Genetic and geographic population structure in the malaria vector, Anopheles farauti, provides a candidate system for pioneering confinable gene-drive releases.

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.

A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Comparative assessment of a novel fan box trap for collecting Anopheles farauti and culicine mosquitoes alive in tropical north Queensland, Australia.

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.

Population structure and invasion history of Aedes aegypti (Diptera: Culicidae) in Southeast Asia and Australasia.

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.

A bacterium against the tiger: further evidence of the potential of noninundative releases of males with manipulated Wolbachia infection in reducing fertility of Aedes albopictus field populations in Italy.

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.

Insecticide resistance compromises the control of Aedes aegypti in Bangladesh.

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.

Seasonal assessment on the effects of time of night, temperature and humidity on the biting profile of Anopheles farauti in north Queensland, Australia using a population naive to malaria vector control pressures.

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.

Wolbachia wAlbB inhibit dengue and Zika infection in the mosquito Aedes aegypti with an Australian background.

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.

Comparisons of chemosensory gene repertoires in human and non-human feeding Anopheles mosquitoes link olfactory genes to anthropophily.

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.

Gene flow between island populations of the malaria mosquito, Anopheles hinesorum, may have contributed to the spread of divergent host preference phenotypes.

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.

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia.

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 wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-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 wMel-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.

Mark-release-recapture of male Aedes aegypti (Diptera: Culicidae): Use of rhodamine B to estimate movement, mating and population parameters in preparation for an incompatible male program.

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.

The impact of sublethal permethrin exposure on susceptible and resistant genotypes of the urban disease vector Aedes aegypti.

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.

The presence of knockdown resistance mutations reduces male mating competitiveness in the major arbovirus vector, Aedes aegypti.

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.

Identifying the fitness costs of a pyrethroid-resistant genotype in the major arboviral vector Aedes aegypti.

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.

Defining the larval habitat: abiotic and biotic parameters associated with Anopheles farauti productivity.

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.

Population genetics of Anopheles koliensis through Papua New Guinea: New cryptic species and landscape topography effects on genetic connectivity.

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.

Waterproof, low-cost, long-battery-life sound trap for surveillance of male Aedes aegypti for rear-and-release mosquito control programmes.

BACKGROUND: Sterile male rear-and-release programmes are of growing interest for controlling Aedes aegypti, including use an "incompatible insect technique" (IIT) to suppress transmission of dengue, Zika, and other viruses. Under IIT, males infected with Wolbachia are released into the suppression area to induce cytoplasmic incompatibility in uninfected populations. These and similar mosquito-release programmes require cost-effective field surveys of both sexes to optimize the locations, timing, and quantity of releases. Unfortunately, traps that sample male Ae. aegypti effectively are expensive and usually require mains power. Recently, an electronic lure was developed that attracts males using a 484 Hz sinusoidal tone mimicking the female wingbeat frequencies, broadcast in a 120 s on/off cycle. When deployed in commercially available gravid Aedes traps (GATs), the new combination, sound-GAT (SGAT), captures both males and females effectively. Given its success, there is interest in optimizing SGAT to reduce cost and power usage while maximizing catch rates. METHODS: Options considered in this study included use of a smaller, lower-power microcontroller (Tiny) with either the original or a lower-cost speaker (lcS). A 30 s on/off cycle was tested in addition to the original 120 s cycle to minimize the potential that the longer cycle induced habituation. The original SGAT was compared against other traps incorporating the Tiny-based lures for mosquito capture in a large semi-field cage. The catch rates in waterproofed versions of this trap were then compared with catch rates in standard [BG-Sentinel 2 (BGS 2); Biogents AG, Regensburg, Germany] traps during an IIT field study in the Innisfail region of Queensland, Australia in 2017. RESULTS: The system with a low-power microcontroller and low-cost speaker playing a 30 s tone (Tiny-lcS-30s) caught the highest proportion of males. The mean proportions of males caught in a semi-field cage were not significantly different among the original design and the four low-power, low-cost versions of the SGAT. During the IIT field study, the waterproofed version of the highest-rated, Tiny-lcS-30s SGAT captured male Ae. aegypti at similar rates as co-located BGS-2 traps. CONCLUSIONS: Power- and cost-optimized, waterproofed versions of male Ae. aegypti acoustic lures in GATs are now available for field use in areas with sterile male mosquito rear-and-release programmes.

Smallest Anopheles farauti occur during the peak transmission season in the Solomon Islands.

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.