## 4 Discussion  
Cellular responses to stressors are an evolutionary, ubiquitous, and essential mechanism for cell survival. HSPs are known as extrinsic chaperons that are involved in certain cellular processes, such as germ cell differentiation, reproduction, development, thermoprotection, mammalian autoimmune defense, and toxic stress responses, and they have even been regarded as a potential marker of environmental stress [36; 37; 38; 39; 40; 41; 42]. HSPs are found in all eukaryotes and are identified based on their size, molecular weight, and functions. _HSP60_, _HSP70_ and _HSP90_ are highly conserved genes and are stress-inducible and multigenic [43]. It has been observed that the _HSP60_ and _HSP70_ family members play significant roles in cell survival, stress, and thermal tolerance in response to various heat shocks [44]. Here, we studied two stress-related genes, _Mj-HSP60_ and _Mj-HSP67B2_, and conducted expression analysis in different tissues of _M. japonicus_ after treatment with the xenobiotic DEHP. _Mj-HSP60_ and _Mj-HSP67B2_ were highly expressed in the gonad and hepatopancreas, respectively. In addition, these molecules are moderately expressed in the gills, muscle, heart, and stomach. Our findings are consistent with the results of an earlier study showing that the hepatopancreas is the main source of immune  
Figure 4: Expression analysis of _HSP60_ in the (**A**) gill and (**B**) hepatopancreas of _Macrophthalmus japonicus_ exposed to 1, 10, and 30 ug L\({}^{-1}\) DEHP after 1, 4, and 7 days. Values were normalized against _GAPDH_. Bars indicate the standard deviation of the mean. Statistically significant differences are represented by asterisks as \({}^{*}P<0.05\) and \({}^{**}P<0.01\), compared to controls (control ratio value = 1). Figure 5: Expression analysis of _HSP67B2_ in the (**A**) gill and (**B**) hepatopancreas of _Macrophthalmus japonicus_ exposed to 1, 10, and 30 ug L\({}^{-1}\) DEHP for 1, 4, and 7 days. The values were normalized against _GAPDH_. Bars indicate the standard deviation of the mean. Statistically significant differences are represented by asterisks as \({}^{*}P<0.05\) and \({}^{**}P<0.01\) as compared to controls (control ratio value = 1). molecules in crustaceansens [45]. The hepatopancreas acts as an essential metabolic center in crustaceansens and performs versatile roles in defense systems, detoxification, reactive oxygen species production, digestion, absorption, and nutrient secretion. Owing to the critical importance of the hepatopancreas in detoxification and immunological activities, it is highly sensitive to xenobiotic exposure. Similarly, increased upregulation of _HSP90_ was noted in the hepatopancreas of _P. mondon_[46]. In addition, three HSPs, namely _MrHSP60, MrHSP70_ and _MrHSP90_, are constitutively expressed in _M. rosenbergii_ during pathogenic infections involving different tissues [47]. Related results were obtained in the Pacific oyster _Crassostrea gigas_, which exhibits highly upregulated _HSP70_ expression in the gill tissue after exposure to Cu2+ [48]. DEHP has been shown to alter the expression of HSPs in _Chironomus riparius_[49; 50]. In this species, _HSP40_ and _HSP90_ mRNA expression levels increased under various DEHP concentrations for 24 h, which caused morphological deformities [49]. In addition, _HSP70_ showed increased expression when treated with low doses of DEHP. Overall, our results indicated that two HSPs, _Mj-HSP60_ and _Mj-HSP67B2_, in _M. japonicus_ are constitutively expressed, owing to DEHP exposure at day 1. Hence, these molecules can be considered as upregulated responses of xenobiotic levels for the early exposure time in _M. japonicus_ crabs. However, at long-term exposure for 7 days, there are different expression patterns between the _Mj-HSP60_ and the _Mj-HSP67B2_ transcripts.