**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 [36–42]. 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

molecules in crustaceans [45]. The hepatopancreas acts as an essential metabolic center in crustaceans 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. monodon* [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. The *Mj-HSP60* expression was downregulated in most crabs after 7 days of DEHP exposure due to reducing cellular immune protection, although expressions of the detoxifying *Mj-HSP67B2* gene [51] were continuously upregulated in DEHP-treated groups compared to the control. *HSP67B2* is significant both in detoxification and in anti-oxidative stress systems, as well as immune protection [26,27,51]. For instance, in *P. trituberculatus*, an important marine and aquaculture species, *Mj-HSP60* displays differential expression patterns in response to environmental salinity stress and exhibits upregulation in the gills [52].

Likewise, *L. vannamei HSP60* mRNA is regulated between 4 and 32 h after the injection of bacteria [53]. *HSP70* is upregulated 24 h after copper exposure in the zebra mussel *Dreissena polymorpha* and midge larvae *Chironomus tentans* [54,55]. In addition, *HSP70* expression is dramatically induced, owing to microbial pathogens in the Chinese shrimp *Fenneropenaeus chinensis* [56]. However, little is known regarding the response of *HSP60* to xenobiotics and stresses in invertebrates such as the sea anemone (*Anemonia viridis*) [29], *D. polymorpha* [54], and the white shrimp (*Litopenaeus vannamei*) [57]. The limited study reported that HSP67B2 acts like a rhodanese homolog with a single RHOD domain, is characterized from the housefly M. domestica, and plays potential roles under oxidative stress conditions [57]. *M. domestica*, and plays potential roles under oxidative stress conditions [51]. In crustaceans, *HSP* expression studies have been conducted on the Asian paddle crab *Charybdis japonica,* with exposure to EDCs (bisphenol A and 4-nonylphenol) [16,58]. To date, this is the first nucleotide and protein sequence information reported regarding *Mj-HSP60* and *Mj-HSP67B2* in the crab species *M. japonicus*. Our gene expression results revealed the potential involvement of the two HSPs in the immune system of crabs. This study highlights the potential importance of these molecules in crustaceans, protecting cells against pathogens as well as in severe cellular and environmental stress conditions.

**Author Contributions:** Conceptualization, K.P., W.-S.K. and I.-S.K; methodology, K.P., W.-S.K. and I.-S.K; formal analysis, K.P., W.-S.K. and I.-S.K; investigation, K.P., W.-S.K. and I.-S.K; resources, K.P., W.-S.K. and I.-S.K; writing—original draft preparation, K.P., W.-S.K. and I.-S.K; supervision, K.P., W.-S.K. and I.-S.K; project administration, I.S.K; funding acquisition, K.P., I.-S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Research Foundation of Korea, South Korea, which is funded by the Korean Government [NRF-2018-R1A6A1A-03024314] and [NRF-2019-R1I1A1A-01056855].

**Conflicts of Interest:** The authors declare that they have no conflicts of interest.
