Ultrastructure of spiracles of Musca domestica and Hydrotaea chalcogaster (Diptera: Muscidae)

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1 Parasitol Res (2006) 100:19 23 DOI /s ORIGINAL PAPER Ultrastructure of spiracles of Musca domestica and Hydrotaea chalcogaster (Diptera: Muscidae) Kabkaew L. Sukontason & Rungkanta Methanitikorn & Worachote Boonsriwong & Somsak Piangjai & Hiromu Kurahashi & Roy C. Vogtsberger & Kom Sukontason Received: 16 April 2006 / Accepted: 20 April 2006 / Published online: 9 June 2006 # Springer-Verlag 2006 Abstract Spiracles are major respiratory openings in the exoskeleton of insects. Oxygen, a necessary gas for cell activity, must pass through the spiracle to enter the respiratory system. In this study, we investigated the fine structure of spiracles of adult females of Musca domestica L. and Hydrotaea chalcogaster (Wiedemann), both medically important fly species in many parts of the world, by utilizing scanning electron microscopy. The mesothoracic spiracle of M. domestica is large and elongate-oval in shape, with its anterior end being gradually tapered. The outer surface is densely covered with slender setae of variable distribution and orientation. The metathoracic spiracle is semicircular or D-shaped, with its rim possessing long, fine, inwardly curved setae. A net-like valve or sieve plate, which has a smooth rim with swollen surface, is located within the atrium of this species. The abdominal spiracles are circular with a symmetrically swollen peritreme surrounding the opening. The inner filtering apparatus is composed of many spiral tubes, each possessing many small spines. As for H. chalcogaster, the tapering mesothoracic spiracle is covered with long setae arranged consistently inward from the peritreme, giving it a K. L. Sukontason (*) : R. Methanitikorn : W. Boonsriwong : S. Piangjai : K. Sukontason Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand klikitvo@mail.med.cmu.ac.th H. Kurahashi Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo , Japan R. C. Vogtsberger Department of Biology, Midwestern State University, Wichita Falls, TX 76308, USA combed appearance. The metathoracic spiracle is similarly arranged but triangularly rounded in shape, with the anterior and posterior rims possessing long fine setae. The net-like valve within the atrium has a smooth, swollen rim, whereas the inner edge of the atrium bears short, slender setae where it meets with the peritreme of the spiracle. The abdominal spiracles of this species look similar to that of M. domestica, with the exception of the filtering apparatus that bears only a few small spines. The function of these spiracles is discussed. Introduction Musca domestica L. and Hydrotaea chalcogaster (Wiedemann) are muscid flies of medical importance in many parts of the world. They are not only known for causing myiasis, mechanically transmitting various pathogens, and being domestic nuisance (Bohart and Gressitt 1951; Zumpt 1965; Greenberg 1971; Sukontason et al. 2000) but are also currently included in forensically associating fly species (Smith 1986; Carvalho et al. 2000). A micro-anatomical investigation of insects enables us to understand some functional morphological points of view. In insects, spiracles are major respiratory openings in the exoskeleton. Oxygen, a necessary gas for cell activity, must pass through the spiracle to enter the respiratory system. The structure of the spiracle or the difference in the thoracic and abdominal spiracle within an individual is morphologically representative of a variety among different groups of insect (Richards and Davies 1977). Considering the fact that some differences in the ultrastructure of the spiracle were observed in some flies of close species (Pape and Arnaud 2001), there have been few published works on the spiracle ultrastructure of flies of medical importance

20 Parasitol Res (2006) 100:19 23 (Fernandes et al. 2004). Therefore, the objective of this study was to determine and compare the ultrastructure of the external morphology of spiracles in adult M.

2 20 Parasitol Res (2006) 100:19 23 (Fernandes et al. 2004). Therefore, the objective of this study was to determine and compare the ultrastructure of the external morphology of spiracles in adult M. domestica and H. chalcogaster. Materials and methods Adult flies of M. domestica 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. The laboratory colonies were maintained in natural temperatures with light/dark photoperiods, and larvae are fed a fresh pork liver diet. Adults were fed on 10% sugar solution mixed with a small amount of multivitamin syrup. Specimens of H. chalcogaster were obtained from a bait-trap used in a fly survey in Chiang Mai, northern Thailand (17 21 N and E) in February They were kept in 70% alcohol for about a year before being prepared for the scanning electron microscopy (SEM) process. For scanning electron microscopy observation, adult flies of both species were washed several times with normal saline solution, fixed in 2.5% glutaraldehyde at 4 C for 24 h, then subjected to postfixation in 1% osmium tetroxide and dehydrated in a graded alcohol series. This was followed by treatment in acetone and critical point drying. Finally, they were mounted on stubs sputter-coated with gold and viewed under a JEOL JSM-5910LV scanning electron microscope. Results The spiracles of adult female M. domestica were located laterally on the mesothorax (Fig. 1), metathorax, and abdominal segments (Fig. 2). The mesothoracic spiracle lay on the dorsal margin of the pleuron, near the junction of the pronotum and mesonotum (Fig. 1). It was large and elongate-oval, with its anterior end tapering gradually (Fig. 3). The outer surface completely covered the finely porous opening with short, slender setae of variable distribution and orientation (Fig. 4). The metathoracic spiracle, located before halter, was semicircular or D-shaped, with its rim possessing long, fine, inwardly curved setae (Fig. 5). This metathoracic spiracle is atriate spiracle, having internal closing apparatus, appearing as a net-like valve or sieve plate, which had a valve lip with a smooth, swollen surface for covering the inside chamber or atrium (Fig. 6). The abdominal spiracles lay at the lateral margin of the tergites and were more or less circular with a symmetrically swollen peritreme surrounding the opening (Fig. 7). The intima was composed of many spiral ridges of cuticular lining or taenidia, each possessing many inwardly curved small spines (Fig. 8). Regarding adult female H. chalcogaster, the tapering mesothoracic spiracle was covered with long, slender setae arranged consistently inward from the peritreme, giving it a combed appearance (Figs. 9 and 10). Some small particles, probably dust, were seen attached to the surface of the setae (Fig. 10, arrowhead). The metathoracic spiracle was arranged similarly to M. domestica but triangularly rounded in shape, with the anterior and posterior rims possessing long, fine setae with ramified bases (Fig. 11). As the half-opened mechanism observed in this study, the valve lip was smooth and swollen. The internal closing apparatus had swollen valve lip. Next to this lip, the surface of closing apparatus is analogous to sieve plate or filter meshwork (Fig. 11, inset), as similarly seen in M. domestica (Fig. 6). The observed atrium had an internally smooth surface, whereas the inner edge of the atrium bore short, slender setae where it met with the peritreme of the spiracle (Fig. 12). The abdominal spiracles looked similar to those of M. domestica, being circular with a symmetri- Fig. 1 Scanning electron micrograph of adult female M. domestica. Lateral view of anterior region showing antenna (a), compound eye (c), anterior spiracle (as), and mouthpart (mp). Bar=500 μm Fig. 2 Scanning electron micrograph of adult female M. domestica. Lateral view of posterior region showing abdominal spiracle (s) on the fourth and fifth segment. Bar=500 μm

Parasitol Res (2006) 100:19 23 21 Fig. 3 Scanning electron micrograph of adult female M. domestica. Large, elongate-oval mesothoracic spiracle. Bar=50 μm Fig.

3 Parasitol Res (2006) 100: Fig. 3 Scanning electron micrograph of adult female M. domestica. Large, elongate-oval mesothoracic spiracle. Bar=50 μm Fig. 4 Scanning electron micrograph of adult female M. domestica. Higher magnification of mesothoracic spiracle densely covered with slender setae of variable distribution and orientation. Arrows indicate perforated pores. Bar=10 μm Fig. 5 Scanning electron micrograph of adult female M. domestica. Semicircular metathoracic spiracle, with its rim possessing long, fine inwardly curved setae. Bar=50 μm Fig. 6 Scanning electron micrograph of adult female M. domestica. Closing mechanism of the metathoracic spiracle showing net-like valve or sieve plate (right), which has a smooth rim with swollen surface (left). Bar=5 μm Fig. 7 Scanning electron micrograph of adult female M. domestica. Abdominal spiracle with swollen peritreme (p) and spiral ridges of taenidia (f). Bar=10 μm Fig. 8 Scanning electron micrograph of adult female M. domestica. Higher magnification of taenidia composed of many inwardly curved small spines. Bar=2 μm cally swollen peritreme surrounding the opening (Fig. 13). The intima was composed of many spiral taenidia, each bearing only a few small spines (Fig. 14). Discussion Respiration in flies is mediated by the multi-branched tracheal system, which has cuticular openings called spiracles located at the thorax and abdomen. This study showed that the mesothoracic, metathoracic, and abdominal spiracles are morphologically different, which is the case in both M. domestica and H. chalcogaster. In the mesothoracic spiracle, the size and orientation of the setae covering the finely porous opening is different in both fly species. The short setae, oriented in variable ways, were observed in M. domestica (see Fig. 3), and this agreed with the blow flies, Chrysomya rufifacies (Macquart) (Chapman 1991) or Chrysomya megacephala (F.) (unpublished data). The long setae, consistently arranged inward in H. chalcogaster (see Fig. 9), corresponded with that previously reported in the bot fly, Dermatobia hominis (L.) (Fernandes et al. 2004) orflesh fly, Parasarcophaga (=Liosarcophaga) dux (Thomson) (unpublished data). However, despite the difference in size and orientation of the setae, the function as a protection from entering the dust or fine particles in the tracheal system is comparable (Chapman 1998). Investigation using flowthrough respirometry, the role of carbon dioxide emission

4 22 Parasitol Res (2006) 100:19 23 Fig. 9 Scanning electron micrograph of adult female H. chalcogaster. Tapering mesothoracic spiracle covered with long setae having a ramified base and combed appearance. Bar=50 μm Fig. 10 Scanning electron micrograph of adult female H. chalcogaster. Higher magnification of setae covering the minute perforated plate (arrows). Dark arrowheads indicating small materials attached to the surface of the setae. Bar=2 μm Fig. 11 Scanning electron micrograph of adult female H. chalcogaster. Rounded triangular metathoracic spiracle, with long fine setae at the anterior and posterior rims. The smooth and swollen valve lip (v) is visible as a half-opened mechanism partially covering the atrium (a). Bar=20 μm. The sieve plate or filter meshwork (inset) is adjacent to the valve. Inset bar=1 μm Fig. 12 Scanning electron micrograph of adult female H. chalcogaster. Short and slender setae at the inner edge of the atrium. Bar=2 μm Fig. 13 Scanning electron micrograph of adult female H. chalcogaster. Abdominal spiracle with circular, swollen peritreme (p) surrounding the opening. Inward of the opening is the spiral taenidia (f) before the atrium (a). Bar=5 μm Fig. 14 Scanning electron micrograph of adult female H. chalcogaster. Few small spines at the taenidia. Bar=2 μm from the mesothoracic spiracle, was reported in the dry habitat species of the dung beetle (Duncan and Byrne 2005). In this study, the inner closing apparatus underneath the dense setae, which acts as filter apparatus of the mesothoracic spiracle in both M. domestica and H. chalcogaster, could not be clearly seen under SEM. However, when the mesothoracic spiracle of M. domestica was examined through light microscopy, the inner closing apparatus was observed (data not shown). This occurrence resembled the anterior prothoracic spiracle of the hawkmoth, Manduca sexta (L.) (Lepidoptera: Sphingidae), demonstrating that the inner closing apparatus is visible after the removal of the filter lamellae (Wasserthal 2001). In phorid fly, Megaselia scalaris (Loew), the inner closing apparatus of the mesothoracic spiracle was apparent under SEM, with the setae existing only at the rim of spiracle (unpublished data). Regarding the metathoracic spiracle, a slight difference was noticeable in its shape in that M. domestica looked D-shaped but somewhat triangularly rounded, contrary to that in H. chalcogaster. Nevertheless, the ultrastructure of the sieve plate in both fly species is similar, as well as the external covering of long setae. Comparing the morphology of the metathoracic spiracle with other flies, a difference was noticed in some species, for example, between Bezzimyia bisecta sp. n. and Bezzimyia barbarista sp. n. (Diptera: Rhinophoridae). When demonstrated by SEM, the metatho-

5 Parasitol Res (2006) 100: racic spiracle of the former species bears two short fringes, while the latter species has no fringe (Pape and Arnaud 2001). This study demonstrated that the short, slender setae inside the atrium of the metathoracic spiracle were clearly visible in H. chalcogaster but could not be seen in M. domestica due to the closing mechanism. The hair inside the atrium helps to prevent the entry of dust (Richards and Davies 1977). However, both fly species are endowed with the inner closing apparatus. Many authors indicated that closing the spiracles by the valve mechanism in a certain phase is a physiological adaptation that reduced water loss in insects (Richards and Davies 1977; Kestler 1985; Borrer et al. 1989; Chapman 1991; Lehmann 2001). Looking at the abdominal spiracle of M. domestica and H. chalcogaster, the closing apparatus could not be externally observed; but the inwardly curved small spines along the taenidia were noticeable. Nevertheless, the inner closing valve was reported internally, as depicted inside the atrium in some textbook (Gullan and Cranston 2000). In assessing between both fly species, a greater number of inwardly curved small spines along the taenidia was observed in M. domestica than in H. chalcogaster. Moreover, compared with other arthropods, the morphology of the abdominal spiracle of both fly species having small spines along the spiral taenidia was analogous to arthropods such as the thoracic spiracle of diplura, Campodea spp. (Order Diplura) (Eisenbeis and Wichard 1987), spiracle on the epinotum of the worker wood ant, Formica polyctena Förster (Order Hymenoptera) (Eisenbeis and Wichard 1987), and blow fly, C. megacephala or flesh fly, P. dux (unpublished data). This taenidia prevents the tube from collapsing under pressure while retaining its flexibility (Chapman 1991). In conclusion, the ultrastructural study of the spiracle in adult female M. domestica and H. chalcogaster in this study allowed us to understand the functional morphological viewpoint of this structure. Acknowledgements This work received support from the Thailand Research Fund. We thank B. Kuntalue for technical assistance. References Bohart GE, Gressitt JL (1951) Filth-inhabiting flies of Guam. Bernice P Bishop Mus Bull 204:1 152 Borrer DJ, Triplehorn CA, Johnson NF (1989) An introduction to the study of insects, 6th edn. Saunders College, USA Carvalho LML, Thyssen PJ, Linhares AX, Palhares FAB (2000) A checklist of arthropods associated with pig carrion and human corpses in Southeastern Brazil. Mem Inst Oswaldo Cruz 95: Chapman RF (1991) General anatomy and function. In: CSIRO Division of Entomology (ed) The insects of Australia, 2nd edn, vol 1. Melbourne University Press, pp Chapman RF (1998) The insects structure and function, 4th edn. Cambridge University Press, UK Duncan FD, Byrne MJ (2005) The role of the mesothoracic spiracles in respiration in flighted and flightless dung beetle. J Exp Biol 208: Eisenbeis G, Wichard W (1987) Atlas on the biology of soil arthropods. Springer, Berlin Heidelberg New York Fernandes FF, Chiarini-Garcia H, Linardi PM (2004) Scanning electron microscopy studies of sensilla and other structures of adult Dermatobia hominis (L. Jr., 1781) (Diptera: Cuterebridae). J Med Entomol 41: Greenberg B (1971) Flies and disease, vol I. Ecology, classification and biotic associations. Princeton University Press, NJ Gullan PJ, Cranston PS (2000) The insects. An outline of entomology, 2nd edn. Blackwell Science, UK Kestler P (1985) Respiration and respiratory water loss. In: Hoffmann K (eds) Environmental physiology and biochemistry of insects. Springer, Berlin Heidelberg New York, pp Lehmann FA (2001) Matching spiracle opening to metabolic need during flight in Drosophila. Science 294: Pape T, Arnaud PH Jr (2001) Bezzimyia a genus of native New World Rhinophoridae (Insecta, Diptera). Zool Scr 30: Richards OW, Davies RG (1977) Imms general textbook of entomology. 10th edn, vol 1. Structure, physiology and development. Chapman and Hall, London Smith KGV (1986) A manual of forensic entomology. Cornell University Press, NY Sukontason K, Boonchu M, Khantawa B, Sukontason K, Piangjai S, Choochote W (2000) Musca domestica as a mechanical carrier of bacteria in Chiang Mai, North Thailand. J Vector Ecol 25: Wasserthal LT (2001) Flight-motor-driven respiratory air flow in the hawkmoth Manduca sexta. J Exp Biol 204: Zumpt F (1965) Myiasis in man and animals in the Old World. Butterworths, London, UK

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

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