Observations of the third instar larva and puparium of Chrysomya bezziana (Diptera: Calliphoridae)

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1 Parasitol Res (2006) 99: DOI /s ORIGINAL PAPER Observations of the third instar larva and puparium of Chrysomya bezziana (Diptera: Calliphoridae) K. L. Sukontason & S. Piangjai & W. Boonsriwong & N. Bunchu & R. Ngern-klun & R. C. Vogtsberger & K. Sukontason Received: 10 January 2006 / Accepted: 2 May 2006 / Published online: 27 May 2006 # Springer-Verlag 2006 Abstract Observations on the ultrastructure of the third instar larva and puparium of the Old World screw-worm fly, Chrysomya bezziana, are presented utilizing both light microscopy (LM) and scanning electron microscopy (SEM). Results of this study indicate that the shape of the intersegmental spines between the pro- and mesothorax markedly differ from other blow fly species (Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes, Lucilia cuprina) in being broad-based with sharp recurved tips. Other characters such as the posterior spiracles, number of papillae on the anterior spiracles, oral grooves, and posterior spiracular hairs also differ. The strong and robust mouthhooks may explain the ability of larvae to penetrate deeply into human tissues. Perforated sieve plates covered with antler-like projections were observed within the anterior spiracles of the puparium of C. bezziana. The posterior spiracular discs each bear three spiracular slits with 2-μm wide openings that were viewed either open or closed by a membrane underneath. This study expands our knowledge of the fine details of the external morphology of both the third instar larva and puparium of C. bezziana, which is an obligatory myiasis-producing species in many regions. A key to differentiate the third instar of C. bezziana from other blow flies in Thailand is given. K. L. Sukontason (*) : S. Piangjai : W. Boonsriwong : N. Bunchu : R. Ngern-klun : K. Sukontason Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand klikitvo@mail.med.cmu.ac.th R. C. Vogtsberger Department of Biology, Midwestern State University, Wichita Falls, TX 76308, USA Introduction Chrysomya bezziana Villeneuve, the Old World screwworm, is of medical, veterinary, and economic importance because its larvae cause myiasis in humans and animals, especially livestock (Papasarathorn and Piyarasana 1962; Zumpt 1965; Alahmed 2004; Mahon et al. 2004). Published data shows that areas of the human body in which myiasis by C. bezziana has occurred include the mastoid (Papasarathorn and Piyarasana 1962), the scalp (Papasarathorn et al. 1967), the subdermis of the throat from a tracheostomy (Vitavasiri et al. 1995), the gangrenous wound (Nacapunchai and Laohavichit 1999), the foot (Prasad et al. 2000), and the mouth (Ng et al. 2003). This species has recently been reported as one of the forensic entomological specimens found associating in human corpses in Malaysia (Lee et al. 2004) and Thailand (Sukontason et al., unpublished data). Geographically, this fly is widely distributed in Africa, the Middle East, the Indian subcontinent, south-eastern Asia, and Australia (Mahon et al. 2004) and was recently recorded as an insect involved in parasitic zoonoses in Papua New Guinea (Owen 2005). Morphological and molecular analyses of C. bezziana populations by Hall et al. (2001) has revealed that there are two races of this fly species, one from sub-saharan Africa and the other from the Gulf region and Asia. General morphology of immature stages of C. bezziana has been described by previous authors (Zumpt 1965; Papasarathorn et al. 1967; Kitching 1976) using either light microscopy (LM) or scanning electron microscopy (SEM). The present paper is intended to describe more detailed morphological characteristics of the third instar larva and puparium of C. bezziana, highlighting those characters that can be used to differentiate among these immature stages of blow fly species of medical importance in Thailand using LM and SEM.

2 670 Parasitol Res (2006) 99: Fig. 1 Light micrograph of third-instar larva of C. bezziana. Anterior region showing the cephalopharyngeal skeleton (c), anterior spiracle (as), and spines between pro- and mesothorax (s) Fig. 2 Light micrograph of third-instar larva of C. bezziana. Anterior spiracle showing palmate shape due to five papillae arranged in single row Fig. 3 Light micrograph of third-instar larva of C. bezziana. Enlarged view of intersegmental spines between pro- and mesothorax highlighting their single, darkened and tapered tips that are recurved toward the body Fig. 4 Light micrograph of third-instar larva of C. bezziana. Caudal view of contiguous posterior spiracles. Dorsal ends of the inner and middle spiracular slits (sl) are slightly convergent; whereas, that of the outer slits is slightly divergent. Peritreme (p) is thick and incomplete Materials and methods Third instar larvae of C. bezziana were obtained by collecting specimens from a myiasis case in the Chiang Mai province of northern Thailand. Specimens were reared to puparia and adults (to confirm identification) in the laboratory of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, using fresh pork liver that was provided as the larval food source. A few third instar larvae of C. bezziana were selected for examination using LM. These specimens were washed several times with normal saline solution and killed by transferring into a beaker containing nearly boiling water for 5 min. The dead larvae were cut at two sites using a sharp blade to result in three body portions. The position of the first cut site was through the mesothorax (third body segment) to produce an anterior body portion for viewing the cephalopharyngeal skeleton, spines between the proand mesothorax, and the anterior spiracles. The position of the second cut site was through the 11th body segment to produce a posterior body portion conducive to positioning for optimal viewing of the morphology of the posterior spiracles. Permanent mounting of the anterior and posterior body portions was carried out by transferring each of them directly into a few drops of Entellan (Merck, Germany) that had been previously placed on clean glass slides. Cover slips were then placed over the specimens, and the examination of morphological characters was made using an Olympus compound microscope equipped with a calibrated ocular micrometer. Images were subsequently recorded with a digital camera (Nikon, Tokyo, Japan). To make observations via SEM, third instar larvae and puparia were first washed three times by shaking 30 min in a shaking bath to remove any pork liver tissue residue from body surfaces. Specimens were prepared for the SEM by blotting water from the body surfaces with filter paper, gently placing them onto double-stick tape on viewing stubs, and then coating them with gold for 30 s in a sputtercoating apparatus (SPI-MODULE Sputter Coater, USA). This process enabled viewing under a JEOL JSM-5910LV scanning electron microscope (Tokyo, Japan). Results The body of the third-instar larva of C. bezziana is of typical muscoid shape that gradually tapers anteriorly. When examined under a light microscope, the black cephalopharyngeal skeleton is easily visible internally through the prothorax in the cephalic region of the body (Fig. 1). Anterior spiracles are palmate in shape with each being composed of four to six papillae arranged in a single row and located at the dorso-posterior margin on each side of the prothorax (Figs. 1 and 2). The cuticular interseg-

3 Parasitol Res (2006) 99: Fig. 5 SEM micrograph of cephalic end of third-instar larva of C. bezziana. Anterolateral view of anterior region showing antenna (an), maxillary palp (mp), oral grooves (og), mouthhook (mh), and labium (l) Fig. 6 SEM micrograph of cephalic end of third-instar larva of C. bezziana. Anterior spiracle displaying four papillae arranged in a single row Fig. 7 SEM micrograph of cephalic end of third-instar larva of C. bezziana. Intersegmental spines between pro-and mesothorax showing their broad bases abruptly tapered to sharply recurved tips Fig. 8 SEM micrograph of cephalic end of third-instar larva of C. bezziana. Ventral view of a pair of trichoid sensilla that each bear three setae and are located on the anterior edge of mesothorax. Inset provides greater detail of trichoid sensillum at higher magnification Fig. 9 SEM micrograph of caudal end of third-instar larva of C. bezziana. Lateral view of the caudal segment showing posterior spiracles (ps) located in a slightly sunken depression Fig. 10 SEM micrograph of caudal end of third-instar larva of C. bezziana. Posterior view of caudal segment showing positioning of posterior spiracles (ps) Fig. 11 SEM micrograph of caudal end of third-instar larva of C. bezziana. Posterior spiracular discs each bearing three spiracular slits (sl) and fringed with posterior spiracular hairs (psh) around the outer periphery. Dorsal ends of inner and middle spiracular slits are slightly convergent, and that of the outer spiracular slit is slightly divergent. A small compressed button (b) is observed at the base of the slits Fig. 12 SEM micrograph of caudal end of third-instar larva of C. bezziana. Cluster of broad and branched flattened spiracular hairs

4 672 Parasitol Res (2006) 99: Fig. 13 SEM micrograph of cephalic end of puparium of C. bezziana. Dorso-lateral view of anterior region showing domed shape of prothorax with anterior spiracle (as) and recurved spines (s) between pro- and mesothorax Fig. 14 SEM micrograph of cephalic end of puparium of C. bezziana. Anterior spiracle with four papillae bearing oval spiracular openings Fig. 15 SEM micrograph of cephalic end of puparium of C. bezziana. Higher magnification within oval spiracular opening of the anterior spiracle showing perforated spiracular plate (or sieve plate) covered by antler-like projections Fig. 16 SEM micrograph of cephalic end of puparium of C. bezziana. Enlarged view of recurved intersegmental spines between the pro- and mesothorax mental spines between the pro- and mesothorax each have a single tapered point that is darkened and recurved toward the body (Fig. 3). At the caudal end, the posterior spiracles of the third instar larva are decidedly contiguous with each bearing three separated straight slits. The dorsal ends of the inner and middle spiracular slits are slightly convergent; whereas, the dorsal end of the outer spiracular slit is slightly divergent from the other two (Fig. 4). These spiracular slits are collectively encircled by a dark, thick peritreme that is incomplete ventro-medially around the compressed button (Fig. 4). Scanning electron micrographs of the third instar larva clearly showed the cephalic segment composed of a pair of nipple-like antennae adjacent to a pair of dome-shaped maxillary palps, a pair of large, robust mouthhooks, a broad area of transverse ridges between oral grooves, and a trilobed labium (Fig. 5). Four to six papillae were observed on each anterior spiracle (Fig. 6). The intersegmental spines between the pro- and mesothorax are very broad-based with each having a very sharply pointed tip that is recurved posteriorly (Fig. 7). While looking at the thoracic segments in ventral view, a pair of trichoid sensilla that were each composed of three setae were observed (Fig. 8). Upon observation of the caudal segment, the pair of posterior spiracles could be seen within a slightly sunken depression (Figs. 9 and 10). As was observed with LM, it was clear that the dorsal ends of the inner and middle spiracular slits are slightly convergent while those of the outer spiracular slits are slightly divergent (Fig. 11). With the use of SEM, it is evident that the spiracular slits are interspaced with bundles of broad and branched flattened spiracular hairs around their periphery (Fig. 12). The scanning electron micrographs of the cephalic end of the puparium of C. bezziana revealed a dome-shaped prothorax (Fig. 13). Anterior spiracles on the puparium consisted of a single row of four papillae with oval-shaped spiracular openings (Fig. 14). Upon closer examination with SEM, it was discovered that within each spiracular opening there is a spiracular plate (or sieve plate) that is perforated by small sized pore-like aeropyles ( μm) and covered with antler-like projections that probably function as filtering devices (Fig. 15). The intersegmental spines between the pro- and mesothorax appear unchanged from those observed on the third instar larva (Fig. 16). Observations on the posterior spiracles of the puparium, including the characteristics of the spiracular slits, posterior spiracular hairs, and button (Fig. 17) all corresponded with those made on the third instar larva of C. bezziana. When greater attention was given to examining the spiracular slits, it was revealed that the opening of the slit measured 2 μm in width (Fig. 18), and some were closed with a membrane-like structure underneath (Fig. 19).

5 Parasitol Res (2006) 99: papillae on anterior spiracle L. cuprina. Posterior spiracular peritreme incomplete 3 3. Four to six papillae on anterior spiracle; cuticular spines between pro- and mesothorax arranged singly; each spine is broad-based with darkened, sharply pointed tip C. bezziana. eight to 13 papillae on anterior spiracle 4 4. Cuticular spines between pro- and mesothorax often in rows; presence of dark patches of spines on dorsolateral area; nine to 13 papillae on anterior spiracle C. nigripes. Cuticular spines between pro- and mesothorax arranged singly; absence of dark patches of spines on dorso-lateral area; eight to 12 papillae on anterior spiracle C. megacephala Discussion Fig. 17 SEM micrograph of caudal end of puparium of C. bezziana. Caudal view of posterior spiracular discs each with the characteristic three spiracular slits (sl) with peripheral posterior spiracular hairs (psh) and basal compressed button (b) Fig. 18 SEM micrograph of caudal end of puparium of C. bezziana. Open posterior spiracular slit Fig. 19 SEM micrograph of caudal end of puparium of C. bezziana. Closed posterior spiracular slit with membrane-like structure underneath A key to simplify the identification of third instar of C. bezziana from other blow flies of medical importance in Thailand was given as follows: 1. Elongate tubercles present on the thoracic and abdominal segments; the tip of tubercles bearing many small spines; nine to 12 papillae on anterior spiracle C. rufifacies. Thoracic and abdominal segments lacking elongate tubercles 2 2. Posterior spiracular peritreme complete; cuticular spines between pro- and mesothorax with broad-based having darkened, sharply pointed tip; four to seven Before the current study, morphological study of the immature stages of C. bezziana has been limited to that by Kitching (1976) in which comparison of some of the morphological features were made with other flies in the genus Chrysomya. In this study, several detailed characteristics of the third instar larva and puparium of C. bezziana are presented. Our observations of the cuticular spine morphology between the pro- and mesothorax of C. bezziana larvae utilizing light and scanning electron microscopy clearly confirm this as a distinguished feature when compared to some other medically important fly species reported by Sukontason et al. (2004). The broad base and sharply pointed recurved spines of C. bezziana differ greatly in appearance when compared to those of other species that have been previously reported (i.e., Chrysomya megacephala, Chrysomya rufifacies, Chrysomya nigripes and Lucilia cuprina) (Sukontason et al. 2004, 2005). Both of these features of C. bezziana larvae vary from that of other blow fly species (Ishijima 1967; Kitching 1976; Sukontason et al. 2004, 2005). The profile of the oral grooves in the cephalic segment and the posterior spiracular hairs on the spiracular discs of the caudal segment of C. bezziana larvae also differ from the following blow fly species: C. megacephala, C. rufifacies, C. nigripes, and L. cuprina (Sukontason et al. 2003a,b, 2004, 2006). Comparison of posterior spiracular hairs was also used by Aspoas (1990) as one of the characteristics used for distinguishing between morphologically similar larvae of flesh fly species. The observations from scanning electron microscopy in this study provide some fine details of the morphology of the third instar larva of C. bezziana which may help explain some of the behavior of this species. For example, the feeding ability of C. bezziana larvae may be explained by the strong mouthhooks that are used for penetrating deeply into many tissues. Prasad et al. (2000) noted that

6 674 Parasitol Res (2006) 99: C. bezziana larvae could burrow into healthy tissue adjacent to wounds and could also penetrate into bones surrounding the nasal sinuses. Other components observed in the third instar larva are used as sensory organs such as the antennae, maxillary palps, and trichoid sensilla. These structures were morphologically similar by comparison to other blow flies such as C. megacephala, C. rufifacies, and C. nigripes (Sukontason et al. 2003a,b, 2004). In studies of the fine structures of house fly larvae (Musca domestica) using transmission electron microscopy (TEM), the domeshaped structure of the antenna was assumed to be an olfactory receptor (Chu-Wang and Axtell 1971); whereas, the maxillary palp may serve a dual function for both contact chemo- and mechanoreception (Chu-Wang and Axtell 1972). Regarding fine morphology of the puparium of C. bezziana, we provide finer details of the palmate anterior spiracles which clearly contain perforated sieve plates within that are covered with antler-like projections. In addition, the minute furrow of the posterior spiracular slits was observed as either open or closed by a membrane underneath. The present study expands our knowledge of the fine details of the external morphology of both the third instar larva and puparium of C. bezziana, which is an obligatory myiasis-producing species in many countries, including Thailand. Acknowledgements We are grateful for the technical assistance given by Budsabong Kuntalue. We thank the Faculty of Medicine, Chiang Mai University, and the Thailand Research Fund for their support. References Alahmed AM (2004) Myiasis in sheep farms in Riyadh Region, Saudi Arabia. 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