Overproduced Esterases Research Articles

Fenitrothion Resistance in Culex pipiens pipiens (Diptera: Culicidae) from Tunisia: Status and Challenges

Culex pipiens is considered the potential vector of West Nile virus outbreaks that have affected Tunisia. The objective of the present study was to evaluate the fenitrothion insecticide resistance status of Culex pipiens pipiens in three populations of insects from three Tunisian states. Standardized method bioassays were conducted to shed light on fenitrothion susceptibility status in field Culex pipiens pipiens populations.The cross-resistance between fenitrothion and propoxur, and the polymorphism of over-produced esterases and AChE 1 were also investigated. Resistances to fenitrothion were moderate and low in sample # 2 and 3 (12.9 and 7.2, respectively). The highest resistance ratio was observed in populations collected in Northern Tunisia (sample # 1) with a rate of 60.9 fold at the LC50 level. The use of a synergist suggested the non-involvement of CYP450, esterases and/or glutathione-S-transferase in resistance of Culex pipiens pipiens to the tested insecticide. However, the biochemical investigation of detoxification enzymes using starch electrophoresis showed the involvement of four esterases, including A2-B2, A4-B4 (and/or A5–B5), B12 and C1. The fenitrothion resistant populations were highly resistant to propoxur indicating the possible involvement of their common target site (AChE 1) in the recorded resistance. Our results are particularly interesting to update the information base for rational deployment of the existing tools for mosquito's control.

Pirimiphos-Methyl Resistance Status of Field Populations of Culex pipiens (Diptera: Culicidae) From Grand Tunis Area, Northeast Tunisia

Mosquito species are responsible for the transmission of many parasitic diseases and their control leads to the phenomenon of resistance to insecticides. Five Culex pipiens samples were collected from various localities of Grand Tunis area, Northeast Tunisia, between June 2003 and November 2005. All the studied samples were resistant to pirimiphos methyl (organophosphorus insecticide). Our results showed the implication of insensitive acetylcholinesterase and overproduced esterases in the resistance of the Tunisian populations of Culex pipiens. Results were discussed in relation to resistance mechanism.

Temephos Resistance in Three Populations of Culex pipiens Collected from Three Districts of Southern Tunisia and Its Significance for the Resistance Mechanism

Objectives: The aim of this study was to investigate the resistance to temephos (OP) in three populations of Culex pipiens collected from three districts of Southern Tunisia and its significance for the resistance mechanism. Methods: Resistance to temephos insecticide was studied on Culex pipiens mosquitoes collected in three localities of Southern Tunisia. Larvae were used for different bioassays and adults were stored in -80°C for esterases identification. Results: Bioassays revealed the susceptibility of sample # 3. The weakest resistance was recorded in sample # 3 (RR50 = 0.68) and the strongest resistance for the sample # 2 (RR50 = 3.6). At LC95, two samples (# 1, and 3) were susceptible. The use of synergists showed the non-involvement of resistance mechanisms inhibited by DEF and Pb. Our investigation reported that temephos resistance could be explained by the two most common mechanisms of resistance to OP (overproduced esterases and AChE 1 mutation). Conclusion: Both detoxification mechanisms and target site alteration were involved in the resistance to temephos as reported in our study. These results are very important for the implementation and development of vector control strategies

Evaluation of Chlorpyrifos Resistance and Biochemical Mechanisms of Culex pipiens in Five Localities of Grand Tunis Area, Northeast Tunisia

Five Culex pipiens samples were collected at preimaginal stages from breeding sites in 5 localities of Grand Tunis area, Northeast Tunisia, between March 2002 and November 2005. Larvae were used for bioassays using ethanol solutions of chlorpyrifos and propoxur insecticides. All samples were resistant to chlorpyrifos (RR>1, p 1,000-fold) were recorded in samples # 2, 4, and 5. Our synergist study showed that the increased detoxification by EST (and/or GST) had only a minor role in the chlorpyrifos resistance, although several overproduced esterases, known to be involved in the OPs resistance, were detected in all the resistant field samples. The mortality due to propoxur was significantly correlated with the LC50 of chlorpyrifos and indicated an insensitive AChE.

Evaluation of Fenitrothion against Culex pipiens (Diptera: Culicidae) Larvae in Grand Tunis Area of Tunisia

Resistance toward 2 insecticides (fenitrothion and propoxur) was analyzed in 5 samples of Culex pipiens populations collected from various localities of Grand Tunis area, Northeast Tunisia. All studied samples were resistant to fenitrothion and propoxur insecticides. The study of esterase’s activities showed the existence of five overproduced esterases: C1, A1, A2-B2, A4-B4 (and/or A5-B5) and B12. One or several esterases were detected in the studied samples. We also showed that the resistance to fenitrothion was correlated with the propoxur resistance indicating that modifications of the target, the AChE 1, can be involved in the OP resistance. These results should be considered in the current mosquitoes control programs in Tunisia.

Study Confirming Resistance to Fenitrothion of Culex pipiens (Diptera: Culicidae) from Central Tunisia

Five natural populations of Culex pipiens were taken as larvae in the central Tunisia to evaluate their resistance level of fenitrothion. Our study showed that all samples were resistant to fenitrothion at LC50. The RR50 ranged from 9.2 in sample # 2 to 59.2 in sample # 5. Starch electrophoresis detected the overproduced esterases in all studied samples. The most frequent esterase A2B2 was detected in samples # 5 with a frequency of 31%. Three other esterases were detected in samples # 1, 2, 3, and 4: A4-B4 and/or A5-B5, A12, and C1. Synergists showed that the involvement of CYTP450 in the resistance to fenitrothion (OP) is not neglected. Cross-resistance of fenitrothion and propoxur was detected indicate the involvement of target site alteration (AChE1) in fenitrothion resistance. It should be noted that study of the polymorphism of AChE 1 will be of great importance.

Distribution and dynamics of esterase alleles in Culex pipiens complex in China

To investigate insecticide resistance and dynamic changes of carboxylesterase polymorphism in mosquitoes with time in the Culex pipiens complex (Diptera: Culicidae), nine field mosquito populations were collected in China. The resistance levels of fourth-instar larvae to organophosphate (dichlorvos, parathion, and chlorpyrifos), carbamate (fenobucarb and propoxur), and pyrethroid (permethrin, deltamethrin and tetramethrin) insecticides were determined by bioassay. Larvae had more resistance to organophosphate insecticides than to carbamate insecticides. A low but significant resistance was observed for carbamate insecticides. The resistance to pyrethroid insecticides varied from sensitive to high. Starch gel electrophoresis revealed the presence of the overproduced esterases B1, A2B2, A8B8, A9B9, B10 and A11B11. The frequency of each overproduced esterases varied depending on its regional localities. Compared with published surveys, the C. pipiens complex, which exhibited a high polymorphism of applied esterase alleles in China, showed dynamic evolution over time under local specific insecticide selection. The results are discussed in the context of recent alterations to insecticide campaigns, and in the evolution of resistance genes in Chinese C. pipiens populations.

High incidence of ace-1 duplicated haplotypes in resistant Culex pipiens mosquitoes from Algeria

The status of genes conferring resistance to organophosphate and carbamate insecticides has been examined in Culex pipiens pipiens mosquitoes sampled in Algeria. Presence of overproduced esterases was sporadic, but acetylcholinesterase-1 resistant alleles were observed in almost all samples. We focused our study on the AChE1 G119S substitution characterized in almost all samples, mostly at the heterozygous state. A genetic test revealed the presence of ace-1 duplication associating a susceptible and a resistant ace-1 copy. Molecular characterization showed a high occurrence of ace-1 duplication with six distinct duplicated alleles out of four samples. The inferred frequency of duplicated allele suggests that it is replacing the single resistant G119S allele. Finally, we discuss the mechanism at the origin of these duplicated haplotypes and their consequences on the management of insecticide resistance.

Dynamics of Esterase Alleles in <I>Culex pipiens</I> Complex Mosquitoes in Beijing

To investigate insecticide resistance levels and dynamic changes of carboxylesterase polymorphism with time in resistant populations of mosquitoes in the Culex pipiens complex, four field populations were collected in September 2006. The resistance levels of fourth-instar larvae to organophosphate (dichlorvos, parathion, and chlorpyrifos), carbamate (fenobucarb and propoxur), and pyrethroid (permethrin and tetramethrin) insecticides were determined by bioassay. Larvae had a low but significant resistance to organophosphate and carbamate insecticides but no significant resistance to pyrethroid insecticides. Starch gel electrophoresis revealed the presence of the overproduced esterases B1, A2-B2, A8-B8, A9-B9, and All-B11. The frequency of each overproduced esterases varied depending on its regional localities. Compared with previous surveys, the polymorphism of amplified esterase alleles in Beijing populations increased, although the resistance level to organophosphate insecticides declined.

Filariasis vector in China: insecticide resistance and population structure of mosquito Culex pipiens complex

Seven field populations of mosquito Culex pipiens complex (Diptera: Culicidae) were collected from four provinces of China. The resistance status of larvae to dichlorvos, parathion, chlorpyrifos, fenobucarb (BPMC) and propoxur were determined by bioassays, disclosing that they were more resistant to organophosphate (moderate or low resistance) than to carbamate (low or no significant resistance) insecticides. Starch gel electrophoresis confirmed the presence and distribution of overproduced esterases B1, A2-B2, A8-B8 and A9-B9, the frequencies of which varied according to their regional origins. Electrophoretic polymorphism at four putatively neutral loci (got-1, got-2, pgi and pgm) showed that the overall genetic differentiation found across all populations was significantly large (Fst = 0.28, P < 10(-4)), and genetic exchange was slightly restricted by distance isolation (P = 0.018).

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

AbstractIn 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.

FITNESS COSTS OF INSECTICIDE RESISTANCE IN NATURAL BREEDING SITES OF THE MOSQUITO CULEX PIPIENS

Genetic changes conferring adaptation to a new environment may induce a fitness cost in the previous environment. Although this prediction has been verified in laboratory conditions, few studies have tried to document this cost directly in natural populations. Here, we evaluated the pleiotropic effects of insecticide resistance on putative fitness components of the mosquito Culex pipiens. Experiments using different larval densities were performed during the summer in two natural breeding sites. Two loci that possess alleles conferring organophosphate (OP) resistance were considered: ace-1 coding for an acetylcholinesterase (AChE1, the OP target) and Ester, a “super locus” including two closely linked loci coding for esterases A and B. Resistance ace-1 alleles coding for a modified AChE1 were associated with a longer development time and shorter wing length. The pleiotropic effects of two resistance alleles Ester1 and Ester4 coding for the overproduced esterases A1 and A4-B4, respectively, were more variable. Both A1 and A4-B4 reduced wing length, although only A1 was associated with a longer preimaginal stage. The fluctuating asymmetry (FA) of the wing did not respond to the presence or to the interaction of resistance alleles at the two loci at any of the density levels tested. Conversely, the FA of one wing section decreased when larval density increased. This may be the consequence of selection against less developmentally stable individuals. The results are discussed in relation to the local evolution of insecticide resistance genes.

SIGNIFICANCE OF PASSIVE MIGRATION IN EVOLUTION OF INSECTICIDE RESISTANCE IN THE MOSQUITO CULEX PIPIENS (DIPTERA: CULICIDAE)

AbstractIn order to determine the combined effects of migration and gene flow on evolution of insecticide resistance in the mosquito Culex pipiens, four samples were collected in China, among which two were collected along the railway from Beijing to Guangzhou. Bioassay data showed that the resistance levels of the four populations to dichlorvos were high and to parathion moderate as compared with the susceptible strain and there was no significant difference among the four populations to the same organophosphate (OP) insecticide. Starch electrophoresis was done to identify the frequency of known overproduced esterases and to analyze genetic diversity among various populations by electrophoretic polymorphism of five presumably neutral loci. The results indicated that the gene flow between populations existed and the number of effective migrants (Nm) was related to collection geography (Nm from 1.67 to 40.07). In contrast with lower genetic differentiation between two nearby populations (between GZ and ZS, ZZ and SQ) and higher genetic differentiation between two distant populations (between GZ and ZZ), there was a significant and inconsistent difference in the distribution of resistance alleles, A2‐B2 when explained only with active migration. This divergent situation could be straightened out when considering passive migration (such as railway transport) which increased the spread of A2‐B2 along the railway, i.e., in GZ and ZZ. The resistance alleles, A2‐B2, dispersing to around areas by active migration suffered from the limitation of gene flow and the speed of invasion.

Tissue Localization of Overproduced Esterases in the MosquitoCulex 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.

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.

An overview of the evolution of overproduced esterases in the mosquito Culex pipiens.

Insecticide resistance genes have developed in a wide variety of insects in response to heavy chemical application. Few of these examples of adaptation in response to rapid environmental change have been studied both at the population level and at the gene level. One of these is the evolution of the overproduced esterases that are involved in resistance to organophosphate insecticides in the mosquito Culex pipiens. At the gene level, two genetic mechanisms are involved in esterase overproduction, namely gene amplification and gene regulation. At the population level, the co-occurrence of the same amplified allele in distinct geographic areas is best explained by the importance of passive transportation at the worldwide scale. The long-term monitoring of a population of mosquitoes in southern France has enabled a detailed study to be made of the evolution of resistance genes on a local scale, and has shown that a resistance gene with a lower cost has replaced a former resistance allele with a higher cost.

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.

A new esterase gene amplification involved in OP resistance in Culex pipiens mosquitoes from China.

Two overproduced esterases (A8 and B8) not previously described were found in southern China. They provide 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 mosquito species. This coamplification is distinct from all other similar events thus far reported. The peculiar situation in southern China, where numerous OP resistance alleles at these two loci were found, is discussed in comparison with the Mediterranean situation, the only one with a similar diversity of overproduced esterases.

Esterases A5-B5 in organophosphate-resistant Culex pipiens from Italy.

Culex pipiens mosquitos from Lignano city, Udine province, northeast Italy, were found to carry over-produced non-specific esterases A1, A2-B2 and A4-B4 or A5-B5, detected by starch gel electrophoresis, giving multiple resistance to organophosphorus insecticides. In order to differentiate between A4-B4 and A5-B5 esterases, the latter known only from Cyprus whereas the former is widespread in Italy and elsewhere, restriction fragment length polymorphism (RFLP) analysis was performed at the esterase B locus. Both B4 and B5 haplotypes were found. This is the first record of A5-B5 esterase-mediated resistance in continental Europe.

An insensitive acetylcholinesterase in Culex pipiens (Diptera:Culicidae) from Portugal.

Resistance mechanisms of a strain (PRAIAS) of northern house mosquito, Culex pipiens L., collected in Portugal in 1993, and highly resistant to organophosphates and carbamates, were investigated by comparing the resistance characteristics to 3 organophosphorous (temephos, chlorpyrifos, malathion) and 1 carbamate (propoxur) insecticides in the presence or absence of synergists; and by determining the possible occurrence of overproduced esterases or insensitive acetylcholinesterase (AChE). The reference strain MSE from southern France, with an insensitive AChE, was included in all analyses for comparison. For organophosphorous insecticides, resistance in PRAIAS was caused by an insensitive AChE and an increase in oxidative metabolism, although the 2nd mechanism has only a marginal effect. For propoxur, the insensitive AChE was the only resistance mechanism detected. Biochemical properties of both the French and Portuguese insensitive AChEs were similar. We cannot exclude the possibility that PRAIAS and MSE strains possess exactly the same insensitive AChE allele.