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OSMOREGULATORY ORGANS OF IMMATURE CULICOIDES SONORENSIS (DIPTERA: CERATOPOGONIDAE) IN NORTH AMERICA1 Will K. Reeves2 ABSTRACT: Aquatic insects must regulate the ion concentrations of their haemolymph, and freshwater insects tend to loose ions to their aquatic environment. The osmoregulatory organs of larvae and pupae of C. sonorensis have not been previously reported. Silver nitrate was used to detect tissues in immature stages of C. sonorensis with active chloride exchange. Larvae of C. sonorensis actively exchanged ions with their anal papillae, cutaneous chloride cells, and to a lesser extent the hindgut. Only the cutaneous chloride cells were stained in their pupae. KEY WORDS: Diptera, Ceratopogonidae, biting midge, Culicoides sonorensis, osmoregulation, larva, pupa, chloride cells
Aquatic insects survive in habitats with fluctuating osmotic conditions and must regulate the ion concentrations of their haemolymph. Freshwater insects tend to loose ions to the environment. The water-saturated soils and wet feces in and around livestock feedlots support a variety of aquatic and semiaquatic Diptera, including Musca spp. and Culicoides spp. (Blanton and Wirth, 1979; Skoda et al., 1993). Larvae of Musca autumnalis and M. domestica can develop in semi-aquatic habitats containing wet dung, and probably use their anal organs to regulate the ion concentrations in their haemolymph (Stoffolano, 1970). The toxicity of chemicals that inhibit the osmoregulation of larval flies has been previously studied (e.g. Broce et al., 1988) and might represent a potential category of pesticides. Larvae of Culicoides spp. presumably use their anal papillae for osmoregulation but some species lack these organs (Murphree and Mullen, 1991). Lawson (1951) described the larvae of C. nubeculosus and used silver nitrate to stain the anal papillae, which he suggested they used for osmoregulation. The osmoregulatory organs of larvae and pupae of C. sonorensis have not been previously reported, but these organs are probably similar to those of C. nubeculosus. Understanding the osmoregulatory organs of C. sonorensis is important because they could possibly be targeted with pesticides. Culicoides sonorensis is a member of the C. variipennis complex that was recently revised by Holbrook et al. (2000). The C. variipennis complex contains three species: C. variipennis, Culicoides sonorensis, and Culicoides occidentalis. Culicoides sonorensis is economically important in North America, where it vectors two major viral diseases, bluetongue and epizootic hemorrhagic disease of deer, to domestic ruminants and wildlife (Mellor et al., 2000). METHODS The osmoregulatory organs of C. sonorensis were detected using the techniques described by Koch (1938). Briefly, 15 live 4th instar larvae, 4 dead larvae, and 7 ______________________________ 1
Received on January 22, 2008. Accepted on February 25, 2008.
2
Arthropod-Borne Animal Diseases Research Laboratory, USDA, 1000 E. University Avenue, Department 3354, Laramie, Wyoming 82071 U.S.A. E-mail:
[email protected]. Mailed on November 12, 2008
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pupae were removed from the VanRyan colony of C. sonorensis and allowed to soak in 150 ml of distilled water for 20 minutes. The colony has been maintained continuously at the USDA Arthropod-Borne Animal Diseases Research Laboratory, as described Hunt (1994). Ten live larvae, 3 dead larvae, and 5 pupae were placed in a Petri dish with 50 ml of 1% aqueous silver nitrate and held in the dark for 30 min. The larvae and pupae were then uncovered and held in bright light on a dissecting microscope for 20 min. Larvae and pupae in distilled water were compared to those in the silver nitrate and photographed (Figs. 1 and 2). Tissues that transport chloride ions were stained black when insoluble silver salts precipitated and blackened under bright light. The experimental larvae were immobilized because, 1% silver nitrate will paralyze and eventually kill immatures of Culicoides (Lawson, 1951). Unstained insects were immobilized with ice. Voucher specimens were deposited in the University of Wyoming Insect Collection. RESULTS Three different organs were stained by silver nitrate in live larvae of C. sonorensis, the anal papillae and cutaneous chloride cells (Fig. 1). The hindgut also turned light brown in some larvae, but there were no darkened tissues in the dead larvae. After staining with silver nitrate, cutaneous chloride cells were visible on the pupae (Fig. 2). DISCUSSION Tissues in dead larvae of C. sonorensis were not stained, which indicates that a physiological process caused the staining. Lawson (1951) noted that the anal papillae of C. nubeculosus were only lightly stained by silver nitrate, and he inferred that they actively reduced the silver nitrate. The anal papillae of C. sonorensis stained dark black (Fig. 1), which indicates that the reaction was a simple reduction of silver chloride. Single chloride cells were scattered over the body of the larvae of C. sonorensis (Fig. 1). Chloride cells were more numerous on the dorsum and near the ends of each segment. They were distributed on all body segments except the head capsule and were bilaterally asymmetrical. Komnick (1977) reviewed the structures of these cells in aquatic insects and noted their presence on larvae of other Nematocera. In addition to these external organs, a portion of the hindgut was lightly stained in some larvae. This indicates that the hindgut actively reduced silver nitrate but at a lower rate than the anal papillae or chloride cells. All of these organs are most likely involved with osmoregulation and the uptake of ions from the environment. Pupae of C. sonorensis float to the surface of their developmental habitat and are slightly hydrophobic. Chloride cells were visible on the pupae and were present on the all surfaces, but were most numerous on the ventral surface of the abdomen (Fig. 2). The chloride cells were generally smaller than those on the larvae; however, these organs must continue to exchange chloride ions with the environment. Previous descriptions of pupae of Culicoides (e.g. Lawson, 1951) did not mention chloride cells.
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Figures 1-2. 1. Silver nitrate stained (top) and unstained (bottom) larvae of Culicoides sonorensis. The black stained anal papillae and chloride cells, indicating the osmoregulatory organs, are marked with arrows. 2. Chloride cells on the abdomen of a pupa of Culicoides sonorensis are stained black with silver nitrate.
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ACKNOWLEDGEMENTS I thank Dr. W. Grogan, Jr. for his helpful review of the manuscript, Dr. A. Broce for his suggestions and help with the silver staining procedure and to L. DeBrey and J. Kempert for maintaining the laboratory colonies of C. sonorensis.
LITERATURE CITED Blanton, F. S. and W. W. Wirth. 1979. The sand flies (Culicoides) of Florida (Diptera: Ceratopogonidae). Arthropods of Florida and Neighboring Land Areas 10: 1-204. Broce, A. B., M. J. Gordowitz, and J. G. Riley. 1988. Effect of the ionophore lasalocid on face fly (Diptera: Muscidae) larval survival and physical and chemical parameters of cattle feces. Journal of the Kansas Entomological Society 61: 471-476. Holbrook, F. R., W. J. Tabachnick, E. T. Schmidtmann, C. N. McKinnon, R. J. Bobian, and W. L. Grogan. 2000. Sympatry in the Culicoides variipennis complex: a taxonomic reassessment. Journal of Medical Entomology 37: 65-76. Hunt, G. J. 1994. A procedural manual for the large-scale rearing of the biting midge, Culicoides variipennis (Diptera: Ceratopogonidae). U.S. Department of Agriculture, Agricultural Research Service, ARS-121. National Technical Information Service. Springfield, Virginia, U.S.A. pp. 1-68. Koch, H. J. 1938. The osmo-regulatory function of anal papillae in Culicidae. Journal of Experimental Biology 15: 152-160. Komnick, H. 1977. Chloride cells and chloride epithelia of aquatic insects. International Review of Cytology 49: 285-329. Lawson, J. W. H. 1951. The anatomy and morphology of the early stages of Culicoides nubeculosus Meigen (Diptera: Ceratopogonidae = Heleidae). Transactions of the Royal Entomological Society of London 102: 511-574. Mellor P. S., J. Boorman, and M. Baylis. 2000. Culicoides biting midges: their role as arbovirus vectors. Annual Review of Entomology 45: 307-340. Murphree, C. S. and G. R. Mullen. 1991. Comparative larval morphology of the genus Culicoides Latreille (Diptera: Ceratopogonidae) in North America with a key to species. Bulletin of the Society of Vector Ecology 16: 269-399. Skoda, S. R., G. D. Thomas, and J. B. Campbell. 1993. Abundance of immature stages of the housefly (Diptera; Muscidae) from five areas in beef cattle feedlot pens. Journal of Economic Entomology 86: 455-461. Stoffolano, J. G., Jr. 1970. The anal organ of larvae of Musca autumnalis, M. domestica, and Orthellia caesarion (Diptera: Muscidae). Annals of the Entomological Society of America 63: 1647-1654.
