## 3 Results
### 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 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.  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Species** & **Gene Name** & \
\begin{tabular}{c} **Accession** \\ **Number** \\ \end{tabular} &
\begin{tabular}{c} **RHOD Superfamily** \\ **Domain Length** \\ \end{tabular} & **Identity (\%)** & **Similarity (\%)** & **Gap (\%)** \\ \hline _Macrophthalmus_ & Heat Shock protein & & & & & \\ _japanics_ & \& \& \& & & \\ _Penaas tsunami_ & Heat Shock protein & & & & & & \\ _Penaas tsunami_ & Heat Shock protein & ROT83326.1 & 103 & 72.8 & 81.6 & 4.9 \\ _Lepepotherius_ & Heat Shock protein & ACO11957.1 & 106 & 43.4 & 60.4 & 7.5 \\ _salmonis_ & Heat Shock protein & XP\_023305341.1 & 101 & 40.0 & 60.0 & 10.5 \\ _Luxilin capriana_ & PREDICTED: heat shock protein & XP\_018026303.1 & 106 & 44.3 & 58.5 & 7.5 \\ _Hyalella marca_ & PREDICTED: heat shock protein & XP\_017840691.1 & 99 & 39.8 & 58.3 & 8.7 \\ _Drosophila bascili_ & Heat Shock protein & EN62322.1 & 103 & 39.8 & 56.3 & 4.9 \\ _Aedes aegypti_ & Heat Shock protein & XP\_021696792.1 & 99 & 35.9 & 54.4 & 8.7 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Percentage identity, similarity, and gaps involving _Macrophthalmus japonicus_ HSP67B2 and HSP67B2 homologs from other species at the amino acid level  
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.