Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans.

Multiple sensory cues emanating from humans are thought to guide blood-feeding female mosquitoes to a host. To determine the relative contribution of carbon dioxide (CO2) detection to mosquito host-seeking behavior, we mutated the AaegGr3 gene, a subunit of the heteromeric CO2 receptor in Aedes aegypti mosquitoes. Gr3 mutants lack electrophysiological and behavioral responses to CO2. These mutants also fail to show CO2-evoked responses to heat and lactic acid, a human-derived attractant, suggesting that CO2 can gate responses to other sensory stimuli. Whereas attraction of Gr3 mutants to live humans in a large semi-field environment was only slightly impaired, responses to an animal host were greatly reduced in a spatial-scale-dependent manner. Synergistic integration of heat and odor cues likely drive host-seeking behavior in the absence of CO2 detection. We reveal a networked series of interactions by which multimodal integration of CO2, human odor, and heat orchestrates mosquito attraction to humans.

A decade of stability for wMel Wolbachia in natural Aedes aegypti populations.

Mosquitoes carrying Wolbachia endosymbionts are being released in many countries for arbovirus control. The wMel strain of Wolbachia blocks Aedes-borne virus transmission and can spread throughout mosquito populations by inducing cytoplasmic incompatibility. Aedes aegypti mosquitoes carrying wMel were first released into the field in Cairns, Australia, over a decade ago, and with wider releases have resulted in the near elimination of local dengue transmission. The long-term stability of Wolbachia effects is critical for ongoing disease suppression, requiring tracking of phenotypic and genomic changes in Wolbachia infections following releases. We used a combination of field surveys, phenotypic assessments, and Wolbachia genome sequencing to show that wMel has remained stable in its effects for up to a decade in Australian Ae. aegypti populations. Phenotypic comparisons of wMel-infected and uninfected mosquitoes from near-field and long-term laboratory populations suggest limited changes in the effects of wMel on mosquito fitness. Treating mosquitoes with antibiotics used to cure the wMel infection had limited effects on fitness in the next generation, supporting the use of tetracycline for generating uninfected mosquitoes without off-target effects. wMel has a temporally stable within-host density and continues to induce complete cytoplasmic incompatibility. A comparison of wMel genomes from pre-release (2010) and nine years post-release (2020) populations show few genomic differences and little divergence between release locations, consistent with the lack of phenotypic changes. These results indicate that releases of Wolbachia-infected mosquitoes for population replacement are likely to be effective for many years, but ongoing monitoring remains important to track potential evolutionary changes.

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.

Local introduction and heterogeneous spatial spread of dengue-suppressing Wolbachia through an urban population of Aedes aegypti.

Dengue-suppressing Wolbachia strains are promising tools for arbovirus control, particularly as they have the potential to self-spread following local introductions. To test this, we followed the frequency of the transinfected Wolbachia strain wMel through Ae. aegypti in Cairns, Australia, following releases at 3 nonisolated locations within the city in early 2013. Spatial spread was analysed graphically using interpolation and by fitting a statistical model describing the position and width of the wave. For the larger 2 of the 3 releases (covering 0.97 km2 and 0.52 km2), we observed slow but steady spatial spread, at about 100-200 m per year, roughly consistent with theoretical predictions. In contrast, the smallest release (0.11 km2) produced erratic temporal and spatial dynamics, with little evidence of spread after 2 years. This is consistent with the prediction concerning fitness-decreasing Wolbachia transinfections that a minimum release area is needed to achieve stable local establishment and spread in continuous habitats. Our graphical and likelihood analyses produced broadly consistent estimates of wave speed and wave width. Spread at all sites was spatially heterogeneous, suggesting that environmental heterogeneity will affect large-scale Wolbachia transformations of urban mosquito populations. The persistence and spread of Wolbachia in release areas meeting minimum area requirements indicates the promise of successful large-scale population transformation.

Reflections from an old Queenslander: can rear and release strategies be the next great era of vector control?

In this perspective, I discuss the great eras of vector control, centring on Aedes aegypti, the primary vector of dengue, Zika and several other viruses. Since the discovery and acceptance of the role of mosquitoes as vectors of disease agents, several significant strategies have been developed and deployed to control them and the diseases they transmit. Environmental management, insecticides and, to a lesser extent, biological control have emerged as great eras of vector control. In the past decade, the release of massive numbers of specifically modified mosquitoes that mate with wild populations has emerged as a significant new strategy to fight vector-borne diseases. These reared and released mosquitoes have been modified by the addition of a symbiont (e.g. Wolbachia bacteria), radiation or introduction of a genetic construct to either sterilize the wild mosquitoes they mate with, crashing the population, or to reduce the wild population's capacity to vector pathogens. Will these new rear and release strategies become the next great era of vector control? From my vantage point as a dengue control manager and researcher involved in two Wolbachia programmes, I will discuss the hurdles that rear and release programmes face to gain widespread acceptance and success.

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.

Advances in Vector Control Science: Rear-and-Release Strategies Show Promise but Don't Forget the Basics.

Both chikungunya and Zika viruses have recently swept from Africa across the Pacific to the Americas, causing major outbreaks of disease in humans. In the meantime, dengue epidemics continue throughout the tropics. Traditional vector control programs based on strategies from 1950s and 1960s have been relatively ineffective in combating recent epidemics. In response, new methods involving the rearing and releasing of large numbers of mosquitoes to eliminate or modify local Aedes populations are being developed, with several currently conducting field releases in high-risk countries. These advances, include the release of Wolbachia-infected Aedes aegypti and Aedes albopictus, for either its virus-blocking capabilities, sterilization by cytoplasmic incompatibility, or both; the release of Aedes carrying dominant lethal genes, such as the OX513A strain of A. aegypti; and other emerging techniques, such as advancing gene-drive technologies, are summarized, as well as current stages of development and primary operational and regulatory hurdles. Although these technologies show great promise, none are ready for widespread rollout for cities of millions of people. Thus, efforts should be made to avoid methods such as space sprays that have failed and improve existing technologies to increase their efficacy.

Discovery of new orbiviruses and totivirus from Anopheles mosquitoes in Eastern Australia.

Three new viruses classifiable within the Totivirus and Orbivirus genera were detected from Anopheles mosquito species collected in Eastern Australia. The viruses could not be isolated in C6/36 mosquito cell cultures but were shown to replicate in their mosquito hosts by small RNA analysis. The viruses grouped phylogenetically with other viruses recently detected in insects. These discoveries contribute to a better understanding of commensal viruses in Australian mosquitoes and the evolution of these viruses.

Predicting Wolbachia invasion dynamics in Aedes aegypti populations using models of density-dependent demographic traits.

BACKGROUND: Arbovirus transmission by the mosquito Aedes aegypti can be reduced by the introduction and establishment of the endosymbiotic bacteria Wolbachia in wild populations of the vector. Wolbachia spreads by increasing the fitness of its hosts relative to uninfected mosquitoes. However, mosquito fitness is also strongly affected by population size through density-dependent competition for limited food resources. We do not understand how this natural variation in fitness affects symbiont spread, which limits our ability to design successful control strategies. RESULTS: We develop a mathematical model to predict A. aegypti-Wolbachia dynamics that incorporates larval density-dependent variation in important fitness components of infected and uninfected mosquitoes. Our model explains detailed features of the mosquito-Wolbachia dynamics observed in two independent experimental A. aegypti populations, allowing the combined effects on dynamics of multiple density-dependent fitness components to be characterized. We apply our model to investigate Wolbachia field release dynamics, and show how invasion outcomes can depend strongly on the severity of density-dependent competition at the release site. Specifically, the ratio of released relative to wild mosquitoes required to attain a target infection frequency (at the end of a release program) can vary by nearly an order of magnitude. The time taken for Wolbachia to become established following releases can differ by over 2 years. These effects depend on the relative fitness of field and insectary-reared mosquitoes. CONCLUSIONS: Models of Wolbachia invasion incorporating density-dependent demographic variation in the host population explain observed dynamics in experimental A. aegypti populations. These models predict strong effects of density-dependence on Wolbachia dynamics in field populations, and can assist in the effective use of Wolbachia to control the transmission of arboviruses such as dengue, chikungunya and zika.

The Siren's Song: Exploitation of Female Flight Tones to Passively Capture Male Aedes aegypti (Diptera: Culicidae).

The need to capture male mosquitoes has intensified recently as a result of a number of male-based sterile insect technique (SIT) and population-modification programs focused on Aedes aegypti (L.) having initiated field releases. Here, we report the results of the successful exploitation of the attraction of male Ae. aegypti to female flight tones to enhance male collections in nonmechanical passive (nonbattery powered) Gravid Aedes Traps (GAT). Prior to field studies, male attraction to female flight tones of 484 and 560 Hz, as well as to a male flight tone of 715 Hz, were assessed in a series of controlled release-recapture and semifield trials. These trials determined that a pure tone of 484 Hz was significantly more attractive to free-flying males than the other flight tones and enabled their collection in sound-baited GATs (ca. 95% capture rate after 2 h; 484 Hz at 65 dB). In contrast, gravid females were unresponsive to male or female flight tones and were evenly distributed among sound-baited and control GATs. Importantly, under normal field conditions sound-baited GATs (484 Hz at 70 dB) captured significantly more male Ae. aegypti per 24-h trap interval (1.3 ± 0.37) than controls (0.2 ± 0.13). Overall, sound-bated GATs captured approximately twice as many Ae. aegypti (male and female; 3.0 ± 0.68 per interval, 30 total) than controls (1.5 ± 0.56 per interval, 15 total). These results reveal that sound-baited GATs are a simple and effective surveillance tool for Ae. aegypti that would allow current male-based SIT and population-modification programs to effectively monitor males in their target populations.

A spatial simulation model for dengue virus infection in urban areas.

BACKGROUND: The World Health Organization estimates that the global number of dengue infections range between 80-100 million per year, with some studies estimating approximately three times higher numbers. Furthermore, the geographic range of dengue virus transmission is extending with the disease now occurring more frequently in areas such as southern Europe. Ae. aegypti, one of the most prominent dengue vectors, is endemic to the far north-east of Australia and the city of Cairns frequently experiences dengue outbreaks which sometimes lead to large epidemics. METHOD: A spatially-explicit, individual-based mathematical model that accounts for the spread of dengue infection as a result of human movement and mosquito dispersion is presented. The model closely couples the four key sub-models necessary for representing the overall dynamics of the physical system, namely those describing mosquito population dynamics, human movement, virus transmission and vector control. Important features are the use of high quality outbreak data and mosquito trapping data for calibration and validation and a strategy to derive local mosquito abundance based on vegetation coverage and census data. RESULTS: The model has been calibrated using detailed 2003 dengue outbreak data from Cairns, together with census and mosquito trapping data, and is shown to realistically reproduce a further dengue outbreak. The simulation results replicating the 2008/2009 Cairns epidemic support several hypotheses (formulated previously) aimed at explaining the large-scale epidemic which occurred in 2008/2009; specifically, while warmer weather and increased human movement had only a small effect on the spread of the virus, a shorter virus strain-specific extrinsic incubation time can explain the observed explosive outbreak of 2008/2009. CONCLUSION: The proof-of-concept simulation model described in this study has potential as a tool for understanding factors contributing to dengue spread as well as planning and optimizing dengue control, including reducing the Ae. aegypti vector population and for estimating the effectiveness and cost-effectiveness of future vaccination programmes. This model could also be applied to other vector borne viral diseases such as chikungunya, also spread by Ae. aegypti and, by re-parameterisation of the vector sub-model, to dengue and chikungunya viruses spread by Aedes albopictus.

Development of the gravid Aedes trap for the capture of adult female container-exploiting mosquitoes (Diptera: Culicidae).

Monitoring dengue vector control by sampling adult Aedes aegypti (L.) recently has been used to replace both larval and pupal surveys. We have developed and evaluated the Gravid Aedes Trap (GAT) through a sequential behavioral study. The GAT does not require electricity to function, and trapped mosquitoes are identified easily during trap inspections. The GAT concept relies on visual and olfactory cues to lure gravid Ae. aegypti and an insecticide to kill trapped mosquitoes. Gravid mosquitoes are lured to a black bucket base containing oviposition attractant (infusion) and are trapped in a translucent chamber impregnated with a pyrethroid insecticide where they are killed within 3-15 min. In semifield observations, the GAT captured a significantly higher proportion of gravid mosquitoes than the double sticky ovitrap. We also demonstrated that the visual cues of the prototype GAT-LgBF (large black base bucket with a black funnel at the top of the translucent chamber) captured a significantly higher proportion of gravid mosquitoes than the other prototypes. The visual contrast created by the addition of a white lid to the top of the black funnel significantly increased the number of captured gravid mosquitoes when compared with the GAT-LgBF in semifield trials. We conclude that the GAT is more efficient in recapturing gravid Ae. aegypti when compared with sticky ovitraps. The GAT is an effective, practical, low cost, and easily transportable trap, features that are essential in large-scale monitoring programs, particularly in areas where funding is limited.

Field validation of the gravid Aedes trap (GAT) for collection of Aedes aegypti (Diptera: Culicidae).

Current surveillance methods for adult Aedes aegypti (L.) are expensive, require electrical power (e.g., the BG-Sentinel trap, BGS), are labor intensive (aspirators), or require difficult to use and costly adhesives (sticky ovitraps). Field trials were conducted in Cairns (Australia) to compare the efficacy of the newly designed Gravid Aedes Trap (GAT) against existing sticky ovitraps (MosquiTRAP and double sticky ovitrap) and the BGS. Latin square design trials confirmed that alarge GAT using a 9.2-liters bucket treated with Mortein Barrier Outdoor Surface Spray ([AI] 0.3 g/kg imiprothrin and 0.6 g/kg deltamethrin) outperformed a smaller 1.2-liters GAT and collected, on average, 3.7x and 2.4X more female Ae. aegypti than the MosquiTRAP and double sticky ovitrap, respectively. Field trials showed that the GAT collected 10-50% less female Ae. aegypti than the BGS trap but 30% more gravid mosquitoes than the BGS. Trials using the BGS and the GAT indicated that there was no difference in capture rates between female Ae. aegypti uninfected and infected with the wMel strain of Wolbachia, and wMel infection rates were nearly identical at >90% to field captured Ae. aegypti. The potential for the GAT to be used for dengue virus surveillance was also demonstrated with dengue virus type 3 RNA detected in five-sixths and six-sixths pools ofAe. aegypti stored in a GAT held at 28 degreeC and 60% relative humidity for 7 and 14 d, respectively. Mosquito knock down in GATs treated with Mortein surface spray set in 30, 70, and 99% shade was comparable for up to 2 mo, with only approximately 10% of adults escaping. The GAT is therefore a useful tool for capturing adult Ae. aegypti and may be suitable for other container-inhabiting species such as Aedes albopictus (Skuse) and Culex quinquefasciatus Say. The low cost and practicality of operation make the GAT suitable for vector surveillance and projects requiring monitoring of mosquitoes for Wolbachia and arboviruses, especially in developing countries.

Implications of dengue outbreaks for blood supply, Australia.

Dengue outbreaks have increased in size and frequency in Australia, and transfusion-transmitted dengue poses a risk to transfusion safety. Using whole blood samples collected during the large 2008-2009 dengue epidemic, we estimated the risk for a dengue-infectious blood donation as ≈1 in 7,146 (range 2,218-50,021).

Novel estimates of Aedes aegypti (Diptera: Culicidae) population size and adult survival based on Wolbachia releases.

The size of Aedes aegypti (L.) mosquito populations and adult survival rates have proven difficult to estimate because of a lack of consistent quantitative measures to equate sampling methods, such as adult trapping, to actual population size. However, such estimates are critical for devising control methods and for modeling the transmission of dengue and other infectious agents carried by this species. Here we take advantage of recent releases of Wolbachia-infected Ae. aegypti coupled with the results of ongoing monitoring to estimate the size of adult Ae. aegypti populations around Cairns in far north Queensland, Australia. Based on the association between released adults infected with Wolbachia and data from Biogents Sentinel traps, we show that data from two locations are consistent with population estimates of approximately 5-10 females per house and daily survival rates of 0.7-0.9 for the released Wolbachia-infected females. Moreover, we estimate that networks of Biogents Sentinel traps at a density of one per 15 houses capture around 5-10% of the adult population per week, and provide a rapid estimate of the absolute population size of Ae. aegypti. These data are discussed with respect to release rates and monitoring in future Wolbachia releases and also the levels of suppression required to reduce dengue transmission.

Residual treatment of Aedes aegypti (Diptera: Culicidae) in containers using pyriproxyfen slow-release granules (Sumilarv 0.5G).

The residual efficacy of pyriproxyfen against Aedes aegypti (L.) was examined by treating 2-liter buckets with a range of rates of Sumilarv 0.5G (100, 10, 1, and 0.1 mg product/liter or nominal dose of 500, 50, 5, and 0.5 ppb active ingredient) under semifield conditions. Approximately every 2 wk, pupal emergence inhibition (EI) was measured by using Cairns colony Ae. aegypti. Pooled water samples from the five replicate buckets were analyzed for active pyriproxyfen by using ultra-high-pressure liquid chromatography with tandem mass spectrometry detection. A strong dose-response in EI was exhibited, with the 0.1 mg/liter giving approximately 50% EI for only the initial week, whereas the 10 and 100 mg/liter doses produced EI > 90% for 8 and 40 wk, respectively. Measurable levels of active ingredient were detected in the 100, 10, and 1 mg/liter treatments, with measured starting concentrations of just 1-2-1.4% of the delivered (active ingredient) dose. Pyriproxyfen was detected in the 100 mg/liter treatment through the entire course of the trial (60 wk).

Operational use of household bleach to "crash and release" Aedes aegypti prior to Wolbachia-infected mosquito release.

Dengue (family Flaviviridae, genus Flavivirus, DENV) remains the leading arboviral cause of mortality in the tropics. Wolbachia pipientis has been shown to interrupt DENV transmission and is presently being trialled as a biological control. However, deployment issues have arisen on methods to temporarily suppress wild mosquito populations before Wolbachia-infected mosquito releases. By suppressing wild populations, fewer Ae. aegypti releases are required to achieve a sustainable Wolbachia density threshold. Furthermore, public distress is reduced. This study tests the application of domestic bleach (4% NaCIO) to temporarily "crash" immature Aedes populations in water-filled containers. Spray application NaClO (215 ppm) resulted in a mean 48-h mortality of 100, 100, 97, and 88% of eggs, second-instar larvae, fourth-instar larvae, and pupae, respectively. In the field, NaClO delayed ovipositing by 9 d in cooler months, and 11 d in hotter months, after which oviposition resumed in treated receptacles. We found bleach treatment of pot-plant bases did not cause wilting, yellowing, or dropping of leaves in two ornamental plants species. Domestically available NaClO could be adopted for a "crash and release" strategy to temporarily suppress wild populations of Ae. aegypti in containers before release of Wolbachia-infected mosquitoes. The "crash and release" strategy is also applicable to other mosquito species, e.g., Aedes albopictus (Skuse), in strategies using released mosquitoes.

A simple non-powered passive trap for the collection of mosquitoes for arbovirus surveillance.

Mosquitoes often are collected as part of an arbovirus surveillance program. However, trapping and processing of mosquitoes for arbovirus detection is often costly and difficult in remote areas. Most traps, such as the gold standard Center for Disease control light trap, require batteries that must be charged and changed overnight. To overcome this issue we have developed several passive traps for collection of mosquitoes that have no power requirements. The passive traps capture mosquitoes as they follow a CO2 plume up a polyvinyl chloride pipe leading to a clear chamber consisting of a plastic crate. We believe the translucent, clear windows created by the crate inhibits escape. Once inside the crate mosquitoes readily feed on honey-treated Flinders Technology Associates cards that then can be processed by polymerase chain reaction for viral ribonucleic acid. Of the two designs tested, the box or crate-based passive trap (passive box trap, PBT) generally caught more mosquitoes than the cylinder trap. In Latin square field trials in Cairns and Florida, PBTs collected mosquitoes at rates of 50 to 200% of Center for Disease Control model 512 light traps. Mosquito collections by PBTs can be increased by splitting the CO2 gas line so it services two traps, or by placing an octenol lure to the outside of the box. Very large collections can lead to crowding at honey-treated cards, reducing feeding rates. Addition of fipronil to the honey killed mosquitoes and did not impact feeding rates nor the ability to detect Kunjin viral ribonucleic acid by polymerase chain reaction; this could be used to minimize crowding affects on feeding caused by large collections. The passive traps we developed are made from inexpensive, commonly available materials. Passive traps may thus be suitable for collection of mosquitoes and potentially other hematophagous dipterans for pathogen surveillance.

Field evaluation of selected traps and lures for monitoring the filarial and arbovirus vector, Aedes polynesiensis (Diptera: Culicidae), in French Polynesia.

The efficacy of the BG-Sentinel (BGS) and the BG-Mosquitito (BGM) mosquito traps for sampling populations of the important filariasis and dengue vector Aedes (Stegomyia) polynesiensis (Marks) was evaluated in French Polynesia against human bait collections (HBC) using a modified Centers for Disease Control and Prevention backpack aspirator. Traps were baited with BG-Lure (a combination of lactic acid, ammonia, and caproic acid) or carbon dioxide plus octenol (1-octen-3-ol) known as attractants to aedine mosquitoes. Mosquito sampling was conducted on two typical islands of French Polynesia: the high, volcanic island of Moorea, and the low, coral island (atoll) of Tetiaroa Sampling efficacy was measured in a randomized Latin Square design. Production of carbon dioxide from yeast-sugar fermentation was used as an alternative source of CO2 because supply via dry ice, gas cylinders, or propane combustion in remote tropical islands is costly and challenging. Although the BGS trap captured the greatest number ofAe. polynesiensis in both island settings, catch rates of BGS or BGM baited with either lure were not significantly different from that of HBC. On Moorea, the number of collected aedes species in the BGS trap baited with either lure was significantly greater than the BGM with BG-lure. On Tetiaroa, BGM trapping was severely hampered by damage from rats, and the traps were removed from the study. Our study confirms the efficiency, comparability, and convenience of the BGS trap, a robust and safe alternative to HBC for sampling Aedes mosquitoes in research and surveillance efforts against filariasis and arboviruses in the South Pacific.

Exploiting mosquito sugar feeding to detect mosquito-borne pathogens.

Arthropod-borne viruses (arboviruses) represent a global public health problem, with dengue viruses causing millions of infections annually, while emerging arboviruses, such as West Nile, Japanese encephalitis, and chikungunya viruses have dramatically expanded their geographical ranges. Surveillance of arboviruses provides vital data regarding their prevalence and distribution that may be utilized for biosecurity measures and the implementation of disease control strategies. However, current surveillance methods that involve detection of virus in mosquito populations or sero-conversion in vertebrate hosts are laborious, expensive, and logistically problematic. We report a unique arbovirus surveillance system to detect arboviruses that exploits the process whereby mosquitoes expectorate virus in their saliva during sugar feeding. In this system, infected mosquitoes captured by CO(2)-baited updraft box traps are allowed to feed on honey-soaked nucleic acid preservation cards within the trap. The cards are then analyzed for expectorated virus using real-time reverse transcription-PCR. In field trials, this system detected the presence of Ross River and Barmah Forest viruses in multiple traps deployed at two locations in Australia. Viral RNA was preserved for at least seven days on the cards, allowing for long-term placement of traps and continuous collection of data documenting virus presence in mosquito populations. Furthermore no mosquito handling or processing was required and cards were conveniently shipped to the laboratory overnight. The simplicity and efficacy of this approach has the potential to transform current approaches to vector-borne disease surveillance by streamlining the monitoring of pathogens in vector populations.