Morphology of puparia of Megaselia scalaris (Diptera: Phoridae), a fly species of medical and forensic importance

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1 Parasitol Res (2006) 98: DOI /s SHORT COMMUNICATION Kabkaew L. Sukontason. Worachote Boonsriwong. Sirisuda Siriwattanarungsee. Somsak Piangjai. Kom Sukontason Morphology of puparia of Megaselia scalaris (Diptera: Phoridae), a fly species of medical and forensic importance Received: 8 April 2005 / Accepted: 28 September 2005 / Published online: 2 December 2005 # Springer-Verlag 2005 Abstract Megaselia scalaris (Loew), a scuttle fly, is a fly species of medical and forensic importance. For use in forensic investigation, fly specimens found to associate with human corpses have to be identified at species level. Herein, we present the morphology of the puparia of the above fly species using scanning electron microscopy. The characteristic of the intersegmental spines along the dorsal and lateral segments and sculpture of the pupal respiratory horn of this puparia may be useful in future studies, to distinguish it from other closely related species. Megaselia scalaris (Loew), a scuttle fly (Diptera: Phoridae), is a fly species of medical importance. The larvae are myiasis-producing agents in humans and animals (Zumpt 1965; Singh and Rana 1989; Disney 1994; Hira et al. 2004), and pestiferous in fruit (Karunaweera et al. 2002), while adults are a predator of other insect colonies (Barre et al. 1991). This fly has been employed as an animal subject in biological and/or medical experiments (Suck and Traut 2000; Harrison and Cooper 2003; Trumble and Jensen 2004). The other viewpoint of medical importance is the involvement with human corpses in forensic investigations or forensic entomology. Many authors have revealed that some stages in the life cycle of M. scalaris (eggs, larvae, pupae, and/or adults) were found to associate with cases of human death (e.g., Lord 1990; Disney 1994; Greenberg and Wells 1998; Goff 2000; Byrd and Castner 2001; Sukontason et al. 2001; Greenberg and Kunich 2002; Campobasso et al. 2004). Toxicological analysis of the K. L. Sukontason (*) Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand klikitvo@mail.med.cmu.ac.th Tel.: Fax: pupal cases of this species was performed by Miller et al. (1994) to determine concentrations of amitriptyline and its metabolite, nortriptyline, in a mummified corpse. M. scalaris is a member of about 3,000 worldwide species of the family Phoridae, and several genera within this family (e.g., Megaselia, Aneurina, Conicera, Diploneura, Dohrniphora, Metopina, Triphleba) were found to relate in vertebrate carrion (Smith 1986). Within the genera of Megaselia per se, in particular the puparia, Zumpt (1965) indicated the difficulty of identifying between species due to their similarity of morphological appearance. Regarding this, and prior to use in forensic investigation, information on their morphologies, particularly those useful in identification, is of initial significance. In this study, we presented a detailed description on the morphology of the puparia of M. scalaris to enlarge the database of this fly in the forensic entomology arena. Puparia of M. scalaris were obtained from a laboratory colony maintained at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, northern Thailand. When the pupal stage was reached, 3-day-old puparia were prepared for the scanning electron microscopy (SEM) process. They were gently placed onto double-stick tape on stubs and coated with gold for 30 s in a sputter-coating apparatus (SPI-MODULE Coater Sputter, USA). This enabled viewing under a JEOL JSM-5910LV SEM (Tokyo, Japan) using a high vacuum mode. The puparia of M. scalaris are dorsoventrally flattened, bearing the striking feature of a pair of long pupal respiratory horns on the dorsum at the end of the 5th segment (or the 1st abdominal segment; Fig. 1). At the ventral view, the cephalic segment is invaginated, showing only some part of the dorsal end and a pair of dome-shaped dorsal organs or antennae (Fig. 2, arrows). When dorsally observed, a pair of anterior spiracles is found dorsolaterally on the prothorax (Fig. 3). These spiracles are more or less rounded, bearing two straight spiracular openings (slits), close together at one end and separated at the other (Fig. 4). Just beneath the anterior spiracle, the intersegmental spines between the pro- and mesothorax are single pointed and appear in five rows

269 Fig. 3 Scanning electron micrograph of the puparia of M. scalaris.

Bar 100 μm Fig. 1 Scanning electron micrograph of Megaselia scalaris puparium, showing a striking pair of long pupal respiratory horns on the dorsum at the end of the 5th segment. Bar 500 μm (Fig. 5).

These respiratory horns are long and slender, with their apex being slightly curved (Fig. 6). The surface of the horns bears numerous spirally arranged papillae from its base through the apex (Fig.

As they are spirally formed, it could be assumed that about 90 papillae of the total occurred in the cuticle of one horn.

2 269 Fig. 3 Scanning electron micrograph of the puparia of M. scalaris. Dorsal view of the anterior region showing a pair of anterior spiracles (as) dorsolaterally found on the prothorax, prior to the intersegmental spines (s) between the pro- and mesothorax. Bar 100 μm Fig. 1 Scanning electron micrograph of Megaselia scalaris puparium, showing a striking pair of long pupal respiratory horns on the dorsum at the end of the 5th segment. Bar 500 μm (Fig. 5). The prominent structure on the anterior region is a pair of pupal respiratory horns, extruding through the posterolateral walls. These respiratory horns are long and slender, with their apex being slightly curved (Fig. 6). The surface of the horns bears numerous spirally arranged papillae from its base through the apex (Fig. 6). The number of papillae could be estimated on only one side in about 45 of them, since only the top view could be clearly seen under SEM. As they are spirally formed, it could be assumed that about 90 papillae of the total occurred in the cuticle of one horn. Using higher magnification, each papilla appears oval, and domed-shaped, and is located on the convex base. Each has a single longitudinal straight opening (Fig. 7). The integument of puparia is covered by spinose setae and has a pointed end, while the dorsal and lateral surfaces between each body segment bear short tubercles (Figs. 1 and 8), with the apex bearing minute spines (Fig. 9). At the posterior end, a pair of posterior spiracles is observed on the dorsal surface of the last segment (Fig. 10). Each spiracular plate is composed of four straight spiracular slits, two of each group in parallel (Fig. 11). The large posterior spiracular hairs appear centrally between the two groups, while the tiny hairs are found adjacent to each group. A large cavity, in the Fig. 2 Scanning electron micrograph of the puparia of M. scalaris. Ventral view of the anterior end indicating an invaginated cephalic segment. A pair of dome-shaped dorsal organs (arrows) was observed. Bar 50 μm Fig. 4 Scanning electron micrograph of the puparia of M. scalaris. Anterior spiracle bearing two straight slits (s), with one end closed while the other is open. Bar 5 μm

270 Fig. 5 Scanning electron micrograph of the puparia of M. scalaris. Intersegmental spines between the pro- and mesothorax are in ~5 rows of single pointed.

Many genera of the Phoridae fly members have been reported to regularly associate with vertebrate carrions (e.g., Anevrina, Conicera, Diploeura, Dohrniphora, Metopina, Triphleba, somemegaselia; Smith 1986).

Liu and Greenberg (1989) investigated immature stages of many fly species of forensic importance, which included M. scalaris; whereas the forensic use of M.

scalaris have been collected from human corpses in forensic investigations in many parts of the world (e.g., Sukontason et al. 2001; Campobasso et al. 2004). Fig.

3 270 Fig. 5 Scanning electron micrograph of the puparia of M. scalaris. Intersegmental spines between the pro- and mesothorax are in ~5 rows of single pointed. Bar 5 μm position most fitted to the ecdysial scar or button, locates near the tiny spiracular hairs (Fig. 11). Many genera of the Phoridae fly members have been reported to regularly associate with vertebrate carrions (e.g., Anevrina, Conicera, Diploeura, Dohrniphora, Metopina, Triphleba, somemegaselia; Smith 1986). As mentioned by this author, some phorids are regarded as forensically important, such as Conicera tibialis Schmitz, the Coffin-fly, and Megaselia rufipes (Meigen). Liu and Greenberg (1989) investigated immature stages of many fly species of forensic importance, which included M. scalaris; whereas the forensic use of M. scalaris and Megaselia abdita Schmitz was reiterated in the case investigations of Greenberg and Wells (1998). Thus far, immature stages of M. scalaris have been collected from human corpses in forensic investigations in many parts of the world (e.g., Sukontason et al. 2001; Campobasso et al. 2004). Fig. 7 Scanning electron micrograph of the puparia of M. scalaris. Higher magnification of papillae showing their oval, dome-shaped and located on the convex base, with each one bearing single longitudinal straight opening. Bar 5 μm Although the form of fly puparia is rather uniformed, examination under SEM reveals many distinguishing details by which they can be identified (Erzinclioglu 2000). Such examination of puparia has been the entomological evidence in forensic investigation, as documented by Erzinclioglu (2000). Liu and Greenberg (1989) represented some characters in the key to identify puparia of forensically important flies, using the size, shape, tubercles along the body, number of papillae on the anterior spiracle, and total of bubble membranes and respiratory horns on the fifth segment. In this study, we showed various structural features that could be used for differentiation from other closely related phorids. Between the intersegments, puparia of M. scalaris bear short tubercles, each with minute spines at the apex. This structure probably differs from those diagrammatically revealed in M. rufipes puparia, bearing long processes (Zumpt 1965), or in Conicera (Smith 1986). The other possibly useful Fig. 6 Scanning electron micrograph of the puparia of M. scalaris. Left pupal respiratory horn being long and slender with an apex slightly curved outward. Numerous papillae form spirally (arrows) at the base through the apex. Bar 50 μm Fig. 8 Scanning electron micrograph of the puparia of M. scalaris. Dorsal integument showing a short tubercle (sp) and dense covering of spinose setae with a pointed end. Bar 50 μm

271 Fig. 9 Scanning electron micrograph of the puparia of M. scalaris. Short tubercle with minute spines at the apex.

In flies of the family Muscidae, the length of the respiratory horn and arrangement of papillae are found to be different in some species, as demonstrated in Ophyra by Skidmore (1985).

) (Siriwattanarungsee et al. 2005). This study showed the morphological features of M.

4 271 Fig. 9 Scanning electron micrograph of the puparia of M. scalaris. Short tubercle with minute spines at the apex. Bar 10 μm feature is the papillae on the respiratory horn, in which their arrangement, number and shape may be species specific. In flies of the family Muscidae, the length of the respiratory horn and arrangement of papillae are found to be different in some species, as demonstrated in Ophyra by Skidmore (1985). Moreover, the arrangement and number of papillae observed on the respiratory horns markedly differ between puparia of the housefly Musca domestica L. and blowfly Chrysomya megacephala (F.) (Siriwattanarungsee et al. 2005). This study showed the morphological features of M. scalaris puparia that may play an important role in differentiating from other fly species, which is the initial requirement in forensic investigations if the species is found to associate with a corpse. Fig. 10 Scanning electron micrograph of the puparia of M. scalaris. Top view of the posterior end indicating a pair of posterior spiracles (ps) seen dorsally at the last segment. Bar 200 μm Fig. 11 Scanning electron micrograph of the puparia of M. scalaris. Right lobe of the posterior spiracle showing four straight spiracular slits (s) arranged as two opposite groups, interspersed with posterior spiracular hairs (psh). The arrow probably indicates the ecdysial scar or button. Bar 10 μm Acknowledgements We thank Budsabong Kuntalue and Anantachai Khumsayyai from the Electron Microscopy Research and Service Center (EMRSC), Faculty of Science, Chiang Mai University, for their technical assistance in using the SEM facilities. The Faculty of Medicine and Chiang Mai University are acknowledged for their financial support. References Barre N, Mauleon H, Garris GI, Kermarree A (1991) Predators of the tick Amblyomma variegatum (Acari: Ixodidae) in Guadeloupe, French West Indies. Exp Appl Acarol 12: Byrd JH, Castner JL (2001) Insects of forensic importance. In: Byrd JH, Castner JL (eds) Forensic entomology: the utility of arthropods in legal investigations. CRC Press, Boca Raton, FL, pp Campobasso CP, Disney RHL, Introna F (2004) A case of Megaselia scalaris (Loew) (Dipt., Phoridae) breeding in a human corpse. Anil Aggrawal s Internet J Forensic Med Toxicol 5:3 5 Disney RHL (1994) Scuttle flies: the Phoridae. Chapman & Hall, London Erzinclioglu Z (2000) Maggots, murder and men. St. Martin s, New York Goff ML (2000) A fly for the prosecution. Harvard University Press, Cambridge, MA Greenberg B, Kunich JC (2002) Entomology and the law: flies as forensic indicators. Cambridge University Press, Cambridge, MA Greenberg B, Wells JD (1998) Forensic use of Megaselia abdita and M. scalaris (Phoridae: Diptera): case studies, development rates, and egg structure. J Med Entomol 35: Harrison DA, Cooper RL (2003) Characterization of development, behavior and neuromuscular physiology in the phorid fly, Megaselia scalaris. Comp Biochem Physiol A 136: Hira PR, Assad RM, Okasha G, Al-Ali FM, Iqbal J, Mutawali KE, Disney RH, Hall MJ (2004) Myiasis in Kuwait: nosocomial infections caused by Lucilia sericata and Megaselia scalaris. Am J Trop Med Hyg 70: Karunaweera ND, Ihalamulla RL, Kumarasinghe SP (2002) Megaselia scalaris (Diptera: Phoridae) can live on ripe bananas a potential health hazard? Ceylon Med J 47:9 10 Liu D, Greenberg B (1989) Immature stage of some flies of forensic importance. Ann Entomol Soc Am 82:80 93

5 272 Lord WD (1990) Case histories of the use of insects in investigations. In: Catts EP, Haskell NH (eds) Entomology and death: a procedural guide. Joyce s Print Shop, Inc., SC, pp 9 37 Miller ML, Lord WD, Goff ML, Donnelly B, McDonough ET, Alexis JC (1994) Isolation of amitryptiline and nortryptiline from fly puparia (Phoridae) and beetle exuviae (Dermestidae) associated with mummified remains. J Forensic Sci 39: Singh TS, Rana D (1989) Urogenital myiasis caused by Megaselia scalaris (Diptera: Phoridae): a case report. J Med Entomol 26: Siriwattanarungsee S, Sukontason KL, Kuntalue B, Piangjai S, Olson JK, Sukontason K (2005) Morphology of the puparia of the housefly, Musca domestica (Diptera: Muscidae) and blowfly, Chrysomya megacephala (Diptera: Calliphoridae). Parasitol Res 96: Skidmore P (1985) The biology of the Musdicae of the world. Dr. W. Junk Publishers, Dordrecht, Netherlands Smith KGV (1986) A manual of forensic entomology. Cornell University Press, Ithaca, NY Suck G, Traut W (2000) TROMB, a new retrotransposon of the gypsy-ty3 group from the fly Megaselia scalaris. Gene 255:51 57 Sukontason K, Sukontason K, Vichairat K, Piangjai S, Lertthamnongtham S, Vogtsberger RC, Olson JK (2001) The first documented forensic entomology case in Thailand. J Med Entomol 38: Trumble JT, Jensen PD (2004) Ovipositional response, developmental effects and toxicity of hexavalent chromium to Megaselia scalaris, a terrestrial detrivore. Arch Environ Contam Toxicol 46: Zumpt F (1965) Myiasis in man and animals in the old world. Butterworth, London, UK

1. Introduction. Forensic Science International 164 (2006)

1. Introduction. Forensic Science International 164 (2006) Forensic Science International 164 (2006) 230 234 www.elsevier.com/locate/forsciint Morphological comparison between Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton (Diptera: Calliphoridae)