Presence of localized elevated metabolic enzymes and kdr mutations in the voltage-gated sodium channel gene indicate early evidence of resistance of Phlebotomus argentipes to alpha-cypermethrin in Bihar, India

BACKGROUND: Visceral leishmaniasis, caused by Leishmania donovani and transmitted by Phlebotomus argentipes, remains a major public health challenge in the Indian subcontinent. Sand fly populations are controlled by using different insecticides, particularly dichlorodiphenyltrichloroethane (DDT) and pyrethroids such as alpha-cypermethrin. Prolonged and irregular use of these insecticides raises concern about the development of resistance in sand fly populations. METHODS: This study aimed to compare metabolic detoxification and knockdown resistance (kdr) mutations in Vgsc and ace-1 between nine sprayed (indoor residual spray, IRS) villages and one unsprayed (non-IRS) village in Muzaffarpur, Bihar, India. A total of 10 Ph. argentipes from each village were used for metabolic detoxification and 10-25 Ph. argentipes were used for target site insensitivity mutations. Homogenized Ph. argentipes aliquots were used for different enzymes activity and DNA was used for the sequence analysis of Vgsc and ace-1 genes to access the presence of kdr mutations. RESULTS: Biochemical assays revealed that levels of detoxification enzymes, including glutathione S-transferase (GST), esterase (PNPA), and cytochrome P450 monooxygenase, were elevated in IRS villages, suggesting localized metabolic resistance. Molecular screening of the voltage-gated sodium channel (Vgsc) gene revealed high frequencies of knockdown resistance (kdr) mutations at codon 1014, with serine (L1014S) (48.5%) being the most prevalent, followed by wild type leucine (L1014) (39.5%). No mutations were detected at codon 119 of the ace-1 gene, indicating the sensitivity to organophosphates in the sand fly population. CONCLUSIONS: The results suggested that continuous and repeated exposure to the synthetic pyrethroid may exert selective pressure, leading to early signs of resistance in Ph. argentipes, mediated through metabolic detoxification mechanisms and mutation in the kdr gene. These findings underscore the importance of ongoing resistance monitoring and the implementation of rotational insecticide strategies to support sustained efforts toward the elimination of visceral leishmaniasis.