Analysis of the molecular mechanism of brain 

tissue of Thamnaconus septentrionalis in response 

to density stress based on transcriptome sequencing

Abstract: Thamnaconus septentrionalis, usually called green fin puffer, is a small to median sized fish in family Monocanthidae, order Tetraodontiformes. It is mainly distributed in the coastal areas of China, Japan, and Korea. In China, it is widely distributed in the Bohai Sea, Yellow Sea, East China Sea, and Taiwan Strait. Among them, East China Sea is one of the most important distribution zones of T. septentrionalis. T. septentrionalis is a relatively common economic fish along the coastal areas of China. After nearly forty years of intensive exploitation, T. septentrionalis resources have declined rapidly, and can not meet the market demand to a great extent at present. Replacing “catching” with “culturing” is one of the most effective way to relieve the pressure of wild resources and ensure the market supply of T. septentrionalis.

High density is one of the most important limiting factors affecting the growth of fish. Density stress can intensify the competition for space, food, oxygen and other resources within the population, and affect gene expression and adaptability. Some studies show that crowding can significantly induce the immune response of fish, and longterm density stress can damage the immune capacity of fish. Under high density conditions,  the composition of fatty acids of fish muscle changes, and the activity of lipid metabolic enzymes can be stimulated with up regulation significantly. Also, high density culture may result in a significant decrease in the activities of various digestive enzymes, which then can affect the immune capacity of organisms. Till now, the molecular response mechanism of brain tissue of T. septentrionalis to density stress has been still unclear.

Transcriptome refers to the collection of all transcripts in a cell under a certain physiological condition. It includes messenger ribonucleic acid (mRNA), ribosomal ribonucleic acid (rRNA), transfer ribonucleic acid (tRNA) and noncoding ribonucleic acid (ncRNA). Transcriptomics is a discipline that studies gene transcription and transcriptional regulation of all kinds of genes and pathways in all cells at the overall level. Transcriptomics is an important means to reveal the molecular mechanism in organisms. When performing transcriptome analysis, we can firstly carry out highthroughput sequencing on aquatic animals, further analyze the expression of genes, annotate the functions of genes, and make clear the expression of relevant genes under specific conditions based on highthroughput sequencing results. Transcriptome analysis has been widely used in studying the biology of some aquatic animals.

In order to understand the molecular response mechanism of the brain tissue of T. septentrionalis under density stress, explore the differentially expressed genes, clarify the regulatory mechanism of differentially expressed genes in the brain tissue of T. septentrionalis, and provide a scientific base for the future studies of intensive artficial aquacuture and molecular breeding of T. septentrionalis, we set up two culturing density groups, a mediumdensity group of 100 ind·m-3 , and a highdensity group of 500 ind·m-3. The brain tissue of the two densities of T. septentrionalis on the 25th and 50th day was obtained, respectively, and the transcriptome sequencing analysis of the brain tissue samples of T. septentrionalis was carried out by using Illumina sequencing platform. A total of 100.3 Gb of highquality sequencing data was obtained. Then, we treated the data in turn with some steps, i.e., quality control, comparison of the obtained data, difference analysis, GO （Gene Ontology）function annotation analysis, KEGG （Kyoto Encyclopedia of Genes and Genomes） function enrichment analysis, and trend analysis. To facilitate the the results, some codes were used in this study: CB means the brain tissue of the control group; D1B represented the brain tissue of the 100 ind·m-3 group on the 25th day; D2B was the brain tissue of the 500 ind·m-3 group on the 25th day; d1B represented the brain tissue of the 100 ind·m-3 group on the 50th day; d2B was brain tissue of the 500 ind·m-3 group on the 50th day. The results were as follows : In CBvsD1B, the expression of 197 genes were upregulated and 418 genes were downregulated; in CBvsD2B, the expression of 114 genes were upregulated and 301 genes were downregulated; in vsd1B, the expression of 317 genes were upregulated and 491 genes were downregulated; in CBvsd2B, the expression of 311 genes were upregulated and 608 genes were downregulated. 2) GO classification analysis showed that differentially expressed genes belonged to 44 categories of biological process, cellular component and molecular function. 3) KEGG enrichment analysis showed that the signal pathways related to the circadian rhythm were significantly enriched in the four different groups. 4) The trend analysis focused on signal pathways such as circadian rhythm, MAPK signaling pathway, calcium ion signaling pathway, and found that some key genes such as clock, cry, bmal1, npas2, bdnf, rps6ka, il1r1, htr5a, ryr2, and pln were differentially expressed in the brain tissue of T. septentrionalis. These differentially expressed genes may play key role in regulating the circadian rhythm and immune ability of T. septentrionalis, which can lay a foundation for studying the regulation mechanism of gene expression of related genes in brain tissue of T. septentrionalis under density stress.

Keywords: Thamnaconus septentrionalis; transcriptome; density stress; gene expression; brain