The microbiome of tardigrades, a phylum of microscopic animals best known for their ability to survive extreme conditions, is poorly studied worldwide and completely unknown in North America. An improved understanding of tardigrade-associated bacteria is particularly important because tardigrades have been shown to act as vectors of the plant pathogen Xanthomonas campestris in the laboratory. However, the potential role of tardigrades as reservoirs and vectors of phytopathogens has not been investigated further. This study analyzed the microbiota of tardigrades from six apple orchards in central Iowa, United States, and is the first analysis of the microbiota of North American tardigrades. It is also the first ever study of the tardigrade microbiome in an agricultural setting. We utilized 16S rRNA gene amplicon sequencing to characterize the tardigrade community microbiome across four contrasts: location, substrate type (moss or lichen), collection year, and tardigrades vs. their substrate. Alpha diversity of the tardigrade community microbiome differed significantly by location and year of collection but not by substrate type. Our work also corroborated earlier findings, demonstrating that tardigrades harbor a distinct microbiota from their environment. We also identified tardigrade-associated taxa that belong to genera known to contain phytopathogens (Pseudomonas, Ralstonia, and the Pantoea/Erwinia complex). Finally, we observed members of the genera Rickettsia and Wolbachia in the tardigrade microbiome; because these are obligate intracellular genera, we consider these taxa to be putative endosymbionts of tardigrades. These results suggest the presence of putative endosymbionts and phytopathogens in the microbiota of wild tardigrades in North America.
The microbiome of tardigrades, a phylum of microscopic animals best known for their ability to survive extreme conditions, is poorly studied worldwide and completely unknown in North America. An improved understanding of tardigrade-associated bacteria is particularly important because tardigrades have been shown to act as vectors of the plant pathogen Xanthomonas campestris in the laboratory. However, the potential role of tardigrades as reservoirs and vectors of phytopathogens has not been investigated further. This study analyzed the microbiota of tardigrades from six apple orchards in central Iowa, USA, and is the first analysis of the microbiota of North American tardigrades. It is also the first ever study of the tardigrade microbiome in an agricultural setting. We utilized 16S rRNA gene amplicon sequencing to characterize the tardigrade community microbiome across four contrasts: location, substrate type (moss or lichen), collection year, and tardigrades versus their substrate. Alpha diversity of the tardigrade community microbiome differed significantly by location and year of collection but not by substrate type. Our work also corroborated earlier findings, demonstrating that tardigrades harbor a distinct microbiota from their environment. We also identified tardigrade-associated taxa that belong to genera known to contain phytopathogens (Pseudomonas, Ralstonia, and the Pantoea/Erwinia complex). Finally, we observed members of the genera Rickettsia and Wolbachia in the tardigrade microbiome; because these are obligate intracellular genera, we consider these taxa to be putative endosymbionts of tardigrades. These results suggest the presence of putative endosymbionts and phytopathogens in the microbiota of wild tardigrades in North America.
The effect of a saccharin-based artificial sweetener was tested on animal performance measures and on the microbial communities associated with the rumen content and with the rumen epithelium during heat stress. Ten cannulated Holstein-Friesian milking dairy cattle were supplemented with 2 grams of saccharin-based sweetener per day, top-dressed into individual feeders for a 7-day adaptation period followed by a 14-day heat stress period. A control group of ten additional cows subjected to the same environmental conditions but not supplemented with sweetener were included for comparison. 16S rRNA gene amplicon sequencing was performed on rumen content and rumen epithelium samples from all animals, and comparisons of rumen content microbiota and rumen epithelial microbiota were made between supplemented and control populations. Supplementation of the saccharin-based sweetener did not affect the rumen content microbiota, but differences in the rumen epithelial microbiota beta-diversity (PERMANOVA, P = 0.003, R 2 = 0.12) and alpha-diversity (Chao species richness, P = 0.06 and Shannon diversity, P = 0.034) were detected between the supplemented and control experimental groups. Despite the changes detected in the microbial community, animal performance metrics including feed intake, milk yield, and short-chain fatty acid (acetic, propionic, and butyric acid) concentrations were not different between experimental groups. Thus, under the conditions applied, supplementation with a saccharin-based sweetener does not appear to affect animal performance under heat stress. Additionally, we detected differences in the rumen epithelial microbiota due to heat stress when comparing initial, pre-stressed microbial communities to the communities after heat stress. Importantly, the changes occurring in the rumen epithelial microbiota may have implications on barrier integrity, oxygen scavenging, and urease activity. This research adds insight into the impact of saccharin-based sweeteners on the rumen microbiota and the responsivity of the rumen epithelial microbiota to different stimuli, providing novel hypotheses for future research.
Many ecdysozoans harbor endosymbiotic bacteria within their microbiota, and these endosymbionts can have a range of positive and negative effects on their hosts. Recent 16S rRNA gene amplicon sequencing studies have provided evidence for endosymbionts within the tardigrade microbiota. In a previous study amplicon study, we determined that sequences corresponding to the endosymbiotic genusRickettsiawere significantly more associated with tardigrades than with the substrate from which they were isolated. Here, we performed fluorescencein situ hybridization(FISH) using aRickettsia-specific probe, RickB1, to determine ifRickettsiacould be found in tardigrades. RickB1 and a probe targeting most bacteria, EUB338, colocalized within the tissues of tardigrades, indicating the presence ofRickettsia. We also performed FISH using RickB1 and a nonsense probe which allowed us to distinguish between false positives and true positives. This method revealed RickB1 signals in tardigrades that were not due to erroneous probe binding, providing further evidence thatRickettsiais present in tardigrades. Future research will be necessary to determine the effects, if any, of this endosymbiont on its tardigrade host.
The aim of this pilot study was to identify changes in both the rumen content and rumen epithelial microbiota in response to the supplementation of Sucram ® , a sodium-saccharin-based sweetener (Pancosma S.A./ADM Groups, Rolle, Switzerland). Rumen microbial communities are essential for animal growth and performance, and changes in these communities can have major effects on these parameters. Little or no research is available regarding how saccharin-based artificial sweeteners, fed to cattle in attempts to increase palatability and encourage feed intake, affect rumen microbial communities. The rumen epithelium and rumen content microbiota of five lactating Holstein-Friesian dairy cattle were compared before (baseline, BL) and after a 28-day supplementation of Sucram ® using Illumina MiSeq-based 16S rRNA gene sequencing. After supplementation of Sucram ® , significant changes in the abundance of specific taxa were detected: an increase in Prevotella and Sharpea species, a decrease in Treponema , Leptospiraceae , Ruminococcus and methanogenic archaea (p<0.05), but Sucram® did not affect the overall rumen microbial community structure. This is the first study to report an effect of Sucram ® on ruminant microbial communities.
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