A Description of the Complete Metamorphosis of the Sea Urchin Lytechinus Variegatus Cultured in Synthetic Sea Water

The urchin Lytechinus variegatus was cultured through metamorphosis, from eggs and sperm collected from mature forms, in synthetic sea water, in the laboratory. A unicellular green alga was used as the nutritional source. A description and photographic record are presented of the complete development from fertilized egg to adult. The general pattern of the metamorphosis of sea urchins has been described in some detail by Hyman (1955), who used larval forms of Psammechinus, Paracentrotus, Echinus, and Anthocidaris as examples. A photographic study of the larval development of Arbacia punctulata under laboratory conditions was published by Harvey (1956), who noted the difficulty of obtaining stages later than those of the newly metamorphosed adult. Tennent (1910), in a study primarily concerned with abnormality in development, reported the culture of Lytechinus variegatus in the laboratory. He did not describe the later metamorphic stages. A paper by Hinegardner (1969) described the development of Lytechinus pictus under laboratory conditions and provided information on culture techniques. The present study describes the culture and development of Lytechinus variegatus in synthetic sea water and includes a photographic record of twenty-three stages from fertilization to adult. MATERIALS AND METHODS Specimens of Lytechinus variegatus were obtained from Florida (The Gulf Specimen Company, Panacea, Florida) and kept at 21°C in ten-gallon aquaria containing synthetic sea water (Instant Ocean, Aquarium Systems, Inc., Wickliffe, Ohio). Specific gravity was maintained at 1.023 (salinity 32°/100). The aquaria were fitted with undergravel filters covered with a three-inch layer of limestone as a filtering medium. The urchins were maintained from four to six months on a diet of frozen shrimp. The fertile period of Lytechinus variegatus is from March to October. Gametes were obtained by injecting 5 ml of 0.5 M KCL into the peristomial membrane with a hypodermic needle. Few urchins survived for more than one to two weeks after injection (Tyler, 1966). Hinegardner (1969) reported that urchins injected with 0.1-0.2 molar acetylcholine-sea water solution are more likely to survive. Sperm were collected by pipette and stored without water in sterile plastic petri dishes. Eggs were collected by inverting the urchin over a beaker containing synthetic sea water. Both eggs and sperm were immediately refrigerated at 5°C to preserve their viability. Sperm suspensions consisted of one to two drops of the concentrated sperm in 10 ml of synthetic sea water. Egg suspensions were prepared in 150-ml Erlenmeyer flasks containing 100 ml of synthetic sea water; a sufficient number of eggs was transferred by pipette from the stock container to form a thin layer of eggs on the bottom of the flasks. Both suspensions were kept in ice baths during their use in the laboratory; sperm suspensions were especially susceptible to deterioration, and the percentage of fertilizations decreased after 15-20 minutes. Egg Supported by a research grant from Baldwin-Wallace College. Manuscript received May 19, 1969. Senior student and Associate Professor of Biology respectively. THE OHIO JOURNAL OF SCIENCE 71(1): 30, January, 1971. No. 1 SEA URCHIN METAMORPHOSIS 31 suspensions and undiluted sperm kept at 5°C remained viable for an average of 24 hours. On one occasion fertilizations were obtained throughout a period of 40 hours. The number of abnormally developed embryos increased with the age of the gametes at the time of fertilization. Fertilization was accomplished by combining two drops of sperm suspension and two drops of egg suspension on a depression slide and mixing quickly with a toothpick. Each slide was microscopically examined to confirm the formation of the fertilization membranes. The fertilized eggs from two slides prepared by this method were immediately transferred to a 125-ml glass jar containing 110 ml of synthetic sea water. The injection of one urchin of each sex provided sufficient eggs and sperm to establish 75 of the 125-ml cultures, each having approximately 100 fertilized eggs. Fertilizations were prepared one or more times at hourly intervals for a period of 24 hours in order to establish a continuing series of developing embryos. The jars were provided with loosely fitted plastic lids and evaporation was minimal, with no significant change in specific gravity occurring during the developmental period. The cultures were maintained at 23°C and were not aerated. After 24 hours at 23°C, the embryos developed to the pluteus stage with a digestive tract. At this point the availability of an algal suspension to the freeswimming pluteus larvae was a critical factor. It was important that the algae be suspended; an initial loss of larvae was observed in cultures where the algae had settled to the bottom or the sides of the containers. A freshwater unicellular green alga (12 n) resembling Chlorella was used as a nutritional source. Algal cultures were established several weeks prior to each series of fertilizations by adding a suspension of mixed freshwater algae from an established ten-gallon aquarium in the laboratory to an aerated one-gallon jar of synthetic sea water to which eight grams of soluble fertilizer had been added (HYPONeX 7-6-19, Hydroponic Chemical Company, Inc., Copley, Ohio). It is significant that a sufficient amount of freshwater algae survived and reproduced each time to maintain a stock marine culture of the suspended Chlorella-like green alga (which has not yet been identified). Five milliliters of this algal suspension were added to each culture container. The jars were placed six inches from a 40-watt cool-white fluorescent lamp fixture, which was positioned on the counter surface in such a way as to illuminate the cultures from the side. The light intensity at six inches was 280 footcandles, and the photoperiod was 12 hours. For purposes of examination, developing embryos and larvae were removed from the containers by pipette and transferred to a drop of synthetic sea water on a depression slide. All photographs were taken of living specimens from slides prepared in the above manner. Coverslips were not used because of their tendency to distort or injure the larvae, especially while attempting to return them to the culture containers.