Observations on first and second-instar larvae of Megaselia scalaris (Loew) (Diptera: Phoridae)

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1 June, 2004 Journal of Vector Ecology 79 Observations on first and second-instar larvae of Megaselia scalaris (Loew) (Diptera: Phoridae) Noppawan Boonchu, Kom Sukontason 1, Kabkaew L. Sukontason, Tarinee Chaiwong, Somsak Piangjai, and Roy C. Vogtsberger 2 Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 2 Department of Biology, Hardin-Simmons University, Abilene, TX 79698, U.S.A. 1 Corresponding author Received 20 March 2003; Accepted 11 June 2003 ABSTRACT: The ultrastructure of the first and second-instar larvae of Megaselia scalaris (Loew) was studied using scanning electron microscopy (SEM). Significant changes in morphological features were observed in the anterior and posterior spiracles, but only minimal changes in the labium and mouthhooks were seen. The ultrastructure of M. scalaris larvae not only provides chronological transformation of their larval instars, but it can also be used to explain their feeding behavior and mode of respiration. In addition, morphological structures useful for specific identification of first or second-instar larvae collected from human corpses may be used in forensic investigations. Journal of Vector Ecology 29(1): Keyword Index: Megaselia scalaris, larvae morphology, forensic entomology, scanning electron microscopy. INTRODUCTION Megaselia scalaris (Loew), commonly known as humpbacked or scuttle fly, is an insect that affects humans in many different ways. It has been recorded not only as an annoyance pest of humans and animals in the natural environment and in laboratory insect colonies, but also as a cause of myiasis (Zumpt 1965, Singh and Rana 1989, Barre et al. 1991, Disney 1994). Maggots of M. scalaris are known to infest certain kinds of fruits (e.g., ripe banana) and may potentially cause intestinal myiasis in humans upon consumption (Karunaweera et al. 2002). In addition, specimens of M. scalaris collected from a corpse may be useful in forensic investigations. Due to its relatively small size (~ 2-3 mm), M. scalaris was the only insect found breeding within a tightly sealed corpse (Greenberg and Wells 1998). Toxicological analysis of pupal cases of this fly species was used to detect concentrations of amitriptyline and its metabolite, nortriptyline, in a corpse by Miller et al. (1994). Knowledge of the morphology of all the immature stages of M. scalaris would provide valuable biological information. To date, most of the morphological studies of this species involve only the egg, third-instar larva, and puparium, while information on the first and secondinstar larvae are lacking (Zumpt 1965, Smith 1986, Liu and Greenberg 1989, Sukontason et al. 2002). Thus, the objective of this present study was to determine if any of the morphological features of the first and secondinstar larvae were different from the mature third-instar. Information such as this may provide answers to questions about the behavior of M. scalaris maggots that feed upon human tissues. MATERIALS AND METHODS First and second-instar larvae of M. scalaris obtained from a laboratory colony maintained at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand were utilized in this study. Larvae were first collected from the colony and washed several times with normal saline solution. They were then pre-fixed with 2.5% glutaraldehyde mixed in phosphate buffer solution (PBS) at a ph of 7.4 at 4 C for 24 hr, rinsed twice with PBS at 10-min intervals, and post-fixed with 1% osmium tetroxide at room temperature for 3-4 d. Specimens were then rinsed twice with PBS and dehydrated with alcohol. The dehydration process sequentially subjected the larvae to the following increased alcohol concentrations: 30%, 50%, 70%, 80%, and 90%. Larvae remained in each concentration of alcohol for 12 hr during each step of the dehydration process. They were placed in absolute alcohol for another two 12 hr periods followed by acetone for two 12 hr

2 80 Journal of Vector Ecology June, 2004 Figures 1-8. Scanning electron micrographs of first-instar larva of M. scalaris. (1) Dorsal view of the entire body with anterior end facing left. (2) Frontal view of cephalic segment showing antenna (A), labrum (LB), mouthhook (MH) and labium (L). Arrow indicates maxillary palp complex. (3) Dome-shape of antenna with six lateral sensory papillae, four in a group and two removed. (4) Papillae on maxillary palp complex or terminal organ. (5) Rudimentary spines along the body integument. (6) Rows of spines on the first thoracic segment. (7) Postero-lateral view of the caudal segment showing the pair of posterior spiracular discs (PS). (8) Posterior spiracular discs each bearing two straight slits (S) coalescing ventrally and interspaced with groups of relatively broad-based posterior spiracular hairs (PSH).

3 June, 2004 Journal of Vector Ecology 81 Figures Scanning electron micrographs of second-instar larva of M. scalaris. (9) Antero-lateral view of cephalic segment showing antenna (A), maxillary palp complex (MPC), mouthhook (MH) and oral grooves (OG). (10) Deeply serrated mouthhooks (MH) and triangular-shaped labium (L). (11) Anterior spiracle evident as a circular structure bearing two straight slits (S) coalesced at one end. (12) Posterior spiracular disc appearing slender and constricted in the middle. Each expanded end of the disc bears two straight slits (S), while the posterior spiracular hairs (PSH) appear relatively slender compared to those of the first-instar larva. periods. Finally, the larvae were subjected to critical point drying, attached to double-stick tape on aluminum stubs and coated with gold in the sputter-coating apparatus in order to be viewed under a JEOL-JSM840A scanning electron microscope. RESULTS AND DISCUSSION The body of the first-instar larva of M. scalaris is cylindrical, tapered toward the head, and is approximately 750 µm in length (Figure 1). The cephalic region is about as wide as its length and is composed of two cephalic lobes (Figure 2). A tuberculate labrum is located between the bases of the deeply serrated mouthhooks, while the labium appears as a flattened, bi-lobed structure that curves ventrally. A pair of antennae (or dorsal organs) is located dorsally on each side of the cephalic region. The antenna is dome-shaped, with six sensory papillae located lateral to the antenna; four are clustered together in a group and two are removed from the cluster (Figure 3). The maxillary palp complex (or terminal organ) is evident as six papillae more or less arranged in a row (Figure 4). Rudimentary spines are present on the body integument both dorsally and laterally (Figure 5). Dense, toothlike spines, each bearing a single, sharp pointed tip, are arranged in several rows on the thoracic segment (Figure 6). A pair of protruding posterior spiracular discs is located dorsally on the caudal segment (Figure 7). Each of these posterior spiracular discs bears two straight slits that coalesce ventrally and are interspaced with groups of relatively broad-based posterior spiracular hairs (Figure 8). Overall, the morphology of the second-instar larva is generally similar to that of the first-instar. Unlike the first-instar, the oral grooves are more well-developed and evident (Figure 9). Each mouthhook appears as a semi-circular lobe that has its peripheral margin deeply serrated, and the labium appears as a large triangularshaped structure that curves ventrally (Figure 10). Each prothoracic spiracle is a circular papilliform structure

4 82 Journal of Vector Ecology June, 2004 that contains two straight slits that coalesce at one end (Figure 11). Conversely, the posterior spiracular discs appear slender and constricted in the middle. The expanded ends of the spiracular plate each contain two straight slits. Spiracular hairs originate at, or near, the constriction of the spiracular plate (Figure 12). This study clearly shows that the early larval stages of M. scalaris share many morphological features during their development, but several morphological changes have been observed in these larvae of the family Phoridae from the first to the second-instar. Far fewer changes have been seen in development from the second to the third-instar. This trend has also been observed in the larval metamorphosis of several blow fly species in the family Calliphoridae, such as Chrysomya rufifacies, Chrysomya bezziana, and Cochliomyia hominivorax (Kitching 1976, Leite and Guevara 1993). With exception of the larger body size seen in transformation to the next larval stage, the most prominent changes observed were in the prothoracic and posterior abdominal spiracles. The prothoracic spiracles appear to be absent in the first-instar larvae of M. scalaris since they could not be found under SEM observation, but are quite apparent as circular structures containing two spiracular slits in both the second and third-instar larvae. The spiracular slits of the posterior abdominal spiracles in the first-instar are rudimentary, but have relatively broad-based posterior spiracular hairs. This arrangement was also observed in C. rufifacies, Chrysomya megacephala, and C. hominivorax by Leite and Guevara (1993). Khole (1977) reported that spiracular hairs (= sun ray structures) are absent in the first-instar larvae of Lucilia cuprina and C. rufifacies, but present in second and third-instars; they are entirely absent in Sarcophaga ruficornis and C. megacephala. According to Khole (1977), sun rays help raise the posterior spiracles above the medium and increase the angle of contact with the medium to This enables the larvae to feed longer than the ones without the sun rays whose angle of contact is A minimal change was observed in the structure of the mouthparts during larval transformation of M. scalaris. In the first-instar larva, the labium is a ventrallycurving, bi-lobed structure; whereas, in the second and third-instars it is triangular and ventrally-curved (Sukontason et al. 2002). The ventral curvature of the labium is believed to help the larva in feeding, particularly in cases of myiasis where it is used to anchor the larva to the vertebrate tissue so it can successfully feed on the host. The mouthhooks showed little change having deeply serrated lateral margins in all larval instars. Of course, this adaptation may also promote successful feeding in the larvae to enable them to accumulate food energy for transformation to the pupal stage and eventual molt to an adult. Revelation of the ultrastructure of larvae of M. scalaris by means of SEM not only provides details of the chronological transformation of features of their larval stages, but it can also be used to help explain their feeding behavior and means of respiration. Use of this ultrastructure for the specific identification of first or second-instar larvae collected from corpses may be helpful in providing information in forensic investigations. Acknowledgments The authors are indebted to Dr. Henry Disney for providing valuable information concerning Megaselia scalaris. We gratefully acknowledge an anonymous reviewer for critically reviewing this manuscript. We thank Budsabong Kuntalue and Natchanart Thijuk for their assistance. This work received support from the Faculty of Medicine Endowment Fund for Medical Research, Faculty of Medicine, Chiang Mai University, Thailand. REFERENCES CITED Barre, N., H. Mauleon, G.I. Garris, and A. Kermarree Predators of the tick Amblyomma variegatum (Acari: Ixodidae) in Guadeloupe, French West Indies. Exp. Appl. Acarol. 12: Disney, R.H.L Scuttle Flies: The Phoridae. Chapman-Hall, London, 467 pp. Greenberg, B. and J.D. Wells Forensic use of Megaselia abdita and M. scalaris (Phoridae: Diptera): case studies, development rates, and egg structure. J. Med. Entomol. 35: Karunaweera, N.D., R.L. Ihalamulla, and S.P.W. Kumarasinghe Megaselia scalaris (Diptera: Phoridae) can live on ripe bananas a potential health hazard? Ceylon Med. J. 47: Khole, V Significance of sun-ray structure in the spiracles of blowfly larvae (Diptera: Calliphoridae). Comp. Physiol. Ecol. 2: Kitching, R.L The immature stages of the Old- World screw-worm fly, Chrysomya bezziana Villenuve, with comparative notes on other Australasian species of Chrysomya (Diptera, Calliphoridae). Bull. Entomol. Res. 66: Leite, A.C.R. and J.D.E. Guevara Scanning electron microscopy of the larval instars of Cochliomyia hominivorax. Med. Vet. Entomol. 7: Liu, D. and B. Greenberg Immature stages of some

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