Developmental Stages of the Fly Phormia regina and their use in Forensic Science Nathan Allison, College of Nursing, M

1. Developmental Stages of the Fly Phormia regina and their use in Forensic Science Nathan Allison, College of Nursing, Marshall University Abstract Forensic investigations utilize many techniques to estimate time of death. When a death occurs, the most obvious post-mortem indicator is the appearance of flies and other insects that feed on the carcass. One common carrion fly is the black blowfly, Phormia regina. This fly is one of the first to appear and lay its eggs in the orifices of the decaying carcass. By examining the larval stages of these flies, an accurate time of death can be determined. In this project, second (four day) and third (six day) instar larval stages were examined using a SEM. Morphological differences were used to differentiate between the second and third larval stages. Specifically, the spiracles (breathing organs) were imaged. Due to the extensive cuticular folding that occurred while preparing the specimens, three processes of preparation were used. The first process involved only the dehydration of the larvae in an ethanol series. The second process involved fixing the larvae in four percent glutaraldehyde followed by dehydration in an ethanol series. The third process involved the use of Karnovsky’s fixative. Karnovsky’s fixative was used because of the cuticular folding that occurred while using the other two methods of preparation. The process with Karnovsky’s fixative began by placing the larvae in the fixative for an hour. Then the larvae were washed with Cocadylate buffer, pH of 7.5. Finally, the larvae where post-fixed in 0.47% OsO4 for an hour and a half. Once this process was completed the larvae were processed through an ethanol series. All larvae were subsequently dried and sputter coated for imaging with the Jeol 5310 SEM. By observing the larvae with the SEM, the morphological differences in spiracle formation were determined. The second instar had two small slits in each spiracle while the third instar had three. Since the spiracles have different features in each larval stage, not only can the investigator look at the size of the larvae but the spiracle formation that has taken place. Therefore, the spiracles can be used as a tool in order to estimate the time of death of a specific carcass. Results and Discussion For over one hundred years blowflies and other Calliphoridae have been recognized as primary decomposers of animal cadavers. In this study we attempt to refine the practice of using fly colonization of cadavers to determine time of death. Although size of larvae can be used to determine which larval stage of development a specific species of fly is in, there is a wide range of sizes that each larval stage can contain. This depends on the amount of food available to the larvae have and the ambient and maggot mass temperatures in which they develop (Joy et al. 2002). This is why spiracular formation is often used to determine the stage of development a larva is in. As can be seen in the low magnification image of the second instar (Figure 5) larva there are two slits in each spiracle. On the other hand, the low magnification image of the third instar (Figure 6) larva shows there are three slits in each of the spiracles. It appears that the number of slits in each spiracle is a more consistent way of classifying larval development then just looking at the size of the larvae itself. ` The high magnification micrographs of the second (Figure 7) and third (Figure 8) instars confirm the results that were obtained by using low magnification. They also show the many other characteristics that are found throughout larval development. These figures show the peritreme and “button” that are seen in Figure 3. Spiracular hairs are also seen surrounding all the slits that are located in the spiracles. The hairs appear to be flattened, most likely caused by drying of the specimen. To conclude, another species of fly (Phaenicia) was examined in order to compare the spiracle morphology between related species. Figure 9 presents the results. The peritreme is seen completelely surrounding each spiracle and the “button” is easier to distinguish. These features that are clearly seen with the SEM are much harder to discern using a light microscope. We therefore believe that the SEM will be a valuable tool for forensic entomologists as the field develops and precision techniques become more refined. Figure 3. (1)Peritreme and (2) “Button” Figure 5. Low magnification image of a second instar (4 day) larvae. Introduction Forensic entomologists are concerned with the many insects that appear at a carcass. Realizing that these insects have their own stories to tell, they can piece together the events that occurred in the mysteries that they are attempting to solve (Schrof 1991). Insects are relatively predictable due to their persistent nature of remaining in the same environment and eating the same food (Fernandez 2001). By examining the crime scene, a forensic entomologist can determine facts that would be otherwise unknown. For example, by examining if the certain species of fly will appear indoors or out, in warm or cool weather, in the shade or the sun, or in daylight or night, a forensic entomologist will be able to recreate the events that occurred prior to the discovery of the body (Schrof 1991). Many insects bombard a body after a death, but the most common post-mortem insect is the blowfly. To distinguish between species of blowflies, forensic entomologists will examine such features like hooks that surround the mouth and the spiracles at the rear of the developing larvae. The spiracles are the breathing organs of the larvae and allow the larvae to place their heads directly into the flesh of the body that they are feeding on (Sachs 1998). A common blowfly that is encountered around fresh corpses is known as Phormia regina (Figure 1) (Joy et al. 2002). These flies will go through three larval developmental stages (instars), a pupa stage and will emerge as adults. In the development of Phormia regina, one oval opening is apparent in each spiracle of the first instar of development. As the larvae mature into the second instar, another opening is developed and each spiracle will contain two oval openings (Figure 2a). When the larvae reach the third instar state of development, a third slit (Figure 2b) will become apparent in each spiracle. As the larvae develop through each stage, an incomplete peritreme and a feature known as a “button” (Figure 3) will be seen surrounding each of the spiracles that are present (Hall 1948). By determining the number of openings in each spiracle, a forensic entomologist is able to accurately determine time of death through the specific larval stage that is present (Sachs 1998). • Objectives • Examine the morphological characteristics of the spiracles of the larvae of Phormia regina. • Distinguish between the differences in spiracular formation of second (4 day) and third (6 day) instar larval stages of Phormia regina. • To compare spiracle morphology in third instar larvae of Phaenicia sp. with that of Phormia regina. Figure 7. High magnification image of a second instar (4 day) larvae. Materials and Methods Sample The larvae of Phormia regina were collected from raccoon carcasses (Joy et al. 2002). Ten four day and ten six day larvae were prepared for use in the SEM. All the larvae were collected during an experiment preformed by Dr. Joy of the Marshall University, Department of Biological Sciences. The single larva of Phaenicia sp. was collected from a hawk carcass in the spring of 2002. Specimen Preparation Due to the delicate nature of the samples, three different preparation techniques were compared to observe which resulted in less deformation of the sample. These included: Procedure 1 Dehydration of the four day and six day larvae in an ethanol series. This involved soaking the larvae in 30, 50, 70, 90 and 100 percent ethanol for intervals of 10 minutes. Procedure 2 Fixing the larvae in four percent glutaraldehyde. Followed by dehydration in an ethanol series (See Procedure 1). Procedure 3 The process with Karnovsky’s fixative began by placing the larvae in the fixative for an hour. Then the larvae were washed with Cocadylate buffer, pH of 7.5. Finally, the larvae where post-fixed in 0.47% OsO4 for an hour and a half. Once this process was completed the larvae were processed through an ethanol series (See Procedure 1). Imaging All the test samples were sputter coated and imaged under the Jeol 5310 SEM that was provided by the staff at Marshall University. Electron energies ranged from 15 to 20kV. Working Distance was relatively long (20-30 mm) to improve depth of field. Magnification ranged from 75x to 500x. Figure 6. Low magnification image of a second instar (4 day) larvae. Figure 8. High magnification image of a third instar (6 day) larvae. Figure 1.Adult Phormia regina Figure 2. Montages of (a) second instar and (b) third instar phormia larvae