Seawater-transferred rainbow trout farming is a developing industry, which has experienced a strong increase in production during the last three decades [9]. However, the incidence of GS fish has hindered its economic growth and is an animal welfare concern. The cause of the development of this unwanted phenotype remains unknown. Understanding the underpinning metabolic traits that characterise this phenotype could be the first step towards optimized rearing strategies and feeding protocols that decrease the incidence of GS. To this end, untargeted –omics approaches were used to study the liver proteome and lipidome of GS fish compared to FG fish, while targeted approaches were employed to measure smoltification status (NKA activity), growth-related (plasma IGF-I abundance and liver igf1, igfbp1b, ghr, and ctsl transcription) and stress-related parameters (plasma cortisol).
Since growth is the attribute that most clearly distinguishes GS and FG fish, it would be expected that growth-related molecular drivers differ between the two phenotypes. IGF-I is a peptide hormone that promotes growth in fish [51–53], along with growth hormone and its receptor GHr1, among others. IGFBP1b inhibits the binding of IGF-I to its receptor and represses growth [54, 55] and CTSL, an endopeptidase, are both induced under catabolic conditions [56, 57]. Indeed, differences between the two phenotypes were found in some of these growth-regulatory molecules. No evidence to confirm changes in the expression of ghr1, igfbp1b or ctsl was obtained, but the involvement of IGF-I was demonstrated. IGF-I was significantly lower in GS fish at both plasma and liver transcription levels. These results reiterate the importance of the somatotropic axis in the regulation of growth and show their involvement in the development of GS [58]. Moreover, while plasma IGF-I increased in FG fish during their time in seawater, levels in GS did not vary. This suggests that GS development might be associated to suboptimal seawater adaptation, which may trigger the development of this phenotype after animals are forcefully transferred to seawater. Another hypothesis for the reduction in growth once fish are transferred could be that GS fish become subordinate fish. Other studies have demonstrated how subordinate social status has a detrimental effect on growth, which is accompanied by a metabolic mobilisation of reserves [18, 59]. However, in such cases, metabolic changes have been, at least in part, driven by chronically high levels of plasma cortisol in the subordinate fish [17, 18, 60]. In the present study, plasma cortisol levels were similar between both groups suggesting that this phenotype is unlikely to be triggered by the development of a subordinate social status when animals are transferred at sea. Furthermore, from a metabolic perspective, subordinate fish have been shown to rely on β-oxidation of circulating free fatty acids rather than on triglycerides for energy [22] a feature not observed in the seawater-transferred GS phenotype.
The liver is a key organ for the accumulation and mobilization of energy reserves [37]. A labelled proteomic approach was used to unravel differences in the liver proteome between FG and GS. From a protein perspective, several proteins from the 19 identified differential proteins consistently pointed towards ER stress in GS livers. The ER is central to the processes of energy production, protein and lipid synthesis and it is closely related to oxidative stress and redox homeostasis [61]. In this study, an upregulation of chaperones known to increase in response to ER stress such as calreticulin and PDI [62, 63] was detected in GS livers. Downregulated differential proteins in the GS phenotype included catalase (I and II), a crucial antioxidant enzyme, the inhibition of which has been reported under ER stress conditions. Other proteins also reported to be closely linked to ER stress and redox homeostasis, such as glycine N-methyltransferase, or involved in lipid and energy homeostasis, such as annexin, SCP-2 and malate dehydrogenase were also differentially expressed in the two phenotypes [64, 65]. The increase, and more recently the proven relocation of stress induced ER cytosol chaperones to the cell plasma membrane have expanded the understanding of the functional role of these proteins, which can modulate immune responses in response to proteotoxic stress [66]. Within this context, ER stress has been shown to induce inflammation. The release of cytokines can be directly induced by ER/unfolded protein response (UPR) pathways or indirectly through the interaction with innate immune cells. In other organisms, the cross-talk of Toll-like receptors and ER/UPR pathways is associated to viral infections due to increased viral protein synthesis and assembly. This hypothesis cannot be discarded, as in the GS phenotype, besides the indication of ER stress, an increase in granzyme M was detected. This protein is a chymotrypsin-like serine protease that is abundantly expressed in innate effector natural killer cells and acts as a first line of defence against virus-infected or transformed tumour cells [67]. With current data, it is not possible to map out if the activation of ER/UPR pathways is a trigger or a consequence of an inflammatory state, a question that has been previously raised [68].
Other proteins upregulated in GS livers included the chaperone HSP90-α1, alpha-2-macroglobulin (broad spectrum protease inhibitor) and other proteins also associated with the ER. These proteins are involved in the translocation of secretory proteins (i.e. translocating chain-associated membrane protein) or involved in providing reducing equivalents to maintain adequate levels of reductive cofactors in the ER (i.e. GDH/6PGL endoplasmic bifunctional protein) [69, 70]. Therefore, our proteomic results in GS point towards ER stress as a key mechanism reflecting a state of functional imbalance.
As already mentioned, the ER is a key organelle of cellular lipid synthesis coordinating the transfer of lipids at the cellular level with ER stress associated with anomalous lipid metabolism [71]. In this respect, ER stressors can disrupt lipid metabolism. Lipidomic data showed important differences in the hepatic lipid composition of the two phenotypes. The biggest differences between FG and GS livers were found in energy reserve species (i.e. TAGs), which were lower in GS fish livers. Other studies have shown how ER stressors such as starvation and nutrient deficiencies can modulate autophagy, which plays a vital role in cell survival under long-term ER stress situations [72]. Autophagy provides cells with energy by mobilising energy stores such as TAGs. This mobilisation of lipid reserves is clearly observed in GS fish livers.
Other lipids which differentiated the two phenotypes were found in the phospholipid fraction. Phospholipids are key structural constituents of cellular membranes and of lipoproteins involved in the transport of dietary lipid from the intestine and liver to the rest of the body [73, 74]. In general terms, glycerophospholipids were elevated in GS fish livers, mainly PC, PE, PS and CL. Perturbations of glycerophospholipids, especially PC and PE levels, can result in lipid bilayer stress, which in turns causes ER stress [75]. Furthermore, being components of membranes, glycerophospholipids are affected by oxidative stress. This is especially the case for CL, due to their almost exclusive location in mitochondrial membranes where the electron-transport chain occurs, and where there is intense ROS production [76]. CL are involved in the biogenesis, dynamics and organization of mitochondrial membranes, controlling their permeability and contributing to the assembly of mitochondrial protein complexes involved in respiration and energy production [77, 78]. These lipids, which were elevated in GS fish livers, can be used as biomarkers for mitochondrial dysfunction [78, 79].
Some interesting associations between the proteomics and lipidomics datasets were also found. For instance, the higher levels of the differential protein SCP-2 in the FG group is likely related to the higher abundance of TAGs found in this group. SCP-2 is thought to transfer cholesterol and phospholipids from the inner ER membrane to the plasma membrane [80], binding both fatty acids and isoprenoids such as dolichol [81] and facilitating the esterification of cholesterol to cholesteryl-esters. This putative SCP-2 was more abundant in FG fish, with C20:3 cholesteryl-ester also found to be elevated within the same condition, albeit at low absolute levels. The abundance of TAG in FG fish livers likely results in the formation of lipoproteins within the blood, resulting in the translocation of cholesterol and phospholipid to the plasma membrane, as well as the upregulation of cholesteryl-esters as a means of storage. Also of interest is the decreased abundance, 21-fold, of dolichol in GS fish livers. GS fish were found to have elevated dolichyl-disphophooligosaccharide-protein glycosyltransferase subunit 1 protein, which correlates with the reduction in the dolichol substrate pool. Decrease in dolichol has been proposed as a marker of aging, as well as of calorie restriction. In calorie restricted mice, dolichol was found to decrease in the liver [82], with this trend appearing to be present within the GS fish, this may indicate that at least to some extent, GS are experiencing caloric restriction.
CL are implicated in the energetic balance [77, 78] and ceramides regulate a wide variety of molecular processes [83, 84]. While they are very different in composition and nature, both of these lipid classes are prone to peroxidation, which can lead to dysfunctional mitochondria in the case of CL [76], and to the induction of apoptosis for ceramides [83, 85]. In this sense, the differential proteins catalase (I and II) and PDI (I, II, and III) are both involved in cell redox homeostasis. Catalase catalyses the decomposition of hydrogen peroxide to water and oxygen and its activity is used as a biomarker of oxidative stress. Therefore, it is a crucial enzyme in protecting the cell from oxidative damage by ROS and has been proposed as a biomarker and potential tool for the treatment of liver diseases like hepatitis and hepatocarcinoma [86]. Moreover, this enzyme may also control bioenergetic metabolism by regulating the activity of the Krebs cycle, respiratory chain, and ATP synthesis [86]. On the other hand, PDI acts as a converging hub for hydrogen peroxide generation pathways, including oxidases and peroxidases [87]. Moreover, it is tightly connected to oxidoreductases, mitochondria, and NADPH oxidases, the three main mechanisms of oxidant generation [87–89]. Therefore, although PDI deficiency results in health conditions [90, 91], it represents a mechanism of oxidative stress regulation. Therefore, these two seemingly opposite differential proteins, in combination with the differences in CL and ceramide lipid composition, seem to indicate that GS might be under higher levels of oxidative stress. In turn, this could be associated with dysfunctional hepatic cellular membranes and mitochondrial membranes and might explain their physiological challenges. Indeed, hepatic oxidative stress induced by diet [92, 93] or chemical exposure has been linked to decreased growth and feed efficiency in fish. Therefore, it is possible that GS fish have different nutritional needs that their current diet is unable to fulfil.
In conclusion, results from this study reveal ER stress as a key mechanism underlying stunted growth in seawater transfer in rainbow trout. While the drivers leading to the activation of ER stress are still unknown, proteomic and lipidomic data point towards an unresolved viral infection or a nutritional deficiency as underlying drivers of this phenotype.