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Background: The mosquito Culex annulirostris Skuse (Diptera: Culicidae) is an important arbovirus vector in Australasia. It is part of the Culex sitiens subgroup that also includes Cx. palpalis and Cx. sitiens. Single locus mitochondrial and nuclear DNA sequencing studies suggest that Cx. annulirostris consists of a complex of at least two species. We tested this hypothesis by analysing both nuclear microsatellite data and additional mitochondrial sequence data to describe the population genetics of Cx. annulirostris through Australia, Papua New Guinea (PNG) and the Solomon Archipelago., Methods: Twelve novel microsatellite markers for Cx. annulirostris were developed and used on over 500 individuals identified as Cx. annulirostris by molecular diagnostics. Ten of the 12 microsatellites then used for analysis using Discriminant Analysis of Principal Components, a Bayesian clustering software, STRUCTURE, along with estimates of Jost's D statistic that is similar to F ST but better suited to microsatellite data. Mitochondrial cytochrome oxidase I (COI) DNA sequence were also generated complementing previous work and analysed for sequence diversity (Haplotype diversity, Hd and Pi, π), Tadjima's D, and pairwise F ST between populations. An allele specific molecular diagnostic with an internal control was developed., Results: We confirm the existence of multiple genetically and geographically restricted populations. Within mainland Australia, our findings show that Cx. annulirostris consists of two genetically and geographically distinct populations. One population extends through northern Australia and into the south-east coast of Queensland and New South Wales (NSW). The second Australian population occurs through inland NSW, Victoria, South Australia, extending west to southern Western Australia. These two Australian populations show evidence of possible admixture in central Australia and far north Queensland. Australia's Great Dividing Range that runs down southeast Australia presents a strong gene-flow barrier between these two populations which may be driven by climate, elevation or river basins. In PNG we find evidence of reproductive isolation between sympatric cryptic species occurring through PNG and Australia's northern Cape York Peninsula. A PCR-based molecular diagnostic was developed to distinguish these two cryptic species., Conclusion: This study adds to the growing body of work suggesting that the taxon presently known as Cx. annulirostris now appears to consist of at least two cryptic species that co-occur in northern Australia and New Guinea and can be distinguished by a ITS1 PCR diagnostic. The Solomon Islands population may also represent a distinct species but in light its geographic isolation and lack of sympatry with other species would require further study. Additionally, the mitochondrial and nuclear DNA evidence of population structure between geographic regions within Australia appears latitudinal and elevational driven and may suggest an additional subspecies in that hybridise where they overlap., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: Not applicable., (© 2024. The Author(s).)

Indoor insecticide applications are the primary tool for reducing malaria transmission in the Solomon Archipelago, a region where Anopheles farauti is the only common malaria vector. Due to the evolution of behavioural resistance in some An. farauti populations, these applications have become less effective. New malaria control interventions are therefore needed in this region, and gene-drives provide a promising new technology. In considering developing a population-specific (local) gene-drive in An. farauti, we detail the species' population genetic structure using microsatellites and whole mitogenomes, finding many spatially confined populations both within and between landmasses. This strong population structure suggests that An. farauti would be a useful system for developing a population-specific, confinable gene-drive for field release, where private alleles can be used as Cas9 targets. Previous work on Anopheles gambiae has used the Cardinal gene for the development of a global population replacement gene-drive. We therefore also analyse the Cardinal gene to assess whether it may be a suitable target to engineer a gene-drive for the modification of local An. farauti populations. Despite the extensive population structure observed in An. farauti for microsatellites, only one remote island population from Vanuatu contained fixed and private alleles at the Cardinal locus. Nonetheless, this study provides an initial framework for further population genomic investigations to discover high-frequency private allele targets in localized An. farauti populations. This would enable the development of gene-drive strains for modifying localised populations with minimal chance of escape and may provide a low-risk route to field trial evaluations., (© 2024. The Author(s).)

During preliminary mosquito surveys at Cowley Beach Training Area in north Queensland, Australia, it was found that the utility of the standard encephalitis virus surveillance (EVS) trap for collecting the malaria vector Anopheles farauti (Laveran) adults was compromised by the harsh tropical conditions. With the aim of increasing the survival rate of mosquitoes, we designed a downdraft fan box trap (FBT) that incorporated a screened fan at the bottom of the trap, so mosquitoes did not have to pass through a fan. The FBT was tested against the EVS and Centers for Disease Control (CDC) light traps, where mosquitoes do pass through a fan, and a nonpowered passive box trap (PBT). We conducted 4 trials to compare the quantity and survival of An. farauti and culicine mosquitoes were collected in these traps. Although not significant, the FBT collected more An. farauti than the EVS trap and PBT and significantly less An. farauti than the CDC light trap. However, the FBT improved on the CDC light trap in terms of the survival of An. farauti adults collected, with a significantly higher percentage alive in the FBT (74.6%) than in the CDC light trap (27.5%). Thus, although the FBT did not collect as many anophelines as the CDC, it proved to be superior to current trap systems for collecting large numbers of live and relatively undamaged mosquitoes. Therefore, it is recommended that FBTs be used for collecting An. farauti adults in northern Australia, especially when high survival and sample quality are important., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Incompatible insect technique (IIT) is a population suppression approach based on the release of males with manipulated Wolbachia infection inducing egg inviability in wild females. We here present results of multiple field releases of incompatible ARwP males carried out in 2019 in a 2.7-ha green area within urban Rome (Italy) to assess the effect on Aedes albopictus egg viability. Data are compared with results obtained in 2018, when the approach was tested for the first time in Europe., Results: An average of 4674 ARwP males were released weekly for 7 weeks, resulting in a mean ARwP:wild male ratio of 1.1:1 (versus 0.7:1 in 2018). Egg-viability dynamics in ovitraps significantly varied between treated and control sites, with an estimated overall reduction of 35% (versus 15% in 2018). The estimated proportion of females classified as mated with ARwP males was 41.8% and the viability rate of eggs laid by these females (9.5%) was on average significantly lower than that of females only mated with wild males (87.8%); however, high variability in fertility was observed. Values of ARwP male competitiveness were 0.36 and 0.73 based on the overall viability rate of eggs in ovitraps and on female fertility, respectively; thus, well above the conventional 0.2 threshold for an effective suppressive impact in the field., Conclusions: Results further support the potential of IIT as a tool to contribute to Ae. albopictus control in the urban context, stressing the need for larger field trials to evaluate the cost-efficacy of the approach in temperate regions. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Background: With no effective drugs or widely available vaccines, dengue control in Bangladesh is dependent on targeting the primary vector Aedes aegypti with insecticides and larval source management. Despite these interventions, the dengue burden is increasing in Bangladesh, and the country experienced its worst outbreak in 2019 with 101 354 hospitalized cases. This may be partially facilitated by the presence of intense insecticide resistance in vector populations. Here, we describe the intensity and mechanisms of resistance to insecticides commonly deployed against Ae. aegypti in Dhaka, Bangladesh., Results: Dhaka Ae. aegypti colonies exhibited high-intensity resistance to pyrethroids. Using CDC bottle assays, we recorded 2-24% mortality (recorded at 24 h) to permethrin and 48-94% mortality to deltamethrin, at 10× the diagnostic dose. Bioassays conducted using insecticide-synergist combinations suggested that metabolic mechanisms were contributing to pyrethroid resistance, specifically multi-function oxidases, esterases, and glutathione S-transferases. In addition, kdr alleles were detected, with a high frequency (78-98%) of homozygotes for the V1016G mutation. A large proportion (≤ 74%) of free-flying and resting mosquitoes from Dhaka colonies survived exposure to standard applications of pyrethroid aerosols in an experimental free-flight room. Although that exposure affected the immediate host-seeking behavior of Ae. aegypti, the effect was transient in surviving mosquitoes., Conclusion: The intense resistance characterized in this study is likely compromising the operational effectiveness of pyrethroids against Ae. aegypti in Dhaka. Switching to alternative chemical classes may offer a medium-term solution, but ultimately a more sustainable and effective approach to controlling dengue vectors is required. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

The dengue mosquito, Aedes aegypti (Linnaeus, 1762), is a highly invasive and medically significant vector of dengue, yellow fever, chikungunya and Zika viruses, whose global spread can be attributed to increased globalization in the 15th through 20th century. Records of the invasion history of Ae. aegypti across Southeast Asia are sparse and there is little knowledge regarding the invasion routes that the species exploited to gain a foothold in the Indo-Pacific. Likewise, a broad and geographically thorough investigation of Ae. aegypti population genetics in the Indo-Pacific is lacking, despite this region being highly impacted by diseases transmitted by this species. We assess 11 nuclear microsatellites and mitochondrial COI sequences, coupled with widespread sampling through the Indo-Pacific region to characterise population structure at a broad geographic scale. We also perform a comprehensive literature search to collate documentation of the first known records of Ae. aegypti at various locations in the Indo-Pacific. We revealed additional spatial population genetic structure of Ae. aegypti in Southeast Asia, the Indo-Pacific and Australasia compared with previous studies and find differentiation between multiple Queensland and Torres Strait Islands populations. We also detected additional genetic breaks within Australia, Indonesia and Malaysia. Characterising the structure of previously unexplored populations through this region enhances the understanding of the population structure of Ae. aegypti in Australasia and Southeast Asia and may assist predictions of future mosquito movement, informing control strategies as well as assessing the risk of new invasion pathways., Competing Interests: Authors have no conflict of interest., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Background: Anopheles farauti is one of the major vectors of malaria in the Southwest Pacific region and is responsible for past outbreaks in Australia. With an adaptable biting profile conducive to behavioural resistance to indoor residual spraying (IRS) and insecticide-treated nets (ITNs), its all-night biting behaviour can switch to biting mostly in the early evening. With limited insight into the biting profile of An. farauti populations in areas that have not encountered IRS or ITNs, the aim of this study was to develop insights on the biting behaviour of a malaria control naive population of An. farauti., Methods: Biting profiles of An. farauti were conducted at Cowley Beach Training Area, in north Queensland, Australia. Initially, encephalitis virus surveillance (EVS) traps were used to document the 24-h biting profile of An. farauti and then human landing collections (HLC) were used to follow the 18.00-06.00 h biting profile. The human landing catches (HLC) were performed at both the end of the wet (April) and dry (October) seasons., Results: Data exploration using a Random Forest Model shows that time of night is the most important variable for predicting An. farauti biting activity. Temperature was found to be the next important predictor, followed by humidity, trip, collector, and season. The significant effect of time of night and peak in time of night biting, between 19.00 and 20.00 h was also observed in a generalized linear model. The main effect of temperature was significant and non-linear and appears to have a positive effect on biting activity. The effect of humidity is also significant but its relationship with biting activity is more complex. This population's biting profile is similar to populations found in other parts of its range prior to insecticide intervention. A tight timing for the onset of biting was identified with more variation with the end of biting, which is likely underpinned by an endogenous circadian clock rather than any light intensity., Conclusion: This study sees the first record of a relationship between biting activity and the decreasing temperature during the night for the malaria vector, Anopheles farauti., (© 2023. Crown.)

Biological control of mosquito vectors using the endosymbiotic bacteria Wolbachia is an emerging strategy for the management of human arboviral diseases. We recently described the development of a strain of Aedes aegypti infected with the Wolbachia strain wAlbB (referred to as the wAlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a wAlbB infected Ae. aegypti strain from the USA. Field releases of male wAlbB2-F4 mosquitoes resulted in the successful suppression of wild populations of mosquitoes in the trial sites by exploiting the strain's Wolbachia-induced cytoplasmic incompatibility. We now demonstrate that the strain is resistant to infection by dengue and Zika viruses and is genetically similar to endemic Queensland populations. There was a fourfold reduction in the proportion of wAlbB2-F4 mosquitoes that became infected following a blood meal containing dengue 2 virus (16.7%) compared to wild type mosquitoes (69.2%) and a 6-7 fold reduction in the proportion of wAlbB2-F4 mosquitoes producing virus in saliva following a blood meal containing an epidemic strain of Zika virus (8.7% in comparison to 58.3% in wild type mosquitoes). Restriction-site Associated DNA (RAD) sequencing revealed that wAlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the wAlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the wAlbB2-F4 strain retains the insecticide susceptible phenotype and genotype of native Australian mosquitoes. We demonstrate that the Wolbachia wAlbB2-F4, in addition to being suitable for population suppression programs, can also be effective in population replacement programs given its inhibition of virus infection in mosquitoes. The ease at which a target mosquito population can be transfected with wAlbB2, while retaining the genotypes and phenotypes of the target population, shows the utility of this strain for controlling the Ae. aegypti mosquitoes and the pathogens they transmit., Competing Interests: The authors have declared that no competing interests exist.

We investigate the genetic basis of anthropophily (human host use) in a non-model mosquito species group, the Anopheles farauti complex from the southwest Pacific. This complex has experienced multiple transitions from anthropophily to zoophily, contrasting with well-studied systems (the global species Aedes aegypti and the African Anopheles gambiae complex) that have evolved to be specialist anthropophiles. By performing tests of selection and assessing evolutionary patterns for >200 olfactory genes from nine genomes, we identify several candidate genes associated with differences in anthropophily in this complex. Based on evolutionary patterns (phylogenetic relationships, fixed amino acid differences, and structural differences) as well as results from selection analyses, we identify numerous genes that are likely to play an important role in mosquitoes' ability to detect humans as hosts. Our findings contribute to the understanding of the evolution of insect olfactory gene families and mosquito host preference as well as having potential applied outcomes., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)