HIF-1-mediated regulation of LDH gene unravels key insights into MCDV-1 pathogenesis in mud crabs Scylla paramamosain.

Hypoxia-inducible factors -1 (HIF-1) is a crucial transcription factor that regulates the expression of glycolytic genes. Our previous study proved that the Mud crab dicistrovirus-1 (MCDV-1) can induce aerobic glycolysis that favors viral replication in mud crab Scylla paramamosain. However, the role of HIF-1 on key glycolytic genes during the MCDV-1 infection has not been examined. In this study, the intricate interplay between HIF-1 and the key glycolysis enzyme, lactate dehydrogenase (LDH), was investigated after MCDV-1 infection. The expression of LDH was significant increased after MCDV-1 infection. Additionally, the expression of HIF-1α was upregulated following MCDV-1 infection, potentially attributed to the downregulation of prolyl hydroxylase domains 2 expression. Subsequent examination of the SpLDH promoter identified the presence of hypoxia response elements (HREs), serving as binding sites for HIF-1α. Intriguingly, experimental evidence demonstrated that SpHIF-1α actively promotes SpLDH transcription through these HREs. To further elucidate the functional significance of SpHIF-1α, targeted silencing was employed, resulting in a substantial reduction in SpLDH expression, activity, and lactate concentrations in MCDV-1-infected mud crabs. Notably, SpHIF-1α-silenced mud crabs exhibited higher survival rates and lower viral loads in hepatopancreas tissues following MCDV-1 infection. These results highlight the critical role of SpHIF-1α in MCDV-1 pathogenesis by regulating LDH gene dynamics, providing valuable insights into the molecular mechanisms underlying the virus-host interaction.

The Pleiotropic Phenotypes Caused by an hfq Null Mutation in Vibrio harveyi.

Hfq is a global regulator and can be involved in multiple cellular processes by assisting small regulatory RNAs (sRNAs) to target mRNAs. To gain insight into the virulence regulation of Hfq in Vibrio harveyi, the hfq null mutant, ∆hfq, was constructed in V. harveyi strain 345. Compared with the wild-type strain, the mortality of pearl gentian sharply declined from 80% to 0% in ∆hfq when infected with a dose that was 7.5-fold the median lethal dose (LD50). Additionally, ∆hfq led to impairments of bacterial growth, motility, and biofilm formation and resistance to reactive oxygen species, chloramphenicol, and florfenicol. A transcriptome analysis indicated that the expression of 16.39% genes on V. harveyi 345 were significantly changed after the deletion of hfq. Without Hfq, the virulence-related pathways, including flagellar assembly and bacterial chemotaxis, were repressed. Moreover, eleven sRNAs, including sRNA0405, sRNA0078, sRNA0419, sRNA0145, and sRNA0097, which, respectively, are involved in chloramphenicol/florfenicol resistance, outer membrane protein synthesis, electron transport, amino acid metabolism, and biofilm formation, were significantly down-regulated. In general, Hfq contributes to the virulence of V. harveyi 345 probably via positively regulating bacterial motility and biofilm formation. It is involved in flagellar assembly and bacterial chemotaxis by binding sRNAs and regulating the target mRNAs.

Identification and functional characterization of activating transcription factor 6 (ATF6) from the mud crab (Scylla paramamosain) in response to hydrogen peroxide and bacterial challenge.

Activating transcription factor 6 (ATF6) is critical for regulation of unfolded protein response (UPR), which is involved in the endoplasmic reticulum (ER) proteostasis maintenance and cellular redox regulation. In the present study, a ATF6 gene from the mud crab (designated as Sp-ATF6) has been cloned and identified. The open reading frame of Sp-ATF6 was 1917 bp, encoding a protein of 638 amino acids. The deduced amino acid sequences of Sp-ATF6 contained a typical basic leucine zipper (BZIP domain). Sp-ATF6 was widely expressed in all tested tissues, with the highest expression levels in the hemocytes and the lowest in the muscle. Subcellular localization showed that Sp-ATF6 was expressed in both nucleus and cytoplasm of S2 cells. The expression level of Sp-ATF6 was induced by hydrogen peroxide and V. parahaemolyticus challenge, indicating that the ATF6 pathway was activated in response to ER stress. In order to know more about the regulation mechanism of the Sp-ATF6, RNA interference experiment was investigated. Knocking down Sp-ATF6 in vivo can decrease the expression of antioxidant-related genes (CAT and SOD) and heat shock proteins (HSP90 and HSP70) after V. parahaemolyticus infection. All these results suggested that Sp-ATF6 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Essential role of the HSC70 in the mud crab Scylla paramamosain in response to Vibrio parahaemolyticus infection.

Heat shock proteins play an important role in host defense, and modulate immune responses against pathogen infection. In this study, a novel HSC70 from the mud crab (designated as SpHSC70) was cloned and characterized. The full length of SpHSC70 contained a 58 bp 5'untranslated region (UTR), an open reading frame (ORF) of 2,046 bp and a 3'UTR of 341 bp. The SpHSC70 protein included the conserved DnaK motif. The mRNA of SpHSC70 was highly expressed in the hemocytes, heart and hepatopancreas, and lowly expressed in the intestine. The subcellular localization results indicated that SpHSC70 was localized in both the cytoplasm and the nucleus. Moreover, SpHSC70 was significantly responsive to bacterial challenge. RNA interference experiment was designed to investigate the roles of SpHSC70 in response to bacterial challenge. V. parahaemolyticus infection induced the expression levels of SpPO, SpHSP70, SpSOD and SpCAT. Knocking down SpHSC70 in vivo can decrease the expression of these genes after V. parahaemolyticus infection. These results suggested that SpHSC70 could play a vital role in defense against V. parahaemolyticus infection via activating the immune response and antioxidant defense signaling pathways in the mud crab.

The role of Nrf2 signaling pathway in the mud crab (Scylla paramamosain) in response to Vibrio parahaemolyticus infection.

The transcription factor Nrf2 plays vital roles in detoxification and antioxidant enzymes against oxidative stress. However, the function of Nrf2 in crustaceans is not well studied. In this study, a novel Nrf2 gene from the mud crab (Sp-Nrf2) was identified. It was encoded 245 amino acids. Sp-Nrf2 expression was ubiquitously expressed in all tested tissues, with the highest expression level in the gill. Sp-Nrf2 protein was mainly located in the nucleus. The expression levels of Sp-Nrf2, and antioxidant-related genes (HO-1 and NQO-1) were induced after Vibrio parahaemolyticus infection, indicating that Nrf2 signaling pathway was involved in the responses to bacterial infection. Over-expression of Sp-Nrf2 could improve cell viability after H2O2 exposure, indicating that Sp-Nrf2 might relieve oxidative stress. Silencing of Sp-Nrf2 in vivo decreased HO-1 and NQO-1 expression. Moreover, knocking down Sp-Nrf2 in vivo can increase malondialdehyde content and the mortality of mud crabs after V. parahaemolyticus infection. Our results indicated that Nrf2 signaling pathway played a significant role in immune response against bacterial infection.

Essential role of the Cytochrome P450 2 (CYP2) in the mud crab Scylla paramamosain antioxidant defense and immune responses.

Cytochrome P450 (CYPs) enzymes are one of the critical detoxification enzymes, playing a key role in antioxidant defense. However, the information of CYPs cDNA sequences and their functions are lacked in crustaceans. In this study, a novel full-length of CYP2 from the mud crab (designated as Sp-CYP2) was cloned and characterized. The coding sequence of Sp-CYP2 was 1479 bp in length and encoded a protein containing 492 amino acids. The amino acid sequence of Sp-CYP2 comprised a conserved heme binding site and chemical substrate binding site. Quantitative real-time PCR analysis revealed that Sp-CYP2 was ubiquitously expressed in various tissues, and it was highest in the heart followed by the hepatopancreas. Subcellular localization showed that Sp-CYP2 was prominently located in the cytoplasm and nucleus. The expression of Sp-CYP2 was induced by Vibrio parahaemolyticus infection and ammonia exposure. During ammonia exposure, ammonia exposure can induce oxidative stress and cause severely tissue damage. Knocking down Sp-CYP2 in vivo can increase malondialdehyde content and the mortality of mud crabs after ammonia exposure. All these results suggested that Sp-CYP2 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Glutaredoxin 2 in the mud crab Scylla paramamosain: Identification and functional characterization under hypoxia and pathogen challenge.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that plays a key role in antioxidant defense. In this study, a novel Grx2 gene (SpGrx2) was identified from the mud crab Scylla paramamosain, which consists of a 196 bp 5' untranslated region, a 357 bp open reading frame, and a 964 bp 3' untranslated region. The putative SpGrx2 protein has a typical single Grx domain with the active center sequence C-P-Y-C. The expression analysis revealed that the SpGrx2 mRNA was most abundant in the gill, followed by the stomach and hemocytes. Both mud crab dicistrovirus-1 and Vibrioparahaemolyticus infection as well as hypoxia could differentially induce the expression of SpGrx2. Furthermore, silencing SpGrx2 in vivo affected the expression of a series of antioxidant-related genes after hypoxia treatment. Additionally, SpGrx2 overexpression significantly increased the total antioxidant capacity of Drosophila Schneider 2 cells after hypoxia, resulting in a reduction of reactive oxygen species and malondialdehyde content. The subcellular localization results indicated that SpGrx2 was localized in both the cytoplasm and the nucleus of Drosophila Schneider 2 cells. These results indicate that SpGrx2 plays a crucial role as an antioxidant enzyme in the defense system of mud crabs against hypoxia and pathogen challenge.

Toxic effects of cadmium exposure on intestinal histology, oxidative stress, microbial community, and transcriptome change in the mud crab (Scylla paramamosain).

Cadmium is one of hazardous pollutants that has a great threat to aquatic organisms and ecosystems. The intestine plays important roles in barrier function and immunity to defend against environmental stress. However, whether cadmium exposure caused the intestine injury is not well studied. Thus, the aim of this study was to explore the potential mechanisms of cadmium toxicity in the intestine of mud crab (Scylla paramamosain) via physiological, histological, microbial community, and transcriptional analyses. Mud crabs were exposed to 0, 0.01, and 0.125 mg/L cadmium. After a 21-day of cadmium exposure, 0.125 mg/L cadmium caused intestine damaged by decreasing superoxide dismutase and catalase activities, and increasing hydrogen peroxide and malondialdehyde levels. Integrated biological index analysis confirmed that the toxicity of cadmium exhibited a concentration-dependent manner. Comparative transcriptional analyses showed that the up-regulations of several genes associated with heat shock proteins, detoxification and anti-oxidant defense, and two key signaling pathways (PI3k-Akt and apoptosis) revealed an adaptive response mechanism against cadmium exposure. Transcriptomic analysis also suggested that cadmium exposure disturbed the expression of ion transport and immune-related genes, indicating that it has negative effects on the immune functions of the mud crab. Furthermore, the intestinal microbial diversity and composition were significantly influenced by cadmium exposure. The abundance of the dominant phyla Fusobacteria and Bacteroidetes significantly changed after cadmium exposure. KEGG pathway analysis demonstrated that cadmium exposure could change energy metabolism and environmental information processing. Overall, we concluded that excessive cadmium exposure could be potentially exerted adverse effects to the mud crab health by inducing oxidative damage, decreasing immune system, disrupting metabolic function, and altering intestinal microbial composition. These results provided a novel insight into the mechanism of cadmium toxicity on crustaceans.

Hypoxia triggers the proliferation of antibiotic resistance genes in a marine aquaculture system.

The transmission of antibiotic resistance genes (ARGs) affects the safety of aquaculture animals. Dissolved oxygen (DO) can affect the transmission of ARGs, but its mechanism of action in this process is unclear. We conducted laboratory breeding experiment with low and control DO groups. Combined quantitative PCR and 16S rRNA sequencing to study the effect of DO on the spread of ARGs. Hypoxia treatment significantly increased the accumulation of ammonium and nitrite in aquaculture water, and it increased the relative abundances of ARGs and mobile genetic elements (MGEs), especially the ARGs resistant to drugs in the categories of sulfonamide, (flor)/(chlor)/(am)phenicol, and MLSB (macrolide, lincosamide and streptogramin B) and the MGE intI-1(clinic), by 2.39-95.69 % in 28 days relative to the control DO treatment. Though the abundance of ARG carries, especially the Rhodocyclaceae, Caldilineaceae, Cyclobacteriaceae, Saprospiraceae, Enterobacteriaceae, Sphingomonadaceae families, showed higher abundance in low DO groups, relating to the vertical transmission of ARGs. Hypoxia treatment is more likely to promote the horizontal gene transfer (HGT)-related pathways, including ABC transporters, two component system, and quorum sensing, thus to induce the HGT of ARGs. The changed bacterial proliferation also altered the abundance of MGEs, especially intI-1(clinic), which induced HGT of ARGs as well. Additionally, pearson correlation results revealed that the succession of bacterial community function played the strongest role in ARG proliferation, followed by bacterial community structure and MGEs. Our results highlight the importance of suitable DO concentration in controlling the spread of ARGs especially the HGT of ARGs. In the context of global attention to food safety, our results provide important information for ensuring the safety of aquatic products and the sustainable development of aquaculture.

Identification and functional characterization of glutaredoxin 5 from the mud crab (Scylla paramamosain) in response to cadmium and bacterial challenge.

Glutaredoxin (Grx) is a class molecule oxidoreductase, which plays a key role in maintaining redox homeostasis and regulating cell survival pathways. However, the expression pattern and function of Grx remain unknown in the mud crab (Scylla paramamosain). In the present study, a novel full-length of Grx 5 from the mud crab (designated as Sp-Grx 5) was cloned and characterized. The open reading frame of Sp-Grx 5 was 441 bp, which encoded a putative protein of 146 amino acids. The amino acid sequence of Sp-Grx 5 contained a typical C-G-F-S redox active motif and several GSH binding sites. Sp-Grx 5 widely existed in all tested tissues with a high-level expression in hepatopancreas. Subcellular localization showed that Sp-Grx 5 was located in the cytoplasm and nucleus. The expression of Sp-Grx 5 was significantly up-regulated after Vibrio parahaemolyticus infection and cadmium exposure, suggesting that Sp-Grx 5 was involved in innate immunity and detoxification. Furthermore, overexpression of Sp-Grx 5 could improve cells viability after H2O2 exposure. All these results indicated that Sp-Grx 5 played important roles in the redox homeostasis and innate immune response in crustaceans.

Characterization of phosphofructokinase (PFK) from mud crab Scylla paramamosain and its role in mud crab dicistrovirus-1 proliferation.

Phosphofructokinase (PFK), the key enzyme of glycolysis, can catalyze the irreversible transphosphorylation of fructose-6-phosphate forming fructose-1, 6-biphosphate. In the present study, a PFK gene from the mud crab Scylla paramamosain, named SpPFK, was cloned and characterized. The full length of SpPFK contained a 5'untranslated region (UTR) of 249 bp, an open reading frame of 2,859 bp, and a 3'UTR of 1,248 bp. The mRNA of SpPFK was highly expressed in the gill, followed by the hemocytes and muscle. The expression of SpPFK was significantly up-regulated after mud crab dicistrovirus-1 (MCDV-1) infection. Knocking down SpPFK in vivo by RNA interference significantly reduced the expression of lactate dehydrogenase after MCDV-1 infection. Furthermore, silencing of SpPFK in vivo increased the survival rate of mud crabs and decreased the MCDV-1 copies in the gill and hepatopancreas after MCDV-1 infection. All these results suggested that SpPFK could play an important role in the process of MCDV-1 proliferation in mud crab.

Nutrients, temperature, and oxygen mediate microbial antibiotic resistance in sea bass (Lateolabrax maculatus) ponds.

Antibiotic resistance genes (ARGs) have drawn increasing attention as novel environmental pollutants because of the threat they impose on human and animal health. The sea bass (Lateolabrax maculatus) is the third most cultured marine fish in China. Therefore, a study of ARG pollution in the sea bass culture environment is of great significance for the healthy and sustainable development of the sea bass industry. Here, we systematic investigated the contents of 23 antibiotic resistance-related genes (ARRGs), including 19 ARGs and four mobile genetic elements, and analyzed bacterial community composition and environmental parameters in sea bass ponds. The relative abundance (ARRG copies/16S ribosomal RNA gene copies) of ARRGs was up to 3.83 × 10-2. Sul1 was the most abundant ARRG, followed by ereA, intI-1, sul2, dfrA1, and aadA. Both the ARRG changes and aquatic microbiota succession were mainly driven by water temperature (WT), dissolved oxygen (DO), and NO3-. WT is positively correlated with the most ARGs and some of the top 38 Operational Taxonomic Units (OTUs) belonging to the orders of Frankiales, Micrococcales, Chitinophagales, and Sphingomonadales. Furthermore, WT is negatively related with some other OTUs of the orders Frankiales, Xanthomonadales, Micrococcales, and Rhizobiales. However, DO and NO3- have the opposite function with WT on specific taxa and ARGs. These results indicate that sea bass ponds are reservoirs of ARGs, and are driven mainly by the nutrient, temperature, and oxygen with inducing specific microbial taxa. The regulation of environmental factors (increasing DO and NO3-) can be conducted to reduce drug resistance risk in aquaculture ponds. Therefore, environmental factors and specific taxa could be the indicators of ARG contamination and can be used to establish an antibiotic elimination system and consequently realize a sustainable aquaculture industry.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi.

Vibrio harveyi can cause infections and diseases in a variety of marine vertebrates and invertebrates, which are harmful to the aquaculture industry. The LuxS quorum-sensing system regulates the expression of virulence factors in a wide variety of pathogenic bacteria. In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. Statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and luxS mutant. However, deletion of luxS decreased the secretion of extracellular protease, while increasing swimming and swarming abilities. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented this defect. Since motility is flagella dependent, 16 V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly upregulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phases. Our results indicate that LuxS plays an important role in controlling motility, flagella biogenesis, and extracellular protease secretion in V. harveyi.

The role of caspase 3 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection.

Functional characterization of VscCD, an important component of the type â ¢ secretion system of Vibrio harveyi.

Vibrio harveyi is a Gram-negative bacterium that occurs widely in the ocean and a kind of pathogenic bacteria associated with vibriosis in grouper. We investigated whether the VscCD protein of the type Ⅲ secretion system (T3SS) was important for pathogenicity of V. harveyi. Mutations to the vscC and vscD genes (ΔvscCD) and complementation of the ΔvscCD mutant (C-ΔvscCD) were created. Moreover, the biological characteristics of the wild-type (WT) and mutant strains of V. harveyi 345 were compared. The results showed that deletion of the vscCD genes had no effect on bacterial growth, swimming/swarming ability, secretion of extracellular protease, or biofilm formation. However, as compared with the V. harveyi 345: pMMB207 (WT+) and complementary (C-ΔvscCD) strains, the ΔvscCD: pMMB207 (ΔvscCD+) mutant displayed decreased resistance to acid stress, H2O2, and antibiotics. In addition, infection of the pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) showed that as compared with the WT+ and C-ΔvscCD strains, the ΔvscCD+ strain significantly reduced cumulative mortality of the host. The colonization ability of the ΔvscCD+ mutant in the spleen and liver tissues of the pearl gentian grouper was significantly lower than that of the WT+ and C-ΔvscCD strains. In the early stage of infection with the ΔvscCD+ strain, the expression levels of IL-1ß, IL-16, TLR3, TNF-α, MHC-Iα, and CD8α were up-regulated to varying degrees. As compared with the WT+ and C-ΔvscCD strains, luxR expression was significantly up-regulated in the ΔvscCD+ strain, while the expression of vcrH and vp1668 was significantly down-regulated. As an important component of the T3SS, VscCD seemed to play a significant role in the pathogenesis of V. harveyi.

Oxidative stress, DNA damage, and cellular response in hydrogen peroxide-induced cell injury of mud crab (Scylla paramamosain).

Oxidative stress is considered as the toxicity mechanism of environmental stressors on aquatic organisms. This study aims to explore the effects of oxidative stress on physiological responses, DNA damage and transcriptional profiles of the mud crabs Scylla paramamosain. In the present study, mud crabs were injected with 0.1% and 1% hydrogen peroxide (H2O2) for 72 h. The results showed that superoxide dismutase and catalase activities significantly decreased after H2O2 injection. Malondialdehyde content, H2O2 content, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activity significantly increased after H2O2 injection. Moreover, DNA damage occurred after H2O2 injection. Transcriptome analysis showed that 531 and 372 differentially expressed genes (DEGs) were identified after 0.1% and 1% H2O2 injection, respectively. These DEGs were mainly involved in the oxidative stress response and immune functions. All these results indicated that oxidative stress could impair both antioxidant defense systems and immune systems. Transcriptome analysis provided valuable information on gene functions associated with the response to oxidative stress in the mud crab.

Hypoxia-induced oxidative stress and transcriptome changes in the mud crab (Scylla paramamosain).

Mud crab (Scylla paramamosain) is an economically important cultured species in China. Hypoxia is a major environmental stressor during mud crab culture. In the present study, we investigated the oxidative stress and transcriptome changes in the gills of mud crab after intermediate hypoxia stress with dissolved oxygen (DO) 3.0 ± 0.2 mg/L (named as "DO3") and acute hypoxia stress with DO 1.0 ± 0.2 mg/L (named as "DO1") for 0, 3, 6, 12 and 24 h. The superoxide dismutase (SOD) activity of DO1 increased significantly at 3, 6 and 24 h after hypoxia stress, while SOD activity of DO3 increased significantly at 6 and 24 h. The total antioxidant capacity (T-AOC) increased significantly at 6, 12 and 24 h after hypoxia stress. The malondialdehyde (MDA) concentration of DO1 increased significantly at 6, 12 and 24 h after hypoxia stress, while MDA concentration of DO3 only increased significantly at 6 h. The lactate dehydrogenase (LDH) activity of DO1 increased significantly at 3, 6, 12 and 24 h after hypoxia stress, while LDH activity of DO3 increased significantly at 12 and 24 h. Transcriptomic analysis was conducted at 24 h of gill tissues after hypoxia stress. A total of 1052 differentially expressed genes (DEGs) were obtained, including 394 DEGs between DO1 and DO3, 481 DEGs between DO1 and control group, 177 DEGs between DO3 and control group. DEGs were enriched in the pathways related to metabolism, immune functions, ion transport, and signal transduction. Transcriptional analysis showed that glycolysis and tricarboxylic acid cycle genes were the key factors in regulating the adaptation of mud crab to hypoxia stress.

What drives putative bacterial pathogens removal within seagrass meadows?

To analyze the mechanism of bacterial pathogen removal in seagrass meadows, we compared bacterial pathogens abundance in trapped particles in different seagrass meadows under different intensities of human activities. We compared the particle deposition rates and abundances of bacterial pathogen in Thalassia hemprichii, Enhalus acoroides stands and adjacent unvegetated patches. The bacterial pathogens abundance was much higher in E. acoroides than in adjacent unvegetated patches, however, the trapped particles under T. hemprichii were lower than in nearby unvegetated areas with the exception of the pristine seagrass meadow. These results indicate that seagrass, at least E. acoroides, can remove bacterial pathogens by trapping particles. What is unknown, nevertheless, is how the trapped bacterial pathogens are removed by T. hemprichii. We put forward that antibacterial chemical compounds release from seagrass was stimulated by stress from human activities for inhibition of bacterial pathogen. This putative mechanism needs to be explored in future studies.

The role of tumor suppressor protein p53 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

The tumor suppressor protein p53 plays important roles in DNA repair, cell cycle and genetic stability. In the present study, a p53 gene in the mud crab (Scylla paramamosain) (designated as Sp-53) was identified and characterized. The open reading frame of Sp-53 was comprised a 1383 bp, which encoded a putative protein of 460 amino acids. Sp-53 is expressed in all examined tissues, with the highest expression in hepatopancreas and hemocytes. Vibrio parahaemolyticus infection induced oxidative stress, and led to DNA damage. The Sp-53 transcriptions in hepatopancreas were significantly up-regulated after V. parahaemolyticus infection. RNA interference (RNAi) experiment was used to understand the roles of Sp-53 in response to V. parahaemolyticus infection. Knocking down Sp-53 in vivo significantly reduced the expression of the Mn-SOD, Gpx3 and caspase 3 after V. parahaemolyticus infection. Moreover, the mortality of mud crabs and DNA damage in Sp-53-silenced mud crab challenged with V. parahaemolyticus were significantly higher than those in the control group. All these results suggested that Sp-53 played an important role in responses to V. parahaemolyticus infection through its participation in regulation of antioxidant defense, DNA repair and apoptosis.

Oxidative stress, cell cycle arrest, DNA damage and apoptosis in the mud crab (Scylla paramamosain) induced by cadmium exposure.

Cadmium is one of the most common heavy metal pollutants in the aquatic environment. Mud crab (Scylla paramamosain) is considered a model organism to monitor the impact of heavy metals. However, knowledge about toxicological mechanism of cadmium in crustaceans still remains limited. In this study, mud crabs were exposed to different concentrations of cadmium (0, 1.25, 2.5, 5 and 10 mg/L) for 72 h. Cadmium exposure significantly decreased superoxide dismutase (SOD) activity, catalase (CAT) activity and total antioxidative capacity (T-AOC), and significantly increased malondialdehyde (MDA) and H2O2 levels. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activity significantly increased after cadmium exposure. Moreover, integrated biological responses version 2 (IBRv2) analysis suggested that cadmium exposure exerted stronger toxicity on mud crab. Furthermore, oxidative stress induced by cadmium exposure could decrease total hemocyte count (THC), interrupt Ca2+ homeostasis, and lead to cytological damage. Cadmium exposure induced DNA damage, which activated DNA damage response signaling ATR-CHK1-p53 pathway. Our results also showed that cadmium exposure significantly increased the apoptosis and caspase-3 mRNA levels, which implied that cadmium induced apoptosis through a caspase-3 pathway.