Trap Location and Premises Condition Influences on Aedes aegypti (Diptera: Culicidae) Catches Using Biogents Sentinel Traps During a 'Rear and Release' Program: Implications for Designing Surveillance Programs.

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.

Establishment of wMel Wolbachia in Aedes aegypti mosquitoes and reduction of local dengue transmission in Cairns and surrounding locations in northern Queensland, Australia.

Background: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and subsequently shown in laboratory studies to reduce transmission of a range of viruses including dengue, Zika, chikungunya, yellow fever, and Mayaro viruses that cause human disease. Here we report the entomological and epidemiological outcomes of staged deployment of Wolbachia across nearly all significant dengue transmission risk areas in Australia. Methods: The  wMel strain of  Wolbachia was backcrossed into the local  Aedes aegypti genotype (Cairns and Townsville backgrounds) and mosquitoes were released in the field by staff or via community assisted methods. Mosquito monitoring was undertaken and mosquitoes were screened for the presence of  Wolbachia. Dengue case notifications were used to track dengue incidence in each location before and after releases. Results: Empirical analyses of the Wolbachia mosquito releases, including data on the density, frequency and duration of Wolbachia mosquito releases, indicate that Wolbachia can be readily established in local mosquito populations, using a variety of deployment options and over short release durations (mean release period 11 weeks, range 2-22 weeks). Importantly, Wolbachia frequencies have remained stable in mosquito populations since releases for up to 8 years. Analysis of dengue case notifications data demonstrates near-elimination of local dengue transmission for the past five years in locations where Wolbachia has been established. The regression model estimate of Wolbachia intervention effect from interrupted time series analyses of case notifications data prior to and after releases, indicated a 96% reduction in dengue incidence in Wolbachia treated populations (95% confidence interval: 84 - 99%). Conclusion: Deployment of the wMel strain of Wolbachia into local Ae. aegypti populations across the Australian regional cities of Cairns and most smaller regional communities with a past history of dengue has resulted in the reduction of local dengue transmission across all deployment areas.

Modifying the Biogents Sentinel Trap to Increase the Longevity of Captured Aedes aegypti.

Aedes aegypti (L.) (Diptera: Culicidae) is a vector of viruses causing dengue, Zika, chikungunya, and yellow fever and subsequently pose a significant global threat to public health. While sampling live mosquitoes is useful for surveillance purposes, most traps targeting Aedes kill captured mosquitoes. The Biogents Sentinel (BGS) trap, the gold standard for capturing Ae. aegypti, is one such trap. In our study, we modified the BGS trap to increase the survival of captured Ae. aegypti by replacing the trap's catch bag with a catch pot that protects mosquitoes from desiccation by airflow. A sucrose-soaked sponge or nucleic acid preservative card can also be placed inside the pot to enhance mosquito survival and augment arbovirus detection. These modifications to the BGS significantly increased the longevity of mosquitoes captured with weekly survivals of 93% in a semifield structure and 86% in the field. These high survival rates resulted in 3.5 times more alive Ae. aegypti captured weekly in the modified BGS compared to the original BGS, despite 40% lower overall catch rates. These cheap and simple trap modifications facilitate easier specimen identification as well as experiments requiring live field-collected samples such as virus detection from mosquito saliva and excreta.

Scaled deployment of Wolbachia to protect the community from dengue and other Aedes transmitted arboviruses.

Background: A number of new technologies are under development for the control of mosquito transmitted viruses, such as dengue, chikungunya and Zika that all require the release of modified mosquitoes into the environment. None of these technologies has been able to demonstrate evidence that they can be implemented at a scale beyond small pilots. Here we report the first successful citywide scaled deployment of Wolbachia in the northern Australian city of Townsville. Methods: The wMel strain of Wolbachia was backcrossed into a local Aedes aegypti genotype and mass reared mosquitoes were deployed as eggs using mosquito release containers (MRCs). In initial stages these releases were undertaken by program staff but in later stages this was replaced by direct community release including the development of a school program that saw children undertake releases. Mosquito monitoring was undertaken with Biogents Sentinel (BGS) traps and individual mosquitoes were screened for the presence of Wolbachia with a Taqman qPCR or LAMP diagnostic assay. Dengue case notifications from Queensland Health Communicable Disease Branch were used to track dengue cases in the city before and after release. Results: Wolbachia was successfully established into local Ae. aegypti mosquitoes across 66 km 2 in four stages over 28 months with full community support.  A feature of the program was the development of a scaled approach to community engagement. Wolbachia frequencies have remained stable since deployment and to date no local dengue transmission has been confirmed in any area of Townsville after Wolbachia has established, despite local transmission events every year for the prior 13 years and an epidemiological context of increasing imported cases. Conclusion: Deployment of Wolbachia into Ae. aegypti populations can be readily scaled to areas of ~60km 2 quickly and cost effectively and appears in this context to be effective at stopping local dengue transmission.

Use of rhodamine B to mark the body and seminal fluid of male Aedes aegypti for mark-release-recapture experiments and estimating efficacy of sterile male releases.

BACKGROUND: Recent interest in male-based sterile insect technique (SIT) and incompatible insect technique (IIT) to control Aedes aegypti and Aedes albopictus populations has revealed the need for an economical, rapid diagnostic tool for determining dispersion and mating success of sterilized males in the wild. Previous reports from other insects indicated rhodamine B, a thiol-reactive fluorescent dye, administered via sugar-feeding can be used to stain the body tissue and seminal fluid of insects. Here, we report on the adaptation of this technique for male Ae. aegypti to allow for rapid assessment of competitiveness (mating success) during field releases. METHODOLOGY/PRINCIPLE FINDINGS: Marking was achieved by feeding males on 0.1, 0.2, 0.4 or 0.8% rhodamine B (w/v) in 50% honey solutions during free flight. All concentrations produced >95% transfer to females and successful body marking after 4 days of feeding, with 0.4 and 0.8% solutions producing the longest-lasting body marking. Importantly, rhodamine B marking had no effect on male mating competitiveness and proof-of-principle field releases demonstrated successful transfer of marked seminal fluid to females under field conditions and recapture of marked males. CONCLUSIONS/SIGNIFICANCE: These results reveal rhodamine B to be a potentially useful evaluation method for male-based SIT/IIT control strategies as well as a viable body marking technique for male-based mark-release-recapture experiments without the negative side-effects of traditional marking methods. As a standalone method for use in mating competitiveness assays, rhodamine B marking is less expensive than PCR (e.g. paternity analysis) and stable isotope semen labelling methods and less time-consuming than female fertility assays used to assess competitiveness of sterilised males.

Residual and pre-treatment application of starycide insect growth regulator (triflumuron) to control Aedes aegypti in containers.

BACKGROUND: Dengue is not endemic in Australia, although the vector mosquito Aedes aegypti is. In order to prevent dengue establishment, Australia maintains tight control of vector mosquitoes. Tight control programs often necessitate regular repeat application of treatments, and this is resource intensive. We sought to investigate the efficacy and longevity of triflurumon Starycide® Insect Growth Regulator under field conditions in tropical Australia against Aedes. We compared two doses (0.48 and 0.96 ppm), applied to water receptacles with delayed flooding by zero, two, and four weeks. RESULTS: We found greater than 90% inhibition of pupae development was achieved for up to 12 weeks. A dose of 0.96 ppm, when delayed for two or four weeks before flooding, provided 50 times inhibition relative to controls, for up to 22 weeks, suggesting triflumuron bound to the plastic substrate and offered a greater residual effect than applications without delayed flooding. CONCLUSION: We conclude that triflurumon offers excellent efficacy and longevity against Aedes in artificial containers. If deployed under standard conditions, re-application may not be required for up to 22 weeks, furthermore, initial applications could be performed under dry conditions, and this application method may improve the residual effects.

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.