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

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.

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses.

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5-10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs.

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.

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.

Molecular characterization of a cDNA encoding Na+/K+/2Cl- cotransporter in the gill of mud crab (Scylla paramamosain) during the molt cycle: Implication of its function in osmoregulation.

Although iono-regulatory processes are critical for survival of crustaceans during the molt cycle, the mechanisms involved are still not clear. The Na+/K+/2Cl- cotransporter (NKCC), a SLC12A family protein that transports Na+, K+ and 2Cl- into cells, is essential for cell ionic and osmotic regulation. To better understand the role of NKCC in the molt osmoregulation, we cloned and characterized a NKCC gene from the mud crab, Scylla paramamosain (designated as SpNKCC). The predicted SpNKCC protein is well conserved, and phylogenetic analysis revealed that this protein was clustered with crustacean NKCC. Expression of SpNKCC was detected in all the tissues examined but was highest in the posterior gills. Transmission electron microscopy revealed that posterior gills had a thick type of epithelium for ion regulation while the anterior gills possessed a thin phenotype related to gas exchange. During the molting cycle, hemolymph osmolality and ion concentrations (Na+ and Cl-) increased significantly over the postmolt period, remained stable in the intermolt and premolt stages and then decreased at ecdysis. Meanwhile, the expression of SpNKCC mRNA was significantly elevated (26.7 to 338.8-fold) at the ion re-establishing stages (postmolt) as compared with baseline molt level. This pattern was consistent with the coordinated regulation of Na+/K+-ATPase α-subunit (NKA α), carbonic anhydrase cytoplasmic (CAc) isoform and Na+/H+ exchanger (NHE) genes in the posterior gills. These data suggest that SpNKCC may be important in mediating branchial ion uptake during the molt cycle, especially at the postmolt stages.

[Effects of Different Planting Years of Dendrocalamus brandisii on Soil Fungal Community].

To explore the effect of soil fungal community under different planting years in Dendrocalamus brandisii, the soil samples from D. brandisii with different planting years (5, 10, 20, and 40 a) were taken as the research object. The soil fungal community structure, diversity, and its functional groups of different planting years were analyzed using high-throughput sequencing technology and the FUNGuild fungal function prediction tool, and the main soil environmental factors influencing the variations in soil fungal community were examined. The results showed that the dominant fungal communities at the phylum level were Ascomycota, Basidiomycota, Mortierellomycota, and Mucoromycota. The relative abundance of Mortierellomycota decreased and then increased with the increase in planting years, and there was a significant difference among different planting years (P<0.05). The dominant fungal communities at the class level were Sordariomycetes, Agaricomycetes, Eurotiomycetes, and Mortierellomycetes. The relative abundance of Sordariomycetes and Dothideomycetes decreased and then increased with the increase in planting years, and there were significant differences among different planting years (P<0.01). The Richness index and Shannon index of soil fungi increased and then decreased with the increase in planting years, and the Richness index and Shannon index in 10 a were significantly higher than those of other planting years. Non-metric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) showed that there were significant differences in soil fungal community structure with different planting years. The functional prediction with FUNGuild showed that the main functional trophic types of soil fungi in D. brandisii were pathotroph, symbiotroph, and saprotroph, and the most dominant functional group was endophyte-litter saprotroph-soil saprotroph-undefined saprotroph. The relative abundance of endophytes gradually increased with the increase in planting years. Correlation analysis showed that pH, total potassium (TK), and nitrate nitrogen (NO-3-N) were the main soil environmental factors affecting the change in fungal community. In summary, the planting year of D. brandisii has changed soil environmental factors and has thus changed the structure, diversity, and functional groups of soil fungal communities.

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.

IKK (inhibitor of NF-κB kinase) is the critical regulator for NF-κB (nuclear factor-κB) 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 SpIKKß, SpIKKε1 and SpIKKε2, were firstly cloned through RT-PCR and RACE methods. This is also the first report about the identification of two IKKε genes in mud crab and even in crustaceans. The SpIKKß 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 SpIKKε isoforms, SpIKKε1 and SpIKKε2, 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 SpIKKß was classified into the IKKs class while SpIKKεs 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 SpIKKß and SpIKKεs 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-κB 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.

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 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.

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, SpPrx5s 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.