Piscidin: Antimicrobial peptide of rock bream, Oplegnathus fasciatus.

The piscidin family consists of antimicrobial peptides (AMPs) that are mainly found in fish and are crucial effectors of fish innate immune responses. The piscidin family typically has broad-spectrum antimicrobial activity and can modulate immune responses. In this study, we cloned rock bream piscidin (Rbpisc) and investigated its gene expression and biological activity (including antimicrobial and cytotoxic activities). The coding region of Rbpisc consisted of 213 base pairs (bp) encoding 70 amino acid residues. The tertiary structure predicted for Rbpisc includes an amphipathic helix-loop-helix structure. The Rbpisc gene was highly expressed in the gills of healthy fish. The gene expression of Rbpisc increased in the gills after pathogen infection, while the expression was down-regulated in other tissues. A synthetic peptide based on the AMP 12 domain amino acid sequence of Rbpisc appeared to have broad-spectrum antimicrobial activity against various bacteria. However, the synthetic peptide exhibited weak haemolytic activity against fish erythrocytes. These results suggest that Rbpisc might play an important role in the innate immune responses of rock bream.

Microarray analysis of gene expression in olive flounder liver infected with viral haemorrhagic septicaemia virus (VHSV).

The most fatal viral pathogen in olive flounder Paralichthys olivaceus, is viral hemorrhagic septicemia virus, which afflicts over 48 species of freshwater and marine fish. Here, we performed gene expression profiling on transcripts isolated from VHSV-infected olive flounder livers using a 13 K cDNA microarray chip. A total of 1832 and 1647 genes were upregulated and down-regulated over two-fold, respectively, after infection. A variety of immune-related genes showing significant changes in gene expression were identified in upregulated genes through gene ontology annotation. These genes were grouped into categories such as antibacterial peptide, antigen-recognition and adhesion molecules, apoptosis, cytokine-related pathway, immune system, stress response, and transcription factor and regulatory factors. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to the microarray data. In conclusion, these results may be useful for the identification of specific genes or for the diagnosis of VHSV infection in flounder.

Liquid Chromatography-Mass Spectrometry-Based Rapid Secondary-Metabolite Profiling of Marine Pseudoalteromonas sp. M2.

The ocean is a rich resource of flora, fauna, and food. A wild-type bacterial strain showing confluent growth on marine agar with antibacterial activity was isolated from marine water, identified using 16S rDNA sequence analysis as Pseudoalteromonas sp., and designated as strain M2. This strain was found to produce various secondary metabolites including quinolone alkaloids. Using high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis, we identified nine secondary metabolites of 4-hydroxy-2-alkylquinoline (pseudane-III, IV, V, VI, VII, VIII, IX, X, and XI). Additionally, this strain produced two novel, closely related compounds, 2-isopentylqunoline-4-one and 2-(2,3-dimetylbutyl)qunoline-4-(1H)-one, which have not been previously reported from marine bacteria. From the metabolites produced by Pseudoalteromonas sp. M2, 2-(2,3-dimethylbutyl)quinolin-4-one, pseudane-VI, and pseudane-VII inhibited melanin synthesis in Melan-A cells by 23.0%, 28.2%, and 42.7%, respectively, wherein pseudane-VII showed the highest inhibition at 8 µg/mL. The results of this study suggest that liquid chromatography (LC)-MS/MS-based metabolite screening effectively improves the efficiency of novel metabolite discovery. Additionally, these compounds are promising candidates for further bioactivity development.

Production of disulfide bond-rich peptides by fusion expression using small transmembrane proteins of Escherichia coli.

Recombinant expression in Escherichia coli allows the simple, economical, and effective production of bioactive peptides. On the other hand, the production of native peptides, particularly those rich in disulfide bonds, is a major problem. Previous studies have reported that the use of carrier proteins for fusion expression can result in good peptide yields, but few are folded correctly. In this study, two transmembrane small proteins in E. coli, YoaJ and YkgR, which both orientate with their N-termini in cytoplasm and their C-termini in periplasm, were used for fusion expression. The recombinant production of two peptides, asteropsin A (ASPA) and ß-defensin (BD), was induced in the periplasm of E. coli using a selected carrier protein. Both peptides were expressed at high levels, at yields of approximately 5-10 mg/L of culture. Mass spectrometry showed that the resulting peptide had the same molecular weight as their natural forms. After purification, single peaks were observed by reversed phase high-performance liquid chromatography (RP-HPLC), demonstrating the absence of isoforms. Furthermore, cytoplasmically expressed fusion proteins with a carrier at their C-termini did not contain disulfide bonds. This study provides new carrier proteins for fusion expression of disulfide bond-rich peptides in E. coli.

Functional characterisation and expression analysis of recombinant serum amyloid P isoform 1 (RbSAP1) from rock bream (Oplegnathus fasciatus).

Lectins are carbohydrate-binding proteins that play important roles in the recognition and elimination of pathogens via the innate immune system. Pentraxins (PTX) are humoral lectins, which are multifunctional proteins in vertebrates. Pentraxins can be divided into two groups based on their primary structure: short (C-reactive protein and serum amyloid P [SAP]) and long pentraxins (PTX3 and neuronal pentraxins). Previously, SAP was shown to have Ca(2+)-dependent binding specificity for various ligands and to be a major acute phase protein. In this study, we identified and characterised the gene encoding SAP isoform 1 in rock bream (Oplegnathus fasciatus) (RbSAP1) and analysed its expression in various tissues after a pathogen challenge. An alignment analysis conducted based on the deduced amino acid sequence of RbSAP1 (1918 bp full-length cDNA with a 699 bp open reading frame encoding 232 amino acids) and SAPs and PTXs isolated from other organisms, revealed that the pentraxin domain and cysteine residues of the deduced protein are conserved. RbSAP1, which was ubiquitously expressed in all tissues examined, was predominantly detected in head kidney, trunk kidney, peripheral blood leukocytes, and gills. RbSAP1 expression was dramatically up-regulated in the kidney and liver after infection with Edwardsiella tarda, Streptococcus iniae, or red seabream iridovirus. Purified rRbSAP1 was able to bind Gram-negative bacteria, Gram-positive bacteria, and pathogen-associated molecular patterns. Interestingly, rRbSAP1 aggregated Gram-negative bacteria in the presence of Ca(2+). The anti-pathogen activity of rRbSAP1 suggests that SAP functions in innate immunity in the rock bream.

Functional analysis of Pacific oyster (Crassostrea gigas) ß-thymosin: Focus on antimicrobial activity.

An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the ß-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas ß-thymosin, we overexpressed a recombinant ß-thymosin (rcgTß) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTß was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures.

Molecular and Functional Characterization of Thioredoxin 1 from Korean Rose Bitterling (Rhodeus uyekii).

Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family. In this study, we cloned and characterized thioredoxin 1 cDNA from the Korean rose bitterling Rhodeus uyekii (RuTrx). The full-length RuTrx cDNA consists of 674 bp with a 324 nt open reading frame (ORF) encoding a 107 aa protein. The deduced RuTrx amino acid sequence indicated a characteristic redox active site, (31)WCGPC(35). Pairwise alignment revealed RuTrx amino acid identity (55.1%-83.2%) with orthologs from various species of mammalia, amphibia, fish and bird. Phylogenetic analysis was conducted to determine the evolutionary position of RuTrx. Expression analysis showed that RuTrx transcripts were present in all of the tissues examined, and was high in the hepatopancreas of R. uyekii. During early development, the expression of RuTrx transcripts was increased. Recombinant RuTrx protein (rRuTrx) was tested for its capacity to serve as an antioxidant enzyme using a metal-catalyzed oxidation (MCO) system. The ability of rRuTrx to protect against supercoiled DNA cleavage due to oxidative nicking increased in a dose-dependent manner. In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx. Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

Cloning, characterisation, and expression analysis of the cathepsin D gene from rock bream (Oplegnathus fasciatus).

Cathepsins are lysosomal cysteine proteases belonging to the papain family, members of which play important roles in normal metabolism for the maintenance of cellular homeostasis. Rock bream (Oplegnathus fasciatus) cathepsin D (RbCTSD) cDNAs were identified by expressed sequence tag analysis of a lipopolysaccharide-stimulated rock bream liver cDNA library. The full-length RbCTSD cDNA (1644 bp) contained an open reading frame of 1191 bp encoding 396 amino acids. Alignment analysis revealed that the active sites and N-glycosylation sites of the deduced protein were well conserved. Phylogenetic analysis revealed that RbCTSD is most closely related to the Mi-iuy croaker (Miichthys miiuy) cathepsin D. RbCTSD was ubiquitously expressed in all the examined tissues, predominantly in muscle and kidneys. RbCTSD mRNA expression was also examined in several tissues under conditions of bacterial and viral challenge. All examined tissues of fish infected with Edwardsiella tarda (E. tarda), Streptococcus iniae (S. iniae), and red sea bream iridovirus (RSIV) showed significant increases in RbCTSD expression compared with the control. In the kidney and spleen, RbCTSD mRNA expression was markedly upregulated following infection with all tested pathogens. These findings indicate that RbCTSD plays an important role in the innate immune response of rock bream. Furthermore, these results provide important information for the identification of other cathepsin D genes in various fish species.

Expression analysis and biological activity of moronecidin from rock bream, Oplegnathus fasciatus.

The piscidin-family, one of antimicrobial peptides (AMPs) mainly distributed in fish, is crucial effectors of fish innate immune response. Piscidin-family typically has broad-spectrum antimicrobial activity and the ability to modulate the immune response. In this study, we identified moronecidin (Rbmoro) included in piscidin-family from rock bream and investigated its gene expression using quantitative real-time PCR and biological activity (including antimicrobial and cytotoxic activity). The coding region of Rbmoro was 204 bp encoding 67 amino acid residues. Tertiary structure prediction of Rbmoro showed an amphipathic α-helical structure. Rbmoro gene was widely expressed in different tissues of healthy fish. Additionally, Rbmoro gene expression was induced in all tested tissues after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. We synthesized mature peptide of Rbmoro based on amino acid sequence of its AMP 12 domain, and the synthetic peptide appeared broad-spectrum antimicrobial activity to various bacteria. However, the synthetic peptide has weak haemolytic activity against fish erythrocytes. These results suggest that Rbmoro might play an important role in innate immune response of rock bream.

Molecular characterization and expression analysis of a peroxiredoxin 1 cDNA from Korean rose bitterling (Rhodeus uyekii).

Peroxiredoxins (Prxs), also known as natural killer cell enhancing factors in fish, role as antioxidant proteins and participate in a variety of biological processes, including H2O2-mediated cell signaling, molecular chaperoning, and mitochondrial function. In this study, we isolated and characterized a Prx 1 cDNA from the Korean rose bitterling Rhodeus uyekii, and designated it RuPrx 1. The RuPrx 1 cDNA encodes a 197-amino-acid polypeptide that belongs to the class of typical 2-Cys Prxs that contain peroxidatic and resolving cysteines. The deduced RuPrx 1 protein shows strong homology (77.38-92.89 %) with Prx 1 proteins from other species, including fish, amphibians, and mammals, and it is most closely related to rainbow smelt Prx 1. RuPrx 1 mRNA was ubiquitously detected in all tested tissues and its expression was comparatively high in the brain, intestine, kidney, liver, ovary, stomach, and testis. Expression of RuPrx 1 mRNA in liver peaked 3 h post-infection with Aeromonas hydrophila and decreased 24 h post-infection while the expression in intestine decreased 24 h post-infection. These results suggest that RuPrx 1 is conserved through evolution and may play roles similar to its mammalian counterparts.

Antimicrobial activity of peptides derived from olive flounder lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI).

We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

Characterization, expression profile, and promoter analysis of the Rhodeus uyekii vitellogenin Ao1 gene.

The fish Vitellogenin (Vg) gene has been applied as a biomarker for exposure to estrogenic compounds in the aquatic environment. In this study, we cloned and characterized Vg cDNA from the Korean rose bitterling Rhodeus uyekii (Ru-Vg). The Ru-Vg cDNA encodes a 1424-amino-acid polypeptide that belongs to the VgAo1 family and contains a putative signal peptide, lipovitellin I, phosvitin, and lipovitellin II, but does not contain the vWFD domain or the C-terminal peptide. The deduced Ru-Vg protein has high amino acid identity (73.97%-32.17%) with fish Vg proteins. Pairwise alignment and phylogenetic analysis revealed that Ru-Vg is most closely related to Acheilognathus yamatsutae Vg. Ru-Vg transcripts were detected using quantitative polymerase chain reaction in all tissues tested, with the highest level of expression observed in the ovary. Ru-Vg mRNA was upregulated in R. uyekii hepatopancreas cells in response to treatment with 17ß-estradiol (E2) or 17α-ethinylestradiol (EE2). Luciferase reporter expression, driven by the 5'-regulatory region of the Ru-Vg gene spanning from -1020 bp to the start codon was induced by the estrogen receptor and was synergistically activated by treatment with E2 or EE2. These results suggest that R. uyekii and the Ru-Vg gene may be useful as biomarkers for exposure to E2 or EE2.

Molecular and functional characterizations of a Kunitz-type serine protease inhibitor FcKuSPI of the shrimp Fenneropenaeus chinensis.

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44-53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNA was expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays.

Molecular and functional analyses of the fast skeletal myosin light chain2 gene of the Korean oily bitterling, Acheilognathus koreensis.

We identified and characterized the primary structure of the Korean oily bitterling Acheilognathus koreensis fast skeletal myosin light chain 2 (Akmlc2f), gene. Encoded by seven exons spanning 3955 bp, the deduced 168-amino acid AkMLC2f polypeptide contained an EF-hand calcium-binding motif and showed strong homology (80%-98%) with the MLC2 proteins of Ictalurus punctatus and other species, including mammals. Akmlc2f mRNA was highly enriched in skeletal muscles, and was detectable in other tissues. The upstream regions of Akmlc2f included a TATA box, one copy of a putative MEF-2 binding site and several putative C/EBPß binding sites. The functional activity of the promoter region of Akmlc2f was examined using luciferase and red fluorescent protein reporters. The Akmlc2f promoter-driven reporter expressions were detected and increased by the C/EBPß transcription factor in HEK293T cells. The activity of the promoter of Akmlc2f was also confirmed in the developing zebrafish embryo. Although the detailed mechanism underlying the expression of Akmlc2f remains unknown, these results suggest the muscle-specific expression of Akmlc2f transcript and the functional activation of Akmlc2f promoter by C/EBPß.

First report of cathepsin E in a teleost (Korean rose bitterling, Rhodeus uyekii): Molecular characterisation and tissue distribution.

We isolated and characterised a cDNA encoding the aspartic protease cathepsin E (CTSE) in Korean rose bitterling, Rhodeus uyekii. The full-length Rhodeus uyekii CTSE (RuCTSE) cDNA (1396 bp) contains an open reading frame of 1218 bp, encoding 405 amino acids. Alignment of multiple CTSE protein sequences revealed that two of the aspartyl protease active site residues and a disulphide bond were well-conserved among the other CTSE sequences. Phylogenetic analysis revealed that RuCTSE is most closely related to freshwater fish cathepsin E. RuCTSE is widely expressed in the liver, spleen, ovary, testis, brain, eye, intestine, muscle, fin, stomach, and kidney. This first report of teleost CTSE will provide important information related to the identification of other cathepsin E genes in various fish species and will serve as a useful molecular tool to help clarify biological activities in other teleosts.

Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus Takifugu.

Pufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

First draft genome sequence of the rock bream in the family Oplegnathidae.

The rock bream (Oplegnathus fasciatus) is one of the most economically valuable marine fish in East Asia, and due to various environmental factors, there is substantial revenue loss in the production sector. Therefore, knowledge of its genome is required to uncover the genetic factors and the solutions to these problems. In this study, we constructed the first draft genome of O. fasciatus as a reference for the family Oplegnathidae. The genome size is estimated to be 749 Mb, and it was assembled into 766 Mb by combining Illumina and PacBio sequences. A total of 24,053 transcripts (23,338 genes) are predicted, and among those transcripts, 23,362 (97%), are annotated with functional terms. Finally, the completeness of the genome assembly was assessed by CEGMA, which resulted in the complete mapping of 220 (88.7%) core genes in the genome. To the best of our knowledge, this is the first draft genome for the family Oplegnathidae.

Genome sequence of pacific abalone (Haliotis discus hannai): the first draft genome in family Haliotidae.

Background: Abalones are large marine snails in the family Haliotidae and the genus Haliotis belonging to the class Gastropoda of the phylum Mollusca. The family Haliotidae contains only one genus, Haliotis, and this single genus is known to contain several species of abalone. With 18 additional subspecies, the most comprehensive treatment of Haliotidae considers 56 species valid [ 1 ]. Abalone is an economically important fishery and aquaculture animal that is considered a highly prized seafood delicacy. The total global supply of abalone has increased 5-fold since the 1970s and farm production increased explosively from 50 mt to 103 464 mt in the past 40 years. Additionally, researchers have recently focused on abalone given their reported tumor suppression effect. However, despite the valuable features of this marine animal, no genomic information is available for the Haliotidae family and related research is still limited. To construct the H . discus hannai genome, a total of 580-G base pairs using Illumina and Pacbio platforms were generated with 322-fold coverage based on the 1.8-Gb estimated genome size of H . discus hannai using flow cytometry. The final genome assembly consisted of 1.86 Gb with 35 450 scaffolds (>2 kb). GC content level was 40.51%, and the N50 length of assembled scaffolds was 211 kb. We identified 29 449 genes using Evidence Modeler based on the gene information from ab initio prediction, protein homology with known genes, and transcriptome evidence of RNA-seq. Here we present the first Haliotidae genome, H . discus hannai , with sequencing data, assembly, and gene annotation information. This will be helpful for resolving the lack of genomic information in the Haliotidae family as well as providing more opportunities for understanding gastropod evolution.

Cytogenetic study of diploid and induced tetraploid in Korean rose bitterling, Rhodeus uyekii.

In this study, we induced tetraploidy in Korean rose bitterling, Rhodeus uyekii, by applying various hydrostatic pressure shock conditions. Tetraploidy was not induced under 4500 psi pressure treatment in any experimental group. Instead, the induction rate of tetraploidy was highest under 7500 psi hydrostatic pressure treatment. As a result, when the processing method was similar and as the process time increased, the induction rate of each experimental group increased; however, there was no significant difference (P > 0.05). The production rate was 3.1 %, which was highest in all experimental groups exposed to 6000 psi for 10 min after being fertilized for 100 min. The production rate was highest in the experimental groups treated with hydrostatic pressure alone, whereas the production rate was lowest in groups treated under hydrostatic pressure with chemical treatment. The abnormal rate of all experimental groups treated with 7500 psi for 20 min was very high, at about 5 %. Based on these studies, only hydrostatic pressure shock was considered effective at inducing tetraploidy based on the calculated hatching, abnormal, and induction rates. The most effective condition for inducing tetraploidy was 6000 psi of hydrostatic pressure shock for 10 min after being fertilized for 100 min. The chromosome number of the induced tetraploid Korean rose bitterling was 4n = 96, while that of the diploid was 2n = 48. In the diploid, there were 1 or 2 nucleoli in the cells, whereas the induced tetraploids contained 1, 2, 3, or 4. The DNA content of tetraploids and diploids were 3.68 ± 0.009 pg/nucleus and 1.84 ± 0.019 pg/nucleus, respectively, according to flow cytometric analysis. The DNA content and chromosome number of the tetraploids were twice that of the diploids.

Complete sequence and polymorphisms of female Ruditapes philippinarum (Mollusca: Bivalvia) mitochondria genome.

Mitogenome of female Ruditapes philippinarum organism was sequenced, and genomic variation and phylogeny were examined in this study. Length of the mitogenome was 22 089 bp showing 94.28% of sequence identity with previously reported sequence. Total 707 single nucleotide polymorphisms, SNPs, were detected and 50 residues were non-synonymous SNPs among the 202 SNPs in protein-coding genes. Deleted genomic fragments with of 265 bp and 322 bp were observed in non-coding regions, ND2 to ND4L and ND4L to tRNA(Ile), respectively. Phylogenic analysis confirmed that used organisms were female R. philippinarum, and the species has closer evolutionary distance with genus Paphia rather than genus Meretrix. Our finding will be help to set an insight for population and evolutionary genomics of Veneroida clams as well as application to marine industry.