Figure 2 . Calliphora vomitoria Members of the Calliphoridae family are easily

1. Figure 1. Blowfly life cycle Initially, adult female flies lay eggs (top) which then hatch into the larval stage, which can be subdivided into developmental stages known as instars. Larvae transition between the three instars (right) through molting. The 3rd instar (bottom right), which is also the largest, is the peak of the feeding stage for a larvae, hence, the reason 3rd instar larvae are used for analysis. The larval stage is followed by a prepupal stage (non- feeding) stage during which the larvae migrate and prepare for pupation. The pupal stage (bottom left) lasts for approximately 10 days. Adult flies ultimately emerge from the pupa under the proper environmental conditions. The duration of the various developmental stages is entirely temperature-dependent. Results Unfortunately, the experiment was not able to be carried out. Although a colony of C. vomitoria was successfully established, it was not able to be sustained during the four weeks of winter break. When the colony was reestablished from refrigerated pupa, the flies that resulted did not produce eggs during the 5 weeks of their lifespan. Despite several unsuccessful attempts, the colony was not able to be reestablished a third time. The protocol for establishing and maintaining a colony was however successfully determined. The carrion plant (Stapelia gigantea) serves as a reliable source from which eggs can be collected during the plant’s flowering season (figure 4). The eggs may then be reared on ground beef at 20ºC with 50% humidity until reaching the third instar. The meat and larvae are then contained in an apparatus depicted in figure 5. The air holes in the lid are covered to prevent larval escape during the prepupal stage. The mouse bedding at the base mimics the soil of the natural environment in which the prepupal larvae may burry themselves prior to pupation. Once the adult flies emerge from the pupa, the eggs that they lay may be used to maintain the colony. This protocol was adapted from Sherman and Wyle from their 1996 article on maggot rearing [6]. Calliphora vomitoria as an indicator species of drug presence and concentration through toxicological analysisLauren Talasnik, advised by Dr. McShaffrey Adapted from www.nlm.nih.gov Figure 2. Calliphora vomitoria Members of the Calliphoridae family are easily distinguishable based on the presence of a blue, green, or purple metallic exoskeleton and their large, red eyes. In addition, more specific characteristics of the vomitoria species, such as the body length, presence of hair on the eyes, the width of the frons, and the length of 3rd antennae, can be used to identify this species. http://www.efabre.net/ Introduction The applications of forensic entomology in criminal investigations - and specifically, of the blowfly life cycle (figure 1) in determining postmortem interval - are well recognized [1]. More recently however, the uses for necrophagous insects have been expanded to include the determination of drug presence and drug concentration in the deceased [1,2]. This branch of forensic entomology, known as entomotoxicology, uses toxicological analysis to detect drugs in carrion-feeding organisms collected from crime scenes [1]. This practice becomes particularly useful in cases where a body is recovered in an advanced state of decomposition where there may not be fleshy or fluid remains available on which to perform analysis. In such cases, larvae may serve as alternative specimens on which to perform drug detection analysis [1,2]. The growing incidence of drug, toxin, and poison-related deaths in recent years reaffirms the importance of entomotoxicology, as it becomes necessary for investigators to develop alternative methods for drug detection within the deceased for matters of forensic importance [1]. Gas chromatography mass spectroscopy (GC-MS) is among the analytical methods which may be used for drug detection and quantification in larvae. Beyer et al.’s 1980 study used GC-MS to detect high concentrations of prescription drugs in Cochiliomyia macellaria [2]. These and other studies demonstrate evidence of greater bioaccumulation of toxins detected within larvae than within the original source of the drug [2,3,4]. However, concentrations detected within Calliphora vomitoria, a more common blowfly species, are anticipated to yield lower concentrations of the drugs acetaminophen, ibuprofen, and aspirin relative to those found within the meat samples themselves. This expectation stems from evidence that the larvae’s active metabolism and continual process of excretion during ingestion may work to diminish the concentrations able to be recovered [5]. The purpose of this study was then to establish a correlation between the concentrations of drugs detected in larvae relative to the spiked meat samples on which they are reared, and to assess the appropriateness of using C. vomitoria for entomotoxicological purposes. Figure 5. Rearing container Figure 4. Carrion Plant (Stapelia gigantea) Methodology Colony Rearing A colony of C. vomitoria was maintained to establish a continual population of larvae for experimentation. Following several failed attempts to attract flies and induce egg laying through meat lures, eggs were ultimately collected from the flowers of carrion plants. The larvae were then reared until adulthood. Upon emerging as adult flies, the fly population was analyzed to determine the fly species through anatomical characteristics (figure 2). The resulting C. vomitoria colony was maintained within the rearing cage (figure 3) at 20ºC with 50% humidity, and followed a 16:8 light-dark cycle, which produced the optimal environmental conditions favoring their development. As a precautionary measure, approximately 20 pupa were collected from the colony and refrigerated to a state of suspended animation so that they could be used to regenerate the colony if necessary. Experimental Setup Portions of minced meat are treated with various amounts of the drugs aspirin, ibuprofen, and acetaminophen to yield final concentrations of 0, 1, 10, 100, and 1000 mg/kg. The drug-treated meat is then analyzed through GC-MS to detect drug presence and quantify the concentration present. Twenty third instar larvae are transferred onto each meat portion, and reared until the prepupal stage. Larvae are then collected and washed through two cycles of distilled water and methanol to remove surface contamination, are then frozen at -20ºC in preparation for analysis. Analysis Frozen larvae are homogenized and centrifuged through a series of cycles. The resulting supernatant is removed and used for analysis. Similarly, the spiked meat portions are homogenized and centrifuged through a series of cycles. The resulting supernatant off the drug-spiked meat is also used to detect the concentration of drug within the meat. Figure 3. The rearing cage Adult C. vomitoria flies were transferred to a rearing cage which consisted of a plastic frame surrounded by netting to contain the flies. The cage was suspended over water to maintain humidity and collect excrement. A water source, protein source, and granular sugar were provided for nutritional purposes. In addition, a meat source was provided as a site for putrefaction, or egg laying. The meat source was contained within a dark canister to both mimic the dark environment preferred by blowflies during putrefaction, and to maintain the moisture content of the meat. Conclusions Although no conclusions could be drawn due to experimental complications, there are modifications which should be made upon a retrial of the experiment. The use of a meat lure, such as ground beef or liver, to attract flies for putrefaction was found to be largely ineffective. Because such lures do not possess any of the physical characteristics or emit the same carrion odor of other natural lures, such as compost pile or decaying animal, they were unable to attract flies and thereby induce putrefaction. Thus, lures such as carrion plants or decaying animals, which serve as natural lures for necrophagous species, are more effective. In addition, the type and size of the meat source used for rearing can be crucial. When rearing flies on a meat source, ground beef seems to be more effective than liver at attracting the flies for feeding. In addition, using smaller portions of ground beef than those which were used (less than 250 grams) would help to ensure that the meat does not spoil prior to consumption, and that the meat does not attract unwanted fly species from the surrounding environment. Smaller portions of granular sugar and protein will also reduce the potential for infestation of other fly species. The most crucial modification to the experiment would be to operate under much higher levels of humidity. Although a pool of water was added to the base of the rearing cage to increase humidity, the room in which the rearing took place never reached humidity levels higher than 21%. Operating under higher levels of humidity would help to ensure optimal growth of the species, especially since their progression through the developmental stages is temperature-dependent. This modification will encourage faster growth and development while lowering the likelihood of fly death due to improper environmental conditions. Literature Cited [1] Introna F., Campobasso C.P., Goff M.L. 2001. Entomotoxicology. Forensic Science International 120: 42-47. [2] Beyer J.C., Enos W.F., Stajic M. 1980. Drug identification through analysis of maggots. Journal of Forensic Science 25: 411-412. [3] Goff M. L. 2000. A fly for the prosecution/ how insect evidence helps solve crimes. Cambridge (MA): Harvard University Press; 225p. [4] Gagliano-Candela R., Aventaggiato L. 2000. The detection of toxic substances in entomological specimens. International Journal of Legal Medicine 114: 197-203. [5] Sadler D.W., Fuke C., Court F., Pounder D.J. 1994. Drug accumulation and elimination in Calliphora vicina larvae. Forensic Science International 71: 191-197. [6] Sherman RA, Wyle FA. 1996. Low-cost, low-maintenance rearing of maggots in hospitals, clinics, and schools. American Journal of Medical Hygiene 54: 38-41. Acknowledgements I would like to acknowledge Dr. McShaffrey, my capstone advisor, for his support and feedback during the entire capstone process. I would also like to thank Dr. Egolf for her assistance with the GC-MS protocol and operation. In addition, I would like to acknowledge Dr. Brown, the entire capstone class, and the Marietta College biology department.