A Multi-Species Simulation of Mosquito Disease Vector Development in Temperate Australian Tidal Wetlands Using Publicly Available Data.

Worldwide, mosquito monitoring and control programs consume large amounts of resources in the effort to minimise mosquito-borne disease incidence. On-site larval monitoring is highly effective but time consuming. A number of mechanistic models of mosquito development have been developed to reduce the reliance on larval monitoring, but none for Ross River virus, the most commonly occurring mosquito-borne disease in Australia. This research modifies existing mechanistic models for malaria vectors and applies it to a wetland field site in Southwest, Western Australia. Environmental monitoring data were applied to an enzyme kinetic model of larval mosquito development to simulate timing of adult emergence and relative population abundance of three mosquito vectors of the Ross River virus for the period of 2018-2020. The model results were compared with field measured adult mosquitoes trapped using carbon dioxide light traps. The model showed different patterns of emergence for the three mosquito species, capturing inter-seasonal and inter-year variation, and correlated well with field adult trapping data. The model provides a useful tool to investigate the effects of different weather and environmental variables on larval and adult mosquito development and can be used to investigate the possible effects of changes to short-term and long-term sea level and climate changes.

Quantitative PCR assay for the detection of Aedes vigilax in mosquito trap collections containing large numbers of morphologically similar species and phylogenetic analysis of specimens collected in Victoria, Australia.

BACKGROUND: Aedes vigilax is one of the most significant arbovirus vector and pest species in Australia's coastal regions. Occurring in multiple countries, this mosquito species occurs as a species complex which has been separated into three clades with two detected in Australia. Until recently, Ae. vigilax has largely been absent from Victoria, only occasionally caught over the years, with no reported detections from 2010 to 2016. Complicating the detection of Ae. vigilax is the shared sympatric distribution to the morphologically similar Ae. camptorhynchus, which can exceed 10,000 mosquitoes in a single trap night in Victoria. Currently, there are no molecular assays available for the detection of Ae. vigilax. We aim to develop a quantitative PCR (qPCR) for the detection of Ae. vigilax, with the specificity and sensitivity of this assay assessed as well as a method to process whole mosquito traps. METHODS: Trapping was performed during the 2017-2020 mosquito season in Victoria in two coastal areas across these 3 consecutive years. A qPCR assay was designed to allow rapid identification of Ae. vigilax as well as a whole mosquito trap homogenizing and processing methodology. Phylogenetic analysis was performed to determine which clade Ae. vigilax from Victoria was closest to. RESULTS: Aedes vigilax was successfully detected each year across two coastal areas of Victoria, confirming the presence of this species. The qPCR assay was proven to be sensitive and specific to Ae. vigilax, with trap sizes up to 1000 mosquitoes showing no inhibition in detection sensitivity. Phylogenetic analysis revealed that Ae. vigilax from Victoria is associated with clade III, showing high sequence similarity to those previously collected in New South Wales, Queensland and Western Australia. CONCLUSIONS: Aedes vigilax is a significant vector species that shares an overlapping distribution to the morphologically similar Ae. camptorhynchus, making detection difficult. Here, we have outlined the implementation of a specific and sensitive molecular screening assay coupled with a method to process samples for detection of Ae. vigilax in collections with large numbers of non-target species.

Response of Mosquitoes Associated with Estuarine Wetlands to Bushfire in Australia.

The response of mosquitoes to bushfire is poorly understood. During the 2019-20 summer, many regions of Australia were impacted by devastating bushfires. An area of estuarine and brackish-water wetlands alongside the Georges River, Sydney, New South Wales, was burned in January 2020. Mosquito populations within the area were monitored as part of the local authority's mosquito management program, providing a unique opportunity to record the response of key mosquitoes of pest and public health concern to bushfire. Ground pools within a tidally influenced swamp oak forest dominated by Casuarina glauca and associated wetlands dominated by Phragmites australis and Bolboschoenus spp. had been identified as suitable habitat for a range of mosquitoes, including Aedes alternans, Ae. vigilax, and Verrallina funerea. Surveys of immature stages of mosquitoes within recently burned habitats inundated by tides demonstrated that mosquito eggs survived the direct and indirect impacts of fire and immature stages successfully completed development as reflected in concomitant changes in adult mosquito populations following the bushfire. This unique observation has implications for mosquito management following bushfire in Australia and internationally.

The Insect-Specific Parramatta River Virus Is Vertically Transmitted by Aedes vigilax Mosquitoes and Suppresses Replication of Pathogenic Flaviviruses In Vitro.

Insect-specific flaviviruses (ISFs) have been isolated from a range of mosquito species from different parts of the world. These viruses replicate efficiently in mosquitoes but do not appear to replicate in vertebrates. There is increasing evidence that ISFs persist in nature through vertical transmission, and that they interfere with the replication and transmission of pathogenic flaviviruses in the mosquito host. A novel ISF species, Parramatta River virus (PaRV), was previously shown to occur at high rates in Aedes (Ae.) vigilax mosquitoes collected from Sydney, Australia. We investigated whether vertical transmission was the mechanism of viral persistence in Ae. vigilax populations and whether PaRV affected replication of the pathogenic flaviviruses, West Nile virus (WNV), and dengue virus type 3 (DENV-3) in cultured mosquito cells. Progeny reared from eggs obtained from field-collected infected females had infection rates as high as 142 and 85 per 1000 for females and males, respectively. In vitro experiments showed that replication of both WNV and DENV-3 was significantly suppressed in Aedes albopictus (C6/36) cells persistently infected with PaRV. Our studies with PaRV support the findings of previous investigations that ISFs persist in nature through vertical transmission and that ISFs can suppress the replication of pathogenic flaviviruses in coinfected mosquito cells.

Field Effectiveness of a Metofluthrin Fan-Based Emanator and Deet as Repellents Against Aedes vigilax in Southeast Queensland, Australia1.

A field study to compare a formulation containing 40% deet (N,N-diethyl-3-methyl benzamide) in ethanol (Bushman™) and a battery-powered fan emanator with a chemical strip containing 31.2% metofluthrin (OFF!® Clip-On™) was conducted at Redcliffe, Queensland, Australia, in February 2016. The 40% deet provided 100% protection against mosquitoes for 5 h until tests ceased, while the OFF! Clip-On device provided only 42.2-60.8% protection against Aedes vigilax during the same period.

Dispersal of the Mosquito Aedes vigilax (Diptera: Culicidae) From Urban Estuarine Wetlands in Sydney, Australia.

Aedes vigilax (Skuse) is a pest and vector species associated with coastal wetlands and the abundance of this mosquito has been identified as contributing to increased risk of mosquito-borne disease outbreaks. As urban development continues to encroach on these coastal wetlands, pest and public health impacts are becoming of increasing concern and in the absence of broadscale mosquito control. Urban planners are looking to buffer zones and other land use planning options to minimize contact between mosquitoes and humans but gaps in the understanding of dispersal ranges of mosquitoes hamper the adoption of these strategies. A mark-release-recapture experiment was conducted to measure the dispersal of this mosquito from an urban estuarine wetland in Sydney, Australia. An estimated total of over 150,000 wild caught female mosquitoes were marked with fluorescent dust and then released. A network of 38 traps was then operated for 5 d within an area of 28 km2. A total of 280 marked mosquitoes was recaptured, representing less than 1% of the estimate 250,000 marked mosquitoes released. Marked mosquitoes were recaptured up to 3 km from the release point, providing an insight into the dispersal range of these mosquitoes. The mean distance traveled by marked mosquitoes was 0.83 km, a result reflecting the greater proportion of marked mosquitoes recaptured near release point. The findings of this study indicate that effective buffer zones between estuarine wetlands and high-density urban developments would be an impractical approach to minimizing pest and public health impacts associated with this mosquito.

Temperature explains broad patterns of Ross River virus transmission.

Thermal biology predicts that vector-borne disease transmission peaks at intermediate temperatures and declines at high and low temperatures. However, thermal optima and limits remain unknown for most vector-borne pathogens. We built a mechanistic model for the thermal response of Ross River virus, an important mosquito-borne pathogen in Australia, Pacific Islands, and potentially at risk of emerging worldwide. Transmission peaks at moderate temperatures (26.4°C) and declines to zero at thermal limits (17.0 and 31.5°C). The model accurately predicts that transmission is year-round endemic in the tropics but seasonal in temperate areas, resulting in the nationwide seasonal peak in human cases. Climate warming will likely increase transmission in temperate areas (where most Australians live) but decrease transmission in tropical areas where mean temperatures are already near the thermal optimum. These results illustrate the importance of nonlinear models for inferring the role of temperature in disease dynamics and predicting responses to climate change.

"Looking over the Backyard Fence": Householders and Mosquito Control.

(1) Background: Vector-borne diseases are a significant public health problem in Western Australia. Mosquitoes are responsible for the transmission of a number of pathogens and may pose a serious nuisance problem. Prevention efforts in the State are multi-faceted and include physical, chemical, and cultural control methods for restricting mosquito breeding. This is less complex where breeding areas are located within public open spaces. In Australia's developed urban areas, breeding sites are, however, frequently located within private residential landholdings, where the scope of public health officials to act is constrained by law and practicality. Consequently, mosquito prevention in these locations is predominantly the responsibility of the residents. This research addressed a gap, both in understanding the degree to which "backyard" mosquito breeding has the potential to contribute to local mosquito problems, and in assessing what residents "think and do" about mosquito control within their home environment. (2) Methods: The study was conducted in the Town of Bassendean, a metropolitan Local Government Area of Perth, Western Australia, in close proximity to two natural, productive mosquito breeding sites, namely Ashfield Flats and Bindaring Park. A total of 150 householders were randomly surveyed during the summer of 2015-2016, to gauge residents' knowledge, attitudes, and practices (KAP (knowledge, attitudes, and practices) Survey) in regards to mosquitoes, their breeding and ecology, and avoidance or minimization strategies. The survey comprised nine questions covering residents' knowledge (3 questions), attitudes (3 questions), and practices (3 questions), as well as additional questions regarding the basic demographics of the resident. Larvae were collected from backyard containers and reared to adults for species identification. A series of Encephalitis Vector Surveillance carbon dioxide (EVS CO2) traps were also deployed, to assess adult mosquito density and species composition. (3) Results: Aedes notoscriptus (Skuse), a known container-inhabiting species, accounted for just over 50% of all mosquitoes identified. Most residents were aware of mosquito-borne disease and its risk in their local area. While the majority (79%) of the sample correctly identified Ross River virus as the most common infection in WA, a significant gap in the general knowledge of residents in regards to mosquito biology and breeding habits, was noted. Furthermore, only 50% of residents reported using personal protective measures to reduce mosquito bites and only one in six residents undertook physical or chemical mosquito control around their home. Additionally, 60% of respondents believed that mosquito control was "a job for the council and the state government", rather than for individual householders. (4) Conclusions: A significant gap in the knowledge of residents in the study area existed in regards to the general knowledge of mosquitoes and their breeding habits; types of treatments that could be employed within the home; and the residents' responsibility for the management of mosquito breeding on their private property. A public education campaign has been deployed to educate the residents.

The insect-specific Palm Creek virus modulates West Nile virus infection in and transmission by Australian mosquitoes.

BACKGROUND: Insect-specific viruses do not replicate in vertebrate cells, but persist in mosquito populations and are highly prevalent in nature. These viruses may naturally regulate the transmission of pathogenic vertebrate-infecting arboviruses in co-infected mosquitoes. Following the isolation of the first Australian insect-specific flavivirus (ISF), Palm Creek virus (PCV), we investigated routes of infection and transmission of this virus in key Australian arbovirus vectors and its impact on replication and transmission of West Nile virus (WNV). METHODS: Culex annulirostris, Aedes aegypti and Aedes vigilax were exposed to PCV, and infection, replication and transmission rates in individual mosquitoes determined. To test whether the virus could be transmitted vertically, progeny reared from eggs oviposited by PCV-inoculated Cx. annulirostris were analysed for the presence of PCV. To assess whether prior infection of mosquitoes with PCV could also suppress the transmission of pathogenic flaviviruses, PCV positive or negative Cx. annulirostris were subsequently exposed to WNV. RESULTS: No PCV-infected Cx. annulirostris were detected 16 days after feeding on an infectious blood meal. However, when intrathoracically inoculated with PCV, Cx. annulirostris infection rates were 100 %. Similar rates of infection were observed in Ae. aegypti (100 %) and Ae. vigilax (95 %). Notably, PCV was not detected in any saliva expectorates collected from any of these species. PCV was not detected in 1038 progeny reared from 59 PCV-infected Cx. annulirostris. After feeding on a blood meal containing 10(7) infectious units of WNV, significantly fewer PCV-infected Cx. annulirostris were infected or transmitted WNV compared to PCV negative mosquitoes. Immunohistochemistry revealed that PCV localized in the midgut epithelial cells, which are the first site of infection with WNV. CONCLUSIONS: Our results indicate that PCV cannot infect Cx. annulirostris via the oral route, nor be transmitted in saliva or vertically to progeny. We also provide further evidence that prior infection with insect-specific viruses can regulate the infection and transmission of pathogenic arboviruses.

A novel insect-specific flavivirus replicates only in Aedes-derived cells and persists at high prevalence in wild Aedes vigilax populations in Sydney, Australia.

To date, insect-specific flaviviruses (ISFs) have only been isolated from mosquitoes and increasing evidence suggests that ISFs may affect the transmission of pathogenic flaviviruses. To investigate the diversity and prevalence of ISFs in Australian mosquitoes, samples from various regions were screened for flaviviruses by ELISA and RT-PCR. Thirty-eight pools of Aedes vigilax from Sydney in 2007 yielded isolates of a novel flavivirus, named Parramatta River virus (PaRV). Sequencing of the viral RNA genome revealed it was closely related to Hanko virus with 62.3% nucleotide identity over the open reading frame. PaRV failed to grow in vertebrate cells, with only Aedes-derived mosquito cell lines permissive to replication, suggesting a narrow host range. 2014 collections revealed that PaRV had persisted in A. vigilax populations in Sydney, with 88% of pools positive. Further investigations into its mode of transmission and potential to influence vector competence of A. vigilax for pathogenic viruses are warranted.

Laboratory evaluation of predation on mosquito larvae by Australian mangrove fish.

A series of laboratory experiments compared predation rates of three native eastern Australian mangrove fish species (Psuedomugil signifer, Hyseleotris galii, Pseudogobius sp.) and the exotic Gambusia holbrooki on 2nd and 4th instar Aedes vigilax larvae, in order to determine their potential as mosquito control agents in mangrove forests. All four species preyed on significant numbers of both 2nd and 4th instar larvae. All showed a similar pattern of larval consumption, gorging on larvae in the first hour of each experiment, before reducing to a relatively constant background feeding rate. Gambusia holbrooki showed the highest larval consumption rates, but is unsuitable as a mosquito control agent due to it being an exotic pest species in Australia. Of the three native species, P. signifer showed the greatest potential as a mosquito control agent, having consumption rates comparable to G. holbrooki, and was the only species that did not show a significant reduction in larval consumption in the night experiments.

Comparing Aedes vigilax Eggshell Densities in Saltmarsh and Mangrove Systems with Implications for Management.

Aedes vigilax (Skuse), a nuisance and disease vector, is prolific in intertidal wetlands in Australia. Aedine mosquitoes oviposit directly onto substrate. The eggshells are relatively stable spatially and temporally, providing an estimate of mosquito larval production. The aims of the research were to compare, at a general level, oviposition in mangroves and saltmarshes, and to compare oviposition between different habitats within mangroves and saltmarshes. The results indicated that there were no significant differences between production in mangrove and saltmarsh overall. However, within each system there were significant differences between habitat classes, with mangrove hummocks being the most productive. All classes, except for fringing mangrove forests, produced sufficient densities of eggshells (>0.05/cc) to warrant concern. While mosquito production in mangroves is known, the significantly higher production rates in the mangrove hummock habitats had not been demonstrated. This warrants improved management strategies that both specifically target these parts of mangrove systems and, secondly, addresses the longer-term potential for mangrove hummock habitats developing in the future; such as, in response to sea level rise and mangrove encroachment into saltmarsh. A strategy to increase tidal flushing within the systems would improve water quality and mitigate adverse impacts while providing a source reduction outcome.

The impact of encroachment of mangroves into saltmarshes on saltwater mosquito habitats.

Will mangrove encroachment into saltmarshes affect saltwater mosquito habitats? To address this, we synthesized information from two perspectives: 1) at a detailed level, the immature mosquito habitat within mangroves; 2) at a more general or regional level, changes due to mangrove expansion into saltmarshes. This is a synthesis of two research projects. One showed that mosquito larval habitats in mangroves are complex, related to the detailed interactions between topography and tidal patterns and that not all parts of a mangrove forest are suitable habitat. The other, based on remote sensing and analysis of rainfall data, showed that mangrove encroachment in eastern Australia is related to both climate and human land use over several decades (1972-2004). An important question emerged: when mangroves encroach into saltmarshes will they displace saltmarsh immature mosquito habitats or will they replace them with mangrove ones? There is no simple answer: it will vary with climate change and sea level scenario and how these affect the system. We conclude that mosquito management, which is locally implemented, needs to be integrated with land use planning systems, which often operate at a more general level.