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Nile tilapia (Oreochromis niloticus) is one of the important economic fish species cultured worldwide. However, Streptococcus agalactiae has emerged as a significant bacterial threat, severely impacting the economy of tilapia industry. The immune response underlying the resistance of tilapia to S. agalactiae are not well understood, hindering the reasonable evaluation of breeding and the formulation of effective strategies. In this study, we investigated the differences in T-cell immunity between S. agalactiae-resistant and -susceptible tilapia. Compared with susceptible tilapia, resistant tilapia exhibited a higher percentage of T cells and BrdU + T cells during infection, indicating a superior proliferative capacity. Whether infected or not, T cells from resistant fish demonstrated a greater ability to resist apoptosis. Additionally, T cell effector genes, including interleukin (IL)-2, interferon (IFN)-γ, perforin A, and granzyme B were expressed at higher levels in resistant tilapia after infection. Along with these T-cell immune responses, resistant fish showed more effective clearance of infection. Our study elucidates the T-cell immune responses in resistant tilapia, which may contribute to the high resistance of tilapia to S. agalactiae, and provide valuable theoretical references for the selection and evaluation of disease-resistant fish strains in the future., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Perforin, produced by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), is one of the effectors of cell-mediated cytotoxicity (CMC) in vertebrates, playing a paramount role in killing target cells. However, whether and how perforin is involved in adaptive immune responses in early vertebrates remains unclear. Using Nile tilapia (Oreochromis niloticus) as a model, we investigated the characteristics of perforin in early vertebrates. Oreochromis niloticus perforin (OnPRF) possesses 2 conserved functional domains, membrane attack complex/perforin (MACPF) and protein kinase C conserved region 2 (C2) domains, although they share low amino acid sequence similarity with other homologs. OnPRF was widely expressed in various immune tissues and could respond to lymphocyte activation and T-cell activation in vitro at both the transcriptional and protein levels, indicating that it may be involved in adaptive immune responses. Furthermore, after infection with Edwardsiella piscicida and Aeromonas hydrophila, the mRNA and protein levels of OnPRF were significantly up-regulated within the adaptive immune response period. Additionally, we revealed that many transcription factors were involved in the transcriptional regulation of OnPRF, including p65, c-Fos, c-Jun, STAT1 and STAT4, and there was a synergy among these transcription factors. Overall, these findings demonstrate the involvement of OnPRF in T-cell activation and adaptive immune response in tilapia, thus providing new evidence for comprehending the evolution of immune response in early vertebrates., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Secreted by natural killer cells and cytotoxic T lymphocytes, Granzyme B is involved in regulating the adaptive immune response in vertebrates and plays a pivotal role in resisting virus invasion and removing pathogens. Although it had been extensively studied in mammals, the involvement of Granzyme B in adaptive immune response of early vertebrates remained elusive. In this study, we investigated the Granzyme B in Oreochromis niloticus (OnGrB), found that its function domain was conserved. Additionally, OnGrB was widely expressed in various tissues and could respond to T-cell activation in vitro at the transcriptional level. Furthermore, we prepared the recombinant OnGrB (rOnGrB) as an immunogen to develop a mouse anti-OnGrB monoclonal antibody (mAb). Using this anti-OnGrB mAb as a tool, we explored the expression of OnGrB in the adaptive immune response of tilapia. Our findings revealed that T cell was a significant source of OnGrB production, the expression of OnGrB at the protein level and the proportion of OnGrB  +  T cells increased after both T cell activation in vitro and infection with Edwardsiella piscicida in vivo. More importantly, our findings also preliminarily illuminated that p65 could regulate the transcriptional activity of OnGrB. These results indicated that OnGrB was involved in the adaptive immunity of tilapia and played a critical role in T cell function in teleost. Our study provided theoretical support and new perspectives for understanding adaptive immunity in teleost., (Copyright © 2024. Published by Elsevier Ltd.)

As one of subunits for interleukin-2 receptor (IL-2R), CD122 can bind to IL-2 and then activate downstream signal transduction to participate in adaptive immune response. Although CD122 has been identified and investigated from several teleost species, studies on its function at T-cell level are still scarce for lack of specific antibodies. In this study, a typical CD122 in Nile tilapia (Oreochromis niloticus) was characterized by bioinformatics analysis, cloned to produce retrovirus infected NIH/3T3 cells for mouse immunization. After cell fusion and screening, we successfully developed a mouse anti-tilapia CD122 monoclonal antibody (mAb), which could specifically recognize CD122 and identify CD122-producing T cells of tilapia. Using the mAb to detect, CD122 was found to widely distribute in immune-related tissues, and significantly elevate post Edwardsiella piscicida infection or T-cell activation. More importantly, the expansion of CD122 + T cells and up-regulation of CD122 occurred both in total T cells and T-cell subsets during T-cell activation upon in vitro stimulation or in vivo infection. These results indicate that CD122 can be used as a T-cell activation marker in tilapia. Notably, CD122 mAb blocking blunted the activation of MAPK/Erk and mTORC1 pathways, and inhibited T-cell proliferation, suggesting a critical role of CD122 in ensuring proper proliferation of tilapia T cells. Therefore, this study enriches the knowledge of T-cell responses in fish and provides new evidence for understanding the evolution of lymphocyte-mediated adaptive immunity., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As a multipotent cytokine, interleukin (IL)-2 plays important roles in activation, differentiation and survival of the lymphocytes. Although biological characteristics and function of IL-2 have been clarified in several teleost species, evidence regarding IL-2 production at the cellular and protein levels is still scarce in fish due to the lack of reliable antibody. In this study, we developed a mouse anti-Nile tilapia IL-2 monoclonal antibody (mAb), which could specifically recognize IL-2 protein and identify IL-2-producing lymphocytes of tilapia. Using this mAb, we found that CD3 + T cells, but not CD3 - lymphocytes, are the main cellular source of IL-2 in tilapia. Under resting condition, both CD3 + CD4-1 + T cells and CD3 + CD4-1 - T cells of tilapia produce IL-2. Moreover, the IL-2 protein level and the frequency of IL-2 + T cells significantly increased once T cells were activated by phytohemagglutinin (PHA) or CD3 plus CD28 mAbs in vitro. In addition, Edwardsiella piscicida infection also induces the IL-2 production and the expansion of IL-2 + T cells in the spleen lymphocytes. These findings demonstrate that IL-2 takes part in the T-cell activation and anti-bacterial adaptive immune response of tilapia, and can serve as an important marker for T-cell activation of teleost fish. Our study has enriched the knowledge regarding T-cell response in fish species, and also provide novel perspective for understanding the evolution of adaptive immune system., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As a pleiotropic cytokine consisting of IL-12p35 and IL-12p40, Interleukin-12 (IL-12) features in inflammation regulation and anti-bacterial immunity. While IL-12 homologs have been identified in non-mammalian species, the precise mechanisms by which IL-12 contributes to early adaptive immune responses in vertebrates remain incompletely understood. Herein, an evolutionary conserved Oreochromis niloticus IL-12 (defined as OnIL-12) was identified by synteny characterization, structural comparisons and phylogenetic pattern of IL-12p35b and IL-12p40a. IL-12p35b and IL-12p40a exhibited widespread expression in lymphoid-related tissues of tilapia, while their mRNA expression in head-kidney demonstrated a significant increase after Edwardsiella piscicida infection. Compared with other lymphocytes, recombinant OnIL-12 (rOnIL-12) displayed stronger affinity binding to T cells. Although stimulation of lymphocytes with the p35b or p40a subunit resulted in a significant induction of IFN-γ expression, rOnIL-12 showed stronger potential to promote IFN-γ expression than these subunits. rOnIL-12 not only elevated the mRNA expression level Th1 cell-associated transcription factor T-bet in lymphocytes, but also increased the proportion of CD4-1 + IFN-γ + lymphocytes. Moreover, the mRNA and phosphorylation levels of STAT1, STAT3, STAT4 and STAT5 were enhanced by rOnIL-12. These findings will offer previous evidence for further exploration into the regulatory mechanisms of Th1 cellular immunity in early vertebrates., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As a pleiotropic cytokine mainly secreted by CD4 + T cells, interleukin (IL)-22 plays an important role in immune regulation and infection elimination. Despite IL-22 homologues have been identified in non-mammal, whether and how IL-22 participates in the adaptive immune response of early vertebrates have not been fully addressed. In this study, we identified an evolutionarily conserved IL-22 from Nile tilapia Oreochromis niloticus (defined as OnIL-22), proved by its properties regarding sequence, gene structure, functional domain, tertiary structure and phylogeny. IL-22 was broadly expressed in lymphoid-related tissues of tilapia, and with relatively higher levels in skin, gill, intestine and liver. The expression of OnIL-22 in spleen lymphocytes was markedly induced at the adaptive immune stage after Streptococcus agalactiae infection. Moreover, once lymphocytes were activated by PMA plus ionomycin or T-cell specific mitogen PHA in vitro, OnIL-22 expression was obviously up-regulated at both mRNA and protein levels. These results thus suggest that activated T cells produce IL-22 to take part in the adaptive immune response of tilapia. Furthermore, treatment of lymphocytes with recombinant OnIL-22 increased the expression of genes related to proliferation and survival, and further promoted the proliferation and reduced the apoptosis of lymphocytes during bacterial infection or T-cell activation. These cellular effects of IL-22 seem to be associated with JAK1/STAT3 axis downstream of IL-22, because IL-22 application not only elevated the mRNA expression of JAK1 and STAT3, but also enhanced their phosphorylation in lymphocytes. Altogether, we suggest that activated T cells produce IL-22 to promote lymphocyte proliferation and survival probability via JAK1/STAT3 signaling pathway, thus participating in adaptive immune response of Nile tilapia. Our study therefore provides helpful perspective for understanding the function and mechanism of adaptive immune system in teleost., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Interleukin-2 inducible T cell kinase (ITK) plays a predominant role in the T-cell receptor (TCR) signaling cascade to ensure valid T-cell activation and function. Nevertheless, whether it regulates T-cell response of early vertebrates remains unknown. Herein, we investigated the involvement of ITK in the lymphocyte-mediated adaptive immune response, and its regulation to T-cell activation in the Nile tilapia Oreochromis niloticus. Both sequence and structure of O. niloticus ITK (OnITK) were remarkably conserved with its homologues from other vertebrates, implying its potential conserved function. OnITK mRNA was extensively expressed in lymphoid-related tissues, and with the relative highest level in peripheral blood. Once Nile tilapia was infected by Edwardsiella piscicida, OnITK in splenic lymphocytes was significantly up-regulated on 7-day post infection at both transcription and translation levels, suggesting that OnITK might involve in the primary adaptive immune response of teleost. Furthermore, upon splenic lymphocytes were stimulated by T-cell specific mitogen PHA, OnITK mRNA and protein levels were dramatically elevated. More importantly, treatment of splenic lymphocytes with specific inhibitor significantly crippled OnITK expression, which in turn impaired the inducible expression of T-cell activation markers IFN-γ, IL-2 and CD122, indicating the critical roles of ITK in regulating T-cell activation of Nile tilapia. Taken together, our results suggest that ITK takes part in the lymphocyte-mediated adaptive immunity of tilapia, and is indispensable for T-cell activation of teleost. Our findings thus provide novel evidences for understanding the mechanism regulating T-cell immunity of early vertebrates, as well as the evolution of adaptive immune system., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Killer cell lectin-like receptor G subfamily 1 (KLRG1) is a receptor generally expressed on effector CD8 + T cells or NK cells at terminal differentiation stage, and it will be highly induced for lymphocyte cytotoxicity upon pathogen infection or lymphocyte activation. However, little is known about the character or function of KLRG1 in lower vertebrates. In present study, we reappraised a molecule that previously defined as KLRG1 in the genomic sequence of Nile tilapia Oreochromis niloticus, and identified it as an atypical KLRG1-like molecule (defined as On-KLRG1-L), and illustrated its potential function serving as a marker representing effector T lymphocytes of fish species. On-KLRG1-L consists of two C-type lectin-like domains (CTLDs) without transmembrane region, and the tertiary structure of the CTLD is highly alike to that in mouse KLRG1. As a CTLD-containing protein, the recombinant On-KLRG1-L could bind PGN and several microbes in vitro. On-KLRG1-L was widely expressed in immune-associated tissues, with the highest expression level in the gill. Once Nile tilapia is infected by Aeromonas hydrophila, mRNA level of On-KLRG1-L in spleen lymphocytes were significantly up-regulated on 5 days after infection. Meanwhile, On-KLRG1-L protein was also induced on 5 or 8 days after A. hydrophila infection. Furthermore, we found both mRNA and protein levels of On-KLRG1-L were dramatically enhanced within several hours after spleen lymphocytes were activated by T cell-specific mitogen PHA in vitro. More importantly, the ratio of On-KLRG1-L + T cells was also augmented after PHA stimulation. The observations suggested that the KLRG1-like molecule from Nile tilapia participated in lymphocyte activation and anti-bacterial adaptive immune response, and could serve as an activation marker of T lymphocytes. Our study thus provided new evidences to understand lymphocyte-mediated adaptive immunity of teleost., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ribosomal protein S6 kinase beta-1 (S6K1) is a serine/threonine kinase downstream of the mechanistic target of rapamycin (mTOR) pathway, and plays crucial roles in immune regulation. Although remarkable progress has been achieved with a mouse model, how S6K1 regulates adaptive immunity is largely unknown in early vertebrates. In this study, we identified an S6K1 from Nile tilapia Oreochromis niloticus (OnS6K1), and further investigated its potential regulatory role on the adaptive immunity of this fish species. Both sequence and structure of OnS6K1 were highly conserved with its homologs from other vertebrates and invertebrates. OnS6K1 was widely expressed in immune tissues, and with a relative higher expression level in the liver, spleen and head kidney. At the adaptive immune stage of Nile tilapia that infected with Aeromonas hydrophila, mRNA expression of OnS6K1 and its downstream effector S6 was significantly up-regulated in spleen lymphocytes. Meanwhile, their phosphorylation level was also enhanced during this process, suggesting that S6K1/S6 axis participated in the primary response of anti-bacterial adaptive immunity in Nile tilapia. Furthermore, after spleen lymphocytes were activated by the T cell-specific mitogen PHA or lymphocytes agonist PMA in vitro, mRNA and phosphorylation levels of S6K1 were elevated, and phosphorylation of S6 was also enhanced. Once S6K1 activity was blocked by a specific inhibitor, both mRNA and phosphorylation levels of S6 were severely impaired. More importantly, blockade of S6K1/S6 axis reduced the expression of T cell activation marker IFN-γ and CD122 in PHA-activated spleen lymphocytes, indicating the essential role of S6K1/S6 axis in regulating T cell activation of Nile tilapia. Together, our study suggests that S6K1 and its effector S6 regulate lymphocyte activation of Nile tilapia, and in turn promote lymphocyte-mediated adaptive immunity. This study enriched the mechanism of adaptive immune response in teleost and provided useful clues to understand the evolution of adaptive immune system., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As an important economic mollusk in coastal areas, Octopus ocellatus dependents on innate immune system to resist the invasion of microorganisms. Lysozyme is a crucial effector owing to its significant lytic activity against bacterial pathogens during the immune responses. In this study, characteristic and immune function of an I-type lysozyme from O. ocellatus (OoLyz) was investigated. OoLyz shared a close relationship with the lysozymes from other bivalve mollusks. The mRNA of OoLyz exhibited a broad transcript in different tissues/organs, and with the greatest expression in hepatopancreas. The expression of OoLyz was significantly raised when O. ocellatus was infected by Vibrio anguillarum or Micrococcus luteus, suggesting OoLyz participated in innate immune response of host. Prokaryotic recombinant OoLyz (rOoLyz) exhibited obvious bacteriolysis ability towards both gram-negative bacteria V. anguillarum and Escherichia coli, and gram-positive bacteria M. luteus and Staphylococcus aureus. The bacteriolysis activities of rOoLyz towards gram-negative but not gram-positive bacteria was heat stable, indicating that OoLyz might clear gram-positive bacterium by enzyme-dependent mechanisms, but eliminate gram-negative microbe via enzymatic activity independent way. Scanning electron microscopy analysis showed that rOoLyz destroyed microbes by damaging cell wall. More importantly, the fact that rOoLyz could directly degrade the peptidoglycan, further revealed its bactericidal mechanism as a muramidase. Our results revealed the essential role of I-type lysozyme in the innate immunity of O. ocellatus, and shed new light to understand the mechanism of immune defense of mollusks., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

H-Ras is a guanosine triphosphatase (GTPase), which acts as a molecular switch and controls multiple important cellular processes including lymphocyte activation and function. However, regulatory mechanism of adaptive immune response by H-Ras remains unclear in non-mammalian animals. In the present study, we investigated the involvement of H-Ras in lymphocyte activation with a teleost model Oreochromis niloticus. H-Ras from O. niloticus (On-H-Ras) is highly conserved with those from other vertebrates. The mRNA of On-H-Ras showed a wide expression pattern in the lymphoid-tissues and with the highest level in liver. After Aeromonas hydrophila infection, transcription of On-H-Ras was significantly induced on day 8 but came back to basal level on day 16, suggesting that On-H-Ras potentially participated in primary response during the adaptive immunity. Furthermore, On-H-Ras mRNA was obviously up-regulated when leukocytes were activated by T lymphocyte mitogen PHA in vitro. Meanwhile, protein level of H-Ras was also augmented once leukocytes were stimulated with lymphocyte receptor signaling agonist PMA and ionomycin. More importantly, once Ras activity was inhibited by specific inhibitor, the up-regulation of lymphocyte activation marker CD122 was obviously impaired during lymphocyte activation process. Therefore, On-H-Ras regulated lymphocyte activation through both mRNA and protein level. Altogether, our results illustrated the involvement of H-Ras in teleost adaptive immunity via controlling lymphocyte activation, and thus provided a novel perspective to understand evolution of the lymphocyte-mediated adaptive immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

RAF proto-oncogene serine/threonine-protein kinase (c-Raf) is a MAP kinase kinase kinase (MAPKKK) that participates in the Erk1/2 pathway and plays an important role in lymphocyte activation. However, the study on how c-Raf regulates adaptive immunity in non-mammal is still limited. In present study, based on analysis of sequence characteristics of c-Raf from Oreochromis niloticus (On-c-Raf), we investigated its regulation roles on teleost lymphocyte activation. The On-c-Raf was highly conserved during evolution, which was composed of a Raf-like Ras-binding domain (RBD), a protein kinase C conserved region 1 (C1) domain and a serine/threonine protein kinase catalytic (S_TKc) domain. Its mRNA showed a wide distribution in tissues of O. niloticus and with the highest expression in gill. After Aeromonas hydrophila infection, during the adaptive immune stage transcription level of On-c-Raf was significantly upregulated on day 8, but came back to original level on day 16 and 30, suggesting the potential involvement of On-c-Raf in primary response but not memory formation. Furthermore, On-c-Raf mRNA in leukocytes of Nile tilapias was obviously induced by in vitro stimulation of T cell mitogen PHA. More importantly, in vitro stimulation of lymphocytes agonist PMA augmented phosphorylation level of On-c-Raf in leukocytes detected by western-blot and immunofluorescent. Thus, c-Raf regulated lymphocyte activation of Nile tilapia on both mRNA and phosphorylation level. Together, our results revealed that the c-Raf from teleost Nile tilapia engaged in adaptive immune response by regulating lymphocytes activation. Since the regulatory mechanism of lymphocyte-mediated adaptive immunity is largely unknown in teleost, our study provided important evidences to understand teleost adaptive immunity, and also shed a novel perspective for the evolution of adaptive immune system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Galactoside-binding lectin (galectin) is a type of pathogen recognition molecule that occupies an important position in the invertebrate innate immunity system. Our previous study has identified a galectin gene in mollusk Solen grandis (SgGal-1) and illustrated its potential roles in innate immunity. By the functional study using recombinant protein and specific antibody, here, we confirmed the pivotal roles of SgGal-1 in immune defense of S. grandis. SgGal-1 protein was expressed in many tested tissues including gill, mantle, hepatopancreas and gonad, except hemocytes and muscle. The recombinant SgGal-1 (rSgGal-1) bound PGN and β-glucan instead of LPS in vitro, and it further caused significant agglutination of five different microbes, suggesting SgGal-1 served as a pattern recognition receptor (PRR) involved in immune defense of mollusk. Furthermore, SgGal-1 recruited hemocytes to encapsulate, which was blocked by anti-rSgGal-1 serum. In the meantime, rSgGal-1 as well as promoted the phagocytosis of hemocytes against Escherichia coli in vitro. All these results suggested that SgGal-1 in S. grandis not only acted as a PRR recognizing microbes but also directly participated in the process of immune opsonization to protect the host from pathogenic infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Peptidoglycan recognition proteins (PGRPs) are indispensable molecules in innate immunity due to their prominent function in sensing and eliminating invading microorganisms. In the present study, a short type PGRP from razor clam Solen grandis (SgPGRP-S1) was recombinantly expressed and purified to investigate its potential function in innate immunity. As a pattern recognition receptor, recombinant SgPGRP-S1 (rSgPGRP-S1) specifically bind Lys-type and Dap-type peptidoglycan in vitro, but not lipopolysaccharide or β-glucan. The peptidoglycan binding ability of rSgPGRP-S1 resulted in significant agglutination activity against Gram-negative Escherichia coli and Listonella anguillarum, as well as Gram-positive Micrococcus luteus. Furthermore, rSgPGRP-S1 was bactericidal, significantly suppressing the growth of both E. coli and Gram-positive Staphylococcus aureus. The protein also exhibited strong amidase activity and degraded bacterial peptidoglycan in the presence of Zn 2+ , suggesting amidase activity might contribute to SgPGRP-S1 antibacterial activity. These results indicate SgPGRP-S1 is multifunctional in innate immunity, mediating both immune recognition and bacteria elimination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sialic acid-binding lectins (SABLs) are ubiquitous ancient molecules with binding properties to N-acetyl or N-glycolyl carbohydrates, and play crucial roles in both adaptive and innate immune responses. In present study, recombinant protein and antibodies of two SABLs from mollusk Solen grandis (SgSABL-1 and SgSABL-2) were prepared to investigate their functions in innate immunity. The recombinant protein of SgSABL-1 (rSgSABL-1) could bind LPS, PGN and β-glucan in vitro, while rSgSABL-2 could only bind PGN rather than LPS and β-glucan. Be coincident with their PAMPs recognition properties, rSgSABL-1 displayed a broad agglutination spectrum towards gram-positive bacteria Micrococcus luteus, gram-negative bacteria Listonella anguillarum and fungi Pichia pastoris, and rSgSABL-2 only showed remarkable agglutinative effect on M. luteus and L. anguillarum. More importantly, after PAMPs recognition, rSgSABL-1 and rSgSABL-2 enhanced phagocytosis as well as encapsulation ability of hemocytes in vitro, and the enhanced encapsulation could be blocked by specific antibodies. All these results indicated that SgSABL-1 and SgSABL-2 functioned as two compensative pattern-recognition receptor (PRRs) with distinct recognition spectrum and involved in the innate immune response of S. grandis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Serine protease inhibitors (SPIs) play a crucial role in regulation of both host and bacterial serine protease. They are classified into several protein families, where Kazal-type inhibitors are one of families with multi-domain. In the present study, the polymorphism of AiSPI from Bay scallop Argopecten irradians was found to be associated with disease resistance of bay scallop against Listonella anguillarum. Nine single nucleotide polymorphisms (SNPs) were identified in the exon region of AiSPI, where five SNPs were non-synonymous mutation. Three of these mutations were located in "kazal-like 3"domain, two SNP loci positioned at +536, +1312 were selected for further association studies. For the locus +536, the genotype frequency of A/G in the resistant stock (12.8%) was significantly lower (p < 0.05) than that in the susceptible stock (35.1%), while, the genotype A/A in the resistant stock (87.2%) was significantly higher in comparison with susceptible stock (64.9%) (p < 0.05). The G allele frequencies were 6.4% and 17.6% in resistant stock and susceptible stock, respectively, and χ 2 -test revealed a significant difference in the frequency distribution between the two stocks (p < 0.05). But there was no significant association between the mutation C-T at locus +1312 with either resistant or susceptible group (p > 0.05). The genotype frequencies of T/T, T/C, C/C at locus +1312 were 94.6%, 2.7% and 2.7% respectively in the susceptible stock, while 100%, 0% and 0% respectively in the resistant stock. The amino acid change for the mutation at locus +536 A-G was from asparagine to serine, and the predicted homology model of this amino acid variation could affect its function as well as the structural integrity of the domain. In vitro elastase inhibition assay of the protein variants at locus +536 was conducted to explicate the effect of SNP. The increasing concentration of protein (0 mmol/L- 2.93 mmol/L) was incubated with 80 nmol/L elastase where the residual enzyme activity values for rAiSPI (N) with A variant and rAiSPI (S) with G variant were started to reduce from 0.40 to 0.215 and 0.435 to 0.356, respectively. The elastase inhibition ability of rAiSPI (N) variant was significantly higher than that of rAiSPI (S) (p < 0.01). The results suggested that the mutation at locus +536A/A significantly associated with disease resistance of bay scallop would shed light for selective breeding program., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

C-type lectin is one important pattern recognition receptor (PRR) that plays crucial roles in multiple immune responses. A C-type lectin from sea cucumber Apostichopus japonicus (AjCTL-1) was characterized in the present study. The amino acid sequence of AjCTL-1 shared high similarities with other C-type lectins from invertebrates and vertebrates. The C-type lectin domain (CTLD) of AjCTL-1 contained a Ca(2+)-binding site 2 and four conserved cysteine residues. AjCTL-1 mRNA expression patterns in tissues and after bacterial challenge were then analysed. Quantitative PCR revealed that AjCTL-1 mRNA was widely expressed in the tested tissues of healthy sea cucumber. The highest expression level occurred in gonad followed by body wall, coelomocytes, tentacle, intestinum and longitudinal muscle, and the lowest expression level was in respiratory tree. AjCTL-1 mRNA expression in coelomocytes was significantly induced by gram-negative Listonella anguillarum and gram-positive Micrococcus luteus, with different up-regulation patterns post-challenge. Recombinant AjCTL-1 exhibited the ability to bind peptidoglycan directly, agglutinate M. luteus, Staphylococcus aureus and Escherichia coli, in a Ca(2+)-dependant manner, and enhance the phagocytosis of coelomocytes against E. coli in vitro. The results indicated that AjCTL-1 could act as a PRR in Apostichopus japonicus and had critical roles in non-self recognition and bacterial clearance against invading microbes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP&#95;941373) and Ixodes scapularis (XP&#95;002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide-induced TNF-α factor (LITAF) is one of the most important transcription factors mediating TNF-α transcription. In the present study, a LITAF gene (designated as SgLITAF) was identified from razor clams Solen grandis. The full-length cDNA of SgLITAF was of 1476 bp, encoding a polypeptide of 130 amino acids showed high similarity to other known LITAFs. SgLITAF encoded a LITAF domain and the Zn(2+)-binding motifs in the domain were well conserved. The mRNA transcripts of SgLITAF were detected in all tested tissues of healthy razor clams, including mantle, gill, gonad, hemocytes, muscle and hepatopancreas, and with the highest expression level in hepatopancreas. The expression level of SgLITAF in hemocytes was significantly up-regulated (P < 0.01) after razor clams were stimulated by LPS or β-1, 3-glucan, but no obvious fluctuation of SgLITAF mRNA expression was observed after PGN stimulation. All the results indicated that there might be a LITAF-regulated TNF-α signaling pathway existing in S. grandis, which involved in the immune response not only against gram-negative bacteria but also towards fungi., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rab GTPase is essential for the control of intracellular membrane trafficking in all eukaryotic cells and further affects the ability of phagocytic cells to scavenge pathogen. In the present study, the cDNAs for two crab Rab proteins (EsRab-1 and EsRab-3) were identified from the Chinese mitten crab Eriocheir sinensis by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNAs of EsRab-1 and EsRab-3 were of 892 bp and 965 bp with ORFs of 615 bp and 630 bp, respectively. The cDNAs encoded two peptides of 204 and 209 amino acid residues with the conserved GTP/Mg(2+) binding sites, Switch I region and Switch II region in RAB domains. The mRNA transcripts of EsRab-1 and EsRab-3 were both highest expressed in hepatopancreas, and marginally expressed in other tissues including hemocytes, muscle, gonad, gill and heart. After the crabs were challenged by bacteria Vibrio anguillarum, the expression levels of both EsRab-1 and EsRab-3 in hemocytes were significantly up-regulated, and reached the highest level at 1.5 h post-stimulation, which was 7-fold (P < 0.05) and 6-fold (P < 0.01) of blank group for EsRab-1 and EsRab-3, respectively. No significant change of mRNA expression was detected for either EsRab-1 or EsRab-3 in crabs stimulated by Pichia pastoris. These results clearly suggested the involvement of Rab proteins in crab anti-bacterial immunity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Serine protease inhibitors (serpins) represent an expanding superfamily of endogenous inhibitors that regulate proteolytic events and involve in a variety of physiological processes. A serine protease inhibitor, namely Esserpin, was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) analysis. The full-length cDNA of Esserpin was of 2367 bp, including an open reading frame (ORF) of 1371 bp encoding a polypeptide of 456 amino acids with estimated molecular mass of 49.95 kDa and theoretical isoelectric point of 6.03. A putative signal peptide of 23 amino acids and a classical serpin domain were identified in Esserpin. The deduced amino acid sequence of Esserpin shared homology with serpins from Fenneropenaeus chinensis and Pacifastacus leniusculus. The mRNA transcripts of Esserpin could be detected in all the examined tissues including heart, gill, hemocytes, muscle, gonad and hepatopancreas, and the highest expression level was present in gonad. After the crabs were challenged by Vibrio anguillarum and Pichia pastoris, the expression levels of Esserpin transcripts in hemocytes were significantly up-regulated, and peaked at 24 h (5.18-fold of blank group, P < 0.05) and 3 h (2.87-fold of blank group, P < 0.05), respectively. The functional activity of Esserpin was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombinant Esserpin (rEsserpin) could inhibit trypsin activities in a dose-dependent manner, and it could lead to 100% inhibition of trypsin activities under the concentration of 873.76 nM, while there was no evident inhibition of chymotrypsin observed with rEsserpin. Moreover, rEsserpin inhibited the growth of E. coli at the final concentration of 1747.52 nM, and it also significantly depressed (P < 0.05) the phenoloxidase activity in the plasma at the final concentration of 873.76 nM. These results indicated that Esserpin was a homologue of serpin in crab and it could be induced after immune stimulation and mediate immune response possibly via the inhibition of bacterial growth and the regulation of prophenoloxidase-activating system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the present study, the expression of some immune-related genes was examined as indicator to understand the development of immune defense system during the ontogenesis of scallop Chlamys farreri. The mRNA transcripts of pattern-recognition receptors (PRRs) including CfPGRP-S1, CfLGBP, CfLec-1 and CfLec-3 were observed at a low level or even undetected at early developmental stages from eggs to blastula, and then began to increase overwhelmingly in trochophore. For the genes of immune effector including CfLYZ, CfLBP/BPI, CfSOD and CfCAT, their mRNA transcripts were higher expressed in embryos, and increased significantly in D-hinged or early veliger larvae. The whole-mount immunofluorescence assay revealed two immunoreactive spots of CfPGRP-S1 were first observed in the mid-ventral region of prototroch in trochophore, and the immunopositive fluorescence of CfLGBP, CfLec-1 and CfLec-3 appeared at the same spots in early D-hinged larvae. Most of the PRRs were located in velum, mouth, esophagus and stomach region in early and mid-veliger larvae, and especially the strong immunopositive fluorescence of CfLec-3 was observed in velum. The immunoreactive areas of CfLYZ, CfLBP/BPI, CfSOD and CfCAT were observed in trochophore and early D-hinged larvae. After D-hinged larvae, they distributed in different tissues from the edge of velum, mouth, esophagus to the region around digestive gland. After bacterial challenge, the mRNA expression of CfLGBP, CfLec-1 and CfLec-3 did not change significantly in trochophore, while a down-regulation of CfPGRP-S1 was observed at 6 h (P < 0.05). The expression of CfPGRP-S1 and CfLGBP decreased or increased inversely in D-hinged and late veliger larvae respectively, whereas CfLec-1 and CfLec-3 increased significantly during 6-24 h after bacterial challenge in the two stages (P < 0.05). In contrast, the expressions of immune effectors in trochophore and late veliger larvae were significant up-regulated at 6 h, 12 h or 24 h after bacterial challenge (P < 0.05). However, in late D-hinged larvae, CfLYZ and CfSOD expressions were significantly down-regulated at 6 h, while CfLBP/BPI expression was up-regulated at 6 h and 24 h post challenge (P < 0.05). These results indicated that the immune defense system of scallop might appear firstly in the mid-ventral region of prototroch in trochophore, and developed maturely after late D-hinged larvae. The developing immune system in the D-hinged and late veliger larvae could respond to the immune stimulation in different manner., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Serine proteinase inhibitor (SPI) serves as a negative regulator in immune signal pathway by restraining the activities of serine proteinase (SP) and plays an essential role in the innate immunity. In the present study, a Kunitz-type SPI was identified from the mollusk razor clam Solen grandis (designated as SgKunitz). The full-length cDNA of SgKunitz was of 1284 bp, containing an open reading frame (ORF) of 768 bp. The ORF encoded four Kunitz domains, and their amino acids were well conserved when compared with those in other Kunitz-type SPIs, especially the six cysteines involved in forming of three disulfide bridges in each domain. In addition, the tertiary structure of all the four domains adopted a typical model of Kunitz-type SPI family, indicating SgKunitz was a new member of Kunitz-type SPI superfamily. The mRNA transcripts of SgKunitz were detected in all tested tissues of razor clam, including muscle, mantle, gonad, gill, hepatopancreas and hemocytes, and with the highest expression level in gill. When the razor clams were stimulated by LPS, PGN or β-1, 3-glucan, the expression level of SgKunitz mRNA in hemocytes was significantly up-regulated (P < 0.01), suggesting SgKunitz might involved in the processes of inhibiting the activity of SPs during the immune responses triggered by various pathogens. Furthermore, the recombinant protein of SgKunitz could effectively inhibit the activities of SP trypsin and chymotrypsin in vitro. The present results suggested SgKunitz could serve as an inhibitor of SP involving in the immune response of S. grandis, and provided helpful evidences to understand the regulation mechanism of immune signal pathway in mollusk., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Peptidoglycan recognition protein (PGRP) is a pattern recognition receptor, playing important roles in the innate immune response against invasive pathogens. The single nucleotide polymorphism (SNP) loci in scallop PGRP gene (CfPGRP) were screened from Chlamys farreri to investigate their association with disease resistance of scallop against Listonella anguillarum. Thirteen SNP sites were identified in PGRP domain of CfPGRP, and two of them at positions 4407 and 4408 which are located in the same codon resulted in a nonsynonymous substitution. The genotype frequency of CG/CG in the resistant stock was significantly lower than that in susceptible stock (0% vs 32.4%), while that of CG/TA in the resistant stock was significantly higher than that in susceptible stock (P < 0.01). The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfPGRP-S1 (R) with CG variant in 4407-4408 site, rCfPGRP-S1 (Y) with TA variant in 4407-4408 site, were elucidated by examining their P/N value at 405 nm with ELISA assay. The in vitro binding activities of the two rCfPGRP-S1 variants to both lipopolysaccharide (LPS) and peptidoglycan (PGN) varied (P < 0.05) in a dose-dependent manner, and rCfPRPP-S1(Y) exhibited significantly higher affinity to PGN and LPS than that of rCfPGRP-S1(R) (P < 0.05). The growth inhibition assay was conducted to find the antibacterial activities of the two variants. Both rCfPGRP-S1(R) and rCfPGRP-S1 (Y) displayed obvious activity to suppress the growth of Escherichia coli, but there was no significant difference in suppression activity of two variants (P > 0.05). The results suggested that the polymorphism at locus 4407-4408 of CfPGRP-S1 considerably affected its PAMP binding activity, and the SNP locus 4407-4408 CG/TA was associated with disease resistance of scallop against L. anguillarum infection, which could be used as a candidate marker for future selection in zhikong scallop breeding program., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, β-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F' as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Glutathione S-transferases (GSTs) are a superfamily of antioxidant enzymes, which play crucial roles in detoxification and protection of tissues from oxidative damage caused by reactive oxygen species (ROS). In this study, a sigma-class GST was identified from razor clam Solen grandis (designated as SgGST-S1), and its expression patterns, both in tissues and toward microorganism glycan as well as organic contaminants stimulation, were then characterized. The full-length cDNA of SgGST-S1 was of 1291 bp, containing a 5' untranslated region (UTR) of 27 bp, and a 3' UTR of 619 bp with a poly (A) tail. The open reading frame (ORF) was of 645 bp, encoding a polypeptide of 214 amino acids with the predicted molecular weight of 24.8 kDa, which shared 47% identity with GST from Ruditapes philippinarum. The analysis of conserved domain and phylogenetic relationship strongly suggested that SgGST-S1 was a member of sigma-class GST. The mRNA of SgGST-S1 was constitutively expressed in all tested tissues of healthy razor clam, including mantle, gill, gonad, hemocytes, muscle, and hepatopancreas, and it was highly expressed in hepatopancreas. The mRNA expression of SgGST-S1 in hemocytes was significantly up-regulated (P < 0.01) after razor clam was stimulated by peptidoglycan (PGN) or β-1, 3-glucan, but not LPS. In addition, the SgGST-S1 transcript level was also significantly (P < 0.01) induced by exposure of benzo[a]pyrene (B[a]P) or Polybrominated Diphenyl Ethers (PBDE). All the results indicated that SgGST-S1 might serve as an antioxidant enzyme involving in the detoxification cause by both microorganism glycan and organic contaminants., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) is a pattern recognition receptor (PRR) recognizing and binding both LPS and β-1, 3-glucan, playing important roles in innate immunity. In the present study, the single nucleotide polymorphisms (SNPs) were assessed in LGBP gene from scallop Chlamys farreri (designated CfLGBP), and eight SNPs were found in its potential LPS and glucanase binding motif. The locus +7679 with the transition of G-A, which produced an amino acid substitution at codon 360 from a non polar Glycine to polar Serine, was selected to inspect their association with disease resistance/susceptibility to Listonella anguillarum. Three genotypes G/G, G/A and A/A, were revealed at locus +7679, and their frequencies were 89.7%, 7.7% and 2.6% in the resistant stock, while 63.2%, 34.2% and 2.6% in the susceptible stock, respectively. The frequency of genotypes G/G and G/A were significantly different (P < 0.05) between the two stocks. The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfLGBP (G) with G variant at locus +7679 and rCfLGBP (S) with A variant at locus +7679, were elucidated by ELISA assay. The binding affinities of both LPS and β-glucan binding affinity were varied in a dose-dependent manner, where the binding affinity of rCfLGBP (G) was significantly higher than that of rCfLGBP (S) (P < 0.05). The results collectively suggested that the polymorphism of +7679 G/G in CfLGBP possibly enhances the binding activity of LPS and β-glucan, and was associated to disease resistance of scallop against L. anguillarum, which could be a potential marker applied in future selection of scallop with enhanced resistance to L. anguillarum., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sialic acid-binding lectin (SABL) plays crucial role in both innate and adaptive immune responses benefiting from its predominant affinity toward glycan. In the present study, two SABLs from razor clam Solen grandis (designated as SgSABL-1 and SgSABL-2) were identified, and their expression patterns, both in tissues and towards microorganism glycan stimulation, were then characterized. The cDNA of SgSABL-1 and SgSABL-2 was 988 and 1281 bp, containing an open reading frame (ORF) of 744 and 570 bp, respectively, and deduced amino acid sequences showed high similarity to other invertebrates SABLs. Both SgSABL-1 and SgSABL-2 encoded a C1q domain. SgSABL-1 and SgSABL-2 were found to be constitutively expressed in a wide range of tissues with different levels, including mantle, gill, gonad, hemocyte, muscle, and hepatopancreas, and both of them were highly expressed in hepatopancreas. SgSABL-1 and SgSABL-2 could be significantly induced after razor clams were stimulated by acetylated subunits-containing glycan LPS and PGN, suggesting the two SgSABLs might perform potential function of glycan recognition. In addition, SgSABL-2 could also be induced by β-1,3-glucan. All these results indicated that SgSABL-1 and SgSABL-2 might be involved in the immune response against microbe infection and contributed to the pathogens recognition., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Peptidoglycan recognition proteins (PGRPs) play crucial role in innate immunity for both invertebrates and vertebrates, owing to their prominent ability in detecting and eliminating invading bacteria. In the present study, two short PGRPs from mollusk Solen grandis (designated as SgPGRP-S1 and SgPGRP-S2) were identified, and their expression patterns, both in tissues and toward three PAMPs stimulation, were then characterized. The full-length cDNA of SgPGRP-S1 and SgPGRP-S2 was 1672 and 1285 bp, containing an open reading frame (ORF) of 813 and 426 bp, respectively, and deduced amino acid sequences showed high similarity to other members of PGRP superfamily. Both SgPGRP-S1 and SgPGRP-S2 encoded a PGRP domain. The motif of Zn(2+) binding sites and amidase catalytic sites were well conserved in SgPGRP-S1, but partially conserved in SgPGRP-S2. The two PGRPs exhibited different tissue expression pattern. SgPGRP-S1 was highly expressed in muscle and hepatopancreas, while SgPGRP-S2 was highly in gill and mantle. The mRNA expression of SgPGRP-S1 could be induced acutely by stimulation of PGN, and also moderately by β-1,3-glucan, but not by LPS, while expression of SgPGRP-S2 was significantly up-regulated (P < 0.01) when S. grandis was stimulated by all the three PAMPs, though the expression levels were relatively lower than SgPGRP-S1. Our results suggested SgPGRP-S1 and SgPGRP-S2 could serve as pattern recognition receptors (PRRs) involved in the immune recognition of S. grandis, and they might perform different functions in the immune defense against invaders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca(2+) binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln(122)-Pro(123)-Asn(124)) in LvLectin-1, but QPD (Gln(128)-Pro(129)-Asp(130)) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P<0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P<0.01) and 12 h (P<0.05), but the expression level of LvLectin-1 down-regulated significantly (P<0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates IL-2 gene at level of transcription, splicing and translation in vertebrates and plays significant roles in immune system. In this study, an ILF2 homolog was identified from Chinese mitten crab Eriocheir sinensis (designated as EsILF) by expressed sequence tag (EST) analysis. The full-length cDNA of EsILF was of 2159bp, containing a 5' untranslated region (UTR) of 90bp, a 3' UTR of 866bp with a poly (A) tail, and an open reading frame (ORF) of 1203bp encoding a polypeptide of 400 amino acids with the predicted molecular weight of 44.3kDa, which shared 59.6-64.5% identities with vertebrate ILF2. There were a conserved N-terminal RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain in the primary structure, strongly suggesting that EsILF was a homolog of vertebrate ILF2. The mRNA of EsILF was constitutively expressed in all tested tissues of untreated crabs, including hepatopancreas, gill, gonad, muscle, heart and hemocytes, with highest expression in muscle and relative lower levels in hemocytes and gonad. The mRNA expression of EsILF in hemocytes was regulated differently after the crabs were stimulated by bacteria Listonella anguillarum and fungi Pichia pastoris GS115. The expression level was significantly (P<0.05) down-regulated to 0.35- and 0.29-fold compared with blank group at 6h and 12h after the stimulation of L. anguillarum, while P. pastoris significantly (P<0.05) up-regulated the expression level to 3.2-fold compared with the blank group at 6h post treatment. The results indicated that EsILF was involved in the immune response of crab toward both L. anguillarum and P. pastoris., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of proteins that can bind pathogen-associated molecular patterns (PAMPs) and microorganisms through the recognition of carbohydrates, thus they are directly involved in innate defense mechanisms as part of the acute-phase response to infection. In this study, the cDNA of a novel C-type lectin (designated as AiCTL-7) was cloned from bay scallop Argopecten irradians by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of AiCTL-7 was of 651 bp containing a 525 bp open reading frame which encoded a signal peptide of 15 residues and a conserved carbohydrate-recognition domain (CRD) of 174 residues with the EPD and WSD motifs instead of the invariant EPN and WND motifs for determining the carbohydrate-binding specificity and constructing Ca(2+)-binding site 2 in vertebrates. The deduced amino acid sequence of AiCTL-7 CRD shared homology not only with the CRDs of C-type lectins in mollusks, but also with the fish lectin CRDs. The mRNA transcripts of AiCTL-7 were mainly detected in the tissue of hepatopancreas and also marginally detectable in kidney, gonad, hemocytes, heart and adductor of health scallop. After challenge with fungi Pichia pastoris GS115 and Gram-negative bacteria Listonella anguillarum, the relative expression level of AiCTL-7 was up-regulated significantly in hepatopancreas and hemocytes. The CRD of AiCTL-7 was recombined and expressed in Escherichia coli, and the recombinant protein (rAiCTL-7) aggregated P. pastoris remarkably in a Ca(2+)-dependent manner, and this agglutination could be inhibited by d-mannose, but not by d-galactose or β-1,3-glucan. However, rAiCTL-7 displayed no obvious agglutinating activity against L. anguillarum. These results collectively indicated that AiCTL-7 was involved in the primitive acute-phase response to microbial invasion as an important pattern recognition receptor (PRR) in the innate immune system of scallops., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel C-type lectin gene from scallop Argopecten irradians (designated as AiCTL-6) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of AiCTL-6 was 1080 bp. The open reading frame encoded a polypeptide of 307 amino acids, including a signal sequence and a C-type lectin-like domain (CTLD) of 150 amino acid residues longer than any usual CTLD. It contained six conserved cysteine residues involved in the formation of three internal disulfide bridges and an EPD (Glu(269)-Pro(270)-Asp(271)) motif at the Ca(2+)-binding site 2. The deduced amino acid sequence of AiCTL-6 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, AiCTL-6 mRNA was found mainly in hepatopancreas and gill, and marginally expressed in other tissues. After the scallops were challenged by Listonella anguillarum for 6 h, the mRNA expression of AiCTL-6 was up-regulated significantly to 7.2-fold compared to the blank group. While at 9 h post Micrococcus luteus challenge, its expression level was 60.1 times higher than that of the blank group. The functional activity of AiCTL-6 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiCTL-6 could agglutinate Gram-negative bacteria E. coli TOP10F', Gram-positive bacteria M. luteus and Staphylococcus aureus. These results collectively suggested that AiCTL-6, as a novel member of C-type lectin family, contributed to the host defense mechanisms against invading microorganism in A. irradians., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

Dopamine beta hydroxylase (DBH) is a critical enzyme in the biosynthesis of catecholamines, and also plays an important role in complex neuroendocrine-immune regulatory network. In the present study, the cDNA encoding dopamine beta hydroxylase (designated CfDBH) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The full-length cDNA of CfDBH was of 2302 bp, containing a 5' untranslated region (UTR) of 32 bp, a 3' UTR of 461 bp with a poly (A) tail, and an open reading frame (ORF) of 1809 bp encoding a polypeptide of 603 amino acids. The deduced amino acid sequence of CfDBH contained a signal peptide, a DOMON domain and a Cu2&#95;monooxygen domain, and it shared 39.4%-42.9% similarity with other reported DBHs. The conserved domains in CfDBH and the amino acid sequence similarity with other DBHs strongly suggested that it was a homologue of DBH in C. farreri. The mRNA expression of CfDBH in various tissues and its temporal expression in haemocytes of scallops stimulated with LPS were ascertained by Quantitative real-time RT-PCR. The mRNA transcripts of CfDBH were detected in all the examined tissues with the highest expression level in hepatopancreas. The expression level of CfDBH in haemocytes was up-regulated after LPS stimulation and increased to hundreds fold higher than that of the control group at 12 h, and then decrease significantly to 0.36-fold and 0.31-fold at 24 h and 48 h respectively. The results suggested pathogen infections significantly induced the expression level of CfDBH, and the activation of DBH could influence the immune response of scallop C. farreri through changing the concentration of catecholamines., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coli, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C. farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks., (Copyright 2010 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity. The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues. The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively. But its expression level did not change significantly during peptidoglycan (PGN) stimulation. The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3). The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way. The agglutinating activity could be inhibited by d-mannose, LPS and glucan, but not by d-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs., (2010 Elsevier Ltd. All rights reserved.)

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans.