Yi-Lian Zhou

Effect of cadmium exposure on hepatopancreas and gills of the estuary mud crab (Scylla paramamosain): Histopathological changes and expression characterization of stress response genes

Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans.

Identification and characterization of atypical 2-cysteine peroxiredoxins from mud crab Scylla paramamosain: The first evidence of two peroxiredoxin 5 genes in non-primate species and their involvement in immune defense against pathogen infection

Peroxiredoxin 5 (Prx5) belongs to a novel family of evolutionarily conserved antioxidant proteins that protect cells against various oxidative stresses. Generally, no more than one Prx5 transcript had been reported in non-primate species. In this study, two Prx5 genes (coined as SpPrx5-1 and SpPrx5-2) were firstly isolated from the mud crab, Scylla paramamosain, through RT-PCR and RACE methods. The open reading frame of SpPrx5-1 and SpPrx5-2 were 561 bp and 429 bp in length, encoding 186 and 142 amino acids polypeptide, respectively. Both the conserved signatures of peroxiredoxin catalytic center and Prx5-specific domain were identified in SpPrx5-1 and SpPrx5-2. Phylogenetic analysis indicated that both SpPrx5 clustered together with other animal Prx proteins and were classified into Prx5 subfamily. Tissue-specific expression analysis revealed that both SpPrx5-1 and SpPrx5-2 were ubiquitously expressed, highest in hepatopancreas, and showed remarkably similar transcription patterns. Quantitative RT-PCR analysis exhibited that both SpPrx5 genes changed dramatically in hepatopancreas, although showing different expression profiles, after virus-analog poly (I:C) or Vibrio alginolyticus challenge. The expression levels of both SpPrx5s were significantly enhanced in hepatopancreas after poly (I:C) stimulation, while SpPrx5-2 exhibited a more prompt response than SpPrx5-1. Nevertheless, the expression levels of both SpPrx5s were significantly reduced in hepatopancreas after Vibrio alginolyticus challenge in which SpPrx5-1 showed a more prompt response than SpPrx5-2. These results suggested the involvement of SpPrx5s in responses against viral and bacterial infections and further highlighted their functional importance in the immune system of Scylla paramamosain.

Identification and characterization of pro-interleukin-16 from mud crab Scylla paramamosain: The first evidence of proinflammatory cytokine in crab species

ABSTRACT IL‐16 is a pro‐inflammatory cytokine originally designated as a lymphocyte chemoattractant factor. In mammal and avian, it has been characterized as an essential regulator of various cellular processes including cell recruitment and activation against pathogen invasion. So far, neither of the full‐length of IL‐16 homologue nor the response mechanism against pathogen was reported in crab species. In the present study, the pro‐IL‐16 homologue was firstly cloned and characterized from mud crab Scylla paramamosain. The full‐length Sp‐pro‐IL‐16 consisted of 4107 bp with an opening reading frame encoding 1369 amino acids. Multiple alignment analysis showed the putative amino acid sequence of Sp‐pro‐IL‐16 had about 73.86% identity with Litopenaeus vannamei pro‐IL‐16. Additionally, two conserved PDZ domains and protein binding sites were found in Sp‐pro‐IL‐16 and showed high similarities about 94.19% and 51.14% with their Litopenaeus vannamei and Mus musculus counterparts. RT‐PCR analysis indicated that Sp‐pro‐IL‐16 transcripts were constitutively expressed in all tissues examined with an extreme high level in hepatopancreas. Moreover, Sp‐pro‐IL‐16 transcripts in hepatopancreas were significantly up‐regulated 15‐fold at 72 h after Vibrio alginolyticus challenge and 3.5‐fold at 12 h after virus‐analog Poly (I:C) challenge. The Western blot analysis revealed that Sp‐pro‐IL‐16 can be cleaved to its bioactive form, an approximately 35 kDa mature IL‐16, and the protein levels of both pro‐IL‐16 and mature IL‐16 increased after Vibrio alginolyticus challenge. It is the first experimental identification of pro‐inflammatory cytokine IL‐16 in arthropods. This study could shed new light on further understanding of the response mechanism of pro‐inflammatory cytokine IL‐16 in Scylla paramamosain against pathogens. Meanwhile, it brought new insight into the origin and evolution of IL‐16 in crab species. HighlightsFirst identified and characterized proinflammatory cytokine IL‐16 in crab species.Confirmation of early divergence during evolutionary process by phylogenetic analysis.Expression pattern analyzed and found to be correlated with the pathogen infection.Cleavage confirmed from pro‐IL‐16 to mature IL‐16 in protein level.Provide new insight into the origin and evolution of proinflammatory cytokine IL‐16 in crabs and even in invertebrates.

Identification and characterization of six peroxiredoxin transcripts from mud crab Scylla paramamosain: The first evidence of peroxiredoxin gene family in crustacean and their expression profiles under biotic and abiotic stresses

HighlightsPrx gene family was firstly identified from the mud crab.The expression profiles of SpPrxs under biotic and abiotic stresses were investigated.The comparative analysis of Prx family in invertebrates and vertebrates was conducted for the first time.The peroxidase activity of SpPrxs was firstly determined. Abstracts The peroxiredoxins (Prxs) define a novel and evolutionarily conserved superfamily of peroxidases able to protect cells from oxidative damage by catalyzing the reduction of a wide range of cellular peroxides. Prxs have been identified in prokaryotes as well as in eukaryotes, however, the composition and number of Prxs family members vary in different species. In this study, six Prxs were firstly identified from the mud crab Scylla paramamosain by RT‐PCR and RACE methods. Six SpPrxs can be subdivided into three classes: (a) three typical 2‐Cys enzymes denominated as Prx1/2, 3, 4, (b) two atypical 2‐Cys enzymes known as Prx5‐1 and Prx5‐2, and (c) a 1‐Cys isoform named Prx6. The evolutionarily conserved signatures of peroxiredoxin catalytic center were identified in all six SpPrxs. Phylogenetic analysis revealed that SpPrx3, SpPrx4, SpPrx5 s and SpPrx6 were clearly classified into Prx3‐6 subclasses, respectively. Although SpPrx1/2 could not be grouped into any known Prx subclasses, SpPrx1/2 clustered together with other arthropods Prx1 or unclassified Prx and could be classified into the typical 2‐Cys class. The comparative and evolutionary analysis of the Prx gene family in invertebrates and vertebrates were also conducted for the first time. Tissue‐specific expression analysis revealed that these six SpPrxs were expressed in different transcription patterns while the highest expression levels were almost all in the hepatopancreas. Quantitative RT‐PCR analysis exhibited that the gene expression profiles of six SpPrxs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and hypoosmotic salinity, suggesting that the SpPrxs might play different roles in response to various stresses. The recombinant proteins including the SpPrx1/2, SpPrx4, SpPrx5‐1 and SpPrx6 were purified and the peroxidase activity assays indicated that all these proteins can reduce H2O2 in a typical DTT‐dependent manner. To our knowledge, this is the first study about the comprehensive characterization of Prx gene family in Scylla paramamosain and even in crustaceans. These results would broaden the current knowledge of the whole Prx family as well as be helpful to understand and clarify the evolutionary pattern of Prx family in invertebrate and vertebrate taxa.

Identification and functional analysis of inhibitor of NF-κB kinase (IKK) from Scylla paramamosain: The first evidence of three IKKs in crab species and their expression profiles under biotic and abiotic stresses

&NA; IKK (inhibitor of NF‐&kgr;B kinase) is the critical regulator for NF‐&kgr;B (nuclear factor‐&kgr;B) pathway against pathogenic invasion in vertebrates or invertebrates. However, the IKK from crab species has not yet been identified. In the present study, three full‐length cDNA sequences of IKKs from mud crab Scylla paramamosain, designated as SpIKK&bgr;, SpIKK&egr;1 and SpIKK&egr;2, were firstly cloned through RT‐PCR and RACE methods. This is also the first report about the identification of two IKK&egr; genes in mud crab and even in crustaceans. The SpIKK&bgr; cDNA was 2824 bp in length with an open reading frame (ORF) of 2382 bp, which encoded a putative protein of 793 amino acids (aa). The ORF of two SpIKK&egr; isoforms, SpIKK&egr;1 and SpIKK&egr;2, were 2400 bp and 2331 bp in length encoding 799 aa and 776 aa, respectively. The crucial conserved residues and functional domains, including the kinase domains (KDs) and leucine zipper (LZ), were identified in all SpIKKs. Phylogenetic analysis suggested that SpIKK&bgr; was classified into the IKKs class while SpIKK&egr;s could be grouped into the IKK‐related kinases class. The qRT‐PCR analysis showed that three SpIKKs were constitutively expressed in all tested tissues and the highest expression levels of SpIKK&bgr; and SpIKK&egr;s were all in hemocyte. The gene expression profiles of SpIKKs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and air exposure, suggesting that the SpIKKs might play different roles in response to pathogens infections, heavy metal and air exposure. Moreover, IKKs from mud crab can significantly activate mammalian NF‐&kgr;B pathway, suggesting the function of IKKs might be evolutionally well‐conserved. Results of the RNAi experiments suggested that SpIKKs might regulate the immune signaling pathway when hemocytes were challenged with V. parahemolyticus or virus‐analog poly (I:C). All of these results indicated that the obtained SpIKKs might be involved in stress responses against biotic or abiotic stresses, and it also highlighted their functional conservation in the innate immune system from crustaceans to mammals.

Effects of both cold and heat stresses on the liver of giant spiny frog Quasipaa spinosa: stress response and histological changes

ABSTRACT Ambient temperature-associated stress can affect normal physiological functions in ectotherms. To assess the effects of cold or heat stress on amphibians, giant spiny frogs (Quasipaa spinosa) were acclimated at 22°C followed by exposure to 5°C or 30°C for 0, 3, 6, 12, 24 and 48 h, respectively. Histological alterations, apoptotic index, generation of mitochondrial reactive oxygen species (ROS), antioxidant activity indices and stress-response gene expression in frog livers were subsequently determined. Results showed that many fat droplets appeared after 12 h of heat stress and the percentage of melanomacrophage centres significantly changed after 48 h at both stress conditions. Furthermore, the mitochondrial ROS levels were elevated in a time-dependent manner up to 6 h and 12 h in the cold and heat stress groups, respectively. The activities of superoxide dismutase, glutathione peroxidase and catalase were successively increased with increasing periods of cold or heat exposure, and their gene expression levels showed similar changes in both stress conditions. Most tested heat shock protein (HSP) genes were sensitive to temperature exposure, and the expression profiles of most apoptosis-related genes was significantly upregulated at 3 and 48 h under cold and heat stress, respectively. Apoptotic index at 48 h under cold stress was significantly higher than that under heat stress. Notably, lipid droplets, HSP30, HSP70 and HSP110 might be suitable bioindicators of heat stress. The results of these alterations at physiological, biochemical and molecular levels might contribute to a better understanding of the stress response of Q. spinosa, and perhaps amphibians more generally, under thermal stress. Summary: Physiological, biochemical and molecular responses to cold and heat stresses on the liver of the giant spiny frog Quasipaa spinosa.

Effects of heat stress on the liver of the Chinese giant salamander Andrias davidianus: Histopathological changes and expression characterization of Nrf2-mediated antioxidant pathway genes

Nuclear factor E2-related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant-relevant genes, and the Nrf2-mediated antioxidant pathway has been regarded as a critical switch in the initiation of cellular defence systems against oxidative damages. In this study, Nrf2 was first identified and characterized in the Chinese giant salamander (Andrias davidianus). A. davidianus was exposed to a high ambient temperature of 30 °C for various periods of time (0, 3, 6, 12, 24, 48 and 72 h). We investigated the effects of heat stress on alterations of the hepatic malondialdehyde (MDA) concentration, the activities of lactic acid dehydrogenase (LDH), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), the histology of the liver, and the mRNA expression patterns of 11 genes involved in the Nrf2-mediated antioxidant pathway in A. davidianus. The results showed that both the hepatic LDH activity and MDA content significantly increased after heat exposure, indicating that heat stress could induce cell injury and oxidative damage. Histological analysis of the liver showed that heat stress caused hepatocyte abnormalities, fat accumulation and ultrastructural alterations of the hepatocytes, endoplasmic reticulum and nuclei. The expression patterns of genes involved in the Nrf2-mediated antioxidant pathway in the liver were distinct when A. davidianus was exposed to heat stress. To the best of our knowledge, this study is the first on the characterization of Nrf2 in A. davidianus and even in amphibians. The results indicated that heat stress could induce oxidative damage, and the Nrf2 antioxidant pathway might play a critical role in the resistance against heat stress in A. davidianus. These findings will deepen and enrich the current knowledge on the evolutionary conserved antioxidant roles and mechanisms of Nrf2 in A. davidianus, or even in amphibians, in the antioxidant defence against heat stress.

Identification and functional analysis of immune deficiency (IMD) from Scylla paramamosain: The first evidence of IMD signaling pathway involved in immune defense against bacterial infection in crab species

&NA; Immune deficiency (IMD) pathway, one of the most essential pattern recognition receptor signaling pathways, plays vital roles in innate immune responses to eliminate pathogen infection in invertebrates. In the present study, an immune deficiency (IMD) gene and two NF‐&kgr;B family members, Relish and Dorsal, were identified and characterized in mud crab Scylla paramamosain for the first time. The deduced SpIMD, SpRelish and SpDorsal protein contained conserved death domain and classical NF‐&kgr;B domains, respectively. Phylogenetic analysis suggested that SpIMD was classified into the invertebrate IMD branch, and SpRelish could be classified into the type I NF‐&kgr;B class while SpDorsal could be grouped into the type II NF‐&kgr;B class. Tissue distribution results showed these three genes were ubiquitously expressed in all tested tissues. The expression patterns of IMD signaling pathway and NF‐&kgr;B genes, including SpIMD, SpIKK&bgr;, SpIKK&egr;, SpRelish and SpDorsal, were distinct when crabs were stimulated with Vibro alginolyticus, indicating that they might be involved in responding to bacterial infection. When SpIMD was silenced by in vivo RNA interference assay, the expression levels of IMD pathway and antimicrobial peptides (AMPs) genes, including SpIKK&bgr;, SpRelish, SpALF1‐6 and SpCrustin, were significantly down‐regulated (p < 0.05). Correspondingly, the bacteria clearance ability of hemolymph was extremely impaired in IMD silenced crabs. Overall, the IMD played vital roles in innate immune response by regulating the expressions of its down‐stream signaling genes and AMPs in S. paramamosain. These findings might pave the way for a better understanding of innate immune system and establish a fundamental network for the IMD signaling pathway in crustaceans. HighlightsThe SpIMD, SpRelish and SpDorsal genes were firstly identified and characterized.IMD signaling pathway existed in crab and responded to bacterial infection.IMD regulated expressions of antimicrobial peptides genes in S. paramamosain.