Effects Of Acute Handling Stress On Coilia Nasus

The Coilia nasus is one of the most important economic fishes. However, the capture production of C. nasus has been diminishing these years, because of the water pollution, over-fishing, coastal construction and many other complex factors. Worse still, C. nasus is strongly sensitive to stress, such as handling. This often results in death, which causes huge losses in aquaculture. In order to deepen the understanding of the mechanism of the acute stress-related neuroendocrine factor after handling in C. nasus, and find out the effective method to relieve the stress, we at first used RIA to detect the cortisol levels in plasma and head-kidney, and then identified the partial sequences of C, nasus CRH, UI, POMC and (3-actin using the primer pairs designed according to homologene sequences in conservative regions. After that, brain CRH, POMC and UI mRNA levels were determined by real-time quantitative PCR (RT-qPCR). Secondly, we used high-throughput transcriptome sequencing to analysis the difference gene expression profiles between the control group (Cl), less sensitive group (S1) and most sensitive group (S2). Eleven stress-related DEGs were chosen for validation using RT-qPCR; and several molecular markers were verified. At last, we detect the cortisol levels, free thyroxin (FT4) content and the liver malondialdehyde (MDA) levels under the conditions of different concentration of SNP and10%o NaCl after handling. Then we used Larva Coilia nasus and primary cell of liver as models to further study the mechanism of SNP to alleviate stress. The main results are as follows:(1) CRH and POMC mRNA expressions were significantly declined after half hour stimulation (P<0.01)(P<0.05). But there is no change in U mRNA level. Taken together, our study reflected the particularity of stress-related-molecule expressions under the handling acute stress in brain of C. nasus through hypothalamic-pituitary-interrenal axis.(2) Through high-throughput transcriptome sequencing, we discovered4072genes were up-regulated and8712genes were down-regulated in the most sensitive group when compared with the control, while the less sensitive group had2283genes expression increased and4297decreased. Eleven stress-related DEGs were chosen for validation using RT-qPCR, and all transcripts had the same trend in expression based on qPCR and RNA-seq respectively. In GO enrichment analysis of DEGs between C1and S2, we found three thyroid hormone-related GO terms were significantly enriched, including abundant thyroid hormone-related DEGs, while we did not find these DEGs between C1and S1. These results suggested thyroid signaling pathways might be associated with the sensitivity of C. nasus to acute stress.(3) Compared with the control group, C. nasus in100μM SNP and10%o NaCl condition had a higher survival rate after handling, and shared a steadier cortisol or FT4level and a less MDA level. These results indicated that100μM and10%o NaCl relieve the stress by maintaining the metabolism balance and reducing the liver lipid peroxide levels.(4) The laser-scan confocal vivo image showed that larva C. nasus, under the stimulation of AAPH (induced oxidative stress), had lower reactive oxygen species (ROS) level in100μM SNP condition, it suggested that100μM SNP is benefit for reducing oxidize stress in larva C. nasus.(5) The result of ROS level and laser-scan confocal image in liver primary cell of C. nasus showed that100μM SNP could relieve the ROS level induced by AAPH, but300μM SNP could caused excessive oxidative reaction.