Diet is a major determinant of intestinal microbiome composition. While studies have evaluated microbiome responses to diet variation, less is understood of how the act of feeding influences the microbiome, independent of diet type. Here, we use the clownfish Premnas biaculeatus, a species reared commonly in ornamental marine aquaculture, to test how the diversity, predicted gene content, and gene transcription of the microbiome vary over a two-day diurnal period with a single daily feeding event. This study used fish fed four times daily, once daily, or every three days prior to the diurnal period, allowing us also to test how feeding frequency affected microbiome diversity. The amount of time between feedings had no affect on baseline diversity of the microbiome. In contrast, the act of feeding itself caused a significant short term change in the microbiome, with microbiome diversity, predicted gene content, and gene transcription varying significantly between time points immediately before and 1.5 hours post feeding. Variation was driven by abundance shifts involving exact sequence variants (ESVs), with one ESV identified as Photobacterium sp. increasing from <0.5% of sequences immediately pre-feeding to 34% at 1.5 hours post-feeding. Other ESVs from a range of microbial groups also increased dramatically after feeding, with the majority also detected in the food. One ESV identified as Clostridium perfringens represented up to 55% of sequences but did not vary significantly over the diurnal period and was not detected in the food. Post-feeding samples were enriched in transcripts and predicted genes for social interactions, cell motility, and coping with foreign DNA, whereas time points farther from feeding were enriched in genes of diverse catabolic and biosynthetic functions. These results confirm feeding as a significant destabilizing force in clownfish intestinal microbiomes, likely due to both input of cells attached to food and stimulation of resident microbes. Microbes such as Photobacterium may episodically transition from environmental reservoirs to growth in the gut, likely in association with food particles. This transition may be facilitated by functions for navigating a new environment and interacting with neighboring microbes and host cells. Other taxa, such as Clostridium, are comparatively stable intestinal members and less likely to be affected by passing food. Conclusions about microbiome ecology may therefore differ based on when samples were collected relative to the last feeding.
Despite extensive study of intestinal microbiome diversity and the role of diet type in structuring gut microbial communities, we know very little about short-term changes in the intestinal microbiome as a result of feeding alone. Sampling microbiomes over a feeding cycle will allow us to differentiate opportunistic, feeding-responsive microbes from resident, potentially commensal members of the gut community. Also, since feeding has the potential to alter microbiome structure, sampling at different points relative to the last feeding event will likely yield different conclusions about microbiome composition and function. This variation should be addressed in comparative microbiome studies. Our study contributes to knowledge of short-term changes in the gut microbiome associated with feeding events.