Resistance to DDT and pyrethroids and increased kdr mutation frequency in An. gambiae after the implementation of permethrin-treated nets in Senegal

IntroductionThe aim of this study was to evaluate the susceptibility to insecticides of An. gambiae mosquitoes sampled in Dielmo (Senegal), in 2010, 2 years after the implementation of Long Lasting Insecticide-treated Nets (LLINs) and to report the evolution of kdr mutation frequency from 2006 to 2010.MethodsWHO bioassay susceptibility tests to 6 insecticides were performed on adults F0, issuing from immature stages of An. gambiae s.l., sampled in August 2010. Species and molecular forms as well as the presence of L1014F and L1014S kdr mutations were assessed by PCR. Longitudinal study of kdr mutations was performed on adult mosquitoes sampled monthly by night landing catches from 2006 to 2010.FindingsNo specimen studied presented the L1014S mutation. During the longitudinal study, L1014F allelic frequency rose from 2.4% in year before the implementation of LLINs to 4.6% 0–12 months after and 18.7% 13–30 months after. In 2010, An. gambiae were resistant to DDT, Lambda-cyhalothrin, Deltamethrin and Permethrin (mortality rates ranging from 46 to 63%) but highly susceptible to Fenitrothion and Bendiocarb (100% mortality). There was significantly more RR genotype among An. gambiae surviving exposure to DDT or Pyrethroids. An. arabiensis represented 3.7% of the sampled mosquitoes (11/300) with no kdr resistance allele detected. An. gambiae molecular form M represented 29.7% of the mosquitoes with, among them, kdr genotypes SR (18%) and SS (82%). An. gambiae molecular form S represented 66% of the population with, among them, kdr genotype SS (33.3%), SR (55.6%) and RR (11.1%). Only 2 MS hybrid mosquitoes were sampled and presented SS kdr genotype.ConclusionBiological evidence of resistance to DDT and pyrethroids was detected among An. gambiae mosquitoes in Dielmo (Senegal) within 24 months of community use of LLINs. Molecular identification of L1014F mutation indicated that target site resistance increased after the implementation of LLINs.     

Pyrethroid Resistance in an Anopheles funestus Population from Uganda

Background

The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.

Methodology/Principal Findings

A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F₁ adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.

Conclusion

This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.     

Time-of-day specific changes in metabolic detoxification and insecticide resistance in the malaria mosquito Anopheles gambiae

Mosquitoes exhibit ∼24 h rhythms in physiology and behavior, regulated by the cooperative action of an endogenous circadian clock and the environmental light:dark cycle. Here, we characterize diel (observed under light:dark conditions) time-of-day changes in metabolic detoxification and resistance to insecticide challenge in Anopheles gambiae mosquitoes. A better understanding of mosquito chronobiology will yield insights into developing novel control strategies for this important disease vector. We have previously identified >2000 rhythmically expressed An. gambiae genes. These include metabolic detoxification enzymes peaking at various times throughout the day. Especially interesting was the identification of rhythmic genes encoding enzymes capable of pyrethroid and/or DDT metabolism (CYP6M2, CYP6P3, CYP6Z1, and GSTE2). We hypothesized that these temporal changes in gene expression would confer time-of-day specific changes in metabolic detoxification and responses to insecticide challenge. An. gambiae mosquitoes (adult female Pimperena and Mali-NIH strains) were tested by gene expression analysis for diel rhythms in key genes associated with insecticidal resistance. Biochemical assays for total GST, esterase, and oxidase enzymatic activities were undertaken on time-specific mosquito head and body protein lysates. To determine for rhythmic susceptibility to insecticides by survivorship, mosquitoes were exposed to DDT or deltamethrin across the diel cycle. We report the occurrence of temporal changes in GST activity in samples extracted from the body and head with a single peak at late-night to dawn, but no rhythms were detected in oxidase or esterase activity. The Pimperena strain was found to be resistant to insecticidal challenge, and subsequent genomic analysis revealed the presence of the resistance-conferring kdr mutation. We observed diel rhythmicity in key insecticide detoxification genes in the Mali-NIH strain, with peak phases as previously reported in the Pimperena strain. The insecticide sensitive Mali-NIH strain mosquitoes exhibited a diel rhythm in survivorship to DDT exposure and a bimodal variation to deltamethrin challenge. Our results demonstrate rhythms in detoxification and pesticide susceptibility in An. gambiae mosquitoes; this knowledge could be incorporated into mosquito control and experimental design strategies, and contributes to our basic understanding of mosquito biology.     

Insecticide susceptibility of Anopheles coluzzii and Anopheles gambiae mosquitoes in Ibadan,Southwest Nigeria

The emergence of insecticide resistance in Anopheles (Diptera: Culicidae) mosquitoes has great implications for malaria control in Nigeria. This study aimed to determine the dynamics of insecticide susceptibility levels and the frequency of knock‐down resistance (kdr) mutations (L1014F) in wild Anopheles coluzzii Coetzee & Wilkerson sp. n. and Anopheles gambiae Giles from the Ojoo and Bodija areas of Ibadan, in southwest Nigeria. Insecticide susceptibility to pyrethroids, organophosphates, carbamates and organochlorines was assessed using World Health Organization (WHO) bioassays. A subset of the mosquitoes exposed to pyrethroids and DDT was used for species and molecular form identification; kdr genotyping was determined using the TaqMan real‐time polymerase chain reaction assay. The mosquitoes were resistant to pyrethroids and DDT but completely susceptible to organophosphates and carbamates. Bodija samples (n = 186) consisted of An. gambiae (91.4%) and An. coluzzii (8.1%) and included one An. coluzzii/An. gambiae hybrid specimen. All mosquitoes screened in Ojoo (n = 26) were An. gambiae. The 1014F kdr mutation was detected at frequencies of 24.5 and 5.8% in Bodija and Ojoo, respectively. No correlation was observed between kdr genotypes and resistance phenotypes. The results indicate that metabolic resistance probably plays an important role in the development of resistance and highlight the need to implement insecticide resistance management strategies.     

A longitudinal study on Anopheles mosquito larval abundance in distinct geographical and environmental settings in western Kenya

Background

Malaria vector control in Africa depends upon effective insecticides in bed nets and indoor residual sprays. This study investigated the extent of insecticide resistance in Anopheles gambiae s.l., Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya where ownership of insecticide-treated bed nets has risen steadily from the late 1990s to 2010. Temporal and spatial variation in the frequency of a knock down resistance (kdr) allele in A. gambiae s.s. was quantified, as was variation in phenotypic resistance among geographic populations of A. gambiae s.l.

Methods

To investigate temporal variation in kdr frequency, individual specimens of A. gambiae s.s. from two sentinel sites were genotyped using RT-PCR from 1996-2010. Spatial variation in kdr frequency, species composition, and resistance status were investigated in additional populations of A. gambiae s.l. sampled in western Kenya in 2009 and 2010. Specimens were genotyped for kdr as above and identified to species via conventional PCR. Field-collected larvae were reared to adulthood and tested for insecticide resistance using WHO bioassays.

Results

Anopheles gambiae s.s. showed a dramatic increase in kdr frequency from 1996 - 2010, coincident with the scale up of insecticide-treated nets. By 2009-2010, the kdr L1014S allele was nearly fixed in the A. gambiae s.s. population, but was absent in A. arabiensis. Near Lake Victoria, A. arabiensis was dominant in samples, while at sites north of the lake A. gambiae s.s was more common but declined relative to A. arabiensis from 2009 to 2010. Bioassays demonstrated that A. gambiae s.s. had moderate phenotypic levels of resistance to DDT, permethrin and deltamethrin while A. arabiensis was susceptible to all insecticides tested.

Conclusions

The kdr L1014S allele has approached fixation in A. gambiae s.s. populations of western Kenya, and these same populations exhibit varying degrees of phenotypic resistance to DDT and pyrethroid insecticides. The near absence of A. gambiae s.s. from populations along the lakeshore and the apparent decline in other populations suggest that insecticide-treated nets remain effective against this mosquito despite the increase in kdr allele frequency. The persistence of A. arabiensis, despite little or no detectable insecticide resistance, is likely due to behavioural traits such as outdoor feeding and/or feeding on non-human hosts by which this species avoids interaction with insecticide-treated nets.     

Exploring mechanisms of multiple insecticide resistance in a population of the malaria vector Anopheles funestus in Benin

Background

The insecticide resistance status of the malaria vector Anopheles funestus and the underlying resistance mechanisms remain uncharacterised in many parts of Africa, notably in Benin, West Africa. To fill this gap in our knowledge, we assessed the susceptibility status of a population of this species in Pahou, Southern Benin and investigated the potential resistance mechanisms.

Methodology/Principal Findings

WHO bioassays revealed a multiple resistance profile for An. funestus in Pahou. This population is highly resistant to DDT with no mortality in females after 1h exposure to 4%DDT. Resistance was observed against the Type I pyrethroid permethrin and the carbamate bendiocarb. A moderate resistance was detected against deltamethrin (type II pyrethroids). A total susceptibility was observed against malathion, an organophosphate. Pre-exposure to PBO did not change the mortality rates for DDT indicating that cytochrome P450s play no role in DDT resistance in Pahou. No L1014F kdr mutation was detected but a correlation between haplotypes of two fragments of the Voltage-Gated Sodium Channel gene and resistance was observed suggesting that mutations in other exons may confer the knockdown resistance in this species. Biochemical assays revealed elevated levels of GSTs and cytochrome mono-oxygenases in Pahou. No G119S mutation and no altered acetylcholinesterase gene were detected in the Pahou population. qPCR analysis of five detoxification genes revealed that the GSTe2 is associated to the DDT resistance in this population with a significantly higher expression in DDT resistant samples. A significant over-expression of CYP6P9a and CYP6P9b previously associated with pyrethroid resistance was also seen but at a lower fold change than in southern Africa.

Conclusion

The multiple insecticide resistance profile of this An. funestus population in Benin shows that more attention should be paid to this important malaria vector for the implementation and management of current and future malaria vector control programs in this country.     

Differential resistance of insect sodium channels with kdr mutations to deltamethrin, permethrin and DDT

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V_50,act) from −17.3 mV (wild-type) to −13.1 and −13.5 mV respectively, whereas L1014S caused no shift in V_50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥1 nM), permethrin (≥30 nM) or DDT (≥1 ??M) resulted in hyperpolarizing shifts in V_50,act. Deltamethrin, permethrin and DDT also produced “tail currents” with EC₅₀s of 0.043, 0.40 and 65 ??M and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V_50,act. We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.     

Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypti

Insecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98-100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and β esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l(-1), 0.2 mg l(-1) and 1.0 mg l(-1) for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80-98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of β-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.     

Impacts of Agricultural Practices on Insecticide Resistance in the Malaria Vector Anopheles arabiensis in Khartoum State,Sudan

Background

Agricultural pesticides may play a profound role in selection of resistance in field populations of mosquito vectors. The objective of this study is to investigate possible links between agricultural pesticide use and development of resistance to insecticides by the major malaria vector Anopheles arabiensis in northern Sudan.

Methodology/Principal Findings

Entomological surveys were conducted during two agricultural seasons in six urban and peri-urban sites in Khartoum state. Agro-sociological data were collected from 240 farmers subjected to semi-structured questionnaires based on knowledge attitude and practice (KAP) surveys. Susceptibility status of An. arabiensis (n=6000) was assessed in all sites and during each season using WHO bioassay tests to DDT, deltamethrin, permethrin, Malathion and bendiocarb. KAP analysis revealed that pesticide application was common practice among both urban and peri-urban farmers, with organophosphates and carbamates most commonly used. Selection for resistance is likely to be greater in peri-urban sites where farmers apply pesticide more frequently and are less likely to dispose of surpluses correctly. Though variable among insecticides and seasons, broad-spectrum mortality was slightly, but significantly higher in urban than peri-urban sites and most marked for bendiocarb, to which susceptibility was lowest. Anopheles arabiensis from all sites showed evidence of resistance or suspected resistance, especially pyrethroids. However, low-moderate frequencies of the L1014F kdr allele in all sites, which was very strongly associated with DDT, permethrin and deltamethrin survivorship (OR=6.14-14.67) suggests that resistance could increase rapidly.

Conclusions

Ubiquitous multiple-resistance coupled with presence of a clear mechanism for DDT and pyrethroids (kdr L1014F) in populations of An. arabiensis from Khartoum-Sudan suggests careful insecticide management is essential to prolong efficacy. Our findings are consistent with agricultural insecticide use as a source of selection for resistance and argue for coordination between the integrated vector control program and the Ministry of Agriculture to permit successful implementation of rational resistance management strategies.     

Sensitivity of the Drosophila para sodium channel to DDT is not lowered by the super-kdr mutation M918T on the IIS4-S5 linker that profoundly reduces sensitivity to permethrin and deltamethrin

DDT inhibits Na channel inactivation and deactivation, promotes Na channel activation and reduces the resting potential of Xenopus oocytes expressing the Drosophila para Na channel. These changes are only marginally influenced by the single mutation M918T (super-kdr) but are reduced approximately 10-fold by either the single mutation L1014F (kdr) or the double mutation L1014F+M918T, both of which confer resistance to the pyrethroids permethrin and deltamethrin. We conclude that DDT binds either to or in the region of L1014 on IIS6 but only weakly to M918 on the IIS4-S5 linker, which is part of a high-affinity binding site for permethrin and deltamethrin.     

Malaria vector composition and insecticide susceptibility status in Guinea Conakry,West Africa

This study provides data on malaria vector species composition and insecticide susceptibility status from three localities in Guinea Conakry. A total of 497 mosquitoes were collected resting indoors and morphologically identified as belonging to the Anopheles gambiae complex. The majority of these were An. gambiae s.s. (99.6%), but a small percentage (0.4%) were identified as Anopheles arabiensis. Thirty‐four Anopheles funestus s.s. were also collected. The molecular S form of An. gambiae s.s. was predominant over the M form in Siguiri (95%) and Boffa (97.4%), whereas at Mt Nimba the M form was more abundant (61.4%) than the S form (38.1%). One hybrid M/S specimen was recorded from Mt Nimba. Siguiri populations showed high levels of resistance to DDT, dieldrin and bendiocarb. Anopheles gambiae from Boffa were largely susceptible to the insecticides tested. At Mt Nimba, resistance to DDT and bendicocarb was detected. Biochemical enzyme analysis showed that an altered acetylcholinesterase is operating in the field at low levels. The frequency of the 1014F kdr allele in the An. gambiae S form was 0.24 at Siguiri and 0.14 at Mt Nimba. A single RR specimen was found in the M form. The heterogeneity in species composition and resistance profiles between sites requires vector control interventions to be tailored to each site based on the data collected from ongoing monitoring and surveillance.     

Three years of insecticide resistance monitoring in Anopheles gambiae in Burkina Faso: resistance on the rise?

ABSTRACT: Background and methods A longitudinal Anopheles gambiae s.l. insecticide-resistance monitoring programme was established in four sentinel sites in Burkina Faso. For three years, between 2008 and 2010, WHO diagnostic dose assays were used to measure the prevalence of resistance to all the major classes of insecticides at the beginning and end of the malaria transmission season. Species identification and genotyping for target site mutations was also performed and the sporozoite rate in adults determined. RESULTS: At the onset of the study, resistance to DDT and pyrethroids was already prevalent in An. gambiae s.l. from the south-west of the country but mosquitoes from the two sites in central Burkina Faso were largely susceptible. Within three years, DDT and permethrin resistance was established in all four sites. Carbamate and organophosphate resistance remains relatively rare and largely confined to the south-western areas although a small number of bendiocarb survivors were found in all sites by the final round of monitoring. The ace-1R target site resistance allele was present in all localities and its frequency exceeded 20% in 2010 in two of the sites. The frequency of the 1014 F kdr mutation increased throughout the three years and by 2010, the frequency of 1014 F in all sites combined was 0.02 in Anopheles arabiensis, 0.56 in An. gambiae M form and 0.96 in An. gambiae S form. This frequency did not differ significantly between the sites. The 1014 S kdr allele was only found in An. arabiensis but its frequency increased significantly throughout the study (P = 0.0003) and in 2010 the 1014 S allele frequency was 0.08 in An. arabiensis. Maximum sporozoite rates (12%) were observed in Soumousso in 2009 and the difference between sites is significant for each year. CONCLUSION: Pyrethroid and DDT resistance is now established in An. gambiae s.l. throughout Burkina Faso. Results from diagnostic dose assays are highly variable within and between rounds of testing, and hence it is important that resistance monitoring is carried out on more than one occasion before decisions on insecticide procurement for vector control are made. The presence of 1014 S in An. gambiae s.l., in addition to 1014 F, is not unexpected given the recent report of 1014 S in Benin but highlights the importance of monitoring for both mutations throughout the continent. Future research must now focus on the impact that this resistance is having on malaria control in Burkina Faso.     

Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa)

An entomological survey was carried out at 15 sites dispersed throughout the three eco-climatic regions of Burkina Faso (West Africa) in order to assess the current distribution and frequency of mutations that confer resistance to insecticides in An. gambiae s.l. populations in the country. Both knockdown (kdr) resistance mutation variants (L1014F and L1014S), that confer resistance to pyrethroid insecticides, were identified concomitant with the ace-1 G119S mutation confirming the presence of multiple resistance mechanisms in the An. gambiae complex in Burkina Faso. Compared to the last survey, the frequency of the L1014F kdr mutation appears to have remained largely stable and relatively high in all species. In contrast, the distribution and frequency of the L1014S mutation has increased significantly in An. gambiae s.l. across much of the country. Furthermore we report, for the first time, the identification of the ace.1 G116S mutation in An. arabiensis populations collected at 8 sites. This mutation, which confers resistance to organophosphate and carbamate insecticides, has been reported previously only in the An. gambiae S and M molecular forms. This finding is significant as organophosphates and carbamates are used in indoor residual sprays (IRS) to control malaria vectors as complementary strategies to the use of pyrethroid impregnated bednets. The occurrence of the three target-site resistance mutations in both An. gambiae molecular forms and now An. arabiensis has significant implications for the control of malaria vector populations in Burkina Faso and for resistance management strategies based on the rotation of insecticides with different modes of action.     

Variation in host preferences of malaria mosquitoes is mediated by skin bacterial volatiles

The host preferences of the anthropophilic mosquito species in the Anopheles gambiae complex (Diptera: Culicidae) are mediated by skin bacterial volatiles. However, it is not known whether these mosquitoes respond differentially to skin bacterial volatiles from non‐human host species. In this study, the responses of two malaria mosquito species in the An. gambiae complex, Anopheles gambiae s.s. (hereafter, An. gambiae) and Anopheles arabiensis, with different host preferences, to volatiles released from skin bacteria were tested. Skin bacteria collected from human, cow and chicken skin significantly increased trap catches; traps containing bacteria collected from human skin caught the highest proportions of An. gambiae and An. arabiensis. Traps with bacteria of human origin caught a significantly higher proportion of An. gambiae than of An. arabiensis, whereas bacterial volatiles from the chicken attracted significantly higher numbers of An. arabiensis than of An. gambiae. Additionally, An. gambiae showed a specialized response to volatiles from four specific bacteria, whereas An. arabiensis responded equally to all species of bacteria tested. Skin bacterial volatiles may therefore play important roles in guiding mosquitoes with different host preferences. The identification of these bacterial volatiles can contribute to the development of an odour blend that attracts mosquitoes with different host preferences.     

Molecular Ecology of Pyrethroid Knockdown Resistance in Culex pipiens pallens Mosquitoes

Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies.     

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F₁ individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.     

Occurrence of L1014F and L1014S mutations in insecticide resistant Culex quinquefasciatus from filariasis endemic districts of West Bengal,India

IntroductionLymphatic filariasis causes long term morbidity and hampers the socio-economic status. Apart from the available treatments and medication, control of vector population Culex quinquefasciatus Say through the use of chemical insecticides is a widely applied strategy. However, the unrestrained application of these insecticides over many decades has led to resistance development in the vectors.MethodsIn order to determine the insecticide susceptibility/resistance status of Cx. quinquefasciatus from two filariasis endemic districts of West Bengal, India, wild mosquito populations were collected and assayed against six different insecticides and presence of L1014F; L1014S kdr mutations in the voltage-gated sodium channel gene was also screened along with the use of synergists to evaluate the role of major detoxifying enzymes in resistance development.ResultsThe collected mosquito populations showed severe resistance to insecticides and the two synergists used–PBO (piperonyl butoxide) and TPP (triphenyl phosphate), were unable to restore the susceptibility status of the vector thereupon pointing towards a minor role of metabolic enzymes. kdr mutations were present in the studied populations in varying percent with higher L1014F frequency indicating its association with the observed resistance to pyrethroids and DDT. This study reports L1014S mutation in Cx. quinquefasciatus for the first time.