Development and application of species ID and insecticide resistance assays, for monitoring sand fly Leismania vectors in the Mediterranean basin and in the Middle East

Authors: BALASKA, SOFIA - University Of Greece - Crete; KHAJEHALI, JAHANGIR - Isfahan University Of Technology; MAVRIDIS, KONSTANTINOS - Foundation For Research And Technology - Hellas; AKINER, MUSTAFA - Recep Tayyip Erdogan University; REMADI, LATIFA - Earthshell; KIOULOS, ILIAS - Agricultural University Of Athens; MIAOULIS, MICHAEL - European Biological Control Laboratory (EBCL); FOTAKIS, ALEXANDROS - Istituto Superiore Di Sanita; CHASKOPOULOU, ALEXANDRA - European Biological Control Laboratory (EBCL); VONTAS, JOHN - Foundation For Research And Technology - Hellas

Submitted to: PLOS Neglected Tropical Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2024
Publication Date: 12/3/2024
Citation: Balaska, S., Khajehali, J., Mavridis, K., Akiner, M., Remadi, L., Kioulos, I., Miaoulis, M., Fotakis, A., Chaskopoulou, A., Vontas, J. 2024. Development and application of species ID and insecticide resistance assays, for monitoring sand fly Leismania vectors in the Mediterranean basin and in the Middle East. PLOS Neglected Tropical Diseases. 18(12):e0012408. https://doi.org/10.1371/journal.pntd.0012408.
DOI: https://doi.org/10.1371/journal.pntd.0012408

Interpretive Summary: Phlebotomine sand flies vector leishmaniasis to human and animals, a neglected tropical disease of the (sub-)tropics, displaying an expanding epidemiological tendency towards previously non-endemic regions. As leishmaniasis management largely relies on vectors’ insecticidal control, regular monitoring of insecticide resistance (IR) is a core element of integrated vector management. IR data are limited in sand fly populations worldwide due to the unavailability of robust molecular diagnostic and phenotyping tools. In this study we characterised the pyrethroid resistance (PyR) status of populations originating from countries of the Mediterranean basin and the Middle East; i.e. Greece, Turkey and Iran. PyR-associated mutations were detected in all three countries, while the populations from Greece exhibited susceptibility upon exposure to deltamethrin in CDC bioassays. We established a novel individual bioassay protocol to enable IR monitoring in settings with multi species population structure, and applied it comparatively among three distinct sand fly species, revealing that they respond differently to insecticide treatment. Our study will facilitate systematic surveillance and evidence-based sand fly control in the endemic regions.

Technical Abstract: Background:Development of insecticide resistance (IR) in sand fly populations is an upcoming issue of public health concern, threatening leishmaniasis mitigation efforts by insecticide-based vector control. There is a major knowledge gap in the IR status of wild populations worldwide, possibly attributed to the unavailability of specialized tools, such as bioassay protocols, species baseline susceptibility to insecticides and molecular markers, to monitor such phenomena in sand flies. Methodology/Principal findings: Several sand fly populations from (semi-)rural regions of Greece, Turkey and Iran were sampled and identified to species, showing populations’ structure in accordance with previously reported data. Genotyping of known pyrethroid resistance-associated loci revealed the occurrence of voltage-gated sodium channel (vgsc) mutations in all surveyed countries. Knock-down resistance (kdr) mutation L1014F was prevalent in Turkish regions and L1014F and L1014S were recorded for the first time in Iran, and in Turkey and Greece, respectively, yet in low frequencies. Moreover, CDC bottle bioassays against pyrethroids in mixed species populations from Greece indicated full susceptibility, using though mosquito discriminating doses. In parallel, we established a novel individual bioassay protocol and applied it comparatively among distinct Phlebotomus species’ populations, to detect any possible divergent species-specific response to insecticide. Indeed, a significantly different knock-down rate between P. simici and P. perfiliewi was observed upon exposure to deltamethrin. Conclusions/Significance: IR in sand flies is increasingly reported in leishmaniasis endemic regions, highlighting the necessity to generate additional monitoring tools, that could be implemented in relevant eco-epidemiological settings, in the context of IR management. Our molecular and phenotypic data add to the IR map in a macroarea with otherwise limited data coverage.