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Transcriptomic studies in fish have considerably contributed to understanding the host responses under viral hemorrhagic septicemia virus (VHSV) infection. However, changes from the perspective of the pathogen and host-pathogen interactions have been relatively underestimated. Given that VHSV genes can be poly-adenylated during replication, this study investigated the global changes from both VHSV and host perspectives using the host transcriptomic data. To achieve this, we utilized transcriptomic data from VHSV infected flounder obtained at different water temperatures (13 °C and 20 °C). We collected the sequence reads belonging to VHSV through an bioinformatic pipeline developed especially for this study. The VHSV reads were used to construct the consensus reference genome and to investigate the expression of VHSV genes and the frequency of variants under different water temperatures. In addition, both linear and logarithmic scales of VHSV transcription levels, along with host transcriptomes, were used to understand pathogen-host interaction through weighted correlation network analysis (WGCNA). The results revealed that VHSV transcription can exceed 5% of host transcriptome during the infection. Single nucleotide variants (SNVs) appeared more frequently in 13 °C groups than in 20 °C groups. While VHSV can replicate at both 13 °C and 20 °C, host transcriptomic responses were notably different, with stronger immune responses and more frequent VHSV genetic changes observed at a lower temperature. This suggests that VHSV infection at low water temperatures significantly influences both the host transcriptional changes and pathogen. Through WGCNA, numerous genes in the module that correlated with VHSV reads on a linear scale were found to be related to cytoskeleton modulation and viral activity. By contrast, the gene module (black) correlated with VHSV reads on a logarithmic scale was strongly associated with host immune responses, such as TNF signaling, necroptosis, and the NF-kappa B signaling pathway. The dual RNA-seq approaches developed in this study will immensely enhance our understanding of host-pathogen interactions across different temperatures. • This study developed a dual RNA-seq pipeline for VHSV and olive flounder using the standard host transcriptome. • VHSV infection at low temperatures may lead to more frequent variants. • VHSV transcription can account for more than 5% of the host transcriptome during infection. • Lower water temperatures had a substantial effect on both host transcription and the pathogen. • The biological pathways, including immune-related pathways, activated by VHSV replication may differ in sensitivity and responsiveness. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) play a pivotal role in activating naïve T-cells and bridging innate and adaptive immunity. This study aimed to identify and characterize CD83 and CD276 as potential markers for DCs in olive flounder (Paralichthys olivaceus). Specific antibodies against these markers were developed and used to analyze their distribution in peripheral blood leukocytes (PBLs) and intestinal tissues. Flow cytometry and immunohistochemistry revealed that CD83 and CD276 are expressed on DCs, with peak expression observed one week after oral administration of an inactivated viral hemorrhagic septicemia virus (VHSV) vaccine. Gene expression analysis further demonstrated significant activation of immune-related genes, including CD3, IgM, and TLRs, indicating that oral vaccine administration effectively activates the intestinal mucosal immune system. These findings provide valuable insights into fish immune responses and establish CD83 and CD276 as promising DC markers, contributing to the development of mucosal vaccine strategies in aquaculture. • An oral vaccine for olive flounder (Paralichthys olivaceus) against VHSV was developed. • It was shown that oral vaccines induce mucosal immunity in olive flounder. • Immune gene expressions were identified as promising markers for assessing mucosal immune response. [ABSTRACT FROM AUTHOR]

Treatments to control infectious diseases in aquacultured fish commonly focus on the use of antibiotics (which are ineffective against viruses) and inorganic disinfectants. Currently, antiviral treatments are not available for use in aquaculture. The use of plasmid vectors to overexpress antiviral proteins in fish has been reported, with positive results against viral infections. The aim of this work was to overexpress the antiviral proteins interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) and natural killer enhancing factor (NKEF) using plasmid vectors (pIFIT5 and pNKEF) as novel therapeutic agents against viral infections. The antiviral activity of pIFIT5 against viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) was evaluated in vitro in epithelioma papulosum cyprinid and Chinook salmon embryo cell lines. Moreover, pNKEF antiviral activity against IPNV was also evaluated. Results showed that pIFIT5 had antiviral activity against both viruses, but pNKEF did not protect against IPNV. In addition, we evaluated the use of pIFIT5 and pNKEF constructs as therapeutics in rainbow trout. Biodistribution assays demonstrated systemic distribution of the IFIT5-TFP1 and NKEF-TFP1 proteins in rainbow trout injected with the plasmids, and pIFIT5 and pNKEF triggered an immune response in the injected fish. In a pilot study, we evaluated the level of protection conferred by pIFIT5 and pNKEF against VHSV in rainbow trout, and we observed 10 % survival for pIFIT5 compared to pTFP. Together, these results suggest the potential of pIFIT5 as therapeutic agent in aquaculture. • Overexpression of proteins IFIT5 and NKEF using plasmid vectors as antivirals. • pIFIT5 exerts antiviral activity against two different kind of viruses in vitro. • pIFIT5 and pNKEF triggered an immune response in vivo. • The antiviral activity of pIFIT5 and pNKEF against VHSV was probed in rainbow trout. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 2 (IRF2) is a member of the IRF family that is specifically involved in diverse immune responses via interferon (IFN)/IRF-dependent signaling pathways. In this study, IRF2 of Epinephelus akaara (EAIRF2) was identified and characterized by evaluating its structural and functional properties. EAIRF2 showed the highest homology with IRF2 of Epinephelus coioides and clustered with teleosts in the phylogenetic tree. The highest level of EAIRF2 mRNA was found in the blood under normal physiological conditions. In the immune challenge experiment, significant transcriptional modulation of EAIRF2 upon lipopolysaccharide (LPS), polyinosinic: polycytidylic acid (poly I:C), and nervous necrosis virus (NNV) challenge were observed. The subcellular localization assay confirmed the role of EAIRF2 as a transcription factor by revealing its specific nuclear localization. To elucidate its functional implications in antiviral defense, EAIRF2 was overexpressed in fathead minnow cells, which were subsequently infected with viral hemorrhagic septicemia virus (VHSV). Notably, cells overexpressing EAIRF2 exhibited a significant reduction in the transcription of VHSV genes. Concurrently, the genes associated with the IFN/IRF signaling pathway were upregulated. Furthermore, the Hoechst and propidium iodide dual staining assay, water-soluble tetrazolium-1 (WST-1) assay, and transcriptional analysis of B-cell lymphoma 2-associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) indicated that EAIRF2 possesses anti-apoptotic properties during viral infection and poly I:C treatment. Additionally, EAIRF2 overexpression in murine macrophages induced M1 polarization and augmented relative marker gene expression. Collectively, these findings suggest that EAIRF2 is a pivotal immune-related gene, specifically implicated in the IFN/IRF-mediated antiviral defense mechanism, apoptotic signaling pathway, and activation of macrophage-mediated immune responses in Epinephelus akaara. The finding of this study enhances our understanding of IRF2's function in teleost immunity and presents potential avenues for developing therapeutic strategies against viral infections and other immune-related conditions in aquaculture species. • EAIRF2 expression was upregulated in response to Poly I:C, LPS, and NNV stimulation. • EAIRF2 demonstrated antiviral activity against VHSV. • EAIRF2 restricted virus-mediated apoptosis. • EAIRF2 overexpression polarized murine macrophage towards M1 phase. [ABSTRACT FROM AUTHOR]

The replication and mortality caused by the viral hemorrhagic septicemia virus (VHSV) in fish vary depending on temperature. VHSV causes mortality at the temperatures below 15 °C, while infection is not established in olive flounder at temperatures above 25 °C. However, how VHSV infection manifests at the cellular level under different temperature conditions is not understood. In this study, we aimed to elucidate the mechanism by which VHSV infection is controlled by comparing viral replication and immune responses in vitro and in vivo. In the in vitro experiment, viral mRNA levels on day 5 post-challenge differed more than 10-fold between temperatures, highest at 15 °C, followed by 20 °C, and lowest at 25 °C, with replication gradually increasing over 5 days. In vivo , replication peaked by day 2 and then declined at all temperatures. VHSV infection showed a controlled tendency in the fish, whereas the virus exhibited a continuously increasing infection pattern for up to 5 days in olive flounder spleen cell cultures at a temperature of 25 °C. The level of viral infection within the cells (25 °C) was lower compared to conditions below 20 °C; however, the expression levels of interferon-related genes and pro-inflammatory cytokine genes were relatively low. The expression of ISG15 and Mx genes in spleen (25 °C) was significantly high at 24 h post challenge in the in vivo experiment. • At 25°C, VHSV infection increased for 5 days in spleen cultures (in vitro) but was controlled in olive flounder (in vivo). • At 25°C, ISG (IFN-1β, ISG15, Mx) and pro-inflammatory cytokine (IL-1β, IL6, TNFα) gene levels were lower than below 20°C in vitro. • ISG15 and Mx gene expression (25°C) was high at 24 h post challenge in olive flounder spleen (in vivo). [ABSTRACT FROM AUTHOR]

Nucleoredoxin (NXN) is a prominent oxidoreductase enzyme, classified under the thioredoxin family, and plays a pivotal role in regulating cellular redox homeostasis. Although the functional characterization of NXN has been extensively studied in mammals, its role in fish remains relatively unexplored. In this study, the NXN gene from Planiliza haematocheilus (PhNXN) was molecularly and functionally characterized using in silico tools, expression analyses, and in vitro assays. The predicted protein sequence of PhNXN contained 418 amino acids with an anticipated molecular mass of 47.53 kDa. It comprised a highly conserved 188CPPC191 catalytic motif in the central NXN domain and two thioredoxin-like domains enriched with conserved Cys residues. PhNXN protein was primarily localized in the cytoplasm and nucleus of the cells. The spatial and temporal expression analyses of PhNXN mRNA showed the highest expression level in the brain under normal physiological conditions, while a significant modulation was detected in the blood and head kidney following immunostimulation with polyinosinic: polycytidylic acid, lipopolysaccharides, and Lactococcus garvieae. Recombinant PhNXN protein exhibited DPPH radical scavenging, thiol-dependent disulfide reduction, and cupric ion reduction activities. Additionally, PhNXN overexpression significantly suppressed oxidative stress-induced cell death, heavy metal cation-induced reactive oxygen species production, and viral hemorrhagic septicemia virus-induced cellular apoptosis in fish cells. Furthermore, PhNXN overexpression in RAW 264.7 cells demonstrated notable nitric oxide scavenging activity, M2-type polarization, and anti-inflammatory effect. Collectively, the overall findings of the study highlight the antioxidant and immunological functions of PhNXN in red-lip mullet. • Nucleoredoxin from P. haematocheilus was molecularly and functionally characterized. • PhNXN was ubiquitously expressed in all the examined tissues. • Relative PhNXN expression was significantly upregulated upon immune challenges. • Recombinant PhNXN exhibited antioxidant and metal cation reduction activities. • Overexpressed PhNXN enhanced anti-inflammatory effects while suppressing ROS production and cellular apoptosis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine with critical roles in inflammation, cell survival, and defense. As a member of the TNF superfamily, it exerts its effects by binding to transmembrane receptors and triggering various downstream signaling pathways. Although TNF-α′s involvement in antiviral responses in mammals is well-established, its role in teleost remains poorly understood. This study investigated the contribution of TNF-α1 to antiviral immunity in zebrafish using a tnf-α1 (−/−) knockout (KO) line. We challenged both wild-type and tnf-α1 (−/−) zebrafish with viral hemorrhagic septicemia virus (VHSV) at both embryonic and adult stages. Mortality was observed at 4 days post-infection (dpi) in tnf-α1 -deficient adult fish challenged with 5 × 106 TCID 50 (VHSV) and at 5 dpi in adult wild fish challenged with the same concentration. In addition, tnf-α1 (−/−) KO adult fish reached the maximum mortality of 100 % at 20 dpi, whereas wild adult fish reached 54 % mortality at the same time point. This increased susceptibility to early mortality was associated with a higher viral burden and altered expression of key immune genes, including the pro-inflammatory cytokines il-6 and il-1β, the anti-inflammatory cytokine il-10 , and interferon-related genes such as irf1 and ifn-γ. Our findings demonstrate the crucial role of TNF-α1 in antiviral defense mechanisms in zebrafish and provide valuable insights into the functional conservation of TNF-α signaling across vertebrate species. This knowledge may contribute to the development of strategies to combat viral diseases in fish. • TNF-α1 knockout zebrafish were significantly more susceptible to VHSV infection than wild-type fish. • Knockout zebrafish showed earlier mortality and higher viral loads, highlighting TNF-α1's role in viral replication control. • TNF-α1 absence altered immune gene expression significantly during viral infection. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a key adaptor molecule in tumor necrosis factor receptor signaling complexes, facilitating downstream immune-related signaling cascades. This study aimed to elucidate its function in teleost fish by characterizing the TRAF2 homolog of the red-spotted grouper (Epinephelus akaara, EaTraf2). The open reading frame of EaTraf2 encodes a putative protein of 520 amino acids, containing several characteristic domains of TRAF2. These structural features of EaTraf2 are conserved across diverse organisms, with a relatively higher sequence identity to TRAF2 orthologs from other bony fish. Transcriptional analysis demonstrated that EaTraf2 was ubiquitously expressed in all examined tissues, with the highest level observed in blood. Upon immune challenge, EaTraf2 expression significantly increased in the early stages of stimulation in both blood and spleen. Subcellular localization analysis revealed that EaTraf2 is predominantly localized in the cytoplasm. Overexpression of EaTraf2 in fathead minnow (FHM) cells resulted in elevated levels of interferon and inflammation-associated genes following viral hemorrhagic septicemia virus (VHSV) infection, along with reduced viral gene expression. This provided compelling evidence that EaTraf2 possesses antiviral properties. Furthermore, EaTraf2 demonstrated the ability to promote cell death induced by oxidative stress. Additionally, luciferase reporter assays revealed that EaTraf2 activates the NF-κB signaling pathway upon poly(I:C) stimulation and the Jun N-terminal kinase (JNK) signaling pathway in response to H 2 O 2 treatment. Overall, our study elucidated the role of EaTraf2 in regulating innate immune responses and mediating stress-induced cell death. These findings enhance our understanding of TRAF2 in fish and may contribute to improved health management strategies in finfish aquaculture. • EaTraf2 mRNA was highly expressed in immune-related tissues and significantly upregulated in response to immune stimulations. • EaTraf2 protein was predominantly localized in the cytoplasm. • Overexpression of EaTraf2 reduced viral replication via NF-κB pathway. • Overexpression of EaTraf2 promoted oxidative stress-induced cell death via JNK pathway. [ABSTRACT FROM AUTHOR]

The tripartite motif-containing (TRIM) superfamily is the largest family of RING-type E3 ubiquitin ligases that is conserved across the metazoan kingdom. Previous studies in mammals have demonstrated that TRIM59 possesses ubiquitin-protein ligase activity and acts as a negative regulator of NF-κB signaling. However, TRIM59 has rarely been characterized in fish. This study aimed to characterize TRIM59 from Epinephelus akaara (Eatrim59) and elucidate its structural features, expression patterns, and functional properties in innate immune responses and in the regulation of apoptosis. Eatrim59 is composed of 406 amino acids with a molecular weight of 45.84 kDa and a theoretical isoelectric point of 5.25. It comprises a conserved RING domain, a B-box motif, and a coiled-coil region. Subcellular localization analysis revealed that Eatrim59 was localized in the endoplasmic reticulum. Eatrim59 was ubiquitously expressed in all tissues examined, with the highest relative expression detected in the blood, followed by the brain and spleen. Temporal expression of Eatrim59 was dynamically regulated in response to in vivo immune stimulation by Toll-like receptor ligands and nervous necrosis virus infection. In FHM cells overexpressing Eatrim59, an increase in viral replication was observed upon infection with the Viral hemorrhagic septicemia virus. This phenomenon is attributed to Eatrim59-mediated downregulation of interferon, pro-inflammatory cytokines, and other antiviral pathways. Moreover, macrophages stably overexpressing Eatrim59 exhibited a decrease in nitric oxide production and the formation of a filamentous actin structure upon lipopolysaccharide stimulation, indicating dampened M1 polarization. Furthermore, a decrease in apoptosis was observed in Eatrim59-overexpressing FHM cells under oxidative stress induced by H 2 O 2. In conclusion, these findings demonstrate the multifaceted role of Eatrim59 as a regulator of innate immune response and apoptosis in E. akaara. • EaTrim59 expression was significantly regulated in response to LPS, poly (I:C), and NNV stimulation. • EaTrim59 overexpression enhanced the viral replication upon VHSV infection and inhibited H 2 O 2 -induced apoptosis. • Eatrim59 overexpression dampened M1 polarization of murine macrophages. [ABSTRACT FROM AUTHOR]

Genomic prediction utilizes relationships between phenotypes and thousands of genetic markers dispersed across the genome of a species under selection to estimate an individual's breeding value. Viral hemorrhagic septicemia (VHS) is a devastating disease that causes high mortality in the olive flounder (Paralichthys olivaceus). Selection of VHS resistant individuals using traditional pedigree-based approaches is slow and ineffective; therefore, we investigated the potential of genomic selection to identify superior VHS-resistant individuals. In this study, various statistical models and algorithms, including the multilayer perceptron (MLP) and convolutional neural network (CNN), were compared for their prediction accuracy of breeding values. Other models assessed include pedigree-based best linear unbiased prediction (PBLUP), genomic best linear unbiased prediction (GBLUP), Bayesian A (BA), Bayesian B (BB), Bayesian C (BC), Bayesian Lasso (BL), Bayesian ridge regression (BRR), elastic net (EN), ridge regression (RR), and random forest (RF). These models were assessed for their ability to predict VHS resistance based on genomic data obtained from high-quality 70 K single nucleotide polymorphism (SNP) Affymetrix® Axiom® myDesign™ Genotyping Array from 865 animals. Furthermore, we investigated the population structure of the selected flounder population using a genomic relatedness matrix, PCA analysis, kinship coefficients, and multidimensional scaling. The results revealed that RF had the highest prediction accuracy for the three VHS-resistance traits (binary survival, days to death, and time of death), followed by BRR and GBLUP. PBLUP exhibits the lowest accuracy for these traits. Machine learning (ML) models, such as RF, outperformed traditional PBLUP and GBLUP, showing the greatest improvement over other Bayesian methods (BA, BB, and BC). The optimal parameters were determined for the best models, including specific marker sizes and sample size recommendations. The selected models and their parameters significantly improved the prediction of VHS resistance, demonstrating the potential of genomic selection to outperform the traditional pedigree-based methods. Our findings indicate that genomic selection can be more effective using ML models than conventional approaches in predicting VHS resistance and offers a means to enhance the genetic resistance of olive flounder in aquaculture to this disease. • Identifying disease resistance traits are necessary for breeding programs to create better animals. • A 70 K high density SNP array was used in the genomic prediction for VHSV traits. • The prediction accuracy ranged from 0.53 to 0.69 (lowest for PBLUP and highest for RF and CNN) • Machine learning models outperformed traditional PBLUP, GBLUP, and Bayesian models. • Increasing marker and population numbers also increase the genomic prediction ability. [ABSTRACT FROM AUTHOR]

In this study, the Peroxiredoxin-5 (PRDX5) gene from starry flounder (Platichthys stellatus , PsPRDX5) was identified through next-generation sequencing (NGS) and verified via cloning and sequencing. The predicted amino acid sequence, derived from the cDNA, was confirmed by multiple sequence alignment and phylogenetic analysis, establishing its homology with related sequences. PsPRDX5 mRNA was expressed across all examined tissues in healthy starry flounder, with distinct tissue-specific expression profiles. Following artificial infection with viral hemorrhagic septicemia virus (VHSV) and Streptococcus parauberis PH0710, significant alterations in PsPRDX5 mRNA expression were observed. VHSV infection resulted in a pronounced downregulation of PsPRDX5 in most major tissues, except for a significant upregulation in the intestine at 7 days post-infection. Conversely, infection with S. parauberis PH0710 led to a significant upregulation of PsPRDX5 in nearly all tissues. These results suggest that the differential expression of PsPRDX5 in response to VHSV and S. parauberis PH0710 reflects pathogen-specific immune and antioxidant defense mechanisms, highlighting the role of PsPRDX5 in the immune response of starry flounder to both viral and bacterial infections. • PRDX5 was identified from starry flounder and its expression was detected in all tissues examined. • Tissue- and gene-specific expression levels of PRDX5 mRNA were confirmed in healthy starry flounder. • PsPRDX5 mRNA expression was significantly regulated by S. parauberis PH0710 and VHSV infections. [ABSTRACT FROM AUTHOR]

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii ; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi , AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H 2 O 2 -treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii. • The membrane-associated Ring-CH5 (MARCH5) from Amphiprion clarkii (AcMARCH5) was identified and characterized. • Temporal expression of AcMARCH5 in blood, spleen and head kidney is drastically modulated by various immune stimuli. • AcMARCH5 overexpression suppressed antiviral gene expression and promoted VHSV viral replication. • AcMARCH5 overexpression reduced pro-inflammatory cytokine production in Poly I:C stimulated macrophages. • AcMARCH5 showed an anti-apoptotic property during H 2 O 2 induced oxidative stress. [ABSTRACT FROM AUTHOR]

Pink salmon (Oncorhynchus gorbuscha , Walbaum 1792) is an invasive alien species in the Fennoscandian rivers, and its growing populations cause concern for their impact on the native salmonids and river ecosystems. One of the major concerns is fish diseases and the potential ability of pink salmon to transmit pathogens to the native salmonid species. In this study, pink salmon were sampled during their spawning migration in the Rivers Tana and Neiden in northern Norway and Finland in 2021. The fish were surveyed for viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmonid alphavirus (SAV), piscine orthoreovirus (PRV), Renibacterium salmoninarum , and Gyrodactylus salaris. No viral diseases or infections with R. salmoninarum or G. salaris were detected. However, 23 % of the fish were found infected with a parasitic roundworm, Hysterothylacium aduncum. Additionally, external anomalies, such as hemorrhages or lesions in the skin, were observed in 35 % of the fish studied, and some of the fish exhibited degenerative myopathy. Based on this study, pink salmon appears not to pose a high infection risk to the native salmonid fish populations in the Rivers Tana and Neiden. Nevertheless, the results demonstrate that pink salmon have the potential to carry salmonid pathogens. More information on the susceptibility of pink salmon to different pathogens and on the overall effect this invasive species has on native fish species is needed. As the number of migrating pink salmon in the Fennoscandian rivers appears to increase, the potential risk of parasites and other pathogens spreading between fish species will also rise. [ABSTRACT FROM AUTHOR]