Scanning Electron Microscopic Studies of the Life Cycle of Ichthyophthirius multifiliis

Abstract

All stages in the life cycle of the holotrichous ciliate Ichthyophthirius multifiliis were examined using the scanning electron microscope. Different procedures were used to prepare the tomite, trophozoite and cyst stages. Photomicrographs of freeze dried and critical point dried organisms fixed with glutaraldehyde are presented along with views of trophozoites and tomites deciliated by dibucaine hydrochloride to reveal the pellicle and buccal cavity normally covered by cilia. The holotrichous ciliate Ichthyophthirius multifiliis Fouquet, 1876 is a worldwide obligate ectoparasite of freshwater teleosts (Paperna, 1972; Nigrelli et al., 1976). Epizootics of ichthyophthiriasis rarely occur in freely flowing water (Allison and Kelley, 1963; Kozel, 1976) but can devastate populations of cultured and feral fishes in close confinement, with younger fish being most susceptible (Pillay, 1968). The life cycle has 3 stages termed tomite, trophozoite, and cyst. The small 15 x 40 ,um fusiform tomite, also termed theront, is the infective stage and can survive only 96 hr without a host (Suzuki, 1935). The narrowed anterior end of the tomite possesses a characteristic filamentous projection from a conical depression in the pellicle. This filament differs from the somatic infraciliature in its 3 times greater length and usually rigid orientation (Kozel and Dobra, 1978). Cytochemical techniques have demonstrated a capsule-like structure, also located near the anterior end of the tomite, which contained acid phosphatase and non-specific esterases. The capsule and anterior filament may be part of a perforatorium as cells partially burrowed into host tissues have never been observed to possess a trailing filament (Kozel, 1980). Beckert (1975) considered the tomite to be able to drill its way into the host epithelium with its pointed anterior end and its cilia. After burrowing into the epidermis of the teleost the tomite becomes spherical and develops its oral apparatus. It is now called a trophozoite. The parasite cilia dislodge surrounding host tissue and the trophozoite ingests the debris through its cytostome. The buccal cavity is a rigid cuplike Received 1 June 1984; revised 19 October 1984; accepted 23 October 1984. * Send reprint requests to GWF. t Emeritus Professor of Biology. organ located near the anterior end and is equipped with long cilia that beat continually inward (MacLennan, 1935). The trophozoite grows at a linear rate, dependent on temperature, and leaves the host when it reaches a diameter of 100-1,000 ,um (MacLennan, 1942). The freed trophozoite swims in slow spirals, compared to the tomite, and eventually adheres to surfaces of its environment. A fine gelatinous exocyst is exuded, the oral apparatus is resorbed and schizogony begins. The first 2-4 daughter cells of the schizont form an endocyst of fine anastomosing fibrils that reinforce the exocyst. The endocyst may be formed in more than 1 layer in larger schizonts (MacLennan, 1937). The endocyst(s) and exocyst are not uniform in thickness and for the ultrastructure of these membranes and of the schizonts, the reader is referred to Ewing et al. (1983), and Brown and Gratzek (1980). A large cyst may release up to 1,000 infective tomites to complete the cycle. The purpose of this paper is to present scanning electron micrographs of the entire life cycle of I. multifiliis and the ultrastructure of several surface features. MATERIALS AND METHODS A culture of I. multifiliis was maintained in the laboratory by serial passage on non-immune 8 to 20 cm channel catfish (Ictalurus punctatus) kept in 80-liter aquaria. Water temperature was maintained at 22 C and aeration provided 7-8 ppm dissolved oxygen. The water pH (6-8) was controlled by adding 3 millequivalents/litre of NaHCO3 as a buffer and adjusting with 1 molar NaOH or HC1. Trophozoites were collected by rubbing infected fish by hand in 18-cm diameter syracuse dishes filled with aquaria water filtered through 0.45-micrometer filters and termed biologically conditioned water (BCW). This filtered water served as a culture medium for I. multifiliis allowing the parasite to complete its life cycle. When a number of trophozoites came off the catfish they were transferred by Pasteur pipet through 2 washes of distilled deionized water, 10 min in each wash.