Transcriptome analysis of the Japanese eel (Anguilla japonica) during larval metamorphosis.

BACKGROUND: Anguillid eels spend their larval period as leptocephalus larvae that have a unique and specialized body form with leaf-like and transparent features, and they undergo drastic metamorphosis to juvenile glass eels. Less is known about the transition of leptocephali to the glass eel stage, because it is difficult to catch the metamorphosing larvae in the open ocean. However, recent advances in rearing techniques for the Japanese eel have made it possible to study the larval metamorphosis of anguillid eels. In the present study, we investigated the dynamics of gene expression during the metamorphosis of Japanese eel leptocephali using RNA sequencing. RESULTS: During metamorphosis, Japanese eels were classified into 7 developmental stages according to their morphological characteristics, and RNA sequencing was used to collect gene expression data from each stage. A total of 354.8 million clean reads were generated from the body and 365.5 million from the head, after the processing of raw reads. For filtering of genes that characterize developmental stages, a classification model created by a Random Forest algorithm was built. Using the importance of explanatory variables feature obtained from the created model, we identified 46 genes selected in the body and 169 genes selected in the head that were defined as the "most characteristic genes" during eel metamorphosis. Next, network analysis and subsequently gene clustering were conducted using the most characteristic genes and their correlated genes, and then 6 clusters in the body and 5 clusters in the head were constructed. Then, the characteristics of the clusters were revealed by Gene Ontology (GO) enrichment analysis. The expression patterns and GO terms of each stage were consistent with previous observations and experiments during the larval metamorphosis of the Japanese eel. CONCLUSION: Genome and transcriptome resources have been generated for metamorphosing Japanese eels. Genes that characterized metamorphosis of the Japanese eel were identified through statistical modeling by a Random Forest algorithm. The functions of these genes were consistent with previous observations and experiments during the metamorphosis of anguillid eels.

A novel jumbo Tenacibaculum maritimum lytic phage with head-fiber-like appendages.

A novel jumbo bacteriophage (myovirus) is described. The lytic phage of Tenacibaculum maritimum, which is the etiological agent of tenacibaculosis in a variety of farmed marine fish worldwide, was plaque-isolated from seawater around a fish aquaculture field in Japan. The phage had an isometric head 110-120 nm in diameter, from which several 50- to 100-nm-long flexible fiber-like appendages emanate, and a 150-nm-long rigid contractile tail. The full genomes of the two representative phages (PTm1 and PTm5) were 224,680 and 226,876 bp long, respectively, both with 29.7% GC content, and the number of predicted open reading frames (ORFs) was 308 and 306, respectively. The average nucleotide sequence identity between PTm1 and PTm5 was 99.95%, indicating they are quite similar to each other. A genetic relationship was found in 15.0-16.6% of the predicted ORFs among the T. maritimum phages PTm1 and PTm5, the Tenacibaculum spp. phage pT24, and the Sphingomonas paucimobilis phage PAU. Phylogenetic analysis based on the terminase large subunit genes revealed that these four phages (PTm1, PTm5, pT24 and PAU) are more closely related than the other 10 jumbo myoviruses that have similar genome sizes. Transmission electron microscopy observations suggest that the head fibers of the T. maritimum phage function as tentacles to search and recognize the host cell surface to facilitate infection.

Complete genome sequence and characterization of virulence genes in Lancefield group C Streptococcus dysgalactiae isolated from farmed amberjack (Seriola dumerili).

Lancefield group C Streptococcus dysgalactiae causes infections in farmed fish. Here, the genome of S. dysgalactiae strain kdys0611, isolated from farmed amberjack (Seriola dumerili) was sequenced. The complete genome sequence of kdys0611 consists of a single chromosome and five plasmids. The chromosome is 2,142,780 bp long and has a GC content of 40%. It possesses 2061 coding sequences and 67 tRNA and 6 rRNA operons. One clustered regularly interspaced short palindromic repeat, 125 insertion sequences, and four predicted prophage elements were identified. Phylogenetic analysis based on 126 core genes suggested that the kdys0611 strain is more closely related to S. dysgalactiae subsp. dysgalactiae than to S. dysgalactiae subsp. equisimilis. The genome of kdys0611 harbors 87 genes with sequence similarity to putative virulence-associated genes identified in other bacteria, of which 57 exhibit amino acid identity (>52%) to genes of the S. dysgalactiae subsp. equisimilis GGS124 human clinical isolate. Four putative virulence genes, emm5 (FGCSD_0256), spg_2 (FGCSD_1961), skc (FGCSD_1012), and cna (FGCSD_0159), in kdys0611 did not show significant homology with any deposited S. dysgalactiae genes. The chromosomal sequence of kdys0611 has been deposited in GenBank under Accession No. AP018726. This is the first report of the complete genome sequence of S. dysgalactiae isolated from fish.

Development and characterization of 19 novel microsatellite markers in the Pacific seaweed pipefish Syngnathus schlegeli using next-generation sequencing.

Syngnathids (pipefishes, seahorses and seadragons) are vulnerable to human-mediated habitat perturbation. The Pacific seaweed pipefish Syngnathus schlegeli has a large distribution in the northwestern Pacific, where deterioration, loss and fragmentation of its seagrass habitat are occurring through coastal development. So far, few studies have been conducted to access the genetic structure and conservation status of S. schlegeli because of the low number of genetic markers currently described. Nineteen polymorphic microsatellite markers were developed for S. schlegeli using next-generation sequencing, and characterized in 32 individuals. The mean number of alleles was 14, with 2-28 alleles per locus. The estimates of observed heterozygosity (HO) and expected heterozygosity (HE) varied depending on the locus, ranging from 0.063 to 1.000, and from 0.062 to 0.969, respectively. Seventeen of the 19 microsatellites conformed to Hardy-Weinberg equilibrium. These new microsatellite markers should provide a wealth of information for studies on conservation genetics and the behavioral ecology of S. schlegeli.

Multilocus sequence analysis of Vibrionaceae isolated from farmed amberjack and the development of a multiplex PCR assay for the detection of pathogenic species.

Since 2011, high mortality rates and symptoms consistent with vibriosis have been observed in farmed amberjack (Seriola dumerili) in Japan. To identify 41 strains isolated from diseased amberjack, a multilocus sequence analysis using nine concatenated genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) was conducted. Twenty-seven strains were identified as Vibrio harveyi, suggesting an epidemic of V. harveyi infection in amberjack farms. Other strains were identified as Vibrio anguillarum, Vibrio owensii and Photobacterium damselae subsp. damselae. To develop an efficient diagnostic method for vibriosis in amberjack, a multiplex PCR system was developed to identify V. anguillarum, V. harveyi and P. damselae subsp. damselae. The method successfully discriminated between these three bacterial species, with amplification products of 350 bp for V. anguillarum, 545 bp for V. harveyi and 887 bp for P. damselae subsp. damselae and can be used for diagnosis in aquaculture farms.

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tuna's RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

Complete genome sequence analysis of two Pseudomonas plecoglossicida phages, potential therapeutic agents.

Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416-1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33-39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents.

Full-genome sequence of a novel myovirus, GF-2, infecting Edwardsiella tarda: comparison with other Edwardsiella myoviral genomes.

Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious infectious diseases in both marine and freshwater fish farms worldwide. Previously, we reported the complete genome sequences of three E. tarda-lytic bacteriophages (two podoviruses and a myovirus), which were isolated from fish tissues and fish-rearing seawater. Further genomic information regarding E. tarda phages is important for understanding phage-host interactions as well as for applications of the phages for the control of disease. Here, we report the complete genome sequence of a novel E. tarda phage (GF-2) of myovirus morphology (family Myoviridae), isolated from tissue homogenates of a cultured Japanese flounder (Paralichthys olivaceus) that succumbed to edwardsiellosis in Japan. The size of the entire genome was 43,129 bp, with a GC content of 51.3 % and containing 82 open reading frames (ORFs). The GF-2 genome possesses lysogeny-related genes that have not been found in the reported Edwardsiella phage genomes. Comparative genomics of Edwardsiella myophages suggest that the C-terminal domains of the tail fiber proteins have relevance to their host specificity. Thus, GF-2 genome information provides a novel resource for our understanding of the molecular mechanisms involved in their host specificity and for detection of E. tarda in aquaculture environments.

A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication.

BACKGROUND: Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. RESULTS: We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. CONCLUSIONS: The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.

Development of a chub mackerel with less-aggressive fry stage by genome editing of arginine vasotocin receptor V1a2.

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

Effective gene collection from the metatranscriptome of marine microorganisms.

BACKGROUND: Metagenomic studies, accelerated by the evolution of sequencing technologies and the rapid development of genomic analysis methods, can reveal genetic diversity and biodiversity in various samples including those of uncultured or unknown species. This approach, however, cannot be used to identify active functional genes under actual environmental conditions. Metatranscriptomics, which is similar in approach to metagenomics except that it utilizes RNA samples, is a powerful tool for the transcriptomic study of environmental samples. Unlike metagenomic studies, metatranscriptomic studies have not been popular to date due to problems with reliability, repeatability, redundancy and cost performance. Here, we propose a normalized metatranscriptomic method that is suitable for the collection of genes from samples as a platform for comparative transcriptomics. RESULTS: We constructed two libraries, one non-normalized and the other normalized library, from samples of marine microorganisms taken during daylight hours from Hiroshima bay in Japan. We sequenced 0.6M reads for each sample on a Roche GS FLX, and obtained 0.2M genes after quality control and assembly. A comparison of the two libraries showed that the number of unique genes was larger in the normalized library than in the non-normalized library. Functional analysis of genes revealed that a small number of gene groups, ribosomal RNA genes and chloroplast genes, were dominant in both libraries. Taxonomic distribution analysis of the libraries suggests that Stramenopiles form a major taxon that includes diatoms. The normalization technique thus increases unique genes, functional categories of genes, and taxonomic richness. CONCLUSIONS: Normalization of the marine metatranscriptome could be useful in increasing the number of genes collected, and in reducing redundancies among highly expressed genes. Gene collection through the normalization method was effective in providing a foundation for comparative transcriptomic analysis.

Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in Japanese flounder Paralichthys olivaceus leucocytes during Edwardsiella tarda and viral hemorrhagic septicemia virus infection.

Transcriptional changes in the peripheral blood leucocytes (PBL) of Japanese flounder Paralichthys olivaceus challenged by Edwardsiella tarda and viral hemorrhagic septicemia virus (VHSV) were investigated using suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis. First, we constructed an SSH cDNA library using mRNA samples isolated from PBL of P. olivaceus that had been experimentally infected with E. tarda. We then examined the transcriptional changes occurring in the PBL due to E. tarda and VHSV infection using a cDNA microarray produced using clones produced from the SSH library. A total of 565 and 180 cDNA sequences corresponding to mRNA species that are either up- or down-regulated by E. tarda infection were isolated by SSH. While host gene expression responses in response to E. tarda and VHSV infection share several response elements, distinct patterns of gene expression were also observed. Specifically, E. tarda infection enhanced the expression of cell adhesion molecules while VHSV enhanced the expression of interferon and proteasome-related genes. In challenge trials of the two infectious agents, expression profiles of chemokines were also observed to differ. The results indicated that distinguishing between viral and bacterial infection is possible based on the RNA expression profiles of PBL from infected fish.

Characterization of lsa(D), a Novel Gene Responsible for Resistance to Lincosamides, Streptogramins A, and Pleuromutilins in Fish Pathogenic Lactococcus garvieae Serotype II.

Aims: Fish pathogenic Lactococcus garvieae serotype II has been isolated from cultured fish species in Japan. This study aimed to investigate the molecular mechanisms of lincomycin (LCM)-resistant L. garvieae serotype II and assess the molecular basis for lincosamides-streptogramins A-pleuromutilins (LSAP)-resistant phenotype. Results: We identified a novel lsa(D)-encoded 497-aa ATP-binding cassette F (ABC-F) protein in the LSAP-resistant strains. Amino acid identities of 41.25-54.73% were obtained between the deduced amino acids from Lsa(D) and other Lsa-type ABC-F proteins. Furthermore, comparative analysis revealed that the allele of lsa(D) with single point mutation at 233 aa position (TGG → TAG; tryptophan→premature termination codon [PTC]) in LSAP-sensitive strains. The minimum inhibitory concentrations of antimicrobials against the lsa(D) complementary strain and lsa(D)-disrupted mutant confirmed that lsa(D) conferred the LSAP-resistant phenotype. The reverse transcription-polymerase chain reaction could not detect the noncoding region of lsa(D) allelic variant in the LSAP-sensitive strains. Additionally, the PTC (TAG) in LCM-sensitive strains was replaced by TGG, CAG, or TAT in the laboratory-induced revertant mutants. Conclusions: The novel lsa(D) conferred the LSAP-resistant phenotype in clinically LCM-resistant L. garvieae serotype II strains. However, the allele of lsa(D) gene containing the PTC was found in L. garvieae serotype II, resulting in the LSAP-susceptible phenotype.

Prediction of the Sex-Associated Genomic Region in Tunas (Thunnus Fishes).

Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex determination gene in Thunnus fishes or at least functions at a crucial point in the sex-differentiation cascade.

Estimation of tuna population by the improved analytical pipeline of unique molecular identifier-assisted HaCeD-Seq (haplotype count from eDNA).

Many studies have investigated the ability to identify species from environmental DNA (eDNA). However, even when individual species are identified, the accurate estimation of their abundances by traditional eDNA analyses has been still difficult. We previously developed a novel analytical method called HaCeD-Seq (Haplotype Count from eDNA), which focuses on the mitochondrial D-loop sequence. The D-loop is a rapidly evolving sequence and has been used to estimate the abundance of eel species in breeding water. In the current study, we have further improved this method by applying unique molecular identifier (UMI) tags, which eliminate the PCR and sequencing errors and extend the detection range by an order of magnitude. Based on this improved HaCeD-Seq pipeline, we computed the abundance of Pacific bluefin tuna (Thunnus orientalis) in aquarium tanks at the Tokyo Sea Life Park (Kasai, Tokyo, Japan). This tuna species is commercially important but is at high risk of resource depletion. With the developed UMI tag method, 90 out of 96 haplotypes (94%) were successfully detected from Pacific bluefin tuna eDNA. By contrast, only 29 out of 96 haplotypes (30%) were detected when UMI tags were not used. Our findings indicate the potential for conducting non-invasive fish stock surveys by sampling eDNA.

Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus.

Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 muM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.

Molecular characterization of the major yolk protein of the Japanese common sea cucumber (Apostichopus japonicus) and its expression profile during ovarian development.

The most abundant protein in the coelomic fluid of the Japanese common sea cucumber (Apostichopus japonicus) was purified through two steps of liquid chromatography. Subsequent peptide sequencing and cDNA cloning demonstrated that the purified fraction contained two similar but distinct proteins. Deduced amino acid sequences of these proteins revealed about 30% identity with those of sea urchin major yolk protein (MYP) and therefore they were designated AjMYP1 and AjMYP2. The full-length cDNAs for AjMYP1 and AjMYP2 consisted of 4600 and 4420bp, with predicted protein lengths of 1365 and 1345 amino acid residues, respectively. RT-PCR detected transcripts for both types of AjMYPs in all the tissues and organs examined. The transcript levels of both AjMYPs in the ovary were apparently elevated at late stages of ovarian development whereas the MYP content of the ovary examined by SDS-PAGE remained stable throughout ovarian development.

A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone.

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).

Author Correction: A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.