Supplementary file 1_Ontogenetic expression of bile and lipid metabolism genes in Atlantic cod (Gadus morhua L.) larvae in relation to hepatic lipid accumulation.docx

Atlantic cod (Gadus morhua) stores excess dietary lipids as triacylglycerol (TAG) in the liver, but substantial accumulation emerges only during the late larval stage, characterized by a sharp increase in hepatic vacuolization (4-35%) between 45 and 61 days post-hatching (dph). To investigate mechanisms underlying this transition, we examined the ontogeny of bile and lipid metabolic pathways in cod larvae (2–60 dph) using RNA sequencing of whole-body larvae and quantitative real-time PCR of digestive tissues for key genes involved in dietary lipid processing. We further tested transcriptomic effects of two dietary phospholipid (PL) levels (ca. 7 and 6% dry matter) with or without bile salt (BS) supplementation (0.04% dry matter) during co-feeding (17–34 dph) and through 60 dph. Ontogenetic expression patterns indicated that hepatic lipid accumulation is not constrained by the capacity for lipid digestion, synthesis or transport. Instead, the onset of hepatic lipid deposition coincided with pronounced upregulation of the TAG-synthesizing enzyme dgat2 and the lipid droplet-stabilizing protein plin2 in digestive tissues, with plin2 also increasing sharply in whole-body larvae. Dietary PL level and BS supplementation elicited only modest and inconsistent transcriptional responses, suggesting limited dietary modulation of the examined pathways under the tested conditions. We propose dgat2 and plin2 as candidate markers of liver development in cod larvae and emphasize the need for multi-omics approaches to clarify the mechanisms regulating hepatic lipid storage.