Although still produced on a small scale [1], meagre (Argyrosomus regius) is a fish species with the potential to expand in the Mediterranean aquaculture industry due to its high flesh quality, fast growth, high feed efficiency, and good adaptation to captivity [[2], [3], [4]]. However, the species' nutritional requirements are still not well known, and developing diets for this species that further promote growth and health is an important step towards increasing production.

Novel diet formulations tend to rely less on fisheries products, which are the traditional ingredients used in carnivorous fish feeds, and use more economical and sustainable ingredients that can still fulfil species' nutritional requirements but often may exert physiological alterations that lead to less than optimal growth, health, and welfare [5]. Although the prolonged effects of such novel diets are mostly unknown, they may render fish more susceptible to diseases and stress.

Studying fish welfare is complex, may cause additional stress to the fish, and often requires animal sacrifice. Therefore, it becomes imperative to find proper techniques and biomarkers that allow a better evaluation of new diets' effects and that are easy and practical to apply in an aquaculture setting. Using non-lethal methods to evaluate fish health and welfare has generated interest as it allows for less animal sacrifice and reduces production losses [6]. These methods usually measure parameters from blood, mucus, skin, and feces samples to monitor fish health and welfare [[7], [8], [9]].

Blood analysis is a routine procedure to assess physiological parameters in humans and other vertebrates. Blood can be easily and quickly obtained, and provides crucial information on various physiological characteristics, from immunological status to stress response and nutritional condition [6,10]. However, it is important to highlight that blood biomarkers must be optimized for each species [11,12]. Ideally, each marker should have a reference interval specific to the studied species, representing the healthy baseline values under production conditions and tailored to the species and age group, thereby enabling a better understanding of how specific alterations, such as changes in nutrition, impact fish well-being. These reference intervals are crucial as biomarker values can be directly and indirectly influenced by both intrinsic factors (such as species, sex, and age) and extrinsic factors (such as temperature, salinity, and population density) [13]. Reference intervals for blood parameters of fish have been obtained for well-known species, such as European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), Atlantic salmon (Salmo salar), among others [10,[12], [13], [14], [15]]. Furthermore, most studies only include haematological parameters, lacking information on other important biochemical and immunological parameters [6].

Skin mucus establishes a critical barrier between the fish and external threats [16,17]. Mucus is composed of different biomolecules that can vary depending on species, endogenous (e.g., sex, developmental stage) and exogenous (e.g., stress, pH, infections, nutrition) conditions [16,18]. Skin mucus has an important role in fish immunity and stress response, as evidenced in studies performed in different species [[19], [20], [21]]. Studies on the effect of nutrition on skin mucus have received some attention in the last decade but remain limited [[22], [23], [24], [25], [26]]. In meagre, skin mucus response to newer feeds has only been recently studied by [27,28].

Proteomics holds significant promise for biomarker discovery in research, offering insights beyond expression levels by also characterising protein post-translational modifications [29]. Moreover, proteomic analysis can be performed on various sample types, including non-lethal ones, helping to minimise animal sacrifice. By enabling the study of multiple proteins simultaneously, and allowing for the study of biochemical pathways, it provides a more specific and accurate understanding of the effects of external factors on an organism [30,31]. This way it can uncover biomarkers that would allow for an early and effective detection of disease [32]. The rise of shotgun proteomics studies in aquaculture nutrition has been relatively recent. Proteomics has a multi-faceted application ranging from reproductive management to species conservation, disease control, and beyond [33] and has been applied in aquaculture to study, for instance, diseases [34] and stress [35,36]. As fish nutrition significantly impacts fish metabolism, growth, health, and welfare, the application of proteomics in this field has been increasing in recent years [33].

Proteomic analysis of plasma and skin mucus has been applied to study the effects of stress [[37], [38], [39]], chemotherapeutics [40], disease [41,42], and nutrition [43,44] in different fish species. So far, however, no studies have dived into the proteomic analysis of skin mucus and plasma of meagre.

This study aimed to analyze the blood and skin mucus proteome of meagre fed with diets designed to induce optimal and suboptimal nutritional status, aiming to identify sensitive non-lethal markers related to malnutrition and explore their potential as biomarkers to predict nutritional status. Additionally, the study sought to evaluate physiological plasma parameters to elucidate their response to malnutrition and their relationship with the proteome.