Tan AL et al. (2025), Microbial communities experienced during early ...

ECB-ART-54474

iScience 2025 Oct 21;2811:113767. doi: 10.1016/j.isci.2025.113767.

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Microbial communities experienced during early development shape the host immune system and epigenome.

Tan AL, Hendricks SF, Carter EE, Buckley KM, Strader ME.

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Shaping of the epigenomic landscape by local environments during early life stages could initiate adaptive developmental plasticity, allowing organisms to optimize their phenotypes for local conditions. Elevated seawater temperature can increase bacterial loads and pathogen spread, making it imperative to understand how physiological and molecular traits of marine organisms are impacted, especially the immune system. Experimentally manipulated temperatures and microbial environments during embryogenesis resulted in changes in larval morphology, immune cell development, and the epigenome of the purple sea urchin (Strongylocentrotus purpuratus). Development amid high microbial richness led to increased chromatin accessibility, predominantly in promoter regions, whereas elevated temperature altered very few chromatin regions but was associated with more differentially expressed genes. Larvae raised in high microbial richness also exhibited delayed immune system development. Overall, we show that early developmental environments strongly influence whole-organism and molecular phenotypes, with early exposure to microbes altering the host immune system and epigenome.

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	Figure 1. Microbial communities are distinct in low and high microbial richness seawater treatments(A) Schematic of the experimental design to create combinatorial treatments of microbial richness and temperature. Gametes from three male and two female adult sea urchins were pooled and embryos were reared in four environmental treatments. Each treatment was replicated in triplicate. Pluteus larvae were collected at 5 days post-fertilization (dpf) prior to the onset of feeding. LMR: low microbial richness artificial seawater, HMR: high microbial richness artificial seawater.(B) Faith’s phylogenetic diversity and Shannon diversity analyses of the microbial communities in seawater samples, based on 16S rRNA amplicon sequencing. For Faith’s, Tukey’s post hoc revealed significant differences between all treatments except 18°C LMR and 14°C LMR (pTukey = 0.626) and 18°C HMR and 14°C HMR (pTukey = 0.926). For Shannon, Tukey’s post hoc revealed significant differences between all treatments except 18°C LMR and 14°C LMR (pTukey = 0.603) and 18°C HMR and 14°C HMR (pTukey = 0.997). Other alpha diversity metrics are available in Figure S1. Letters indicate statistical significance; groups with different letters are significantly different. Boxes represent the median and first and third quartiles. Whiskers represent 1.5× the interquartile range.(C) Mean absolute microbial load of LMR and HMR seawater conditions based on qPCR for the 16S rRNA gene (pT-test = 0.009). Letters indicate statistical significance; groups with different letters are significantly different.(D) Principal coordinates analysis (PCoA) using weighted Unifrac distances of microbial communities in seawater samples. Full combinatorial design of the two temperatures and two microbial communities explained, in total, 90% of the variance.(E) Relative abundance of the top six microbial classes across all samples. “Other” contains classes contributing less than 1% relative abundance. Each column is an individual sample.
	Figure 2. Larval size is shaped by temperature and environmental microbial content(A) Representative larvae at the time of collection (5 days post-fertilization). Scale bar = 100 μm. LMR: low microbial richness artificial seawater, HMR: high microbial richness artificial seawater.(B and C) Quantification of larval size based on body length (B) and area (C). Larval arm length was calculated by measuring from the apex to each arm tip and averaged across all four arms. Body area was measured by outlining the body at a mid-point cross-section. The overall effect of microbial richness was significant (length and area: pANOVA < 0.001) but the effect of temperature was not (length: pANOVA = 1; area: pANOVA = 0.23). The effect of 18°C HMR was significantly different from 14°C HMR (length: pANOVA = 0.022; area: pANOVA 0.006). Individual dots represent individual larval sizes. Letters indicate statistical significance; groups with different letters are significantly different. Boxes represent the median and first and third quartiles. Whiskers represent 1.5× the interquartile range.(D) Correlation of Sulfitobacter (family Rhodobacteraceae) counts and mean larval length in the two microbial conditions (p = 0.004, adj. R2 = 0.5300). The gray shaded area represents a 95% confidence interval.
	Figure 3. Temperature and microbial content differentially affect larval chromatin accessibility and gene expression(A and B) Differentially accessible regions based on ATAC-seq. HMR was compared to LMR (A) and 18°C was compared to 14°C (B). Chromatin was considered differentially accessible if the FDR/adjusted p-value ≤ 0.05 (represented by the horizontal red line) and absolute log2 fold change ≥ 0.20 (vertical red lines); numbers represent how many sites met these criteria. LMR: low microbial richness artificial seawater, HMR: high microbial richness artificial seawater.(C) Principal coordinates analysis of ATAC-seq results. Full combinatorial design of the two temperatures and two microbial communities explained, in total, 73% of the variance.(D and E) Differentially expressed genes (DEGs) based on RNA-seq. HMR was compared to LMR (D) and 18°C was compared to 14°C (E). Genes were considered differentially expressed if FDR/adjusted p-value ≤ 0.05 and absolute log2 fold change ≥ 0.5 (represented by the horizontal vertical red lines, respectively); numbers represent how many genes met these criteria. DEGs induced by interactions of terms are shown in Figure S2.(F) Principal coordinates analysis (PCoA) of RNA-seq results. Full combinatorial design of the two temperatures and two microbial communities explained, in total, 74% of the variance.
	Figure 4. Environmental conditions cause extensive functional changes in gene expression(A) Venn diagram shows the total number of differentially expressed genes (DEGs) responding to HMR, 18°C, or both environmental conditions. LMR: low microbial richness artificial seawater, HMR: high microbial richness artificial seawater.(B) All DEGs plotted based on the log2 fold change due to HMR and 18°C. The box in the center marks the absolute log2 fold change ≥ 0.5 cut-off for significance. Position on the x axis shows expression change based on HMR vs. LMR while y axis position shows expression change based on 18°C vs. 14°C. Color coding indicates whether the DEG responded to microbial content, temperature, both, or neither environmental condition.(C) Venn diagram shows the total number of gene ontology (GO) terms enriched in HMR, 18°C, or both environmental conditions, following RNA-seq analysis.(D) GO terms enriched by both HMR and 18°C (see Figure S3 for GO terms enriched by only one environmental condition). Direction of delta rank indicates whether associated DEGs had higher or lower expression (positive and negative delta ranks, respectively) in HMR (compared to LMR) and 18°C (compared to 14°C). MR: microbial richness.
	Figure 5. Relationship between chromatin state and gene expression(A–C) Gene expression plotted against the distance of the corresponding ATAC-seq peak, if present, from the transcription start site (TSS) for all genes (A), microbial content-associated differentially expressed genes (DEGs) (B), or temperature-associated DEGs (C).(D and H) Distribution of microbial content-associated (D) or temperature-associated (H) differentially accessible regions of chromatin across genic regions.(E–G) Comparison of gene expression based on whether their promoter was open or closed for all genes (E), microbial content-associated DEGs (F), or temperature-associated DEGs (G). Across all genes in the RNA-seq dataset, the fold change was 1.2× higher when the promoter was open (E; pT-test < 0.001). For microbial content-associated DEGs, the fold change was 1.2× lower when the promoter was open (F; pT-test < 0.001). For temperature-associated DEGs, the fold change was 2.2× lower when the promoter was open (G; pT-test < 0.001).(I–L) Expression change of genes with significantly enriched gene ontology (GO) terms related to chromosome structure: chromosome organization, chromosomal region, and structural constituent of chromatin (I); nucleic acid metabolism: nucleic acid metabolic process, DNA metabolic process, and RNA metabolic process (J); transcription or translation: translation, protein-DNA complex assembly, ribosome, structural constituent of ribosome, ribosomal subunit, ribonucleoprotein complex, protein-DNA complex, and RNA binding (K); and transcription factors based on ontology of the S. purpuratus genome annotation60 (L). DEGs are plotted based on the log2 fold change due to HMR and 18°C. The box in the center marks the absolute log2 fold change ≥ 0.5 cut-off for significance. Position on the x axis shows expression change based on HMR vs. LMR while y axis position shows expression change based on 18°C vs. 14°C. Gene symbols are included for genes that had a significant expression change and verified orthologs (i.e., genes with LOC ID symbols were not labeled). LMR: low microbial richness artificial seawater, HMR: high microbial richness artificial seawater.
	Figure 6. Expression of developmental, metabolism, and transcription factor genes are altered by environmental conditions(A and B) Expression change of developmental (A) and metabolism-associated genes (B) in larvae at 5 days post-fertilization. Differentially expressed genes (DEGs) are plotted based on the log2 fold change due to HMR and 18°C. The box in the center marks the absolute log2 fold change ≥ 0.5 cut-off for significance. Position on the x axis shows expression change based on HMR vs. LMR while y axis position shows expression change based on 18°C vs. 14°C. Gene symbols are included for genes that had a significant expression change and verified orthologs (i.e., genes with LOC ID symbols were not labeled). LMR: low microbial richness artificial seawater, HMR: high microbial richness artificial seawater.(C) Motifs with known homology enriched in open chromatin regions, based on MEME-Suite analysis. Zinc-finger family members were also present in every treatment but were excluded from C and D for ease of data visualization.(D) Expression change of transcription factor genes with significantly enriched motifs. Zinc-finger family members were excluded because their functions are largely unknown in echinoderms. DEGs are plotted based on the log2 fold change due to HMR and 18°C. The box in the center marks the absolute log2 fold change ≥ 0.5 cut-off for significance. Position on the x axis shows expression change based on HMR vs. LMR while y axis position shows expression change based on 18°C vs. 14°C. The points are labeled according to the transcription factor family to which the genes belong.
	Figure 7. Immune system development is delayed by elevated temperature or high microbial richness(A) Representative image of 5 days post-fertilization (dpf) larva showing pigment cells (indicated by arrow) and midgut epithelium thickness measurements (red dotted lines). Three measurements were taken from around the midgut (see red dotted lines for example) and averaged to find the midgut epithelium thickness of each larva. Scale bar = 100 μm.(B and D) Quantification of pigment cells (B) and midgut epithelium thickness among treatments (D). Pigment cell counts and average midgut epithelium thickness were adjusted for body length. Pigment cell counts (B) were significantly effected by microbial content (pDunn's < 0.01) but temperature only had a significant effect in the LMR treatment (14°C compared to 18°C, pDunn's = 0.02). The larval midgut epithelium (D) was significantly thicker in the larvae at 14°C LMR compared to all other environmental treatments (pDunn's < 0.03). Letters indicate statistical significance; groups with different letters are significantly different. Boxes represent the median and first and third quartiles. Whiskers represent 1.5× the interquartile range. Boxplots of the uncorrected values are shown in Figure S5. LMR: low microbial richness artificial seawater, HMR: high microbial richness artificial seawater.(C and E) Correlation of adjusted pigment cell counts (C; p = 0.006, adj. R2 = 0.5348) and midgut epithelium thickness (E; p = 0.021, adj. R2 = 0.4050) with the total counts of Sulfitobacter (family Rhodobacteraceae) in the two microbial conditions. The gray shaded area represents 95% confidence interval.(F and G) Expression change in immune system genes (F) and known pigment cell markers and precursors (G). Differentially expressed genes are plotted based on the log2 fold change due to HMR and 18°C. The box in the center marks the absolute log2 fold change ≥ 0.5 cut-off for significance. Position on the x axis shows expression change based on HMR vs. LMR while y axis position shows expression change based on 18°C vs. 14°C. Gene symbols are included for genes that had a significant expression change and LOC IDs are listed for genes without verified orthologs.