Development and field-evaluation of vector surveillance tools for identifying arbovirus circulation, mosquito species and insecticide resistance

Vector surveillance builds the foundation for effective planning of mosquito-borne disease prevention. Data, on the species composition of vector populations alongside their pathogen infection and insecticide resistance status, are crucial for informed decisions on vector control strategies. The overarching goal of this PhD thesis was to contribute to novel and improved vector surveillance tools for the characterisation of mosquito field populations.
Continental Europe is facing an emerging threat from mosquito-transmitted viral diseases as several locally transmitted recent outbreaks have demonstrated. The main obstacles for launching large-scale arbovirus surveillance in Switzerland are the laborious processing of thousands of mosquitoes, the need for a constant cold chain to preserve viral RNA in infected specimens and the high costs associated with these factors. An alternative approach is using honey-baited nucleic acid preserving cards (FTA cards) to sample saliva from potentially infectious mosquitoes that feed on the sugar bait. During my PhD, I evaluated honey-baited FTA cards as an innovative method for detecting arbovirus circulation in mosquito field populations (Part I).
The importance of early warning through mosquito surveillance is equally applicable in the African context, where the raise and rapid spread of insecticide resistance threatens the success of malaria control programmes. Molecular diagnostics is increasingly important for effective insecticide resistance management strategies. Currently, several highly complex protocols for detecting molecular resistance mechanisms in malaria vectors exist. To facilitate the use of molecular diagnostics in endemic countries, I was engaged in the Diagnosis Management Communication-Malaria Vector Control (DMC-MALVEC) project that aims to develop an automated sample-to-answer diagnostic tool (LabDisk) to screen Anopheles gambiae s.l. mosquitoes simultaneously for pathogen infection and insecticide resistance markers as well as their species composition (Part II).
Part I – Surveillance of arboviruses using honey-baited FTA cards
The objective of Chapter 2 was to evaluate whether honey-baited FTA cards, in combination with an efficient mosquito trap, are a sufficiently sensitive tool to detect virus circulation in an area of low arbovirus prevalence. In a field trial in southern Switzerland, we measured the efficacy of three mosquito traps that were equipped with honey-baited FTA cards, and we found that the Box gravid trap caught 11× more target specimens than the BG-GAT and the BG-Sentinel 2 trap. Screening a total of 507 Aedes and 500 Culex females as well as 240 FTA cards for the presence of arboviruses, we detected Usutu virus in a pool of six Culex pipiens/torrentium mosquitoes and on the FTA card from the same trap. In conclusion, we found honey-baited FTA cards in combination with the Box gravid trap to be a highly sensitive early-warning tool for mosquito-borne virus circulation.
Part II – Surveillance of malaria vectors and insecticide resistance
The second part of this PhD thesis aimed at developing and optimising (RT-)qPCR assays that meet the technical requirements of the Anopheles gambiae LabDisk and the associated reader. The assays were then validated using field-collected specimens and compared with transcriptome sequencing with a particular focus on detecting genes involved in insecticide resistance.
In Chapter 3, we developed a method for estimating the allele frequency of knockdown resistance (kdr) mutations in a pool of ten Anopheles mosquitoes. Linear regression models can predict the allelic frequency in pooled specimens as a function of the ΔCt values (Ct mutant – Ct wild type probe) with comparable accuracy to genotyping individuals. In Chapter 4, we developed one-step multiplex RT-qPCR assays de novo to quantify expression levels of seven cytochrome P450-dependent monooxygenase (P450), including CYP6M2, CYP6P4, CYP6P3, CYP6P1, CYP6Z1, CYP9K1 and CYP4G16, and the glutathione S-transferase GSTE2. All (RT-)qPCR assays for detecting target-site (Chapter 3) and metabolic (Chapter 4) insecticide resistance are efficient, sensitive, specific and reproducible using the same chemistry and thermal cycle parameters. This qualifies them for integration into the sophisticated Anopheles gambiae LabDisk or into simpler malaria vector diagnostic kits with lyophilised reagents that can be used on conventional qPCR instruments.
In Chapter 5, we successfully applied the new (RT-)qPCR assays on multi-insecticide resistant malaria vectors collected in southern Côte d’Ivoire. In this study, we found that highly pyrethroid resistant Anopheles coluzzii from Tiassalé were still fully susceptible to malathion, despite high frequencies of the acetylcholinesterase mutation Ace1-G280S that would typically confer malathion resistance. By sequencing the transcriptomes of individual mosquitoes, we found numerous P450s that were highly overexpressed. This could indicate negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate the pro-insecticide malathion, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathione S-transferases, and other genes putatively involved in insecticide resistance and highlighted several candidates for functional validation.
In conclusion, my PhD projects contributed to the development, evaluation and implementation of innovative, flexible and reliable vector surveillance tools that describe relevant characteristics of mosquito field populations. In particular, I contributed (I) to the implementation of honey-baited FTA cards as a novel arbovirus surveillance tool in Switzerland; and (II) to improved malaria vector diagnostics with novel protocols for more effective and efficient surveillance of molecular insecticide resistance mechanisms.