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.

First report of canine cutaneous myiasis caused by Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) in Thailand.

Although myiasis caused by the blow fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), has been reported in Thailand, all of the cases were human. This study described three cutaneous myiasis cases caused by C. bezziana in dogs in Chiang Mai province, northern Thailand. The removal of fly larvae together with specific treatment to cure all cases in this study was represented.

Short Communication: First report of canine cutaneous myiasis caused by Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) in Thailand

@#Although myiasis caused by the blow fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), has been reported in Thailand, all of the cases were human. This study described three cutaneous myiasis cases caused by C. bezziana in dogs in Chiang Mai province, northern Thailand. The removal of fly larvae together with specific treatment to cure all cases in this study was represented.

Orbital ophthalmomyiasis caused by Chrysomya bezziana in Thailand.

Orbital ophthalmomyiasis occurs infrequently in Thailand. Herein, we report a case in Chiang Mai, Thailand, of orbital ophthalmomyiasis due to larvae of the blow fly Chrysomya bezziana (Diptera: Calliphoridae). A 94-year-old woman was admitted to Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, with a swollen and ulcerated right upper eyelid. The lesion in the eyelid had multiple holes around the ulcer site; bleeding was accompanied by pus and necrotic tissue - the site was filled with dipteran larvae. Eleven larvae were removed from the patient, of which five were killed for microscopic examination and six were reared in the laboratory under ambient temperature and natural relative humidity until they metamorphose into adult. Five third instars and one adult were morphologically identified as C. bezziana. The predisposing factors were probably chronic immobility, inability of the patient to perform daily activities, and presumably neglected and/or poor personal hygiene. To our knowledge, this case represents the first reported case of orbital ophthalmomyiasis caused by C. bezziana in Thailand.

Three Sarcophagid species (Diptera: Sarcophagidae) newly recorded in Thailand.

This study contributed new records of three flesh fly species (Diptera: Sarcophagidae) to the fauna of Thailand - Miltogramma tibita Chao & Zhang (subfamily Miltogrammatinae), Myorhina situliformis (Zhong, Wu & Fan, 1982), and Iranihindia martellata (Senior-White, 1924) (subfamily Sarcophaginae). Collections of these species were performed using a sweep net and one-day old beef offal as bait. Miltogramma tibita differs from other known Miltogramma by having a fine long seta on the dorsal surface of tarsomeres 2-4. With this new record, the number of species belonging to the genus Miltogramma known from Thailand has increased to three which includes Miltogramma angustifrons (Townsend, 1933) and Miltogramma iberica Villeneuve, 1912. The new record of My. situliformis makes a total of three species for Myorhina and these include Myorhina otiophalla (Fan & Chen, 1981) and Myorhina caudagalli (Böttcher, 1912). Regarding Iranihindia, the recording of I. martellata makes a total of two species, the other being Iranihindia martellatoides (Baranov, 1931). This study provides a revised key of each genus where these newly recorded species were recorded, with their re-descriptions, illustrations, photographs, and scanning electron micrographs focusing on the male genitalia. The findings of these newly recorded species means that a total of 86 species of flesh flies have been recorded from Thailand.

Aural myiasis caused by Parasarcophaga (Liosarcophaga) dux (Thomson) in Thailand.

Herein is reported the first case in Thailand of aural myiasis caused by the flesh fly, Parasarcophaga (Liosarcophaga) dux (Thomson). A 5-day-old infant was taken to hospital with a slightly bloody ear. Two fly larvae exiting the ear and another recovered by a physician were alive, and confirmed as P. dux species from adult examination results. This case brought attention to the need for protection against synanthropic flies, particularly for infants and/or hearing impaired patients.

The blow fly, Chrysomya megacephala, and the house fly, Musca domestica, as mechanical vectors of pathogenic bacteria in Northeast Thailand.

The Oriental latrine fly, Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and the house fly, Musca domestica L., (Diptera: Muscidae) are synanthropic flies which are adapted to live in close association with human habitations, thereby making them likely mechanical vectors of several pathogens to humans. There were two main aims of this study. The first aim was to determine the prevalence of these two fly species from five types of human habitations including: fresh-food markets, garbage piles, restaurants, school cafeterias and paddy fields, in the Muang Ubon Ratchathani and Warinchamrap districts of Ubon Ratchathani province of Northeast Thailand. Flies collection were conducted monthly from September 2010-October 2011 using a reconstructable funnel trap, containing 1 day-tainted beef offal as bait. A total of 7 750 flies (6 401 C. megacephala and 1 349 M.domestica) were collected. The second aim was to examine the potential of these flies to carry pathogenic bacteria. Bacteria were isolated from 994 individual flies collected using a sweep net (555 C. megacephala and 439 M. domestica). A total of 15 bacterial genera were isolated from the external surfaces, comprising ten genera of gram-negative bacteria and five gram-positive bacteria. The most common bacteria isolated from both species were coagulase-negative staphylococci, followed by Streptococcus group D non-enterococci. Human pathogenic enteric bacteria isolated were Salmonella sp., Shigella sp., Escherichia coli O157:H7, Salmonella typhi, Bacillus sp., and Enterococcus sp., of which S. typhi is the first report of isolation from these fly species. Other human pathogens included Staphylococcus aureus and Pseudomonas aeruginosa. Not only were the number of C. megacephala positive for bacteria significantly higher than for M. domestica, but they were also carrying ~11-12 times greater bacterial load than M. domestica. These data suggest that both fly species should be considered potential mechanical vectors of bacterial pathogens associated with human habitations year-round in this region of Northeast Thailand.

Morphology of puparia of flesh flies in Thailand.

Puparia of five flesh fly species were investigated for forensic study. Boettcherisca nathani (Lopes, 1961), Boettcherisca peregrina (Robineau-Desvoidy, 1830), Lioproctia pattoni (Senior-White, 1924), Liopygia ruficornis (Fabricius, 1794) and Parasarcophaga (Liosarcophaga) dux (Thomson, 1869) were examined with a scanning electron microscopy (SEM). Differences between species were found in the number and arrangement of papillae in the anterior spiracle, the shape of intersegmental spines between the prothorax and mesothorax and the pattern of spiracular tufts at the posterior spiracle. The anterior spiracle of B. nathani had two rows, comprising 21-27 papillae; while those of B. peregrina and L. pattoni had one or two irregular rows with 24-26 and 20-28 papillae, respectively. Anterior spiracle of L. ruficornis and P. dux had one row of 10-15 papillae. Intersegmental spines between the prothorax and mesothorax and pattern of spiracular tufts at the posterior spiracle are morphologically different. L. ruficornis and P. dux puparia are similar, but the position of the interslit plate between the inner and middle spiracular slits was found to be an important attribute to separate both species. Morphometric analysis on the length and width of puparia of these species revealed statistically different among them. The key for identifying puparia of forensically important flesh flies has been provided.

Ontogenensis and developmental rate of the blow fly, Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae).

Blow flies of the genus Hypopygiopsis are considered forensically important. In Thailand, four Hypopygiopsis species coexist, i.e., Hypopygiopsis fumipennis, Hypopygiopsis infumata, Hypopygiopsis violacea and Hypopygiopsis tumrasvini. In this study, the ontogeny and developmental rate of H. tumrasvini eggs, larvae and pupae were determined in the laboratory chamber reared at 25.0 ± 2.0°C and 80.0 ± 5.0% RH. Larvae emerged from eggs 10-12 h after deposition. Mean length of the first, second, third (feeding phase), third (post-feeding phase) instars and puparia were 3.5 ± 1.1, 7.2 ± 1.1, 13.5 ± 1.8, 12.5 ± 0.5 and 9.0 ± 0.7 mm, respectively. The median development time for first, second, third instar (feeding phase), third instar (post-feeding phase) and pupariation period was 8 h, 10 h, 34 h, 22 d and 9-10 d, respectively. Developmental curve of the larval length indicated the rapid progression from 0 until 40 h from the first instar until the feeding third instar. Video recording of pupariation revealed the development of pupal respiratory horn beneath the larval integument at 27.0 h; whereas it protruded through the orifice of the integument at 27.5 h.

Parasarcophaga (Liopygia) ruficornis (Diptera:Sarcophagidae): a flesh fly species of medical importance.

Parasarcophaga (Liopygia) ruficornis is a well-known flesh fly species of medical importance, both as a myiasis-producing agent and fly seen in a forensic entomology context. This study performed a comprehensive literature review of this fly species, dealing with morphology, bionomics and medical involvement. Important characteristics used to identify P. ruficornis have been provided for both its third instar and adult for identification purpose in the future.

Morphology and identification of fly eggs: application in forensic entomology.

Fly eggs found in corpses can be used as entomological evidence in forensic investigation. This study aims to investigate the morphology of forensically important fly eggs. Eggs of Chrysomya rufifacies, Chrysomya megacephala, Chrysomya pinguis, Chrysomya nigripes, Hypopygiopsis tumrasvini, Lucilia cuprina, Lucilia porphyrina and Musca domestica were examined using 1% potassium permanganate solution for 1 min. Morphometric analysis revealed that the mean length of Hy. tumrasvini (1.63 mm) and C. pinguis (1.65 mm) eggs was the longest, followed by that of L. porphyrina (1.45 mm), C. rufifacies (1.34 mm). The egg length, width of median area and darkness staining of hatching pleats were distinctive features. Four categories of median area were proposed, based on width; (1) distinctly wide (Megaselia scalaris, Synthesiomyia nudiseta); (2) wide (C. nigripes, M. domestica); (3) slightly widening (Hy. tumrasvini, L. cuprina, L. porphyrina); and (4) narrow (C. rufifacies, C. albiceps, C. megacephala, C. pinguis). Four species were examined using SEM, i.e., C. megacephala, C. pinguis, Hy. tumrasvini and L. porphyrina. The eggs of C. megacephala demonstrated swollen hatching pleats. Inside, the hexagon of the chorion appeared as a sponging bumpy feature. The egg of C. pinguis was similar to C. megacephala, except for the sponging bumpy feature on the outer surface of the hatching pleats. Regarding Hy. tumrasvini and L. porphyrina, their island structure was apparent at the inner surface of the upright hatching pleats. The key for identifying these eggs together with other reported species in Thailand has been updated.

Morphology and identification of first instars of the European and Mediterranean blowflies of forensic importance. Part I: Chrysomyinae.

Light microscopy photographs, line illustrations and scanning electron micrographs are provided for first instars of Chrysomya albiceps (Wiedemann), Chrysomya megacephala (Fabricius) and Protophormia terraenovae (Robineau-Desvoidy) (all: Diptera: Calliphoridae). The following morphological structures are documented: pseudocephalon; antennal complex; maxillary palpus; facial mask; thoracic and abdominal spinulation; spiracular field; posterior spiracles, and cephaloskeleton. New diagnostic features of the cephaloskeleton and the spinulation of abdominal segments are described. Verification of earlier descriptions revealed major discrepancies among published data. The present results allow for the clarification, correction and, especially, complementation of existing information provided by numerous authors. Finally, an identification key for first-instar larvae of European and Mediterranean Chrysomyinae of forensic importance is presented.

Observations of the third instar larva and puparium of Chrysomya bezziana (Diptera: Calliphoridae).

Observations on the ultrastructure of the third instar larva and puparium of the Old World screw-worm fly, Chrysomya bezziana, are presented utilizing both light microscopy (LM) and scanning electron microscopy (SEM). Results of this study indicate that the shape of the intersegmental spines between the pro- and mesothorax markedly differ from other blow fly species (Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes, Lucilia cuprina) in being broad-based with sharp recurved tips. Other characters such as the posterior spiracles, number of papillae on the anterior spiracles, oral grooves, and posterior spiracular hairs also differ. The strong and robust mouthhooks may explain the ability of larvae to penetrate deeply into human tissues. Perforated sieve plates covered with antler-like projections were observed within the anterior spiracles of the puparium of C. bezziana. The posterior spiracular discs each bear three spiracular slits with approximately 2-microm wide openings that were viewed either open or closed by a membrane underneath. This study expands our knowledge of the fine details of the external morphology of both the third instar larva and puparium of C. bezziana, which is an obligatory myiasis-producing species in many regions. A key to differentiate the third instar of C. bezziana from other blow flies in Thailand is given.

Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina (Diptera: Calliphoridae), and flesh fly, Liosarcophaga dux (Diptera: Sarcophagidae).

Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina, and flesh fly, Liosarcophaga dux, are presented utilizing scanning electron microscopy (SEM). Emphasis was focused on characteristic features of the puparia that could be used for differentiation from other forensically important fly species. The puparium of L. cuprina typically measures 6.2+/-0.2 mm in length and 2.3+/-0.1 mm in width. Each anterior spiracle contains a single row of 5-7 papillae. The intersegmental spines between the prothorax and mesothorax are triangular with constricted tips. A clustered bubble membrane, comprising about 30 mammillate structures, is positioned dorsolaterally on each side of the first abdominal segment in young puparia but is replaced by short, tubular respiratory horns in aged puparia. The posterior end of the puparium is broadly truncate and bears a pair of medially positioned posterior spiracles that each contains three straight spiracular slits. The puparium of L. dux is larger in comparison to L. cuprina and typically measures 9.9+/-0.3 mm in length and 3.8+/-0.2 mm in width. An anterior spiracle of this species contains 14-17 papillae. The intersegmental spines between the prothorax and mesothorax are broad and triangular. A convoluted structure of unknown function was observed at the dorsolateral edge of segments 5-11. In L. dux, the caudal segment of the puparium is slightly tapered and abruptly truncated and contains a pair of posterior spiracles that are located within a deep concavity in the terminal end. Each posterior spiracular disc appears D-shaped, with a pronounced medial projection and three vertically oriented long, narrow spiracular slits. The anatomical features presented herein may be useful for identification of fly puparia of these two species in future forensic entomological investigations.

Ultrastructure of adhesive device in fly in families calliphoridae, muscidae and sarcophagidae, and their implication as mechanical carriers of pathogens.

The ultrastructure of adhesive device or the pulvilli, pad-like structure between the tarsal claws of the legs, is presented in the blowfly (Calliphoridae), housefly and relatives (Muscidae), and flesh fly (Sarcophagidae) through scanning electron microscopy. The blowfly species were Chrysomya chani, Chrysomya nigripes, Chrysomya pinguis, and Chrysomya villeneuvi; while those of the housefly and relatives were Musca domestica and Hydrotaea chalcogaster, respectively. Fresh fly species included Boettcherisca peregrina and Liosarcophaga dux. Numerous tenent setae were observed on the ventral side of the pulvilli. Two features of the tip of the tenent setae were found as a spatula-like (in C. chani, C. pinguis, C. nigripes, M. domestica, H. chalcogaster, B. peregrina, and L. dux) and spoon-like tip (in C. villeneuvi). Transmission electron microscopy of the tenent setae in M. domestica revealed the electron-lucent centrally located, suggesting an adhesive substance. These results provided anatomical information that allow us to not only understand the successful attachment of flies to smooth surfaces but also clarify their role as a mechanical carrier of microorganisms.

Isolation and in vitro antimalarial activity of hexane extract from Thai Picrasma javanica B1 stembark.

The in vitro antimalarial activities against Plasmodium falciparum K1 of four extracts from the stembark of Picrasma javanica B1; ie water, methanol, chloroform and hexane extracts were studied using a modification of the [3H]hypoxanthine incorporation method. It was found that the hexane extract showed in vitro antimalarial activity with IC50 of 3.3 microg/ml. The extract was further fractionated using quick column chromatography, resulting in ten fractions. Fraction V was the most effective against P. falciparum K1 with IC50 of 4.4 microg/ml. Further isolation of fraction V using a column chromatographic technique provided six fractions. According to 1H- and 13C-NMR spectra, it could be concluded that the major compound in fraction V-3 was beta-sitosterol. Unfortunately, the antimalarial activity of beta-sitosterol could not be determined because of its low solubility in DMSO. However, fractions V-2 and V-4 still showed in vitro antimalarial activities with IC50 of 2.8 and 3.4 microg/ml, respectively. The further fractionation of these two active fractions could lead to promising candidates as antimalarial agents.

Cystatin capture enzyme-linked immunosorbent assay for serodiagnosis of human clonorchiasis and profile of captured antigenic protein of Clonorchis sinensis.

Enzyme-linked immunosorbent assay (ELISA) with crude extracts of adult Clonorchis sinensis has been reported to have a high degree of sensitivity with a moderate degree of specificity for the serodiagnosis of clonorchiasis. The cystatin capture ELISA was investigated for its usefulness for the serodiagnosis of human clonorchiasis. Cystatin bound specifically to cysteine proteinases in crude extracts of adult C. sinensis worms, and its binding capacity was not hindered competitively by the other proteinase inhibitors tested. The cystatin capture ELISA for clonorchiasis showed a higher degree of specificity than the conventional ELISA, which produced some cross-reactivities to sera from patients with cysticercosis, sparganosis, and opisthorchiasis. Immunoglobulin G antibodies to C. sinensis cysteine proteinases were produced in experimental rabbits at week 3, and their levels increased rapidly and remained at a plateau after 8 weeks of infection. Of the proteins from the C. sinensis crude extract captured with cystatin, seven proteins were reactive with the serum from patients with clonorchiasis. The cystatin capture ELISA is indicated to be a sensitive and highly specific immunodiagnostic assay for serodiagnosis of human clonorchiasis.

The first documented forensic entomology case in Thailand.

The forensic entomology case described herein is the first such case documented in Thailand. A mummified corpse of a 32-yr-old man was discovered in a forested habitat, with the larvae of six species of flies (Diptera) found in association with the corpse at the time of its discovery, i.e., those of Hydrotaea (=Ophyra) spinigera Stein (family Muscidae), Piophila casei (L.) (family Piophilidae), Megaselia scalaris (Loew) (family Phoridae), Sagus sp. (family Stratiomyidae), and larvae of two unidenitified flesh fly species (family Sarcophagidae). The presence and age of the larval specimens of P. casei, M. scalaris, and H. spinigera gave entomological evidence that the postmortem interval for the corpse was 3-6 mo. This report also documets some of the forensically important fly species that occur in Thailand.