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 aquac

Insects 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. • Diets with increasing levels of insect meal inclusion modify Atlantic salmon fry feeding and activity behaviour. • Atlantic salmon fry fed diets with low inclusion of insect meal exhibit more natural feeding behaviours. • High inclusion level develops feeding aversion. • Central serotonergic turnover is downregulated by high insect meal inclusions.