Molecular characterization and developmental expression patterns of apolipoprotein A-I in Senegalese sole (Solea senegalensis Kaup).

The apolipoprotein A-I (ApoA-I) is an essential component of the high density lipoproteins (HDL). In this study, the cDNA and genomic sequences of this apolipoprotein were characterized for first time in Solea senegalensis. The predicted polypeptide revealed conserved structural features including ten repeats in the lipid-binding domain and some residues involved in cholesterol interaction and binding. The gene structure analysis identified four exons and three introns. Moreover, the synteny analysis revealed that apoA-I did not localize with other apolipoproteins indicating a divergent evolution with respect to the apoA-IV and apoE cluster. The phylogenetic analyses identified two distinct apoA-I paralogs in Ostariophysi (referred to as Ia and Ib) and only one (Ib) in Acanthopterygii. Whole-mount in situ hybridization located the apoA-I signal mainly in the yolk syncytial layer in lecitotrophic larval stages. Later at mouth opening, the mRNA signals were detected mainly in liver and intestine compatible with its role in the HDL formation. Moreover, a clear signal was detected in some regions of the brain, retina and neural cord suggesting a role in local regulation of cholesterol homeostasis. After metamorphosis, apoA-I was also detected in other tissues such as gills, head kidney and spleen suggesting a putative role in immunity. Expression analyses in larvae fed two diets with different triacylglycerol levels indicated that apoA-I mRNA levels were more associated to larval size and development than dietary lipid levels. Finally, qPCR analyses of immature and mature transcripts revealed distinct expression profiles suggesting a posttranscriptional regulatory mechanism.

Genomic characterization and expression analysis of four apolipoprotein A-IV paralogs in Senegalese sole (Solea senegalensis Kaup).

The apolipoprotein A-IV (ApoA-IV) plays a key role in lipid transport and feed intake regulation. In this work, four cDNA sequences encoding ApoA-IV paralogs were identified. Sequence analysis revealed conserved structural features including the common 33-codon block and nine repeated motifs. Gene structure analysis identified four exons and three introns except for apoA-IVAa1 (with only 3 exons). Synteny analysis showed that the four paralogs were structured into two clusters (cluster A containing apoA-IVAa1 and apoA-IVAa2 and cluster B with apoA-IVBa3 and apoA-IVBa4) linked to an apolipoprotein E. Phylogenetic analysis clearly separated the paralogs according to their cluster organization as well as revealed four subclades highly conserved in Acanthopterygii. Whole-mount analyses (WISH) in early larvae (0 and 1day post-hatch (dph)) showed that the four paralogs were mainly expressed in yolk syncytial layer surrounding the oil globules. Later, at 3 and 5dph, the four paralogs were mainly expressed in liver and intestine although with differences in their relative abundance and temporal expression patterns. Diet supply triggered the intensity of WISH signals in the intestine of the four paralogs. Quantification of mRNA abundance by qPCR using whole larvae only detected the induction by diet at 5dph. Moreover, transcript levels increased progressively with age except for apoA-IVAa2, which appeared as a low-expressed isoform. Expression analysis in juvenile tissues confirmed that the four paralogs were mainly expressed in liver and intestine and secondary in other tissues. The role of these ApoA-IV genes in lipid transport and the possible role of apoA-IVAa2 as a regulatory form are discussed.

Characterization of the genomic responses in early Senegalese sole larvae fed diets with different dietary triacylglycerol and total lipids levels.

The aim of this work was to evaluate the genomic responses of premetamorphic sole larvae (9 days post-hatching, dph) fed diets with different lipid and triacylglycerol (TAG) content. For this purpose, two diets with high (rotifers enriched with a fish oil-based emulsion; referred to as HTAG) and low (rotifers enriched with a krill oil-based emulsion; LTAG) levels of total lipids and TAG were evaluated. Lipid class and fatty acid (FA) profiles, histological characterization of intestine, liver and pancreas and expression patterns using RNA-seq were determined. Discriminant analysis results showed that larvae could be clearly differentiated on the basis of their FA profile as a function of the diet supplied until 9dph although no difference in growth was observed. RNA-seq analysis showed that larvae fed HTAG activated coordinately the transcription of apolipoproteins (apob, apoa4, apoc2, apoe, and apobec2) and other related transcripts involved in chylomicron formation, likely to facilitate proper lipid absorption and delivery. In contrast, larvae fed LTAG showed higher mRNA levels of several pancreatic enzymes (try1a, try2, cela1, cela3, cela4, chym1, chym2, amy2a and pnlip) and appetite modulators (agrp1) and some intra- and extracellular lipases. Moreover, KEGG analysis also showed that several transcripts related to lipid metabolism and glycolysis were differentially expressed with a higher abundance in larvae fed LTAG diet. All these data suggest that early larvae were able to establish compensatory mechanisms for energy homeostasis regulating key molecules for FA and TAG biosynthesis, FA uptake and intracellular management of TAG and FA to warrant optimal growth rates.