Tardigrade community microbiomes in North American orchards include putative endosymbionts and plant pathogens

Abstract

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.

FluorescenceIn SituHybridization Provides Evidence for the Presence of the Endosymbiotic Bacterial GenusRickettsiain Tardigrades

“…The copyright holder for this preprint this version posted December 1, 2022. ; https://doi.org/10.1101/2022.12.01.518763 doi: bioRxiv preprint that tardigrades infected with Rickettsia or other endosymbionts may exhibit altered reproductive physiology (Guidetti et al, 2019b;Kaczmarek et al, 2020;Tibbs-Cortes et al, 2022;Vecchi et al, 2018). It is possible that the putative Rickettsia identified in this work may affect the reproduction of the host Paramacrobiotus sp.…”

“…Contamination is a major concern in microbiome studies of low microbial biomass samples such as tardigrades (Davis et al, 2018; Eisenhofer et al, 2019; Karstens et al, 2019; Tibbs-Cortes et al, 2022). Notably, our recent survey of tardigrade microbiomes identified and removed 986 contaminant taxa using in silico methods, including five of what would otherwise have the top ten most abundant bacterial taxa in the study, despite the use of recommended sterile laboratory techniques.…”

“…We previously analyzed the microbiota of tardigrade communities collected from apple orchards in central Iowa, USA. The OTU classified as Rickettsia was most abundant in the tardigrades from lichen samples collected in 2020 at Location 1 (Tibbs-Cortes et al, 2022).…”

“…More recent studies have used 16S rRNA gene amplicon sequencing to survey the tardigrade microbiome in at least 13 tardigrade species collected from locations in Antarctica, Curaçao, Ecuador, Madagascar, and the United States, as well as in Italy, Poland, Sweden, and the United Kingdom. These surveys found that tardigrade microbiota are altered by laboratory culture and are distinct from and usually less diverse than the microbiota of their substrate (Boscaro et al, 2022; Kaczmarek et al, 2020; Tibbs-Cortes et al, 2022; Vecchi et al, 2018; Zawierucha et al, 2022). Proteobacteria has been consistently reported as the most abundant bacterial phylum associated with tardigrades, while Firmicutes, Bacteroidetes , and Actinobacteria have each been reported as highly abundant in at least three previous surveys of the tardigrade microbiota (Kaczmarek et al, 2020; Mioduchowska et al, 2021; Tibbs-Cortes et al, 2022; Vecchi et al, 2018).…”

“…The recent analysis by our group also yielded sequencing-based evidence suggesting the presence of endosymbionts in tardigrades. Specifically, an OTU (OTU 180) classified as a member of the genus Rickettsia was significantly more abundant in the tardigrade community microbiota than in the substrate (Tibbs-Cortes et al, 2022). Rickettsia is a diverse genus consisting of obligate intracellular symbionts with a wide variety of hosts including mammals, leeches, cnidarians, and insects (Davison et al, 2022; McGinn & Lamason, 2021).…”