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Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance.

ABSTRACT: Several Glutathione S-transferases (GSTs) enzymes, in insects, have previously been implicated in resistance developed against DDT and other insecticides. The GST enzyme particularly sigma class have important physiological role in detoxification of lipid peroxidation by-products in insects. Phlebotomus argentipes has been intensely exposed to DDT over years due to Indoor Residual Spray (IRS) programme for Kala-azar elimination in Bihar, India. However, in P. argentipes, role of GSTs in DDT resistance have not been elucidated. Here, sigma class GST of P. argentipes (Parg-GST?) was successfully cloned, expressed and purified by affinity chromatography. The recombinant Parg-GST? was found to be highly active towards cumene hydroperoxide and 4-HNE having specific activity 92.47 & 203.92?µM/min/mg of protein, respectively and exhibited low activity towards universal substrate CDNB i.e., 8.75?µM/min/mg of protein. RT-PCR and immunoblot analysis showed at least 2 and 1.8 fold overexpression of Parg-GST? in the single exposed and non exposed DDT resistant P. argentipes as compared to susceptible, implicating Parg-GST? also involved in DDT resistance probably by imparting enhanced stress tolerance. The DDT, H2O2 and temperature induction assays demonstrated stress-dependent induction of Parg-GST? expression indicating its important role in oxidative stress redressal.

SUBMITTER: Hassan F

PROVIDER: S-EPMC6928345 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance.

Hassan Faizan F Singh Krishn Pratap KP Ali Vahab V Behera Sachidananda S Shivam Pushkar P Das Pradeep P Dinesh Diwakar Singh DS

Scientific reports 20191223 1

Several Glutathione S-transferases (GSTs) enzymes, in insects, have previously been implicated in resistance developed against DDT and other insecticides. The GST enzyme particularly sigma class have important physiological role in detoxification of lipid peroxidation by-products in insects. Phlebotomus argentipes has been intensely exposed to DDT over years due to Indoor Residual Spray (IRS) programme for Kala-azar elimination in Bihar, India. However, in P. argentipes, role of GSTs in DDT resi ...[more]

PMID: 31873171

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Project description:BackgroundLeishmania donovani-induced and sand fly-transmitted leishmaniasis is a growing health problem in Sri Lanka. Limited knowledge on biological and behavioral characteristics of probable vector Phlebotomus argentipes hinders disease control. Here, insecticide susceptibility patterns of P. argentipes were investigated with exploration of probable underlying resistance mechanisms.MethodsAdult sand flies were collected using standard cattle baited net traps and CDC light traps from selected sites in four districts. Adult F1 progeny of P. argentipes were exposed to different concentrations of DDT, malathion, deltamethrin and propoxur using WHO susceptibility bioassay kits. Post-1-h knockdown and post-24-h mortality were recorded and analyzed. Metabolic enzyme activity and the sensitivity of the acetylcholinesterase target-site were determined by biochemical assays using wild-caught flies. Extracted fly DNA samples were tested for the presence of knockdown-resistance (kdr) type mutations.ResultsThe LC100 values for DDT, malathion, propoxur and deltamethrin were 0.8-1.5%, 0.9-2.0%, 0.017-0.03% and 0.007% respectively. Insecticide-susceptibility levels were higher than the discriminating dosages established for Aedes mosquitoes, except for malathion. The lowest susceptibility levels (except for deltamethrin) were detected in the Mamadala population, whereas the highest levels were detected in the Mirigama population. The percentage of knocked-down sand flies was < 75% at any tested concentration, including those, which exhibited 100% mortality after 24 h. Elevated activity levels of glutathione S-transferase (3%, 7%, 12.5% and 14%) and esterase (2%, 5%, 5.5% and 6.5%) were detected in flies that originated from Mirigama, Pannala, Thalawa and Mamadala respectively, while monooxygenase quantities remained below the cut-off level. Ten to 34.5% of flies were heterozygous for acetylcholinesterases target-site insensitivity, associated with organophosphate and carbamate resistance. Pyrethroid-resistance-associated L1014F kdr-type mutation in the voltage gated sodium channel gene was detected in 30/53 flies.ConclusionsPopulations of P. argentipes in Sri Lanka are largely susceptible to common insecticides, except for malathion (used extensively in the past for malaria control). Their insecticide susceptibility appears negatively associated with past malaria endemicity of the study sites, with signs of early insecticide tolerance. Presence of insecticide target site insensitivity in a notable proportion of flies and enhanced insecticide metabolizing enzyme activities imply potential future challenges for leishmaniasis control, with a call for urgent proactive measures for its containment.

Project description:BackgroundThe sand fly Phlebotomus argentipes is arguably the most important vector of leishmaniasis worldwide. As there is no vaccine against the parasites that cause leishmaniasis, disease prevention focuses on control of the insect vector. Understanding reproductive behaviour will be essential to controlling populations of P. argentipes, and developing new strategies for reducing leishmaniasis transmission. Through statistical analysis of male-female interactions, this study provides a detailed description of P. argentipes courtship, and behaviours critical to mating success are highlighted. The potential for a role of cuticular hydrocarbons in P. argentipes courtship is also investigated, by comparing chemicals extracted from the surface of male and female flies.Principal findingsP. argentipes courtship shared many similarities with that of both Phlebotomus papatasi and the New World leishmaniasis vector Lutzomyia longipalpis. Male wing-flapping while approaching the female during courtship predicted mating success, and touching between males and females was a common and frequent occurrence. Both sexes were able to reject a potential partner. Significant differences were found in the profile of chemicals extracted from the surface of males and females. Results of GC analysis indicate that female extracts contained a number of peaks with relatively short retention times not present in males. Extracts from males had higher peaks for chemicals with relatively long retention times.ConclusionsThe importance of male approach flapping suggests that production of audio signals through wing beating, or dispersal of sex pheromones, are important to mating in this species. Frequent touching as a means of communication, and the differences in the chemical profiles extracted from males and females, may also indicate a role for cuticular hydrocarbons in P. argentipes courtship. Comparing characteristics of successful and unsuccessful mates could aid in identifying the modality of signals involved in P. argentipes courtship, and their potential for use in developing new strategies for vector control.

Project description:BackgroundA number of studies on visceral leishmaniasis (VL) vector control have been conducted during the past decade, sometimes came to very different conclusion. The present study on a large sample investigated different options which are partially unexplored including: (1) indoor residual spraying (IRS) with alpha cypermethrin 5WP; (2) long lasting insecticide impregnated bed-net (LLIN); (3) impregnation of local bed-nets with slow release insecticide K-O TAB 1-2-3 (KOTAB); (4) insecticide spraying in potential breeding sites outside of house using chlorpyrifos 20EC (OUT) and different combinations of the above.MethodsThe study was a cluster randomized controlled trial where 3089 houses from 11 villages were divided into 10 sections, each section with 6 clusters and each cluster having approximately 50 houses. Based on vector density (males plus females) during baseline survey, the 60 clusters were categorized into 3 groups: (1) high, (2) medium and (3) low. Each group had 20 clusters. From these three groups, 6 clusters (about 300 households) were randomly selected for each type of intervention and control arms. Vector density was measured before and 2, 4, 5, 7, 11, 14, 15, 18 and 22 months after intervention using CDC light traps. The impact of interventions was measured by using the difference-in-differences regression model.ResultsA total of 17,434 sand flies were collected at baseline and during the surveys conducted over 9 months following the baseline measurements. At baseline, the average P. argentipes density per household was 10.6 (SD = 11.5) in the control arm and 7.3 (SD = 8.46) to 11.5 (SD = 20.2) in intervention arms. The intervention results presented as the range of percent reductions of sand flies (males plus females) and rate ratios in 9 measurements over 22 months. Among single type interventions, the effect of IRS with 2 rounds of spraying (applied by the research team) ranged from 13% to 75% reduction of P. argentipes density compared to the control arm (rate-ratio [RR] ranged from 0.25 to 0.87). LLINs caused a vector reduction of 9% to 78% (RR, 0.22 to 0.91). KOTAB reduced vectors by 4% to 73% (RR, 0.27 to 0.96). The combination of LLIN and OUT led to a vector reduction of 26% to 86% (RR, 0.14 to 0.74). The reduction for the combination of IRS and OUT was 8% to 88% (RR, 0.12 to 0.92). IRS and LLIN combined resulted in a vector reduction of 13% to 85% (RR, 0.15 to 0.77). The IRS and KOTAB combination reduced vector densities by 16% to 86% (RR, 0.14 to 0.84). Some intermediate measurements for KOTAB alone and for IRS plus LLIN; and IRS plus KOTAB were not statistically significant. The bioassays on sprayed surfaces or netting materials showed favourable results (>80% mortality) for 22 months (IRS tested for 12 months). In the KOTAB, a gradual decline was observed after 6 months.ConclusionsLLIN and OUT was the best combination to reduce VL vector densities for 22 months or longer. Operationally, this is much easier to apply than IRS. A cost analysis of the preferred tools will follow. The relationship between vector density (males plus females) and leishmaniasis incidence should be investigated, and this will require estimates of the Entomological Inoculation Rate.

Dataset Information

Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance.

Publications

Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance.

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