Insufficient duration of insecticidal efficacy of Yahe® insecticide-treated nets in Papua New Guinea.

BACKGROUND: Insecticide-treated nets (ITNs) are the backbone of anti-malarial vector control in Papua New Guinea (PNG). Over recent years the quality and performance of ITNs delivered to PNG decreased, which has likely contributed to the stagnation in the malaria control effort in the country. The present study reports results from the first 24 months of a durability study with the ITN product Yahe LN® in PNG. METHODS: The durability study was conducted in four villages on the northern coast of PNG, in an area with high malaria parasite transmission, following WHO-recommended methodology adapted to the local scenario. A cohort of n = 500 individually identifiable Yahe® ITNs was distributed by the PNG National Malaria Control Programme from October to December 2021. Insecticidal efficacy of the ITNs was tested using cone bioassays with fully pyrethroid susceptible Anopheles farauti colony mosquitoes at baseline and at 6 months intervals, alongside evaluation of physical integrity and the proportion of ITNs lost to follow-up. A questionnaire was used to collect information on ITN end user behaviour, such as the frequency of use and washing. The observations from the durability study were augmented with simulated laboratory wash assays. RESULTS: Gradual uptake and replacement of previous campaign nets by the communities was observed, such that at 6 months 45% of all newly distributed nets were in use in their designated households. Insecticidal efficacy of the Yahe® nets, expressed as the percent 24 h mortality in cone bioassays decreased from 91 to 45% within the first 6 months of distribution, even though > 90% of study nets had never been washed. Insecticidal efficacy decreased further to < 20% after 24 months. ITNs accumulated physical damage (holes) at a rate similar to previous studies, and 35% were classified as 'too torn' by proportional hole index after 24 months. ITNs were lost to follow-up such that 61% of cohort nets were still present after 24 months. Laboratory wash assays indicated a rapid reduction in insecticidal performance with each consecutive wash such that average 24 h mortality was below 20% after 10 washes. CONCLUSION: Yahe® ITNs are not performing as per label claim in an area with fully pyrethroid susceptible vectors, and should be investigated more comprehensively and in other settings for compliance with currently recommended durability and efficacy thresholds. The mass distribution of low quality ITN products with variable performance is one of the major ongoing challenges for global malaria control in the last decade.

Large-scale releases and establishment of wMel Wolbachia in Aedes aegypti mosquitoes throughout the Cities of Bello, Medellín and Itagüí, Colombia.

BACKGROUND: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and has been shown to reduce the transmission of dengue and other Aedes-borne viruses. Here we report the entomological results from phased, large-scale releases of Wolbachia infected Ae. aegypti mosquitoes throughout three contiguous cities located in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Local wMel Wolbachia-infected Ae. aegypti mosquitoes were generated and then released in an initial release pilot area in 2015-2016, which resulted in the establishment of Wolbachia in the local mosquito populations. Subsequent large-scale releases, mainly involving vehicle-based releases of adult mosquitoes along publicly accessible roads and streets, were undertaken across 29 comunas throughout Bello, Medellín and Itagüí Colombia between 2017-2022. In 9 comunas these were supplemented by egg releases that were undertaken by staff or community members. By the most recent monitoring, Wolbachia was found to be stable and established at consistent levels in local mosquito populations (>60% prevalence) in the majority (67%) of areas. CONCLUSION: These results, from the largest contiguous releases of wMel Wolbachia mosquitoes to date, highlight the operational feasibility of implementing the method in large urban settings. Based on results from previous studies, we expect that Wolbachia establishment will be sustained long term. Ongoing monitoring will confirm Wolbachia persistence in local mosquito populations and track its establishment in the remaining areas.

Uncovering the genetic diversity within the Aedes notoscriptus virome and isolation of new viruses from this highly urbanised and invasive mosquito.

The Australian backyard mosquito, Aedes notoscriptus, is a highly urbanised pest species that has invaded New Zealand and the USA. Importantly, Ae. notoscriptus has been implicated as a vector of Ross River virus, a common and arthritogenic arbovirus in Australia, and is a laboratory vector of numerous other pathogenic viruses, including West Nile, yellow fever, and Zika viruses. To further explore live viruses harboured by field populations of Ae. notoscriptus and, more specifically, assess the genetic diversity of its virome, we processed 495 pools, comprising a total of 6,674 female Ae. notoscriptus collected across fifteen suburbs in Brisbane, Australia, between January 2018 and May 2019. Nine virus isolates were recovered and characterised by metagenomic sequencing and phylogenetics. The principal viral family represented was Flaviviridae. Known viruses belonging to the genera Flavivirus, Orbivirus, Mesonivirus, and Nelorpivirus were identified together with two novel virus species, including a divergent Thogoto-like orthomyxovirus and an insect-specific flavivirus. Among these, we recovered three Stratford virus (STRV) isolates and an isolate of Wongorr virus (WGRV), which for these viral species is unprecedented for the geographical area of Brisbane. Thus, the documented geographical distribution of STRV and WGRV, both known for their respective medical and veterinary importance, has now been expanded to include this major urban centre. Phylogenies of the remaining five viruses, namely, Casuarina, Ngewotan, the novel Thogoto-like virus, and two new flavivirus species, suggested they are insect-specific viruses. None of these viruses have been previously associated with Ae. notoscriptus or been reported in Brisbane. These findings exemplify the rich genetic diversity and viral abundance within the Ae. notoscriptus virome and further highlight this species as a vector of concern with the potential to transmit viruses impacting human or animal health. Considering it is a common pest and vector in residential areas and is expanding its global distribution, ongoing surveillance, and ecological study of Ae. notoscriptus, together with mapping of its virome and phenotypic characterisation of isolated viruses, is clearly warranted. Immanently, these initiatives are essential for future understanding of both the mosquito virome and the evolution of individual viral species.

Effects of attachment substrate, larval diet, and temperature on development and survival of immature Coquillettidia linealis (Skuse) and Coquillettidia xanthogaster (Edwards).

A sustainable colony allowed investigations into attachment substrates, diet and temperature in the development of the immature stages of Cq. linealis and its local congener Cq. xanthogaster. As immatures, these mosquitoes attach to submerged plants for respiration, and various field-collected and laboratory-raised plants were compared with non-living substrates. Hydroponically-grown wheat plant seedlings provided the most suitable attachment substrate. Development and survival of immatures to eclosion were investigated with three types and three quantities of a standardized diet. Development and survival at rearing temperatures between 10° and 30° C were also investigated. Diet type appeared to have little influence on development. However, at the lowest diet quantity, development to pupation was significantly reduced while the highest diet quantity impacted on the eclosion of pupae. Mean duration of immature development for Cq. linealis increased as temperature decreased. Time from 1st instar to median eclosion did not differ significantly between 23° C (6.0 weeks) and 25° C (5.3 weeks), although it was 10 weeks at 20° C, and 30° C was lethal. For Cq. xanthogaster, similarly, mean duration of immature development increased as temperature decreased, with development time from 1st instar to median eclosion significantly longer at 20° C (7.5 weeks) compared to 23° C (5.0 weeks) and 30° C (4.0 weeks).

Colonization of Coquillettidia linealis (Skuse) with reference to other Coquillettidia and Mansonia species.

The immature stages of Coquillettidia and Mansonia mosquitoes are cryptic and spend the duration of their development attached to the tissues of subsurface aquatic plants. This obligatory association makes them difficult to collect and has precluded detailed investigation of the biology of Coquillettidia linealis, a species of significant pest and vector status in Australia, as well as other species in the genus Coquillettidia in Australia and elsewhere. This paper describes the first successful establishment and maintenance of a colony of Cq. linealis. Blood-seeking wild adults were collected and induced to oviposit under laboratory conditions, where methods were developed to rear larval populations to adult maturity in a colony that was maintained through eight successive generations. Colonies of Coquillettidia xanthogaster and Coquillettidia sp. nr crassipes were also established and reared through at least six generations and five generations, respectively, while two species of Mansonia, Mansonia uniformis and Mansonia septempunctata, were also reared successfully for six and two generations, respectively.

Field evaluation of the establishment potential of wMelPop Wolbachia in Australia and Vietnam for dengue control.

BACKGROUND: Introduced Wolbachia bacteria can influence the susceptibility of Aedes aegypti mosquitoes to arboviral infections as well as having detrimental effects on host fitness. Previous field trials demonstrated that the wMel strain of Wolbachia effectively and durably invades Ae. aegypti populations. Here we report on trials of a second strain, wMelPop-PGYP Wolbachia, in field sites in northern Australia (Machans Beach and Babinda) and central Vietnam (Tri Nguyen, Hon Mieu Island), each with contrasting natural Ae. aegypti densities. METHODS: Mosquitoes were released at the adult or pupal stages for different lengths of time at the sites depending on changes in Wolbachia frequency as assessed through PCR assays of material collected through Biogents-Sentinel (BG-S) traps and ovitraps. Adult numbers were also monitored through BG-S traps. Changes in Wolbachia frequency were compared across hamlets or house blocks. RESULTS: Releases of adult wMelPop-Ae. aegypti resulted in the transient invasion of wMelPop in all three field sites. Invasion at the Australian sites was heterogeneous, reflecting a slower rate of invasion in locations where background mosquito numbers were high. In contrast, invasion across Tri Nguyen was relatively uniform. After cessation of releases, the frequency of wMelPop declined in all sites, most rapidly in Babinda and Tri Nguyen. Within Machans Beach the rate of decrease varied among areas, and wMelPop was detected for several months in an area with a relatively low mosquito density. CONCLUSIONS: These findings highlight challenges associated with releasing Wolbachia-Ae. aegypti combinations with low fitness, albeit strong virus interference properties, as a means of sustainable control of dengue virus transmission.

Body size and wing shape measurements as quality indicators of Aedes aegypti mosquitoes destined for field release.

There is increasing interest in rearing modified mosquitoes for mass release to control vector-borne diseases, particularly Wolbachia-infected Aedes aegypti for suppression of dengue. Successful introductions require release of high quality mosquitoes into natural populations. Potential indicators of quality are body size and shape. We tested to determine if size, wing/thorax ratio, and wing shape are associated with field fitness of Wolbachia-infected Ae. aegypti. Compared with field-collected mosquitoes, released mosquitoes were larger in size, with lower size variance and different wing shape but similar in wing-thorax ratio and its associated variance. These differences were largely attributed to nutrition and to a minor extent to wMel Wolbachia infection. Survival potential of released female mosquitoes was similar to those from the field. Females at oviposition sites tended to be larger than those randomly collected from BG-Sentinel traps. Rearing conditions should thus aim for large size without affecting wing/thorax ratios.

Operational use of household bleach to "crash and release" Aedes aegypti prior to Wolbachia-infected mosquito release.

Dengue (family Flaviviridae, genus Flavivirus, DENV) remains the leading arboviral cause of mortality in the tropics. Wolbachia pipientis has been shown to interrupt DENV transmission and is presently being trialled as a biological control. However, deployment issues have arisen on methods to temporarily suppress wild mosquito populations before Wolbachia-infected mosquito releases. By suppressing wild populations, fewer Ae. aegypti releases are required to achieve a sustainable Wolbachia density threshold. Furthermore, public distress is reduced. This study tests the application of domestic bleach (4% NaCIO) to temporarily "crash" immature Aedes populations in water-filled containers. Spray application NaClO (215 ppm) resulted in a mean 48-h mortality of 100, 100, 97, and 88% of eggs, second-instar larvae, fourth-instar larvae, and pupae, respectively. In the field, NaClO delayed ovipositing by 9 d in cooler months, and 11 d in hotter months, after which oviposition resumed in treated receptacles. We found bleach treatment of pot-plant bases did not cause wilting, yellowing, or dropping of leaves in two ornamental plants species. Domestically available NaClO could be adopted for a "crash and release" strategy to temporarily suppress wild populations of Ae. aegypti in containers before release of Wolbachia-infected mosquitoes. The "crash and release" strategy is also applicable to other mosquito species, e.g., Aedes albopictus (Skuse), in strategies using released mosquitoes.

Bacillus thuringiensis var. israelensis misting for control of Aedes in cryptic ground containers in north Queensland, Australia.

In Australia, dengue is not endemic, although the vector mosquito Aedes aegypti is established in far north Queensland (FNQ). Aedes albopictus has recently invaded the Torres Strait region, but is not established on mainland Australia. To maintain dengue-free, public health departments in FNQ closely monitor introduced dengue infections and confine outbreaks through rigorous vector control responses. To safeguard mainland Australia from Ae. albopictus establishment, pre-emptive strategies are required to reduce its breeding in difficult to access habitats. We compare the residual efficacy of VectoBac WDG, Bacillus thuringiensis var. israelensis (Bti) formulation, as a residual treatment when misted across a typical FNQ bushland using a backpack mister (Stihl SR 420 Mist Blower) at two dose rates up to 16 m. Semi-field condition results, over 16 weeks, indicate that Bti provided high mortality rates (> 80%) sustained for 11 weeks. Mist application penetrated 16 m of dense bushland without efficacy decline over distance.

Effects of Beauveria bassiana on survival, blood-feeding success, and fecundity of Aedes aegypti in laboratory and semi-field conditions.

The fungus Beauveria bassiana reduces Aedes aegypti longevity in laboratory conditions, but effects on survival, blood-feeding behavior, and fecundity in realistic environmental conditions have not been tested. Adult, female Ae. aegypti infected with B. bassiana (FI-277) were monitored for blood-feeding success and fecundity in the laboratory. Fungal infection reduced mosquito-human contact by 30%. Fecundity was reduced by (mean ± SD) 29.3 ± 8.6 eggs per female per lifetime in the laboratory; egg batch size and viability were unaffected. Mosquito survival, blood-feeding behavior, and fecundity were also tested in 5 meter × 7 meter × 4 meter semi-field cages in northern Queensland, Australia. Fungal infection reduced mosquito survival in semi-field conditions by 59-95% in large cages compared with 61-69% in small cages. One semi-field cage trial demonstrated 80% reduction in blood-feeding; a second trial showed no significant effect. Infection did not affect fecundity in large cages. Beauveria bassiana can kill and may reduce biting of Ae. aegypti in semi-field conditions and in the laboratory. These results further support the use of B. bassiana as a potential biocontrol agent against Ae. aegypti.

A secure semi-field system for the study of Aedes aegypti.

BACKGROUND: New contained semi-field cages are being developed and used to test novel vector control strategies of dengue and malaria vectors. We herein describe a new Quarantine Insectary Level-2 (QIC-2) laboratory and field cages (James Cook University Mosquito Research Facility Semi-Field System; MRF SFS) that are being used to measure the impact of the endosymbiont Wolbachia pipientis on populations of Aedes aegypti in Cairns Australia. METHODOLOGY/PRINCIPAL FINDINGS: The MRF consists of a single QIC-2 laboratory/insectary that connects through a central corridor to two identical QIC-2 semi-field cages. The semi-field cages are constructed of two layers of 0.25 mm stainless steel wire mesh to prevent escape of mosquitoes and ingress of other insects. The cages are covered by an aluminum security mesh to prevent penetration of the cages by branches and other missiles in the advent of a tropical cyclone. Parts of the cage are protected from UV light and rainfall by 90% shade cloth and a vinyl cover. A wooden structure simulating the understory of a Queenslander-style house is also situated at one end of each cage. The remainder of the internal aspect of the cage is covered with mulch and potted plants to emulate a typical yard. An air conditioning system comprised of two external ACs that feed cooled, moistened air into the cage units. The air is released from the central ceiling beam from a long cloth tube that disperses the airflow and also prevents mosquitoes from escaping the cage via the AC system. Sensors located inside and outside the cage monitor ambient temperature and relative humidity, with AC controlled to match ambient conditions. Data loggers set in the cages and outside found a <2 °C temperature difference. Additional security features include air curtains over exit doors, sticky traps to monitor for escaping mosquitoes between layers of the mesh, a lockable vestibule leading from the connecting corridor to the cage and from inside to outside of the insectary, and screened (0.25 mm mesh) drains within the insectary and the cage. A set of standard operating procedures (SOP) has been developed to ensure that security is maintained and for enhanced surveillance for escaping mosquitoes on the JCU campus where the MRF is located. A cohort of male and female Aedes aegypti mosquitoes were released in the cage and sampled every 3-4 days to determine daily survival within the cage; log linear regression from BG-sentinel trapping collections produced an estimated daily survival of 0.93 and 0.78 for females and males, respectively. CONCLUSIONS/SIGNIFICANCE: The MRF SFS allows us to test novel control strategies within a secure, contained environment. The air-conditioning system maintains conditions within the MRF cages comparable to outside ambient conditions. This cage provides a realistic transitional platform between the laboratory and the field in which to test novel control measures on quarantine level insects.

Field validation of a transcriptional assay for the prediction of age of uncaged Aedes aegypti mosquitoes in Northern Australia.

BACKGROUND: New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. METHODOLOGY/PRINCIPAL FINDINGS: We produced "free-range" test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R(2) value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. CONCLUSIONS/SIGNIFICANCE: The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where mosquito survivorship or lifespan reduction are crucial to their success. The approximate cost of the method was US$7.50 per mosquito and 60 mosquitoes could be processed in 3 days. The assay is based on conserved genes and modified versions are likely to support similar investigations of several important mosquito and other disease vectors.

Rapid estimation of Aedes aegypti population size using simulation modeling, with a novel approach to calibration and field validation.

New approaches for control of the dengue vector Aedes aegypti (L.) are being developed, including the potential introduction of life-shortening symbiont bacteria into field populations and the release of transgenic strains with reduced vector competency. With these new approaches comes the need for rapid estimations of existing field population size. Here, we describe the use of simulation modeling with container-inhabiting mosquito simulation (CIMSiM) for estimation of Ae. aegypti pupal crop size in north Queensland, Australia. CIMSiM was calibrated for local conditions by deploying "sentinel key containers" (tire, 2-liter plastic bucket, 0.6-liter pot plant base, and tarpaulin indentation) in which water flux and pupal productivity were studied for 72 d. Iterative adjustment of CIMSiM parameters was used to fit model outputs to match that of sentinel key containers. This calibrated model was then used in a blind field validation, in which breeding container and local meteorological data were used to populate CIMSiM, and model outputs were compared with a field pupal survey. Actual pupae per ha during two 10-d periods in 2007 fell within 95% confidence intervals of simulated pupal crop estimates made by 10 replicate simulations in CIMSiM, thus providing a successful field validation. Although the stochasticity of the field environment can never be wholly simulated, CIMSiM can provide field-validated estimates of pupal crop in a timely manner by using simple container surveys.

Expectoration of Flaviviruses during sugar feeding by mosquitoes (Diptera: Culicidae).

Biological transmission of arboviruses to a vertebrate host occurs when virions are expelled along with saliva during blood feeding by a hematophagous arthropod. We undertook experiments to determine whether mosquitoes expectorate flaviviruses in their saliva while sugar feeding. Batches of Culex annulirostris Skuse and Culex gelidus Theobald (Diptera: Culicidae) were orally infected with Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV), Kunjin (family Flaviviridae, genus Flavivirus, KUNV; a subtype of West Nile virus), and Murray Valley encephalitis (family Flaviviridae, genus Flavivirus, MVEV) viruses. After a 7-d extrinsic incubation, these mosquitoes were offered sucrose meals via cotton pledgets, which were removed daily and processed for viral RNA by using real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assays. JEV, MVEV, and KUNV RNA was detected in all pledgets removed from batches of Cx. gelidus on days 7-14 postexposure. In contrast, detection rates were variable for Cx. annulirostris, with KUNV detected in 0.3 M sucrose pledgets on all days postexposure, and JEV and MVEV detected on 57 and 50% of days postexposure, respectively. Higher concentrations of sucrose in the pledget did not increase virus detection rates. When individual JEV-infected Cx. gelidus were exposed to the sucrose pledget, 73% of mosquitoes expectorated virus with titers that were detectable by TaqMan RT-PCR. These results clearly show that flaviviruses are expectorated by infected mosquitoes during the process of sugar feeding on artificial pledgets. Potential applications of the method for arboviral bioassays and field surveillance are discussed.

Infection and dissemination of dengue virus type 2 in Aedes aegypti, Aedes albopictus, and Aedes scutellaris from the Torres Strait, Australia.

To determine their relative roles in transmission of dengue virus (DENV) in the Torres Strait region of northern Australia, we examined infection and dissemination of a sympatric strain of dengue virus type 2 (DENV-2) in Aedes scutellaris, Ae. albopictus, and Ae. aegypti. In experiments using membrane feeders for virus exposure, infection rates were 83% and 43% for Ae. scutellaris and Ae. aegypti, respectively. Salivary gland infection rates for both species were 43%. In experiments using pledgets for virus exposure, infection rates for Ae. aegypti, Ae. scutellaris, and Ae. albopictus were 68%, 55%, and 37%, respectively. Aedes albopictus exhibited the greatest barriers to infection with only 7% tested developing a salivary gland infection, compared to 42% and 24% of Ae. aegypti and Ae. scutellaris, respectively. These results suggest that Ae. scutellaris may have been responsible for DENV transmission on Torres Strait islands, where Ae. aegypti does not occur. In contrast, Ae. albopictus may not be an important vector of DENV-2 from the Torres Strait.