Molecular characterization of insecticide resistance and transmission competence in sand flies, major vectors of leismaniasis

Informazioni relative al progetto

LeVec

ID dell’accordo di sovvenzione: 101152599

DOI

10.3030/101152599

Progetto chiuso

Data della firma CE 18 Aprile 2024

Data di avvio 1 Maggio 2024

Data di completamento 30 Aprile 2026

Finanziato da

• Marie Skłodowska-Curie Actions (MSCA)

Costo totale

Nessun dato

Contributo UE

€ 153 486,72

Coordinato da

IDRYMA TECHNOLOGIAS KAI EREVNAS
Greece

Periodic Reporting for period 1 - LeVec (Molecular characterization of insecticide resistance and transmission competence in sand flies, major vectors of leismaniasis)

Periodo di rendicontazione: 2024-05-01 al 2026-04-30

Sintesi del contesto e degli obiettivi generali del progetto

Leishmaniasis is a neglected tropical disease, causing up to one million new infections every year. Transmitted through the bite of infected phlebotomine sand flies. Leishmaniasis is endemic in more than 90 countries across tropical, subtropical and Mediterranean regions. Depending on Leishmania parasite species, leishmaniasis can cause severe skin lesions, destructive mucosal disease or life-threatening visceral infections. Countries in the Eastern Mediterranean region, including Tunisia, continue to experience a substantial disease burden, making effective vector control a public health priority. As no widely accessible human vaccine is currently available, reducing contact between humans and infected sand flies remains the most effective approach to limiting disease transmission. This relies largely on insecticide-based interventions, such as indoor residual spraying, insecticide-treated materials and environmental vector control. However, the intensive use of insecticides in both public health and agriculture is increasing the risk that sand fly populations will develop resistance, potentially reducing the effectiveness of these control measures. Unlike mosquitoes, where resistance mechanisms have been extensively investigated, the biological and molecular processes responsible for insecticide resistance in sand flies are still poorly understood. Another major challenge is that not all sand flies’ species transmit Leishmania parasites. Understanding the molecular interactions that determine vector competence is essential for improving our knowledge of disease transmission and identifying new opportunities for vector control.
The LeVec project addressed these two important knowledge gaps by combining molecular biology, genomics and functional validation approaches. The project investigated the genetic mechanisms associated with pyrethroid insecticide resistance in sand flies collected from Tunisia and explored the molecular factors influencing their ability to transmit Leishmania parasites. By generating new knowledge on both insecticide resistance and vector competence, LeVec contributes to the development of molecular surveillance tools, supports evidence-based vector control programmes and strengthens preparedness against the spread of leishmaniasis in endemic regions. The project also reinforces European and international efforts to combat neglected tropical diseases through innovative, sustainable and science-based vector management strategies.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

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.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

LeVec considerably advances the understanding of insecticide resistance and vector competence in sand flies by combining field ecology, molecular biology, comparative transcriptomics and functional genomics in a disease vector for which molecular information remains limited.
The project identified novel candidate genes associated with pyrethroid resistance, including overexpressed cytochrome P450 monooxygenases, providing new insights into the metabolic mechanisms potentially responsible for reduced insecticide susceptibility. The establishment of functional validation pipelines, including heterologous protein expression, insecticide metabolism assays and RNA interference, represents an important methodological advance that will facilitate future studies of resistance mechanisms in sand flies.
The complementary mosquito study significantly broadened the impact of the project by providing one of the first comprehensive molecular surveys of insecticide resistance markers in Tunisian mosquito populations. The results establish an important baseline for national resistance monitoring programmes, demonstrate emerging pyrethroid resistance in major mosquito vectors, and support the continued effectiveness of diflubenzuron for vector control. Together with the sand fly findings, these results contribute to a better understanding of insecticide resistance across medically important vectors sharing the same environments.
The project's results have direct relevance for public health by supporting the development of molecular tools for resistance surveillance, improving evidence-based insecticide resistance management and contributing to more sustainable integrated vector management programmes in Tunisia, the Mediterranean region and other endemic countries. The generated datasets, candidate molecular markers and experimental methodologies constitute valuable resources for the scientific community and create opportunities for future collaborative research. Continued functional validation of the identified candidate genes, together with their incorporation into routine surveillance programmes, will further enhance the long-term impact and uptake of the project's results.