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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 w Mel 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

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. 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. 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.

Background: The w Mel 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  w Mel 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 w Mel 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.

Transgenerational epigenetic inheritance (TEI) describes the process whereby distinct epigenetic states are transmitted between generations, resulting in heritable gene expression and phenotypic differences that are independent of DNA sequence variation. Chromatin modifications have been demonstrated to be important in TEI; however, the extent to which they require other signals to establish and maintain epigenetic states is still unclear. Here we investigate whether small non-coding RNAs contribute to different epigenetic states of the Fab2L transgene in Drosophila triggered by transient long-range chromosomal contacts, which requires Polycomb complex activity to deposit the H3K27me3 modification for long-term TEI. By analysing mutants deficient in small non-coding RNAs, high-throughput sequencing data, long-range chromosomal contacts and gene expression, we demonstrate that small non-coding RNAs do not contribute directly to initiation or maintenance of silencing. However, we uncover an indirect role for microRNA expression in transgene silencing through effects on the Polycomb group gene Pleiohomeotic . Additionally, we show that a commonly used marker gene, Stubble ( Sb ), affects Pleiohomeotic expression, which may be important in interpreting experiments assaying Polycomb function in Drosophila development. By ruling out a plausible candidate for TEI at the Fab2L transgene, our work highlights the variability in different modes of TEI across species.

Background:The Royal College of Surgeons Basic Surgical Skills (BSS) course is ubiquitous among UK surgical trainees but is geographically limited and costly. The COVID-19 pandemic has reduced training quality. Surveys illustrate reduced logbook completion and increased trainee attrition. Local, peer-led teaching has been shown to be effective at increasing confidence in surgical skills in a cost-effective manner. Qualitative data on trainee well-being, recruitment, and retention are lacking.Objective:This study aims to evaluate the impact of a novel program of weekly, lunchtime BSS sessions on both quantitative and qualitative factors.Methods:A weekly, lunchtime BSS course was designed to achieve the outcomes of the Royal College of Surgeons BSS course over a 16-week period overlapping with 1 foundation doctor rotation. All health care workers at the study center were eligible to participate. The study was advertised via the weekly, trust-wide information email. Course sessions included knot tying, suturing, abscess incision and drainage, fracture fixation with application of plaster of Paris, joint aspirations and reductions, abdominal wall closure, and basic laparoscopic skills. The hospital canteen sourced unwanted pig skin from the local butcher for suturing sessions and pork belly for abscess and abdominal wall closure sessions. Out-of-date surgical equipment was used. This concurrent, nested, mixed methods study involved descriptive analysis of perceived improvement scores in each surgical skill before and after each session, over 4 iterations of the course (May 2021 to August 2022). After the sessions, students completed a voluntary web-based feedback form scoring presession and postsession confidence levels on a 5-point Likert scale. Qualitative thematic analysis of voluntary semistructured student interview transcripts was also performed to understand the impact of a free-to-attend, local, weekly, near-peer teaching course on perceived well-being, quality of training, and interest in a surgical career. Students consented to the use of feedback and interview data for this study. Ethics approval was requested but deemed not necessary by the study center’s ethics committee.Results:There were 64 responses. Confidence was significantly improved from 47% to 73% (95% CI 15%-27%; P<.001; t13=5.3117) across all surgical skills over 4 iterations. Among the 7 semistructured interviews, 100% (7/7) of the participants reported improved perceived well-being, value added to training, and positivity toward near-peer teaching and 71% (5/7) preferred local weekly teaching. Interest in a surgical career was unchanged.Conclusions:This course was feasible around clinical workloads, resourced locally at next to no cost, environmentally sustainable, and free to attend. The course offered junior doctors not only a weekly opportunity to learn but also to teach. Peer-led, decentralized surgical education increases confidence and has a positive effect on perceptions about well-being and training. We hope to disseminate this course, leading to reproduction in other centers, refinement, and wide implementation.

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.

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.

We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts.

Background The surveillance and control of mosquito-borne diseases is dependent upon understanding the bionomics and distribution of the vectors. Most studies of mosquito assemblages describe species abundance, richness and composition close to the ground defined often by only one sampling method. In this study, we assessed Australian mosquito species near the ground and in the sub-canopy using two traps baited with a variety of lures. Methods Mosquitoes were sampled using a 4 × 4 Latin square design at the Cattana Wetlands, Australia from February to April 2020, using passive box traps with octenol and carbon dioxide and three variations of a sticky net trap (unbaited, and baited with octenol or octenol and carbon dioxide). The traps were deployed at two different heights: ground level (≤ 1 m above the ground) and sub-canopy level (6 m above the ground). Results In total, 27 mosquito species were identified across the ground and sub-canopy levels from the different traps. The abundance of mosquitoes at the ground level was twofold greater than at the sub-canopy level. While the species richness at ground and sub-canopy levels was not significantly different, species abundance varied by the collection height. Conclusions The composition of mosquito population assemblages was correlated with the trap types and heights at which they were deployed. Coquillettidia species, which prefer feeding on birds, were mainly found in the sub-canopy whereas Anopheles farauti , Aedes vigilax and Mansonia uniformis , which have a preference for feeding on large mammals, were predominantly found near the ground. In addition to trap height, environmental factors and mosquito bionomic characteristics (e.g. larval habitat, resting behaviour and host blood preferences) may explain the vertical distribution of mosquitoes. This information is useful to better understand how vectors may acquire and transmit pathogens to hosts living at different heights. Graphical abstract

Background: The w Mel 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  w Mel 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 w Mel 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.