Abstract :
[en] Long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), are indispensable nutrients for human health and for optimal growth and development in fish. Although some fish species possess the capacity to biosynthesize n-3 LC-PUFA endogenously from α-linolenic acid (ALA, C18:3n-3), this biosynthesis potential varies widely among species and is strongly constrained by trophic level and feeding ecology. As a major freshwater carnivorous species in global aquaculture, largemouth bass Micropterus salmoides exhibits a high dependence on dietary EPA and DHA and a limited capacity for endogenous biosynthesis, which has become a critical bottleneck for fish oil (FO) replacement and the sustainable development of aquaculture. Therefore, elucidating its biosynthetic capacity, regulatory mechanisms and nutritional modulation of n-3 LC-PUFA is of fundamental importance for optimizing lipid nutrition strategies and developing sustainable alternative lipid resources.
In this thesis, we addressed the central question of “the endogenous biosynthetic capacity and nutritional regulation of n-3 LC-PUFA in largemouth bass” by integrating molecular, cellular and whole-animal approaches, including heterologous expression, primary hepatocyte culture, stable isotope tracing and multi-factor feeding trials. Through this multi-level strategy, we systematically clarified the existence, limitations and nutritional modulation of the biosynthetic pathway.
At the molecular level, heterologous expression in yeast identified two key desaturase genes: fads2 (fatty acid desaturase 2), which exhibited Δ6 desaturase activity catalyzing the conversion of ALA to C18:4n-3, and delta4 fads (acyl-CoA Delta4 desaturase-like), which possessed dual Δ5 and Δ4 activities, converting C20:4n-3 to EPA and C22:5n-3 to DHA, respectively. These results provide direct enzymatic evidence that largemouth bass possesses a complete set of key desaturation steps required for n-3 LC-PUFA biosynthesis. At the cellular level, primary hepatocyte experiments showed that exogenous ALA was efficiently incorporated into cells; however, ALA incubation did not lead to a significant increase in DHA content (P > 0.05), suggesting that downstream elongation and terminal desaturation steps constitute major metabolic constraints. In vivo stable isotope tracing further demonstrated the physiological relevance of this pathway by detecting 13C-labelled C20:4n-3 derived from 13C-ALA in plasma of largemouth bass. Taken together, these molecular, cellular and in vivo results indicate that largemouth bass possesses a “functionally complete but tightly constrained” endogenous n-3 LC-PUFA biosynthesis system, in which substrate availability alone is insufficient to ensure effective DHA accumulation.
Building on this mechanistic foundation, we conducted a series of feeding trials to further clarify how nutritional factors modulate this inherently limited biosynthetic capacity. A 2 × 2 factorial feeding trial was designed with oil source (soybean oil vs. cottonseed oil) and inclusion level (50% vs. 100% vegetable oil replacement) as experimental factors, with all diets formulated to contain 30% fish meal as the basal protein source. Although the soybean oil (SO) groups exhibited higher dietary ALA and increased desaturase gene expression, neither tissue n-3 LC-PUFA levels nor hepatic desaturase abundances were significantly enhanced (P > 0.05), indicating that transcriptional upregulation alone is insufficient to overcome downstream metabolic bottlenecks. In contrast, cottonseed oil (CO) significantly reduced final body weight, weight gain and specific growth rate while increasing feed conversion ratio (P < 0.05), while inducing enhanced lipid catabolism, as indicated by elevated atgl (Adipose triglyceride lipase) expression and p-AMPK (Phosphorylated AMP-activated protein kinase) levels (P < 0.05). Collectively, these results suggest that the combination of ALA deficiency and accelerated lipid breakdown represents a key mechanism underlying CO-induced growth depression. Moreover, the findings demonstrate that when exogenous n-3 LC-PUFA supply is sufficient to support growth, additional ALA supplementation does not further stimulate endogenous n-3 LC-PUFA biosynthesis.
In the 2 × 2 factorial trial based on purified diets completely free of fish meal and FO, the complementary roles of ALA and DHA were clearly demonstrated. Fish fed linseed oil (LO) exhibited significantly higher growth indicators and tissue n-3 PUFA levels than those fed CO (P < 0.05), whereas delta4 fads and elovl5 expression were lower in the LO-fed groups than in the CO-fed groups (P < 0.05). Dietary DHA inclusion significantly improved growth performance and tissue DHA levels in CO-fed fish (P < 0.05), while reducing liver crude lipid and triglyceride contents (P < 0.05). At the same time, DHA significantly downregulated hepatic elovl 8a expression and reduced Δ5 and Δ4 Fads protein abundances (P < 0.05), indicating a strong feedback inhibition of endogenous n-3 LC-PUFA biosynthesis. These results indicate that while adequate dietary ALA supported growth and DHA biosynthesis, severe ALA deficiency upregulated partial n-3 LC-PUFA biosynthesis-related genes but still reduced tissue DHA, with dietary DHA inclusion alleviating growth constraints under ALA-deficient conditions.
At the level of feed resource innovation, this study further utilized black soldier fly Hermetia illucens (BSF) larvae as a functional lipid carrier. Dietary fish processing by-products significantly increased larval ALA and enabled the accumulation of EPA and DHA, which were undetectable in control larvae (P < 0.05). In the feeding trial, largemouth bass fed the DHA-enriched BSF oil (BSFO) diet exhibited significantly higher final body weight and weight gain and a lower feed conversion ratio than those fed the control BSFO diet (P < 0.05). DHA inclusion markedly increased DHA, total n-3 PUFA and the n-3/n-6 PUFA ratio in muscle and liver, while reducing n-6 PUFA accumulation (P < 0.05). Plasma lipid profiles were significantly improved, with higher HDL-c (High-density lipoprotein cholesterol) and lower LDL-c (Low-density lipoprotein cholesterol) levels (P < 0.05). Meanwhile, the protein abundances of Δ6, Δ5 and Δ4 Fads were significantly decreased by dietary DHA (P < 0.05), further confirming feedback inhibition of hepatic n-3 LC-PUFA biosynthesis.
In conclusion, largemouth bass possesses a low-efficiency but conditionally sufficient pathway for endogenous n-3 LC-PUFA biosynthesis. Under practical aquaculture conditions, ALA functions as a key essential fatty acid, supporting tissue EPA and DHA accretion and growth when supplied at adequate levels, whereas dietary DHA compensates for insufficient precursor availability. Furthermore, DHA deposition is regulated by both dietary supply and endogenous metabolism. Overall, SO, LO, moderately supplemented CO, and n-3 LC-PUFA-enriched BSFO represent promising sustainable alternatives to fish oil in largemouth bass diets.
