Genome-wide identification of the NHE gene family in Coilia nasus and its response to salinity challenge and ammonia stress

Abstract

Background: In aquatic environments, pH, salinity, and ammonia concentration are extremely important for aquatic animals. NHE is a two-way ion exchange carrier protein, which can transport Na⁺ into cells and exchange out H⁺, and also plays key roles in regulating intracellular pH, osmotic pressure, and ammonia concentration.

Results: In the present study, ten NHEs, the entire NHE gene family, were identified from Coilia nasus genome and systemically analyzed via phylogenetic, structural, and synteny analysis. Different expression patterns of C. nasus NHEs in multiple tissues indicated that expression profiles of NHE genes displayed tissue-specific. Expression patterns of C. nasus NHEs were related to ammonia excretion during multiple embryonic development stages. To explore the potential functions on salinity challenge and ammonia stress, expression levels of ten NHEs were detected in C. nasus gills under hypotonic stress, hypertonic stress, and ammonia stress. Expression levels of all NHEs were upregulated during hypotonic stress, while they were downregulated during hypertonic stress. NHE2 and NHE3 displayed higher expression levels in C. nasus larvae and juvenile gills under ammonia stress.

Conclusions: Our study revealed that NHE genes played distinct roles in embryonic development, salinity stress, and ammonia exposure. Syntenic analysis showed significant difference between stenohaline fish and euryhaline fishes. Our findings will provide insight into effects of C. nasus NHE gene family on ion transport and ammonia tolerance and be beneficial for healthy aquaculture of C. nasus.

Keywords: Chinese tapertail anchovy; Gene expression; High environmental ammonia (HEA); Hypertonic stress; Hypotonic stress; Na+-H+ exchangers.

Fig. 1

Chromosomal location of NHE gene family. The chromosomes were characterized by yellow bars. Chromosome numbers are shown on the left of the chromosomes. NHE genes are marked in red on the right of the chromosomes. LG:the name of chromosomes in C. nasus genome

Fig. 2

Phylogenetic analyses of NHE proteins from representative vertebrates. The tree was contributed by the neighbor-joining (NJ) method in MEGA X with 1000 bootstrap replications. Bootstrapping values were showed via circles on each branch. C. nasus NHEs were highlighted by the red star. The plasmalemmal subgroup (NHEβ, NHE1–5) and the intracellular subgroup (NHE6–9) were differentiated by red and green branches

Fig. 3

Structural analysis of C. nasus NHE genes. A gene structure, (B) conversed domains and (C) motifs. The plasmalemmal subgroup (NHEβ, NHE1–5), and the intracellular subgroup (NHE6–8) were differentiated by pink and green

Fig. 4

Syntenic analysis of NHE genes between C. nasus and five other fish species, including Ictalurus Punetaus (A), Oreochromis niloticus (B), Cyprinus carpio (C), Salmo salar (D), and Clupea harengus (E). The gray lines indicate the collinear blocks within C. nasus and other fish genomes. The red lines indicate the pairs of NHE genes

Fig. 5

Expression profiles of multiple tissues (A) and embryonic development stages (B). B: brain, E: eye, G: gill, H: heart, HK: head kidney, K: kidney, I: intestine, L: liver, M: muscle, S: spleen. The expression data were processed by log scale. The branches on the left represent the cluster analysis of different genes from different samples based on their expression patterns. The HK sample of NHE2 was used as the reference sample in tissues expression, and the 2-cell sample of NHE1 was used as the reference sample in embryonic development expression

Fig. 6

Expression profiles of NHE genes in C. nasus gills in response to hypotonic (FW vs Control) and hypertonic (SW vs Control) stress at multiple time points. Different capital letters indicate significant difference among different time points in the same groups at P < 0.05. Different lower-case letters indicate significant difference between different groups at the same time point at P < 0.05. FW: salinity ~ 1 ppt, Control: salinity 10 ppt, SW: salinity 30 ppt. The 0 h sample of Control group was used as the reference sample

Fig. 7

Expression patterns of NHE genes in C. nasus larvae and juvenile gills responding to ammonia stress. Significant differences were noted by asterisk (P < 0.05). The samples of larvae and juveniles in control group were used as reference sample, respectively