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.