Full-Length Transcriptome Sequences Provide Insight Into Hermaphroditism of Freshwater Pearl Mussel Hyriopsis schlegelii.

The freshwater mussel Hyriopsis schlegelii is a cultured bivalve in China, and the quality of the pearls produced is affected by the type of gonads. However, because of the lack of a published genome and the complexity of sex determination, research on sex reversal and development of this species is limited. In this study, Illumina RNA-seq and PacBio Isoform Sequencing (Iso-Seq) were combined to analyze the gonads of H. schlegelii. A total of 201,481 high-quality transcripts were generated. The study identified 7,922 differentially expressed genes in three comparison group (females versus males, hermaphrodites versus females, and hermaphrodites versus males). Twenty-four genes were identified as potential sex-related genes, including sox9 and wnt4 involved in sex determination, and vtg, cyp17a1 and 17ß-hsd2 involved in gonadal development. We also speculated a possible pathways for the formation of hermaphroditism in H. schlegelii. The data provide a clear view of the transcriptome for H. schlegelii gonads and will be valuable in elucidating the mechanisms of gonad development.

Molecular characterization of an inhibitor of apoptosis protein (IAPs) in freshwater pearl mussel, Hyriopsis schlegelii.

The inhibitor of apoptosis proteins (IAPs) played important roles in inhibiting the apoptosis of tumor cells by regulating caspase activity in mammals. In this study, we first cloned the full-length cDNA sequence of IAPs gene (designated as Hs-IAPs) in Hyriopsis schlegelii. The Hs-IAPs gene contained an open reading frame of 1719 nucleotides, encoding a predicted protein of 572 amino acids. qRT-PCR assay indicated that the Hs-IAPs gene was ubiquitously expressed in different tissues, and the highest expression level was in gills. Furthermore, we purified and obtained the recombinant protein of Hs-IAPs which showed a molecular weight of 82.5 kDa. We used H2O2 stimulation experiment to explore the possible function of Hs-IAPs. The results showed that the percentage of viable cells significantly increased following the Hs-IAPs concentration. These indicated that the Hs-IAPs may play a role in anti-oxidation causing by H2O2, and its anti-oxidative may be crucial in the process of apoptosis.

Complete F mitochondrial genomes of the Biwa pearl mussel, Hyriopsis schlegelii: the first report from the species' native lake in Japan.

We determined two complete mitochondrial sequences of female-transmitted (F) mitogenomes of two Hyriopsis schlegelii specimens from this species' original habitat, Lake Biwa. The mitogenomes were both 15,951 bp in length, and the gene contents and orders agreed with those of the typical F mitogenome of Hyriopsis. Molecular phylogenetic analysis based on the previously identified 13 partial sequences confirmed that the two mitogenomes both belonged to H. schlegelii and not to a closely related Chinese species, Hyriopsis cumingii. The same analysis revealed that two mitogenomes (HQ641406 and FJ529186) previously published as H. schlegelii and H. cumingii might be misidentified.

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.

Comparative proteomics analysis of spermary and ovary in Hyriopsis schlegelii.

We provide the first large-scale quantitative proteomics analysis in Hyriopsis schlegelii. To investigate the proteins expressed in the gonads, a quantitative proteomics approach has been utilized to analyze differentially expressed proteins between the spermary and ovary. In this study, we identified and quantified 2416 proteins in the gonads of Hyriopsis schlegelii. Of these, 559 proteins showed significantly different expression between the spermary and ovary. Some specific proteins expressed in either the spermary or ovary were identified in Hyriopsis schlegelii. In addition, a series of proteins related to gametogenesis were also identified. Compared with previous reports, many proteins in Hyriopsis schlegelii identified here have different expression patterns between the spermary and ovary. The special hermaphroditism in Hyriopsis schlegelii may contribute to these inconsistent results. The provided proteomics data could be considered as a starting point for subsequent studies focusing on the proteins involved in sexual gland development and maturity.

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.

De novo transcriptome sequencing to identify the sex-determination genes in Hyriopsis schlegelii.

This study presents the first analysis of expressed transcripts in the spermary and ovary of Hyriopsis schlegelii (H. schlegelii). A total of 132,055 unigenes were obtained and 31,781 of these genes were annotated. In addition, 19,511 upregulated and 25,911 downregulated unigenes were identified in the spermary. Ten sex-determination genes were selected and further analyzed by real-time PCR. In addition, mammalian genes reported to govern sex-determination pathways, including Sry, Dmrt1, Dmrt2, Sox9, GATA4, and WT1 in males and Wnt4, Rspo1, Foxl2, and ß-catenin in females, were also identified in H. schlegelii. These results suggest that H. schlegelii and mammals use similar gene regulatory mechanisms to control sex determination. Moreover, genes associated with dosage compensation mechanisms, such as Msl1, Msl2, and Msl3, and hermaphrodite phenotypes, such as Tra-1, Tra-2α, Tra-2ß, Fem1A, Fem1B, and Fem1C, were also identified in H. schlegelii. The identification of these genes indicates that diverse regulatory mechanisms regulate sexual polymorphism in H. schlegelii.

Molecular cloning, sequence analysis, and cadmium stress-rated expression changes of BTG1 in freshwater pearl mussel (Hyriopsis schlegelii).

The B cells translocation gene 1 (BTG1) is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among verteates. Here, for the first time we cloned the full-length cDNA sequence of Hyriopsis schlegelii (Hs-BTG1), an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of cDNA ends (RACE) together with splicing the EST sequence from a haemocyte cDNA liary, we found that Hs-BTG1 contains a 525 bp open reading frame (ORF) encoding a 174 amino-acid polypeptide, a 306 bp 5' untranslated region (5' UTR), and a 571 bp 3' UTR with a Poly(A) tail as well as a transcription termination signal (AATAAA). Homologue searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the inverteate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas (2.03-fold), mantle (2.07-fold), kidney (2.2-fold) and haemocyte (2.5-fold) were enhanced by cadmium (Cd²âº) stress, suggesting that Hs-BTG1 may have played a significant role in H. schlegelii adaptation to adverse environmental conditions.