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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 .

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 w AlbB (referred to as the w AlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a w AlbB infected Ae . aegypti strain from the USA. Field releases of male w AlbB2-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 w AlbB2-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 w AlbB2-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 w AlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the w AlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the w AlbB2-F4 strain retains the insecticide susceptible phenotype and genotype of native Australian mosquitoes. We demonstrate that the Wolbachia w AlbB2-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 w AlbB2, 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.

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.

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. Graphical Abstract

Within the last century, increases in human movement and globalization of trade have facilitated the establishment of several highly invasive mosquito species in new geographic locations with concurrent major environmental, economic and health consequences. The Asian tiger mosquito, Aedes albopictus, is an extremely invasive and aggressive daytime-biting mosquito that is a major public health threat throughout its expanding range. We used 13 nuclear microsatellite loci (on 911 individuals) and mitochondrial COI sequences to gain a better understanding of the historical and contemporary movements of Ae. albopictus in the Indo-Pacific region and to characterize its population structure. Approximate Bayesian computation (ABC) was employed to test competing historical routes of invasion of Ae. albopictus within the Southeast (SE) Asian/Australasian region. Our ABC results show that Ae. albopictus was most likely introduced to New Guinea via mainland Southeast Asia, before colonizing the Solomon Islands via either Papua New Guinea or SE Asia. The analysis also supported that the recent incursion into northern Australia's Torres Strait Islands was seeded chiefly from Indonesia. For the first time documented in this invasive species, we provide evidence of a recently colonized population (the Torres Strait Islands) that has undergone rapid temporal changes in its genetic makeup, which could be the result of genetic drift or represent a secondary invasion from an unknown source. There appears to be high spatial genetic structure and high gene flow between some geographically distant populations. The species' genetic structure in the region tends to favour a dispersal pattern driven mostly by human movements. Importantly, this study provides a more widespread sampling distribution of the species' native range, revealing more spatial population structure than previously shown. Additionally, we present the most probable invasion history of this species in the Australasian region using ABC analysis.

The range of the Asian tiger mosquito Aedes albopictus is expanding globally, raising the threat of emerging and re-emerging arbovirus transmission risks including dengue and chikungunya. Its detection in Papua New Guinea's (PNG) southern Fly River coastal region in 1988 and 1992 placed it 150 km from mainland Australia. However, it was not until 12 years later that it appeared on the Torres Strait Islands. We hypothesized that the extant PNG population expanded into the Torres Straits as an indirect effect of drought-proofing the southern Fly River coastal villages in response to El Nino-driven climate variability in the region (via the rollout of rainwater tanks and water storage containers). Examination of the mosquito's mitochondrial DNA cytochrome oxidase I (COI) sequences and 13 novel nuclear microsatellites revealed evidence of substantial intermixing between PNG's southern Fly region and Torres Strait Island populations essentially compromising any island eradication attempts due to potential of reintroduction. However, two genetically distinct populations were identified in this region comprising the historically extant PNG populations and the exotic introduced population. Both COI sequence data and microsatellites showed the introduced population to have genetic affinities to populations from Timor Leste and Jakarta in the Indonesian region. The Ae. albopictus invasion into the Australian region was not a range expansion out of PNG as suspected, but founded by other, genetically distinct population(s), with strong genetic affinities to populations sampled from the Indonesian region. We now suspect that the introduction of Ae. albopictus into the Australian region was driven by widespread illegal fishing activity originating from the Indonesian region during this period. Human sea traffic is apparently shuttling this mosquito between islands in the Torres Strait and the southern PNG mainland and this extensive movement may well compromise Ae. albopictus eradication attempts in this region.

Anopheles hinesorum is a mosquito species with variable host preference. Throughout New Guinea and northern Australia, An. hinesorum feeds on humans (it is opportunistically anthropophagic) while in the south‐west Pacific's Solomon Archipelago, the species is abundant but has rarely been found biting humans (it is exclusively zoophagic in most populations). There are at least two divergent zoophagic (nonhuman biting) mitochondrial lineages of An. hinesorum in the Solomon Archipelago representing two independent dispersals. Since zoophagy is a derived (nonancestral) trait in this species, this leads to the question: has zoophagy evolved independently in these two populations? Or conversely: has nuclear gene flow or connectivity resulted in the transfer of zoophagy? Although we cannot conclusively answer this, we find close nuclear relationships between Solomon Archipelago populations indicating that recent nuclear gene flow has occurred between zoophagic populations from the divergent mitochondrial lineages. Recent work on isolated islands of the Western Province (Solomon Archipelago) has also revealed an anomalous, anthropophagic island population of An. hinesorum. We find a common shared mitochondrial haplotype between this Solomon Island population and another anthropophagic population from New Guinea. This finding suggests that there has been recent migration from New Guinea into the only known anthropophagic population from the Solomon Islands. Although currently localized to a few islands in the Western Province of the Solomon Archipelago, if anthropophagy presents a selective advantage, we may see An. hinesorum emerge as a new malaria vector in a region that is now working on malaria elimination.

New Guinea is a topographically and biogeographically complex region that supports unique endemic fauna. Studies describing the population connectivity of species through this region are scarce. We present a population and landscape genetic study on the endemic malaria‐transmitting mosquito, Anopheles koliensis (Owen). Using mitochondrial and nuclear sequence data, as well as microsatellites, we show the evidence of geographically discrete population structure within Papua New Guinea (PNG). We also confirm the existence of three rDNA ITS2 genotypes within this mosquito and assess reproductive isolation between individuals carrying different genotypes. Microsatellites reveal the clearest population structure and show four clear population units. Microsatellite markers also reveal probable reproductive isolation between sympatric populations in northern PNG with different ITS2 genotypes, suggesting that these populations may represent distinct cryptic species. Excluding individuals belonging to the newly identified putative cryptic species (ITS2 genotype 3), we modeled the genetic differences between A. koliensis populations through PNG as a function of terrain and find that dispersal is most likely along routes with low topographic relief. Overall, these results show that A. koliensis is made up of geographically and genetically discrete populations in Papua New Guinea with landscape topography being important in restricting dispersal. In a population genetic study of the malaria mosquito Anopheles koliensis through Papua New Guinea (PNG), we use ribosomal DNA genotyping, nuclear microsatellites, along with mitochondrial and nuclear DNA sequencing to reveal genetic and geographically district populations through PNG and one reproductively isolated cryptic species was identified in northern PNG. Using landscape genetic methods, we show that this geographic structure observes through PNG is best explained by landscape topography, with slope (or elevation) presenting as a significant factor.

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.

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.