Nematocidal effect of Piper retrofractum Vahl on morphology and ultrastructure of Strongyloides stercoralis third-stage infective larvae.

In a previous research work aimed at discovering natural helminthicides as alternatives to conventional synthetic drugs, Piper retrofractum fruit hexane extract (PHE) has been shown to possess promising nematocidal activity against the third-stage infective larvae of Strongyloides stercoralis. Thus, this study was designed to evaluate the chemical composition and the impact of PHE on symptom and structural alterations of S. stercoralis. Chemical analysis of PHE by gas chromatography-mass spectrometry demonstrated 26 different compounds, constituting 100% of the total composition. The main components were 4-acetylphenyl (4-benzoylphenoxy) acetate (14.86%) and octyl methoxycinnamate (12.72%). Nematocidal bioassays revealed promising potential of PHE against S. stercoralis larvae, with an LC50 value of 0.059 mg/ml, while the reference drug ivermectin exerted higher efficacy, with an LC50 value of 0.020 µg/ml. Behavioural observations under light microscopy revealed that PHE-treated S. stercoralis larvae moved slowly, became paralysed and eventually died during 24 h of incubation. The dead larvae appeared under light microscope as straight worms with unknown vacuoles of different sizes inside their internal bodies. Morphological alterations of the PHE-treated S. stercoralis larvae, such as straight bodies with swollen cuticle, faded transverse annulations and faded longitudinal striations, as well as shallow and smooth lateral longitudinal grooves, were seen clearly under scanning electron microscopy. Ultrastructural changes in the treated larvae, such as protruded lateral longitudinal grooves, loose muscle with vacuolation, dissociation between the hypodermis and cuticle and marked intracellular disorganization with vacuolation, were detected under transmission electron microscopy. The results of this study provide evidence that PHE is toxic against S. stercoralis and also a potential new alternative for anti-Strongyloides chemotherapy.

Identification of medically and forensically relevant flies using a decision treelearning method.

Blow flies, flesh flies, and house flies can provide excellent evidence for forensic entomologists and are also essential to the fields of public health, medicine, and animal health. In all questions, the correct identification of fly species is an important initial step. The usual methods based on morphology or even molecular approaches can reach their limits here, especially when dealing with larger numbers of specimens. Since machine learning already plays a major role in many areas of daily life, such as education, business, industry, science, and medicine, applications for the classification of insects have been reported. Here, we applied the decision tree method with wing morphometric data to construct a model for discriminating flies of three families [Calliphoridae, Sarcophagidae, Muscidae] and seven species [Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart), Chrysomya (Ceylonomyia) nigripes Aubertin, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), Musca domestica Linneaus, and Parasarcophaga (Liosarcophaga) dux Thomson]. One hundred percent overall accuracy was obtained at a family level, followed by 83.33% at a species level. The results of this study suggest that non-experts might utilize this identification tool. However, more species and also samples per specimens should be studied to create a model that can be applied to the different fly species in Thailand.

Mitochondrial DNA variation in the malaria vector Anopheles minimus across China, Thailand and Vietnam: evolutionary hypothesis, population structure and population history.

The effects of Pleistocene environmental fluctuations on the distribution and diversity of organisms in Southeast Asia are much less well known than in Europe and North America. In these regions, the combination of palaeoenvironmental reconstruction and inferences about population history from genetic data has been very powerful. In Southeast Asia, mosquitoes are good candidates for the genetic approach, with the added benefit that understanding the relative contributions of historical and current processes to population structure can inform management of vector species. Genetic variation among populations of Anopheles minimus was examined using 144 mtDNA COII sequences from 23 sites in China, Thailand and Vietnam. Haplotype diversity was high, with two distinct lineages that have a sequence divergence of over 2% and exhibit different geographical distributions. We compare alternative hypotheses concerning the origin of this pattern. The observed data deviate from the expectations based on a single-panmictic population with or without growth, or a stable but spatially structured population. However, they can be readily accommodated by a model of past fragmentation into eastern and western refugia, followed by growth and range expansion. This is consistent with the palaeoenvironmental reconstructions currently available for the region.

A new species and four newly recorded species of black flies (Diptera: Simuliidae) from Bhutan.

Simulium (Simulium) thimphuense sp. nov. is described from a pupa and a mature larva in Bhutan. This new species is placed in the S. multistriatum species-group, and is characterized by the pupal gill with eight thread-like filaments divergent basally at an acute angle when viewed laterally and the cocoon slipper-shaped with several small openings anterolaterally. Four species of Simulium (Simulium) are newly recorded from Bhutan: S. barraudi Puri in the S. multistriatum species-group, S. nodosum Puri in the S. nobile species-group, S. chiangmaiense Takaoka & Suzuki in the S. striatum species-group and S. himalayense Puri in the S. variegatum species-group. Our study increases the number of black fly species known from Bhutan from 18 to 23.

The efficacy of pyriproxyfen-treated resting boxes on the reproductivity of Aedes aegypti (Diptera: Culicidae) in the laboratory.

Pyrethroid resistance is a problem for controlling the dengue vector Aedes aegypti worldwide. One strategy to cope with resistance is to use another insecticide with a different mode of action. Pyriproxyfen (PPF), an insect growth regulator, is normally used at very low concentrations for controlling the immature stages of mosquitoes. At high concentrations, it has a reproductive effect on exposed female mosquitoes. In this study, we demonstrated by using CDC bottle and cone bioassays that tarsal contact with 333 mg AI PPF/m2 for 1 min was sufficient to cause over 95% emergence inhibition (EI) in the progeny of exposed Ae. aegypti females. Exposure for 5 min completely inhibited fecundity. As Ae. aegypti adult mosquitoes are generally drawn towards darker areas, we evaluated the efficacy of a resting box (35x35x55 cm) treated with PPF aimed at disrupting reproductivity of free-flying mosquitoes in the laboratory. We found that the resting box led to 94% EI of exposed females, either before or after blood feeding. The resting box was also attractive for male mosquitoes. Exposed males could transfer sufficient PPF to virgin females via copulation to cause about 90% EI. Additionally, PPF-exposed gravid females from the treated resting box were able to disseminate sufficient PPF to small larvaecontaining cups to reduce adult emergence by 50%. Based on 10 min exposure, the residual effect of PPF-treated resting boxes (over 80% EI) was observed over a 4 month-period. PPF-treated resting boxes may potentially be useful in dengue vector control programs, however further evaluation under natural field conditions are needed.

A method for distinguishing the important malaria vectors Anopheles dirus and An. cracens (Diptera: Culicidae) based on antennal sensilla of adult females.

Some species of the Anopheles dirus species complex are considered to be highly competent malaria vectors in Southeast Asia. Anopheles dirus is the primary vector of Plasmodium falciparum and P. vivax while An. cracens is the main vector of P. knowlesi. However, these two species are difficult to distinguish and identify based on morphological characters. Hence, the aim of this study was to investigate the potential use of antennal sensilla to distinguish them. Large sensilla coeloconica borne on the antennae of adult females were counted under a compound light microscope and the different types of antennal sensilla were examined in a scanning electron microscope. The antennae of both species bear five types of sensilla: ampullacea, basiconica, chaetica, coeloconica and trichodea. Observations revealed that the mean numbers of large sensilla coeloconica on antennal flagellomeres 2, 3, 7, 10 and 12 on both antennae of both species were significantly different. This study is the first to describe the types of antennal sensilla and to discover the usefulness of the large coeloconic sensilla for distinguishing the two species. The discovery provides a simple, reliable and inexpensive method for distinguishing them.

Complex population history of two Anopheles dirus mosquito species in Southeast Asia suggests the influence of Pleistocene climate change rather than human-mediated effects.

Anopheles dirus and Anopheles baimaii are closely related species which feed on primates, particularly humans, and transmit malaria in the tropical forests of mainland Southeast Asia. Here, we report an in-depth phylogeographic picture based on 269 individuals from 21 populations from mainland Southeast Asia. Analysis of 1537 bp of mtDNA sequence revealed that the population history of A. baimaii is far more complex than previously thought. An old expansion (pre-300 kyr BP) was inferred in northern India/Bangladesh with a wave of south-eastwards expansion arriving at the Thai border (ca 135-173 kyr BP) followed by leptokurtic dispersal very recently (ca 16 kyr BP) into peninsular Thailand. The long and complex population history of these anthropophilic species suggests their expansions are not in response to the relatively recent (ca 40 kyr BP) human expansions in mainland Southeast Asia but, rather, fit well with our understanding of Pleistocene climatic change there.

Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA.

The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are either connected by gene flow and/or share a recent common history.

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.

Cloning, expression and characterization of an insect class I glutathione S-transferase from Anopheles dirus species B.

Insect class I glutathione S-transferases (GSTs) were expressed from cDNA obtained from larvae of the Thai malaria vector. Anopheles dirus in a PCR RACE (rapid amplification of cDNA ends) reaction using a primer to the conserved N-terminal region of An. gambiae class I GSTs and a synthetic oligo d(T)-adaptor primer. Seven different plasmids, resulting from sub-cloning of an original single 0.7 Kb PCR band, were picked at random and sequenced. Four of these were clearly GSTs on the basis of putative amino acid sequence conservation. All the sequences had a conserved N-terminal region, but were highly divergent at the C-terminus. The variability in the PCR products suggests that there is a high level of GST class I isoenzyme variability in larval An. dirus. One of the subclones from the PCR reaction contained a full coding region of the cDNA for GST. This had a putative amino acid sequence which was 76 and 91% identity to the An. gambiae GST class I, agGST 1-5 and agGST 1-6 respectively, but only 48% identity to agGST 1-2. The catalytically active enzyme, expressed in Escherichia coli, was strongly immuno-cross reactive with antisera raised against the two An. gambiae class I GSTs. The expressed enzyme was purified to homogeneity from an E. coli cell lysate by S-hexylglutathione agarose affinity chromatography. The enzyme had a high specific activity with CDNB, and also used DCNB and ethacrynic acid as substrates. In addition, it had peroxidase and DDTase activity and its activity with CDNB, was strongly inhibited by a range of organophosphorus and pyrethroid insecticides. This is consistent with the predicted role of this GST class in insecticide resistance.

Isoenzymes of glutathione S-transferase from the mosquito Anopheles dirus species B: the purification, partial characterization and interaction with various insecticides.

Previously we have purified and characterized a major glutathione S-transferase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a model mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koottathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996) Insect Biochem. Mol. Biol. 26:3, 277-285]. In this report we have purified an isoenzyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST-4b, GST-5 and GST-6, were also partially purified and characterized for comparison. All of the Anopheles GST isoenzymes preferred 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V(max) of GST-4c was 24.38 micromole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K(m)s for CDNB and glutathione. Despite being only partially pure GST-4b had nearly a four-fold greater V(max) for CDNB than GST-4c. In contrast, GST-4c possessed the greatest DDT-dehydrochlorinase specific activity among the purified insect GST isoenzymes and no activity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of inhibiting CDNB conjugating activity to different extents for the different isoenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inhibitors. The inhibition studies demonstrate different degrees of interaction of the An. dirus isoenzymes with various insecticides. The GSTs were inhibited more readily by organochlorines and pyrethroids than by the phosphorothioates and carbamate. In a comparison between An. dirus and previous data from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of three GST classes in anopheline insects.

Detection of sporozoites of Plasmodium vivax and Plasmodium falciparum in mosquitoes by ELISA: false positivity associated with bovine and swine blood.

Blood samples from cows and pigs were tested for possible cross-reactivity with a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) kit designed for detection of human malaria sporozoites in mosquitoes. The results revealed that 4 of 16 cows (25%) reacted positively with both Plasmodium falciparum (2A10) and P. vivax (NSV3) monoclonal antibodies and 8 (50%) were positive with NSV3 only. One of 12 pigs (8.33%) was positive with both antibodies, and 2 (16.6%) were positive with NSV3 only. The positivity was associated with plasma, but not with the blood cell fraction. Antigenic extracts of Sarcocystis, Toxoplasma gondii and Trypanosoma evansi gave negative ELISA results, suggesting that these were not the factors in animal blood which gave positive results. Laboratory Anopheles dirus A fed on blood of a positive cow by membrane feeding also gave a positive ELISA result. Furthermore, some blood-fed culicine mosquitoes collected directly from a positive cow were ELISA-positive. The cross-reactive factor(s) in plasma has (have) not yet been identified. These false positive ELISA results could complicate the assessment of sporozoite rate in mosquito populations if the study were carried out by ELISA only, especially in areas where cattle and swine are present.

Entomological evaluation of community-wide use of lambdacyhalothrin-impregnated bed nets against malaria in a border area of north-west Thailand.

This paper reports 2 studies. (i) After a year of baseline data collection, lambdacyhalothrin-treated bed nets were introduced into 3 of 5 villages in north-west Thailand, the remaining 2 being treated with placebo. Human bait collections were carried out in each village on 2 nights per month, for 8 months of each year, and the biting densities were compared between the first year and the second year. The treated bed nets did not have any significant impact on the density or parous rates of Anopheles sawadwongporni and A. maculatus s.s. populations. The results for A. dirus s.l. were not conclusive because of the low number caught. Significant reductions in biting and parous rates of A. minimus species A were observed in only one of the 3 treated villages, and there was no overall difference between treated and control groups. However, the trial suffered from the washing of nets by villagers and the low rate of reimpregnation. (ii) A short-term study involved 4 villages in a cross-over design, and lasted 48 d. For the first 24 d, residents of 2 villages were given new treated nets while the other 2 villages retained their own untreated nets. For the second 24 d, this situation was reversed. Daily light-trapping revealed no significant difference in the indoor densities or parous rates of A. minimus species A between the periods with treated or untreated nets.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of the specific status of Anopheles flavirostris (Diptera: Culicidae).

Anophelesflavirostris (Ludlow) from Lombok Island, Indonesia, was crossed with An. minimus species A (CM strain) from Thailand and species E (ISG strain), a new sibling species, from Japan, to determine genetic compatibility. We also compare the scanning micrographs of female cibarial armature of these three species. Both An. minimus CM and ISG females crossed with An. flavirostris males produced eggs without embryos. One An. flavirostris female crossed with an An. minimus CM male deposited unhatchable eggs with an 18% embryonation rate. The scanning micrographs of the cibarial armature clearly show significant differences among An. flavirostris and the two sibling species of the An. minimus complex. These results support the specific status of An. flavirostris. Previous records of An. minimus Theobald in this country are also discussed.

Evidence for a new sibling species of Anopheles minimus from the Ryukyu Archipelago, Japan.

The Anopheles minimus complex is known to comprise at least 2 sibling species (A and C) in Thailand and Vietnam. This study investigated the specific status of An. minimus on Ishigaki Island, the Ryukyu Archipelago, Japan using morphological and genetic analyses. Morphological studies revealed that almost all (99.5%) of the adult mosquitoes are characterized by the humeral pale spot on the costa of their wings, a character that partially differentiates species A and C elsewhere. A high frequency (81.4%) have a pale fringe spot at the tip of vein 1A, a character rarely observed in other An. minimus populations. Significant seasonal variation in the size of wild An. minimus mosquitoes on the island was observed, with the largest size in the winter. Scanning micrographs of the cibarial armature of females from Ishigaki Island revealed that over 90% had cone filaments clearly differing in shape from those of species A or C. The Giemsa-stained metaphase karyotypes of larval brain cells were somewhat similar to those of species A, with a few exceptions, but were very different from those reported for species C. Crossing experiments between species A (CM strain) from Thailand and the progeny of An. minimus from Ishigaki Island (ISG strain) revealed postzygotic genetic incompatibility, although no prezygotic isolation. Hybrid progeny were only obtained from CM female x ISG male. F2 hybrid progeny were not obtained, since the hybrid males were sterile or almost sterile with atrophied testes or abnormal spermatozoa, although the polytene chromosomes of hybrid larvae showed synapsis. The hybrid females backcrossed with either CM or ISG males laid eggs with significantly lowered fertility and viability. The sequence for the D3 region of the 28S gene of ribosomal DNA of the ISG strain differed from those of species A and C. In addition, sequence data from Vietnamese mosquitoes suggest that the An. minimus complex may contain additional species. The morphological, cytogenetic, molecular, and hybridization evidence together suggest the existence of another sibling species of the An. minimus complex on Ishigaki Island, which is provisionally designated An. minimus species E.

Susceptibility of Thai zoophilic Anophelines and suspected malaria vectors to local strains of human malaria parasites.

Wild caught zoophilic Anopheles and suspected malaria vector species collected in northwest Thailand were experimentally infected with local human malaria parasites using a membrane feeding. One week post-feeding a number of mosquitos were dissected for oocyst examination. The remainder were kept for another one week or more, and then the salivary glands were examined for the presence of sporozoites. The results revealed that An. vagus, An. kochi and An. annularis were susceptible to both Plasmodium falciparum and P. vivax whereas An. barbirostris and An. sinensis were susceptible to only P. vivax. The non-susceptibility to P. falciparum of these two mosquito species may indicate their poor vector status of this malaria species in the field.

Acute human isosporiasis in Thailand: a case report.

A case of acute human isosporiasis in Thailand was reported. A 57-year-old man developed diarrhoea after prednisolone administration for the management of nephrotic syndrome. Stool examination revealed numerous Isospora oocysts. On the basis of parasite morphology and sporulation time, it was identified as Isospora belli Wenyon, 1923. The patient recovered for the diarrhoea without specific treatment.

Entomological and epidemiological investigations of malaria transmission in relation to population movements in forest areas of north-west Thailand.

Transmission of forest-related malaria was observed entomologically and epidemiologically for 2 transmission seasons in 1990 and 1991 in 5 villages of Mae Sariang district, Mae Hong Son Province, north-west Thailand. The entomological study included collections of mosquitos and determination of infection rate by using enzyme-linked immunosorbent assay in the residential villages and the farm huts. The epidemiological study included fortnightly visits to 30% of the households to interview and record movement activities and illness of villagers. Circumsporozoite proteins, in most cases of Plasmodium falciparum, were detected in Anopheles minimus species A, An. dirus s.l., An. maculatus s.s. and An. sawadwongporni in residential villages and/or farm huts, suggesting transmission could occur there. Movement of people away from their residences occurred throughout the year for several reasons with a sharp peak in July for agricultural activity, mainly ploughing and planting for rice cultivation. The relative risk of infection for people engaged in agricultural activity was 3 times that of people living in the residential villages. Although a higher biting density of vectors was generally evident at the farm huts, the estimated inoculation rates in the 2 settings were similar. Movement for forest activity increased after harvesting rice in the cool dry season and carried the highest malaria risk, suggesting different epidemiological and probably entomological conditions which need further investigation. The significance is discussed of discrepancies between the case classification system used by this study and that used by malaria sector staff.

Filariasis in Tak Province, northwest Thailand: the presence of subperiodic variant Wuchereria bancrofti.

The microfilariae found in carriers at Tak Province, Northwestern Thailand were morphologically and morphometrically studied. It was found that the parasites conformed to that of W. bancrofti microfilaria. The microfilarial periodicity as determined from four carriers was found to be nocturnally (early evening) subperiodic type showing a distinct peak at 1800 hours.