Schuh NW et al. (2019), Bacterial Exposure Mediates Developmental Plast...

ECB-ART-48615

Front Immunol 2019 Jan 01;10:3014. doi: 10.3389/fimmu.2019.03014.

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Bacterial Exposure Mediates Developmental Plasticity and Resistance to Lethal Vibrio lentus Infection in Purple Sea Urchin (Strongylocentrotus purpuratus) Larvae.

Schuh NW , Carrier TJ , Schrankel CS , Reitzel AM , Heyland A , Rast JP .

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Exposure to and colonization by bacteria during development have wide-ranging beneficial effects on animal biology but can also inhibit growth or cause disease. The immune system is the prime mediator of these microbial interactions and is itself shaped by them. Studies using diverse animal taxa have begun to elucidate the mechanisms underlying the acquisition and transmission of bacterial symbionts and their interactions with developing immune systems. Moreover, the contexts of these associations are often confounded by stark differences between "wild type" microbiota and the bacterial communities associated with animals raised in conventional or germ-free laboratories. In this study, we investigate the spatio-temporal kinetics of bacterial colonization and associated effects on growth and immune function in larvae of the purple sea urchin (Strongylocentrotus purpuratus) as a model for host-microbe interactions and immune system development. We also compare the host-associated microbiota of developing embryos and larvae raised in natural seawater or exposed to adult-associated bacteria in the laboratory. Bacteria associated with zygotes, embryos, and early larvae are detectable with 16S amplicon sequencing, but 16S-FISH indicates that the vast majority of larval bacterial load is acquired after feeding begins and is localized to the gut lumen. The bacterial communities of laboratory-cultured embryos are significantly less diverse than the natural microbiota but recapitulate its major components (Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes), suggesting that biologically relevant host-microbe interactions can be studied in the laboratory. We also demonstrate that bacterial exposure in early development induces changes in morphology and in the immune system. In the absence of bacteria, larvae grow larger at the 4-arm stage. Additionally, bacteria-exposed larvae are significantly more resistant to lethal infection with the larva-associated pathogen Vibrio lentus suggesting that early exposure to high levels of microbes, as would be expected in natural conditions, affects the immune state in later larvae. These results expand our knowledge of microbial influences on early sea urchin development and establish a model in which to study the interactions between the developing larval immune system and the acquisition of larval microbiota.

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Genes referenced: LOC100887844

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	Figure 1. Filtration/antibiotic treatment and addition of adult-exposed water modify bacterial load and diversity in culture seawater. 0.22 Î¼m-filtered FSW with 100 U/mL penicillin/and 100 Î¼g/mL streptomycin (P/S), normal FSW, and FSW with 20% 40 Î¼m-filtered adult-exposed tank water (AEW) were plated on marine broth agar and incubated at 14â15Â°C. (A) plates photographed after 7 days. (B,C) Number (B) and diversity (C) of colonies counted after ~3 days. Error bars represent mean Â± SEM. nd, not detected; p < 0.05; p < 0.01; **p < 0.001, 1-tailed 1-sample t-tests vs. mean = 0 (vs. P/S, gray) or 2-tailed t-tests (FSW vs. TW, black). Figures represent 13 independent experiments.
	Figure 2. Bacterial microbiota are localized to the larval mouth and gut lumen. Bacterial colonization was visualized in 4-arm larvae using fluorescent 16S in situ hybridization. (A) 4 dpf laboratory-raised larvae grown in FSW + 20% 40 Î¼m adult-exposed tank water (top, AEW) or 0.2 Î¼m-filtered FSW + P/S (bottom, P/S). (B) 9 dpf larva raised in natural seawater. All scale bars = 50 Î¼m. hg, hindgut; mg, midgut; mo, mouth. In the literature, the midgut and hindgut are also referred to as the stomach and intestine. Images are representative of >5 independent experiments.
	Figure 3. The natural seawater and laboratory microbiomes in early larval development. (A,B) UniFrac-weighted principal coordinate analysis of operational taxonomic units (OTUs) associated with zygotes, 4 dpf larvae, and 11 dpf larvae raised in natural seawater (NSW) and in the laboratory (AEW). (C) Relative class-level abundance profiles across early development in NSW- and AEW-exposed larvae. (D) Venn diagram illustrating NSW-specific, AEW-specific, and overlapping larva-associated bacterial OTUs. (E) Differential phylogenetic diversity (Faith's index) over early development in NSW- and AEW-reared zygotes and larvae.
	Figure 4. Bacterial load perturbation induces morphological plasticity in S. purpuratus larvae. (A) Schematic diagram of a 4-arm pluteus larva indicating the morphological measurements made in this study. (B) DIC images of 6â11 dpf AEW- and P/S-raised larvae. Scale bars = 50 Î¼m. (C) Embryo size at 24 hpf. (D) Morphological measurements of P/S- FSW-, and AEW- raised larvae from 6â11 dpf. Statistics represent the results of a 2-tailed t test (C) or 2-way ANOVA with post-test Bonferroni testing between treatment pairs at each time point (D). Error bars represent mean Â± SD. ns, not significant (p > 0.05); ***p < 0.001.
	Figure 5. Cellular immune response and larval pathology during lethal V. lentus infection. (A) Unexposed larva. (B) Surviving larva at 2â3 days post exposure with residual immune activity. The midgut epithelium is hypertrophied and activated pigment cells are present at the ectoderm, pharyngeal/midgut epithelium (a.k.a. the esophageal and gastric epithelia), and coelomic pouch. A globular cell is patrolling the blastocoel. (C) Clumping and adherence of V. lentus to the larval ectoderm. Several globular cells and activated pigment cells are present at the ectoderm. (D) Skeletoectodermal penetration and larval lysis. The apical tips of the body rods have punctured the ectoderm. Many globular cells and activated pigment cells are present. Dashed white lines indicate the margins of lysed larval contents. (EâG) Quantification of changes in immune cell behavior in response to 105, 106, or 107 V. lentus cells/mL at 6 and 24 h post exposure. (H) Larval mortality in response to 105, 106, or 107 V. lentus cells/mL at 6 and 24 h post exposure. Lysed or skeletonized larvae were scored as dead. Scale bars = 50 Î¼m. ap, apex; mg, midgut; red asterisks, activated pigment cells; black arrows, aggregated V. lentus; white arrows, globular cells. Error bars represent mean Â± SEM. Statistics represent the results of KruskalâWallis with post-hoc Dunn's tests between experimental groups and unexposed controls. nd, not detected; ns; not significant, p < 0.05; p < 0.01, **p < 0.001.
	Figure 6. Bacterial exposure increases larval resistance to lethal V. lentus infection. AEW and P/S larvae were raised to 10 dpf and exposed to 107 V. lentus/ mL for 24 h. (A) DIC images of 10 dpf AEW- (left) and P/S- (right) raised larvae at 24 h post-exposure. Dashed lines indicate margins of lysed ectodermal contents and bacterial invasion. Arrows indicate clumps of adherent V. lentus. White asterisks indicate globular cells. ap, apex; mg, midgut. (B) Larval mortality at 24 h post-exposure to V. lentus. Mortality was scored based on lysed ectoderm and/or exposed skeleton. Scale bars = 50 Î¼m. Figures represent ~20 larvae/group observed in two independent experiments.

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