Chuanling Qiao

The same esterase B1 haplotype is amplified in insecticide-resistant mosquitoes of the Culex pipiens complex from the Americas and China.

In Culex pipiens, overproduction of nonspecific esterases is a common mechanism of resistance to organophosphate insecticides. The esterases are attributed to closely linked loci named A and B, and overproduction of all esterases B is due to gene amplification. In order to determine if the esterase B1 identified by electrophoretic studies in Culex pipiens mosquitoes from different countries is overproduced due to the amplification of the same DNA haplotype, the amplified region encompassing the structural esterase B1 gene was characterized by restriction mapping and RFLP. The same amplified haplotype was found in mosquitoes with an esterase B1 protein, independently of their geographical origin: French Guiana, Venezuela, Puerto Rico, California and China. Large variations in amplification levels were observed. It is concluded that B1 amplification has a unique origin, either in America or in Asia, and has subsequently spread by migration. This migration is more limited than that of A2-B2 esterases, since B1 is confined to the Americas, the Carribean and part of China, whereas the A2-B2 distribution now includes the Americas, the Carribean, Asia, Africa, the Pacific islands and Europe.

Two single mutations commonly cause qualitative change of nonspecific carboxylesterases in insects

Carboxylesterases provide key mechanisms of resistance to insecticides, particularly organophosphates (OPs), in insects. One resistance mechanism is a qualitative change in the properties of a carboxylesterase. Two mutant forms, G151D and W271L, have been observed, mostly in dipteran species, to affect substrate specificity of enzymes. But whether these two single mutations can commonly change character of insect carboxylesterases is unknown. In our study carboxylesterase genes from seven insects distributed among four orders were cloned, mutated at position 151 or 271 and expressed in Escherichia coli. The kinetics of the purified recombinant proteins was examined towards an artificial carboxylester and two OP insecticides. The G/A151D and W271L mutation significantly reduced carboxylesterase activity in 87.5% and 100% cases, respectively, and at the same time conferred OP hydrolase activities in 62.5% and 87.5% cases, respectively. Thus, the change at position 271 is more effective to influence substrate specificity than that at position 151. These results may suggest that these two mutations have the potential to cause insecticide resistance broadly in insects.

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.

Different Amino-Acid Substitutions Confer Insecticide Resistance Through Acetylcholinesterase 1 Insensitivity in Culex vishnui and Culex tritaeniorhynchus (Diptera: Culicidae) from China

Abstract Insecticide resistance owing to insensitive acetylcholinesterase (AChE)1 has been reported in several mosquito species, and only two mutations in the ace-1 gene have been implicated in resistance: 119S and 331W substitutions. We analyzed the AChE1 resistance status of Culex vishnui (Theobald) and Culex tritaeniorhynchus Giles sampled in various regions of China. These two species displayed distinct mutations leading to AChE1 insensitivity; the 119S substitution in resistant C. vishnui mosquitoes and the 331W substitution in resistant C. tritaeniorhynchus. A biochemical test was validated to detect the 331W mutation in field samples. The comparison of the recombinant G119S and 331W mutant proteins produced in vitro with the AChE1 extracted from resistant mosquitoes indicated that the AChE1 insensitivity observed could be specifically attributed to these substitutions. Comparison of their biochemical characteristics indicated that the resistance conferred by these mutations depends on the insecticide used, regardless of its class. This resistance seemed to be fixed in the Cx. tritaeniorhynchus populations sampled in a 2,000-km transect, suggesting a very high level of insecticide application or a low fitness cost associated with this 331W mutation.

Do mosquitoes acquire organophosphate resistance by functional changes in carboxylesterases

Carboxylesterase‐based metabolic resistance to organophosphates (OPs) in insects has been shown to originate either from mutations in esterase‐encoding sequences or from amplification of esterase genes. This study aimed to test the hypothesis that mosquitoes can acquire OP resistance by functional changes in carboxylesterases. Mutations were introduced into the esterase B1 of mosquito Culex pipiens by site‐directed mutagenesis at positions 110 and 224. Three single mutants (G110D, W224L, and W224S) and two double mutants (G110D/W224L and G110D/ W224S) were expressed and purified. All five mutants lost native carboxylesterase activity. Mutation W224L converted esterase B1 to an OP hydrolase and increased its malathion carboxylesterase activity. No obvious OP hydrolysis was observed by G110D or W224S. Our data strongly support our hypothesis and suggest that mutation W224L might occur in natural populations of mosquitoes. Sequence comparison shows that the site 224 is especially highly conserved among various insect carboxylesterases. This leads to another hypothesis: that the position 224 plays a key role in insect carboxylesterases’ switching from their native physiological functions to other functional niches un‐der selection pressure exerted by insecticides.—Cui, F., Qu, H., Cong, J., Liu, X.‐L., Qiao, C. L. Do mosquitoes acquire organophosphate resistance by functional changes in carboxylesterases? FASEB J. 21, 3584–3591 (2007)

Structure, Function and Applications of Carboxylesterases from Insects for Insecticide Resistance

Carboxylesterases (EC 3.1.1.1) distribute broadly in insects, and play an important role in the metabolism with various functions. This paper reviews the insect carboxylesterases including the definitions and reaction mechanism, classification, structural context, functions especially on insecticide resistance, and its application.

Transcriptomic and phylogenetic analysis of Culex pipiens quinquefasciatus for three detoxification gene families

BackgroundThe genomes of three major mosquito vectors of human diseases, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus, have been previously sequenced. C. p. quinquefasciatus has the largest number of predicted protein-coding genes, which partially results from the expansion of three detoxification gene families: cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST), and carboxyl/cholinesterases (CCE). However, unlike An. gambiae and Ae. aegypti, which have large amounts of gene expression data, C. p. quinquefasciatus has limited transcriptomic resources. Knowledge of complete gene expression information is very important for the exploration of the functions of genes involved in specific biological processes. In the present study, the three detoxification gene families of C. p. quinquefasciatus were analyzed for phylogenetic classification and compared with those of three other dipteran insects. Gene expression during various developmental stages and the differential expression responsible for parathion resistance were profiled using the digital gene expression (DGE) technique.ResultsA total of 302 detoxification genes were found in C. p. quinquefasciatus, including 71 CCE, 196 P450, and 35 cytosolic GST genes. Compared with three other dipteran species, gene expansion in Culex mainly occurred in the CCE and P450 families, where the genes of α-esterases, juvenile hormone esterases, and CYP325 of the CYP4 subfamily showed the most pronounced expansion on the genome. For the five DGE libraries, 3.5-3.8 million raw tags were generated and mapped to 13314 reference genes. Among 302 detoxification genes, 225 (75%) were detected for expression in at least one DGE library. One fourth of the CCE and P450 genes were detected uniquely in one stage, indicating potential developmentally regulated expression. A total of 1511 genes showed different expression levels between a parathion-resistant and a susceptible strain. Fifteen detoxification genes, including 2 CCEs, 6 GSTs, and 7 P450s, were expressed at higher levels in the resistant strain.ConclusionsThe results of the present study provide new insights into the functions and evolution of three detoxification gene families in mosquitoes and comprehensive transcriptomic resources for C. p. quinquefasciatus, which will facilitate the elucidation of molecular mechanisms underlying the different biological characteristics of the three major mosquito vectors.

Development of an autofluorescent Pseudomonas nitroreducens with dehydrochlorinase activity for efficient mineralization of γ-hexachlorocyclohexane (γ-HCH)

Biodegradation or bioremediation is a more efficient and environmental friendly method for detoxification of hexachlorocyclohexane (HCH) residues compared to physical and chemical methods. Here, we report the functional expression of dehydrochlorinase (LinA) and enhanced green fluorescent protein (EGFP) in Pseudomonas nitroreducens for efficient biodegradation of gamma-HCH. The broad-host-range plasmid pVAG33, harboring dehydrochlorinase gene (linA) and enhanced green fluorescent protein gene (egfp), was constructed and transformed into the soil-isolated 1,2,4-trichlorobenzene (1,2,4-TCB)-degrading strain P. nitroreducens J5-1. Functional expression of LinA and EGFP was confirmed in the recombinant strain by Western-blotting analysis and by determining their enzymatic activities and fluorescence intensity. The recombinant strain could rapidly degrade 10microg ml(-1) gamma-HCH in 28h determined by GC-ECD analysis. It could completely mineralize gamma-HCH via gamma-HCH through 1,2,4-TCB and 3,4,6-trichlorocatechol and eventually entered the TCA cycle as determined by GC-MS analysis. The engineered strain can be applied in the form of a biocatalyst in a bioreactor for rapid degradation of HCH and chlorobenzene residues. Meanwhile, it can be easily monitored on-line by fluorescence of EGFP for its activity and fate.

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.

Carboxylesterase-mediated insecticide resistance: Quantitative increase induces broader metabolic resistance than qualitative change

Carboxylesterases are mainly involved in the mediation of metabolic resistance of many insects to organophosphate (OP) insecticides. Carboxylesterases underwent two divergent evolutionary events: (1) quantitative mechanism characterized by the overproduction of carboxylesterase protein; and (2) qualitative mechanism caused by changes in enzymatic properties because of mutation from glycine/alanine to aspartate at the 151 site (G/A151D) or from tryptophan to leucine at the 271 site (W271L), following the numbering of Drosophila melanogaster AChE. Qualitative mechanism has been observed in few species. However, whether this carboxylesterase mutation mechanism is prevalent in insects remains unclear. In this study, wild-type, G/A151D and W271L mutant carboxylesterases from Culex pipiens and Aphis gossypii were subjected to germline transformation and then transferred to D. melanogaster. These germlines were ubiquitously expressed as induced by tub-Gal4. In carboxylesterase activity assay, the introduced mutant carboxylesterase did not enhance the overall carboxylesterase activity of flies. This result indicated that G/A151D or W271L mutation disrupted the original activities of the enzyme. Less than 1.5-fold OP resistance was only observed in flies expressing A. gossypii mutant carboxylesterases compared with those expressing A. gossypii wild-type carboxylesterase. However, transgenic flies universally showed low resistance to OP insecticides compared with non-transgenic flies. The flies expressing A. gossypii W271L mutant esterase exhibited 1.5-fold resistance to deltamethrin, a pyrethroid insecticide compared with non-transgenic flies. The present transgenic Drosophila system potentially showed that a quantitative increase in carboxylesterases induced broader resistance of insects to insecticides than a qualitative change.