Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research

Abstract

Background: Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the wholeanimal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators. Results: A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus. Conclusions: Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.

Mid-infrared spectroscopic screening of metabolic alterations in stress-exposed gilthead seabream (Sparus aurata)

“…In the HYP trial, fish, with an initial body weight of 397.99 ± 16.56 g, experienced low levels of saturated oxygen over 48 h. Nitrogen was injected in the water according to the experimental groups' requirements: HYP CTRL -100% saturated oxygen, HYP 30 -30% saturated oxygen, and HYP 15 -15% saturated oxygen. Data regarding zootechnical parameters were previously published by the authors 4 . At the end of each experimental period, three fish per tank (n = 9 per treatment) were lethally anaesthetized using tricaine methanesulfonate (MS-222; Sigma Aldrich, St. Louis, Missouri, USA).…”

“…These potential changes in these absorption bands appear to be all correlated and suggest a metabolic reprogramming in the fish system to deal with the increase of energy demand during adverse situations. The plasma levels of specific metabolites associated with the physiological response to stress were assessed in these fish in a previous study and published elsewhere 4 . Activation of the HPI-axis was previously suggested and supports the hypothesis of a potential metabolic reprogramming to deal with the stressors that fish were exposed to 4 .…”

“…Stress in fish has been extensively studied 3 , but only recently, more modern and sensitive techniques started to be applied in this endeavour. High-throughput technologies have been gaining popularity to unveil the main changes occurring in farmed fish metabolism caused by stressful rearing conditions 4 . Among them is metabolomics, which allows for the non-selective chemical analysis of metabolites in a given biological system 5 .…”

“…In the present work, gilthead seabream ( Sparus aurata ) adults, a widely cultured species in the European aquaculture, were submitted to three different stressful rearing conditions, namely overcrowding, net handling and hypoxia. These stressors were demonstrated before to induce significant changes in the levels of specific metabolites, known to be associated with the physiological stress response in fish, and stress-related proteins, in the blood plasma of farmed gilthead seabream ( Sparus aurata ) 4 . In this work, potential metabolic changes were investigated in the liver of the challenged fish, since this organ plays a key role in the metabolic responses triggered by the stress response, mainly in the supply of energy for the animal to cope with adverse situations 2 .…”

Proteomic Strategies to Evaluate the Impact of Farming Conditions on Food Quality and Safety in Aquaculture Products

“…The effects of stress on growth have been studied extensively in animal production and aquaculture [ 54 ]. Different chronic stress conditions, such as confinement, overcrowding, repetitive handling, deficient water and diet and hypoxia, affect the welfare and stress response of aquaculture organisms and several proteomic studies have identified robust protein signatures for chronic stress in fish [ 55 ]. Elevated cortisol due to long-term stress conditions has a strong impact on the entire organism and is directly linked to the inhibition of muscle growth by inhibiting protein synthesis and increasing protein catabolism to obtain energy from amino acids [ 56 ].…”