Biology subjects

Devine, G. J.

Publications and source records attributed to Devine, G. J..

13 recordsLinked to original sources

bioRxiv2024

Fitness compatibility and dengue virus inhibition in a Bangladeshi strain of Aedes aegypti infected with the Wolbachia strain wAlbB

Dengue cases in Bangladesh have surged in recent years. The existing insecticide-based control program, implemented in parts of the country, is challenged by issues of insufficient household coverage and high levels of insecticide resistance in the primary dengue virus (DENV) vector, Aedes aegypti. A more sustainable, effective alternative could be the implementation of a Wolbachia-mediated disease management strategy. Infecting mosquitoes with Wolbachia can change their reproductive compatibility and their ability to transmit DENV. These new phenotypes can be exploited to suppress or replace wild-type Ae. aegypti populations. Such strategies require the development of well-characterised Wolbachia-infected strains with biological characteristics that are comparable with local mosquitoes. We created and characterised a Wolbachia-infected Ae. aegypti strain with a Dhaka wild-type genetic background, and compared its reproductive compatibility, maternal inheritance, fitness, and virus-blocking ability to the parental strains (Dhaka wild-type and wAlbB2-F4). The new Ae. aegypti strain wAlbB2-Dhaka demonstrated complete cytoplasmic incompatibility with the wild-type strain and complete maternal transmission, retaining levels of pyrethroid resistance of the Dhaka wild-type (70% survival to 10 times the dose of permethrin expected to kill susceptible mosquitoes). No significant fitness costs were detected during laboratory comparisons of fecundity, fertility, survival, mating competitiveness, or desiccation tolerance. Compared to the wild-type strain, wAlbB2-Dhaka mosquitoes had a significantly reduced number of DENV genome copies in the bodies (44.4%, p = 0.0034); two-fold reduction in dissemination to legs and wings (47.6%, p < 0.0001); and >13-fold reduction of DENV in saliva expectorates (proxy of transmission potential) (92.7%, p < 0.0001) 14 days after ingesting dengue-infected blood. Our work indicates that the wAlbB2-Dhaka strain could be used for Ae. aegypti suppression or replacement strategies for dengue management in Bangladesh. Author summaryBangladesh is currently grappling with a series of severe dengue outbreaks, resulting in more than 300 thousand infections, 1700 fatalities, and a significant influx of patients requiring hospitalisation in 2023. These outbreaks coincide with the emergence of high levels of insecticide resistance in the Ae. aegypti population, which is contributing to the failure of conventional, insecticidal control strategies. In search of more sustainable, effective dengue control strategies, approaches using the endosymbiotic bacteria Wolbachia that render mosquitoes resistant to arbovirus infection are being trialled in a number of countries. We transinfected Dhaka wild-type Ae. aegypti with the Wolbachia strain wAlbB2 by backcrossing it with a Wolbachia wAlbB2-infected Ae. aegypti strain and assessed the new strains suitability for field release. We compared the fitness and virus-blocking ability of the backcrossed strain with its parental strains. We demonstrated that the new strain, wAlbB2-Dhaka, is as fit as the Dhaka wild-type strain and blocked [~]92% of dengue virus transmission. This strain has great potential for Wolbachia-mediated dengue management strategies in Bangladesh.

zoology↗

bioRxiv2024

Long term co-circulation of multiple arboviruses in southeast Australia revealed by xeno-monitoring of mosquitoes and metatranscriptomics

Arbovirus surveillance of wild-caught mosquitoes is an affordable and sensitive means of monitoring virus transmission dynamics at various spatial-temporal scales, and emergence and re-emergence during epidemic and interepidemic periods. A variety of molecular diagnostics for arbovirus screening of mosquitoes (known as xeno-monitoring) are available, but most provide limited information about virus diversity. PCR-based screening coupled with metatranscriptomics is an increasingly affordable and sensitive pipeline for integrating complete viral genome sequencing into surveillance programs. This enables large-scale, high-throughput arbovirus screening from diverse samples. We collected mosquitoes in CO2-baited light traps from five urban parks in Brisbane from March 2021 to May 2022. Mosquito pools of [≤]200 specimens were screened for alphaviruses and flaviviruses using virus genus-specific primers and reverse transcription quantitative PCR (qRT-PCR). A subset of virus-positive samples was then processed using a mosquito-specific ribosomal RNA depletion method and then sequenced on the Illumina NextSeq. Overall, 54,670 mosquitoes, representing 26 species were screened in 382 pools. Thirty detections of arboviruses were made in 28 pools. Twenty of these positive pools were further characterised using meta-transcriptomics generating 18 full-length genomes. These full-length sequences belonged to four medically relevant arboviruses: Barmah Forest, Ross River, Sindbis-like and Stratford viruses. Phylogenetic and evolutionary analyses revealed the evolutionary progression of arbovirus lineages over the last 100 years, highlighting long-distance dispersal across the Australian continent and continuous circulation characterised by constant turnover of virus lineages.

genomics↗

bioRxiv2022

Investigation on key aspects of mating biology in the mosquito Aedes koreicus

Aedes koreicus is a mosquito (Diptera: Culicidae) from Northeast Asia with a rapidly expanding presence outside its original native range. Over the years the species has been discovered in several new countries, either spreading after first introduction or remaining localised to limited areas. Notably, recent studies have demonstrated the ability of the species to transmit zoonotic parasites and viruses both in the field and in laboratory settings. Combined with its invasive potential, the possible role of Ae. koreicus in pathogen transmission highlights the public health risks of its invasion. In this study, we used a recently established population from Italy to investigate aspects of biology that influence reproductive success in Ae. koreicus: autogeny, mating behaviour, mating disruption by the sympatric invasive species Aedes albopictus, and the presence of the endosymbiont Wolbachia pipientis. Our laboratory population did not exhibit autogenic behaviour and required a blood meal to complete its ovarian cycle. When we exposed Ae. koreicus females to males of Ae. albopictus, we observed repeated attempts at insemination and an aggressive, disruptive mating behaviour initiated by males. Despite this, no sperm was identified in Ae. koreicus spermathecae. Wolbachia was not detected in this Ae. koreicus population and therefore had no effect on Ae. koreicus reproduction.

ecology↗

bioRxiv2021

A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros)

BackgroundAn optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetles mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. ResultsHigh-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.11%). These quality metrics place our assembly as the most complete of the published Coleopteran genomes. The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes showing BUSCO completeness of 92.09%, as well as the expected number of non-coding RNAs and the number and structure of paralogous genes in a gene family like Sigma GST. ConclusionsThe genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

genomics↗

bioRxiv2020

Temperature alters gene expression in mosquitoes during arbovirus infection

Arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya constitute a significant proportion of the global disease burden. The principal vector of these pathogens is the mosquito Aedes (Ae.) aegypti, and its ability to transmit virus to a human host is influenced by environmental factors such as temperature. However, exactly how ambient temperature influences virus replication within mosquitoes remains poorly elucidated, particularly at the molecular level. Here, we use chikungunya virus (CHIKV) as a model to understand how the host mosquito transcriptome responds to arbovirus infection under different ambient temperatures. We exposed CHIKV-infected mosquitoes to 18 {degrees}C, 28 {degrees}C and 32 {degrees}C, and found higher temperature correlated with higher virus replication levels, particularly at early time points post-infection. Lower ambient temperatures resulted in reduced virus replication levels. Using RNAseq, we found that temperature significantly altered gene expression levels in mosquitoes, particularly components of the immune response. The highest number of significantly differentially expressed genes in response to CHIKV was observed at 28 {degrees}C, with a markedly more muted effect observed at either lower (18 {degrees}C) or higher (32 {degrees}C) temperatures. At the higher temperature, the expression of many classical immune genes, including Dicer-2 in the RNAi pathway, was not substantially altered in response to CHIKV. Upregulation of Toll, IMD and JAK-STAT pathways was only observed at 28 {degrees}C. Time post infection also led to substantially different gene expression profiles, and this effect varied depending upon the which temperature mosquitoes were exposed to. Taken together, our data indicate temperature significantly modulates mosquito gene expression in response to infection, potentially leading to impairment of immune defences at higher ambient temperatures.

microbiology↗

bioRxiv2020

Performance of aerial Bacillus thuringiensis var. israelensis applications in mixed saltmarsh-mangrove systems and use of affordable unmanned aerial systems to identify problematic levels of canopy cover

BACKGROUNDIn the Australian southeast, the saltmarsh mosquito Aedes vigilax (Skuse) is the focus of area-wide larviciding campaigns employing the biological agent Bacillus thuringiensis var. israelensis (Bti). Although generally effective, frequent inundating tides and considerable mangrove cover can make control challenging. Here, we describe the efficacy and persistence of an aqueous Bti suspension (potency: 1200 International Toxic Units; strain AM65-52) within a mixed saltmarsh-mangrove system and the use of affordable unmanned aerial systems (UAS) to identify and map problematic levels of mangrove canopy cover. RESULTSHigh mangrove canopy density (>40% cover) reduced product deposition by 74.5% (0.013{+/-} 0.002 l/cm2 vs. 0.051{+/-} 0.006 l/cm2), larval mortality by 27.7% (60.7{+/-} 4.1% vs. 84.0{+/-} 2.4%), and ground level Bti concentrations by 32.03% (1144 {+/-} 462.6 vs. 1683 {+/-} 447.8 spores ml-1) relative to open saltmarsh. Persistence of product post-application was found to be low (80.6% loss at 6 h) resulting in negligible additional losses to tidal inundation 24 h post-application. UAS surveys accurately identified areas of high mangrove cover using both standard and multispectral imagery, although derived index values for this vegetation class were only moderately correlated with ground measurements (R2=0.17-0.38) at their most informative scales. CONCLUSIONThese findings highlight the complex operational challenges that affect coastal mosquito control in heterogeneous environments. The problem is exacerbated by continued mangrove transgression into saltmarsh habitat in the region. Emerging UAS technology can help operators optimize treatments by accurately identifying and mapping challenging canopy cover using both standard and multispectral imaging.

microbiology↗

bioRxiv2020

The complete mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) based on long-read nanopore sequencing

The coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is a severe and invasive pest of coconut and other palms throughout Asia and the Pacific. The biocontrol agent, Oryctes rhinoceros nudivirus (OrNV), has successfully suppressed O. rhinoceros populations for decades but new CRB invasions started appearing after 2007. A single-SNP variant within the mitochondrial cox1 gene is used to distinguish the recently-invading CRB-G lineage from other haplotypes, but the lack of mitogenome sequence for this species hinders further development of a molecular toolset for biosecurity and management programmes against CRB. Here we report the complete circular sequence and annotation for CRB mitogenome, generated to support such efforts. Sequencing data were generated using long-read Nanopore technology from genomic DNA isolated from a CRB-G female. The mitochondrial genome was assembled with Flye v.2.5, using the short-read Illumina sequences to remove homopolymers with Pilon, and annotated with MITOS. Independently-generated transcriptome data were used to assess the O. rhinoceros mitogenome annotation and transcription. The aligned sequences of 13 protein-coding genes (PCGs) (with degenerate third codon position) from O. rhinoceros, 13 other Scarabaeidae taxa and two outgroup taxa were used for the phylogenetic reconstruction with the Maximum likelihood (ML) approach in IQ-TREE and Bayesian (BI) approach in MrBayes. The complete circular mitochondrial genome of O. rhinoceros is 20,898 bp-long, with a gene content canonical for insects (13 PCGs, 2 rRNA genes, and 22 tRNA genes), as well as one structural variation (rearrangement of trnQ and trnI) and a long control region (6,204 bp). Transcription was detected across all 37 genes, and interestingly, within three domains in the control region. ML and BI phylogenies had the same topology, correctly grouping O. rhinoceros with one other Dynastinae taxon, and recovering the previously reported relationship among lineages in the Scarabaeidae. In silico PCR-RFLP analysis recovered the correct fragment set that is diagnostic for the CRB-G haplogroup. These results validate the high-quality of the CRB mitogenome sequence and annotation.

genomics↗

bioRxiv2020

What happens on islands, doesn't stay on islands: Patterns of synchronicity in mosquito nuisance and host-seeking activity between a mangrove island and adjacent coastal development

Coastal development is expanding globally in response to mass human migration, yet urban planning guidelines often overlook the problems that human encroachment on or near coastal mosquito habitat may cause. This study aimed at elucidating the frequency and magnitude of dispersal of highly vagile saltmarsh mosquitoes from productive off-shore bay islands to adjacent coastal human developments. Inter-population dynamics and daily host-seeking activity of saltmarsh mosquitoes were monitored daily at 15-minute intervals within a productive bay island and adjacent coastal development in southeast Queensland, Australia, using emerging smart trap technology over a 2-month period of high mosquito activity. The regulation of mosquito dispersal and host-seeking activity by local environmental factors, e.g. temperature, relative humidity and hourly wind patterns, were also investigated. The data show that the primary saltmarsh mosquitoes Aedes vigilax (Skuse) and Culex sitiens (Wiedemann) disperse from offshore breeding sites to neighboring mainland areas in high numbers and in highly synchronized waves despite unfavorable wind patterns and the need to traverse a considerable expanse (ca. 1.4 km) of open water. Patterns of host-seeking activity within each site were also remarkably similar despite notable differences in the local environment demonstrating a consistency in host-seeking activity across disparate habitats. These findings demonstrate that distant saltmarsh habitats, including offshore breeding sites, are likely to be primary sources of mosquito nuisance for coastal housing developments. This observation highlights the need to develop new planning and regulatory guidelines that alert urban planners to the risks of encroaching on habitats close to the sources of highly vagile mosquito species.

ecology↗

bioRxiv2020

Using spatial genetics to quantify mosquito dispersal for control programs

BackgroundHundreds of millions of people get a mosquito-borne disease every year, of which nearly one million die. Mosquito-borne diseases are primarily controlled and mitigated through the control of mosquito vectors. Accurately quantified mosquito dispersal in a given landscape is critical for the design and optimization of the control programs, yet the field experiments that measure dispersal of mosquitoes recaptured at certain distances from the release point (mark-release-recapture MRR studies) are challenging for such small insects and often unrepresentative of the insects true field behavior. Using Singapore as a study site, we show how mosquito dispersal patterns can be characterized from the spatial analyses of genetic relatedness among individuals sampled over a short time span without interruption of their natural behaviors. Methods and FindingsWe captured ovipositing females of Aedes aegypti, a major arboviral disease vector, across floors of high-rise apartment blocks and genotyped them using thousands of genome-wide SNP markers. We developed a methodology that produces a dispersal kernel for distance that results from one generation of successful breeding (effective dispersal), using the distances separating full siblings, 2nd and 3rd degree relatives (close kin). In Singapore, the estimated dispersal distance kernel was exponential (Laplacian), giving the mean effective dispersal distance (and dispersal kernel spread {sigma}) of 45.2 m (95%CI: 39.7-51.3 m), and 10% probability of dispersal >100 m (95%CI: 92-117 m). Our genetic-based estimates matched the parametrized dispersal kernels from the previously reported MRR experiments. If few close-kin are captured, a conventional genetic isolation-by-distance analysis can be used, and we show that it can produce {sigma} estimates congruent with the close-kin method, conditioned on the accurate estimation of effective population density. We also show that genetic patch size, estimated with the spatial autocorrelation analysis, reflects the spatial extent of the dispersal kernel tail that influences e.g. predictions of critical radii of release zones and Wolbachia wave speed in mosquito replacement programs. ConclusionsWe demonstrate that spatial genetics (the newly developed close-kin analysis, and conventional IBD and spatial autocorrelation analyses) can provide a detailed and robust characterization of mosquito dispersal that can guide operational vector control decisions. With the decreasing cost of next generation sequencing, acquisition of spatial genetic data will become increasingly accessible, and given the complexities and criticisms of conventional MRR methods, but the central role of dispersal measures in vector control programs, we recommend genetic-based dispersal characterization as the more desirable means of parameterization.

ecology↗

bioRxiv2020

A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: a useful tool for inferring transmission pathways

IntroductionMany arboviruses of public health significance are maintained in zoonotic cycles with complex transmission pathways. The presence of serum antibody against arboviruses in vertebrates provides evidence of their historical exposure but reveals nothing about the vector-reservoir relationship. Moreover, collecting blood or tissue samples from vertebrate hosts is ethically and logistically challenging. We developed a novel approach for screening the immune status of vertebrates against Ross River virus that allows us to implicate the vectors that form the transmission pathway for this commonly notified Australian arboviral disease. MethodsA micro-plaque reduction neutralisation test (micro-PRNT) was developed and validated on koala (Phascolarctos cinereus) sera against a standard PRNT. The ability of the micro-PRNT to detect RRV antibodies in mosquito blood meals was then tested using some convenient mosquito models. Laboratory-reared Aedes aegypti were fed, via a membrane, on sheep blood supplemented with RRV seropositive and seronegative human sera. Aedes notoscriptus were fed on RRV seropositive and seronegative human volunteers. Blood-fed mosquitoes were harvested at various time points after feeding and their blood meals analysed for the presence of RRV neutralizing antibodies using the micro-PRNT. ResultsThere was significant agreement of the plaque neutralization resulting from the micro-PRNT and standard PRNT techniques (R2=0.65; P<0.0001) when applied to RRV antibody detection in koala sera. Sensitivity and specificity of the micro-PRNT assay were 88.2% and 96%, respectively, in comparison with the standard PRNT. Blood meals from mosquitoes fed on sheep blood supplemented with RRV antibodies neutralised RRV by [≥]50% until 60 hr post feeding. Similarly, mosquito blood meals from RRV seropositive human volunteers neutralised the virus by [≥]50% until 48 hr post-blood feeding. ConclusionsThe small volumes of blood present in mosquito abdomens can be used to identify RRV antibodies and therefore host exposure to arbovirus infection. In tandem with the accurate identification of the mosquito, and diagnostics for the host origin of the blood meal, this technique has tremendous potential for exploring RRV transmission pathways. It can clearly be adapted for similar studies on other mosquito borne zoonoses.

microbiology↗

bioRxiv2019

Complete genome sequence of Oryctes rhinoceros Nudivirus isolated from Coconut Rhinoceros Beetle in the Solomon Islands

Oryctes Nudivirus (OrNV) has been an effective biocontrol agent against the insect pest Oryctes rhinoceros (Coleoptera: Scarabaeidae) for decades, but there is evidence that resistance could be evolving in some host populations. We detected OrNv infection in O. rhinoceros from the Solomon Islands and used Oxford Nanopore Technologies (ONT) long-read sequencing to determine the full length of the virus genomic sequence isolated from an individual belonging to a mitochondrial lineage (CRB-G) that was previously reported as resistant to OrNV. The complete circular genome of the virus consisted of 125,917 nucleotides, encoding 130 open reading frames (ORFs) for proteins in the range ~5 kDa (51aa) to ~140 kDa (1280aa). This Solomon Islands isolate has high (99.85%) nucleotide sequence identity to the previously sequenced isolate PV505 from Southern Luzon, Philippines, and has 138 amino acid modifications when compared with the originally described full genome sequence of the Ma07 strain from Malaysia.

genomics↗

bioRxiv2019

Spatial and epidemiologic features of dengue in Sabah, Malaysia

In South East Asia, dengue epidemics have increased in size and geographical distribution in recent years. Most studies investigating dengue transmission and control have had an urban focus, while less consideration is currently given to rural settings, or where urban and rural areas overlap. We examined the spatiotemporal distribution and epidemiological characteristics of reported dengue cases in the predominantly rural state of Sabah, in Malaysian Borneo - an area where sylvatic and urban circulation of pathogens are known to intersect. We found that annual dengue incidence rates were spatially variable over the 7-year study period from 2010-2016 (state-wide mean annual incidence of 21 cases/100,000 people; range 5-42/100,000), but were highest in rural localities in the western districts of the state (Kuala Penyu, Nabawan, Tenom and Kota Marudu). The eastern districts exhibited lower overall dengue rates; however, we noted a concentration of severe (haemorrhagic) dengue cases (44%) in Sandakan and Tawau districts. Dengue incidence was slightly higher for males than females, and was significantly higher for both genders aged between 10 and 29 years (24/100,000; p=0.029). The largest ever recorded outbreaks occurred during 2015-2016, with the vector Aedes albopictus found to be most prevalent in both urban and rural households (House Index of 64%), compared with Ae. Aegypti (15%). These findings suggest that dengue outbreaks in Sabah are driven by the sporadic expansion of dengue virus in both urban and rural settings. This may require tailoring of preventative strategies to suit different transmission ecologies across Sabah. Further studies to better understand the drivers of dengue in Sabah may aid dengue control efforts in Malaysia, and more broadly in South East Asia.\n\nAuthor summaryIn order to combat the rising regional incidence of dengue in South East Asia, the drivers of transmission must be better characterised across different environmental settings. We conducted the first retrospective analysis of dengue epidemiology in the predominantly rural state of Sabah, Malaysia, where both urban and sylvatic transmission cycles exist. Human notification data over a 7-year period were reviewed and spatiotemporal and demographic risk factors identified. We found: O_LIUrban habitats and population density are not the only determinants mediating the spread of epidemic dengue in Sabah. Case from both urban and rural localities contributed equally to dengue outbreaks.\nC_LIO_LIHuman demographic risk factors included being aged between 10 and 29 years, and being male.\nC_LIO_LIHigh incidence areas for dengue do not predict the occurrence of severe dengue. Severe dengue was largely localised to lower incidence districts in the east of the state.\nC_LIO_LIThe sole presence of Aedes albopictus in and around the majority of urban and rural case households suggests that this vector may play a major role in facilitating outbreaks.\nC_LI\n\nA complex interplay of risk factors likely mediates dengue transmission in Sabah, influenced by both regional climate trends and localised human and ecological influences. This study emphasises that the increasing spread of dengue in urban South East Asia is also mirrored in more rural areas, and suggests a need for control strategies that address both urban and rural dengue risk.

epidemiology↗

bioRxiv2019

Life on the margin: rainwater tanks facilitate overwintering of the dengue vector, Aedes aegypti, in a sub-tropical climate.

A key determinant of insect persistence in marginal habitats is the ability to tolerate environmental extremes such as temperature. Aedes aegypti is highly invasive and little is known about the physiological sensitivity of the species to fluctuating temperature regimes at the lower critical threshold. This has implications that limit establishment and persistence of the species in sub-optimal regions. Daily winter temperatures were measured in common Australian larval habitats, replicated in environmental chambers, and used to investigate the effect of fluctuating temperatures on the development and survival of tropical and subtropical strains of Australian Ae. aegypti. Development was slow for all treatments but both strains were able to complete development to the adult stage, suggesting previous models underestimate the potential for the species to persist in eastern Australia. Results suggested that thermal buffering in large volume habitats, and water that persists for greater than 32 days, will facilitate completion of the life cycle during sub-tropical winters. Furthermore, we provide a non-linear estimate of the lower critical temperature of Ae. aegypti that suggests the current threshold may be incorrect. Our study demonstrates that the current re-introduction of water storage containers such as rainwater tanks, into major Australian population centres will increase the risk of Ae. aegypti establishment by permitting year-round development south of its current distribution.

zoology↗