Eukaryotic genome sequencing and de novo assembly, once the exclusive domain of well-funded international consortia, have become increasingly affordable, thus fitting the budgets of individual ...research groups. Third-generation long-read DNA sequencing technologies are increasingly used, providing extensive genomic toolkits that were once reserved for a few select model organisms. Generating high-quality genome assemblies and annotations for many aquatic species still presents significant challenges due to their large genome sizes, complexity, and high chromosome numbers. Indeed, selecting the most appropriate sequencing and software platforms and annotation pipelines for a new genome project can be daunting because tools often only work in limited contexts. In genomics, generating a high-quality genome assembly/annotation has become an indispensable tool for better understanding the biology of any species. Herein, we state 12 steps to help researchers get started in genome projects by presenting guidelines that are broadly applicable (to any species), sustainable over time, and cover all aspects of genome assembly and annotation projects from start to finish. We review some commonly used approaches, including practical methods to extract high-quality DNA and choices for the best sequencing platforms and library preparations. In addition, we discuss the range of potential bioinformatics pipelines, including structural and functional annotations (e.g., transposable elements and repetitive sequences). This paper also includes information on how to build a wide community for a genome project, the importance of data management, and how to make the data and results Findable, Accessible, Interoperable, and Reusable (FAIR) by submitting them to a public repository and sharing them with the research community.
Continuous monitoring of the present genetic status is essential to preserve the genetic resource of wild populations. In this study, we sequenced regional Pacific abalone Haliotis discus samples ...from three different locations around the Korean peninsula to assess population structure, utilizing Genotyping-by-Sequencing (GBS) method. Using PstI enzyme for genome reduction, we demonstrated the resultant library represented the whole genome region with even spacing, and as a result 16,603 single nucleotide variants (SNVs) were produced. Genetic diversity and population structure were investigated using several methods, and a strong genetic heterogeneity was observed in the Korean abalone populations. Additionally, by comparison of the variant sets among population groups, we were able to discover 26 Korean abalone population-specific SNVs, potentially associated with phenotype differences. This is the first study demonstrating the feasibility of GBS for population genetic study on H. discus. Our results will provide valuable data for the genetic conservation and management of wild abalone populations in Korea and help future GBS studies on the marine mollusks.
MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, ...but its biological functions in fish are unknown. We isolated and characterized
from the olive flounder
(
). The coding region of
comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates.
mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The
mRNA level increased during early development. In addition, the
transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of
in VHSV infection, single-cell-derived
knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of
were selected.
disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish
, which may play a role in the response to viral infection.
Haliotis discus hannai, a species of Pacific abalone, is a highly valuable food source throughout Northeast Asia. As H. discus hannai primarily feed on brown algae and largely extract their energy ...from algal polysaccharides, understanding the mechanisms by which they digest algal polysaccharides is essential for elucidating their energy metabolism. Gut microbes, as well as the host animal, are involved in the process of polysaccharide degradation. To identify algal polysaccharide-digestion mechanisms and their origin, we analyzed the metagenome and metatranscriptome of abalone visceral extracts. Microbial communities were characterized using the 16S rRNA gene sequences in the metagenome and our results differed significantly from those of previous studies using traditional microbiological methods such as bacterial cultivation and cloning. A greater diversity of bacterial taxa was identified here than was previously identified using cultivation methods. Furthermore, the most abundant bacterial taxa also differed from previous studies, which is not common when comparing the results of bacterial culturing with those of molecular methodologies. Based on the metatranscriptome, overall functions were identified and additional analyses were performed on the coding sequences of algal polysaccharide-digestive enzymes, including alginate lyase. Results of the transcriptomic analyses suggest that alginate lyase in the visceral extracts of H. discus hannai was produced by the host itself, not by visceral bacteria. This is the first next-generation sequencing study performed on abalone to characterize the visceral microbiota and the source of the ability to digest algal polysaccharides by analyzing the metagenome and metatranscriptome together.
We isolated and purified an antimicrobial peptide (AMP) from the mantle of the hard-shelled mussel, Mytilus coruscus. The peptide was purified through C18 reversed-phase high-performance liquid ...chromatography, and displayed antibacterial activity. Total molecular mass of 11,182 Da was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry. The N-terminal 23-amino acid sequence of its purified peak was obtained through Edman degradation, revealing 82% identity with myticusin-1 of M. coruscus. Complete sequence of the target peptide was determined through cDNA cloning and rapid amplification of cDNA ends. The complete sequence comprised 574 bp with a 387-bp open reading frame (ORF) encoding 24 amino acids of a signal peptide and 104 amino acids of a mature peptide, which was named myticusin-beta. Furthermore, we discovered two novel isoforms of myticusin-beta. We constructed and expressed recombinant myticusin-beta, which displayed antimicrobial activity against gram-positive (Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, Streptococcus iniae, Streptococcus mutans) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, Klebsiella pneumoniae). Purified recombinant myticusin-beta also showed anti-parasitic activity at various concentrations. A short AMP analog was designed and synthesized based on the sequence of myticusin-beta, with markedly improved antimicrobial activity. Expression of myticusin-beta was detected in the mantle at the highest level, followed by hemocytes. The results obtained in this work suggest that myticusin-beta is an immune-related AMP of M. coruscus and an effective alternative to antibiotics.
•We purified 11,182 Da of a novel antimicrobial peptide from mantle tissue extract of Mytilus coruscus.•TrxA-fused recombinant myticusin-beta exhibited antibacterial and anti-scuticociliate activity without causing hemolysis.•Designed and synthesized myticusin-beta analog revealed markedly improved antimicrobial activity.•Myticusin-beta is an immune-related antimicrobial peptide of Mytilus coruscus and an effective alternative to antibiotics.
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.
Toll-like receptors (TLRs) are well-known pattern recognition receptors that play key immunological roles in a diverse range of organisms. In this study, two novel invertebrate TLRs from disk abalone ...(designated as AbTLR-A and AbTLR-B) were identified and functionally characterized for the first time. AbTLR-A and AbTLR-B comprised the typical TLR domain architecture containing an extracellular leucine-rich repeat domain, transmembrane domain, and Toll/interleukin-1 receptor domain. Expressional analysis revealed that both TLRs were constitutively expressed at all the early embryonic stages of disk abalone analyzed, with the highest level of AbTLR-A found at the 16-cell stage and AbTLR-B at the trochophore stage. According to tissue distribution analysis, prominent mRNA expression of AbTLR-A and AbTLR-B was detected in the hemocytes and gills, respectively. AbTLR-A and AbTLR-B mRNAs were significantly up-regulated in response to Gram-negative Vibrio parahemolyticus, Gram-positive Listeria monocytogenes, and viral hemorrhagic septicemia virus injections in abalone hemocytes and gills. Overexpression of AbTLR-A and AbTLR-B in HEK293T cells directly activated nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) responsive reporters. Neither TLRs showed a high response to pathogen-associated molecular patterns in vitro. Co-expression of AbTLR-A and AbTLR-B with AbMyD88-2 and AbMyD88-X activated NF-κB-responsive reporters in a synergetic manner. These findings demonstrate the involvement of AbTLR-A and AbTLR-B in abalone innate immunity.
•Two novel Toll Like Receptors (TLRs) were identified from Disk abalone (AbTLR-A and AbTLR-B).•Both abalone TLRs comprised with typical TLR structural features.•Constitutive expressed mRNA of AbTLR-A and AbTLR-B was detected at early embryonic developmental stages of disk abalone.•AbTLR-A and AbTLR-B expression was modulated upon immune challenge.•Overexpression of both TLRs in HEK293T cells directly activated NF-κB and AP-1.
In this study, we describe the identification and characterization of a proto type galectin, galectin-1, from rock bream Oplegnathus fasciatus (OfGal-1). Galectins are evolutionarily conserved ...carbohydrate binding lectins that show a wide range of functions related to development and immune physiology. They have been identified as pattern recognition receptors of innate immune system that recognize a broad range of microbes. OfGal-1 cDNA comprised of 993 bp with an open reading frame of 408 bp that encodes 135 amino acids. A single carbohydrate recognition domain was present in the OfGal-1 amino acid sequence. The sequence comparison by multiple and pairwise alignments and the phylogenetic tree emphasized the strong evolutionary conservation of Gal-1. The typical β-sandwich structure was identified from the predicted tertiary structure. The constitutive expression of mRNA transcripts was detected in a wide range of tissues examined, with the highest expression in the heart. Immune challenges with live bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus, and mitogens (lipopolysaccharide and poly I:C) modulated the expression of OfGal-1 mRNAs in the gills, head kidney, and liver. The recombinant OfGal-1 (rOfGal-1) strongly agglutinatinated the human erythrocytes, and this hemagglutination was inhibited by lactose and d-galactose. A wide range of bacteria (S. iniae, S. parauberis, Escherichia coli, Edwardsiella tarda, Vibrio anguillarum, Vibrio harveyi, and Vibrio tapetis) and a ciliate (Miamiensis avidus) were also effectively recognized by rOfGal-1. Significant antiviral activity against rock bream irido virus was also demonstrated by rOfGal-1. Collectively, results from the present study indicate that OfGal-1 can recognize a wide range of microbes and is a vital pattern recognition receptor in the innate immune system of rock bream.
•Galectin-1 was cloned and characterized from Rock bream Oplegnathus fasciatus (OfGal-1).•OfGal-1 was ubiquitously expressed in all examined tissues.•OfGal-1 transcripts were significantly induced upon bacterial and viral stimulations.•Recombinant OfGal-1showed agglutination as well as binding with microbial pathogens.•Antiviral activity of OfGal-1 against RBIV and VHSV was determined.
Climate change is one of the most important threats to farmed abalone worldwide. Although abalone is more susceptible to vibriosis at higher water temperatures, the molecular mode of action ...underlying this has not been fully elucidated. Therefore, this study aimed to address the high susceptibility of Halitotis discus hannai to V. harveyi infection using abalone hemocytes exposed to low and high temperatures. Abalone hemocytes were divided into four groups, 20C, 20 V, 25C, and 25 V, depending on co-culture with (V)/without (C) V. harveyi (MOI = 12.8) and incubation temperature (20 °C or 25 °C). After 3 h of incubation, hemocyte viability and phagocytic activity were measured, and RNA sequencing was performed using Illumina Novaseq. The expression of several virulence-related genes in V. harveyi was analyzed using real-time PCR. The viability of hemocytes was significantly decreased in the 25 V group compared to cells in the other groups, whereas phagocytic activity at 25 °C was significantly higher than at 20 °C. Although a number of immune-associated genes were commonly upregulated in abalone hemocyte exposed to V. harveyi, regardless of temperature, pathways and genes regarding pro-inflammatory responses (interleukin-17 and tumor necrosis factor) and apoptosis were significantly overexpressed in the 25 V group compared to the 25C group. Notably, in the apoptosis pathway, genes encoding executor caspases (casp3 and casp7) and pro-apoptotic factor, bax were significantly up-regulated only in the 25 V group, while the apoptosis inhibitor, bcl2L1 was significantly up-regulated only in the 20 V group compared to the control group at the respective temperatures. The co-culture of V. harveyi with abalone hemocytes at 25 °C up-regulated several virulence-related genes involved in quorum sensing (luxS), antioxidant activity (katA, katB, and sodC), motility (flgI), and adherence/invasion (ompU) compared to those at 20 °C. Therefore, our results showed that H. discus hannai hemocytes exposed to V. harveyi at 25 °C were highly stressed by vigorously activated inflammatory responses and that the bacterial pathogen overexpressed several virulence-related genes at the high temperature tested. The transcriptomic profile of both abalone hemocytes and V. harveyi in the present study provide insight into differential host-pathogen interactions depending on the temperature conditions and the molecular backgrounds related to increased abalone vulnerability upon global warming.
•Pacific abalone (Haliotis discus hannai) hemocytes at 25 °C showed higher phagocytic activity than 20 °C.•Hemocyte viability was significantly decreased only when exposed to V. harveyi at 25 °C, but not at 20 °C.•V. harveyi exposure induced overexpression of pro-inflammatory and apoptosis-related genes in abalone hemocytes only at 25 °C.•V. harveyi up-regulated several key virulence factors including quorum sensing at 25 °C compared to 20 °C.•Temperature upshift increased Vibrio virulence and abalone stress response, indicating their vulnerability to global warming.
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