Yu-Yin Chen

Classification and phagocytosis of circulating haemocytes in Chinese mitten crab (Eriocheir sinensis) and the effect of extrinsic stimulation on circulating haemocytes in vivo.

Eriocheir sinensis (Henri Milne Edwards 1854) is one of the most important aquaculture species in China. In this investigation, we characterised the different types of haemocytes of E. sinensis using light and electron microscopy combined with cytochemical analysis and determined the in vivo phagocytic ability of different haemocyte types by injecting polystyrene beads. The haemocytes of E. sinensis were divided into three types: hyalinocytes, semigranulocytes and granulocytes. The hyalinocytes had no or few cytoplasmic granules; the semigranulocytes contained abundant small granules and a few large refractile cytoplasmic granules; and the granulocytes contained numerous large refractile cytoplasmic granules. The hyalinocytes were demonstrated to be the most abundant circulating haemocytes and the most avid phagocytic haemocytes, accounting for approximately 88.7% of the total phagocytes. The haemocyte-containing granules displayed limited phagocytic ability, with approximately 5.0% of granulocytes and 6.3% of semigranulocytes displaying positive phagocytic ability against the invading polystyrene beads in vivo. After injection with Aeromonas hydrophila, Bacillus subtilis and different concentrations of lipopolysaccharide for 0.25, 0.5, 1, 2, 4, 6 and 8 h, all three types of haemocytes experienced dramatic decline and then rapid recovery to their initial levels. A high concentration of lipopolysaccharide and A. hydrophila were extremely toxic to the crabs, as they induced a more serious loss of haemocytes compared with a low concentration of lipopolysaccharide and B. subtilis. Overall, the results obtained in this study indicate that a small proportion of the haemocytes of E. sinensis contributed to the phagocytic process, and the migration of haemocytes and haemocyte lysis were most likely a prominent pathway for pathogen elimination.

Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis.

Pelodiscus sinensis is the most common turtle species that has been raised in East and Southeast Asia. However, there are still limited studies about the immune defense mechanisms in its small intestine until now. In the present research, histological analysis and transcriptome analysis was performed on the small intestine of P. sinensis after intragastric challenge with LPS to explore its mechanisms of immune responses to pathogens. The result showed the number of intraepithelial lymphocytes (IELs) and goblet cells (GCs) in its intestine increased significantly at 48xa0h post-challenge with LPS by intragastrical route, indicating clearly the intestinal immune response was induced. Compared with the control, a total of 748 differentially expressed genes (DEGs) were identified, including 361 up-regulated genes and 387 down-regulated genes. Based on the Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG), 48 immune-related DEGs were identified, which were classified into 82 GO terms and 14 pathways. Finally, 18 DEGs, which were randomly selected, were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide valuable information for further analysis of the immune defense mechanisms against pathogens in the small intestine of P. sinensis.

Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis

In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 µg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.中文概要目 的评估细菌脂多糖(LPS)在体外环境下对中华绒螯蟹血细胞凋亡的影响,并探讨其诱导血细胞凋亡的机制。创新点首次在体外条件下实验证明了LPS 可诱导中华绒螯蟹血细胞的凋亡,并确定了活性氧自由基在细胞凋亡过程中的变化和作用。方 法在体外26 °C 条件下,培养中华绒螯蟹血细胞。用浓度1 µg/ml 的LPS 处理血细胞0、4、8 和16 h后,利用4’,6-二脒基-2-苯基吲哚(DAPI)染色、荧光显微镜和透射电子显微镜观察血细胞的形态变化,用DNA Ladder 试剂盒检测脱氧核糖核酸(DNA)的损伤程度。分别用浓度1 和10 µg/ml的LPS 处理血细胞0、1 和2 h,经膜联蛋白 V-异硫氰酸荧光素(V-FITC)/碘化丙啶(PI)染色后,用流式细胞仪检测血细胞凋亡数量。收集血细胞制成细胞悬浮液,加入LPS 至终浓度为10 µg/ml,同时加入2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)探针至终浓度为1 µmol/L;对照组加入等量生理盐水和DCFH-DA 探针。在26 °C孵育不同时间后,用流式细胞仪和激光共聚焦显微镜检测血细胞活性氧的产生情况。结 论LPS 可诱导中华绒螯蟹血细胞出现典型的凋亡特征,包括染色质浓缩和边聚、核膜破裂、线粒体肿胀、凋亡小体形成等(图1 和2),形成明显的DNA 梯形条带(图3),且细胞凋亡数量与LPS 浓度呈正相关,大多数凋亡细胞处于凋亡晚期(图4)。活性氧检测结果显示:经LPS 刺激后,细胞内活性氧自由基信号显著增强,随后又迅速减弱(图5 和6)。综上所述,体外LPS 可诱导中华绒螯蟹血细胞产生明显的凋亡反应,且凋亡过程伴随着显著的活性氧爆发。

Immune response of peroxinectin of Chinese mitten crab Eriocheir sinensis to exterior stimulation

Peroxinectin possesses the features of both peroxidase activity and adhesive property and plays important roles in innate immune system of crustaceans. In this study, the sequence of peroxinectin of Eriocheir sinensis (EsPX) was analyzed and its expression in response to exterior stimulation was detected in both in vivo and in vitro examination. We showed that the full-length cDNA sequence was composed of 2701u2009bp and owned a molecular mass of 85.2u2009kDa and a theoretical pI (isoelectric point) of 6.91. Real-time PCR revealed that the EsPX was mainly distributed in the muscle, hemocytes and stomach. Furthermore, the EsPX was verified to be located in hyalinocytes, semigranulocytes and granulocytes, and was distributed throughout the cytoplasm and nucleus, especial in cytoplasm. After injected with beads, lipopolysaccharide (LPS) and Aeromonas hydrophila, the EsPX mRNA expression was significantly up-regulated and peaked up at 4, 2 and 16u2009h respectively (Pu2009<0.05). In the in vitro experiment, the stimulation of LPS and beads also induced a prominent boost of EsPX protein in primary cultured hemocytes. The expression of EsPX was peaked up at 4 and 8u2009h for LPS and beads challenged groups respectively, followed by remarkable release of the incremental EsPX into the extracellular matrix. These findings suggested that the expression of EsPX was susceptible to exterior stimulation, and that the highly expressional EsPX would be released into extracellular matrix by the exterior stimulus.

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

ABSTRACT 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. HighlightsHemocytes from S. paramamosain were characterized by cytochemical staining, electron microscopy and flow cytometry.The cell count and the proportion of hemocytes varied differently after different pathogen infections.Phagocytosis of bacteria was primarily executed by granulocytes and semigranulocytes.L‐15 medium supplemented with 10% FBS was the optimal condition for semigranulocytes primary culture.Adenovirus could be employed to transfer foreign genes into crustacean hemocytes.

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 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.