## 3 Results
### Basal Levels of M. Japonicus VTG Expression in Multiple Tissues  
The basal expression levels of VTG were observed in six tissues (gill, hepatopancreas, muscle, ovaries, heart, and stomach) (Figure 3). The fold change of each VTG mRNA expression in each tissue was based on the VTG expression level in gill tissue (when the set value of the gill = 1). The _M. japonicus_ VTG gene expression was expressed differentially in all investigated tissues. The high level of _M. japonicus_ VTG expression was evident in the ovaries and hepatopancreas, whereas relatively low levels were observed in the gill, muscle, stomach, and heart. The _M. japonicus_ VTG mRNA expression varied significantly between the crab gill and ovaries, or hepatopancreas (\(p<0.05\)). Figure 2: Phylogenetic tree of _M. japonicus_ VTG gene constructed by neighbor-joining analysis (bootstrap value 1000). The numbers at the nodes are the percentage bootstrap values. GenBank accession numbers for VTGs are: _M. japonicus_ VTG (Acc **), _Ericoheir sinensis_ (KC699915), _Longpotamon honanense_ (AKI23633), _Callinectes sapidus_ vitellin precursor (AEI59132), _C. sapidus_ VTG (ABC41925), _Portunus trituberculosisatus_ (AAX94762), _Scylla paramamosain_ (ACO36035), _Charybdis feriata_ (AAU93694), _Homarus americanus_ (ABO09863), _Upogebia major_ (BAF91417), _Penaeu sjaponicus_ (BAD98732), _Penaeu s chinensis_ (ABC86571), _Penaeu s monodon_ (ABB89953), _Penaeu s vannamei_ (AAP76571), and _Metapaneus ensis_ (AAN40700). **_Accession #s to be deposited in GenBank_. VTG Gene Expressions in M. Japonicus Ovaries and Hepatapancreas after DEHP, BPA, or Irgard Exposures  
After an exposure of 24 h, VTG mRNA expression generally increased in _M. japonicus_ hepatopancreas exposed to DEHP, BPA or irgard (Figure 4A). In particular, exposure to 1 and 30 \(\upmu\)g L\({}^{-1}\) DEHP significantly induced VTG gene expression (\(p<0.05\)) relative to the control (when the value of the control = 1). In the ovaries, transcriptional levels of VTG increased slightly after BPA exposure (Figure 4B). In contrast, irgard slightly decreased VTG expression at a relatively low concentration of 1 \(\upmu\)g L\({}^{-1}\), although VTG response was somewhat induced at a relatively high irgard concentration of 30 \(\upmu\)g L\({}^{-1}\). After exposure to different concentrations of DEHP, VTG gene expression was significantly upregulated at 10 (2.8-fold) and 30 \(\upmu\)g L\({}^{-1}\) (3.3-fold) of DEHP in a concentration-dependent manner (\(p<0.05\)). The VTG gene response increased in hepatopancreas exposed to DEHP and BPA for 96 h, whereas a decrease in VTG expression resulted from irgard exposure (Figure 5A). The up-regulation of VTG gene expression was significantly different at a relatively high concentration of BPA (30 \(\upmu\)g L\({}^{-1}\)) (\(p<0.05\)). In addition, DEHP exposure significantly induced the up-regulation of the VTG expression in the hepatopancreas. The highest VTG expression was observed at 30 \(\upmu\)g L\({}^{-1}\) (4.9-fold) DEHP (\(p<0.01\)). In contrast, after 96 h of irgard exposure, VTG decreased in a dose-dependent manner. In the _M. japonicus_ ovaries, BPA and DEHP exposure significantly upregulated transcriptional levels of VTG following exposure to all concentrations (\(p<0.05\)) (Figure 5B). Similar to the VTG gene response in the hepatopancreas, the VTG expression in the _M. japonicus_ ovaries downregulated after irgard exposure for 96 h. The highest level of VTG expression was found at 30 \(\upmu\)g L\({}^{-1}\) (5.1-fold) of DEHP (\(p<0.01\)). Figure 3: Basal expression of _M. japonicus_ VTG transcripts among different tissues (Gi, gill; Hp, hepatopancreas; Ms, muscle; Ov, ovaries; Ht, heart; St, stomach). Each tissue was collected from 10 mud crabs. The experiments were performed in triplicate. The data are represented as the mean \(\pm\) SD. The relative expression levels of each gene in each tissue were compared with the respective gene expression levels in gills (relative to set value of gill = 1).