Source tracking and transmission of antibiotic resistance genes mediated by core microbiota in black soldier fly larvae bioconversion of doxycycline‐contaminated hen manure

Black soldier fly larvae (BSFL) bioconversion of doxycycline-contaminated hen manure raises concerns about antibiotic resistance gene (ARG) transmission. Although core microbiota in gut may participate in ARG dynamics, the relative contributions of microbiota from manure and baseline larval gut to the BSFL gut resistome remain unclear. Using metagenomics, amplicon sequencing, and cultivable bacteria analysis, we found that: (1) Providencia, Klebsiella, Enterococcus, and Escherichia-Shigella dominated the BSFL gut and served as primary ARG hosts; (2) Fecal filtrate intervention drastically altered gut microbiota and ARG profiles, suppressing Klebsiella (by 95.56%) and Escherichia-Shigella (to < 0.5%) while enriching Providencia (3.53 fold increase) and Enterococcus, with increased ARGs such as tet(59) and qnrD1. Long-read metagenomic analysis indicated that viable manure-borne bacteria, rather than cell-free fecal filtrate, delivered structurally intact ARG-MGE units as key carrier of mobile resistance cassettes; (3) Source tracking (FEAST) assigned the majority of ARBs (> 90%) and their ARGs in the BSFL gut to the baseline BSFL gut source, with manure-derived ARBs contributing 4.80%. Culture-based 16S rRNA gene homology provided evidence consistent with possible manure-to-gut transfer of selected Escherichia-Shigella and Enterococcus. The BSFL gut resistome was more strongly associated with the baseline BSFL microbiota than with manure, but manure still represented a detectable source of ARB and ARG introduction. FEAST source assignments should be interpreted as composition-based bioinformatic estimates rather than definitive proof of origin, due to lack of direct functional tracing. These findings have critical implications for assessing the ecological safety of BSFL-based waste valorization.