Introduction
Ontogenesis is a process of growth, differentiation and maturation of cells, tissues, organs, and systems (Chambers and Leggett, 1987), where fish larvae undergo dramatic changes in body shape, swimming ab ility, metabolism and behavior. Nutrition is one of the most important factors influencing the ontogeny of the organs in the digestive tract (Chambers and Leggett 1987). Copepods have the advantage that the fatty acids d ocosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are found in the phospholipids of cell membranes. Copepods do not need enrichment like rotifers and Artemia metanauplii and so their use does not increase the problem with the oily surface of the rearing tanks (Conceição et al. 2010) . In addition, copepods are very rich in free amino acids, which stimulate the sense of smell of fish larvae and enhance feeding behavior and food search in the tank (Rønnestad et al. 2013).
Materials & Methods
In a marine hatchery (Galaxidi Marine Farms A/S) , four cylindroconical tanks of 2,7 00 were stocked with 150,000 greater amberjack larvae ( Seriola dumerili ) in each, which were reared until 45 days post-hatching (dph ). The larvae were initially fed from 3-17 dph in two tanks with copepod nauplii and rotifers (Brachionus sp.) (Copepods group), while in the other two tanks, the larvae were fed only with rotifers (Control group). A ll the tanks were fed with rotifers (3-27 dph ), Artemia nauplii (12-22 dph ), enriched Artemia metanauplii (20-30 dph ), and formulated diet (25-45 dph).
A second experiment with gilthead seabream was carried out at the facilities of the Department of Biology at the U niversity of P atras. S ix cylindroconical tanks (three tanks – Control and three tanks – Copepods group) were stocked with 10,000 seabream eggs ( Sparus aurata) in 100 L tanks and these were reared until 25 dph . The feeding protocol was similar to the greater amberjack experiment with the only difference being that the Copepods group fed with Acartia tonsa nauplii days 3 to 14 after hatching. In both experiments, s amples were taken every three days anaesthetized and transferred to a fixative (4% formaldehyde solution) . Fixed samples were rinsed in distilled water, dehydrated in graded ethanol 70, 80 and 100% and then embedded in methacrylate resin (Technovit 7100 Heraeus Kulzer, Germany). Serial sections of 5 mm were obtained with a microtome (Leica SM200R, Germany), stained according to Bennett et al. (1976) and observed through a light microscope . The factors that we choose to study to determine the effect of the copepods in the ontogeny of the intestine and the liver were the length of the villi , the abundance of goblet cells (intestine), and the percentage of area covered with lipid vacuoles - ACLV (liver).
Results
Villi and goblet cells in the intestine of Sparus aurata 25 dph are shown i n Figure 1a and in ACLV in the liver of Seriola dumerili juveniles 36 dph are shown in Figure 1b.
Conclusions
In conclusion, the use of copepods in the rearing of both species, Seriola dumerili and Sparus aurata affected positively the ontogeny and development of the intestine (length of the villi and abundance of goblet cells) as well as the lipid accumulation in the liver.
Acknowledgements
This study was supported through the research project “Improvement of broodstock management and fingerling production methods for greater amberjack (Seriola dumerili)” with grant number MIS 5045873, funded by the General Secretariat for Research and Innovation (GSRI) in Greece.
References
Bennett, HS, Wyrick, AD, Lee, SW, McNeil, JH, 1976. Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives, and simple stains. Stain Technology 51:71-94.
Chambers, R. C., Leggett, W. C., 1987. Size and Age at Metamorphosis in Marine Fishes: An Analysis of Laboratory-Reared Winter Flounder (Pseudopleutonectes americanus ) with a Review of Variation in Other Species. Canadian Journal of Fisheries and Aquatic Sciences. 44, 1936-1947.
Conceição, L.E.C., Yúfera, M., Makridis, P., Morais, S., Dinis, M. T. 2010, Live feeds for early stages of fish rearing. Aquaculture Research. 41, 613-640.
Rønnestad, I., Yufera, M., Ueberschär, B., Ribeiro, L., Sæle, Ø, Boglione C., 2013. Feeding behavior and digestive physiology in larval fish: current knowledge, and gaps, and bottlenecks in research. Reviews in Aquaculture 5(1), 59-98.