## 3 Discussion  
In this study, allatostatin (_AST_) and juvenile hormone acid O-methyltransferasearase (_JHAMT_) genes identified in _D. armandi_ were analyzed for their expression and function. The PISCF gene encodes a biological peptide that appears to be highly conserved compared with other heterologous proteins. For example, the peptide identified in _T. castaneum_ (Trica-PISCF) also seems to be identical to the _Tenebro molitor_ sequence (Tenno-PISCF) [43, 66]. _DaJHAMT_ displays the characteristic sequence of a SAM-dependent methyltransferase,  
Figure 10: Knockdown of _DaAST_ and _DaJHAMT_ affected frontalin biosynthesis. Frontalin levels in male adults injected with dsRNA for the _DaAST_ and _DaJHAMT_ genes. Different letters above the bars indicate a significant difference (one-way ANOVA, \(p\) < 0.05, with [PERSON]’s test for multiple comparisons). Figure 9: Biological morphology of _D. armandi_ from the pupa to adult stage. **(A)** Arrows point to the place of pupa injection; **(B1,B2)** normal adult morphology in the control group; **(C1,C2)** abnormal morphology of adult _D. armandi_ that were treated with dsAST; **(D1,D2)** abnormal morphology of adult _D. armandi_ after dsJHAMT treatment. which shows the structure of the active site for binding SAM and transferring the methyl group to FA or JHA [67]. _DaAST_ was highly expressed after each molt in the pupal stage and during adult migration, while _DaJHAMT_ was highly expressed on the third day of the larval stage. Because of their role in regulating JH biosynthesis, food uptake and visceral muscle contraction, _AST_ peptides are also known as \"brain-gut peptides\" [68]. Consistent with its function, _DaAST_ is usually mainly expressed in the brain. Real-time PCR showed that _DaAST_ was abundantly expressed in the brains of pupae; lower expression values were found in the heads of larvae and adults, but increased expression was found in the intestines of larvae and adults. Similarly, _AST_ of _L. decemlineata_ and _T. castaneum_ were also predominantly distributed in the brain + CC/CA complex and intestines [42; 43]. This study found that after the application of JHA, the mRNA levels of several key genes involved in JH biosynthesis in insects, such as _AST_ and _JHAMT_, were regulated by JH biosynthesis in insects after JHA administration. _JHAMT_ is the most important regulator of the JH biosynthesis process [69]. The qRT-PCR results showed that the application of low concentrations of JHA stimulated the expression of _DaAST_ in _D. armandi_ larvae but suppressed the expression of _DaJHAMT_ in _D. armandi_ pupae. Meanwhile, low concentrations of JHA stimulated _DaJHAMT_ expression in _D. armandi_ larvae but suppressed _DaJHAMT_ expression in pupae, which is consistent with observations in _Galeruca daurica_[70] and _Colaphellus bouringi_[71]. Thus, a negative feedback loop appears to control the relation between hemolymph JH titers and _AST_ in _Diplotera punctata_: a higher JH titer in hemolymph stimulates _AST_ gene expression, and then functional AS peptides inhibit JH biosynthesis and release in CA [72]. JH negatively feeds back to regulate the endocrine glands, which induce their products themselves. The direct or indirect inhibition of JH biosynthesis by CA first appeared in studies evaluating the effects of JH analogs. Topical application of such analogs inhibited JH biosynthesis in a dose-dependent manner, as did topical treatment with JH itself [72]. These results indicated that exogenous JH analogs paracrine release of _AST_ in intact calcium and act via the hemolymph in unconnected calcium to inhibit JH biosynthesis in the brain [73; 74]. RNAi was introduced to assess the possible effects of _DaAST_ and _DaJHAMT_ on JH biosynthesis in _D. armandi_. After injection with dsAST or dsJHAMT, the expression of the target gene dsAST/dsJHAMT was knocked down in larvae, pupae and adults, indicating the success of RNAi. When _D. armandi_ larvae and adults ingested ds_DaAST_, the mRNA level of _DaJHAMT_ increased significantly.