Identification of Fasciola species based on mitochondrial and nuclear DNA reveals the co-existence of intermediate Fasciola and Fasciola gigantica in Thailand.

Molecular techniques were used to identify Fasciola species collected from Chiang Mai Thailand. Morphometrically, 65 stained and 45 fresh worms collected from cattle suggested the possible occurrence of both F. gigantica and F. hepatica. Twenty-two worms comprising 15 from cattle and 7 from human patients, were identified subsequently based on three genetic markers: mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2). All of them presented the F. gigantica type in maternally inherited mitochondrial sequences (nad1 and cox1), with six types in each sequence (FgNDI-CM1 to FgNDI-CM6 and FgCOI-CM1 to FgCOI-CM6, respectively). Remarkably, the predominant nad1 type, FgNDI-CM6, was identical to that of aspermic Fasciola sp. formerly reported from Thailand, Japan, Korea, China, Vietnam, and Myanmar. ITS2 sequences were analyzed successfully in 20 worms. Fifteen worms showed the F. gigantica type and five (including one worm from a patient) had mixed ITS2 sequences of both F. gigantica and F. hepatica in the same worms, with additional heterogeneity within both ITS2 types. This study revealed the intermediate form of Fasciola coexisting with F. gigantica for the first time in Thailand.

Morphology of immature stages of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) for use in forensic entomology applications.

In forensic investigations, all immature stages of flies (egg, larvae, and puparium) can serve as entomological evidence at death scenes. These insects are primarily used to estimate the post mortem interval (PMI), but can also be involved in the analysis of toxic substances, determining manner of death, and in indicating relocation of a corpse in homicide cases. In this study, we present the morphology of the egg, larvae, and puparium of Hemipyrellia ligurriens, a blow fly species of forensic importance in Thailand. Examination was conducted using scanning electron microscopy (SEM). The egg stage was found to display a relatively wide plastron region (or median hatch line area) that spans almost the entire length of the egg. The median hatch line is oriented in an upright position. External chorionic sculpture of the egg is present in a hexagonal pattern whose reticular boundaries are slightly elevated. In the larval stages, the most prominent morphological changes were detected upon comparison of the first to the second instar; whereas, the differences between second and third instar larvae were less obvious outside of the increase in number of posterior spiracular slits. Most of the major differences involve body size and structure of the anterior and posterior spiracles. Each anterior spiracle in both the second and third instars projects five to seven papillae apically. Each posterior spiracular disc of a third instar exhibits a complete peritreme, three spiracular slits, and a prominent button that is ventromedially located. The puparium is coarctate and features a clustered bubble membrane comprised of approximately 57 mammillate structures positioned dorsolaterally on each side of the first abdominal segment in young puparia. This feature is replaced by short, tubular respiratory horns in aged puparia. This study provides more detailed exposure of important morphological features that can be used for accurate identification of immature stages of H. ligurriens. Information presented can aid in forensic investigations involving this fly species.

Ommatidia of blow fly, house fly, and flesh fly: implication of their vision efficiency.

This work aims to elucidate the number of ommatidia or facets (the outwardly visible units of each ommatidium) for compound eyes in blow flies [Chrysomya megacephala (F.), Chrysomya rufifacies (Macquart), Chrysomya nigripes (Aubertin), Lucilia cuprina (Wiedemann)], house flies (Musca domestica L.), and flesh flies (Liosarcophaga dux Thomson) by manual counts of the corneal spreads. The head of the fly in each species was soaked in 20% potassium hydroxide solution at room temperature for 7 days, and the clear compound eye was dissected into six small parts, each of which was placed onto a slide and flattened using a coverslip. Images of each part were obtained using a microscope connected to a computer. The printed images of each part were magnified, and the total number of ommatidia per eye was manually counted. For males, the mean number of ommatidia was statistically different among all flies examined: L. dux (6,032) > C. rufifacies (5,356) > C. nigripes (4,798) > C. megacephala (4,376) > L. cuprina (3,665) > M. domestica (3,484). Likewise, the mean number of facets in females was statistically different: L. dux (6,086) > C. megacephala (5,641) > C. rufifacies (5,208) > C. nigripes (4,774) > L. cuprina (3,608) > M. domestica (3433). Scanning electron microscopy analysis of adult flies revealed the sexual dimorphism in the compound eye. Male C. megacephala had large ommatidia in the upper two thirds part and small ommatidia in the lower one third part, whereas only small ommatidia were detected in females. Dense postulate appearance was detected in the external surface of the corneal lens of the ommatidia of C. megacephala, C. rufifacies, and C. nigripes, while a mix of dense postulate appearance and variable groove array length was detected in L. cuprina and M. domestica. The probable functions of ommatidia are discussed with reference to other literature.

Morphology and developmental rate of blowflies Chrysomya megacephala and Chrysomya rufifacies in Thailand: application in forensic entomology.

The larval morphology and developmental rate of Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), the two most forensically important blowfly species in Thailand, are presented. Morphological comparison of the third instar of both species revealed different characteristics (e.g., body appearance, cephalopharyngeal skeleton, dorsal cuticular spines between the prothorax and mesothorax, and feature of the posterior spiracle), thereby, allowing correct identification. A data analysis was conducted in Chiang Mai province, Northern Thailand during 2000-2001 on the developmental rate of both flies under natural ambient temperature and a natural light-dark photoperiod. The results indicated that larvae of C. megacephala developed more rapidly in April, pupariation initiated at 84 h at temperatures averaging 31.4 degrees C, and the larvae grew slower in the rainy season and winter. Similarly, rapid development of C. rufifacies larvae appeared in the summer, with a pupariation period as short as 96 h in June (average temperature 27.4 degrees C). Analysis of the median body length of C. megacephala and C. rufifacies larvae in different seasons of the years 2000-2001 in Thailand revealed that both species developed rapidly in the summer; pupariation of C. rufifacies initiated at 144 h, while C. megacephala initiated pupariation at 156 h. This information is potentially useful for estimating the postmortem interval of a corpse in forensic investigations, where the corpse becomes infesting with these fly species.

Forensic entomology cases in Thailand: a review of cases from 2000 to 2006.

This paper presents and discusses 30 cases of cadavers that had been transferred for forensic entomology investigations to the Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, northern Thailand, from 2000 to 2006. Variable death scenes were determined, including forested area and suburban and urban outdoor and indoor environments. The fly specimens found in the corpses obtained were the most commonly of the blow fly of family Calliphoridae, and consisted of Chrysomya megacephala (F.), Chrysomya rufifacies (Macquart) Chrysomya villeneuvi Patton, Chrysomya nigripes Aubertin, Chrysomya bezziana Villeneuve, Chrysomya chani Kurahashi, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), and two unknown species. Flies of the family Muscidae [Hydrotaea spinigera Stein, Synthesiomyia nudiseta (Wulp)], Piophilidae [Piophila casei (L.)], Phoridae [Megaselia scalaris (Loew)], Sarcophagidae [Parasarcophaga ruficornis (F.) and three unknown species], and Stratiomyiidae (Sargus sp.) were also collected from these human remains. Larvae and adults of the beetle, Dermestes maculatus DeGeer (Coleoptera: Dermestidae), were also found in some cases. Chrysomya megacephala and C. rufifacies were the most common species found in the ecologically varied death scene habitats associated with both urban and forested areas, while C. nigripes was commonly discovered in forested places. S. nudiseta was collected only from corpses found in an indoor death scene.

Comparison between Musca domestica and Chrysomya megacephala as carriers of bacteria in northern Thailand.

A comparison between the common house fly, Musca domestica, and the Oriental latrine fly, Chrysomya megacephala, was assessed for their potential as carriers of bacteria in urban areas of Chiang Mai Province, northern Thailand. C. megacephala was significantly more likely to carry bacterial species than M. domestica; however, no significant difference was found between the number of positive male and female flies within the same species. A total of 42 bacterial species were isolated. The most common bacterium isolated from M. domestica was coagulase-negative staphylococci (n=57) followed by Escherichia coli (n=10) and Viridans streptococci (n=10), while that of C. megacephala was non-fermentative gram-negative bacilli (n=59) followed by coagulase-negative staphylococci (n=54).

Ultrastructure of spiracles of Musca domestica and Hydrotaea chalcogaster (Diptera: Muscidae).

Spiracles are major respiratory openings in the exoskeleton of insects. Oxygen, a necessary gas for cell activity, must pass through the spiracle to enter the respiratory system. In this study, we investigated the fine structure of spiracles of adult females of Musca domestica L. and Hydrotaea chalcogaster (Wiedemann), both medically important fly species in many parts of the world, by utilizing scanning electron microscopy. The mesothoracic spiracle of M. domestica is large and elongate-oval in shape, with its anterior end being gradually tapered. The outer surface is densely covered with slender setae of variable distribution and orientation. The metathoracic spiracle is semicircular or D-shaped, with its rim possessing long, fine, inwardly curved setae. A net-like valve or sieve plate, which has a smooth rim with swollen surface, is located within the atrium of this species. The abdominal spiracles are circular with a symmetrically swollen peritreme surrounding the opening. The inner filtering apparatus is composed of many spiral tubes, each possessing many small spines. As for H. chalcogaster, the tapering mesothoracic spiracle is covered with long setae arranged consistently inward from the peritreme, giving it a "combed" appearance. The metathoracic spiracle is similarly arranged but triangularly rounded in shape, with the anterior and posterior rims possessing long fine setae. The net-like valve within the atrium has a smooth, swollen rim, whereas the inner edge of the atrium bears short, slender setae where it meets with the peritreme of the spiracle. The abdominal spiracles of this species look similar to that of M. domestica, with the exception of the filtering apparatus that bears only a few small spines. The function of these spiracles is discussed.

Morphological comparison between Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton (Diptera: Calliphoridae) puparia, forensically important blow flies.

In Thailand, the hairy maggots of the blow flies, Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton, are of forensic importance. Both flies are closely related species, not only in the morphological appearance of their larvae and puparia, but also on the aggressive feeding habit of the former. In our continuing studies of forensically important flies, identification of immature ones needs particular attention. In this study, we reported the morphological comparison between the puparia of these two blow fly species using scanning electron microscopy (SEM). Observation revealed that the cuticular sculpture of tubercles along the dorsal and lateral segments had markedly different features: with C. rufifacies having many sharp spines assembling only at the tip, while of C. villeneuvi bore stout spines throughout the tubercle. A larger number of globules at the bubble membrane on the dorsolateral border of the fifth segment was found in C. villeneuvi (average 225) than in C. rufifacies (average 35), and more papillae was observed on the anterior spiracle in C. villeneuvi (13-15) than in C. rufifacies (9-12). However, the morphology of distinct net-like patches of the integument and structure of the posterior spiracle of both species were almost identical. Morphological comparison in this study permitted identification of the puparia of both fly species, particularly in areas where they co-exist.

Morphology of puparia of Megaselia scalaris (Diptera: Phoridae), a fly species of medical and forensic importance.

Megaselia scalaris (Loew), a scuttle fly, is a fly species of medical and forensic importance. For use in forensic investigation, fly specimens found to associate with human corpses have to be identified at species level. Herein, we present the morphology of the puparia of the above fly species using scanning electron microscopy. The characteristic of the intersegmental spines along the dorsal and lateral segments and sculpture of the pupal respiratory horn of this puparia may be useful in future studies, to distinguish it from other closely related species.

Morphological observation of puparia of Chrysomya nigripes (Diptera: Calliphoridae) from human corpse.

This article presents Chrysomya nigripes Aubertin as a blow fly species of forensic importance in Thailand, and morphological observation of fly puparia using scanning electron microscopy (SEM). Morphologically, we focused on the characteristics of puparia used to accurately identify fly species. Numerous puparia of C. nigripes were found aggregated, adhering side by side, on the tibia of a skeletonized corpse, which was recovered from a forested area of Chiang Mai, northern Thailand. In the triangular shape of the anterior end of the puparia, three thoracic segments and broad hairy patches beginning dorsolaterally at the sixth segments were distinguishing characteristics. This study, showing pupariation of the flies along the bone of a corpse as well as morphological features, provides important guidance in identifying C. nigripes puparia. A key to differentiate puparia of C. nigripes from the other flies of forensic importance in Thailand is given.

Ultrastructure of eggs of Paracapillaria (Crossicapillaria) philippinensis and evidence related to its life cycle.

The varied ultrastructure of the eggshell of Paracapillaria (Crossicapillaria) philippinensis, collected from a human sample, is reported from a scanning electron microscopy study. Two distinct egg shapes were identified: typical peanut-shaped and swollen peanut-shaped. Both thick and thin eggshells were detected. Thick eggshells are either fairly smooth or bear a beam-like network in relation to the pillars in their surface ultrastructure. Thin eggshells are transparent allowing visibility of the coiled larva within. Presence of the thin shell provides supportive evidence of autoinfection involved in the life cycle of this medically important parasite.

Susceptibility of Musca domestica and Chrysomya megacephala to Permethrin and deltamethrin in Thailand.

Susceptibility to permethrin and deltamethrin was assessed in the house fly, Musca domestica L., and blow fly Chrysomya megacephala (F.). The laboratory strain of M. domestica was captured in Muang District (downtown), Chiang Mai, northern Thailand, and the field strains were collected from Muang District, two agricultural areas of Hang Dong District and San Khampaeng District. These strains displayed susceptibility to both permethrin and deltamethrin insecticides, with LD50 values of 0.0049, 0.0019, 0.0003, and 0.0005 microg/fly and 0.1058, 0.0185, 0.1251, and 0.0981 microg/fly, respectively. Tests on the laboratory strain of C. megacephala and the field strains collected from four areas of Chiang Mai (Muang District [downtown], two agricultural areas of Hang Dong District, and San Khampaeng District) and the wooded area near the National Forest revealed slightly lower susceptibility compared with house fly, with LD50 values of permethrin equal to 0.0028,0.0027,0.0024, 0.0007, and 0.0034 microg/fly and of deltamethrin equal to 0.0461, 0.0267,0.1327,0.1151, and 0.0244 microg/fly. These results indicated that the adult populations of both fly species were susceptible to both insecticides.

Effect of microwave irradiation on the blow fly Chrysomya megacephala (F.) (Diptera: Calliphoridae).

Chrysomya megacephala (F) is a blow fly species of medical importance, being pestiferous and a mechanical carrier of various microorganisms to human food. Various strategies to reduce the fly population in endemic areas are needed. We investigated the effects of microwaves at 2,450 MHz, with third-instar exposure times of 7, 10, 15, 30 and 60 seconds on the larvae. Survival of the larvae was monitored by their emergence. About 60% of the larvae lived after 7 seconds of exposure, most died by 15 seconds, and all were dead at 30 and 60 seconds. The number of survivals decreased with increasing exposure times. This indicates the effect of microwave irradiation on fly larval survival.

Morphology of second and third instars of Chrysomya villeneuvi Patton (Diptera: Calliphoridae), a fly species of forensic importance.

The morphology of the second and third instars of Chrysomya villeneuvi Patton, a fly species of forensic importance, was presented by use of light microscopy. Both instars were of hairy appearance, bearing elongated tubercles along the abdominal and caudal segments. The anterior spiracle had 13-15 papillae. Minute dark spots were observed to thoroughly cover the tubercle's surface, with 4-6 strong dark tips. Regarding the third instar, the intersegmental spines between the prothorax and mesothorax were heavily pigmented. The posterior spiracle had a thick and heavily pigmented incomplete peritreme. The surface and tip of the tubercles was covered with heavily pigmented sharp spines. The integument of the body was covered with numerous distinct net-like patches. A comparison with another well-known hairy maggot, Chrysomya rufifacies (Macquart), was discussed.

First report of human myiasis caused by Chrysomya megacephala and Chrysomya rufifacies (Diptera: Calliphoridae) in Thailand, and its implication in forensic entomology.

We report a forensic entomology case associated with human myiasis in Chiang Mai Province, northern Thailand. The remains of a 53-yr-old-male were concurrently infested with third instars of the two blow fly species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), near a severe tumor lesion presented on the lower right leg. The presence of third instars, approximately 5 d old, on the day following postmortem indicated that myiasis occurred before death. This is the first report of both fly species acting as a myiasis-producing agent in Thailand. Unsynchronized data between the age of fly larvae due to myiasis premortem and verified age/ condition of the corpse suggest a potential complication and error in the estimation of postmortem interval if other predisposition fly infestations are not considered.

Evidence of Haplorchis taichui infection as pathogenic parasite: three case reports.

Haplorchis taichui is the minute intestinal fluke which parasitizes the small intestine of humans and other mammals. We present the pathology in the small intestine of 3 human cases caused by H. taichui. Microscopic examination revealed mucosal ulceration, mucosal and submucosal haemorrhages, fusion and shortening villi, chronic inflammation, and fibrosis of the submucosa. This finding clearly indicated H. taichui as pathogenic; thus prevention of infection and treatment should be paramount.

Surface ultrastructure of prestomal teeth of some flies in the families Calliphoridae, Muscidae and Sarcophagidae.

Prestomal teeth of flies in the families Calliphoridae (Chrysomya pinguis, Chrysomya villeneuvi, and Chrysomya chani), Muscidae (Hydrotaea (= Ophyra) chalcogaster) and Sarcophagidae (Boettcherisca peregrina) were presented using scanning electron microscopy. The slight to deep bifurcation of the tips of their teeth were similarly observed in all species examined. The anatomical feature of prestomal teeth of the flies examined may contribute to the understanding of their feeding behavior and the medical importance of their role in the future.

Larval morphology of Chrysomya nigripes (Diptera: Calliphoridae), a fly species of forensic importance.

The morphology of all instars of Chrysomya nigripes Aubertin, a blow fly species of forensic importance, is presented with the aid of both light microscopy and scanning electron microscopy (SEM). Morphological features of the cephalopharyngeal skeleton, anterior spiracle, posterior spiracle, and dorsal spines between the prothorax and mesothorax are highlighted. No consistent features were found, even using SEM, for distinguishing the first instar of C. nigripes from that of Chrysomya megacephala (F.) or Chrysomya rufifacies (Macquart), two other commonly associated blow fly species in corpses in Thailand. Several features observed in second and third instars proved to be valuable characteristics for separating these species.

Morphology of the puparia of the housefly, Musca domestica (Diptera: Muscidae) and blowfly, Chrysomya megacephala (Diptera: Calliphoridae).

Examination of the puparia of the housefly, Musca domestica L. and blowfly Chrysomya megacephala (F.), through scanning electron microscopy (SEM), revealed many differences in the profile of their morphology. Special attention was focused on puparial characteristics used to differentiate between the two fly species studied, and between other forensically important flies. Results of this study indicate that the housefly puparia are almost evenly rounded at both ends and the anterior spiracle bears six papillae. A pair of pupal respiratory horns is found laterally before the posterior boundary of the first abdominal segment, bearing numerous papillae that have a longitudinal opening along the oval convex base. The peritreme of each posterior spiracle forms a crude forward or reverse D-shape, encircling three sinuous slits. The blowfly pupariums anterior spiracle contains 8-12 papillae. The pupal respiratory horns protrude slightly and in some specimens a group of approximately 38 globules on the bubble-like membrane may be observed. Each of the posterior spiracles is more or less an oval- shaped peritreme, encircling three straight spiracular slits. The anatomical features presented herein allow for the differentiation of puparia of the two fly species studied and could prove useful in future forensic entomological assessments.

Ultramorphology of eggshell of flesh fly Liosarcophaga dux (Diptera: Sarcophagidae).

Eggs of Liosarcophaga dux (Thomson) (Diptera: Sarcophagidae) are elongated and slightly bean-shaped, measuring approximately 1.5 mm in length. Each is covered externally by an eggshell comprised of polygonal patterns. In this study, scanning and transmission electron microscopy were used to examine the eggshell of this species of flesh fly for the first time. No plastron region or median area was detected. Ultrathin sectioning of the eggshell revealed multiple layers in the shell that could be observed using transmission electron microscopy.