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.
A transmission electron microscopy (TEM) study was conducted to characterize the helium bubble distributions in tritium-charged-and-aged 304L and 21Cr-6Ni-9Mn stainless steel fusion welds containing approximately 150 appm helium-3. TEM foils were prepared from C-shaped fracture toughness test specimens containing delta (δ) ferrite levels ranging from 4 to 33 volume percent. The weld microstructures in the low ferrite welds consisted mostly of austenite and discontinuous, skeletal δ ferrite. In welds with higher levels of δ ferrite, the ferrite was more continuous and, in some areas of the 33 volume percent sample, was the matrix/majority phase. The helium bubble microstructures observed were similar in all samples. Bubbles were found in the austenite but not in the δ ferrite. In the austenite, bubbles had nucleated homogeneously in the grain interiors and heterogeneously on dislocations. Bubbles were not found on any austenite/austenite grain boundaries or at the austenite/δ ferrite interphase interfaces. Bubbles were not observed in the δ ferrite because of the combined effects of the low solubility and rapid diffusion of tritium through the δ ferrite which limited the amount of helium present to form bubbles and/or the limited resolution of TEM technique used to image the bubbles.
The effect of hydrogen on the fracture toughness properties of Types 304L, 316L and 21-6-9 forged stainless steels was investigated. Fracture toughness samples were fabricated from forward-extruded forgings. Samples were uniformly saturated with hydrogen after exposure to hydrogen gas at 34 MPa or 69 and 623 K prior to testing. The fracture toughness properties were characterized by measuring the J-R behavior at ambient temperature in air. The results show that the hydrogen-charged steels have fracture toughness values that were about 50-60% of the values measured for the unexposed steels. The reduction in fracture toughness was accompanied by a change in fracture appearance. Both uncharged and hydrogen-charged samples failed by microvoid nucleation and coalescence, but the fracture surfaces of the hydrogen-charged steels had smaller microvoids. Type 316L stainless steel had the highest fracture toughness properties and the greatest resistance to hydrogen degradation.
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