In insect-microbe symbiosis, understanding the diversity of associated bacteria is crucial. DNA-dependent sequence methods are widely used to assess microbial diversity in insects, but they cannot distinguish between live and dead microbes. In contrast, RNA-dependent sequencing can identify alive bacterial communities, making them more suitable for evaluating alive microbiota diversity. However, its practical reliability in insect-microbe symbiosis remains poorly validated. This study investigated larval gut bacteria diversity of Delia antiqua, a major pest of Liliaceae crops, by employing both DNA- and RNA-dependent 16S rRNA amplicon sequencing. The reliability of both sequencing methods was evaluated by comparing the effects of synthetic communities (SynComs, constructed according to DNA- or RNA-dependent sequencing) and bacterial communities from wild larvae on axenic larvae. Results revealed significant differences in bacterial community between DNA- and RNA-dependent sequence samples. Compared to bacterial communities from wild larvae, the SynCom constructed based on RNA-dependent sequencing exhibited inhibition effects on D. antiqua larvae survival and body weight, while DNA-dependent SynCom did not, suggesting that DNA-dependent methods were superior for assessing symbiotic microbiota in D. antiqua. This work will provide insights into microbial diversity detection in D. antiqua and offer a framework for other insect-microbe studies.