all insecticides except temephos (1.8x) and permethrin (6.7x). To determine whether Ae. polynesiensis had developed resistance to these insecticides in Tahiti, a geographical survey covering 12 islands of the Society, Tuamotu, Tubuai, Marquesas, and Gambier archipelagoes was undertaken with three insecticides (temephos, deltamethrin, and permethrin). Two- to threefold variations in LC50S were observed among collections. Results are discussed in relationship to the level of insecticide exposure on the different islands.

Resistance monitoring in Culex pipiens (Diptera: Culicidae) from central-eastern France.

Insensitive acetylcholinesterase (AceR) and five over-produced esterases (A1, A2 and B2, and A4 and B4) involved in detoxification are responsible for resistance to organophosphorous insecticides (OPs) in Culex pipiens L. from the Rhône-Alpes region, where C. pipiens control is mainly accomplished with the OPs temephos and chlorpyrifos using 0.15 mg/liter doses. The strong linkage disequilibria observed between esterases A1 and Est-20(0.64), esterases A4 and B4, and esterases A2 and B2 indicate that these genes were introduced in the Rhône-Alpes region. AceR and esterase A1, which appeared in the south of France 3 yr before the start of mosquito control in Rhône-Alpes, had the highest frequencies. All resistant genotypes were shown to be killed by 0.15 mg/liter temephos in natural breeding sites, but not by 0.15 mg/liter chlorpyrifos. These results are discussed in relation with mosquito control strategies.

Resistance to organophosphorus and pyrethroid insecticides in Culex pipiens (Diptera: Culicidae) from Tunisia.

Resistance to the organophosphates temephos and chlorpyrifos, the carbamate propoxur, the pyrethroid permethrin, and the organochloride DDT was investigated in Tunisian populations of Culex pipiens pipiens (L.) collected between 1990 and 1996. Resistance to temephos was uniformly low, reaching 10-fold in the most resistant population. In contrast, resistance to chlorpyrifos was highly variable, reaching the highest level (> 10,000-fold) recorded worldwide. The chlorpyrifos-resistant populations also were highly resistant to propoxur. Some populations also showed high resistance to permethrin (up to 5,000-fold) and moderate resistance to DDT (approximately 20-fold). Bioassays conducted in the presence of synergists showed that increased detoxification had only a minor role in resistance, although several over-produced esterases known to be involved in organophosphate resistance were detected. To better understand the factors influencing the distribution of resistance in Tunisia, the polymorphism of genes involved in organophosphate resistance (i.e., over-produced esterases and insensitive acetylcholinesterase) was investigated in relation to the genetic structure of populations studied by analyzing the electrophoretic polymorphism of "neutral" genes. Over the area studied, and despite a high level of gene flow, resistance genes showed a patchy distribution. Results are discussed in relation to the selection pressure caused by insecticide treatments.

Cross-resistance to pyrethroid and organophosphorus insecticides in the southern house mosquito (Diptera:Culicidae) from Cuba.

A sample of the southern house mosquito, Culex pipiens quinquefasciatus Say, from Cuba was subjected to lambda-cyhalothrin selection to evaluate the usefulness of this pyrethroid insecticide for mosquito control. High resistance developed after 6 generations of selection. Little or no cross-resistance was observed to other pyrethroids (deltamethrin and cypermethrin), to a carbamate (propoxur) and to some organophosphates (chlorpyrifos and pirimiphos-methyl), but high cross-resistance was found to malathion (organophosphate). Possible resistance mechanisms responsible for this phenomenon are discussed.

Esterases A2 and B2 in Culex quinquefasciatus (Diptera: Culicidae): role in organophosphate resistance and linkage.

Two highly active esterases, A2 and B2, were isolated in SeLax, a strain of Culex quinquefasciatus Say from California which demonstrated resistance levels of 19, 14, and 4.3 times to the organophosphate insecticides chlorpyrifos, temephos, and malathion, respectively. Selection of SeLax by temephos during 31 generations increased resistance to this insecticide to 65 times. This resistance was shown to be DEF-suppressible, monofactorial, and strongly associated with the presence of esterases A2 and B2. Although these two esterases are encoded by distinct structural genes, no disjunction was observed when SeLax was crossed to S-Lab, a susceptible strain, or to S54, a strain resistant to organophosphates by means of esterase A1. However, when SeLax was crossed to strain Tem-R, which is resistant to organophosphates because of a highly active esterase B1, all possible recombinants occurred. These results were discussed in relation to the recent discovery that increased activity of B esterases in the genus Culex is caused by gene amplification.

A new mechanism conferring unprecedented high resistance to chlorpyrifos in Culex pipiens (Diptera: Culicidae).

The cause of high resistance to chlorpyrifos observed in Tunisian Culex pipiens (L.) was investigated by comparing a Tunisian strain G (> 10,000-fold resistance), a French strain T (approximately 50-fold resistance), and a susceptible reference strain S. Strains G and T had the same level of propoxur resistance (approximately 1,000-fold) and were homozygous for an autosomal propoux-insensitive acetylcholinesterase (AChE-1). In G and T strains, as well as in the offspring of different F1s and backcrosses using these F1s and the S strain, the effect of DEF and Pb synergists on chlorpyrifos resistance was low or absent, indicating that increased detoxification by enzymes inhibited by these chemicals had a minor role. Chlorpyrifos resistance in the G strain was caused by a major gene (or group of genes) tightly linked to the Ace-1 gene (coding AChE-1 enzyme). The possibility of allelism between this gene and the Ace-1R allele present in the T strain was rejected by showing that AChE-1 inhibition by chlorpyrifos-oxon was not different between G and T mosquitoes.

Overproduced esterases in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Vietnam.

The electrophoretic polymorphism of loci encoding for 10 enzymes was studied in Culex p. quinquefasciatus Say from six localities of Vietnam. The analysis of 11 "neutral genes" showed that differentiation among samples was low, but significant (Fst = 0.06), and significantly related to geographic distance between sample sites. These results are similar to those observed in other countries (Europe and west Africa). A single type of overproduced esterases (A2-B2) was observed, and its frequency was high (60-100%) in all samples. This situation is in sharp contrast with that observed in other countries of South East Asia (China, South Korea and Japan), where two or more types of overproduced esterases have been reported. A map summarizing the geographic distribution of Asian Cr. p. quinquefasciatus with overproduced esterases is provided.

Distribution of organophosphate and carbamate resistance in Culex pipiens quinquefasciatus (Diptera: Culicidae) in West Africa.

The distribution of organophosphate and carbamate resistance was investigated in 33 samples of Culex pipiens quinquefasciatus Say from 25 cities in Côte d'Ivoire and Burkina Faso. Organophosphate resistance levels were higher in Côte d'Ivoire than in Burkina Faso. Chlorpyrifos resistance ratios at LC95 ranged from 4 to 30 times in Côte d'Ivoire and from 3 to 6 times in Burkina Faso. For temephos, ratios ranged from 3 to 18 and from 1 to 2, respectively. Of 27 samples from Côte d'Ivoire, 25 also displayed cross resistance to carbamates as shown by a mortality plateau in bioassays with propoxur and carbosulfan (similar to chlorpyrifos). Cross resistance to organophosphates and carbamates was caused by an insensitive acetylcholinesterase allele (AceR). This gene was absent from Burkina Faso, except in Niangoloko near the Côte d'Ivoire border. Organophosphate resistance also was associated with the presence of A2-B2 overproduced esterases which had higher frequencies in Côte d'Ivoire (75-100%) than in Burkina Faso (40-50%). Two other esterases with the same electrophoretic mobility as C2 from Puerto Rico and B1 from California were identified for the 1st time in West Africa. "C2" was widespread, whereas "B1" was present in only a few mosquitoes from Côte d'Ivoire. These differences in resistance patterns should be taken into consideration in planning urban mosquito control strategies within 2 countries.

Resistance to Bacillus sphaericus involves different mechanisms in Culex pipiens (Diptera:Culicidae) larvae.

Field Culex pipiens pipiens (L.) mosquitoes that were collected after a control failure with Spherimos in southern France developed high resistance (> 10,000-fold) to Bacillus sphaericus crystal toxin after < 8 generations of laboratory selection. We show that this resistance is encoded by a single major recessive gene on linkage group I at 22.1 recombination units from the sex locus, and that it is not associated with any loss of binding affinity between brush border membrane fractions and the B. sphaericus radiolabeled toxin. Thus, in Southern France, resistance differs from the high B. sphaericus resistance developed after laboratory selection of Californian C. p. quinquefasciatus. This demonstrates that at least 2 different mechanisms may confer high levels of resistance to B. sphaericus crystal toxin in mosquitoes of the C. pipiens complex. These results have important implications for mosquito control strategies.