Geometric morphometrics to distinguish the cryptic species Anopheles minimus and An. harrisoni in malaria hot spot villages, western Thailand.

Anopheles minimus Theobald 1901 and An. harrisoni Harbach & Manguin 2007 belong to the same species complex. They are morphologically similar and can exist in sympatry but have blood host preferences. The most accurate method for their identification is based on molecular techniques. Here, we measure the level of interspecific discrimination by geometric morphometry. Sixty-seven An. minimus and 22 An. harrisoni specimens were selected based on their morphological integrity and confirmed by identification polymerase chain reaction of internal transcribed spacer 2. These samples were used as reference data allowing for a morphometric identification based on geometric shape. Despite size overlap between the two species, there was a significant shape divergence allowing for differentiation of An. minimus and An. harrisoni with 90% accuracy. An intraspecific study of An. minimus showed a summer period associated to the reducing of wing size, which did not influence the shape-based differentiation of An. harrisoni. Wing venation geometry can be used to distinguish between these cryptic species mainly based on shaped divergence. This study suggests that geometric morphometrics represent a convenient low-cost method to complement morphological identification, especially concerning damaged specimens, i.e., insects having accidentally lost the anatomical features allowing a reliable morphological identification.

Geometric morphometric analysis of the effect of temperature on wing size and shape in Aedes albopictus.

Wing geometry helps to identify mosquito species, even cryptic ones. On the other hand, temperature has a well-known effect on insect metric properties. Can such effects blur the taxonomic signal embedded in the wing? Two strains of Aedes albopictus (laboratory and field strain) were examined under three different rearing temperatures (26, 30 and 33 °C) using landmark- and outline-based morphometric approaches. The wings of each experimental line were compared with Aedes aegypti. Both approaches indicated similar associations between wing size and temperature. For the laboratory strain, the wing size significantly decreased as the temperature increased. For the field strain, the largest wings were observed at the intermediate temperature. The two morphometric approaches describing shape showed different sensibilities to temperature. For both strains and sexes, the landmark-based approach disclosed significant wing shape changes with temperature changes. The outline-based approach showed lesser effects, detecting significant changes only in laboratory females and in field males. Despite the size and shape changes induced by temperature, the two strains of Ae. albopictus were always distinguished from Ae. aegypti. The present study confirms the lability of size. However, it also suggests that, despite environmentally-induced variation, the architecture of the wing still provides a strong taxonomic signal.

Species composition and population dynamics of phlebotomine sand flies in a Leishmania infected area of Chiang Mai, Thailand.

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.

Didilia sp. infecting Phlebotomus stantoni in Thailand.

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.

Seasonal distribution of phlebotomine sand flies (Diptera: Psychodidae) in Tham Phra Phothisat temple, Saraburi province, Thailand.

Phlebotomine sand flies have long been incriminated as vectors of leishmaniasis in various parts of both the Old and New World. Prompted by recent indigenous cases of leishmaniasis in Thailand, a bionomic study of sand flies was undertaken in Tham Phra Phothisat temple, Saraburi province. In this study, sand flies were collected using Centers for Disease Control (CDC) light traps, to clarify the activity patterns and species composition of the sand flies. Traps were laid from August 2005 to July 2006. The insects were collected monthly between 1800-0600 hours. A total of 8,131 sand flies were collected with a female:male ratio of 1.9:1. Sixteen species were identified, of which 5 belonged to the genus Phlebotomus, 9 to Sergentomyia and 1 to Chinius. Species comprised the abundant species (Sergentomyia silvatica 35.6%, Sergentomyia barraudi 18.1%, Sergentomyia anodontis, 17.1%, Sergentomyia iyengari 11.9%, and Sergentomyia gemmea 11.2%); the less common species (<2%) were Sergentomyia dentata 1.8%, Phlebotomus stantoni 1.1%, Sergentomyia indica 1.0%, Phlebotomus argentipes 0.8%, Sergentomyia perturbans 0.4%, Chinius barbazani 0.3%, Phlebotomus asperulus 0.2%, Phlebotomus philippinensis gouldi 0.1%, Phlebotomus major major 0.1%, Sergentomyia quatei 0.1% and Sergentomyia bailyi 0.1%. The results revealed seasonal variation in sand fly prevalence, with the highest peak in July. Soil samples collected were characterized by alkaline (pH 7.6).

Seasonal distribution of phlebotomine sand flies (Diptera: Psychodidae) in Tham Phra Phothisat temple, Saraburi province, Thailand

Phlebotomine sand flies have long been incriminated as vectors of leishmaniasis in various parts of both the Old and New World. Prompted by recent indigenous cases of leishmaniasis in Thailand, a bionomic study of sand flies was undertaken in Tham Phra Phothisat temple, Saraburi province. In this study, sand flies were collected using Centers for Disease Control (CDC) light traps, to clarify the activity patterns and species composition of the sand flies. Traps were laid from August 2005 to July 2006. The insects were collected monthly between 1800-0600 hours. A total of 8,131 sand flies were collected with a female:male ratio of 1.9:1. Sixteen species were identified, of which 5 belonged to the genus Phlebotomus, 9 to Sergentomyia and 1 to Chinius. Species comprised the abundant species (Sergentomyia silvatica 35.6%, Sergentomyia barraudi 18.1%, Sergentomyia anodontis, 17.1%, Sergentomyia iyengari 11.9%, and Sergentomyia gemmea 11.2%); the less common species (<2%) were Sergentomyia dentata 1.8%, Phlebotomus stantoni 1.1%, Sergentomyia indica 1.0%, Phlebotomus argentipes 0.8%, Sergentomyia perturbans 0.4%, Chinius barbazani 0.3%, Phlebotomus asperulus 0.2%, Phlebotomus philippinensis gouldi 0.1%, Phlebotomus major major 0.1%, Sergentomyia quatei 0.1% and Sergentomyia bailyi 0.1%. The results revealed seasonal variation in sand fly prevalence, with the highest peak in July. Soil samples collected were characterized by alkaline (pH 7.6).

Autochthonous visceral leishmaniasis: a report of a second case in Thailand.

Visceral leishmaniasis (VL) is a rare disease in Thailand. Only one previous case has been reported in which transmission was likely autochthonous. We conducted an investigation of a case of VL, which included serological and symptom surveys of people who lived near the case in Nan Province and Bangkok, serological surveys of domestic animals in his home village and sand fly surveys in his home village and in Bangkok. No humans interviewed met our case definition for possible VL. One hundred thirty-one villagers were seronegative for Leishmania antibodies. We found three cows and one cat that had positive direct agglutination tests for Leishmania spp, but we were unable to confirm current infection by PCR. Sand fly surveys showed that most of the flies were of the Sergentomyia genus, which has not previously been reported as a competent vector in Thailand. Nonetheless, we conclude, based on the patient's lack of travel outside Thailand and the presence of seropositive domestic animals in his home village, that he was most likely infected by the bite of a sand fly in Thailand. We believe this is the second case of autochthonously transmitted VL in Thailand.

Seasonal variation of cave-dwelling phlebotomine sandflies (Diptera:Psychodidae) in Phra Phothisat Cave, Saraburi Province, Thailand.

Phlebotomine sandflies are widely distributed in Thailand. In view of recent occurrence of indigenous cases of leishmaniasis in Thailand, a bionomic study of sandflies was undertaken in Phra Phothisat cave, Saraburi Province, Thailand from August 2005 to July 2006. The insects were collected monthly by CDC light traps between 06:00 PM and 06:00 AM. They were preserved in 80% alcohol and mounted with Hoyer's medium for species identification. A total of 5,514 sandflies were collected with a female:male ratio of 2.5:1. The collected sandflies belong to 13 species in the genera Phlebotomus and Sergentomyia, namely S. silvatica (43.5%), S. anodontis (31.5%), S. dentata (15.3%), S. barraudi (3.3%), P. argentipes (2.0%), P. philippinensis gouldi (1.0%), P. stantoni (0.5%), S. gemmea (0.5%), P. major major (0.1%), S. perturbans (0.1%), S. iyengari (0.1%), S. bailyi (0.1%), and P. teshi (0.1%). The results revealed seasonal variation in sandflies with the highest peak in July (436 sandflies/trap-night). Some of the sandflies could not be identified and were assumed to be new species. Soil samples inside the cave were analyzed for chemical characteristics. The soil was characterized by moderate acidity (pH 5.8) with various amount of chemicals and nutrients.

Anopheles barbirostris/campestris as a probable vector of malaria in Aranyaprathet, Sa Kaeo Province.

As a result of dramatic increase in malaria cases in Sa Kaeo Province from 666 cases in 1995 to 4,381 in 1997, a brief entomological study was carried out during January 1998 to December, 1999 in Pa Rai subdistrict where most malaria cases were reported. Of fourteen species of mosquitos found, only Anopheles barbirostris group was the most abundant species throughout the year. Adult identification was not able to confirm species within An. barbirostris group, particularly between An. barbirostris and An. campestris because of morphological resemblance. Therefore, the barbirostris group captured in this study is reported to be either An. barbirostris or An. campestris. The seasonal prevalence of barbirostris/campestris was bimodal in distribution (September and November) and coincided well with malaria occurrence in this area. Human landing collections revealed high adult densities with 20 bites/person-night for mean indoor density and 53.5 bites/person-night for mean outdoor density. The biting peak was during 21.00-24.00 hours. Among 223 barbirostris/campestris dissected for oocysts and sporozoites only one gut from the outdoor collection in November was infected with oocysts. There were no sporozoites detected in salivary glands of all mosquitos collected. This appears to indicate that in the absence of major vectors local species may serve as potential transmitters of malaria in Thailand.

Thanaka (Limonia acidissima) and deet (di-methyl benzamide) mixture as a mosquito repellent for use by Karen women.

The prevention and treatment of drug-resistant malaria is becoming increasingly difficult. On the Thai-Myanmar border multi-drug resistant strains of falciparum malaria are increasing and, because the malaria vector Anopheles bite outdoors during early evening, insecticide house-spraying or impregnated bednets provide only limited protection. Therefore, the protective efficacy of repellent formulations containing di-methyl benzamide (deet) and permethrin against local vectors was estimated, when applied to the skin, and their acceptability amongst pregnant Karen women who are at relatively high risk from malaria was assessed. Human landing catches of mosquitoes showed that almost complete protection was achieved using different formulations of 20% deet and 0.5% permethrin for up to 6 h. All-night collections from human subjects indicated that this repellent combination reduced exposure to malaria parasites by at least 65 and 85% for those transmitted by Anopheles minimus and An. maculatus, respectively, the two principal vectors in this area. Pregnant women in the camps preferred repellents which were mixed with 'thanaka', a root paste made from pulp of the wood apple tree, Limonia acidissima, used locally as a cosmetic. Apart from a temporary warming sensation where repellent thanaka was applied to the skin, the repellents were well tolerated. An intervention trial is currently in progress to determine whether deet mixed with thanaka can protect pregnant women against malaria in this part of the world. Bioassays using a laboratory strain of Aedes aegypti demonstrated that thanaka is itself slightly repellent at high dosages and the mixture with deet provides protection for over 10 h. This treatment would therefore also provide some personal protection against dengue, which is increasing locally, transmitted by Ae. aegypti and Ae. albopictus biting during the daytime.