Transcriptomic response to three osmotic stresses in gills of hybrid tilapia (Oreochromis mossambicus female × O. urolepis hornorum male)

Abstract

Background: Osmotic stress is a widespread phenomenon in aquatic animal. The ability to cope with salinity stress and alkaline stress is quite important for the survival of aquatic species under natural conditions. Tilapia is an important commercial euryhaline fish species. What's more tilapia is a good experimental material for osmotic stress regulation research, but the molecular regulation mechanism underlying different osmotic pressure of tilapia is still unexplored.

Results: To elucidate the osmoregulation strategy behind its hyper salinity, alkalinity and salinity-alkalinity stress of tilapia, the transcriptomes of gills in hybrid tilapia (Oreochromis mossambicus ♀ × O. urolepis hornorum ♂) under salinity stress (S: 25‰), alkalinity stress(A: 4‰) and salinity-alkalinity stress (SA: S: 15‰, A: 4‰) were sequenced using deep-sequencing platform Illumina/HiSeq-2000 and differential expression genes (DEGs) were identified. A total of 1958, 1472 and 1315 upregulated and 1824, 1940 and 1735 downregulated genes (P-value < 0.05) were identified in the salt stress, alkali stress and saline-alkali stress groups, respectively, compared with those in the control group. Furthermore, Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted in the significant different expression genes. In all significant DEGs, some of the typical genes involved in osmoregulation, including carbonic anhydrase (CA), calcium/calmodulin-dependent protein kinase (CaM kinase) II (CAMK2), aquaporin-1(AQP1), sodium bicarbonate cotransporter (SLC4A4/NBC1), chloride channel 2(CLCN2), sodium/potassium/chloride transporter (SLC12A2 / NKCC1) and other osmoregulation genes were also identified. RNA-seq results were validated with quantitative real-time PCR (qPCR), the 17 random selected genes showed a consistent direction in both RNA-Seq and qPCR analysis, demonstrated that the results of RNA-seq were reliable.

Conclusions: The present results would be helpful to elucidate the osmoregulation mechanism of aquatic animals adapting to saline-alkali challenge. This study provides a global overview of gene expression patterns and pathways that related to osmoregulation in hybrid tilapia, and could contribute to a better understanding of the molecular regulation mechanism in different osmotic stresses.

Keywords: Osmoregulation; Osmotic stress; Tilapia; Transcriptome.

Fig. 1

Hierarchical cluster analysis of differentially expressed genes based on the data of log ratio fold change. Heat map showing differentially expressed mRNAs upon osmotic stress in hybrid tilapia. The expression heat map was generated with the value of log₁₀^FPKM. The color scale shows the levels of differentially expressed genes: red color indicates enhanced expression of mRNA and blue color indicates decrease in expression levels of mRNA

Fig. 2

The number of differentially expressed DEGs in different groups

Fig. 3

DEGs number and venn diagram of overlap of the different groups. a: DEGs number and venn diagram of overlap of the C vs. S, C vs. A and C vs. SA groups. b: DEGs number and venn diagram of overlap of the C vs. S, C vs. A, C vs. SA, S vs. SA and A vs. SA groups

Fig. 4

Gene Ontology (GO) classification of assembled unigenes. a: C vs. S; b: C vs. A; c: C vs. SA; d: S vs. SA; e: A vs. SA

Fig. 5

Gene Ontology (GO) classification of differential expression genes in group S, A, SA, S & A, S & SA, A & SA S, and A & SA. a: S; b: A; c: SA; d: S & A; e: S & SA; f: A & SA; g: S, A & SA

Fig. 6

Validation of RNA-seq data by qPCR. To validate the RNA-seq data, the relative mRNA levels of 17 randomly selected DEGs in the gills of hybrid tilapia were examined by qPCR. The mRNA levels by qPCR are presented as the fold change compared with the mock-treated control after normalization against β -actin. The relative expression levels from the RNA-seq analysis were calculated as log₂FC values. The gene nkai1 was calculated in two different groups. (Note: nkain1–1 was in a group C vs. A, nkain1–2 was in group C vs. SA; trpv4–1 was in group S vs. SA, trpv4–2 was in group C vs. S)