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.     

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.     

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.     

Larval competition of Chrysomya megacephala and Chrysomya rufifacies (Diptera: Calliphoridae): behavior and ecological studies of two blow fly species of forensic significance

Chrysomya megacephala and Chrysomya rufifacies are two predominant necrophagous species in Taiwan. Larvae of the latter can prey on other maggots, including that of their own species as facultative food. This facultative characteristic of C. rufifacies may enhance its competitive advantage over other maggots and could also change the situation of other coexisting colonies. In this study, these two species were colonized in the laboratory, and the main objective was to try to understand the effect of competition on larval development. According to our results, intraspecific competition mostly occurred as competition for food; when the rearing density was increased, larvae pupated earlier, resulting in a lighter adult dry weight. The tendencies were similar in both species, but C. megacephala developed smaller viable adults and had higher survivorship at high densities. Although C. rufifacies could use the food resource of cannibalism, its survivorship was still low. Our results also showed there were significant interactions between intraspecific competition and the density factor. However, with interspecific competition, the first-instar larvae of C. rufifacies invaded maggot masses of C. megacephala to feed together. The third instars of C. rufifacies were able to expel C. megacephala larvae from food by using a fleshy protrusion on their body surface; C. megacephala was usually forced to pupate earlier by shortening its larval stages. The results indicated that a temporary competitive advantage could only be obtained by C. rufifacies under a proper larval density. In addition, the effects on different larval stages, the responses to different competition intensities, and the temperature-dependent effects on interspecific competition are also discussed. In general, under mixed-species rearing at different temperatures and densities, larval duration, adult dry weight, and survivorship of both species decreased. However, our results did not completely agree with previous studies, and we suspect that the difference was partially caused by different experimental designs and different biological characters of different blow fly colonies. Our results also suggest that both the predation ability and defense or escape activity should be taken into account when evaluating larval competitive advantages. The durations of larval stages of these two species could be decreased by approximately 54 h when a single species was reared alone and food was limited; the largest reduction in larval duration, approximately 25 h in C. megacephala and 34 h in C. rufifacies, caused by interspecific competition was under a high larval density. In conclusion, competition decreased the larval duration of these two species by up to 2 d; this also draws attention to justifying the postmortem interval estimation of using larval developmental data when larval competition exists.     

Effect of nandrolone decanoate on the development of three species of Chrysomya (Diptera:Calliphoridae), flies of forensic importance in Brazil

Necrophagous insects are valuable tools for postmortem interval (PMI) estimation or for determining the cause of death. Due to the increase in deaths related to drug abuse, it is crucial to know how these substances affect the development of flies that feed on corpses, to avoid errors in the PMI estimates. This study evaluated the effect of nandrolone decanoate, an anabolic androgenic steroid, on the development ofimmatures of Chrysomya megacephala (F.), Chrysomya putoria (Wiedemann), and Chrysomya albiceps (Wiedemann) (Diptera:Calliphoridae) when added to an artificial rearing diet. Four experimental groups were delineated: three of them were given diets containing 4.5, 22.5, or 45 mg/kg nandrolone decanoate; and a drug-free control group. Weights were recorded at 12-h intervals from larval eclosion to pupation. No statistically significant differences were observed in mean larval weights, emergence interval, or emergence rates for all groups. However, differences in the three species were observed during the larval development. Initially, C. putoria reared in the highest concentration of decanoate showed greater weight gain. However, at older ages, immatures reached lower mean weights than the control group. For C. albiceps, the highest concentration of decanoate contributed to an effective lack of weight gain during almost the entire course of larval development. Therefore, the influence of any drug on development should always be considered.     

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.     

Scanning electron microscopy of the third-instar Piophila casei (Diptera: Piophilidae), a fly species of forensic importance

The morphology of third-instar Piophila casei (L.) is described by means of scanning electron microscopy. Features of the anterior cephalic region and creeping welts that are used in larval skipping and creeping, respectively, are highlighted. Morphological features classically used for larval identification are also illustrated.     

Ultrastructure of immature stages of Cochliomyia macellaria (Diptera: Calliphoridae), a fly of medical and veterinary importance

Cochliomyia macellaria (Diptera: Calliphoridae) is known as the secondary screwworm because it causes secondary or facultative myiasis when the larvae feed on necrotic tissues. This fly has a significant medical and veterinary importance since it has been reported to transport eggs of Dermatobia hominis (human botfly), which can cause significant economic losses to livestock. Since this screwworm has been collected colonizing both pig carcasses and human cadavers, it is considered one of the most important species for forensic entomology studies. Scanning electron microscopy (SEM) gives detailed information on the morphological characteristics which can help identify the immature forms of the flies. The aim of this study was to describe and analyze the morphological characteristics of the eggs, all the larval instars, and the puparia of Cochliomyia macellaria using SEM. The egg is ellipsoid and the dorsal surface is concave. The islands inside the median area had no anastomosis, but some perforations could be observed. From the second larval instar onwards, besides the intersegmental spines, other bands of spines were observed at the abdominal segments. Two spiracular openings were visible on the first and second larval instars, which were not expected. These characteristics are specific to Cochliomyia genus. The number and the general aspect of the spine tips in the cephalic region, the intersegmental bands on the abdomen, and the number of the spiracular openings could together help identify C. macellaria.     

Oviposition preferences of two forensically important blow fly species, Chrysomya megacephala and C. rufifacies (Diptera: Calliphoridae), and implications for postmortem interval estimation

Necrophagous blow fly species (Diptera: Calliphoridae) are the most important agents for estimating the postmortem interval (PMI) in forensic entomology. Nevertheless, the oviposition preferences of blow flies may cause a bias of PMI estimations because of a delay or acceleration of egg laying. Chrysomya megacephala (F.) and C. rufifacies (Macquart) are two predominant necrophagous blow fly species in Taiwan. Their larvae undergo rather intense competition, and the latter one can prey on the former under natural conditions. To understand the oviposition preferences of these two species, a dual-choice device was used to test the choice of oviposition sites by females. Results showed when pork liver with and without larvae of C. rufifacies was provided, C. megacephala preferred to lay eggs on the liver without larvae. However, C. megacephala showed no preference when pork liver with and without conspecific larvae or larvae of Hemipyrellia ligurriens (Wiedemann) was provided. These results indicate that females of C. megacephala try to avoid laying eggs around larvae of facultatively predaceous species of C. rufifacies. However, C. rufifacies showed significant oviposition preference for pork liver with larvae of C. megacephala or conspecific ones when compared with pork liver with no larvae. These results probably imply that conspecific larvae or larvae of C. megacephala may potentially be alternative food resources for C. rufifacies, so that its females prefer to lay eggs in their presence. When considering the size of the oviposition media, pork livers of a relatively small size were obviously unfavorable to both species. This may be because females need to find sufficient resources to meet the food demands of their larvae. In another experiment, neither blow fly species showed an oviposition preference for pork livers of different stages of decay. In addition, the oviposition preferences of both species to those media with larvae were greatly disturbed in a dark environment. If we removed the larvae that had previously fed on the pork liver and let the females choose, no oviposition preference was observed; but both species still showed a preference for the larger media in the dark. This suggests that female blow flies can use visual cues to recognize larvae on the media and decide on their oviposition site. Our studies point out the effects of some biotic and abiotic factors which were previously overlooked, and remind us to reevaluate these effects on oviposition, especially when using insect developmental data to estimate PMIs.     

Differential replication of heterochromatin in polytene chromosomes of the Old World screw-worm fly,Chrysomya bezziana (Diptera: Calliphoridae), analysed by fluorescence staining

The distribution and replication of heterochromatin in polytene trichogen chromosomes of the Old World screw-worm fly,Chrysomya bezziana, were studied using fluorescent staining techniques. Quinacrine and distamycin-DAPI, which selectively stain AT-rich DNA, and chromomycin, specific for GC-rich sequences, were used. Bright quinacrine and DA-DAPI fluorescence was found in the sex chromosome body and in all autosomal centromere regions. Chromomycin (CMA) staining results in very little bright fluorescence of the sex chromosome body and autosomal centromeric regions, but many bright bands of varying morphology are distributed in autosomal arms. The expected negative CMA staining of quinacrine and DA-DAPI bright regions was not found. The lack of reciprocal staining patterns may result from changes in the higher order chromatin structure of polytene chromosomes, or intercalation of divergent heterochromatic sequences. Comparison of the different staining techniques in mitotic and polytene cells shows that heterochromatin is differentially under-replicated, so that the proportions of the distinct fluorescent-specific chromatin changes during polytenization. CMA staining within autosomal arms suggests that repeated sequences intercalated in euchromatin are co-replicated during polytenization. The numerous fluorescent markers described also provide further morphological features for use in comparative cytological analysis ofC. bezziana.     

Interdisciplinary workshop yields new entomological data for forensic sciences: Chrysomya rufifacies (Diptera: Calliphoridae) established in North Carolina

ABSTRACT Workshops are used for educating law enforcement personnel on the application of entomological, anthropological, and botanical techniques to gather forensically important information from a body recovery site. From 8 to 11 June 2004, such a workshop was conducted in the metropolitan area of Charlotte, NC, by the American Academy of Applied Forensics at Central Piedmont Community College. For this workshop, three pig carcasses weighing individually between 40 and 60 kg were placed in the field 4 June, whereas three pigs similar in size were placed in the field 7 June. During the afternoon session on 11 June, workshop participants collected three Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) third instar larvae from a pig carcass placed in the field on 7 June. These data represent the first records of this forensically important insect in North Carolina; furthermore, these data provide evidence of this species expansion into new geographic regions of the United States. This finding is an example of the benefits derived from an experiential and interdisciplinary approach to educating death scene investigators. Learners acquired new knowledge, put it into practice through the "body" recovery exercise, and ultimately contributed to science by way of the discovery and first documentation of a forensically important insect previously not known to inhabit North Carolina.     

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.     

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.     

Molecular phylogeny of Chrysomya albiceps and C. rufifacies (Diptera: Calliphoridae).

Mitochondrial DNA was used to infer the phylogeny and genetic divergences of Chrysomya albiceps (Wiedemann) and C. rufifacies (Maquart) specimens from widely separated localities in the Old and New World. Analyses based on a 2.3-kb region including the genes for cytochrome oxidase subunits I and II indicated that the 2 species were separate monophyletic lineages that have been separated for > 1 million years. Analysis of DNA, in the form of either sequence or restriction fragment-length polymorphism (RFLP) data, will permit the identification of problematic specimens.     

Sterilization of blow fly eggs,Chrysomya megacephala and Lucilia cuprina, (Diptera: Calliphoridae) for maggot debridement therapy application

Parasitology Research - Maggot debridement therapy (MDT) is an application of sterile laboratory-reared blow fly larvae to remove necrotic tissue and disinfect wounds for medical conditions. For...