**1.2 First feeding**

One of the key restrictions in larval rearing is first feeding at early stages of development. This is a major bottleneck for larval culture, due primarily to their small size and often poorly developed digestive system (Person Le Ruyet, et al., 1993). Many marine fish larvae require motile prey organisms (Pedersen *et al*., 1987; Pedersen and Hjelmeland, 1988). Visual skill is not only important for feeding but also for orientation, schooling and eluding predators (Blaxter, 1986, Batty 1987). Larval survival clearly depends on their ability to feed successfully (Heath, 1992). During the endo-exotrophic phase (Mani-Ponset *et al.,* 1996), larvae utilize nutrients from both yolk sac and their surrounding environment. This phase starts soon after hatching, especially in larvae with a small yolk sac (Calzada *et al.,* 1998). This first feeding phase is critical for larval survival; therefore, successful synchronization between exhaustion of endogenous reserves and first feeding must occur.

Larval mouth size at first feeding is also an important factor for larval survival. The mouth size of first-feeding larvae mechanically restricts the size of the food particles that can be ingested. In general, mouth size is correlated with body size, which in turn is influenced by egg diameter and the period of endogenous feeding (i.e., yolk sac consumption period). For example, Atlantic salmon eggs are usually at least four times larger than Gilthead sea bream eggs and consequently on hatching yield large salmon larvae with large yolk sac supplies (i.e., sufficient endogenous feed reserves for the first three weeks of their development). Whereas first-feeding Gilthead sea bream larvae are very small with limited yolk sac reserves, and consequently can only feed endogenously for about three days (Jones & Houde, 1981).

#### **1.2.1 Background phytoplankton ('Green water')**

Most marine fish larvae are visual feeders and feeding success of larvae at various developmental stages depends on the provision of suitable food, the rearing environment, and on the visibility and adequate density of the prey (Ina *et al*., 1979, Hunter, 1980). Publications on the rearing of marine fish larvae indicate that phytoplankton cultures enhance survival rates (May, 1971; Al-Abdul-Elah, 1984; Hernandez-Cruz, *et al*., 1994; Marliave, 1994). Furthermore, several papers have discussed the beneficial effect of adding microalgae to larval rearing tanks in order to improve larval growth and survival (Howell 1979; Scott & Middleton 1979; Jones & Houde, 1981; Bromley & Howell 1983; Vasquez-Yeomans, *et al.,* 1990; Naas, *et al*., 1992; Hernandez-Cruz *et al*., 1994; Marliave 1994; Tamaru, *et al*., 1994). These papers discuss the effect of micro-algae on the nutritional and behavioural aspects of fish larvae. Some fish larvae take up substantial amounts of micro-algae during the initial days after hatching (Van der Meeren, 1991; Reitan, *et al*., 1991) which maybe used as a food source. In recent years, the benefits of culturing larvae in `green water' is considered to be optical rather than nutritional to fish larvae (Marliave 1994).

#### **1.2.2 Rotifers and their nutritional value**

Live food organisms are an important food source for the first feeding of early larval stages. The most widely used starter live-food organism in fish larviculture is the marine rotifer *Brachionus plicatilis*. The successful development of commercial fish farms in the Mediterranean has been made possible by several improvements in production techniques for rotifers (Candreva *et al*., 1996; Dehasque *et al*., 1998). Rotifers are an ideal link in the food chain for different stages of fish and shrimp larvae. Rotatoria (=Rotifera) belong to the smallest metazoan of which over 1000 species have been described, 90% of which inhabit freshwater habitats. They seldom reach 2 mm in body length. Males have reduced sizes and are less developed than females; some measuring only 60 µm. The body of all species consists of a constant number of cells, with various *Brachionus* species containing approximately 1000 cells, which should not be considered as single identities, but as a plasma area. Growth of the animal is achieved by plasma volume increase and not by cell division. The epidermis contains a densely packed layer of keratin-like proteins and is called the lorica. The shape of the lorica and the profile of the spines and ornaments allow determination of different species and morphotypes. A rotifer body is differentiated into three distinct parts consisting of the head, trunk and foot. The head carries the rotatory organ or corona, which is easily recognized by its annular ciliation and is the characteristic that led to the name Rotatoria (bearing wheels). The retractable corona assures locomotion and a whirling water movement, for the uptake of small food particles (mainly algae and detritus). The trunk contains the digestive tract, the excretory system and the genital organs. A characteristic organ of rotifers is the mastax (a calcified apparatus in the mouth region) that is very effective in grinding ingested particles. The foot is a ring-type retractable structure without segmentation ending in one or four toes. *B. plicatilis,* a cosmopolitan inhabitant of inland saline and coastal brackish waters. It has a lorica length of 100 to 340 µm, with the lorica ending in 6 occipital spines (Fukusho, 1989).

The nutritional value of *B. plicatilis* is dependent on the nutritional value of its food source, which can influence its suitability as a starter feed for marine larvae, and is determined by the concentrations of highly unsaturated fatty acids ((n-3) HUFA), such as docosahexaenoic acid (DHA, 22:6(n-3)) and eicosapentaenoic acid (EPA, 20:5(n- 3)). Low dietary HUFA levels can lead to high mortality in fish larviculture. Koven *et al.* (1990) suggested that HUFAs function as essential components of bio-membranes, and that their levels in the tissue phospholipid fraction are associated with larval growth. Rainuzzo *et al*. (1997) emphasized the importance of DHA in the development of neural tissues such as brain and retina, considering that the larval head constitutes a significant part of the body mass, and that predatory fish larvae rely on vision to capture their food. Sorgeloos *et al*., (1988) reported a strong correlation between dietary EPA content and survival, and between DHA and growth of Asian sea bass larvae. Watanabe (1993) concluded that DHA and EPA increased survival and growth of several marine fish larvae. At the same time, Kanazawa (1993) observed that high DHA levels increased the tolerance of red sea-bream larvae to various stressful conditions.
