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.

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.

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.

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.

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.

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.

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.

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.

Metaphase karyotypes of Anopheles paraliae (Diptera:Culicidae) in Thailand and evidence to support five cytological races.

Sixteen isoline colonies of Anopheles paraliae were established from wild-caught females collected from cow-baited traps at 4 locations in Thailand. They showed 3 types of X (X1, X2, X3) and 5 types of Y (Y1, Y2, Y3, Y4, Y5) chromosomes based on the number and amount of major block(s) of heterochromatin present in the heterochromatic arm, and were designated as Forms A (X3, Y1), B (X1, X2, X3, Y2), C (X3, Y3), D (X1, X2, X3, Y4) and E (X3, Y5). Form A was found in Songkhla Province, Form B was obtained in Ratchaburi, Nakhon Si Thammarat and Songkhla Provinces, Form C was acquired in Chanthaburi Province, Form D was recovered in Ratchaburi and Songkhla Provinces, and Form E was encountered in Ratchaburi Province. Hybridization experiments among the 7 isoline colonies, which represented the 5 karyotypic forms of An. paraliae, revealed genetic compatibility in providing viable progenies and synaptic salivary gland polytene chromosomes through F2-generations, and thus suggest the conspecific nature of these karyotypic forms. These results were supported by the very low intraspecific sequence divergence (mean genetic distance = 0.000-0.002) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the 5 forms.

Relative developmental and reproductive fitness associated with F1534C homozygous knockdown resistant gene in Aedes aegypti from Thailand.

The effect of permethrin resistance, conferred by a homozygous mutation (F1534C) in the voltage-gated sodium channel protein, upon the reproductive fitness of Aedes aegypti (PMD-R strain) from Thailand was evaluated by comparing with a pyrethroid-susceptible sub colony (PMD strain). The parameters evaluated included larval development time, pupation success, adult emergence, egg production and hatchability, mating ability, female wing length and adult longevity. Larval development times were similar with very low mortality of larvae, pupae and emerging adults among either strain. However, PMD produced significantly fewer females than PMD-R. The mean numbers of eggs laid by PMD (54.2±15.9) and PMD-R (54.6±14.5) strains were not significantly different but the hatchability of PMD eggs (53.7%) was lower than PMD-R eggs (71.2%). The mean wing length of PMD females (2.85±0.15 mm) was longer than PMD-R females (2.74±0.09 mm). The insemination rates for both strains were 100%. The longevity of both strains was mostly not significantly different, over 90% of both sexes surviving at day 30. Our results suggest that the presence of the homozygous F1534C mutation does not lead to fitness reductions. This is in accordance with the high frequency of this allele found among wild populations of Ae. aegypti in many countries. These results also suggest that the removal of pyrethroid insecticide selection pressure may not lead to a regression of 1534C alleles in pyrethroid resistant Ae. aegypti.

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.

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.

High levels of population structure caused by habitat islands in the malarial vector Anopheles scanloni.

The genetic structure of four populations of the malarial vector Anopheles scanloni in Thailand was studied using mitochondrial DNA sequences. Four highly divergent lineages were observed, all with signals of population expansion. Since An. scanloni is restricted to 'islands' of limestone karst habitat, this suggests there is a metapopulation-type dynamic in this species, with restricted gene flow, extinctions and drift all contributing to lineage divergence. Historical environmental change and marine transgressions may also have contributed to population extinction, expansion and divergence. Although there is some current gene flow inferred between nearby populations, it is extremely restricted between the northern and southern populations, which also differed by one fixed polymorphism at the ITS2 rDNA locus. Crossing experiments showed no post-mating barriers existing between the north and the south, but the lack of gene flow between these populations could ultimately result in speciation and has implications for malaria control strategies.

Molecular identification of mosquito species in the Anopheles annularis group in southern Asia.

The Anopheles annularis group of subgenus Cellia Theobald (Diptera: Culicidae) includes five currently recognized species in southern Asia: An. annularis Van der Wulp, Anopheles nivipes (Theobald) and Anopheles philippinensis Ludlow, which are widespread in the region, Anopheles pallidus Theobald, which is known in Sri Lanka, India and Myanmar, and Anopheles schueffneri Stanton, which occurs in Java and Sumatra. Identification of the four mainland species based on morphology is problematic. In view of the fact that the three widespread species are variously involved in malaria transmission in different parts of the region, we developed a species-specific polymerase chain reaction assay based on rDNA internal transcribed spacer 2 (ITS2) sequences to facilitate entomological and epidemiological studies of the four species. The method proved to be reliable when tested over a wide geographical area.

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.