Pin Huan

Transcriptomic Analysis of the Clam Meretrix meretrix on Different Larval Stages

The clam Meretrix meretrix (Mollusca: Bivalvia) is an important commercial species in China. The deficiency of genomic and transcriptomic data is becoming the bottleneck of further researches on its complex and unique developmental processes. To improve this situation, in this study, a large-scale RNA sequencing was conducted on M. meretrix on larval stages. In particular, mRNAs of trochophore, D-veliger, pediveliger, and postlarva were purified, reverse transcribed, and sequenced through 454 sequencing technology. A total of 704,671 reads were obtained and assembled into 124,737 unique sequences (35,205 contigs and 89,532 singletons). Further analysis showed that 118,075 (94.66%) of these sequences were low-expression-level transcripts. Fifteen thousand two hundred fifteen (12.20%) of the unique sequences were annotated by searching against Uniprot Protein Knowledgebase, while the others (109,522, 87.80%) were left as novel sequences. Gene ontology analysis of the annotated sequences showed that most of them were assigned to certain gene ontology terms. By analyzing the depth of each unique sequence, a preliminary quantification analysis was conducted. An amount of sequences that showed a dramatic transcript discrepancy among the four larval stages were screened, which were related to development, growth, shell formation, and immune responses etc. As the first attempt on large-scale RNA sequencing of marine bivalve larvae, this work would enrich the knowledge of larval development of marine bivalves and provide fundamental support for further researches.

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.

Identification of a tyrosinase gene potentially involved in early larval shell biogenesis of the Pacific oyster Crassostrea gigas

The larval shell emerges early in embryogenesis of mollusks, but the detailed mechanisms of its biogenesis remain to be determined. In this study, we cloned a tyrosinase gene (cgi-tyr1) that potentially functioned in larval shell biogenesis from the Pacific oyster Crassostrea gigas, a worldwide bivalve species. Sequence analysis of cgi-tyr1 revealed that it had typical copper-binding domains and a signal peptide. Through whole mount in situ hybridization and an electron scanning microscopic observation, we detected the expression of cgi-tyr1 firstly in the saddle-shaped shell field in trochophores, indicating that cgi-tyr1 might participate in the biogenesis of the initial non-calcified shell of trochophores. In the following development to early D-veliger, cells in the central region of shell field exhibited no detectable cgi-tyr1 expression, and cgi-tyr1 expression was sustained only in the edge of the shell field and the hinge region, indicating that cgi-tyr1 might function fundamentally in shell growth from trochophore to early D-veliger. Unexpectedly, cgi-tyr1 expression was not detected after the D-veliger stage. This indicated that other molecules might function in later shell development. Our results suggested a role for a tyrosinase gene that specifically functioned in the initial phase of the larval shell biogenesis of C. gigas. This work would shed light on future studies on larval shell development and might be helpful to understand how the molluscan shell emerged during evolution.

A BAC-Based Physical Map of Zhikong Scallop (Chlamys farreri Jones et Preston)

Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (∼5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.

Comparative proteomic analysis of challenged Zhikong scallop (Chlamys farreri): A new insight into the anti-Vibrio immune response of marine bivalves

The current studies on molecular mechanism of bivalves interacting with bacteria are mainly on mRNA and recombinant protein levels. These works provide little information on natural proteins, which limit further understandings. In this study, we conducted a pioneer work to promote researches on the anti-Vibrio immune response of Zhikong Scallop Chlamys farreri through proteomic techniques. Firstly a reference map was constructed for the hepatopancreas of C. farreri. Totally 65 protein spots were included in the reference map, while 46 of them were identified. Gene ontology analysis revealed high activities of metabolism and immunity in hepatopancreas. Furthermore, hepatopancreas of C. farreri injected with Vibrio harveyi at 24 h post-injection (hpi) were used for comparative proteomic analysis. Totally 27 differentially expressed proteins spots after challenge were screened; and 15 were successfully identified. These proteins include some immune-related proteins, metabolism enzymes, and new molecules which were not paid attentions in previous immunity studies in C. farreri. The results indicated that molecular chaperons and the antioxidant system are key elements in the anti-Vibrio immune response of hepatopancreas of C. farreri. The identification of new molecules provides indications for further studies. The results of this work provide a new insight into the anti-Vibrio immune response of marine bivalves.

Multiple ferritin subunit genes of the Pacific oyster Crassostrea gigas and their distinct expression patterns during early development

Multiple ferritin subunit genes are reported in mollusks, but they have not been systematically classified. Based on the recently published whole genome sequence, we screened out the four ferritin subunit genes (cgi-fer1-cgi-fer4) from the Pacific oyster Crassostrea gigas. The four genes were predicted to encode two non-secretory and two secretory peptides. Further phylogenetic analyses revealed two groups of non-secretory and secretory ferritin subunits in mollusks. This differs dramatically from the situation in mammals or insects, which contain only non-secretory or secretory ferritin subunits. These results emphasize the evolution of molluscan ferritin subunit genes. The expression patterns of the four genes during early development exhibited dramatic differences, indicating the functional diversity of these genes. Among them, cgi-fer2 was the only gene expressed prevalently and is thus suggested to be the major house-keeping ferritin subunit gene. The expression of the other three genes was tissue-specific beginning in the D-veliger stage. Based on their expression patterns, we inferred important functions of cgi-fer2 in ciliated tissues and of the other three genes in the digestive system. Moreover, our results indicated potentially different roles of ferritin subunit genes during larval shell formation in gastropods and bivalves, which may be helpful in understanding the molecular mechanisms that cause the different shells of gastropods and bivalves. In addition, we conducted a further semi-quantitative analysis of the four genes in four major developmental stages and five adult tissues. The results also revealed dramatically different expression patterns of the genes, which brought additional functional indications. This work may promote studies on molluscan ferritins and shed light on the evolution of ferritin subunit genes among different animal groups.

Chromosomal localization and molecular marker development of the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallop Chlamys farreri (Jones et Preston) (Pectinoida, Pectinidae)

Zhikong scallop Chlamys farreri (Jones et Preston) is an economically important species in China. Understanding its immune system would be of great help in controlling diseases. In the present study, an important immunity-related gene, the Lipopolysaccharide and Beta-1,3-glucan Binding Protein (LGBP) gene, was located on C. farreri chromosomes by mapping several lgbp-containing BAC clones through fluorescence in situ hybridization (FISH). Through the localization of various BAC clones, it was shown that only one locus of this gene existed in the genome of C. farreri, and that this was located on the long arm of a pair of homologous chromosomes. Molecular markers, consisting of eight single nucleotide polymorphism (SNPs) markers and one insertion-deletion (indel), were developed from the LGBP gene. Indel marker testing in an F1 family revealed slightly distorted segregation (p = 0.0472). These markers can be used to map the LGBP gene to the linkage map and assign the linkage group to the corresponding chromosome. Segregation distortion of the indel marker indicated genes with deleterious alleles might exist in the surrounding region of the LGBP gene.

The sea cucumber genome provides insights into morphological evolution and visceral regeneration

Apart from sharing common ancestry with chordates, sea cucumbers exhibit a unique morphology and exceptional regenerative capacity. Here we present the complete genome sequence of an economically important sea cucumber, A. japonicus, generated using Illumina and PacBio platforms, to achieve an assembly of approximately 805 Mb (contig N50 of 190 Kb and scaffold N50 of 486 Kb), with 30,350 protein-coding genes and high continuity. We used this resource to explore key genetic mechanisms behind the unique biological characters of sea cucumbers. Phylogenetic and comparative genomic analyses revealed the presence of marker genes associated with notochord and gill slits, suggesting that these chordate features were present in ancestral echinoderms. The unique shape and weak mineralization of the sea cucumber adult body were also preliminarily explained by the contraction of biomineralization genes. Genome, transcriptome, and proteome analyses of organ regrowth after induced evisceration provided insight into the molecular underpinnings of visceral regeneration, including a specific tandem-duplicated prostatic secretory protein of 94 amino acids (PSP94)-like gene family and a significantly expanded fibrinogen-related protein (FREP) gene family. This high-quality genome resource will provide a useful framework for future research into biological processes and evolution in deuterostomes, including remarkable regenerative abilities that could have medical applications. Moreover, the multiomics data will be of prime value for commercial sea cucumber breeding programs.

A Label-Free Proteomic Analysis on Competent Larvae and Juveniles of the Pacific Oyster Crassostrea gigas

Current understandings on the molecular mechanisms underlying bivalve metamorphosis are still fragmentary, and a comprehensive description is required. In this study, using a large-scale label-free proteomic approach, we described and compared the proteomes of competent larvae (CL) and juveniles (JU) of the Pacific oyster, Crassostrea gigas. A total of 788 proteins were identified: 392 in the CL proteome and 636 in the JU proteome. Gene Ontology analysis of the proteome from each sample revealed active metabolic processes in both stages. Further quantitative analyses revealed 117 proteins that were differentially expressed between the two samples. These proteins were divided into eight groups: cytoskeleton and cell adhesion, protein synthesis and degradation, immunity and stress response, development of particular tissues, signal regulation, metabolism and energy supply, transport, and other proteins. A certification experiment using real-time PCR assay confirmed 20 of 30 examined genes exhibited the same trends at the mRNA and protein levels. The differentially expressed proteins may play roles in tissue remodeling, signal transduction, and organ development during and after metamorphosis. Novel roles were proposed for some differentially expressed proteins, such as chymotrypsin. The results of this work provide an overview of metamorphosis and post-metamorphosis development of C. gigas at the protein level. Future studies on the functions of the differentially expressed proteins will help to obtain a more in-depth understanding of bivalve metamorphosis.

A GATA2/3 gene potentially involved in larval shell formation of the Pacific oyster Crassostrea gigas.

Shells are one of the most notable features of the majority of mollusks. In addition, the shell is also considered a key characteristic during molluscan evolution and development. However, although the morphological changes during larval shell formation have been well described, the underlying molecular mechanisms remain poorly understood. In this study, we focused on the potential involvement of a GATA gene in shell formation because GATA genes are often downstream genes of BMP (bone morphogenetic protein) signaling pathways, which have been suggested to participate in molluscan shell formation. In the Pacific oyster Crassostrea gigas, we observed that the expression of a GATA2/3 homolog (cgi-gata2/3) was clearly restricted to the edge of the shell field in early larval stages (trochophore and D-veliger). This expression pattern supports the notion that cgi-gata2/3 gene plays conserved roles in bilaterian ectodermal development. It is possible that cgi-gata2/3 is one shell-formation gene under the regulation of BMP signaling pathways. In addition, cgi-gata2/3 was also detected in the ventral side of embryos. The expression of cgi-gata2/3 away from the shell field may be involved in hematopoiesis. Our results provide fundamental support for studies into the molecular mechanisms of larval shell formation and the functions of molluscan GATA genes.