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• Cloning, characterization, and expression analysis of a thioredoxin from orange-spotted grouper (Epinephelus coioides).

  Authors: Jingguang Wei, Minglan Guo, Huasong Ji, Yang Yan, Zhengliang Ouyang, Xiaohong Huang, Youhua Hang, Qiwei Qin

  Developmental and comparative immunology.

  Thioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spottedThioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spotted grouper, Epinephelus coioides (designated as Ec-TRX). The full-length cDNA of Ec-TRX was comprised of 767 bp with a 327 bp open reading frame that encodes a putative protein of 108 amino acids. Quantitative real-time PCR analysis revealed that the Ec-TRX mRNA was distributed abundantly in grouper, E. coioides skin and liver, and the expression in liver was up-regulated after viral challenge with Singapore grouper iridovirus (SGIV). Recombinant Ec-TRX (rEc-TRX) was expressed in E. coli BL21 (DE3) and purified for mouse anti-Ec-TRX serum preparation. The rEc-TRX fusion protein was demonstrated to possess the expected redox activity in enzymatic analysis, and scavenge free radicals and protect supercoiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. Subcellular localization revealed that Ec-TRX was distributed in both cytoplasm and nucleus. Overexpression of Ec-TRX in grouper spleen (GS) cells could promote the growth of GS cells and inhibit the replication of SGIV. These results suggest that Ec-TRX could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to viral challenge.

• Specific amino acids affecting Drosophila melanogaster prophenoloxidase activity in vitro.

  Authors: Yang Chen, Fei Liu, Bing Yang, Anrui Lu, Shiwei Wang, Jingfang Wang, Qing-Zhi Ling, Xuan Li, Brenda T Beerntsen, Erjun Ling

  Developmental and comparative immunology.

  Insect prophenoloxidase (PPO) is a key enzyme that induces melanization around invading pathogens and at wounds to prevent further infection. Drosophila melanogaster has three PPO genes which haveInsect prophenoloxidase (PPO) is a key enzyme that induces melanization around invading pathogens and at wounds to prevent further infection. Drosophila melanogaster has three PPO genes which have different biochemical properties following over-expression in S2 cells. As shown by automatic melanization of S2 cells, recombinant PPO3 (rPPO3) became activated upon Cu(2+) addition (Cu(2+)-aided cells melanization without ethanol activation and substrate addition: +Cu(2+); -DOPA, -Ethanol). The exact reasons for this phenomenon are still unknown. In this study, using site-directed mutagenesis and over-expression methods, we found that the place holder, two independent amino acids (equal to Manduca sexta amino acid residues: F218 and S393 in MsPPO1, F224 and E395 in MsPPO2) in the active site pocket and a missing fragment (similar to (565)RPGDPGT(571) in MsPPO1 and (571)QGSDPRR(577) in MsPPO2) at the C-terminus of PPO3, affect rPPO3-S2 cells Cu(2+)-aided auto-melanization. Some mutations nearly rescued rPPO3 Cu(2+)-aided auto-activation, which suggests that the auto-activation of wild type rPPO3 was not due to cleavage by serine proteases. We also found that the corresponding amino acids in the active site pocket have similar effect on PPO1 as on PPO3. PPO1 staining activity (Cu(2+) added or not during PPO transfection; cells melanized after ethanol activation and substrate addition: +/-Cu(2+); +DOPA, +Ethanol) has a positive relationship with the active site pocket size as does rPPO3. The fragment of rPPO1corresponding to the one missing from the C-terminus of PPO3 has no influence on rPPO1 staining activity after it is deleted. However, the staining activities of rPPO2 mutants decreased after deletion of those same amino acid sequences. When the corresponding fragments from PPO1 or PPO2 were inserted into PPO3, the mutant rPPO3 had no influence on staining activity, but had a significantly lowered Cu(2+)-aided auto-activation. Thus, we found that some amino acids are important for rPPO3 Cu(2+)-aided auto-activation as well as other PPO staining activity in vitro.

• Litopenaeus vannamei inhibitor of apoptosis protein 1 (LvIAP1) is essential for shrimp survival.

  Authors: Jiann-Horng Leu, Yen-Chen Chen, Li-Li Chen, Kuan-Yu Chen, Huai-Ting Huang, Jan-Ming Ho, Chu-Fang Lo

  Developmental and comparative immunology.

  The members of the inhibitor of apoptosis protein (IAP) family are involved in the regulation of diverse cellular processes, including apoptosis, signal transduction and mitosis. Here, we report theThe members of the inhibitor of apoptosis protein (IAP) family are involved in the regulation of diverse cellular processes, including apoptosis, signal transduction and mitosis. Here, we report the cloning and characterization of three IAP genes from Pacific white shrimp Litopenaeus vannamei: LvIAP1, LvIAP2 and LvSurvivin. LvIAP1, the orthologue of Penaeus monodon IAP (PmIAP), consists of three BIR domains and one RING domain; LvIAP2 consists of two BIR domains and LvSurvivin has only one BIR domain. Expression profiling by absolute quantitative real-time RT-PCR revealed that of the three IAP genes, LvIAP1 had the highest expression levels in almost all examined tissues and LvSurvivin had the lowest expression levels. Furthermore, among the examined tissues, the lymphoid organs most strongly expressed all three genes. When LvIAP1 expression was silenced by injection of its corresponding dsRNA, the shrimp died within 48 h after injection, whereas injection of the other two dsRNAs did not cause shrimp death. In LvIAP1-silenced shrimp, the number of circulating haemocytes decreased dramatically because of extensive apoptosis. This suggested that LvIAP1 is central to the regulation of shrimp haemocyte apoptosis.

• A single immunoglobulin-domain IgSF protein from Sciaenops ocellatus regulates pathogen-induced immune response in a negative manner.

  Authors: Shun-Feng Cheng, Yong-Hua Hu, Bo-Guang Sun, Min Zhang, Heng Chi, Li Sun

  Developmental and comparative immunology.

  The immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that include various immunoregulatory receptors such as novel immune type receptors (NITRs), which are a family ofThe immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that include various immunoregulatory receptors such as novel immune type receptors (NITRs), which are a family of diversified proteins found exclusively in bony fish. In this study, we identified and analyzed an IgSF protein, SoIgSF1, from red drum (Sciaenops ocellatus). SoIgSF1 is composed of 225 amino acid residues and moderately related to teleost NITRs. In silico analysis indicated that SoIgSF1 is a type I transmembrane glycoprotein and contains an N-terminal signal peptide sequence, a single extracellular immunoglobulin V domain, a transmembrane region, and a cytoplasmic region. However, unlike most NITRs, the cytoplasmic region of SoIgSF1 exhibits no consensus inhibitory or stimulatory signaling sequences but has two tyrosine-containing motifs that conform to the right-half sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM). Quantitative real time RT-PCR analysis showed that SoIgSF1 expression occurred mainly in immune organs and was drastically induced by viral and bacterial infection. Immunofluorescence microscopy indicated that viral infection of head kidney (HK) leukocytes induced surface expression of SoIgSF1, which was able to interact with antibodies against recombinant SoIgSF1. Antibody cross-linking of SoIgSF1 on HK leukocytes inhibited the expression of immune relevant genes and promoted viral and bacterial infection. Taken together, these results indicate that SoIgSF1, though lacking canonical intracellular signaling motifs, is involved in regulation of host immune response during pathogen infection possibly by functioning as a negative signaling receptor through a novel mechanism.

• Spawning stress triggers WSSV replication in brooders via the activation of shrimp STAT.

  Authors: Shin-Jen Lin, Hui-Lan Hsia, Wang-Jing Liu, Jiun-Yan Huang, Kuan-Fu Liu, Wei-Yu Chen, Ying-Chun Yeh, Yun-Tzu Huang, Chu-Fang Lo, Kuang-Hsiung Kou, Han-Ching Wang

  Developmental and comparative immunology.

  In the early days of shrimp aquaculture, wild-captured brooders usually spawned repeatedly once every 2 to 4 days. However, since the first outbreaks of white spot disease (WSD) nearly 20 years ago,In the early days of shrimp aquaculture, wild-captured brooders usually spawned repeatedly once every 2 to 4 days. However, since the first outbreaks of white spot disease (WSD) nearly 20 years ago, captured female brooders often died soon after a single spawning. Although these deaths were clearly attributable to WSD, it has always been unclear how spawning stress could lead to an outbreak of the disease. Using real-time qPCR, we show here that while replication of the white spot syndrome virus (WSSV; the causative agent of WSD) is triggered by spawning, there was no such increase in the levels of another shrimp DNA virus, IHHNV (infectious hypodermal and hematopoietic necrosis virus). We also show that levels of activated STAT are increased in brooders during and after spawning, which is important because shrimp STAT is known to transactivate the expression of the WSSV immediate early gene ie1. Lastly, we used dsRNA silencing experiment to show that both WSSV ie1 gene expression and WSSV genome copy number were reduced significantly after shrimp STAT was knocked-down. This is the first report to demonstrate in vivo that shrimp STAT is important for WSSV replication and that spawning stress increases activated STAT, which in turn triggers WSSV replication in WSSV-infected brooders.

• The binding spectra of carp C3 isotypes against natural targets independent of the binding specificity of their thioester.

  Authors: Satoko Ichiki, Yoko Kato-Unoki, Tomonori Somamoto, Miki Nakao

  Developmental and comparative immunology.

  The central component of complement, C3, plays a versatile role in innate immune defense of vertebrates and some invertebrates. A notable molecular characteristic of this component is an intra-chainThe central component of complement, C3, plays a versatile role in innate immune defense of vertebrates and some invertebrates. A notable molecular characteristic of this component is an intra-chain thioester site that enables C3 to bind covalently to its target. It has been reported that the binding preference of the thioester to hydroxyl or amino groups is primarily defined by presence or absence of the catalytic histidine residue at position 1126 in human C3. In teleosts, a unique C3 (non-His type) has been found, in addition to the common His type C3. These distinct C3 isoforms may provide diversity in the target-binding attributable to the different binding specificities of their thioesters. In the present study, we examine the hypothesized correlation of the catalytic histidine with the binding spectra of two major C3 isotypes of carp towards various model and natural targets. The results reveal that non-His type C3, rather than His type C3, has a wider range of binding spectrum, despite the binding specificity of its thioester being limited to amino groups. It is therefore hypothesized that the binding spectra of C3 isotypes are not defined by the binding specificity of the thioester but is more affected by differences in microbe-associated molecular patterns that activate complement. Overall, the present data imply that non-His type C3 plays a significant role against bacterial infections in the fish defense system.

• The role of Nfil3 in zebrafish hematopoiesis.

  Authors: Fränze Progatzky, Harriet Taylor, Laurence Bugeon, Sorcha Cassidy, Andreas Radbruch, Margaret J Dallman, Jonathan R Lamb

  Developmental and comparative immunology.

  Nfil3, a transcription factor that has an array of functions in immune cells, has been described as key regulator of CD8α(+) dendritic cell and natural killer cell development in mice. In this reportNfil3, a transcription factor that has an array of functions in immune cells, has been described as key regulator of CD8α(+) dendritic cell and natural killer cell development in mice. In this report we show that Nfil3 is enriched in the myeloid compartment of adult zebrafish including eosinophils. Knockdown of Nfil3 in pu.1:GFP embryos resulted in a reduced number of myeloid cells as early as 24 hours post-fertilization, while erythropoiesis was unaffected. Using mpx and fms-fluorescent transgenic fish we found that all myeloid cell lineages, and in particular macrophages, had reduced numbers at 4 days post-fertilization. This was reflected by less myeloid cells accumulating at a wound site. Pu.1, l-plastin, csf1r and mpx had reduced expression in Nfil3 morphants while runx1, gata1 and rag1 were unaffected. Collectively, these results describe a conserved expression pattern of Nfil3 in evolutionarily divergent species and indicate that Nfil3 is central to myeloid lineage commitment.

• Diversity and multiple functions of lectins in shrimp immunity.

  Authors: Xian-Wei Wang, Jin-Xing Wang

  Developmental and comparative immunology.

  Lectins play important roles in many biological processes, including protein trafficking, cell signaling, pathogen recognition, as effector molecules, and so on, because of their capacity to bindLectins play important roles in many biological processes, including protein trafficking, cell signaling, pathogen recognition, as effector molecules, and so on, because of their capacity to bind carbohydrates. Presently, seven groups of lectins have been identified in shrimp: C-type, L-type, P-type, M-type, fibrinogen-like domain lectins, galectins, and calnexin/calreticulin. These lectins have different structures, diverse expression patterns, and multiple functions in the shrimp immune response. This review summarizes the research progress and analyzes the diversity of shrimp lectins, focusing mainly on the C-type lectin family. Shrimp C-type lectins show considerable diversity in their domain architectures, sugar substrates, tissue distributions, expression patterns responding to pathogen challenge and different functions in shrimp immunity.

• Identification and Characterization of Inositol-requiring enzyme-1 and X-box Binding Protein 1, Two Proteins Involved in the Unfolded Protein Response of Litopenaeus vannamei.

  Authors: Yi-Hong Chen, Li Zhao, Li-Ran Pang, Xiao-Yun Li, Shao-Ping Weng, Jian-Guo He

  Developmental and comparative immunology.

  The inositol-requiring enzyme-1 (IRE1)-X-box binding protein 1 (IRE1-XBP1) pathway is the key branch of the unfolded protein response (UPR). To investigate the role of the IRE1-XBP1 pathway inThe inositol-requiring enzyme-1 (IRE1)-X-box binding protein 1 (IRE1-XBP1) pathway is the key branch of the unfolded protein response (UPR). To investigate the role of the IRE1-XBP1 pathway in reducing environmental stress and increasing anti-viral immunity in Litopenaeus vannamei, homologues of IRE1 (designated as LvIRE1) and XBP1 (designated as LvXBP1) were identified and characterized. The full-length cDNA of LvIRE1 is 4908bp long, with an open reading frame (ORF) that encodies a putative 1174 amino acid protein. The full-length cDNA of LvXBP1 is 1746bp long. It contains two ORFs that encode putative 278 amino acid and 157 amino acid proteins, respectively. LvXBP1 mRNA has the predicted IRE1 splicing motifs CNG'CNGN located within the loop regions of two short hairpins. Sequencing of the splicing fragment induced by endoplasmic reticulum (ER)-stress showed a 3bp or 4bp frame shift from the predicted sites. The spliced form LvXBP1 (LvXBP1s) contained an ORF encodes a putative 463 amino acid protein. The reporter gene assays indicated that LvXBP1s activates the promoter of L. vannamei immunoglobulin heavy chain binding protein (LvBip), an important UPR effector. RT-PCR showed that LvXBP1 was spliced during the experiments. For heat shock treatment, the total LvXBP1expression was increased and peaked at about 36h, whereas the percentages of the two isoforms were relatively stable. For the WSSV challenge, LvXBP1 was upregulated during the experiment and the percentage of the spliced form continuously declined after 18h of infection. Knock-down of LvXBP1 by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Furthermore, the expression profiles of LvIRE1 and LvXBP1 in the gills, hemocytes, intestines, and hepatopancreas of the WSSV-challenged shrimp were detected using real-time RT-PCR. Taken together, these results confirm that the IRE1-XBP1 pathway is important for L. vannamei environmental stress resistance, suggest that L. vannamei IRE1-XBP1 may activated by WSSV and be annexed to serve the virus.

• Immune Gene Expression in Trout Cell Lines Infected with the Fish Pathogenic Oomycete Saprolegnia Parasitica.

  Authors: Irene de Bruijn, Rodrigo Belmonte, Vicky L Anderson, Marcia Saraiva, Tiehui Wang, Pieter van West, Christopher J Secombes

  Developmental and comparative immunology.

  The oomycete Saprolegnia parasitica causes significant losses in the aquaculture industry, mainly affecting salmon, trout and catfish. Since the ban of malachite green, effective control measures areThe oomycete Saprolegnia parasitica causes significant losses in the aquaculture industry, mainly affecting salmon, trout and catfish. Since the ban of malachite green, effective control measures are currently not available prompting a re-evaluation of the potential for immunological intervention. In this study, the immune response of salmonid cells is investigated at the transcript level, by analysis of a large set of immune response genes in four different rainbow trout cell lines (RTG-2, RTGill, RTL and RTS11) upon infection with S. parasitica. Proinflammatory cytokine transcripts were induced in all four cell lines, including IL-1β1, IL-8, IL-11, TNF-α2, as well as other components of the innate defences, including COX-2, the acute phase protein serum amyloid A and C-type lectin CD209a and CD209b. However, differences between the four cell lines were found. For example, the fold change of induction was much higher in the epithelial RTL and macrophage-like RTS11 cell lines compared to the fibroblast cell lines RTG-2 and RTGill. Several antimicrobial peptides (AMPs) were also up-regulated in response to Saprolegnia infection, including hepcidin and cathelicidin 1 (rtCATH1) and 2 (rtCATH2). An rtCATH2 peptide was synthesised and tested for activity and whilst it showed no killing activity for zoospores, it was able to delay sporulation of S. parasitica. These results demonstrate that particular immune genes are up-regulated in response to S. parasitica infection and that AMPs may play a crucial role in the first line of defence against oomycetes in fish.

• Expression profiles of six novel C-type lectins in response to bacterial and 20E injection in the cotton bollworm (Helicoverpa armigera).

  Authors: Jia-Lin Wang, Xu-Sheng Liu, Qi Zhang, Hao-Bin Zhao, Yu-Feng Wang

  Developmental and comparative immunology.

  C-type lectins can act as pattern recognition receptors (PRRs) and play an important role in innate immunity. Two C-type lectins (HaCTL1 and HaCTL2) have been previously identified in the cottonC-type lectins can act as pattern recognition receptors (PRRs) and play an important role in innate immunity. Two C-type lectins (HaCTL1 and HaCTL2) have been previously identified in the cotton bollworm (Helicoverpa armigera). Here we isolate six C-type lectins from H. armigera (HaCTL3, 4, 5, 6, 7 and 8). All six new HaCTLs encode a signal peptide (or partial signal peptide) and complete tandem carbohydrate-recognition domains (CRDs). HaCTL4, 5, 6, 7 and 8 mRNA increased in the fat body after injection with both killed Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, whereas HaCTL3 mRNA was upregulated following E. coli injection only. Recombinant HaCTL3 exhibited agglutinating activity against both Gram-negative and Gram-positive bacteria in a calcium-dependent manner. Agglutination inhibitory analysis indicated that rHaCTL3 recognizes maltose, trehalose, peptidoglycan and lipopolysaccharides. HaCTL3 and HaCTL8 mRNA showed upregulation while HaCTL4, 5, and 6 mRNA downregulation post 20-hydroxyecdysone (20E) injection. Our results indicate that the six novel C-type lectins of H. armigera may play important roles in defending against bacteria as PRRs and the hormone 20E can function in regulating immunity through lectins.

• Molecular cloning and functional study of calreticulin from a lepidopteran pest, Pieris rapae.

  Authors: Lei Wang, Qi Fang, Jiaying Zhu, Fei Wang, Zunnuraen Akhtar, Gongyin Ye

  Developmental and comparative immunology.

  Insects have an effective innate immune system to protect themselves from exogenous invaders. Calreticulin is a multifunctional protein mainly involved in directing proper conformation of proteins,Insects have an effective innate immune system to protect themselves from exogenous invaders. Calreticulin is a multifunctional protein mainly involved in directing proper conformation of proteins, controlling calcium level, and participating in immune responses. Previous suppression subtractive hybridization assay showed that the expression of Pieris rapae calreticulin (PrCRT) was suppressed after injection of Pteromalus puparum venom. In this study, we obtained a full length cDNA of PrCRT and expressed recombinant wild type and the N-domain deleted mutant PrCRT in bacteria. Real time quantitative PCR and western blot analyses showed that PrCRT mRNA and protein were expressed in hemocytes, Malpighian tubule, midgut, epidermis and fat body, with a higher level in hemocytes. PrCRT was probably located in endoplasmic reticulum distributing in the cytoplasm of hemocytes. Recombinant PrCRT was first able to attach and then enter the hemocytes by endocytosis. PrCRT mRNA in hemocytes was significantly induced after injection of yeast or beads, but did not change noticeably after injection of Escherichia coli or Micrococcus lysodeikticus. Recombinant PrCRT enhanced cellular encapsulation by P. rapae hemocytes in vitro, and the N-domain of PrCRT was required for encapsulation. RNAi of PrCRT by dsRNA injection impaired the ability of hemocytes to encapsulate beads. After parasitization by P. puparum, PrCRT mRNA and protein levels in P. rapae pupal hemocytes were significantly suppressed compared to non-parasitized control. Our results suggest that PrCRT is involved in cellular encapsulation and the pupal parasitoid P. puparum can decrease PrCRT expression to impair host cellular immune response.

• Transcriptional analysis of orange-spotted grouper reacting to experimental grouper iridovirus infection.

  Authors: Ming-Shan Wu, Chien-Wen Chen, Yu-Cheng Liu, Hseng-Hsiang Huang, Chih-Hung Lin, Chyng-Shyan Tzeng, Chi-Yao Chang

  Developmental and comparative immunology.

  Disease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture. Thirty-eight up- and 48 down-regulated known entities have been identified using aDisease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture. Thirty-eight up- and 48 down-regulated known entities have been identified using a GIV-infected grouper kidney cDNA microarray chip. Further quantitative validation was executed in the head-kidney and spleen for 24 candidate genes and 7 immune factors following GIV inoculation. Significant induction with various patterns could be seen in 30 tested genes in the spleen. However, only 23 genes had induction in the head-kidney and meanwhile 5 genes showed reduction. Transcriptional expression profiles of selected genes in response to lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (PIC) were also established to compare with the GIV-stimulated expression. The results indicated that the responses of most genes facing GIV invasion have more similarities to PIC treatment than LPS. Seven genes are thought to be interferon-related factors: RNA helicase DHX58, ISG15, viperin, HECT E3 ligase (HECT), CD9, urokinase plasminogen activator surface receptor (PLAUR) and Mx-1. Following immunization with inactivated GIV, significant induction could be seen in DHX58, viperin, IL-1β, IL-8, COX-2, HECT, PLAUR, IgM, Mx-1, very large inducible GTPase-1 (VLIG1) and TNF-α in the head-kidney or spleen, and the latter 6 genes also had a gradual increasing pattern by a boosting immunization. These factors might play important roles in adaptive antiviral protection. Thus, we have characterized the temporal response patterns of virus responsive genes and have also identified several potential immune markers to further investigate host antiviral defense mechanisms.

• A review of complementary mechanisms which protect the developing marsupial pouch young.

  Authors: M J Edwards, L A Hinds, E M Deane, J E Deakin

  Developmental and comparative immunology.

  Marsupials are born without a functioning adaptive immune system, into a non-sterile environment where they continue to develop. This review examines the extent of exposure of pouch young toMarsupials are born without a functioning adaptive immune system, into a non-sterile environment where they continue to develop. This review examines the extent of exposure of pouch young to microorganisms and describes the protective mechanisms that are complementary to adaptive immunity in the developing young. Complementary protective mechanisms include the role of the innate immune system and maternal protection strategies, such as immune compounds in milk, prenatal transfer of immunoglobulins, antimicrobial compounds secreted in the pouch, and chemical or mechanical cleaning of the pouch and pouch young.

• A novel acute phase reactant, serum amyloid A-like 1, from Oplegnathus fasciatus: Genomic and molecular characterization and transcriptional expression analysis.

  Authors: Kasthuri Saranya Revathy, Navaneethaiyer Umasuthan, Ilson Whang, Youngdeuk Lee, Sukkyoung Lee, Myung-Joo Oh, Sung-Ju Jung, Cheol Young Choi, Choul-Ji Park, Hae-Chul Park, Jehee Lee

  Developmental and comparative immunology.

  Acute phase response is a significant component of innate immunity, playing a vital role in the signaling processes and elimination of invading pathogens. Acute phase proteins are synthesized inAcute phase response is a significant component of innate immunity, playing a vital role in the signaling processes and elimination of invading pathogens. Acute phase proteins are synthesized in liver and secreted into the blood for transportation to an infection site, where the defense function is exerted. Serum amyloid A (SAA) and C-reactive proteins are the major positive acute phase proteins. In this study, we have identified and characterized a novel SAA related gene from rock bream (Oplegnathus fasciatus), designated OfSAAL1. Genomic characterization revealed the presence of 13 exons and 12 introns, similar to SAAL1 in zebrafish. Multiple protein sequence alignment revealed high conservation with other SAAL1 homologues. Phylogenetic analysis showed that OfSAAL1 clustered with another fish homologue, and pairwise alignment revealed highest identity and similarity at the amino acid level with zebrafish SAAL1. Promoter region analysis revealed the presence of immunologically significant transcription factor binding sites. Tissue distribution profiling to indicate physiological relevance showed the highest levels occur in blood, followed by liver, suggesting a positive immune role in rock bream. Transcriptional analysis by reverse transcription polymerase chain reaction to understand OfSAAL1 responsiveness to immune challenge with poly I:C, Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus, revealed a significant level of elevation from 12 h to 48 h post-infection in blood, spleen, head kidney, and liver. To our knowledge, OfSAAL1 is the first characterized SAAL1 homologue from teleosts. We anticipate that its identification will prove inspiring for further studies of SAAL1 homologues as biomarkers of the acute phase response.

• A Monoclonal Antibody for the CD45 Receptor in the Teleost Fish Dicentrarchus Labrax.

  Authors: Catia Marozzi, Fabrizio Bertoni, Elisa Randelli, Francesco Buonocore, Anna Maria Timperio, Giuseppe Scapigliati

  Developmental and comparative immunology.

  The CD45 tyrosine phosphatase plays an important role in regulating T lymphocyte activation in vertebrate species. In this study we describe some molecular and functional features of the CD45The CD45 tyrosine phosphatase plays an important role in regulating T lymphocyte activation in vertebrate species. In this study we describe some molecular and functional features of the CD45 receptor molecule from the European sea bass Dicentrarchus labrax. Following immunization with fixed sea bass thymocytes, we obtained a murine monoclonal antibody (mAb) able to stain fish leukocytes both alive, by immunofluorescence of thymus and mucosal tissues, and fixed, by in situ immunohistochemistry of tissue sections. The selected IgG(2) mAb (DLT22) was able to recognize by western blots polypeptides mainly at 180 kDa and 130 kDa in thymus, spleen, intestine and gill leukocyte. Accordingly, a 130 kDa polypeptide immunoprecipitated with DLT22 from thymocytes and analysed by nano-RP-HPLC-ESI-MS/MS, gave peptide sequences homologous to Fugu CD45, that were employed for the homology cloning of a partial sea bass CD45 cDNA sequence. This cDNA sequence was employed to measure by quantitative PCR the transcription of the CD45 gene both in unstimulated and in in vitro stimulated leukocytes, showing that the gene transcription was specifically modulated by LPS, ConA, PHA, IL-1, and poly I:C. When splenocytes were stimulated in vitro with ConA and PHA, a cell proliferation paralleled by an increase of DLT22-positive leukocytes was also observed. These data indicate that the DLT22 mAb recognises a putative CD45 molecule in sea bass, documenting the presence of CD45-like developing lymphocytes in thymus and CD45-associated functional stages of lymphocytes in this species, thus dating back to teleost fish the functional activities of these cell populations in vertebrates.

• Advances in research of fish immune-relevant genes: a comparative overview of innate and adaptive immunity in teleosts.

  Authors: Lv-Yun Zhu, Li Nie, Guan Zhu, Li-Xing Xiang, Jian-Zhong Shao

  Developmental and comparative immunology.

  Fish is considered to be an important model in comparative immunology studies because it is a representative population of lower vertebrates serving as an essential link to early vertebrateFish is considered to be an important model in comparative immunology studies because it is a representative population of lower vertebrates serving as an essential link to early vertebrate evolution. Fish immune-relevant genes have received considerable attention due to its role in improving understanding of both fish immunology and the evolution of immune systems. In this review, we discuss the current understanding of teleost immune-relevant genes for both innate and adaptive immunity, including pattern recognition receptors, antimicrobial peptides, complement molecules, lectins, interferons and signaling factors, inflammatory cytokines, chemokines, adaptive immunity relevant cytokines and negative regulators, major histocompatibility complexes, immunoglobulins, and costimulatory molecules. The implications of these factors on the evolutionary history of immune systems were discussed and a perspective outline of innate and adaptive immunity of teleost fish was described. This review may provide clues on the evolution of the essential defense system in vertebrates.

• Jagged1 and Notch1 help edit M cell patterning in Peyer's patch follicle epithelium.

  Authors: En-Hui Hsieh, David D Lo

  Developmental and comparative immunology.

  Mucosal epithelium M cells are dispersed across Peyer's patch follicle associated epithelium (PPFAE) with minimal clustering. Since Notch signaling can influence patterning in epithelia, we examinedMucosal epithelium M cells are dispersed across Peyer's patch follicle associated epithelium (PPFAE) with minimal clustering. Since Notch signaling can influence patterning in epithelia, we examined its influence on PPFAE M cell distribution. Conditional deletion of Notch1 in intestinal epithelium increased PPFAE M cells and also increased M cell clustering, implying a role for Notch in both M cell numbers and lateral inhibition. By contrast, conditional deletion of the ligand Jagged1 also increased M cell clustering, but with a paradoxical decrease in M cell density. In vitro, inhibition of Notch signaling reduced expression of an M cell associated gene CD137, consistent with cis-promoting effects on M cell development. Thus, Jagged1 may have a cis-promoting role in committed M cells, but a trans- inhibitory effect on neighboring cells. In sum, Jagged1-Notch signaling may edit the pattern of M cells across the PPFAE, which may help optimize mucosal immune surveillance.

• Why Spawning Salmon Return To Their Natal Stream: The Immunological Imprinting Hypothesis.

  Authors: Patty Zwollo

  Developmental and comparative immunology.

  The immune system of salmonids is remarkably similar to that of mammals, including the presence of B and T lymphocytes and a highly diverse antibody repertoire. However, fish lack bone marrow.The immune system of salmonids is remarkably similar to that of mammals, including the presence of B and T lymphocytes and a highly diverse antibody repertoire. However, fish lack bone marrow. Instead, development of immune cells takes place in the anterior kidney, which also houses long-lived, immunoglobulin-secreting plasma cells (LLPCs). These LLPCs should protect adult salmon against pathogens that are encountered upon return to their natal grounds for spawning. Here I present a hypothesis which links immune memory in the form of LLPCs to chemical imprinting and the highly accurate return rates of adults to their natal streams.

• Proteomics and its applications to aquaculture in China: Infection, immunity, and interaction of aquaculture hosts with pathogens.

  Authors: Xuan-Xian Peng

  Developmental and comparative immunology.

  China is the largest fishery producer worldwide in term of its aquaculture output, and plays leading and decisive roles in international aquaculture development. To improve aquaculture output furtherChina is the largest fishery producer worldwide in term of its aquaculture output, and plays leading and decisive roles in international aquaculture development. To improve aquaculture output further and promote aquaculture business development, infectious diseases and immunity of fishes and other aquaculture species must be studied. In this regard, aquaculture proteomics has been widely carried out in China to get a better understanding of aquaculture host immunity and microbial pathogenesis as well as host-pathogen interactions, and to identify novel disease targets and vaccine candidates for therapeutic interventions. These proteomics studies include development of novel methods, assays, and advanced concepts in order to characterize proteomics mechanisms of host innate immune defense and microbial pathogenesis. This review article summarizes some recently published technical approaches and their applications to aquaculture proteomics with an emphasis on the responses of aquaculture animals to bacteria, viruses, and other aqua-environmental stresses, and development of broadly cross-protective vaccine candidates. The reviewed articles are those that have been published in international peer reviewed journals.

• Recent advances in researches on the innate immunity of shrimp in China.

  Authors: Fuhua Li, Jianhai Xiang

  Developmental and comparative immunology.

  The annual production of shrimp culture in mainland of China has been over one million tons for several years. The major cultivated penaeidae species are Litopenaeus vannamei, FenneropenaeusThe annual production of shrimp culture in mainland of China has been over one million tons for several years. The major cultivated penaeidae species are Litopenaeus vannamei, Fenneropenaeus chinensis, Penaeus monodon and Marsupenaeus japonicus. Due to the importance of shrimp aquaculture in China, researchers have paid more attention to the molecular mechanism of shrimp disease occurrence and tried to develop an efficient control strategy to control disease. This paper summarizes the research progress related to innate immunity of penaeid shrimp made in the last decade in China. Several pattern recognition receptors, such as lectin, toll, lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and tetraspanin were identified. The major signal transduction pathways, including Toll pathway, IMD pathway, which might be involved in the immune response of shrimp, were focused on and most of the components in Toll pathway were identified. Also, cellular immune responses such as phagocytosis and apoptosis were regarded playing very important roles in anti-WSSV infection to shrimp. The molecules involved in the maintenance of the immune homeostasis of shrimp and the progress on molecular structure and pathogenic mechanism of WSSV were summarized. Therefore, the brief outline about the immune system of shrimp is drawn based on the recent data which will help us to understand the immune responses of shrimp to different pathogens.

• The arginine kinase in Zhikong scallop Chlamys farreri is involved in immunomodulation.

  Authors: Xiaowei Shi, Lingling Wang, Zhi Zhou, Chuanyan Yang, Yang Gao, Leilei Wang, Linsheng Song

  Developmental and comparative immunology.

  Arginine kinase (AK) catalyzes the reversible phosphorylation of l-arginine to form phosphoarginine, and plays a critical role in energy metabolism in invertebrates. In the present study, a scallopArginine kinase (AK) catalyzes the reversible phosphorylation of l-arginine to form phosphoarginine, and plays a critical role in energy metabolism in invertebrates. In the present study, a scallop AK gene was identified from Chlamys farreri with an open reading frame (ORF) of 1101bp encoding for a protein of 366 amino acids (designed as CfAK). An ATP-gua PtransN domain which was described as a guanidine substrate specificity domain (GS domain) and an ATP-gua Ptrans domian which was responsible for binding ATP, were both identified in CfAK. The mRNA transcripts of CfAK were detectable in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in the muscle and the lowest level in the hemocytes. The expression level of CfAK mRNA increased from fertilized eggs to eyebot, and reached the highest in the trochophore stage. The relative expression level of CfAK mRNA in muscle was up-regulated significantly after LPS (0.5mg/mL) stimulation, and reached the peak at 6h (5.2-fold, P<0.05). The activity of inducible nitric oxide synthase (iNOS) in the supernatant of muscle homogenate increased significantly from 3.2U/mg at 0h to 9.7U/mg at 12h after LPS stimulation, while the concentration of nitric oxide (NO) in the supernatant of muscle homogenate began to increase at 3h (21.55μmol/L), and reached the top concentration at 24h (42.27μmol/L), then recovered to the normal level after 48h. The recombinant protein of CfAK (rCfAK) expressed in Escherichia coli displayed Arginine kinase activity, and its apparent K(m) was 0.82±0.11 and 1.24±0.13mM for l-arginine and ATP-Na, respectively. The results indicated that the CfAK was involved in energy production and utilization during the whole life process, and might refer to the immunomodulation process via altering the NO concentration and iNOS activity in scallop Chlamys farreri.

• Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias).

  Authors: Lauren E Smith, Kathryn Crouch, Wei Cao, Mischa R Müller, Leeying Wu, John Steven, Michael Lee, Musen Liang, Martin F Flajnik, Heather H Shih, Caroline J Barelle, Janet Paulsen, Davinder S Gill, Helen Dooley

  Developmental and comparative immunology. 36(4):665-79.

  The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscriptThe cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.

• Teleost IL-6 promotes antibody production through STAT3 signaling via IL-6R and gp130.

  Authors: Mitsuru Kaneda, Tomoyuki Odaka, Hiroaki Suetake, Daisuke Tahara, Toshiaki Miyadai

  Developmental and comparative immunology.

  Teleost IL-6 is upregulated after antigen stimulation; therefore, we hypothesized that fish IL-6 contributes to antibody production during immune responses against infections. To verify thisTeleost IL-6 is upregulated after antigen stimulation; therefore, we hypothesized that fish IL-6 contributes to antibody production during immune responses against infections. To verify this hypothesis, we first cloned IL-6R and gp130 in fugu (Takifugu rubripes) in the present study. The membrane and soluble forms of IL-6R were identified by the identification of cDNA clones of IL-6R homologues. Three STAT3-docking sites were found in the intracellular region of fugu gp130. Expression analysis showed that fugu IL-6R and gp130 were expressed in mIgM(+) B cells, suggesting that fugu B cells are stimulated by IL-6. Recombinant fugu IL-6 (rfIL-6) increased the gene expression of secretory antibodies by mIgM(+) B cells in vitro. The rfIL-6 and soluble form of rfIL-6R activated STAT3 phosphorylation in the B cells and a cultured cell line transfected with fugu gp130. These results indicate that fugu IL-6 enhances antibody production in the B-cell lineage via gp130 and STAT3 signaling.

• Genomic structure, molecular characterization and functional analysis of Pekin duck interleukin-10.

  Authors: Qingxia Yao, Karl P Fischer, D Lorne Tyrrell, Klaus S Gutfreund

  Developmental and comparative immunology.

  Here we describe the cloning and expression of Pekin duck IL-10 (duIL-10) and a six exon-5 intron structure of an IL-10 gene. Two transcripts encoding duIL-10 with an alternately spliced 3'UTR, and aHere we describe the cloning and expression of Pekin duck IL-10 (duIL-10) and a six exon-5 intron structure of an IL-10 gene. Two transcripts encoding duIL-10 with an alternately spliced 3'UTR, and a transcript lacking exon 5 with a novel coding sequence for its C-terminus (duIL-10ΔE5) were isolated from splenocytes. The duIL-10 protein has an amino acid identity of 79% and 47% with chicken and human IL-10, respectively. The duck IL-10 gene shares a similar structure of the respective exons 1-5 with the IL-10 genes of other vertebrates but has an alternate exon. The duIL-10 3D structure by homology modeling was similar to that of the human IL-10 monomer, whereas the predicted duIL-10ΔE5 protein lacks helix F. DuIL-10 and duIL-10ΔE5 transcripts were most abundant in primary and secondary immune organs and lung. Recombinant duIL-10 suppressed duck IL-2 transcripts in mitogen-activated PBMCs. Our observation suggests evolutionary conservation of structure and function of the duIL-10 protein but the roles of the novel IL-10 splice variants in the regulation of duck immune responses and evolution of vertebrate immunity remain to be elucidated.

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