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

: 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 e ﬀ ects 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 e ﬀ ects on their expression levels after exposure to DEHP. Putative HSP60 and small HSP67B2 proteins had conserved HSP-family protein sequences with di ﬀ erent 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 suggest that cellular immune protection could be disrupted by DEHP toxicity through transcriptional changes to HSPs in crustaceans. Small and large HSPs might be di ﬀ erentially involved in responses against environmental stressors and in detoxiﬁcation in M. japonicus crabs. K.P., W.-S.K. K.P., W.-S.K. K.P., W.-S.K.


Introduction
Artificial chemical additives have come to the fore as one of the main environmental pollution triggers. Plasticizers, which assign flexibility and durability to plastic, have been heavily utilized, owing to the widespread application of plastic products. As the most common plasticizer, di-2-ethylhexyl phthalate (DEHP) has contributed to the manufacture of flexible products from solid plastics such as polyvinyl chloride [1]. Owing to its widespread use, DEHP is ubiquitously released into the aquatic environment [2,3]. A recent study showed that the main source of DEHP is emissions from household sewage and sludge disposal activities [2]. DEHP is detected at high levels in all sediment samples taken from coastal bays, indicating ubiquitous contamination of the marine environment [3]. DEHP concentrations were found to range from 3020 to 3970 ng/g in sediments from the Kuwait Coast, Pearl River Delta in China, and Kaohsiung Harbor in Taiwan [4][5][6]. In addition, in the northwestern

Preparation of M. japonicus Individuals
Crabs used in this study were collected from the Yeosu marine products market in Korea. All individuals involved were 3 ± 0.5 cm in shell height, 3.5 ± 0.8 cm in shell width, and 7.5 ± 3.5 g in body weight. We prepared glass tanks (45.7 × 35.6 × 30.5 cm) filled with seawater at 18 • C, with 25% salinity and a photoperiod of 12 h. Crabs were stabilized in glass tanks for 1 day prior to exposure to DEHP solutions. After 1 day, healthy, undamaged crabs were selected for DEHP exposure experiments (below).

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 −1 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 −1 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.

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 reagent (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.

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 −∆∆Ct method [29].

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.

Statistical Analysis
The Statistical Package for the Social Sciences (SPSS) 12.0 KO (SPSS Inc., Chicago, IL, USA) was used for statistical analysis in this study. Data are presented as the mean ± standard deviation. Two-way analysis of variance was conducted to identify the statistical effects of the exposure period and each DEHP dose on Mj-HSP60 and Mj-HSP67B2 mRNA expression. Significant differences were presented as *P < 0.05 and **P < 0.01.

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.

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.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 13   Pairwise identity percentage was calculated using the EMBOSS alignment program. 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.

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 −1 DEHP (P < 0.01), 3.2-fold higher for 10 µg L −1 (P < 0.05), and 9.4-fold higher for 30 µg L −1 (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 −1 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 −1 (0.3-fold) and 30 µg L −1 (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 L −1 , 2.6-fold for 10 µg L −1 , and 2.9-fold for 30 µg L −1 DEHP (P < 0.05). By days 4 and 7, Mj-HSP60 expression levels returned to control levels for the 1 µg L −1 group. In the 10 µg L −1 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.

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 −1 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 Appl. Sci. 2020, 10, 2766 8 of 12 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 −1 (P < 0.05), 5.48-fold for 10 µg L −1 (P < 0.01), and 5.88-fold for 30 µg L −1 (P < 0.01). 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 −1 (0.3-fold) and 30 μg L −1 (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 L −1 , 2.6-fold for 10 μg L −1 , and 2.9-fold for 30 μg L −1 DEHP (P < 0.05). By days 4 and 7, Mj-HSP60 expression levels returned to control levels for the 1 μg L −1 group. In the 10 μg L −1 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. 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).

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 −1 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 −1 (P < 0.05), 5.48-fold for 10 μg L −1 (P < 0.01), and 5.88-fold for 30 μg L −1 (P < 0.01).

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 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 Figure 5. Expression analysis of HSP67B2 in the (A) gill and (B) hepatopancreas of Macrophthalmus japonicus exposed to 1, 10, and 30 µg 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).

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 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 Cu 2+ [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.