Accurate identification of Australian mosquitoes using protein profiling.

Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.

Anncaliia algerae Microsporidial Myositis, New South Wales, Australia.

We describe the successful management of Anncaliia algerae microsporidial myositis in a man with graft versus host disease after hemopoietic stem cell transplantation. We also summarize clinical presentation and management approaches and discuss the importance of research into the acquisition of this infection and strategies for prevention.

New genotypes of Liao ning virus (LNV) in Australia exhibit an insect-specific phenotype.

Liao ning virus (LNV) was first isolated in 1996 from mosquitoes in China, and has been shown to replicate in selected mammalian cell lines and to cause lethal haemorrhagic disease in experimentally infected mice. The first detection of LNV in Australia was by deep sequencing of mosquito homogenates. We subsequently isolated LNV from mosquitoes of four genera (Culex, Anopheles, Mansonia and Aedes) in New South Wales, Northern Territory, Queensland and Western Australia; the earliest of these Australian isolates were obtained from mosquitoes collected in 1988, predating the first Chinese isolates. Genetic analysis revealed that the Australian LNV isolates formed two new genotypes: one including isolates from eastern and northern Australia, and the second comprising isolates from the south-western corner of the continent. In contrast to findings reported for the Chinese LNV isolates, the Australian LNV isolates did not replicate in vertebrate cells in vitro or in vivo, or produce signs of disease in wild-type or immunodeficient mice. A panel of human and animal sera collected from regions where the virus was found in high prevalence also showed no evidence of LNV-specific antibodies. Furthermore, high rates of virus detection in progeny reared from infected adult female mosquitoes, coupled with visualization of the virus within the ovarian follicles by immunohistochemistry, suggest that LNV is transmitted transovarially. Thus, despite relatively minor genomic differences between Chinese and Australian LNV strains, the latter display a characteristic insect-specific phenotype.

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.

EFFECTS OF SUGAR CONCENTRATION ON FECUNDITY, BITING BEHAVIOR AND SURVIVABILITY OF FEMALE AEDES (STEGOMYIA) ALBOPICTUS (SKUSE).

This study was conducted to better understand the effect of different sucrose concentrations on Aedes albopictus fecundity, biting behavior and survival. Laboratory strain Ae. albopictus females were raised at four different sucrose concentrations (10%, 30%, 50%, and 70%) and their fecundity, host biting on and survival rates were determined. Mosquitoes fed on high (50% or 70%) showed higher mean fecundity rate compared to those on low (10% or 30%) sucrose concentration, and had higher daily biting rate. On the other hand, mosquitoes fed on the low (10% or 30%) sucrose concentrations recorded higher survival rate. These results suggest female mosquitoes deficient in nutrient intake during sugar feeding may regain nutrients needed during blood feeding, whereas those fed on high sucrose concentration have high fecundity due to high biting rate but have low survivability due to low sucrose intake during sugar feeding. Thus, Ae. albopictus females have a capability to regulate their metabolic needs based on sugar nutrient availability.

Anncaliia algerae microsporidial myositis.

The insect microsporidian Anncaliia algerae was first described in 2004 as a cause of fatal myositis in an immunosuppressed person from Pennsylvania, USA. Two cases were subsequently reported, and we detail 2 additional cases, including the only nonfatal case. We reviewed all 5 case histories with respect to clinical characteristics, diagnosis, and management and summarized organism life cycle and epidemiology. Before infection, all case-patients were using immunosuppressive medications for rheumatoid arthritis or solid-organ transplantation. Four of the 5 case-patients were from Australia. All diagnoses were confirmed by skeletal muscle biopsy; however, peripheral nerves and other tissues may be infected. The surviving patient received albendazole and had a reduction of immunosuppressive medications and measures to prevent complications. Although insects are the natural hosts for A. algerae, human contact with water contaminated by spores may be a mode of transmission. A. algerae has emerged as a cause of myositis, particularly in coastal Australia.

A scoping review of evidence of naturally occurring Japanese encephalitis infection in vertebrate animals other than humans, ardeid birds and pigs.

Japanese encephalitis virus (JEV) is the leading cause of human encephalitis in Asia. JEV is a vector-borne disease, mainly transmitted by Culex mosquitoes, with Ardeidae birds as maintenance hosts and pigs as amplifying hosts. Other vertebrate animal hosts have been suggested to play a role in the epidemiology of JEV. This scoping review followed PRISMA guidelines to identify species in which evidence of naturally occurring JEV infection was detected in vertebrates other than ardeid birds, pigs and people. Following systematic searches, 4372 records were screened, and data were extracted from 62 eligible studies. Direct evidence (virus, viral antigen or viral RNA) of JEV infection was identified in a variety of mammals and birds (not always identified to the species level), including bats, passerine birds (family Turdidae), livestock (cattle [Bos taurus] and a goat [Capra hircus]), carnivores (two meerkats [Suricata suricatta]), and one horse (Equus caballus). Bat families included Pteropodidae, Vespertilionidae, Rhinolophidae, Miniopteridae, Hipposideridae. Indirect evidence (antibodies) was identified in several mammalian and avian orders, as well as reported in two reptile species. However, a major limitation of the evidence of JEV infection identified in this review was diagnostic test accuracy, particularly for serological testing. Studies generally did not report diagnostic sensitivity or specificity which is critical given the potential for cross-reactivity in orthoflavivirus detection. We hypothesise that bats and passerine birds could play an underappreciated role in JEV epidemiology; however, development of diagnostic tests to differentiate JEV from other orthoflaviviruses will be essential for effective surveillance in these, as well as the companion and livestock species that could be used to evaluate JEV control measures in currently endemic regions.

An efficient and biodegradable alginate-gelatin hydrogel beads as bait against Aedes aegypti and Aedes albopictus.

The effective control of Aedes mosquitoes using traditional control agents is increasingly challenging due to the presence of insecticide resistance in many populations of key mosquito vectors. An alternative strategy to insecticides is the use of toxic sugar baits, however it is limited due to short-term efficacy. Alginate-Gelatin hydrogel beads (AGHBs) may be an effective alternative by providing longer periods of mosquito attraction and control, especially of key vectors of dengue viruses such as Aedes aegypti and Aedes albopictus. Sodium alginate (ALG) and gelatin (GLN) are natural polymers, which can be a potential candidate to develop the AGHBs baits due to their biodegradability and environmental safety. Here we provide an assessment of the preparation of AGHBs optimized by varying the concentrations of ALG, GLN, and its cross-linking time (TIME). Fourier transform infrared spectroscopy (FTIR) analysis results in the determination of liquid bait loaded in the AGHBs. The evaluation of AGHBs' effectiveness as the potential baiting tool based on the mortality rate of mosquitoes after the bait consumption. The 100 % percent mortality of Aedes mosquitoes was obtained within 72 h of bait consumption. The field evaluation also justifies the applicability of AGHBs for outdoor applications. We conclude that the AGHBs are applicable as a baiting tool in carrying liquid bait in achieving mosquito mortality.

Does the monomolecular film aquatain mosquito formula provide effective control of container-breeding mosquitoes in Australia?

The mosquito control potential of the silicone-based monomolecular film Aquatain Mosquito Formula (AMF) was investigated in field tests against the backyard mosquitoes Aedes notoscriptus and Culex quinquefasciatus. Plastic tubs, with and without emergent aquatic vegetation (Cyperus alternifolius), were sampled weekly for 2 wk prior to an application of Aquatain and up to 6 wk postapplication. The mean abundance of mosquito larvae and pupae was compared between pre- and postapplication periods as well as between treatment and control tubs. There was a significant reduction in the abundance of immature stages of both Ae. notoscriptus and Cx. quinquefasciatus within 48 h of application, and the mean weekly abundance of larvae of both species was significantly lower in treatment tubs compared with control tubs for up to 6 wk postapplication. Egg rafts, larvae, and pupae were not detected in treatment tubs until 5 wk postapplication. The results indicate that AMF holds great potential for mosquito control in backyard habitats.

The importance of males: larval diet and adult sugar feeding influences reproduction in Culex molestus.

Culex molestus is an obligatory autogenous mosquito that is closely associated with subterranean habitats in urban areas. The objective of our study was to investigate the influence of larval and adult nutrition on the role of males in determining the expression of autogeny in Cx. molestus. Mosquitoes raised at low and high larval diets had sex ratio, wing length, mating rates, autogenous egg raft size, and hatching rates recorded. There was a higher ratio of males to females when raised at a low larval diet. Mean wing lengths of both males and females were significantly greater when raised at the high larval diet regime. Regardless of larval or adult diet, males mated with only a single female. Mosquitoes raised at the higher larval diet regimes developed significantly more autogenous eggs. However, the egg raft size was reduced when adult females were denied access to sugar. The results of this study indicate that the performance of males in the reproductive process is influenced by both larval diet and adult sugar feeding.

Japanese Encephalitis Virus Emergence in Australia: Public Health Importance and Implications for Future Surveillance.

Japanese encephalitis virus (JEV) continues to cause significant numbers of human infections and fatalities despite the availability of efficacious vaccines. It is regarded as an emerging mosquito-borne pathogen with the potential of introduction into many countries. In 2022, JEV was detected in Australia on a hitherto unprecedented scale, with local transmission by indigenous mosquitoes to amplifying swine hosts and to humans. In this study, we review this recent disease activity, propose possible routes of virus movement, ecological drivers of activity, and consider possible future transmission scenarios. Measures to enhance current surveillance systems and potential strategies for health authorities to minimize future risks are discussed.

Citizen Science Mosquito Surveillance by Ad Hoc Observation Using the iNaturalist Platform.

Citizen science mosquito surveillance has been growing in recent years due to both increasing concern about mosquito-borne disease and the increasing popularity of citizen science projects globally. Health authorities are recognising the potential importance of citizen science to expanding or enhancing traditional surveillance programs. Different programs have shown success in engaging communities to monitor species of medical importance through low-cost methods. The Mozzie Monitors project was established on iNaturalist-an open citizen science platform that allows participants to upload photos (i.e., observers) and assist identification (i.e., identifiers). This article describes the likelihood of citizen scientists submitting photos of mosquitoes, assesses user submission behaviour, and evaluates public health utility from these citizen science-derived data. From October 2018 to July 2021, the Mozzie Monitors project on iNaturalist received 2118 observations of 57 different species of mosquitoes across Australia. The number of observers in the system increased over time with more than 500 observers and 180 identifiers being active in the project since its establishment. Data showed species bias with large-bodied and colourful mosquitoes being over-represented. Analyses also indicate regional differentiation of mosquito fauna per state, seasonality of activity, and ecological information about mosquitoes. The iNaturalist citizen science platform also allows connectedness, facilitated communication and collaboration between overall users and expert entomologists, of value to medical entomology and mosquito management.

Supporting urban ecosystem services across terrestrial, marine and freshwater realms.

The terrestrial, freshwater and marine realms all provide essential ecosystem services in urban environments. However, the services provided by each realm are often considered independently, which ignores the synergies between them and risks underestimating the benefits derived collectively. Greater research collaboration across these realms, and an integrated approach to management decisions can help to support urban developments and restoration projects in maintaining or enhancing ecosystem services. The aim of this paper is to highlight the synergies and trade-offs among ecosystem services provided by each realm and to offer suggestions on how to improve current practice. We use case studies to illustrate the flow of services across realms. In our call to better integrate research and management across realms, we present a framework that provides a 6-step process for conducting collaborative research and management with an Australian perspective. Our framework considers unifying language, sharing, and understanding of desired outcomes, conducting cost-benefit analyses to minimise trade-offs, using multiple modes of communication for stakeholders, and applying research outcomes to inform regulation. It can be applied to improve collaboration among researchers, managers and planners from all realms, leading to strategic allocation of resources, increased protection of urban natural resources and improved environmental regulation with broad public support.

Assessing the Risk of Exotic Mosquito Incursion through an International Seaport, Newcastle, NSW, Australia.

Exotic mosquitoes, especially container-inhabiting species such as Aedes aegypti and Aedes albopictus, pose a risk to Australia as they bring with them potentially significant pest and public health concerns. Notwithstanding the threat to public health and wellbeing, significant economic costs associated with the burden of mosquito control would fall to local authorities. Detection of these mosquitoes at airports and seaports has highlighted pathways of introduction but surveillance programs outside these first ports of entry are not routinely conducted in the majority of Australian cities. To assist local authorities to better prepare response plans for exotic mosquito incursions, an investigation was undertaken to determine the extent of habitats suitable for container-inhabiting mosquitoes in over 300 residential properties adjacent to the Port of Newcastle, Newcastle, NSW. More than 1500 water-holding containers were recorded, most commonly pot plant saucers, roof gutters, and water-holding plants (e.g., bromeliads). There were significantly more containers identified for properties classified as untidy but there was no evidence visible that property characteristics could be used to prioritise property surveys in a strategic eradication response. The results demonstrate that there is potential for local establishment of exotic mosquitoes and that considerable effort would be required to adequately survey these environments for the purpose of surveillance and eradication programs.

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.

Reflections on a highly unusual summer: bushfires, COVID-19 and mosquito-borne disease in NSW, Australia.

Mosquitoes and mosquito-borne disease are a normal part of the Australian summer but the 2019-2020 summer was anything but normal. Above average temperatures and below average rainfall resulted in drought across many parts of New South Wales (NSW), Australia, which then contributed to catastrophic bushfires. However, by late summer, above average rainfall resulted in a dramatic increase in mosquito abundance. While the coronavirus disease 2019 (COVID-19) pandemic unfolded, NSW experienced increased activity of mosquito-borne Ross River virus. All these extreme events created many challenges for managing the pest and the public health risks associated with mosquitoes, from maintenance of mosquito monitoring and control programs through to unique challenges of communicating mosquito bite prevention advice to local communities. There are important lessons to be learned in situations where extreme weather events may influence the risk of mosquito-borne disease through driving changes in the abundance and diversity of mosquito populations, while also influencing the abundance and distribution of native wildlife that represents important local reservoirs of arboviruses. Similarly, supporting the maintenance of mosquito monitoring and management programs while local authorities face competing priorities due to extreme natural disasters and/or public health events is critical.

Citizen science and smartphone e-entomology enables low-cost upscaling of mosquito surveillance.

Mosquito surveillance remains a cornerstone of pest and disease control operations globally but is strongly limited in scale by resources. The use of citizen science to upscale scientific data collection is commonplace, and mosquito surveillance programs have begun to make use of citizen scientists in several countries, particularly for exotic species detection. Here we report on a proof of concept trial in southern Australia for a citizen science mosquito surveillance program characterised by fixed point trapping with BG GAT devices and remote mosquito identification through emailed images, which we term 'e-entomology'. In a study with 126 participants, we detected mosquito seasonality with peak abundance in mid-summer (1.78 mosquitoes per trap per day), weather correlations (positive correlation with maximum temperature, r = 0.41) and a diversity of species (15 of 22 known species in the region) in a metropolitan setting. Whilst we demonstrated that the costs of a citizen science program is only about 20% of a comparable professional surveillance program, the mosquito community sampled by citizen scientists was biased towards container-inhabiting species, particularly Aedes notoscriptus. This is the first time fixed-point mosquito trapping has been combined with citizen science e-entomology to deliver comprehensive surveillance of urban mosquitoes.

Integrating statistical and mechanistic approaches with biotic and environmental variables improves model predictions of the impact of climate and land-use changes on future mosquito-vector abundance, diversity and distributions in Australia.

Changes to Australia's climate and land-use patterns could result in expanded spatial and temporal distributions of endemic mosquito vectors including Aedes and Culex species that transmit medically important arboviruses. Climate and land-use changes greatly influence the suitability of habitats for mosquitoes and their behaviors such as mating, feeding and oviposition. Changes in these behaviors in turn determine future species-specific mosquito diversity, distribution and abundance. In this review, we discuss climate and land-use change factors that influence shifts in mosquito distribution ranges. We also discuss the predictive and epidemiological merits of incorporating these factors into a novel integrated statistical (SSDM) and mechanistic species distribution modelling (MSDM) framework. One potentially significant merit of integrated modelling is an improvement in the future surveillance and control of medically relevant endemic mosquito vectors such as Aedes vigilax and Culex annulirostris, implicated in the transmission of many arboviruses such as Ross River virus and Barmah Forest virus, and exotic mosquito vectors such as Aedes aegypti and Aedes albopictus. We conducted a focused literature search to explore the merits of integrating SSDMs and MSDMs with biotic and environmental variables to better predict the future range of endemic mosquito vectors. We show that an integrated framework utilising both SSDMs and MSDMs can improve future mosquito-vector species distribution projections in Australia. We recommend consideration of climate and environmental change projections in the process of developing land-use plans as this directly impacts mosquito-vector distribution and larvae abundance. We also urge laboratory, field-based researchers and modellers to combine these modelling approaches. Having many different variations of integrated (SDM) modelling frameworks could help to enhance the management of endemic mosquitoes in Australia. Enhanced mosquito management measures could in turn lead to lower arbovirus spread and disease notification rates.