## 3 Results and Discussion  
### Expression of proPO System-Related Genes in Gonads After Exposure to EDCs  
The nucleotide sequence for the _M. japonicus AK_ gene was 1074 bp. Meanwhile, phylogenetic analysis identified two major phylogenetic groups (Figure 1b). The _M. japonicus AK_ gene was highly similar to those in _Macrophthalmus milloti, Ilygrapsus paluducla, Thalassograpsus harpax, Cyclograpsus intermedius, Ericoheir sinensis, Pseudograpsus crassus, Chasmagathus granuloma,_ and _Hemiplax hirtipes_ within a same clade. The presence of crustaceans belonging to different clusters was observed, including _Scylla olivacea, Scylla paramamosain, Portuus trituberculatus, Callinectes actuatus_, _Callinectes bellicosus_, and _Cherax quadricarinatus_. The _AK_ gene was noted in another clade group, which included many insect clades, such as _Phyllotreta striolata, Bombyx mori, Solenopsis invcita, Triatoma vitticeps, Apis mellifera, Cissites cephalotes, Locusta migratoria manilensis, Schistocera americana, Periplaneta americana_, _Battella germanica_, and _Periplaneta americana_. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Gene** & **Primer Sequence** & **Amplification** & **Efficiency (\%)** & **Accession Number** \\  & & **Size (bp)** & & \\ \hline _AK_ & TACGACATCTCCAACAAGCG & 75 & 96 & KJ653259 \\  & AAGAATACCGTCCTGATCTCC & & & \\ _GAPDH_ & TGCTGATCCCATGTTTG & 147 & 105 & KJ653265 \\  & AGGCCCTGACAATCTCAAAG & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Primers used for gene amplification. The phylogenetic distribution of \(AK\) has been the focus of extensive research to elucidate its evolutionary history and functional significance across a diverse range of taxonomic groups. \(AK\) has exhibited a complex evolutionary trajectory whereby it has undergone multiple independent duplications and diversification along taxonomic lineages. This has resulted in the emergence of two distinct patterns: Those with a single \(AK\) gene and those displaying complex distribution patterns characterized by expansion and diversification. This evidence indicates that the \(AK\) gene is integral to adapting diverse organisms, particularly marine invertebrates, to specific environmental and physiological requirements [28,29]. In this study, phylogenetic tree analysis of \(AK\) showed that the studied species, \(M\). _japonicus_, clustered with \(M\). _milloti_ and \(I\). _paludicola_. \(M\). _japonicus_ and \(M\). _milloti_ belong to the same genus, which can be explained by the high morphological and ecological similarities observed between the two species. Meanwhile, \(I\). _paludicola_ belongs to the Macrophthalmidae but is in the same clade despite being a different genus, suggesting that these may be phylogenetically related at a molecular level rather than classified by morphological characters. Furthermore, the arginine binding site and ADP binding site within the \(AK\) gene exhibited the same homology with \(M\). _milloti_ and \(I\). _paludicole_, which are members of the same clade. This indicates that the \(AK\) gene is highly functionally conserved among intertidal species. The \(AK\) gene is widely distributed within insects and various crustaceans, suggesting that the \(AK\) gene in \(M\). _japonicus_ identified in this study is phylogenetically well conserved within Arthropoda. Several studies have indicated that \(AK\)s may be susceptible to numerous stressful environmental factors. As an illustration, the \(AK\) gene expression in freshwater giant prawn (_Macrobrachium rosenbergii_) is enhanced following exposure to infectious subcutaneous and hematopoietic necrosis viruses and has been identified as a potential contributor to crustacean immune responses [30]. Moreover,  
Figure 1: Schematic diagram of the arginine pathway (**a**) and phylogenetic analysis (**b**) of the \(AK\) gene in \(M\). _japonicus_.