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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.