BioMed Research International

BioMed Research International / 2014 / Article
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Fish and Crayfish Toxicology

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Review Article | Open Access

Volume 2014 |Article ID 563131 |

Sijun Dong, Mei Kang, Xinlong Wu, Ting Ye, "Development of a Promising Fish Model (Oryzias melastigma) for Assessing Multiple Responses to Stresses in the Marine Environment", BioMed Research International, vol. 2014, Article ID 563131, 17 pages, 2014.

Development of a Promising Fish Model (Oryzias melastigma) for Assessing Multiple Responses to Stresses in the Marine Environment

Academic Editor: Zhi-Hua Li
Received05 Nov 2013
Revised08 Jan 2014
Accepted23 Jan 2014
Published03 Mar 2014


With the increasing number of contaminants in the marine environment, various experimental organisms have been “taken into labs” by investigators to find the most suitable environmentally relevant models for toxicity testing. The marine medaka, Oryzias melastigma, has a number of advantages that make it a prime candidate for these tests. Recently, many studies have been conducted on marine medaka, especially in terms of their physiological, biochemical, and molecular responses after exposure to contaminants and other environmental stressors. This review provides a literature survey highlighting the steady increase of ecotoxicological research on marine medaka, summarizes the advantages of using O. melastigma as a tool for toxicological research, and promotes the utilization of this organism in future studies.

1. Introduction

Estuaries and coastal waters are contaminated by high levels of anthropogenic pollutants [1], creating an urgent need for ecotoxicological studies of marine pollution. The ecotoxicological characteristics of pollutants in saltwater and freshwater environments are different. The parameters of seawater are significantly different from those of freshwater (i.e., salinity, density, buoyancy, pH, ionic strength, and dissolved oxygen (DO)), and these differences impact the ecotoxicological characteristics of the pollutants, such as the packing fraction and size, the distribution of the contaminants in liquid and solid phases, and the bioaccumulation of the contaminants [24].

In addition, the studies of the organisms living in the two different environments have also presented different results. Although Oryzias latipes (freshwater fish) and Oryzias melastigma (seawater fish) are closely related, their branchial FXYD domain-containing ion transport regulator (FXYD) proteins exhibit divergent expression patterns [5]. Kif7 is not expressed in O. melastigma but is highly expressed in the brain of zebrafish, which is a freshwater fish [6]. There is an inverse correlation between the muscle water contents (MWC) and salinity in O. latipes; however, the two parameters are not related in O. melastigma [7]. Exposure to perfluorooctane sulfonates (PFOS) shortened the hatching time and increased the hatching rate of O. melastigma but had the opposite effects in zebrafish [810]. These differences illustrate that ecotoxicological results from freshwater environments cannot be directly applied to the marine environment. At present, aquatic toxicological research is largely carried out under freshwater environmental conditions, and research in the marine environment is urgently needed.

The biologic impact of toxic pollutants on fish is an important area of study in ecotoxicology. Fish models, such as zebrafish (Danio rerio), tilapia (Oreochromis niloticus), and rainbow trout (Oncorhynchus mykiss), have been widely used for ecotoxicological studies in the freshwater environment. Although some estuarine species, for example, Corophium acherusicum, Enteromorpha linza, and Ctenogobius giurinus, can be used for the study of ecotoxicology in marine environments, the research still lags well behind that in freshwater environments, and problems such as species specificity and the lack of genetic information in these species do exist.

O. melastigma, also named O. dancena or Indian medaka, has many advantages as a fish model in marine toxicological research. This review summarizes the advantages and research findings of marine toxicological studies using O. melastigma and encourages further investigation of ecotoxicology in the marine environment using this fish model.

2. Advantages of O. melastigma as a Research Model in Toxicological Studies

O. melastigma originates from the coastal waters and fresh waters of Pakistan, India, Myanmar, and Thailand. In classification, O. melastigma and O. latipes belong to the order Beloniformes, family Adrianichthyidae and genera Oryzias. The embryo of this species has been identified as an important tool for toxicology investigations by the regime of ILSI Health and Environmental Sciences Institute (HESI). As a fish model, it shares many advantages as follows.(1)O. melastigma is small in size (4.5 to 23 mm) and has a short generation time (2-3 months). These characteristics make it available to culture on a large scale under laboratory conditions ( artificial seawater, 28 ± 1°C, and in a 14 h light: 10 h dark cycle). The relatively large eggs and transparent color simplify experimental observations and operations, such as observing developmental changes during each stage of growth [11].(2)O. melastigma has distinct sexual dimorphism, and the morphology of the anal fin is very prominent approximately 1 month after hatching, rendering it highly desirable for gender studies [12]. Researchers have recommended that future risk evaluation of immune-modulatory chemicals must include parallel assessment of both genders. This makes O. melastigma, owing to its characteristics of distinct gender dimorphism and the presence of sex-determining Dmy gene of its homologous species O. latipes, suitable for toxicity evaluation [13].(3)O. melastigma possesses strong environmental tolerance. This organism is capable of adapting to a wide range of temperatures; thus, mutants can be derived that are conveniently temperature sensitive [14]. O. melastigma has the ability to survive in aquatic environments with a wide range of salinity. Although O. latipes can adapt to varying salinity environments to some degree, the adaptive capacity of O. latipes is lower than that of O. melastigma, which can thrive in water of varying salinity ranging from 0 to 35 ppt [1].(4)The eggs and larvae of O. melastigma are sensitive to many environmental pollutants. If the specific sensitive gene responding to pollutants or other environmental stresses can be identified at the molecular level, then environmental pollution can be quickly identified. The molecular staging of O. melastigma embryos, focusing on the heart, pectoral fin, brain, eye, pancreas, muscle, liver, and neuron system, has been fully described [15].(5)Studies of O. latipes in anatomy, physiology, and other aspects have been increasingly extensive and systematic, and the genome sequences of O. latipes have been completed. Many common characteristics exist between O. latipes and O. melastigma in phylogeny; thus, the brackish O. melastigma can serve as a good marine fish model for developmental studies by utilizing the resources developed from O. latipes. The corresponding genetic chip information of O. melastigma has been acquired which makes it convenient for the study of O. melastigma [1, 14, 16]. Additionally, homologous species could be fully used for comparative biology, in a similar manner to Drosophila, for which the genome analysis of multiple species has greatly promoted the study of comparative biology [14, 17].

All of these advantages enhance the potential of O. melastigma to be a competent model organism in marine ecotoxicology.

3. The Research Background of O. melastigma in Molecular Biology

Sharing a high degree of similarity, most of the research findings of the congeneric species of O. melastigma, such as O. latipes, could be applied to O. melastigma mostly. Notably, even though O. melastigma is similar to the other medaka species, some differences still exist. For example, omChgh is characterized by eight exons and seven introns, while the second isoform of the Chgh gene has only seven exons in the O. latipes genome [6, 18, 19]. Dlx2 is expressed only in the telencephalon and diencephalon of O. melastigma, while it is also expressed in the rhombencephalon of O. latipes [1]. O. latipes and O. melastigma share completely identical peptide sequences but bear very different glycan structures [19]. This phenomenon suggests that further exploration of the marine medaka genome and proteome is needed [20].

3.1. The Research Background of O. melastigma Genes

A substantial number of molecular biological studies for O. melastigma are being conducted. The complete mitochondrial genome of O. melastigma has been obtained from the genome data sequenced by next-generation sequencers [21]. A batch of organ-specific molecular markers have also been identified, such as the makers for brain, eyes, heart, liver, and muscle [15]. These markers can be used to indicate the developmental status of specific organs, and their abnormal expression can be used to indicate the toxicity of pollutants on organ development. Chen et al. [1] analyzed the expression of 11 organ-specific expression genes during each period of embryonic development by in situ hybridization (ISH) and determined that 8 of the 11 genes are similar to those expressed during the embryonic development of zebrafish and O. latipes.

In addition to the above specified genes of organ development, some functional genes in different tissues have been analyzed as well (Table 1). Some immune-related genes, including complement-related genes and inflammation-related genes, have been analyzed. Bo et al. used suppression subtractive hybridization (SSH) to identify differentially expressed immune genes in the liver of O. melastigma infected with Vibrio parahaemolyticus [30]. Based on an NCBI BLAST search of the 1279 sequenced clones in the SSH libraries, 396 genes were identified, and 38 were involved in the immune process. Additionally, genes involved in cellular metabolism, biological regulations, general response to stimuli, transport processes, signal transduction, and cellular component organization were obtained [30]. Some genes related to metabolism, osmoregulatory and cardiac development in O. melastigma have also been submitted. Whole omCyp genes were registered at the GenBank database. To date, various Cyp gene families have been identified. The transcript profiling of whole omCyp genes has been finished for O. melastigma exposed to water accommodated fractions (WAFs) of Iranian crude oil [25, 33].

FunctionsGenesExposed tissuesExposed toReferences

Reference genes 18S, Rpl7, and β-actin

Hypoxia-responsiveTelomerase reverse transcriptase (Tert) Ovary, liver, testis, kidney, gill, brain, spleen, intestine, eye, muscle, and skinHypoxia[16, 22]
Hypoxia-inducible factor-1α (Hif  1α) Liver, testisHypoxia[22]
Erythropoietin (Epo) Liver, testis, and embryosHypoxia, PFOS[22, 23]
Leptin receptor (Lepr) Liver, gill, heart, kidney, gill, brain, spleen, intestine, eye, muscle, ovary, and testisHypoxia[24]
Hemoxygenase-1 (Ho) Liver, gill, and heartWAFs, Hypoxia[24, 25]

Immune toxicityGlutathione peroxidase (Gpx) EmbryosPFOS[26]
Catalase (Cat) EmbryosPFOS[26]
Uncoupling protein 2 (Ucp2) EmbryosPFOS [26, 27]
Cyclooxygenase-2 (Cox2) EmbryosPFOS[26]
Peroxisome proliferator-activated receptors(Ppars): Pparα, Pparβ, and PparγEmbryos, whole fishPFOS, WAFs[25, 26, 28]

Complement-related genesLectin, mannose-binding 2 (Mbl2) LiverPBDE-47[29]
Cyan fluorescent protein (Cfp) LiverPBDE-47[29]
Complement component: C1r/s, C3, C9, C3-2, C4, C1q, C5, C8, C1 inhibitor LiverPBDE-47, Vibrio parahaemolyticus [29, 30]
Prothrombin (F2) Liver[30]
Complement factor: Hf, Bf LiverVibrio parahaemolyticus [30]
Hepcidin (Hep): Hep1, Hep2 Liver, spleen, gill, intestine, ovary, testis, brain, and embryosVibrio parahaemolyticus [30]
Mannose-binding lectin-associated serine protease (Masp) LiverVibrio parahaemolyticus [30]

Inflammation-related genesTumor necrosis factor-α (Tnfα) EmbryosPFOS, BPA[26, 31]
Interleukin (Il): Il1β, Il8 EmbryosPFOS, BPA[26, 31]
CC chemokine eotaxin-1 (Ccl11) EmbryosBPA[31]
Superoxide dismutase (Sod) EmbryosPFOS, BPA[26, 31]

Osmoregulatory mechanismNa+/K+-ATPase (Nka) Gill, embryos BPA, SW (35 ), BW (15 ), and FW (0)[7, 31, 32]
Na+, K+, 2Cl cotransporter (Nkcc): Nkcc1a, Nkcc1b, and Nkcc2 Gill, liver, testis, intestine, ovary, brain, muscle, kidney, heart, Fin, and eyeSW (35 ), BW (15 ), and FW (0)[32]
FXYD domain-containing ion transport regulator (Fxyd): Fxyd5, Fxyd6, Fxyd7, Fxyd8, Fxyd9, Fxyd11, and Fxyd12 Gill, intestine, kidney, brain, eye, liver, and caudal finSW (35 ), BW (15 ), FW (0)[5]

Cardiac development-related genes NK2 transcription factor related 5 (Nkx2.5) EmbryosPFOS, BPA[23, 31]
Cyclooxygenase (Cox): Cox1, Cox2 EmbryosPFOS, BPA[23, 31]
ATP synthase EmbryosPFOS[23, 27]
Bone morphogenetic protein (Bmp4) EmbryosPFOS, BPA[23, 31]
Fibroblast growth factor 8 (Fgf8) EmbryosPFOS, BPA[23, 31]
GATA-binding protein 4 (Gata4) EmbryosPFOS, BPA[23, 31]
Leptin receptor (Lerp) EmbryosBPA[31]
SET and MYND domain containing 1 (Smyd1) EmbryosPFOS[23]

MetabolismsCytochrome P450 (Cyps) Liver, gill, embryos, intestine, and ovaryPFOS, WAFs [25, 28, 33]
Aldehyde dehydrogenase (Aldh) WAFs[25]
Glutathione S-transferase (Gst): Gsta, Gstk, Gsto, Gstt, and Gstz WAFs[25]
Sulfotransferase (Sult): Sult1, Sult2 cytosolic, Sult2b1, Sult2b2, Sult3 cytosolic, Sult3-like, Sult4a1, and Sult6b1 WAFs[25]
UDP-glucuronyltransferases (Ugts): Ugt1b, Ugt2a, Ugt2a2, Ugt2a3, Ugt2b33, Ugt2b3-like, Ugt5a1, and Ugt5g1 WAFs[25]
Hydroxysteroid dehydrogenase (Hsd): 3β-Hsd, 11 β-Hsd, and 17β-Hsd WAFs[25]
Aryl Hydrocarbon Receptor (Ahr): Ahr1, Ahr2 Embryos, whole fishPFOS, WAFs [25, 28]
5 α-reductase (Srd5a) WAFs[25]
Steroidogenic acute regulatory protein (Star) WAFs[25]
ATP-binding cassette (Abc): Abcb1, Abcc2, Abcc3, Abcc4, and Abcg2 WAFs[25]
Heat shock protein (Hsp): Hsp10, Hsp22, Hsp27, Hsp30, Hsp60, Hsp70, Hsp75, Hsp90a, Hsp90β, Hspβ7, and Hspβ11 WAFs[25]
Choriogenin H and L (Chgh and Chgl) Liver, embryos, and larvaePFOS, E2, EE2, BPA, and NP[28, 34]
Kinesin superfamily7 (Kif7) Brain, kidney, liver, muscle, ovary, and testicle[6]
Aryl hydrocarbon receptor nuclear translocator (Arnt) EmbryosPFOS[28]
Vitellogenin (Vtg) Embryos, liver, gill, intestinePFOS[28]
Estrogen receptor (Er) EmbryosPFOS[28]
Horiolysin H and L (Hce and Lce) EmbryosPFOS[10]

Notes: 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47), bisphenol A (BPA), polycyclic aromatic hydrocarbons (PAHs), sea water (SW), fresh water (FW), brackish water (BW), 17β-Estradiol (E2), 17α-ethinylestradiol (EE2), 4-nonylphenol (NP).

Second generation high-throughput sequencing technology has greatly enhanced the ability to obtain genetic information. Huang et al. extracted RNA from O. melastigma following exposure to pollutants during various developmental periods and used Illumina high-throughput sequencing to obtain 6 GB data. They performed bioinformatics analyses and identified a large number of toxicology-related genes, thus providing a broad molecular basis for further toxicological investigations [27].

Differentially expressed genes can be largely obtained in fish after exposure to pollutants using gene chip technology. Chinese scholars have constructed a dedicated gene chip for O. melastigma, which contains 180 genes related to cell division, detoxification reactions, hypoxia response, oxidative stress, apoptosis, growth, sex determination, gonadal differentiation, and reproductive hormone secretion [35]. This chip includes the most common marker genes for toxicological studies and can be used effectively for gene screening with differential expression. Using newly developed sequencing technology (Illumina RNA-Seq) and digital gene expression (DGE) technology, a total of approximately 145 thousand unigenes were obtained with 565 bp of unigene N50 [27], which were further enriched in various molecular pathways involved in the response to PFOS exposure and related to neurobehavioral defects, mitochondrial dysfunction, and the metabolism of proteins and fats.

3.2. The Research Foundation of O. melastigma Proteins

The detection of protein expression levels requires corresponding antibodies. Because of the conservation of homologous proteins, antibodies have certain commonalities in allied species. The antibody library of zebrafish has been relatively completed; thus we can use them to directly screen for the specific antibody that reacts with the homologous protein in O. melastigma, avoiding the tedious processes of antibody preparation. Through immunohistochemical assay (IHCA) screening of whole embryos, 17 types of zebrafish antibodies can cause specific immune reactions with O. melastigma. These antibodies have a close relationship with the development of nerve, heart, and brain, providing a basis for toxicological research on protein levels [15, 16]. In addition, mouse anti-human TERT monoclonal antibody mAb476 can specifically combine with the TERT protein of O. melastigma [16].

The tissue distribution of the protein expression in O. melastigma under various environment stresses has been partly finished intuitively by WB, IHCA, and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) (Table 2). The expression of the TERT protein in the cytoplasm and nucleus of O. melastigma can be quantified by Western blotting (WB) [41]. Kong et al. observed the TERT protein expression of O. melastigma in the testis, ovary, muscle, brain, gill, intestine, kidney, and liver of adult fish using IHCA [16]. Proliferating Cell Nuclear Antigen (PCNA) is the protein marker reflecting cell proliferation, which can be detected by means of IHCA in O. melastigma. Experimental results showed a significant correlation between PCNA and TERT in transcriptional and translational expression levels [11]. PCNA detection can also reflect the spatial and temporal characteristics of O. melastigma embryonic development [15].

Related functionsProteinsExpression tissues and exposure conditionReferences

Cell structureHistone-binding protein RBBP4Gill (Br)[36]
GelsolinGill, brain (Br)[36]
Krt4 proteinGill (Br)[36]

Oxidative stress responseHemoglobin beta chainGill (Br)[36]
Histone H3Gill (Br)[36]
Glial fibrillary acidic proteinBrain (Br)[36]
Keratin 15 [KRT15]Brain (Br), liver (Hg) [36, 37]
Zgc: 65851Brain (Br)[36]
Type I cytokeratin, enveloping layer [CYT1]Brain (Br), liver (Hg)[36, 37]
Myosin light chain 2Brain (Br)[36]
Tropomyosin alpha-3 chainBrain (Br)[36]
α-Tubulin 1Liver (Hg)[37]
Keratin 8Liver (Hg)[37]
α-ActinLiver (Hg)[37]
Keratin 18Liver, brain (Hg)[37]
β-ActinLiver, brain (Hg)[37]
Type I keratin-like proteinLiver (Hg)[37]
Lamin type BLiver (Hg)[37]
Krt5 proteinBrain (Hg)[37]
Type II basic cytokeratinBrain (Hg)[37]
Keratin K10 [KRT10]Liver (Hg)[38]
Novel protein similar to vertebrate plectin 1 [PLEC]Liver (Hg)[38]
Peroxiredoxin 4Liver (Hg)[38]
Peroxiredoxin 6Liver (Hg)[38]
Glutathione S-transferase [GSTR]Liver (Hg)[38]
SOD [Cu-Zn]Liver (Hg)[38]
Aldehyde dehydrogenase 1 family, member A2Brain (Hg)[38]
Aldehyde dehydrogenase, mitochondrialBrain (Hg)[38]
Peroxiredoxin-2 [PRDX2]Liver (Hg)[38]
Natural killer enhancing factorLiver (Hg)[37]
Peroxiredoxin-1 [PRDX1]Liver (Hg)[38]
DJ-1 protein [DJ-1]Liver (Hg)[38]
Cathepsin D [CTSD]Liver (Hg)[38]
proliferating cell nuclear antigen [PCNA]Testis, muscle, kidney, liver Cheek, brain, intestine, and ovary embryo during each development period (H)[16, 39]
Telomerase Reverse Transcriptase [TERT]Testis, brain, muscle, gill, intestine, kidney (N), and liver (H)[16]
superoxide dismutase [SOD]Whole fish (Z)[40]
Metallothionein [MT]Whole fish (Z)[40]
heat shock protein 70 [HSP70]Whole fish (Z)[40]

MetabolismApoA-IV4Gill (Br)[36]
Aldose reductaseGill, brain (Br)[36]
Pyruvate carboxylaseBrain (Br)[36]
Dpysl5a proteinBrain (Br)[36]
Triosephosphate isomeraseBrain (Br)[36]
EnolaseBrain (Br)[36]
Glutamine synthetaseBrain (Br, Hg)[36, 37]
Isovaleryl coenzyme A dehydrogenaseBrain (Br)[36]
Glyceraldehyde 3-phosphate dehydrogenaseBrain (Br)[36]
Homogentisate 1,2-dioxygenaseLiver (Hg)[37]
Alanyl-tRNA synthetase, cytoplasmicLiver (Hg)[37]
Dihydrolipoamide S-acetyltransferaseLiver (Hg)[37]
AdenosylhomocysteinaseLiver (Hg)[37]
Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrialLiver (Hg)[37]
Brain-type fatty acid binding proteinLiver (Hg)[37]
Methionine adenosyltransferase-likeLiver (Hg)[37]
S-formylglutathione hydrolaseLiver (Hg)[37]
Apolipoprotein A1Brain (Hg)[37]
Pyruvate kinaseBrain (Hg)[37]
Dihydropyrimidinase-related protein 5Brain (Hg)[37]
Dihydropyrimidinase-like 2Brain (Hg)[37]
Enolase 1, (alpha)Brain (Hg)[37]
Creatine kinase, brain bBrain (Hg)[37]
Total glutathione [GSH]Whole fish (W)[25]
Glutathione S-transferase [GST] Whole fish (W)[25]
Sulfotransferase [SULT]Whole fish (W)[25]
Superoxide dismutase [SOD]Whole fish (W)[25]
Glutathione reductase [GR]Whole fish (W)[25]
Glutathione peroxidase [GPx]Whole fish (W)[25]
Catalase, CATWhole fish (W)[25]
ATP synthase subunit d, mitochondrial [ATP5H]Liver (Hg)[38]
Electron-transferring-flavoprotein dehydrogenase [ETFDH]Liver (Hg)[38]
Electron transferring flavoprotein subunit alpha, mitochondrial [ETFA]Liver (Hg)[38]
Pyruvate dehydrogenase (lipoamide) beta [PDHB]Liver (Hg)[38]
Phytanoyl-CoA dioxygenase domain-containing protein 1 [PHYD1]Liver (Hg)[38]
Delta3,5-delta2,4-dienoyl-CoA isomerase, mitochondrial [ECH1]Liver (Hg)[38]
Phosphorylase [PYGB]Liver (Hg)[38]
Formimidoyltransferase-cyclodeaminase [FTCD]Liver (Hg)[38]

Signal transductionPutative transient receptor protein 2Gill (Br)[36]
Myosin regulatory light chain 2Gill (Br)[36]
FXYD domain-containing ion transport regulatorGill (S)[5]
NKCC1a-like proteinGill (S)[32]
NKA α-subunitGill (S)[7, 32]
GrancalcinGill (Br)[36]

Protein modificationMyosin light chain 2Gill (Br)[36]
Calreticulin, like 2Gill (Br)[36]
Transforming protein RhoABrain (Br, Hg)[36, 37]
CalmodulinBrain (Br)[36]
Annexin 4Liver (Hg)[37]
14-3-3E1 proteinLiver (Hg)[37]
14-3-3 proteinLiver (Hg)[37]
Annexin A13Brain (Hg)[37]
Cytosolic nonspecific dipeptidaseLiver (Hg)[37]
Proteasome alpha 1 subunitLiver (Hg)[37]
HSP-90Brain (Hg)[37]

Other function relatedChaperonin containing TCP1, subunit 8 (theta)Brain (Hg)[37]
Beta-synucleinBrain (Br)[36]
SH3-domain GRB2-like endophilin B2Brain (Br)[36]
Complement component C3-1Liver (Hg)[37]
Carbonic anhydrase 1Brain (Hg)[37]
ATPase, H+ transporting, V0 subunit D isoform 1Brain (Hg)[37]
TransferrinBrain (Hg)[37]
Eukaryotic translation initiation factor 3, subunit 2 beta [EIF3S2]Liver (Hg)[38]
Histone H4Liver (Hg)[38]
Ependymin [EPD]Liver (Hg)[38]
GammaN1 crystallin [CRYGN1]Liver (Hg)[38]

Notes: the abbreviations in parentheses mean the protein expression in the environment of exposure to normal (N), hypoxia (H), brevetoxins (Br), HgCl2 (Hg), salinity (S), nZnO (Z), and WAFs of Iranian crude oil (W).

Proteomics refers to the research method of identifying protein characteristics on the large-scale level, and it has become one of the hot spots of aquatic toxicology [39]. Quantitative proteomic analysis demonstrated that hepatotoxicity caused by Hg might involve oxidative stress, cytoskeleton impairment, and energy metabolism alteration, highlighting that the fish liver might be an important target for Hg attack. And proteins such as cathepsin D, GST, and peroxiredoxin-1 responding to Hg treatment in a dose-dependent manner could be used as potential biomarkers of aquatic Hg monitoring [38]. Exposure to PbTx-1 resulted in the alteration of the protein expression involved in cell structure, macromolecule metabolism, neurotransmitter release, and the distribution of signal transduction which may help explain the damage mechanisms of aquatic toxins in fish [36].

4. Utilization of O. melastigma in Toxicological Studies

O. melastigma has been used as a research model for assessing multiple responses to stresses of organic chemicals, inorganic chemicals, detrimental organisms, and environmental stress (Table 3). The toxicity responses of O. melastigma are different from some species under environmental stresses, which may even have a totally opposite effect (Table 4).

Responsive toToxicological research aboutAge of fishExposure concentration and timeMain worksMain conclusionsReferences

Organic chemicals
WAFsCYP1A-involved detoxification mechanism3-week-old fish and adults 2.5, 5, 10, 20, 40, 60, 80, and 100% WAF for 24 h; 5% for 6, 12, 24, 48, 72, and 96 h Transcript profiling of whole omCyp genes, enzyme activity and steroid hormones assay, omCyp1a mRNA expression in different tissues during different developmental stages, and effects of β-NF, BaP, and WAF on expression of omCyp1a WAF induced CYP-involved detoxification mechanism but reduced steroidogenic metabolism; omCyp1a would be associated with the initiation of the cellular defense systems.[25, 33]
PBDE-47Immune-modulatory effectsThree-month-old290 and 580 ng/day from 2 dpf to hatchingCorrelation between BDE-47 body burden and complement gene expression (RT-PCR) in different gendersGenes studied were gender dependent (males > females); BDE-47 is not biotransformed in marine medaka.[29]
Maternal transfer2- and 3-month-old μg/day for 21 daysAccumulation of PBDE 47 in 2-month-old fish and maternal transfer of PBDE 47 from adult female medaka to eggsPBDE 47 transfer is associated with lipid mobilization during egg production.[42]
PFOSMitochondrial dysfunctionEmbryos0.25 and 1 mg/L from 2 dpf to 6 dpfSequence the RNA mixtures using Solexa/Illumina RNA-Seq at various developmental stages and after various types of exposure, and DGE and qRT-PCR analysis for relative gene expressionThe mitochondrial dysfunction appears to be involved in multiple toxicological effects of PFOS on O.  melastigma embryos.[27]
Precocious hatching Embryos1, 4, and 16 mg/L from 2 dpf to hatchingRecord the time for hatching, hatching rate and mortality of fry hatched within a week, and hatching enzymatic activity and RT-PCR analysis for gene expressionPFOS induced the hatching enzyme, leading to the precocious hatching of embryos and the decrease of larvae survival.[10]
Endocrine-disruptive effectEmbryos1, 4, and 16 mg/L for 2 dpf, 4 dpf, and 10 dpf, respectivelyThe mortality and malformation rates, the transcriptional responses of the ER, AHR, and PPAR pathways to PFOS by RT-PCR, and quantification of PFOS in exposure solutions and medaka embryos PFOS has estrogenic activity and endocrine-disruptive properties and could elicit gene responses in a stage-specific manner.[28]
Cardiac toxicityEmbryos1, 4, and 16 mg/L for from 2 dpf to hatchingCardiac morphology, heart rates and the SV-BA distance of the heart was measured; RT-PCR analysis of gene expression profiles was conducted.PFOS affected the development and function of the heart in the marine medaka embryos.[23]
ImmunotoxicityEmbryos0, 1, 4, and 16 mg/L from 2 dpf to hatchingPFOS body burden, survival rates, and growth parameters of fish larvae during 17 dph, liver histological examination, and gene expression in fish larvae after LPS exposure for 12 h at 27 dphThe immunosuppression effects caused by PFOS could lead to functional dysfunction or weakness of the immune system in the fish larvae.[26]
BPACardiac toxicity Embryos200 μg/L for 2 dpf-incubationHeart beat rate, SV-BA distance of embryos, body length and width, histology, and BPA-induced inflammation-related genes and heart-related genesBPA induced cardiac toxicity of the O.  melastigma embryos.[31]
PAHs (ANF, Pyr, Phe, and BaP)Developmental malformations EmbryosDifferent PAHs for 18 daysDeformity assessment, heart rate, heart elongation, hatch rate, and EROD and Caspase-3/7 activity assays of embryos exposed to PAHs with or without 100 μg/L ANFInhibition of CYP1A, EROD, and Caspase-3/7 activities can be used as indicator in the ecological early warning and PAHs detection.[43, 44]
Estrogen (E2, EE2, NP, and BPA)Estrogenic pollutantsSexually matureE2, EE2 (1, 10, 100, and 500 ng/L); NP, BPA (1, 10, 100, and 200 μg/L) for 7 daysE2-inducible choriogenins expression in embryos and yolk-sac larvae by end-point PCR; effects of EE2, BPA, and NP, respectively, on omChgh and omChgl expression by RT-PCRThe rapid inducibility (within 24 h) of omChgh by E2 during early developmental stages was found to be more estrogen sensitive than omChgl. [34]
BenzotriazoleReproductive effect3-month-old0.01, 0.1, and 1 mg/L for 4 and 35 daysBenzotriazole can induce Vtg and Cyp19a gene expression but inhibits the Cy1a1 gene expression (qPCR analysis).Benzotriazole had adverse potential on the endocrine system.[45]

Inorganic chemicals
DWNTs Ecotoxicity data of DWNTs48 h posthatching10, 50, and 100 mg/L for 14 daysMortality and total length of medaka fish larvae over 14 days exposed to different concentrations of stirred and sonicated double-walled carbon nanotubes. So-DWNTs are more toxic than st-DWNTs; the dispersion method and size of aggregations should be considered in DWNT toxicity testing.[46]
nZnOSublethal toxicities<24 h4 and 40 mg/L ZnO for 96 hStress responses in fish after acute exposure (SDS-PAGE) nZnO did not display the same toxicity as ZnO towards the fish.[40]
HgCl2Hepatotoxicity and neurotoxicityWeighing 0.5 ± 0.05 g1000 μg/L for 8 h; 1 or 10 μg/L for 60 dProtein expression profile in liver and brain exposed to HgCl2 (MALDI-TOF/TOF MS) and mercury accumulation and damaged liver ultrastructure in medakaHg hepatotoxicity might involve oxidative stress, cytoskeleton impairment, and a dysfunction in metabolism.[37, 38]
Cd2+, Hg2+, Cr6+, and Pb2+Toxic effects of heavy metalsEmbryos and larvae96 h and 14 dThe mortality, heart beat rate, and malformation rates The fish species has relatively high sensitivity to heavy metal stress.[47]

Detrimental organisms
Vibrio parahaemolyticus Immunotoxicity5-month old6 × 105 cfu/fish for 6 h, 24 h and 48 hqPCR analysis of the complement genes in liver; age-, tissue-, and gender-differences in the expression of hepcidin; hepcidin expression in hepatocyte by ISHO.  melastigma can serve as a model to understand the basic biological processes related to immune function.[30, 48]
K. brevis: PbTx-1Neurotoxicity Adult0, 6, 8, 10, 12, 16 and 18 μg/L for 24 h; 6 μg/L for 2 daysAlgal toxicity (toxic symptoms, 24 hour mortality, 1/LT50) and its supernatant, MeOH and TCM extracts of O.  melastigma; changes in protein profiles in medaka gill and brain exposed to PbTx-1K. brevis-induced hypoventilation response in medaka; the down-regulation of several proteins involved in cell protection.[36, 49]
C. marina Ichthyotoxins of C.  marina 4–8 months-old10,000 cells/mL for 0, 24, 48 and 60 hAlgal cell density, growth rate, their toxicity (toxic symptoms, 24-hour mortality, 1/LT50) and its supernatant, MeOH and TCM extracts to O.  melastigma Fish susceptibility to C.  marina is related to its growth rate, but not to cell density; C. marina developed the hyperventilation response of the fish.[49, 50]

Environmental stress
HypoxiaHypoxia-responsive4-week old adult1.8 ± 0.2 mg O2/L for 3 months; 12 weeks 1.8 mg O2/L for 24, 48 and 96 hAdult male fish were processed for ISH and IHC; volume density indices of omTERT mRNA and protein, PCNA and TUNEL signals in liver hepatocytes after chronic exposure to hypoxia; expression of Tert, Hif  1α, Epo, Lepr, and Ho in tissues by RT-PCRHypoxia upregulates omTERT expression via omHIFhif-1 in liver and testis and the omLepR omLEPR expression demonstrated its independent control in endocrine and peripheral tissues.[16, 22, 24]
Ichthyotoxins of C.  marina 4–8 months old7 mg/L, 6.0  mg/L and 1  mg/L DO for 60 hOxygen consumption rate, threshold lethal DO and correlation between body weight and survival time of marine medaka inside the sealed syringeFish susceptibility to C. marina is related to the susceptibility of the fish to hypoxia.[50]
SalinityOsmoregulatory mechanism2.50 ± 0.30 cmSW (35 ), BW (15 ), FW (0) for Three weeks or 1 monthPlasma osmolality, MWC, Na+/Cl concentration, time course, NKCC1a-like protein expression, NKA activity, NKA-IR cell activity, NKA α-subunit mRNA and protein expression in gills in response to hypoosmotic challenge; salinity effects on multiple Fxyd mRNA and FXYD11 protein abundance; co-immunoprecipitation of NKA with FXYD11 and the localization of Fxyd11 mRNA in gill sections in freshwater-acclimated marine medakaThe expression pattern of branchial Fxyd11 was similar to that of Nkaα in the O.  latipes, but non-correlated expression patterns were observed in the O.  melastigma at both the mRNA and protein levels; the lowest NKA activities were found in the environments with salinities similar to their natural habitats.[5, 7, 32]
Ichthyotoxins of C. marina 4–8 months old70 seawater and natural seawater. The LT50 of marine medaka at different ages (4–8 months-old) exposed to 70 hypersalinity (70 -SW) Fish susceptibility to C. marina is not related to its tolerance to hypersalinity stress.[50]

Notes: days postfertilization (dpf); days posthatching (dph); sinus venosus-bulbus arteriosus (SV-BA); lipopolysaccharides (LPS); β-naphthoflavone (β-NF); benzo[a]pyrene (BaP); phenanthrene (Phe); pyrene (Pyr); methanol (MeOH); chloroform (TCM); quantitative polymerase chain reaction (qPCR).

Exposing toO.  melastigma Other speciesReferences

PFOSHatched in advance and hatching rate increased.Hatch was delayed and hatching rate was not affected or decreased in zebrafish.[810]
Ke in the larvae ranged from 0.04/d to 0.07/d.Ke ranged from 0.053 to 1.700 L/kg/d in blood, kidney, liver and gall bladder and from 0.02 to 0.23/d in carcass and liver concentrations in rainbow trout (Oncorhynchus  mykiss).[5153]
Did not alter Epo Led to high mortality in zebrafish[23]

Phe, Pyr, and BaPNOEC values were 50, 25, and 10 μg/L, respectively.NOEC values were 10, 50, and 1.8 μg/L, respectively, in the water flea (Tigriopus  japonicus).[54]

E2The mRNA level of 3Bhsd (steroidogenic enzymes) was increased.Decreased the production of 11-KT and mRNA levels of steroidogenic enzymes in zebrafish and decreased the production of testosterone in human[5557]

DWNTsGrowth inhibition was observed at 10 mg/L for so-DWNTs but not for st-DWNTs.Population growth was reduced to 0.1 mg/L for so-DWNTs and 10 mg/L for st-DWNTs in the water flea.[23]

nZnOLack of change was observed in the SOD activities. SOD activities were decreased for the first few days but recovered soon in O.  latipes and were also significantly depleted in mouse embryo fibroblast cells, more toxic in Skeletonema costatum and Thalassiosira pseudonana, and less toxic in Elasmopus rapax and the water flea.[40]

CercariaeDid not infectInfected in liver and kidneys of Channa  punctatus, infected in the muscles of Cliona  orientalis, and did not infect in Puntius  sophore and Gambusia  affinis [58]

SalinityPrefers hypoosmotic conditionsPrefers hyperosmotic conditions in Javanese medaka (Oryzias Javanicus)[11, 59]
MWC was constant with the increase of salinity in O.  melastigma. MWC was decreased with the increase of salinity in O.  latipes.[7]

Hypoxia HAS was not present.HAS was identified in zebrafish and Fugu.[60]
3Bhsd and Cyp19a mRNA expression upregulation3Bhsd and Cyp19a mRNA expression was reduced in zebrafish.[55]

Notes: the elimination rate constant (Ke); No Observed Effect Concentration (NOEC); 11-ketotestostrone (11-KT); HIF-1 ancillary sequence (HAS).

4.1. Toxicological Studies for Organic Chemicals

The choriogenin of teleost fish is considered to be part of the structural interlayer of chorionic precursor cells, which are sensitive to estrogenic contaminants. It increased the expression of the egg-shell precursor protein gene in the liver when exposed to a high concentration of 17β-Glycol and 17α-ethinyl estradiol [34]. The Chgh and Chgl of O. melastigma are sensitive to exposure to estradiol and nonylphenol, and the response of the male fish is more sensitive compared to the female. This indicates that the two genes can be used as sensitive biomarkers to detect pollution levels of estrogen contaminants in the marine environment [34].

The WAF exposure induced CYP-involved detoxification effects but reduced CYP-involved steroidogenic metabolism in the marine medaka. As well-characterized biomarkers of toxicants exposures, omCyp1a and omCyp1b were highly induced following WAF exposure [25, 43]. Some previous studies have shown potentially synergistic effects after coexposure of O. melastigma embryos to CYP1A inhibitors and PAH-type CYP1A inducers [43]. The acute aquatic toxicity of some seawater organisms exposed to polycyclic aromatic hydrocarbons (PAHs) in the laboratory is summarized in Table 5. Distinctly, O. melastigma showed high tolerance to PAHs compared to other species. The heart elongation (heart tube) of O. melastigma embryos and heart deformities of these juvenile fishes have been recommended as potential biomarkers of the existence of PAH pollution by Mu et al. [43].

Scientific nameLC50/EC50PAHs (μg/L)References

Corophium acherusicum LC5031049[54]
Neomysis awatschensis LC50130[54]
Tigriopus japonicus LC505461743.46[54]
Neomysis awatschensis LC5015.2[54]
Nitzschia closterium EC5071.556.851[54]
Enteromorpha linza EC502070209286[54]
Oncorhynchus mykiss LC5032002000[64]
Acanthogobius lactipes LC50295[54]
Ctenogobius giurinus LC5013.1[54]
O. melastigma LC50639931275705[54]
Sparus macrocephalus LC50800[54]
Strongylocentrotus intermedius LC50520[54]
Hemicentrotus pulcherrimus LC501.0561.56[54]

Notes: median lethal concentration (LC50); median effective concentration (EC50).

Studies quantified the endogenous expression of all six complement system genes including C1r/s, Mbl2, CfpF2, C3, and C9, in the liver of marine medaka and found that the expression levels were higher in males than in females. BDE-47 exposure downregulates the expression of all six genes in males, while in females the expression of Mbl2, Cfp, and F2 mRNAs was upregulated and C3 and C9 remained stable with exposure time and dose. These results indicate that the future direction for fish immunotoxicology should include parallel assessment for both genders [29]. Two hepcidins in O. melastigma play a complementary role in the innate defense system. Gender specificity should be taken into consideration in immunotoxicological studies in time and extent of induction of the two hepcidin genes in infected O. melastigma [48].

PFOS has estrogenic activity and endocrine-disruptive properties that elicit transcriptional responses on POPs-related pathways in a stage-specific manner [6163]. The marine biological toxicity of PFOS was systematically studied by Dong et al. using O. melastigma [10, 23, 2628]. Their results showed that exposure to PFOS could induce the hatching enzyme both at transcriptional and enzymatic activity levels and further lead to decreases of average hatching time and increases of the average hatchability of O. melastigma embryos, which in turn induced the mortality of the larvae hatched from exposed embryos. All of these effects were dose dependent [10]. They also found that PFOS is toxic to the development of the cardiovascular system of O. melastigma, affecting the expression of cardiac development-related genes, morphological development, and function of the heart in the marine medaka [23].

Some research has also been conducted in their laboratory with embryos exposed to low concentrations of bisphenol A (BPA). The result showed that the expression of heart development-related genes and inflammation-related genes in O. melastigma was altered, the body length and width decreased, and the larvae exhibited inflammation foci in the heart ventricles [31].

4.2. Toxicological Studies for Inorganic Chemicals

Subacute toxicity experiments with ambient concentrations of pollutants are often closer to environmental value and thus have great significance in toxicological evaluation. In evaluating the toxicity of ZnO, researchers evaluated the subacute toxicity of two zinc oxides on the expression of SOD, MT, and HSP70 in O. melastigma and found that the two zinc oxides show differences in the induction of three proteins [40]. In the toxicity assessing, double-wall carbon nanotubes (DWNTs) (10 mg/L) following ultrasonic treatment inhibited the growth of O. melastigma larvae [46].

O. melastigma is also used in the evaluation of heavy metal toxicity. The 96 h LC50s of this fish following exposure to Cu2+, Cd2+, Hg2+, Cr6+, Pb2+, and Zn2+ are shown in Table 6, from which we could determine that O. melastigma has a strong sensitivity to metal stress compared to other marine species. Toxicity detection of O. melastigma for copper, tributyltin (TBT), and five commonly used antifouling fungicides, including s-triazine, diuron, pyrithione zinc, copper pyrithione, and chlorothalonil, indicate that the 96 h LC50 of this fish’s tolerance for copper, s-triazine, and diuron is at the level of mg/L, while others are μg/L [91]. Exposing fertilized eggs and newly hatched O. melastigma juveniles to Cd2+, Hg2+, Cr6+, and Pb2+ significantly reduced the hatching ability of the embryos and the heart rates above a certain concentration [47]. Metal accumulation of inorganic mercury in the liver and brain of O. melastigma induced oxidative stress, cytoskeletal reorganization and/or disruption, dysfunction in metabolism, protein modification, signal transduction, and other related functions [37, 38].

SpeciesExposure agesLC50 (mg/L)References

Argopecten ventricosus Juvenile0.3960.8303.430[65]
Chironomus furens Larvae52.[66]
Chironomus plumosus Larvae42.[66]
Chironomus riparius Adult0.0430.021[67]
Cynoglossus semilaevis Postlarvae0.0250.1781.0260.0451.18[68]
Duttaphrynus melanostictus Larvae0.[69]
Echinogammars olivii Adult0.250.621.30[70]
Farfantepenaeus paulensis Postlarvae0.833.31[71]
Fundulus heteroclitus Postlarvae1.718.21880.068129.5[7274]
Hyalella azteca Adult0.210.013[67]
Hexagenia spp.Adult0.0737.82[67]
Liza vaigiensis Postlarvae3.71380.0835[72]
Lutjanus argentimaculatus Juvenile9820.10.38[75]
Menidia menidia Juvenile6.3910.112[75, 76]
Oreochromis niloticus Juvenile0.800.82[77]
O. latipes Postlarvae5.612.4[72]
O. melastigma Postlarvae7.31.12>201.4560.09743 [78]
Pagrosomus major Postlarvae0.315.63.6[79, 80]
Palaemon elegans Adult2.525.8812.3[70]
Penaeus indicus Postlarvae0.82047.22[81, 82]
Penaeus monodon Postlarvae2.285.77–7.283.02[83, 84]
Penaeus penicillatus Larvae3.0254.267[85]
Poecilia reticula Juvenile2.3617.7143.4[86]
Penaeus setiferus Postlarvae0.03080.017[87, 88]
Priopidichthys marianus Juvenile140310. 35[72]
Rivulus marmoratus Postlarvae1.421.185.314.3147.9[72, 74]
Sphaeroma serratum Adult1.984.616.12[70]
Sparus macrocephalus Larvae0.20.31.8[80]
Stenocypris major Adult0.02520.01310.52621.1898[89]
Tubifex tubifex Adult0.160.87[67]
Zebrafish Adult0.1746.497116.432181.090.1444.48[86, 90]

4.3. Toxicological Researches for Detrimental Organisms

The median lethal time (LT50) of O. melastigma is treated as an indicator of pollutant toxicity for toxicological comparison and correlation analysis. In addition, the acute toxicity test can provide the appropriate dose for the study of molecular toxicological mechanisms, such as the determination of the 24 h LC50 value in O. melastigma exposed to brevetoxins (PbTxs). These concentrations of PbTxs can be determined in follow-up proteomics studies [36].

ISH showed that Vibrio parahaemolyticus would induce the expression of hepcidin genes in the nuclei and cytoplasm of liver cells of O. melastigma [48]. It is also used to characterize the toxicity of the toxins generated by Chattonella marina and Karenia brevis. Test fishes exposed to the toxins generated by C. marina exhibit hyperventilation, while those exposed to the toxins generated by K. brevis exhibit hypoventilation [49]. With the assistance of the proteomic approach combined with other methods, the toxicological mechanism of aquatic toxins in marine organisms will be elucidated easily and conveniently [36].

4.4. Toxicological Studies for Environmental Stress

O. melastigma can serve as a marine fish model for assessing molecular responses to stresses in the marine environment. Hypoxia upregulates omTert expression via omHIF-1 in the liver and testis of nonneoplastic fish [22]. Significant changes were observed in the transcription, translation, cell proliferation, and apoptosis level of TERT in the liver of O. melastigma exposed to hypoxic conditions for 3 months [16]. Anoxic conditions can increase the expression of Tert in the liver and testicular tissue of O. melastigma, which is mediated by anoxia-induced factor-1 [24]. The expression of leptin receptor gene exhibits tissue specificity when exposed to hypoxia, and this gene was identified as a sensitive marker gene for a hypoxic environment [22].

The experimental animal, marine medaka, is suitable for studying the mechanism of hypoosmoregulatory. Studies show that branchial omNkcc1a mRNA levels are induced significantly with an increase in environmental salinities. Salinity-dependent expression of Nkcc1a is in the branchial mitochondria-rich (MR) cells of O. melastigma, which suggests a critical role in hypoosmoregulatory endurance of this fish [32]. Studies have also indicated that O. latipes exhibited better hypoosmoregulatory ability, while O. melastigma exhibited better hyperosmoregulatory ability. These results support the hypothesis that the lowest branchial NKA activities of these two species were found in the environments that have similar salinities to their natural habitats [7].

5. Conclusion

O. melastigma have biological characteristics such as small size, high fecundity, short life cycle, sexual reproduction, and distinctive life stages that would allow their use as a marine fish model. Additionally, their ease of cultivation facilitates the use of O. melastigma in independent laboratories. The availability of knowledge on their sensitivity towards inorganic and organic compounds and the increasingly complete knowledge on their genes and proteins will also enhance the potential of O. melastigma as suitable models in marine aquatic ecotoxicology and toxicogenomics. Researchers have demonstrated the potential application of O. melastigma as an ideal marine test fish for marine pollution assessments and ecotoxicological studies of organic chemicals, inorganic chemicals, microorganism, and environmental stress in relation to cardiac toxicity, hepatotoxicity, neurotoxicity, ecotoxicity, immunotoxicity, and so forth.

O. melastigma can also serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment. O. melastigma showed high tolerance to PAHs and strong sensitivity to metal stress compared to other species. The heart elongation of O. melastigma embryo and omChgh and omLepr expression are used as potential biomarkers to indicate PAH mixtures contamination or an oil spill, estrogenic chemicals in the marine environment, and growth and/or endocrine disruption in this marine fish, respectively. The expression of the leptin receptor gene, which was identified as a sensitive marker gene for hypoxia environment, exhibits tissue specificity in O. melastigma. We may be able to develop biomarkers for more specific adverse effects that can be used for both ecotoxicology and human health risk assessment because of the high degree of evolutionary conservation among vertebrates [92].

Although some toxicological research has been conducted using this small fish species as a model, there is still much to be studied. Fortunately, transcriptome analyses and proteomic approaches, along with new methodologies in O. melastigma, such as gene knockdown, gene overexpression, gene chip technology, second-generation high-throughput sequencing technology, RNA-Seq, and DGE technology, can be expected to further accelerate the knowledge of the toxicological mechanisms of aquatic toxins in marine animals in the future. Demonstrating and understanding toxicity mechanisms in O. melastigma that are common between humans and fish and wildlife are necessary if we are to integrate findings from laboratory and ecotoxicology studies with human health risk assessment.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


This work was supported by the National Natural Science Foundation of China (21207127, 21277137). The authors thank Qiansheng Huang and Kevin Francesconi for critical discussion and correction of the manuscript.


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