10.1007/s00436-005-0100-0

K. L. Sukontason¹, N. Bunchu¹, R. Methanitikorn¹, T. Chaiwong¹, B. Kuntalue² and K. Sukontason¹

(1)	Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand

(2)	Electron Microscopy Research and Service Center (EMRSC), Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand

Received: 20 November 2005 Accepted: 28 November 2005 Published online: 14 January 2006

Abstract The ultrastructure of adhesive device or the pulvilli, pad-like structure between the tarsal claws of the legs, is presented in the blowfly (Calliphoridae), housefly and relatives (Muscidae), and flesh fly (Sarcophagidae) through scanning electron microscopy. The blowfly species were Chrysomya chani, Chrysomya nigripes, Chrysomya pinguis, and Chrysomya villeneuvi; while those of the housefly and relatives were Musca domestica and Hydrotaea chalcogaster, respectively. Fresh fly species included Boettcherisca peregrina and Liosarcophaga dux. Numerous tenent setae were observed on the ventral side of the pulvilli. Two features of the tip of the tenent setae were found as a spatula-like (in C. chani, C. pinguis, C. nigripes, M. domestica, H. chalcogaster, B. peregrina, and L. dux) and spoon-like tip (in C. villeneuvi). Transmission electron microscopy of the tenent setae in M. domestica revealed the electron-lucent centrally located, suggesting an adhesive substance. These results provided anatomical information that allow us to not only understand the successful attachment of flies to smooth surfaces but also clarify their role as a mechanical carrier of microorganisms.

Several species of flies in the families Calliphoridae (blowflies), Muscidae (housefly and its relative), and Sarcophagidae (flesh flies) have been incriminated as a public health concern in many parts of the world. Due to their life cycle of synanthropic behavior, which is closely associated with the human environment, along with their feeding habit, adult flies in these groups could be mechanical carriers of numerous pathogenic microorganisms from filth to human food, thereby causing diseases to humans (e.g., Greenberg 1971; Levine and Levine 1991; Sukontason et al. 2000; Sulaiman et al. 2000). These pathogens (bacteria, virus, protozoa cyst, helminth ova, or larva) have been mechanically carried by flies via the external integument, particularly organs such as the mouth parts, wings, legs, or adhesive device that come into contact with filth (Tan et al. 1997). Study using scanning electron microscopy (SEM) revealed that a large number of bacteria, Escherichia coli 0157:H7, adhered to the surface of housefly mouth parts and proliferated in the minute spaces of the labellum, thereby suggesting a none simple mechanical carrier (Kobayashi et al. 1999). The adhesive device of the terminal structure between the tarsal claws of flies has also been demonstrated through SEM as a mechanical transporter of microorganisms. In this publication, we present the ultrastructure of the adhesive device in eight fly species, commonly found in downtown Chiang Mai, north Thailand, to reveal the functional morphology of this organ as a mechanical carrier of microorganisms.

Three fly species used in this study were from laboratory colonies maintained at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. They belonged to the family Calliphoridae (Chrysomya nigripes), Muscidae (Musca domestica), and Sarcophagidae (Liosarcophaga dux). The other five species were obtained from a fly collection in Chiang Mai Province, northern Thailand. They belonged to the family Calliphoridae (Chrysomya chani, Chrysomya pinguis, and Chrysomya villeneuvi), Muscidae (Hydrotaea chalcogaster), and Sarcophagidae (Boettcherisca peregrina).

Adult flies of each species were killed by placing them in a plastic bag put in a freezer that is set at 4°C for approximately 30 min. Flies killed individually were prepared for SEM processing, as described by Sukontason et al. (2005). Only M. domestica was selected as a subject model for inspection of the ultrathin section using transmission electron microscopy (TEM); the procedure of Sukontason et al. (2005) was followed.

Results

Basically, each fly leg was composed of a coxa, trochanter, femur, tibia, and five tarsal segments. Dorsally viewed, the pretarsus of the blowfly C. chani consisted of a pair of claws with sharp apices and a pair of pulvilli underneath each claw, by which, the empodium and unguitractor plate were not clearly seen (Fig. 1). For the housefly M. domestica, numerous tenent setae were seen on the ventral side of the pulvilli and claws (Fig. 2). In the sarcophagid B. peregrina, the dorsal view of the pulvilli represented a remarkably large, smooth pad (Fig. 3), while the ventral surface illustrated heavily packed tenent setae (Fig. 4).

Fig. 1 SEM of the adhesive device of flies. Top view of the tarsomere of the blowfly, C. chani, showing the tarsal claw (CL) and pulvilli (PU)

Fig. 2 SEM of the adhesive device of flies. Ventro-oblique view of the tarsomere of the housefly M. domestica showing the tarsal claw and pulvilli. Arrow indicates small attached material

Fig. 3 SEM of the adhesive device of flies. Top view of the tarsomere of the fresh fly B. peregrina showing the tarsal claw (CL) and pulvilli (PU)

Fig. 4 SEM of the adhesive device of flies. Ventral view of the tarsomere of fresh fly B. peregrina showing the densely packed setae of the pulvilli (PU) and empodium (EP)

In the examination using higher magnification of the pulvilli, the terminal setae of C. chani (Fig. 5), C. pinguis (Fig. 6), and C. nigripes (Fig. 7) were as morphologically bent outward as a spatula-like tip; however, those of C. villeneuvi were as bent inwardly as a spoon-like tip (Fig. 8). The terminal setae of M. domestica, H. chalcogaster, B. peregrina, and L. dux were similar to spatula-like tips (Figs. 9, 10, 11, and 12, respectively). However, some pointed tips were observed in M. domestica (Fig. 9).

Fig. 5 SEM of the tenent setae on the ventral surface of fly pulvilli. Spatula-like tip of the tenent setae of C. chani

Fig. 6 SEM of the tenent setae on the ventral surface of fly pulvilli. Spatula-like tip of the tenent setae of C. pinguis

Fig. 7 SEM of the tenent setae on the ventral surface of fly pulvilli. Spatula-like tip of the tenent setae of C. nigripes

Fig. 8 SEM of the tenent setae on the ventral surface of fly pulvilli. Spoon-like tip of the tenent setae of C. villeneuvi

Fig. 9 SEM of the tenent setae on the ventral surface of fly pulvilli. Tenent setae of M. domestica showing spatula-like and pointed-like tip

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Fig. 10 SEM of the tenent setae on the ventral surface of fly pulvilli. Spatula-like tip of tenent setae of H. chalcogaster

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Fig. 11 SEM of the tenent setae on the ventral surface of fly pulvilli. Spatula-like tip of tenent setae of B. peregrina

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Fig. 12 SEM of the tenent setae on the ventral surface of fly pulvilli. Spatula-like tip of tenent setae of L. dux

An ultrathin cross section through the tenent setae of M. domestica revealed an electron-dense thick wall, in contrast with the central electron-lucent (Figs. 13 and 14).

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Fig. 13 Ultrathin cross section through the tenent setae of the hind legs of M. domestica. Serial position of the tenent setae

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Fig. 14 Ultrathin cross section through the tenent setae of the hind legs of M. domestica. Higher magnification of tenent seta showing an electron-dense thick wall, with the central electron-lucent

The ultrastructure of the adhesive device has been investigated using SEM in several insect groups, such as Coleoptera (Betz 1996, 2003; Betz and Mumm 2001), Hymenoptera (Federle et al. 2001; Federle and Endlein 2004), Strepsiptera (Pohl and Beutel 2004), Diptera (McAlpine 1991; Gorb 1998; Niederegger et al. 2002; Niederegger and Gorb 2003; Gaume et al. 2004), and Heteroptera (Gorb and Gorb 2004). In insects, the adhesive device is not only used principally in locomotion and attachment on a smooth surface but also in catching and as a defense against predators (Betz and Mumm 2001; Betz and Kölsch 2004; Gorb 2004). In the blowfly Calliphora vicina, the contact behavior role of the tenent setae in attachment has been revealed using a high-speed video recording (Niederegger et al. 2002; Niederegger and Gorb 2003).

In this study, the SEM investigation showed two remarkable features of the tip of the tenent setae: the spatula-like tip (as seen in C. chani, C. pinguis, C. nigripes, M. domestica, H. chalcogaster, B. peregrina, and L. dux) and spoon-like tip (as seen in C. villeneuvi). The spatula-like tip in this study was similar to type I setae in the syrphid fly Episyrphus balteatus (Syrphidae) (Gorb 1998), kelp fly Amma blancheae (Coelopidae) (McAlpine 1991), or blowfly Calliphora vimitoria (Gaume et al. 2004). However, either the bearing spatula-like or spoon-like tip of the tenent setae enabled flies to increase the number of contact points for attachment to a surface (Gorb and Gorb 2004).

The electron-lucent at the central region of the tenent setae of M. domestica, as seen in TEM, was consistent with E. balteatus (Gorb 1998). It has been suggested that the electron-lucent areas may be due to lipid-containing substances, most of which are dissolved during the TEM procedure (Gorb 1998). Investigation in Stenus (Coleoptera: Staphylinidae) showed that the beetle appears to release tarsal secretion through the tenent setae, within which lipid and proteinaceous fraction are contained (Betz 2003). In C. vicina, the tenent setae were reported as filled with secretion (Gorb 1998). Likewise, an adhesive fluid was secreted by the fly pad of C. vomitoria (Gaume et al. 2004). Investigation on another blowfly species Calliphora erythrocephala showed that the location of the secretion released opened at the end of the tenent setae (Gorb 1998). Regarding this, the electron-lucent area in the tenent setae of M. domestica would imply that the substance secreted through their tip. Such substances appeared to be important not only for the successful attachment to smooth surfaces (Gorb 2004), but, in view of the mechanical vector, also as a glue for attaching microorganisms (see Fig. 2, arrow).

No remarkable differences in the microtopography of the adhesive device were observed between sexes of the fly species examined. These similarities would, therefore, be involved by similar functions and/or roles displayed by each adult sex. Investigations in northern Thailand showed that there was no difference between either sex of adult M. domestica in the role played as a mechanical carrier of 42 bacterial species; for example, coagulase-negative staphylococci, E. coli, Viridans streptococci, nonfermentative gram-negative bacilli, Klebsiella pneunoniae, Morganella morganii, Enterobacter cloacae, Providencia stuartii, Proteus mirabilis, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio cholerae non-01, and Staphylococcus aureus (Sukontason et al., unpublished data).

In conclusion, this investigation revealed the morphological features of adhesive devices in flies in the families Calliphoridae, Muscidae, and Sarcophagidae, which are commonly found in Thailand. These results provide anatomical information that allows us to clarify the role of these flies as a mechanical carrier of many microorganisms.

Original Paper

References