Oncospheres of the shark tapeworm, Lacistorhynchus tenuis, were hatched in vitro from coracidia, by diluting the seawater with distilled water. Experiments with varying dilutions of seawater showed that a 50% dilution (about 600 milliosmoles per liter) was optimal, resulting in hatching of 85% of the oncospheres within 15 min. Per cent hatching was less with higher or lower dilutions. Urea or sucrose solutions of an osmolarity comparable to that of seawater did not induce hatching. Environmental conditions which induce hatching of cestode oncospheres in vitro have been determined for several cyclophyllidean species (Silverman, 1954; Bertzen and Voge, 1965; Voge, 1967). In tetrarhynchid cestodes, the oncosphere is surrounded by a ciliary envelope which must be shed in the first intermediate host before development can proceed. Riser (1956) established that copepods served as the first intermediate host for the shark tapeworm, Lacistorhynchus tenuis. Conditions which stimulate the hatching of oncospheres of this tapeworm from their ciliary envelope are described below. MATERIALS AND METHODS Gravid proglottids of Lacistorhynchus tenuis were obtained from a naturally infected female leopard shark (Triakis semifasciata) collected in the coastal waters off Los Angeles, California, and from one brown smooth hound (Rhinotriakis henlei) obtained by Dr. John Simmons at Berkeley Pier, Alameda County, California. Proglottids were washed briefly in Simmons' shark saline (Simmons, 1961), modified to contain 355 mM sucrose (to raise the osmolarity to that found in the shark gut). The proglottids were then transferred to petri dishes containing Millipore-filtered seawater, and stored at 9 C for 5 days. After 5 days, most of the eggs had undergone development but did not contain recognizable oncospheres. At this time, half the petri dishes containing eggs were brought to room temperature (i.e., 23 C) while the others were left at 9 C. To retard bacterial growth, antibiotics were added to the eggs at 23 C in a final concentration of 600 uAg streptomycin and 600 units of penicillin per ml seawater. After an additional 5 days, little further development was observed in the 9 C group, while those eggs kept at room temperature had become fully embryonated; nearly all of the coracidia had escaped from Received for publication 6 December 1968. * Supported in part by USPHS Research Grant 5-ROI-AI-07332. the egg on the 5th day. These coracidia were used in hatching experiments. The results presented below and shown in Figure 1 are based on weighted averages from three separate experiments performed with each solution, with an average number of 15 coracidia in each experiment. Using calibrated pipettes, 2to 3-day-old coracidia were placed in seawater in wells of depression slides, and test solutions were added as follows: freshly boiled, deionized glass-distilled water in 0.01-ml increments from 0.01 ml to 0.09 ml, to sufficient coracidial suspension to make a final volume of 0.10 ml. Coracidia were also transferred to large volumes of 950 milliosmoles/liter artificial seawater (Altman and Dittmer, 1964, p. 541), 950 milliosmolar sucrose solution, or 1,060 milliosmolar urea, and observed for 30 min or longer. Hatching experiments were performed as follows: First, an aliquot of coracidial suspension was placed in a depression slide, and the coracidia counted; experimental hatching solutions were then added and, after 15 min, the number of coracidia was again counted, the difference between the two counts being attributed to hatched oncospheres. The criterion for hatching was complete emergence from, or at least rupture of, the immobilized ciliary coat with partial emergence of the oncosphere. After the completion of the seawater dilution experiments, larger volumes (1 to 2 ml) of these solutions were prepared and their osmolarities determined either by freezing point depression on a Fiske Freezing Point Depression Osmometer, or by thermal resistance on a Mechrolab Vapor Pressure Osmometer. For the freezing point depression measurements, bottled standards were used at 500 and 1,000 milliosmoles/liter. With the vapor pressure machine, primary standard urea was dried to constant weight and diluted with boiled, glass-distilled water to make a 0.900 M solution. When the 900 milliosmole standard was checked against the bottled standards on the freezing point apparatus it was found to differ by only 20 milliosmoles.
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Mar 17, 2015
Chris L Chabot + 3
Open Access
