Methanogenic activities signatures in broilers fed black soldier fly (Hermetia illucens) larvae-based diets

Methanogenic archaea in the avian gut contribute to hydrogen turnover and thus play a role in fermentative efficiency and greenhouse gas emission. While black soldier fly larvae meal (BSFLM) has been explored as a replacement for fishmeal in broiler diets, less is known about how varying BSFLM inclusion levels affect methanogenesis pathways and enzyme activities in the gut microbial community. This study investigated the presence, relative abundance, and shifts in methanogenesis enzyme pathways in broilers fed diets with increasing levels of BSFLM. Arbor Acre Plus broiler chicks were allocated to five dietary treatments over 8 weeks. Diets replaced fishmeal with BSFLM at 0 % (control), 25 %, 50 %, 75 %, and 100 % levels. Cecal samples were collected post-mortem, DNA extracted, and the bacterial 16S rRNA (V1-V9) region sequenced on PacBio Sequel IIe. Functional predictions via PICRUSt2 were used to identify KEGG orthologs associated with methanogenesis (e.g. K00200-K00205 for FMD, K00399 etc. for MCR, methyltransferases, etc.). Enzyme detection across treatments was assessed qualitatively and semi-quantitatively (e.g. "low", "moderate", "strong") based on relative abundance. Key methanogenic enzymes (including FMD, MCH, MTD, F420-dependent enzymes, methyltransferases, and MCR) were profiled, and their activity compared across treatments. Correlations were examined and predicted functional capacity via PICRUSt2. Methanogenesis-related pathways were detectable in all dietary treatments. In the control (0 % BSFLM), enzyme levels were minimal, reflecting low background methanogenic potential. At 25 % BSFLM inclusion (T2), there was strong detection of hydrogenotrophic pathway enzymes (FMD, coenzyme F420 hydrogenase, MCR) and moderate presence of methylotrophic methyltransferases, suggesting dominance of hydrogenotrophic methanogenesis. In the 50 % BSFLM treatment (T3), enzyme levels declined somewhat: hydrogenotrophic enzyme activity was moderate, and methylotrophic components were weaker. With 100 % BSFLM (T5), methylotrophic pathway enzymes (methanol- and methylamine-corrinoid protein Co-methyltransferases) were strongly detected, while hydrogenotrophic enzymes persisted but at low levels. These shifts have implications for gut fermentation efficiency, substrate (hydrogen and methyl compound) availability, nitrogen metabolism, and possibly GHG emission. Optimization of BSFLM inclusion levels may help balance production performance and environmental sustainability.