Stress initiates adaptive processes that allow the organism to cope with real or perceived threats. However, the initially “adaptive” stress responses can arise as “maladaptive” because of a persistent activation of stress effectors. This undesirable situation is controlled by the process of stress habituation, which refers to the physiological adaptation to recurrent exposure to persis tent or intermittent stressors. T here is now evidence in farmed gilthead sea bream that relatively high stocking den sity triggers a number of adaptive responses in nature, including changes in physical activity and social behaviour, metabolic homeostasis, muscle growth-regulatory mechanisms and microbial communities on the gut and skin surfaces (Holhorea et al., 2023: doi.org/10.3389/fphys.2023.1272267) . Moving forward, the aim of this study was the behavioural testing of the stress habituation of fish kept at different stocking densities , using a compression test as a recurrent stressor with focus on the stress recovery phase.
Two -year old fish (425-440 g) were grown up from June to August at two different stocking densities (CTRL 10-15 kg/m3 ; HD, 18-24 kg/m3 ) in a flow-through system (3,000 l tanks , IATS aquaculture infrastructure), following the natural changes in day-length and temperature that increased from 21ºC to 29 ºC over the course of the trial. Water [O2 ] was maintained always regulated, varying between 5.2-4.2 ppm and 4.2-3.2 ppm in CTRL and HD fish, respectively. At the end of the trial, randomly selected fish from CTRL and HD fish were taken for blood haematology and skeletal muscle transcriptomic analyses. Fish images were also taken for scoring external tissue damage. Then, four days later , fish were subjected to a compression test in two consecutive days with a 75% reduction of the available space for fish free displacements during 45 min (https://vimeo.com/927085326) . Behaviour was continuously monitored by means of the AEFishBIT data-logger, externally attached to the operculum for the simultaneous individual measurements of physical activity and respiration.
HD fish showed a reduc tion of voluntary feed intake associated to a significant reduction of growth rates and haematopoietic activity, as a result of a n inferred hypo-meta bolic state that is prone to use metabolic fuels for exercise rather than growth in comparison to CTRL fish . Otherwise, HD f ish showed signs of skin erosion and epidermal/muscle injuries that would promote a shift of the muscle transcriptome pattern towards muscle reparation rather than muscle protein accretion by itself. In line with all this, the recovery phase for the behavioural measurements of activity and secondly respiration rates after stress testing w ere shorte ned in HD fish. This became especially evident after the second stressor exposure , with a shortened stress recovery time after the repeated stressor in HD fish (2 h) , while it remained almost invariant in CTRL fish (6-8 h) . Altogether, this study provides new insights to infer the complex interplay of stress habituation and adaptive responses to high stocking densities in farmed gilthead sea bream.
This work was supported by AQUAEXCEL3.0 ( H2020; 871108) and Next-Generation GVA-ThinkInAzul ( THINKINAZUL/2021/024) projects.