## 3 Results  
### Metabolomics  
Metabolites identified in samples were composed of those derived from porcine carcasses ingested by maggots and metabolites of maggots. The identification of analytes was based on the MS2 database but limited to classification by species because the structure was matched based on the elemental composition levels of small molecules. To build a list of differential metabolites (DMs) for each group, pelletized proteins were denatured through a focused lysis procedure and the initial protein concentration was determined using the BCA assay. Concentrations of metabolites were estimated in proportion to concentrations of proteins. Metabolites were dried, resuspended, and used for analysis. Using LC-MS/MS analysis, 212, 222, 368, and 194 metabolites were identified on day 3, day 5, day 9, and day 11, respectively (Table S1). By filtering using annotation implemented with the Metabolomics Standards initiative (MSI) of the Metabolomics Society [42], 134, 153, 258, and 194 metabolites were identified with level 2 and 33, 37, 57, and 31 metabolites were identified with level 3 on day 3, day 5, day 9, and day 11, respectively. Figure S1 presents peak area of the top 20 metabolites. These are average values of triplication results. These metabolites identified at different sampling time points were visualized by PCA plot (Figure 2a), showing differences among the four time points. A heatmap was generated for 573 metabolites (Figure 2b). According to the time of sampling given in this study, Figure 3 shows down- and up-regulated metabolites indicated by green and red, respectively. First, in the comparison of day 5 vs. day 3, 309 metabolites were differentially expressed (210 up- and 99 down-regulated). Subsequently, 366 metabolites (315 up- and 51 down-regulated) and 457 (41 up- and 416 down-regulated) metabolites were differentially expressed in the comparison of day 9 vs. day 5 and the comparison of day 11 vs. day 9, respectively. DMs are listed in Table S2.  
Figure 2: Metabolomics analysis results. (**a**) PCA plot, (**b**) Heatmap.  
Among 267 pathways identified from the metabolite dataset, top 20 pathways are listed in Table 1. Quantitative metabolites were significantly matched and potentially correlated with nucleotide-related pathways such as purine nucleotides degradation II (aerobic) and purine nucleotide salvage in Compound Discoverer through the KEGG database. Quantitative values for metabolites in each pathway are shown in Figure 4. Changes in quantitative values of day 5 vs. day 3, day 9 vs. day 5, and day 11 vs. day 9 are shown in Figure 5.  
Figure 3: Differentially expressed metabolites. (**a**) Day 5 vs. day 3, (**b**) Day 9 vs. day 5, (**c**) Day 11 vs. day 9.  
shown on the y-axis as log2 fold change. In the purine nucleotides degradation II pathway, 16 referenced compounds among 27 compounds in the pathway were matched, with adenosine 5'-monophosphate (AMP), guanosine monophosphate (GMP), and xanthine commonly increased until day 5, which is the initial stage of growth. They showed a tendency to decrease until day 11, the time of being an adult insect. In the superpathway of purine nucleotide salvage pathway, 14 referenced compounds out of 54 compounds in the pathway were matched, of which purine nucleotide compounds such as inosine, adenosine, and guanine matched with a similar tendency.