## 3 Results and Discussion
### NGS Analysis  
Table 5 illustrates the changes in alpha diversity indices for OM and DM during storage at various temperatures. The Goods coverage index was 100.0%, indicating sufficient data to characterize the microbial communities. The alpha diversity indices for OM and DM decreased under all storage conditions except for the Simpson index of DM at certain temperatures. Similar decreases in alpha diversity have been reported for other fish species [32; 43; 44]. Figure 1 illustrates the bacterial composition at the genus level, emphasizing the dominance of _Pseudomonas_ during later stages of storage. On day 0, the most abundant genera were _Staphylococcus_ (OM: 11.9%; DM: 15.4%) and _Pseudomonas_ (OM: 9.8%; DM: 11.7%). By day 14 of refrigerated storage (Figure 1A), _Pseudomonas_ was predominant, accounting for 88.9% in OM and 90.9% in DM. Under ice storage conditions on day 21 (Figure 1B), _Pseudomonas_ remained the dominant genus (90.4% in OM and 91.7% in DM). On day 50 of SC storage (Figure 1C), _Pseudomonas_ continued to dominate (91.8% in OM and 94.9% in DM). The increased abundance of _Pseudomonas_ was consistently associated with spoilage progression under all storage conditions. The spoilage-causing bacteria _Acinetobacter_, _Brochothrix_, and _Shewanella_ were present in the later stages of storage under all storage conditions, although in lower proportions than that of _Pseudomonas_. The relative abundance of these bacteria was lower in SC than in the other storage conditions. In the VCC culture, the _Aeromonas_ counts exceeded 7.5 Log CFU/g on the final sampling date under all conditions. Enterobacteriaceae displayed colony counts similar to those of _Pseudomonas_ on all final sampling dates. These bacteria were not detected using NGS. These results align with the finding that traditional culture methods can detect spoilage bacteria that cannot be identified using NGS [45; 46]. Similarly, Enterobacteriaceae, which have been identified as spoilage agents in freshwater fish [47], were observed in traditional cultures but were not detected by NGS. These discrepancies highlight the limitations of culture-dependent methods. In the VCC results, although H\({}_{2}\)S-producing bacteria multiplied significantly in refrigerated or iced OM and DM, these counts did not exceed the initial spoilage level (7 Log CFU/g) (Table 1) and the proportion did not increase significantly in the NGS results (Figure 1). _Shewanella putrefaciens_ is considered the main spoilage bacterium in marine seafood stored at low temperatures. A study that used iced marine fish (cod, plaice, and flounder) found that the main H\({}_{2}\)S-producing organism was _S. bultica_[48]. Combined with the results for _Pseudomonas_, this reflects the competition between microflora and the dynamics of survival of the fittest in resource utilization. _Shewanella_ is an important spoilage bacterium; however, its contribution to spoilage was not significant under SC conditions in the present study. The observations in the present study reinforce previous findings that culture-dependent and culture-independent methods capture complementary aspects of microbial communities, underscoring the need for approaches that accurately assess spoilage [49]. The PCoA results for the bacterial communities in OM and DM during storage at different temperatures are shown in Figure 2. The PCoA scores of the first three principal components (PCo1, PCo2, and PCo3) accounted for 40.5, 17.5, and 9.5% of the total variation, respectively. Across all storage conditions, the PCoA scores of the samples shifted rightward on the PC1 axis over the storage time, leading to the convergence of spoiled samples within a specific area. This suggests that the microbial communities in both muscle types evolved towards similar communities during storage, with the transition occurring more slowly at lower temperatures. Figure 3 shows the distinct clustering patterns, emphasizing the dominance of _Pseudomonas_ under all storage conditions. The samples were grouped into two main clusters.