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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).)

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the "Debug" Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia w AlbB from Aedes albopictus with a local strain, we generated a w AlbB2-F4 strain incompatible with both the wild-type (no Wolbachia ) and w Mel- Wolbachia Ae. aegypti now extant in North Queensland. The w AlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and w Mel- Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable., Competing Interests: Competing interest statement: Verily Life Sciences assisted with project funding., (Copyright © 2021 the Author(s). Published by PNAS.)

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., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Background: In urban environments, some of the most common control tools used against the mosquito disease vector Aedes aegypti are pyrethroid insecticides applied as aerosols, fogs or residual sprays. Their efficacy is compromised by patchy deployment, aging residues, and the evolution and invasion of pyrethroid-resistant mosquitoes. A large proportion of mosquitoes in a given environment will therefore receive sublethal doses of insecticide. The potential impact of this sublethal exposure on the behaviour and biology of Ae. aegypti carrying commonly reported resistance alleles is poorly documented., Results: In susceptible insects, sublethal exposure to permethrin resulted in reductions in egg viability (13.9%), blood avidity (16.7%) and male mating success (28.3%). It caused a 70% decrease in the lifespan of exposed susceptible females and a 66% decrease in the insecticide-resistant females from the parental strain. Exposure to the same dose of insecticide in the presence of the isolated kdr genotype resulted in a smaller impact on female longevity (a 58% decrease) but a 26% increase in eggs per female and a 37% increase in male mating success. Sublethal permethrin exposure reduced host-location success by 20-30% in all strains., Conclusion: The detrimental effects of exposure on susceptible insects were expected, but resistant insects demonstrated a less predictable range of responses, including negative effects on longevity and host-location but increases in fecundity and mating competitiveness. Overall, sublethal insecticide exposure is expected to increase the competitiveness of resistant phenotypes, acting as a selection pressure for the evolution of permethrin resistance. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Rapid advances in biological and digital support systems are revolutionizing the population control of invasive disease vectors such as Aedes aegypti. Methods such as the sterile and incompatible insect techniques (SIT/IIT) rely on modified males to seek out and successfully mate with females, and in doing so outcompete the wild male population for mates. Currently, these interventions most frequently infer mating success through area-wide population surveillance and estimates of mating competitiveness are rare. Furthermore, little is known about male Ae. aegypti behaviour and biology in field settings. In preparation for a large, community scale IIT program, we undertook a series of mark- release-recapture experiments using rhodamine B to mark male Ae. aegypti sperm and measure mating interactions with females. We also developed a Spatial and Temporally Evolving Isotropic Kernel (STEIK) framework to assist researchers to estimate the movement of individuals through space and time. Results showed that ~40% of wild females captured daily were unmated, suggesting interventions will need to release males multiple times per week to be effective at suppressing Ae. aegypti populations. Males moved rapidly through the landscape, particularly when released during the night. Although males moved further than what is typically observed in females of the species, survival was considerably lower. These unique insights improve our understanding of mating interactions in wild Ae. aegypti populations and lay the foundation for robust suppression strategies in the future., Competing Interests: The authors have declared that no competing interests exist.

Background: The development of insecticide resistance in mosquitoes can have pleiotropic effects on key behaviours such as mating competition and host-location. Documenting these effects is crucial for understanding the dynamics and costs of insecticide resistance and may give researchers an evidence base for promoting vector control programs that aim to restore or conserve insecticide susceptibility., Methods and Findings: We evaluated changes in behaviour in a backcrossed strain of Aedes aegypti, homozygous for two knockdown resistance (kdr) mutations (V1016G and S989P) isolated in an otherwise fully susceptible genetic background. We compared biting activity, host location behaviours, wing beat frequency (WBF) and mating competition between the backcrossed strain, and the fully susceptible and resistant parental strains from which it was derived. The presence of the homozygous kdr mutations did not have significant effects on blood avidity, the time to locate a host, or WBF in females. There was, however, a significant reduction in mean WBF in males and a significant reduction in estimated male mating success (17.3%), associated with the isolated kdr genotype., Conclusions: Our results demonstrate a cost of insecticide resistance associated with an isolated kdr genotype and manifest as a reduction in male mating success. While there was no recorded difference in WBF between the females of our strains, the significant reduction in male WBF recorded in our backcrossed strain might contribute to mate-recognition and mating disruption. These consequences of resistance evolution, especially when combined with other pleiotropic fitness costs that have been previously described, may encourage reversion to susceptibility in the absence of insecticide selection pressures. This offers justification for the implementation of insecticide resistance management strategies based on the rotation or alternation of different insecticide classes in space and time., Competing Interests: The authors have declared that no competing interests exist.

Background: Effective vector control measures are essential in a world where many mosquito-borne diseases have no vaccines or drug therapies available. Insecticidal tools remain the mainstay of most vector-borne disease management programmes, although their use for both agricultural and public health purposes has resulted in selection for resistance. Despite this, little is known about the fitness costs associated with specific insecticide-resistant genotypes and their implications for the management of resistance. In Aedes aegypti, the primary vector of dengue, chikungunya, and Zika, the best-characterised resistance mechanisms are single-point mutations that protect the voltage-gated sodium channel from the action of pyrethroids., Methods: We evaluated the fitness cost of two co-occurring, homozygous mutations (V1016G and S989P) by back-crossing a resistant strain of A. aegypti from Timor-Leste into a fully susceptible strain from Queensland. The creation of the backcross strain allowed us to isolate these kdr mutations in an otherwise susceptible genetic background., Results: In comparison to the susceptible strain, the backcrossed colony exhibited longer larval development times (5 days, P < 0.001), 24% fewer mosquitoes reached the adult stage (P = 0.005), had smaller wing lengths (females, P = 0.019 and males, P = 0.007) and adult female mosquitoes had a shorter average lifespan (6 days, P < 0.0006)., Conclusions: These results suggest specific and significant fitness costs associated with the double homozygous V1016G/S989P genotype in the absence of insecticides. The susceptibility of a population may recover if the fitness costs of resistant genotypes can be emphasised through the use of insecticide rotations and mosaics or the presence of untreated spatial or temporal refuges.

Background: In the Solomon Island, the dominant malaria vector, Anopheles farauti, is highly anthropophagic and increasingly exophilic and early biting. While long-lasting insecticide-treated nets remain effective against An. farauti, supplemental vector control strategies will be needed to achieve malaria elimination. Presently, the only World Health Organization recommended supplemental vector control strategy is larval source management (LSM). Effective targeted larval source management requires understanding the associations between abiotic, chemical and biological parameters of larval habitats with the presence or density of vector larvae., Methods: Potential and actual An. farauti larval habitats were characterized for presence and density of larvae and associated abiotic, chemical and biological parameters., Results: A third of all sampled potential habitats harboured An. farauti larvae with 80% of An. farauti positive habitats being in three habitat classifications (swamps/lagoons, transient pools and man-made holes). Large swamps were the most abundant positive habitats surveyed (43% of all An. farauti positive habitats). Habitats with An. farauti larvae were significantly associated with abiotic (pH, nitrate, ammonia and phosphate concentrations and elevated temperature) and biotic (predators) parameters., Conclusion: Large swamps and lagoons are the largest and most abundant An. farauti habitats in the Solomon Islands. Positive habitats were more frequently associated with the presence of predators (vertebrates and invertebrates) and higher water temperatures. Cohabitation with predators is indicative of a complex habitat ecosystem and raises questions about the potential of biological control as an effective control strategy. Increased presence of An. farauti with higher water temperature suggests a potential explanation for the coastal distribution of this species which is not found inland at elevated altitudes where temperatures would be cooler.

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., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

As the incidence of arboviral diseases such as dengue, Zika, chikungunya, and yellow fever increases globally, controlling their primary vector, Aedes aegypti (L.) (Diptera: Culicidae), is of greater importance than ever before. Mosquito control programs rely heavily on effective adult surveillance to ensure methodological efficacy. The Biogents Sentinel (BGS) trap is the gold standard for surveilling adult Aedes mosquitoes and is commonly deployed worldwide, including during modern 'rear and release' programs. Despite its extensive use, few studies have directly assessed environmental characteristics that affect BGS trap catches, let alone how these influences change during 'rear and release' programs. We assessed male and female Ae. aegypti spatial stability, as well as premises condition and trap location influences on BGS trap catches, as part of Debug Innisfail 'rear and release' program in northern Australia. We found similar trends in spatial stability of male and female mosquitoes at both weekly and monthly resolutions. From surveillance in locations where no males were released, reduced catches were found at premises that contained somewhat damaged houses and unscreened properties. In addition, when traps were located in areas that were unsheltered, more than 10 m from commonly used sitting areas or more visually complex catches were also negatively affected. In locations where males were released, we found that traps in treatment sites, relative to control sites, displayed increased catches in heavily shaded premises and were inconsistently influenced by differences in house sets and building materials. Such findings have valuable implications for a range of Ae. aegypti surveillance programs., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Malaria transmission varies in intensity amongst Solomon Island villages where Anopheles farauti is the only vector. This variation in transmission intensity might be explained by density-dependent processes during An. farauti larval development, as density dependence can impact adult size with associated fitness costs and daily survivorship., Methods: Adult anophelines were sampled from six villages in Western and Central Provinces, Solomon Islands between March 2014 and February 2017. The size of females was estimated by measuring wing lengths, and then analysed for associations with biting densities and rainfall., Results: In the Solomon Islands, three anopheline species, An. farauti, Anopheles hinesorum and Anopheles lungae, differed in size. The primary malaria vector, An. farauti, varied significantly in size among villages. Greater rainfall was directly associated with higher densities of An. farauti biting rates, but inversely associated with body size with the smallest mean sized mosquitoes present during the peak transmission period. A measurable association between body size and survivorship was not found., Conclusions: Density dependent effects are likely impacting the size of adult An. farauti emerging from a range of larval habitats. The data suggest that rainfall increases An. farauti numbers and that these more abundant mosquitoes are significantly smaller in size, but without any reduced survivorship being associated with smaller size. The higher malaria transmission rate in a high malaria focus village appears to be determined more by vector numbers than size or survivorship of the vectors.

Background: Species in the Anopheles farauti complex are major malaria vectors in the Asia Pacific region. Anopheline mosquitoes exhibit circadian and diel rhythms in sugar- and blood-feeding (biting), flight activity, oviposition, and in some species, a short-lived dusk/early night associated swarming behaviour during which mating occurs. A behavioural study of wild-caught mosquitoes from Queensland, Australia was conducted to investigate the differences in diel rhythmic flight activity between two cryptic species in several reproductive states., Results: The 24-hour flight activity of individual adult female mosquitoes under light:dark cycle conditions were monitored with a minute-to-minute time resolution using an infrared beam break method. Mosquitoes were analyzed for reproductive state (insemination and parity) and identified to species [An. farauti (s.s.) Laveran and An. hinesorum Schmidt] by PCR analysis. We compared daily total flight activity, timing of activity onset, the peak in early nocturnal activity, and patterns of activity during the scotophase (night). Species-specific differences between An. farauti and An. hinesorum were observed. Compared to An. farauti, An. hinesorum had an earlier onset of dusk activity, an earlier peak in nocturnal activity, and a higher level of activity at the onset of darkness. Small differences between species were also observed in the pattern of the dusk/early-night bouts of activity. A second nocturnal peak in inseminated nulliparous An. hinesorum was also observed during the middle of the scotophase., Conclusions: The behavioural differences between these two sympatric species of the An. farauti complex might contribute to subtle differences in habitat adaptation, the timing of host-seeking and/or sugar-feeding activity. This study provides baseline data for analysis of populations of mosquitoes from other geographical regions where these species are malaria vectors, such as in the Solomon Islands and Papua New Guinea. This is important as selective pressures due to long-term use of indoor residual spraying of insecticides and insecticide-treated bed nets are shifting the nocturnal profile of biting behaviour of these vectors to earlier in the night.

Background: Decisions on when vector control can be withdrawn after malaria is eliminated depend on the receptivity or potential of an area to support vector populations. To guide malaria control and elimination programmes, the potential of biting rates, sporozoite rates, entomological inoculation rates and parity rates to estimate malaria receptivity and transmission were compared within and among geographically localised villages of active transmission in the Western Province of the Solomon Islands., Results: Malaria transmission and transmission potential was heterogeneous in both time and space both among and within villages as defined by anopheline species composition and biting densities. Biting rates during the peak biting period (from 18:00 to 00:00 h) of the primary vector, Anopheles farauti, ranged from less than 0.3 bites per person per half night in low receptivity villages to 26 bites per person in highly receptive villages. Within villages, sites with high anopheline biting rates were significantly clustered. Sporozoite rates provided evidence for continued transmission of Plasmodium falciparum, P. vivax and P. ovale by An. farauti and for incriminating An. hinesorum, as a minor vector, but were unreliable as indicators of transmission intensity., Conclusions: In the low transmission area studied, sporozoite, entomological inoculation and parity rates could not be measured with the precision required to provide guidance to malaria programmes. Receptivity and potential transmission risk may be most reliably estimated by the vector biting rate. These results support the meaningful design of operational research programmes to ensure that resources are focused on providing information that can be utilised by malaria control programmes to best understand both transmission, transmission risk and receptivity across different areas.

Mosquitoes' importance as vectors of pathogens that drive disease underscores the importance of precise and comparable methods of taxa identification among their species. While several molecular targets have been used to study mosquitoes since the initiation of PCR in the 1980s, its application to mosquito identification took off in the early 1990s. This review follows the research's recent journey into the use of mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI or COX1) as a DNA barcode target for mosquito species identification - a target whose utility for discriminating mosquitoes is now escalating. The pros and cons of using a mitochondrial genome target are discussed with a broad sweep of the mosquito literature suggesting that nuclear introgressions of mtDNA sequences appear to be uncommon and that the COI works well for distantly related taxa and shows encouraging utility in discriminating more closely related species such as cryptic/sibling species groups. However, the utility of COI in discriminating some closely related groups can be problematic and investigators are advised to proceed with caution as problems with incomplete lineage sorting and introgression events can result in indistinguishable COI sequences appearing in reproductively independent populations. In these - if not all - cases, it is advisable to run a nuclear marker alongside the mtDNA and thus the utility of the ribosomal DNA - and in particular the internal transcribed spacer 2 - is also briefly discussed as a useful counterpoint to the COI.

Background: 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., Methodology/principal Findings: 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., Conclusions/significance: 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.

Dengue fever is the most common arboviral disease worldwide. It is caused by dengue viruses (DENV) and the mosquito Aedes aegypti is its primary vector. One of the most powerful determinants of a mosquito's ability to transmit DENV is the length of the extrinsic incubation period (EIP), the time it takes for a virus to be transmitted by a mosquito after consuming an infected blood meal. Here, we repeatedly measured DENV load in the saliva of individual mosquitoes over their lifetime and used this in combination with a breeding design to determine the extent to which EIP might respond to the evolutionary forces of drift and selection. We demonstrated that genetic variation among mosquitoes contributes significantly to transmission potential and length of EIP. We reveal that shorter EIP is genetically correlated with reduced mosquito lifespan, highlighting negative life-history consequences for virus-infected mosquitoes. This work highlights the capacity for local genetic variation in mosquito populations to evolve and to dramatically affect the nature of human outbreaks. It also provides the impetus for isolating mosquito genes that determine EIP. More broadly, our dual experimental approach offers new opportunities for studying the evolutionary potential of transmission traits in other vector/pathogen systems., (© 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.)

Background: There is an urgent need for vector control tools to supplement long-lasting insecticidal nets (LLINs) and indoor residual spraying; particularly in the Solomon Islands where the primary vector, Anopheles farauti, is highly anthropophagic and feeds mainly outdoors and early in the evening. Currently, the only supplementary tool recommended by the World Health Organization is larval source management (LSM). The feasibility and potential effectiveness of LSM requires information on the distribution of anophelines, the productivity of larval habitats and the potential impacts of larval control on adult fitness., Methods: The distribution of anophelines in Central and Western Provinces in the Solomon Islands was mapped from cross-sectional larval habitat surveys. The composition and micro-distribution of larval instars within a large permanent river-mouth lagoon was examined with a longitudinal survey. Density-dependent regulation of An. farauti larvae was investigated by longitudinally following the development and survival of different densities of first instars in floating cages in a river-mouth lagoon., Results: Five anopheline species were molecularly identified from a range of fresh and brackish water habitats: An. farauti s.s., An. hinesorum, An. lungae, An. nataliae and An. solomonis. The most common habitats used by the primary malaria vector, An. farauti, were coastal lagoons and swamps. In the detailed study of lagoon micro-productivity, An. farauti was non-uniformly distributed with highest densities found at collections sites most proximal and distal to the mouth of the lagoon. The survival of An. farauti larvae was more than twofold lower when larvae were held at the highest experimental density (1 larva per 3.8 cm(2)) when compared with the lowest density (1 larva per 38 cm(2))., Conclusions: The only documented major malaria vector collected in larval surveys in both Central and Western Provinces was An. farauti. Lagoons and swamps, the most common, largest and (potentially) most productive larval sites of this malaria vector, were "few, fixed and findable" and theoretically, therefore, amenable to successful LSM. However, the immense scale and complexity of these ecosystems in which An. farauti larvae are found raises questions regarding the ability to effectively control the larvae, as incomplete larviciding could trigger density dependent effects resulting in increased larval survivorship. While LSM has the potential to significantly contribute to malaria control of this early and outdoor biting vector, more information on the distribution of larvae within these extensive habitats is required to maximize the effectiveness of LSM.

Background: The effectiveness of vector control on malaria transmission by long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) depends on the vectors entering houses to blood feed and rest when people are inside houses. In the Solomon Islands, significant reductions in malaria have been achieved in the past 20 years with insecticide-treated bed nets, IRS, improved diagnosis and treatment with artemisinin combination therapies; despite the preference of the primary vector, Anopheles farauti, to feed outdoors and early in the evening and thereby avoid potential exposure to insecticides. Rational development of tools to complement LLINs and IRS by attacking vectors outdoor requires detailed knowledge of the biology and behaviours of the target species., Methods: Malaria transmission in Central Province, Solomon Islands was estimated by measuring the components comprising the entomological inoculation rate (EIR) as well as the vectorial capacity of An. farauti. In addition, the daily and seasonal biting behaviour of An. farauti, was examined and the duration of the feeding cycle was estimated with a mark-release-recapture experiment., Results: Anopheles farauti was highly exophagic with 72% captured by human landing catches (HLC) outside of houses. Three-quarters (76%) of blood feeding on humans was estimated to occur before 21.00 h. When the hourly location of humans was considered, the proportion of exposure to mosquito bites on humans occurring indoors (πi) was only 0.130 ± 0.129. Peak densities of host seeking An. farauti occurred between October and January. The annual EIR was estimated to be 2.5 for 2012 and 33.2 for 2013. The length of the feeding cycle was 2.1 days., Conclusions: The short duration of the feeding cycle by this species offers an explanation for the substantial control of malaria that has been achieved in the Solomon Islands by LLINs and IRS. Anopheles farauti is primarily exophagic and early biting, with 13% of mosquitoes entering houses to feed late at night during each feeding cycle. The two-day feeding cycle of An. farauti requires females to take 5-6 blood meals before the extrinsic incubation period (EIP) is completed; and this could translate into substantial population-level mortality by LLINs or IRS before females would be infectious to humans with Plasmodium falciparum and Plasmodium vivax. Although An. farauti is primarily exophagic, the indoor vector control tools recommended by the World Health Organization (LLINs and IRS) can still provide an important level of control. Nonetheless, elimination will likely require vector control tools that target other bionomic vulnerabilities to suppress transmission outdoors and that complement the control provided by LLINs and IRS.