Metamorphosis of Aplysia californica in Laboratory Culture

Abstract

To utilize the advantages offered by the large identified nerve cells of the marine mollusc Aplysia californica for cellular biological studies of development, we have devised simple techniques for growing this species in the laboratory in large number with a generation time as short as nineteen weeks. We have used the cultured animals to study the life cycle from fertilized egg to reproductive adult. The major developmental and behavioral changes occur at metamorphosis, when the larvae settle on the seaweed Laurencia pacifica and the locomotor and feeding behaviors are transformed into their adult forms. We have examined the timetable for the abandonment of larval behaviors and the emergence of adult ones and found that the transition from swimming to crawling occurs first and marks the onset of metamorpbosis. The change from ciliary feeding to radular feeding occurs later and signals the end of metamorphosis. Other adult behaviors, such as the reflex responses and fixed-action patterns of the mantle organs, appear after metamorphosis.The large nerve cells in the marine gastropod mollusc Aplysia are useful for studying the cellular biology of identified neurons and for relating individual cells to behavior (1-3). These features could also be advantageous for studying the development of the nervous system and of the neural control of behavior. It has not been possible to use Aplysia for developmental studies, however, because the animal could not be successfully cultured through metamorphosis. In this paper we describe techniques for culturing the most extensively used species: Aplysia californica.* Since each animal lays more than 105 eggs, and mortality is low with these techniques, an almost unlimited number of larvae can be cultured through metamorphosis and inbred strains can be established.With modifications, these techniques may be applicable to other Aplysia species and to related opisthobranchs used in neurobiological studies, such as Navanax, Hermissenda, and Pleurobranchaea.We also describe the development of Aplysia californica from fertilized egg to reproductive adult. The most dramatic changes occur at metamorphosis. Particularly instructive for analyzing the life cycle are the changes in locomotion which characterize the onset of metamorphosis and the changes in feeding which mark its termination. We have examined these transformations and provide a timetable for the transition of this behavioral repertoire from larva to adult. This timetable provides normative data for subsequent studies on the development of the nervous system. RESULTSMethods of laboratory culture We collected egg masses from four sexually mature adults (obtained from Dr. Rimmon C. Fay of the Pacific BioMarine Supply Co., Venice, Calif.), kept in a holding tank of aerated sea water (from the Marine Biological Laboratory, Woods Hole, Mass.), and fed daily with sea lettuce (Ulva sp.). Animals were induced to lay eggs throughout the year by keeping the temperature at 180 (4).Egg masses were transferred into 1-liter glass c...

Larvae of the sea hare Aplysia californica settle and metamorphose on an assortment of macroalgal species

“…Cultures were stirred with a jet of 25ml of seawater once each day. Using these techniques, larvae exhibited growth rates comparable to those observed in other studies (Kriegstein et al 1974, Paige 1986.…”

“…Contrary to previous reports (Kriegstein et al 1974, Kriegstein 1977, larvae of Aplysia californica did not settle and metamorphose specifically on Laurencia pacifica (Fig. 1).…”

“…Kriegstein et al (1974) found that laboratory-reared larvae would settle and metamorphose on L a u r e n c i a pacifica, but not on species of Plocarnium, Polysiphonia, Daysia, C h o n d r u s or Ulva. Subsequently, Capo et al (1979) reported that 2 red algae from the New England coast, N e o a g a rdheilla baileyl and Gracilaria sp., would also induce settlement and metamorphosis of A .…”

“…However, few naturally-occurring chemical inducers of larval settlement and metamorphosis have been isolated and identified (Kato et al 1975, Cuomo 1985, Pawlik 1986 Marine molluscs of the genus Aplysia are among the most intensively studied animals on earth and have been the subject of research on development, growth and energetics, circadian rhythms, and the neural basis for learning, memory and behavior (reviewed in Kandel 1979, Carefoot 1987. Research interest in one of the best studied sea hares, A. califol-nica, from the coast of California, prompted the laboratory cultivation of the planktotrophic larvae of these animals through metamorphosis (Kriegstein et al 1974). In the field, young recruits of these herbivorous snails were most frequently found eating red algae, primarily of the genus L a u r e n c i a (Kupfermann & Carew 1974).…”

Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization

“…Abdominal and cerebral ganglia from l-3 gm animals raised in the laboratory (Kriegstein, 1977;Kriegstein et al, 1974) were bathed in proteolytic enzyme for 2 hr, and adult abdominal ganglia excised from loo-150 gm animals (Sea Life Supply, Sand City, CA) were exposed to the same proteolytic digest for 2.5 hr. The ganglia were pinned in Sylgard-(Dow Coming, Midland, MI) coated dishes and desheathed.…”