Easy-to-Use CRISPR-Cas9 Genome Editing in the Cultured Pacific Abalone (Haliotis discus hannai).

The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene ß-tubulin and light-sensitive transmembrane protein r-opsin as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of ß-tubulin and r-opsin genes from injected larvae identified various genomic long-fragment deletions. In situ hybridization showed gene expression patterns of ß-tubulin and r-opsin were significantly altered in the mosaic mutants. Knocking out ß-tubulin in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of r-opsin in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.

Host-microbiota interactions play a crucial role in oyster adaptation to rising seawater temperature in summer.

Climate change, represented by rising and fluctuating temperature, induces systematic changes in marine organisms and in their bacterial symbionts. However, the role of host-microbiota interactions in the host's response to rising temperature and the underlying mechanisms are incompletely understood in marine organisms. Here, the symbiotic intestinal microbiota and transcriptional responses between diploid and triploid oysters that displayed susceptible and resistant performance under the stress of rising temperature during a summer mortality event were compared to investigate the host-microbiota interactions. The rising and fluctuating temperatures triggered an earlier onset and higher mortality in susceptible oysters (46.7%) than in resistant oysters (17.3%). Correlation analysis between microbial properties and environmental factors showed temperature was strongly correlated with indices of α-diversity and the abundance of top 10 phyla, indicating that temperature significantly shaped the intestinal microbiota of oysters. The microbiota structure of resistant oysters exhibited more rapid changes in composition and diversity compared to susceptible oysters before peak mortality, indicating that resistant oysters possessed a stronger ability to regulate their symbiotic microbiota. Meanwhile, linear discriminant analysis effect size (LefSe) analysis found that the probiotics Verrucomicrobiales and Clostridiales were highly enriched in resistant oysters, and that potential pathogens Betaproteobacteriales and Acidobacteriales were enriched in susceptible oysters. These results implied that the symbiotic microbiota played a significant role in the oysters' adaptation to rising temperature. Accompanying the decrease in unfavorable bacteria before peak mortality, genes related to phagocytosis and lysozymes were upregulated and the xenobiotics elimination pathway was exclusively expressed in resistant oysters, demonstrating the validity of these immunological functions in controlling proliferation of pathogens driven by rising temperature. Compromised immunological functions might lead to proliferation of pathogens in susceptible oysters. This study might uncover a conserved mechanism of adaptation to rising temperature in marine invertebrates from the perspective of interactions between host and symbiotic microbiota.

Integrated application of transcriptomics and metabolomics provides insights into condition index difference mechanisms in the Pacific oyster (Crassostrea gigas).

The condition index (CI) is an economically important tool for assessing the quality of oysters, such as the Pacific oyster Crassostrea gigas. However, little is known about the mechanisms that underlie differences in CI between different C. gigas populations. In this study, we integrated transcriptomic and metabolomic profiling to investigate the mechanisms that underlie the differences between high- and low-CI groups in one- and two-year-old populations of C. gigas. The results indicate that differences in CI were associated with the regulation of growth-related genes, the FoxO signaling pathway, and the complex regulation of carbohydrate, lipid, amino acid, and energy metabolism. Moreover, the mechanisms underlying these differences differed between the populations. This study is the first to elucidate the molecular and chemical mechanisms associated with CI, and the results will be helpful for breeding higher quality oysters.

Comparative and evolutionary analyses reveal conservation and divergence of the notch pathway in lophotrochozoa.

Lophotrochozoan species exhibit wide morphological diversity; however, the molecular basis underlying this diversity remains unclear. Here, we explored the evolution of Notch pathway genes across 37 metazoan species via phylogenetic and molecular evolutionary studies with emphasis on the lophotrochozoans. We displayed the components of Notch pathway in metazoans and found that Delta and Hes/Hey-related genes, as well as their functional domains, are duplicated in lophotrochozoans. Comparative transcriptomics analyses allow us to pinpoint sequence divergence of multigene families in the Notch signalling pathway. We identified the duplication mechanism of a mollusc-specific gene, Delta2, and found it displayed complementary expression throughout development. Furthermore, we found the functional diversification not only in expanded genes in the Notch pathway (Delta and Hes/Hey-related genes), but also in evolutionary conservative genes (Notch, Presenilin, and Su(H)). Together, this comprehensive study demonstrates conservation and divergence within the Notch pathway, reveals evolutionary relationships among metazoans, and provides evidence for the occurrence of developmental diversity in lophotrochozoans, as well as a basis for future gene function studies.

Characterization of Free Fatty Acid Receptor 4 and Its Involvement in Nutritional Control and Immune Response in Pacific Oysters (Crassostrea gigas).

Free fatty acid receptor 4 (FFAR4) has various physiological functions, including energy regulation and immunological homeostasis. We examined the only FFAR4 homologue in the Pacific oyster Crassostrea gigas (CgFFAR4), which functions as a sensor of long-chain fatty acids. CgFFAR4 is 1098 bp long and contains a seven-transmembrane G protein-coupled receptor domain. CgFFAR4 expression was high in the hepatopancreas, but it was downregulated after fasting, indicating that it plays an essential role in food digestion. Lipopolysaccharide stimulation downregulated CgFFAR4 level, probably as an immune response of the animal. Reduced glycogen level alongside decreased insulin receptor, insulin receptor substrate, and C. gigas glycogen synthase transcription levels after CgFFAR4 knockdown revealed that CgFFAR4 was involved in the regulation of fatty acid and glycogen levels via the insulin pathway. Accordingly, this is the first study on an invertebrate FFAR and provides new insights into the role of this receptor in immune response and nutritional control.

Divergence and plasticity shape adaptive potential of the Pacific oyster.

The interplay between divergence and phenotypic plasticity is critical to our understanding of a species' adaptive potential under rapid climate changes. We investigated divergence and plasticity in natural populations of the Pacific oyster Crassostrea gigas with a congeneric oyster Crassostrea angulata from southern China used as an outgroup. Genome re-sequencing of 371 oysters revealed unexpected genetic divergence in a small area that coincided with phenotypic divergence in growth, physiology, heat tolerance and gene expression across environmental gradients. These findings suggest that selection and local adaptation are pervasive and, together with limited gene flow, influence population structure. Genes showing sequence differentiation between populations also diverged in transcriptional response to heat stress. Plasticity in gene expression is positively correlated with evolved divergence, indicating that plasticity is adaptive and favoured by organisms under dynamic environments. Divergence in heat tolerance-partly through acetylation-mediated energy depression-implies differentiation in adaptive potential. Trade-offs between growth and survival may play an important role in local adaptation of oysters and other marine invertebrates.

The effects of feeding Lactobacillus pentosus on growth, immunity, and disease resistance in Haliotis discus hannai Ino.

To study the effects of probiotic-added food on the survival and growth of abalone (Haliotis discus hannai Ino), the expression levels of nonspecific immune genes and the anti-Vibrio parahaemolyticus infection were examined. During an 8-week culturing experiment in an indoor aquarium and a 2-week V. parahaemolyticus artificial infection experiment, the control group was fed with untreated food once a day, while the experimental groups (L1, L2 and L3) were fed with Lactobacillus pentosus added food. The concentration of probiotics in the experimental food was 103 cfu/g (L1), 105 cfu/g (L2) and 107 cfu/g (L3), respectively. The results showed that the survival rate, shell length-specific growth rate, and the food conversion rate (FCR) of abalones in L1 and L2 were significantly higher than the control group. The food intake of abalones in L3 was significantly lower than that in L1, L2 and the control group, but there was no significant difference in FCR identified between L1, L2 and L3. In the L. pentosus-added groups, the total number of blood lymphocytes, lysozyme activity, acid phosphatase, superoxide dismutase, and expression levels of Mn-superoxide dismutase (Mn-SOD) and thioredoxin peroxidase (TPx) were significantly higher than the control group, while the malondialdehyde (MDA) content was significantly lower than the control group. The phagocytic activity of blood lymphocytes, catalase activity and the expression levels of heat shock protein 70 (HSP70) of abalones in the control group were significantly lower than that in L1 and L2, but there was no significant difference when compared with L3. The levels of O2-, NO produced by respiratory burst of blood lymphocytes and the expression levels of catalase (CAT) in L1 and L2 were significantly higher than both L3 and the control group. Seven days after infection with V. parahaemolyticus, all abalones in the control group were dead. After 14 days the cumulative mortality rate of abalones in the L. pentosus-added groups was significantly lower than that in the control group. Therefore, the 103 cfu/g and 105 cfu/g L. pentosus-added food not only promoted food intake and growth of abalones, but also improved their non-specific immunity and reduced V. parahaemolyticus infection, indicating that this strain is a good potential candidate for probiotic added food in the aquaculture industry.

Effects of a probiotic (Bacillus licheniformis) on the growth, immunity, and disease resistance of Haliotis discus hannai Ino.

To study the effects of a probiotic (Bacillus lincheniformis) on the survival and growth of Haliotis discus hannai Ino, the expression levels of nonspecific immune genes and the resistance to Vibrio parahaemolyticus infection were assessed. Abalones (shell length: 27.64 ±â€¯1.59 mm, body weight: 4.17 ±â€¯0.32 g) were selected for use in an 8-week culture experiment and a 2-week V. parahaemolyticus artificial infection experiment. In both experiments, the control group (C) was fed with a basal feed and the experimental groups were fed with experimental food prepared by spraying the probiotic on the basal feed at different concentrations: 103 (B1), 105 (B2), and 107 (B3) cfu/mL. The survival rate, total number of blood lymphocytes, activity of acid phosphatase, and expression level of heat shock protein 70 were significantly higher in B1, B2, and B3 than in C (P < 0.05). The specific growth rate of shell length, food intake, food conversion rate, phagocytic activity of blood lymphocytes, activities of myeloperoxidase and catalase (CAT), and expression levels of CAT and thioredoxin peroxidase of abalones in B2 were significantly higher than those in B1 and C (P < 0.05). Although the level of O2- produced by the respiratory burst of blood lymphocytes in B2 was not significantly different from those in B1 and B3, they were significantly higher than that in C (P < 0.05). The activity of superoxide dismutase (SOD), the nitric oxide levels produced by the respiratory burst of blood lymphocytes, and the expression levels of Mn-SOD in B1 and B3 were significantly higher than those in C but significantly lower than those in B2 (P < 0.05). Fourteen days after infection with V. parahaemolyticus, the cumulative mortality of abalones in B2 was significantly lower than those in B1 and C (P < 0.05). These results indicate that the food containing 105 cfu/mL Bacillus licheniformis promoted food intake and growth of abalones and also improved their resistance to V. parahaemolyticus infection. Thus, B. licheniformis is a good potential probiotic.

Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation.

BACKGROUND: Studies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc. RESULTS: Homologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster's mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5'-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms. CONCLUSIONS: Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these 'relatively young' genes was critical for the origin and radiation of eukaryotes.

The oyster genome reveals stress adaptation and complexity of shell formation.

The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster's adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.

Molecular characterization of a mollusk chicken-type lysozyme gene from Haliotis discus hannai Ino, and the antimicrobial activity of its recombinant protein.

A chicken-type lysozyme was obtained and characterized from the abalone Haliotis discus hannai Ino (HdLysC). The full-length cDNA of HdLysC was 586 bp, and it contained an open reading frame of 441 bp, encoding a 147-amino acid protein with a calculated molecular mass of 15.64 kDa and an isoelectric point of 4.87. The amino acid sequence of HdLysC possessed all conserved features critical for the fundamental structure and function of c-type lysozymes, including the two catalytic residues, Glu54 and Asp70. The genomic length of HdLysC was 2865 bp, with four exons interrupted by three introns. The genomic structure of HdLysC was more similar to vertebrates than invertebrates. Many putative transcription factor binding sites involved in the immune system and cancer were found in the promoter region of HdLysC. Quantitative real-time RT-PCR detected HdLysC expression in all examined tissues, as well as in an expression profile of abalone gills challenged with bacteria Vibrio anguillarum. HdLysC transcripts were found to be most abundantly expressed in mantles, and weakly expressed in hemocytes, and increased expression of HdLysC also observed after bacterial stimulation. HdLysC was expressed in Escherichia coli, and the recombinant protein showed bacteriolytic activity against both Gram-positive and Gram-negative bacteria. Of the Vibrio species tested, more effective activity was detected against V. anguillarum.

Development of gene-targeted SNP markers for genomic mapping in Pacific abalone Haliotis discus hannai Ino.

Useful and novel DNA markers are needed for aquaculture genetics and breeding. In this study, we report the discovery and development of gene-targeted single nucleotide polymorphisms (SNPs) for genomic mapping in the Pacific abalone Haliotis discus hannai Ino. Single EST or EST-contigs from 66 genes that had positive BLASTx matches (E-value ≤ 1e-8) were used for polymerase chain reaction (PCR) amplification. PCR products from the two parents of one mapping family were directly sequenced, and 83 SNP loci were found from 17 genes. Allele-specific PCR (AS-PCR) was developed and optimized for genotyping of 11 SNP loci in 120 progeny of the mapping family. Nine of the loci conformed to the expected Mendelian ratio of 1:1 based on the χ(2) test (P > 0.05) and could potentially be used for linkage map construction. Our data also indicate that the sequencing of two parents may be a practical strategy for the discovery of informative SNPs for linkage mapping in a particular mapping population.