## 3 Results and Discussion
### Expression Analysis of \(Ak\) Genes in M. japonicus Following Irgarol Exposure
Furthermore, an earlier study indicated a direct correlation between arginine activity and ovarian development in the Chinese nitten crab (_Eriocheir sinensis_), suggesting its involvement in crustacean reproduction [49]. This indicates that _AK_ may directly or indirectly affect energy metabolism and other physiological processes such as reproduction, feeding, and movement. In addition, irgarol exposure may be involved in \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Source** & **Country** & **Type** & **Concentration (ng L\({}^{-1}\))** & **Reference** \\ \hline \multirow{6}{*}{Water} & Peninsular Malaysia & Coast, Marine & 2021.00 & [8] \\  & California & Coast & 254.00 & [3] \\  & South Korea & Coast, Marine & 14.10 & [39] \\  & Iran, Bushehr & Coast, Marine & 63.40 & [40] \\  & Zanzibar & Coast & 15.44 & [9] \\  & Panama & Coast, Marine & 5.00 & [41] \\ \hline \multirow{6}{*}{Sediment} & California & Coast & 9.00 & [3] \\  & Iran, Bushehr & Coast, Marine & 35.40 & [40] \\ \cline{1-1}  & Japan & Ocean & 61.00 & [42] \\ \cline{1-1}  & South Korea & Coast & 7.79 & [43] \\ \cline{1-1}  & Japan & Coast, Marine & 128.00 & [44] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The environmentally relevant concentration of irgarol. arginine metabolism, which may be involved in a wide range of molecular-level stresses, including oxidative stress and cellular activity (Figure 1a). Previous studies have shown that arginine kinase is highly conserved in marine crabs, such as _Callinectes sapidus_ and _Carcinus maenas_, and that its enzymatic activity doubles when exposed to low-salinity environments [50]. This suggests that arginine kinase functions as an efficient energy buffer when the ATP/ADP ratio is low, responding to physiological mechanisms that support ATP supply during external stress responses. Nevertheless, to date, studies on _AK_ in crustaceans have been largely confined to molecular characterizations and effects in response to salinity or bacterial infections. Consequently, there is a pressing need for further studies on cellular responses at different substance levels to provide a comprehensive picture of the ecotoxicological effects. The gills and hepatopancreas of crustaceans are important tissues for biological processes as a defense response to external stress. In this study, an exposure time-dependent increase in expression was observed in gill tissue at low concentrations (1 ug L\({}^{-1}\)), while hepatopancreas tissue demonstrated a concentration-dependent increase in _AK_ gene expression across all exposure groups (Figure 4). Additionally, the hepatopancreas exhibited tissue-specific differences, with relatively higher _AK_ gene expression than the gills. The observed tissue-specific differences in gene expression align with trends reported in previous studies. For instance, in _M. japonicus_ exposed to irgard, the expression of the Chitinase 5 gene was significantly higher in the hepatopancreas compared to the gills [10]. Similarly, exposure to the di-(2-ethylhexyl) phthalate (DEHP) revealed heightened sensitivity of the hepatopancreas, as evidenced by increased expression of the ryanodine receptor gene, which is involved in energy metabolism [26]. The observed differences in expression patterns across different tissues in response to irgard exposure can be attributed to variations in biological functional groups. The gills are important in homeostasis, osmotic control, and ion transport between the external medium and the hemolymph [51]. The gills are also used as a marker for stress response, as they show direct contact with exogenous substances. Hepatopancreas tissue performs typical liver functions in vertebrates and is involved in a variety of molecular functions such as the activation of the prophenoloxidase system, the main immune mechanism in crustaceans, nutrient absorption, and the production of diges  
Figure 4: Heatmap of relative mRNA levels in the _M. japonicus_ gill and hepatopancreas after exposure to 1, 10, and 30 ug L\({}^{-1}\) irgard on days 1, 4, and 7.