## 3 Results and Discussion
### Expression Analysis of \(Ak\) Genes in M. japonicus Following Irgarol Exposure  
We observed changes in \(AK\) gene expression in \(M\). _japonicus_ gill tissue upon irgarol exposure (Figure 2). Here, upon 4 and 7 days of irgarol exposure, the \(AK\) gene showed the highest gene expression at the high concentration of 30 ug L\({}^{-1}\), while lower gene expression was observed at 1 ug L\({}^{-1}\) irgarol exposure, compared to the control. On day 1, an irgarol exposure of 1 ug L\({}^{-1}\) exhibited relatively high gene expression compared to the control, but 10 ug L\({}^{-1}\) irgarol exhibited significantly low \(AK\) gene expression (\(p<0.05\)) (Figure 2a). At 4 days, 1 ug L\({}^{-1}\) and 10 ug L\({}^{-1}\) irgarol exposures produced lower \(AK\) expressions than the controls (\(p<0.05\)), but increased \(AK\) gene expression was observed in gills exposed to 30 ug L\({}^{-1}\) irgarol (\(p<0.05\)) (Figure 2b). After 7 days of irgarol exposure, a sharp decrease in \(AK\) gene expression was observed at 1 ug L\({}^{-1}\) irgarol (\(p<0.001\)); however, expression increased to levels similar to controls as the irgarol exposure concentration rose (Figure 2c). In addition, exposure to the higher concentration of 30 ug L\({}^{-1}\) irgarol produced a significant increase in expression in the gills compared to controls (\(p<0.05\)). Irgarol exposure induced higher \(AK\) gene expression in all \(M\). _japonicus_ hepatopancreas exposure groups compared to the control group (Figure 3). At day 1 of exposure, \(AK\) gene expression was statistically significantly increased in response to 1 ug L\({}^{-1}\) (\(p<0.05\)) and 10 ug L\({}^{-1}\) (\(p<0.001\)) irgarol exposures; however, a decrease was observed at the higher irgarol exposure concentration of 30 ug L\({}^{-1}\) (Figure 3a). In addition, hepatopancreas tissues exposed to 1 ug L\({}^{-1}\) and 10 ug L\({}^{-1}\) irgarol for 4 days exhibited similar gene expression although expressions were higher for both compared to the control (\(p<0.001\)). Figure 2: Change in \(M\). _japonicus_ gill \(AK\) mRNA expression levels on days 1 (**a**), 4 (**b**), and 7 (**c**) after exposure to 1, 10, and 30 ug L\({}^{-1}\) irgarol. The values were normalized against _GAPDH_ expression and are presented as the mean \(\pm\) SE. The expression level of the control was set as 1. Significant differences are indicated with asterisks: \({}^{**}\)\(p<0.05\), \({}^{***}\)\(p<0.001\). Comparatively, \(AK\) expression decreased after exposure to 30 ug L\({}^{-1}\) irgarol (\(p<0.05\)), similar to the expression pattern observed on day 1 of exposure (Figure 3b). Nevertheless, \(AK\) gene expression was significantly increased in all irgarol-exposed groups on day 7 (\(p<0.001\)) (Figure 3c). Irgarol is a common antifouling paint that functions as a photosynthesis inhibitor and is well known for its ability to induce a decrease in phytoplankton productivity. As antifouling paints are released into the environment, they can cause toxic effects on a variety of marine organisms, disrupting normal metabolic functions and leading to disturbances in growth, development, reproduction, and behavior [24; 32]. Irgarol is a major threat to biodiversity due to its wide distribution within the marine and estuarine environment (Table 2). Prior research has demonstrated that \(M\). _japonicus_ exposed to irgarol exhibited a concentration-dependent reduction in exoskeleton surface roughness, accompanied by a time-concentration-dependent increase in chitinase gene expression, which plays a role in exoskeleton formation [10]. Furthermore, the coral Acropora tenuis exhibited alterations in heat shock protein gene expressions, accompanied by a notable decline in the photosynthetic efficiency of its symbiotic dinoflagellates following elevated concentrations of irgarol exposure [33]. Irgarol, a widely used antifouling agent, has been shown to exert significant physiological and behavioral effects on crustaceans.