The first Toll receptor from the triangle-shell pearl mussel Hyriopsis cumingii

Toll receptor was first discovered in Drosophila and has an important function in the innate immunity of invertebrates. In this study, the Toll receptor HcToll1 from Hyriopsis cumingii with a full length of 3810 bp consisting of a 3687 bp ORF that encodes a total of 1228 amino acids protein was selected for further study. The HcToll1 protein consisted of a signal peptide, 17 LRR domains, 2 LRRCT domains, 1 LRRNT domain, 1 TM domain, and 1 TIR domain. Phylogenetic analysis results showed that HcToll1 was clustered in one group together with other mollusca tolls. RT-PCR analysis results showed that HcToll1 was expressed in all tested tissues such as hemocytes, hepatopancreas, gills, and mantle. qRT-PCR analysis results showed that HcToll1 expression was increased by the presence of Escherichia coli, Vibrio anguillarum, Staphyloccocus aureus, and White Spot Syndrome Virus (WSSV). Over-expression of HcTIR could up-regulate expression of drosomycin gene in Drosophila S2 cells. The results of our study indicated that HcToll1 is a functional Toll and it has an important function in the generation of innate immune responses of H. cumingii against microbial challenge.     

Molecular characterization and expression of metallothionein from freshwater pearl mussel,Hyriopsis schlegelii

Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys–Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.     

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.     

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.     

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.     

Eleven polymorphic microsatellites isolated from red swamp crayfish, Procambarus clarkii

Eleven microsatellites were isolated from a genomic DNA library enriched for CA- and GA-repeats, and were characterized in 48 individuals of red swamp crayfish Procambarus clarkii. All 11 microsatellites were polymorphic with an average of allele number of 6.6 per locus. Genotypic distributions of these 11 markers were found to be in conformance with the expectations of Hardy-Weinberg equilibrium. All 11 markers were unlinked. These markers are being used to study the invasion routine, genetic diversity and population structure of the species P. clarkii.     

Isolation and characterization of polymorphic microsatellites from Chinese mitten crab,Eriocheir sinensis

Chinese mitten crab (Eriocheir sinensis) has great commercial significance in Asia, but is an invasive organism in Europe and America. We isolated five novel microsatellites for E. sinensis. All five loci were polymorphic. The average allele number was 16.8 per locus with a range of 13 to 21, while the expected heterozygosity ranged from 0.82 to 0.91 with an average of 0.88. These markers could be applicable to studies of the genetic diversity and population structure of E. sinensis.     

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.     

Physicochemical stream bed characteristics and recruitment of the freshwater pearl mussel (Margaritifera margaritifera)

1. The freshwater pearl mussel (Margaritifera margaritifera) is endangered and of conservation importance. We used its survival/mortality during the critical post‐parasitic phase as a biological indicator for the habitat quality of the stream substratum. 2. We established and tested biological, physical and chemical methods of assessing the stream bed in 26 streams from seven European countries. We analysed penetration resistance, texture, the concentrations and ratios of C, N, S, P, Fe, Mn in fine material <100 μm, and redox, pH and electric conductivity at the surface and at 5 and 10 cm into the substratum. 3. Sites with high stream bed quality (promoting pearl mussel populations with good juvenile recruitment) had coarser and better sorted substrata with significantly lower quantities of fines, and a higher Mn concentration in the fines, than poor quality sites. Redox potential (Eh) at sites without recruitment differed markedly between the free‐flowing water at the surface and at 5 and 10 cm in the bed, whereas no differences were detectable at good quality sites. This was also true of electric conductivity and, to a lesser extent, pH. The stream bed at sites lacking pearl mussel recruitment had a more variable and higher penetration resistance, indicating clogging of the interstitial macropore system by the deposition of mud and compaction of the stream bed. 4. Our results show that habitat quality for pearl mussels depends strongly on the exchange between the surface and the interstices, which is governed by physicochemical characteristics of the stream substratum. Combined measurements of penetration resistance, depth gradients of Eh and texture were most suitable for assessing stream bed quality, while water chemistry was insufficient because of the decoupling of interstitial and free‐flowing water at poor quality sites.     

Physical microhabitat requirements of freshwater pearl mussels,Margaritifera margaritifera (L.)

Surface sediment diatoms from the east coast of Lake Tanganyika were analysed using ordination and classification techniques, and compared with assemblages previously described from the northern part of the lake. Grain-size analyses were performed on subsamples. Four groups of diatom assemblages were recognised. The first group clusters samples taken in the north, far from the Rusizi river mouth. The second group comprises samples taken on silty sediment along the Tanzanian coast, including one sample taken near the mouth of the Malagarazi river and those from the northernmost part of the lake. The third group comprises surface sediments along the Burundian coast (near Ramba and Magara), and the fourth is characterised by epipsammic taxa. A sample taken near the central arm of the Malagarazi river is included in the latter group. The impact of small rivers on the diatom assemblages in the surface sediments is restricted to the mouth area.     

Cloning of highly polymorphic microsatellites in the horse

We have isolated equine microsatellites by screening a genomic library with (TG)n and (TC)n probes. TG microsatellites were found to be more abundant than TC repeats, with an estimated frequency of one per 100,000bp. Sequence analysis of eight TG-positive clones revealed varying structures of the repeat regions; perfect stretches of TG repeats, imperfect stretches of TG repeats and compound regions of TG and TC repeats. Five loci were analysed by PCR and showed extensive polymorphism; three to seven alleles and heterozygosities of 0.40-0.76 were observed when screening 20-30 unrelated individuals. The high degree of polymorphism, their abundance and the possibility of automating the typing procedure make these loci ideal for standardized paternity testing in the horse. Furthermore, we demonstrate that single hairs can be used as starting material for the PCR analysis.     

Twelve polymorphic microsatellites in Oriental river prawn, Macrobrachium nipponense

Oriental river prawn, Macrobrachium nipponense, is a commercially important freshwater prawn species in China, Japan, Korea and Vietnam. Due to overfishing for food, the wild stocks M. nipponense are endangered. Twenty microsatellite loci were isolated from the M. nipponense. Twelve of these loci were polymorphic (seven to 16 alleles per locus), with expected heterozygosity ranging from 0.68 to 0.86 (n = 48). These polymorphic loci provide a valuable tool for assessing genetic diversity of wild and cultured populations.     

Isolation and characterization of 17 polymorphic microsatellites in grass carp

Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp (Ctenopharyngodon idellus) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy–Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks.     

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.     

The immunostimulating effect of bacterial genomic DNA on the innate immune responses of bivalve mussel, Hyriopsis cumingii Lea

The genomic DNA of Escherichia coli, which contains the unmethylated CpG motif, was used to evaluate the immunostimulating effect of bacterial DNA on innate immune responses in the bivalve mussel Hyriopsis cumingii Lea. The results showed that the E. coli DNA had no significant effect on the production of superoxide anion (O(2-)) or acid phosphatase (AP) by haemocytes in vitro. However, the bactericidal activity of the haemocytes was significantly increased when the cells were incubated with 50 or 100mug/ml bacterial DNA for 12 and 24h. Antibacterial activity, lysozyme activity, and prophenoloxidase (proPO) production of haemolymph were also increased, when the bivalve molluscs were injected with 50 or 100mug/ml of bacterial DNA for 12 and 24h. These activities returned to the control level after 48h. This work showed the bacterial DNA with unmethylated CpG motif could activate some parameters of the immune system of bivalve molluscs in vivo and in vitro.