Molecular identification of forensically important blowfly species (Diptera: Calliphoridae) from Germany

Forensic entomology applies knowledge about the behaviour and ecology of insects associated to corpses to homicide investigations. It is possible to calculate a minimum post-mortem interval by determining the age of the oldest blowfly larvae feeding on a corpse. The growth rate of the larvae is highly dependent on temperature and also varies between the different blowfly species infesting a corpse. It is, thus, crucial to correctly identify the species collected from a crime scene. To increase the quality of species identification, molecular methods were applied to 53 individuals of six different species sampled in Bonn, Germany: Calliphora vicina, Calliphora vomitoria, Lucilia caesar, Lucilia sericata, Lucilia illustris, and Protophormia terraenovae. We extracted DNA and checked a 229 bp sequence within the mitochondrial cytochrome oxidase subunit I. The sequences of the local flies were aligned to published data of specimens from other countries. We also studied the practical value of the analysed DNA region for their differentiation. All species were matched correctly by a Basic Local Alignment Search Tool (BLAST) search apart from L. caesar and L. illustris. Although molecular methods are very useful—especially if it is necessary to identify small fragments of insect material or very young larvae—we propose to use it only in addition to the conventional methods.     

Light-induced variability in development of forensically important blow fly Phormia regina (Diptera: Calliphoridae)

The use of the postmortem interval (PMI) in practical applications of forensic entomology is based upon developmental data of blow flies (Diptera: Calliphoridae) generated under controlled environmental conditions. Careful review of the published forensic entomology data sets showed that experimental (environmental) parameters differed between studies. Despite the differences in study design, there are no empirical data on the effect of photoperiod on blow fly development; yet, photoperiod has been shown to alter some insect development and behavior among noncalliphorids. Consequently, will differences in photoperiod alter the developmental times of calliphorids, and thereby alter PMI estimates? To answer this question, we used a replicated design with precise temperature measurement to examine the effects of photoperiod on the forensically important blow fly Phormia regina (Meigen). We concluded that inaccurate temperature recordings by using set-chamber temperatures over rearing-container temperatures would have overshadowed any affect light had on development. Second, constant light increased variation in overall adult developmental time and significantly delayed development compared with cyclic light. Finally, not accounting for delayed development induced by photoperiod underestimated the initial empirical estimate of the PMI. These sources of variation need to be included in forensic estimates because this variation can compromise predictions of PMI based upon current data sets. Without pinpointing optimal photoperiods with which to test development, we must assume that potentially large sources of variability exist within current estimates of the PMI.     

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.     

Sensilla on the antenna of blow fly,Triceratopyga calliphoroides Rohdendorf (Diptera: Calliphoridae)

Blow fly, Triceratopyga calliphoroides Rohdendorf, is a common and synanthropic species of medical and forensic significance in eastern Asia. Field monitoring studies have indicated that olfaction system plays an important role in guiding the behavior of insect species. To further our understanding of fly olfaction, scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) are applied to examine the sensillar morphology of adults, with an emphasis on sensory pit and sacculus. Both microtrichiae and several mechanoreceptors are detected on antennal scape and pedicel. Except for these two structures, pedicellar buttons are also found in antennal pedicellar depression after the separation of antennal pedicel and funiculus. Eight types of antennal sensilla are observed on external surface of antennal funiculus (one type of trichoid sensilla, three types of basiconic sensilla, three types of coeloconic sensilla, and one type of clavate-like sensilla), while two types (one type of basiconic sensilla and one type of coeloconic-like sensilla) are detected at the inner surface of sensory pits or sacculus. As the first to investigate cuticular invaginations of blow flies via paraffin section, the internal structure of abundant sensory pits and an excessively complex sacculus on antennal funiculus are revealed. After comparison to other species previously studied, this phenomenon is proved to be the most unique feature of T. calliphoroides, armed with a discussion on its morphology, function, and possible evolutionary implications.     

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.     

Nocturnal oviposition behavior of blow flies (Diptera: Calliphoridae)

It is widely held that blow flies, in general, and Phaenicia sericata (Meigen), in particular, are not active at night and do not lay eggs during that time. P. sericata is thought to require sunlight and warmth for oviposition. Three common and forensically important flies--Calliphora vicina (Robineau-Desvoidy), P. sericata, and Phormia regina (Meigen)--oviposited during the dark hours of the night during the summers of 1988 and 1989. Nocturnal oviposition can alter the usual estimate of the postmortem interval in homicide cases by as much as 12 h. Cases are presented that serve to change our concept of P. sericata from an obligate heliophile to a facultative heliophile that has a willingness to enter dim or dark places to oviposit.     

How photoperiods affect the immature development of forensically important blowfly species Chrysomya albiceps (Calliphoridae)

The purpose of this study was to verify the influence of different photoperiods on larval body weight, post-embryonic development, and viability of Chrysomya albiceps. The bioassays were performed in acclimatized chamber at 27?±?1?°C and 60?±?10?% relative humidity regulated by three different light and dark cycles for a period of 24?h-12:12, 24:00, and 00:24. Four replications with 50 newly hatched larvae each were used per photoperiod. The larvae were placed in container containing bovine ground meat (50?g). When mature larvae spontaneously abandoned the diet, they were individually weighed and separated into glass tubes until emergence. The larvae that did not abandon the diet were not weighed. The larvae from 24?h of photophase did not abandon the diet and pupated inside, so these larvae were not weighed and the larval and pupal developments were not registered. The mean of larval body weight did not vary between the 24?h scotophase and 12?h photophase. The mean duration of larval developmental time varied significantly when comparing the 24?h scotophase and the 12?h photophase, and the pupal developmental time also varied for the same photoperiods; in both stages, the duration of development was faster in the 24?h scotophase. The developmental time of neo-larvae to adult was verified in all photoperiods and gradually increased as photophase increased. There was a trend to augment of viability with the reduction of photophase length.     

Thermoregulation in larval aggregations of carrion-feeding blow flies (Diptera: Calliphoridae)

The growth and development of carrion-feeding calliphorid (Diptera: Calliphoridae) larvae, or maggots, is of great interest to forensic sciences, especially for estimation of a postmortem interval (PMI). The development rate of calliphorid larvae is influenced by the temperature of their immediate environment. Heat generation in larval feeding aggregations (=maggot masses) is a well-known phenomenon, but it has not been quantitatively described. Calculated development rates that do not include internally generated temperatures will result in overestimation of PMI. Over a period of 2.5 yr, 80 pig, Sus scrofa L., carcasses were placed out at study sites in north central Florida and northwestern Indiana. Once larval aggregations started to form, multiple internal and external temperatures, and weather observations were taken daily or every few days between 1400 and 1800 hours until pupation of the larvae. Volume of each aggregation was determined by measuring surface area and average depth. Live and preserved samples of larvae were taken for species identification. The four most common species collected were Lucilia coeruleiviridis (=Phaenicia) (Macquart) (77%), Cochliomyia macellaria (F.) (8.3%), Chrysomya rufifaces (Macquart) (7.7%), and Phormnia regina (Meigen) (5.5%). Statistical analyses showed that 1) volume of a larval mass had a strong influence on its temperature, 2) internal temperatures of masses on the ground were influenced by soil temperature and mass volume, 3) internal temperatures of masses smaller than 20 cm3 were influenced by ambient air temperature and mass volume, and 4) masses larger than 20 cm3 on the carcass had strongly regulated internal temperatures determined only by the volume of the mass, with larger volumes associated with higher temperatures. Nonsignificant factors included presence of rain or clouds, shape of the aggregation, weight of the carcass, species composition of the aggregation, time since death, or season.     

The use of COI barcodes for molecular identification of forensically important fly species in Germany

Deoxyribonucleic acid (DNA)-based insect identification has become a routine and accurate tool in forensic entomology. In the present study, we demonstrate the utility of the mitochondrial DNA cytochrome oxidase I gene "barcoding region" as a universal marker for molecular identification of forensically important Diptera. We analyzed 111 specimens belonging to 13 species originating from Frankfurt am Main, Germany (Calliphoridae: Calliphora vicina, Calliphora vomitoria, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Lucilia sericata, Lucilia silvarum, Phormia regina, Protophormia terraenovae; Piophilidae: Parapiophila vulgaris; Muscidae: Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis). Intraspecific variation ranged from 0 to 1.17% and interspecific variation occurred between 1.17% and 15.21%. Although differences within species were generally less than among species, divergence percentages overlapped due to low interspecific nucleotide divergence of the recently separated sister species L. caesar and L. illustris. However, all species formed distinct monophyletic clades and thus the cytochrome oxidase 1 (COI) barcode has been shown suitable for clear differentiation and identification of forensically relevant Diptera in Germany.     

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.     

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.     

A comparison of blow fly (Diptera: Calliphoridae) and beetle (Coleoptera) activity on refrigerated only versus frozen-thawed pig carcasses in Indiana

To have large numbers of replicates in forensic entomology research, it is often necessary to freeze carcasses and thaw them before use in experiments. Research on the effects of freezing on decomposition is minimal in the literature and the effect that it has on insect (e.g., blow fly, beetle) activity is virtually unknown. The objective of this study was to compare the differences in insect activity occurring on refrigerated only versus frozen-thawed pig carcasses, and to characterize the associated dipteran fauna. Seven pigs, frozen for 2 mo and then thawed before the experiment, were compared with seven pigs killed and stored in a walk in refrigerator approximately 12 h before the start of the experiment. No significant differences in time to the appearance of adult flies, eggs, larvae, or the initiation and conclusion of larval migration were observed between refrigerated only and frozen-thawed pig carcasses. Beetles from the family Staphylinidae also did not show a significant difference in arrival times. Phormia regina (Meigen), Lucilia coeruleiviridis (Macquart), Cochliomyia macellaria (F.), Pollenia rudis (F.), and Hydrotaea leucostoma (F.) comprised the dipteran taxa present on the carcasses. Results suggest that freezing pigs before exposure in the field does not significantly alter blow fly life events or the appearance of staphylinid beetles.     

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...     

Rehydration of forensically important larval Diptera specimens

Established procedures for collecting and preserving evidence are essential for all forensic disciplines to be accepted in court and by the forensic community at large. Entomological evidence, such as Diptera larvae, are primarily preserved in ethanol, which can evaporate over time, resulting in the dehydration of specimens. In this study, methods used for rehydrating specimens were compared. The changes in larval specimens with respect to larval length and weight for three forensically important blow fly (Diptera: Calliphoridae) species in North America were quantified. Phormia regina (Meigen), Cochliomyia macellaria (F.), and Chrysomya rufifacies (Macquart) third-instar larvae were collected from various decomposing animals and preserved with three preservation methods (80% ethanol, 70% isopropyl alcohol, and hot-water kill then 80% ethanol). Preservative solutions were allowed to evaporate. Rehydration was attempted with either of the following: 80% ethanol, commercial trisodium phosphate substitute solution, or 0.5% trisodium phosphate solution. All three methods partially restored weight and length of specimens recorded before preservation. Analysis of variance results indicated that effects of preservation, rehydration treatment, and collection animal were different in each species. The interaction between preservative method and rehydration treatment had a significant effect on both P. regina and C. macellaria larval length and weight. In addition, there was a significant interaction effect of collection animal on larval C. macellaria measurements. No significant effect was observed in C. rufifacies larval length or weight among the preservatives or treatments. These methods could be used to establish a standard operating procedure for dealing with dehydrated larval specimens in forensic investigations.     

Interaction effects of temperature and food on the development of forensically important fly, Megaselia scalaris (Loew) (Diptera: Phoridae)

In forensic entomology, breeding of fly larvae in a controlled laboratory environment using animal tissue is a common technique to obtain insect developmental time for the estimation of postmortem interval. Previous studies on growth media are mostly on the effect of different diets on fly development. However, the interaction effects between temperature and food type used have not been explored. The objective of this study was to compare the use of cow's liver agar and raw liver on the development of a forensically important fly, Megaselia scalaris (Loew). This study also determined the interaction between different temperatures and different food types on the growth of this species. A total of 100 M. scalaris eggs were transferred into each of the two media mentioned above. Liver agar was prepared by adding dried ground liver into nutrient agar, whilst raw liver was naturally prepared from the same animal source. This experiment was conducted at 27, 30 and 33?°C in an incubator in a continuously dark condition. Length and weight of larvae, puparia and adult samples were determined. Total developmental times for larvae feeding on liver agar at each temperature were approximately 7–15?h slower than those feeding on raw liver. Survival rates were almost equal in both diets but were lower at 33?°C. Mean larva length in both diets did not differ significantly at all temperatures, but larvae feeding on liver agar had lower mean weight values than those in raw liver at 30 and 33?°C. The effect of temperature was significant in female puparia weight and male adult weight whereas the effect of diet types was significant in both male and female puparia size and weight. Interaction effects of temperature and food type on M. scalaris puparium size and adult weight were significant, indicating that puparium size and adult weight depended on both food type and temperature. This experiment highlighted the use of cow's liver agar as an alternative diet to breed M. scalaris in the laboratory and the importance of considering the interaction effect between temperatures and food types when deciding the most suitable medium in fly larva rearing.     

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.     

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.