**E**ff**ects of di-(2-ethylhexyl) phthalate on Transcriptional Expression of Cellular Protection-Related** *HSP60* **and** *HSP67B2* **Genes in the Mud Crab** *Macrophthalmus japonicus*

### **Kiyun Park 1, Won-Seok Kim 2 and Ihn-Sil Kwak 1,2,\***


Received: 10 March 2020; Accepted: 14 April 2020; Published: 16 April 2020

**Abstract:** Di-2-ethylhexyl phthalate (DEHP) has attracted attention as an emerging dominant phthalate contaminant in marine sediments. *Macrophthalmus japonicus*, an intertidal mud crab, is capable of tolerating variations in water temperature and sudden exposure to toxic substances. To evaluate the potential effects of DEHP toxicity on cellular protection, we characterized the partial open reading frames of the stress-related heat shock protein 60 (*HSP60*) and small heat shock protein 67B2 (*HSP67B2*) genes of *M. japonicus* and further investigated the molecular effects on their expression levels after exposure to DEHP. Putative *HSP60* and small *HSP67B2* proteins had conserved HSP-family protein sequences with different C-terminus motifs. Phylogenetic analysis indicated that *M. japonicus HSP60* (*Mj-HSP60*) and *M. Japonicus HSP67B2* (*Mj-HSP67B2)* clustered closely with *Eriocheir sinensis HSP60* and *Penaeus vannamei HSP67B2*, respectively. The tissue distribution of Heat shock proteins (HSPs) was the highest in the gonad for *Mj-HSP60* and in the hepatopancreas for *Mj-HSP67B2*. The expression of *Mj-HSP60* Messenger Ribonucleic Acid (mRNA) increased significantly at day 1 after exposure to all doses of DEHP, and then decreased in a dose-dependent and exposure time-dependent manner in the gills and hepatopancreas. *Mj-HSP67B2* transcripts were significantly upregulated in both tissues at all doses of DEHP and at all exposure times. These results sugges<sup>t</sup> that cellular immune protection could be disrupted by DEHP toxicity through transcriptional changes to HSPs in crustaceans. Small and large HSPs might be differentially involved in responses against environmental stressors and in detoxification in *M. japonicus* crabs.

**Keywords:** di(2-ethylhexyl) phthalate (DEHP); crustacean; heat shock proteins (HSPs); gene expression; environmental risk assessment
