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Food-handling practices were studied in 119 and 158 households, respectively, in an urban and a rural community in Peninsular Malaysia. Hazard analyses, including microbiological analysis of foods, were carried out in two households in each community and in a house that prepared food for distribution in the urban area. Kitchen hygiene was generally acceptable, although rated "poor" in some instances in the rural area. Food prepared for lunch was usually sufficient for dinner also, the leftover items being stored at ambient temperature until required. In the house that prepared food for distribution, breakfast was prepared during the evening, stored at ambient temperature overnight, and reheated before sale the next morning. There was a local preference for cooking food at temperatures close to boiling point; this reduced the numbers of vegetative cells but not those of spores. In some stored foods the populations of Staphylococcus aureus, Bacillus cereus and mesophilic aerobic bacteria increased, the last-mentioned reaching spoilage levels. Reheating reduced the populations of proliferating bacteria in most foods to acceptable levels but would not have destroyed heat-resistant enterotoxins. Because of their importance in combating acute bacterial foodborne disease, the control of the temperature and time factors during the cooking and storage of food should receive special attention in education on health and food safety., Introduction Foodborne illness, one of the most widespread kinds of disease (1), remains uncontrolled in both developed and developing countries. Its etiology, impact on public health, and epidemiology vary and [...]

Leishmaniasis, a sandfly-transmitted protozoan infection, is a neglected health threat in Thailand and the information on its vector is scarce. This study aimed to identify sandfly distribution, abundance, and environmental conditions of natural breeding sites in the cave areas of Satun Province, where previous cases of leishmaniasis were reported. Sandflies were collected during a six-month period using CDC light traps and modified emergence traps. Species distribution, relative abundance, and environmental conditions of potential breeding sites were determined. Our survey of 12,790 sandflies found the highest female abundance in April-May. We identified six known species, the most prevalent being Sergentomyia anodontis . We also found S. barraudi , a potential Leishmania spp. vector, distributing in this area. Most male sandflies had partially rotated genitalia, indicating the breeding site proximity to our trap locations. Potential resting/breeding sites were discovered outside the cave during February-March, and inside during May-June. The environmental parameters showed warm climate, moderate humidity, moderately alkaline pH, moderate-to-high macronutrients, and low-to-high organic matters. In summary, our study provided the spatiotemporal distribution and environmental condition of sandfly potential breeding sites in the cave areas of Satun Province. This data may contribute to more effective vector surveillance programs in the future.

The vectors of scrub typhus are the larval stage of trombiculid mites, termed "chiggers". These vectors are very small - the larvae are approximately 0.2 mm in size - and therefore their morphological identification is difficult. Trombiculid mites are widely distributed across Asia and they can be identified at the genus level by the shape, size and setae/sensilla distribution of their dorsal chitin plate (scutum = shield), while morphological identification at the species level requires more mite characteristics. We recently developed a methodology to ascertain paired matched genotype and morphotype of individual chiggers, based on autofluorescence and brightfield microscopy with subsequent molecular identification using the COI gene (approximately 640bp length). However, based on 20 chigger specimens characterised by paired genotypic and morphological data consisting of the four species [Walchia ewingi with 2 subspecies]: Walchia ewingi lupella (n = 9), W. ewingi ewingi (n = 2), W. alpestris (n = 2), W. kritochaeta (n = 5) and W. minuscuta (n = 2) we found evidence of genetic polymorphism and morphological plasticity within the genus Walchia. The phylogenetic inference of the intra-genus relationships within the Walchia spp., based on COI gene (Blankaartia spp. served as outgroup), delineated the five included species by an average interspecific divergence of mean distance 0.218 (0.126 - 0.323). We therefore applied landmark-based and outline-based geometric morphometric (GM) approaches to differentiate Walchia species using scutum measurements. A total of 261 scutum images of Walchia spp. were examined by landmark-based GM (140 chigger specimens) and outline-based GM (121 specimens) techniques. All Walchia spp. showed significant differences in scutum size and shape. W. minuscuta showed the smallest mean scutum size in both techniques. The largest scutum was found in W. ewingi lupella and W. ewingi ewingi by landmark-based and outline-based GM analysis, respectively. The scutum shapes of W. alpestris and W. minuscuta were clearly distinguished from the other species. Cross-validated classification scores were different depending on species and digitizing techniques and landmark-based GM showed better scores than outline-based GM. We conclude that the morphologically closely-related trombiculid mite species can be further differentiated by their scutum features alone, using GM approaches. This technique is a promising tool for the much-needed characterization studies of chiggers and needs evaluation using matched morphometric and genotyping data for other genera of trombiculids., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Climate change and human activity affect the geographical and annual distribution and population abundance of mosquitoes. As natural habitats are reduced, it is hypothesized that mosquitoes may seek refuge in more stable environments such as cave habitats. Therefore, we explored the species diversity of mosquitoes exploiting cave habitats in Thailand. Ten species belonging to 4 genera were collected, of which none were considered to be true cave-dwelling species (Troglobiont). The known cavernicolous species, Aedes cavaticus , was observed to oviposit outside of the cave and therefore should be categorized as a subtroglophilic species. Other species were also oviposited inside the cave but should be regarded as trogloxenic species. There was no clear association between environmental factors and mosquito abundance inside the 4 limestone caves, except for Ae. cavaticus , which was positively correlated with rainfall. This study indicates that different biotic or abiotic factors may be involved in mosquito oviposition site selection inside caves.

We investigated the nocturnal activity of cave-dwelling sand flies at different time intervals and determined their species composition and seasonal variation. Sand flies were captured on one night each month using CDC light traps from 18:00-06:00 with the collecting bag being changed every two h between February, 2010 and January, 2011. A total of 18,709 individuals, including 10,740 males and 7,969 females, was collected. The overall ratio between male and female specimens was 1:0.74. The collected specimens included 14 species from four genera, Chinius, Idiophlebotomus, Phlebotomus, and Sergentomyia. Sergentomyia phadangensis was the most abundant species (comprising 31.9% of the collected individuals), followed by Se. anodontis (22.8%) and Ph. mascomai (18.2%). The highest number of specimens was collected in July (15.6%), followed by May (15.5%) with the peak of collection recorded at the time interval of 00:01-02:00, followed by 22:01-00:00. However, there were no significant differences observed among time intervals of sand fly collections (p=0.154). Observations of the nocturnal activity of male and female sand flies throughout the night suggest that phlebotomine sand flies show the greatest activity level after midnight., (© 2018 The Society for Vector Ecology.)

Nematode infection in wild caught Phlebotomine sand flies was investigated in Thailand. Light microscopy (LM) and scanning electron microscopy (SEM) were used to detect and morphologically characterize entomopathogenic nematodes that presented in the sand flies. Didilia sp. nematodes were found for the first time in the body cavity of wild caught male Phlebotomus stantoni sand flies. The Didilia sp. was identified based on the morphology of the adult nematodes, from their stylet and teeth at the anterior tip, body length, and egg shell sculpture. It was noted that every infected male sand fly had unrotated genitalia, which would not allow them to mate, thus leading to the loss of their offspring. This finding provided information that might lead to study on whether or not the Didilia sp. has the potential to control sand fly population.

Phlebotomine sand flies are established vectors of leishmaniasis in humans. In Thailand, Leishmania martiniquensis and "Leishmania siamensis" have been described as causative agents of leishmaniasis. In this study, a survey of sand flies in the Leishmania infected area of Hang Dong district, Chiang Mai, Thailand was performed using CDC light traps for eight consecutive months, from January to August 2016. A total of 661 sand flies were collected, and of 280 female sand flies, four species of the genus Sergentomyia including Sergentomyia gemmea, S. barraudi, S. indica, and S. hivernus and one species of the genus Phlebotomus, Phlebotomus stantoni, were identified. S. gemmea and S. hivernus were found in Chiang Mai for the first time. The density of captured female sand flies was high in warm and humid periods from June to August, with temperatures of around 26°C and relative humidity about 74%. In addition, S. gemmea was the most predominant species in the area. Further studies as to whether or not these sand fly species could be a vector of Leishmaniasis in Thailand are required.

Geographic populations of the two main sandflies genera present in Thailand were studied for species and population identification. Size and shape of Phlebotomus stantoni and Sergentomyia hodgsoni from different island and mainland locations were examined by landmark-based geometric morphometrics. Intraspecific and interspecific wing comparison was carried out based on 12 anatomical landmarks. The wing centroid size of P. stantoni was generally larger than that of S. hodgsoni. Within both species, wings from the continent were significantly larger than those from island populations. Size variation could be significant between geographic locations, but could also overlap between genera. The wing venation geometry showed non-overlapping differences between two species. The within-species variation of geometric shape between different geographical locations was highly significant, but it could not interfere with the interspecies difference. The lack of species overlapping in shape, and the high discrimination between geographic populations, make geometric shape a promising character for future taxonomic and epidemiological studies., (Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Increasing evidence of autochtonous leishmaniasis in Thailand has raised concern to understand how transmission of leishmaniasis occurs and determine its epidemiology for disease control. However knowledge of the vectorial capacity for Leishmania has been limited by difficulties and failure of sand fly breeding in the laboratory. In this study, a colony of Phlebotomus major major and Phlebotomus stantoni were established under laboratory conditions. Both colonies were started with a single gravid female and allowed to observe for all developmental stages. We reported their biological characteristics for the first time to be a baseline data for planning vector control measures. The life cycle of P. major major and P. stantoni are commonly completed in 66 days (range 48-76 days) and 48 days (range 39-49 days), respectively. Eggs hatched within 10 days after being laid. Survival rate of entire life cycle for P. major major was 38% and 100% for P. stantoni. Our results suggest that attempts to determine biological characteristics of the sand fly species and their vector potential remain vital if special attention is given to successful colonization.

Aedes aegypti (Diptera: Culicidae) (L.), Ae. albopictus (Skuse), and Ae. scutellaris (Walker) are important mosquito vectors of dengue and chikungunya viruses. They are morphologically similar and sympatric in some parts of their distribution; therefore, there is a risk of incorrect morphological identification. Any confusion could have a negative impact on epidemiological studies or control strategies. Therefore, we explored two modern tools to supplement current morphological identification: DNA barcoding and geometric morphometric analyses. Field larvae were reared to adults and carefully classified based on morphological traits. The genetic analysis was based on the 658bp each of 30COI sequences. Some Culex spp., Mansonia bonneae, were included as outgroups, and inclusion of a few other Aedes spp. facilitated phylogenetic inference of the relationship between Ae. albopictus and Ae. scutellaris. The two species were separated by an average interspecific divergence of 0.123 (0.119-0.127). Morphometric examination included landmark- (392 specimens) and outline-based (317 specimens) techniques. The shape of the wing showed different discriminating power based on sex and digitizing technique. This is the first time that Ae. scutellaris and Ae. albopictus have been compared using these two techniques. We confirm that these morphologically close species are valid, and that geometric morphometrics can considerably increase the reliability of morphological identification., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Since 1996, there are emerging autochthonous cases of leishmaniasis in Thailand due to Leishmania "siamensis" and to L. martiniquensis explaining a recent interest for the sand fly fauna where Sergentomyia gemmea and Se. barraudi have been considered possible vectors in the country., Methods: Field studies were undertaken in a cave of Phitsanulok Province, Thailand. Phlebotomine sandflies have been studied morphologically and some have been processed for molecular biology (sequencing of cytB rDNA)., Results: A new species of sand fly, belonging to the genus Sergentomyia: Se. phadangensis n. sp., is described. The association of the male and female is supported by the homology of the sequences of cytochrome b rDNA., Conclusions: The description of a new species in Thailand is of importance in view of the existence of autochthonous leishmaniases.

Bat bugs are blood-feeding insects of bats or warm blooded animals and humans. Since 2011, Leptocimex spp (Heteroptera: Cimicidae) has been reported in Thailand. However, microscopic examination of Leptocimex spp is complicated, especially when the entire body of the specimen is not available. To confirm the phenotypic identification of L. inordinatus from a limestone bat cave in Thailand, partial fragments of mitochondrial cytochrome c oxidase subunit 1 (COI) gene and 16S mitochondrial ribosomal DNA were PCR amplified and sequenced, which revealed 97% sequence identity with Cimicidae family members, being most similar to Cacodminae gen. sp. and C. vicinus, both bat bugs. Phylogenetic tree construction showed that L. inordinatus has a separate genetic lineage from that of with human bed bugs (Cimex hemipterus or C. lectularius), swallow bugs and other tick species. The presence of L. inordinatus in a bat-dwelling cave frequented by humans presents a potential public health problem requiring attention in particular regarding the possibility of zoonotic transmission of pathogens.

A survey of biting midges in animal sheds, mangroves and beaches along the Andaman coastal region in southern Thailand between April 2012 and May 2013 collected 10 species of Culicoides which were not previously known from Thailand. These new records are C. arenicola, C. flavipunctatus, C. hui, C. kinari, C. kusaiensis, C. parabubalus, C. quatei, C. spiculae, C. pseudocordiger and C. tamada. An updated checklist of species of Culicoides reported from Thailand is provided.

The present study reported for the first time phlebotomine sandfly species inhabiting edible-nest swiftlet cave of the isolated island, based on field collections made during June 2010-May 2011. The insect diversity was relatively lower to that of mainland caves. All species, Phlebotomus stantoni, Sergentomyia anodontis, Sergentomyia bailyi, Sergentomyia gemmea, Sergentomyia hodgsoni and Sergentomyia punjabensis were either endemic island species or native elsewhere in Thailand. Sergentomyia hodgsoni was the most prevalent species accounted for 94.7% and classified as a troglophile species. Seasonal pattern of the phlebotomine abundance and some aspects of their population characteristics were described and discussed. Two ectoparasites, Ornithodorus and Paracimex sp. were also incidentally collected from the swiftlet cave.

Fleas are the vectors of many communicable diseases that are normally found in oriental rats. Climate and environmental changes influence the habitat and migration patterns of vectors. In this study, the oriental rat flea abundance, represented as total flea index, was determined in correlation to host specificity and various environmental factors. The number of hosts and fleas calculated from 3 specific habitats (shipping area, decayed area, and market area) from July 2010 to June 2011. The results showed that the common hosts in the shipping area and decayed area were Rattus rattus and R. exulans, with the total flea indexes of 3.36 and 1.58. R. norvegicus was the most common host identified in the market area. Fleas were virtually absent in rat hosts collected from the market area. Both the density of reservoir hosts and the total flea index were positively correlated with the mean annual rainfall and temperature. These data could be useful for control of rat populations in each specific habitat.

Evidence on the effectiveness of insecticide-treated curtains (ITCs) for reducing densities of Aedes mosquitoes, the principal vectors of dengue, is scarce. In Laem Chabang southeast of Bangkok, Thailand, the Breteau Index (BI) (number of positive containers/100 houses) was 45 in October 2006. In March 2007, we distributed long-lasting ITCs in 22 clusters (2,032 houses) and selected 66 control clusters (661 houses). Routine control activities continued in all clusters. Six months after distribution, the BI was 25.8 and 77.6 in intervention and control areas, respectively (P < 0.001). Eighteen months after distribution, the BI was 21.8 and 23.8, respectively (P = 0.28). The average number of ITCs/house at cluster level was associated with the BI (P < 0.01) after six months, when 70.5% of households still used ITCs, but not at 18 months, when ITC coverage had decreased to 33.2%. Deployment of ITCs can result in considerable reductions in Aedes infestation levels, but the effect is coverage dependent.

The efficacy of insecticide-treated window curtains (ITCs) for dengue vector control was evaluated in Thailand in a cluster-randomized controlled trial. A total of 2,037 houses in 26 clusters was randomized to receive the intervention or act as control (no treatment). Entomological surveys measured Aedes infestations (Breteau index, house index, container index, and pupae per person index) and oviposition indices (mean numbers of eggs laid in oviposition traps) immediately before and after intervention, and at 3-month intervals over 12 months. There were no consistent statistically significant differences in entomological indices between intervention and control clusters, although oviposition indices were lower (P < 0.01) in ITC clusters during the wet season. It is possible that the open housing structures in the study reduced the likelihood of mosquitoes making contact with ITCs. ITCs deployed in a region where this house design is common may be unsuitable for dengue vector control.

The large and recent migrations of the main vector of the Chikungunya virus, Aedes albopictus, raise questions about the epidemiological impact of possible microevolutionary changes in new territories. Quantitative traits are suitable to detect such changes as induced by environmental adaptations, local competition and founder effects. Using landmark-based geometric morphometrics, we compared the size and shape of 22 populations (1572 females) of Ae. albopictus. The collection sites covered five countries around the world, with special emphasis on Asia, which is assumed to be the origin of the vector. Some collections came from places where an important epidemic outbreak of Chikungunya has recently occurred. Quantitative comparisons were based on 18 anatomical landmarks on the wing. To contrast geographic and possible interspecific shape variation, close species were introduced in the sample, namely five Aedes riversi and nine Ae. flavopictus from Japan. The three species had similar size, but they were clearly separated by shape. Within Ae. albopictus, there was general agreement on size variation with the available climatic data. Shape variation was less related to climatic data than to either geography or a known itinerary of past territorial expansion. Thus, two main clusters were distinguished by wing shape variation: the first one composed of the Southeast Asian sample, the second one grouping Japan, Florida, Hawaii and the Reunion Island samples. The Southeast Asian countries, assumed to be at the origin of the geographic expansion of the mosquito, had similar wings and constituted a distinct group where localities clustered into northern and southern localities. Contrasting with this homogeneous group, very distant localities such as United States (US) and Japan shared a common shape pattern. The US Ae. albopictus samples (Hawaii and Florida) were indeed very similar to the Japanese samples, with Florida behaving exactly like a northern Japanese locality, close to Tokyo and Yokohama. Shape proximity among these distant areas could be explained by a common and relatively recent ancestor, as generally suggested in the literature. The three Reunion Island samples conformed a group external to the Southeast Asian countries, loosely connected to the remaining localities. Thus, areas where recent epidemics took place, the Reunion Island and the southernmost localities of Thailand, did not cluster together, but represented separate groups. In sum, the size of the wing was apparently under the influence of climatic factors, while its shape could contribute information on species and geographic differences. Apparent departures from expectations based on geography were explained by known past and present migratory routes. These patterns of shape variation were compatible with genetic drift, suggesting microevolutionary changes probably induced by the expansion of the Ae. albopictus mosquito., (Copyright © 2012 Elsevier B.V. All rights reserved.)

One of the mechanisms responsible for pyrethroid resistance in mosquitoes is mutations in domain IIS6 of voltage-gated sodium channel gene (kdr). Aedes aegypti larvae were collected from the central provinces of Thailand (Bangkok, Prachin Buri and Ratchaburi) and colonized until they became adults. Partial fragment of kdr of permethrin-resistant mosquitoes were amplified by RT-PCR and sequenced. Among the four nucleotide mutations detected, two mutations resulted in two amino acid substitutions, S(TCC) 989 P(CCC) and V(GTA)1016 G(GGA). Among 94 permethrin-resistant mosquitoes, the SS genotype (SS/VV) was found to predominate (n = 74), followed by SR (SP/VG) (n = 15) and RR (PP/ GG) genotypes (n = 5), with the resistant allele frequency ranging from 0.03 to 0.17. As pyrethroid insecticides are currently being advocated for use in Thailand, investigations of pyrethroid resistance in other regions of the country are needed to prevent potential cross-resistance among different types of insecticides.

Stomoxyini flies (Diptera: Muscidae) include species of parasitic flies of medical and veterinary importance. The adult flies feed on the blood of mammals and may transmit several parasites and pathogens. We conducted an entomological survey of Stomoxyini flies from different sites in Thailand. Stomoxyini flies were collected at four major types of sites: zoos, livestock farms, wildlife conservation areas and a national park using vavoua traps between November 2010 and April 2011. A total of 3,314 Stomoxyini flies belonging to the genera Stomoxys, Haematobosca, Haematostoma and Haematobia were collected. Eight species were identified: S. calcitrans (46.6%), S. uruma (26.8%), S. pulla (4.3%), S. indicus (0.7%), S. sitiens (0.1%), H. sanguinolenta (11.2 %), H. austeni (0.5%) and H. irritans exigua (9.8%). The diversity of Stomoxyini flies in the livestock farms was higher than the other sites. Altitude correlated with the number of flies. This study provides information that may be useful for Stomoxyini flies control.

Understanding specific gene regulation during responses to malaria infection is key to dissecting the mosquito defense mechanisms and host/parasite interactions. A full-length serine protease cDNA was isolated from the fat body of female Anopheles dirus, a major malaria vector in Thailand. The predicted amino acid sequence of SERF4 identifies it as a member of the serine protease family containing a single trypsin-like protease domain. Digestive trypsins function in the female mosquito midgut and are inducible in two phases in this tissue upon blood intake. However, the gene was highly upregulated in the midgut at day 3 postinfection by Plasmodium falciparum. In situ hybridization confirmed that SERF4 transcripts were located in the midgut epithelial cells rather than hemocytes or other tissues associated with the midgut. SERF4 was also strongly downregulated in the whole insects at day 16 after infection in comparison with the blood-fed control. Changes in the expression of the SERF4 gene in response to infection with this human malaria parasite suggest a role in parasite-specific innate immunity., (© 2012 Wiley Periodicals, Inc.)

We reviewed the literature for surveys of parasitoid of filth flies in Thailand. We found 5 families, with 9 genera and 14 species identified in Thailand. We describe the ecological niches and biology of common species, including Spalangia cameroni, S. endius, S. nigroaenea and Pachycrepoideus vindemmiae.

A tsunami affected area in Phang Nga province, Thailand was explored randomly as some freshwater sites had changed into brackish-water sites. A survey of four areas found Culex sitiens to be the most dominant mosquito species.This mosquito prefers to breed in putrefied water with garbage and it was found in almost every stagnant, brackish-water site in full sunlight. The larval density was more than 300 larvae/dip/250 ml water. Its biting cycle, determined by human landing catch, was nocturnal, with a single peak at 19.00-20.00 hr. The maximum rate was 108 mosquitoes per person/hour. The biology of the mosquito was studied by colonization in natural water under laboratory conditions. The mean number of eggs per raft was 158.1 ± 31.7, hatchability 96.6 ± 4.1%, development from 1st instar larvae to adult was 8.8-11.7 days, and longevity of adult males was 7.3-41.3 days and females 11.0-52.7 days. The ratio of adult males to adult females was 1:1.1 ± 0.2.

In the South of Thailand, six Mansonia species are recorded as filariasis vectors, among which Ma. bonneae and Ma. dives. These two species are distributed in the same breeding place, mainly the swamp forest, but appear to be of problematic identification using traditional morphological characters. Because of the risk of wrong identification during epidemiological or biological studies, complementary techniques are needed to distinguish the two species. We used on the same field collected specimens both genetic (DNA barcoding) and phenetic (geometric morphometrics) techniques. Both methods converged to identify two separate entities in accordance with morphological differences and geographic origins. Shape divergence between species was more pronounced in males than in females. Notably, the amount of within species sexual shape dimorphism was much larger than shape divergence as recorded between species. In spite of these two species of Mansonia being evolutionary very close, simple DNA barcoding was resolutive. Geometric morphometrics, because it is a fast and low-cost procedure, appeared as an interesting complement to modern diagnostic techniques applied in medical entomology. It also was able to provide information relevant to the ecology of the two species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An entomological survey of sand flies was conducted in Naresuan Cave in Noen Maprang District, Phitsanulok Province, during November 2009 to December 2010. A total of 10,115 cave-dwelling sand flies were collected with CDC light traps nocturnally (06:00 AM and 06:00 PM) and diurnally (06:00 PM and 06:00 AM). The ratio between male and female sand flies was 1:1.3 (4,363:5,752). The ratio between the number of sand flies caught nocturnally and diurnally was 2.6:1 (7,268:2,847). In this study, 13 species belonging to 4 genera were identified, of which 4 belonged to the genus Phlebotomus, 7 to Sergentomyia, 1 to Nemopalpus and 1 to Chinius. An abundance of species were observed: Nemopalpus vietnamensis (49.15%), P. argentipes (20.15%), C. barbazani (15.79%), P. teshi (9.53%), and S. anodontis (3.21%). Less common species (<1%) were S. barraudi (0.63%), P. stantoni (0.57%), S. dentata (0.49%), S.quatei (0.17%), P. philippinensis gouldi (0.12%), S.silvatica (0.10%), S. gemmea (0.05%), and S. iyengari (0.04%). The predominant species in the Naresuan Cave was Nemopalpus vietnamensis (49.15%). The data demonstrates variability in sand fly prevalence, species composition, and relative abundance in caves. P. argentipes was found throughout the day in the caves, which is important because it is believed to be the Leishmania spp vector. This study highlights the diurnal activity of the sand fly and the day-time risk of leishmaniasis. In conclusion, although leishmaniasis has not been reported in Phitsanulok, there should be heightened awareness of infection in these areas with vectors of the protozoa.

DNA-based identification system using the mitochondrial cytochrome oxidase subunit I (COI) gene has enabled validation of many species in certain taxonomic groups. These primer combinations were able to work universally across Insecta. Here, a set of three primer pairs were successful in amplifying COI of Mansonia annulata mosquito, a potential vector of Brugia malayi. By merging all three amplicons the whole COI was obtained. Primer pair TY-J-1460/C1N2087 amplified 5' region of COI, LepF1/LepR1 the central and C1J2090/TL2N3014 the 3' region, generating COI amplicons of 650, 700 and 950 base pairs, respectively. When Ma. annulata sequences were compared with those from online sources, they formed a cluster group that is clearly distinct from other allied species.

During 2008-2009 2,401 Phlebotomine sand flies were collected in 14 limestone caves in Kanchanaburi Province, Thailand to determine the prevalence and type of cavernicolous species that have the potential to be leishmaniasis vectors. Twenty species belonging to the genera Chinius, Nemopalpus, Phlebotomus and Sergentomyia were identified. An additional man-biting species, P. major major was recorded for the first time in Thailand. Ecological observations of the habitats were made. It is expected the diversity of cavernicolous sand flies is more than currently known. An updated list of 26 phlebotomine species for Thailand is provided.

A longitudinal entomological survey was conducted to provide in-depth information on An. epiroticus and determine whether ecological and entomological factors could influence malaria transmission in Rayong Province, Thailand. The mosquitoes were collected monthly from May 2007 to April 2008 by human landing catch technique from 6:00-12:00 PM for 2 consecutive nights, at 3 collection sites. A total of 3,048 mosquitoes within 5 species were captured: An. epiroticus, Culex quinquefasciatus Say, Cx. sitiens Wiedemann, Aedes aegypti (L.) and Ae. albopictus Skuse. PCR was used for molecular identification of An. sundaicus complex, by determination of COI, ITS2, and D3 genes. The target mosquitoes were An. epiroticus, which was the predominant species, accounting for 43.8% of specimens collected. The biting cycle pattern increased during 6:00-8:00 PM and reached a maximum of 6.6 bites/person/hour by 12:00 PM. The mosquitoes varied in population density throughout the year. The highest biting rate was 37.6 bites/person/ half night in September and the lowest (10.2 bites/person/half night) in January. Nested PCR and real-time PCR techniques were used to detect the malaria parasite in An. epiroticus adult females. Nine of 926 (0.97%) mosquitoes tested were malaria parasite positive: 6 P. falciparum and 3 P. vivax. The infective mosquitoes were found in the dry and early rainy seasons. The overall annual entomological inoculation rate (EIR) in the village was 76.6. The overall parity rate was 74%. A total of 38 cement tanks were used to characterize the nature of the breeding places of An. epiroticus. An. epiroticus larvae coexisted with Aedes and Culex larvae; the maximum larval density was more than 140 larvae per dip in May. Breeding places included fresh, brackish and salt water, typically with full sunlight and mats of green algae on the water surface. The salinity of the water ranged from 0.5 to 119.4 g/l, with a narrow pH range of 8.2-8.7. Dissolved oxygen was highest in November (6.27 mg/l) and lowest in March (3.46 mg/l). The water temperature varied between 24.6 and 32.8 degrees C.

Entomological surveys (2001-2005) were carried out in Narathiwat Province to determine mosquito fauna of the peat swamp forest. Fifty-four species belonging to 13 genera were identified from 837 larval specimens and 3,982 adult mosquitoes. These included the major vectors for Brugian fillariasis: Mansonia annulata, Ma. bonneae, Ma. dives, Ma. uniformis and Ma. indiana. Ma. annulata and An. letifer were reported for the first time in Thailand as lymphatic filariasis vectors. Three species inhabiting Nepenthes pitchers (N. mirabilis): Tripteroides tenax, Toxorhynchites manopi and Uranotaenia edwardsi, were recorded for the first time in Thailand; Zeugnomyia gracilis was also found common in the peat swamp forest.

ITS2 DNA sequences of 42 isoline colonies of Anopheles barbirostris species A1 and A2 were analyzed and a new genetic species, temporarily designated as species A4 (Chiang Mai), was revealed. The large sequence divergences of the ITS2 (0.116-0.615), COI (0.023-0.048), and COII (0.030-0.040) genes between A. barbirostris species A4/A1 (Chiang Mai), A4/A2 (Phetchaburi), A4/A3 (Kanchanaburi), and A4/Anopheles campestris-like Form E (Chiang Mai) provided good supporting evidence. Species A1, A2, A3, and A4 share a mitotic karyotype of Form A (X(1), X(2), Y(1)). Crossing experiments between species A4 and the other four species yielded strong reproductive isolation producing few and/or non-hatched eggs and inviable and/or abnormal development of the reproductive system of F(1) progenies. Moreover, available F(1) hybrid larvae showed asynaptic polytene chromosome arms. Hence, molecular and cytogenetic evidence strongly support the existence of A. barbirostris species A4, which is more closely related to A. campestris-like Form E than to species A1, A2, and A3. Additionally, crossing experiments among 12 and seven isolines of different cytological forms of species A1 (A, B, C, D) and A2 (A, B), respectively, yielded fertile and viable F(1) progenies. Thus, different karyotypic forms occurring in natural populations of species A1 and A2 merely represent intraspecies variation of sex chromosomes due to the extra blocks of heterochromatin.

Dengue fever and its more serious complications dengue haemorrhagic fever and dengue shock syndrome are growing public health problems in tropical and subtropical countries. In the absence of a vaccine, most dengue control programmes rely heavily on the use of insecticides to target the Aedes mosquito vectors. As a limited number of insecticides are routinely used in control, monitoring for the presence of resistance is an essential component of dengue prevention programmes. The pyrethroid insecticides target the voltage-gated sodium channel on the insects' neurons. Substitutions at residue 1016 of this protein have been associated with pyrethroid and DDT resistance in Aedes aegypti populations from Latin America and Asia. Here we report on the development of a simple colorimetric assay to detect these mutations in individual mosquitoes. Evaluation of this diagnostic assay on 180 Ae. aegypti individuals from Thailand revealed the presence of high frequencies of the Val1016Gly mutation throughout the country. The assay requires no specialised equipment and will enable monitoring for insecticide resistance associated alleles to be routinely incorporated into dengue surveillance operations.