The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). High-carbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC2.7.1.11), pyruvate kinase (EC2.7.1.40), glucose-6-phosphate dehydrogenase (EC1.1.1.49) and 6-phosphogluconate dehydrogenase (EC1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish likeSparus auratacan tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

The small GTPase Rab4 is involved in endocytosis through sorting and recycling early endosomes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for effectors that specifically interact with Rab4‐Q67L, the GTP‐bound form of Rab4. We cloned an ubiquitous 80‐kDa protein, identical to CD2‐associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS), that interacts with Rab4‐Q67L in the yeast two‐hybrid system and in vitro. CD2AP/CMS expressed in mammalian cells was localized to punctate structures and along actin filaments. None of the known markers of early endosomes [Early Endosomes Antigen 1 (EEA1), Rab5 and Rab11] colocalized with the CD2AP/CMS‐positive vesicles. However, coexpression of Rab4‐Q67L with CD2AP/CMS induces a significant enlargement of EEA1‐positive early endosomes. Rab4, CD2AP/CMS and Rab7 colocalized in these modified endosomes. Coexpression of c‐Cbl and CD2AP/CMS also resulted in an enlargement of early endosomes. Using various truncated forms of CD2AP/CMS, we demonstrate that early endosomes enlargement requires that CD2AP/CMS interacts with both Rab4 and c‐Cbl. The expression of a truncated form of CD2AP/CMS that retains the ability to interact with Rab4 but not c‐Cbl inhibits ligand‐induced PDGF receptor degradation. We propose that CD2AP/CMS, through interactions with Rab4 and c‐Cbl, controls early endosome morphology and may play a role in traffic between early and late endosomes, and thus in the degradative pathway.

Chitosan is increasingly used for safe nucleic acid delivery in gene therapy studies, due to well-known properties such as bioadhesion, low toxicity, biodegradability and biocompatibility. Furthermore, chitosan derivatization can be easily performed to improve the solubility and stability of chitosan–nucleic acid polyplexes, and enhance efficient target cell drug delivery, cell uptake, intracellular endosomal escape, unpacking and nuclear import of expression plasmids. As in other fields, chitosan is a promising drug delivery vector with great potential for the fish farming industry. This review highlights state-of-the-art assays using chitosan-based methodologies for delivering nucleic acids into cells, and focuses attention on recent advances in chitosan-mediated gene delivery for fish biotechnology applications. The efficiency of chitosan for gene therapy studies in fish biotechnology is discussed in fields such as fish vaccination against bacterial and viral infection, control of gonadal development and gene overexpression and silencing for overcoming metabolic limitations, such as dependence on protein-rich diets and the low glucose tolerance of farmed fish. Finally, challenges and perspectives on the future developments of chitosan-based gene delivery in fish are also discussed.