## 2 Results
### DaAST and DaJHAMT Transcript Levels in D. armandi at Different Life Stages and in Different Tissues  
The transcript levels of _DaAST_ throughout the developmental stages were determined using qRT-PCR. Relative to larvae, one-way ANOVA showed statistically significant differences in transcript expression levels among different development stages (_F_ = 6.128, df = 14, \(p\) < 0.0001) (Figure 3). The expression of _DaAST_ showed a downward trend in larvae, beginning with small larvae and reaching the lowest value in mature larvae. The expression then decreased in the pupae and reached a peak on the second day of the pupal stage, then dropped to a second trough that appeared at the teneral stage. In adults, the expression reached a maximum in emergent females and decreased to some extent, beginning in feeding females. The expression levels in females were higher than those in males (Figure 3A). \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Gene Name** & **Accession** & **Full Length** & **ORF Size** & & & \\  & **No.** & **(bp) 1** & **(aa/bp) 1** & **Mw (kDa) 1** & **I.P. 1** & **Signal Peptide Prediction 2** \\ \hline _DaAST_ & MW645339 & 471 & 114/345 & 13.28 & 8.66 & SP 0.766 mTP 0.043 other 0.194 \\ _DaJHAMT_ & MW645341 & 1137 & 275/828 & 15.11 & 9.04 & SP 0.069 mTP 0.062 other 0.932 \\ \hline \hline \end{tabular}  
* As predicted by ProtoParam [63]. 2 As predicted by Target P 1.1 [64]. I.P.: isoelectric point; Mw: molecular weight; ORF: open reading frame; SP: secretory pathway signal peptide; mTP: mitochondrial targeting peptide.  
\end{table} 
Table 2: Physicochemical properties and cellular localization of _DaAST_ and _DaJHAMT_ of _D. armandi_. The transcript levels of _DaJHAMT_ throughout the developmental stages were determined using qRT-PCR. Relative to larvae, a one-way ANOVA showed statistically significant differences in transcript expression levels among different development stages (\(F\) = 2.402, df = 14, \(p\) = 0.022) (Figure 3B). In larvae, the expression of _DaJHAMT_ first increased and then decreased. From pupae to the teneral stage, the expression patterns of _DaJHAMT_ were similar to those of _AST_. In adults, the expression levels of _DaJHAMT_ remained constant from the teneral stage to the feeding adult stage but were lower than the levels in pupae (Figure 3B). The tissue distribution of _DaAST_ and _DaJHAMT_ transcripts in various tissues from three developmental stages (larva, pupa, adult) were investigated by real-time RT-PCR analysis. For _DaAST_, at the larva stage, statistically significant differences were not found among different tissues of _DaAST_ (Figure 4A); in the pupa, the expression of _DaAST_ in the head reaches the highest level (\(F\) = 11.562, df = 3, \(p\) = 0.003); During the period of adult emergence, the highest expression level of _DaAST_ was observed in female and male anterior midguts (female: \(F\) = 7.038, df = 6, \(p\) = 0.001; male: \(F\) = 19.926, df = 6, \(p\) < 0.0001) (Figure 4C). For _DaJHAMT_, quantitative RT-PCR (qRT-PCR) analysis of _DaJHAMT_ showed that statistically significant differences were found among different tissues in all developmental stages. _DaJHAMT_ was highly expressed in larval and pupal heads, and the highest expression was found in the larval and pupal guts (larva: \(F\) = 4.386, df = 3, \(p\) = 0.042; pupa: \(F\) = 15.022, df = 3, \(p\) = 0.001) (Figure 4B). During the period of adult emergence, statistically significant differences were not found among the different tissues of females (\(F\) = 1.703, df = 6, \(p\) = 0.193). Meanwhile, the highest expression level of _DaJHAMT_ was observed in male Malpighian tubules (\(F\) = 15.369, df = 6, \(p\) < 0.0001); the anterior midgut and hindgut had higher expression volumes than the other tissues (Figure 4D). Figure 3: Relative expression of _DaAST and DaJHAMT_ genes in different stages of _D. armandi_. (**A**) _DaAST_; (**B**) _DaJHAMT_.