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.     

Effects of temperature on development of Phormia regina (Diptera: Calliphoridae) and use of developmental data in determining time intervals in forensic entomology

Precise developmental data for forensic indicator blow fly species are essential for accuracy in the estimate of the post-mortem interval (PMI). Why, then, does the determination of the PMI result in conflicting time frames when published conspecific developmental data are used? To answer this question, we conducted constant temperature trials between the developmental minimum temperature and upper threshold temperatures (8-32 degrees C) on the forensically important blow fly species Phormia regina (Meigen) (Diptera: Calliphoridae). Flies were reared using two designs to quantify sources of variation. We measured rearing container temperatures and internal growth chamber temperatures by using thermocouples to accurately record temperatures experienced by larvae and to construct a degree-day model. Differences in experimental design, as seen across temperature studies for this fly species, did not significantly impact larval development. We also found that using set chamber temperatures rather than rearing container temperatures altered the final degree-day model. Using any minimum threshold (including an empirically determined true minimum) other than that from linear interpolation (x-intercept) violated degree-day assumptions and invalidated estimates of the PMI. We observed the minimum developmental temperature to be higher (14 degrees C) than that generated under the x-intercept method (5.46 degrees C) by using data from oviposition to adult emergence. This difference along with the noted difference in accumulated degree-days (using different base temperatures) suggests a need for additional experimentation on other forensically important fly species at low temperature thresholds to help with development of curvilinear models. Former and current estimates of the PMI may be inaccurate if the process to determine the time frame ignored degree-day model assumptions or was based upon questionable data sets.     

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.     

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.     

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.     

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.     

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.     

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.     

Permeabilization of Cochliomyia hominivorax (Diptera: Calliphoridae) embryos

Embryos of the primary screwworm, Cochliomyia hominivorax (Coquerel), were successfully permeabilized for use in subsequent cryopreservation studies. Mortality was greater for eggs incubated for < 5 h before treatment. The mean survival of embryos to first instars was 55.7, 61.1, and 62.6% when the embryos were incubated for 5, 5.5, and 6 h before treatment, respectively. The survival to the pupal and adult stages was low. An improved media for culturing the embryos during and immediately after treatment needs to be devised and the procedure for rearing the larval stages also needs to be altered to improve survival for emerging adults.     

Developmental rate,size, and sexual dimorphism of Megaselia scalaris (Loew) (Diptera: Phoridae): its possible implications in forensic entomology

Cosmopolitan scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae) is one of the commonest forensic species recorded colonizing human corpse indoors and in concealed environment. The occurrence of this species in such environments provides a higher evidential value to assist estimation of postmortem interval (PMI) compared to other forensically important dipterans. However, developmental and size data of M. scalaris are still lacking and they are derived from a limited range of thermal values. The objective of this study is to develop the growth model of M. scalaris by emphasizing the size range of larvae and puparia at different constant temperatures. This species was reared in six replicates at eight varying constant temperatures ranging from 23 to 36 °C and cow’s liver was provided as food source. Larvae and puparia were sampled at set time intervals and measured by their length and weight. Because interpretation of forensic entomological evidence is subject to application of different techniques, development of M. scalaris is expressed herein by using developmental table, length/morphological stage diagrams and linear/nonlinear estimation methods. From the findings, it is very important to highlight that sexual dimorphism of M. scalaris during post feeding larva and pupa stage could be observed based on size and developmental periods. Mean length and weight ratios of male to female puparia are approximately 0.8 and 0.3–0.5, respectively, indicating sexual dimorphism of this species. Developmental period in female are 4.0–11.4 h (post feeding larval stage), 3.7–24.0 h (pupal stage), and 3.0–20.1 h (total developmental period) longer in male. Due to this dimorphism, PMI estimation using M. scalaris post feeding larva or puparium specimens must be carried out carefully by to avoid inaccuracy and misinterpretation.     

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.     

Classification of screwworms (Diptera: Calliphoridae) by larval spine morphology

The morphology of screwworm, Cochliomyia hominivorax (Coquerel), larval spines was affected by larval substrate and geographic effects on variation. Spine morphology of individual larvae was used to determine criteria for discrimination analysis classification. Classification criteria for types of wounds and host species were determined for larvae from central and southern Mexico. Larvae from marginal populations (United States, Caribbean Islands, Costa Rica) were classified by these criteria. Classifications were 27% accurate for wound type and 20% correct for animal species. When single egg masses were split and larvae were reared on laboratory diet and animal substrates, analysis of spine types showed significant rearing environment and sibship (egg mass) effects. Cluster analysis of geographic groups showed that larvae from central populations were closely clustered, whereas marginal groups were morphologically dissimilar from each other and from the central populations. These results indicate that larval spine morphology has limited value in taxonomic or systematic studies of screwworms and that mating compatibility was not related to spine similarity among populations. The significant differences between wound-reared and artificial media-reared larvae indicate that differences in spine morphology could be used to verify that samples from the field were indeed taken from animal wounds.