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-GSTσ) was successfully cloned, expressed and purified by affinity chromatography. The recombinant Parg-GSTσ 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-GSTσ in the single exposed and non exposed DDT resistant P. argentipes as compared to susceptible, implicating Parg-GSTσ also involved in DDT resistance probably by imparting enhanced stress tolerance. The DDT, H2O2 and temperature induction assays demonstrated stress-dependent induction of Parg-GSTσ expression indicating its important role in oxidative stress redressal.

objectives Indoor residual spraying (IRS) with insecticides is the main vector control intervention for the elimination of visceral leishmaniasis in India. After a change in IRS policy in 2015 due to widespread resistance of Phlebotomus argentipes to DDT, IRS with DDT was replaced with alphacypermethrin IRS in 2016. The objective of the present study was to evaluate the susceptibility of P. argentipes to DDT and its alternatives, namely malathion and pirimiphos-methyl (organophosphates); alpha-cypermethrin, deltamethrin, lambda-cyhalothrin and permethrin (pyrethroids), and bendiocarb and propoxur (carbamates), in support of visceral leishmaniasis elimination in India.methods Phlebotomus argentipes sandflies were collected from the visceral-leishmaniasis endemic states of Bihar, Jharkhand and West Bengal. In the WHO tube tests, the phenotypic susceptibility of F1, 2-day old, non-blood fed females were determined against filter papers impregnated with DDT 4%, malathion 5%, pirimiphos-methyl 0.25%, alpha-cypermethrin 0.05%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, permethrin 0.75%, bendiocarb 0.1% and propoxur 0.1%, which were sourced from Universiti Sains Malaysia. The knockdown of sandflies after 1-h exposure and mortality at 24 h after the 1-h exposure period were scored.results Mean mortality of P. argentipes 24 h after exposure in tube tests was 22.6% for DDT and ≥ 98% for other insecticide-impregnated papers tested.conclusion Phlebotomus argentipes continues to be highly resistant to DDT with no reversal of resistance after DDT's withdrawal from IRS. P. argentipes was fully susceptible to pyrethroid, organophosphate and carbamate insecticides tested. Regular monitoring is warranted for insecticide resistance management in sandfly vectors.

A nuclear polyhedrosis virus isolated from the Egyptian cotton leafworm, Spodoptera littoralis, was found to infect termite castes of Kalotermes flavicollis. Laboratory studies indicated that no specific trend toward mortality responses among the different individuals of termites was noted. All test castes of termites, young, middle-sized, old and reproductive nymphs, and soldiers, were quite equal in their response to the virus infection, regardless of whether the virus concentration was high or low; a concentration of 6.4 x 10 8 polyhedra/ml, killed only 64% of the treated individuals, while the dosage of 6.4 x 10 7 polyhedra/ml produced over 90% mortality. Also, the same trend of response was recorded with the survivors which received challenge doses of NPV suspension each 9 or 10 days. On the other hand, these results revealed that if the NPV is used to control termites, periodic applications would be more efficacious than one treatment. Virus symptoms were recorded in all affected insects.

Phlebotomus argentipes is an established vector for Visceral leishmaniasis prevalent in the Indian subcontinent. Insect Glutathione S-transferases (GST) enzyme plays a pivotal role in the metabolism of xenobiotics and chemical insecticides. We report herein the identification and characterization of a delta class GST from the sandfly, P. argentipes. The resulting clone (rParg-GSTδ) is successfully sequenced, which revealed 76.43% and 66.32% gene identity with GST from Phlebotomus papatasi (Scopoli; Diptera: Psychodidae) and Lutzomiya longipalpis (Lutz and Neiva; Diptera: Psychodidae), respectively. The identified rParg-GST amino acid Blast results revealed 82.6% homology to delta class GST of Phlebotomus papatasi and more than 50% homology to Lepidoptera which comprises butterflies and moths. The Phylogenetic analysis of Parg-GST with different classes of Insect GSTs further supported its classification as delta class. A functional recombinant Parg-GSTδ protein (rParg-GSTδ) was expressed in Escherichia coli (Migula; Enterobacterales: Enterobacteriaceae) cells in a soluble form, purified to homogeneity and found to be active against a substrate 1-chloro-2,4-dintrobenzene (CDNB) and lipid peroxidation by-product 4-Hydrxynonenal (4-HNE). Interestingly, rParg-GSTδ demonstrates high dehydrochlorination activity against dichlorodiphenyltrichloroethane (DDT) i.e., 16.27 nM/µg in high performance liquid chromatography (HPLC) assay. These results provide evidence of direct DDT metabolism property exhibited by P. argentipes GST and set the foundation to decipher the metabolic resistance mechanism in P. argentipes against insecticides.

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