Effects of low salinity stress on osmoregulation and gill transcriptome in different populations of mud crab Scylla paramamosain.

Animals living in estuaries suffer from rapid and continuous salinity fluctuations, while the global warming and extreme precipitation aggravate this situation. Osmoregulation is important for estuarine animals adapt to salinity fluctuations. The present study investigated the effects of low salinity stress on osmoregulation and gill transcriptome in two populations of mud crab from Hangzhou Bay and Zhangzhou Bay of China, respectively. Crabs were transferred from salinity 25 ppt to 5 ppt for 96 h. Edematous swelling in gill filaments was caused by low salinity stress and was more serious in Zhangzhou Bay population. Gill Na+/K+-ATPase activity increased (p < 0.01) in both populations under the low salinity stress and was significantly higher (p < 0.01) in Hangzhou Bay population than in Zhangzhou Bay population. According to transcriptome analysis, there were 191 genes differentially expressed under the low salinity stress in gill tissue of both populations. Several ion transport and energy metabolism related pathways, as well as the arginine and proline metabolism pathway, were enriched by these genes. On the other hand, 272 genes were identified to differentially express between two populations under the low salinity stress, but not under the control salinity. The enrichment analysis showed that these genes were mainly related to ion transport, energy metabolism, osmolytes metabolism and methyltransferase activity. In conclusion, the present study suggested that mud crab exploited a combination of extracellular anisosmotic regulation and intracellular isosmotic regulation for osmoregulation under the low salinity stress. Hangzhou Bay population showed a greater osmoregulatory capacity, which is probably due to the enhanced ion transport, energy supply, and osmolytes regulation. Meanwhile, epigenetic modification might also contribute to an inherent osmoregulation ability for Hangzhou Bay population to response to salinity fluctuation rapidly.

The first identification of three AdIRAK2 genes from an evolutionarily important amphibian Andrias davidianus and their involvement in NF-κB activation and inflammatory responses.

Interleukin-1 receptor associated kinases (IRAK) is the most important downstream kinases of TLRs/IL-1R signaling pathway for signal transduction and activation of inflammatory response against pathogen infections. However, the molecular identification and function characterization of IRAK2 homologs in lower vertebrate remains obscure. In this study, three IRAK2 genes (AdIRAK2a, AdIRAKb and AdIRAK2c) and their respective transcripts were identified from the Chinese giant salamander Andrias davidianus. This is the first evidence that three different IRAK2 genes exist in an ancient amphibian species, which has never been reported in other vertebrates. The complete open reading frames (ORFs) of AdIRAK2a, AdIRAK2b and AdIRAK2c were 2112 bp, 1917 bp and 816 bp encoding deduced proteins of 703 amino acids (aa), 628 aa and 271 aa, respectively. All three AdIRAK2 proteins contained two predicted conserved functional domains, including a death domain (DD) and a serine/threonine protein kinases domain (KD). Phylogenetic analysis showed that the three AdIRAK2s clustered together with other known IRAK2 of vertebrates. The three AdIRAK2s were ubiquitously expressed in all tested tissues with a similar tissues distribution pattern. After challenge of Aeromonas hydrophila (A. hydrophila), Staphylococcus aureus (S.aureus), giant salamander iridovirus (GSIV, belonging to the genus Ranavirus in the family Iridoviridae) and polyinosinic:polycytidylic acid (poly(I:C)), the expression levels of all AdIRAK2s in blood were significantly altered, however, they exhibited distinct response patterns. Moreover, the results of over-expression and RNAi of AdIRAK2s implied the involvement of AdIRAK2s in triggering NF-κB-mediated signaling pathways and inflammatory responses. This study might provide a better understanding of the presence and immune regulation function of IRAK2 in amphibians and even in vertebrates.

Identification and functional analysis of epidermal growth factor receptor (EGFR) from Scylla paramamosain: The first evidence of two EGFR genes in animal and their involvement in immune defense against pathogen infection.

The epidermal growth factor receptor (EGFR) is a pleiotropic glycoprotein which plays a role in regulating cell proliferation, migration and differentiation. However, the genetic diversity of EGFR in crustaceans as well as its function, such as whether it is involved in immune regulation, remains obscure. In this study, two EGFR genes, including EGFR1 and EGFR2, and three transcripts were identified and characterized in Scylla Paramamosain for the first time. To our knowledge, this is the first time that more than one EGFR gene was identified in a single species. The complete open reading frames (ORFs) of SpEGFR1, SpEGFR2a and SpEGFR2b were 4377 bp, 4404 bp and 4341 bp encoding deduced proteins of 1458 amino acids (aa), 1467 aa and 1446 aa, respectively. All EGFR had a signal peptide region and two Recep_L_domain region, followed by a transmembrane region and a conserved tyrosine kinase domain (TyrKc), and phylogenetic analysis demonstrated three SpEGFRs clustered together with invertebrate EGFR branch. Tissue specific expression analysis depicted that all SpEGFRs presented similar transcription patterns. The expression levels of SpEGFR1 and SpEGFR2s in hepatopancreas and gills were significantly altered after the stimulation of bacterial and viral pathogens including Staphylococcus aureus, Vibrio alginolyticus, White spot syndromre virus and Polycytidylinic acid. The in vivo RNA interference assays demonstrated that expression levels of SpIKK, two members of NF-κB (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF1-5) were significantly reduced when SpEGFR1 or SpEGFR2 was silenced, respectively. The transcription patterns of SpIKK, SpRelish, SpDorsal and AMPs exhibited similar down- or up-regulation trend when the primary cultured hemocytes were treated with EGFR antagonist or agonist for 24 h. These results suggested that SpEGFR might play an important role in innate immune responses to bacterial and viral infections by regulating the NF-κB pathway. It also provided a better understanding of the origin or evolution of EGFR in crustaceans and even invertebrates.

Identification and function analysis of two fibroblast growth factor receptor (FGFR) from Scylla paramamosain: The evidence of FGFR involved in innate immunity in crustacean.

The fibroblast growth factor receptor (FGFR) belongs to the tyrosine kinase family consisting of four members (FGFR1-4). This study involved identification and characterization of FGFR1 and FGFR3 from mud crab Scylla paramamosain for the first time. The obtained cDNAs of SpFGFR1 and SpFGFR3 were 2,380 bp and 2,982 bp in length with a 1,503 bp and 2,310 bp open reading frame, respectively. The predicted SpFGFR1 protein included three immunoglobulin domains and a transmembrane region, while SpFGFR3 protein possessed a typical TyrKc (Tyrosine kinase, catalytic) domain. Real-time PCR analysis showed that SpFGFR1 and SpFGFR3 were highly expressed in the hepatopancreas. Furthermore, the expression levels of SpFGFR1 and SpFGFR3 in the hepatopancreas were enhanced following challenges with Vibro alginolyticus, Staphylococcus aureus, Poly (I:C) and White spot syndrome virus, which shows the involvement of SpFGFR1 and SpFGFR3 in innate immune response to infections from bacteria and virus. There was significant suppression of six antimicrobial peptide genes (SpALF1-5 and SpCrustin) and three NF-κB members (SpDorsal, SpIKK and SpRelish) when SpFGFR1 and SpFGFR3 was interfered in vivo. Also, treatment of the hemocytes with specific inhibitor of SpFGFR for 24 h consistently down-regulated SpDorsal, SpRelish and AMPs. These results suggested that SpFGFR1 and SpFGFR3 played important roles in regulating the Toll signaling pathway and immune deficiency (IMD) pathway through NF-κB signaling pathway. These findings may provide new insights into the role of FGFRs in the innate immune function of crustaceans.

The health risk for consumers under heavy metal scenarios: Reduce bioaccumulation of Cd in estuary mud crab (Scylla paramamosain) through the antagonism of Se.

Heavy metal pollution has gained increasing attention over past years, and notably, cadmium (Cd) is a non-essential heavy metal that can be toxic to human and wildlife. Furthermore, selenium (Se) is a component of the selenoproteins and influences the toxicity of Cd in different organisms, and protect organisms as a kind of heavy metal antagonist. This study exposed mud crab to 5.0 mg/L Cd for 28 days, and investigated whether different concentrations (0.1, 0.2, 0.3 mg/kg) of selenite (Na2SeO3) or selenomethionine (SeMet) affect the bioaccumulation of Cd, serum biochemical index, antioxidant and stress-response genes of S. paramamosain. The results showed that the Cd concentration in Cd group was significantly higher than the organic or inorganic Se group. Serum biochemical index demonstrated that Se might relieve the damage or dysfunction of hepatopancreas caused by both Cd accumulation and toxicity. Furthermore, Se improved CAT, GPx T-AOC and SOD activity, and decreased MDA concentrations and the lipid peroxidation levels, antagonistic to Cd. Then, this study analyzed the expression of 26 stress-related genes, the results indicated that the inorganic and organic Se might reduce the damage of cell and the toxicity of heavy metals in the hepatopancreas after Cd exposure. Therefore, this study indicated that Se might alleviate Cd toxicity via the different antioxidative mechanisms, and increased the understanding of environmental toxins on estuary crustaceans.

The effects of salinities stress on histopathological changes, serum biochemical index, non-specific immune and transcriptome analysis in red swamp crayfish Procambarus clarkii.

This study aimed to investigate whether different salinity stresses (0, 2, 4, 6, or 8 ppt NaCl) affect the histoarchitecture, serum biochemical indices, antioxidant responses, and transcriptome analysis of the red swamp crayfish Procambarus clarkii. Transmission electron microscopy results showed that the degree of damage to the nuclei and mitochondria in the hepatopancreas increased with increasing salinity. Scanning electron microscopy results showed that the degree of gill wrinkles was enhanced under salinity stress. Serum biochemical indices demonstrated that the cholesterol significantly decreased while the triglyceride, aspartate transaminase, and alanine transaminase contents significantly increased with increasing salinity. The antioxidant parameters, including catalase, total antioxidant capacity, and glutathione peroxidase, significantly decreased, while the malondialdehyde content significantly increased under salinity stress. Transcriptome analysis revealed that the expression pattern of immune-related genes showed a downward trend. These findings enrich our knowledge about the salinity stress response of farmed organisms and provide a theoretical base for salinity domestication and saline soil cultivation of P. clarkii, which might contribute to income improvement, employment generation, food security, and environmental safety.

Identification and functional analysis of drosophila mothers against decapentaplegic protein gene 1 (Smad1) from the red swamp crayfish Procambarus clarkii: The first evidence of Smad1 involved in immunity in invertebrates.

Smads, part of signaling cascades that represent downstream pathways of the TGF-ß super family proteins, are pleiotropic cytokines with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, the specific functions of Smads remain unknown in crustaceans. In the present study, the drosophila mothers against decapentaplegic protein gene 1 (Smad1) was firstly identified and characterized from the Red Swamp Crayfish Procambarus clarkii. The obtained cDNA sequence of pcSmad1was 2, 503 bp long with a 1, 488 bp open reading fame, which encoded a putative protein of 496 amino acids. Furthermore, pcSmad1 responded to both Aeromonas hydrophila and WSSV challenge, suggesting the involvement of pcSmad1 in innate immune responses. Knockdown of pcSmad1 in vivo dramatically increased the expressions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. Additionally, overexpression of pcSmad1 in HEK293T cells could markedly activate NF-κB signaling. Taken together, these results indicated that pcSmad1 played an immune-regulatory role in crayfish's innate immunity, which may provide a better understanding of TGF-ß superfamily members in crustacean.

Effects of air exposure stress on crustaceans: Histopathological changes, antioxidant and immunity of the red swamp crayfish Procambarus clarkii.

Air exposure stress may result in oxidative damage and ultimately disease or death in crustaceans. Using the Procambarus clarkia, one of the main commercial aquaculture species in China, as a study model, the molecular mechanism including histopathological changes, antioxidant capacity and immunity response under the air exposure stress were firstly evaluated. Results showed that the surfaces of gill were wrinkled while the morphologies of the nuclei and mitochondria in the hepatopancreas were altered after 48 h of air exposure stress, and the damage of mitochondria was more serious after additional bacterial infection. Moreover, the activity of antioxidant enzymes increased at first and then decreased along with increasement of air exposure time. The concentration of malondialdehyde (MDA) in hepatopancreas was significantly increased under the air exposure stress, while the bacterial infection further aggravated such oxidative damage. The transcriptome analysis exhibited that the stress- and immunity-related genes in hepatopancreas altered when response to the air exposure stress. This study could help uncover the mechanisms of aerial exposure stress responses in Procambarus clarkii.

Functional differences in the products of two TRAF3 genes in antiviral responses in the Chinese giant salamander, Andrias davidianus.

Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.

Identification and functional analysis of transforming growth factor-ß type III receptor (TßR3) from Scylla paramamosain: The first evidence of TßR3 involved in development and innate immunity in invertebrates.

Transforming growth factor-ß type III receptor (TßR3), as a co-receptor of TGF-ß superfamily, plays critical roles in development and growth as well as some disease pathogeneses by presenting ligands to other receptors in vertebrates. However, the identification and functional characterization of TßR3 had not been reported yet in invertebrates. In the present study, TßR3 was first identified and characterized in mud crab Scylla paramamosain. The obtained cDNA length of SpTßR3 was 2, 424 bp with a 1, 854 bp open reading frame, which encoded a putative peptide of 617 amino acids containing a typical transmembrane region and a Zona pellucida (ZP) domain. Real-time PCR results showed that SpTßR3 was predominantly expressed at early embryonic development stage and early postmolt stage, suggesting its participation in development and growth. We report, for the first time in invertebrates, the challenge of both Vibro alginolyticus and Poly (I:C) could alter the expression patterns of SpTßR3. Notably, the expression levels of SpIKK, two NF-κB members (SpRelish and SpDorsal), and five antimicrobial peptide genes (SpCrustin and SpALF1-4) were significantly suppressed when SpTßR3 was interfered in vivo. Secondly, the overexpression of SpTßR3 in vitro could activate NF-κB signaling through the dual-luciferase reporter assays. Furthermore, the bacterial clearance assay after SpTßR3 was silenced in vivo highlighted the potential of SpTßR3 in activating the innate immune responses. These results implied the involvement of SpTßR3 in the innate immune responses by regulating the NF-κB pathway. This study first indicated that TßR3 was present in invertebrate, and it participated in not only the development and growth but also the innate immunity of S. paramamosain. It also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.