Symbiotic associations of metazoans with bacteria strongly influence animal biology since bacteria are ubiquitous and virtually no animal is completely free from them. Tardigrades are micrometazoans famous for their ability to undergo ametabolic states (cryptobiosis) but very little information is available on potential microbial associations. We characterized the microbiomes of six limnoterrestrial tardigrade species belonging to several phylogenetic lines in tandem with the microbiomes of their respective substrates. The experimental design enabled us to determine the effects of both the environment and the host genetic background on the tardigrade microbiome; we were able to define the microbial community of the same species sampled from different environments, and the communities of different species from the same environment. Our 16S rRNA gene amplicon approach indicated that the tardigrade microbiome is species-specific and well differentiated from the environment. Tardigrade species showed a much lower microbial diversity compared to their substrates, with only one significant exception. Forty-nine common OTUs (operational taxonomic units) were classified into six bacterial phyla, while four common OTUs were unclassified and probably represent novel bacterial taxa. Specifically, the tardigrade microbiome appears dominated by Proteobacteria and Bacteroidetes. Some OTUs were shared between different species from geographically distant samples, suggesting the associated bacteria may be widespread. Putative endosymbionts of tardigrades from the order Rickettsiales were identified. Our results indicated that like all other animals, tardigrades have their own microbiota that is different among species, and its assembly is determined by host genotype and environmental influences.

Tardigrades represent one of the most abundant groups of Antarctic metazoans in terms of abundance and diversity, thanks to their ability to withstand desiccation and freezing; however, their biodiversity is underestimated. Antarctic tardigrades from Dronning Maud Land and Victoria Land were analysed from a morphological point of view with light microscopy and scanning electron microscopy, and from a molecular point of view using two genes (18S, 28S) analysed in Bayesian inference and maximum-likelihood frameworks. In addition, indel-coding datasets were used for the first time to infer tardigrade phylogenies. We also compared Antarctic specimens with those from Italy and Greenland. A combined morphological and molecular analysis led to the identification of two new evolutionary lineages, for which we here erect the new genera Acanthechiniscus, gen. nov. (Echiniscidae, Echiniscoidea) and Mesobiotus, gen. nov. (Macrobiotidae, Macrobiotoidea). Moreover, two species new to science were discovered: Pseudechiniscus titianae, sp. nov. (Echiniscidae : Echiniscoidea) and Mesobiotus hilariae, sp. nov. (Macrobiotidae : Macrobiotoidea). This study highlights the high tardigrade diversity in Antarctica and the importance of an integrated approach in faunal and taxonomic studies. http://zoobank.org/urn:lsid:zoobank.org:pub:8AAB42BF-B781-4418-A385-DC80C18EC31D

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Data from a previous study showed that microbiomes of six tardigrade species are species-specific and distinct from associated environmental microbes. We here performed a more in-depth analyses of those data, to identify and characterize new potential symbionts. The most abundant bacterial operational taxonomic units (OTUs) found in tardigrades are classified, and their prevalence in other environments is assessed using public databases. A subset of OTUs was selected for molecular phylogenetic analyses based on their affiliation with host-associated bacterial families in tardigrades. Almost 22.6% of the most abundant OTUs found do not match any sequence at 99% identity in the IMNGS database. These novel OTUs include four putative tardigrade endosymbionts from Alphaproteobacteria (Anaplasmataceae and Candidatus Tenuibacteraceae), which are characterized by 16S rRNA gene analysis and investigated for their infection rates in: Echiniscus trisetosus, Richtersisus coronifer and Macrobiotus macrocalix. These putative endosymbionts have an infection prevalence between 9.1% and 40.0%, and are, therefore, likely secondary symbionts, not essential for tardigrade survival and reproduction. Using fluorescence in situ hybridization (FISH), we detected bacteria on the cuticle and within the ovary of E. trisetosus, suggesting possible vertical transmission. This study highlights the great contribution in biodiversity discovery that neglected phyla can provide in microbiome and symbiosis studies.