Identification of Perna viridis contaminated with diarrhetic shellfish poisoning toxins in vitro using NIRS and a discriminative non-negative representation-based classifier.

Diarrhetic shellfish poisoning (DSP) toxins are one of the most widespread marine biotoxins that affect aquaculture and human health, and their detection has become crucial. In this study, near-infrared reflectance spectroscopy (NIRS) with non-destructive characteristics was used to identify DSP toxins in Perna viridis. The spectral data of the DSP toxin-contaminated and non-contaminated Perna viridis samples were acquired in the 950-1700 nm range. To solve the discrimination of spectra with crossover and overlapping, a discriminative non-negative representation-based classifier (DNRC) has been proposed. Compared with collaborative and non-negative representation-based classifiers, the DNRC model exhibited better performance in detecting DSP toxins, with a classification accuracy of 99.44 %. For a relatively small-scale sample dataset in practical applications, the performance of the DNRC model was compared with those of classical models. The DNRC model achieved the best results for both identification accuracy and F-measure, and its detection performance did not significantly decrease with decreasing sample size. The experimental results validated that a combination of NIRS and the DNRC model can facilitate rapid, convenient, and non-destructive detection of DSP toxins in Perna viridis.

Solution structure of recombinant Pvfp-5ß reveals insights into mussel adhesion.

Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5ß, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5ß has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5ß is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials.

Genetic stock identification in Perna viridis (Linnaeus1758) from the Indian Peninsula by using microsatellite markers.

BACKGROUND: Perna viridis (Linnaeus, 1758), the Asian green mussel, is native to the Asia-Pacific region. The species is extensively distributed in the Indian subcontinent and is a candidate species for aquaculture in the Southeast Asian region. Availability of genetic information on wild populations is essential for the effective conservation and management of Perna species. The present study assessed the genetic variation and population structure across the distribution range of this species from the Indian peninsula by using microsatellite markers to determine the genetic structuring among the species. METHODS: A total of 15 microsatellite loci with M13 labeling were used for the genetic characterization of P. viridis along Indian waters. Genotyped data were analyzed using analytical software to determine the genetic stocks and understand the genetic variability across the populations. RESULTS: We identified 15 polymorphic markers to understand the genetic stocks and variability across Perna populations. The mean value of the observed heterozygosity (Hobs: 0.741) for all populations was closer to the expected heterozygosity (Hexp: 0.75). The pairwise Fst values between the west and east coasts of India varied significantly, indicating the existence of significant genetic structure between the populations. CONCLUSIONS: Genetic stock identification using software analysis exhibited two distinct stocks, one along the west coast (Arabian Sea) and another along the east coast (Bay of Bengal). Bottleneck analysis indicated the genetic stability of species in the wild. P. viridis is a commercially vital species in Indian peninsular regions. The present study suggests the adoption of stock-specific relaying programs of the species from Indian waters in future studies.

Molecular cloning and expression analysis of mytilin-like antimicrobial peptides from Asian green mussel Perna viridis.

Mytilin is one of the most important CS-αß peptides involved in innate immune response in Mytilidae. In this study, we successfully identified four mytilin-like antimicrobial peptides (pernalins) from Asian green mussel Perna viridis by aligning the P. viridis transcriptome with 186 mytilins and myticins related sequences collected from the transcriptome data of six Mytilus species. Analysis on gene structure showed that pernalin genes had high conservation with mytilin B of Mediterranean mussel Mytilus galloprovincialis. Interestingly, all pernalin genes have a similar tissue expression feature, evidenced by the highest transcription level observed in the hemocytes and followed by the mantle. The lowest transcription level was observed in the foot and gills. qRT-PCR analysis showed that all pernalin genes were significantly down-regulated at each time points from 3 h to 48 h after Vibrio parahaemolyticus infection, suggesting their timely immune responses after bacterial infection.

Small RNA analysis of Perna viridis after exposure to Prorocentrum lima, a DSP toxins-producing dinoflagellate.

Diarrheic shellfish poisoning toxins (DSP toxins) are a set of the most important phycotoxins produced by some dinoflagellates. Studies have shown that DSP toxins have various toxicities such as genotoxicity, cytotoxicity, and immunotoxicity to bivalve mollusks. However, these toxicities appear decreasing with exposure time and concentration of DSP toxins. The underlying mechanism involved remains unclear. In this study, small RNA sequencing was performed in the digestive gland of the mussel Perna viridis after exposure to DSP toxins-producing dinoflagellate Prorocentrum lima for different time periods. The potential roles of miRNAs in response and detoxification to DSP toxins in the mussel were analyzed. Small RNA sequencing of 12 samples from 72 individuals was conducted by BGISEQ-500. A total of 123 mature miRNAs were identified, including 90 conserved miRNAs and 33 potential novel miRNAs. After exposure to P. lima, multiple important miRNAs displayed some alterations. Further miRNA target prediction revealed some important genes involved in cytoskeleton, apoptosis, complement system and immune stress. qPCR demonstrated that miR-71_5, miR-750_1 and novel_mir4 were significantly up-regulated at 6 h after exposure to P. lima, while miR-100_2 was significantly down-regulated after 96 h of exposure. Accordingly, putative target genes of these differentially expressed miRNAs experienced some changes. After 6 h of DSP toxins exposure, NHLRC2 and C1q-like were significantly down-regulated. After 96 h of DSP toxins exposure, NHLRC2 was significantly up-regulated. It is reasonable to speculate that the mussel P. viridis might respond to DSP toxins through miR-750_1, novel_mir4 and miR-71_5 regulating the expression of relevant target genes involved in apoptosis, cytoskeleton, and immune response, etc. This study might provide new clues to uncover the toxic response of bivalve to DSP toxins and lay a foundation for revealing the roles of miRNAs in the environmental adaptation in shellfish.

Transcriptomics reveal triphenyltin-induced molecular toxicity in the marine mussel Perna viridis.

Triphenyltin (TPT) is widely used as an active ingredient in antifouling paints and fungicides, and continuous release of this highly toxic endocrine disruptor has caused serious pollution to coastal marine ecosystems and organisms worldwide. Using bioassays and transcriptome sequencing, this study comprehensively investigated the molecular toxicity of TPT chloride (TPTCl) to the marine mussel Perna viridis which is a commercially important species and a common biomonitor for marine pollution in Southeast Asia. Our results indicated that TPTCl was highly toxic to adult P. viridis, with a 96-h LC10 and a 96-h EC10 at 18.7 µg/L and 2.7 µg/L, respectively. A 21-day chronic exposure to 2.7 µg/L TPTCl revealed a strong bioaccumulation of TPT in gills (up to 36.48 µg/g dry weight) and hepatopancreas (71.19 µg/g dry weight) of P. viridis. Transcriptome analysis indicated a time course dependent gene expression pattern in both gills and hepatopancreas. Higher numbers of differentially expressed genes were detected at Day 21 (gills: 1686 genes; hepatopancreas: 1450 genes) and at Day 28 (gills: 628 genes; hepatopancreas: 238 genes) when compared with that at Day 7 (gills: 104 genes, hepatopancreas: 112 genes). Exposure to TPT strongly impaired the endocrine system through targeting on nuclear receptors and putative steroid metabolic genes. Moreover, TPT widely disrupted cellular functions, including lipid metabolism, xenobiotic detoxification, immune response and endoplasmic-reticulum-associated degradation expression, which might have caused the bioaccumulation of TPT in the tissues and aggregation of peptides and proteins in cells that further activated the apoptosis process in P. viridis. Overall, this study has advanced our understanding on both ecotoxicity and molecular toxic mechanisms of TPT to marine mussels, and contributed empirical toxicity data for risk assessment and management of TPT contamination.

A novel ferritin subunit gene from Asian green mussel, Perna viridis (Linnaeus, 1758).

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5' untranslated region (UTR) of 112 bp and 3' UTR of 165 bp. The 5' UTR and 3' UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3' loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.

Genomics and Transcriptomics of the green mussel explain the durability of its byssus.

Mussels, which occupy important positions in marine ecosystems, attach tightly to underwater substrates using a proteinaceous holdfast known as the byssus, which is tough, durable, and resistant to enzymatic degradation. Although various byssal proteins have been identified, the mechanisms by which it achieves such durability are unknown. Here we report comprehensive identification of genes involved in byssus formation through whole-genome and foot-specific transcriptomic analyses of the green mussel, Perna viridis. Interestingly, proteins encoded by highly expressed genes include proteinase inhibitors and defense proteins, including lysozyme and lectins, in addition to structural proteins and protein modification enzymes that probably catalyze polymerization and insolubilization. This assemblage of structural and protective molecules constitutes a multi-pronged strategy to render the byssus highly resistant to environmental insults.

Population connectivity and genetic structure of Asian green mussel, Perna viridis along Indian waters assessed using mitochondrial markers.

Perna viridis (Linnaeus, 1758), the Asian green mussel, belonging to the family Mytilidae is widely distributed along the Indian coast. The species is majorly found in southeastern countries and is considered an ideal candidate for aquaculture due to its high nutritional value and growth rate. Obtaining their genetic information is essential for their sustainable capture-based production. In the present study, genetic variation, population structure, and demographic processes of the populations across the distribution of this species were assessed using the mitochondrial DNA ATPase6 and cytb gene. In total, we selected 170 samples from five localities across the Indian subcontinent including Andaman Sea. Sequence analysis of partial cytb (885 bp) and ATPase6 (714 bp) genes revealed 45 and 58 haplotypes, respectively. The significant coefficient of genetic differentiation (FST: 0.255 for cytb and 0.252 for ATPase6) and analyses of molecular variance indicated three varieties of stocks, namely Arabian Sea, Bay of Bengal, and Andaman Sea. All the populations showed low nucleotide diversity, suggesting severe historical bottleneck events and high haplotype diversity, indicating population expansion. The genetic variation and demographic process reported in this study will form the baseline information for framing policies, which can be adopted while planning stock specific ranching and relaying programmes in the Indian subcontinent with view to enhance and manage the fishery.

De novo transcriptome analysis of the mussel Perna viridis after exposure to the toxic dinoflagellate Prorocentrum lima.

Diarrheic shellfish poisoning (DSP) toxins are produced by harmful microalgae and accumulate in bivalve mollusks, causing various toxicity. These toxic effects appear to abate with increasing DSP concentration and longer exposure time, however, the underlying mechanisms remain unclear. To explore the underlying molecular mechanisms, de novo transcriptome analysis of the digestive gland of Perna viridis was performed after Prorocentrum lima exposure. RNA-seq analysis showed that 1886 and 237 genes were up- and down-regulated, respectively after 6 h exposure to P. lima, while 265 genes were up-regulated and 217 genes were down-regulated after 96 h compared to the control. These differentially expressed genes mainly involved in Nrf2 signing pathways, immune stress, apoptosis and cytoskeleton, etc. Combined with qPCR results, we speculated that the mussel P. viridis might mainly rely on glutathione S-transferase (GST) and ABC transporters to counteract DSP toxins during short-term exposure. However, longer exposure of P. lima could activate the Nrf2 signaling pathway and inhibitors of apoptosis protein (IAP), which in turn reduced the damage of DSP toxins to the mussel. DSP toxins could induce cytoskeleton destabilization and had some negative impact on the immune system of bivalves. Collectively, our findings uncovered the crucial molecular mechanisms and the regulatory metabolic nodes that underpin the defense mechanism of bivalves against DSP toxins and also advanced our current understanding of bivalve defense mechanisms.

Rejection of the genetic implications of the "Abundant Centre Hypothesis" in marine mussels.

The 'Abundant-Centre Hypothesis' is a well-established but controversial hypothesis stating that the abundance of a species is highest at the centre of its range and decreases towards the edges, where conditions are unfavourable. As genetic diversity depends on population size, edge populations are expected to show lower intra-population genetic diversity than core populations, while showing high inter-population genetic divergence. Here, the genetic implications of the Abundant-Centre Hypothesis were tested on two coastal mussels from South Africa that disperse by means of planktonic larvae, the native Perna perna and the invasive Mytilus galloprovincialis. Genetic structure was found within P. perna, which, together with evidence from Lagrangian particle simulations, points to significant reductions in gene flow between sites. Despite this, the expected diversity pattern between centre and edge populations was not found for either species. We conclude that the genetic predictions of the Abundant-Centre Hypothesis are unlikely to be met by high-dispersal species with large population sizes, and may only become evident in species with much lower levels of connectivity.

Comparative influence of genetics, ontogeny and the environment on elemental fingerprints in the shell of Perna canaliculus.

The trace elemental composition of biogenic calcium carbonate (CaCO3) structures is thought to reflect environmental conditions at their time of formation. As CaCO3 structures such as shell are deposited incrementally, sequential analysis of these structures allows reconstructions of animal movements. However, variation driven by genetics or ontogeny may interact with the environment to influence CaCO3 composition. This study examined how genetics, ontogeny, and the environment influence shell composition of the bivalve Perna canaliculus. We cultured genetically distinct families at two sites in situ and in the laboratory. Analyses were performed on shell formed immediately prior to harvest on all animals as well as on shell formed early in life only on animals grown in the laboratory. Discriminant analysis using 8 elements (Co, Ti, Li, Sr, Mn, Ba, Mg, Pb, Ci, Ni) classified 80% of individuals grown in situ to their family and 92% to growth site. Generalised linear models showed genetics influenced all elements, and ontogeny affected seven of eight elements. This demonstrates that although genetics and ontogeny influence shell composition, environmental factors dominate. The location at which shell material formed can be identified if environmental differences exist. Where no environmental differences exist, genetically isolated populations can still be identified.

High-throughput identification of heavy metal binding proteins from the byssus of chinese green mussel (Perna viridis) by combination of transcriptome and proteome sequencing.

The Byssus, which is derived from the foot gland of mussels, has been proved to bind heavy metals effectively, but few studies have focused on the molecular mechanisms behind the accumulation of heavy metals by the byssus. In this study, we integrated high-throughput transcriptome and proteome sequencing to construct a comprehensive protein database for the byssus of Chinese green mussel (Perna viridis), aiming at providing novel insights into the molecular mechanisms by which the byssus binds to heavy metals. Illumina transcriptome sequencing generated a total of 55,670,668 reads. After filtration, we obtained 53,047,718 clean reads and subjected them to de novo assembly using Trinity software. Finally, we annotated 73,264 unigenes and predicted a total of 34,298 protein coding sequences. Moreover, byssal samples were analyzed by proteome sequencing, with the translated protein database from the foot transcriptome as the reference for further prediction of byssal proteins. We eventually determined 187 protein sequences in the byssus, of which 181 proteins are reported for the first time. Interestingly, we observed that many of these byssal proteins are rich in histidine or cysteine residues, which may contribute to the byssal accumulation of heavy metals. Finally, we picked one representative protein, Pvfp-5-1, for recombinant protein synthesis and experimental verification of its efficient binding to cadmium (Cd2+) ions.

Up-regulation of Nrf2-dependent antioxidant defenses in Perna viridis after exposed to Prorocentrum lima.

It is well documented that diarrhetic shellfish poisoning (DSP) toxins have strong genetic toxicity, cytotoxicity and oxidative damage to bivalve species. However, these toxic effects seem to decrease with the extension of exposure time and the increment of the toxin concentration, the mechanism involved remained unclear, though. In this paper, we found that expression of the genes related to cytoskeleton and Nrf2 signaling pathway displayed different changes over time in the gill of Perna viridis after exposure to DSP toxins-producing microalga Prorocentrum lima. During the short-term exposure (3 h and 6 h), KEAP1 gene expression was significantly up-regulated, coupled with up-regulation of MRP, ABCB1 and CAT transcriptions and down-regulation of GPx1 and NQO1 mRNA. After longer exposure to high density of P. lima, Nrf2 was significantly up-regulated, accompanied with up-regulation of Nrf2 pathway related genes such as NQO1, SOD, GST-ω and ABCB1, whereas KEAP1 was down-regulated. TUBA1C and TUBB1 transcripts were significantly down-regulated after short-term exposure of P. lima, but both of them were up-regulated at 96 h after exposure to high density of P. lima. Paraffin section demonstrated that P. lima had a strong damage on the gill of mussels during the short-term exposure. However, the negative effect to the gill decreased, and the gill restored after longer exposure (96 h). Taking together, we proposed that P. lima had a negative impact on cytoskeleton of mussel gill tissue, could cause oxidative damage to the gills. However, longer exposure of P. lima in high density could activate Nrf2 signaling pathway, thereby reducing the influence of toxin on mussel. Our study might provide a novel clue for the resistance mechanism of shellfish to DSP toxins.

Marine contamination and cytogenotoxic effects of fluoxetine in the tropical brown mussel Perna perna.

Concerns are growing about the presence of fluoxetine (FLX) in environmental matrices, as well as its harmful effects on non-target organisms. FLX in aquatic ecosystems has been detected in a range varying from pg/L to ng/L, while adverse effects have been reported in several organisms inhabiting freshwater and marine environments. The present study quantifies FLX concentrations in seawater samples from Santos Bay, Brazil and assesses metabolic responses and sublethal effects on the tropical brown mussel Perna perna. Levels of ethoxyresorufin­O­deethylase, dibenzylfluorescein dealkylase, glutathione S-transferase, glutathione peroxidase, cholinesterase, lipoperoxidation, and DNA damage were assessed in the gills and digestive gland of these animals, and lysosomal membrane stability was also assessed in hemocytes. FLX altered phase I and II enzyme activities, caused cytogenotoxic effects, and negatively impacted the overall health of mussels exposed to environmentally relevant concentrations. These findings contribute to characterize the risks of introducing this drug into the marine environment.

Genotoxicity Biomonitoring Along a Coastal Zone Under Influence of Offshore Petroleum Exploration (Southeastern Brazil).

Offshore oil exploration creates threats to coastal ecosystems, including increasing urbanization and associated effluent releases. Genotoxicity biomarkers in mussels were determined across a gradient of coastal zone influences of offshore petroleum exploration in southeastern Brazil. Coastal ecosystems such as estuaries, beaches and islands were seasonally monitored for genotoxicity evaluation using the brown mussel Perna perna. The greatest DNA damage (5.2% ± 1.9% tail DNA and 1.5‰  ± 0.8‰ MN) were observed in urban estuaries, while Santana Archipelago showed levels of genotoxicity near zero and is considered a reference site. Mussels from urban and pristine beaches showed intermediate damage levels, but were also influenced by urbanization. Thus, mussel genotoxicity biomarkers greatly indicated the proposed oil exploration and urbanization scenarios that consequently are genetically affecting coastal organisms.

Identification and characterization of a novel defensin from Asian green mussel Perna viridis.

Defensin is one of the most diversified groups of antimicrobial peptides in invertebrate. In the present study, a novel defensin member referred as Pv-Def was identified and characterized from Asian green mussel Perna viridis. Using in silico survey of several EST databases released from diverse tissues of P. viridis, a single peptide referred as Pv-Def was predicted as defensin homologue with Mytilus counterparts. Further analysis on gene structure revealed that Pv-Def was 1001 nt in length and consisted of 3 exons and 2 introns. The precursor of Pv-Def was composed of a signal peptide of 19 amino acids and a mature peptide of 45 amino acids. The mature Pv-Def peptide contains 6 cysteines which formed 3 disulfide bonds at 27C1- 54C4, 40C2- 60C5 and 44C3- 62C6. Like most of the defensin family members, mature Pv-Def peptide included an alpha helix and 2 beta strands. Pv-Def showed significantly tissue-specific expression pattern, while highest transcription level was observed in hepatopancreas, which was about 900 folds to that in hemocytes. Moreover, the expression of Pv-Def mRNA in hemocytes was significantly and accurately up-regulated at different time intervals by Vibrio parahaemolyticus challenge. Interestingly, phylogenetic analysis suggested that the Pv-Def possesses closest relationships with arthropods counterparts rather than other mollusk defensins. To our knowledge, this is the first time that a defensin member was reported in Asian green mussel P. viridis.

Evidence for rangewide panmixia despite multiple barriers to dispersal in a marine mussel.

Oceanographic features shape the distributional and genetic patterns of marine species by interrupting or promoting connections among populations. Although general patterns commonly arise, distributional ranges and genetic structure are species-specific and do not always comply with the expected trends. By applying a multimarker genetic approach combined with Lagrangian particle simulations (LPS) we tested the hypothesis that oceanographic features along northeastern Atlantic and Mediterranean shores influence dispersal potential and genetic structure of the intertidal mussel Perna perna. Additionally, by performing environmental niche modelling we assessed the potential and realized niche of P. perna along its entire native distributional range and the environmental factors that best explain its realized distribution. Perna perna showed evidence of panmixia across >4,000 km despite several oceanographic breaking points detected by LPS. This is probably the result of a combination of life history traits, continuous habitat availability and stepping-stone dynamics. Moreover, the niche modelling framework depicted minimum sea surface temperatures (SST) as the major factor shaping P. perna distributional range limits along its native areas. Forthcoming warming SST is expected to further change these limits and allow the species to expand its range polewards though this may be accompanied by retreat from warmer areas.

Antifouling biocides: Impairment of bivalve immune system by chlorothalonil.

Marine ecosystems are subjected to a variety of contaminants. Antifouling paints, for example, have been extensively used to protect ship surfaces from marine biofouling, but their toxicity has generated great concern. Thus, we evaluated the effect of the biocide chlorothalonil on the immune system of Perna perna mussels. The mussels were exposed to 0 (control), 0.1µg/L and 10µg/L of chlorothalonil for up to 96h. After 24h and 96h of exposure, the following immune-related parameters were analyzed in the hemolymph of mussels: total hemocyte count, cell adhesion, phagocytic activity, level of reactive oxygen species, cell viability and comet assay. After 24h and 96h of chlorothalonil exposure, cellular adhesion increased and the hemocyte viability reduced. Moreover, an increase in phagocytic activity was also observed after 96h of exposure to cholorothalonil. The exposure to 10µg/L of chlorothalonil for 96h reduced the air survival capacity of mussels. Total hemocyte count, ROS generation and DNA damage were not affected by the contaminant exposure. Our results indicate that chlorothalonil affected important immune responses of the bivalves, demonstrating that this biocide has effects on non-target species. This modulation of immune system reduced the health status of mussels, which could compromise their ability to survive in the environment.

De novo assembly and comparative transcriptome analysis of the foot from Chinese green mussel (Perna viridis) in response to cadmium stimulation.

The Chinese green mussel, Perna viridis, is a marine bivalve with important economic values as well as biomonitoring roles for aquatic pollution. Byssus, secreted by the foot gland, has been proved to bind heavy metals effectively. In this study, using the RNA sequencing technology, we performed comparative transcriptomic analysis on the mussel feet with or without inducing by cadmium (Cd). Our current work is aiming at providing insights into the molecular mechanisms of byssus binding to heavy metal ions. The transcriptome sequencing generated a total of 26.13-Gb raw data. After a careful assembly of clean data, we obtained a primary set of 105,127 unigenes, in which 32,268 unigenes were annotated. Based on the expression profiles, we identified 9,048 differentially expressed genes (DEGs) between Cd treatment (50 or 100 µg/L) at 48 h and the control, suggesting an extensive transcriptome response of the mussels during the Cd stimulation. Moreover, we observed that the expression levels of 54 byssus protein coding genes increased significantly after the 48-h Cd stimulation. In addition, 16 critical byssus protein coding genes were picked for profiling by quantitative real-time PCR (qRT-PCR). Finally, we reached a primary conclusion that high content of tyrosine (Tyr), cysteine (Cys), histidine (His) residues or the special motif plays an important role in the accumulation of heavy metals in byssus. We also proposed an interesting model for the confirmed byssal Cd accumulation, in which biosynthesis of byssus proteins may play simultaneously critical roles since their transcription levels were significantly elevated.