Changes in circulating insulin-like growth factor-1 and its binding proteins in yearling rainbow trout during spring under natural and manipulated photoperiods and their relationships with gill Na+, K+-ATPase and body size.

Smoltification in salmonids occurs during spring in response to increasing photoperiod to prepare for marine life. Smoltification is associated with increased hypo-osmoregulatory ability and enhanced growth potential, mediated by growth hormone and insulin-like growth factor (IGF)-1. Rainbow trout is uniquely insensitive to the induction of smoltification-associated changes by photoperiod, such as the activation of gill Na+,K+-ATPase (NKA). We measured the circulating IGF-1 and IGF-binding protein (IGFBP)-2b levels in yearling rainbow trout exposed to natural and manipulated photoperiods during spring and correlated these with gill NKA activity and body size. Although the effect of photoperiod manipulation on body size and circulating IGF-1 and IGFBP-2b was negligible, they were positively correlated with gill NKA activity in fish under simulated natural photoperiod. We next pit-tagged yearling rainbow trout and fed them a restricted ration or to satiation under a natural photoperiod. In April, gill NKA activity was higher in the satiation group than in the restricted group and positively correlated with body size and growth rate. In addition, circulating IGFBP-2b was positively correlated with gill NKA, size and growth, whereas circulating IGF-1 was correlated only with size and growth. The relationship between circulating IGF-1 and growth intensified from May to June, suggesting that the IGF-1-growth relationship was disrupted in April when gill NKA was activated. Two additional IGFBPs were related to growth parameters but not to gill NKA activity. The present study suggests that circulating IGFBP-2b and IGF-1 mediate the size-dependent activation of gill NKA in yearling rainbow trout during spring.

Endoplasmic reticulum stress as a key mechanism in stunted growth of seawater rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. RESULTS: No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. CONCLUSION: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.

Measuring activity-rest rhythms under different acclimation periods in a marine fish using automatic deep learning-based video tracking.

Most organisms synchronize to an approximately 24-hour (circadian) rhythm. This study introduces a novel deep learning-powered video tracking method to assess the stability, fragmentation, robustness and synchronization of activity rhythms in Xyrichtys novacula. Experimental X. novacula were distributed into three groups and monitored for synchronization to a 14/10 hours of light/dark to assess acclimation to laboratory conditions. Group GP7 acclimated for 1 week and was tested from days 7 to 14, GP14 acclimated for 14 days and was tested from days 14 to 21 and GP21 acclimated for 21 days and was tested from days 21 to 28. Telemetry data from individuals in the wild depicted their natural behavior. Wild fish displayed a robust and minimally fragmented rhythm, entrained to the natural photoperiod. Under laboratory conditions, differences in activity levels were observed between light and dark phases. However, no differences were observed in activity rhythm metrics among laboratory groups related to acclimation period. Notably, longer acclimation (GP14 and GP21) led to a larger proportion of individuals displaying rhythm synchronization with the imposed photoperiod. Our work introduces a novel approach for monitoring biological rhythms in laboratory conditions, employing a specifically engineered video tracking system based on deep learning, adaptable for other species.

Polarized Trout Epithelial Cells Regulate Transepithelial Electrical Resistance, Gene Expression, and the Phosphoproteome in Response to Viral Infection.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

Liver colour scoring index, carotenoids and lipid content assessment as a proxy for lumpfish (Cyclopterus lumpus L.) health and welfare condition.

Ensuring lumpfish health and welfare in salmon farms is vital to reduce the high mortality rates reported and to guarantee a high delousing efficiency. Recent observations of farmed lumpfish livers have shown colours ranging from pale (colours 1 and 2), through bright orange (colours 3 and 4), to dark reddish-brown (colours 5 and 6), some of which may be related to welfare condition. To characterize the status of lumpfish deployed in four Faroese salmon farms, several welfare indicators were assessed: a weight-length relationship, scoring of external physical damage, and after dissection, stomach content and liver colour scoring. Liver samples were weighed, stored and analysed for lipid content, lipid classes, total pigments, fatty acid profile and histopathology to explain the differences between the mentioned liver colours. Bright orange livers, liver colours 3 and 4, were related to increased levels of carotenoid pigments rather than levels of lipids and appear to reflect good fish welfare. However, dark reddish-brown colours, liver colours 5 and 6, were associated with very low levels of triacyl glycerides in the liver, indicating use of lipid reserves and poor welfare condition. Histopathology confirmed that the dark reddish-brown livers, liver colours 5 and 6, formed a distinct group. Thus, liver colour was shown to be a good welfare indicator and should be monitored in farms.

Innovation in Nucleotide-Binding Oligomerization-Like Receptor and Toll-Like Receptor Sensing Drives the Major Histocompatibility Complex-II Free Atlantic Cod Immune System.

The absence of MHC class II antigen presentation and multiple pathogen recognition receptors in the Atlantic cod has not impaired its immune response however how underlying mechanisms have adapted remains largely unknown. In this study, ex vivo cod macrophages were challenged with various bacterial and viral microbe-associated molecular patterns (MAMP) to identify major response pathways. Cytosolic MAMP-PRR pathways based upon the NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) were identified as the critical response pathways. Our analyses suggest that internalization of exogenous ligands through scavenger receptors drives both pathways activating transcription factors like NF-kB (Nuclear factor-kappa B) and interferon regulatory factors (IRFs). Further, ligand-dependent differential expression of a unique TLR25 isoform and multiple NLR paralogues suggests (sub)neofunctionalization toward specific immune defensive strategies. Our results further demonstrate that the unique immune system of the Atlantic cod provides an unprecedented opportunity to explore the evolutionary history of PRR-based signaling in vertebrate immunity.

Plasma proteome profiling of freshwater and seawater life stages of rainbow trout (Oncorhynchus mykiss).

The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.