Dahui Yu

Highly expressed EGFR in pearl sac may facilitate the pearl formation in the pearl oyster, Pinctada fucata.

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, proliferation, differentiation and migration. Yet whether it functions in pearl formation or not is not reported. In this study, EGFR was cloned from the pearl oyster Pinctada fucata (named as Pf-EGFR) and its expression profiles were investigated. The cDNA was 2156bp long with an ORF of 1017bp encoding 338 amino acid residues. The deduced polypeptide contained an L domain and a cysteine-rich domain, consistent with the characteristics of ErbB family. The sequence of Pf-EGFR shared 22.78-56.71% identity with other EGFRs. The genomic sequence of Pf-EGFR consisted of six exons and five introns, being 5190bp in total length, and expressed in all the tested tissues with a higher expression level in the pearl sac (P<0.05). In situ hybridization showed that Pf-EGFR was specifically expressed on both the inner side of the outer fold and the outer side of the inner fold of the mantle, as well as on the whole pearl sac including the connective tissues. In addition, Pf-EGFR was markedly increased at larval metamorphosis, significantly higher than other development periods (P<0.05). These results indicated that the Pf-EGFR may facilitate pearl formation as well as larval metamorphosis.

Differentially expressed immune-related genes in hemocytes of the pearl oyster Pinctada fucata against allograft identified by transcriptome analysis

ABSTRACT The pearl oyster Pinctada fucata is commonly cultured for marine pearls in China. To culture pearls, a mantle piece from a donor pearl oyster is grafted with a nucleus into a receptor. This transplanted mantle piece may be rejected by the immune system of the recipient oyster, thus reducing the success of transplantation. However, there have been limited studies about the oysters immune defense against allograft. In this study, hemocyte transcriptome analysis was performed to detect the immune responses to allograft in P. fucata at 0 h and 48 h after a transplant. The sequencing reaction produced 92.5 million reads that were mapped against the reference genome sequences of P. fucata. The Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to identify all immune‐related differentially expressed genes (DEGs). Compared with patterns at 0 h, a total of 798 DEGs were identified, including 410 up‐regulated and 388 down‐regulated genes at 48 h. The expression levels of interleukin receptor and toll‐like receptor in hemocytes were increased significantly 48 h post‐transplant, indicating that the oyster immune response was induced. Finally, altered levels of 18 randomly selected immune‐related DEGs were confirmed by quantitative real‐time PCR (qRT‐PCR). Our results provide the basis for further analysis of the immune rejection of allotransplantation. HighlightsImmune molecules was identified in pearl oyster Pinctada fucata after transplantation.Compared with patterns at 0 h, a total of 798 DEGs were identified, at 48 h post transplantation.Acute Phase Proteins, like HSPs and Mts were expressed in the 0 h‐48 h after transplantation.Toll‐like receptor, Endocytosis and Apoptosis signal pathway play significant roles in the allograft immune mechanism.

Co-expression of march5b and tlr7 in large yellow croaker Larimichthys crocea in response to Cryptocaryon irritans infection

In this study, molecular characteristics of march5b and co-expression of march5b and tlr7 in response to the infection of Cryptocaryon irritans in the large yellow croaker Larimichthys crocea were investigated. The full-length complementary (c)DNA of march5b was 1314 bp, including an open reading frame of 846 bp encoding a polypeptide of 281 amino acids, and the full-length genomic sequence was composed of 23,577 nucleotides, including six exons and five introns. The putative March5b protein contained a RINGv motif and four transmembrane domains. The march5b transcripts were broadly distributed in all detected tissues, with a strong expression in blood, brain and gills, and a weak expression in kidney by quantitative PCR analysis. The expression of march5b and tlr7 in the skin, gills, spleen and head kidney changed in the same manner at most time points post-primary infection with C. irritans. Significant increase was observed in the skin with march5b at days 2 and 3 by 26.10 and 6.88 fold, respectively, and with tlr7 at day 3 by 57.68 fold, when compared with the control. Their expressions, however, were decreased in the gills, especially at day 3 (march5b by 8.9%, tlr7 by 22.06%). In the spleen and head kidney, march5b and tlr7 transcripts were up-regulated early, then noticeably declined at day 3. These results suggested that march5b and tlr7 are co-expressed in response to parasite infection and March5b probably catalyses ubiquitination of some proteins of TLR7 signalling pathway.

Serum immune response of pearl oyster Pinctada fucata to xenografts and allografts

ABSTRACT The mantle piece from the donor pearl oyster would be rejected by the immune system of recipient oyster in pearl culture practice, especially in the case that the donor and receptor are different species. Thus, investigation of the immune response of recipient oyster to grafted mantle pieces, particularly to xenografts, is of importance in creating xenograft transplantation technology for pearl culture industry. The humoral immune responses of P. fucata to allograft (mantle piece of P. fucata) and xenografts (mantle pieces of P. maxima and P. margaritifera, respectively) were studied in this paper. The oysters receiving no transplantations were served as the control group. The serum was collected from recipient P. fucata at 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d, respectively after transplantation, and the serum antibacterial activity, lysozyme activity (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (TAC), and agglutination to rabbit red blood cells were investigated. The result indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells, with variation between groups and between time points, respectively. The antibacterial activity in the experimental group was significantly higher than that in the control group at 2–4 d, but lower at 5–11 d and returned back to normal at 15 d, with significant differences among experimental groups (P < 0.05). The LZM in the experimental group was significantly higher than that in the control group at 3–7 d, with significant differences in bacteriolytic activity among various groups (P < 0.05). Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2–9 d, with significant differences among various groups at 3–9 d (P < 0.05). The TAC level in the experimental groups was higher than that in the control group at 1–7 d, with significant differences among various groups at 4–7 d (P < 0.05). The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts, with no specific to any of them. Thus, it is necessary to further screen immune rejection factors specific to xenografts, including cellular immune components. HighlightsFew research studys on the xenotransplantation mechanism of pearl culture up to now.Enzymes activities and aggregation effect of the recipient oyster serum were studied.P. fucata have immune response to both xenografts and allografts in varying degree.

Differential Gene Expression during Larval Metamorphic Development in the Pearl Oyster, Pinctada fucata, Based on Transcriptome Analysis

P. fucata experiences a series of transformations in appearance, from swimming larvae to sessile juveniles, during which significant changes in gene expression likely occur. Thus, P. fucata could be an ideal model in which to study the molecular mechanisms of larval metamorphosis during development in invertebrates. To study the molecular driving force behind metamorphic development in larvae of P. fucata, transcriptomes of five larval stages (trochophore, D-shape, umbonal, eyespots, and spats) were sequenced using an Illumina HiSeq™ 2000 system and assembled and characterized with the transcripts of six tissues. As a result, a total of 174,126 unique transcripts were assembled and 60,999 were annotated. The number of unigenes varied among the five larval stages. Expression profiles were distinctly different between trochophore, D-shape, umbonal, eyespots, and spats larvae. As a result, 29 expression trends were sorted, of which eight were significant. Among others, 80 development-related, differentially expressed unigenes (DEGs) were identified, of which the majority were homeobox-containing genes. Most DEGs occurred among trochophore, D-shaped, and UES (umbonal, eyespots, and spats) larvae as verified by qPCR. Principal component analysis (PCA) also revealed significant differences in expression among trochophore, D-shaped, and UES larvae with ten transcripts identified but no matching annotations.

Transcriptome analysis of the immune reaction of the pearl oyster Pinctada fucata to xenograft from Pinctada maxima

Abstract The pearl oyster Pinctada maxima exhibits great difficulty to culture pearls through nuclear insertion with an allograft, but it is easy for P. fucata to culture pearls after allografting. If P. fucata could be used as a surrogate mother to culture P. maxima pearls, it would benefit the pearl culture industry of P. maxima. However, this is blocked by the immune rejection of P. fucata against P. maxima mantle grafts. In this study, the immune responses of P. fucata hemocyte to allograft and xenograft were investigated after transplantation by transcriptome analysis. In total, 107.93 Gb clean reads were produced and assembled using the reference genome of P. fucata. Gene Ontology Term enrichment and KEGG enrichment analyses indicated that apoptosis, hippo signaling pathway, oxidation‐reduction, MAPK signaling pathway, ribosome, protein processing in endoplasmic reticulum, purine metabolism, NF‐kappa B signaling pathway, oxidative phosphorylation, Ras signaling pathway, and ubiquitin mediated proteolysis were involved in response to transplantation. Many genes related to oxidation‐reduction reactions, the MAPK signaling pathway, and apoptosis were identified by comparison of the allograft group and the xenograft group at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h post‐transplantation. Among them, the expression levels of NADH dehydrogenase, succinate dehydrogenase and other dehydrogenases were increased significantly in the xenograft groups compared with allograft groups at 0 h post transplantation, indicating that a respiratory burst of neutrophils occurred immediately after xenograft transplantation. Additionally, HSP70 was highly expressed from 0 h to 96 h in the xenograft groups, indicating an oyster immune response to the xenograft. The genes enriched in the ribosome and hippo‐signaling pathways were also identified, and expression patterns of these DEGs were different as compared between transplantation and control groups. Finally, altered expression levels of 10 randomly selected immune‐related DEGs were confirmed by quantitative real‐time PCR. These results indicated that oxidation‐reduction is likely the key factor responsible for immune rejection to transplantation. The findings should provide some new insight into the molecular mechanism of immune rejection of the host against xenograft, and thus benefit to development of immunosuppressive reagents to facilitate effective xenograft pearling. HighlightsImmune molecules were identified in P. fucata after transplanting xenograft from P. maxima.Oxidation‐reduction, MAPK and apoptosis were the key terms in comparing allografting and xenografting.Some factors like NADH dehydrogenase showed altered expression in comparison of allograft with xenograft.

Identification and Differential Expression of Biomineralization Genes in the Mantle of Pearl Oyster Pinctada fucata

A series of proteins are involved in shell formation of the pearl oyster Pinctada fucata, but the involved mechanisms and the relative expression levels of these proteins have not been elucidated. In this study, we sequenced and characterized the transcriptome of P. fucata mantle tissue. A total of 100,679 unique transcripts were assembled, 43687 Unigenes were annotated, and 48654 CDSs were determined. Of these, GO annotated 16353 Unigenes, COG defined 11585 unigenes into 25 categories, and KEGG sorted 25053 unigenes into 258 pathways. In total, 67 biomineralization-related genes were identified, of which 23 genes were newly described in P. fucata. These genes included ones that expressed shell matrix proteins, regulatory factors, and uncharacterized genes. Differential expression of these 67 genes and 9 other biomineralization-related genes was confirmed using qPCR. Of the 8 nacreous layer-related genes, MSI60 (774.00) was expressed at a much higher level than the others. KRMP2-4 and MSI31 were the most highly expressed of the 13 prismatic layer-related genes and KRMP2 was expressed at nearly 10000 times of the level of the 18S gene. For genes related to both layers, shematrin 2 (3977.84), nacrein (2404.75), PFMG 10 (2113.93), and PFMG 4 (1015.89) were highly expressed, and ferritin-like protein (877.54) and PFMG 8 (516.48) were highly expressed among the 16 undefined genes. The expression levels of regulation factors were generally low, and the highest level was 324.09 (EF-hand) and the lowest occurred in the BMP and wnt families. The expression levels of the prismatic matrix proteins were much higher than those of nacreous ones, consistent with a thicker prismatic layer. MSI60 and nacrein are likely the main components of the nacreous layer, and KRMP2-4, MSI31, shematrin 2, and PFMG 10 gene products are the main components of the prismatic layer. This is the first report of transient expression levels of a large number of biomineralization-related genes at the same time in mantle tissue of P. fucata. These findings provide a novel perspective to understand the molecular mechanisms of shell formation and will be beneficial to genetic improvement of P. fucata for the production of high-quality pearls as well.

Characterization of transcriptome and identification of biomineralization genes in winged pearl oyster (Pteria penguin) mantle tissue

The winged pearl oyster Pteria penguin is a commercially important marine pearl oyster species, with pearls that are quite different from those of other pearl oysters. Among such species, mantle tissue is the main organ responsible for shell and pearl formation, a biomineralization process that is regulated by a series of genes, most of which remain unknown. In this study, we sequenced and characterized the transcriptome of P. penguin mantle tissue using the HiSeq 2000 sequencing platform. A total of 93,204 unique transcripts were assembled from 51,580,076 quality reads, with a mean length of 608bp, and 40,974 unigenes were annotated. The sequence data enabled the identification of 79,702 potential single nucleotide polymorphism loci and 4345 putative simple sequence repeat loci. A total of 71 unique transcripts were identified homologous to known biomineralization genes, including mantle gene, nacrein, pearlin, pif, chitinase, and shematrin, of which only 3 were previously reported in P. penguin. qPCR analysis indicated that 10 randomly selected biomineralization genes were much more highly expressed in mantle tissue than in the other tissues. In addition, 30 unique sequences were identified as highly expressed, with FPKM values of >3000, and most of these were biomineralization-related genes, including shematrin family genes, a jacalin-related lectin synthesis gene, calponin-2, and paramyosin. These findings will be useful for future studies of biomineralization in P. penguin, as well as in other Pteria species.

Characterization of the bay scallop (Argopecten irradians concentricus Say) transcriptome and identification of growth-related genes.

The bay scallop Argopecten irradians concentricus is a commercially important bivalve species for aquaculture in China. However, the scarcity of transcripotomic and genomic resources of the bay scallop has hindered the progress of genetic improvement of the animal. To change this situation, de novo transcriptome sequencing with Illumina HiSeq™2000 sequencing platform was carried out for the bay scallop. Through de novo assembly, 107,145 high quality unigenes were obtained. Totally, 44,901 and 34,673 unigenes were annotated in the Nr database and Swiss-Prot database, respectively. With gene ontology (GO) annotations, 77,834 (51.58%), 49,367 (32.72%) and 23,695 (15.70%) were involved in biological processes, cellular components and molecular functions, respectively. One hundred and thirty seven unigenes putatively involved in growth were identified through blastx. More than 2706 simple sequence repeats (SSRs) and 82,116 single nucleotide polymorphisms (SNPs) were also detected. These genes and markers may serve as new valuable resources for functional genomic and genetic studies on the scallop A. i. concentricus.

Identification of twenty novel polymorphic microsatellite DNA markers from transcripts of the pearl oyster Pinctada fucata using next-generation sequencing approach

The pearl oyster, Pinctada fucata (Gould 1850) is a commercially important marine shellfish cultured for producing saltwater pearls mainly in China and Japan (Yu and Chu 2006). It is common in most areas of tropical and subtropical oceans and seas in the Pacific and Indian regions. In 1965, this specie was successfully propagated and reared under artificial conditions in Guangxi province in southern China and expanded rapidly to the neighbouring Guangdong and Hainan provinces subsequently (Meng et al. 1996). The pearls produced by the animals are referred to as ‘South China Sea Pearl’, accounting for over 90% production of the total marine pearls produced in China. For the last few years, some traits of P. fucata appear to have degenerated, due to overfishing, coastal water pollution and artificial propagation of years without recording their background, which hampered the advance of the pearl industry. Genetic improvement and culture of elite varieties should be carried out to prevent slowdown of the growth rate because of inbreeding depression and deterioration of the pearl quality. Microsatellite DNA markers have proved to be a useful tool for evaluating the level of genetic variation of natural populations in many fishery animals because of the high polymorphism, abundance, neutrality and codominance (Liu and Cordes 2004). Polymorphicmicrosatellite loci have been frequently applied in the analysis of genetic diversity of populations. In spite of some microsatellite loci in this species were reported (Tong et al. 2007; Kuang et al. 2009; Shi et al. 2009; Qu et al. 2010; You et al. 2012; Wu et al.