Evidence of the Autophagic Process during the Fish Immune Response of Skeletal Muscle Cells against Piscirickettsia salmonis.

Autophagy is a fundamental cellular process implicated in the health of the cell, acting as a cytoplasmatic quality control machinery by self-eating unfunctional organelles and protein aggregates. In mammals, autophagy can participate in the clearance of intracellular pathogens from the cell, and the activity of the toll-like receptors mediates its activation. However, in fish, the modulation of autophagy by these receptors in the muscle is unknown. This study describes and characterizes autophagic modulation during the immune response of fish muscle cells after a challenge with intracellular pathogen Piscirickettsia salmonis. For this, primary cultures of muscle cells were challenged with P. salmonis, and the expressions of immune markers il-1ß, tnfα, il-8, hepcidin, tlr3, tlr9, mhc-I and mhc-II were analyzed through RT-qPCR. The expressions of several genes involved in autophagy (becn1, atg9, atg5, atg12, lc3, gabarap and atg4) were also evaluated with RT-qPCR to understand the autophagic modulation during an immune response. In addition, LC3-II protein content was measured via Western blot. The challenge of trout muscle cells with P. salmonis triggered a concomitant immune response to the activation of the autophagic process, suggesting a close relationship between these two processes.

Transcriptomic analysis reveals a Piscirickettsia salmonis-induced early inflammatory response in rainbow trout skeletal muscle.

Skeletal muscle is the most abundant tissue in teleosts and is essential for movement and metabolism. Recently, it has been described that skeletal muscle can express and secrete immune-related molecules during pathogen infection. However, the role of this tissue during infection is poorly understood. To determine the immunocompetence of fish skeletal muscle, juvenile rainbow trout (Oncorhynchus mykiss) were challenged with Piscirickettsia salmonis strain LF-89. P. salmonis is the etiological agent of piscirickettsiosis, a severe disease that has caused major economic losses in the aquaculture industry. This gram-negative bacterium produces a chronic systemic infection that involves several organs and tissues in salmonids. Using high-throughput RNA-seq, we found that 60 transcripts were upregulated in skeletal muscle, mostly associated with inflammatory response and positive regulation of interleukin-8 production. Conversely, 141 transcripts were downregulated in association with muscle filament sliding and actin filament-based movement. To validate these results, we performed in vitro experiments using rainbow trout myotubes. In myotubes coincubated with P. salmonis strain LF-89 at an MOI of 50, we found increased expression of the proinflammatory cytokine il1b and the pattern recognition receptor tlr5s 8 and 12 h after infection. These results demonstrated that fish skeletal muscle is an immunologically active organ that can implement an early immunological response against P. salmonis.

Effects of crowding on the three main proteolytic mechanisms of skeletal muscle in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Skeletal muscle is one of the tissues most affected by stress conditions. The protein degradation in this tissue is vital for the supply of energy mediated by different proteolytic pathways such as the ubiquitin-proteasome (UPS), autophagy-lysosome (ALS) and the calpain/calpastatin system (CCS). Nevertheless, the regulation of this proteolytic axis under stress conditions is not yet completely clear. Chile is the main producer of rainbow trout (Oncorhynchus mykiss) in the world. This intensive fish farming has resulted in growing problems as crowding and stress are one of the major problems in the freshwater stage. In this context, we evaluated the crowding effect in juvenile rainbow trout kept in high stocking density (30 kg/m3) for 15, 45 and 60 days, using a control group of fish (10 kg/m3). RESULTS: Plasmatic cortisol and glucose were evaluated by enzyme immunoassay. The mRNA levels of stress-related genes (gr1, gr2, mr, hsp70, klf15 and redd1), markers of the UPS (atrogin1 and murf1) and CCS (capn1, capn1, cast-l and cast-s) were evaluated using qPCR. ALS (LC3-I/II and P62/SQSTM1) and growth markers (4E-BP1 and ERK) were measured by Western blot analysis. The cortisol levels increased concomitantly with weight loss at 45 days of crowding. The UPS alone was upregulated at 15 days of high stocking density, while ALS activation was observed at 60 days. However, the CCS was inactivated during the entire trial. CONCLUSION: All these data suggest that stress conditions, such as crowding, promote muscle degradation in a time-dependent manner through the upregulation of the UPS at early stages of chronic stress and activation of the ALS in long-term stress, while the CCS is strongly inhibited by stress conditions in the rainbow trout muscle farmed during freshwater stage. Our descriptive study will allow perform functional analysis to determine, in a more detailed way, the effect of stress on skeletal muscle physiology as well as in the animal welfare in rainbow trout. Moreover, it is the first step to elucidate the optimal crop density in the freshwater stage and improve the standards of Chilean aquaculture.

Chronic stress inhibits growth and induces proteolytic mechanisms through two different nonoverlapping pathways in the skeletal muscle of a teleost fish.

Chronic stress detrimentally affects animal health and homeostasis, with somatic growth, and thus skeletal muscle, being particularly affected. A detailed understanding of the underlying endocrine and molecular mechanisms of how chronic stress affects skeletal muscle growth remains lacking. To address this issue, the present study assessed primary (plasma cortisol), secondary (key components of the GH/IGF system, muscular proteolytic pathways, and apoptosis), and tertiary (growth performance) stress responses in fine flounder ( Paralichthys adspersus) exposed to crowding chronic stress. Levels of plasma cortisol, glucocorticoid receptor 2 ( gr2), and its target genes ( klf15 and redd1) mRNA increased significantly only at 4 wk of crowding ( P < 0.05). The components of the GH/IGF system, including ligands, receptors, and their signaling pathways, were significantly downregulated at 7 wk of crowding ( P < 0.05). Interestingly, chronic stress upregulated the ubiquitin-proteasome pathway and the intrinsic apoptosis pathways at 4wk ( P < 0.01), whereas autophagy was only significantly activated at 7 wk ( P < 0.05), and meanwhile the ubiquitin-proteasome and the apoptosis pathways returned to control levels. Overall growth was inhibited in fish in the 7-wk chronic stress trial ( P < 0.05). In conclusion, chronic stress directly affects muscle growth and downregulates the GH/IGF system, an action through which muscular catabolic mechanisms are promoted by two different and nonoverlapping proteolytic pathways. These findings provide new information on molecular mechanisms involved in the negative effects that chronic stress has on muscle anabolic/catabolic signaling balance.