Background: Scylla paramamosain (Crustacea: Decapoda: Portunidae: Syclla De Hann) is a commercially important mud crab distributed along the coast of southern China and other Indo-Pacific countries (Lin Z, Hao M, Zhu D, et al, Comp Biochem Physiol B Biochem Mol Biol 208-209:29–37, 2017; Walton ME, Vay LL, Lebata JH, et al, Estuar Coast Shelf Sci 66(3–4):493–500, 2006; Wang Z, Sun B, Zhu F, Fish Shellfish Immunol 67:612–9, 2017). While S. paramamosain is a euryhaline species, a sudden drop in salinity induces a negative impact on growth, molting, and reproduction, and may even cause death. The mechanism of osmotic regulation of marine crustaceans has been recently under investigation. However, the mechanism of adapting to a sudden drop in salinity has not been reported. Methods: In this study, transcriptomics analysis was conducted on the gills of S. paramamosain to test its adaptive capabilities over 120 h with a sudden drop in salinity from 23 ‰ to 3 ‰. Results: At the level of transcription, 135 DEGs (108 up-regulated and 27 down-regulated) annotated by NCBI non-redundant (nr) protein database were screened. GO analysis showed that the catalytic activity category showed the most participating genes in the 24 s-tier GO terms, indicating that intracellular metabolic activities in S. paramamosain were enhanced. Of the 164 mapped KEGG pathways, seven of the top 20 pathways were closely + + related to regulation of the Na /K -ATPase. Seven additional amino acid metabolism-related pathways were also found, along with other important signaling pathways. Conclusion: Ion transport and amino acid metabolism were key factors in regulating the salinity adaptation of S. paramamosain in addition to several important signaling pathways. Keywords: Scylla paramamosain, Gill, Osmoregulation, Transcriptional profiling, Differentially expressed gene Background osmoregulatory capacities to adjust to the shifting salin- Salinity as a key abiotic parameter that influences the ities of estuary and wetland waters within limits. In crusta- distribution, abundance, physiology, and well-being of ceans, the adaptability of osmoregulation is primarily crustaceans. [3, 4, 15, 34]. Salinity is also an important achieved by the gills [7, 12, 16, 28, 34, 36]. Since regulation factor in the production of crustacean aquaculture , of osmotic pressure in marine animals involves energy which can affect growth, survival, molting, oogenesis, consumption, drastic changes in salinity can lead to death embryogenesis and larval quality [6, 14, 21, 30, 32, 35, 38]. of the organism. The salinity of crustacean aquaculture can easily be af- Recent studies have shown that low salinity influences fected by a local torrential rain . Fortunately, most ion channel activity [33, 37, 44, 45] and L-type free marine species which have been studied have amino acids [1, 25, 39, 42, 43], which are tightly involved + + with osmoregulation. In particular, the Na /K -ATPase, a well-known ion channel, is the main ion * Correspondence: firstname.lastname@example.org transport enzyme of post-larvae in crustaceans. Its func- School of Marine Science, Ningbo University, Ningbo 315211, Zhejiang, China tion in the organism is to enhance adaptability to salinity Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, Zhejiang, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Genomics (2018) 19:421 Page 2 of 12 changes through osmoregulation [20, 22, 26, 27]. Chung the salinity of 23‰ and 3‰ filtered sea water was used in and Lin  cloned the full-length α-subunit of the Na / the C and LS groups, respectively. K -ATPase cDNA, indicating the osmoregulatory role of the channel via both mRNA and protein expres- Total RNA isolation and gene expression analysis sion. Likewise, Lu et al.  completed the cDNA Total RNA was isolated from gill tissue using sqRT-PCR cloning of glutamate dehydrogenase and its expression, RNAiso Plus (TaKaRa, Dalian, China). The cDNA was indicating that GDH played an important role in control- synthesized using the Perfect Real Time version of the ling osmoregulation through free amino acids in S. PrimerScriptTM RT reagent kit with gDNA Eraser paramamosain. (Perfect Real Time) (TaKaRa,) according to manufacturer’s Although a great deal of progress had been made in instructions. Then, sq-RT-PCR were chosen to analyze the study of osmotic adjustment in crustaceans, there genes, and performed in a total reaction volume of 25 μl have been relatively fewer studies on S. paramamosain. according to the manufacturers’ instructions. The S. para- In particular, the molecular mechanism of adaptation to mamosain beta-actin gene and 18S ribosomal RNA gene sudden salinity drop has not yet been reported. Since S. were selected as the internal control. Primers used in this paramamosain is a euryhaline species , it is easy to study are listed in Additional file 1: Table S1. overlook the impact of salinity on the organism’s physi- ology. In the environment, a sudden drop in salinity Transcriptome sequencing caused by heavy rain over a short period of time (drop For Illumina paired-end sequencing, equivalent quan- by > 10‰) may lead to death. In this study, we simulated tities of total RNA isolated from the three mud crabs a drastic reduction in salinity from 23‰ to 3‰. Then, were pooled as one sample, and eventually there were the molecular mechanism of adapting to the salinity three samples in each group, CK and LS. After poly (A) drop was analyzed by transcriptome analysis. mRNA was purified and fragmented into small pieces, we used random hexamer primers and reverse tran- Methods scriptase (Invitrogen) to carry out first-strand cDNA Experimental animals and sectionalization synthesis. Second-strand cDNA synthesis was per- A total of 300 randomly selected crabs with a body formed with RNase H (Invitrogen) and DNA polymer- weight of ~ 30 g was selected and kept in a natural water ase I (New England BioLabs, Beijing, China). A cDNA environment with a salinity of 23 ‰ and a temperature library was constructed with average insert sizes of of approximately 20 °C. Every 50 crabs were randomly 200–500 bp and cDNA sequencing was conducted selected (weight ~ 30 g) as a group, with a total of six using the Illumina HiSeqTM 4000 system according groups, housed in six cement pools under identical to the manufacturer’s protocols, with read length of physical and chemical conditions. The salinity of the sea- 150 bp Transcriptome Quantification analyses two in- water for three of the groups was adjusted to 3‰ from dependent cDNA libraries were constructed for the 23‰, which dropped by 20‰. These three groups were two organs in parallel according to the Transcriptome defined as the LS (low salinity) group. The other three protocol. The transcriptome sequencing was per- groups were defined as the CK groups, where the salinity formed by BGI (BGI, Shenzhen, China). of seawater was kept at 23‰. All other conditions were the same as the LS group. It should be noted that the space of a pond in the ex- Analysis of differentially expressed genes periment was big enough for juvenile crabs, and we Because the annotated genome of S. paramamosain has added a few tiles in ponds as shelter, which could effect- not been published, de novo assembly was used here as ively avoid fighting and killing each other. reference for further analysis. Firstly, the raw reads were filtered to remove adaptor and low quality sequences. HE staining Afterfiltration, clean reads were assembled into unigenes Gill morphology and ultrastructure of S. paramamosain using Trinity de novo assembler, followed by TGICL were observed using light microscopy after hematoxylin clustering tool. The reads from control (CK) and experi- (HE) staining. HE staining was conducted according to mental group (LS) were mapped against the assembled the method of Wang et al. . First, gills were set in Unigene using HISAT. The FPKM (fragments per kb per paraffin and sliced into sections with a thickness of up million reads) method was used to calculate the expres- to 5–10 μm. Then, the sections were de-waxed using xy- sion abundance. Each unigene was subjected to a lene and rehydrated in an ethanol series. Sections were BLASTX search against the NCBI non-redundant (nr) stained with eosin and HE which purchased from Invitro- protein database with an e-value threshold of 10–3. R gen (Carlsbad, CA, USA). Preparation of 4% paraformalde- package DESeq2 were performed to identify the differ- hyde solution were made using filtered isotonic sea water: entially expressed genes (DEGs). DEG was considered Wang et al. BMC Genomics (2018) 19:421 Page 3 of 12 as unigene with greater than 2-fold change and p-value concentrated within 24, 48, and 72 h. In addition, the LS < 0.05. Gene ontology (GO) terms and KEGG pathway group showed hyperactivity within 48 h. As time went annotation were achieved using the Blast2GO program by, the motility was diminished and normalized (Fig. 1a). and kaas (KEGG Automatic Annotation Server) on-line The LS group did not have food over 72 h, and gradually program (http://www.genome.jp/kaas-bin/kaas_main), started to eat over time. Conditions returned to normal respectively. after 120 h. The gills are an important organ in osmoregulation of Results marine crustaceans, with the gill filament serving as the Adaptive phenotype of S. paramamosain to sudden drop basic units of function. According to the results of gill in salinity slicing, under normal conditions, the gill filaments of There were four deaths in the CK group within 7 days the crab in the CK group were regular (Fig. 1b). Gills of and 24 deaths in the LS group. The LS death time was crabs in the LS group became shorter and thicker from 6 h and reached the shortest at 72 h, which was less than half that of gills observed in the CK group (Fig. 1a & b). Gills then gradually became longer with time and returned to normal after 120 h (Fig. 1b & c). The changes in gill filament anatomy was consistent with those of the physiological activities mentioned above, suggesting that changes in gill filaments play an import- ant regulatory role in adaptation of S. paramamosain to decrease in salinity. Differentially expressed genes (DEGs) in the gill of S. paramamosain Gills of marine crustaceans play an important role in osmoregulation. In our study, the mud crabs had ad- justed to a salinity of 3‰ in 120 h after a sudden drop in salinity. In order to study the molecular mechanism underlying this adaptation, we performed transcriptional profiling at 120 h. Approximately 39.6 Gb bases were generated in total on the BGISEQ-500 sequencing platform. Because the genome sequencing of S. paramamosain has not yet been elucidated, after reads filtering, Trinity was used to perform de novo assembly with clean reads (Additional file 1: Table S2). Tgicl ( Pertea et al., 2003) was used on cluster transcripts to remove abun- dance and get Unigenes (Additional file 1: Table S3). The proportion of bases with low quality (< 20) was very low in all samples, indicating a high quality of sequences. Finally, high-quality transcripts were obtained (Table 1) and used as reference sequences. Genes were annotated using Unigenes by aligning with seven functional data- base as follows 36,376 (NR: 34.59%), 38,958 (NT: Fig. 1 Food intake and morphology change of gills in response to 37.04%), 26,425 (Swissprot: 25.13%), 26,056 (KOG: the sudden drop in salinity in S. paramamosain. a, Food intake change in Scylla paramamosain. The juvenile crab with a body 24.77%), 28,890 (KEGG: 27.47%), 4859(GO: 4.62%), and weight of ~ 30 g eat about two Sinonovacula constricta (Lamarck) in 19,756 (InterPro: 18.78%) (Additional file 1: Figure S2). 24 h, so three Sinonovacula constricta (Lamarck) per crab were feed For functional annotation results, we detected 32,627 every day and cleaned up the leftovers regularly to prevent the water CDS by Transdecoder. We also detected 74,041 SSR going bad. b, the phenotypic changes of gill filaments within 120 h distributed on 44,075 unigenes, and predicted 12,623 after the salinity drop: 23‰ in CK group, others were the phenotypes of 12, 24, 48, 72, 96, and 120 h after salinity dropped to 3‰. c, changes transcription factor (TF) coding unigenes. of gills in length; “0” indicated CK group, and the others were the gill A total of 249 genes was differentially expressed in the length at 12, 24, 48, 72, 96, 120 h after salinity dropped to 3‰.Barsare LS Group and the CK group, including 207 up-regulated 300 μmin b genes and 42 down-regulated genes (fold change> = 2.00 Wang et al. BMC Genomics (2018) 19:421 Page 4 of 12 Table 1 Quality metrics of transcripts in the gill of Scylla paramamosain Sample Total Number Total Length (bp) Mean Length N50 N70 N90 GC(%) CK_1 93,877 54,675,298 582 952 445 232 45.50 CK_2 100,075 57,680,722 576 938 436 230 45.61 CK_3 97,185 52,573,783 540 803 400 225 45.75 LS_1 87,582 49,398,653 564 885 424 229 45.75 LS_2 97,564 51,685,899 529 777 390 222 45.88 LS_3 81,111 58,440,218 720 1354 617 262 46.89 Sample: Sample name; Total Number: The total number of transcripts; Total Length: The read length of transcripts; Mean Length: The average length of transcripts; N50: The N50 length was used to determine the assembly continuity such that the higher the better. N50 is a weighted median statistic such that 50% of the totallength is contained in the unigenes that are equal to or larger than this value. N70: Similar to the N50; N90: Similar to the N50. GC (%): the percentage of G and C bases in all transcripts and adjusted p value <= 0.05) (Fig. 2). Of the 249 DEGs, Malacostraca (5 DEGs), and Myriapoda (1 DEG) (Fig. 3). 217 were annotated at least one of the following: Nr The remaining 60 DEGs (44%) belonged to the Phylum (192), Nt (160), Swissprot (154), KEGG (163), KOG Chordata (26 DEGs), Protozoa (12 DEGs), Mollusca (10 (152), Interpro (130) and GO (32. No description was DEGs), Ciliata (5 DEGs), Nematoda (3 DEGs), Echinoder- found for 3 DEGs (3 of which are down-regulated mata (2 DEG), Coelenterata (1 DEG), and Platyhelminthes genes). Of the 192 differential genes annotated in the Nr (1 DEG) (Fig. 3). database, 57 were described as “hypothetical” in the Gen- Bank database and were also excluded from further ana- Functional annotation lysis. Finally, 135 DEGs that were annotated with Nr were Gene Ontology (GO) analysis was performed on DEGs screened, out of which 108 were up-regulated and 27 were using Blast2GO [9, 17]. According to the second-tier down-regulated genes (Additional file 1: Table S4). GO terms, 32 DEGs were assigned 24 GO annotations Of these 135 DEGs, BUD13 homolog (ID: CL3353.Con which represented three main GO categories: biological tig2) was the highest-upregulated gene (90.13 fold) followed process (18), cellular component (18), and molecular by serine/threonine-protein kinase (ID: CL90.Contig3, function (23) (Fig. 4 & Additional file 1: Figure S3). 71.65 fold) and beta-1, 4-N-acetylgalactosaminyl transferase Among them, 24 GO terms were assigned to the (ID: CL2951.Contig2; 67.99 fold) (Additional file 1:Table up-regulated group and 17 to the down-regulated group. S4). The three most highly down-regulated genes were Twelve processes were identified in the biological uncharacterized proteins LOC107039269 (ID: CL94.Con process category, with 14 DEGs involved in cellular pro- tig5; − 34.61 fold), kinesin light chain (ID: CL643.Contig10; cesses, 12 involved in metabolic processes, and 11 in- − 34.03 fold) and aquaporin-12 (ID: CL5376.Contig4; − volved in single-organism processes. This identified 27.99 fold) (Additional file 1:Table S4).75 (56%) showed three biological processes as the most strongly affected the highest similarity to genes belonging to phylum Arthro- in the gill of S. paramamosain by the sudden drop in poda (Fig. 3). Of the 75 DEGs, 52 showed highest similarity salinity (Fig. 4). In the cellular component category, to genes from class Insecta, and the remainder to genes membrane (14), membrane part (10), cell parts (7), and from class Crustacea (9 DEGs), Merostomata (8 DEGs), cell (7) were most involved (Fig. 4). In the molecular Fig. 2 Differentially expressed genes (DEGs) from the gill of S. paramamosain. a, Volcano plot of DEGs; b, Heatmap of DEGs. Fold change> = 2.00 and adjusted p value <= 0.05 Wang et al. BMC Genomics (2018) 19:421 Page 5 of 12 suggested that the salinity of the water environment dropped sharply. In order to maintain life activity, the metabolic activity of the cells in S. paramamosain was strengthened, in particular the catalytic function of enzymes regulating ion changes and osmotic pressure, processes involved in adapting to the new environment with dropped salinity. In addition, among all the 24 s-tier GO terms, localization, response to stimulus, and multi-organization processes from the biological process category, as well as the extracellular region, membrane-enclosed lumen from cellular component, transporter activity, and electron car- rier activity from molecular function categories were all upregulated DEGs with no downregulated DEGs. Fig. 3 Class distribution diagram of annotations form Nr database of DEGs pathway analysis 135 differentially expressed genes (DEGs) in response to the sudden drop in salinity in the gill of S. paramamosain. These 135 DEGs showed With DEGs, KEGG pathway classification was performed a certain degree of similarity to genes belonging to phylum (Fig. 5) according to the KEGG database website (http:// Arthropoda (75 DEGs), Chordata (26), Protozoa (12), Mollusca (10), www.genome.jp/kegg/pathway.html). Of all DEGs, 119 Ciliata (5), Nematoda (3), Echinodermata (2), Coelenterata (1), and were mapped in 164 KEGG pathways which were graded Platyhelminthes (1 DEG). The pie chart on the lower side of the into six categories (level 1) according to their biological image shows taxonomic groups of Phylum, and each fan represented a phylum. The upper pie chart showed taxonomic groups of Class function, including organismal systems (51), human dis- whichcontainsix classes. Numbers and proportion of DEGs are eases (47), environmental information processing (40), counted in each end of the bar genetic information processing (28), metabolism (27), cel- lular processes (22) (Fig. 5a & b). function category, only four items contained the catalytic Of the 164 pathways, 51 (42.86%) were related to the activity (16), binding (11), transporter activity (3) and organismal systems category which was subdivided into electron carrier activity (1) (Fig. 4). It is worth noting nine subsets (level 2): aging, circulatory system, develop- that the catalytic activity category (14) had the most par- ment, digestive system, endocrine system, excretory sys- ticipating genes in the 24 s-tier GO terms. The results tem, immune system, nervous system, sensory system (Fig. 5a and Table 2). The detailed KEGG pathways of organismal systems and DEGs involved are presented in Table 2. 47 (39.50%) were related to human diseases (Fig. 5a & b). These genes contained Unigene45614 (toxo- plasmosis, ko05145) annotated cytochrome C (Marsupe- naeus japonicus) CL979. Contig1 (insulin resistance, ko04931) annotated solute carrier family 2, Unigene 44,862 (Staphylococcus aureus infection, ko05150) annotated kera- tin, type I cytoskeletal 9 (Marmota marmota marmota), CL979. Contig1 (pathways in cancer, ko05200) annotated solute carrier family 2, facilitated glucose transporter mem- ber 3 (Zootermopsis nevadensis)(Additional file 1: Table S5). It is worth mentioning, these genes were not disease genes, but they were relevant in certain pathologies. Forty genes (33.61%) were related to environment in- formation processing which was subdivided into three subsets: signal transduction, signaling molecules and interaction, and membrane transport. Of 164 pathways, 39 genes were most abundant in the signal transduc- Fig. 4 GO annotation of differentially expressed genes (DEGs) in tion category, which contained 20 KEGG pathways response to the sudden drop in salinity in the gill of S. paramamosain. DEGs were assigned to second-tier GO categories associated with three (Additional file 1: Table S6): AMPK signaling pathway parent terms: biological process, cellular component, and molecular (ko04152), PI3K-Akt signaling pathway (ko04151), function. Blue bars indicate up-regulated DEGs and orange bars indicate HIF-1 signaling pathway (ko04066), cGMP-PKG sig- down-regulated DEGs. Numbers of DEGs are counted in each end of bar naling pathway (ko04022), phosphatidylinositol Wang et al. BMC Genomics (2018) 19:421 Page 6 of 12 Fig. 5 Pathway classification (a) and differentially expressed genes (DEGs) distribution (b). B, OS: Organismal Systems, HD: Human Disease, EIP: Environmental Information Processing, GIP: Genetic Information Processing, M: Metabolism, CP: Cellular Processes. X axis represents the number of DEGs. Y axis represented functional classification of KEGG. There were seven branches for KEGG pathways: Cellular Processes, Environmental Information Processing, Genetic Information Processing, Human Disease (For animals only), Metabolism, Organismal Systems and Drug Develop (all of the DEGs did not involve) signaling system (ko04070), hedgehog signaling path- environment information processing were involved, such way in fly (ko04341), hedgehog signaling pathway as ABC transporters (ko02010) which belonged to mem- (ko04340), sphingolipid signaling pathway (ko04071), brane transport, ECM-receptor interaction (ko04512), cell cAMP signaling pathway (ko04024), notch signaling adhesion molecules (CAMs) (ko04514), and neuroactive pathway (ko04330), MAPK signaling pathway in fly ligand-receptor interaction (ko04080), which belonged to (ko04013), hippo signaling pathway (ko04390), FoxO signaling molecules and interaction. Throughout all of en- signaling pathway (ko04068), MAPK signaling pathway vironment information processing pathways, the results (ko04010), calcium signaling pathway (ko04020), Wnt sig- indicated gill as an important organ for salinity regulation naling pathway (ko04310), mTOR signaling pathway for S. paramamosain. After the gills detected the salinity (ko04150), hippo signaling pathway in fly (ko04391), Ras drop, the salinity drop transformed into stress signals signaling pathway (ko04014), and phospholipase D signal- transmitted to other parts of the body, thereby initiating ing pathway (ko04072). In addition, the other pathways of regulation mechanism used by the gills. A complex Wang et al. BMC Genomics (2018) 19:421 Page 7 of 12 Table 2 Organismal Systems pathways and DEGs involved Pathway DEGs genes (51) Pathway ID Level 2 Longevity regulating pathway - multiple species Unigene32292, Unigene35344, Unigene35501 ko04213 Aging Longevity regulating pathway Unigene32292, Unigene35501, CL3443.Contig1 ko04211 Aging Longevity regulating pathway - worm CL3933.Contig2 ko04212 Aging Cardiac muscle contraction Unigene11331 ko04260 Circulatory system Adrenergic signaling in cardiomyocytes Unigene11331, Unigene44161 ko04261 Circulatory system Vascular smooth muscle contraction Unigene44161 ko04270 Circulatory system Osteoclast differentiation Unigene19465, Unigene31660 ko04380 Development Axon guidance Unigene40814 ko04360 Development Dorso-ventral axis formation Unigene34154 ko04320 Development Protein digestion and absorption Unigene12705, CL6154.Contig2, Unigene38622, ko04974 Digestive system CL922.Contig5, Unigene11331, CL922.Contig9, CL452.Contig3, CL922.Contig7, Unigene17070, CL3933.Contig2 Bile secretion Unigene12705, Unigene33785, Unigene38622, ko04976 Digestive system Unigene11331, CL979.Contig3, CL979.Contig1, Unigene17070 Mineral absorption Unigene12705, Unigene15443, Unigene38622, ko04978 Digestive system Unigene11331, Unigene17070 Carbohydrate digestion and absorption Unigene11331 ko04973 Digestive system Vitamin digestion and absorption CL4600.Contig3 ko04977 Digestive system Salivary secretion Unigene10578, Unigene11331 ko04970 Digestive system Gastric acid secretion Unigene11331 ko04971 Digestive system Pancreatic secretion Unigene11331 ko04972 Digestive system Thyroid hormone signaling pathway Unigene33785, Unigene111, Unigene113, ko04919 Endocrine system Unigene41655, Unigene36212, Unigene31721, Unigene11331, Unigene112, CL979.Contig3, CL979.Contig1, Unigene17270 Adipocytokine signaling pathway Unigene33785, CL3443.Contig1, CL979.Contig3, ko04920 Endocrine system CL979.Contig1 Insulin secretion Unigene33785, Unigene11331, CL979.Contig3, ko04911 Endocrine system CL979.Contig1, CL5287.Contig1 Thyroid hormone synthesis Unigene10157, Unigene27634, Unigene11857, ko04918 Endocrine system Unigene11331 PPAR signaling pathway CL1683.Contig3, CL1683.Contig5, CL2638.Contig2 ko03320 Endocrine system Estrogen signaling pathway Unigene10157, Unigene35344, Unigene27634 ko04915 Endocrine system Progesterone-mediated oocyte maturation Unigene10157, Unigene27634 ko04914 Endocrine system Glucagon signaling pathway Unigene33785, CL979.Contig3, CL979.Contig1 ko04922 Endocrine system Renin-angiotensin system Unigene49236 ko04614 Endocrine system Insulin signaling pathway Unigene32292, Unigene35501, Unigene44161 ko04910 Endocrine system Oxytocin signaling pathway Unigene44161 ko04921 Endocrine system GnRH signaling pathway CL6599.Contig1 ko04912 Endocrine system Proximal tubule bicarbonate reclamation Unigene12705, Unigene15693, Unigene38622, ko04964 Excretory system Unigene11331, Unigene17070 Aldosterone-regulated sodium reabsorption Unigene11331 ko04960 Excretory system Endocrine and other factor-regulated calcium Unigene11331 ko04961 Excretory system reabsorption Antigen processing and presentation Unigene10157, Unigene35344, Unigene27634, ko04612 Immune system Unigene11857, Unigene41750 NOD-like receptor signaling pathway Unigene10157, Unigene27634 ko04621 Immune system Wang et al. BMC Genomics (2018) 19:421 Page 8 of 12 Table 2 Organismal Systems pathways and DEGs involved (Continued) Pathway DEGs genes (51) Pathway ID Level 2 Hematopoietic cell lineage Unigene15456, Unigene47891l, Unigene15457 ko04640 Immune system B cell receptor signaling pathway Unigene15456, Unigene47891, Unigene15457 ko04662 Immune system Platelet activation Unigene44161 ko04611 Immune system Fc gamma R-mediated phagocytosis CL6599.Contig1, CL4852.Contig1 ko04666 Immune system Long-term potentiation Unigene44161 ko04720 Nervous system GABAergic synapse CL3399.Contig2 ko04727 Nervous system Dopaminergic synapse Unigene44161 ko04728 Nervous system Glutamatergic synapse CL6599.Contig1, Unigene5252, CL3399.Contig2 ko04724 Nervous system Phototransduction CL6217.Contig3, CL41.Contig2 ko04744 Sensory system Olfactory transduction CL6217.Contig3, CL41.Contig2 ko04740 Sensory system Inflammatory mediator regulation of TRP channels Unigene44161 ko04750 Sensory system molecular signal feedback mechanism for regulation could synthesis (ko0491), and the regulatory genes of Na / + + ultimately achieve osmotic pressure balance of as an adap- K -ATPase were all up-regulated。Crustacean Na / tive response to a sudden salinity drop. K -ATPase plays an important role in the regulation of Functional enrichment was also performed on DEGs hematopoietic osmotic pressure at different salinities + + according to the above KEGG pathway classification. . Current research on crustacean Na /K -ATPase The top 20 pathways (Fig. 6a & Table 3) showed that has been extensively reported [10, 23]suggestingthat it seven pathways were directly related to the active regula- is a widespread P-type ATPase that plays an important + + + + tion of the Na /K -ATPase enzyme: Proximal tubule bi- role in maintaining Na ,K homeostasis . As all carbonate reclamation (ko04964), protein digestion and known, free amino acids [1, 25, 39, 42, 43] also played absorption (ko04974), bile secretion (ko04976), thyroid an important role in osmoregulation. In addition, sev- hormone signaling pathway (ko04919), mineral absorption eral pathways connected with amino acid metabolism (ko04978), insulin secretion (ko04911), thyroid hormone were detected, such as for arginine biosynthesis Fig. 6 Pathway functional enrichment of differentially expressed genes (DEGs) (a) and pathway of Proximal tubule bicarbonate reclamation (ko04964)(b). A, X axis represents enrichment factor. Y axis represents pathway name. The color indicates the q value (high: white, low: blue), the lower q value indicates the more significant enrichment. Point size indicates DEG number (The larger dots refer to larger amount). Rich Factor refers to the value of enrichment factor, which is the quotient of foreground value (the number of DEGs) and background value (total Gene amount). The larger the value, the more significant the enrichment. b, Pathways were mapped using the KEGG Mapper (http://www.genome.jp/kegg/mapper.html). Up-regulated DEGs are boxed in red, DEG ID numbers are shown outside the box in blue Wang et al. BMC Genomics (2018) 19:421 Page 9 of 12 Table 3 The greatest functional classification differences between CK and LS NO Pathway DEGs genes (119) / All genes P value Pathway ID (28890) with pathway annotation 1 Proximal tubule bicarbonate reclamation 4 (4.2%) / 53 (0.18%) 1.239607e-05 ko04964 2 Fructose and mannose metabolism 5 (4.2%) / 98 (0.34%) 0.000239203 ko00051 3 Protein digestion and absorption 10 (8.4%) / 456 (1.58%) 0.000280083 ko04974 4 Bile secretion 7 (5.9%) / 226 (0.78%) 0.0003098698 ko04976 5 Amoebiasis 17 (14.3%) / 1195 (4.14%) 0.0004429313 ko05146 6 Antigen processing and presentation 5 (4.2%) / 113 (0.39%) 0.0004620034 ko04612 7 Protein processing in endoplasmic reticulum 9 (7.6%) / 455 (1.57%) 0.001166426 ko04141 8 Glycosphingolipid biosynthesis - lacto and neolacto series 4 (3.4%) / 94 (0.33%) 0.002007803 ko00601 9 Thyroid hormone signaling pathway 11 (9.2%) / 746 (2.58%) 0.003509184 ko04919 10 AMPK signaling pathway 8 (6.7%) / 465 (1.61%) 0.005048467 ko04152 11 Nitrogen metabolism 2 (1.7%) / 19 (0.07%) 0.005079646 ko00910 12 Vibrio cholerae infection 14 (11.8%) / 1170 (4.05%) 0.006652947 ko05110 13 Mineral absorption 5 (4.2%) / 214 (0.74%) 0.007464861 ko04978 14 Adipocytokine signaling pathway 4 (3.4%) / 153 (0.53%) 0.01119608 ko04920 15 ECM-receptor interaction 7 (5.9%) / 440 (1.52%) 0.01272333 ko04512 16 Toxoplasmosis 4 (3.4%) / 172 (0.6%) 0.01655378 ko05145 17 Insulin secretion 5 (4.2%) / 264 (0.91%) 0.01723485 ko04911 18 Thyroid hormone synthesis 4 (3.4%) / 195 (0.67%) 0.02489002 ko04918 19 PI3K-Akt signaling pathway 14 (11.8%) / 1386 (4.8%) 0.0255091 ko04151 20 Insulin resistance 4 (3.4%) /214 (0.74%) 0.0333897 ko04931 (ko00220) (Additional file 1: Table S8), alanine, aspartate Discussion and glutamate metabolism (ko00250) (Additional file 1: The mechanism of osmotic regulation of aquatic crusta- Table S8), lysine degradation (ko00310) (Additional file 1: ceans has received attention in recent years. Extensive Table S8), valine, leucine and isoleucine degradation work and quite a few important results have been reported (ko00280) (Additional file 1: Table S8), amino sugar and nu- on the morphological structure of osmotic regulatory or- cleotide sugar metabolism (ko00520) (Additional file 1: gans [5, 24], ion transport regulation [10, 23, 33], regula- Table S8), biosynthesis of amino acids (ko01230) (Add- tion of hemolymph osmoregulation (;Huong et al., itional file 1: Table S8), pyrimidine metabolism (ko00240) Table 4 Validity of DEGs in Transcriptomic data (Additional file 1: Table S8). In addition, several im- Gene ID Transcriptome results verification results portant signal pathways were found, cAMP signaling Gene.7653 up up pathway (ko04024) (Additional file 1:Table S6), MAPK signaling pathway (ko04013, ko04010) (Add- Gene.16162 up up itional file 1: Table S6), Wnt signaling pathway Gene.54888 up up (ko04310) (Additional file 1: Table S6), mTOR signaling Gene.22643 up up pathway (ko04150) (Additional file 1: Table S6), Ras sig- Gene.6925 up up naling pathway (ko04014) (Additional file 1: Table S6). Gene. 803 down down The results implied these pathways might all take part in Gene. 18,132 down down osmoregulation. Gene. 1196 down down Gene. 397 down down Validity of DEGs in transcriptomic data Gene. 2748 down down Ten differentially expressed genes were randomly sam- Gene. 7653: CL1096.Contig1_All, Gene. 16,162: CL2951.Contig2_All, Gene. pled for verification by transcriptional level experiments, 54,888: Unigene41750_All, Gene. 22,643: CL4861.Contig2_All, Gene. 6925: CL979.Contig3_All, Gene. 803: CL94.Contig5_All, Gene. 18,132: CL3482.Contig1_All, and the results were consistent with those of DEG ana- Gene. 1196:CL4395.Contig1_All, Gene.397:CL41.Contig2_All, Gene.2748: lyses (Table 4 & Additional file 1: Figure S3), indicating CL358.Contig1_All. The S. paramamosain beta-actin gene and 18S rRNA that the results of DEG were reliable. gene were selected as the internal control Wang et al. BMC Genomics (2018) 19:421 Page 10 of 12 2001, ), and neuroendocrine regulation [13, 29]. For To date, reports on the regulation of infiltration of both euryhaline and stenohaline species, changes in salin- S. paramamosain are still quite limited to cloning + + ity will result in the organism adapting through the regula- and expression of the Na /K -ATPase and clon- tion of the neuroendocrine system, osmotic regulatory ing and expression of glutamate dehydrogenase organs (mainly gills), hematopoietic osmotic pressure, and (GDH) . GHD is an important enzyme for the ion transport. A series of changes will occur to adapt to metabolism of glycine, proline, and alanine, which the changing external environment in order to maintain serve as general osmolytes in aquatic animals. In this normal physiological and metabolic activity. However, few study, GO annotation of DEG analysis showed that studies on neuroendocrine regulation, regulation of ion the most involved genes were derived from the cat- transport enzymes, or osmotic regulation are reported for egory of catalytic activity of molecular function (Fig. S. paramamosain. Thus, this paper focuses on osmotic 4), suggesting that thepossibleroleof freeamino regulation in aquatic crustaceans. Since sequencing of the acids in osmotic regulation is by enzymolysis. In + + genome of S. paramamosain has not been completed yet, addition, we found that Na /K -ATPaseis veryac- much of the information obtained still depends on tive through the KEGG pathway classification and transcriptomics. functional enrichment of DEGs (Figs. 5 and 6). The + + S. paramamosain is a euryhaline species, and espe- results showed that the Na /K -ATPase strength- cially loves living in shallow sea and estuary near- ened the ion exchange function necessary to maintain shore. In China, the salinity of seawater in S. the osmotic balance required for normal survival after paramamosain ponds on the farm is between 25‰ salinity drop from 23‰ to 3‰. In addition, there and 3‰ in most area, and the minority such as in were many other KEGG pathways and differentially Shanghai is below 3‰. There is a production experi- expressed genes, which might directly or indirectly ence in the actual production process that amplitude participate in the regulation of osmotic adjustment of of variation in salinity exceeding 10‰ would cause S. paramamosain, providing a valuable data source death for mud crabs (This only refers to sudden sal- for subsequent studies. inity drop). 23‰ is a normal salinity of the seawater S. paramamosain normally lives in estuary areas, for juvenile crabs S. paramamosain, which was living in where the environment is significantly different from the salinity before our treatment. We had made a freshwater, brackish water and seawater. Sometimes the preliminary experiment to select a salinity for treat- salinity changes constantly, and this change requires a ment with 10 juvenile crabs as a group, including response in behavior, morphology, and biochemical 13‰,8‰,5‰,3‰,and 1‰,and thedegreeofsalin- physiology. In production, the sudden drop in salinity ity drop was 10‰,15‰,18‰,20‰, and 22‰,re- usually results from heavy rainfall in strong convective spectively. In the end, we found some individuals in weather. For example, two typhoons hit Zhuhai in salinity 3‰ begin to die, and most individuals nearly August 2017 in one week in Guangdong Province, caus- died in salinity 1‰.So3‰ might be the optimal ing huge losses to the crab farming industry. In this choice as a critical point for the research of adaptive study, the cumulative salvage rate in six days was 16% mechanism responding to sudden drop in salinity, (24/150) in the salinity sag test but may be higher in ac- and we finally selected the 3‰ in our study. More- tual production. Because of the more complicated water over, the LS death time was concentrated within 24, environment system in production, the sudden drop in 48, and 72 h. The LS group showed hyperactivity salinity is often accompanied by a decrease in water within 48 h, and as time went by, the motility was di- temperature. The comprehensive factors led to a de- minished and normalized. The LS group did not have crease in crab immunity. As a result, there was an in- food over 72 h, and gradually started to eat over crease in pathogenic microorganisms in the water and time. Conditions returned to normal after 120 h. To an increase in crab mortality. In this study, only the sal- be honest, the mechanism of adaptive process is very inity was changed and the rest of the environmental fac- complex, and need more in-depth research in the fu- tors were controlled. For the first time, the molecular ture work.The studyaimed at theadaptivemechan- mechanism of S. paramamosain adapting to the salinity isminresponsetosuddensalinitydrop. So we drop was studied. Through the research presented here, compared the CK with 120 h_group (a state of we had discovered a large number of potential genes complete adaptation) to discover the difference of be- that are related to the salinity adaptation in S. parama- tween normal condition and the adaptive status after mosain. The possible KEGG pathway provided a basis sudden salinity drop from 23‰ to 3‰, and eventually for further research. In addition, this study was an im- reveal the adaptive mechanisms in response to sudden sal- portant supplement to the physiological study of aquatic inity drop in the mud crab, S. paramamosain at the level crustacean infiltration, but also provided a scientific of transcription. basis for the regulation of crustacean aquaculture. Wang et al. BMC Genomics (2018) 19:421 Page 11 of 12 Conclusions Abbreviations GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; In conclusion, we analyzed transcriptomic changes in LS: Low salinity; S. paramamosain: Scylla paramamosain; sqRT-PCR: semiquantitative the gills after a sudden drop in salinity in S. parama- reverse-transcription PCR mosain. One hundred thirty-five DEGs annotated by Acknowledgments Nr were screened, of which 108 were up-regulated Aside from funding support, we also thank BGI (BGI, Shenzhen, China) for and 27 were down-regulated. GO analysis showed sequencing consultation and support. that catalytic activity (14) had the most participating Funding genes in the 24 s-tier GO terms, indicating that intra- This work was supported by the Major Sci & Tech Special Project of Zhejiang cellular metabolic activities in S. paramamosain were Province (no: 2016C02055–8), Ministry of Agriculture of China & China Agriculture enhanced. Based on KEGG pathway and biological Research System (no: CARS-48), the K. C. Wong Magna Fund in Ningbo University. The funders had no role in study design, data collection and analysis, decision to functional enrichment on DEGs, the top 20 pathways publish, or preparation of the manuscript. showed that seven pathways were directly related to + + the active regulation of the Na /K ATP enzyme: Availability of data and materials Proximal tubule bicarbonate reclamation (ko04964), Raw Illumina sequences were deposited in the National Center for Biotechnology Information (NCBI) and our SRA records will be accessible with the following link protein digestion and absorption (ko04974), bile se- after the indicated release date: https://www.ncbi.nlm.nih.gov/sra/SRP129841,SRA cretion (ko04976), thyroid hormone signaling pathway accession: SRP129841; Temporary Submission ID: SUB3501735. (ko04919), mineral absorption (ko04978), Insulin se- Authors’ contributions cretion (ko04911), thyroid hormone synthesis CW conceived and designed the study. HW and JL performed the cultivation + + (ko0491), and the regulatory genes in Na /K ATPase of experimental animals, HW, JL, LT, HW, and CM performed and analyzed all were all up-regulated. Additionally, several amino acid the other experiments. HW wrote the manuscript with support from all authors. All authors read and approved the final manuscript. metabolism pathways were detected: arginine biosyn- thesis (ko00220), alanine, aspartate and glutamate me- Ethics approval tabolism (ko00250), lysine degradation (ko00310), The animal subjects used in the present study are crabs, which are invertebrates valine, leucine and isoleucine degradation (ko00280), and are exempt from this requirement. amino sugar and nucleotide sugar metabolism Competing interests (ko00520), biosynthesis of amino acids (ko01230), pyr- The authors declare that they have no competing interests. imidine metabolism (ko00240). In addition, some fam- ous signal pathways were found, such as cAMP Publisher’sNote signaling pathway (ko04024), MAPK signaling path- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. way (ko04013, ko04010), Wnt signaling pathway (ko04310), mTOR signaling pathway (ko04150), Ras Received: 9 January 2018 Accepted: 18 May 2018 signaling pathway (ko04014). Our findings suggest + + that not only Na /K ATPase and amino acids played References a key role in osmoregulation, but also some import- 1. Abe H, Okuma E, Amano H, et al. Role of free d- and l-alanine in the ant signal pathways participated in osmoregulation. Japanese mitten crab Eriocheir japonicus to intracellular osmoregulation Ultimately, survival of S. paramamosain maybesus- during downstream spawning migration. Comp Biochem Phys A. 1999; 123(1):55–9. tained in new surroundings with a sudden drop in 2. Ahearn GA, Duerr JM, Zhuang Z, et al. 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