Review : ToWARD The eSTABLIShMenT oF The TooLS FoR MonIToRInG coASTAL envIRonMenTS UTILIZInG Gene ReSPonSe In oRYZIAS FISHES

Southeast Asia is known as a center of biodiversity of the earth. As economic growth of this region is remarkable, conservation of biodiversity is a top priority issue. For this purpose, it is important to monitor the environmental condition in effective ways. We are trying to detect pollutants in coastal and estuary waters through the expression level of pollutant-responsive genes of rice fishes of the genus Oryzias, widely distributed in Asia. Japanese medaka O. latipes is a useful model because whole genome sequence is available. Javanese medaka O. javanicus and Indian medaka O. dancena, both of which are widely distributed in Southeast Asia and adaptable to seawater, are also potential models. One possible method for pollution monitoring is the use of transgenic fish bearing artificial gene construct containing the pollutant-responsive promoter and a reporter gene. For example, transgenic strains that can detect estrogen-like substances have already been established. It is also probable to detect pollutants by quantifying mRNA or proteins expressed from the pollutant-responsive gene. In any case, the most important point is to identify the gene that responds to specific pollutants. Comprehensive transcriptomic analyses are powerful tool for this purpose. Organotin-responsive genes are being screened at present.


InTRoDUcTIon
Southeast Asia, containing Indochina and Malay Peninsula and many large and small islands with different geological background, is known to be "the center of biodiversity".For terrestrial ecosystems, specific fauna and flora reflecting the geological history of each area have been reported.Southeast Asia also has a variety of coastal and marine environments, where unique ecosystems with high biodiversity, such as coral reefs, mangroves, and seagrass beds, are observed.On the other hand, Southeast Asia is a region whose economy is growing rapidly.Thus, special care must be taken for conservation of the biodiversity, the precious treasure of the earth.
To minimize the negative impact on the ecosystems caused by economic growth, it is important to monitor the status of the environments effectively and continuously.In the case of the monitoring of the marine environment, one way is to measure the level of pollutants in water by chemical analyses.However, chemical analyses of water require expensive equipment and reagents as well as skilled techniques.In addition, it is difficult to evaluate the environmental status correctly from the analysis on limited amount and number of samples.
A possible solution of such problems is the use of bioindicators.Blue and green mussels, for example, have been utilized as the sample for chemical analyses of pollutants (Goldberg et al., 1978;Monirith et al., 2003).As the mussels attach themselves to a substrate and do not move frequently, the result of analyses indicates the pollution status of the point where the mussels were collected.In addition, the mussels bio-concentrate the pollutants and thus the data represent the long-term status of the sampling points.Their characteristics to bio-concentrate pollutants also increase the sensitivity of the analysis.Thus, the use of the mussels has become popular for pollution analyses.However, even using mussels, problems accompanying chemical analyses still remain.
We are trying to detect the pollution through the expression of genes that respond to pollutants.For this purpose, possible models are small fish species, of which genomic information is abundant.Unlike direct chemical analyses of pollutants, the small size is not an obstacle for mRNA analyses; it is rather advantageous as it is easy to catch and handle such fish.Here we propose that small medaka fishes (rice fishes) are ideal models for environmental monitoring through gene expression.

MeDAKA FISheS
Medaka fishes of the genus Oryzias inhabit fresh and salt water in Asia (Parenti 2008;Roberts, 1998).About 20 species have been recorded in Asia; all species are small, even adult fish are usually less than 4 cm.They prefer shallow still water or slow stream near shore or bank and often found in rice paddy, ponds, irrigation channels and small creeks.Most species are surface swimmer, and relatively large eyes at sides of the slightly flattened head are easily recognizable when looking down the swimming fish from above.
Another two species, Javanese medaka O. javanicus (Fig. 1) and the Indian medaka O. dancena are becoming popular especially in the field of environmental sciences.For scientists in Southeast Asia, these two species are easily obtainable because of their wide distribution in the region (Parenti, 2008;Roberts 1998;Yusof et al., 2012).They can adapt to both fresh and salt water (Inoue andTakei, 2002, 2003) although the former prefers hyperosmotic environment and the latter hypoosmotic (Yusof et al., 2012).Therefore, these species can be used for experiments in seawater.

PoLLUTAnT DeTecTIon USInG TRANSGENIC FISH
"Transgenic fish" is the fish to which foreign gene(s) are introduced.It is commonly used to examine regulation and function of genes.It also becomes a powerful tool to detect pollutants in water by introducing a construct of pollutantresponsive promoter with appropriate reporter gene.The possibility of the use of transgenic fish has been proposed in 1990s (Inoue et al., 1992;Kinoshita et al., 1994).Recently, one of the authors of this article (MK) established a transgenic Japanese medaka strain bearing the construct of the choriogenin H (Chg-H) promoter, which is responsive to estrogens, with green fluorescence protein (GFP) gene; and demonstrated that it can detect estrogens in the water in a dose-dependent manner (Kurauchi et al., 2005;2008).Using the strain, we surveyed the pollution status of some areas in Thailand and Malaysia (Kinoshita et al., 2010).As Japanese medaka can adapt to seawater after adequate acclimation process, it is possible to use it for the assay of estrogenic substances in seawater.
As the endogenous Chg-H promoter was used in this transgenic strain, it is expected that the promoter of the transgene is regulated by the same manner as the original Chg-H gene.It implies that the transgenic strain detect all the substances that activate the Chg-H gene even if the pollutants were unknown substances.In addition, it is also visualize the process of bio-concentration of pollutants by the host fish.So, it may be possible to detect low concentration of pollutants in the case of long-term exposure.
For the use of the transgenic strains, the simplest way is the direct exposure to the water samples.The pollution status becomes visible just putting the fish into the water and monitor the fluorescence by a fluorescent microscope.However, as transgenic fish are genetically modified organisms (GMOs), it may cause serious problems on natural ecosystem if they escaped to natural environment, and thus the use of transgenic animals in non-enclosed environment is pro hibited in most countries under The Convention on Biological Diversity.Therefore, we do not intend in situ use of the transgenic strains in the field.Instead, the sample water should be brought to the laboratory where transgenic fish are maintained.Transportation of water sample is not a problem when the sampling point is close to the laboratory for transgenic assay.However, when it is distant, transportation of water is costly, and the chemical composition may change during transportation.To solve these, mini-columns such as a Sep-Pak C-18 cartridge are used to trap the pollutants (Kurauchi et al., 2005;Kinoshita et al., 2010).The use of the columns reduces the volume of samples and the possibility of the degradation of the components.The use of the column is also advantageous as the pollutants can be concentrated.It increases sensitivity of the assay when the concentration of the pollutants is low.

DeTecTIon oF exPReSSIon WIThoUT USInG TRAnSGenIcS
Transgenic strains are very convenient tools for detection of pollutants.However, as mentioned above, their use is limited to the laboratory where the biological containment is possible.Thus, we are also considering the way of pollutant detection without using transgenics.In pollution-detecting transgenic fish, a foreign reporter gene (e.g., GFP) is used to make the detection of gene expression easier but the promoter of the transgene is derived from the host genome sequence.It means that, when GFP is expressed in the transgenic fish, the transcription of the original gene also occurs.For example, the Chg-H-GFP transgenic fish express fluorescence, the transcripts of the original Chg-H gene also increases.By detecting Chg-H mRNA, the estrogenic pollutants can be detected.In fact, Yu et al. (2006) and Chen et al. (2008) reported that Chg-H mRNA increased after estrogen exposure in Javanese and Indian medaka, respectively.Thus, the detection of mRNA is a possible solution although the equipment for quantitative real-time PCR is necessary.
Another possible solution is the detection of translation products, i.e., proteins.For quantitative detection of proteins, enzyme-linked immunosolbent assay (ELISA) may be useful as it does not special equipment except for a microplate reader.For example, ELISA for Chg-H detection has been established in several fish species to detect estrogenic activities in environment (Fujita et al. 2004;Prakash et al. 2007;Brander et al. 2012)

ScReenInG oF PoLLUTAnT ReSPonSIve GeneS
For establishing the systems to detect pollutants mentioned above, the most important point is to identify the gene that respond to the pollutant.Although some pollution-responsive genes have been discovered, for example, metallothioneins for metals (Inoue et al., 1992;Kinoshita et al., 1994;Woo et al., 2006), cytochrome P450 (CYP) proteins for many organic pollutants (see Uno et al., 2012 for a review).For discovery of new genes respond to pollutants, comprehensive transcriptomic analyses may be the most efficient way.For such analyses, several techniques have been used such as differential display, (Liang and Pardy, 1992), microarray (Schena et al., 1995), high coverage expression profiling (HiCEP) (Fukumura et al., 2003), but the "next generation" sequencing (Mardis, 2008), the new technologies rapidly developed recently, will accelerate the research further.Screening of the genes that respond to organotin compounds, which have been included in antifouling paints (Harino et al., 2013), is in progress.We expect that a variety of systems that can detect various pollutants will be established by using appropriate model species such as Oryzias fishes, which are easily obtainable and culturable, and gene expression profiling techniques.

AcKnoWLeDGMenTS
This study is supported by Asian CORE program of Japan Society for the Promotion of Sciences (JSPS).It is also supported by JSPS and LIPI under Japan -Indonesia Research Cooperative Program.

Figure 1 .
Figure 1.Javanese medaka Oryzias javanicus.It was obtained from National Bioresource Project Medaka (NBRP) in National Institute for Basic Biology, Okazaki, Japan.Most Oryzias species have relatively flat back, perhaps reflecting their surface swimming habitat, and a small dorsal fin locates near the tail fin.

Figure 2 .
Figure 2. Principle of the detection of the expression of a pollutant-responsive gene in transgenic and wild fish.Choriogenin H (Chg-H) gene that responds to estrogen-like endocrine disrupting chemicals (EDCs) was used as an example.A, transgene consisting of Chg-H promoter and green fluorescence protein (GFP), which was introduced into transgenic fish.The translated protein is detected by fluorescence.B, normal (wild type) gene.The transcribed mRNA and translated protein are detected by quantitative reversetranscription PCR (q-PCR) and enzyme-linked immunosolvent assay (ELISA), respectively.