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Wolbachia MLST gene GenBank accession numbers. Wolbachia strain names Wolbachia wsp, and MLST gene sequence Genbank accession numbers.

Wolbachia 16S rRNA gene qRT PCR representative standard curve generated with a synthetic oligonucleotide standard (a) to measure gene copies per μL (b).

BACKGROUND: Resistance to major public health insecticides in Côte d'Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions. METHODS: This study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from Southeast Côte d'Ivoire in 2019. RESULTS: Phenotypic resistance was intense: >25% of mosquitoes survived exposure to 10 times the doses of pyrethroids required to kill susceptible populations. Similarly, the 24-hour mortality rate with deltamethrin-only LLINs was very low and not significantly different from that with an untreated net. Sublethal pyrethroid exposure did not induce significant delayed vector mortality effects 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide, or new insecticides clothianidin and chlorfenapyr, were highly toxic to A. coluzzii. Pyrethroid-susceptible A. coluzzii were significantly more likely to be infected with malaria, compared with those that survived insecticidal exposure. Pyrethroid resistance was associated with significant overexpression of CYP6P4, CYP6P3, and CYP6Z1. CONCLUSIONS: Study findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating piperonyl butoxide, chlorfenapyr, or clothianidin in areas of high resistance intensity in Côte d'Ivoire.

Additional file 2: Figure S1. Environmental data. Temperature (a, c, e), relative humidity (b, d, f) and presence of rain (blue drops) in each study site are shown. Graphs represent the temperature and relative humidity in each sampling point (a-e in Maferinyah Centre One, f-j in Senguelen and k-o in Fandie) across 10 collection intervals (5 days and 5 nights) per site. Note that each interval starts with a night collection followed by a day collection except day 10 in Senguelen, which starts with a day collection due to an interval repetition.

Additional file 1: Table S1. Coordinates and description of the sampling points in Maferinyah Centre One, Senguelen and Fandie. Latitude and longitude were obtained using GPS (eTrex 10, Garmin). Table S2. Number of mosquitoes collected per site, sampling point, time period and trap. Note that 150 collections were performed in total: 50 collections per site (× 3 sites); 30 collections per trap (× 5 traps); 10 collections per sampling point (× 15 sampling points). Table S3. PCR assays. Primers, final volumes and conditions of each PCR assay are shown. Table S4. Species confirmed by molecular analysis. Sequencing, or a combination of sequencing and species-specific end-point PCR were used to confirm species. A representative specimen from each species is shown, with GenBank accession numbers for sequences generated in this study provided. Table S5. Statistical differences between the abundance of mosquitoes captured by the five traps. Table showing the results of the final Generalised Linear Mixed Model: Abundance ~ Site + (1|Point) + Time + Trap for the difference in the abundance of mosquitoes captured by the 5 traps. Table S6. Mosquitoes used for molecular identification. Number and proportion of mosquitoes used for molecular ID within each genus (A), each trap and each site (B).

Background Wolbachia , a widespread bacterium that can influence mosquito-borne pathogen transmission, has recently been shown to infect Anopheles ( An .) species that are malaria vectors in Sub-Saharan Africa. Although there are studies reporting strains in the An. gambiae complex, the apparent low density and low prevalence rates requires further investigation. In contrast, strains in other species appear higher density allowing a greater understanding of phylogenetics and more accurate determination of prevalence rates in wild mosquito populations. Methods Anopheles mosquitoes were collected in the Faranah and Maferinyah regions of Guinea in June-July 2018. RNA was extracted from 542 females and reverse transcribed to determine Wolbachia prevalence rates, demonstrate gene expression and estimate relative strain densities using quantitative PCR. Molecular confirmation of mosquito species and Wolbachia multilocus sequence typing (MLST) was carried out to analyse phylogenetic relationships of newly discovered strains. Results Low prevalence rates were detected in An. gambiae s.s. (0.0: 2.9%) and novel Wolbachia strains were discovered in two species: the w AnMe strain in An. melas (18/168: prevalence rate of 10.7%) and the w AnsX strain in a previously unidentified Anopheles species we have termed ‘ An . species X’ (1/1). Novel strains were phylogenetically diverse, with w AnMe clustering with Wolbachia Supergroup A strains, and w AnsX closest to Supergroup B strains. Significantly higher density strains were present in An. species X and An. melas compared to An. gambiae s.s. and were comparable in density to a novel Wolbachia strain discovered in Culex watti termed w Wat. Conclusions The discovery of novel Wolbachia strains provides further insight into the strain diversity within Anopheles species. Confirmation of gene expression for all MLST and wsp genes for w AnsX and w AnMe strains and qPCR analysis indicates these are candidate strains for transinfection to create stable infections in other Anopheles species. They have the potential for use in Anopheles biocontrol strategies either through population replacement or suppression control strategies.

Background: The Recife Metropolitan Region (RMR), north-eastern Brazil, was the epicentre of the 2015 Zika virus (ZIKV) epidemic, which was followed by a 2016 chikungunya virus (CHIKV) epidemic. It historically has amongst the highest incidence of dengue virus (DENV) infections and is the only remaining focus of lymphatic filariasis (LF) in Brazil. In early 2015, a molecular xenomonitoring surveillance project focused on Culex (Cx.) quinquefasciatus commenced to inform LF elimination activities. Aedes (Ae.) aegypti mosquitoes were also collected, concurrent with the first microcephaly cases detected in the RMR. In terms of the 2015 ZIKV epidemic, these are the earliest known field-collected mosquitoes, preserved for potential RNA virus detection, when ZIKV was known to be circulating locally. Methods: Adult mosquitoes were collected in two sites (0.4 km 2) of Sítio Novo, Olinda, RMR, from July 22 to August 21, 2015. Mosquitoes were morphologically identified, sorted by physiological status, and pooled (up to 10 mosquitoes per house per day or week). RNA was extracted, reverse transcribed and the cDNA tested by real-time PCR. Results: A total of 10,139 adult female Cx. quinquefasciatus and 939 adult female Ae. aegypti were captured. All female Ae. aegypti specimens were included within 156 pools and screened for ZIKV, DENV and CHIKV. In addition, a sub-set of 1,556 Cx. quinquefasciatus adult females in 182 pools were screened for ZIKV. No evidence of infection with any of the three arboviruses was found. Conclusions: The absence of arbovirus detection may have been expected given the extremely restricted geographic area and collection of mosquitoes during a very short time period of peak mosquito abundance (July-September), but low arbovirus circulation (November-March). However, this study demonstrates the potential to retrospectively screen for additional unexpected pathogens in situations of rapid emergence, such as occurred during the outbreak of ZIKV in the RMR.

Background The biting behaviour and dispersal of insect vectors in the field underlies the transmission of many diseases. Here, a novel collection methodology coupled with the molecular analysis of blood-meal sources and digestion rates is introduced with the aim of aiding the understanding of two critical and relatively understudied mosquito behaviours: plasticity in blood-host choice and vector dispersal. Results A collection strategy utilising a transect of mosquito traps placed at 50 m intervals allowed the collection of blood-fed Anopheles coluzzii from a malaria-endemic village of southern Ghana where human host availability ranged from zero (a cattle pen), increasing until humans were the dominant host choice (the middle of the village). Blood-meal analysis using PCR showed statistically significant variation in blood-meal origins for mosquitoes collected across the 250 m transect: with decreasing trend in Bovine Blood Index (OR = 0.60 95% CI: 0.49–0.73, P

Mosquitoes represent the major arthropod vectors of human disease worldwide transmitting malaria, lymphatic filariasis, and arboviruses such as dengue virus and Zika virus. Unfortunately, no treatment (in the form of vaccines or drugs) is available for most of these diseases and vector control is still the main form of prevention. The limitations of traditional insecticide-based strategies, particularly the development of insecticide resistance, have resulted in significant efforts to develop alternative eco-friendly methods. Biocontrol strategies aim to be sustainable and target a range of different mosquito species to reduce the current reliance on insecticide-based mosquito control. In this review, we outline non-insecticide based strategies that have been implemented or are currently being tested. We also highlight the use of mosquito behavioural knowledge that can be exploited for control strategies.