## 3 Results and Discussion
### Expression Analysis of \(Ak\) Genes in M. japonicus Following Irgarol Exposure
From a physiological perspective, irgarol exposure disrupts cellular homeostasis by inducing mitochondrial dysfunction and increasing reactive oxygen species (ROS) production in crustaceans [34]. In addition, irgarol exposure is closely linked to developmental delays and increased mortality rates during the larval stages of crustaceans such as _Tigriopus japonicus_, highlighting its high neurotoxic potential and its capacity to impair normal behavioral functions [35]. Chronic exposure to irgarol has also been reported to reduce hatching success and negatively affect early developmental stages in _Artenia salina_[36]. The endocrine systems of crustaceans are diverse and intricate, regulating several critical biological processes, including molting, growth, and reproduction [10; 37; 38]. As a result, these systems render crustaceans highly susceptible  
Figure 3: Change in \(M\). _japonicus_ hepatopancreas \(AK\) 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_ and are presented as the mean \(\pm\) SE. The expression level of the control was set as 1. Significant differences are indicated with asterisks: **\(p<0.05\), ***\(p<0.001\). to disruption by exogenous substances. Furthermore, crustacean immune systems, which rely predominantly on innate immunity, may be vulnerable to disruption by anthropogenic chemicals. Consequently, examining the cellular-level alterations observed in _M. japonicum_s following irgarol exposure is paramount from an ecotoxicological perspective within estuarine ecosystems. In the present study, mRNA expression of _AK_ in gill tissues was higher in 30 \(\mu\)g L\({}^{-1}\) irgarol-exposed individuals at days 4 and 7 compared to controls, confirming a concentration-dependent trend. Notably, hepatopancreas exhibited higher _AK_ expression compared to controls (1.0-fold) at all exposure concentrations, with the highest _AK_ gene expression observed after 7 days of exposure alongside a clear exposure time-dependent trend (Figure 4). _AK_ plays a role in mediating physiological stress responses in marine organisms, and its activity and expression can be highly sensitive to exposure to toxins and other harmful chemicals [45; 46]. Previous studies have demonstrated a time- and concentration-dependent reduction in the survival rate of _M. japonicus_ upon exposure to irgarol. Additionally, significant time-dependent upregulation of genes associated with exoskeleton formation (e.g., Chitinase 1, 4, 5), endocrine regulation (Ecdysteroid receptor), and immune response (serine proteinase) was observed [10]. Irgarol exposure also induced a significant time-dependent increase in p38 MAPK gene expression in the gill and hepatopancreas tissues of _M. japonicus_[27]. These findings suggest that irgarol exerts toxicological effects on _M. japonicus_, promoting increased expression of endocrine and immune-related genes while impacting exoskeleton formation processes. Consistent with these prior observations, our study also revealed a similar trend of increased _AK_ gene expression in _M. japonicus_ under irgarol exposure, further supporting its role in mediating molecular responses to environmental stressors. In addition, previous studies have characterized the molecular features of the _AK_ gene in mud crabs and found that acute toxic effects were observed in the hepatopancreas tissue upon infection with the bacterium _Vibrio alginolyticus_[47]. Moreover, concentration- and time-dependent alterations in the _AK_ gene expression were observed in the gills and hepatopancreas of _M. japonicus_ in response to changes in salinity (low salinity: 10 PSU, control: 25 PSU, high salinity: 30 PSU) [14]. Therefore, the _AK_ gene expression observed in _M. japonicus_ gills and hepatopancreas in this study was found to be highly sensitive to external agents, a result that is consistent with those of previous studies [33; 48].