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.

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

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

Antibody profiling using a recombinant protein-based multiplex ELISA array accelerates recombinant vaccine development: Case study on red sea bream iridovirus as a reverse vaccinology model.

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme-linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack (Seriola quinqueradiata). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack (S. dumerili). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents.

High degree of genetic differentiation in marine three-spined sticklebacks (Gasterosteus aculeatus).

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three-spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene-based genome-scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection-as determined with several outlier detection methods-was low (F(ST) = 0.033 or 0.011, respectively), whereas average FST for directionally selected markers was significantly higher (F(ST) = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes.

Quantitative genetics of body size and timing of maturation in two nine-spined stickleback (Pungitius pungitius) populations.

Due to its influence on body size, timing of maturation is an important life-history trait in ectotherms with indeterminate growth. Comparison of patterns of growth and maturation within and between two populations (giant vs. normal sized) of nine-spined sticklebacks (Pungitius pungitius) in a breeding experiment revealed that the difference in mean adult body size between the populations is caused by differences in timing of maturation, and not by differential growth rates. The fish in small-sized population matured earlier than those from large-sized population, and maturation was accompanied by a reduction in growth rate in the small-sized population. Males matured earlier and at smaller size than females, and the fish that were immature at the end of the experiment were larger than those that had already matured. Throughout the experimental period, body size in both populations was heritable (h(2) = 0.10-0.64), as was the timing of maturation in the small-sized population (h(2) = 0.13-0.16). There was a significant positive genetic correlation between body size and timing of maturation at 140 DAH, but not earlier (at 80 or 110 DAH). Comparison of observed body size divergence between the populations revealed that Q(ST) exceeded F(ST) at older ages, indicating adaptive basis for the observed divergence. Hence, the results suggest that the body size differences within and between populations reflect heritable genetic differences in the timing of maturation, and that the observed body size divergence is adaptive.

High degree of sex chromosome differentiation in stickleback fishes.

BACKGROUND: Studies of closely related species with different sex chromosome systems can provide insights into the processes of sex chromosome differentiation and evolution. To investigate the potential utility of molecular markers in studying sex chromosome differentiation at early stages of their divergence, we examined the levels and patterns of genetic differentiation between sex chromosomes in nine-spined (Pungitius pungitius) and three-spined sticklebacks (Gasterosteus aculeatus) using microsatellite markers. RESULTS: A set of novel microsatellite markers spanning the entire length of the sex chromosomes were developed for nine-spined sticklebacks using the sequenced genomes of other fish species. Sex-specific patterns of genetic variability and male-specific alleles were identified at most of these loci, indicating a high degree of differentiation between the X and Y chromosomes in nine-spined sticklebacks. In three-spined sticklebacks, male-specific alleles were detected at some loci confined to two chromosomal regions. In addition, male-specific null alleles were identified at several other loci, implying the absence of Y chromosomal alleles at these loci. Overall, male-specific alleles and null alleles were found over a region spanning 81% of the sex chromosomes in three-spined sticklebacks. CONCLUSIONS: High levels but distinct patterns of sex chromosome differentiation were uncovered in the stickleback species that diverged 13 million years ago. Our results suggest that the Y chromosome is highly degenerate in three-spined sticklebacks, but not in nine-spined sticklebacks. In general, the results demonstrate that microsatellites can be useful in identifying the degree and patterns of sex chromosome differentiation in species at initial stages of sex chromosome evolution.

Global analysis of genes involved in freshwater adaptation in threespine sticklebacks (Gasterosteus aculeatus).

Examples of parallel evolution of phenotypic traits have been repeatedly demonstrated in threespine sticklebacks (Gasterosteus aculeatus) across their global distribution. Using these as a model, we performed a targeted genome scan--focusing on physiologically important genes potentially related to freshwater adaptation--to identify genetic signatures of parallel physiological evolution on a global scale. To this end, 50 microsatellite loci, including 26 loci within or close to (<6 kb) physiologically important genes, were screened in paired marine and freshwater populations from six locations across the Northern Hemisphere. Signatures of directional selection were detected in 24 loci, including 17 physiologically important genes, in at least one location. Although no loci showed consistent signatures of selection in all divergent population pairs, several outliers were common in multiple locations. In particular, seven physiologically important genes, as well as reference ectodysplasin gene (EDA), showed signatures of selection in three or more locations. Hence, although these results give some evidence for consistent parallel molecular evolution in response to freshwater colonization, they suggest that different evolutionary pathways may underlie physiological adaptation to freshwater habitats within the global distribution of the threespine stickleback.

A high incidence of selection on physiologically important genes in the three-spined stickleback, Gasterosteus aculeatus.

Genome scan approaches to detect footprints of directional selection in the genomes of wild animal and plant populations have become popular tools to study local adaptation and speciation at the molecular level. Most studies thus far have used random molecular markers and found footprints of directional selection at, on average, 5% (range: 1-15%) of the examined loci. We focused on physiologically important genes that exhibit transcriptional responses to specific environmental or developmental conditions and assessed if these genes have been subject to directional selection and are responsible for local adaptation in the three-spined stickleback (Gasterosteus aculeatus). Using microsatellite markers located within or closely linked to (<6 kb) target genes, we investigated footprints of directional selection for 157 genes with known physiological functions in three marine and six freshwater populations. A high incidence (16.6%) of footprints of directional selection for these genes was revealed by four different outlier tests. In a subset of four populations screened with both physiologically important and random genes, footprints of directional selection were more frequent in physiologically important genes (13.4%) as compared with random genes (2.4%). In general, our findings indicate strong selective pressures on physiologically important genes, suggesting that these genes have significant functions in evolutionary adaptation to environmental heterogeneity.

Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius).

BACKGROUND: Identification of genes involved in adaptation and speciation by targeting specific genes of interest has become a plausible strategy also for non-model organisms. We investigated the potential utility of available sequenced fish genomes to develop microsatellite (cf. simple sequence repeat, SSR) markers for functionally important genes in nine-spined sticklebacks (Pungitius pungitius), as well as cross-species transferability of SSR primers from three-spined (Gasterosteus aculeatus) to nine-spined sticklebacks. In addition, we examined the patterns and degree of SSR conservation between these species using their aligned sequences. RESULTS: Cross-species amplification success was lower for SSR markers located in or around functionally important genes (27 out of 158) than for those randomly derived from genomic (35 out of 101) and cDNA (35 out of 87) libraries. Polymorphism was observed at a large proportion (65%) of the cross-amplified loci independently of SSR type. To develop SSR markers for functionally important genes in nine-spined sticklebacks, SSR locations were surveyed in or around 67 target genes based on the three-spined stickleback genome and these regions were sequenced with primers designed from conserved sequences in sequenced fish genomes. Out of the 81 SSRs identified in the sequenced regions (44,084 bp), 57 exhibited the same motifs at the same locations as in the three-spined stickleback. Di- and trinucleotide SSRs appeared to be highly conserved whereas mononucleotide SSRs were less so. Species-specific primers were designed to amplify 58 SSRs using the sequences of nine-spined sticklebacks. CONCLUSIONS: Our results demonstrated that a large proportion of SSRs are conserved in the species that have diverged more than 10 million years ago. Therefore, the three-spined stickleback genome can be used to predict SSR locations in the nine-spined stickleback genome. While cross-species utility of SSR primers is limited due to low amplification success, SSR markers can be developed for target genes and genomic regions using our approach, which should be also applicable to other non-model organisms. The SSR markers developed in this study should be useful for identification of genes responsible for phenotypic variation and adaptive divergence of nine-spined stickleback populations, as well as for constructing comparative gene maps of nine-spined and three-spined sticklebacks.

History vs. habitat type: explaining the genetic structure of European nine-spined stickleback (Pungitius pungitius) populations.

The genetic structure of contemporary populations can be shaped by both their history and current ecological conditions. We assessed the relative importance of postglacial colonization history and habitat type in the patterns and degree of genetic diversity and differentiation in northern European nine-spined sticklebacks (Pungitius pungitius), using mitochondrial DNA (mtDNA) sequences and 12 nuclear microsatellite and insertion/deletion loci. The mtDNA analyses identified - and microsatellite analyses supported - the existence of two historically distinct lineages (eastern and western). The analyses of nuclear loci among 51 European sites revealed clear historically influenced and to minor degree habitat dependent, patterns of genetic diversity and differentiation. While the effect of habitat type on the levels of genetic variation (coastal > freshwater) and differentiation (freshwater > coastal) was clear, the levels of genetic variability and differentiation in the freshwater sites were independent of habitat type (viz. river, lake and pond). However, levels of genetic variability, together with estimates of historical effective population sizes, decreased dramatically and linearly with increasing latitude. These geographical patterns of genetic variability and differentiation suggest that the contemporary genetic structure of freshwater nine-spined sticklebacks has been strongly impacted by the founder events associated with postglacial colonization and less by current ecological conditions (cf. habitat type). In general, the results highlight the strong and persistent effects of postglacial colonization history on genetic structuring of northern European fauna and provide an unparalleled example of latitudinal trends in levels of genetic diversity.

Chromatophore distribution and inferior performance of albino Japanese flounder Paralichthys olivaceus with special reference to different chromatophore expression between albinism and pseudo-albinism.

Albinism with a large variation in body color was found in a hatchery population of Japanese flounder. In addition to albinism, ambicoloration and pseudo-albinism were simultaneously observed in some individuals. Albinos had a remarkably lower number of melanophores on the scales of ocular side than wild-type individuals did, although no significant difference was observed in the numbers of xanthophores and iridophores. The intensity of body color significantly correlated with the number of melanophores among the albinos. No significant differences were observed in the intensity of body color and the number of melanophores between the ocular side and the ambicoloration area. Pseudo-albinism was accompanied by the reductions of melanophores and xanthophores, indicating the different expression patterns of chromatophores between albinism and pseudo-albinism. The combined effects of albinism and pseudo-albinism caused the disappearances of melanophores and xanthophores in the pseudo-albinism area of albinos. In addition to chromatophores, the different characteristics of several phenotypic traits were observed between albinos and wild-type individuals. Growth-related traits of the albinos were inferior to those of the wild-type individuals. Furthermore, the albinos had a larger pseudo-albinism area and a higher vertebral deformed rate than the wild-type individuals did. Individual multilocus heterozygosity and inbreeding coefficient measured by microsatellite loci did not show any indication that the albinos had higher inbreeding coefficient than the wild-type individuals did. This study demonstrated the expression patterns of chromatophores in the body color abnormalities of a flatfish species and the potential pleiotropic effects of an albinism gene on some phenotypic traits.