Mitochondrial and microbial diversity of the invasive mosquito vector species Culex tritaeniorhynchus across its extensive inter-continental geographic range.

Background: Culex (Cx.) tritaeniorhynchus is an invasive mosquito species with an extensive and expanding inter-continental distribution, currently reported across Asia, Africa, the Middle East, Europe and now Australia. It is an important vector of medical and veterinary pathogens which cause significant morbidity and mortality in human and animal populations. Across regions endemic for Japanese encephalitis virus (JEV), Cx. tritaeniorhynchus is considered the major vector and has also been shown to contribute to the transmission of several other zoonotic arboviruses including Rift Valley fever virus (RVFV) and West Nile virus (WNV). Methods: In this study, we used laboratory vector competence experiments to determine if Cx. tritaeniorhynchus from a Southern European population were competent JEV vectors. We also obtained samples from multiple geographically dispersed Cx. tritaeniorhynchus populations from countries within Europe, Africa, Eurasia and Asia to perform phylogenetic analysis to measure the level of mitochondrial divergence using the cytochrome oxidase subunit 1 ( CO1) gene. We also undertook bacterial 16S rRNA gene amplicon sequencing to determine microbial diversity and used multi-locus sequence typing (MLST) to determine any evidence for the presence of strains of the naturally occurring endosymbiotic bacterium Wolbachia. Results: Cx. tritaeniorhynchus from a Greek population were shown be be competent vectors of JEV with high levels of virus present in saliva. We found a signficant level of mitochondrial genetic diversity using the mosquito CO1 gene between geographically dispersed populations. Furthermore, we report diverse microbiomes identified by 16S rRNA gene amplicon sequencing within and between geographical populations. Evidence for the detection of the endosymbiotic bacteria Wolbachia was confirmed using Wolbachia-specific PCR and MLST. Conclusions: This study enhances our understanding of the diversity of Cx. tritaeniorhynchus and the associated microbiome across its inter-continental range and highlights the need for greater surveillance of this invasive vector species in Europe.

The mosquito species Culex (Cx.) tritaeniorhynchus is expanding its range and is now present in over 50 countries across Asia, Africa, the Middle East, Europe and now Australasia. It can transmit human and animal pathogens, resulting in significant morbidity and mortality. This species transmits Japanese encephalitis virus in endemic areas of Asia, and it has also been shown to contribute to the transmission of several other viruses that can infect humans, including Rift Valley fever virus and West Nile virus. In this study, we firstly undertook some lab experiments to show that Cx. tritaeniorhynchus from a Southern European population are competent vectors of Japanese encephalitis virus. We also obtained field mosquitoes from countries within Europe, Africa, Eurasia and Asia and used phylogenetic analysis to demonstrate a high level of mitochondrial divergence within and between populations. In addition, we analysed the bacteria present within mosquitoes and found a high level of microbial diversity. Finally, we present evidence for the presence of Wolbachia endosymbiotic bacteria in some populations of this mosquito species. This study highlights the need for greater surveillance of this invasive vector species ­ particularly in Europe.

High-throughput barcoding method for the genetic surveillance of insecticide resistance and species identification in Anopheles gambiae complex malaria vectors.

Surveillance of malaria vector species and the monitoring of insecticide resistance are essential to inform malaria control strategies and support the reduction of infections and disease. Genetic barcoding of mosquitoes is a useful tool to assist the high-throughput surveillance of insecticide resistance, discriminate between sibling species and to detect the presence of Plasmodium infections. In this study, we combined multiplex PCR, custom designed dual indexing, and Illumina next generation sequencing for high throughput single nucleotide polymorphism (SNP)-profiling of four species from the Anopheles (An.) gambiae complex (An. gambiae sensu stricto, An. coluzzii, An. arabiensis and An. melas). By amplifying and sequencing only 14 genetic fragments (500 bp each), we were able to simultaneously detect Plasmodium infection; insecticide resistance-conferring SNPs in ace1, gste2, vgsc and rdl genes; the partial sequences of nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and intergenic spacers (IGS), Short INterspersed Elements (SINE), as well as mitochondrial genes (cox1 and nd4) for species identification and genetic diversity. Using this amplicon sequencing approach with the four selected An. gambiae complex species, we identified a total of 15 non-synonymous mutations in the insecticide target genes, including previously described mutations associated with resistance and two new mutations (F1525L in vgsc and D148E in gste2). Overall, we present a reliable and cost-effective high-throughput panel for surveillance of An. gambiae complex mosquitoes in malaria endemic regions.

Evidence of extrinsic factors dominating intrinsic blood host preferences of major African malaria vectors.

One of the key determinants of a haematophagous vector's capacity to transmit pathogens is its selection of which host to secure a blood meal from. This choice is influenced by both intrinsic (genetic) and extrinsic (environmental) factors, but little is known of their relative contributions. Blood fed Anopheles mosquitoes were collected from a malaria endemic village in Ghana. Collections were conducted across a range of different host availabilities and from both indoor and outdoor locations. These environmental factors were shown to impact dramatically the host choice of caught malaria vectors: mosquitoes caught indoors were ten-fold more likely to have sourced their blood meal from humans; and a halving in odds of being human-fed was found for mosquitoes caught only 25 m from the centre of the village. For the first time, we demonstrate that anthropophagy was better explained by extrinsic factors (namely, local host availability and indoor/outdoor trapping location) than intrinsic factors (namely, the (sibling) species of the mosquito caught) (respective Akaike information criterion estimates: 243.0 versus 359.8). Instead of characterizing biting behaviour on a taxonomic level, we illustrate the importance of assessing local entomology. Accounting for this behavioural plasticity is important, both in terms of measuring effectiveness of control programmes and in informing optimal disease control strategies.

The relationship between insecticide resistance, mosquito age and malaria prevalence in Anopheles gambiae s.l. from Guinea.

Insecticide resistance across sub-Saharan Africa may impact the continued effectiveness of malaria vector control. We investigated the association between carbamate and pyrethroid resistance with Anopheles gambiae s.l. parity, Plasmodium falciparum infection, and molecular insecticide resistance mechanisms in Guinea. Pyrethroid resistance was intense, with field populations surviving ten times the insecticidal concentration required to kill susceptible individuals. The L1014F kdr-N1575Y haplotype and I1527T mutation were significantly associated with mosquito survival following permethrin exposure (Prevalence Ratio; PR = 1.92, CI = 1.09-3.37 and PR = 2.80, CI = 1.03-7.64, respectively). Partial restoration of pyrethroid susceptibility following synergist pre-exposure suggests a role for mixed-function oxidases. Carbamate resistance was lower and significantly associated with the G119S Ace-1 mutation. Oocyst rates were 6.8% and 4.2% among resistant and susceptible mosquitoes, respectively; survivors of bendiocarb exposure were significantly more likely to be infected. Pyrethroid resistant mosquitoes had significantly lower parity rates than their susceptible counterparts (PR = 1.15, CI = 1.10-1.21). Our findings emphasize the need for additional studies directly assessing the influence of insecticide resistance on mosquito fitness.

Correction: Alternative strategies for mosquito-borne arbovirus control.

[This corrects the article DOI: 10.1371/journal.pntd.0006822.].

Investigating the blood-host plasticity and dispersal of Anopheles coluzzii using a novel field-based methodology.

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 < 0.01) and correspondingly, an increasing trend in Human Blood Index (OR = 1.50 95% CI: 1.05-2.16, P = 0.028) as the transect approached the village. Using qPCR, the host DNA remaining in the blood meal was quantified for field-caught mosquitoes and calibrated according to timed blood digestion in colony mosquitoes. Time since blood meal was consumed and the corresponding distance the vector was caught from its blood-host allowed the estimation of An. coluzzii dispersal rates. Within 7 hours of feeding, mosquitoes typically remained within 50 m of their blood-host but at 60 hours they had dispersed up to 250 m. CONCLUSIONS: Using this methodology the remarkably small spatial scale at which An. coluzzii blood-host choice can change was demonstrated. In addition, conducting qPCR on host blood from field-caught mosquitoes and calibrating with timed experiments with colonised mosquitoes presents a novel methodology for investigating the dispersal behaviour of vectors. Future adaptations to this novel method to make it broadly applicable to other types of setting are also discussed.

Alternative strategies for mosquito-borne arbovirus control.

BACKGROUND: Mosquito-borne viruses-such as Zika, chikungunya, dengue fever, and yellow fever, among others-are of global importance. Although vaccine development for prevention of mosquito-borne arbovirus infections has been a focus, mitigation strategies continue to rely on vector control. However, vector control has failed to prevent recent epidemics and arrest expanding geographic distribution of key arboviruses, such as dengue. As a consequence, there has been increasing necessity to further optimize current strategies within integrated approaches and advance development of alternative, innovative strategies for the control of mosquito-borne arboviruses. METHODS AND FINDINGS: This review, intended as a general overview, is one of a series being generated by the Worldwide Insecticide resistance Network (WIN). The alternative strategies discussed reflect those that are currently under evaluation for public health value by the World Health Organization (WHO) and represent strategies of focus by globally recognized public health stakeholders as potential insecticide resistance (IR)-mitigating strategies. Conditions where these alternative strategies could offer greatest public health value in consideration of mitigating IR will be dependent on the anticipated mechanism of action. Arguably, the most pressing need for endorsement of the strategies described here will be the epidemiological evidence of a public health impact. CONCLUSIONS: As the burden of mosquito-borne arboviruses, predominately those transmitted by Aedes aegypti and A. albopictus, continues to grow at a global scale, new vector-control tools and integrated strategies will be required to meet public health demands. Decisions regarding implementation of alternative strategies will depend on key ecoepidemiological parameters that each is intended to optimally impact toward driving down arbovirus transmission.

Using the human blood index to investigate host biting plasticity: a systematic review and meta-regression of the three major African malaria vectors.

BACKGROUND: The proportion of mosquito blood-meals that are of human origin, referred to as the 'human blood index' or HBI, is a key determinant of malaria transmission. METHODS: A systematic review was conducted followed by meta-regression of the HBI for the major African malaria vectors. RESULTS: Evidence is presented for higher HBI among Anopheles gambiae (M/S forms and Anopheles coluzzii/An. gambiae sensu stricto are not distinguished for most studies and, therefore, combined) as well as Anopheles funestus when compared with Anopheles arabiensis (prevalence odds ratio adjusted for collection location [i.e. indoor or outdoor]: 1.62; 95% CI 1.09-2.42; 1.84; 95% CI 1.35-2.52, respectively). This finding is in keeping with the entomological literature which describes An. arabiensis to be more zoophagic than the other major African vectors. However, analysis also revealed that HBI was more associated with location of mosquito captures (R2 = 0.29) than with mosquito (sibling) species (R2 = 0.11). CONCLUSIONS: These findings call into question the appropriateness of current methods of assessing host preferences among disease vectors and have important implications for strategizing vector control.

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa.

Background:  Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the  Anopheles (An.) genera, but has recently been found in  An. gambiae s.l. populations in West Africa.  As there are numerous  Anopheles species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine  Wolbachia prevalence rates, characterise novel  Wolbachia strains and determine any correlation between the presence of  Plasmodium,  Wolbachia and the competing bacterium  Asaia. Methods:  Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017.  Molecular analysis was undertaken using quantitative PCR, Sanger sequencing,  Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial  16S rRNA gene.  Results: Novel  Wolbachia strains were discovered in five species:  An. coluzzii,  An. gambiae s.s.,  An. arabiensis,  An. moucheti and  An. species A, increasing the number of  Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with  Wolbachia supergroup B strains.  We also provide evidence for resident strain variants within  An. species A.  Wolbachia is the dominant member of the microbiome in  An. moucheti and  An. species A but present at lower densities in  An. coluzzii.  Interestingly, no evidence of  Wolbachia/Asaia co-infections was seen and  Asaia infection densities were shown to be variable and location dependent.  Conclusions: The important discovery of novel  Wolbachia strains in  Anopheles provides greater insight into the prevalence of resident  Wolbachia strains in diverse malaria vectors.  Novel  Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other  Anopheles mosquito species, which could be used for population replacement or suppression control strategies.

Personal Protection of Permethrin-Treated Clothing against Aedes aegypti, the Vector of Dengue and Zika Virus, in the Laboratory.

BACKGROUND: The dengue and Zika viruses are primarily transmitted by Aedes aegypti mosquitoes, which are most active during day light hours and feed both in and outside of the household. Personal protection technologies such as insecticide-treated clothing could provide individual protection. Here we assessed the efficacy of permethrin-treated clothing on personal protection in the laboratory. METHODS: The effect of washing on treated clothing, skin coverage and protection against resistant and susceptible Ae. aegypti was assessed using modified WHO arm-in-cage assays. Coverage was further assessed using free-flight room tests to investigate the protective efficacy of unwashed factory-dipped permethrin-treated clothing. Clothing was worn as full coverage (long sleeves and trousers) and partial coverage (short sleeves and shorts). Residual permethrin on the skin and its effect on mosquitoes was measured using modified WHO cone assays and quantified using high-pressure liquid chromatography (HPLC) analysis. RESULTS: In the arm-in-cage assays, unwashed clothing reduced landing by 58.9% (95% CI 49.2-66.9) and biting by 28.5% (95% CI 22.5-34.0), but reduced to 18.5% (95% CI 14.7-22.3) and 11.1% (95% CI 8.5-13.8) respectively after 10 washes. Landing and biting for resistant and susceptible strains was not significantly different (p<0.05). In free-flight room tests, full coverage treated clothing reduced landing by 24.3% (95% CI 17.4-31.7) and biting by 91% (95% CI 82.2-95.9) with partial coverage reducing landing and biting by 26.4% (95% CI 20.3-31.2) and 49.3% (95% CI 42.1-59.1) respectively with coverage type having no significant difference on landing (p<0.05). Residual permethrin was present on the skin in low amounts (0.0041mg/cm2), but still produced a KD of >80% one hour after wearing treated clothing. CONCLUSION: Whilst partially covering the body with permethrin-treated clothing provided some protection against biting, wearing treated clothing with long sleeves and trousers provided the highest form of protection. Washing treated clothing dramatically reduced protection provided. Permethrin-treated clothing could provide protection to individuals from Ae. aegypti that show permethrin resistance. Additionally, it could continue to provide protection even after the clothing has been worn. Field trials are urgently needed to determine whether clothing can protect against dengue and Zika.

Permethrin-Treated Clothing as Protection against the Dengue Vector, Aedes aegypti: Extent and Duration of Protection.

INTRODUCTION: Dengue transmission by the mosquito vector, Aedes aegypti, occurs indoors and outdoors during the day. Personal protection of individuals, particularly when outside, is challenging. Here we assess the efficacy and durability of different types of insecticide-treated clothing on laboratory-reared Ae. aegypti. METHODS: Standardised World Health Organisation Pesticide Evaluation Scheme (WHOPES) cone tests and arm-in-cage assays were used to assess knockdown (KD) and mortality of Ae. aegypti tested against factory-treated fabric, home-dipped fabric and microencapsulated fabric. Based on the testing of these three different treatment types, the most protective was selected for further analysis using arm-in cage assays with the effect of washing, ultra-violet light, and ironing investigated using high pressure liquid chromatography. RESULTS: Efficacy varied between the microencapsulated and factory dipped fabrics in cone testing. Factory-dipped clothing showed the greatest effect on KD (3 min 38.1%; 1 hour 96.5%) and mortality (97.1%) with no significant difference between this and the factory dipped school uniforms. Factory-dipped clothing was therefore selected for further testing. Factory dipped clothing provided 59% (95% CI = 49.2%- 66.9%) reduction in landing and a 100% reduction in biting in arm-in-cage tests. Washing duration and technique had a significant effect, with insecticidal longevity shown to be greater with machine washing (LW50 = 33.4) compared to simulated hand washing (LW50 = 17.6). Ironing significantly reduced permethrin content after 1 week of simulated use, with a 96.7% decrease after 3 months although UV exposure did not reduce permethrin content within clothing significantly after 3 months simulated use. CONCLUSION: Permethrin-treated clothing may be a promising intervention in reducing dengue transmission. However, our findings also suggest that clothing may provide only short-term protection due to the effect of washing and ironing, highlighting the need for improved fabric treatment techniques.