Yesso Scallop Patinopecten Yessoensis Research Articles

Genome-wide identification and expression profiling of the SOX gene family in a bivalve mollusc Patinopecten yessoensis

SOX family is composed of transcription factors that play vital roles in various developmental processes. Comprehensive understanding on evolution of the SOX family requires full characterization of SOX genes in different phyla. Mollusca is the second largest metazoan phylum, but till now, systematic investigation on the SOX family is still lacking in this phylum. In this study, we conducted genome-wide identification of the SOX family in Yesso scallop Patinopecten yessoensis and profiled their tissue distribution and temporal expression patterns in the ovaries and testes during gametogenesis. Seven SOX genes were identified, including SOXB1, B2, C, D, E, F and H, representing the first record in protostomes with SOX members identical to that proposed to exist in the last common ancestor of chordates. Genomic structure analysis identified relatively conserved exon-intron structures, accompanied by intron insertion. Quantitative real-time PCR analysis revealed possible involvement of scallop SOX in various functions, including neuro-sensory cell differentiation, hematopoiesis, myogenesis and gametogenesis. This study represents the first systematic characterization of SOX gene family in Mollusca. It will assist in a better understanding of the evolution and function of SOX family in metazoans.

Disease and mortality among Yesso scallops Patinopecten yessoensis putatively caused by infection with Francisella halioticida.

During the fall of 2015, up to 40% mortality occurred in juvenile Yesso scallops Patinopecten yessoensis at an aquaculture site in Baynes Sound, British Columbia, Canada. Macroscopic lesions were present in 11% of the scallops, and histopathology consisting of multifocal and diffuse haemocyte infiltration was observed in 44% of the specimens examined. Histologically, small Gram-negative intracellular bacteria-like particles were observed within necrotic haemocytes of the lesions, suggesting a bacterial aetiology. DNA was extracted from adductor muscle lesions of diseased scallops, and the 16s rDNA gene as well as the DNA-directed RNA polymerase beta subunit (rpoB) were amplified by PCR. Sequence analyses of the resulting 413 and 925 bp fragments were a 100% match to the reference sequence for Francisella halioticida, originally described as the cause of mortality in abalone from Japan. Isolation and culture of the bacteria was not possible at the time, as no further diseased specimens were available. Results from in situ hybridization assays as well as examination by transmission electron microscopy provide further evidence supporting the hypothesis that F. halioticida was the most probable causative agent of the lesions and mortality.

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.

Cloning of invertebrate gonadotropin-releasing hormone receptor (GnRHR)-like gene in Yesso scallop, Patinopecten yessoensis

Although the invGnRH peptide was characterized from the nerve ganglia in bivalves, its signaling mechanism is nevertheless unclear. The objective of this paper was to identify the invGnRHR cDNA from Yesso scallop as a first step for understanding of invGnRH in bivalve neuroendocrine system. We performed PCR cloning mediated with transcriptome survey and expression analysis with various tissues of the scallop. Our results showed that we identified an invGnRHR-like cDNAs from not only Yesso scallop Patinopecten yessoensis but also Pacific oyster Crassostrea gigas. In addition, we subsequently identified an adipokinetic hormone receptor (AKHR)-like and AKH-like cDNA pair from the scallop. Comparison of the tissue distributions of both receptor mRNAs suggested functional divergence of two homologous neuropeptides. In brief, py-GnRHR-like mRNA showed broad distribution in various tissues including the nerve ganglia, while py-AKHR-like mRNA was expressed in the nerve ganglia and restricted to some limited peripheral tissues. The findings suggested that both py-GnRH and py-AKH signals were utilized via their own receptors. qPCR assays revealed their receptor mRNA expression in the gonads during a maturation, showing that py-GnRHR-like mRNA in the pre-mature gonads was higher than other mature stages.

Physical mapping of immune-related genes in Yesso scallop (Patinopecten yessoensis) using fluorescent in situ hybridization.

The innate immune system plays a pivotal role in defending invasion of microorganisms for scallops. Previous studies on immune-related genes in the Yesso scallop, Patinopecten yessoensis (Jay, 1857) have mainly focused on characterization and expression pattern in response to bacterial challenge, no research has been carried out on the cytogenetic level yet. In the present study, eight fosmid clones containing the sequences of key immune-related genes (PyNFkB, PyTRAF2, PyTRAF4, PyTRAF7, PyMyd88-1, PyMyd88-3, PyMKK-7 and PyTNFR) were isolated and seven of them were successfully mapped on chromosomes of Patinopecten yessoensis utilizing fluorescence in situ hybridization. Wherein, PyMyd88-1, PyMyd88-3 and PyMKK-7 located on the same chromosome pair with adjacent positions and the other genes were mapped on four non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of such genes of the Yesso scallop will lay a foundation for studies such as assignment of interested genes to chromosomes, construction cytogenetic maps and so on.

Transcriptome-wide analysis reveals candidate genes responsible for the asymmetric pigment pattern in scallop Patinopecten yessoensis

Yesso scallop Patinopecten yessoensis is an economically important marine bivalve species in aquaculture and fishery in Asian countries. The colors of the left and right shells are obviously distinct, typically having reddish-brown for the left and white for the right. This left-right asymmetric pigment pattern is a very unique phenomenon among invertebrates, whereas the molecular mechanisms that control regional differences in pigmentation are not clear. To better understand the left-right asymmetric pigment pattern, we apply Illumina digital gene expression (DGE) to characterize the gene expression profiles in left and right mantle tissues, and identify five differentially expressed genes, including Cytochrome P450 and other four unknown genes. Among the five genes, one gene shows significantly higher expression in the right mantle, while other four exhibit significantly higher expression in the left mantle. We further validate the DGE results by using quantitative real-time PCR for P450, resulting in approximately 32-fold higher expression in the left mantle than that in the right mantle. These findings will not only help assist our understanding of the sophisticated processes of shell pigmentation in scallops, but also provide new insights into the adaptive evolution of phenotypes to maximize survival that underlie the left-right asymmetric pigment pattern in molluscs.

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.

Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis.

Mitogen-activated protein kinases (MAPKs) are protein Ser/Thr kinases that play a vital role in innate immune responses by converting extracellular stimuli into a wide range of cellular responses. Although MAPKs have been extensively studied in various vertebrates and invertebrates, our current understanding of MAPK signaling cascade in scallop is in its infancy. In this study, three MAPK genes (PyERK, PyJNK, and Pyp38) were identified from Yesso scallop Patinopecten yessoensis. The open reading frame of PyERK, PyJNK, and Pyp38 was 1104, 1227, and 1104bp, encoding 367, 408, and 367 amino acids, respectively. Conservation in some splicing sites was revealed across the three PyMAPKs, suggesting the common descent of MAPKs genes. The expression profiles of PyMAPKs over the course of ten different developmental stages showed that they had different expression patterns. In adult scallops, PyMAPKs were primarily expressed in muscles, hemocytes, gill, and mantle. To gain insights into their role in innate immunity, we investigated their expression profiles after infection with Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Vibrio anguillarum). Significant difference in gene expression was only found in PyERK and PyJNK, but not Pyp38, suggesting Pyp38 may not participate in immune response to bacterial infection. Besides, PyERK and PyJNK exhibited more drastic change against the invasion of V.anguillarum than M.luteus, suggesting they could be more sensitive to Gram-negative bacteria than Gram-positive bacteria. This study provides valuable resource for elucidating the role of MAPK signal pathway in bivalve innate immune response.

Chromosomal mapping of tandem repeats in the Yesso Scallop, Patinopecten yessoensis (Jay, 1857), utilizing fluorescence in situ hybridization.

Construction of cytogenetic maps can provide important information for chromosome identification, chromosome evolution and genomic research. However, it hasn’t been conducted in many scallop species yet. In the present study, we attempted to map 12 fosmid clones containing tandem repeats by fluorescence in situ hybridization (FISH) in the Yesso scallop Patinopecten yessoensis (Jay, 1857). The results showed 6 fosmid clones were successfully mapped and distributed in 6 different pairs of chromosomes. Three clones were respectively assigned to a pair of metacentric chromosomes, a pair of submetacentric chromosomes and a pair of telocentric chromosomes and the remaining 3 clones showed their loci on three different pairs of subtelocentric chromosomes by co-hybridization. In summary, totally 8 pairs of chromosomes of the Yesso scallop were identified by 6 fosmid clones and two rDNA probes. Furthermore, 6 tandem repeats of 5 clones were sequenced and could be developed as chromosome specific markers for the Yesso scallop. The successful localization of fosmid clones will undoubtedly facilitate the integration of linkage groups with cytogenetic map and genomic research for the Yesso scallop.

The Rho GTPase Family Genes in Bivalvia Genomes: Sequence, Evolution and Expression Analysis.

BackgroundRho GTPases are important members of the Ras superfamily, which represents the largest signaling protein family in eukaryotes, and function as key molecular switches in converting and amplifying external signals into cellular responses. Although numerous analyses of Rho family genes have been reported, including their functions and evolution, a systematic analysis of this family has not been performed in Mollusca or in Bivalvia, one of the most important classes of Mollusca.ResultsIn this study, we systematically identified and characterized a total set (Rho, Rac, Mig, Cdc42, Tc10, Rnd, RhoU, RhoBTB and Miro) of thirty Rho GTPase genes in three bivalve species, including nine in the Yesso scallop Patinopecten yessoensis, nine in the Zhikong scallop Chlamys farreri, and twelve in the Pacific oyster Crassostrea gigas. Phylogenetic analysis and interspecies comparison indicated that bivalves might possess the most complete types of Rho genes in invertebrates. A multiple RNA-seq dataset was used to investigate the expression profiles of bivalve Rho genes, revealing that the examined scallops share more similar Rho expression patterns than the oyster, whereas more Rho mRNAs are expressed in C. farreri and C. gigas than in P. yessoensis. Additionally, Rho, Rac and Cdc42 were found to be duplicated in the oyster but not in the scallops. Among the expanded Rho genes of C. gigas, duplication pairs with high synonymous substitution rates (Ks) displayed greater differences in expression.ConclusionA comprehensive analysis of bivalve Rho GTPase family genes was performed in scallop and oyster species, and Rho genes in bivalves exhibit greater conservation than those in any other invertebrate. This is the first study focusing on a genome-wide characterization of Rho GTPase genes in bivalves, and the findings will provide a valuable resource for a better understanding of Rho evolution and Rho GTPase function in Bivalvia.

Characterization of GnRH-like peptides from the nerve ganglia of Yesso scallop, Patinopecten yessoensis

There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides of Yesso scallop Patinopecten yessoensis (i.e., peptide identification, immunohistochemical localization, and immunoquantification) in order to reveal their bioactive form in bivalves. Using mass spectrometry (MS), we identified two invGnRH (py-GnRH) peptides from the scallop nerve ganglia: a precursor form of py-GnRH peptide (a non-amidated dodecapeptide; py-GnRH12aa-OH) and a mature py-GnRH peptide (an amidated undecapeptide; py-GnRH11aa-NH2). Immunohistochemical staining allowed the localization of both py-GnRH peptides in the neuronal cell bodies and fibers of the cerebral and pedal ganglia (CPG) and the visceral ganglion (VG). We found that the peptides showed a dimorphic distribution pattern. Notably, the broad distribution of mature py-GnRH in neuronal fibers elongating to peripheral organs suggests that it is multi-functional. Time-resolved fluorescent immunoassays (TR-FIA) enabled the quantification of each py-GnRH form in the single CPG or VG tissue obtained from one individual. In addition, we observed greater abundance of mature py-GnRH in VG compared with its level in CPG, suggesting that VG is the main producing organ of mature py-GnRH peptide and that py-GnRH may play a central regulatory role in neurons of scallops. Our study provides evidence, for the first time, for the presence of precursor and mature forms of invGnRH peptides in the nerve ganglia of an invertebrate.

The genome-wide identification of mitogen-activated protein kinase kinase (MKK) genes in Yesso scallop Patinopecten yessoensis and their expression responses to bacteria challenges.

Mitogen-activated protein kinase kinases (MKK) are the essential components of the evolutionarily conserved MAPK signaling cascade, which regulates a variety of cellular activities and innate immune responses. Although MKK genes have been extensively studied in various vertebrate and invertebrate species, they have not been systematically characterized in bivalves. In this study, we identified and characterized five MKK genes (PyMKK1/2, PyMKK4, PyMKK5, PyMKK3/6 and PyMKK7) in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine their identities and evolutionary relationships. To gain insights into the possible roles of MKK genes during scallop innate immune responses, quantitative real-time PCR (qRT-PCR) was used to investigate their expression profiles during different developmental stages in samples taken from healthy adult tissues and hemocytes after Micrococcus luteus and Vibrio anguillarum bacterial infections. The Yesso scallop MKKs (PyMKKs) were found to have highly conserved structural features compared to the MKK genes from other invertebrate species. Using qRT-PCR analysis, three distinct expression patterns were detected among the PyMKKs over the course of ten different developmental stages. In adult scallops, the majority of the PyMKKs were highly expressed in mantle, gill, muscle and hemocytes. The differential expression patterns of the five PyMKKs after M. luteus (Gram-positive) and V. anguillarum (Gram-negative) bacterial infections suggested their possible involvement in the innate immune response and provide the foundation and resource for the further study on innate immune response of MAPK signal pathway in mollusk.

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.

Effects of Delayed First Feeding on Larval Growth and Survival of Yesso Scallop (Patinopecten yessoensis)

The time of first feeding is an important factor for establishing successful initial feeding in molluscan hatcheries. The effects of delayed first feeding on larval growth and survival in the larvae of Yesso Scallop Patinopecten yessoensis were investigated in this study. Groups of larvae were fed at early stage of D-shaped larvae or delayed for 24, 48, 72, 96, 120 and 144 h. When first feeding was delayed for less than 72 h, the P. yessoensis larvae grew as rapidly as those non-starved larvae. When delayed first feeding for more than 72 h, the growth rates of larvae were significantly reduced (less than 2 µm day-1), half or less than those of the non-starved group. The survival rates decreased sharply with the prolonged starvation period and the extremely low survival rates were observed in the groups that were delayed first feeding for more than 72 h. The results indicate that the prolonged starvation had a deleterious impact on the larval growth and survival when delayed first feeding for 96 h or more. To avoid mass mortality and obtain adequate growth and survival, food availability within 72 h after early D-shaped larvae is critical important for the successful initial feeding in commercial culture of this species.

Identification of two secreted ferritin subunits involved in immune defense of Yesso scallop Patinopecten yessoensis

As an important iron storage protein, ferritin plays a crucial role in the iron-withholding defense system. In this study, two secreted ferritin subunits (PyFerS1 and PyFerS2) were identified from the Yesso scallop, Patinopecten yessoensis. The complete DNA sequences of the two ferritins are 7101 and 5359 bp, consisting of seven and five exons, respectively. The full-length cDNAs of PyFerS1 and PyFerS2 are 960 and 956 bp in length, encoding 228 and 220 amino acids, respectively. They have typical ferritin structures, with four long α-helices, one short α-helix and an L-loop. Signal peptides were found at the N-terminus of both ferritins, and phylogenetic analysis showed that they both clustered with secreted mollusc ferritins. PyFerS1 possesses all seven conserved residues of the ferroxidase center, whereas PyFerS2 only has two. Real-time PCR analysis indicated high expression level of PyFerS2 in the D-shaped larvae, and PyFerS1 in both D-shaped larvae and fertilized eggs. In adult scallops, PyFerS1 was only detected in the hepatopancreas, whereas PyFerS2 was detected in both hepatopancreas and mantle. After the scallops were challenged by iron ion or bacteria Vibrio anguillarum, the expression of both PyFerS1 and PyFerS2 was significantly elevated, suggesting they may play a role in scallop innate immune defense. For the first time, secreted ferritins were cloned and comprehensively characterized in bivalve molluscs. It will assist in better understanding of the role of secreted ferritins in bivalve innate immunity.

Identification and characterisation of pathogenic Vibrio splendidus from Yesso scallop (Patinopecten yessoensis) cultured in a low temperature environment

A fatal disease with apparent lesions on the adductor muscles of Yesso scallop Patinopecten yessoensis has occurred for three consecutive years from 2009 to 2011 in the northeastern China. This disease has been suspected to be closely associated with bacterial infections. In the present study, 96 bacteria were isolated from the lesions of moribund scallops. They were grouped into Vibrio (88 strains), Moritella (5 strains), Pseudomonas (2 strains) and Shewanella (1 strain) by 16S rDNA sequencing. In the Vibrio group, Vibrio splendidus was the predominant species, consisting of 43 strains. A strain of V. splendidus, named JZ6, was identified as a potential pathogen because it possessed two important virulence factors, Vsm and OmpU. Stain JZ6 was cold-adapted, and could surviveand exhibit haemolytic activity from 4°C to 32°C, with the highest activity observed at 10°C. The pathogenicity of JZ6 was further confirmed by injection and immersion challenges to scallops at 10°C. The cumulative mortality rates in the two experiments were 80% and 45%, respectively, which were significantly higher (P<0.05) than those of control and blank groups. The disorganisation of muscle fibres in challenged scallops was observed to be histopathologically identical to that in moribund Yesso scallop collected from the same farm. The results indicate that V. splendidus is the predominantly culturable bacteria from the lesions of diseased Yesso scallop, and JZ6 probably is the pathogenic agent to Yesso scallop in this low temperature environment.

Mollusc gonadotropin-releasing hormone directly regulates gonadal functions: A primitive endocrine system controlling reproduction

Gonadotropin-releasing hormone (GnRH) is central to the control of vertebrate reproductive cycles and since GnRH orthologs are also present in invertebrates, it is likely that the common ancestor of bilateral animals possessed a GnRH-like peptide. In order to understand the evolutionary and comparative biology of GnRH peptides we cloned the cDNA transcripts of prepro GnRH-like peptides from two species of bivalve molluscs, the Yesso scallop Patinopecten yessoensis and the Pacific oyster Crassostrea gigas. We compared their deduced uncleaved and mature amino acid sequences with those from other invertebrates and vertebrates, and determined their sites of expression and biological activity. The two molluscan GnRH sequences increased the number of known protostome GnRHs to six different forms, indicating the current classification of protostome GnRHs requires further revision. In both molluscs, RT-PCR analysis showed that the genes were highly expressed in nervous tissue with lower levels present in peripheral tissues including the gonads, while immunocytochemistry, using anti-octopus GnRH-like peptide, demonstrated the presence of GnRH-like peptide in neural tissue. Putative scallop GnRH-like peptide stimulated spermatogonial cell division in cultured scallop testis, but the scallop GnRH-like peptide did not stimulate LH release from cultured quail pituitary cells. This is the first report of the cloning of bivalve GnRH-like peptide genes and of molluscan GnRH-like peptides that are biologically active in molluscs, but not in a vertebrate.