Research Product

Rivulus marmoratus is a naturally self-fertilizing cyprinodontid fish inhabiting mangrove marshes throughout the caribbean. As a result of internal self-fertilization this oviparous species is composed of a number of isogenic, homozygous lines (clones), several of which have been identified by histocompatibility experiments and maintained in laboratory culture for over 30 years. Simplified culture and handling methods are given and data are presented on the reproduction, growth and development of rivulus under laboratory culture as a prelude to the evaluation of its potential as a bioassay animal. Several types of bioassays were run and evaluated using rivulus: behavioral, carcinogenicity,teratogenicity, toxicity, and mutagenicity. Advantages and disadvantages of using rivulus for such bioassays are discussed. Behaviorally, rivulus is capable of detecting and avoiding water contaminated with H2S. They respond (EC50 = 123.6 ppb H2S) by leaping from the water and remaining emergent for various periods of time while respiring cutaneously. Hepatocellular carcinoma among other pathologic changes were observed in livers of rivulus a year after exposure of adults and larvae to diethylnitrosamine (45, 30, and 15 ppm in water) for 5 weeks and 12 weeks, respectively. No pathologic changes were found in embryos exposed similarly. High rates of various skeletal malformations resulted in offspring of adults exposed to dibutyl phthalate (DBP) and 2,3,4,6-tetrachlorophenol (TECP) at concentrations of 20, 10 and 5% (DBP - 0.740, 0.370, 0.185 mg/l; TECP - 0.220, 0.110, 0.055 mg/l) of the larval 96-hour LC50. No dose-response relationships of skeletal malformations were found for similar exposures to pentachlorophenol, 2,3,5-trichlorophenol or bromoform, however, chronic exposure of developing hatchlings to TECP resulted in fin and gill erosion and chronic exposure to bromoform produced dorsal fin abnormalities. As part of a mutagenesis bioassay 14 enzyme systems representing 28 loci were screened for the three laboratory clones and one wild-caught clone but no electrophoretic differences were found. Attempts to culture rivulus cells failed. Also, the karyotype of rivulus is not suitable for short-term cyctogenetic assays such as the sister chromatid exchange (SCE) assay. Alternatively, however, the toadfish (Opsanus tau) possesses a suitable karyotype for SCE analysis and we have been successful in culturing toadfish cells to fourth passage. Increased rates of SCE were obtained when toadfish cells were exposed in vitro to the mutagen ethyl methanesulfonate but not bromoform. Another set of experiments is presented which involves characterization of the nature of the toadfish cytochrome P450 system.