Risk assessment of pesticide compounds: IPT and TCZ cause hepatotoxicity, activate stress pathway and affect the composition of intestinal flora in red swamp crayfish (Procambarusclarkii).

Isoprothiolane (IPT) and tricyclazole (TCZ) are widely used in rice farming and recently in combined rice-fish farming. However, co-cultured animals are affected by these pesticides. To investigate the organismal effects and toxicity of pesticides, crayfish were exposed to 0, 1, 10, or 100 ppt TCZ or IPT for 7 days. Pesticide bioaccumulation, survival rate, metabolic parameters, structure of intestinal flora, and antioxidant-, apoptosis-, and HSP-related gene expression were determined. Pesticide exposure caused bioaccumulation of IPT or TCZ in the hepatopancreas and muscles of crayfish; however, IPT bioaccumulation was higher than that of TCZ. Both groups showed significant changes in hepatopancreatic serum biochemical parameters. Mitochondrial damage and chromosomal agglutination were observed in hepatopancreatic cells exposed to 100 ppt IPT or TCZ. IPT induced more significant changes in serum biochemical parameters than TCZ. The results of intestinal flora showed that Vibro, Flavobacterium, Anaerorhabdus and Shewanella may have potential for use as a bacterial marker of TCZ and IPT. Antioxidant-, apoptosis-, and HSP-related gene expression was disrupted by pesticide exposure, and was more seriously affected by IPT. The results suggest that IPT or TCZ induce hepatopancreatic cell toxicity; however, IPT or TCZ content in dietary crayfish exposed to 1 ppt was below the food safety residue standard. The data indicated that IPT exposure may be more toxic than TCZ exposure in hepatopancreas and intestines and toxicity of organism are alleviated by activating the pathway of stress-response, providing an understanding of pesticide compounds in rice-fish farming and food safety.

Toxic effects and molecular mechanisms of estuarian crustaceans (Scylla paramamosain) exposed to five commonly used benzophenones.

Benzophenones (BPs) are commonly used in personal care products like sunscreens and are increasingly being released into the environment, raising concerns about their potential ecotoxic effects. BPs as emerging environmental contaminants, little is known about their toxic effects on estuarine organisms. This study firstly investigated the toxic effects of five commonly used BPs on mud crabs (Scylla paramamosain). The crabs were exposed to varying concentrations of BPs for 14 days. The results showed that BPs caused damage to antioxidant systems in crabs. Transcriptome sequencing revealed that BP-3 and BP-1 had a greater impact on the crabs compared to the other BPs. Specifically, BP-1 and BP-3 caused severe damage to organelles and ribosomes. BP affected catalytic activity and hydrolase activity, BP-2 affected phosphoenolpyruate carboxykinase activity, and BP-4 affected tRNA aminoacylation and hydrolase activity. These findings can enhance our understanding of the ecotoxicity of BPs and may help to protect estuarine ecosystems.

The first identification and functional analysis of two drosophila mothers against decapentaplegic protein genes (SpSmad1 and SpSmad2/3) and their involvement in the innate immune response in Scylla paramamosain.

Smad，a member of the TGF-ß superfamily，controls cell proliferation，growth and guiding cell differentiation, thus playing a crucial role in diseases. However, the presence as well as specific function of Smad in crabs is still unknown. In this study, two Smads (Smad1 and Smad2/3) were identified for the first time from the mud crab Scylla paramamosain. The complete open reading frames of SpSmad1 and SpSmad2/3 were 1,497bp and 1,338bp, encoding deduced proteins of 498 and 445 amino acids respectively. Moreover, under the administration of Vibrio alginolyticus and WSSV, the relative expression levels of SpSmad1 and SpSmad2/3 were significantly increased, indicating their involvement in the innate immune response of mud crabs. Knockdown of SpSmad1 and SpSmad2/3 in vivo not only led to the increasement of the expressions of NF-κB signaling genes and antimicrobial peptides genes, but also significantly affected the bacterial clearance process of mud crabs. Additionally, overexpression of SpSmad1 and SpSmad2/3 in HEK293T cells could markedly activate NF-κB signaling. These results indicated that Smad1 and Smad2/3 participated in the innate immunity of Scylla paramamosain, and might provide a better understanding of the presence and immune regulatory functions of Smad1 and Smad2/3 in crabs and even invertebrates.

Identification and functional analysis of endoplasmic reticulum oxidoreductase 1 (ERO1) from the green mud crab Scylla paramamosain: The first evidence of ERO1 involved in invertebrate immune response.

Endoplasmic reticulum oxidoreductase 1 (ERO1) is an important mediator in regulating disulfide bond formation and maintaining endoplasmic reticulum homeostasis. Its activity is transcriptionally regulated by the unfolded protein response (UPR) in the endoplasmic reticulum, which is known to be essential in immunity. However, whether ERO1 is involved in innate immunity in invertebrates remains unclear. In the present study, two subtypes of ERO1 from Scylla paramamosain were first identified and characterized. Sequence analysis revealed the conserved ERO1 domain and the oxidative capacity assay verified the oxidative capacity of SpERO1 recombinant protein. Moreover, SpERO1s were found to be ubiquitously expressed in all the tested tissues, with the highest expression observed in hemocytes. Two SpERO1s exhibited distinct expression patterns in response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV). Importantly, the downregulation of the expression of immune factors upon bacterial challenge in SpERO1-silenced crabs was observed. These results provided an initial foundation for further investigations into the role of ERO1 in the innate immunity of invertebrates.

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.

Characterization of fibroblast growth factor receptor 4 (FGFR4) from the red swamp crayfish Procambarus clarkii and its role in antiviral and antimicrobial immune responses.

FGFRs involved multiple physiological processes, such as endocrine homeostasis, wound repair, and cellular behaviors including proliferation, differentiation and survival. In the present study, the homologs of fibroblast growth factor receptor 4 (FGFR4) were identified and characterized from the red swamp crayfish Procambarus clarkii for the first time. The full-length cDNAs of pcFGFR4 were 2878 bp with 2451 bp open reading frame (ORF), respectively. The deduced pcFGFR4 protein contained an immunoglobulin, two immunoglobulin C-2 Type, a transmembrane region and a catalytic domain. Real-time PCR analysis showed that pcFGFR4 were highly expressed in muscle and hemocyte. Moreover, the expression levels of pcFGFR4 in the hepatopancreas and hemocyte were positively stimulated after challenge with Aeromonas hydrophila and WSSV, implying the involvement of pcFGFR4 against bacterial and viral infections in innate immune responses. While pcFGFR4 were silenced in vivo, the expression levels of antimicrobial peptide (AMP) genes (pcALF1-5,8 and pcCrustin1-2) and NF-κB signaling components (pcDrosal and pcRelish) were significantly reduced. Additionally, NF-κB signaling could be markedly activated by overexpression of pcFGFR4 in HEK293T cells. Finally, our results indicated that pcFGFR4 regulated crayfish's innate immunity by modulating NF-κB signaling. These findings may provide new insights into pcFGFR4-mediated signaling cascades in crustaceans and provide a better understanding of crustacean innate immune system.

Bioaccumulation of Cd and comparative transcriptome analysis after the antagonism of Se in the hepatopancreas of estuary mud crab (Scylla paramamosain).

Cadmium (Cd) is a heavy metal contaminant and can be toxic to environment. What's more, Selenium (Se) protects organism as heavy metal antagonist. The present study aimed to investigate whether inorganic (Na2SeO3) or organic (L-SeMc) Se have an effect on the Cd bioaccumulation, antioxidant and immunity of the mud crab (Scylla paramamosain) under Cd exposure. The study showed that the concentration of Cd in hepatopancreas under Cd exposure was higher than the inorganic or organic Se group (P < 0.05), notably, Cd concentration of hepatopancreas in organic Se treatment is less than that in inorganic Se treatment (P < 0.05). Furthermore, this study analyzed 28 gene expression about antioxidant and immune from transcriptome, the result indicated that L-SeMc (organic Se) can reduced intracellular ROS production and oxidative damage. Furthermore, apoptosis was enhanced after Cd exposure, but Se could protect against apoptosis via expression of cathepsin B. Consequently, Organic Se may have a better effect than inorganic Se on reducing Cd toxicity. This study could provide the molecular basis that Se might alleviate Cd toxicity and increases the understanding of the environmental contaminant on crustaceans.

Identification and functional analysis of two drosophila mothers against decapentaplegic protein(Smad)genes and their involvement in immune responses in the red swamp crayfish Procambarus clarkii.

Drosophila mothers against decapentaplegic proteins (Smads), the crucial signal transducers in activating downstream gene transcription through transforming growth factor beta (TGF-ß) receptors, are the pleiotropic factors with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, whether Smads are involved in immune response in crustaceans remains unexplored. In the present study, the Smad3 and Smad4 were firstly identified and functionally characterized from the Red Swamp Crayfish Procambarus clarkii. The full-length cDNAs of pcSmad3 and pcSmad4 were 1, 670 bp and 3, 060 bp with 1, 326 bp and 1, 875 bp open reading frame (ORF), respectively. Real-time PCR analysis of the expression profiles demonstrated that pcSmad3 and pcSmad4 were predominantly expressed at in stomach, heart, and hemocytes. Notably, the expression levels of pcSmad3 and pcSmad4 both Aeromonas hydrophila and WSSV challenges were significantly altered, suggesting the involvement of pcSmad3 and pcSmad4 in innate immune responses. Knockdown of pcSmad3 and pcSmad4 in vivo dramatically activated the transcriptions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. The overexpression of pcSmad3 and pcSmad4 could significantly activate NF-κB signaling in HEK293T cells. Meanwhile, the clearance of bacteria was significantly reduced with knockdown of pcSmad3 and pcSmad4 in vivo. Results indicated that pcSmad3 and pcSmad4 played an immune-regulatory role in crayfish's innate immunity, which might pave the for a better understanding of the TGF-ß superfamily members in crustacean.

Cdc42 Promotes Axonogenesis of Primary Hippocampal Neurons by Inhibiting Glycogen Synthase Kinase-3ß.

BACKGROUND: Progressive axon degeneration is a common pathological feature of neurodegenerative diseases. Cdc42 is a member of the Rho GTPase family that participates in axonogenesis. GSK-3ß is a serine/threonine kinase highly implicated in neuronal development and neurodegeneration. This study aimed to examine whether cdc42 promotes axonogenesis by regulating GSK-3ß activity. METHODS: Hippocampal neurons were isolated from neonatal Sprague-Dawley rats and transfected with designated plasmid vectors to alter the activities of cdc42 and GSK-3ß. LiCl treatment was used to inhibit the GSK-3ß activity in primary neurons. GSK-3ß activity was determined by an enzyme activity assay kit. Immunofluorescence staining was used to detect axons stained with anti-Tau-1 antibody and dendrites stained with anti-MAP2 antibody. RESULTS: Transfection with an active cdc42 mutant (cdc42F28L) decreased the activity of GSK-3ß and induced axonogenesis in primary rat hippocampal neurons, while transfection with a negative cdc42 mutant (cdc42N17) resulted an opposite effect. Moreover, transfection with plasmid vectors carrying wild-type GSK-3ß or a constitutively active GSK3ß mutant (GSK-3ß S9A) increased the activity of GSK-3ß and attenuated axonogenesis of primary hippocampal neurons with excessive cdc42 activity, whereas inhibition of GSK-3ß by LiCl abolished the inhibitory effect of the negative cdc42 mutant on axonogenesis. CONCLUSIONS: This study suggests that cdc42 induces axonogenesis of primary rat hippocampal neurons via inhibiting GSK-3ß activity. These findings support further investigation into the mechanisms of cdc42/GSK-3ß-mediated axonogenesis.

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 Inactivated gE/TK Gene-Deleted Vaccine Against Pseudorabies Virus Type II Confers Effective Protection in Mice and Pigs.

The highly virulent and antigenic variant of Pseudorabies virus (PRV) that emerged from classical Bartha-K61-vaccinated pig herds has caused substantial economic losses to the swine industry in China since 2011. A safe and more effective vaccine is most desirable. In this study, a gE/TK gene-deficient PRV, namely, HD/c, was constructed based on a PRV type II DX strain isolated from a commercial vaccine-immunized farm and the HD/c-based inactivated vaccine was formulated and evaluated for its safety, immunogenicity, and protective efficacy in mice and piglets. The resulting PRV HD/c strain has a similar growth curve to the parental DX strain. After vaccination, the inactivated HD/c vaccine did not cause any visible gross pathological or histopathological changes in the tissues of mice and piglets and provided rapid and potent protection against the challenge of the classical and variant PRVs at day 21 post-vaccination in mice. A single immunization of 108.5TCID50 inactivated PRV HD/c strain-elicited robust immunity with high titer of neutralizing antibody and provided complete protection from the lethal challenge of PRV DX strain in piglets. These results indicated that the inactivated PRV HD/c vaccine with the deletion of gE/TK genes was a safe and effective PRV vaccine candidate for the control of PRV.

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.

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.

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.

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.

Cdc42 Facilitates Axonogenesis by Enhancing Microtubule Stabilization in Primary Hippocampal Neurons.

The establishment of polarity is an essential process in early neuronal development. Cdc42, a GTPase of the Rho family, is a key regulator of cytoskeletal dynamics and neuronal polarity. However, the mechanisms underlying the action of cdc42 in regulating axonogenesis have not been elucidated. Here, we expressed wild-type cdc42, a constitutively active cdc42 mutant (cdc42F28L) and a dominant negative cdc42 mutant (cdc42N17), respectively, in the primary hippocampal neurons to alter the activity of cdc42. We found that cdc42 activities were paralleled with the capacities to promote axonogenesis in the cultured neurons. Cdc42 also enhanced microtubule stability in the cultured neurons. Pharmacologically stabilizing microtubules significantly abrogated the defective axonogenesis induced by cdc42 inhibition. Moreover, cdc42 promoted the dephosphorylation of collapsing response mediator protein-2 (CRMP-2) at Thr514 by increasing GSK-3ß phosphorylation at Ser9 in the cultured neurons. These findings suggest that cdc42 may facilitate axonogenesis by promoting microtubule stabilization in rat primary hippocampal neurons.

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.