Identification of functional genes involved in Cd2+ response of Chinese surf clam (Mactra chinensis) through transcriptome sequencing

doi:10.1016/j.etap.2015.11.006

Environmental Toxicology and Pharmacology

Volume 41, January 2016, Pages 113-120

https://doi.org/10.1016/j.etap.2015.11.006 Get rights and content

Highlights

•
Totally 39.9 M raw reads and 56,712 unigenes were obtained.
•
21,305 unigenes were annotated.
•
Conducted an acute test of Cd²⁺.
•
Six selected function genes were linked to Cd²⁺exposure.
•
This study enriched the molecule genetics data of Mactra chinensis.

Abstract

The Chinese surf clam Mactra chinensis is an economically important bivalve species in the coastal waters of Liaoning and Shandong Province, China. In this study, we carried out transcriptome sequencing to develop molecular resources for M. chinensis and conducted an acute test of Cd²⁺ stimulation through quantitative real-time PCR (qRT-PCR) to analyze the relative expression of six functional genes. A total of 100,839 transcripts and 56,712 unigenes were obtained from 39.9 million filtered reads and 21,305 unigenes were annotated by hitting against NCBI database. According to the results of qRT-PCR, heat shock protein 22 (Hsp22) and cytochrome P450 (CYP450(2C31)) were inhibited in the low concentration, and induced in the high concentration of Cd²⁺; thioredoxin peroxidase (TPx-A) was at normal level in low concentration, but induced in high concentration of Cd²⁺; glutathione peroxidase A (GPA), glutathione peroxidase 1 (GPA1) and Mn superoxide dismutase gene (MnSOD) were down-regulated when exposed to any treatment groups. Expression levels of the six functional genes following Cd²⁺ exposure indicated that these genes were linked to environmental stress. Moreover, the present work enriched the molecule genetic data of M. chinensis.

Introduction

The Chinese surf clam Mactra chinensis is a familiar mudflat species in the coastal waters of Liaoning, Shandong province of China and also distributes along the coast of Japan and Korea (Qi, 1998). As filter-feeding sedentary species are prone to accumulate pollutants, the Chinese surf clam may be considered as a model species in environmental toxicology (Rittschof and McClellan-Green, 2005). M. chinensis is also an economically important species. However, owing to overfishing and coastal environment deterioration, the natural populations of M. chinensis have considerably declined over the past decades.

Next-generation sequencing (NGS) platforms, including the Illumina/Solexa Genome Analyzer, Roche/454 FLX, and Applied Biosystems SOLID system, are popular in genomic studies. The products of it, named as transcriptome, make the scientific research more operability and interesting. Always the model organisms was thoroughly researched, but now the non-model organisms have an opportunity to be understood. At the same time, the advent of NGS bring a new insight into the biological method for the research of environmental toxicology, population genetics and other research of biological mechanism. As Illumina features outstanding advantages, such as high accuracy, high-throughput, high sensitivity, and low operating cost, it has been widely used to sequence the transcriptome of many species, such as Japanese scallop Mizuhopecten yessoensis (Meng et al., 2013), Asian clam Corbicula fluminea (Chen et al., 2013), the eastern oyster Crassostrea virginica (Zhang et al., 2014), pearl oyster Pinctada martensii (Zhao et al., 2012), the king scallop Pecten maximus (Pauletto et al., 2014) and bay scallop Argopecten irradians concentricus Say (Fan et al., 2015).

Heavy metal pollution in marine ecosystems is a global environmental problem, particularly in the east coast of China where the water has been considerably polluted by industrial development (Wang et al., 2013, Wu et al., 2012). As a non-essential metal for organisms, Cd is highly toxic to wildlife, teratogenic and carcinogenic to humans (Eisler, 2000) except for newly discovered biological role in marine diatoms (Lane and Morel, 2000), and highly accumulated by marine organisms (Pan and Wang, 2012). Zhang and Sun (2010) identified that the safe level of Cd²⁺ concentration for the Chinese surf clam is 0.04 mg/L; when the Cd²⁺ concentration reaches 5.52 mg/L, half clams would be killed within 72 h. The uptake of Cd by living cells can lead to reactive oxygen species (ROS) generation and eventually cell death, depending on metal dose and exposure time (Benavides et al., 2005, Leonard et al., 2004). ROS is naturally produced during oxygen metabolism and includes superoxides (O²⁻), hydrogen peroxide (H₂O₂), hydroxyl radicals (HO⁻), and singlet oxygen (1O₂); certain parts of excess ROS are removed by antioxidant systems to maintain redox homeostasis (Choi et al., 2007, Zhao et al., 2013). The antioxidant defense system, which is a detoxification process against oxide stress, consists of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione, heat shock proteins (Hsp), and cytochrome P450 (CYP450) (Pan and Zhang, 2006, Piano et al., 2004).

In this study, de novo transcriptome sequencing was performed in the gills of the Chinese surf clam by using the Illumina HiSeqTM 2000 platform to obtain a reliable database. An acute toxicity experiment was also performed under Cd²⁺ exposure to analyze the relative expression of six functional genes through quantitative real-time polymerase chain reaction (qRT-PCR). The results could not only provide valuable and reliable data for the Chinese surf clam aquaculture and environmental monitoring management, but also elucidate the toxicological mechanism of marine pollutants in marine filter-feeders.

Section snippets

M. chinensis sample collection

Freshly healthy M. chinensis samples for transcriptome sequencing were collected from the seafood market in November, 2013 in Dalian. The gill was collected and immersed into RNA-EZ Reagents D RNA-Be-Locker A, then stored at −80 °C and sent to Biomarker technologies Co., Ltd.

Adult Chinese surf clams were collected randomly except the same size from Changxing Market (Dalian, Liaoning Province, China) in June, 2014 and cultivated in 4 × 100 L tanks (40 clams/tank) for one week before treatment.

Sequencing of short expressed reads and de novo assembly from M. chinensis transcriptome

Approximately 39.9 M raw reads from M. chinensis were generated using Illumina/Hiseq-2000 RNA-seq. All sequences with raw read data were deposited at the National Center for Biotechnology Information Sequence Read Archive under Accession No. SRX528175. The GC content, Q30, and total nucleotides were 38.13%, 83.55%, and 8,057,791,380, respectively (Table 1). The assembly of the 39.9 M reads produced 4,206,190 contigs with a N50 length of 49 bp and a mean length of 52.57 bp. The assembly of the

Discussion

We have constructed the gill tissue expressed gene profiles and sequenced 39.9 M reads from M. chinensis using Illumina/Hiseq-2000 RNAseq technology. A total of 21,305 unigenes were hit against the databases. Annotation information could not be assigned for a large percentage (only 37.7%) of these unigenes. The same problem also existed in other bivalvia transcriptome, such as 28.9% of the Asian clam (C. fluminea) unigenes (Chen et al., 2013), 36.19% of the pearl oyster (P. martensii) unigenes (

Acknowledgments

This research was supported by Grants from National Natural Science Foundation of China (31101899 and 31572595) and the Key Laboratory for Ecological Environment in Coastal Areas, State Oceanic Administration (201511).

References (38)

C.Y. Choi et al.
Cadmium affects the expression of metallothionein (MT) and glutathione peroxidase (GPX) mRNA in goldfish, Carassius auratus
Comp. Biochem. Physiol. C. Toxicol. Pharmacol.
(2007)
F.J. Gonzalez
Role of cytochromes P450 in chemical toxicity and oxidative stress: studies with CYP2E1
Mutat. Res.
(2005)
S.S. Leonard et al.
Metal-induced oxidative stress and signal transduction
Free Radic. Biol. Med.
(2004)
Y.S. Lim et al.
Removals of hydrogen peroxide and hydroxyl radical by thiol-specific antioxidant protein as a possible role in vivo
Biochem. Biophys. Res. Commun.
(1993)
K.F. Liu et al.
Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages
Dev. Comp. Immunol.
(2010)
K.J. Livak et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method
Methods
(2001)
K. Pan et al.
Trace metal contamination in estuarine and coastal environments in China
Sci. Total. Environ.
(2012)
L.Q. Pan et al.
Metallothionein, antioxidant enzymes and DNA strand breaks as biomarkers of Cd exposure in a marine crab, Charybdis japonica
Comp. Biochem. Physiol. C. Toxicol. Pharmacol.
(2006)
M. Pauletto et al.
Deep transcriptome sequencing of Pecten maximus hemocytes: a genomic resource for bivalve immunology
Fish Shellfish Immunol.
(2014)
W.A. Pushpamali et al.
Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis
Fish Shellfish Immunol.
(2008)

D. Rittschof et al.

Molluscs as multidisciplinary models in environment toxicology

Mar. Pollut. Bull.

(2005)

H.C. Schroder et al.

Induction of DNA damage and expression of heat shock protein HSP70 by polychlorinated biphenyls in the marine sponge Suberites domuncula Olivi

J. Exp. Mar. Biol. Ecol.

(1999)

S.L. Wang et al.

Heavy metal pollution in coastal areas of South China: a review

Mar. Pollut. Bull.

(2013)

I.N. Zelko et al.

Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression

Free Radic. Biol. Med.

(2002)

C.H. Zhao et al.

Identification and characterisation of ROS modulator 1 in Lampetra japonica

Fish Shellfish Immunol.

(2013)

J.R. Arthur

The glutathione peroxidases

Cell Mol. Life Sci.

(2000)

M.P. Benavides et al.

Cadmium toxicity in plants

Braz. J. Plant Physiol.

(2005)

H.H. Chen et al.

Sequencing and De novo assembly of the Asian clam (Corbicula fluminea) transcriptome using the Illumina GAllx method

Plos One

(2013)

A. De Maio

Heat shock proteins: facts, thoughts, and dreams

Shock

(1999)

Cited by (12)

The acute neurotoxicity of inorganic mercury in Mactra chinensis philippi
2024, Aquatic Toxicology
Inorganic mercury (IHg) is hazardous to marine organisms especially resulting in neurotoxicity, bivalves are sensitive to pollutants as “ocean sentinel”, but data on the neurotoxicity of IHg in bivalves are sparse. So we chosed M. chinensis philippi with typical neural structures in bivalves to investigate the neurotoxicity of IHg, which could be helpful to understand the specificity of neural regulation and the response characteristics of bivalves. After acute exposed to IHg (HgCl₂) for 24 h, the metabolites of ganglion tissues in M. chinensis philippi were evaluated using ¹H-nuclear magnetic resonance based metabolomics; Ca²⁺, neurotransmitters (nitric oxide, glutamate, acetylcholine) and related enzymes (calcineurin, nitric oxide synthase and acetylcholinesterase) were measured using biochemical detection. Compared to the control group, the levels of the nitric oxide (81.04 ± 12.84 μmol/g prot) and acetylcholine (30.93 ± 12.57 μg/mg prot) in M. chinensis philippi of IHg-treated were decreased, while glutamate (2.11 ± 0.61 mmol/L) increased significantly; the activity of nitric oxide synthase (679.34 ± 135.33 U/mg prot) was increased, while acetylcholinesterase (1.39 ± 0.44 U/mg prot) decreased significantly, and the activity of calcineurin (0.52 ± 0.02 U/mg prot) had a statistically insignificant increasing tendency. The concentration of Ca²⁺ (0.92 ± 0.46 mmol/g prot) in the IHg-treated group was significantly higher than that in the control group. OPLS-DA was performed to reveal the difference in metabolites between the control and IHg-challenged groups, the metabolites of glucose, glutamine, inosine, succinate, glutamate, homarine, and alanine were sensitive to IHg, subsequently metabolic pathways that were affected including glucose metabolism, glutamine metabolism, nucleotide metabolism, Krebs cycle, amino acid metabolism and osmotic regulation. In our study, IHg interfered with metabolites in M. chinensis philippi, thus the corresponding metabolic pathways were changed, which influenced the neurotransmitters subsequently. Furthermore, Ca²⁺overload affected the synthesis or degradation of the neurotransmitters, and then the altered neurotransmitters involved in changes in metabolic pathways again. Overall, we hypothesized that the neurotoxic effects of IHg on bivalve were in close contact with metabolism, neurotransmitters, related enzymes and Ca²⁺, which could be effective neurotoxic biomarkers for marine environmental quality assessment, and also provide effective data for the study of the regulatory mechanism of the nervous system in response to IHg in bivalves.
Genome-wide identification and expression profiling revealed tissue-specific inducible expression of cytochrome P450s conferring cadmium tolerance in the Pacific oyster, Crassostrea gigas
2021, Aquaculture Reports
Citation Excerpt :
However, relatively fewer CYP gene families have been determined in marine mollusks compared to vertebrates and terrestrial invertebrates. Only a few species, including Chlamys farreri, Ruditapes philippine, Mactra chinensis, Mytilus edulis, Mytilus coruscus, Crassostrea brasiliana have identified partial CYP families, e.g., CYP1-like, CYP2, CYP3-like, CYP4 and CYP356 (Pan et al., 2011; Tian et al., 2014; Toledo-Silva et al., 2008; Zanette et al., 2013; Zhang et al., 2010, 2019; Zhang et al., 2016). Oysters are worldwide distributed mollusks, known to bioaccumulate and tolerate high concentrations of heavy metals.
Cytochrome P450s (CYPs) are present in almost all organisms and play critical roles in metabolism of endogenous substrates and detoxification of xenobiotic compounds. Oysters are a group of bivalves that are important in marine ecosystem and aquaculture program, adapting to the harsh environment in intertidal zones. The studies on CYPs and their potential involvement in dealing with toxic xenobiotics in oysters were limited. In the present study, we performed a multiple omics analyses and identified a full-set of 123 CYP genes in the Pacific oyster, Crassostrea gigas. These CYP genes were classified into 5 clans and 16 families. The phylogenetic tree constructed with other species showed extensive gene expansion of CYPs in the clan 2 and mitochondrial clan in the C. gigas. The amino acid sequence analysis showed conserved typical motifs of CYP genes. The expression profiles of CYPs quantified across different tissues (gill and digestive gland) at various time points (0d, 0.5d, 7d, 14d, 28d) revealed their roles in response to Cd exposure. Notably, CYP17A1-like_6 and CYP2C50 were expressed at significantly higher levels compared with other CYP genes, suggesting their crucial roles in the detoxification of Cd in the C. gigas. This work provides valuable information toward understanding of CYP-mediated detoxification of cadmium in mollusks, and will be important for surveillance of marine environment pollution and seafood safety.
Transcriptome analysis and histopathological observations of Geloina erosa gills upon Cr(VI) exposure
2020, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
Citation Excerpt :
Which was longer than that reported in fat greenling (Hexagrammos otakii) (548 bp) (Diao et al., 2019) and olive flounder (Paralichthys olivaceus) (563 bp) (Hwang et al., 2018). The GO analysis of unigenes showed that biological regulation, cellular component organization or biogenesis and cellular process were the major subcategories in the biological processes category, while cell and membrane part were the dominant groups in the cellular components category, which was consistent with previous studies in other mollusks and crustaceans (Han et al., 2019; Diao et al., 2019; Zhang et al., 2016). Combining the KOG and KEGG analysis, a large majority of unigenes were involved in signal transduction.
The heavy metal contamination like Cr(VI) has been increased by human activities and that threats the ecosystem health of mangrove areas. Bioindicator is an emerging tool in the environmental contamination assessment. The objective of this study was to investigate the Geloina erosa response mechanisms and sensitivities of several biomarkers in the Cr(VI) exposure and identify the G. erosa capability of being used as heavy metals bioindicator. In this study, G. erosa was exposed to 100 μmol·L⁻¹ Cr(VI) for 48 h. After transcriptome sequencing, a total of 134,817 unigenes were obtained, including 12,555 up-regulated and 18,829 down-regulated differentially expressed genes and were validated through quantitative real-time PCR. In addition, a total of 12,185 SSRs and 1,428,214 candidate SNPs were identified from all the G. erosa transcriptome libraries. Histopathology of the gill indicated the Cr(VI) exposure induced damage of the organ leading to its immunization, detoxification or apoptosis reactions. Among eight genes of the selected biomarkers, Calm, HSP70, CYP450, ATG5, TLR2, MYD88 and CASP8 were up-regulated, while TLR4 was down-regulated in response to the Cr(VI) exposure.
Ovarian transcriptome analysis of Mactra chinensis provides insights into genes expressed during the intermediate and ripening stages
2019, Animal Reproduction Science
Citation Excerpt :
There have been, however, few studies of bivalve gonad development-related molecular mechanisms and these previous studies have mainly focused on the pacific oyster (Crassostrea gigas) (Naimi et al., 2009), Lymnaea stagnalis (Feng et al., 2009), pearl oyster (Pinctada matensii) (Yu et al., 1998) and species in which the genes involved were confined to several more classic homologs of vertebrate development genes (Matsumoto et al., 2003; Fleury et al., 2008; Meistertzheim et al., 2009; Liu et al., 2012). Most of the research focused on M. chinensis breeding has been on fertilization (Chung, 2007), developmental histology, cytology (Chung et al., 1987; Li et al., 2011), and larval nutrition (Shen, 2005; Tyurin and Drozdov, 2005; Zhang et al., 2016a,b). The regulatory mechanism of development, however, has been reported only a few times (Reunov et al., 2014; Kim et al., 2016), and there has been no research about the genes related to ovarian development.
Enhancing the production of aquatic animals is very important for fishery management and aquaculture applications. Ovaries have important functions in producing oocytes and hormones. The Chinese clam (Mactra chinensis) is a nutritious saltwater shellfish. Significant biochemical changes take place during the sexual maturation of M. chinensis; however, the genetic mechanisms of this process are unclear. Transcriptome sequencing can determine gene expression changes as development occurrs. In the present study, transcriptome sequencing was used to produce a comprehensive transcript dataset for the ovarian development of M. chinensis. The different ovarian developmental stages were determined using hematoxylin-eosin staining. There was identification of 54,172 unigenes at the intermediate stage and 63,081 at the ripening stage, and 80,141 all-unigenes were assembled to determine the molecular mechanism of ovarian development in M. chinensis. Quantitative real-time PCR for nine mRNAs confirmed the RNA-seq data. Functional annotation of the transcripts indicated there were important pathways in ovarian development, such as those involving the vitellogenin gene. Six pathways associated with ovarian development were identified: estrogen signaling pathway, GnRH signaling pathway, progesterone-mediated oocyte maturation, ovarian steroidogenesis, steroid hormone biosynthesis, and steroid biosynthesis. Significant upregulation of protein kinase alpha (PKA) and calmodulin (CAM) in four of the pathways indicates that PKA and CAM are active in M. chinensis ovarian development during maturation. Results of the present study provide the first comprehensive transcriptomic resource for M. chinensis ovaries, which will increase understanding of the molecular mechanisms underlying sexual maturation and promote molecular nutritional studies of M. chinensis.
De novo transcriptome sequencing of an estuarine amphipod Grandidierella japonica exposed to zinc
2018, Marine Genomics
Citation Excerpt :
Among them, 4935 unigenes were hit against the nt database, 6186 against the COG database and 20,241 against the Swissprot database. The low proportion of annotated unigenes (25.8%) was due to the limited length of assembled sequences (Zhang et al., 2016). The proportion of unigenes with homologies to known sequences was 13% for unigenes of 200–1000 bases in length, but was increased with increasing length of unigenes, and reached 73% for unigenes of > 1300 bases in length (Fig. 1).
Despite the ecological and ecotoxicological importance of the amphipod Grandidierella japonica, there is little molecular information on this species. We performed de novo transcriptome sequencing of G. japonica under zinc exposure to develop toxicant responsive transcriptome resources. Using the Ion Torrent platform we obtained 11 million raw reads, which were assembled into 105,166 transcripts (N50: 511 bases). The obtained sequences include many stress- and defence-related genes such as antioxidant genes and members of the cytochrome P450 family as well as potential reference genes. Our transcriptome data of G. japonica should be a useful reference for future ecological and ecotoxicological studies.
Application of cDNA-AFLP to biomarker exploration in a non-model species Grandidierella japonica
2017, Ecotoxicology and Environmental Safety
Citation Excerpt :
These disadvantages make it challenging to derive multiple novel biomarkers for a non-model species, for which no information on the genome is available. A few NGS studies have succeeded in discovering biomarkers for non-model species exposed to contaminants (Olsvik et al., 2015; Zhang et al., 2016), but most of the biomarkers found in these studies are well-known and relatively abundant transcripts such as heat shock protein and cytochrome P450. This study focused on the use of cDNA-amplified fragment length polymorphism (AFLP) to identify biomarkers with low expression level for non-model species.
Biomarkers of exposure can be used to identify specific contaminants that are adversely affecting aquatic organisms. However, it remains prohibitively costly to investigate multiple novel biomarkers of exposure in a non-model species, despite the development of next-generation sequencing technology. In this study, we focused on the use of cDNA-amplified fragment length polymorphism (AFLP) as a cost-effective biomarker discovery tool to test whether it could identify biomarkers of exposure in the non-model amphipod species Grandidierella japonica. Loci were identified that were differentially expressed in amphipods exposed to reference chemicals (Cu, Zn, and nicotine) and to an environmental sample (road dust) at sublethal concentrations. Eight loci were shown to respond consistently to nicotine at different concentrations, but not to Cu or Zn. Some of the loci also responded to an environmental road dust sample containing nicotine. These findings suggest that loci identified using cDNA-AFLP could be used as biomarkers of nicotine exposure in environmental samples with complex matrices. Further studies with other organisms and toxicants are needed, but we have demonstrated that the use of cDNA-AFLP to identify biomarkers for ecotoxicological studies of non-model species is at least feasible.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Identification of functional genes involved in Cd2+ response of Chinese surf clam (Mactra chinensis) through transcriptome sequencing

Highlights

Abstract

Introduction

Section snippets

M. chinensis sample collection

Sequencing of short expressed reads and de novo assembly from M. chinensis transcriptome

Discussion

Acknowledgments

Comp. Biochem. Physiol. C. Toxicol. Pharmacol.

Mutat. Res.

Free Radic. Biol. Med.

Biochem. Biophys. Res. Commun.

Dev. Comp. Immunol.

Methods

Sci. Total. Environ.

Comp. Biochem. Physiol. C. Toxicol. Pharmacol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Mar. Pollut. Bull.

J. Exp. Mar. Biol. Ecol.

Mar. Pollut. Bull.

Free Radic. Biol. Med.

Fish Shellfish Immunol.

The glutathione peroxidases

Cell Mol. Life Sci.

Cadmium toxicity in plants

Braz. J. Plant Physiol.

Sequencing and De novo assembly of the Asian clam (Corbicula fluminea) transcriptome using the Illumina GAllx method

Plos One

Heat shock proteins: facts, thoughts, and dreams

Shock

Identification of functional genes involved in Cd²⁺ response of Chinese surf clam (Mactra chinensis) through transcriptome sequencing