Diet is a major determinant of intestinal microbiome composition. While studies have evaluated microbiome responses to diet variation, less is understood about 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 2-day diurnal period with a single daily feeding event. This study used fish fed four times daily, once daily, or every 3 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 effect 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 postfeeding. 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 prefeeding to 34% at 1.5 h postfeeding. 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. Postfeeding 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.IMPORTANCE 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.
Experiment 1
Subjects
- Location of subjects
- Georgia
- Host species Species from which microbiome was sampled. Contact us to have more species added.
- Premnas biaculeatus
- Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology
- Digestive tract Alimentary canal,Alimentary tract,Digestive canal,Digestive tube,Enteric tract,Gut,Gut tube,Digestive tract,digestive tract
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Pre-fed group at 1100 h
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Post-fed group at 1230 h
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- The post-fed group consists of fish fed at 1230 hours. (1.5 hours postfeeding)
- Group 0 sample size Number of subjects in the control (unexposed) group
- 17
- Group 1 sample size Number of subjects in the case (exposed) group
- 18
Lab analysis
- Sequencing type
- 16S
- 16S variable region One or more hypervariable region(s) of the bacterial 16S gene
- V3-V4
- Sequencing platform Manufacturer and experimental platform used for quantifying microbial abundance
- Illumina
Statistical Analysis
- Data transformation Data transformation applied to microbial abundance measurements prior to differential abundance testing (if any).
- relative abundances
- Statistical test
- ANCOM
- Significance threshold p-value or FDR threshold used for differential abundance testing (if any)
- 0.05
- MHT correction Have statistical tests be corrected for multiple hypothesis testing (MHT)?
- No
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- increased
Signature 1
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: increased abundance
in
Post-fed group at 1230 h
| NCBI | Quality Control | Links |
|---|
| Alcaligenaceae | | |
| Anoxybacillus | | |
| Arcobacter | | |
| Azoarcus | | |
| Bacillales | | |
| Bacillus | | |
| Bacteroidales | | |
| Balneolaceae | | |
| Beijerinckiaceae | | |
| Cetobacterium | | |
| Chromatiaceae | | |
| Citrobacter | | |
| Cryomorphaceae | | |
| Erwinia | | |
| Eubacteriales | | |
| Geobacillus | | |
| Hydrogenophaga | | |
| Jeotgalicoccus | | |
| Lactobacillaceae | | |
| Lactobacillales | | |
| Lactobacillus | | |
| Lactococcus | | |
| Leuconostoc | | |
| Moritella | | |
| Nocardiopsaceae | | |
| Opitutae | | |
| Paracoccus | | |
| Pasteurellaceae | | |
| Peptostreptococcaceae | | |
| Peptostreptococcus | | |
| Photobacterium | | |
| Planococcaceae | | |
| Prevotella | | |
| Proteus | | |
| Pseudidiomarina | | |
| Psychrilyobacter | | |
| Psychrobacter | | |
| Psychromonas | | |
| Rhodobaca | | |
| Sporanaerobacter | | |
| Sporosarcina | | |
| Staphylococcaceae | | |
| Staphylococcus | | |
| Streptococcus | | |
| Tepidimicrobium | | |
| Thermus | | |
| Ureibacillus | | |
| Vagococcus | | |
| Veillonellaceae | | |
| Vibrionaceae | | |
| Virgibacillus | | |
| Xanthomonadaceae | | |
| Yersinia | | |
| MarinicellaceaeMarinicellaceae | | |
Revision editor(s): Muqtadirat,
Victoria
Signature 2
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: decreased abundance
in
Post-fed group at 1230 h
| NCBI | Quality Control | Links |
|---|
| Alteromonadales | | |
Revision editor(s): Muqtadirat,
Victoria
Experiment 2
Differences from previous experiment shown
Subjects
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Post-fed group at 1400 h
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- The post-fed group consists of fish fed at 1400 hours. (3 hours post-feeding)
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
Signature 1
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: increased abundance
in
Post-fed group at 1400 h
| NCBI | Quality Control | Links |
|---|
| Photobacterium | | |
| Vibrionaceae | | |
| Arcobacter | | |
| Paracoccus | | |
| Psychromonas | | |
| Staphylococcus | | |
| Lactobacillus | | |
| Psychrobacter | | |
| Cetobacterium | | |
| Streptococcus | | |
| Bacillus | | |
| Lactococcus | | |
| Geobacillus | | |
| Thermus | | |
| MarinicellaceaeMarinicellaceae | | |
| Pasteurellaceae | | |
| Bacillales | | |
| Hydrogenophaga | | |
| Citrobacter | | |
| Alcaligenaceae | | |
| Tepidimicrobium | | |
| Lactobacillales | | |
| Opitutae | | |
| Planococcaceae | | |
| Staphylococcaceae | | |
| Cryomorphaceae | | |
| Peptostreptococcaceae | | |
| Prevotella | | |
| Xanthomonadaceae | | |
| Veillonellaceae | | |
| Proteus | | |
| Anoxybacillus | | |
| Peptostreptococcus | | |
| Chromatiaceae | | |
| Vagococcus | | |
| Leuconostoc | | |
| Jeotgalicoccus | | |
| Erwinia | | |
| Bacteroidales | | |
| Yersinia | | |
| Moritella | | |
| Psychrilyobacter | | |
| Lactobacillaceae | | |
| Virgibacillus | | |
| Eubacteriales | | |
| Sporosarcina | | |
| Nocardiopsaceae | | |
| Balneolaceae | | |
| Ureibacillus | | |
| Azoarcus | | |
| Pseudidiomarina | | |
| Rhodobaca | | |
| Sporanaerobacter | | |
| Beijerinckiaceae | | |
Revision editor(s): Victoria
Signature 2
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: decreased abundance
in
Post-fed group at 1400 h
| NCBI | Quality Control | Links |
|---|
| Alteromonadales | | |
Revision editor(s): Victoria
Experiment 3
Differences from previous experiment shown
Subjects
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Post-fed group at 1600 h
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- The post-fed group consists of fish fed at 1600 hours. (5 hours postfeeding)
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- increased
Signature 1
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: increased abundance
in
Post-fed group at 1600 h
| NCBI | Quality Control | Links |
|---|
| Yersinia | | |
| Bacteroidales | | |
| Erwinia | | |
| Jeotgalicoccus | | |
| Leuconostoc | | |
| Vagococcus | | |
| Chromatiaceae | | |
| Peptostreptococcus | | |
| Anoxybacillus | | |
| Proteus | | |
| Veillonellaceae | | |
| Xanthomonadaceae | | |
| Prevotella | | |
| Peptostreptococcaceae | | |
| Staphylococcaceae | | |
| Planococcaceae | | |
| Opitutae | | |
| Lactobacillales | | |
| Bacillales | | |
| Tepidimicrobium | | |
| Alcaligenaceae | | |
| Citrobacter | | |
| Hydrogenophaga | | |
| Pasteurellaceae | | |
| MarinicellaceaeMarinicellaceae | | |
| Thermus | | |
| Geobacillus | | |
| Lactococcus | | |
| Bacillus | | |
| Streptococcus | | |
| Cetobacterium | | |
| Psychrobacter | | |
| Lactobacillus | | |
| Staphylococcus | | |
| Psychromonas | | |
| Paracoccus | | |
| Arcobacter | | |
| Vibrionaceae | | |
| Photobacterium | | |
| Moritella | | |
| Psychrilyobacter | | |
| Lactobacillaceae | | |
| Virgibacillus | | |
| Eubacteriales | | |
| Sporosarcina | | |
| Nocardiopsaceae | | |
| Balneolaceae | | |
| Ureibacillus | | |
| Azoarcus | | |
| Pseudidiomarina | | |
| Rhodobaca | | |
| Sporanaerobacter | | |
| Beijerinckiaceae | | |
Revision editor(s): Victoria
Signature 2
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: decreased abundance
in
Post-fed group at 1600 h
| NCBI | Quality Control | Links |
|---|
| Cryomorphaceae | | |
| Alteromonadales | | |
Revision editor(s): Victoria
Experiment 4
Differences from previous experiment shown
Subjects
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Post-fed group at 2000 h
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- The post-fed group consists of fish fed at 2000 hours. (9 hours post-feeding)
- Group 1 sample size Number of subjects in the case (exposed) group
- 17
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- decreased
Signature 1
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: increased abundance
in
Post-fed group at 2000 h
| NCBI | Quality Control | Links |
|---|
| Alcaligenaceae | | |
| Anoxybacillus | | |
| Arcobacter | | |
| Azoarcus | | |
| Bacillales | | |
| Bacillus | | |
| Bacteroidales | | |
| Balneolaceae | | |
| Beijerinckiaceae | | |
| Cetobacterium | | |
| Chromatiaceae | | |
| Citrobacter | | |
| Erwinia | | |
| Eubacteriales | | |
| Geobacillus | | |
| Hydrogenophaga | | |
| Jeotgalicoccus | | |
| Lactobacillaceae | | |
| Lactobacillales | | |
| Lactobacillus | | |
| Lactococcus | | |
| Leuconostoc | | |
| Moritella | | |
| Nocardiopsaceae | | |
| Paracoccus | | |
| Pasteurellaceae | | |
| Peptostreptococcaceae | | |
| Peptostreptococcus | | |
| Photobacterium | | |
| Planococcaceae | | |
| Prevotella | | |
| Proteus | | |
| Pseudidiomarina | | |
| Psychrilyobacter | | |
| Psychrobacter | | |
| Psychromonas | | |
| Rhodobaca | | |
| Sporanaerobacter | | |
| Sporosarcina | | |
| Staphylococcaceae | | |
| Staphylococcus | | |
| Tepidimicrobium | | |
| Thermus | | |
| Ureibacillus | | |
| Vagococcus | | |
| Veillonellaceae | | |
| Vibrionaceae | | |
| Virgibacillus | | |
| Yersinia | | |
| Xanthomonadaceae | | |
Revision editor(s): Victoria
Signature 2
Source: Supplemental Table S2
Description: The table shows the diverse microbial groups that fluctuated in abundance over the 2-day sampling period.
Abundance in Group 1: decreased abundance
in
Post-fed group at 2000 h
| NCBI | Quality Control | Links |
|---|
| Alteromonadales | | |
| Cryomorphaceae | | |
| Opitutae | | |
| Streptococcus | | |
| MarinicellaceaeMarinicellaceae | | |
Revision editor(s): Victoria
