Geometric morphometric methods for identification of oyster species based on morphology.

Both genetic and environmental factors affect the morphology of oysters. Molecular identification is currently the primary means of species identification, but it is inconvenient and costly. In this research, we evaluated the effectiveness of geometric morphometric (GM) techniques in distinguishing between two oyster species, Crassostreagigas and C.ariakensis. We used traditional morphometric and GM methods, including principal component analysis (PCA), thin-plate spline analysis (TPS) and canonical variable analysis (CVA), to identify specific features that distinguish the two species. We found that differences in shape can be visualised using GM methods. The Procrustes analysis revealed significant differences in shell morphology between C.gigas and C.ariakensis. The shells of C.ariakensis are more prominent at the widest point and are more scattered and have a greater variety of shapes. The shells of C.gigas are more oval in shape. PCA results indicated that PC1 explained 45.22%, PC2 explained 22.09% and PC3 explained 10.98% of the variation between the two species, which suggests that the main morphological differences are concentrated in these three principal components. Combining the TPS analysis function plots showed that the shell shape of C.ariakensis is mainly elongated and spindle-shaped, whereas the shell shape of C.gigas is more oval. The CVA results showed that the classification rate for the two species reached 100% which means that C.ariakensis and C.gigas have distinct differences in shell morphology and can be completely separated, based on morphological characteristics. Through these methods, a more comprehensive understanding of the morphological characteristics of different oyster populations can be obtained, providing a reference for oyster classification and identification.

Transcriptome analysis reveals core lncRNA-mRNA networks regulating melanization and biomineralization in Patinopecten yessoensis shell-infested by Polydora.

BACKGROUND: Patinopecten yessoensis, a large and old molluscan group, has been one of the most important aquaculture shellfish in Asian countries because of its high economic value. However, the aquaculture of the species has recently been seriously affected by the frequent outbreaks of Polydora disease, causing great economic losses. Long non-coding RNAs (lncRNAs) exhibit exhibit crucial effects on diverse biological processes, but still remain poorly studied in scallops, limiting our understanding of the molecular regulatory mechanism of P. yessoensis in response to Polydora infestation. RESULTS: In this study, a high-throughput transcriptome analysis was conducted in the mantles of healthy and Polydora-infected P. yessoensis by RNA sequencing. A total of 19,133 lncRNAs with 2,203 known and 16,930 novel were identified. The genomic characterizations of lncRNAs showed shorter sequence and open reading frame (ORF) length, fewer number of exons and lower expression levels in comparison with mRNAs. There were separately 2280 and 1636 differentially expressed mRNAs and lncRNAs (DEGs and DELs) detected in diseased individuals. The target genes of DELs were determined by both co-location and co-expression analyses. Functional enrichment analysis revealed that DEGs involved in melanization and biomineralization were significantly upregulated; further, obviously increased melanin granules were observed in epithelial cells of the edge mantle in diseased scallops by histological and TEM study, indicating the crucial role of melanizaiton and biomineralization in P. yessoensis to resist against Polydora infestation. Moreover, many key genes, such as Tyrs, Frizzled, Wnts, calmodulins, Pifs, perlucin, laccase, shell matrix protein, mucins and chitins, were targeted by DELs. Finally, a core lncRNA-mRNA interactive network involved in melanization and biomineralization was constructed and validated by qRT-PCR. CONCLUSIONS: This work provides valuable resources for studies of lncRNAs in scallops, and adds a new insight into the molecular regulatory mechanisms of P. yessoensis defending against Polydora infestation, which will contribute to Polydora disease control and breeding of disease-resistant varieties in molluscs.

Quantitative proteomic analysis reveals the molecular mechanism of the Yesso scallop (Patinopecten yessoensis) in response to Polydora infection.

The Yesso scallop is a large and ancient molluscan group with great economic value; however, it has recently suffered severe cases of Polydora infection. Polydora parasitizes the shells of scallops, badly damaging shell structures and affecting growth and mortality. To investigate the molecular mechanism of Yesso scallops' response to Polydora infection, proteomic profiling changes in the mantle tissues of Polydora-infected (diseased) and healthy scallops were systematically analysed by tandem mass tags (TMT) labelling technology in this study. A total of 519 differentially expressed proteins (DEPs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed most innated immune-related functions and pathways were significantly downregulated in diseased scallops, except the phagocytosis pathway, indicating an important role of phagocytosis in response to Polydora infection. DEPs involved in the phagocytosis pathway were associated with phagocytic receptor recognition, phagosome biogenesis and pathogen degradation, and they were further verified by quantitative real-time PCR. The results elucidate the molecular components of phagocytosis in molluscs for the first time. Polydora can be encapsulated by melanization with an obvious appearance in shells; indeed, melanization-related DEPs were upregulated in diseased scallops. Inhibition of apoptosis and nervous modulation may be also involved in the response mechanism, with some highly associated proteins significantly differentially expressed. Finally, a protein-protein interaction network was constructed to provide a global view of the interaction relationships of the DEPs. The study predicts the molecular response mechanism of Yesso scallops to Polydora infection, and lays a theoretical foundation for Polydora disease control.

Isolation, Identification, and Function of Rhodotorula mucilaginosa TZR2014 and Its Effects on the Growth and Health of Weaned Piglets.

A red yeast isolated from orange and grape soil and identified by the 26S rDNA sequence analysis revealed that it was Rhodotorula mucilaginosa and named TZR2014. Its biomass and carotenoid production reached a maximum when using the fermentation medium with pH 6.0, containing 5% glucose, 1% peptone, and 1.5% yeast powder. TZR2014 was resistant to 55°C for 15 min, 0.2% pig bile salts for 4 h, and artificial gastric and intestinal fluids. A total of thirty 28-day weaned pigs were divided into three groups, and the piglets were fed a basal diet (CON), a basal diet and orally administered 1 ml 1.0 × 1010 CFU/ml Candida utilis DSM 2361 three times (C. utilis), or a basal diet and orally administered 1 ml 1.0 × 1010 CFU/mL TZR2014 three times daily (R. mucilaginosa) for 4 weeks. Compared with the piglets in the CON group, those in the C. utilis or R. mucilaginosa group reported an increased average daily weight gain and average daily feed intake (P < 0.05) and a decreased feed/gain (P < 0.05). The diarrhea rate of piglets in the R. mucilaginosa group was lower than that in the CON and C. utilis groups (P < 0.05). Compared with that in the CON and C. utilis groups, the R. mucilaginosa group reported an increased ileum villus height (P < 0.05), serum concentration of total antioxidant content, total superoxide dismutase, and glutathione peroxidase and pepsin and lipase activities in the intestinal content, while it reported a decreased serum concentration of malondialdehyde and pH of the intestinal tract (P < 0.05). The relative abundances of Proteobacteria and Megasphaera of caecum in the R. mucilaginosa group were lower than those in the CON and C. utilis groups (P < 0.05). The relative abundances of Prevotella, Ruminococcaceae, Succinivibrio, Rikenellaceae RC9 gut group, and Roseburia of caecum in the R. mucilaginosa group were higher than those in the CON and C. utilis groups (P < 0.05). R. mucilaginosa TZR2014 can produce carotenoids and adapts to the animal's gastrointestinal environment. Oral R. mucilaginosa TZR2014 improved growth performance, enhanced antioxidant capacity, strengthened gastrointestinal digestion, and maintained the intestinal microbiological balance of piglets.

Genome-wide DNA methylation profile changes associated with shell colouration in the Yesso scallop (Patinopecten yessoensis) as measured by whole-genome bisulfite sequencing.

BACKGROUND: Mollusca, a phylum of highly rich species, possess vivid shell colours, but the underlying molecular mechanism remains to be elucidated. DNA methylation, one of the most common epigenetic modifications in eukaryotes, is believed to play a vital role in various biological processes. However, analysis of the effects of DNA methylation on shell colouration has rarely been performed in molluscs, limiting the current knowledge of the molecular mechanism of shell colour formation. RESULTS: In the present study, to reveal the role of epigenetic regulation in shell colouration, WGBS, the "gold standard" of DNA methylation analysis, was first performed on the mantle tissues of Yesso scallops (Patinopecten yessoensis) with different shell colours (brown and white), and DNA methylomes at single-base resolution were generated. About 3% of cytosines were methylated in the genome of the Yesso scallop. A slight increase in mCG percentage and methylation level was found in brown scallops. Sequence preference of nearby methylated cytosines differed between high and low methylation level sites and between the brown- and white-shelled scallops. DNA methylation levels varied among the different genomic regions; all the detected regions in the brown group exhibited higher methylation levels than the white group. A total of 41,175 DMRs (differentially methylated regions) were detected between brown and white scallops. GO functions and pathways associated with the biosynthesis of melanin and porphyrins were significantly enriched for DMRs, among which several key shell colour-related genes were identified. Further, different correlations between mRNA expression levels and DNA methylation status were found in these genes, suggesting that DNA methylation regulates shell colouration in the Yesso scallop. CONCLUSIONS: This study provides genome-wide DNA methylation landscapes of Yesso scallops with different shell colours, offering new insights into the epigenetic regulatory mechanism underlying shell colour.

Genome-wide identification, characterisation and expression analysis of the ALAS gene in the Yesso scallop (Patinopecten yessoensis) with different shell colours.

Porphyrins, one of the most common shell pigments, are by-products of the haem pathway. 5-Aminolaevulinate synthase (ALAS) is the first and rate-limiting enzyme in this pathway and has been well studied in vertebrate species. However, the function of ALAS in shell colouration has been poorly studied in molluscs, which are renowned for their colourful shells. In the present study, an ALAS gene, named PyALAS, was identified through whole-genome scanning in the Yesso scallop (Patinopecten yessoensis), an economically and evolutionarily important bivalve species in which the shell colour represents polymorphism. Two conserved domains were detected in the PyALAS protein sequence, including a Preseq-ALAS domain and a 5-ALAS domain, confirming the identification of PyALAS. Phylogenetic analysis of the ALAS proteins among various invertebrate and vertebrate species revealed a high consistency between the molecular evolution of ALAS and the species taxonomy. PyALAS was ubiquitously expressed in most adult tissues of the Yesso scallop. The left mantle expressed a significantly higher level of PyALAS than the right side in brown scallops, whereas there was no significant difference in white scallops. Significantly different expression levels of PyALAS was also detected between the two different shell colour strains. These data indicate that PyALAS plays an important role in shell colouration in Yesso scallops and the present study provides new insights into the molecular mechanism of shell colouration in molluscs.

Histological and Expression Differences Among Different Mantle Regions of the Yesso Scallop (Patinopecten yessoensis) Provide Insights into the Molecular Mechanisms of Biomineralization and Pigmentation.

The molecular mechanisms of shell formation and pigmentation are issues of great interest in molluscan studies due to the unique physical and biological properties of shells. The Yesso scallop, Patinopecten yessoensis, is one of the most important maricultural bivalves in Asian countries, and its shell color shows polymorphism. To gain more information about the underlying mechanisms of shell formation and pigmentation, this study presents the first analyses of histological and transcriptional differences between different mantle regions of the Yesso scallop, which are thought to be responsible for the formation of different shell layers. The results showed major microstructural differences between the edge and central mantles, which were closely associated with their functions. Different biomineralization-related GO functions, which might participate in the formation of different shell layers, were significantly enriched in the different mantle regions, indicating the different molecular functions of the two mantle regions in shell formation. The melanogenesis pathway, which controls melanin biosynthesis, was the most significantly enriched pathway in the DEGs between the two mantle regions, indicating its important role in shell pigmentation. Tyr, the key and rate-limiting gene in melanogenesis, was expressed at a remarkably high level in the central mantle, while the upstream regulatory genes included in melanogenesis were mainly upregulated in the edge mantle, suggesting the different molecular functions of the two mantle regions in shell pigmentation.

Genome-wide identification, characterization and expression analysis of the MITF gene in Yesso scallops (Patinopecten yessoensis) with different shell colors.

The microphthalmia-associated transcription factor (MITF) is the center of the regulator network of melanin synthesis in vertebrates. However, the role of MITF in shell color formation is poorly studied in mollusks. In the present study, an MITF gene, PyMITF, was first identified at the whole-genome level in Yesso scallop (Patinopecten yessoensis), an evolutionarily and economically important species, the shell color of which shows polymorphism. The PyMITF is a large gene spanning ~37 kb in the genome with 7 introns and 8 exons. A basic helix-loop-helix leucine zipper (bHLH-LZ) domain was detected in the PyMITF protein sequence, which can bind the canonical E-box sequence in the promoter region of the downstream genes. Phylogenetic analysis of the MITFs among vertebrates and invertebrates revealed that the molecular evolution of MITFs was consistent with the species taxonomy. Different expression levels of PyMITF were detected among different shell color strains, indicating the important role of PyMITF involved in shell pigmentation. Besides, PyMITF was expressed at a significantly higher level in the central mantle than that in the edge mantle, proving the participation of the central mantle in shell color formation in molecular level for the first time. The work provides valuable information for the molecular mechanism study of shell color formation.

Transcriptional changes in the Japanese scallop (Mizuhopecten yessoensis) shellinfested by Polydora provide insights into the molecular mechanism of shell formation and immunomodulation.

The Japanese scallop (Mizuhopecten yessoensis) is one of the most important aquaculture species in Asian countries; however, it has suffered severe infection by Polydora in northern China in recent years, causing great economic losses. The Polydora parasitizes the shell of scallops, badly destroying the shell's structure. To investigate the molecular response mechanism of M. yessoensis to Polydora infestion, a comprehensive and niche-targeted cDNA sequence database for diseased scallops was constructed. Additionally, the transcriptional changes in the edge mantle, central mantle and hemocytes, tissues directly related to the disease, were first described in this study. The results showed that genes involved in shell formation and immunomodulation were significantly differentially expressed due to the infestation. Different transcriptional changes existed between the two mantle regions, indicating the different molecular functions likely responsible for the formation of different shell layers. The differential expression of genes for immune recognition, signal transduction and pathogen elimination presented an integrated immune response process in scallops. Moreover, neuromodulation and glycometabolism involved in the regulation process with relevant function significantly enriched. The study provides valuable information for mechanism study of shell formation and immunomodulation in scallops.

Scallop genome provides insights into evolution of bilaterian karyotype and development.

Reconstructing the genomes of bilaterian ancestors is central to our understanding of animal evolution, where knowledge from ancient and/or slow-evolving bilaterian lineages is critical. Here we report a high-quality, chromosome-anchored reference genome for the scallop Patinopecten yessoensis, a bivalve mollusc that has a slow-evolving genome with many ancestral features. Chromosome-based macrosynteny analysis reveals a striking correspondence between the 19 scallop chromosomes and the 17 presumed ancestral bilaterian linkage groups at a level of conservation previously unseen, suggesting that the scallop may have a karyotype close to that of the bilaterian ancestor. Scallop Hox gene expression follows a new mode of subcluster temporal co-linearity that is possibly ancestral and may provide great potential in supporting diverse bilaterian body plans. Transcriptome analysis of scallop mantle eyes finds unexpected diversity in phototransduction cascades and a potentially ancient Pax2/5/8-dependent pathway for noncephalic eyes. The outstanding preservation of ancestral karyotype and developmental control makes the scallop genome a valuable resource for understanding early bilaterian evolution and biology.

MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes.

Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the 'perfect solution', and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost.

Construction of a High-Density Genetic Map and Quantitative Trait Locus Mapping in the Sea Cucumber Apostichopus japonicus.

Genetic linkage maps are critical and indispensable tools in a wide range of genetic and genomic research. With the advancement of genotyping-by-sequencing (GBS) methods, the construction of a high-density and high-resolution linkage maps has become achievable in marine organisms lacking sufficient genomic resources, such as echinoderms. In this study, high-density, high-resolution genetic map was constructed for a sea cucumber species, Apostichopus japonicus, utilizing the 2b-restriction site-associated DNA (2b-RAD) method. A total of 7839 markers were anchored to the linkage map with the map coverage of 99.57%, to our knowledge, this is the highest marker density among echinoderm species. QTL mapping and association analysis consistently captured one growth-related QTL located in a 5 cM region of linkage group (LG) 5. An annotated candidate gene, retinoblastoma-binding protein 5 (RbBP5), which has been reported to be an important regulator of cell proliferation, was recognized in the QTL region. This linkage map represents a powerful tool for research involving both fine-scale QTL mapping and marker assisted selection (MAS), and will facilitate chromosome assignment and improve the whole-genome assembly of sea cucumber in the future.

High-resolution linkage and quantitative trait locus mapping aided by genome survey sequencing: building up an integrative genomic framework for a bivalve mollusc.

Genetic linkage maps are indispensable tools in genetic and genomic studies. Recent development of genotyping-by-sequencing (GBS) methods holds great promise for constructing high-resolution linkage maps in organisms lacking extensive genomic resources. In the present study, linkage mapping was conducted for a bivalve mollusc (Chlamys farreri) using a newly developed GBS method-2b-restriction site-associated DNA (2b-RAD). Genome survey sequencing was performed to generate a preliminary reference genome that was utilized to facilitate linkage and quantitative trait locus (QTL) mapping in C. farreri. A high-resolution linkage map was constructed with a marker density (3806) that has, to our knowledge, never been achieved in any other molluscs. The linkage map covered nearly the whole genome (99.5%) with a resolution of 0.41 cM. QTL mapping and association analysis congruously revealed two growth-related QTLs and one potential sex-determination region. An important candidate QTL gene named PROP1, which functions in the regulation of growth hormone production in vertebrates, was identified from the growth-related QTL region detected on the linkage group LG3. We demonstrate that this linkage map can serve as an important platform for improving genome assembly and unifying multiple genomic resources. Our study, therefore, exemplifies how to build up an integrative genomic framework in a non-model organism.

RADtyping: an integrated package for accurate de novo codominant and dominant RAD genotyping in mapping populations.

Genetic linkage maps are indispensable tools in genetic, genomic and breeding studies. As one of genotyping-by-sequencing methods, RAD-Seq (restriction-site associated DNA sequencing) has gained particular popularity for construction of high-density linkage maps. Current RAD analytical tools are being predominantly used for typing codominant markers. However, no genotyping algorithm has been developed for dominant markers (resulting from recognition site disruption). Given their abundance in eukaryotic genomes, utilization of dominant markers would greatly diminish the extensive sequencing effort required for large-scale marker development. In this study, we established, for the first time, a novel statistical framework for de novo dominant genotyping in mapping populations. An integrated package called RADtyping was developed by incorporating both de novo codominant and dominant genotyping algorithms. We demonstrated the superb performance of RADtyping in achieving remarkably high genotyping accuracy based on simulated and real mapping datasets. The RADtyping package is freely available at http://www2.ouc.edu.cn/mollusk/ detailen.asp?id=727.

Development of a rapid and efficient method for non-lethal DNA sampling and genotyping in scallops.

Non-lethal DNA sampling has long appealed to researchers studying population and conservation genetics, as it does not necessitate removing individuals permanently from their natural environment or destroying valuable samples. However, such an approach has not yet been well established in bivalves. In this study, we demonstrate that the gill represents a good source of tissue for non-lethal sampling in scallops. Removal of a few gill filaments caused no noticeable behavioral abnormalities or increased mortality rates in Zhikong scallop (Chlamys farreri) during a three-month period of observation. To facilitate rapid gill-based DNA extraction, six methods (MA-MF) were designed and evaluated, each requiring less than one hour of processing time. The optimal method was identified as MF, in terms of maintaining DNA integrity and genotyping accuracy. Further optimization of MF method by orthogonal experimental design suggested that the utilization of gills could be limited to 2 mg of sample, which is sufficient for performing up to 20,000 PCR reactions. We also demonstrate the excellent cross-species utility of MF in two additional scallop species, Yesso scallop (Patinopecten yessoensis) and bay scallop (Argopecten irradians). Taken together, our study provides a rapid and efficient approach for applying non-lethal DNA sampling in bivalve species, which would serve as a valuable tool for maintaining bivalve populations and conservation genetics, as well as in breeding studies.

Genomic characterization of interspecific hybrids between the scallops Argopecten purpuratus and A. irradians irradians.

The Peruvian scallop (Argopecten purpuratus) has been introduced to China and has successfully been hybridized with the bay scallop (A. irradians irradians). The F1 hybrids of these two scallops exhibited a large increase in production traits and some other interesting new characteristics. To understand the genetic basis of this heterosis, nuclear gene and partial mtDNA sequences, and genomic in situ hybridization (GISH) were employed to analyze the genomic organization of the hybrids. Amplification of the ribosomal DNA internal transcribed spacer (ITS) showed that the parental ITS sequences were present in all the hybrid individuals, illustrating that the hybrid offspring inherited nuclear DNA from both parents. Sequence analyses of the ITS region further confirmed that the hybrids harbored alleles from their parents; some recombinant variants were also detected, which revealed some alterations in the nuclear genetic material of the hybrids. The analysis of mitochondrial 16S rDNA showed that the hybrids possessed sequences that were identical to the 16S rDNA of the female parents, proving a matrilineal inheritance of mitochondrial genes in scallops. In addition, GISH clearly discriminated between the parental chromosomes and indicated a combination of haploid genomes of duplex parents in the hybrids. The genetic analyses in our study illustrated that the F1 hybrids inherited nuclear material from both parents and cytoplasmic genetic material maternally, and some variations occurred in the genome, which might contribute to a further understanding of crossbreeding and heterosis in scallop species.