The simple ascidian Ciona intestinalis [Fig. 1] belongs to the largest class of tunicates, the Ascidiacea, which are exclusively marine sessile animals. C. intestinalis is probably the most cosmopolitan ascidian species with an annual life cycle (Millar, 1953, 1971; Dybern, 1965). It is euryhaline and epibenthic and has no specific substrate preferences; it can be abundant on rocks, shells and other solid formations, but can also be found on algae and in fouling communities (Kawahara, 1962; Dybern, 1963; Yamaguchi, 1975). C. intestinalis feeds by pumping the surrounding water through a continuously produced mucus net that traps the suspended particles. It is a hermaphrodite with separate ovary and testis. The species is oviparous and the eggs and spermatozoa are expelled from the atrial cavity. Cross fertilization is the rule, because C. intestinalis exhibits a high degree of self-sterility (Morgan, 1940). Development is of the mosaic type: the egg has special localized regions that give rise to particular tissues in development (Conklin, 1905). The larva, which usually hatches within a day after fertilization, resembles a tadpole and is divided into two regions: the anterior trunk (a head) and a posterior tail [Fig. 8; Fig. 9A]. The tail contains a notochord, muscle cells, and a dorsal nervous system. Metamorphosis transforms the non-feeding, mobile larva into a filter feeding, fixed juvenile (Cloney, 1982). The notochord is lost at metamorphosis in all forms of Tunicates except the Larvacea. General ascidian biology, with some insights into reproduction and development, has been treated by Berrill (1975) and Cloney (1990). An exhaustive reference work on ascidian embryogenesis is given by Satoh (1994), and details on the structure of ascidian tissues and organs and their physiology are given by Goodbody (1974). Much of the theoretical importance of the tunicates concerns their larva, which is considered as a prototype of the ancestral chordate (see the review by Satoh and Jeffery, 1995). The Tunicate subphylum (or Urochordata) belongs to the phylum Chordata and is more closely related to the Cephalochordata (es: Branchiostoma) than to the Vertebrata. Because of their phylogenetic location at the boundary between invertebrates and vertebrates, ascidians have the potential to provide important information on the evolutionary origins of the chordate body plan. Utilized for research in developmental and cell biology since the early 1900s (Conklin, 1905), they have now become increasingly important as research models for developmental studies at the molecular level.
Ciona intestinalis is endowed with many favorable characteristics to make it a model system for biological research. It is transparent enough to allow the study of a great part of the internal anatomy, feeding processes, and blood circulation, without resorting to dissection. In addition, thanks to its size, dissection can be performed easily. As type ascidian for developmental and molecular studies, its essential features are hermaphroditism, induction of self-fertilization, very rapid embryonic development, possibility of embryo manipulation (by intracellular injection or electroporation), a reasonably rapid life cycle, and a small and compact genome (approximately 162 Mbp), corresponding to an estimated gene number of 15,000 ± 3,700 (Simmen et al.,1998). Various methods have been used to exploit the unique advantages of these simple chordates. Transgenesis has been performed by using electroporation and microinjection methods for analyzing genes responsible for the specification of chordate tissues (Di Gregorio and Levine, 1998). Chemical mutagenesis methods have been developed to perform genetic screens (Moody et al., 1999; Nakatami et al., 1999; Sordino et al., 2000). Thus, the existence of cultured stock populations of C. intestinalis maintained in laboratory conditions would greatly enhance research endeavors in developmental and genetic fields, as noted recently by Kano et al. (2001) and Sordino et al. (2001). Early studies on the rearing of this ascidian to sexual maturity, without cultivation purposes, were performed by Berrill (1937, 1947), Just (1934), and Yamaguchi (1975). In the present work, we describe the culture conditions that are necessary for successful growth and reproduction of C. intestinalis. In our experience, this system could also be used for rearing other sessile filter-feeding invertebrates that are used for research.
We recognize that topics similar to those in several sections of this paper have been discussed previously by others. For example, Ciona intestinalis is one of the species covered in Methods for Obtaining and Handling Marine Eggs and Embryos (Costello and Henley, 1971). However, the literature on methods for this species is somewhat scattered and varies greatly in the level of detail. Our objective in bringing it together in this paper, with added observations and considerable detail, is to make the material useful to colleagues who wish to utilize this model system, reported as "biology’s rising star" in a recent issue of Science (Pennisi, 2002).
Ciona intestinalis is a hermaphroditic animal, and the gonads consist of a separate ovary and testis. Distinct gonoducts (oviduct and spermiduct) are located along the dorsal side of the intestine [Fig. 1]. The oviduct lies dorsal to the spermiduct and terminates at the same level. The end of the genital ducts is usually marked by an orange-red spot, which can be seen through the open atrial siphon. Freshly collected adults are submerged in tanks in suspended baskets surrounded by circulating seawater. The floating fretworked baskets are suspended far from the bottom of the tanks in order to maintain the adult ascidians as close as possible to their condition in the wild, where they are sessile and attached to a substrate. In this way, adults can be kept in good condition for 3-4 weeks. Mature specimens are usually detected by the fullness of the gonoduct, as observed through the transparent test ([Fig. 2]. Eggs (and full oviduct) are reddish or brownish; sperm (and spermiduct) are white. Continuous illumination, from the time of collection until the utilization of the animals, can be applied in order to prevent spontaneous spawning (See Appendix 1 for the induced-spawning method). Eggs and sperm are removed separately from the gonoducts. The simplest way to collect eggs is by making a longitudinal incision through the test, on the dorsal side, starting just above the atrial siphon ([Fig. 3]. Care should be taken in cutting the layers underneath the test, since too deep a cut can excise the gonoducts, causing loss of gametes. II. Methods for obtaining embryos and larvae
For certain experimental procedures, it may be important to induce spawning in order to collect gametes several times from live ascidians. This method is described in Appendix 1.
B. Fertilization procedures and embryo culture
Fertilization can be obtained by adding a few drops of the diluted sperm preparation to the petri dishes containing the eggs. Gentle agitation helps the mixing of gametes. The dishes should be kept at a temperature in the range of 18 - 20 °C, to bring about development within the next 18 hours. Water should be changed 1 h after insemination, in order to remove surplus sperm. For general purposes, it is not essential to change water at other times during development. (See Fig. 7 and Fig. 8 for images of some developmental stages, and Fig. 19 for the timetable of development.)
Swimming larvae are transferred to clean plastic petri dishes (150 mm diameter) containing fresh seawater. According to the amount of larvae, it may be necessary to transfer them into several dishes.
III. Culture of metamorphic and post-metamorphic stages.
As observed by Cloney (1982), most ascidian larvae settle spontaneously over a period of minutes or hours in laboratory culture vessels, but the percentage may be low with some species. According to our experience with C. intestinalis, most larvae suddenly cling to the clean plastic of dishes, which likely acts as an environmental cue in inducing the settlement and metamorphosis of young larvae (Chia, 1978). That is why we transfer the young larvae a short time after hatching.
For larval transfer and care, the following steps are recommended: (1) first, put a layer of water on the dish; (2) add the larvae (so that they tend to attach mainly onto the bottom of the dish); (3) wait 30 min before adding the remaining water; (4) incubate dishes at a temperature kept at 18 to 22 °C.; (5) during the next three days, check larval metamorphosis through the dissecting microscope; (6) renew seawater daily; (7) during the second day, transfer the larvae that keep swimming to new dishes, where contact with clean plastic helps induce settlement.
B. Metamorphosis
The sequence of major steps of metamorphosis, as described below, is given in Fig. 9.
Metamorphosis transforms the non-feeding, mobile larva into a filter-feeding, fixed juvenile (Cloney, 1982). After settlement, rapid morphogenetic movements, involving physiological changes, lead to the opening of two lateral siphons and the first pair of functional stigmata. At this stage, the ascidians initiate feeding and require an external food source. This is the "first ascidian stage" (FAS) of Berrill (1947). He noted that it was a prolonged developmental phase that terminated more or less suddenly at a critical size. We confirm his correlation with size, identifying the critical size as 1 ± 0.3 mm. The first ascidian stage ends with the fusion of the two peribranchial siphons to form the single mid-dorsal atrial siphon. This typical ascidian condition, the "second ascidian stage" (SAS) of Berrill (1947), is that in which the definitive stigmata forming the branchial sac are found. At this point metamorphosis terminates and the ascidians are considered as juveniles.
IV. Experimental setup for culturing juvenile stages
The setup for C. intestinalis cultures used in our study acts as a sort of chemostat ensuring a constant presence of suspended food in the environment surrounding the ascidians. Two essential requisites characterize this system: the holding apparatus, which ensures the maintainance of these sessile animals in a natural position, and the feeding system, which is ideal for the growth of filter feeders. See the simplified scheme of the setup shown in Fig. 10, and Appendix 2 for functional details on the culture system.
Some notes on the physico-chemical conditions applied to the system are given in Appendix 3.
Since this species is sessile, an important need is for the support for growing animals. We have selected plastic petri dishes as the most appropriate support. Their advantages are evident: they are suitable during larval and post-larval steps of development, which occur out of the tank, and, subsequently, during growth of the animals, because of easy removal from tanks for examination of animals and cleaning [Fig. 11].
We designed special supports to hold the dishes inside the tank [Fig. 12]. These are made of PVC (polyvinylchloride) and are designed to hold pairs of dishes horizontally with the animals growing downwards [Fig. 13], a position closer to the natural one. This position has the additional advantage of preventing massive covering of the animals' surfaces with suspended matter, which is very dangerous during early phases of growth. This system also allows easy visual inspection of the growing animals.
The feeding system employed is shown in Fig. 14. Since undisturbed ascidians filter water continuously at constant rates, we expected that a continuous feeding system would work better than a series of discrete food introductions to achieve the constant presence of suspended food around the animals. For this purpose, we used peristaltic pumps to introduce food into the water. In general, the quality of the suspended particulate load is of considerable importance for the growth of filter-feeding animals. Ascidians are mainly non-selective filter-feeders and do not discriminate between inorganic and organic particles, the latter playing, of course, the essential role in growth. For these reasons, we developed an environmental culture medium very rich in suspended organic particles, and completed it with dried algae. We followed the rule that use of a multicomponent plant-animal diet lessens the danger of malnutrition when specific nutritional requirements are not completely defined. We use a mixture of liquid and powdered food developed for aquaculture of larval stages of crustaceans and fish, produced by Salt Creek Inc., South Salt Lake City, Utah [Fig. 15].
The size range of the powders is 5 to 20 microns. Food is introduced into the aquaria continuously, mixed with seawater [Fig. 14]. As we control the seawater flow rate, we adjust the concentration of food, according to the size and filtration rate of the ascidians. In Appendix 4 the schedule of the animals' daily diet is given together with the details for food preparation.
In summary, the final nutritional conditions ensure the presence of sufficient particulate vegetable and animal matter in the water for the growth of cultured ascidians.
To estimate their growth, the cultured animals can be measured starting from the post-larval stage in which they have open siphons and functional stigmata (first ascidian stage or FAS). For juveniles up to 10 mm we measure the length (excluding the stalk) of relaxed animals every 5 days using an ocular micrometer at the dissecting microscope. For the larger animals, a small ruler is used [Fig. 11]. In Fig. 18] a typical growth curve is shown.
The swiftest way to verify the health of growing animals is to control their filtration efficiency, which is indicated by gut fullness and the release of fecal strings from the atrial siphon [Fig. 16]. To avoid overcrowding in the dishes, which causes filtration competition and disparity of growth, we remove a number of individuals by hand when they reach the size of about 2 cm. Good results in terms of growth time and animal health are obtained with densities ranging from 60 to 100 individuals per 150mm petri dish.
Ciona intestinalis reared in the laboratory reach sexual maturity, with sperm and eggs in the gonoducts, in about 2 months. Sexual development of animals is followed by observing the development of gonoducts and gonads under the dissecting microscope. Individuals are considered sexually mature when gametes are observed within the gonoducts [Fig. 17]. Gametes are mature when they appear in the gonoducts and can be used to produce embryos. In particular, the eggs gather and are first evident in the distal tip of oviduct. When they are present in sufficient number, it is possible to collect the eggs with a pipette (a thin-tipped pipette can be used according to the oviduct size). Eggs are usually fertilizable when they first appear in the oviduct, but fertilization failures occur occasionally with some animals.
The laboratory size of mature individuals ranges from 5 to 8 cm. The size range and rate of animal growth are almost constant (apart from minor individual differences), but they are strictly related to the laboratory conditions applied in our cultivation protocol. Therefore, it is not possible to compare growth rates of cultured Ciona intestinalis with those of wild populations, which are affected by seasonal changes. Needless to say, the latter rates increase during the favorable seasons (spring to early summer, and early autumn in the Thyrrenian Sea) and decrease during the adverse seasons.
Based on these results, it is theoretically possible to obtain up to four generations per year. According to our experience, however, for long-term projects (e.g., inbred lines), it is more realistic to assume production of three generations per year. In fact, there are individual phase differences in the growth and sexual maturation within a population, because the production of gametes is cyclical. On average, 50% of the animals of a single population typically reach maturity simultaneously. Furthermore, starting from a given generation, several trials are sometimes required to obtain offspring successfully.
We thank the SZN Director Dr. Lucio Cariello for giving us the opportunity to carry on this research project. We also thank Prof. Roberto Di Lauro and Dr. Rosaria De Santis for their warm support. Thanks are finally due to Giuseppe Gargiulo for the art work and to Rosamaria Sole for her useful comments.
| Appendix 1: Induced spawning method |
| Appendix 2: Details on the functioning of the Ciona system |
| Appendix 3: Notes on the physico-chemical conditions |
| Appendix 4: Schedule of animals' daily diet and food preparation |
| Figure 1: Adult Ciona intestinalis |
| Figure 2: Living adult C. intestinalis with full gonoducts. |
| Figure 3: Gamete collection |
| Figure 4: Egg collection |
| Figure 5: Egg suspension culture |
| Figure 6: Sperm collection |
| Figure 7: Early embryonic stages |
| Figure 8: Hatching of the larva |
| Figure 9: The main stages of metamorphosis from larva to juvenile |
| Figure 10: Scheme of the setup for culturing C. intestinalis |
| Figure 11: Control procedures on cultivated C. intestinalis |
| Figure 12: Holding supports for dishes |
| Figure 13: Holding apparatus inside the experimental aquarium |
| Figure 14: A view of the feeding system |
| Figure 15: Food "mix-bottles" |
| Figure 16: Filtering juveniles of cultivated C. intestinalis |
| Figure 17: Mature laboratory-cultivated C. intestinalis |
| Figure 18: Ciona intestinalis growth curve |
| Figure 19: Timetable of development |