*2.3. DEHP Exposure Experiments*

DEHP solutions were made from a solid compound (99%, Junsei Chemical Co. Ltd., Tokyo, Japan). For preparation of a 10 mg L−<sup>1</sup> stock solution of DEHP, we dissolved DEHP in 99% acetone at room temperature. This stock solution was diluted with seawater for DEHP solutions with concentrations of 1, 10, and 30 μg L−1. A concentration of <0.5% acetone was used as a solvent control. For the DEHP exposure experiments, a total of 40 crabs were randomly divided into four experimental groups (1, 10, and 30 μg L−<sup>1</sup> DEHP solutions and solvent control). Ten crabs were placed in each glass tank and exposed to one of the three doses of DEHP over days 1, 4, and 7, respectively. Three individuals were selected for tissue extraction at each time interval from the DEHP treatment and control groups. Food was not provided for the crabs, but seawater with equivalent concentrations of DEHP was added every day during the experiments. The experiments were conducted in triplicate with independent samples.

### *2.4. Total RNA Extraction and cDNA Synthesis*

Crab gill and hepatopancreatic tissues were acquired from the exposure and control groups. Total RNA was extracted using TRIzol reagen<sup>t</sup> (Life Technologies, Rockville, MD, USA) with Recombinant DNase I (Takara, Otsu, Japan) according to the manufacturers' protocols. The concentration of each RNA sample was measured using a Nano-Drop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was checked by 1% agarose gel electrophoresis. Single-stranded Complementary Deoxyribonucleic Acid (cDNA) synthesis was carried out with 1000 ng of total RNA using an oligo dT primer (50 μM) for reverse transcription in 20 μL reactions (PrimeScript ™ 1st strand cDNA synthesis kit, Takara) according to the manufacturer's protocol.

### *2.5. Gene Expression Analysis Using Quantitative Reverse-Transcription PCR (RT-PCR) Amplification*

To confirm the expression patterns of *Mj-HSP60* and *Mj-HSP67B2* in various tissues of *M. japonicus*, and in the control and DEHP-exposed samples, quantitative RT-PCR was carried out on an ExicyclerTM96 instrument (Bioneer, Daejeon, Korea). Each reaction was conducted in a final volume of 20 μL containing 10 μL of Accuprep ®2 × Greenstar qPCR Master Mix (Bioneer, Daejeon, Korea), 6 μL of DEPC-treated water, 0.5 μL each of sense primer and antisense primer (10 pM), and 3 μL of 30-fold diluted cDNA sample as a template. Quantitative RT-PCR of two genes was carried out for 40 cycles of 95 ◦C for 15 s and 60 ◦C for 45 s using the following primer pairs: *Mj-HSP60* forward 5-CCCTGAAGGATGAGCTTGAG-3 ; *Mj-HSP60* reverse 5-GCTGGGATGATGGA CTGAAT-3 ; *Mj-HSP67B2* forward 5-GAGCCGCGGTAGATTCTAT G-3 ; *Mj-HSP67B2* reverse 5-CTGGACAAGGAGGGTTTCAA-3; Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) forward 5-TGCTGATGCACCCATGTTT G-3; and *GAPDH* reverse 5-AGGCCCTGGACAATCTCAA AG-3. Melting curves were determined by increasing the temperature from 68 ◦C to 94 ◦C. All samples were amplified in triplicate to ensure reproducibility. The relative expression level of each transcript was determined using *M. japonicus GAPDH* as an internal reference gene and employing the 2−ΔΔCt method [29].

### *2.6. M. japonicus Hsp Identification and Bioinformatics Analysis*

Two HSP genes (*Mj-HSP60* and *Mj-HSP67B2*) were identified by screening a previously generated 454 GS-FLX transcriptome database. Sequences were analyzed based on nucleotide and protein databases using the BLASTN and BLASTX programs (National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, MD, USA), respectively [30]. Two domains, the chaperonin-like super family of *Mj-HSP60* and Rhodonase (RHOD) superfamily of *Mj-HSP67B2*, were identified by PROSITE profile analysis [31]. A phylogenetic tree for the two HSPs was generated by the neighbor joining method using Molecular Evolutionary Genetic Analysis (MEGA X, Pennsylvania State University, State College, PA, USA) [32] with 1000 bootstrap replications.
