**1. 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 [4–6]. In addition, in the northwestern Mediterranean Sea, the range of DEHP concentrations was 42–802 ng L−<sup>1</sup> and 130–924 ng L−<sup>1</sup> in the surface seawater (depth 0.5 m) and bottom seawater (depth 30 m), respectively [7]. DEHP concentrations were found to range from 62 to 4352 ng L−<sup>1</sup> from the bottom to the surface seawater of the Bohai Sea and the Yellow Sea, China [8]. DEHP, an endocrine-disrupting chemical (EDC), exhibits a perturbing e ffect on steroidogenesis activities [9,10].

*Macrophthalmus japonicus* is one of the main benthic species ubiquitously detected in tidal flats and shows high distribution rates in estuarine regions of Korea and Japan [11,12]. As a main member of the tidal flat food chain, this species contributes to the maintenance of biodiversity in estuarine ecosystems. Because of their dominant distribution, crabs might be a good candidate organism to sense changes in the condition of the surrounding environment, as well as changes involving food reserves, as they have abundant nutrients and are of high economic value in commercial fisheries. However, crab habitats are easily exposed to grea<sup>t</sup> hazards, such as plastic waste pollutants and chemicals that are transported into mud flats through rivers or from the ocean. The e ffects of various stress conditions, such as salinity and heavy metal and biocide contaminants, have been reported following expression analysis of immune-related or stress-related genes in crabs [11,13–16]. A recent study showed the relationships between EDCs and gene expression alterations involving crab innate immune systems [17], but there have been no studies of the relationship between stress-related gene expression and EDC exposure. Despite its biological importance as a nutritional resource, few studies have been conducted on the *M. japonicus* genomic DNA sequence.

Heat shock proteins (HSPs) are ubiquitous proteins secreted in cells after exposure to stressful conditions and are classified into six major groups (*HSP27, HSP60, HSP70, HSP90*, and large HSPs) based on their molecular weights [18,19]. HSPs function as molecular chaperones to prevent the formation of denatured proteins during high temperature stress and exhibit upregulation in their expression patterns under such stress conditions [18,20]. In addition, these stress proteins play an important role in the maintenance of normal polypeptide structures and in the promotion of correct refolding of cellular proteins in response to various external stimuli, such as anoxia, heavy metals, or chemicals, which cause protein denaturation [20–22]. HSPs assist in protecting cellular homeostasis from such stress. *HSP60* is well known as a pro-apoptotic molecule, which induces apoptosis and acts as a chaperone for proteins transcribed from mitochondrial DNA [23–25]. *HSP60* is a highly immunogenic protein, which is implicated in a variety of autoimmune diseases [26,27]. The upregulation of *HSP60* indicates its involvement in crucial functions mediating immune responses in the Chinese mitten crab, *Eriocheir sinensis*, after crustacean pathogen infection [27]. *HSP67B2* was characterized as a Relish-regulated gene in the innate immunity of the Chinese shrimp (*Fenneropenaeus chinensis*) [28]. However, there is limited information about the molecular characterization and expression responses involving the crustacean *HSP67B2*.

In the present study, we identify two stress-related genes, *Mj-HSP60* and *Mj-HSP67B2,* in *M. japonicus* crabs to evaluate the toxic e ffects of DEHP on cellular immune protection in crustaceans. We investigate the genomic structure, phylogenetic relationships with other homologous HSPs, and transcriptional responses of HSPs under DEHP stress. We seek to provide molecular information regarding the influence of EDCs on stress-related gene expression in *M. japonicus*.
