Diptera: Calliphoridae), a ﬂy Species of Forensic Importance Kom Sukontasona,*, Kabkaew L

Forensic Science International 154 (2005) 195–199 www.elsevier.com/locate/forsciint

Morphology of second and third instars of Chrysomya villeneuvi Patton (Diptera: Calliphoridae), a ﬂy species of forensic importance Kom Sukontasona,*, Kabkaew L. Sukontasona, Somsak Piangjaia, Paitoon Narongchaib, Wirachai Samaib, Noppawan Boonchua, Duanghatai Sripakdeea, Radchadawan Ngern-kluna, Sirisuda Siriwattanarungseea

aDepartment of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand bDepartment of Forensic Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

Received 23 April 2004; accepted 20 October 2004 Available online 8 December 2004

Abstract

The morphology of the second and third instars of Chrysomya villeneuvi Patton, a ﬂy species of forensic importance, was presented by use of light microscopy. Both instars were of hairy appearance, bearing elongated tubercles along the abdominal and caudal segments. The anterior spiracle had 13–15 papillae. Minute dark spots were observed to thoroughly cover the tubercle’s surface, with 4–6 strong dark tips. Regarding the third instar, the intersegmental spines between the prothorax and mesothorax were heavily pigmented. The posterior spiracle had a thick and heavily pigmented incomplete peritreme. The surface and tip of the tubercles was covered with heavily pigmented sharp spines. The integument of the body was covered with numerous distinct net-like patches. A comparison with another well-known hairy maggot, Chrysomya ruﬁfacies (Macquart), was discussed. # 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Chrysomya villeneuvi; Fly larvae; Identiﬁcation; Forensic entomology

1. Introduction body are distinctive [2]. Since anatomical studies on immature stages of C. villeneuvi are minimal particularly in the Chrysomya villeneuvi Patton is a forensically important larvae, we herein describe additional features of the second blowﬂies species because its larvae have been found in and third instars, while highlighting the most important human corpses [1,2]. Morphologically, the larvae were of diagnostic features to identify this species. This is the hairy appearance, bearing conspicuous tubercles along the principal requirement before the species is used for subse- body segments. Although its third instar is generally similar quent forensic analysis. to another well-known hairy maggot in the Oriental region [Chrysomya ruﬁfacies (Macquart)], the tubercles along their 2. Materials and methods * Corresponding author. Tel.: +66 53 945342; fax: +66 53 217144. The second and third instars of C. villeneuvi were E-mail address: [email protected] obtained from two unknown human male remains that were (K. Sukontason). transferred for investigation to the Department of Forensic

Table 1 Comparison of the distinctive features of the second and third instars of Chrysomya villeneuvi and Chrysomya ruﬁfacies Character C. villeneuvi C. ruﬁfaciesa Second instar Number of papillae of anterior spiracle 13–15 9–12 Tubercles along the body segment Distinct circular rows Indistinct or absence of minute dark spots Tip of tubercles 4–6 strong black crowns Three rows of numerous, minute dark crowns Third instar Number of papillae of anterior spiracle 13–15 9–12 Tubercles along the body segment Sharp spines regularly found Knobs encircling mostly over the entire surface lower half of surface Tip of tubercles Few crowns Three rows of numerous crowns a Source: Sukontason et al. [4].

Medicine, Chiang Mai University, Thailand, on 4 February When highlighting the elongated tubercles along the body 2004. These remains were discovered in the forested area of segments, minute dark spots were observed to thoroughly Muang Pan District, Lampang province, northern Thailand. cover the surface in rows, with 4–6 strong black tips existing During examination, numerous fly larvae (maggots) were at the apex (Fig. 5). In order to compare with the second found under their heads and bodies. Some larval specimens instar of C. rufifacies (laboratory strain) clearly, the tubercle were collected and killed by transferring them into a beaker apex of this species was also shown. The inset in Fig. 5 containing near boiling water [3] for a few minutes. The displays the tubercle apex of C. rufifacies, which had three dead larvae were cut using a sharp blade at two sites to obtain rows of many minute dark tips. As observed in some speci- three body portions (Fig. 1). Each part was mounted by mens of C. villeneuvi, the cuticle of third instar, which transferring them onto a clean glass slide after a few drops of contained broad spines thoroughly covering the tubercle, Entellan1 (Merck: Germany) had been placed on it. A cover was detected beneath the cuticle of the second instar during slip was placed over the specimens and an examination for the molting process (Fig. 6). morphological characters was made under a light micro- Regarding the third instar of C. villeneuvi, the hairy body scope (Olympus1, Japan) equipped with a calibrated eye still existed, but some structures were found to be different. piece micrometer. The images were recorded with a digital The anterior spiracle had 13–15 marginal papillae. The camera (Olympus Camedia, C-4040Zoom1, Japan). More- intersegmental spines between the prothorax and over, some larval specimens were reared to confirm the mesothorax were much more heavily pigmented, particu- species of C. villeneuvi in the laboratory at the Department larly at their ends. The shape of each spine was slender, with of Parasitology, Faculty of Medicine, Chiang Mai Univer- most of them entirely pigmented (Fig. 7). At the caudal sity, using pork liver as food source. segment, elongated tubercles were distinct and regularly distributed (Fig. 8). The posterior spiracle bore three wide slits, with the incomplete peritreme being thick and heavily 3. Results pigmented (Fig. 9). The ends of the peritreme, which had minute projections, were close together and they encircled The second instar of C. villeneuvi was muscoid-shaped, the poorly pigmented button (Fig. 9). The surface and tip of with the cephalic region being tapered anteriorly (Fig. 1). It the tubercles of the third instar were covered with heavily was hairy in appearance, bearing prominent tubercles encir- pigmented sharp spines, which had longer tips (Fig. 10). The cling the abdominal and caudal segments. While focusing on integument of the body was covered with numerous distinct the anterior region, a dark, heavily pigmented cephalophar- net-like patches, with each patch comprising a single large yngeal skeleton was located internally (Fig. 1). The fan- denticle located centrally (Fig. 11). A comparison of the shaped anterior spiracle was located at the posterior margin distinctive features to differentiate the second and of the prothorax (Fig. 1), appearing in a single row with 13– third instars of C. villeneuvi and C. rufifacies is shown in 15 marginal papillae (Fig. 2). The intersegmental spines Table 1. located between prothorax and mesothorax (Figs. 1 and 3) were fairly round at the base, and bore 1–4 heavily pigmented ends. At the posterior end of the body, a pair of posterior 4. Discussion spiracles was located centrally at the hairy caudal segment (Fig. 4). An incomplete peritreme, with wide ends, was The morphology of C. villeneuvi, particularly at the observed encircling two separated spiracular slits (Fig. 4). larval stage, has been studied by a few authors [1,2]. This K. Sukontason et al. / Forensic Science International 154 (2005) 195–199 197

Figs. 1–6. Light micrographs of the second instar of Chrysomya villeneuvi. (1) Lateral view of the whole body. Cephalic region on the left showing a pigmented cephalopharyngeal skeketon (C), anterior spiracle (AS), and intersegmental spines (S) between the prothorax and mesothorax. T: tubercles, PS: posterior spiracles. Bar = 100 mm. (2) Anterior spiracle appearing as a single row, having 14 papillae. Bar = 50 mm. (3) Intersegmental spines. Bar = 50 mm. (4) Posterior spiracle bearing two separated spiracular slits (S). Incomplete peritreme with wide ends (arrows). Bar = 50 mm. (5) Tubercles thoroughly covered with minute dark spots, with 4–6 strong black tips at the apex. Inset displaying many smaller tips of tubercle of C. rufifacies. Bar = 50 mm. (6) Cuticle of third instar, with broad spines (arrow), also detected beneath the cuticle of the second instar. Bar = 50 mm. study indicated that the second and third instars of this fly [4]. In comparison between these two species on the second species were of hairy appearance, but each one held some instar, the crowns or tips at the tubercle apex were strikingly difference in structures. This phenomenon was found in the distinctive; those of C. villeneuvi were stout with fewer second and third instars of C. rufifacies previously described spines (see Fig. 5), but those of C. rufifacies were slender 198 K. Sukontason et al. / Forensic Science International 154 (2005) 195–199

Figs. 7–11. Light micrographs of the third instar of Chrysomya villeneuvi. (7) Heavily pigmented intersegmental spines between the prothorax and mesothorax. Bar = 100 mm. (8) Caudal view showing distinct and regularly distributed elongated tubercles. Posterior spiracle (PS) located centrally. Bar = 100 mm. (9) Posterior spiracle bearing three slits (S), with thick and heavily pigmented, incomplete peritreme (P). Arrows indicate ends of peritreme, with minute projections, encircling poorly pigmented button. Bar = 100 mm. (10) Tubercle indicating heavily pigmented sharp spines and longer spines at the tip. Bar = 50 mm. (11) Integument showing numerous distinct net-like patches, with each patch comprising a single large denticle located centrally. Bar = 50 mm. with more spines [4]. In addition, a number of papillae at the Comparing the third instar of C. villeneuvi with that of C. anterior spiracle had distinctive features. The anterior spira- rufifacies, the ornament of tubercles was found to have cle of C. villeneuvi bore 13–15 papillae (which correlated distinctive features, with the former bearing sharp-end with Greenberg and Kunich [1]), whereas 9–12 papillae were spines that encircled the entire tubercle (see Fig. 10). How- found in C. rufifacies [1,4–6]. ever, the latter bore rounded-knobs around the lower half of K. Sukontason et al. / Forensic Science International 154 (2005) 195–199 199 each tubercle [4]. Since the number of papillae at the anterior References spiracle of the third instar was similar to that of the second instar, it was a distinctive feature. [1] B. Greenberg, J.C. Kunich, Entomology and the Law: Flies as In addition to the different morphological features Forensic Indicators, Cambridge University Press, Cambridge, previously described, the third instar of C. villeneuvi and 2002. C. rufifacies shared some features that could not be differ- [2] K. Sukontason, K.L. Sukontason, T. Chaiwong, N. Boonchu, S. Chrysomya villeneuvi entiated. For example, the surface integument architecture of Piangjai, H. Kurahashi, Hairy maggot of Patton (Diptera: Calliphoridae), a fly species of forensic distinct net-like patches (see Fig. 11), which corresponded to importance, J. Med. Entomol. 40 (2003) 983–984. those described in C. rufifacies by means of scanning [3] T.I. Tantawi, B. Greenberg, The effect of killing and preser- electron microscope [4], or those in another hairy maggot, vative solutions on estimates of maggot age in forensic cases, J. Chrysomya albiceps (Wiedemann) [7]. The morphology of Forensic Sci. 38 (1993) 702–707. posterior spiracles, having an incomplete and a heavily [4] K.L. Sukontason, K. Sukontason, S. Lertthamnongtham, B. pigmented peritreme (see Fig. 9), resembled C. rufifacies Kuntalue, N. Thijuk, R.C. Vogtsberger, J.K. Olson, Surface studied by light microscopy [5,8]. 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