1. Introduction

The demand for animal protein for human consumption is rising as a result of an exponential increase in the world population. Aquaculture is becoming an increasingly important source of protein available for human consumption since is an industry capable of providing solutions to feed a rapidly growing human population and reduce poverty in many countries [1,2,3]. To achieve that, the scale of aquaculture production and the range of farmed species has increased dramatically over the last two decades [4]. Live production always comprises a risk for loss due to infectious diseases [5], with farmed fish, due to husbandry practices in aquaculture, being more vulnerable than wild fish to diseases from a wide range of bacterial, viral, parasitic, and fungal infections [6]. Also, the tendency to higher density production systems, the perturbations in ecological systems balance related to pollution and climatic changes, and the expected increase in international transactions of aquaculture products and their derivatives contributed to alterations in the dynamics of interaction between organisms, infectious agents, and people. This influences pathogen rates of replication and proliferation, leading to a broader geographic distribution of pathogenic agents and an increase in species affected by disease outbreaks [7,8]. This makes disease outbreaks an important constraint to this industry, with a significant impact on the quality, safety, and volume of the fish produced throughout the world [9,10,11,12], that can lead to market access exclusion and major economic loss or costs to the producer [8,13,14].

For several authors, disease outbreaks in aquaculture are the result of a complex network of interactions on aquatic systems between the produced organism, several environmental and zootechnical aspects, and possible pathogenic agents, that present a series of unique challenges in aquatic organism’s health [15,16,17,18,19], as represented in Figure 1.

To address infectious pathologies in farmed fish approaches like epidemiological studies on main areas of aquatic animal health as transboundary and emerging aquatic animal diseases, animal health surveillance and biosecurity program development should be performed. These are crucial to disease prevalence monitorization, early detection of emerging exotic and new diseases, and quality management improvement of aquaculture operations [15,18,19,21].

Nevertheless, to obtain proper epidemiological models, animal health surveillance and biosecurity programs must integrate environmental information and information from different areas like pathogenesis, disease diagnosis, disease resistance, physiological response to pathogens, pathogen characterization, host immune system responses characterization, disease biomarkers, and organism response to disease treatment products [22,23].

The amount of data from different origins and an increase in the reported frequency and severity of marine diseases demands that new diagnostic tools should be implemented for a more rapid and effective diagnosis [24,25,26]. Thus, several scientific advances in aquatic health continue to close the gap to veterinary medicine, and new optical, analytical chemistry, molecular biology [27], and Omics techniques are becoming a reality that offers a vast array of benefits to the aquaculture industry [12,28]. Proteomics techniques are one of those new tools, and one of the most interesting approaches for health management, epidemiology, and fish disease research [3,22,23,29,30]. Proteomics refers to the methodology that addresses the study of the entire complement of proteins expressed in a specific state of an organism or a cell population [31,32]. The proteome, or the full protein complement of the genome, is a highly structured entity, where proteins exert their cellular functions with specificity in time and location, in physical or functional association with other proteins or biomolecules [33,34]. High-throughput proteomics methods based on mass spectrometry (MS) allow the measurement of multiple properties for thousands of proteins, including their abundance, tissue distribution, subcellular localization, post-translational modifications, and protein-protein interactions [34]. Proteomics-based approaches can therefore offer unique insights into fish cellular regulation in response to pathogens and during disease progression, besides enabling fast and sensitive pathogen detection and identification.

2. Fish Health, Stress, and Welfare

Despite being the most consumed animal, fish are seldom afforded the same level of concern regarding their welfare as other vertebrates. The scientific research around fish welfare is at an early stage compared with other land animals produced for human consumption [35]. In part, this lack of consideration is due to the gap between public perception of their intelligence and the scientific evidence [36], along with the absence of a unified definition of the concept [37]. Nevertheless, most definitions consider mainly a feelings-based and a function-based approach [38]. The first gives regard to the emotional-like state of the animal, while good welfare is defined as the absence of negative feelings and the presence of positive feelings [39]. The second definition is more focused on the biological, physiological, and health perspective of the animal, while good welfare is defined as the fish’s ability to cope and adapt to its environment while maintaining homeostasis [40]. Although the fish’s health state offers objective criteria as part of a welfare assessment, it does not provide the complete picture. Good health is essential to ensure good welfare, however, it does not necessarily indicate that the fish is in a good welfare state [37]. On the other hand, poor health i.e., the reduced ability of the animal to normal functioning, to cope with stressful conditions, and to prevent disease, generally implies/leads to a bad welfare status in a variety of contexts. For example, deceased fish, as a consequence of disease, constitute a source of infection and compromise water quality [41]. Additionally, chemical treatments for specific outbreaks can also trigger some level of disturbance on the fish [42,43]. Importantly, a healthy animal in an optimal welfare environment can also be suddenly struck by an acute infection reducing its welfare. For instance, in the case of fish produced in cages, pathogens are naturally embedded in the environment [44]. In most cases, it is often the lousy welfare status itself, due to poor husbandry conditions, which translates into impaired health. Thus, in summary, health and welfare are intimately linked, and poor welfare can be interpreted both as a cause and a consequence of poor health. This section focuses on health as a cornerstone for fish welfare assessment and the effects of stressors on disease resistance, reviewing the most recent approaches employed to study the relationship between certain diseases/pathologies and welfare.

In aquaculture, inappropriate husbandry conditions, or even standard farming practices, are everyday stressors in culture systems [45]. The allostatic load imposed on the animals can reduce functioning immune mechanisms, consequently favoring diseases and threatening fish welfare (Figure 2). For instance, drastic changes in water temperature (from 27 °C to either 19–23 °C or 31–35 °C) decreased the immune response and resistance to pathogens in Mozambique tilapia (Oreochromis mossambicus) [46]. More recently, using a transcriptomics approach, the rearing density in Nile tilapia (Oreochromis niloticus) was shown to significantly impact the susceptibility to the oomycete Saprolegnia parasitica [47]. However, the association between husbandry-induced stress and disease is not that straightforward. For example, acute stressors have been reported to enhance [48,49,50,51] or decrease [52,53] some innate immune responses in fish. On the contrary, chronic stressors have mainly been indicated as immunosuppressors [54,55,56,57,58]. From a productivity perspective, the health of the fish is often interpreted as “absence of disease”, since, from either an ethical or an economic point of view, any disease state is unacceptable for the industry [44]. Therefore, disease prevention and eradication are crucial aspects of a fish farm to ensure the product’s sustainability. Providing optimal welfare conditions, monitoring the health parameters routinely, and alleviating stress are necessary steps towards this goal.

Stress is considered a state of threatened homeostasis [59], which is re-established by a complex network of changes in the physiological systems (allostasis) [60]. As in all other vertebrates, in the face of a perceived stressor, fish launch a widespread reaction, the so-called physiological stress response, which allows the individual to adjust and cope with the predictable and unpredictable changes in its surroundings (eustress) [61]. As a primary response, cortisol and catecholamines are released into the bloodstream, which will induce a series of downstream reactions [62]. In fact, stress is not necessarily detrimental nor immediately equates to compromised welfare. Instead, in the short term, it is an essential adaptation to ensure the best chances of survival [37]. However, when reaching an allostatic overload, usually as a result of a prolonged, repeated, and/or unavoidable stressor, maladaptive effects such as impaired growth and/or reproductive and immune functions, arise (Figure 2) [63,64]. In this case, these are largely associated with diminished welfare and may jeopardize fish health and survival (distress) [65]. The questions raised here are the cost of this acclimation and why stress increases diseases’ susceptibility in fish. First, in terms of energetic costs, the adaptive physiological response needed to counteract the disrupted homeostasis requires a significant amount of energy. This means that if part of the fish’s energy is allocated to face the challenge, then fewer resources will be available for other energy-demanding biological functions, such as some mechanisms of the defense repertoire: the epithelial barriers and the immune system [44]. In terms of immune responses, several mechanisms are immediately activated to respond directly to the challenge. These include an increase of inflammatory markers, the release of hormones, and the expression of acute-phase proteins [66]. Even if a fish has managed to adapt to the stressor for a certain period, these energy stores will eventually be depleted if the stressor persists. Consequently, the total consumption of energy reserves gives rise to the allostatic overload, and the fish may no longer be able to adapt, which can lead to immunosuppression, disease, and in the case of more severe disturbances, even death (Figure 2) [63]. Moreover, several studies also demonstrated the impact of stressful husbandry conditions on the functioning of the epithelial barriers, i.e., the mucus and the epidermal surfaces of the skin, gills, and intestine, which constitute the primary lines of defense against pathogens and harmful substances, showing that injury of these barriers, inevitably leads to impaired disease resistance [67]. Changes in these barriers have been reported in Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua), and rainbow trout (Oncorhynchus mykiss) subjected to different acute stressors [68,69]. Moreover, in Atlantic salmon reared under low dissolved oxygen levels, impaired intestinal barrier function was also observed [70]. These disturbances have mainly been associated with high cortisol levels, though various other hormones, such as catecholamines, endogenous opioids, pituitary hormones, and serotonin, intervene here [71]. Indeed, it is known that cortisol plays an immunomodulatory role, inhibiting specific constituents of the immune system and enhancing others, such as induction of apoptosis, change of differentiation patterns, inhibition of cytokine release, and inhibition of immunocyte migration [72,73,74,75]. Nevertheless, the cortisol response may vary among different species and even among individuals (coping styles) [76] and be affected by several other parameters (e.g., domestication level, age, nutritional state, stressor severity, among others) [53,77,78,79], which may obscure the relationship between stress and immune status. A detailed description of how the endocrine-immune response is mounted and the mechanisms behind these immunoregulatory changes is out of the scope of this review, for this, the authors refer to recent publications [66,80].

Deepening our scientific knowledge on the mechanisms relating to stress, fish health, and welfare, is paramount for the sustainable aquaculture industry. In recent years, more advanced high-throughput technologies, as the case of proteomics, started to be successfully employed in aquaculture research, including for the study of fish diseases and welfare, providing a holistic understanding of the molecular events underlying the physiological stress response and valuable insights on the differential proteins involved in inflammatory processes and immune responses [30,58,81]. Proteomic studies on fish target mainly the liver, however, blood plasma and mucus are taking crescent importance, mainly from an immunological point of view, as skin mucus is one of the primary barriers of defense in fish [82,83,84,85,86] and plasma acts as a mirror/reporter of physiological or pathological conditions [87,88]. Important applications of proteomics in this field concern the study of the effects of certain diseases and parasites on the proteins’ abundance and modifications and the investigation of the host-pathogen interactions [88,89,90,91,92,93,94,95].