Speciation patterns of Aedes mosquitoes in the Scutellaris Group: a mitochondrial perspective.

The Scutellaris Group of Aedes comprises 47 mosquito species, including Aedes albopictus. While Ae. albopictus is widely distributed, the other species are mostly found in the Asia-Pacific region. Evolutionary history researches of Aedes species within the Scutellaris Group have mainly focused on Ae. albopictus, a species that raises significant public health concerns, neglecting the other species. In this study, we aimed to assess genetic diversity and estimate speciation times of several species within the Scutellaris Group. Mosquitoes were therefore collected from various Asia-Pacific countries. Their mitochondrial cytochrome c oxidase subunit 1 (cox1) and subunit 3 (cox3) sequences were analyzed alongside those of other Scutellaris Group species available in the GenBank database. To estimate the divergence time, we analyzed 1849 cox1 gene sequences from 21 species, using three species (Aedes aegypti, Aedes notoscriptus and Aedes vigilax) as outgroups. We found that most of the speciation dates occurred during the Paleogene and the Neogene periods. A separation between the Scutellaris Subgroup and the Albopictus Subgroup occurred approximately 64-61 million years ago (MYA). We also identified a split between species found in Asia/Micronesia and those collected in Melanesia/Polynesia approximately 36-35 MYA. Our findings suggest that the speciation of Aedes species within the Scutellaris Group may be driven by diversity in mammalian hosts, climate and environmental changes, and geological dynamics rather than human migration.

Metabolic Resistance and Not Voltage-Gated Sodium Channel Gene Mutation Is Associated with Pyrethroid Resistance of Aedes albopictus (Skuse, 1894) from Cambodia.

(1) Background: In Cambodia, Aedes albopictus is an important vector of the dengue virus. Vector control using insecticides is a major strategy implemented in managing mosquito-borne diseases. Resistance, however, threatens to undermine the use of insecticides. In this study, we present the levels of insecticide resistance of Ae. albopictus in Cambodia and the mechanisms involved. (2) Methods: Two Ae. albopictus populations were collected from the capital, Phnom Penh city, and from rural Pailin province. Adults were tested with diagnostic doses of malathion (0.8%), deltamethrin (0.03%), permethrin (0.25%), and DDT (4%) using WHO tube assays. Synergist assays using piperonyl butoxide (PBO) were implemented before the pyrethroid assays to detect the potential involvement of metabolic resistance mechanisms. Adult female mosquitoes collected from Phnom Penh and Pailin were tested for voltage-gated sodium channel (VGSC) kdr (knockdown resistance) mutations commonly found in Aedes sp.-resistant populations throughout Asia (S989P, V1016G, and F1534C), as well as for other mutations (V410L, L982W, A1007G, I1011M, T1520I, and D1763Y). (3) Results: The two populations showed resistance against all the insecticides tested (<90% mortality). The use of PBO (an inhibitor of P450s) strongly restored the efficacy of deltamethrin and permethrin against the two resistant populations. Sequences of regions of the vgsc gene showed a lack of kdr mutations known to be associated with pyrethroid resistance. However, four novel non-synonymous mutations (L412P/S, C983S, Q1554STOP, and R1718L) and twenty-nine synonymous mutations were detected. It remains to be determined whether these mutations contribute to pyrethroid resistance. (4) Conclusions: Pyrethroid resistance is occurring in two Ae. albopictus populations originating from urban and rural areas of Cambodia. The resistance is likely due to metabolic resistance specifically involving P450s monooxygenases. The levels of resistance against different insecticide classes are a cause for concern in Cambodia. Alternative tools and insecticides for controlling dengue vectors should be used to minimize disease prevalence in the country.

Bionomics of malaria vectors in Lao PDR, 2018-2020: entomological surveillance as a key tool for malaria elimination.

BACKGROUND: The Lao PDR National Strategic Plan for malaria control and elimination for year 2021-2025 emphasizes the importance of routine entomological surveillance being conducted in areas with high transmission and in active malaria foci in elimination targeted areas. The collection of entomological surveillance data that is closely linked to recent epidemiological data is crucial for improving impact, as it contributes to the evidence package that supports operational and strategic decision-making of national malaria programmes, as they accelerate their last mile of elimination. METHODS: The Center for Malariology Parasitology and Epidemiology (CMPE) entomology team conducted entomological surveillance activities at 13 sentinel sites in 8 provinces and at active transmission foci sites from 2018 to 2020. The techniques used for the mosquito collection were indoor and outdoor human landing collections (from houses and from cultivation areas) and cattle baited net trap collections. RESULTS: There were 5601 Anopheles mosquito females captured and identified throughout the study, on both human and cow bait. They represented 15 different species or species complexes. The primary malaria vectors as well as the secondary vectors were present in all collection sites in the south, indicating that people living in these rural areas with high malaria incidence are exposed to the vectors. The vectors were highly zoophilic, but they still bite humans throughout the night with a high peak of activity before midnight, both indoors and outdoors. Overall, 17% of the malaria vectors were collected indoors when the people are sleeping. This confirms the importance of bed net use during the night. Thirty-two percent of primary and secondary vectors were collected outdoors at times when people are usually awake and outdoors, which shows that people are exposed to potentially infectious mosquitoes and the importance of personal protection at these times. The findings showed that residual transmission may occur outdoors in the villages, and outside the villages in cultivation fields and forested areas. Epidemiological data showed that transmission was higher in surveillance sites which were targeted as part of a malaria response rather than sentinel sites. CONCLUSIONS: Understanding where and how transmission is persisting, monitoring and mapping vector species distribution in areas with active transmission, monitoring biting trends, and designing evidence based and effective vector control interventions are critical to accelerating progress toward malaria elimination. In this context, the role of entomological surveillance combined with epidemiological data should be considered as a cornerstone in achieving malaria elimination.

Genetic Diversity of Dengue Vector Aedes albopictus Collected from South Korea, Japan, and Laos.

Aedes albopictus is native to Southeast Asia and has emerged as a major vector for vector-borne diseases that are spreading rapidly worldwide. Recent studies have shown that Ae. albopictus populations have different genetic groups dependent on their thermal adaptations; however, studies on Korean populations are limited. In this study, we analyzed the genetic diversity and structure of two mitochondrial genes (COI and ND5) and sixteen microsatellites in mosquitoes inhabiting Korea, Japan, and Laos. The results indicate that the Korean population has low genetic diversity, with an independent cluster distinct from the Laos population. Mixed clusters have also been observed in the Korean population. On the basis of these findings, two hypotheses are proposed. First, certain Korean populations are native. Second, some subpopulations that descended from the metapopulation (East Asian countries) were introduced to Japan before migrating to Korea. Furthermore, we previously demonstrated that Ae. albopictus appears to have been imported to Korea. In conclusion, the dengue-virus-carrying mosquitoes could migrate to Korea from Southeast Asian epidemic regions, where they can survive during the severe winter months. The key findings can be used to establish an integrated pest management strategy based on population genetics for the Korean Ae. albopictus population.

Predicting the Effects of Climate Change on Dengue Vector Densities in Southeast Asia through Process-Based Modeling.

BACKGROUND: Aedes aegypti and Ae. albopictus mosquitoes are major vectors for several human diseases of global importance, such as dengue and yellow fever. Their life cycles and hosted arboviruses are climate sensitive and thus expected to be impacted by climate change. Most studies investigating climate change impacts on Aedes at global or continental scales focused on their future global distribution changes, whereas a single study focused on its effects on Ae. aegypti densities regionally. OBJECTIVES: A process-based approach was used to model densities of Ae. aegypti and Ae. albopictus and their potential evolution with climate change using a panel of nine CMIP6 climate models and climate scenarios ranging from strong to low mitigation measures at the Southeast Asian scale and for the next 80 y. METHODS: The process-based model described, through a system of ordinary differential equations, the variations of mosquito densities in 10 compartments, corresponding to 10 different stages of mosquito life cycle, in response to temperature and precipitation variations. Local field data were used to validate model outputs. RESULTS: We show that both species densities will globally increase due to future temperature increases. In Southeast Asia by the end of the century, Ae. aegypti densities are expected to increase from 25% with climate mitigation measures to 46% without; Ae. albopictus densities are expected to increase from 13%-21%, respectively. However, we find spatially contrasted responses at the seasonal scales with a significant decrease in Ae. albopictus densities in lowlands during summer in the future. DISCUSSION: These results contrast with previous results, which brings new insight on the future impacts of climate change on Aedes densities. Major sources of uncertainties, such as mosquito model parametrization and climate model uncertainties, were addressed to explore the limits of such modeling. https://doi.org/10.1289/EHP11068.

MALDI-TOF MS: An effective tool for a global surveillance of dengue vector species.

Dengue, Zika and chikungunya viruses cause significant human public health burdens in the world. These arboviruses are transmitted by vector mosquito species notably Aedes aegypti and Aedes albopictus. In the Pacific region, more vector species of arboviruses belonging to the Scutellaris Group are present. Due to the expansion of human travel and international trade, the threat of their dispersal in other world regions is on the rise. Strengthening of entomological surveillance ensuring rapid detection of introduced vector species is therefore required in order to avoid their establishment and the risk of arbovirus outbreaks. This surveillance relies on accurate species identification. The aim of this study was to assess the use of the Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) as a tool for an international identification and surveillance of these mosquito vectors of arboviruses. Field-mosquitoes belonging to 8 species (Ae. aegypti, Ae. albopictus, Aedes polynesiensis, Aedes scutellaris, Aedes pseudoscutellaris, Aedes malayensis, Aedes futunae and Culex quinquefasciatus) from 6 countries in the Pacific, Asian and Madagascar, were included in this study. Analysis provided evidence that a MALDI-TOF database created using mosquitoes from the Pacific region allowed suitable identification of mosquito species from the other regions. This technic was as efficient as the DNA sequencing method in identifying mosquito species. Indeed, with the exception of two Ae. pseudoscutellaris, an exact species identification was obtained for all individual mosquitoes. These findings highlight that the MALDI-TOF MS is a promising tool that could be used for a global comprehensive arbovirus vector surveillance.

Small-scale field assessment against the dengue vector Aedes aegypti using the auto-dissemination approach in an urban area of Vientiane, Lao PDR.

BACKGROUND: In Lao PDR, dengue fever is the most important vector borne disease and vector control remains the principal method to fight against Aedes aegypti the primary transmitter mosquito species. Vector control management programs need new strategies in addition to conventional larviciding and adulticiding interventions in the country. In this study, we examined the In2Care® Mosquito Trap's efficacy using insecticide auto-dissemination strategy. The insecticide pyriproxyfen, present in powder form inside the trap station, contaminates the body of gravid female mosquitoes visiting the traps and is later on disseminated via the mosquitoes in breeding sites surrounding the traps. We tested the attractiveness of the Traps, their efficacy to reduce the larval and adult abundance, and the impact on emergence rates. Specifically, we tested if the servicing interval of the In2Care® Mosquito Trap could be extended to 12 weeks. METHODS: Two black plastic ovitrap buckets and two BG® sentinel traps were placed in the premises of the Science campus of Vientiane Capital located in an urban area to measure weekly the larval and adult relative abundance of Aedes mosquitoes from 2017 to 2019. Twenty-five In2Care® Mosquito Traps were evenly distributed in this area and two studies of 12 weeks were implemented during January and April 2018 and, July to October 2018 (dry and rainy season, respectively). Every 2 weeks, water samples from 5 In2Care® Traps were randomly selected and tested at the laboratory with Ae. aegypti larvae to measure the larval and pupal mortality. The relative abundance of Aedes mosquitoes in the BG traps® with the presence of In2Care® Traps in 2018, was compared with the surveillance results obtained in 2017 and 2019 without In2Care® Traps. Every week, water samples from the ovitrap buckets were tested for Emergence Inhibition (EI). RESULTS: The In2Care® Traps were very attractive to gravid Ae. aegypti mosquitoes specifically during the rainy seasons with 96% of the traps colonized with larvae/pupae within four weeks. The bioassays showed 100% mortality in the water samples from the traps during the twelve weeks studies showing the good efficacy over time of the pyriproxyfen without additional servicing in the 12 week period. In addition, the larvicide was successfully disseminated into the ovitrap buckets placed in the treated area where 100% of EI during all weeks of intervention was measured. There was no significant effect of the treatment on adult abundance reduction in the treated area, probably due to recolonization of adult mosquitoes surrounding the field experiment. CONCLUSIONS: The observed potential of the In2Care® Mosquito Trap using the auto-dissemination strategy could lead to the use of this new tool in combination with conventional control methods against Dengue vectors in urban tropical areas. Large scale field trials should be implemented in Lao PDR to prove its efficacy for Public Health programs.

Genomic Shifts, Phenotypic Clines, and Fitness Costs Associated With Cold Tolerance in the Asian Tiger Mosquito.

Climatic variation is a key driver of genetic differentiation and phenotypic traits evolution, and local adaptation to temperature is expected in widespread species. We investigated phenotypic and genomic changes in the native range of the Asian tiger mosquito, Aedes albopictus. We first refine the phylogeographic structure based on genome-wide regions (1,901 double-digest restriction-site associated DNA single nucleotide polymophisms [ddRAD SNPs]) from 41 populations. We then explore the patterns of cold adaptation using phenotypic traits measured in common garden (wing size and cold tolerance) and genotype-temperature associations at targeted candidate regions (51,706 exon-capture SNPs) from nine populations. We confirm the existence of three evolutionary lineages including clades A (Malaysia, Thailand, Cambodia, and Laos), B (China and Okinawa), and C (South Korea and Japan). We identified temperature-associated differentiation in 15 out of 221 candidate regions but none in ddRAD regions, supporting the role of directional selection in detected genes. These include genes involved in lipid metabolism and a circadian clock gene. Most outlier SNPs are differently fixed between clades A and C, whereas clade B has an intermediate pattern. Females are larger at higher latitude yet produce no more eggs, which might favor the storage of energetic reserves in colder climate. Nondiapausing eggs from temperate populations survive better to cold exposure than those from tropical populations, suggesting they are protected from freezing damages but this cold tolerance has a fitness cost in terms of egg viability. Altogether, our results provide strong evidence for the thermal adaptation of A. albopictus across its wide temperature range.

Detection of pyrethroid resistance mutations and intron variants in the voltage-gated sodium channel of Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus mosquitoes from Lao People's Democratic Republic.

In Lao People's Democratic Republic, Aedes aegypti (Linnaeus 1762) and Aedes albopictus (Skuse 1894) mosquitoes (Diptera: Culicidae) are vectors of arboviral diseases such as dengue. As the treatment for these diseases is limited, control of the vectors with the use of pyrethroid insecticides is still essential. However, mutations in the voltage-gated sodium channel (vgsc) gene giving rise to pyrethroid resistance are threatening vector control programs. Here, we analysed both Ae. aegypti and Ae. albopictus mosquitoes, which were collected in different districts of Laos (Kaysone Phomvihane, Vangvieng, Saysettha and Xaythany), for vgsc mutations commonly found throughout Asia (S989P, V1016G and F1534C). Sequences of the vgsc gene showed that the F1534C mutation was prevalent in both Aedes species. S989P and V1016G mutations were detected in Ae. aegypti from each site and were always found together. In addition, the mutation T1520I was seen in Ae. albopictus mosquitoes from Saysettha district as well as in all Ae. aegypti samples. Thus, mutations in the vgsc gene of Ae. aegypti are prevalent in the four districts studied indicating growing insecticide resistance throughout Laos. Constant monitoring programmes and alternative strategies for controlling Aedes should be utilized in order to prolong the effectiveness of pyrethroids thereby maximizing vector control.

Monitoring insecticide resistance of adult and larval Aedes aegypti (Diptera: Culicidae) in Phnom Penh, Cambodia.

BACKGROUND: Dengue fever is a major public health concern in Cambodia, with thousands of cases every year in urban, suburban and rural areas of the country. The main vector of dengue fever in Cambodia is Aedes aegypti. The organophosphate larvicide temephos and adulticides belonging to the pyrethroid family have been widely used for decades by public health authorities to fight dengue vectors, but resistance of Ae. aegypti to these insecticides has been previously described for Cambodia. METHODS: In order to adapt the vector control strategy presently used in Cambodia, we tested 14 adulticides belonging to the carbamate, organochlorine, organophosphate, and pyrethroid insecticide families and three larvicides [temephos, spinosad and Bacillus thuringiensis ser. israelensis (Bti)] belonging to three different insecticide families (organophosphates, spinosyns and entomopathogenic bacteria). The standard procedures used here to test the adults and larvae of an Ae. aegypti population from Phnom Penh followed World Health Organization guidelines. RESULTS: For adults, high mortality rates were observed with carbamate, organophosphate and organochlorine (with the exception of dichlorodiphenyltrichloroethane) insecticides (i.e. between 87.6 and 100%), while low mortality rates were observed with all of the tested pyrethroid insecticides (i.e. between 1 and 35%). For larvae, no resistance against Bti was detected [resistance ratio (RR90 < 1.6)], but moderate resistance was observed for temephos and spinosad (RR90 < 5.6). CONCLUSIONS: The results of this study indicate that (i) Bti should be considered a serious alternative to temephos for the control of Ae. aegypti larvae; and (ii) the carbamate adulticides propoxur and bendiocarb should be employed instead of the widely used pyrethroid insecticides for the control of adult Ae. aegypti on land under mosaic farming and crop rotation in Cambodia, as the insects were found to be resistant to the latter types of insecticide. Research focusing on insecticide resistance and innovative and effective vector control strategies should be undertaken as a public health priority in Cambodia.

Low Transmission of Chikungunya Virus by Aedes aegypti from Vientiane Capital, Lao PDR

In 2012−2013, chikungunya virus (CHIKV) was the cause of a major outbreak in the southern part of Lao People's Democratic Republic (Lao PDR). Since then, only a few imported cases, with isolates belonging to different lineages, were recorded between 2014 and 2020 in Vientiane capital and few autochthonous cases of ECSA-IOL lineage were detected in the south of the country in 2020. The CHIKV epidemiological profile contrasts with the continuous and intensive circulation of dengue virus in the country, especially in Vientiane capital. The study's aim was to investigate the ability of the local field-derived Aedes aegypti population from Vientiane capital to transmit the Asian and ECSA-IOL lineages of CHIKV. Our results revealed that, for both CHIKV lineages, infection rates were low and dissemination rates were high. The transmission rates and efficiencies evidenced a low vector competence for the CHIKV tested. Although this population of Ae. aegypti showed a relatively modest vector competence for these two CHIKV lineages, several other factors could influence arbovirus emergence such as the longevity and density of female mosquitoes. Due to the active circulation of CHIKV in Southeast Asia, investigations on these factors should be done to prevent the risk of CHIKV emergence and spread in Lao PDR and neighboring countries.

A genomic amplification affecting a carboxylesterase gene cluster confers organophosphate resistance in the mosquito Aedes aegypti: From genomic characterization to high-throughput field detection.

By altering gene expression and creating paralogs, genomic amplifications represent a key component of short-term adaptive processes. In insects, the use of insecticides can select gene amplifications causing an increased expression of detoxification enzymes, supporting the usefulness of these DNA markers for monitoring the dynamics of resistance alleles in the field. In this context, the present study aims to characterize a genomic amplification event associated with resistance to organophosphate insecticides in the mosquito Aedes aegypti and to develop a molecular assay to monitor the associated resistance alleles in the field. An experimental evolution experiment using a composite population from Laos supported the association between the over-transcription of multiple contiguous carboxylesterase genes on chromosome 2 and resistance to multiple organophosphate insecticides. Combining whole genome sequencing and qPCR on specific genes confirmed the presence of a ~100-Kb amplification spanning at least five carboxylesterase genes at this locus with the co-existence of multiple structural duplication haplotypes. Field data confirmed their circulation in South-East Asia and revealed high copy number polymorphism among and within populations suggesting a trade-off between this resistance mechanism and associated fitness costs. A dual-color multiplex TaqMan assay allowing the rapid detection and copy number quantification of this amplification event in Ae. aegypti was developed and validated on field populations. The routine use of this novel assay will improve the tracking of resistance alleles in this major arbovirus vector.

New insights into malaria vector bionomics in Lao PDR: a nationwide entomology survey.

BACKGROUND: In Laos, the malaria burden remains high despite a significant reduction of cases during the last decade. In the context of the disease elimination by 2030, a nationwide entomological survey was conducted to better understand the distribution, abundance and behaviour of major malaria vectors (Anopheles spp.) in the country. METHODS: Mosquito collections were implemented in ten villages from ten provinces during the rainy and dry seasons of 2014 and 2015 by using human landing catch (HLC) and cow bait collection (CBC) methods. After morphological identification in the field, molecular identification of the sibling species of Anopheles mosquitoes from the Funestus, Leucosphyrus, and Maculatus groups were determined using PCR specific alleles. A screening of Plasmodium falciparum and Plasmodium vivax infections in the vectors was carried out by quantitative PCR assays. RESULTS: A total of 14,146 adult mosquitoes representing 25 different Anopheles species were collected and morphologically identified. Molecular identification revealed the presence of 12 sibling species within the main primary vector groups, including Anopheles maculatus, Anopheles rampae, Anopheles sawadwongporni, Anopheles pseudowillmori, Anopheles dravidicus, Anopheles minimus, Anopheles aconitus, Anopheles pampanai, Anopheles harrisoni, Anopheles dirus, Anopheles baimaii, Anopheles nemophilous. Anopheles maculatus and An. minimus were predominant during both the dry and rainy seasons, but showed highly zoophilic preferences (Zoophilic index of 98% and 95%, respectively). Overall, 22% of the total malaria vectors were collected between 10:00 PM and 5:00 AM indoors when people are sleeping. Twenty-seven percent of primary and secondary vectors were collected outdoors before 10:00 PM or after 5:00 AM, times when people are usually awake and outdoors. Only two specimens were positive for P. falciparum, one An. aconitus from Phongsaly and one An. minimus from Vientiane Province CONCLUSIONS: The results indicate that people living in rural areas in Laos are constantly exposed to malaria vectors throughout the year and specifically outdoors. The use of LLINs/IRS remains important but innovative tools and new strategies are needed to address locally, the early and outdoor malaria transmission. Lack of expertise in general entomological methods may further exacerbate the situation.

Trends of the Dengue Serotype-4 Circulation with Epidemiological, Phylogenetic, and Entomological Insights in Lao PDR between 2015 and 2019.

Dengue outbreaks have regularly been recorded in Lao People's Democratic Republic (PDR) since the first detection of the disease in 1979. In 2012, an integrated arbovirus surveillance network was set up in Lao PDR and an entomological surveillance has been implemented since 2016 in Vientiane Capital. Here, we report a study combining epidemiological, phylogenetic, and entomological analyzes during the largest DENV-4 epidemic ever recorded in Lao PDR (2015-2019). Strikingly, from 2015 to 2019, we reported the DENV-4 emergence and spread at the country level after two large epidemics predominated by DENV-3 and DENV-1, respectively, in 2012-2013 and 2015. Our data revealed a significant difference in the median age of the patient infected by DENV-4 compared to the other serotypes. Phylogenetic analysis demonstrated the circulation of DENV-4 Genotype I at the country level since at least 2013. The entomological surveillance showed a predominance of Aedesaegypti compared to Aedesalbopictus and high abundance of these vectors in dry and rainy seasons between 2016 and 2019, in Vientiane Capital. Overall, these results emphasized the importance of an integrated approach to evaluate factors, which could impact the circulation and the epidemiological profile of dengue viruses, especially in endemic countries like Lao PDR.

High diversity of mosquito vectors in Cambodian primary schools and consequences for arbovirus transmission.

Only few data exist in Cambodia on mosquito diversity and their potential role as vectors. Many arboviruses, such as dengue and Japanese encephalitis, are endemic and mostly affect children in the country. This research sets out to evaluate vector relative abundance and diversity in primary schools in Cambodia in an attempt to explain the apparent burden of dengue fever, severe dengue (DEN), Japanese encephalitis (JE), other arboviral diseases and malaria among children, 15 years and under, attending selected primary schools through vector surveys. Entomological surveys were implemented in primary schools in two provinces of Cambodia to assess the potential risk of exposure of schoolchildren to mosquito vector species. Light traps and BG traps were used to collect adult mosquitoes in 24 schools during the rainy and dry seasons of 2017 and 2018 in Kampong Cham and Tboung Khmum provinces. A total of 61 species were described, including Aedes, Culex and Anopheles species. The relative abundance and biodiversity of mosquito species were dependent on the month and school. Of the 37,725 mosquitoes caught during the study, three species accounted for three-quarters of the relative abundance: Culex vishnui, Anopheles indefinitus and Culex quinquefasciatus. More importantly, nearly 90% of the mosquitoes caught in the schools were identified as potential vectors of pathogens including Japanese encephalitis, dengue, and malaria parasites. Our results showed that schools in Cambodia represent a risk for vector-borne disease transmission and highlight the importance of implementing vector control in schools in Cambodia to decrease the risk of transmission.

Risk of arbovirus emergence via bridge vectors: case study of the sylvatic mosquito Aedes malayensis in the Nakai district, Laos.

Many emerging arboviruses of global public health importance, such as dengue virus (DENV) and yellow fever virus (YFV), originated in sylvatic transmission cycles involving wild animals and forest-dwelling mosquitoes. Arbovirus emergence in the human population typically results from spillover transmission via bridge vectors, which are competent mosquitoes feeding on both humans and wild animals. Another related, but less studied concern, is the risk of 'spillback' transmission from humans into novel sylvatic cycles. We colonized a sylvatic population of Aedes malayensis from a forested area of the Nakai district in Laos to evaluate its potential as an arbovirus bridge vector. We found that this Ae. malayensis population was overall less competent for DENV and YFV than an urban population of Aedes aegypti. Olfactometer experiments showed that our Ae. malayensis colony did not display any detectable attraction to human scent in laboratory conditions. The relatively modest vector competence for DENV and YFV, combined with a lack of detectable attraction to human odor, indicate a low potential for this sylvatic Ae. malayensis population to act as an arbovirus bridge vector. However, we caution that opportunistic blood feeding on humans by sylvatic Ae. malayensis may occasionally contribute to bridge sylvatic and human transmission cycles.

Malaria and Dengue Mosquito Vectors from Lao PDR Show a Lack of the rdl Mutant Allele Responsible for Cyclodiene Insecticide Resistance.

The gamma-aminobutyric acid (GABA) receptor, RDL, plays important roles in neuronal signaling and is the target of highly effective insecticides. A mutation in RDL, commonly A296S, underlies resistance to several insecticides such as cyclodienes. Even though the use of cyclodienes has been banned, the occurrence of mutations substituting A296 is notably high in mosquitoes from several countries. Here, we report a survey investigating the prevalence of the Rdl mutant allele in mosquitoes from Laos, a country where mosquito-borne diseases such as malaria and dengue fever are health concerns. Anopheles and Aedes mosquitoes were collected from 12 provinces in Laos. Adult bioassays on Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) showed that all the populations tested were susceptible to dieldrin (4%) following WHO protocols. Exon 7 from a total of 791 mosquitoes was sequenced to identify the amino acid encoded for at 296 of RDL. Only one of these mosquitoes, Anopheles maculatus rampae Harbach and Somboon (Diptera: Culicidae) from Attapeu, carried the mutant allele being heterozygous for A296S. We therefore found a general lack of the Rdl mutant allele indicating that mosquitoes from Laos are not exposed to insecticides that act on the GABA receptor compared to mosquitoes in several other countries. Identifying the prevalence of the Rdl mutation may help inform the potential use of alternative insecticides that act on the GABA receptor should there be a need to replace pyrethroids in order to prevent/manage resistance.

Distribution of insecticide resistance and mechanisms involved in the arbovirus vector Aedes aegypti in Laos and implication for vector control.

BACKGROUND: The yellow fever mosquito Aedes aegypti is the major vector of dengue, yellow fever, Zika, and Chikungunya viruses. Worldwide vector control is largely based on insecticide treatments but, unfortunately, vector control programs are facing operational challenges due to mosquitoes becoming resistant to commonly used insecticides. In Southeast Asia, resistance of Ae. aegypti to chemical insecticides has been documented in several countries but no data regarding insecticide resistance has been reported in Laos. To fill this gap, we assessed the insecticide resistance of 11 Ae. aegypti populations to larvicides and adulticides used in public health operations in the country. We also investigated the underlying molecular mechanisms associated with resistance, including target site mutations and detoxification enzymes putatively involved in metabolic resistance. METHODS AND RESULTS: Bioassays on adults and larvae collected in five provinces revealed various levels of resistance to organophosphates (malathion and temephos), organochlorine (DDT) and pyrethroids (permethrin and deltamethrin). Synergist bioassays showed a significant increased susceptibility of mosquitoes to insecticides after exposure to detoxification enzyme inhibitors. Biochemical assays confirmed these results by showing significant elevated activities of cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST) and carboxylesterases (CCE) in adults. Two kdr mutations, V1016G and F1534C, were detected by qPCR at low and high frequency, respectively, in all populations tested. A significant negative association between the two kdr mutations was detected. No significant association between kdr mutations frequency (for both 1534C and 1016G) and survival rate to DDT or permethrin (P > 0.05) was detected. Gene Copy Number Variations (CNV) were detected for particular detoxification enzymes. At the population level, the presence of CNV affecting the carboxylesterase CCEAE3A and the two cytochrome P450 CYP6BB2 and CYP6P12 were significantly correlated to insecticide resistance. CONCLUSIONS: These results suggest that both kdr mutations and metabolic resistance mechanisms are present in Laos but their impact on phenotypic resistance may differ in proportion at the population or individual level. Molecular analyses suggest that CNV affecting CCEAE3A previously associated with temephos resistance is also associated with malathion resistance while CNV affecting CYP6BB2 and CYP6P12 are associated with pyrethroid and possibly DDT resistance. The presence of high levels of insecticide resistance in the main arbovirus vector in Laos is worrying and may have important implications for dengue vector control in the country.

Population genetics of Aedes albopictus (Diptera: Culicidae) in its native range in Lao People's Democratic Republic.

BACKGROUND: The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse) is an important worldwide invasive species and can be a locally important vector of chikungunya, dengue and, potentially, Zika. This species is native to Southeast Asia where populations thrive in both temperate and tropical climates. A better understanding of the population structure of Ae. albopictus in Lao PDR is very important in order to support the implementation of strategies for diseases prevention and vector control. In the present study, we investigated the genetic variability of Ae. albopictus across a north-south transect in Lao PDR. METHODS: We used variability in a 1337-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), to assess the population structure of Ae. albopictus in Lao PDR. For context, we also examined variability at the same genetic locus in samples of Ae. albopictus from Thailand, China, Taiwan, Japan, Singapore, Italy and the USA. RESULTS: We observed very high levels of genetic polymorphism with 46 novel haplotypes in Ae. albopictus from 9 localities in Lao PDR and Thailand populations. Significant differences were observed between the Luangnamtha population and other locations in Lao PDR. However, we found no evidence of isolation by distance. There was overall little genetic structure indicating ongoing and frequent gene flow among populations or a recent population expansion. Indeed, the neutrality test supported population expansion in Laotian Ae. albopictus and mismatch distribution analyses showed a lack of low frequency alleles, a pattern often seen in bottlenecked populations. When samples from Lao PDR were analyzed together with samples from Thailand, China, Taiwan, Japan, Singapore, Italy and the USA, phylogenetic network and Bayesian cluster analysis showed that most populations from tropical/subtropical regions are more genetically related to each other, than populations from temperate regions. Similarly, most populations from temperate regions are more genetically related to each other, than those from tropical/subtropical regions. CONCLUSIONS: Aedes albopictus in Lao PDR are genetically related to populations from tropical/subtropical regions (i.e. Thailand, Singapore, and California and Texas in the USA). The extensive gene flow among locations in Lao PDR indicates that local control is undermined by repeated introductions from untreated sites.

A peridomestic Aedes malayensis population in Singapore can transmit yellow fever virus.

The case-fatality rate of yellow fever virus (YFV) is one of the highest among arthropod-borne viruses (arboviruses). Although historically, the Asia-Pacific region has remained free of YFV, the risk of introduction has never been higher due to the increasing influx of people from endemic regions and the recent outbreaks in Africa and South America. Singapore is a global hub for trade and tourism and therefore at high risk for YFV introduction. Effective control of the main domestic mosquito vector Aedes aegypti in Singapore has failed to prevent re-emergence of dengue, chikungunya and Zika viruses in the last two decades, raising suspicions that peridomestic mosquito species untargeted by domestic vector control measures may contribute to arbovirus transmission. Here, we provide empirical evidence that the peridomestic mosquito Aedes malayensis found in Singapore can transmit YFV. Our laboratory mosquito colony recently derived from wild Ae. malayensis in Singapore was experimentally competent for YFV to a similar level as Ae. aegypti controls. In addition, we captured Ae. malayensis females in one human-baited trap during three days of collection, providing preliminary evidence that host-vector contact may occur in field conditions. Finally, we detected Ae. malayensis eggs in traps deployed in high-rise building areas of Singapore. We conclude that Ae. malayensis is a competent vector of YFV and re-emphasize that vector control methods should be extended to target peridomestic vector species.