Characterization of cyclophilin D in freshwater pearl mussel (Hyriopsis schlegelii)

Cyclophilin D (referred to as HsCypD) was obtained from the freshwater pearl mussel (Hyriopsis schlegelii).The full-length cDNA was 2 671 bp,encoding a protein consisting of 367 amino acids.HsCypD was determined to be a hydrophilic intracellular protein with 10 phosphorylation sites and four tetratricopeptide repeat (TPR) domains,but no signal peptide.The core sequence region YKGCIFHRIIKDFMVQGG is highly conserved in vertebrates and invertebrates.Phylogenetic tree analysis indicated that CypD from all species had a common origin,and HsCypD had the closest phylogenetic relationship with CypD from Lottia gigantea.The constitutive mRNA expression levels of HsCypD exhibited tissue-specific patterns,with the highest level detected in the intestines,followed by the gonads,and the lowest expression found in the hemocytes.     

Molecular characteristics and expression of calmodulin cDNA from the freshwater pearl mussel, Hyriopsis schlegelii

Calmodulin (CaM) is a multifunctional intracellular calcium ion receptor protein that participates in a range of cellular processes, including calcium metabolism in mussels. To investigate the role of CaM in freshwater mollusk shell calcium metabolism, the full-length CaM cDNA was isolated from the freshwater pearl mussel, Hyriopsis schlegelii (referred to as hsCaM) using SMART RACE technology. The full-length hsCaM was 855 bp in size, containing a 70-bp 5'-untranslated sequence, a 447-bp open reading frame, a 309-bp 3'-untranslated sequence, and a 26-nucleotide long poly(A) tail. The hsCaM mRNA expression in different mussel tissues was examined using real-time PCR. The hsCaM mRNA was found to be ubiquitously expressed, but far more abundant in the gill, foot, and mantle than in the posterior adductor muscle. Real-time PCR was also used to determine hsCaM mRNA expression levels in mantle tissues of H. schlegelii at different ages. No significant differences between one-, two-, and three-year-old mussels were detected, but expression increased in four-year-old mussels and then decreased in five-year-old mussels. CaM appears to be involved in calcium regulation of the mantle in four-year-old mussels, which may secrete more mother of pearl during pearl culture.     

Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel,Hyriopsis schlegelii

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5′-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.     

池蝶蚌线粒体基因组双单性遗传现象分析

部分双壳贝类的线粒体遗传方式不同于标准的母系遗传(SMI),被称为双单性遗传现象(DUI)。池蝶蚌(Hyriopsis schlegelii)是淡水双壳贝类,是否存在双单性遗传现象?本文采用普通PCR扩增、SHOT-GUN测序及软件拼接获得了雄性池蝶蚌线粒体基因组(以下简称Hs-mtDNA)全序列,并与本实验室已报道的雌性池蝶蚌线粒体基因组全序列进行差异性分析。结果表明,雄性和雌性Hs-mtDNA全长分别为15961 bp和15939 bp,雄性比雌性长22 bp,雌雄线粒体基因组成与排列顺序一致。各蛋白编码基因的碱基数目均一致,碱基转换率为1.01%～7.34%,颠换率为0.00%～0.62%,氨基酸差异率为0.00%～9.35%;其中,COX1基因变异率为2.72%;COX2基因碱基变异率最高,达7.50%,雄性COX2的3'末端没有出现编码延伸区。雄性12S rRNA基因发生5 bp的碱基转换,差异率为0.6%;16S rRNA基因比雌性长9 bp,碱基差异率仅为1.2%。雌雄tRNA-His均位于H链上,介于COX2与ND3之间,没有出现位置的差异性。雌雄Hs-mtDNA的非编码区共有28个1～393 bp的片段,但未见控制区。在tRNA-Glu与tRNA-Tyr间有一段长393 bp的非编码区存在蛋白质翻译功能,但非雄性特异性蛋白。以COX1基因建立系统进化树,池蝶蚌和三角帆蚌(H.cumingii)聚在一起,而含有双单性遗传现象的无齿蚌属的Pyganodon grandis、小方蚌亚科的Venustaconcha ellipsiformis及小方形蚌属的Quadrula quadrula三者雄性聚为一支,雌性聚为一支。因此,雌雄池蝶蚌线粒体存在一定的差异性,但其差异要比其他具有双单性遗传现象的淡水双壳类小得多,且池蝶蚌线粒体遗传可能不存在双单性遗传现象。     

Ten polymorphic microsatellites from freshwater pearl mussel,Hyriopsis cumingii

Freshwater pearl mussel, Hyriopsis cumingii, is the most economically important mussel species in China. Due to overexploitation and changes of water quality, H. cumingii is facing serious population decline and local extinction. We have isolated and characterized 10 polymorphic microsatellite loci from a genomic DNA library enriched for CA and AG repeats with the aim of developing a set of codominant DNA markers for analysing genetic diversity and population structure of this species. The average allele number of the 10 markers was 7.8 per locus, ranging from two to 16 in 24 unrelated individuals. Nine of 10 markers conformed to Hardy–Weinberg equilibrium and inherited independently, suggesting these microsatellites could be useful for studying population genetics, designing conservation strategies and developing breeding programs.     

Molecular cloning and characterization of a sigma‐class glutathione S‐Transferase from the freshwater mussel Hyriopsis cumingii

A full‐length cDNA of a sigma‐like glutathione S‐transferase (GST) was identified from Hyriopsis cumingii (HcGSTS). The deduced amino acid sequence of HcGSTS was found to comprise 203 amino acid residues and to contain the distinct highly conserved glutathione binding site of N‐terminal and the relatively diverse substrate binding site of C‐terminal. Alignment analysis and phylogenetic relationship suggested that the HcGSTS is a sigma‐class GST. The mRNA of HcGSTS was constitutively expressed in all tested tissues, the strongest expression being in the hepatopancreas. The mRNA expression of HcGSTS was significantly up‐regulated (P < 0.05) in all assessed tissues after stimulation of the mussels with peptidoglycan (PGN) and LPS, the only exception being when the gills were challenged with PGN. The expression of HcGSTS mRNA in kidney and foot was also significantly up‐regulated (P < 0.05) by microcystin‐LR. Recombinant HcGSTS exhibited high activity towards the substrate 1‐chloro‐2,4‐dinitrobenzene. The optimal pH was 8.0 and temperature 35 °C.     

不同放养和管理模式下三角帆蚌养殖水体中的浮游生物和初级生产力

通过围隔实验研究了3种放养(单养三角帆蚌,蚌与鲢、鳙混养,蚌与银鲫混养)和2种管理(施肥、投饵兼施肥)模式下养殖水体的浮游生物和初级生产力.结果表明:围隔内浮游植物主要为＜20 μm粒级的种类,其中蓝藻、绿藻和裸藻占优势,硅藻、隐藻、黄藻和甲藻的数量和质量较低;实验期间,围隔内浮游植物数量和质量平均值分别为0.35×109～1.26×109 ind·L-1和32～86 mg·L-1,其中三角帆蚌与鲢、鳙混养的围隔内浮游植物数量和质量较高,蓝藻和硅藻数量和质量明显高于其它围隔;围隔内浮游动物数量以原生动物最多,其质量则以轮虫占优势,枝角类和桡足类等大型浮游动物的数量较少;单养蚌的围隔内浮游动物质量最高;与混养围隔相比,单养蚌的围隔内轮虫质量在浮游动物中的比例较低,桡足类的比例相对较高;蚌与鲢、鳙混养围隔内浮游动物数量最多,原生动物数量在浮游动物中的比例分别高于单养蚌和蚌、银鲫混养的围隔,而轮虫的比例低于后者;实验期间,围隔内初级生产力平均值为4.11～5.45 g O2·m-2·d-1,水呼吸为2.39～4.12 g O2·m-2·d-1,初级生产力与水呼吸的比值为1.25～1.99;单养蚌的围隔内初级生产力最高,蚌、银鲫混养的围隔内初级生产力最低.结果表明:三角帆蚌养殖水体内浮游植物和浮游动物均表现出小型化的趋势;较高的浮游植物数量和质量,特别是较高的硅藻数量和质量,可能是三角帆蚌与鲢、鳙混养围隔内二龄蚌生长较好的原因之一;浮游植物种类组成较初级生产力对蚌生长的影响更大.     

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a “QPS” and an invariant “WND” motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.     

Structural characteristics of gonadal development and hermaphroditic phenomenon in freshwater pearl mussel,Sinohyriopisis schlegelii (Hyriopisis schlegelii)

The period of gonads development was first studied from one to five years in the freshwater pearl mussel Hyriopisis schlegelii. It lasted for 36 months and was divided into three main stages: initiation of gonad formation, a stable growth phase, and a reproductive cell development phase. Each reproductive cycle consisted of five stages: proliferative stage (from late January to late February), growth stage (from late February to late March), maturation stage, spawning stage (from early April to late October) and recovery stage (from early November to late January). Interestingly, a hermaphroditic phenomenon was observed in this mussel for the first time, which appears during the development stage from 26 to 32 months. Male and female follicular tissues coexisted in hermaphrodite individuals with the male follicular tissue accounting for more than 90% of the whole gonad tissue. No hermaphroditic phenomenon was observed in matured gonad. We thus speculate that self-fertilization does not exist in H. schlegelii.     

Morphological development of glochidia in artificial media through early juvenile of freshwater pearl mussel,Hyriopsis (Hyriopsis) bialatus Simpson, 1900

The morphological development and the sequence of organogenesis from glochidium to the early juvenile stage of the freshwater pearl mussel, Hyriopsis bialatus, were observed. Larvae of H. bialatus were cultured in standard tissue culture medium (M199) supplemented with common carp (Cyprinus carpio) plasma and they showed transformation within 10 days. Larval samples were collected every 2 days during glochidia development and subjected to histological processing. Three types of cell masses were developed during this period: the ventral plate (the foot rudiment), lateral pits (the gill rudiment), and the oral plate or endodermic sac (the origin of the digestive tract). The ventral plate gave rise to two foot lobes which fused into a single lobe. The gills were developed from the lateral pits next to the ventral plate, forming a pair of gill buds that became elongated and turned into gill bars. The digestive tract began with the formation of mouth by invagination of the oral plate (or endodermic sac) and formation of a tube underneath the growing foot. Several controversial aspects of organogenesis have been inferred, e.g., de novo formation of the anterior and posterior juvenile adductors, the fate of the mushroom body structure, and foot lobe formation from two separate precursor lobes. A mushroom body protruded into the mantle cavity and remained there throughout the transformation period. Moreover, the evidence of a supporting band (mucoid structure) in the mature glochidium of H. bialatus has never been reported in other freshwater mussel species, and its function and composition need to be further investigated.     

Molecular characterization and mRNA expression of catalase from pearl oyster Pinctada fucata

Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, a catalase cDNA of peal oyster Pincatada fucata (designated as PoCAT) is cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) methods. PoCAT is 2428 bp long and consists of a 5′-UTR of 140 bp, an unusually long 3′-UTR of 749 bp, and an open reading frame (ORF) of 1539 bp. The ORF of PoCAT encodes a polypeptide of 512 amino acids with molecular weight of 58.1 kDa and the theoretical isoelectric point of 8.4. PoCAT shares 62.3–82.2% identity and 73.0–92.0% similarity to other catalase amino acid sequences. Sequence alignment indicates that PoCAT contains the proximal heme-ligand signature sequence (R³⁵¹LFSYSDT³⁵⁸), the proximal active site signature (F⁶¹NRERIPERVVHAKGGGA⁷⁸), and the three catalytic amino acid residues (His⁷², Asn¹⁴⁵, and Tyr ³⁵⁵). PoCAT has two potential glycosylation sites (N⁴³⁶YS⁴³⁸ and N⁴⁷⁸FS⁴⁸⁰) and a peroxisome targeting signal (ASL). PoCAT mRNA was ubiquitously expressed in all detected tissues, and the expression level of PoCAT mRNA was higher in intestine and mantle. The expression profile analysis showed that the expression level of PoCAT mRNA in intestine was significantly up-regulated at 2, 4 and 12 h after Vibrio alginolyticus stimulation. These results demonstrated that PoCAT is a typical member of catalase family and might be involved in innate immune responses of pearl oyster.     

Molecular cloning and copy number variation of a ferritin subunit (Fth1) and its association with growth in freshwater pearl mussel Hyriopsis cumingii

Iron is one of the most important minor elements in the shells of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell growth. A novel ferritin subunit (Fth1) cDNA from the freshwater pearl mussel (Hyriopsis cumingii) was isolated and characterized. The complete cDNA contained 822 bp, with an open reading frame (ORF) of 525 bp, a 153 bp 5' untranslated region (UTR) and a 144 bp 3' UTR. The complete genomic DNA was 4125 bp, containing four exons and three introns. The ORF encoded a protein of 174 amino acids without a signal sequence. The deduced ferritin contained a highly conserved motif for the ferroxidase center comprising seven residues of a typical vertebrate heavy-chain ferritin. It contained one conserved iron associated residue (Try27) and iron-binding region signature 1 residues. The mRNA contained a 27 bp iron-responsive element with a typical stem-loop structure in the 5'-UTR position. Copy number variants (CNVs) of Fth1 in two populations (PY and JH) were detected using quantitative real-time PCR. Associations between CNVs and growth were also analyzed. The results showed that the copy number of the ferritin gene of in the diploid genome ranged from two to 12 in PY, and from two to six in JH. The copy number variation in PY was higher than that in JH. In terms of shell length, mussels with four copies of the ferritin gene grew faster than those with three copies (P<0.05), suggesting that CNVs in the ferritin gene are associated with growth in shell length and might be a useful molecular marker in selective breeding of H. cumingii.     

外来种池蝶蚌与本地种三角帆蚌及其杂交种对三种生态因子的耐受力

为了探索外来种池蝶蚌引入的生态风险,对池蝶蚌(C)与三角帆蚌(S)及其杂交种正交F1(三角帆蚌♀×池蝶蚌♂,SC)、反交F1(池蝶蚌♀×三角帆蚌♂,CS)幼蚌的低氧、干露和重金属汞耐受力进行了比较研究.结果表明:4种幼蚌对低氧、干露和重金属汞均有不同程度的耐受能力.其中CS 的窒息点最低(0.92 mg·L-1),对低氧的耐受力最强,其次是C和SC,窒息点分别为1.04和1.10 mg·L-1,S窒息点最高(1.31 mg·L-1),方差分析表明S与其它3种差异均极显著(P＜0.01),耐低氧能力最弱.C、SC和CS的干露耐受力无明显差异,但要显著强于S(P＜0.01).根据4种幼蚌72 h和96 h对汞的半致死浓度的耐受力由强至弱依次为C＞CS＞SC＞S.表明池蝶蚌的耐受力显著强于三角帆蚌,今后应加强对池蝶蚌的监控和防范力度,以避免对中国的特有种三角帆蚌构成生存威胁.     

Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20

Metallothioneins are rather ubiquitous metal-binding proteins induced by stressing or physiological stimuli. Two major metallothionein isoforms have been identified in mussel: MT10 and MT20. Nevertheless the high sequence homology, the two isoforms exhibit different expression and inducibility in vivo. We cloned and produced in Escherichia coli the MT20 isoform from Mytilus galloprovincilis. cDNA was subcloned into pGEX-6P.1 vector, in frame with a sequence encoding a glutathione-S-transferase (GST) tail. Recombinant protein was purified to electrophoretic homogeneity by affinity chromatography. After enzymatic cleavage of the GST tail the MT moiety was recovered with a final yield of about 5 mg of protein per litre of bacterial culture. The metal-binding ability of MT20 was assessed by absorption spectroscopy upon addition of cadmium equivalents and the metal release was checked as a function of the environment pH. Moreover the protein was analysed for the propensity to polymerization, typical of this class of protein, before and after exposure to reducing and alkylating agents.     

Scanning electron microscopy of glochidia and juveniles of the freshwater mussel,Hyriopsis myersiana

Summary

The ultrastructure of early stages of the mussel, Hyriopsis (Limnoscapha) myersiana (Lea, 1856), was observed by scanning electron microscopy from the glochidial period until the onset of the juvenile stage 10 days later. Further observations were performed for an additional 13 days to assess juvenile development. Glochidia extracted from the brood chambers have a hookless, semi-oval and equivalve calcareous shell with numerous pores in the internal surface, pits in the external surface and cuticular spines in the ventral region. Keratin fibers with a random arrangement in the cuticle of the glochidial shell were also detected. The appearance of the foot within 10 days of in vitro glochidial culture was considered the main feature of metamorphosis to the juvenile stage. Another change during the following 13 days was the formation of a new periostracum exhibiting growth lines under the old glochidial shell. This development occurs mainly in the anterior region and is followed by hardening of the periostracum matrix by calcium deposition. Periostracum growth gradually became apparent in the lateral and posterior regions at the end of this period. The retraction of spines and the alteration of the external surface of the old shell are also described. It is speculated that transcuticular filaments identified in the juvenile stage may have sensory or metabolic exchange functions. The prominent foot, gradually covered by long dense cilia, shows rhythmical movements which suggest a role in feeding. Similarly, cilia present in the mantle may also be involved in the capture of food, while microvilli may facilitate absorption of dissolved materials. Longer cilia, sparsely distributed in the mantle, may function as chemo- or tactile sensors.