Introduction
The early life stages of marine fish are characterised by rapid growth and profound morphological and physiological changes. In Senegalese sole (Solea senegalensis), larval development involves a marked metamorphosis from a pelagic, bilaterally symmetric larva to a benthic post-larval stage, accompanied by extensive digestive tract reorganisation and high metabolic demands. During this critical developmental window, both the immature digestive system and the elevated production of reactive oxygen species (ROS), increase larval susceptibility to oxidative stress, potentially compromising digestive development, nutrient utilisation and overall robustness.
Dietary protein hydrolysates are widely incorporated into larval microdiets due to their high digestibility and the presence of bioactive peptides, which have been shown to enhance digestive function, intestinal maturation, antioxidant capacity and nutrient utilisation in marine fish larvae. In addition to the level of hydrolysate inclusion, the physiological effects of these ingredients are also likely to depend on their peptide molecular weight profile, as peptide weight influences their digestion, intestinal uptake and subsequent physiological responses (Richard et al., 2015; Canada et al., 2017). However, despite increasing evidence supporting the use of protein hydrolysates in larval nutrition, the role of peptide molecular weight profile has received comparatively little attention. Therefore, this study evaluated whether diets containing identical levels of peptide hydrolysates but differing in peptide molecular weight profile modulate growth performance, oxidative status and intestinal molecular responses in Senegalese sole larvae.
Materials and Methods
Senegalese sole larvae were fed from 2 to 17 days after hatching (DAH) with one of two experimental microdiets containing the same inclusion level of peptide hydrolysates but differing in peptide molecular weight profile: a lower-molecular-weight peptide profile (LMP) or an intermediate-molecular-weight peptide profile (IMP). Diets were isonitrogenous and isolipidic and randomly assigned to triplicate tanks.
At 17 DAH, key performance indicators were evaluated through dry weight (DW), total length (TL), condition factor (K), relative growth rate (RGR) and survival rate. Oxidative status was assessed by measuring superoxide dismutase (SOD) and catalase (CAT) activities, and the reduced-to-oxidised glutathione ratio (GSH:GSSG), as well as lipid peroxidation (LPO). Gene expression analysis included antioxidant defence (sod2, cat, gpx4 and nrf2), digestive enzymes (ialp, ampn, lap3, tryp and ctrb), apettite and digestive regulation (cck and npy), nutrient transporters (pept1, lat1 and fabp2), and intestinal barrier markers (cldn15, tjp1 and muc2).
Results and Discussion
At 17 DAH, larvae reached an average dry weight of 0.67 mg, total length of 7.15 mm, and condition factor of 0.92. Mean survival was 48%, while the relative growth rate averaged 26.8% day-1. None of these performance parameters differed between dietary treatments, indicating that both peptide molecular weight profiles similarly supported early larval growth and survival. Likewise, no dietary differences were observed in SOD or CAT activities, the GSH:GSSG ratio, or LPO, indicating that peptide molecular weight profile did not alter basal redox status.
Despite the absence of phenotypic differences, the peptide molecular weight profile significantly influenced intestinal gene expression. Larvae fed the LMP diet exhibited higher expression of the antioxidant-related genes cat, gpx4 and nrf2, suggesting enhanced transcriptional regulation of endogenous antioxidant defences. In parallel, the coordinated upregulation of lap3, cck, pept1 and fabp2 suggests enhanced digestive function and nutrient assimilation, while increased muc2 expression indicates improved intestinal mucosal protection. In contrast, the expression of sod2, ialp, ampn, tryp, ctrb, npy, lat1, cldn15 and tjp1 remained unaffected by dietary treatment.
The coordinated modulation of genes involved in antioxidant regulation, digestion, nutrient transport and mucosal protection suggests that dietary peptide molecular weight profile influences intestinal physiological programming during early ontogeny. Although these transcriptional responses were not reflected in growth performance or redox status at this developmental stage, they indicate that a lower-molecular-weight peptide profile may promote greater physiological readiness of the larval intestine during a period of intense developmental plasticity.
Conclusions
Dietary peptide molecular weight profile modulated the intestinal transcriptional response of Senegalese sole larvae without affecting growth performance or basal oxidative status. A lower-molecular-weight peptide profile induced coordinated upregulation of genes associated with antioxidant defence, digestion, nutrient absorption and mucosal protection, highlighting peptide molecular weight distribution as an important nutritional characteristic to consider when formulating larval microdiets.
Acknowledgments
This work is part of project E!4876 FlatFIRST_1171, supported by EUROSTARS-3 program, and by Portugal and the European Union through ERDF, Algarve 2030, and COMPETE 2030, in the framework of Portugal 2030 and Portuguese national funds from FCT - Foundation for Science and Technology through contracts UID/04326/2025, UID/PRR/04326/2025 and LA/P/0101/2020 (DOI:10.54499/LA/P/0101/2020), and from the operational programmes CRESC Algarve 2020 and COMPETE 2020 through contract EMBRC.PT ALG-01-0145-FEDER-022121 to CCMAR.
References
Canada, P., Concei����o, L. E., Mira, S., Teodosio, R., Fernandes, J. M., Barrios, C., Mill��n, F., Pedroche, J., Valente, L.M.P., & Engrola, S. (2017). Dietary protein complexity modulates growth, protein utilisation and the expression of protein digestion-related genes in Senegalese sole larvae. Aquaculture, 479, 273-284. https://doi.org/10.1016/j.aquaculture.2017.05.028
Richard, N., Engrola, S., Palma, P. S., Simes, D. C., & Concei����o, L. E. (2015). Assessment of protein digestive capacity and metabolic utilisation during ontogeny of Senegalese sole larvae: a tracer study using in vivo produced radiolabelled polypeptide fractions. Aquaculture, 441, 35-44. https://doi.org/10.1016/j.aquaculture.2015.02.003