Differentiation of the Third Instar of Forensically Important Fly Species in Thailand

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1 FORUM Differentiation of the Third Instar of Forensically Important Fly Species in Thailand KOM SUKONTASON, 1 KABKAEW L. SUKONTASON, RADCHADAWAN NGERN-KLUN, DUANGHATAI SRIPAKDEE, AND SOMSAK PIANGJAI Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Ann. Entomol. Soc. Am. 97(6): 1069Ð1075 (2004) ABSTRACT Differentiation of the third instar of forensically important ßy species in Thailand was performed using light microscopy, based on their morphological criteria for ßy identiþcation. Four species of the family Calliphoridae [Chrysomya rufifacies (Macquart), Chrysomya megacephala (F.), Chrysomya nigripes Aubertin, and Lucilia cuprina (Wiedemann)] and two species of the family Muscidae [Musca domestica L. and Hydrotaea ( Ophyra) spinigera Stein] were examined in this study, with the features of the anterior spiracles, dorsal spines between the prothorax and mesothorax, and posterior spiracles being emphasized. The comparisons, presented herein, should be helpful for forensic practitioners to readily distinguish the third instars of ßy species found associated with human cadavers, before their use for further forensic investigations. KEY WORDS differentiation, ßy larvae, forensic entomology, Thailand 1 ksukonta@mail.med.cmu.ac.th. FLY SPECIMENS FOUND ON human corpses are currently important as entomological evidence used in forensic investigations. Although they are mostly used to estimate the post-mortem interval, they are sometimes used to analyze drug substances consumed from the remains or to determine the manner of death (Smith 1986, Goff and Catts 1990, Goff 2000, Byrd and Castner 2001, Greenberg and Kunich 2002). In such cases, these entomological specimens have to be initially identiþed, before being used for further forensic analysis. Among the immature stages in the life cycle of ßies, the larvae of species of the families Calliphoridae (blow ßies) or Muscidae are among those most frequently found associated with corpses. The third instar, which is the ultimate stage of larval development, and has prolonged duration, is the stage most often used for identiþcation, if the appropriate taxonomic key is available. The adult ßy, reared from the third instar, also is often used to conþrm identity. Intensive investigation of forensically important ßy larvae was reported for 17 species by Liu and Greenberg (1989). Wells et al. (1999) presented a key to the third instars of the common carrion-feeding Chrysomyinae in the United States, some of which also have been recorded as forensically important species elsewhere. In Asia, little information is available for identiþcation of forensically important ßy larvae, especially in the form of taxonomic keys (Ishijima 1967, Omar 2002). We report herein the important morphological features of the third instar of ßies that are forensically relevant species in Thailand, by using light microscopy to distinguish between them. The simple procedure, together with the essential features of larvae provided, should allow forensic practitioners to carry out this initial step in forensic entomology. Materials and Methods The third instar of four species of blow ßies (family Calliphoridae) were obtained from laboratory colonies maintained at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. These species were Chrysomya rufifacies (Macquart), Chrysomya megacephala (F.), Chrysomya nigripes Aubertin, and Lucilia cuprina (Wiedemann). Two species of the family Muscidae were included: Musca domestica L. and Hydrotaea ( Ophyra) spinigera Stein. These ßy colonies were maintained at ambient temperature (24Ð28 C) and natural light/dark photoperiod in a cabinet in the rearing room of the Department of Parasitology. Adults were reared on two kinds of food: 1) a mixture of 10% (wt:vol) multivitamin syrup solution and 2) fresh pork liver (used as both a food source and oviposition site). Small pieces of fresh pork liver were changed daily; the mixture of 10% sugar solution, multivitamin syrup, and the supplementary food were changed every 2 d. Subsequently, the oviposition sites were observed daily for the presence of eggs; if present, the eggs /04/1069Ð1075$04.00/ Entomological Society of America

2 1070 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 6 Fig. 1. Light micrograph showing where the third instars of ßies were cut into three portions: anterior region, body, and posterior region. as, anterior spiracle; ps, posterior spiracle; s, spine between the prothorax and mesothorax. were transferred into a 12 by 15 by 6-cm transparent plastic box, and 40 g of fresh pork liver was provided as larval food. To prevent overpopulation, each box housed only 30Ð40 larvae. The lid of each box was rectangular, cut to three-fourths of the total area and the hole covered with Þne material for ventilation and the prevention of other small insects entering the box to oviposit in it. The lid was sealed tightly with adhesive tape to prevent the larvae from crawling out. Liver was replaced daily until third instars developed into prepupae, during the nonfeeding period. Boxes containing pupae were covered and tightly sealed until the emergence of adults, after which the boxes were placed into a rearing cage and the adults released. Approximately 30 third instars of each ßy species were collected from the colony and washed several times with normal saline solution to remove surface artifacts or liver tissue. They were killed by transferring into a beaker containing near boiling water (Tantawi and Greenberg 1993) for a few minutes. The dead larvae were dissected using a sharp blade at two sites to obtain three body portions. The Þrst cutting position was across the mesothorax (the third segment) to view the anterior spiracle, and the spines between proand mesothorax (Fig. 1). The second cutting position was across the 11th body segment, so that the morphology of the posterior spiracle could be clearly viewed (Fig. 1). The anterior and posterior parts were mounted by transferring them onto a clean glass slide after placing a few drops of Entellan (Merck, Darmstadt, Germany) on it. A coverslip was placed over the specimens and the examination of morphological characters was made under a light microscope by using magniþcation of 4 to 40 (Olympus, Tokyo, Japan) equipped with a calibrated eyepiece micrometer. The images were recorded with a digital camera (Nikon, Tokyo, Japan). Results The body of the third instar, represented by C. megacephala, is a typical muscoid-shaped vermiform larva, which is pointed anteriorly and blunt at the posterior end (Fig. 1). The anterior spiracles are located laterally at the distal end of the prothorax (or second segment). The number of papillae of the anterior spiracles differed between species. For C. rufifacies, the number ranges from nine to 12, with 10 papillae being the most common (64%; 16/25), followed by 11 or 12 (16%; 4/25), and nine being the least common (4%; 1/25) (Fig. 2A). For C. megacephala, the number ranges from eight to 12, with 10 or 11 papillae being the most common (40%; 12/30), followed by 12 (13.3%; 4/30), and eight or nine papillae was the least common (3.3%; 1/30) (Fig. 2B). Regarding C. nigripes, the number ranges from nine to 13, with 10 papillae being the most common (53.3%; 16/30), followed by 11 (23.3%; 7/30) and nine (16.7%; 5/30), whereas 12 or 13 were the least common (3.3%; 1/30) (Fig. 2C). By contrast, L. cuprina larvae bore four to seven papillae per spiracle, with Þve papillae being the most common (47.7%; 21/44), followed by six papillae (27.3%; 12/44), four papillae (13.6%; 6/44), and seven papillae (11.4%; 5/44) (Fig. 2D). In M. domestica, Þve to seven papillae per spiracle were found; the most common was seven papillae (57.1%; 12/21), followed by six (33.3%; 7/21) and Þve (9.5%; 2/21) (Fig. 2E). With regard to H. spinigera, the most common number of papillae was Þve (60%; three-þfths), followed by six (40%; two-þfths) (Fig. 2F). The structure and distribution of the dorsal cuticular spines between the prothorax and mesothorax differed greatly in the ßy larvae included in this investigation. Those of third instars of C. rufifacies were arranged singly; each having one to three dark pointed tips (Fig. 3A). The spines of C. megacephala also were arranged singly, but the base of the pigmented part of each spine was concave (Fig. 3B). Although the shape of the spines of C. nigripes was similar to that of C. rufifacies, the spines were smaller, closer together and sometimes occurred in a row (Fig. 3C). The dorsal spines of L. cuprina differed markedly from those of Chrysomya and were arranged in distinct rows, each row having two to six dark spines with single pointed tips (Fig. 3D). The spines of M. domestica (Fig. 3E) and H. spinigera (Fig. 3F) were similar in appearance, triangular, pale pigmented, and arranged in rows. However, each row contained more spines in H. spinigera than in M. domestica. Regarding the posterior spiracles, the peritreme, a dark structure encircling the three relatively straight spiracular openings (slits), was incomplete in all species of Chrysomya examined (Fig. 4AÐC). However, the peritreme was complete in L. cuprina and the button was small (Fig. 4D, arrow). In M. domestica, the peritreme of each spiracle formed a crude forward or reverse D-shape, surrounding three sinuous spiracular slits and an obvious ecdysial scar (button) (Fig. 4E). The shape of the peritreme in H. spinigera was markedly different to that of M. domestica, in that the spiracular slits in H. spinigera were straight and subparallel to each other. The button of H. spinigera was large (Fig. 4F, arrow). A key to simplify the identiþcation of these third instars was summarized for morphological comparison as follows.

3 November 2004 SUKONTASON ET AL.: DIFFERENTIATION OF FLY LARVAE OF FORENSIC IMPORTANCE 1071 Fig. 2. (AÐF) Light micrographs of the anterior spiracle of the third instars of each ßy species. (A) C. rufifacies. (B) C. megacephala. (C) C. nigripes. (D) L. cuprina. (E) M. domestica. (F) H. spinigera. Bar, 40 m for all Þgures. 1. Large, elongate tubercles present on each abdominal segment; the tip of tubercles bearing numerous small spines; thick and dark incomplete posterior spiracular peritreme (Fig. 4A)... C. rufifacies Abdominal segments lacking large, elongate tubercles Posterior spiracular peritreme incomplete (Fig. 4AÐC)...3 Posterior spiracular peritreme complete (Fig. 4DÐF) Dorsal spines between prothorax and mesothorax arranged singly (Fig. 3B); upper end of peritreme normal (Fig. 4B).. C. megacephala Dorsal spines between prothorax and mesothorax often in rows (Fig. 3C); end of upper peritreme gradually enlarged (Fig. 4C, arrowhead)...c. nigripes

4 1072 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 6 Fig. 3. (AÐF) Light micrographs of the dorsal spines between the prothorax and mesothorax of the third instars of each ßy species. (A) C. rufifacies. (B) C. megacephala. (C) C. nigripes. (D) L. cuprina. (E) M. domestica. (F) H. spinigera. Bar, 40 m for all Þgures. 4. Straight spiracular slits (Fig. 4D and F)... 5 Sinuous spiracular slits; thick and dark reverse D-shaped peritreme (Fig. 4E); spines between prothorax and mesothorax triangular, with little pigmentation, arranged in rows (Fig. 3E)... M. domestica 5. Small button (Fig. 4D, arrow); spines between prothorax and mesothorax arranged in rows, with single pointed tip; much pigmentation at the end of pointed tip (as seen in Fig. 3D)... L. cuprina Large button area (Fig. 4F, arrow); spines between prothorax and mesothorax arranged in rows, each is triangular tip; no pigmentation at the end of the tip (Fig. 3F)... H. spinigera

5 November 2004 SUKONTASON ET AL.: DIFFERENTIATION OF FLY LARVAE OF FORENSIC IMPORTANCE 1073 Fig. 4. (AÐF) Light micrographs of the structure of the posterior spiracle of the third instars of each ßy species. (A) C. rufifacies. (B) C. megacephala. (C) C. nigripes. Arrowhead indicates gradually enlarged upper incomplete peritreme. (D) L. cuprina. (E) M. domestica. (F) H. spinigera. p, peritreme; s, slit. Arrows in DÐF indicate button. Bar, 40 m for all Þgures. Discussion IdentiÞcation of ßy larvae for forensic investigations or medical importance may be accomplished by means of several methodologies: by molecular analysis (Stevens and Wall 2001, Wallman and Donnellan 2001, Wells and Sperling 2001, Harvey et al. 2003), scanning electron microscopy (Kitching 1976, Liu and Greenberg 1989, Sukontason et al. 2003), or light microscopy (Shinonaga and Kano 1974, Smith 1986, Erzinclioglu 1987, Wallman 2001). Each method provides unique advantages, and the application depends largely on the availability of scientiþc equipment and personnel; however, identifying the ßy species is the ultimate aim. Herein, we showed that the differentiation of the third instars of ßies in this study could be accomplished by using light microscopy. Although three

6 1074 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 6 morphological features were used for comparison in this investigation, the most distinctive features among these species were associated with the posterior spiracles and dorsal spines between the prothorax and mesothorax, whereas the number of papillae on the anterior spiracles may be used as supplementary feature. A similar conclusion has been previously documented (Shinonaga and Kano 1974). Regarding the features of the dorsal spines, some variation of the shape, position, and orientation exists; thus, comparison of the dorsal spines should always be performed in the same area of the particular larval segment (Colwell and OÕConnor 2000). In this study, the dorsal spines between the prothorax and mesothorax were chosen, because this region is the most adjacent to the head and prothorax, which bear the anterior spiracles. The cutting position, at approximately the position of the mesothorax (Fig. 1), enabled efþcient examination under a light microscope of the two features located at the head and thorax. The use of dorsal spines as comparison features for ßy larvae also has been recorded by other workers, but different larval segments were used. Shinonaga and Kano (1974)) distinguished the third instars of four species of muscid ßy larvae [Morellia saishuensis Ôuchi, Morellia hortensia (Wiedemann), Morellia hortorum (Fallén), and Morellia aenescens R.-D.] by using the shape and size of the spines on the sixth segment. The spines in the dorsal region of the anterior spine band on Þrst abdominal segment (the Þfth segment overall) have been included as distinguishing features in the taxonomic keys among many species of blow ßy larvae in South Australia [i.e., Calliphora hilli hilli Patton, Calliphora dubia (Macquart), Calliphora augur (F.), Calliphora vicina Robineau-Desvoidy, Calliphora stygia (F.), Calliphora maritima Norris, and Calliphora albifrontalis Malloch; Wallman 2001]. Spine characters also may be used to differentiate the larvae of the screwworm, Cochliomyia hominivorax (Coquerel), and secondary screwworm, Cochliomyia macellaria (F.) (Erzinclioglu 1987). Shape and degree of pigmentation of the spines are useful characters for the differentiation of ßy larvae (Wallman 2001). The posterior spiracles of the third instar of the six ßy species examined in this study were morphologically distinctive in character. This feature has been known for the differentiation of ßy larvae (Zumpt 1965; Smith 1986, 1989; Liu and Greenberg 1989). However, the use of posterior spiracles for identiþcation is unreliable, due to their variability, especially when comparing closely related species, as occurring between hairy maggot of C. rufifacies and Chrysomya villeneuvi Patton (K. L. Sukontason et al., unpublished data). In conclusion, the identiþcation of ßy specimens, particularly the third instar, which is the immature stage most frequently found in corpses, is initially essential for their use in further forensic analysis. The comparisons presented herein conþrm that the posterior spiracles and spines between the prothorax and mesothorax provide the two most distinctive characters in the differentiation of the third instars of C. rufifacies, C. megacephala, C. nigripes, L. cuprina, M. domestica, and H. spinigera, with the anterior spiracle providing supplementary characteristics. 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