1. Introduction. Forensic Science International 164 (2006)

Transcription

1 Forensic Science International 164 (2006) Morphological comparison between Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton (Diptera: Calliphoridae) puparia, forensically important blow flies Kabkaew L. Sukontason a, *, Paitoon Narongchai b, Chaturong Kanchai b, Karnda Vichairat b, Somsak Piangjai a, Worachote Boonsriwong a, Nophawan Bunchu a, Duanghatai Sripakdee a, Tarinee Chaiwong a, Budsabong Kuntalue c, Sirisuda Siriwattanarungsee a, Kom Sukontason a a Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand c Electron Microscopy Research and Service Center (EMRSC), Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Received 28 April 2005; accepted 4 February 2006 Available online 9 March 2006 Abstract In Thailand, the hairy maggots of the blow flies, Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton, are of forensic importance. Both flies are closely related species, not only in the morphological appearance of their larvae and puparia, but also on the aggressive feeding habit of the former. In our continuing studies of forensically important flies, identification of immature ones needs particular attention. In this study, we reported the morphological comparison between the puparia of these two blow fly species using scanning electron microscopy (SEM). Observation revealed that the cuticular sculpture of tubercles along the dorsal and lateral segments had markedly different features: with C. rufifacies having many sharp spines assembling only at the tip, while of C. villeneuvi bore stout spines throughout the tubercle. A larger number of globules at the bubble membrane on the dorsolateral border of the fifth segment was found in C. villeneuvi (average 225) than in C. rufifacies (average 35), and more papillae was observed on the anterior spiracle in C. villeneuvi (13 15) than in C. rufifacies (9 12). However, the morphology of distinct netlike patches of the integument and structure of the posterior spiracle of both species were almost identical. Morphological comparison in this study permitted identification of the puparia of both fly species, particularly in areas where they co-exist. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Chrysomya rufifacies; Chrysomya villeneuvi; Puparia; Identification; Forensic entomology 1. Introduction Fly specimens found in corpses and/or death scenes can be used as entomological evidence in forensic investigations of death, i.e. estimation of the postmortem interval, determination of toxic substances, antemortem trauma and verify relocation [1 4]. Not only are the eggs and larvae of flies important as evidence, but puparia is also found to provide valuable information long after the remains of the body have decomposed. Such examples of fly puparia used in investigations of a case scenario by either viable puparia or the empty puparial cases have been recorded [5]. Detection of toxic * Corresponding author. Tel.: ; fax: address: klikitvo@mail.med.cmu.ac.th (K.L. Sukontason). substances (e.g. drugs, pesticides, environmental pollutants) in fly puparia have also been reported [6 9]. However, prior to their use in forensic investigation, correct species identification of fly specimens collected from a corpse is mandatory, since the developmental rate of each species may be different by either temperature or toxic substance. Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton are blow flies of forensic importance in many countries of the world including Thailand [1,4,5,10 13]. Both species share several similarities, for example the anatomical features of their larvae as hairy maggot, geographical distribution, and aggressive feeding habit of the second and third instars [13,14]. Morphology of their larvae have been compared [14,15], however no comparison between their puparia has been found in the literature. We report, herein, the comparison of the morphological features between puparia of C. rufifacies and C /$ see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi: /j.forsciint

2 K.L. Sukontason et al. / Forensic Science International 164 (2006) villeneuvi, highlighting their important characteristics to identify them by SEM. This information will be beneficial for identification before puparia of these species are found in a corpse and/or at a death scene and used for further forensic analysis. 2. Materials and methods Puparia of C. rufifacies were obtained from a laboratory colony maintained at the Department of Parasitology, Faculty of Medicine, Chiang Mai University. As for C. villeneuvi, puparia derived from the metamorphosis of numerous larvae collected from two unknown male human remains, which had been transferred to the Department of Forensic Medicine, Chiang Mai University, Thailand, for forensic investigation on 4 February These remains of unknown age were discovered in forested area in Lampang Province, northern Thailand (north latitude 188 and east latitude 998). For SEM observation, puparia of each species, which had been kept in 70% alcohol, were washed by shaking 20 min in a shaking bath. They were prepared for the SEM by mopping up water on the surface with filter paper and then gently placing onto double-stick tape on stubs and coating with gold for 30 s in a sputter-coating apparatus (SPI-MODULE TM Coater Sputter, Figs Scanning electron micrographs of puparia of C. rufifacies and C. villeneuvi. (1) Anterior end of C. rufifacies showing anterior spiracle (as) and tubercles (t); bar = 500 mm. (2) Anterior spiracle of C. rufifacies having a single row of 10 papillae; bar = 50 mm. (3) Integument of C. rufifacies covered with distinct net-like patches; bar = 50 mm. (4) Anterior end of C. villeneuvi showing anterior spiracle (as) and tubercles (t); bar = 200 mm. (5) Anterior spiracle of C. villeneuvi having a single row of 14 papillae; bar = 50 mm. (6) Integument of C. villeneuvi covered with distinct net-like patches; bar = 50 mm.

3 232 K.L. Sukontason et al. / Forensic Science International 164 (2006) USA). This enabled viewing under a JEOL JSM-5910LV scanning electron microscope (SEM; Tokyo, Japan) using a high vacuum mode. 3. Results Puparia of C. rufifacies and C. villeneuvi were hairy in appearance; pointed anteriorly and blunt posteriorly. The former species measured mm in length (n = 12) and not statistically different from the latter, measured mm in length (n = 12) ( p > 0.05; Mann Whitney U test). At the anterior end, a pair of anterior spiracles was laterally prominent at the second segment (prothorax). Strong tubercles were strikingly noticeable, beginning at the fourth segment (Figs. 1 and 4). A single row of 10 papillae could be observed at the anterior spiracle of C. rufifacies (Fig. 2), in contrast to a single row of 14 papillae in C. villeneuvi (Fig. 5). The integument of the puparia of both species resembled each other, covered with distinct net-like patches (Figs. 3 and 6). Focusing on the tubercles along dorsal and lateral segments of the puparia, elongated tubercles were obviously different; those of C. rufifacies having slender spines assembling only at the tips (Fig. 7), and in contrast, those of C. villeneuvi bearing stout spines throughout the surface (Fig. 9). At the dorso-lateral margins of the fifth segment of the puparia, a group of globular structures called bubble membrane was detected. The number of globules was markedly distinct, with C. rufifacies having 35 globules (Fig. 8); while C. villeneuvi had 225 globules (n = 2)(Fig. 10). At the caudal segment, six pairs of elongated tubercles were similarly located marginally (Figs. 11 and 14). However, when focusing on the surface of these tubercles, a similar appearance to that seen in the third instar was observed. Tubercles of C. rufifacies possessed slender spines assembling only at the tips (Fig. 12); while short, stout spines existed throughout the surface in C. villeneuvi (Fig. 15). A pair of posterior spiracles was located centrally at the caudal segments (Figs. 11 and 14); nevertheless, no distinction was detected in the morphology characteristic of the posterior spiracles (e.g. shape and arrangement of posterior spiracular slits, position of button, arrays of posterior spiracular hairs) between C. rufifacies (Fig. 13) and C. villeneuvi (Fig. 16). 4. Discussion Differentiation between puparia of C. rufifacies and C. villeneuvi is not simple, according to their hairy appearance and similar size. As morphological features previously described, puparia of both blow fly species shared some features that could not be differentiated, even when using SEM. These characteristics were the integument architecture of the net-like patches (see Figs. 3 and 6) and the morphology of the posterior spiracles (see Figs. 13 and 16). Geographically, C. rufifacies and C. Figs Scanning electron micrographs of puparia of C. rufifacies and C. villeneuvi. (7) Elongate tubercle of C. rufifacies showing slender spines assembled only at the tip; bar = 20 mm. (8) Group of 35 globules of bubble membrane at the dorso-lateral margins of the fifth segment of C. rufifacies; bar = 10 mm. (9) Elongate tubercle of C. villeneuvi bearing stout spines throughout the surface; bar = 50 mm. (10) Group of 225 globules of bubble membrane of C. villeneuvi; bar = 20 mm.

4 K.L. Sukontason et al. / Forensic Science International 164 (2006) Figs Scanning electron micrographs of puparia of C. rufifacies and C. villeneuvi. (11) Caudal segment of C. rufifacies showing a pair of posterior spiracles (ps) and six pairs of tubercles along the margin. Idt, inner dorsal tubercle; mdt, median dorsal tubercle; odt, outer dorsal tubercle; ovt, outer ventral tubercle; mvt, median ventral tubercle; ivt, inner ventral tubercle, bar = 500 mm. (12) Inner dorsal tubercle of C. rufifacies having slender spines assembled only at the tip, bar = 100 mm. (13) Posterior spiracle of C. rufifacies indicating three slits(s). Arrow indicated button, bar = 50 mm. (14) Caudal segment of C. villeneuvi showing a pair of posterior spiracles and six pairs of tubercles along the margin, bar = 500 mm. (15) Inner dorsal tubercle of C. villeneuvi, bearing short, stout spines throughout the surface, bar = 100 mm. (16) Posterior spiracle of C. villeneuvi indicating three slits(s). Arrow indicated button, bar = 50 mm. villeneuvi overlap in territories. C. rufifacies expand in distribution throughout tropical and subtropical areas, and North America [20], whereas C. villeneuvi are found in many Asian countries [China (Yunnan, Hainan Is.), Vietnam, Laos, Thailand, Malaysia, Indonesia (Sumatra), Nepal, India, and Sri Lanka] [21]. The results of this study clearly showed that puparia of C. rufifacies and C. villeneuvi could be morphologically differentiated. The most distinctive characteristic observed using SEM was the ornament of elongated tubercles of the puparia (see Figs. 7, 9, 12, and 15). The presence of slender spines assembling only at the tips of C. rufifacies tubercles (Figs. 7 and 12) corresponded with those seen in the third instar observed using SEM [13,16] or light microscopy [14]. Likewise, the short, stout spines existing throughout the surface in C. villeneuvi puparia (Figs. 9 and 15) agreed with those in third instar using light microscopy [14]. Another characteristic difference between the puparia of both species was the number of globules at the bubble membrane on the dorso-lateral border of the fifth segment, and this could only be observed by SEM. A larger number of globules was found in C. villeneuvi (Fig. 10) thaninc. rufifacies (Fig. 8). Difference in the number of globules was one of the

5 234 K.L. Sukontason et al. / Forensic Science International 164 (2006) characteristics used for differentiating puparia of forensically important fly species [17]. The final characteristic was the number of papillae on the anterior spiracle of the puparia. This character might be seen using light microscopy. Nine to 12 papillae were found in C. rufifacies, which agreed with the third instar previously reported [13,15,18,19], while papillae were found in C. villeneuvi, which also agreed with the third instar previously reported [14,15]. The results presented herein enable identification of the puparia of both species, which have a hairy appearance. Acknowledgements We thank the Faculty of Medicine and Chiang Mai University, for publication cost. References [1] K.G.V. Smith, A Manual of Forensic Entomology, British Museum (Natural History), London, United Kingdom, [2] R.D. Hall, Medicocriminal entomology, in: E.P. Catts, N.H. Haskell (Eds.), Entomology and Death, A Procedural Guide, Joyce s Print Shop, Clemson, SC, 1990, pp [3] E.P. Catts, Problems in estimating the postmortem interval in death investigations, J. Agric. Entomol. 9 (1992) [4] B. Greenberg, J.C. Kunich, Entomology and the Law: Flies as Forensic Indicators, Cambridge University Press, United Kingdom, [5] W.D. Lord, Case histories of the use of insects in investigations, in: E.P. Catts, N.H. Haskell (Eds.), Entomology and Death, A Procedural Guide, Joyce s Print Shop, Clemson, SC, 1990, pp [6] M.L. Miller, W.D. Lord, M.L. Goff, B. Donnelly, E.T. McDonough, J.C. Alexis, Isolation of amitryptiline and nortryptiline from fly puparia (Phoridae) and beetle exuviae (Dermestidae) associated with mummified remains, J Forensic Sci. 39 (1994) [7] M.L. Goff, M.L. Miller, J.D. Paulson, W.D. Lord, E. Richards, A.I. Omori, Effects of 3,4-methylenedioxymethamphetamine in decomposing tissues on the development of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and detection of the drug in postmortem blood, liver tissue, larvae, and puparia, J. Forensic Sci. 42 (1997) [8] R. Gagliano-Candela, L. Aventaggiato, The detection of toxic substances in entomological specimens, Int. J. Legal Med. 114 (2001) [9] M. Wood, M. Laloup, K. Pien, N. Samyn, M. Morris, R.A.A. Maes, E.A. de Bruijn, V. Maes, G. De Boeck, Development of a rapid and sensitive method for the quantitation of benzodiazepines in Calliphora vicina larvae and puparia by LC-MS-MS, J. Anal. Toxicol. 27 (2003) [10] M. Barreto, M.E. Burbano, P. Barreto, Flies (Calliphoridae, Muscidae) and beetles (Silphidae) from human cadavers in Cali, Colombia, Mem. Inst. Oswaldo Cruz 97 (2002) [11] K.L. Sukontason, K. Sukontason, P. Narongchai, S. Lertthamnongtham, S. Piangjai, J.K. Olson, Chrysomya rufifacies (Macquart) as a forensicallyimportant fly species in Thailand: A case report, J. Vector Ecol. 26 (2001) [12] K. Sukontason, K.L. Sukontason, T. Chaiwong, N. Boonchu, S. Piangjai, H. Kurahashi, Hairy maggot of Chrysomya villeneuvi Patton (Diptera: Calliphoridae), a fly species of forensic importance, J. Med. Entomol. 40 (2003) [13] K.L. Sukontason, K. Sukontason, S. Lertthamnongtham, B. Kuntalue, N. Thijuk, R.C. Vogtsberger, J.K. Olson, Surface ultrastructure of Chrysomya rufifacies (Macquart) larvae (Diptera: Calliphoridae), J. Med. Entomol. 40 (2003) [14] K. Sukontason, K.L. Sukontason, S. Piangjai, P. Narongchai, W. Samai, N. Boonchu, D. Sripakdee, R. Ngern-klun, S. Siriwattanarungsee, Morphology of second and third instars of Chrysomya villeneuvi Patton (Diptera: Calliphoridae), a fly species of forensic importance, Forensic Sci. Int. 154 (2005) [15] B. Omar, Key to third instar larvae of flies of forensic importance in Malaysia, in: B. Greenberg, J.C. Kunich (Eds.), Entomology and the Law: Flies as Forensic Indicators, Cambridge University Press, Cambridge, 2002, pp [16] J.D. Wells, J.H. Byrd, T.I. Tantawi, Key to third-instar Chrysomyinae (Diptera: Calliphoridae) from carrion in the Continental United States, J. Med. Entomol. 36 (1999) [17] D. Liu, B. Greenberg, Immature stage of some flies of forensic importance, Ann. Entomol. Soc. Am. 82 (1989) [18] H. Ishijima, Revision of the third stage larvae of synanthropic flies of Japan (Diptera: Anthomyiidae, Muscidae, Calliphoridae and Sarcophagidae), Jpn. J. Sanit. Zool. 18 (1967) [19] R.L. Kitching, The immature stages of the Old-World screw-worm fly, Chrysomya bezziana Villeneuve, with comparative notes on other Australasian species of Chrysomya (Diptera, Calliphoridae), Bull. Entomol. Res. 66 (1976) [20] D.L. Baumgartner, Review of Chrysomya rufifacies (Diptera: Calliphoridae), J. Med. Entomol. 30 (1993) [21] H. Kurahashi, L. Chowanadisai, Blow flies (Insecta: Diptera: Calliphoridae) from Indochina, Species Divers 6 (2001)