Larval Morphology of Chrysomya nigripes (Diptera: Calliphoridae), a Fly Species of Forensic Importance

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1 MORPHOLOGY, SYSTEMATICS, EVOLUTION Larval Morphology of Chrysomya nigripes (Diptera: Calliphoridae), a Fly Species of Forensic Importance KABKAEW L. SUKONTASON, 1 ROY C. VOGTSBERGER, 2 NOPPAWAN BOONCHU, 1 TARINEE CHAIWONG, 1 DUANGHATAI SRIPAKDEE, 1 RADCHADAWAN NGERN-KLUN, 1 SOMSAK PIANGJAI, 1 AND KOM SUKONTASON 1,3 J. Med. Entomol. 42(3): 233Ð240 (2005) ABSTRACT The morphology of all instars of Chrysomya nigripes Aubertin, a blow ßy species of forensic importance, is presented with the aid of both light microscopy and scanning electron microscopy (SEM). Morphological features of the cephalopharyngeal skeleton, anterior spiracle, posterior spiracle, and dorsal spines between the prothorax and mesothorax are highlighted. No consistent features were found, even using SEM, for distinguishing the Þrst instar of C. nigripes from that of Chrysomya megacephala (F.) or Chrysomya rufifacies (Macquart), two other commonly associated blow ßy species in corpses in Thailand. Several features observed in second and third instars proved to be valuable characteristics for separating these species. KEY WORDS Chrysomya nigripes, larvae, identiþcation, forensic entomology Chrysomya nigripes Aubertin is a blow ßy species that is currently considered a forensically important ßy species (Greenberg and Kunich 2002). In Thailand, presence of their larvae was discovered in human corpses being transferred for investigation to the Department of Forensic Medicine at Chiang Mai University in northern Thailand. One corpse was in the early stages of decomposition and the other was in a mummiþed stage, but both were from mountainous areas at relatively high altitudes (K.S., unpublished data). When initial sampling of larval specimens from these corpses was performed, the larvae of C. nigripes could not be differentiated from the other blow ßy species. DeÞnitive identiþcation of larvae of this species could not be made due to the lack of morphological information in the literature. Successful rearing of larval specimens to adulthood was required to con- Þrm identiþcation as C. nigripes. The accurate identiþcation of ßy specimens, particularly the larval stages that are commonly collected from corpses, is imperative in forensic entomology. Correct identiþcations provide more accurate estimations of the postmortem interval. Analysis of toxic substances has been performed in different ßy species to see the effects on differing developmental rates of particular species (Goff and Catts 1990, Byrd and Castner 2001, Greenberg and Kunich 2002). C. nigripes has a fairly wide distribution in the Australasian region including Taiwan, the Philippines, China (Yunnan, 1 Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. 2 Department of Biology, Hardin-Simmons University, Abilene, TX 79698Ð Corresponding author, ksukonta@mail.med.cmu.ac.th. Hainan), Vietnam, Laos, Thailand, Malaysia, Indonesia, Nepal, India, Sri Lanka, Pakistan, New Guinea, and Australia (Kitching 1976, Kurahashi and Afzal 2002), but information pertaining to aspects of forensic importance of this species is limited (Kitching 1976, OÕFlynn 1980, Greenberg and Kunich 2002). We report herein the morphology of each instar of this ßy species, focusing primarily on characteristics that will allow identiþcation of larval specimens of C. nigripes. Materials and Methods All instars of C. nigripes used in this study were obtained from a laboratory colony located at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. Laboratory colonies are maintained at natural temperature and light/ dark photoperiod for the area by using the method described by Haskell (1990), and larvae are fed a fresh pork liver diet. Adults are fed a 10% sugar solution mixed with a small amount of multivitamin syrup. For the compound microscopic examination, 25 larvae of each instar were collected from the colony and washed several times with normal saline solution to remove surface artifacts and liver tissue. Specimens were killed by transferring them into a beaker of nearly boiling water for 5 min. The stiffened, dead larvae were then cut at two different sites by using a sharp blade to section the body into three portions. The Þrst cut site is at the mesothorax (third segment) to provide a body portion conducive to viewing the cephalopharyngeal skeleton, anterior spiracle, and spines between the pro-and mesothorax. The second cut site is at the 11th body segment so that the caudal /05/0233Ð0240$04.00/ Entomological Society of America

2 234 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 segment of the body can be positioned for more adequate viewing of the overall morphology of the posterior spiracles. Mounting of the larval bodies was carried out by transferring each portion (cephalic region, abdominal trunk, and caudal segment) into a few drops of Entellan (Merck, Germany) on a clean glass slide. A coverslip was then placed over the specimens and examination of morphological characters of the specimens was made using a Olympus compound microscope equipped with a calibrated ocular micrometer. Images were subsequently recorded with a digital camera (Nikon, Tokyo, Japan). For scanning electron microscopy (SEM) observation, larvae of each instar were collected from the colony and washed several times with normal saline solution to remove the surface artifacts and liver tissue. Specimens were killed by transferring them into a beaker containing water near the boiling point for 5 min. The dead larvae were then preþxed with a 2.5% glutaraldehyde mixture in phosphate-buffered saline (PBS), ph 7.4, at 4 C for 24 h. In addition, larvae were rinsed twice with PBS at 10-min intervals, and postþxed with 1% osmium tetroxide at room temperature for 3Ð4 d. Specimens were then rinsed twice with PBS and dehydrated with alcohol. The dehydration process involved the larvae being sequentially subjected to the following increased alcohol concentrations: 30, 50, 70, 80, and 90%. Larvae remained in each concentration of alcohol for 12 h during each step of the dehydration process. After treatment in the alcohol concentrations, they were placed in absolute alcohol for another two 12-h periods followed by treatment in acetone for two 12-h periods. Finally, the larvae were subjected to critical point drying, attached to double-stick tape on aluminum stubs and coated with gold in a sputter-coating apparatus for them to be viewed under a JEOL-JSM840A scanning electron microscope. Results The body of the Þrst instar is the typical muscoidshaped vermiform larva that is pointed anteriorly and blunt in the posterior end. The most obvious feature in the cephalic region is the cephalopharyngeal skeleton, which is completely internal. It is dark and has a characteristic morphological shape that can be seen in Fig. 1. The cephalopharyngeal skeleton of the second instar is more developed, and its morphological shape (Fig. 2) is more linear and very different from that of the Þrst instar. However, the cephalopharyngeal skeleton is most developed in the third instar, with all portions of the apparatus being heavily pigmented, resulting in its very dark to black coloration. The entire margin of this structure is smooth, clear, and well deþned. The mouthhooks, located at the anterior end of the segment, are strongly bent downward (Fig. 3) and are Þtted for raking food into the mouth opening. The integument of all instars of C. nigripes lacks prominent tubercles along the body segments. Spines in the dorsal region of the body, particularly between the prothorax and mesothorax, are features that have been useful for larval identiþcation in other ßies; therefore, this feature was investigated in this study. Spines in this region of the Þrst instar all have a single point that is abruptly tapered and darkened at the tip (Fig. 4), whereas most of these have two, three, or multiple tips in the second instar (Fig. 5). In the third instar, spines in this dorsal region have a clustered distribution. They are arranged in straight groups of two to Þve spines (Fig. 6) instead of being arranged singly as in the Þrst and second instars. Each spine has one to three points and is darkened at the tip. Examination under the light microscope revealed some characteristic dark bands in the dorsal region of the middle part of the body of the second instar. These dark bands are made up of dense patches of setae restricted primarily to the anterior portion of each segment (Fig. 7, arrowheads). These dark bands are not found in the Þrst and third instars. The anterior spiracles are situated posterolaterally in the prothorax (second segment) and are important features for identifying ßy larvae. The anterior spiracles of the Þrst instar are either rudimentary or absent. In the second and third instars, they are prominent and look fan-shaped with a single row of nine to 13 papillae (10 being the most common) along their distal edge (Fig. 5). The last abdominal segment bears the posterior spiracles, which are rudimentary in the Þrst instar. Two posterior spiracular slits that coalesce ventrally are located in prominences of each spiracle (Fig. 8). Two completely separated slits are found in the second instar, with the slits in the medial position being shorter than those laterally positioned (Fig. 9). The posterior spiracles of the third instar each bear three prominent and separated slits encircled by a dark, thick peritreme that is incomplete ventromedially (Fig. 10). The scanning electron micrographs showed that muscoid and vermiform shape of the Þrst instar is comprised of 12 obvious body segments (Fig. 11). In the cephalic region, a pair of maxillary palp is located beneath the antennae and the ventral organs are situated in line beneath them (Fig. 12). The underdeveloped oral groove is apparent as a small suture between two swollen ridges (Fig. 12). Viewed posteriorly, it can be seen that the Þrst instar bears a pair of posterior spiracular disks in the dorsal half of the caudal segment and a pair of shallow concavities beneath each disk (Fig. 13). At higher magniþcation, each posterior spiracular disk bears two rudimentary slits that coalesce ventrally and are interspaced with extensively branched spiracular hairs (Fig. 14). As would be expected, the second instar is larger in size than the Þrst instar (Fig. 15). In the cephalic region of the second instar, not much has changed in the appearance of the antennae and maxillary palp (Fig. 16). Regarding its thoracic segments, the anterior spiracles arise posterolaterally on the prothorax and are obvious as fan-shaped structures bearing a single row of nine to 13 papillae along the distal margin of each (Fig. 17). Just beneath the anterior spiracle,

3 May 2005 SUKONTASON ET AL.: MORPHOLOGY OF C. nigripes LARVAE 235 Figs Light micrographs of C. nigripes larvae. (1) Cephalopharyngeal skeleton of Þrst instar. (2) Cephalopharyngeal skeleton of second instar. (3) Cephalopharyngeal skeleton of third instar. (4) Spines between prothorax and mesothorax of Þrst instar. (5) Spines between prothorax and mesothorax of second instar. (6) Spines between prothorax and mesothorax of third instar. as, anterior spiracle. Bar, 50 m for all Þgures. the morphology of the thoracic spines that are emphasized in this article can be seen in Fig. 17 where each spine bears multiple tips that project backwards. Trichoid sensilla, pit sensilla, and dome-shaped sensilla can all be found on the ventral surface of the thoracic segments (Fig. 18). Focusing on the abdominal trunk segments, those of the second instar are distinct from the Þrst instar. On the anterodorsal surface of the sixth segment, a smaller assemblage of minute setae can be detected, but a thick, wooly patch of these setae is obvious in this position on all of segments 7Ð11 (Fig. 19). In sharp contrast, the posterodorsal surface of these segments is completely devoid of these setae. The caudal segment bears a pair of posterior spiracles, with each posterior spiracular disk containing two spiracular slits that, unlike in the Þrst instar, are now more developed and entirely separated (Fig. 20). The overall morphology of the third instar is similar to that of the second instar but is much larger. In Fig. 21, the fully developed antennae, maxillary palp,

4 236 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 Figs Light micrographs of C. nigripes larvae. (7) Lateral view of the second instar showing the dark patches of spines on the dorsal surface (arrowheads) (8) Posterior spiracles of Þrst instar with each spiracle bearing two rudimentary slits (s). (9) Posterior spiracles of second instar with each spiracle bearing two separated slits (s). (10) Posterior spiracles of third instar with each spiracle bearing three separated slits encircled by a dark peritreme. Bar, 100 m for all Þgures. mouthhooks, and oral grooves can be clearly seen. Higher magniþcation of the maxillary palp reveals its complexity and its arrangement into a cluster of papillae and two disjunct papillae (Fig. 22). The number and shape of the papillae on the fan-shaped anterior spiracles are similar to that of the second instar (Fig. 23). The intersegmental spines located between the prothorax and mesothorax and just beneath the anterior spiracle are arranged in distinctive straight groups of two to Þve spines with each spine having one to three points at its tip (Fig. 24). The integument of the abdominal trunk segments of the third instar differs from the second instar in lacking the characteristic patches of dorsal setae seen in the second instar. Segments 6 and 7 have two parallel, lateral ridges Þt closely together on each side and each ridge has an irregular cuticular surface (Fig. 25). The following segments also bear the ridges but differ in being covered with backward-projecting setae on their cuticular surfaces (Fig. 25). Bubble membranes were located near the posterior lateral edges of the Þfth segment (Fig. 25, arrow). A group of 35Ð50 globular structures were discovered within a shallow cavity of each bubble membrane (Fig. 26). In viewing the caudal segment in posterior view, the pair of posterior spiracles was prominent (Fig. 27). Each posterior spiracular disk contains three slits that are entirely separated and interspaced with Þne branches of highly branched spiracular hairs (Fig. 28). The ecdysial scar or button (Fig. 28, arrow) is situated ventromedially on each disk. Discussion To properly identify ßy larvae of Muscomorpha to the species level, morphological features such as the anterior spiracles, posterior spiracles, and internal cephalopharyngeal skeleton are usually used (Liu and Greenberg 1989; Smith 1986, 1989; Goff and Catts 1990; Omar 2002). Moreover, characteristics of the spines along the body, particularly in the third instar, have sometimes been reported as useful taxonomic features. Spines are morphologically variable among body segments; therefore, when comparing characteristics of spines, only those from the same particular region of a body segment should be compared. Wallman (2001) clearly illustrated, using light micrographs, the differences in spine morphology and arrangement in the dorsal region of the anterior spine band on the Þrst abdominal segment of third instar of various Calliphora species [i.e., Calliphora hilli hilli

5 May 2005 SUKONTASON ET AL.: MORPHOLOGY OF C. nigripes LARVAE 237 Figs SEM micrographs of Þrst instar of C. nigripes. (11) Lateral view of entire larva showing tapered anterior end (a) and truncate posterior end (p). Bar, 100 m. (12) Ventral view of cephalic region showing antenna (an), maxillary palp (mp), ventral organ (vo) and oral groove (og). Bar, 10 m. (13) Posterior view of caudal segment showing a pair of posterior spiracular disks (psd) and a pair of shallow concavities beneath. Bar, 10 m. (14) Higher magniþcation of the pair of posterior spiracles, each bearing two slits (s) that coalesce ventrally and are interspaced with extensively-branched spiracular hairs (psh). Bar, 10 m. Patton, Calliphora dubia (Macquart), Calliphora augur (F.), Calliphora vicina Robineau-Desvoidy, Calliphora stygia (F.), Calliphora maritima Norris, and Calliphora albifrontalis Malloch]. The characteristic spine arrangement on the caudal segment also can be used to distinguish between the larvae of the screwworms, Cochliomyia hominivorax (Coquerel) and Cochliomyia macellaria (F.) (Erzinclioglu 1987). In C. nigripes, the spine morphology between the prothorax and mesothorax of the third instar, in conjunction with the morphology of their posterior spiracles, has proven to be extremely valuable in differentiating larvae of this species from those of C. megacephala in cases involving mixed populations within a human corpse (Sukontason et al. 2004). Characteristics of other features such as the anterior spiracles, cephalopharyngeal skeleton, and laterodorsal spines on body segments have previously been noted in taxonomic keys by Omar (2002) to distinguish between third instar of C. nigripes and C. megacephala. Several morphological features of the Þrst instars of C. nigripes shown in this study look similar to those of other species of Chrysomya, such as C. rufifacies or C. megacephala, frequently found associated with each other in corpses in Thailand (Sukontason et al. 2003a, b). This includes structures at both the anterior and posterior ends, but in particular, the similarities of the posterior spiracles and the pair of shallow concavities on the ventral half of the caudal segment (Figs. 13 and 14). Rearing to the second instar of these species would allow them to be distinguished using the presence of the dorsal patches of spines on body segments 6Ð11 of C. nigripes or the presence of the distinct, stout tubercles encircling the body segments of C. rufifacies (Sukontason et al. 2003a). Further rearing of second instar to the third instar would certainly allow accurate identiþcation of these Chrysomya species based on their speciþc morphologies. This study clearly shows that the second and third instars of C. nigripes possess distinguishing morphological characteristics. One major feature is the patch of minute setae on the anterior portion of the dorsal surface of segments 6Ð11 in second instar. This feature is fairly difþcult to observe using only light microscopy (Fig. 7), but is obvious when using SEM (Figs. 15 and 19). These patches of setae are lost from the dorsal surface in the third instar and are replaced by two parallel ridges Þt closely together on the lateral edges

6 238 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 Figs SEM micrographs of second instar of C. nigripes. (15) Dorsal view of entire larva showing tapered anterior end (a) and truncate posterior end (p). Bar, 1 mm. (16) Ventral view of cephalic region showing dome-shaped antenna (an) and clustered maxillary palp (mp). Bar, 10 m. (17) Lateral view of area between the prothorax and mesothorax showing anterior spiracle (as) and rows of spines, with each spine bearing from one to four points at the tip. Bar, 10 m. (18) Ventral surface of thoracic segment showing trichoid sensilla (black arrow), pit sensilla (arrowhead) and dome-shaped sensilla (white arrow). Bar, 10 m. (19) Dorsal view of the seventh to ninth abdominal trunk segments showing thick patch of setae on anterior portion of each segment. Bar, 100 m. (20) Posterolateral view of caudal segment showing the pair of posterior spiracular disks (psd). Bar, 100 m. of the abdominal trunk segments (Fig. 25). These ridges are covered with backward-projecting setae on all but segments 6 and 7. Some aspects of these characteristics have been previously used to separate third instar of C. nigripes from those of Chrysomya saffranea (Bigot) and C. megacephala in Australia by Kitching (1976). During metamorphosis of the third instar into the prepupa and then pupa, the cuticle of the third instar is tanned to form a rigid ovoid structure called the puparium. Many cuticular structures of the old third instar still remain on the surface of the puparium and may be useful morphological features for differenti-

7 May 2005 SUKONTASON ET AL.: MORPHOLOGY OF C. nigripes LARVAE 239 Figs SEM micrographs of third instar of C. nigripes. (21) Oblique lateral view of cephalic region showing antenna (an), maxillary palp (mp), oral grooves (og) and the pair of mouthhooks (mh). Bar, 100 m. (22) Higher magniþcation of maxillary palp, comprised of a cluster of papillae and two disjunct papillae. Bar, 10 m. (23) Anterior spiracle. Bar, 10 m. (24) Spines between prothorax and mesothorax. Bar, 10 m. (25) Lateral view of the right side of abdominal trunk segments showing the position of the bubble membrane (arrow) toward the posterior edge of the segment and the two parallel, lateral ridges Þt closely together on each of the following segments. Ridges of the sixth and seventh segments are devoid of setae unlike the following segments whose ridges are covered with setae. Bar, 100 m. (26) Higher magniþcation of the bubble membrane comprised of 35 globules. Bar, 10 m. ating puparia of different ßy species. Liu and Greenberg (1989) compared the bubble membranes, a group of globular structures on the dorsolateral border of the Þfth segment, of the puparia of 10 different species of ßies of forensic importance. In the current study, SEM was used to observe that the bubble membranes of the third instar of C. nigripes have 35Ð50 globules in each. In comparison with the puparia of other blow ßy species in the study by Liu and Greenberg (1989), this number of globules in the bubble membrane is comparable with that of Phormia regina (Meigen) ( 40 globules) and Phaenicia coeruleiviridis (Macquart) ( 50 globules), but it is higher than that of C. macellaria (absent of globules), C. vicina ( 20 glob-

8 240 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 Figs SEM micrographs of posterior view of third instar of C. nigripes. (27) Caudal segment showing the pair of posterior spiracular disks (psd). Bar, 100 m. (28) Higher magniþcation of a posterior spiracular disk, bearing three separated slits and highly-branched spiracular hairs encircled by a peritreme. Arrow indicates the button or ecdysial scar. Bar, 10 m. ules), Calliphora livida Hall ( 25 globules), Phaenicia sericata (Meigen) ( 23 globules), and Lucilia illustris (Meigen) ( 30 globules). However, the number of globules in C. nigripes is much less than that of Calliphora peruviana (Robineau-Desvoidy) ( 70 globules). In Thailand, the identiþcation of C. nigripes larvae from corpses was initially hard to discriminate from the other blow ßy species, especially the commonly collected C. megacephala. This study provides information concerning some important morphological features of all the larval stages of C. nigripes that should be useful in future forensic investigations in the geographical areas where this ßy species occurs. Acknowledgments We appreciate the suggestions of three anonymous reviewers. This work received support from the Faculty of Medicine Endowment Fund for Medical Research, Faculty of Medicine, Chiang Mai University. We thank the Chiang Mai University for funding reprint costs. References Cited Byrd, J. H., and J. L. Castner Insects of forensic importance, pp. 43Ð79. In J. H. Byrd and J. L. Castner [eds.], Forensic entomology: the utility of arthropods in legal investigations. CRC Press, Boca Raton, FL. Erzinclioglu, Y. Z The larvae of some blowßies of medical and veterinary importance. Med. Vet. Entomol. 1: 121Ð125. Goff, M. L., and E. P. Catts Arthropod basics structure and biology, pp. 38Ð71. In E. P. Catts and N. H. Haskell [eds.], Entomology and death: a procedural guide. JoyceÕs Print Shop, Inc., Clemson, SC. Greenberg, B., and J. C. Kunich Entomology and the law: ßies as forensic indicators. Cambridge University Press, Cambridge, United Kingdom. Haskell, N. H Procedures in the entomology laboratory, pp. 111Ð123. In E. P. Catts and N. H. Haskell [eds.], Entomology and death: a procedural guide. JoyceÕs Print Shop, Inc., Clemson, SC. Kitching, R. L The immature stages of the Old-World screw-worm ßy, Chrysomya bezziana Villeneuve, with comparative notes on other Australasian species of Chrysomya (Diptera, Calliphoridae). Bull. Entomol. Res. 66: 195Ð203. Kurahashi, H., and M. Afzal The blow ßies recorded from Pakistan, with the description of one new species (Diptera: Calliphoridae). Med. Entomol. Zool. 53: 213Ð230. Liu, D., and B. Greenberg Immature stage of some ßies of forensic importance. Ann. Entomol. Soc. Am. 82: 80Ð93. O Flynn, M. A IdentiÞcation of early immature stages of some common Queensland carrion ßies. J. Aust. Entomol. Soc. 19: 53Ð61. Omar, B Key to third instar larvae of ßies of forensic importance in Malaysia, pp. 143Ð147. In B. Greenberg and J. C. Kunich [eds.], Entomology and the law: ßies as forensic indicators. Cambridge University Press, Cambridge, United Kingdom. Smith, K.G.V A manual of forensic entomology. British Museum (Natural History), London, United Kingdom. Smith, K.G.V An introduction to the immature stages of British ßies. Royal Entomological Society of London, London, United Kingdom. Sukontason, K., K. L. Sukontason, R. Ngern-klun, D. Sripakdee, and S. Piangjai Differentiation of the third instar of forensically important ßy species in Thailand. Ann. Entomol. Soc. Am. 97: 1069Ð1075. Sukontason, K. L., K. Sukontason, S. Lertthamnongtham, B. Kuntalue, N. Thijuk, R. C. Vogtsberger, and J. K. Olson. 2003a. Surface ultrastructure of Chrysomya rufifacies (Macquart) larvae (Diptera: Calliphoridae). J. Med. Entomol. 40: 259Ð267. Sukontason, K. L., K. Sukontason, S. Piangjai, N. Boonchu, T. Chaiwong, R. C. Vogtsberger, B. Kuntalue, N. Thijuk, and J. K. Olson. 2003b. Larval morphology of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) using scanning electron microscopy. J. Vector Ecol. 28: 47Ð52. Wallman, J. F Third-instar larvae of common carrionbreeding blowßies of the genus Calliphora (Diptera: Calliphoridae) in South Australia. Invertebr. Taxon. 15: 37Ð51. Received 24 November 2004; accepted 18 February 2005.