Changes in behaviour and serotonergic system of Atlantic salmon (Salmo salar) fry related to different levels of black soldier fly larvae meal inclusion in the diet: Exploring the use of nutritional enrichment for its use as positive welfare in aquaculture.

Abstract

nsects are gaining attention for its efficiency in converting low-value substrates into high-quality protein, aligning with principles of the circular economy. This study investigates black soldier fly larvae (Hermetia illucens, BSFL) as a protein source. BSFL meal offers a balanced nutritional profile and functional compounds that may enhance fish welfare. However, limited research exists on its effects on fish behaviour and it´s use as an operational welfare indicator (OWI), a crucial aspect for ensuring high welfare standards and understanding the impact on salmon performance. This study evaluated the feeding behaviour of Atlantic salmon (Salmo salar) fry conditioned from their first feeding stage with four isocaloric and isoproteic diets containing increasing levels of defatted BSFL meal (0, 8, 16, and 24 %), being continuously fed for 24 hours a day, over 21 days. Daily, four 30-minute video recordings (00, 06, 12, and 18 h) were analysed to observe fish activity using a group scan sampling method. The data were fitted to a sinusoidal model to identify circadian rhythm patterns. In addition, brain samples were collected at the end of the experiment for serotonergic activity analysis. Absolute real-time quantitative polymerase chain reaction (qPCR) was used to measure the gene expression of rate-limiting serotonin-synthesis enzymes (tryptophan hydroxylase 1a and tryptophan hydroxylase 2), while high-performance liquid chromatography with electrochemical detection (HPLC-EC) quantified serotonin (5-hydroxytryptamine, 5HT) and its primary oxidative metabolite, 5-hydroxyindolacetic acid (5HIAA). The results show that black soldier fly larvae meal (BSM) diets significantly affect fish behaviour. Low inclusion levels (8 %) emerged as promising for salmon nutritional enrichment, promoting natural behaviour patterns that could be associated with positive welfare. However, higher inclusion levels (24 %) negatively impacted welfare, as evidenced by reduced feed intake and anxiety-like behaviours linked to lower serotonergic turnover. These findings highlight the importance of incorporating behavioural measures when designing feeding protocols. Further research is essential to assess the long-term effects of BSM inclusion on salmon welfare and key performance parameters