Aiguo Yang

Molecular cloning, expression, purification and characterization of vitellogenin in scallop Patinopecten yessoensis with special emphasis on its antibacterial activity.

Vitellogenin (Vg), the major precursor of the egg-yolk proteins, has been found to play an immune role in fish and protochordate amphioxus, however, no study on the immune function of Vg in invertebrates has ever been studied before. In this study, the complete cDNA of Vg was identified from the scallop Patinopecten yessoensis (termed PyVg). The cDNA contained an open reading frame (ORF) of 6888 bp, encoding a polypeptide of 2295 amino acid protein, which had an N-terminal signal peptide followed by the mature Vg. The mature Vg had the domains Vitellogenin_N, domain of unknown function 1943 (DUF1943) and von Willebrand factor type D domain (VWD) as well as the consensus cleavage site (R-X-R/K-R) and conserved motif (KTIGNAG). Tissue distribution assay revealed that PyVg transcripts were predominantly present in the ovary and hepatopancreas, and its expression profile in ovary well reflected the annual cycle of vitellogenesis. Interestingly, bacterial challenge caused a significant change in PyVg expression, hinting an involvement of PyVg in the acute phase response in P. yessoensis. Consistently, recombinant DUF1943 and VWD domains both could interact with LTA and LPS on bacterial wall, and purified native PyVg displayed a broad-spectrum antibacterial activity against both Gram-negative (Escherichia coli and Vibrio anguillarum) and Gram-positive bacteria (Staphylococcus aureus). Overall, these data indicate that Vg is a pattern recognition molecule with bacterial growth-inhibiting activity in the scallop.

Characterization of the Mantle Transcriptome of Yesso Scallop (Patinopecten yessoensis): Identification of Genes Potentially Involved in Biomineralization and Pigmentation

The Yesso scallop Patinopecten yessoensis is an economically important marine bivalve species in aquaculture and fishery in Asian countries. However, limited genomic resources are available for this scallop, which hampers investigations into molecular mechanisms underlying their unique biological characteristics, such as shell formation and pigmentation. Mantle is the special tissue of P. yessoensis that secretes biomineralization proteins inducing shell deposition as well as pigmentation on the shells. However, a current deficiency of transcriptome information limits insight into mechanisms of shell formation and pigmentation in this species. In this study, the transcriptome of the mantle of P. yessoensis was deeply sequenced and characterized using Illumina RNA-seq technology. A total of 86,521 unique transcripts are assembled from 55,884,122 reads that passed quality filters, and annotated, using Gene Ontology classification. A total of 259 pathways are identified in the mantle transcriptome, including the calcium signaling and melanogenesis pathways. A total of 237 unigenes that are homologous to 102 reported biomineralization genes are identified, and 121 unigenes that are homologous to 93 known proteins related to melanin biosynthesis are found. Twenty-three annotated unigenes, which are mainly homologous to calmodulin and related proteins, Ca2+/calmodulin-dependent protein kinase, adenylate/guanylate cyclase, and tyrosinase family are potentially involved in both biomineralization and melanin biosynthesis. It is suggested that these genes are probably not limited in function to induce shell deposition by calcium metabolism, but may also be involved in pigmentation of the shells of the scallop. This potentially supports the idea that there might be a link between calcium metabolism and melanin biosynthesis, which was previously found in vertebrates. The findings presented here will notably advance the understanding of the sophisticated processes of shell formation as well as shell pigmentation in P. yessoensis and other bivalve species, and also provide new evidence on gene expression for the understanding of pigmentation and biomineralization not only in invertebrates but also probably in vertebrates.

Isolation and characterization of melanin pigment from yesso scallop Patinopecten yessoensis

Melanin is one of the essential compounds in the pigments of molluscan shells. However, the effects of melanin on color variations in molluscs are largely unknown. Our previous study suggests that Yesso scallop Patinopecten yessoensis might contain melanin pigment in the dark brown shell. We therefore isolated melanin from the pigmented shells using hydrochloric acid method, and characterized the types of melanin pigments by spectrophotometry. The purified melanin, which was verified by spectrophotometry scanning and HPLC analysis, showed the typical characteristics of melanin absorption spectra and HPLC chromatograms. The contents of pheomelanin and eumelanin in pigmented shells, which were determined by the linear standard curve of melanin at 405 nm and 350 nm absorbance, were 48.23 ± 1.350 and 157.65 ± 5.905 mg, respectively. The present results indicate that the brown-pigmented shells of scallops comprise approximately 76.6% of eumelanin and 23.4% of pheomelanin, which supports the presence of eumelanin-rich pigment in scallop shells. Therefore, the combination of hydrochloric acid extraction and spectrophotometric quantification is a rapid and efficient method to isolate and quantify melanin in shells. This will facilitate the melanin studies related to shell color polymorphism and the selective breeding of bivalves with different shell colors.

Integration of Next Generation Sequencing and EPR Analysis to Uncover Molecular Mechanism Underlying Shell Color Variation in Scallops

The Yesso scallop Patinopecten yessoensis displays polymorphism in shell colors, which is of great interest for the scallop industry. To identify genes involved in the shell coloration, in the present study, we investigate the transcriptome differences by Illumina digital gene expression (DGE) analysis in two extreme color phenotypes, Red and White. Illumina sequencing yields a total of 62,715,364 clean sequence reads, and more than 85% reads are mapped into our previously sequenced transcriptome. There are 25 significantly differentially expressed genes between Red and White scallops. EPR (Electron paramagnetic resonance) analysis has identified EPR spectra of pheomelanin and eumelanin in the red shells, but not in the white shells. Compared to the Red scallops, the White scallops have relatively higher mRNA expression in tyrosinase genes, but lower expression in other melanogensis-associated genes. Meantime, the relatively lower tyrosinase protein and decreased tyrosinase activity in White scallops are suggested to be associated with the lack of melanin in the white shells. Our findings highlight the functional roles of melanogensis-associated genes in the melanization process of scallop shells, and shed new lights on the transcriptional and post-transcriptional mechanisms in the regulation of tyrosinase activity during the process of melanin synthesis. The present results will assist our molecular understanding of melanin synthesis underlying shell color polymorphism in scallops, as well as other bivalves, and also help the color-based breeding in shellfish aquaculture.

Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro

Abstract The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alservers solution (AS), modified Alservers solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alservers solution (AS), modified Alservers solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright‐Giemsa staining, micro‐examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell‐surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response. HighlightsWe studied Changes in hemolymph characteristics of shellfish hemolymph challenged with Vibrio anguillarum under various anticoagulants.All three types of anticoagulants may be used for ark shell blood preservation.The size and permeability of hemocytes changed when treated with the anticoagulants.Thrombus cells of S. brouthtonii may change in various anticoagulants solutions.

Studies on the hemocytes types and their immunological functions in bloody clam(Scapharca broughtonii)

It was the first time to study the types and immunological functions of blood cells in bloody clam(Scapharca broughtonii)by the means of microscopic observation and immunostimulation with pathogenic Vibrio anguillarum,and three types of hemocytes were recognized:red blood cells,white blood cells and thrombus cells.Red blood cells had four developmental states:freshmen,mature,aging and death.White blood cells were divided into acidophils,neutuophils,basophils,lymphocytes,and megalokaryocytic.Acidophils,neutuophils,basophils and thrombus cells were all involved in the blood coagulation.Both red blood cells and white blood cells had phagocytic function,red cells showed the passive phagocytic function and immune adherence function,while white cells showed the active phagocytic function.Acidophils and neutuophils cells were principal in phagocytizing pathogens,and basophils cells could be found phagocytizing large size foreign materials,an increase in the number of white cell and inflammation took place after the bloody clam was infected by pathogen.It was also the first time to show the thrombus cells and its coagulation function in bloody clam(Scapharca broughtonii).The results showed that the blood cells had systemic immunologic defense mechanisms against the mass pathogens invasion into the body of bloody clam(Scapharca broughtonii).

ASSORTATIVE FERTILIZATION IN CHLAMYS FARRERI AND PATINOPECTEN YESSOENSIS AND ITS IMPLICATION IN SCALLOP HYBRIDIZATION

Abstract Assortative mating and reduced hybrid fitness are typically invoked to explain the stability of hybrid zones and the specification of two closely related taxa. Such examples of mate preferences in nature, especially for broadcast free-spawning marine invertebrates, and the understanding of the underlying mechanisms responsible for this positive selection are still lacking. This study documents another example of assortative fertilization by genome in situ hybridization in reciprocal crosses of scallops Chlamys farreri and Patinopecten yessoensis. Females produced eggs selected sperm nonrandomly but showed significant gamete preference of conspecifics over heterospecifics, with Chlamys farrei females using average 89.1% of conspecific sperms and the Patinopecten yessoensis using average 94.3% in natural spawning conditions (temperature 17°C, salinity 31.5‰). This conspecific gamete advantage may be related to the coordinated communication between interacting gametes, and the conspecific bindin genotype selection during the communicational process may also be involved. Two environmental factors, temperature and salinity were also tested to determinate to what extent they affect the patterns of assortative fertilization between these two species. The result showed that temperature seemed to have more influence on assortative fertilization than salinity did. At the temperatures of 11°C, 17°C, 22°C and 26°C, the average conspecific sperms preference of Chlamys farreri eggs was 80.0%, 89.1%, 95.1% and 95.5% respectively, whereas at the salinity of 24‰, 28‰, 31.5‰ and 35.5‰, the average mating preference was 97.2%, 91.2%, 89.1% and 99.5% respectively. These results may have some bearings on the condition dependent alteration in the inter gamete recognition system. Alternatively, it may be simply the byproduct of different sperm tolerance ability under different conditions. The implication it may provide to hybrid production was also discussed.

Exceptionally large mitochondrial fragments to the nucleus in sequenced mollusk genomes

Abstract The available genome sequences of three mollusks (Biomphalaria glabrata, Aplysia californica and Crassostrea gigas) were first used to investigate the nuclear mitochondrial DNAs (NUMTs) in mollusks. The analysis showed that the NUMT contents were high in B. glabrata (17.738 Kb) and C. gigas (17.192 Kb), of which all or almost all mtDNA sequences were transferred to the nucleus, whereas NUMTs are rare (584 bp) in A. californica. The length of NUMTs was 61 to 5492 bp for B. glabrata, 1711 to 15,481 bp for C. gigas, and 124 to 460 bp for A. californica. The largest C. gigas NUMT covered 84.9% (15,481 bp) of its mitochondrial genome, which is rarely found in invertebrates so far. No correlation was found between NUMT content and genome size in the three sequenced mollusk genomes.

Complete mitochondrial genome of Ark shell Scapharca subcrenata

Abstract Complete mitochondrial genome of Scapharca subcrenata was determined in this report. It is 48,161 bp in length, being the largest mitochondrial genome among reported shellfish at present. The entire mitochondrial genome consists of 57 genes including 12 protein-coding genes, 2 ribosomal RNAs and 41 transfer RNAs.

The complete mitochondrial genome of the pen shell Atrina pectinata (Mollusca: Bivalvia: Pinnidae): The first representative from the family Pinnidae

In this study, we described the complete mitochondrial genome of the pen shell Atrina pectinata. It was 16,811 bp in length and consisted of 35 genes including 12 protein-coding genes, 2 ribosomal RNAs, and 21 transfer RNAs (trnS2 and atp8 lacked). All genes were encoded on the same strand. In gene order, there was no completely identical block compared to those of Mytilus edulis, Crassostrea gigas, Chlamys farreri and Venerupis philippinarum. It may be a ground pattern of Pinnidae mitochondrial genomes.