Cheong Huat Tan

Mosquito pupae are conventionally believed to require water for survival and subsequent emergence into adult mosquitoes. This study assessed emergence of Ae. aegypti mosquitoes from four substrates: (1) water; (2) moist filter paper; (3) moist filter paper with a wet cotton pad below it; and (4) double-meshed wire surface with a wet tissue below it. High levels of emergence (at least 90%) were obtained for all four substrates. These mosquitoes displayed comparable fitness levels as represented by their flight ability and mating success. The median survival curve of male mosquitoes showed no significant differences, but significant differences were observed between groups of female mosquitoes that emerged from different substrates. The results will be helpful for programmes that intend to improve the logistical handling of mosquitoes for field releases.

In Singapore, indoor space spraying, such as ultra-low-volume (ULV) treatment, is used to control adult mosquitoes during vector-borne disease outbreaks. This study assessed the efficacy of Mirakn^® GX, a pyrethroid-based insecticide formulation combining d,d-trans-cyphenothrin with desiccated carbon dioxide as a propellant. The implications of these findings for operational use will be discussed. Mirakn^® GX was evaluated against confined field and laboratory strains of Aedes aegypti, Ae. albopictus and Culex quinquefasciatus under semi-field conditions, following World Health Organization (WHO) guidelines. This study was conducted within an occupied residential apartment unit. An additional evaluation focusing on Ae. aegypti, designed to mimic operational practice, was performed using a reduced exposure duration of 10 min. At an application rate of 1 g/m³ with 60 min exposure, 100% knockdown and 99.79–100% mortality were observed across all three mosquito species. A shortened 10 min exposure also resulted in complete knockdown and mortality in both laboratory and field strains of Ae. aegypti, supporting operational feasibility. Under semi-field conditions, the insecticide formulation showed good penetration into enclosed spaces, indicating its potential use for scenarios where timely management of indoor adult mosquitoes is required, including during outbreaks to kill infected mosquitoes.

Background: Wild-type female Aedes aegypti which mates with male Wolbachia-infected Aedes aegypti mosquito produces infertile offspring due to cytoplasmic incompatibility. Sustained releases of Wolbachia-infected males can potentially suppress wild-type mosquito populations. Methods: We conducted a large-scale cluster-randomised trial using Wolbachia-mediated incompatible insect technique-sterile insect technique (IIT-SIT) for the control of dengue in Singapore. This involved releases of male wAlbB-infected Ae. aegypti mosquitoes which were irradiated prior to releases to sterilise residual females to avoid an establishment of wAlbB-infected Ae. aegypti mosquitoes in the field. Fifteen clusters, comprising an estimated 730,328 residents, were randomised into two arms: 8 clusters received deployments of male wAlbB-infected Ae. aegypti (intervention clusters) and 7 clusters received no deployments (control clusters). All clusters practised baseline vector control measures. We assessed the impact of Wolbachia releases on Ae. aegypti and Ae. albopictus abundance using a differences-in-differences framework. The interaction-weighted estimator was used to account for the staggered adoption setting and the dynamic effect of releasing male wAlbB-infected Ae. aegypti on wild-type Ae. aegypti and Ae. albopictus abundance. Results: Intervention clusters which received male wAlbB-infected Ae. aegypti had increasing reductions in Ae. aegypti abundance when subject to longer periods of intervention. In the 0–25 weeks following release, intervention efficacies (IE) were moderate (0-12 weeks: 29.15% [21.80%, 35.24%], 13-25 weeks: 60.35% [50.85%, 66.77%]). Whereas one year post intervention, reductions in Ae. aegypti abundance in intervention clusters were sustained at around 90% (52-103 weeks range: 90.20%–91.26%). Concomitantly, 78-weeks post intervention to the end of intervention, there was an increase in Ae. albopictus abundance in intervention clusters (78-103 weeks range: -32.43% – -63.10%). However, the average weekly increase in the Ae. albopictus abundance due to male wAlbB-infected Ae. aegypti releases was low and estimated to range between 0.0103 – 0.0482. Conclusion: Our results demonstrate the high efficacy of Wolbachia-mediated IIT-SIT in reducing Ae. aegypti abundance.

Background: Phlebotomine sand flies (Diptera: Psychodidae) are tiny, blood-sucking insects that are of significant public and veterinary health importance for their role in the transmission of Leishmania parasites, bacteria, and arboviruses. Although sand flies have been documented in most Southeast Asian countries, there are no published records confirming their presence in Singapore. Here, we present this fauna with descriptions of new species. Methods: Sand fly species identification was confirmed using an integrative taxonomic approach that combines morphological analysis with DNA barcoding of the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit I (COI) genes. Results: We identified eight sand fly species, including four newly described species: Phlebotomus seowpohi n. sp., Sergentomyia leechingae n. sp., Sergentomyia gubleri n. sp., and Sergentomyia retrocalcarae n. sp. Phylogenetic analyses suggest that the new Phlebotomus species, belonging to subgenus Euphlebotomus, is closely related to Phlebotomus argentipes, an important vector of Leishmania donovani from the South Asian region. Conclusions: The potential risk of leishmaniasis in Singapore is compounded by the recent detection of antibodies to Leishmania infantum in local free-roaming dogs. Therefore, continuous monitoring is essential to assess and manage the risk of disease agent transmission, support the development of an early warning system, and enable timely and targeted public health interventions. The findings of this study contribute to the global knowledge on sand flies and enhance our understanding of local fauna diversity and distribution. Graphical

Insect-specific flaviviruses (ISFs) are a group of orthoflaviviruses that can replicate efficiently in arthropods but are unable to replicate in vertebrate hosts. This contrasts with medically important orthoflaviviruses, such as dengue virus (DENV) and West Nile virus (WNV). Using the established monoclonal antibody against viral RNA intermediates in cell assay, we report the first isolation of an ISF, Quang Binh virus (QBV), in Singapore from three pools of Culex gelidus mosquitoes. To determine the spatiotemporal prevalence of QBV, a total of 17,070 mosquitoes, represented as 721 pools, were screened using a QBV-specific reverse transcriptase quantitative PCR assay, revealing 36 QBV-positive pools of mosquitoes detected mainly along the northern coastal regions of Singapore. Repeated detections over 12 months in a north-western nature reserve suggest local stable establishment of the virus. Phylogenetic and molecular analyses show that QBV sequences from the Singapore group together with other Southeast Asian sequences and that Cx. gelidus-derived sequences are phylogenetically distinct from those derived from Culex tritaeniorhynchus. We also present in vitro evidence that QBV is able to suppress DENV2 and WNV_KUN in C6/36 cells by 2.9 logs and 1.8 logs, respectively. This report represents the first known spatiotemporal study of an ISF and highlights QBV’s potential as a biological control against medically important orthoflaviviruses.

Phlebotomine sand flies (Diptera: Psychodidae) are small, blood-sucking insects that are of significant public and veterinary health importance for their role in the transmission of Leishmania parasites, bacteria and arboviruses. Although sand flies have been documented in most Southeast Asian countries, there are no published records confirming their presence in Singapore. Here, we provide the first documented evidence on the presence of sand flies in Singapore. Using an integrated taxonomic approach that combines morphological analysis with DNA barcoding of the mitochondrial cytochrome b ( cytb ) and cytochrome c oxidase subunit I ( COI ) genes, we identified eight sand fly species, including four newly described species – Phlebotomus seowpohi n. sp., Sergentomyia leechingae n. sp., Sergentomyia gubleri n. sp., and Sergentomyia retrocalcarae n. sp.. Phylogenetic analyses suggest that the new Phlebotomus species, belonging to subgenus Euphlebotomus , is closely related to Phlebotomus argentipes , an important vector of Leishmania donovani from the South Asian region. The discovery of phlebotomine sand flies in Singapore underscores the importance of biosurveillance of biting arthropods. Given Singapore’s status as a major travel hub, there is a potential risk of leishmaniasis being introduced either by residents returning, or visitors arriving from endemic regions. This risk is compounded by the recent detection of local canine leishmaniasis. Thus, continuous monitoring is essential to assess and manage the risk of disease transmission, support the development of an early warning system, and enable timely and targeted public health interventions. The findings from this study contributes to the global knowledge on sand fly and enhance our understanding of local sand fly diversity and distribution. Author Summary: Phlebotomine sand flies are small, blood-sucking insects recognized as significant vectors for various diseases including leishmaniasis. This study provides the first evidence of their presence in Singapore. Using an integrated taxonomic approach combining morphological analysis with DNA barcoding, we identified eight sand fly species that include four new species: Phlebotomus seowpohi n. sp., Sergentomyia leechingae n. sp., Sergentomyia gubleri n. sp., and Sergentomyia retrocalcarae n. sp. The presence of sand flies in Singapore highlights an important biosurveillance need, as the region’s status as a major travel hub increases the risk of leishmaniasis being introduced. Importantly, recent findings of canine leishmaniasis in local dogs amplify the threat of disease transmission. Continuous monitoring and understanding of sand fly diversity and ecology are essential for developing effective public health strategies and early warning systems to manage this risk.

Matings between male Aedes aegypti mosquitoes infected with wAlbB strain of Wolbachia and wildtype females yield non-viable eggs. We evaluated the efficacy of releasing wAlbB-infected Ae. aegypti male mosquitoes to suppress dengue. We specified the protocol of a two-arm cluster-randomized test-negative controlled trial (cRCT) and emulated it using a nationally representative test-negative/positive database of individuals reporting for febrile illness to any public hospital, general practitioner or polyclinic. We retrospectively built a cohort of individuals who reside in Wolbachia locations vs a comparator control group who do not reside in Wolbachia locations, using a nationally representative database of all individuals whom report for febrile illness and were tested for dengue at the Environmental Health Institute/hospital laboratories/commercial diagnostic laboratories, through general practitioner clinic, polyclinic or public/private hospital from epidemiological week (EW) 1 2019 to EW26 2022. We emulated a constrained randomization protocol used in cRCTs to balance dengue risk between intervention and control arms in the pre-intervention period. We used the inverse probability weighting approach to further balance the intervention and control groups using a battery of algorithmically selected sociodemographic, environmental and anthropogenic variables. Intention-to-treat analyses were conducted to estimate the risk reduction of dengue given Wolbachia exposure. Intention-to-treat analyses revealed that, compared with controls, Wolbachia releases for 3, 6 and ≥12 months was associated to 47% (95% confidence interval: 25–69%), 44% (33–77%) and 61% (38–78%) protective efficacy against dengue, respectively. When exposed to ≥12 months of Wolbachia releases, protective efficacies ranged from 49% (13–72%) to 77% (60–94%) across years. The proportion of virologically confirmed dengue cases was lower overall in the intervention arm. Protective efficacies were found across all years, age and sex subgroups, with higher durations of Wolbachia exposure associated to greater risk reductions of dengue. Results demonstrated that Wolbachia-mediated sterility can strengthen dengue control in tropical cities, where dengue burden is the greatest.

The sterile insect technique is based on the overflooding of a target population with released sterile males inducing sterility in the wild female population. It has proven to be effective against several insect pest species of agricultural and veterinary importance and is under development forAedesmosquitoes. Here, we show that the release of sterile males at high sterile male to wild female ratios may also impact the target female population through mating harassment. Under laboratory conditions, male to female ratios above 50 to 1 reduce the longevity of femaleAedesmosquitoes by reducing their feeding success. Under controlled conditions, blood uptake of females from an artificial host or from a mouse and biting rates on humans are also reduced. Finally, in a field trial conducted in a 1.17 ha area in China, the female biting rate is reduced by 80%, concurrent to a reduction of female mosquito density of 40% due to the swarming of males around humans attempting to mate with the female mosquitoes. This suggests that the sterile insect technique does not only suppress mosquito vector populations through the induction of sterility, but may also reduce disease transmission due to increased female mortality and lower host contact.

Background: Aedes aegypti (Ae. aegypti) is the major vector that transmits many diseases including dengue, Zika, and filariasis in tropical and subtropical regions. Due to the growing resistance to chemical-based insecticides, biological control methods have become an emerging direction to control mosquito populations. The sterile insect technique (SIT) deploys high doses of ionizing radiation to sterilize male mosquitoes before the release. The Wolbachia-based population suppression method of the incompatible insect technique (IIT) involves the release of Wolbachia-infected males to sterilize uninfected field females. Due to the lack of perfect sex separation tools, a low percentage of female contamination is detected in the male population. To prevent the unintentional release of these Wolbachia-infected females which might result in population replacement, a low dose of X-ray irradiation is deployed to sterilize any female escapees. However, it remains unclear whether these irradiation-induced male and female sterilizations share common mechanisms. Results: In this work, we set out to define the minimum dose of X-ray radiation required for complete female sterilization in Ae. aegypti (NEA-EHI strain). Further results showed that this minimum dose of X-ray irradiation for female sterilization significantly reduced male fertility. Similar results have been reported previously in several operational trials. By addressing the underlying causes of the sterility, our results showed that male sterility is likely due to chromosomal damage in the germ cells induced by irradiation. In contrast, female sterility appears to differ and is likely initiated by the elimination of the somatic supporting cells, which results in the blockage of the ovariole maturation. Building upon these findings, we identified the minimum dose of X-ray irradiation on the Wolbachia-infected NEA-EHI (wAlbB-SG) strain, which is currently being used in the IIT-SIT field trial. Compared to the uninfected parental strain, a lower irradiation dose could fully sterilize wAlbB-SG females. This suggests that Wolbachia-carrying mosquitoes are more sensitive to irradiation, consistent with a previous report showing that a lower irradiation dose fully sterilized Wolbachia-infected Ae. aegypti females (Brazil and Mexican strains) compared to those uninfected controls. Conclusions: Our findings thus reveal the distinct mechanisms of ionizing X-ray irradiation-induced male or female sterility in Ae. aegypti mosquitoes, which may help the design of X-ray irradiation-based vector control methods.

The sterile insect technique (SIT) is based on the overflooding of a target population with released sterile males inducing sterility in the wild female population. The SIT has proven to be effective against several insect pest species of agricultural and veterinary importance and is under development for Aedes mosquitoes. Here, we show that the release of sterile males in high sterile male to wild female ratios may also impact the target female population through mating harassment. Under laboratory conditions, male to female ratios above 50 to 1 reduced the longevity of female Aedes mosquitoes by reducing their feeding success. Under semi-field conditions, blood uptake of females from an artificial host and biting rates on humans were also strongly reduced. Finally, in a field SIT trial conducted in a 1.17 ha area in China, the female biting rate was reduced by 80%, concurrent to a reduction of female mosquito density of 40% due to the swarming of males around humans attempting to mate with the female mosquitoes. This suggests that the SIT does not only suppress mosquito vector populations through the induction of sterility, but might also reduce disease transmission due to increased female mortality and lower host contact.