Mating harassment may boost the effectiveness of the sterile insect technique for Aedes mosquitoes.

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 for Aedes mosquitoes. 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 female Aedes mosquitoes 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.

Efficacy of Wolbachia-mediated sterility to reduce the incidence of dengue: a synthetic control study in Singapore.

BACKGROUND: Due to the absence of available therapeutics and good vaccines, vector control solutions are needed to mitigate the spread of dengue. Matings between male Aedes aegypti mosquitoes infected with the wAlbB strain of Wolbachia and wildtype females yield non-viable eggs. We evaluated the efficacy of releasing wAlbB-infected A aegypti male mosquitoes to suppress dengue incidence. METHODS: In this synthetic control study, we conducted large-scale field trials in Singapore involving release of wAlbB-infected A aegypti male mosquitoes for dengue control via vector population suppression, from epidemiological week (EW) 27, 2018, to EW 26, 2022. We selected two large towns (Yishun and Tampines) to adopt an expanding release strategy and two smaller towns (Bukit Batok and Choa Chu Kang) to adopt a targeted-release approach. Releases were conducted two times a week in high-rise public housing estates. All intervention and control locations practised the same baseline dengue control protocol. The main outcome was weekly dengue incidence rate caused by any dengue virus serotype. We used incidence data collected by the Singapore Ministry of Health to assess the efficacy of the interventions. To compare interventions, we used the synthetic control method to generate appropriate counterfactuals for the intervention towns using a weighted combination of 30 control towns between EW 1, 2014 and EW 26, 2022. FINDINGS: Our study comprised an at-risk population of 607 872 individuals living in intervention sites and 3 894 544 individuals living in control sites. Interventions demonstrated up to 77·28% (121/156, 95% CI 75·81-78·58) intervention efficacy despite incomplete coverage across all towns until EW 26, 2022. Intervention efficacies increased as release coverage increased across all intervention sites. Releases led to 2242 (95% CI 2092-2391) fewer cases per 100 000 people in intervention sites during the study period. Secondary analysis showed that these intervention effects were replicated across all age groups and both sexes for intervention sites. INTERPRETATION: Our results demonstrated the potential of Wolbachia-mediated incompatible insect technique for strengthening dengue control in tropical cities, where dengue burden is the greatest. FUNDING: Singapore Ministry of Finance, Ministry of Sustainability, and the National Environment Agency, and the Singapore National Robotics Program.

Different mechanisms of X-ray irradiation-induced male and female sterility in Aedes aegypti.

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.

Assessing the efficacy of male Wolbachia-infected mosquito deployments to reduce dengue incidence in Singapore: study protocol for a cluster-randomized controlled trial.

BACKGROUND: Dengue is a severe environmental public health challenge in tropical and subtropical regions. In Singapore, decreasing seroprevalence and herd immunity due to successful vector control has paradoxically led to increased transmission potential of the dengue virus. We have previously demonstrated that incompatible insect technique coupled with sterile insect technique (IIT-SIT), which involves the release of X-ray-irradiated male Wolbachia-infected mosquitoes, reduced the Aedes aegypti population by 98% and dengue incidence by 88%. This novel vector control tool is expected to be able to complement current vector control to mitigate the increasing threat of dengue on a larger scale. We propose a multi-site protocol to study the efficacy of IIT-SIT at reducing dengue incidence. METHODS/DESIGN: The study is designed as a parallel, two-arm, non-blinded cluster-randomized (CR) controlled trial to be conducted in high-rise public housing estates in Singapore, an equatorial city-state. The aim is to determine whether large-scale deployment of male Wolbachia-infected Ae. aegypti mosquitoes can significantly reduce dengue incidence in intervention clusters. We will use the CR design, with the study area comprising 15 clusters with a total area of 10.9 km2, covering approximately 722,204 residents in 1713 apartment blocks. Eight clusters will be randomly selected to receive the intervention, while the other seven will serve as non-intervention clusters. Intervention efficacy will be estimated through two primary endpoints: (1) odds ratio of Wolbachia exposure distribution (i.e., probability of living in an intervention cluster) among laboratory-confirmed reported dengue cases compared to test-negative controls and (2) laboratory-confirmed reported dengue counts normalized by population size in intervention versus non-intervention clusters. DISCUSSION: This study will provide evidence from a multi-site, randomized controlled trial for the efficacy of IIT-SIT in reducing dengue incidence. The trial will provide valuable information to estimate intervention efficacy for this novel vector control approach and guide plans for integration into national vector control programs in dengue-endemic settings. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT05505682 . Registered on 16 August 2022. Retrospectively registered.

Standardization of the FAO/IAEA Flight Test for Quality Control of Sterile Mosquitoes.

Successful implementation of the sterile insect technique (SIT) against Aedes aegypti and Aedes albopictus relies on maintaining a consistent release of high-quality sterile males. Affordable, rapid, practical quality control tools based on the male's flight ability (ability to escape from a flight device) may contribute to meeting this requirement. Therefore, this study aims to standardize the use of the original FAO/IAEA rapid quality control flight test device (FTD) (version 1.0), while improving handling conditions and reducing the device's overall cost by assessing factors that could impact the subsequent flight ability of Aedes mosquitoes. The new FTD (version 1.1) is easier to use. The most important factors affecting escape rates were found to be tube color (or "shade"), the combined use of a lure and fan, mosquito species, and mosquito age and density (25; 50; 75; 100 males). Other factors measured but found to be less important were the duration of the test (30, 60, 90, 120 min), fan speed (normal 3000 rpm vs. high 6000 rpm), and mosquito strain origin. In addition, a cheaper version of the FTD (version 2.0) that holds eight individual tubes instead of 40 was designed and successfully validated against the new FTD (version 1.1). It was sensitive enough to distinguish between the effects of cold stress and high irradiation dose. Therefore, the eight-tube FTD may be used to assess Aedes' flight ability. This study demonstrated that the new designs (versions 1.1 and 2.0) of the FTD could be used for standard routine quality assessments of Aedes mosquitoes required for an SIT and other male release-based programs.

Lineage Replacement Associated with Fitness Gain in Mammalian Cells and Aedes aegypti: A Catalyst for Dengue Virus Type 2 Transmission.

Shifting of virus serotypes and clade replacement events are known to drive dengue epidemics. However, only a few studies have attempted to elucidate the virus attributes that contribute to such epidemics. In 2007, Singapore experienced a dengue outbreak affecting more than 8000 individuals. The outbreak ensued with the shuffling of dominant clades (from clade I to clade II) of Dengue virus 2 (DENV-2) cosmopolitan genotype, at a time when the Aedes premise index was significantly low. Therefore, we hypothesized that clade II had higher epidemic potential and fitness than clade I. To test this hypothesis, we tested the replication and apoptotic qualities of clade I and II isolates in mammalian cells and their ability to infect and disseminate in a field strain of Ae. Aegypti. Our findings indicated that clade II replicated more efficiently in mammalian cells than clade I and possessed higher transmission potential in local vectors. This could collectively improve the epidemic potential of clade II, which dominated during the outbreak in 2007. The findings exemplify complex interactions between the emergence, adaptation and transmission potential of DENV, and testify the epidemiological importance of a deeper understanding of virus and vector dynamics in endemic regions.

Strategies to Mitigate Establishment under the Wolbachia Incompatible Insect Technique.

The Incompatible Insect Technique (IIT) strategy involves the release of male mosquitoes infected with the bacterium Wolbachia. Regular releases of male Wolbachia-infected mosquitoes can lead to the suppression of mosquito populations, thereby reducing the risk of transmission of vector-borne diseases such as dengue. However, due to imperfect sex-sorting under IIT, fertile Wolbachia-infected female mosquitoes may potentially be unintentionally released into the environment, which may result in replacement and failure to suppress the mosquito populations. As such, mitigating Wolbachia establishment requires a combination of IIT with other strategies. We introduced a simple compartmental model to simulate ex-ante mosquito population dynamics subjected to a Wolbachia-IIT programme. In silico, we explored the risk of replacement, and strategies that could mitigate the establishment of the released Wolbachia strain in the mosquito population. Our results suggest that mitigation may be achieved through the application of a sterile insect technique. Our simulations indicate that these interventions do not override the intended wild type suppression of the IIT approach. These findings will inform policy makers of possible ways to mitigate the potential establishment of Wolbachia using the IIT population control strategy.

Wolbachia wAlbB remains stable in Aedes aegypti over 15 years but exhibits genetic background-dependent variation in virus blocking.

The ability of the maternally transmitted endosymbiotic bacterium Wolbachia to induce cytoplasmic incompatibility (CI) and virus blocking makes it a promising weapon for combatting mosquito-borne diseases through either suppression or replacement of wild-type populations. Recent field trials show that both approaches significantly reduce the incidence of dengue fever in humans. However, new questions emerge about how Wolbachia-mosquito associations will co-evolve over time and whether Wolbachia-mediated virus blocking will be affected by the genetic diversity of mosquitoes and arboviruses in the real world. Here, we have compared the Wolbachia density and CI expression of two wAlbB-infected Aedes aegypti lines transinfected 15 years apart. We have also assessed wAlbB-mediated virus blocking against dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) viruses and examined whether host genetic backgrounds modulate viral blocking effects by comparing ZIKV infection in mosquitoes with a Mexican genetic background to those with a Singaporean background. Our results show that over 15 years, wAlbB maintained the capacity to form a stable association with Ae. aegypti in terms of both density and CI expression. There were variations in wAlbB-induced virus blocking against CHIKV, DENV, and ZIKV, and higher inhibitory effects on ZIKV in mosquitoes on the Singaporean genetic background than on the Mexican background. These results provide important information concerning the robustness and long-term stability of Wolbachia as a biocontrol agent for arbovirus disease control.

Transient transmission of Chikungunya virus in Singapore exemplifies successful mitigation of severe epidemics in a vulnerable population.

OBJECTIVES: Singapore experienced two major outbreaks of chikungunya in 2008-09 and 2013-14. Despite repeated virus introductions, fresh local outbreaks have not emerged after 2014. The present study reviews the success of chikungunya control in Singapore, despite repeated introduction of virus strains, presence of competent vectors and an immunologically naïve population. METHODS: Chikungunya virus (CHIKV) sequences (421 envelope 1 genes and 56 polyproteins) were analysed to distinguish the indigenous virus groups from 2008 to 2020. Vector surveillance data was used to incriminate the vector/s associated with local outbreaks. The population exposure to CHIKV was determined by assessing the seroprevalence status in three cohorts of sera collected in 2009 (n=2,008), 2013 (n=2,000) and 2017 (n=3,615). RESULTS: Four distinct groups of CHIKV of East, Central and South African genotype have mainly circulated since 2008, transmitted primarily by Aedes albopictus. The age weighted CHIKV IgG prevalence rates were low (1-5%) and showed a non-significant increase from 2009 to 2013, but a significant decrease in 2017. In contrast, the prevalence of CHIKV neutralising antibodies in the population increased significantly from 2009 to 2013, with no significant change in 2017, but the levels remained below 2%. CONCLUSIONS: The evidence suggested that surveillance and vector control strategies implemented were robust to avert severe epidemics, despite repeated introduction of virus strains, presence of competent vectors and an immunologically naïve population.

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes.

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain's fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.  Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.

Prevalence of simian malaria parasites in macaques of Singapore.

Plasmodium knowlesi is a simian malaria parasite currently recognized as the fifth causative agent of human malaria. Recently, naturally acquired P. cynomolgi infection in humans was also detected in Southeast Asia. The main reservoir of both parasites is the long-tailed and pig-tailed macaques, which are indigenous in this region. Due to increased urbanization and changes in land use, there has been greater proximity and interaction between the long-tailed macaques and the general population in Singapore. As such, this study aims to determine the prevalence of simian malaria parasites in local macaques to assess the risk of zoonosis to the general human population. Screening for the presence of malaria parasites was conducted on blood samples from 660 peridomestic macaques collected between Jan 2008 and Mar 2017, and 379 wild macaques collected between Mar 2009 and Mar 2017, using a Pan-Plasmodium-genus specific PCR. Positive samples were then screened using a simian Plasmodium species-specific nested PCR assay to identify the species of parasites (P. knowlesi, P. coatneyi, P. fieldi, P. cynomolgi, and P. inui) present. All the peridomestic macaques sampled were tested negative for malaria, while 80.5% of the 379 wild macaques were infected. All five simian Plasmodium species were detected; P. cynomolgi being the most prevalent (71.5%), followed by P. knowlesi (47.5%), P. inui (42.0%), P. fieldi (32.5%), and P. coatneyi (28.5%). Co-infection with multiple species of Plasmodium parasites was also observed. The study revealed that Singapore's wild long-tailed macaques are natural hosts of the five simian malaria parasite species, while no malaria was detected in all peridomestic macaques tested. Therefore, the risk of simian malaria transmission to the general human population is concluded to be low. However, this can be better demonstrated with the incrimination of the vectors of simian malaria parasites in Singapore.

Using spatial genetics to quantify mosquito dispersal for control programs.

BACKGROUND: Hundreds of millions of people get a mosquito-borne disease every year and nearly one million die. Transmission of these infections is primarily tackled through the control of mosquito vectors. The accurate quantification of mosquito dispersal is critical for the design and optimization of vector control programs, yet the measurement of dispersal using traditional mark-release-recapture (MRR) methods is logistically challenging and often unrepresentative of an insect's true behavior. Using Aedes aegypti (a major arboviral vector) as a model and two study sites in Singapore, we show how mosquito dispersal can be characterized by the spatial analyses of genetic relatedness among individuals sampled over a short time span without interruption of their natural behaviors. RESULTS: Using simple oviposition traps, we captured adult female Ae. aegypti across high-rise apartment blocks and genotyped them using genome-wide SNP markers. We developed a methodology that produces a dispersal kernel for distance which results from one generation of successful breeding (effective dispersal), using the distance separating full siblings and 2nd- and 3rd-degree relatives (close kin). The estimated dispersal distance kernel was exponential (Laplacian), with a mean dispersal distance (and dispersal kernel spread σ) of 45.2 m (95% CI 39.7-51.3 m), and 10% probability of a dispersal > 100 m (95% CI 92-117 m). Our genetically derived estimates matched the parametrized dispersal kernels from previous MRR experiments. If few close kin are captured, a conventional genetic isolation-by-distance analysis can be used, as it can produce σ estimates congruent with the close-kin method if effective population density is accurately estimated. Genetic patch size, estimated by spatial autocorrelation analysis, reflects the spatial extent of the dispersal kernel "tail" that influences, for example, the critical radii of release zones and the speed of Wolbachia spread in mosquito replacement programs. CONCLUSIONS: We demonstrate that spatial genetics can provide a robust characterization of mosquito dispersal. With the decreasing cost of next-generation sequencing, the production of spatial genetic data is increasingly accessible. Given the challenges of conventional MRR methods, and the importance of quantified dispersal in operational vector control decisions, we recommend genetic-based dispersal characterization as the more desirable means of parameterization.

Identification key to the genera of adult female mosquitoes (Diptera: Culicidae) of Singapore.

Accurate identification of mosquitoes is crucial for the management and control of vector species. Although Singapore is a tropical country with high mosquito diversity, there are no identification keys specific to its mosquito fauna. An illustrated identification key to the genera of the adult female mosquitoes of Singapore is presented. Twenty genera are characterized and two genera found in Malaysia, Culiseta and Topomyia, are also included. The key will aid non-taxonomically trained operational officers in adult mosquito identification. The key to the genera provides the first crucial step in the process to species identification.

Viral and Antibody Kinetics, and Mosquito Infectivity of an Imported Case of Zika Fever Due to Asian Genotype (American Strain) in Singapore.

We report a case of a Singaporean who acquired Zika virus (ZIKV) during a visit to Cuba. The infection was confirmed using molecular and serological methods. This report highlights potential drawbacks of using IgG serology for diagnosis of flavivirus infections in endemic regions. The low viremia detected during the early phase of this case resulted in low mosquito infectivity rates, suggesting the possibility of ZIKV transmission prior to clinical onset. The report also emphasizes the challenges of public health interventions for Zika fever and the importance of sustaining a low vector population to reduce the risk of arbovirus transmission in vulnerable regions.

wMel limits zika and chikungunya virus infection in a Singapore Wolbachia-introgressed Ae. aegypti strain, wMel-Sg.

BACKGROUND: Zika (ZIKV) and Chikungunya (CHIKV) viruses are emerging Aedes-borne viruses that are spreading outside their known geographic range and causing wide-scale epidemics. It has been reported that these viruses can be transmitted efficiently by Ae. aegypti. Recent studies have shown that Ae. aegypti when transinfected with certain Wolbachia strains shows a reduced replication and dissemination of dengue (DENV), Chikungunya (CHIKV), and Yellow Fever (YFV) viruses. The aim of this study was to determine whether the wMel strain of Wolbachia introgressed onto a Singapore Ae. aegypti genetic background was able to limit ZIKV and CHIKV infection in the mosquito. METHODOLOGY/PRINCIPAL FINDINGS: Five to seven-day old mosquitoes either infected or uninfected with wMel Wolbachia were orally infected with a Ugandan strain of ZIKV and several outbreak strains of CHIKV. The midgut and salivary glands of each mosquito were sampled at days 6, 9 and 13 days post infectious blood meal to determine midgut infection and salivary glands dissemination rates, respectively. In general, all wild type Ae. aegypti were found to have high ZIKV and CHIKV infections in their midguts and salivary glands, across all sampling days, compared to Wolbachia infected counterparts. Median viral titre for all viruses in Wolbachia infected mosquitoes were significantly lower across all time points when compared to wild type mosquitoes. Most significantly, all but two and one of the wMel infected mosquitoes had no detectable ZIKV and CHIKV, respectively, in their salivary glands at 14 days post-infectious blood meal. CONCLUSIONS: Our results showed that wMel limits both ZIKV and CHIKV infection when introgressed into a Singapore Ae. aegypti genetic background. These results also strongly suggest that female Aedes aegypti carrying Wolbachia will have a reduced capacity to transmit ZIKV and CHIKV.

Diagnostic challenges and case management of the first imported case of Plasmodium knowlesi in Sri Lanka.

BACKGROUND: Sri Lanka has achieved 'malaria-free' status and is now in the phase of prevention of re-introduction of malaria. Imported malaria remains a challenge to resurgence of the disease. The diagnostic challenges encountered and the rapid response initiated to manage a Plasmodium infection, which was later confirmed as Plasmodium knowlesi, the first reported case from Sri Lanka, is discussed. CASE PRESENTATION: An army officer who returned from Malaysia in October 2016 was found to be positive for Plasmodium both by microscopy and rapid diagnostic test (RDT) by the Anti Malaria Campaign Sri Lanka (AMC) during his third visit to a health care provider. Microscopy findings were suspicious of P. knowlesi infection as the smears showed parasite stages similar to both Plasmodium malariae and Plasmodium falciparum. Nested PCR at AMC confirmed Plasmodium genus, but not the species. In the absence of species confirmation, the patient was treated as a case of P. falciparum. The presence of P. knowlesi was later confirmed by a semi-nested PCR assay performed at the Environmental Health Institute, National Environmental Agency in Singapore. The parasite strain was also characterized by sequencing the circumsporozoite gene. Extensive case investigation including parasitological and entomological surveillance was carried out. CONCLUSIONS: Plasmodium knowlesi should be suspected in patients returning from countries in the South Asian region where the parasite is prevalent and when blood smear results are inconclusive.

The queenslandensis and the type Form of the Dengue Fever Mosquito (Aedes aegypti L.) Are Genomically Indistinguishable.

BACKGROUND: The mosquito Aedes aegypti (L.) is a major vector of viral diseases like dengue fever, Zika and chikungunya. Aedes aegypti exhibits high morphological and behavioral variation, some of which is thought to be of epidemiological significance. Globally distributed domestic Ae. aegypti have often been grouped into (i) the very pale variety queenslandensis and (ii) the type form. Because the two color forms co-occur across most of their range, there is interest in understanding how freely they interbreed. This knowledge is particularly important for control strategies that rely on mating compatibilities between the release and target mosquitoes, such as Wolbachia releases and SIT. To address this question, we analyzed nuclear and mitochondrial genome-wide variation in the co-occurring pale and type Ae. aegypti from northern Queensland (Australia) and Singapore. METHODS/FINDINGS: We typed 74 individuals at a 1170 bp-long mitochondrial sequence and at 16,569 nuclear SNPs using a customized double-digest RAD sequencing. 11/29 genotyped individuals from Singapore and 11/45 from Queensland were identified as var. queenslandensis based on the diagnostic scaling patterns. We found 24 different mitochondrial haplotypes, seven of which were shared between the two forms. Multivariate genetic clustering based on nuclear SNPs corresponded to individuals' geographic location, not their color. Several family groups consisted of both forms and three queenslandensis individuals were Wolbachia infected, indicating previous breeding with the type form which has been used to introduce Wolbachia into Ae. aegypti populations. CONCLUSION: Aedes aegypti queenslandensis are genomically indistinguishable from the type form, which points to these forms freely interbreeding at least in Australia and Singapore. Based on our findings, it is unlikely that the presence of very pale Ae. aegypti will affect the success of Aedes control programs based on Wolbachia-infected, sterile or RIDL mosquitoes.

Membrane feeding of dengue patient's blood as a substitute for direct skin feeding in studying Aedes-dengue virus interaction.

BACKGROUND: Understanding the interaction between Aedes vectors and dengue viruses (DENV) has significant implications in determining the transmission dynamics of dengue. The absence of an animal model and ethical concerns regarding direct feeding of mosquitoes on patients has resulted in most infection studies using blood meals spiked with laboratory-cultured DENV. Data obtained from such studies may not reflect the natural human-mosquito transmission scenario. This study explored the potential of using membrane feeding of dengue patient's blood as a substitute for direct skin feeding. METHODS: Four to six-day old female Ae. aegypti were provided the opportunity to feed via direct exposure to a patient's forearm for 15 min or via exposure to EDTA-treated blood from the same patient through an artificial membrane for 30 min. Mosquitoes from both feeding methods were incubated inside environmental chambers. Mosquitoes were sampled at day 13 post-feeding. Midgut and salivary glands of each mosquito were dissected to determine DENV infection by RT-qPCR and viral titration, respectively. RESULTS: Feeding rates: Direct skin feeding assay (DSFA) consistently showed higher mosquito feeding rates (93.3-100%) when compared with the membrane feeding assay (MFA) (48-98.2%). Midgut infection: Pair-wise comparison between methods showed no significant difference in midgut infection rates between mosquitoes exposed via each method and a strong correlation was observed in midgut infection rates for both feeding methods (r = 0.89, P < 0.0001). Overall midgut viral titers (n = 20) obtained by both methods were comparable (P ≥ 0.06). Salivary gland infection: Pair-wise comparison between both methods revealed no significant difference in salivary gland infection rate. Strong correlation in salivary gland infection was observed between DSFA and MFA (r = 0.81, P < 0.0001). In general, mosquitoes fed directly on dengue patients and those on patients' blood (n = 11) had comparable virus titer (P ≥ 0.09). CONCLUSION: DENV midgut and salivary gland infection rates showed good concordance between DSFA and MFA blood meal exposure methods. Freshly-obtained venous blood in EDTA from dengue patients for MFA can be used as a substitute to DSFA, especially in circumstances where bioethics approval or patient recruitment is difficult to obtain for vector competence studies. Nevertheless, mosquito numbers will need to be increased to compensate for lower feeding rate in MFA.