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Abstract Background The widespread use of pyrethroid insecticides in Africa has led to the development of strong resistance in Anopheles mosquitoes. Introducing new active ingredients can contribute to overcome this phenomenon and ensure the effectiveness of vector control strategies. Transfluthrin is a polyfluorinated pyrethroid whose structural conformation was thought to prevent its metabolism by cytochrome P450 monooxygenases in malaria vectors, thus representing a potential alternative for managing P450-mediated resistance occurring in the field. In this study, a controlled selection was used to compare the dynamics of resistance between transfluthrin and the widely used pyrethroid deltamethrin in the mosquito Anopheles gambiae. Then, the associated molecular mechanisms were investigated using target-site mutation genotyping and RNA-seq. Methods A field-derived line of An. gambiae carrying resistance alleles at low frequencies was used as starting material for a controlled selection experiment. Adult females were selected across 33 generations with deltamethrin or transfluthrin, resulting in three distinct lines: the Delta-R line (selected with deltamethrin), the Transflu-R line (selected with transfluthrin) and the Tiassale-S line (maintained without selection). Deltamethrin and transfluthrin resistance levels were monitored in each selected line throughout the selection process, as well as the frequency of the L1014F kdr mutation. At generation 17, cross-resistance to other public health insecticides was investigated and transcriptomes were sequenced to compare gene transcription variations and polymorphisms associated with adaptation to each insecticide. Results A rapid increase in resistance to deltamethrin and transfluthrin was observed throughout the selection process in each selected line in association with an increased frequency of the L1014F kdr mutation. Transcriptomic data support a broader response to transfluthrin selection as compared to deltamethrin selection. For instance, multiple detoxification enzymes and cuticle proteins were specifically over-transcribed in the Transflu-R line including the known pyrethroid metabolizers CYP6M2, CYP9K1 and CYP6AA1 together with other genes previously associated with resistance in An. gambiae. Conclusion This study confirms that recurrent exposure of adult mosquitoes to pyrethroids in a public health context can rapidly select for various resistance mechanisms. In particular, it indicates that in addition to target site mutations, the polyfluorinated pyrethroid transfluthrin can select for a broad metabolic response, which includes some P450s previously associated to resistance to classical pyrethroids. This unexpected finding highlights the need for an in-depth study on the adaptive response of mosquitoes to newly introduced active ingredients in order to effectively guide and support decision-making programmes in malaria control.

Abstract Mosquitoes represent the most important pathogen vectors and are responsible for the spread of a wide variety of poorly treatable diseases. Wolbachia are obligate intracellular bacteria that are widely distributed among arthropods and collectively represents one of the most promising solutions for vector control. In particular, Wolbachia has been shown to limit the transmission of pathogens, and to dramatically affect the reproductive behavior of their host through its phage WO. While much research has focused on deciphering and exploring the biocontrol applications of these WO-related phenotypes, the extent and potential impact of the Wolbachia mobilome remain poorly appreciated. Notably, several Wolbachia plasmids, carrying WO-like genes and Insertion Sequences (IS), thus possibly interrelated to other genetic units of the endosymbiont, have been recently discovered. Here we investigated the diversity and biogeography of the first described plasmid of Wolbachia in Culex pipiens (pWCP) in several islands and continental countries around the world—including Cambodia, Guadeloupe, Martinique, Thailand, and Mexico—together with mosquito strains from colonies that evolved for 2 to 30 years in the laboratory. We used PCR and qPCR to determine the presence and copy number of pWCP in individual mosquitoes, and highly accurate Sanger sequencing to evaluate potential variations. Together with earlier observation, our results show that pWCP is omnipresent and strikingly conserved among Wolbachia populations within mosquitoes from distant geographies and environmental conditions. These data suggest a critical role for the plasmid in Wolbachia ecology and evolution, and the potential of a great tool for further genetic dissection and possible manipulation of this endosymbiont.

Abstract The introduction of neonicotinoids for managing insecticide resistance in mosquitoes is of high interest as they interact with a biochemical target not previously used in public health. In this concern, Bayer developed a combination of the neonicotinoid clothianidin and the pyrethroid deltamethrin (brand name Fludora Fusion) as a new vector control tool. Although this combination proved to be efficient against pyrethroid-resistant mosquitoes, its ability to prevent the selection of pyrethroid and neonicotinoid resistance alleles was not investigated. In this context, the objective of this work was to study the dynamics and the molecular mechanisms of resistance of An. gambiae to the separated or combined components of this combination. A field-derived An. gambiae line carrying resistance alleles to multiple insecticides at low frequencies was used as a starting for 33 successive generations of controlled selection. Resistance levels to each insecticide and target site mutation frequencies were monitored throughout the selection process. Cross resistance to other public health insecticides were also investigated. RNA-seq was used to compare gene transcription variations and polymorphisms across all lines. This study confirmed the potential of this insecticide combination to impair the selection of resistance as compared to its two separated components. Deltamethrin selection led to the rapid enrichment of the kdr L1014F target-site mutation. Clothianidin selection led to the over-transcription of multiple cytochrome P450s including some showing high homology with those conferring neonicotinoid resistance in other insects. A strong selection signature associated with clothianidin selection was also observed on a P450 gene cluster previously associated with resistance. Within this cluster, the gene CYP6M1 showed the highest selection signature together with a transcription profile supporting a role in clothianidin resistance. Modelling the impact of point mutations selected by clothianidin on CYP6M1 protein structure showed that selection retained a protein variant with a modified active site potentially enhancing clothianidin metabolism. In the context of the recent deployment of neonicotinoids for mosquito control and their frequent usage in agriculture, the present study highlights the benefit of combining them with other insecticides for preventing the selection of resistance and sustaining vector control activities.

Abstract Background Mosquito vectors cause a significant human public health burden through the transmission of pathogens. Due to the expansion of international travel and trade, the dispersal of these mosquito vectors and the pathogens they carry is on the rise. Entomological surveillance is therefore required which relies on accurate mosquito species identification. This study aimed to optimize the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for mosquito identification. Methods Aedes aegypti of the Bora-Bora strain and 11 field-sampled mosquito species were used in this study. Analyses were performed to study the impact of the trapping duration on mosquito identification with MALDI-TOF MS. The best preservation methods to use for short, medium and long-term preservation before MALDI-TOF MS analysis were also assessed. In addition, the number of specimens per species required for MALDI-TOF MS database creation was determined. The first MALDI-TOF database of New Caledonian mosquitoes was assembled and the optimal threshold for mosquito species identification according to the sensitivity and specificity of this technique was determined. Results This study showed that the identification scores decreased as the trapping duration increased. High identification scores were obtained for mosquitoes preserved on silica gel and cotton at room temperature and those frozen at − 20 °C, even after two months of preservation. In addition, the results showed that the scores increased according to the number of main spectrum patterns (MSPs) used until they reached a plateau at 5 MSPs for Ae. aegypti. Mosquitoes (n = 67) belonging to 11 species were used to create the MALDI-TOF reference database. During blind test analysis, 96% of mosquitoes tested (n = 224) were correctly identified. Finally, based on MALDI-TOF MS sensitivity and specificity, the threshold value of 1.8 was retained for a secure identification score. Conclusions MALDI-TOF MS allows accurate species identification with high sensitivity and specificity and is a promising tool in public health for mosquito vector surveillance.

BackgroundBiological control programs involving Wolbachia-infected Aedes aegypti are currently deployed in different epidemiological settings. New Caledonia (NC) is an ideal location for the implementation and evaluation of such a strategy as the only proven vector for dengue virus (DENV) is Ae. aegypti and dengue outbreaks frequency and severity are increasing. We report the generation of a NC Wolbachia-infected Ae. aegypti strain and the results of experiments to assess the vector competence and fitness of this strain for future implementation as a disease control strategy in Noumea, NC.Methods/principal findingsThe NC Wolbachia strain (NC-wMel) was obtained by backcrossing Australian AUS-wMel females with New Caledonian Wild-Type (NC-WT) males. Blocking of DENV, chikungunya (CHIKV), and Zika (ZIKV) viruses were evaluated via mosquito oral feeding experiments and intrathoracic DENV challenge. Significant reduction in infection rates were observed for NC-wMel Ae. aegypti compared to WT Ae. aegypti. No transmission was observed for NC-wMel Ae. aegypti. Maternal transmission, cytoplasmic incompatibility, fertility, fecundity, wing length, and insecticide resistance were also assessed in laboratory experiments. Ae. aegypti NC-wMel showed complete cytoplasmic incompatibility and a strong maternal transmission. Ae. aegypti NC-wMel fitness seemed to be reduced compared to NC-WT Ae. aegypti and AUS-wMel Ae. aegypti regarding fertility and fecundity. However further experiments are required to assess it accurately.Conclusions/significanceOur results demonstrated that the NC-wMel Ae. aegypti strain is a strong inhibitor of DENV, CHIKV, and ZIKV infection and prevents transmission of infectious viral particles in mosquito saliva. Furthermore, our NC-wMel Ae. aegypti strain induces reproductive cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, supporting field-releases in Noumea, NC.