IDENTIFICATION OF BAT ECTOPARASITE LEPTOCIMEX INORDINATUS FROM BAT-DWELLING CAVE, KANCHANABURI PROVINCE, THAILAND.

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.

CORRELATION OF HOST SPECIFICITY, ENVIRONMENTAL FACTORS AND ORIENTAL RAT FLEA ABUNDANCE.

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.

Cloning of a trypsin-like serine protease and expression patterns during Plasmodium falciparum invasion in the mosquito, Anopheles dirus (Peyton and Harrison).

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.

Biology of Culex sitiens, a predominant mosquito in Phang Nga, Thailand after a tsunami.

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.

Literature review of parasitoids of filth flies in Thailand: a list of species with brief notes on bionomics of common species.

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.

Field-collected permethrin-resistant Aedes aegypti from central Thailand contain point mutations in the domain IIS6 of the sodium channel gene (KDR).

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.

Distribution and abundance of Stomoxyini flies (Diptera: Muscidae) in Thailand.

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.

Utility of a set of conserved mitochondrial cytochrome oxidase subunit I gene primers for Mansonia annulata identification.

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.

Cavernicolous species of phlebotomine sand flies from Kanchanaburi Province, with an updated species list for Thailand.

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.

Bionomic status of Anopheles epiroticus Linton & Harbach, a coastal malaria vector, in Rayong Province, Thailand.

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.

Distribution of Phlebotomine Sandflies in the Cave Area of Satun Province, Thailand.

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.

Mosquito fauna of "Toh Daeng" swamp forest, Thailand.

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.

Geometric morphometrics of the scutum for differentiation of trombiculid mites within the genus Walchia (Acariformes: Prostigmata: Trombiculidae), a probable vector of scrub typhus.

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.

Expression of three serine protease genes from the South East Asian malaria vector, Anopheles dirus, in relation to blood feeding and parasite infection.

Three serine protease cDNA clones were isolated from Anopheles dirus, a major vector of malaria in Southeast Asia. Transcript abundance was examined following infection by Plasmodium falciparum by RT-PCR analysis. SerF3 exhibited increased transcript abundance in the whole body at 10 days post-infection with P. falciparum. All three genes are candidates for further investigation to determine their roles in mosquito immune responses.

Diversity Of Mosquito Species Ovipositing In Different Zones of Light Intensity Within Limestone Caves In Thailand.

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.

Species composition and nocturnal activity of phlebotomine sand flies (Diptera: Psychodidae) inhabiting a limestone cave in Thailand.

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.

Wing geometry of Phlebotomus stantoni and Sergentomyia hodgsoni from different geographical locations in Thailand.

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.

Laboratory colonization of Mansonia mosquitoes with an emphasis on Ma. annulata and Ma. bonneae.

The present study records the first successful colonization of Mansonia annulata and describes colony maintenance with modification of rearing medium and host plants. Three species of Mansonia mosquitoes (Ma. uniformis, Ma. indiana and Ma annulifera) were successfully reared in ambient environments with adult emergence rates > 50%, while Ma. bonneae and Ma. dives yielded emergence rates > 30%. Colonization of Ma. annulata was modified and improved so that they were successfully raised to adult with emergence rates of 23%. Tube sedge, Lepironia articulata, was utilized as a host plant and peat swamp water was used as a rearing medium. Yeast and small lizard droppings were added daily to the larval medium to maintain microorganisms and pH in the infusion. However, identifying suitable culture medium remains an obstacle to establishing colonies of Ma. annulata, as the culture medium is difficult to mimic in the laboratory. Further study, focusing particularly on larval attachment substrates and rearing medium, is needed to develop a standardized and practical rearing technique for Mansonia mosquitoes.

Formulation of tablets from the crude extract of Rhinacanthus nasutus (Thai local plant) against Aedes aegypti and Culex quinquefasciatus larvae: a preliminary study.

Dried root powder of Rhinacanthus nasutus, Thong Phan Chang (Thai name) were extracted with methanol (MeOH) in a Soxhlet apparatus and made into 2 formulations of tablet containing the extract at 5% and 10% concentration. Due to the viscous and poor flow properties of the crude MeOH extract obtained, a wet granulation method was conducted in developing the tablets. Lactose was used as a filler. Polyvinyl pyrrolidone (PVP) K30 (15% w/w solution in alcohol) was used as the binding agent, while stearic acid (2% w/w) was used as a lubricant. Both formulas of prepared tablets had a smooth shiny surface with a round shape. Other physical properties of the tablets, such as weight variation, friability and disintegration time, met the requirements of the USP XX standard. The mosquito larvicidal activity of prepared tablets containing 5% and 10% R. nasutus extract against Aedes aegypti were not significantly different from each other (p > 0.05), with 48-hour LC50 values of 13.6 and 14.2 mg/I for the 5% and 10% tablets, respectively, while their activities against Culex quinquefasciatus were similar (p > 0.05) with LC50 values of 18.7 and 17.3, respectively. The larvicidal activity levels against Ae. aegypti and Cx. quinquefasciatus were also not significantly different from each other (p > 0.05). No larval mortality was observed in the two control groups: lactose solution and dechlorinated water. Toxicity to female and male fish (Poecilia reticulata) was tested with the prepared tablets. The toxicity of tablets containing 5% and 10% extracts were not significantly different from each other for the P. reticulata females with 48-hour LC50 values of 105.2 and 110.8 mg/I, respectively, and for P. reticulata males with LC50 values of 99.1 and 103.4 mg/I, respectively. Female and male P. reticulata were sensitive to the same dose of the extract. No fish died in the two control groups, with lactose solution and dechlorinated water. Acute-toxicity bioassay with fish showed that with an exposure of 48 hours the LC50 values of the tablets containing 5% and 10% were 5- to 10-fold higher than the LC50 of R. nasutus against mosquito larvae. These prepared tablets could possibly used to control mosquito vectors and be introduced into the mosquito control program.

Bionomics studies of Mansonia mosquitoes inhabiting the peat swamp forest.

The present study was conducted in the years 2000-2002 to determine the bionomics of Mansonia mosquitoes, vectors of nocturnally subperiodic Brugia malayi, inhabiting the peat swamp forest, "Phru Toh Daeng", Narathiwat Province, Thailand. Fifty-four species of mosquitoes belonging to 12 genera were added, for the first time, to the list of animal fauna in the peat swamp forest. Mansonia mosquitoes were the most abundant (60-70%) by all collection methods and occurred throughout the year with a high biting density (10.5-57.8 bites per person-hour). Ma. bonneae was most prevalent (47.5%) and fed on a variety of animal hosts, including domestic cats, cows, monkeys, and man with a maximum biting density of 24.3 bites per person-hour in October. The infective bites were found for the first time in Ma. annulata collected at Ban Toh Daeng (13 00-14 00 hours) and also Ma. bonneae at forest shade (16 00-17 00 hours) and in a village (20 00-21 00 hours) with rates of 0.6, 1.1 and 1.0%, respectively. The biting activities of these two species occurred in both the day and night time, with two lower peaks at 10 00 hours (18.5 bites per person-hour) and 13 00-15 00 (8.5-10.0 bites per person-hour) hours, but the highest peak was 19 00-21 00 hours (31.5-33.0 bites per person-hour) The biting activity patterns corresponded with the periodicity found in man and domestic cats and may play an important role in either transmission or maintenance of the filarial parasites in the peat swamp forest. The relative role of Ma. bonneae and Ma. uniformis in different environmental settings (primary swamp forest and open swamp) on the transmission of nocturnally subperiodic B. malayi merits further study.