Sepia pharaonis is of great commercial value for aquaculture. However, Sepia pharaonis is sensitive to salinity fluctuations. After heavy rain falls, salinity decreased sharply, making a threat to survival of this species. Understanding the molecular mechanisms is an important step in the discovery of signaling pathways that participate in osmoregulation. Given that little is known about Sepia pharaonis, we investigated it with transcriptome sequencing. In our study, a total of 203,852,818 raw reads were produced, and 130,857 unigenes were assembled. It is expected that this will yield key information concerning Sepia pharaonis in salinity stress.
Genes and Processes involved in salinity stress of Sepia pharaonis
Herein, we performed transcriptome sequencing to reveal the genes regulating salinity stress. The deep sequencing technique was employed for denovo assembly of the transcriptome of Sepia pharaonis gill. The development of Sepia pharaonis osmoregulation includes a highly regulated interplay between receptors and effectors.
In Sepia pharaonic, perception of ionic and osmotic imbalances is drive by receptors on the cell membrane. Ion channels (Cl-, Ca2+, Na+, and K+ channels) and AQP(Aquaporin TIP4-1) play key roles in the reception of such signals. Studies have recognized that phospholipase A2 (PLA2) modulates the production of osmolytes by finetuning the mobilization of arachidonic acid, which activates cell swelling [12]. Arachidonic acidmetabolism was enriched in KEGG pathway and PLAa was also annotated.
This led to the identification of transduction pathways for salt stress signals. In GO term, response to stimulus (GO:0050896) was identified. In KEGG , calcium signaling pathway(ko04020) , MAPK signaling pathway (ko04010).This results implies that salt stress changes membrane fluidity, inducing cytosolic Ca2+ cycling [13].
The mitogen-activated protein kinase (MAPK) cascade modulates many cellular processes such as migration, differentiation and cell proliferation. It is reported that MAPK cascade acts when plant in salt stress status[14].
We uncovered many stress factors at low osmotic stress. (A) water channels, osmolyte transporters, and ion channels (including Na+, K+, Ca2+ and Cl- channels), were activated altering the osmotic balances. For example, electron carrier activity (GO:0009055), enzyme regulator activity (GO:0030234) were observed. (B) Degradation or denovo synthesis of free amino acids (FAA) which may play a role of compensating or alleviating the effects of ion influx during salinity stress conditions [15]. For example, arginine and proline metabolism ko00330, alanine, aspartate and glutamate metabolism ko00250, glycine, serine and threonine metabolism ko00260, tryptophan metabolism ko00380, cysteine valine, leucine and isoleucine degradation ko00280, and methionine metabolism ko00270, histidine metabolism ko00340, Tyrosine metabolism ko00350, phenylalanine metabolism ko00360, soleucine, leucine, valine, and biosynthesis ko00290, phenylalanine, tyrosine and tryptophan biosynthesis ko00400,Lysine biosynthesis ko00300 were enriched. As shown in Table S1, FAAs metabolic pathways were activated, which changed osmotic status in the Sepia pharaonis and this allowed them to adjust to osmotic stress environment. (C) ROS-related pathways and genes were enriched. Antioxidant activity (GO:0016209), peroxisome(ko04146), glutathione metabolism (ko00480), glutathione peroxidase 3, superoxide dismutase [Mn], mitochondrial, superoxide dismutase [Cu-Zn], peroxisomal membrane protein11A , peroxisomal biogenesis factor3 were observed. (D) Moreover, many other types of metabolism, including immune responses and endocrine system, were shown to be enriched in the KEGG pathway analysis. We found platelet activation (ko04611), peukocyte transendothelial migration (ko04670), B cell receptor signaling pathway (ko04662), Toll-like receptor signaling pathway (ko04620,T cell receptor signaling pathway (ko04660), and complement and coagulation cascades (ko04610). For endocrine system, thyroid hormone signaling pathway (ko04919), Thyroid hormone synthesis (ko04918), Prolactin signaling pathway (ko04917) were observed. These various regulatory pathways may exist in oysters to allow them to cope with salt stress.
Finally, amino acid metabolism, ion currents and water currents were observed to be balanced to maintain isoosmotic conditions.
DEGs between Treatment Group and Control Group 500
Differential analysis of gene expression between salinity22 and salinity28 provide an opportunity to understand the critical genes in the osmoregulation, including the regulation to low salty stress. As a result, 6153 genes were found to be significantly differentially expressed (p<0.05), including 3340 up regulated and 2813 down regulated genes. 665 (10.81%) of DEGs were annotated successfully to at least one database. To understand the functions of these DEGs, GO and KEGG analyses were performed. All DEGs were assigned to 491 GO terms and 226 pathways through the KEGG database (Table S4).
Of these genes, there were 67 with little to no expression in salinity28 but high expression in salinity22, while 49 with little to no expression in salinity22 but high expression in salinity28.
Zinc finger protein 92 homolog and Zinc finger protein 300 were down-regulated. Zinc finger protein 41, Zinc finger protein 333, AN1-type zinc finger protein 4 and Zinc finger protein 26 were up-regulated. That means zinc finger protein affect each other in salinity stress. Zinc fingers were first identified in a study of transcription in the African clawed frog, Xenopus laevis [16],widely distributed in animals, plants and microorganisms. Proteins that contain zinc fingers (zinc finger proteins) is mostly as interaction modules that bind DNA, RNA, proteins, or other small, useful molecules, and variations in structure serve primarily to alter the binding specificity of a particular protein. In plants, like Oryza sativa [5, 17], Arabidopsis thaliana [18], zinc finger proteins were found to play an important role when plants faced with drought, high salt and low temperature. Histone-lysine N-methyltransferase SETMAR is another one. SETMAR (alternative name: Metnase) is a domesticated primate transposase that enhances DNA repair, replication, and decatenation[19, 20]. It has a histone methyltransferase activity and methylates 'Lys-4' and 'Lys-36' of histone H3, Specifically, it mediates dimethylation of H3 'Lys-36' at sites of DNA double-strand break and may recruit proteins required for efficient DSB repair through non-homologous end-joining [21, 22]. Histones modifications, part of epigenetic processes, influence the efficiency of stress-induced gene expression in plant [23]. All of these implys the possible function of SETMAR in sepia to salinity stress.
To understand the functions of these DEGs, GO and KEGG enrichment were done. For up-regulated genes, adipocytokine signaling pathway, fatty acid metabolism, PPAR signaling pathway, fatty acid degradation, and insulin resistance are the top ones. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, activated by fatty acids and their derivatives. PPAR has three subtypes (PPA Ralpha, beta/delta, and gamma), showing different expression patterns in vertebrates. Each of them is encoded in a separate gene and binds fatty acids and eicosanoids. Additionally, it is reported that during long term salinity exposure, fatty acid composition of gill membrane in Gammarus duebeni changes, being a highly effective method to reduce ion diffusion and water inox [32]. Genes (CPT-1,CPT-2) are both up-regulated in PPAR signaling pathway and fatty acid degradation. Further study should be done to help understand the function of CPT-1,CPT-2 in the pathway. For down-regulated genes, protein processing in endoplasmic reticulum, tyrosine metabolism, betalain biosynthesis, isoquinoline alkaloid biosynthesis, riboflavin metabolism are the top ones. During culture, Sepia pharaonis gives out ink when salinity suddenly changes. Tyrosine metabolism is the biological processes behind this phenomenon. 8 genes down-regulated in tyrosine metabolism may play an important role in osmoregulation in Sepia pharaonis. Down-regulated genes also enriched in Genes oxygen transport, oxygen transporter activity, response to stress, and oxidation−reduction process (Go term). We found related genes: hemocyanin G-type, units Oda to Odg (ODHCY), hemocyanin A-type, units Ode to Odg (Fragment), hemocyanin, units G and H (Fragments), and superoxide dismutase [Cu-Zn]. Osmoregulation influences respiratory gas exchange and transport [24]. The answer to gas exchange and transport, involves mechanisms of salt and water balance. Potassium channel subfamily K member 16 (KCNK16), voltage-gated hydrogen channel 1 (HVCN1),and sodium channel protein 1 brain were identified. Besides, sodium- and chloride-dependent GABA transporter 2 (SLC6A13), sodium-and chloride-dependent glycine transporter 2 (SLC6A5 ), solute carrier family 35 member B1 (slc35b1), solute carrier family 22 member 21 (Slc22a21) and electroneutral sodium bicarbonate exchanger 1 (SLC4A8) were also observed. Especially, SLC6A5 has little to no expression in salinity22 but high expression in salinity 28. GlyT2, a protein that in humans is encoded by the SLC6A5 gene, is a specific marker of glycinergic neurons and a member of the Na+ and Cl−-coupled transporter family SLC6. These ion channel and SLC provides an view to study salt and water balance in Sepia pharaonis.
SSR discovery
SSRs are useful molecular markers for genetic and breeding studies[25]. Next-generation sequencing has recently been used to discover SSRs in aquaculture species, and is considered a time-saving, highly efficient approach. In total, 101576 SSRs were recognized in 130857sequences, and 24737sequences contained more than one SSR. These microsatellites are expected to be useful for genetic linkage mapping and other genetic studies.