Risk Assessment Model System for Aquatic Animal Introduction Based on Analytic Hierarchy Process (AHP).

The spread of invasive species (IS) has the potential to upset ecosystem balances. In extreme cases, this can hinder economical utilization of both aquatic (fisheries) and terrestrial (agricultural) systems. As a result, many countries regard risk assessment of IS as an important process for solving the problem of biological invasion. Yet, some IS are purposefully introduced for what is seen as their potential economic benefits. Thus, conducting IS risk assessments and then formulating policies based on scientific information will allow protocols to be developed that can reduce problems associated with IS incursions, whether occurring purposefully or not. However, the risk assessment methods currently adopted by most countries use qualitative or semiquantitative methodologies. Currently, there is a mismatch between qualitative and quantitative assessments. Moreover, most assessment systems are for terrestrial animals. What is needed is an assessment system for aquatic animals; however, those currently available are relatively rudimentary. To fill this gap, we used the analytic hierarchy process (AHP) to build a risk assessment model system for aquatic IS. Our AHP has four primary indexes, twelve secondary indexes, and sixty tertiary indexes. We used this AHP to conduct quantitative risk assessments on five aquatic animals that are typically introduced in China, which have distinct biological characteristics, specific introduction purposes, and can represent different types of aquatic animals. The assessment results show that the risk grade for Pterygoplichthys pardalis is high; the risk grade for Macrobrachium rosenbergii, Crassostrea gigas, and Trachemys scripta elegans is medium; and the grade risk for Ambystoma mexicanum is low. Risk assessment of the introduction of aquatic animals using our AHP is effective, and it provides support for the introduction and healthy breeding of aquatic animals. Thus, the AHP model can provide a basis for decision-making risk management concerning the introduction of species.

Insulin-like growth factor binding protein 5b of Trachinotus ovatus and its heparin-binding motif play a critical role in host antibacterial immune responses via NF-κB pathway.

Introduction: Insulin-like growth factor binding protein 5 (IGFBP5) exerts an essential biological role in many processes, including apoptosis, cellular differentiation, growth, and immune responses. However, compared to mammalians, our knowledge of IGFBP5 in teleosts remains limited. Methods: In this study, TroIGFBP5b, an IGFBP5 homologue from golden pompano (Trachinotus ovatus) was identified. Quantitative real-time PCR (qRT-PCR) was used to check its mRNA expression level in healthy condition and after stimulation. In vivo overexpression and RNAi knockdown method were performed to evaluate the antibacterial profile. We constructed a mutant in which HBM was deleted to better understand the mechanism of its role in antibacterial immunity. Subcellular localization and nuclear translocation were verified by immunoblotting. Further, proliferation of head kidney lymphocytes (HKLs) and phagocytic activity of head kidney macrophages (HKMs) were detected through CCK-8 assay and flow cytometry. Immunofluorescence microscopy assay (IFA) and dual luciferase reporter (DLR) assay were used to evaluate the activity in nuclear factor-κB (NF-κß) pathway. Results: The TroIGFBP5b mRNA expression level was upregulated after bacterial stimulation. In vivo, TroIGFBP5b overexpression significantly improved the antibacterial immunity of fish. In contrast, TroIGFBP5b knockdown significantly decreased this ability. Subcellular localization results showed that TroIGFBP5b and TroIGFBP5b-δHBM were both present in the cytoplasm of GPS cells. After stimulation, TroIGFBP5b-δHBM lost the ability to transfer from the cytoplasm to the nucleus. In addition, rTroIGFBP5b promoted the proliferation of HKLs and phagocytosis of HKMs, whereas rTroIGFBP5b-δHBM, suppressed these facilitation effects. Moreover, the in vivo antibacterial ability of TroIGFBP5b was suppressed and the effects of promoting expression of proinflammatory cytokines in immune tissues were nearly lost after HBM deletion. Furthermore, TroIGFBP5b induced NF-κß promoter activity and promoted nuclear translocation of p65, while these effects were inhibited when the HBM was deleted. Discussion: Taken together, our results suggest that TroIGFBP5b plays an important role in golden pompano antibacterial immunity and activation of the NF-κß signalling pathway, providing the first evidence that the HBM of TroIGFBP5b plays a critical role in these processes in teleosts.

Molecular characterization and functional analysis of tartrate-resistant acid phosphatase (ACP5) gene in red drum (Sciaenops ocellatus).

Tartrate-resistant acid phosphatase (ACP5) plays an important biological function in immune defense and is highly expressed in activated macrophages, osteoclasts and dendritic cells. In teleost, the functionality of ACP5 remains to be revealed. In this study, we cloned and identified SoACP5 from red drum (Sciaenops ocellatus) and analyzed its function in vivo and in vitro. The open reading frame of SoACP5 is 1002 bp in length, encoding 333 amino acids. SoACP5 shares high sequence identities (96.70%-49.25%) with ACP5 of other species. The SoACP5 mRNA was widely distributed in collected tissues of healthy red drum, and with the maximum in gills. The expression of SoACP5 increased significantly in vivo following challenge with Edwardsiella tarda. Moreover, the recombinant SoACP5 protein (rSoACP5) was purified with his-tag band resin columns, and confirmed to have phosphatase activity which was optimal at pH 5 and 55 °C. Various metal ions (K+, Zn2+, Mn2+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+) have different effects on phosphatase activity. rSoACP5 induced the cellular proliferation of peripheral blood leukocytes. The over-expression and knockdown of SoACP5 in vivo had a significant effect on bacterial proliferation. Furthermore, both of the antibacterial activity and phosphatase activity were decreased when the reducedSoACP5 was oxidized by H2O2. In summary, the present study indicated that SoACP5 is likely involved in host defense against bacterial infection in S. ocellatus.

Bactericidal permeability-increasing protein/LPS-binding protein (BPI/LBP) enhances resistance of golden pompano Trachinotus ovatus against bacterial infection.

Antimicrobial peptides are crucial components of innate immunity against microbial invasions. As a kind of antimicrobial peptides, bactericidal permeability-increasing protein (BPI)/lipopolysaccharide-binding protein (LBP) play vital roles in defending the host against gram-negative bacteria. In the current study, a novel BPI/LBP from Trachinotus ovatus (TroBPI/LBP) was characterized. The full length of TroBPI/LBP cDNA sequence is 1434 bp, which contained 477 amino acids. Multiple amino acid alignments of TroBPI/LBP shows 34.07%-84.49% identity with other fish BPI/LBP. Similar to other BPI/LBP, TroBPI/LBP also possesses an N-terminal signal peptide, a BPI/LBP/CETP N-terminal domain, and a BPI/LBP/CETP C-terminal domain. In vitro, the recombinant protein of TroBPI/LBP showed effective bacterial depression activity and binding activity to gram-negative bacteria. In vivo, TroBPI/LBP was constitutively expressed in tested tissues, and the highest expression level was in liver. Following Vibrio alginolyticus stimulation, the mRNA expression of TroBPI/LBP was significantly upregulated in immune-related tissues, and peaked at 12 h post-infection, which confirmed that TroBPI/LBP was highly sensitive to V. alginolyticus stimuli. Furthermore, functional analyses showed that the overexpression of TroBPI/LBP could enhance the ability of fish to against V. alginolyticus infection, and the knockdown of TroBPI/LBP significantly diminished bacterial clearance capacity post-infection. Therefore, these results suggest that TroBPI/LBP may play an important role in host defense against bacterial infection.

Cromileptes altivelis microRNA Transcriptome Analysis upon Nervous Necrosis Virus (NNV) Infection and the Effect of cal-miR-155 on Cells Apoptosis and Virus Replication.

MicroRNAs (miRNAs) could regulate various biological processes. Nervous necrosis virus (NNV) is one of the primary germs of the Humpback grouper (Cromileptes altivelis), a commercial fish of great importance for Asian aquaculture. However, there is limited available information on the host-virus interactions of C. altivelis. miRNAs have been shown to play key roles in the host response to infection by a variety of pathogens. To better understand the regulatory mechanism of miRNAs, we constructed miRNA transcriptomes and identified immune-related miRNAs of C. altivelis spleen in response to NNV infection. Reads from the three libraries were mapped onto the Danio rerio reference genome. As a result, a total of 942 mature miRNAs were determined, with 266 known miRNAs and 676 novel miRNAs. Among them, thirty-two differentially expressed miRNAs (DEmiRs) were identified compared to the PBS control. These DEmiRs were targeted on 895 genes, respectively, by using miRanda v3.3a. Then, 14 DEmiRs were validated by qRT-PCR and showed consistency with those obtained from high-throughput sequencing. In order to study the relationship between viral infection and host miRNA, a cell line from C. altivelis brain (CAB) was used to examine the expressions of five known DEmiRs (miR-132-3p, miR-194a, miR-155, miR-203b-5p, and miR-146) during NNV infection. The results showed that one miRNA, cal-miRNA-155, displayed significantly increased expression in response to the virus infection. Subsequently, it was proved that overexpression of cal-miR-155 enhanced cell apoptosis with or without NNV infection and inhibited virus replication in CAB cells. Oppositely, the cal-miRNA-155 inhibitor markedly suppressed apoptosis in CAB cells. The results of the apoptosis-related genes mRNA expression also showed the regulation of cal-miR-155 on the apoptosis process in CAB cells. These findings verify that miR-155 might exert a function as a pro-apoptotic factor in reply to NNV stimulation in CAB cells and help us further study the molecular mechanisms of the pathogenesis of NNV in C. altivelis.

Cathepsin C (CTSC) contributes to the antibacterial immunity in golden pompano (Trachinotus ovatus).

Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys251, His397, and Asn419). It shares high homology (69.47%-90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn2+ and Ca2+ but inhibited by Fe2+ and Cu2+. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1ß), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

Interferon regulatory factor 7 contributes to the host response during Vibrio harveyi infection in the golden pompano Trachinotus ovatus.

Vibrio harveyi is regarded as serious pathogen for marine fishes. However, host defense mechanisms involved in V. harveyi infection remain incompletely defined. The transcription factor IFN regulatory factor 7 (IRF7) is largely associated with host defense against viral infections, and the role of IRF7 during V. harveyi infection in fish has not been well illuminated previously. In this study, IRF7 from golden pompano (Trachinotus ovatus) was characterized (TroIRF7). The TroIRF7 gene is 1323 bp, which encodes 440 amino acid residues. Multiple amino acid alignments of TroIRF7 shows 30.37%-80.18% identity with other fish IRF7s, including Epinephelus coioides (80.18%), Larimichthys crocea (79.72%), Collichthys lucidus (79.26%), Miichthys miiuy (79.26%), Channa argus (78.77%), Cynoglossus semilaevis (72.67%), and Gadus morhua (65.23%). Like other IRF7s, TroIRF7 also contains 3 conserved domains: an N-terminal DNA-binding domain (DBD), an IRF association domain (IAD), and a C-terminal serine-rich domain (SRD). In the DBD, 4-5 conserved tryptophans were observed, which is a characteristic unique to all fish IRF7 members. TroIRF7 was constitutively expressed, with high levels in gill, head kidney, spleen, skin, and intestine. V. harveyi infection-induced TroIRF7 transcripts significantly up-regulation and translocation to the nucleus. TroIRF7 overexpression promote the fish to inhibit the replication of V. harveyi. And TroIRF7 knockdown led to decreased bacterial clearance in fish tissue. Furthermore, over-expression of TroIRF7 resulted in an increased production of interferon a3 and IFN signaling molecule in the spleen, suggesting that V. harveyi activates the IRF7- IFN pathway. These results suggest that TroIRF7 is an important component of immune responses against V. harveyi infection.

Major histocompatibility complex class I (MHC Iα) of Japanese flounder (Paralichthys olivaceus) plays a critical role in defense against intracellular pathogen infection.

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda, which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection.

Granulocyte colony stimulating factor (GCSF) of Japanese flounder (Paralichthys olivaceus): Immunoregulatory property and anti-infectious function.

Granulocyte colony stimulating factor (GCSF) is a key regulator of neutrophil production, and plays a vital role in immune response of mammals and teleost against pathogen. Sequences of GCSF were identified in several teleost species, however, the function and activity of GCSF in teleost remain largely unknown. In this study, we examined the biological activity and the immunomodulatory property of a GCSF homologue, PoGCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoGCSF possesses conserved structural characteristics of GCSF proteins, including a signal peptide and a typical IL-6 domain. The expression of PoGCSF was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoGCSF increased the capability of host cells to defense against pathogen infection and enhanced the expression of immune related genes. The knockdown of PoGCSF attenuated the ability of host cells to eliminate pathogenic bacteria. In vivo results showed that overexpression of PoGCSF promoted the host defense against invading pathogenic microorganism. Collectively, this study is the first report about the immunoregulatory property and anti-infectious immunity of GCSF in teleost. These findings suggested that PoGCSF serves as an immune-related cytokine and plays an important role in the immune defense system of Japanese flounder.

High mobility group box 2 of black rockfish Sebastes schlegelii: Gene cloning, immunoregulatory properties and antibacterial effect.

High-mobility group box 2 (HMGB2) is a non-histone chromosomal protein that involved diverse functions such as transcriptional regulation and innate immune responses in mammalian. In teleost, very limited studies on HMGB2 proteins have been documented. Black rockfish (Sebastes schlegelii) is an economic fish species and cultured worldwide. However, the study of black rockfish about immunology is very scarce. In the present study, a HMGB2 homologue gene (SsHMGB2) was identified and characterized in black rockfish. The open reading frame of SsHMGB2 is 648 bp, and the deduced amino acid sequence of SsHMGB2 shares 74.4%-91.2% overall sequence identities with the HMGB2 proteins of several fish species. In silico analysis identified several conserved features, including two basic HMG boxes and an acidic C-terminal tail composed of 24 Asp/Glu residues. Expression of SsHMGB2 occurred in multiple tissues and was upregulated during pathogens infection. Recombinant SsHMGB2 (rSsHMGB2) exhibited apparent binding activities against DNA. In vivo studies showed that the expressions of multiple immune-related genes in head kidney were significantly enhanced when black rockfish were treated with rSsHMGB2. Furthermore, rSsHMGB2 reduced pathogen dissemination and replication in fish kidney and spleen. Taken together, these results suggest that SsHMGB2 possesses apparent immunoregulatory properties and played a role in fighting bacterial infection.

Global discovery of small RNAs in the fish pathogen Edwardsiella piscicida: key regulator of adversity and pathogenicity.

Recently, bacterial small RNA (sRNA) has been shown to be involved as a key regulator in stress responses. sRNAs of Edwardsiella piscicida, an important aquatic pathogen, are not well characterized to date. In this study, using RNA-seq technology, we globally found and identified sRNA candidates expressed from E. piscicida grown in normal LB medium, acid pressure, iron deficiency stress, and oxidation pressure. A total of 148 sRNAs were found, including 19 previously annotated sRNAs and 129 novel sRNA candidates by searching against the Rfam database. Compared in normal condition, the expression of 103 sRNAs (DEsRNA, differentially expressed sRNA) and 1615 mRNAs (DEmRNAs, differentially expressed mRNA) showed significant differences in three stress sample. Based on the prediction by IntaRNA and relational analysis between DEsRNAs and DEmRNAs, 103 DEsRNAs were predicted to regulate 769 target mRNAs. Pleiotropic function of target DEmRNAs indicated that sRNAs extensively participated in a variety of physiological processes, including response to adversity and pathogenicity, the latter was further confirmed by infection experiment. A large number transcription factors appeared in target genes of sRNAs, which suggested that sRNAs likely deeply interlaced within complex gene regulatory networks of E. piscicida. Moreover, 49 Hfq-associated sRNAs were also identified in this study. In summary, we globally discovered sRNAs for the first time in pathogenic bacteria of fish, and our findings indicated that sRNAs in E. piscicida have important roles in adaptation to environmental stress and pathogenicity. These results also provide clues for deciphering regulation mechanism of gene expression related to physiological response and pathogenicity.