The project combined field surveys, laboratory bioassays, molecular analyses, transcriptomics and functional genomics to investigate insecticide resistance and vector competence in sand flies.
Sand flies were collected from several endemic regions of Tunisia and tested for susceptibility to pyrethroid insecticides using WHO tube bioassays and CDC bottle bioassays. While WHO assays indicated full susceptibility, CDC bottle bioassays identified surviving individuals suitable for molecular investigations. Comparative RNA sequencing between resistant and susceptible sand flies identified numerous differentially expressed genes associated with insecticide resistance, including several cytochrome P450 monooxygenases, notably CYP6A8 and CYP6ACJ, as promising candidate resistance genes. Functional validation is currently being completed through heterologous protein expression, enzymatic activity measurements, insecticide metabolism assays and in vivo validation using transgenic approaches.
To complement the sand fly investigations, mosquito populations were collected from the same Tunisian localities. This work expanded the impact of the project by providing one of the first comprehensive molecular investigations of insecticide resistance in Tunisian mosquito vectors. A total of 210 mosquitoes representing the genera Culex, Aedes and Anopheles were identified using molecular methods and screened for resistance-associated mutations affecting pyrethroids, organophosphates, carbamates and diflubenzuron. The study detected emerging pyrethroid resistance in Culex pipiens and Aedes albopictus, moderate amplification of detoxification genes associated with organophosphate resistance in Aedes albopictus, while no molecular evidence of resistance to diflubenzuron was detected. These findings provide valuable baseline data for insecticide resistance surveillance and support evidence-based vector control in Tunisia. The resulting manuscript has been submitted for publication.
The second objective investigated the molecular determinants of vector competence. Candidate genes potentially involved in Leishmania establishment and transmission were selected. During a four-month research secondment, functional validation experiments were established using RNA interference (RNAi). Double-stranded RNA targeting selected candidate genes was synthesised, injected into sand flies, and gene silencing was confirmed by quantitative PCR. Experimentally infected sand flies were subsequently dissected during the early stages of infection to evaluate the effects of gene silencing on parasite development. These experiments provide a robust framework for identifying genes that influence parasite transmission.
Overall, LeVec generated new transcriptomic resources, identified candidate genes involved in insecticide resistance and vector competence, established functional genomics methodologies for sand flies, and produced important molecular surveillance data for mosquito vectors in Tunisia. The project also strengthened international collaboration through advanced training and technology transfer.
