Diet Outweighs Vertical Transmission in Shaping Dung Beetle Larval Gut Microbiomes

The microbiome is central to host development and adaptation, yet the balance between vertical and environmental acquisition, and how hosts shape surrounding microbial communities, remains poorly understood. Dung beetles rely on microbial symbionts to extract nutrients from vertebrate dung, with part of their microbiome vertically inherited via a maternal faecal pellet. However, the relative importance of vertical versus horizontal transmission is unclear. We examined this in the gazelle dung beetle (Digitonthophagus gazella), rearing larvae on brood balls made of dung from grass-(high-quality), hay-(low-quality) or silage-fed (a novel fermentable energy-rich diet) cattle, with or without maternal microbes. We integrated measures of gut morphology with 16S rRNA amplicon sequencing to assess host development and the gut microbiome. Diet significantly influenced overall size, hindgut area, and microbiome composition. Silage-dung fed larvae had more even and taxonomically rich microbiomes, with higher microbial diversity in individuals reared with maternal microbes. Diet explained ~26% of the variation in microbial composition, while the vertical transmission of microbes only explained 3%. Vertical transmission only slightly increases microbial species richness and relative hindgut area but did not influence overall microbial diversity. The larval brood ball contributed 40%-50% of the hindgut microbiome, while maternal microbes contributed < 0.05%. These findings demonstrate that horizontal acquisition through diet is the dominant force shaping larval gut microbiomes, while vertical inheritance plays a minor but detectable role in enhancing richness and gut development. More broadly, this work reinforces the importance of examining host-microbiome-environment interactions in ecological and evolutionary contexts.