## 4 Discussion  
To understand VTG gene responses following exposure to environmental chemical pollutants, the cDNA sequence of _M. japonicus_ VTG was identified and analyzed using phylogenetic analysis. The three functional domains of the crustacean VTG gene are the lipoprotein domain at the N-terminus (LPD_N), the DUF1943 domain, and the von Willebrand factors type-D domain (VWD) [2; 23; 33]. In this study, we characterized the _M. japonicus_ VTG with 3700 bp ORF, which encodes a protein of 1202 amino acids. The deduced protein region of the _M. japonicus_ VTG was predicted as the LPD_N of VTG, which is a conserved region found in various lipid transport proteins [34]. In multiple alignments of the amino acid sequences, _M. japonicus_ VTG showed high homology with the protein from _E. sinensis_. The phylogenetic analysis revealed two clusters representing shrimps or crabs among the crustaceans. In these clusters, three clades were represented by crabs, including _M. japonicus_, other crabs, and _H. americanus_. In this study, it was observed that _M. japonicus_ mainly synthesizes VTG in the hepatopancreas and ovaries. In crustaceans, the hepatopancreas is a critical storage system of nutrients and energy in the forms of carbohydrates, proteins, and lipids, which are essential in regulating an energy balance during the reproductive cycle [35]. The hepatopancreas was also reported to be the main source of VTG activity in decapod species, although VTG expression was also found in the ovaries [5]. The primary expression site of the VTG gene in crustaceans has been a controversial topic. VTG production was only observed in the hepatopancreas of _Macrobrachium rosenbergii_ (freshwater prawn), _Cherenkov quadricarinatus_ (red claw crayfish), _Pandalus hypsinotus_ (coonstired shrimp) and _P. trittuberculatus_[2; 36; 37; 38]. In contrast, the VTG gene was expressed in both the hepatopancreas and ovaries of _Upogeba major_ (mud shrimp) and marine crabs such as _S. paramamosain_, _Carcinus maenas_ (green crab), _C. sapidus_, _C. japonica_ and _E. sinensis_[9; 23; 39; 40; 41]. We identified that the major sites for VTG synthesis in _M. japonicus_ are the hepatopancreas and ovaries. The endocrine disruptor BPA is presently considered a ubiquitous emerging pollutant in aquatic environments. In this study, exposures to BPA triggered up-regulation of _M. japonicus_ VTG expression in the hepatopancreas and ovary tissues for different exposure periods. BPA exposure increased VTG levels in various fish, such as fathead minnows, goldfish, and bluegills [42]. BPA toxicity at lower concentrations (<15 ug L\({}^{-1}\)) showed induced ovarian expression of a steroidogenic gene involving VTG in _G.arus_[43]. In addition, different modulations of VTG were reported in two VTG genes in the gilthead bream _Sparus aurata_ and _G.arus_[43; 44]. These results indicate that induction of VTG following BPA exposure is a major response to the stressful effects of endocrine-disruption. The endocrine effects of DEHP, a distinctive EDC, are noticeable in marine ecosystems where high concentrations of DEHP have been found in river estuaries and coastal areas [45]. In our study, DEHP exposure generally increased _M. japonicus_ VTG expression in the tested tissues. The up-regulation of VTG gene expression in _M. japonicus_ was more sensitive to DEHP exposure than to exposure to the other chemicals tested. In addition, transcriptional responses of the VTG gene in the hepatopancreas were found to be significant under relatively low concentrations of DEHP for the different exposure times, whereas VTG was expressed in the ovaries under all concentrations of DEHP tested. [PERSON] reported that DEHP exposure induced time- and concentration-dependent increases of VTG levels in _C. gariepinus_[17]. Long exposure to low doses of DEHP reduced oocyte maturation [46] and increased the levels of hepatic VTG transcripts in _G.arus_[16]. In addition, VTG levels in marine medaka livers increased after DEHP exposure [47].