**3. Results**

### *3.1. Characterization of Mj-HSP60 and Mj-HSP67B2 in M. japonicus*

We identified two HSP genes (*Mj-HSP60* and *Mj-HSP67B2*) in our 454 GS-FLX transcriptome analysis [33] that were composed of 1360 nucleotides (nt) and 511 nt, which comprised open reading frames encoding 330 and 149 amino acids, respectively (Figures 1A and 2A). *Mj-HSP60* encoded a mature protein of 330 amino acids, 75 bp of 5 untranslated region (UTR) and 57 bp of 3 UTR, with a putative methionine initiation codon (ATG) beginning at 58 nt and a stop codon ending at 1224 nt. The SignalP Server (ExPASy) [34] predicted that the first 28 amino acids in the N-terminal region of the polypeptide chain would form a signal peptide sequence. We found that *Mj-HSP60* included a chaperonin-like super family main domain, whereas a RHOD superfamily motif was detected in *Mj-HSP67B2* (Figure 2A). The predicted molecular mass of the deduced amino acid sequence was 61 kDa, with an estimated isoelectric point (pI) of 5.74. *Mj-HSP60* was identified by a BLAST search of the National Center for Biotechnology Information (NCBI) non-redundant (nr) database. To understand the evolutionary position of the *Mj-HSP60*, we undertook phylogenetic analysis using another 11 species of crustaceans. As shown in Figure 1B, the phylogenetic tree consisted of two clades involving 12 crustacean species. The *Mj-HSP60* formed one main clade with other crabs (*Eriocheir sinensis, Scylla paramamosain,* and *Portunus trituberculatus*) and crayfish (*Cherax cainii*, *Cherax quadricarinatus,* and *Cherax destructor*). The other clade was composed of shrimp species (*Macrobrachium nipponense*, *Macrobrachium rosenbergii, Penaeus japonicus, Penaeus monodon,* and *Penaeus vannamei*). For clear annotation of *Mj-HSP67B2*, we examined the RHOD superfamily domain sequence (98 amino acids) using BLASTN searches of the nr database to detect sequences of other species with high similarity. We carried out pairwise alignment of *Mj-HSP67B2* using EMBOSS alignment (EMBL-EBI, Cambridgeshire, UK) [35] with sequences identified in BLAST searches. The results showed 35.9–72.8% sequence identity, 54.4–81.6% similarity, and 4.9–10.5% gap percentage when compared with *HSP67B2* from other species (Table 1). The *Mj-HSP67B2* sequence revealed considerable identity (72.8%), similarity (81.6%), and gap percentage (4.9%) with *Penaeus vannamei HSP67B2*. In addition, phylogenetic analysis of the *Mj-HSP67B2* was carried out using data from various arthropod species, owing to deficient genomic information regarding the *HSP67B2* in crustaceans (Figure 2B). The results showed that the two main clades were divided into Crustacea and Insecta, including mosquito and fly species. The *Mj-HSP67B2* showed the closest phylogenetic relationship to *Penaeus vannamei HSP67B2*. Given these results from analysis of phylogenetic and pairwise sequence alignment comparisons, our transcript sequence from the transcriptome database was identified as *Mj-HSP67B2*.

**Figure 1.** Genomic information of *Macrophthalmus japonicus HSP60* sequences identified in this study. (**A**) *Mj-HSP60* structure was represented using the BioEdit program (North Carolina State University, Raleigh, NC, USA). The open reading frame (ORF) of *Mj-HSP60* was predicted using the ExPASy tool and is shown as a black box. The yellow box indicates the chaperonin-like super family domain. (**B**) Phylogenetic analysis of *Mj-HSP60* with known *HSP60* sequences from 11 Crustacean species. The phylogenetic tree is based on amino acid sequences translated from *Mj-HSP60* ORF by the neighbor joining method (bootstrap value 1000) using MEGA X software. The numbers at the nodes represent the bootstrap majority consensus values for 1000 replicates. GenBank accession numbers are shown with scientific and common names of each species.

### *3.2. Expression Analysis of Mj-HSP60 and Mj-HSP67B2 in Various Tissues of M. japonicus*

To better understand the expression patterns of *Mj-HSP60* and *Mj-HSP67B2*, quantitative RT-PCR was carried out for six tissue sources (gill, hepatopancreas, muscle, gonad, heart, and stomach) of *M. japonicus*. The highest level of *Mj-HSP60* expression was found in the gonad, while *Mj-HSP67B2* was predominantly expressed in the hepatopancreas (Figure 3). In the gonad, *Mj-HSP60* was expressed 3.7-fold higher than *Mj-HSP67B2*. In contrast, *Mj-HSP67B2* exhibited a higher expression level than *Mj-HSP60* in the gills (1.7-fold) and hepatopancreas (3.1-fold). Relatively low levels of *Mj-HSP60* and *Mj-HSP67B2* expression were observed in the muscle, heart, and stomach tissues.

**Figure 2.** Sequence information for *Macrophthalmus japonicus HSP67B2* identified in this study. (**A**) An open reading frame (ORF) of *Mj-HSP67B2* was predicted using the ExPASy tool and is represented by a black box. The yellow box indicates a RHOD superfamily domain. (**B**) Phylogenetic analysis of *Mj-HSP67B2* with known *HSP67B2* sequences from seven Arthropoda species. The phylogenetic tree is based on amino acid sequences translated from *Mj-HSP67B2* ORF by the neighbor joining method (bootstrap value 1000) using MEGA X software. The numbers at the nodes represents the bootstrap majority consensus values for 1000 replicates. GenBank accession numbers are shown with the scientific and common names of each species.



Pairwise identity percentage was calculated using the EMBOSS alignment program.

**Figure 3.** Relative mRNA expression levels of *HSP60* and *HSP67B2* in various *Macrophthalmus japonicus* tissues. Six tissues were used in this experiment. Quantitative reverse-transcription (RT)-PCR was conducted in triplicate. Bars indicate the standard deviation of the mean. mRNA expression was normalized against *GAPDH*. Abbreviations: Gill (Gi), Hepatopancreas (Hp), Muscle (Ms), Gonad (Gn), Heart (Ht), and Stomach (St).

### *3.3. M. japonicus Mj-HSP60 Expression Changes after DEHP Exposure*

To confirm the effects of DEHP exposure on *Mj-HSP60* expression, we conducted quantitative RT-PCR analysis using mRNA acquired from the gill and hepatopancreas samples after exposure to DEHP for 1, 4, and 7 days. *Mj-HSP60* was expressed approximately 8.2-fold higher after exposure to 1 μg L−<sup>1</sup> DEHP (*P* < 0.01), 3.2-fold higher for 10 μg L−<sup>1</sup> (*P* < 0.05), and 9.4-fold higher for 30 μg L−<sup>1</sup> (*P* < 0.01) in the gill tissue on day 1 (Figure 4A). With the passage of time, expression levels gradually decreased in all DEHP concentration groups. By day 4, for the 10 and 30 μg L−<sup>1</sup> treatment groups, expression levels were restored to control levels. By day 7, *Mj-HSP60* expression levels were lower than those of the control. In particular, sharp decreases in expression levels were found in 10 μg L−<sup>1</sup> (0.3-fold) and 30 μg L−<sup>1</sup> (0.21-fold) (*P* < 0.05) groups. In the hepatopancreatic tissue, expression levels of *Mj-HSP60* exhibited an overall increased pattern compared to the expression levels in the controls on day 1 (Figure 4B). Expression levels significantly increased by 2.4-fold for 1 μg <sup>L</sup>−1, 2.6-fold for 10 μg <sup>L</sup>−1, and 2.9-fold for 30 μg L−<sup>1</sup> DEHP (*P* < 0.05). By days 4 and 7, *Mj-HSP60* expression levels returned to control levels for the 1 μg L−<sup>1</sup> group. In the 10 μg L−<sup>1</sup> DEHP group, *Mj-HSP60* expression decreased to <0.5-fold on day 4, and then recovered slightly toward that of control levels by day 7.

### *3.4. Variation in Expression of Mj-HSP67B2 after DEHP Exposure in M. japonicus*

Expression of *Mj-HSP67B2* consistently increased in the gill and hepatopancreatic tissues for 4 days after DEHP exposure at all concentrations (Figure 5). After a peak in expression at day 4, *Mj-HSP67B2* levels somewhat decreased. These *Mj-HSP67B2* expression patterns were found in the two tissues, regardless of DEHP exposure concentration. Although expression levels of *Mj-HSP67B2* decreased after day 4, the expression was still maintained in the gill tissue at higher levels than those of the controls for all concentration groups, except on day 7 (0.86-fold) for the 1 μg L−<sup>1</sup> group (Figure 5A). Similar changes in *Mj-HSP67B2* expression levels were noted in the hepatopancreas tissue. *Mj-HSP67B2* was strongly overexpressed for 4 days in response to exposure to all concentrations of DEHP (*P* < 0.05), and its expression levels displayed dose-dependent and time-dependent increases for 4 days (Fig. 5B). The highest expression levels were noted on day 4 in each DEHP concentration group (3.9-fold for 1 μg L−<sup>1</sup> (*P* < 0.05), 5.48-fold for 10 μg L−<sup>1</sup> (*P* < 0.01), and 5.88-fold for 30 μg L−<sup>1</sup> (*P* < 0.01).

**Figure 4.** Expression analysis of *HSP60* in the (**A**) gill and (**B**) hepatopancreas of *Macrophthalmus japonicus* exposed to 1, 10, and 30 μg L−<sup>1</sup> 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 μg L−<sup>1</sup> 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).
