Malathion and pyrethroid resistance in Culex quinquefasciatus from Cuba: efficacy of pirimiphos-methyl in the presence of at least three resistance mechanisms

Use of malathion for mosquito control in Cuba for 7 years up to 1986 has selected for elevated non-specific esterase and altered acetylcholinesterase (AChE) resistance mechanisms in populations of the pest mosquito Culex quinquefasciatus Say. These mechanisms are still present in relatively high frequencies in the Havana area, despite the replacement of malathion by pyrethroid insecticides for the last 3 years in the mosquito control programme. Samples of Culex quinquefasciatus populations from within a 100 km radius of Havana had high levels of resistance to malathion and lower levels of resistance to propoxur, but there was little or no cross-resistance to the organophosphorus insecticide pirimiphos-methyl. Selection with malathion for twenty-two consecutive generations in the laboratory increased the level of malathion resistance to 1208-fold and propoxur level to 1002-fold, but the maximum level of pirimiphos-methyl resistance was only 11-fold. Pirimiphos-methyl is still operationally effective, despite the resistance mechanisms segregating, so this insecticide if used for control is unlikely to select either of the known resistance factors directly in the field population. Since 1986, pyrethroids have been used extensively, and low levels of pyrethroid resistance were detected in two of five field population samples tested. Malathion selection did not increase the level of pyrethroid resistance, which indicates that one or more distinct pyrethroid resistance factors are now being selected in the field populations of Culex quinquefasciatus.     

Insecticide susceptibility status in two medically important mosquito vectors,Anopheles gambiae,and Culex quinquefasciatus to three insecticides commonly used in Niger State,Nigeria

High resistance ability on insecticides among major mosquito vectors of diseases in Nigeria is of growing concern for severe control strategies. The objective of this study was to assess the susceptibility status of females Anopheles gambiae and Culex quinquefasciatus complexes mosquitoes to permethrin (21.5 μg/bottle-pyrethroids), propoxur(12.5 μg/bottle-carbamate) and malathion (50 μg/bottle organophosphate), in Niger State, North-Central, Nigeria. Anopheline and Culecine larvae were collected from the larval habitats of the studied sites (Bosso, Katcha, Lapai, and Shiroro) larvae and pupae were identified guided by standard keys and reared to adults in troughs. Insecticide susceptibility bioassays were performed according to the CDC bottle bioassay standard operating procedures on 3 days old, sugar-fed female Anopheles and Culex mosquitoes. Post-exposure mortality after 24hr and knockdown values for KDT₅₀ were calculated. Knock-down at 1-hour insecticide exposure ranged (84–96 %) permethrin, (94–100 %) propoxur and (100 %) malathion for An. gambiae and (86–97 %) permethrin, (92–100 %) propoxur and (96–100 %) malathion for Cx. quinquefasciatus. Mortality, after 24hr post-exposure was 100 % in malathion, indicating the high effect of the insecticide. Tested samples were found potentially resistant to permethrin recorded against mosquitoes collected from all study sites, in two locations of the study sites to propoxur and one location site to malathion. All the tested mosquitoes were found to be potentially resistant to permethrin, however, mosquitoes tested in Katcha and Shiroro resist potentially to propoxur. Except, Culex quinqufasciatus from Lapai that partially resist malathion, all the tested mosquitoes were found to be susceptible to malathion, across the study sites.     

Changes in malathion and pyrethroid resistance after cypermethrin selection of Culex quinquefasciatus field populations of Cuba

Abstract. Use. of the organophosphorus insecticide malathion for mosquito control in Cuba, for 7 years up to 1986, selected elevated non-specific esterase and altered acetylcholinesterase (AChE) resistance mechanisms in Culex quinquefasciatus. In central Havana space-spraying of malathion was replaced by the pyrethroid cypermethrin in 1987: alternate cycles of malathion and cypermethrin were applied in some of the more rural areas of Havana district during 1987-91. Consequently, populations of Cx quinquefasciatus in the central area of Havana developed resistance to cypermethrin, but there is no evidence of pyrethroid resistance in the outlying areas. Malathion resistance levels declined significantly after 1986, measured both by bioassay and the frequency of the elevated esterase resistance mechanism, and then stabilized with no measurable decline during 1990 in any of the populations tested. These populations had less than 10% frequency of susceptible homozygotes for both the esterase and AChE resistance mechanisms, indicating that organophosphate resistance is still prevalent in Cuban Cx quinquefasciatus. These two mechanisms appear to be in linkage equilibrium, suggesting that current selection for double resistance is not strong. In the central Havana region, pirimiphos-methyl, an organophosphorus insecticide unaffected by the two common malathion resistance mechanisms, is now being used in a resistance management strategy designed to avoid pyrethroid resistance spreading.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

Multiple Cytochrome P450 genes: their constitutive overexpression and permethrin induction in insecticide resistant mosquitoes, Culex quinquefasciatus

Four cytochrome P450 cDNAs, CYP6AA7, CYP9J40, CYP9J34, and CYP9M10, were isolated from mosquitoes, Culex quinquefasciatus. The P450 gene expression and induction by permethrin were compared for three different mosquito populations bearing different resistance phenotypes, ranging from susceptible (S-Lab), through intermediate (HAmCq(G0), the field parental population) to highly resistant (HAmCq(G8), the 8(th) generation of permethrin selected offspring of HAmCq(G0)). A strong correlation was found for P450 gene expression with the levels of resistance and following permethrin selection at the larval stage of mosquitoes, with the highest expression levels identified in HAmCq(G8), suggesting the importance of CYP6AA7, CYP9J40, CYP9J34, and CYP9M10 in the permethrin resistance of larva mosquitoes. Only CYP6AA7 showed a significant overexpression in HAmCq(G8) adult mosquitoes. Other P450 genes had similar expression levels among the mosquito populations tested, suggesting different P450 genes may be involved in the response to insecticide pressure in different developmental stages. The expression of CYP6AA7, CYP9J34, and CYP9M10 was further induced by permethrin in resistant mosquitoes. Taken together, these results indicate that multiple P450 genes are up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, thus increasing the overall expression levels of P450 genes.     

Synergism between permethrin and propoxur against Culex quinquefasciatus mosquito larvae

To see if synergism occurs between carbamate and pyrethroid insecticides, we tested permethrin and propoxur as representatives of these two classes of compounds used for mosquito control. Larvicidal activity of both insecticides was assessed separately and together on a susceptible strain of the mosquito Culex quinquefasciatus (Diptera: Culicidae) by two methods. When mixed at a constant ratio (permethrin : propoxur 1 : 60 based on LC50) and tested at serial concentrations to plot dose/mortality regression, significant synergy occurred between them (co-toxicity coefficient = 2.2), not just an additive effect. For example, when the mixture gave 50% mortality, the same concentrations of permethrin and propoxur alone would have given merely 2 x 1% mortality. When a sublethal dose (LC0) of permethrin or propoxur was added to the other (range LC10-LC95), synergism occurred up to the LC80 level. Synergistic effects were attributed to the complementary modes of action by these two insecticide classes acting on different components of nerve impulse transmission. Apart from raising new possibilities for Culex control, it seems appropriate to consider using such mixtures or combinations for insecticide-treated mosquito nets in situations with insecticide-resistant Anopheles malaria vectors.     

Genome analysis of cytochrome P450s and their expression profiles in insecticide resistant mosquitoes, Culex quinquefasciatus

Here we report a study of the 204 P450 genes in the whole genome sequence of larvae and adult Culex quinquefasciatus mosquitoes. The expression profiles of the P450 genes were compared for susceptible (S-Lab) and resistant mosquito populations, two different field populations of mosquitoes (HAmCq and MAmCq), and field parental mosquitoes (HAmCq(G0) and MAmCq(G0)) and their permethrin selected offspring (HAmCq(G8) and MAmCq(G6)). While the majority of the P450 genes were expressed at a similar level between the field parental strains and their permethrin selected offspring, an up- or down-regulation feature in the P450 gene expression was observed following permethrin selection. Compared to their parental strains and the susceptible S-Lab strain, HAmCq(G8) and MAmCq(G6) were found to up-regulate 11 and 6% of total P450 genes in larvae and 7 and 4% in adults, respectively, while 5 and 11% were down-regulated in larvae and 4 and 2% in adults. Although the majority of these up- and down-regulated P450 genes appeared to be developmentally controlled, a few were either up- or down-regulated in both the larvae and adult stages. Interestingly, a different gene set was found to be up- or down-regulated in the HAmCq(G8) and MAmCq(G6) mosquito populations in response to insecticide selection. Several genes were identified as being up- or down-regulated in either the larvae or adults for both HAmCq(G8) and MAmCq(G6); of these, CYP6AA7 and CYP4C52v1 were up-regulated and CYP6BY3 was down-regulated across the life stages and populations of mosquitoes, suggesting a link with the permethrin selection in these mosquitoes. Taken together, the findings from this study indicate that not only are multiple P450 genes involved in insecticide resistance but up- or down-regulation of P450 genes may also be co-responsible for detoxification of insecticides, insecticide selection, and the homeostatic response of mosquitoes to changes in cellular environment.     

Insecticide resistance spectra and resistance mechanisms in populations of Japanese encephalitis vector mosquitoes, Culex tritaeniorhynchus and Cx. gelidus, in Sri Lanka

Culex tritaeniorhynchus Giles and Cx. gelidus Theobald (Diptera: Culicidae), both vectors of Japanese encephalitis, were collected in 1984 and 1998 from two disease endemic localities in Sri Lanka: Anaradhapura and Kandy. Using wild-caught adult mosquitoes from light traps, log dosage-probit mortality curves for insecticide bioassays were obtained for three insecticides: malathion (organophosphate), propoxur (carbamate) and permethrin (pyrethroid). LD50 values showed that, in 1998, Cx. tritaeniorhynchus was -100-fold more resistant to malathion and 10-fold more resistant to propoxur than was Cx. gelidus. This difference was attributed to Cx. tritaeniorhynchus breeding mostly in irrigated rice paddy fields, where it would have been exposed to pesticide selection pressure, whereas Cx. gelidus breeds in other types of aquatic habitats less prone to pesticide applications. Resistance in Cx. tritaeniorhynchus increased between 1984 and 1998, whereas Cx. gelidus remained predominantly susceptible. Propoxur inhibition of acetylcholinesterase (AChE) activity (the target site of organophosphates and carbamates) indicated that in 1998, frequencies of insensitive AChE-based resistance were 9% in Cx. gelidus and 2-23% in Cx. tritaeniorhynchus, whereas in 1984 this resistance mechanism was detected only in 2% of the latter species from Anaradhapura. The AChE inhibition coefficient (ki) with propoxur was 1.86+/-0.24 x 10(5) M(-)1 min(-1) for Cx. tritaeniorhynchus from Anaradhapura in 1998. Both species were tested for activity levels of detoxifying glutathione S-transferases (GSTs) and malathion-specific as well as general carboxylesterases. High activities of GSTs and carboxylesterases were detected in Cx. tritaeniorhynchus but not Cx. gelidus. Malathion-specific carboxylesterase was absent from both species. Native polyacrylamide gel electrophoresis resolved two elevated general carboxylesterases, CtrEstbeta1 and CtrEstalpha1, from Cx. tritaeniorhynchus and none from Cx. gelidus. CtrEstbeta1 was the most intensely staining band. Gel inhibition experiments showed that both elevated esterases were inhibited by organophosphates and carbamates but not by pyrethroids. The major elevated esterase CtrEstbeta1 was partially purified (15-fold) by sequential Q-Sepharose and phenyl Sepharose column chromatography. The bimolecular rate constant (ka) and the deacylation rate constant (k3) for the malaoxon/enzyme interaction were 9.9+/-1.1 x 10(3) M(-1) min(-1) and 3.5+/-0.05 x 10(-4) M(-1) min(-1), respectively, demonstrating that the role of this enzyme in organophosphorus insecticide resistance is sequestration.     

Evaluation of insecticide resistance and biochemical mechanisms in a population of Culex quinquefasciatus (Diptera: Culicidae) from São Paulo, Brazil

To establish an insecticidal resistance surveillance program, Culex quinquefasciatus mosquitoes from S?o Paulo, Brazil, were colonized (PIN95 strain) and analyzed for levels of resistance. The PIN95 strain showed low levels of resistance to organophosphates [malathion (3.3-fold), fenitrothion (11.2-fold)] and a carbamate [propoxur (3.0-fold)]. We also observed an increase of 7.4 and 9.9 in alpha and beta esterase activities, respectively, when compared with the reference IAL strain. An alteration in the sensitivity of acetylcholinesterase to insecticide inhibition was also found in the PIN95 mosquitoes. The resistant allele (Ace.1R), however, was found at low frequencies (0.12) and does not play an important role in the described insecticide resistance. One year later, Cx. quinquefasciatus mosquitoes were collected (PIN96 strain) at the same site and compared to the PIN95 strain. The esterase activity patterns observed for the PIN96 strain were similar to those of the PIN95 mosquitoes. However the occurrence of the Ace.1R allele was statistically higher in the PIN96 strain. The results show that esterase-based insecticide resistance was established in the PIN95 Cx. quinquefasciatus population and that an acethylcholinesterase based resistant mechanism has been selected for. A continuous monitoring of this phenomenon is fundamental for rational mosquito control and insecticide application programs.     

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Although cytochrome P450 (CYP450) enzymes are frequently up-regulated in mosquitoes resistant to insecticides, no regulatory motifs driving these expression differences with relevance to wild populations have been identified. Transposable elements (TEs) are often enriched upstream of those CYP450s involved in insecticide resistance, leading to the assumption that they contribute regulatory motifs that directly underlie the resistance phenotype. A partial CuRE1 (Culex Repetitive Element 1) transposable element is found directly upstream of CYP9M10, a cytochrome P450 implicated previously in larval resistance to permethrin in the ISOP450 strain of Culex quinquefasciatus, but is absent from the equivalent genomic region of a susceptible strain. Via expression of CYP9M10 in Escherichia coli we have now demonstrated time- and NADPH-dependant permethrin metabolism, prerequisites for confirmation of a role in metabolic resistance, and through qPCR shown that CYP9M10 is >20-fold over-expressed in ISOP450 compared to a susceptible strain. In a fluorescent reporter assay the region upstream of CYP9M10 from ISOP450 drove 10× expression compared to the equivalent region (lacking CuRE1) from the susceptible strain. Close correspondence with the gene expression fold-change implicates the upstream region including CuRE1 as a cis-regulatory element involved in resistance. Only a single CuRE1 bearing allele, identical to the CuRE1 bearing allele in the resistant strain, is found throughout Sub-Saharan Africa, in contrast to the diversity encountered in non-CuRE1 alleles. This suggests a single origin and subsequent spread due to selective advantage. CuRE1 is detectable using a simple diagnostic. When applied to C. quinquefasciatus larvae from Ghana we have demonstrated a significant association with permethrin resistance in multiple field sites (mean Odds Ratio?=?3.86) suggesting this marker has relevance to natural populations of vector mosquitoes. However, when CuRE1 was excised from the allele used in the reporter assay through fusion PCR, expression was unaffected, indicating that the TE has no direct role in resistance and hence that CuRE1 is acting only as a marker of an as yet unidentified regulatory motif in the association analysis. This suggests that a re-evaluation of the assumption that TEs contribute regulatory motifs involved in gene expression may be necessary.     

不同地理种群尖音库蚊复组抗性动态和遗传多样性

通过生物测定、蛋白质电泳和等位酶分析等方法对5个不同地区的尖音库蚊复组蚊虫Culex pipiens complex的抗性水平、种群中非特异性酯酶基因表型分布和种群遗传多样性进行了研究。不同地理种群的抗性检测结果表明：5个种群分别对敌敌畏、对硫磷、氯菊酯和溴氰菊酯的抗性较高,对残杀威、巴沙和胺菊酯的抗性较低;朝阳种群对敌敌畏抗性最高（55.7倍）,武汉种群次之;佛山种群对氯菊酯和溴氰菊酯的抗性比率高达123倍和23.9倍。酯酶电泳结果显示：5个种群间酯酶多态性存在差异,广州和佛山两个库蚊种群酯酶表型多态性最高,有B1,A2-B2,A8-B8,A9-B9,B10和A11-B11等6种酯酶表型,提示高活性酯酶是主要的抗性机制。群体遗传学研究表明：每位点平均等位基因数（A）为2.76,平均多态位点百分率（P）为64.45％,平均预期杂合度（He）为0.1943,种群间遗传分化系数（Fst）值为0.10,平均基因流(Nm)＝2.57,说明5个种群有较丰富的遗传多样性,种群内遗传多样性高于种群之间。据此推测,种群间可以通过迁徙等方式进行基因交流,使得遗传结构、抗性水平朝一致性方向变化。本研究对我国尖音库蚊复组蚊虫的综合治理有一定指导意义。     

Pyrethroid resistance in Culex quinquefasciatus from West Africa

Laboratoire de Lutte contre les Insectes Nuisibles, ORSTOM, Montpellier, France, *OCCGE, Institut Pierre Richet, BP 1500, Bouaké 01, Côte d'Ivoire, †INRA, Laboratoire de Biologie des Invertébrés, 123 Bd Meilland, 06606 Antibes, France, and ‡CNRS UMR 5554, Laboratoire de Génétique et Environnement, Institut des Sciences de l'Environnement, Université de Montpellier II (CC 065), 34095 Montpellier, France
Abstract .Pyrethroid resistance was investigated in thirty-three samples of Culex quinquefasciatus Say from twenty-five cities in Côte d'Ivoire and Burkina Faso. Permethrin resistance ratios at LC₅₀ ranged from 9.5- to 82-fold in Côte d'Ivoire and from 17- to 49-fold in Burkina Faso. For deltamethrin, resistance ratios were lower and ranged from nine to thirty-eight in both countries. A strain was selected with permethrin to investigate resistance mechanisms. After forty-two generations of selection, permethrin resistance level reached 3750-fold, but deltamethrin resistance remained unexpectedly unchanged. This indicated that a specific mechanism was involved in permethrin resistance. Synergist assays and biochemical tests indicated that resistance was partly due to P450-dependent oxidases. A target site insensitivity ( kdr ) was also involved, associated with DDT cross resistance and a dramatic loss of permethrin knockdown effect on adults. This resistance should be taken into consideration when planning the use of pyrethroid-impregnated materials in urban areas, as Culex is by far the main source of nuisance. Any failure in nuisance control due to resistance is likely to demotivate people in using impregnated materials.     

Permethrin resistance variation and susceptible reference line isolation in a field population of the mosquito,Culex quinquefasciatus (Diptera: Culicidae)

This study examines the genetic variations and mechanisms involved in the development of permethrin resistance in individual mosquitoes from a field population of Culex quinquefasciatus, HAmCq^G0, and characterizes susceptible reference lines of mosquitoes with a similar genetic background to the field HAmCq^G0 strain. Six upregulated cytochrome P450 genes, CYP9M10, CYP9J34, CYP6P14, CYP9J40, CYP6AA7, and CYP4C52v1, previously identified as being upregulated in the larvae of resistant HAmCq^G8 mosquitoes were examined in the larvae of 3 strains (susceptible S‐Lab, parental HAmCq^G0 and permethrin‐selected highly resistant HAmCq^G8) and 8 HAmCq^G0 single‐egg raft colonies, covering a range of levels of susceptibility/resistance to permethrin and exhibiting different variations in the expression of A and/or T alleles at the L‐to‐F kdr locus of the sodium channel. The 2 lines with the lowest tolerance to permethrin and bearing solely the susceptible A allele at the L‐to‐F kdr locus of the sodium channels, from colonies Cx_SERC5 and Cx_SERC8, showed lower or similar levels of all 6 of the P450 genes tested compared with the S‐Lab strain, suggesting that these 2 lines could be used as the reference mosquitoes in future studies characterizing insecticide resistance in HAmCq mosquitoes. This study also provides a detailed investigation of the mechanisms involved in insecticide resistance in individuals within a population: individuals with elevated levels of resistance to permethrin all displayed one or more potential resistance mechanisms–either elevated levels of P450 gene expression, or L‐to‐F mutations in the sodium channel, or both.     

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

Permethrin Induction of Multiple Cytochrome P450 Genes in Insecticide Resistant Mosquitoes,Culex quinquefasciatus

The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.     

Distribution and spread of pyrethroid and DDT resistance among the Anopheles gambiae complex in Tanzania

The development of insecticide resistance is a threat to the control of malaria in Africa. We report the findings of a national survey carried out in Tanzania in 2011 to monitor the susceptibility of malaria vectors to pyrethroid, organophosphate, carbamate and DDT insecticides, and compare these findings with those identified in 2004 and 2010. Standard World Health Organization (WHO) methods were used to detect knock‐down and mortality rates in wild female Anopheles gambiae s.l. (Diptera: Culicidae) collected from 14 sentinel districts. Diagnostic doses of the pyrethroids deltamethrin, lambdacyhalothrin and permethrin, the carbamate propoxur, the organophosphate fenitrothion and the organochlorine DDT were used. Anopheles gambiae s.l. was resistant to permethrin in Muleba, where a mortality rate of 11% [95% confidence interval (CI) 6–19%] was recorded, Muheza (mortality rate of 75%, 95% CI 66–83%), Moshi and Arumeru (mortality rates of 74% in both). Similarly, resistance was reported to lambdacyhalothrin in Muleba, Muheza, Moshi and Arumeru (mortality rates of 31–82%), and to deltamethrin in Muleba, Moshi and Muheza (mortality rates of 28–75%). Resistance to DDT was reported in Muleba. No resistance to the carbamate propoxur or the organophosphate fenitrothion was observed. Anopheles gambiae s.l. is becoming resistant to pyrethoids and DDT in several parts of Tanzania. This has coincided with the scaling up of vector control measures. Resistance may impair the effectiveness of these interventions and therefore demands close monitoring and the adoption of a resistance management strategy.     

Biochemical characterization of hydrolytic and oxidative enzymes in insecticide resistant and susceptible strains of the German cockroach (Dictyoptera: Blattellidae).

We have identified resistance mechanisms in the German cockroach, Blattella germanica (L.), for propoxur and chlorpyrifos in strains of cockroaches that display multiresistance to several organophosphate and carbamate insecticides. The resistance mechanisms involve the combined effects of increased oxidative and hydrolytic metabolism and both strains are resistant to chlorpyrifos and propoxur. Experiments designed to test for similarity in metabolic enzymes suggest that, although the mechanisms involve similar processes, the enzymes responsible for insecticide detoxification are different in the two strains. Both resistant strains exhibited enhanced activity toward alpha-naphtholic esters relative to a standard susceptible strain; however, analysis of the progeny from resistant X susceptible crosses suggests that this general esterase activity is inherited differently than propoxur or chlorpyrifos resistance. Hybrids of the propoxur-resistant strain displayed the highest activity of all cockroaches tested, in contrast to hybrids of the chlorpyrifos-resistant strain, which were similar to the susceptible strain. Native gel electrophoresis of cytosolic preparations provided further evidence for differences in the pattern of hydrolytic enzymes and inheritance of resistance in the two strains. Analysis of components of the cytochrome P450-dependent monooxygenase system and activities toward model substrates indicate that the two resistance mechanisms also involve different oxidative processes. The propoxur-resistant strain displayed significantly higher levels of total cytochrome P450, but no other components were correlated with resistance. In contrast with the chlopyrifos-resistant strain, which was similar to the susceptible strain in all parameters measured, activity toward model substrates was higher in the propoxur-resistant strain than in any of the other strains and hybrids tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility and avoidance behavior by Culex quinquefasciatus Say to three classes of residual insecticides.

The behavioral responses ofthree colonized strains of Culex quinquefasciatus, two from recent field collections in Thailand (Nonthaburi and Mae Sot) and one from a long-established colony from the National Institute of Health (NIH), Ministry of Public Health, Thailand, were compared during and after exposure to deltamethrin (0.02 g/m2), propoxur (0.2 g/m2), and fenitrothion (0.2 g/m2) using an excito-repellency escape chamber system. We observed striking differences in behavioral response and excito-repellency between mosquito strains and test compounds. Greater escape responses were observed in the NIH strain during direct contact with deltamethrin and fenitrothion compared with the two field populations. Deltamethrin was the most irritant, followed by fenitrothion. Escape responses with propoxur were significantly delayed but increased slightly towards the end of the 30-min exposure period, more notably in the Nonthaburi strain (P < 0.05). Non-contact repellent responses were generally much weaker than irritancy, with the greatest escape response seen with NIH and Nonthaburi. Deltamethrin showed the weakest repellent response overall (< 10% escape), while propoxur again demonstrated a delayed effect (NIH and Mae Sot) before escape occurred. We conclude that irritant and repellent behavioral responses by Cx. quinquefasciatus are important components for assessing the impact of residual spraying in mosquito control programs. A better understanding of chemical properties that elicit behavioral responses in mosquitoes should be considered in formulating control strategies designed to control mosquitoes or mitigate disease transmission risk.     

Identification and classification of detoxification enzymes from Culex quinquefasciatus (Diptera: Culicidae)

Molecular characterization of the insecticide resistance has become a hot research topic ever since the first disease transmitting arthropod (Anopheles gambiae) genome sequence has unveiled in 2002. A recent publication of the Culex quinquefasciatus genome sequence has opened up new opportunities for molecular and comparative genomic analysis of multiple mosquito genomes to characterize the insecticide resistance. Here, we utilized a whole genome sequence of Cx. quinquefasciatus to identify putatively active members of the detoxification supergene families, namely cytochrome P450s (P450s), glutathione-S-transferases (GSTs), and choline/carboxylesterases (CCEs). The Culex genome analysis revealed 166 P450s, 40 GSTs, and 62 CCEs. Further, the comparative genomic analysis shows that these numbers are considerably higher than the other dipteran mosquitoes. These observed speciesspecific expansions of the detoxification super gene family members endorse the popular understanding of the involvement of these gene families in protecting the organism against multitudinous classes of toxic substances during its complex (aquatic and terrestrial) life cycle. Thus, the generated data set may provide an initial point to start with to characterize the insecticide resistance at a molecular level which could then lead the development of an easy to use molecular marker to monitor the incipient insecticide resistance in field environs.     

Insecticide Resistance Profiles and Synergism of Field Aedes aegypti from Indonesia

Information on the insecticide resistance profiles of Aedes aegypti in Indonesia is fragmentary because of the lack of wide-area insecticide resistance surveillance. We collected Ae. aegypti from 32 districts and regencies in 27 Indonesian provinces and used WHO bioassays to evaluate their resistance to deltamethrin, permethrin, bendiocarb, and pirimiphos-methyl. To determine the possible resistance mechanisms of Ae. aegypti, synergism tests were conducted using piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioates (DEF). The Ae. aegypti from all locations exhibited various levels of resistance to pyrethroids. Their resistance ratio (RR₅₀) to permethrin and deltamethrin ranged from 4.08× to 127× and from 4.37× to 72.20×, respectively. In contrast with the findings of other studies, most strains from the highly urbanized cities on the island of Java (i.e., Banten, Jakarta, Bandung, Semarang, Yogyakarta, and Surabaya) exhibited low to moderate resistance to pyrethroids. By contrast, the strains collected from the less populated Kalimantan region exhibited very high resistance to pyrethroids. The possible reasons are discussed herein. Low levels of resistance to bendiocarb (RR₅₀, 1.24–6.46×) and pirimiphos-methyl (RR₅₀, 1.01–2.70×) were observed in all tested strains, regardless of locality. PBO and DEF synergists significantly increased the susceptibility of Ae. aegypti to permethrin and deltamethrin and reduced their resistance ratio to less than 16×. The synergism tests suggested the major involvement of cytochrome P450 monooxygenases and esterases in conferring pyrethroid resistance. On the basis of our results, we proposed a 6-month rotation of insecticides (deltamethrin + synergists ➝ bendiocarb ➝ permethrin + synergists ➝ pirimiphos-methyl) and the use of an insecticide mixture containing pyrethroid and pyrimiphos-methyl to control Ae. aegypti populations and overcome the challenge of widespread Ae. aegypti resistance to pyrethroid in Indonesia.