## 4 Discussion
These results strongly emphasize the suitability of using the VTG gene as a biomarker to assess the environmental presence of DEHP. Since the use of rigarol as an algaecide has increased, rigarol has been detected frequently in coastal environments [48]. However, there is not enough information about the potential effects of rigarol exposure on the endocrine system of crustaceans. In our study, the responses of VTG gene expression following exposure to rigarol were different. After a 24 h exposure to rigarol, the _M. japonicus_ VTG gene was expressed in a dose-dependent manner. The up-regulation of VTG relative to the control decreased in the hepatopancreas and ovaries when exposed to rigarol for 96 h. VTG gene expression recoveredafter 7 d of irgarol exposure. These results are the first report of the endocrine effects of irgarol exposure in crustaceans. A recent study reported that irgarol exposure induced changes in thyroid endpoints in the inland silverside fish (_Mentidia beryllina_) under salinity and temperature changes [28]. In _A. tenuis_, exposure to irgarol for 7 d resulted in body color changes and reduced transcriptional expression of the HSP90 gene [29]. In a previous study, irgarol exposure induced expression changes of digestion related genes and chitinase genes [17]. Thus, VTG gene expression was affected by environmental chemical pollutants such as BPA, DEHP, and irgarol. After EDC exposures, the expression pattern of the _M. japonica_ VTG gene was correlated with short-term EDC exposure in the hepatopancreas (\(p=0.791\)) and long-term EDC exposure in ovary tissue (\(p=0.927\)). In conclusion, our results demonstrate that _M. japonica_ is sensitive to several chemical pollutants, as measured by changes of the VTG gene expression in the hepatopancreas and ovaries. The results suggest that the molecular balance of VTG expression levels of intertidal mud crabs appear to be affected by EDCs such as BPA, DEHP, and irgarol at even relatively low concentrations of 1 ug L\({}^{-1}\). The expression levels of the VTG gene are important factors to be considered for evaluating xenoestrogenic responses. The change of the VTG responses are related with disturbed processes of reproductive physiology such as egg yolk generation in _M. japonica_, because of the up-regulation of VTG expression in the ovaries after EDC exposures. The crab hepatopancreas is an organ for VTG synthesis and storage of energy accumulation, which can support lipid transportation for oogenesis [49]. EDC exposure also impaired the molecular balance of the VTG level through the disturbance of energy metabolisms in _M. japonica_s hepatopancreas. Up or down-regulation of VTG gene may result in sensitive defense activities of _M. japonicus_ crabs to potential EDCs pollutants, given the major function of VTG in stabilizing the endocrine process and triggering lipid uptake for energy storage during crustacean embryogenesis. Our findings suggest that _M. japonica_s VTG may be a useful key to estimate the endocrine effects of antifoulants such as irgarol, BPA, and DEHP. In further studies, we will need to demonstrate the effects of one or more combined exposures of these EDCs in VTG protein synthesis. The following are available online at [[http://www.mdpi.com/2076-3417/9/7/1401/s1](http://www.mdpi.com/2076-3417/9/7/1401/s1)]([http://www.mdpi.com/2076-3417/9/7/1401/s1](http://www.mdpi.com/2076-3417/9/7/1401/s1)), Figure S1: The relative weight (g) in _M. japonicus_ crabs exposed to each dose of BPA, DEHP, and irgarol for 7 d. Figure S2: Transcriptional expression of VTG gene in _M. japonicus_ hepatopancreas exposed to each dose of BPA, DEHP, and irgarol for each exposure period. Figure S3: Transcriptional expression of VTG gene in _M. japonicus_ ovary exposed to each dose of BPA, DEHP, and irgarol for each exposure period. Conceptualization: [PERSON] and I.-S.K. ; Methodology: [PERSON] and I.-S.K. ; Formal Analysis: [PERSON], H.J. and [PERSON]-K.K. ; Investigation: [PERSON], [PERSON]-K.K. and H.J. ; Resources: [PERSON] and I.-S.K.