Maintenance of Genetic Diversity of Black Sea Bream despite Unmonitored and Large-Scale Hatchery Releases.

Stock enhancement, used for replenishing depleted wild finfish populations, is an aggressive approach. Stock enhancement projects in Taiwan involve black sea bream (Acanthopagrus schlegelii), a major commercial species. During 2004-2015, even management agencies conducted stock enhancement projects, leading to numerous private releases that have not been recorded. Stock enhancement by a private hatchery without accurate genetic records may lead to a genetic structure change in wild populations. Using allele frequencies at nine microsatellite loci, we studied the genetic effects of stock enhancement in 19 samples collected from populations in the hatcheries and the wild. In 458 individuals from nine hatchery samples, most populations showed weak but significant genetic differences and complex clusters in structure analysis, indicating dramatic stock change within and among hatcheries. The 10 wild populations (n = 773) also had a complex genetic composition and were genetically different among sampling sites and times. However, a simple and clear cluster in structure analysis was found for only one sampling site, which had no release history. Thus, stock enhancement with complex genetic sources helps maintain genetic diversity but dramatically changes the genetic structure within and among wild populations, especially when stock enhancement is successful.

Functional Adaptation of Vocalization Revealed by Morphological and Histochemical Characteristics of Sonic Muscles in Blackmouth Croaker (Atrobucca nibe).

Sound production in the blackmouth croaker (Atrobucca nibe) was characterized using acoustic, morphological, and histochemical methods. Their calls consisted of a train of two to seven pulses; the frequency ranged from 180 to 3000 Hz, with a dominant frequency of 326 ± 40 Hz. The duration of each call ranged from 80 to 360 ms. Male A. nibe possess a pair of bilaterally symmetric sonic muscles attached to the body wall adjacent to the swim bladder. The average diameter of the sonic muscle fibers was significantly shorter than that of the abdominal muscle fibers. Semithin sections of the sonic muscle fibers revealed a core-like structure (central core) and the radial arrangement of the sarcoplasmic reticulum and myofibrils. Numerous mitochondria were distributed within the central core and around the periphery of the fibers. Most of the fibers were identified as Type IIa on the basis of their myosin adenosine triphosphatase activities, but a few were identified as Type IIc fibers. All sonic muscle fibers exhibited strong oxidative enzyme activity and oxidative and anaerobic capabilities. The features suggest that the sonic muscles of A. nibe are morphologically and physiologically adapted for fast twitching and fatigue resistance, which support fish vocalization.

Environmental DNA (eDNA) Metabarcoding in the Fish Market and Nearby Seafood Restaurants in Taiwan Reveals the Underestimation of Fish Species Diversity in Seafood.

Seafood, especially the traditional one in Taiwan, is rarely sourced from a fixed species and routinely from similar species depending on their availability. Hence, the species composition of seafood can be complicated. While a DNA-based approach has been routinely utilized for species identification, a large scale of seafood identification in fish markets and restaurants could be challenging (e.g., elevated cost and time-consuming only for a limited number of species identification). In the present study, we aimed to identify the majority of fish species potentially consumed in fish markets and nearby seafood restaurants using environmental DNA (eDNA) metabarcoding. Four eDNA samplings from a local fish market and nearby seafood restaurants were conducted using Sterivex cartridges. Nineteen universal primers previously validated for fish species identification were utilized to amplify the fragments of mitochondrial DNA (12S, COI, ND5) of species in eDNA samples and sequenced with NovaSeq 6000 sequencing. A total of 153 fish species have been identified based on 417 fish related operational taxonomic units (OTUs) generated from 50,534,995 reads. Principal Coordinate Analysis (PCoA) further showed the differences in fish species between the sampling times and sampling sites. Of these fish species, 22 chondrichthyan fish, 14 Anguilliformes species, and 15 Serranidae species were respectively associated with smoked sharks, braised moray eels, and grouper fish soups. To our best knowledge, this work represents the first study to demonstrate the feasibility of a large scale of seafood identification using eDNA metabarcoding approach. Our findings also imply the species diversity in traditional seafood might be seriously underestimated and crucial for the conservation and management of marine resources.

Polyculture of Juvenile Dog Conch Laevistrombus canarium Reveals High Potentiality in Integrated Multitrophic Aquaculture (IMTA).

Laevistrombus canarium, also known as dog conch, is a marine gastropod mollusk widely distributed in the Indo-Pacific region. It is an economically crucial species; however, its population has been declining due to overfishing and overexploitation. In this study, the suitable salinity for juvenile L. canarium was between 20 and 35‰. Diatoms and biological detritus by using flow-water from the fish pool were the most favorable diets for newly metamorphosed and 10 mm juveniles. In the polyculture experiment, L. canarium was cultured with whiteleg shrimp, tilapia, small abalone, purple sea urchin, and collector urchin. Better growth was found in all co-culture groups except with whiteleg shrimp. We also found that the polyculture system with or without substrates significantly affected the growth of juveniles. Additionally, we observed that water temperature was the most crucial factor for growth and survival; a water temperature of less than 10 °C might cause the death of L. canarium. We have proposed a novel polyculture and water-flow method for mass production of L. canarium and evaluated the feasibility and benefits of polyculture with other species. The findings from this work reveal the potentiality of L. canarium in integrated multitrophic aquaculture (IMTA) and its implication for aquaculture and resource restoration.

The complete mitochondrial genome of Metasepia tullbergi (Cephalopoda: Sepiidae).

The first complete mitochondrial genome of Metasepia tullbergi has been characterized in this study. The circular mitogenome is 16182 bp in length and comprises 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The organization of these genes is highly consistent with that of other Sepiidae. The overall base composition of mitogenome is 39.20% A, 36.07% T, 8.98% G, and 15.75% C, with 75.27% AT. Phylogenetic analysis further suggests that M. tullbergi is placed within the Sepiidae and is closely related to Sepia latimanus and S. apama.

The complete mitochondrial genome of Laevistrombus canarium (Gastropoda: Stromboidae).

Laevistrombus canarium is a marine gastropod species with high economical value. The complete mitochondrial genome of L. canarium has been characterized in this study. The circular mitogenome is 15626 bp in length and comprises 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA (rRNA) genes. The organization of these genes is consistent with that of other stromboidae species. The overall base composition of mitochondrial genome is 30.87% A, 38.99% T, 15.54% G, and 14.60% C, with 69.86% AT. Phylogenetic analysis further implies that L. canarium is placed within the Stromboidae.

Development of EST-Molecular Markers from RNA Sequencing for Genetic Management and Identification of Growth Traits in Potato Grouper (Epinephelus tukula).

The accuracy and efficiency of marker-assisted selection (MAS) has been proven for economically critical aquaculture species. The potato grouper (Epinephelus tukula), a novel cultured grouper species in Taiwan, shows large potential in aquaculture because of its fast growth rate among other groupers. Because of the lack of genetic information for the potato grouper, the first transcriptome and expressed sequence tag (EST)-derived simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were developed. Initially, the transcriptome was obtained from seven cDNA libraries by using the Illumina platform. De novo transcriptome of the potato grouper yielded 51.34 Gb and 111,490 unigenes. The EST-derived SSR and SNP markers were applied in genetic management, in parentage analysis, and to discover the functional markers of economic traits. The F1 juveniles were identified as siblings from one pair of parents (80 broodstocks). Fast- and slow-growth individuals were analyzed using functional molecular markers and through their association with growth performance. The results revealed that two SNPs were correlated with growth traits. The transcriptome database obtained in this study and its derived SSR and SNP markers may be applied not only for MAS but also to maintain functional gene diversity in the novel cultured grouper.

Stac3 is required for myotube formation and myogenic differentiation in vertebrate skeletal muscle.

Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.

Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes.

We investigated the effects of embryonic temperature (ET) treatments (22, 26 and 31 degrees C) on the life-time recruitment of fast myotomal muscle fibres in zebrafish Danio rerio L. reared at 26/27 degrees C from hatching. Fast muscle fibres were produced until 25 mm total length (TL) at 22 degrees C ET, 28 mm TL at 26 degrees C ET and 23 mm TL at 31 degrees C ET. The final fibre number (FFN) showed an optimum at 26 degrees C ET (3600) and was 19% and 14% higher than for the 22 degrees C ET (3000) and 31 degrees C ET (3100) treatments, respectively. Further growth to the maximum TL of approximately 48 mm only involved fibre hypertrophy. Microarray experiments were used to determine global changes in microRNA (miRNA) and mRNA expression associated with the transition from the hyperplasic myotube-producing phenotype (M(+), 10-12 mm TL) to the hypertrophic growth phenotype (M(-), 28-31 mm TL) in fish reared at 26-27 degrees C over the whole life-cycle. The expression of miRNAs and mRNAs obtained from microarray experiments was validated by northern blotting and real-time qPCR in independent samples of fish with the M(+) and M(-) phenotype. Fourteen down-regulated and 15 up-regulated miRNAs were identified in the M(-) phenotype together with 34 down-regulated and 30 up-regulated mRNAs (>2-fold; P<0.05). The two most abundant categories of down-regulated genes in the M(-) phenotype encoded contractile proteins (23.5%) and sarcomeric structural/cytoskeletal proteins (14.7%). In contrast, the most highly represented up-regulated transcripts in the M(-) phenotype were energy metabolism (26.7%) and immune-related (20.0%) genes. The latter were mostly involved in cell-cell interactions and cytokine pathways and included beta-2-microglobulin precursor (b2m), an orthologue of complement component 4, invariant chain-like protein 1 (iclp), CD9 antigen-like (cd9l), and tyrosine kinase, non-receptor (tnk2). Five myosin heavy chain genes that were down-regulated in the M(-) phenotype formed part of a tandem repeat on chromosome 5 and were shown by in situ hybridisation to be specifically expressed in nascent myofibres. Seven up-regulated miRNAs in the M(-) phenotype showed reciprocal expression with seven mRNA targets identified in miRBase Targets version 5 (http://microrna.sanger.ac.uk/targets/v5/), including asporin (aspn) which was the target for four miRNAs. Eleven down-regulated miRNAs in the M(-) phenotype had predicted targets for seven up-regulated genes, including dre-miR-181c which had five predicted mRNA targets. These results provide evidence that miRNAs play a role in regulating the transition from the M(+) to the M(-) phenotype and identify some of the genes and regulatory interactions involved.

Genomic, evolutionary, and expression analyses of cee, an ancient gene involved in normal growth and development.

The cee (conserved edge expressed protein) gene was recently identified in a genome-wide screen to discover genes associated with myotube formation in fast muscle of pufferfish. Comparative genomic analyses indicate that cee arose some 1.6-1.8 billion years ago and is found as a single-copy gene in most eukaryotic genomes examined. The complexity of its structure varies from an intronless gene in yeast and tunicates to nine exons and eight introns in vertebrates. cee is particularly conserved among vertebrates and is located in a syntenic region within tetrapods and between teleosts and invertebrates. Low dN/dS ratios in the cee coding region (0.02-0.09) indicate that the Cee protein is under strong purifying selection. In Atlantic salmon, cee is expressed in the superficial layers of developing organs and tissues. These data, together with functional screens in yeast and Caenorhabditis elegans, indicate that cee has a hitherto uncharacterized role in normal growth and development.