Fish and Crayfish ToxicologyView this Special Issue
Development of a Promising Fish Model (Oryzias melastigma) for Assessing Multiple Responses to Stresses in the Marine Environment
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.
Estuaries and coastal waters are contaminated by high levels of anthropogenic pollutants , 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 [2–4].
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 . Kif7 is not expressed in O. melastigma but is highly expressed in the brain of zebrafish, which is a freshwater fish . 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 . Exposure to perfluorooctane sulfonates (PFOS) shortened the hatching time and increased the hatching rate of O. melastigma but had the opposite effects in zebrafish [8–10]. 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 .(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 . 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 .(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 . 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 .(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 .(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 . O. latipes and O. melastigma share completely identical peptide sequences but bear very different glycan structures . This phenomenon suggests that further exploration of the marine medaka genome and proteome is needed .
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 . A batch of organ-specific molecular markers have also been identified, such as the makers for brain, eyes, heart, liver, and muscle . 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.  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 . 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 . 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].
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 .
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 . 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 , 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 .
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) . 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 . 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 . PCNA detection can also reflect the spatial and temporal characteristics of O. melastigma embryonic development .
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 . 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 . 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 .
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).
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 . 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 .
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 . 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. .
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 . 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 .
PFOS has estrogenic activity and endocrine-disruptive properties that elicit transcriptional responses on POPs-related pathways in a stage-specific manner [61–63]. The marine biological toxicity of PFOS was systematically studied by Dong et al. using O. melastigma [10, 23, 26–28]. 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 . 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 .
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 .
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 . In the toxicity assessing, double-wall carbon nanotubes (DWNTs) (10 mg/L) following ultrasonic treatment inhibited the growth of O. melastigma larvae .
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 . 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 . 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].
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 .
ISH showed that Vibrio parahaemolyticus would induce the expression of hepcidin genes in the nuclei and cytoplasm of liver cells of O. melastigma . 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 . 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 .
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 . 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 . 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 . 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 .
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 . 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 .
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 .
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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