Fine structure of Chrysomya nigripes (Diptera: Calliphoridae), a fly species of medical importance

The fine structure of Chrysomya nigripes Aubertin, a blow fly species of medical importance, is presented using scanning electron microscopy (SEM) to contribute information on the morphology of the adult of this fly species. The surface of the dome-shaped ommatidia exhibits a microscopic granulose appearance. The palpus is equipped with small sensilla basiconica and sensilla chaetica, which provide sensory reception for detecting environmental information. At the apex of the mouthparts, the labellum is endowed with large numbers of sensilla trichodea and basiconic-like sensilla of variable length. The anterior (mesothoracic) spiracle is elliptical in shape and covered with extensively ramified setae except for a small dorsal aperture. The posterior (metathoracic) spiracle is shaped like a rounded isosceles triangle and covered by two valves of unequal size. The larger valve covers the upper ≈2/3 of the spiracular opening, whereas the smaller valve covers the lower ≈1/3 of the opening. Extensively ramified setae line and cover the valves over the entire spiracle. SEM analyses of the haltere knob and the prosternal organs, located adjacent to the cervical sclerites, revealed a striking resemblance of the morphological features of their sensilla. Each sensillum emanates from a cuticular ring, is ≈12–15 μm in length, has a smooth surface, and terminates in a sharp tip. Various types of sensilla were associated with the ovipositor including sensilla trichodea, sensilla basiconica, sensilla placodea and probably sensilla styloconica. The possible function of sensilla distributed in particular regions of the fly integument is discussed.     

Identifying fly puparia by clearing technique: application to forensic entomology

In forensic investigations, immature stages of the fly (egg, larva, or puparia) can be used as entomological evidence at death scenes, not only to estimate the postmortem interval (PMI), analyze toxic substances, and to determine the manner of death but also to indicate the movement of a corpse in homicide cases. Of these immature stages, puparia represent the longest developmental time, which makes them of useful. However, in order for forensic entomologists to use puparia effectively, it is crucial that they are able to accurately identify the species of fly found in a corpse. Typically, these puparia are similar in general appearance, being coarctate and light brown to dark brown in color, which makes identification difficult. In this study, we report on the clearing technique used to pale the integument of fly puparia, thereby allowing observation of the anterior end (second to fourth segments) and the profile of the posterior spiracle, which are important clues for identification. We used puparia of the blowfly, Chrysomya megacephala (F.), as the model species in this experiment. With placement in a 20% potassium hydroxide solution daily and mounting on a clearing medium (Permount(R), New Jersey), the profile of the posterior spiracle could be clearly examined under a light microscope beginning on the fifth day after pupation, and the number of papillae in the anterior spiracle could be counted easily starting from the ninth day. Comparison of morphological features of C. megacephala puparia with those of other blowflies (Chrysomya nigripes [Aubertin], Chrysomya rufifacies [Macquart], Chrysomya villeneuvi [Patton], Lucilia cuprina [Wiedemann], and Hemipyrellia ligurriens [Wiedemann]) and a housefly (Musca domestica L.) revealed that the anterior ends and the profiles of the posterior spiracles had markedly distinguishing characteristics. Morphometric analysis of the length and width of puparia, along with the length of the gaps between the posterior spiracles of seven fly species, displayed differences among them. This provides a key to identifying the puparia of these seven fly species.     

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.     

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 ~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.     

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 ≈2-μm 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.     

Observations on morphology of immature Lucilia porphyrina (Diptera: Calliphoridae), a fly species of forensic importance

Lucilia porphyrina (Walker) is a blow fly of forensic importance, and shares its geographical distribution with a related forensically important species, Lucilia cuprina (Wiedemann). The immature stages of both species are similar in general appearance; therefore, correct identification should be given special consideration. This study highlighted the main features of L. porphyrina larvae, as observed under light microscopy and scanning electron microscopy. Particular attention is given to the anterior and posterior spiracles, cephalopharyngeal skeleton, and characteristics of the dorsal spines between the prothorax and mesothorax. In the third instar specifically, morphological information on L. porphyrina showed several features that are shared by L. cuprina, and therefore need certain identification to distinguish between them. Such key features are (1) greater posterior spiracle, (2) apparent inner projection between the middle and lower slits of the posterior spiracle, and (3) strongly sclerotized peritreme. The number of papillae on the anterior spiracle may be a supplement, five to nine and three to six in L. porphyrina and L. cuprina, respectively. The key for identifying third instar of forensically important flies in Thailand has been updated to include L. porphyrina.     

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.     

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.     

Molecular identification of forensically important blow fly species (Diptera: Calliphoridae) in Taiwan

Forensic entomology is a discipline that mainly uses insects collected in and around corpses to estimate the post-mortem interval in medicocriminal investigations. Among all scavenger and necrophagous insect groups that are related to corpses, blow flies (Diptera: Calliphoridae) are probably most important, not only because they occur in abundant numbers but also because they are one of the earliest groups to find corpses. However, most entomological evidence is strongly dependent on accurate species identification. Because identification allows the proper developmental data and distribution ranges to be applied in criminal investigations, species in Taiwan were surveyed from early 2000 and were identified using molecular data. Currently, eight species have been identified: Chrysomya megacephala (Fabricius), Chrysomya pinguis (Walker), Chrysomya rufifacies (Macquart), Hemipyrellia ligurriens (Wiedemann), Lucilia bazini Séguy, Lucilia cuprina (Wiedemann), Lucilia hainanensis Fan, and Lucilia prophyrina (Walker). We focused on classifying these blow fly species to establish a knowledge basis for further forensic entomological research in Taiwan. Because molecular data are helpful in identifying insect specimens, especially when no specimen of suitable condition for morphological identification is obtained, we extracted mitochondrial cytochrome oxidase subunit I (COI) DNA of the preceding blow fly species to study its application value for their differentiation. The cloning and sequencing of the COI gene (approximately 1,588 base pairs) of these eight species were completed, and the data were analyzed. Preliminary results revealed the high support of congeneric groupings of species by using COI data; these sequences were also shown to be highly conserved within the same species. To actually use the database of COI sequences under various specimen conditions, specific primers were also applied for different insect stages, different segments of adults, and specimens preserved for various times. A molecular primer key was ultimately constructed for the purpose of rapid and accurate species identification at the molecular level regardless of which stage or which part of a blow fly specimen is collected.     

Differentiation between Lucilia cuprina and Hemipyrellia ligurriens (Diptera: Calliphoridae) larvae for use in forensic entomology applications

Lucilia cuprina and Hemipyrellia ligurriens are forensically important blow flies in many countries. The immature stage, in particular the third instar, of both species are generally similar in morphological appearance. In this study, the use of three main characteristics, the posterior spiracle, the dorsal spines between the first and second thoracic segments, and the cephalopharyngeal skeleton, allows for identification of both fly species, with the number of papillae on anterior spiracle being a supportive characteristic in some cases. Comparison of these characteristics is illustrated in detail.     

Hairy maggot of Chrysomya villeneuvi (Diptera: Calliphoridae), a fly species of forensic importance

We reported on the hairy maggot of Chrysomya villeneuvi Patton, collected from a human corpse in Thailand. Although the general morphology of the third instar closely resembled the more common hairy maggot blow fly, Chrysomya rufifacies (Macquart), the spines along the tubercles of each body segment could be used as a feature to distinguish between these species, with those of C. villeneuri bearing sharp-ended spines that encircle the entire tubercle. Not only has the resemblance of a hairy appearance between C. villeneuvi and C. rufifacies larvae been documented but also their similarity in predacious feeding behavior. The differentiation between these two species is necessary for post-mortem interval estimation in the corpse.     

Fine structure of the reproductive system of <Emphasis Type="Italic">Chrysomya megacephala</Emphasis> (Diptera: Calliphoridae): the external sexual organ

The fine structure of the reproductive system of Chrysomya megacephala (Fabricius), a blow fly species of medical importance, was studied using scanning electron microscopy (SEM) to contribute detailed morphological information on the external sexual organs or genitalia of this species. Analysis of the male genitalia or hypopygium revealed that the epandrium is a broad organ resembling a crescent shape, while the ejaculatory apodeme and aedeagal apodeme show similarity in their lengths. The cercus is significantly longer than the surstylus with its apical end being more or less rounded. Dense patches of long bristles, morphologically similar to the sensilla chaetica and sensilla trichodea, were found along the lower half of the cercus. The surstylus has a stout triangular shape, and the proximal half is greatly endowed with sensilla thought to be sensilla chaetica and sensilla trichodea. The aedeagus per se is prominent and has a clavate shape formed by the base theca and elongated phallus. The vesica of the phallus is a smooth bilobed structure, which curves inward. The juxta and juxta process are armed with many rows of strong spines resembling thorns in their appearance. The harpe is slender, recurved anteriorly, and distally pointed like a sickle. Regarding the female genitalia or ovipositor, SEM observation of the supraanal plate revealed it to be sclerotized and more or less triangular-shaped, lying between a pair of short cerci. Several types of sensilla were found on the supraanal plate, subanal plate and cercus, including the sensilla trichodea, sensilla basiconica, sensilla placodea, and probably, sensilla styloconica. The possible functions of sensilla distributed in particular regions of the fly genitalia are discussed.     

Surface ultrastrucure of larva and puparia of blow fly Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae)

Flies of the genus Hypopygiopsis are forensically important, as their larvae are found to associate with human corpses. In this study, the ultrastructure of larvae and puparia of Hypopygiopsis tumrasvini Kurahashi is presented using scanning electron microscopy (SEM). The larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures, i.e., antenna, maxillary palpus, and ventral organ. Two tufts of fresh outgrowths (or cerri) were observed along the dorsal margin of the mouth opening of the first instar; whereas a strong mouth hook was apparent in the second and third instars. Keilin??s organ, the sensory structure, was noticeable on the ventral surface of the thoracic segments in all instars. In the second and third instars, six conspicuous tubercles were present along the peripheral rims of the last abdominal segment. The puparia were relatively large, measuring 7.77?C9.51?mm in length and 3.10?C3.97?mm in width. At the latero-dorsal edge of the first abdominal segment, a cluster of ??125 bubble membranes was seen in young puparia; whereas a minute pupal respiratory horn was observed in old one. An SEM image revealed antler-like projections lined within a chamber of a broken pupal respiratory horn. Comparison on number of the bubble membranes of the other blow fly species was shown and the role of pupal respiratory horn compared with other dipterans was discussed.     

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.     

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.     

Initial study of arthropod succession on pig carrion in a central European urban habitat

We conducted a carrion succession study within a restricted urban backyard in the city of Vienna, Austria (16 degrees 22'E, 48 degrees 12'N) from May to November 2001 to analyze sequence and composition of the local carrion visiting fauna. Two medium sized clothed domestic pig carcasses (Sus scrofa Linnaeus), were used as surrogate human models. In total, 42 arthropod species from the families Calliphoridae, Sarcophagidae, Sepsidae, Piophilidae, Muscidae, Fanniidae, Sphaeroceridae, Phoridae, Drosophilidae, Anthomyiidae, and Lauxaniidae (Diptera), Formicidae, Braconidae, Pteromalidae, and Vespidae (Hymenoptera), Silphidae, Staphylinidae, Histeridae, Cleridae, and Dermestidae (Coleoptera), as well as species from the orders Isopoda and Acari were collected during the decomposition of these carcasses. A significant feature in this study was the high abundance of Calliphora vomitoria (L.) and Chrysomya albiceps (Wiedemann). In the experiment conducted May to June, larvae and adults of C. vomitoria outnumbered all other blow fly species, followed by Protophormia terraenovae (Robineau-Desvoidy), C. vicina Robineau-Desvoidy, and Lucilia sericata (Meigen). C. vomitoria is generally considered to be rural in distribution, where it prefers shaded locations. The presence of this species in rural as well as in urban habitats in Austria precludes this species as biogeographic indicator. In the study beginning in August large numbers of female adults of the nonindigeous blow fly C. albiceps began oviposition at day 3 after placement of the cadaver. The predatory second and third instars of C. albiceps larvae subsequently almost monopolized the cadaver. C. albiceps is generally described as tropical and subtropical species. The observed northward expansion of its range beyond southern Europe obviously decreases the value of C. albiceps in estimating place of death, in that it is no longer exclusive to southern regions. Our results clearly show, that caution must be used when drawing conclusions from succession data generated in different geographic areas. Moreover, this study demonstrates, that arthropod mediated decomposition of a 44 kg exposed pig carcass in a central European urban habitat can be completed within 3 wk.