Bacterial community structure of Anopheles hyrcanus group, Anopheles nivipes, Anopheles philippinensis, and Anopheles vagus from a malaria-endemic area in Thailand.

Bacterial content of mosquitoes has given rise to the development of innovative tools that influence and seek to control malaria transmission. This study identified the bacterial microbiota in field-collected female adults of the Anopheles hyrcanus group and three Anopheles species, Anopheles nivipes, Anopheles philippinensis, and Anopheles vagus, from an endemic area in the southeastern part of Ubon Ratchathani Province, northeastern Thailand, near the Lao PDR-Cambodia-Thailand border. A total of 17 DNA libraries were generated from pooled female Anopheles abdomen samples (10 abdomens/ sample). The mosquito microbiota was characterized through the analysis of DNA sequences from the V3-V4 regions of the 16S rRNA gene, and data were analyzed in QIIME2. A total of 3,442 bacterial ASVs were obtained, revealing differences in the microbiota both within the same species/group and between different species/group. Statistical difference in alpha diversity was observed between An. hyrcanus group and An. vagus and between An. nivipes and An. vagus, and beta diversity analyses showed that the bacterial community of An. vagus was the most dissimilar from other species. The most abundant bacteria belonged to the Proteobacteria phylum (48%-75%) in which Pseudomonas, Serratia, and Pantoea were predominant genera among four Anopheles species/group. However, the most significantly abundant genus observed in each Anopheles species/group was as follows: Staphylococcus in the An. hyrcanus group, Pantoea in the An. nivipes, Rosenbergiella in An. philippinensis, and Pseudomonas in An. vagus. Particularly, Pseudomonas sp. was highly abundant in all Anopheles species except An. nivipes. The present study provides the first study on the microbiota of four potential malaria vectors as a starting step towards understanding the role of the microbiota on mosquito biology and ultimately the development of potential tools for malaria control.

Development and Comparison of Dengue Vulnerability Indices Using GIS-Based Multi-Criteria Decision Analysis in Lao PDR and Thailand.

Dengue is a continuous health burden in Laos and Thailand. We assessed and mapped dengue vulnerability in selected provinces of Laos and Thailand using multi-criteria decision approaches. An ecohealth framework was used to develop dengue vulnerability indices (DVIs) that explain links between population, social and physical environments, and health to identify exposure, susceptibility, and adaptive capacity indicators. Three DVIs were constructed using two objective approaches, Shannon's Entropy (SE) and the Water-Associated Disease Index (WADI), and one subjective approach, the Best-Worst Method (BWM). Each DVI was validated by correlating the index score with dengue incidence for each spatial unit (district and subdistrict) over time. A Pearson's correlation coefficient (r) larger than 0.5 and a p-value less than 0.05 implied a good spatial and temporal performance. Spatially, DVIWADI was significantly correlated on average in 19% (4-40%) of districts in Laos (mean r = 0.5) and 27% (15-53%) of subdistricts in Thailand (mean r = 0.85). The DVISE was validated in 22% (12-40%) of districts in Laos and in 13% (3-38%) of subdistricts in Thailand. The DVIBWM was only developed for Laos because of lack of data in Thailand and was significantly associated with dengue incidence on average in 14% (0-28%) of Lao districts. The DVIWADI indicated high vulnerability in urban centers and in areas with plantations and forests. In 2019, high DVIWADI values were observed in sparsely populated areas due to elevated exposure, possibly from changes in climate and land cover, including urbanization, plantations, and dam construction. Of the three indices, DVIWADI was the most suitable vulnerability index for the study area. The DVIWADI can also be applied to other water-associated diseases, such as Zika and chikungunya, to highlight priority areas for further investigation and as a tool for prevention and interventions.

Ecological, Social, and Other Environmental Determinants of Dengue Vector Abundance in Urban and Rural Areas of Northeastern Thailand.

Aedes aegypti is the main vector of dengue globally. The variables that influence the abundance of dengue vectors are numerous and complex. This has generated a need to focus on areas at risk of disease transmission, the spatial-temporal distribution of vectors, and the factors that modulate vector abundance. To help guide and improve vector-control efforts, this study identified the ecological, social, and other environmental risk factors that affect the abundance of adult female and immature Ae. aegypti in households in urban and rural areas of northeastern Thailand. A one-year entomological study was conducted in four villages of northeastern Thailand between January and December 2019. Socio-demographic; self-reported prior dengue infections; housing conditions; durable asset ownership; water management; characteristics of water containers; knowledge, attitudes, and practices (KAP) regarding climate change and dengue; and climate data were collected. Household crowding index (HCI), premise condition index (PCI), socio-economic status (SES), and entomological indices (HI, CI, BI, and PI) were calculated. Negative binomial generalized linear models (GLMs) were fitted to identify the risk factors associated with the abundance of adult females and immature Ae. aegypti. Urban sites had higher entomological indices and numbers of adult Ae. aegypti mosquitoes than rural sites. Overall, participants' KAP about climate change and dengue were low in both settings. The fitted GLM showed that a higher abundance of adult female Ae. aegypti was significantly (p < 0.05) associated with many factors, such as a low education level of household respondents, crowded households, poor premise conditions, surrounding house density, bathrooms located indoors, unscreened windows, high numbers of wet containers, a lack of adult control, prior dengue infections, poor climate change adaptation, dengue, and vector-related practices. Many of the above were also significantly associated with a high abundance of immature mosquito stages. The GLM model also showed that maximum and mean temperature with four-and one-to-two weeks of lag were significant predictors (p < 0.05) of the abundance of adult and immature mosquitoes, respectively, in northeastern Thailand. The low KAP regarding climate change and dengue highlights the engagement needs for vector-borne disease prevention in this region. The identified risk factors are important for the critical first step toward developing routine Aedes surveillance and reliable early warning systems for effective dengue and other mosquito-borne disease prevention and control strategies at the household and community levels in this region and similar settings elsewhere.

Systematic studies of Anopheles (Cellia) kochi (Diptera: Culicidae): Morphology, cytogenetics, cross-mating experiments, molecular evidence and susceptibility level to infection with nocturnally subperiodic Brugia malayi.

Anopheles kochi DÓ§nitz (Diptera: Culicidae) is a malaria vector in some countries in South and Southeast Asia. This is the first report to provide clear evidence that two different cytological forms of An. kochi are conspecific based on systematic studies. Two karyotypic forms, i.e., Form A (X1, X2, Y1) and a novel Form B (X1, X2, Y2) were obtained from a total of 15 iso-female lines collected from five provinces in Thailand. Form A was common in all provinces, whereas Form B was restricted to Ubon Ratchathani province. This study determined whether the two karyotypic variants of An. kochi exist as a single or cryptic species by performing cross-mating experiments in association with the sequencing of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA), and cytochrome c oxidase subunit I (COI) of mitochondrial DNA (mtDNA). Cross-mating experiments between the two karyotypic forms revealed genetic compatibility by providing viable progenies through F2 generations. The two forms showed a high sequence similarity of those two DNA regions (average genetic distances: ITS2 = 0.002-0.005, COI = 0.000-0.009). The phylogenetic trees based on ITS2 and COI sequences also supported that four strains (from Bhutan, Cambodia, Indonesia, and Thailand) were all of the same species. Five sensilla types housed on the antennae of female An. kochi were observed under scanning electron microscopy (SEM). In addition, this study found that An. kochi was a refractory vector, revealed by 0% susceptibility rates to infection with nocturnally subperiodic Brugia malayi. The cibarial armature was a resistant mechanism, as it killed the microfilariae in the foregut before they penetrated into the developmental site.

Natural Plasmodium vivax infections in Anopheles mosquitoes in a malaria endemic area of northeastern Thailand.

There was recently an outbreak of malaria in Ubon Ratchathani Province, northeastern Thailand. In the absence of information on malaria vector transmission dynamics, this study aimed to identify the anopheline vectors and their role in malaria transmission. Adult female Anopheles mosquitoes were collected monthly by human-landing catch in Na Chaluai District of Ubon Ratchathani Province during January 2014-December 2015. Field-captured mosquitoes were identified to species using morphology-based keys and molecular assays (allele-specific polymerase chain reaction, AS-PCR), and analysed for the presence of Plasmodium falciparum and Plasmodium vivax using an enzyme-linked immunosorbent assay (ELISA) for the detection of circumsporozoite proteins (CSP). A total of 1,229 Anopheles females belonging to 13 species were collected. Four anopheline taxa were most abundant: Members of the Anopheles barbirostris complex, comprising 38% of the specimens, species of the Anopheles hyrcanus group (18%), Anopheles nivipes (17%) and Anopheles philippinensis (12%). The other nine species comprised 15% of the collections. Plasmodium infections were detected in two of 668 pooled samples of heads/thoraces, Anopheles dirus (1/29) and An. philippinensis (1/97). The An. dirus pool had a mixed infection of P. vivax-210 and P. vivax-247, whereas the An. philippinensis pool was positive only for the latter protein variant. Both positive ELISA samples were confirmed by nested PCR. This study is the first to incriminate An. dirus and An. philippinensis as natural malaria vectors in the area where the outbreak occurred. This information can assist in designing and implementing a more effective malaria control programme in the province.

Evidence to support a conspecific nature of allopatric cytological races of Anopheles nitidus (Diptera: Culicidae) in Thailand.

Metaphase karyotype investigation on two allopatric strains of Anopheles nitidus Harrison, Scanlon, and Reid (Diptera: Culicidae) was conducted in Thailand during 2011-2012. Five karyotypic forms, i.e., Form A (X1, Y1), Form B (X1, Y2), Form C (X2, Y3), Form D (X1, X3, Y4), and Form E (X1, X2, X3, Y5) were obtained from a total of 21 isofemale lines. Forms A, B, and C were confined to Phang Nga Province, southern Thailand, whereas Forms D and E were restricted to Ubon Ratchathani Province, northeastern Thailand. Cross-mating experiments among the five isofemale lines, which were representative of five karyotypic forms of An. nitidus, revealed genetic compatibility by providing viable progenies and synaptic salivary gland polytene chromosomes through F2 generations. The results suggest that the forms are conspecific, and An. nitidus comprises five cytological races. The very low intraspecific sequence variations (average genetic distances = 0.002-0.008) of the nucleotide sequences in ribosomal DNA (internal transcribed spacer 2) and mitochondrial DNA (cytochrome c oxidase subunits I and II) among the five karyotypic forms were very good supportive evidence.