Microbial Ecology (2018) 76:467–481 https://doi.org/10.1007/s00248-017-1134-4

INVERTEBRATE MICROBIOLOGY

The Microbial Community of : Environmental Influence and Species Specificity of Microbiome Structure and Composition

Matteo Vecchi1 & Irene L.G. Newton2 & Michele Cesari1 & Lorena Rebecchi1 & Roberto Guidetti1

Received: 4 May 2017 /Accepted: 19 December 2017 /Published online: 15 January 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Symbiotic associations of metazoans with strongly influence 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 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 , tardigrades have their own microbiota that is different among species, and its assembly is determined by host genotype and environmental influences.

Keywords Endosymbiont . Microbiome . Rickettsiales . Symbiosis . Tardigrada

Introduction

Tardigrada (water bears; Fig. 1) is a of poorly studied known phylum has recently increased as a consequence of ecdysozoan animals, ubiquitous in their distribution (present some of their biological properties (see [3]). Thanks to their worldwide in marine and continental environments) and fa- abilities to withstand physical and chemical extremes, tardi- mous for their ability to survive strong environmental grades have become model organisms for space research stressors such as desiccation (anhydrobiosis) and freezing [4–9], and for the study of the molecular mechanisms behind (cryobiosis) (for a review see [1, 2]). Interest in this scarcely the anhydrobiotic process [10–13]. Moreover, due to their position in the tree of life, tardigrades are recognized as a key taxon in the study of animal phylogeny and evolutionary Electronic supplementary material The online version of this article – (https://doi.org/10.1007/s00248-017-1134-4) contains supplementary developmental biology [14 18]. Lastly, a recent controversy material, which is available to authorized users. has arisen about the tardigrade genome, specifically regarding the number of genes derived from horizontal gene transfer * Matteo Vecchi (HGT) between bacteria and the model tardigrade [email protected] dujardini (Doyère, 1840) [19–24]. There are other important reasons to be interested in the 1 Department of Life Sciences, University of Modena and Reggio microbiota of the tardigrades. Bacterial associations with Emilia, Via Campi 213/D, 41125 Modena, Italy metazoans are currently recognized as a factor influencing 2 Department of Biology, Indiana University, Jordan Hall 221, 1001 E. many different aspects of animal biology such as metabolism 3rd St., Bloomington, IN 47405, USA [25], immunity [26], behavior [27], and perhaps speciation 468 Vecchi M. et al.

influence their microbial community, perhaps constraining bacterial species that could develop a stable association with tardigrades, similarly to what was observed for [40]. Here, we characterized the tardigrade microbiota, paying particular attention to the following research questions: (i) Is the microbial community of tardigrades distinct from that of their habitat? (ii) Do tardigrades have a species-specific mi- crobial community? (iii) Do tardigrades have putative endo- symbionts? (iv) Is there a host-influenced phylogenetic signal in the microbial community composition of tardigrades? and (v) Does laboratory rearing influence the microbiome compo- sition of tardigrades?

Materials and Methods

Experimental Design and Analyzed Tardigrade Species

Fig. 1 Scanning electron microscopy microphotograph of the For this study, six limnoterrestrial tardigrade species were cho- Macrobiotus macrocalix on moss leaflets sen. Along with the tardigrade microbiomes, we also character- ized the microbiome of the corresponding substrates colonized [28]. Indeed, many animals seem to harbor their own specific by tardigrades. To answer our research questions, we character- communities of bacteria and these have been characterized for ized the microbiome associated with the same tardigrade spe- a wide range of animal taxa, including , cnidarians, cies (always along with the microbiome of its substrate) extract- insects, copepods, roundworms, and various spe- ed from different samples, and the microbiomes associated with cies [29–34]. different tardigrade species extracted from the same sample. However, little is known about microbial communities in The seven analyzed samples (S1–S7) were grouped according- the so-called minor phyla, including tardigrades. Microbial ly to the substrates and/or tardigrade species of interest in four communities associated with tardigrades are scarcely or spo- experiments (Exp. 1: S1–S3, Exp. 2: S4–S5, Exp. 3: S6, Exp. 4: radically documented, and prior informations are found main- S7) for a better representation of bacterial abundance and data ly as marginal notes in ecological or taxonomic papers (for a analysis. Sampling details are reported in Table 1. review see [35]). One of the few experimental studies on the The tardigrade species considered for this study belong to bacteria associated with tardigrades [36] suggested a non- different evolutionary lineages: trisetosus Cuénot, random association between tardigrades and bacteria. In that 1932 (, , ); work, phytopathogenic bacteria (Xanthomonas sp. and Acutuncus antarcticus (Richters, 1904) (Eutardigrada, Serratia marcescens Bizio, 1823) differed in their ability to Hypsibioidea, ); Ramazzottius oberhaeuseri persist in, or on, the tardigrade body. Generally, microbial (Doyère, 1840) (Eutardigrada, Hypsibioidea, symbionts can be both transmitted vertically from parents to Ramazzottiidae); Richtersius coronifer (Richters, 1903) offspring and acquired horizontally from the environment or (Eutardigrada, Macrobiotoidea, Richtersiidae); Macrobiotus from other individuals [37]. Therefore, microbial communi- macrocalix Bertolani & Rebecchi, 1993; and ties associated with tardigrades could comprise host-specific Paramacrobiotus areolatus (Murray, 1907) (Eutardigrada, bacteria, such as endosymbionts (as are often observed in Macrobiotoidea, ). The sampled population of many ; see [38]), and/or bacteria belonging to the E. trisetosus contained a mixture of different morphotypes Bcore microbiome^ (as observed in [39]). But the (E. trisetosus, E. medianthus, E. canadensis) belonging to bacterial communities of tardigrades would likely also contain the Echiniscus blumi-canadensis series (see [41]). The se- environmental and food-associated microbes. These hypothe- quencing of a fragment of the cox1 gene (DNA was extracted ses are not mutually exclusive, as the tardigrade microbiome from the animals with a modified HotSHOT protocol, see could consist of a mixture of lineages, including host-specific Supplementary Methods in Supporting Information and [42], microbes and environmentally derived ones, in different pro- and primers were taken from [43]) revealed that individuals of portions, and the number of which could vary for each tardi- the studied population of Echiniscus trisetosus (from S7) be- grade species. In addition, one might suppose that the longing to the three different morphotypes (canadensis, anhydrobiotic and cryobiotic capabilities of tardigrades could medianthus, trisetosus), all belong to the same species (for The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome... 469

Table 1 Experimental design and materials used in the study

Experiments Sample Sample detailsa Tardigrade Number of Replicates code species/ replicates group codes substratesb

Exp. 1 S1 Freshwater sediment frozen after collection Acutuncus 6S1_Acu1-6 Same tardigrade species Edmonson Point, Victoria Land, Antarctica antarcticus in different samples Lat. S 74.330733; Lon. E 165.135883 Substrate 5 S1_Sub 1-5 35 m a.s.l. (Lab. code C3647) S2 Dry freshwater sediment frozen after collection Acutuncus 6S2_Acu1-6 Terranova Bay, Victoria Land, Antarctica antarcticus Lat. S 74.709667; Lon. E 164.101433 Substrate 5 S2_Sub 1-5 69 m a.s.l. (Lab. code C3414) S3 Laboratory culture Acutuncus 5S3_Acu1-5 antarcticus Substrate 5 S3_Sub 1-5 Exp. 2 S4 Lichen (Xanthoria parietina) on tree dried after collection Ramazzottius 5S4_Ram1-5 Same tardigrade species Modena, Italy oberhaeuseri in different samples Lat. N 44.622366; Lon. E 10.943552 Substrate 5 S4_Sub 1-5 34 m a.s.l. (Lab. code C3970) S5 Lichen (Xanthoria parietina) on tree dried after collection Ramazzottius 5S5_Ram1-5 Monte Cenere, Modena, Italy oberhaeuseri Lat. N 44.312667; Lon. E 10.759817 Substrate 5 S5_Sub 1-5 797 m a.s.l. (Lab. code C3973) Exp. 3 S6 Moss (Orthotrichum cupulatum) on rock dried after collection Macrobiotus 5S6_Mac1–5 Different species in the Öland, Sweden macrocalix same substrate Lat. N 56.528867; Lon. E 16.491233 Richtersius 5S6_Ric1-5 35 m a.s.l. (Lab. code C3583) coronifer Substrate 5 S6_Sub 1-5 Exp. 4 S7 Moss (community composed by Grimmia montana, Grimmia Echiniscus 5S7_Ech1-5 Different species in the laevigata,andSyntrichia ruralis) on rock dried after collection trisetosus same substrate Sassomorello, Modena, Italy Paramacrobiotus 5S7_Par1-5 Lat. N 44.424787; Lon. E 10.738364 areolatus 670 m a.s.l. (Lab. code C3902) Substrate 5 S7_Sub 1-5 a Collected substrates containing tardigrades b Matrix (tardigrades or substrate) for which the microbiome has been characterized sequences accession numbers see Table S1 in Supporting the samples were desiccated and sealed in plastic bags under Information), corresponding to the lineage A in [41]. vacuum (S4–S7; for sample code see Table 1), or directly The reproductive mode and the sex ratio are known for the frozen (S1, S2), then stored at − 80 °C at the Department of populations of A. antarcticus [44, 45]andR. coronifer [46, Life Sciences at the University of Modena and Reggio Emilia 47]. Both are made up only of females that reproduce by (Italy) before processing. In addition, a sample (S3) from a parthenogenesis. The gender identification (presence of males laboratory culture was utilized (Table 1). This sample (S3) and females) of the populations of M. macrocalix, comprised specimens of A. antarcticus originally extracted P. areolatus,andR. oberhaeuseri was done by observing the from sample S2 and reared in flasks in the laboratory for type of germinal elements within the gonad of in toto animals 4 years and fed with axenically cultured algae stained with orcein according to the method indicated in [48]. (Chlorococcum sp.); for detailed culturing conditions see The gender identification of the population of E. trisetosus [44]. Samples S1, S2, and S4–S7 were rehydrated or thawed was done by observing the shape of the gonopore on in toto in tap water for half an hour and then sieved (sieves mesh of specimens mounted on slides in Faure mounting medium 500 and 37 μm). The debris collected from the 37 μmsieve [49]. Data about gender identification of these populations was resuspended in distilled water and live (moving) tardi- are reported in Table S2 of Supporting Information. grades were then collected with a clean glass pipette under a stereomicroscope. For the laboratory culture (S3), the sedi- Sampling ment with the animals was directly inspected under a stereo- microscope without any previous sieving. The tardigrade fau- Samples of mosses, lichens, and freshwater sediments were na present in each sample was examined microscopically after collected in Europe and Antarctica (Table 1). After collection, mounting tardigrade specimens on slides in Faure mounting 470 Vecchi M. et al. medium. Mounted tardigrades were then classified to species on Quant-iT™ PicoGreen® dsDNA Assay Kit DNA level by comparing them with the original species descrip- quantification. tions. The samples used in this study contained only one (sam- ples S1–S5) or two (samples S6–S7) tardigrade species easily Amplicon Sequencing and Bioinformatics differentiated from each other. Pooled amplicons were sequenced at the Indiana University Center for Genomics and Bioinformatics core facility DNA Extraction and Amplicon Library Generation (Bloomington, IN, USA) using an Illumina MiSeq and 250 paired-end cycles. Adapter sequences were removed from all In order to prepare tardigrades for DNA extraction, animals reads before raw processing of data using the program suite were washed twice in sterile distilled water after collection. mothur 1.36.1 [51]. Briefly, contigs were generated using the DNA was extracted from pools of 50 specimens (representing make.contigs command, and sequences were screened for am- one replicate; the number of replicates was according to biguous base pairs and length using screen.seqs (maxambig = species and sample; Table 1), after removal of as much water 0, maxlength = 300). Unique sequences were aligned on the as possible with a sterile micropipette. In order to prepare SILVA 16S reference alignment [52], trimmed to homologous substrates for DNA extraction, 500 μl of the substrate debris regions, and preclustered based on two nucleotide differences. suspension without tardigrades was pelleted and all the water Chimeras were detected and removed using the was removed with a micropipette. The number of substrate chimera.uchime command, and lineages found in blank sam- replicates according to the sample is presented in Table 1. ples and identified as potential contaminants DNA was extracted from each replicate of both tardigrades (Acidobacteria_Gp4_family_incertae_sedis, Gemmata, and substrate using a modified Epicenter MasterPure DNA Zavarzinella, Gp4-Armatimonadetes_gp5, Purification Kit (Epicenter, Madison, WI, USA) protocol. In Armatimonas_Armatimonadetes_gp1, Haliscomenobacter, brief, to lyse the microbial community efficiently, samples Roseomonas) were removed, as were sequences classified as were ground in liquid nitrogen, and then resuspended in chloroplasts, mitochondria, , or . Resulting 60 μl of lysis solution (supplied) with the addition of protein- sequences were clustered in OTUs (operational taxonomic ase K (0.66 μg/μl) and RNAse (0.16 μg/μl), before incubation units) with a 97% identity threshold and classified at a confi- at 65 °C for half an hour. The proteins were precipitated with dence threshold of 60 using the SILVA training set. MPC protein precipitation reagent. The DNA in the superna- tant was precipitated with isopropanol overnight at − 20 °C and then centrifuged at 4 °C at 10,000 g for 15 min. The DNA Data Analysis pellet was rinsed with 70% cold ethanol, dried and resuspend- ed in 30 μl of Tris-EDTA Buffer (TE). Extracted DNAs were A representative sequence for each OTU was extracted from then quantified using a Quant-iT™ PicoGreen® dsDNA the mothur-generated alignment and a maximum likelihood Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) phylogeny was generated with the software FastTree [53]on and a NanoDrop ND-1000 UV-vis (Thermo Fisher the CIPRES Science Gateway [54] with default parameters. Scientific). Due to differences in total amounts of DNA ex- Subsequent analyses were performed with the R software tracted from each sample, DNA from animals and controls packages Bphyloseq^ [55], Bvegan^ [56], and Bphytools^ (range 0.6–50.5 ng/μl) was utilized without dilution, while [57]. All samples were rarified to 10,000 sequences (about DNA from substrates (range 6.6–311.7 ng/μl) was diluted to the size of the smallest library), alpha-diversity parameters 4 ng/μl. This decision was made after preliminary tests to were estimated (for a complete list of the calculated indexes determine the best template concentration for amplification. see Table S3 in supporting information), and distance matrixes Additionally, due to the presence of PCR inhibitors, some (weighted Unifrac, unweighted Unifrac, Bray-Curtis, Jaccard) DNA samples (S4_Sub 1, S5_Sub 1, S5_Sub 5, S6_Sub 1, of the samples were computed. The distance matrixes were S7_Sub 5) were cleaned with a PowerClean-Pro DNA Clean- then used for the clustering with the Ward D2 method and Up Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) before ordination using non-metric multi-dimensional scaling. A dilution and amplification. PCR using barcoded Illumina two-way ANOVA on the calculated alpha-diversity indexes primers was performed by following the Earth Microbiome and a PERMANOVA on the distance matrixes were per- protocols [50], with the following differences: HF Phusion formed in order to test differences in microbiome diversity polymerase mix (New England BioLabs, Ipswich, MA) was and composition between the different samples. Statistical used and 3% dimethylsulfoxide (DMSO) was added to the tests beyond those performed in R were implemented using reaction mixtures before cycling at 98 °C for 45 s, 50 °C for mothur (Metastats [58]). The source of the microbial OTUs in 60 s, and 72 °C for 90 s. Amplifications for each sample were the animal replicates was computed with the R script performed in triplicate and pooled before normalization based Bsourcetracker-1.0.1^ [59]. This script calculates the fraction The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome... 471 of a Bsink^ microbiome (animals) that was derived from a six tardigrade species were obtained. The MiSeq 250PE run Bsource^ microbial community (substrates). resulted in a total of 10,657,127 reads, 53% of which The phylogenetic signal (i.e., the tendency of related species (5,740,721 with a range of 10,844–179,816 reads per repli- to resemble each other more than species drawn at random from cate; Table S4 in supporting information) passed quality con- thesametree[60]) of three bacterial community diversity index- trols (see BMethods^). The dataset was rarefied (i.e., subsam- es (D, H′ and λ) with respect to the tardigrade phylogeny was pled to about the size of the smallest library) to 10,000 se- measured with the Blomberg’s K-statistic [61]andtestedfor quences, and 16,795 OTUs (at 97% identity) were identified significance with the R package Bphytools^ using a tardigrade across the rarefied dataset. Due to the high number of OTUs obtained as follows. The 18S sequences of the found, a subset was chosen to be analyzed more in depth and six tardigrade species were downloaded from GenBank to be tested regarding its eventual association with tardigrades. (Accession Numbers: R. oberhaeuseri, AY582122; These selected OTUs, defined as Bcommon OTUs,^ had an E. trisetosus, JX114896; A. antarcticus, AB753790; abundance higher than 5% in at least one of the replicates for R. coronifer, AY582123; M. macrocalix, HQ604976; each experiment. The remaining OTUs were defined as Blow P. areolatus, DQ839602) and aligned on the MAFFT Web frequency OTUs.^ A total of 53 common OTUs were identi- Server [62] using the default parameters. A calibrated tardigrade fied. Forty-nine identified common OTUs were ascribed to six phylogeny was constructed with MrBayes 3.2.6 [65] (parameters bacteria phyla (Proteobacteria, Bacteroidetes, 5×107 generations, burnin 25%, two runs with four chains each, Verrucomicrobia, Actinobacteria, Gemmatimonadetes, and nst = 6, rates = invgamma, brlenspr = clock:uniform, clockvarpr Chloroflexi), while four common OTUs were unclassified. = igr) constraining the topology to the one found by [63]. The abundances of common OTUs are shown in Figs. 2 and A Bayesian tree of the common OTUs (defined as OTUs 3. In addition, a heatmap representation of the common OTUs present with an abundance of 5% in at least one of the repli- abundances is also presented in Fig. S1 of the supporting cates in the rarefied dataset, separately for each experiment) information. Richness estimation of the dataset showed that was obtained after aligning the sequences with the software the sequencing effort attained was 77.4–94.2% of the estimat- SSU-ALIGN (ssu-align and ssu-mask scripts as default pa- ed richness in the original dataset, and 34.8–88.6% in the rameters) [64] and MrBayes 3.2.6 [65] (parameters 5 × 107 rarefied dataset (Table S4). Rarefaction curves generated generations, burnin 25%, two runs with four chains each, nst (Fig. S2 in supporting information) were in agreement with = 2, rates = invgamma, statefreqpr = fixed (equal)). Model these estimates. parameters for all the phylogenetic reconstructions were esti- mated with the software JModelTest2 [66]. Both MrBayes and Alpha Diversity JModelTest2 were run on the CIPRES Science Gateway. For the alpha and beta diversity analyses, only the rarefied dataset In the rarefied dataset, the most abundant bacterial phylum was used. P values adjustment were performed when needed was the Proteobacteria, with an average abundance of 46.0% with the R package function p.adjust according to the of the reads, followed by Bacteroidetes (25.5%), Benjamini-Hochberg method. Actinobacteria (7.5%), Verrucomicrobia (4.5%), and Gemmatimonadetes (2.0%) (Fig. 4A). The remaining 11.5% Data Availability The 16S rDNA Illumina libraries are stored of reads belonged to other bacterial phyla or were unclassified in the DDBJ Sequence Read Archive under the BioProject (unable to be taxonomically assigned, given the training set). number PRJDB5471 (BioSamples SAMD00071624 to The microbiome of tardigrades tended to have a higher and SAMD00071633 and SAMD00071652 to SAMD00071659; statistically significant abundance of Proteobacteria (49.8%, runs DRR083827 to DRR083910). The commands used in p = 0.013; Table S5) with respect to their substrate (41.1%). mothur, the R script used, the .Rdata file containing a Similarly, the phylum Bacteroidetes was found to be more phyloseq object (with the OTU table, the table, abundant in the tardigrade microbiome (29.0%, p = 0.007; and the OTU phylogenetic tree), and the fasta file containing Table S5) than in the substrate microbiome (21.0%). a mothur generated alignment of a representative sequence Inversely, the bacterial phylum Actinobacteria was more (the most abundant) for each OTU are stored in Figshare un- abundant in the substrate (10.0%, p < 0.001; Table S5)than der the accession identifier doi https://doi.org/10.6084/m9. in the animal microbiomes (5.6%). The same pattern was ob- figshare.4546834. served for the bacteria phylum Gemmatimonadetes (4.6% in substrate microbiome vs 0.4% in tardigrade microbiome, p < 0.001; Table S5). Results The two-way ANOVA on the alpha-diversity measures [observed bacterial species diversity calculated as number of To identify the microbiota associated with tardigrades, 16S OTUs (observed diversity; D), Shannon diversity index (H′) rRNA sequences of 82 replicates across seven substrates and and Simpson diversity index (λ); Fig. 5; Table S3]revealed 472 Vecchi M. et al. The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome... 473

ƒFig. 2 Microbial communities found in the Antarctic tardigrade in wild and culture, in which A. antarcticus was found, com- Acutuncus antarcticus across three different substrates (Experiment 1) pared to those of the other species (for detailed two-way and in the tardigrade Ramazzottius oberhaeuseri across two different substrates (Experiment 2). Common OTUs are shown individually, ANOVA results and diversity indexes see Table S6 in while low frequency OTUs were grouped according to their maximum supporting information). abundance. For samples codes (S1-S5) see Table 1. Each column repre- Not one of the examined indexes (Supporting Information sents the average between 5 or 6 biological replicates (Table 1) Fig. S3) showed a statistically significant phylogenetic signal with respect to tardigrade phylogeny (D, K =0.700,p =0.374; significant differences between animal and substrate replicates H′, K =0.772,p =0.270;λ, K =0.994,p =0.121). for all the analyzed indexes (D, p <0.001;H′, p <0.001; λ, p < 0.001; Table S6). In contrast, comparing the different sam- ples (S1, S2, etc.), only the Simpson diversity index was sig- Beta Diversity nificant (D, p =0.890;H′, p =0.634;λ, p = 0.002; Table S6). A two-way ANOVA performed on the diversity indexes of Multivariate statistics (non-metric multi-dimensional scal- only the animal replicates revealed a significant effect of the ing—NMDS, clustering, PERMANOVA) identified a signifi- tardigrade species for all the indexes (D, p < 0.001; H′, cant difference between microbiomes of animals and their p < 0.001; λ, p < 0.001; Table S7), whereas for the sample, substrate microbiomes (PERMANOVA p < 0.001; Table S8 only D and H′ showed a significant effect (D, p <0.001;H′, in supporting information). The comparison among tardigrade p =0.003;λ, p = 0.563; Table S7). species confirmed statistical differences among them in In the tardigrades R. oberhaeuseri, R. coronifer, microbiome compositions (PERMANOVA, p < 0.001; M. macrocalix, P. areolatus,andE. trisetosus, the microbiota Table S9 in supporting information). Specifically, 68.3% of diversity, expressed with the abovementioned indexes, was the variance observed among the tardigrade microbiomes was found to be always lower in animals than that of their respec- explained by the host tardigrade species when the community tive substrates (Table S3;Fig.5). Only in the Antarctic species structure was taken into account (weighted Unifrac; Table S9). A. antarcticus (both wild and cultured specimens), the diver- In contrast, only 27.7% of the variance was explained by the sity of animal-associated microbiota tended to be equal to, if host tardigrade species when the community membership was not higher than, that of the substrates (Table S3; Fig. 5). This considered (Unifrac; Table S9). The microbiomes associated outlier was due to both a higher microbiome diversity of with animals clustered distinctly from those associated with A. antarcticus animals than that of the other tardigrade spe- substrates (Fig. 4A), and replicates clustered in separate cies, and a lower microbiome diversity of the substrates both groups according to their type (animals vs substrates) and

Fig. 3 Microbial communities found in different tardigrade species shown individually, while low frequency OTUs were grouped according collected from the same substrate: Macrobiotus macrocalix and to their maximum abundance. For sample codes (S6-S7) see Table 1. Richtersius coronifer from S6 (Experiment 3), Echiniscus trisetosus and Each column represents the average between 5 biological replicates Paramacrobiotus areolatus from S7 (Experiment 4). Common OTUs are (Table 1) 474 Vecchi M. et al.

Fig. 4 Tardigrade microbiota cluster separately from the substrate simplicity; bar plots show average composition in bacteria phyla for microbiota. (a) Clustering of replicates based on weighted Unifrac each cluster. (b) Ordination plot of Non-metric Multi-Dimensional distances of bacterial communities and ward.D2 clustering algorithm. Scaling of the samples based on the weighted Unifrac distances of bac- Experimental replicates clustered together and were collapsed for terial communities. For replicate group codes see Table 1

species. This pattern persisted regardless of the ecological which the tardigrades were sampled (compare A. antarcticus distance measure used (Fig. S4 in supporting information). and R. oberhaeuseri, for example) (Fig. 4B). The only excep- Similarly, the microbiomes of the animals clustered separately tion to this trend was the A. antarcticus sampled from the from those of their substrates (using NMDS ordination, along laboratory culture, in which the microbiome was divergent the second dimension), and the microbiomes grouped accord- from the microbiome of the wild A. antarcticus (Fig. 4B). ing to host species, and not according to the substrate on Again, this separation of replicates based on their type The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome... 475

Fig. 5 Boxplot of alpha-diversity indexes of bacterial communities among different replicates of tardigrades and substrates. For replicate group codes see Table 1

(animals vs substrates) and associated species was maintained six OTUs were present in all tardigrade species (Table S12 in also when NMDS was performed with different ecological supporting information). distance measures (Fig. S5 in supporting information). In R. oberhaeuseri, three OTUs (30, 166, 246) classified as The Bayesian microbial source tracking (Sourcetracker Rickettsiales (based both on mothur taxonomic assignation script) of the microbial OTUs in the tardigrade microbiome and phylogenetic inference, Fig. 6)werefound,althoughwith estimated that the contribution of the substrate to the tardi- different abundances between tardigrades from the two ana- grade microbiomes ranged from a minimum of ~ 2% to a lyzed samples (S4 and S5). These three OTUs are OTU30 (S4 maximum of ~ 65% with marked differences among the dif- average abundance 1.2%, p = 0.015; S5 average abundance ferent species and samples (Fig. S6 in supporting 1.5%, p = 0.182), OTU166 (S4 average abundance 4.3%, information). In this analysis, as in the analysis of alpha diver- p = 0.178; S5 average abundance < 0.1%, p = 0.420), and sity, A. antarcticus stood out when compared to the other OTU246 (S4 average abundance 5.4%, p =0.092;S5average species, with a higher proportion of the microbiome derived abundance < 0.1%, p =0.499)(TableS12). In M. macrocalix from the substrate in the wild animals from samples S1 and S2 from S6, the same Rickettsiales OTU30 was present with an (average 49.5% ± 8.7 SD) compared to the laboratory- average abundance of 37.1% and was significantly associated cultured animals of sample S3 (average 11.1% ±3.2 SD). In with tardigrades (Metastats p < 0.001; Table S12). In comparison, all the other tardigrade species (R. oberhaeuseri, E. trisetosus from sample S7, OTUs 6 and 7 were present with R. coronifer, M. macrocalix, P. areolatus, E. trisetosus) an average abundance of 24.0 and 25.7%, respectively. For showed a low contribution for the origin of microbiomes from these two OTUs, a strong association with E. trisetosus with their respective substrate, and identified microbiota was uni- respect to the substrate was found (OTU6 p <0.001;OTU7 form between different species and different samples (average p < 0.001; Table S12). In P. areolatus (still from S7), four 15.4% ± 7.1 SD; Table S10 in supporting information). OTUs (6, 7, 166, 1352) classified as Rickettsiales were pres- ent. Although these Rickettsiales OTUs made up a smaller Common Bacterial OTUs Associated with Tardigrade fraction of the P. areolatus microbiome, compared to what Species we observed for E. trisetosus, OTU6 and OTU7 were signif- icantly associated with P. areolatus (OTU6 average abun- The association of all common OTUs with tardigrade species dance 3.5%, p = 0.008; OTU7 average abundance 3.3%, p = or their substrates in the context of each experiment was tested 0.0161; Table S12). Additionally, in P. areolatus, two other with Metastats (Table S11 in supporting information). Among Rickettsiales OTUs (OTU166, average abundance 2.0%; common OTUs, six (OTUs 6, 7, 30, 166, 246, 1352) were OTU1352, average abundance 3.3%) were identified, al- classified by mothur or by phylogenetic inference as though a significant association with the host was not detected Rickettsiales (Alphaproteobacteria; Fig. 6). Reads from these (OTU166, p =0.354;OTU1352,p = 0.422; Table S12). 476 Vecchi M. et al.

Fig. 6 Bayesian phylogenetic tree of common OTUs of bacteria detected in tardigrades and their substrates. Nodes with support values > 0.50 are represented. OTUs in bold are unclassified at phylum level. The taxonomic affiliation and association with tardigrades or substrate (according to Metastats) are shown. Scale bar indicates number of changes per site

Other than Rickettsiales, we found other bacterial OTUs Discussion to be significantly associated with a tardigrade species or with different species from different samples. Some exam- This first in-depth analysis of the tardigrade-associated micro- ples of these OTUs worthy of mention are OTU2 classified biota allowed us to both characterize the host-associated bac- as Pseudomonas and found associated with A. antarcticus terial community and identify putative tardigrade (both wild and cultured, S1, S2, S3), R. oberhaeuseri (S4, endosymbionts. S5), and P. areolatus (S7) (Figs. 2 and 3;TableS12); OTU12, classified as Luteolibacter and associated with The Tardigrade Microbiota Is Distinct A. antarcticus (S1, S2), R. oberhaeuseri (S4), and from the Microbial Community of their Substrate R. coronifer (S6) (Figs. 2 and 3;TableS12); OTU22, un- (Habitat) classified at phylum level, but associated with M. macrocalix (S6), R. coronifer (S6), and P. areolatus We identified a significant difference between tardigrade (S7) (Fig. 3;TableS12); OTU26 classified at family level microbiomes and their substrate microbial communities in (Neisseriaceae) and found associated with P. areolatus (S7) terms of diversity and abundance of OTUs, as supported by (Fig. 3;TableS12); and lastly, OTU45 classified as member alpha and beta diversity metrics and Bayesian microbial com- of the family Oxalobacteraceae that was associated with munity source tracking. Two different patterns in the diversity R. coronifer (S6) (Fig. 3;TableS12). between tardigrades and their substrates were detected. A The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome... 477 much lower microbial diversity in tardigrades compared to observed in other metazoans [68, 69]. Based on composition, their substrates was detected in Ramazzottius oberhaeuseri, the effect of rearing on the microbiome of the tardigrade Macrobiotus macrocalix, Richtersius coronifer, Echiniscus A. antarcticus consists mainly of a decrease of Bacteroidetes trisetosus,andParamacrobiotus areolatus. This could be ex- and Verrucomicrobia coupled with an increase in plained by tardigrade selection of their associated microbial Proteobacteria with respect to wild specimens. The extreme community, inhibiting the growth of some bacterial species reduction of the fraction of the microbiome of cultured and promoting the growth of others. Another hypothesis is A. antarcticus derived from their cultured environment could that substrates, being complex matrixes and having a large indicate a coevolution of A. antarcticus with bacteria that are surface and volume, can host a high bacterial biomass and, present in its wild habitat. This association between some consequently, a numerous and diverse microbial community. bacteria species and A. antarcticus could be due to its selective In contrast, tardigrades, especially due to their small size, can feeding on peculiar bacteria taxa present in its wild environ- host a lower bacterial biomass, consequently affecting its di- ment. Once A. antarcticus was brought in laboratory culture versity. However, it has to be noted that these two hypotheses (see [44]), these bacteria are or could not be present in the (a selective host vs a small host) are not mutually exclusive. culturing environment and the tardigrades must rely on differ- Determining to what extent these two hypotheses explain the ent food sources (cultured algae; see [44]), leading to a differ- obtained data means, in part, to define which components of ent microbiome composition of reared specimens of the microbiota are under the host and/or under environmental A. antarcticus with respect to wild specimens. control. However, it should be noted that also in well-studied Identifying the effect of culturing procedures on tardigrade- systems, such as humans and mice [67], this task has been associated bacteria can inform us as to the stability and spec- challenging. The Bayesian source tracking analyses evidenced ificity of tardigrade microbiomes, can provide clues as to how that a low fraction (from 12.1 to 22.1%) of these tardigrade much of the results based on cultured animals can be translat- species microbiomes comes from the environment microbial ed to the wild environment, and can help us to manipulate the community. This fraction could represent bacteria ingested by microbiota in order to apply experimental approaches. For the tardigrades during their feeding activity, or bacteria that instance, determining which bacterial species inhabit tardi- are associated with tardigrades (e.g., on the cuticular surface) grades could help in the development of better quality control that can be also present as free living in the substrate, or it in sequencing reads before assembly, in order to establish the could have been carried on during the laboratory procedures. true fraction of the tardigrade genome derived from horizontal It must be stated that this estimated fraction could be gene transfer (HGT). Recent studies [19, 22] claimed that a underestimated as during the sample rarefaction,bacteria pres- surprising 17.5% of genes in the tardigrades were derived ent in the tardigrades that come from the substrate (where they from HGT with bacteria, and that these genes may contribute are in low abundance) may have been removed. to the tardigrade high stress tolerance. Several other authors, Notably, A. antarcticus in all the three samples (two wilds in contrast, claimed that this high value was due to an artifact and one cultured animal samples; Fig. 2) had comparable, if in the bacterial sequence filtering [20, 21, 23, 24]. not higher, diversity, with respect to its substrates (Fig. 5), Characterization of the bacteria closely associated with tardi- diverging from the general trend observed in all the other grades could help to sort out which genes are derived from studied tardigrade species. Moreover, the A. antarcticus authentic HGT events, and which may be contaminants from microbiome showed a strikingly higher diversity with respect the microbiota. to all the other analyzed species. These peculiar patterns of A. antarcticus could be due to species-specific features of this The Tardigrade Microbiome Is Species-Specific taxon or to its colonized habitats. For example, with respect to the other analyzed species, which exclusively colonize terres- This study demonstrates that the tardigrade microbiome is trial habitats (moss and lichen), A. antarcticus colonizes true species-specific, as supported by multivariate analyses of beta aquatic environments [44], other than mosses. The high diver- diversity (Fig. 4A–B). sity of the microbial community of cultured animals despite It can be hypothesized that the differences in the the low diversity of their culturing substrate could indicate an microbiomes among tardigrade species are due to the presence inherent high diversity of the A. antarcticus microbiome that of taxa-specific symbionts as could be the six Rickettsiales is independent from its habitat. OTUs (6, 7, 30, 166, 246, 1352). This is especially true for the tardigrade species (M. macrocalix and E. trisetosus)witha Laboratory Rearing Influences the Tardigrade high abundance of these bacteria. The genetic control of the Microbiota Composition host over the microbiome could be also a driver of this species specificity. For instance, this control could be determined (i) This study shows that laboratory rearing affects the by the production of antimicrobial peptides (AMPs), as ob- microbiome composition of tardigrades, similarly to that served in hydras [70]; (ii) by the selective feeding of the host 478 Vecchi M. et al. on different bacteria, as found in some nematodes [33]; and carnivorous diet [73], and the OTUs in question are rarely (iii) or by the specific physical attributes of the tardigrade present in the substrate colonized by P. areolatus. cuticle, such as the presence of pores, folds, and structures The potential presence of endosymbionts in tardigrades that can provide a sheltered environment for bacteria. All opens the door to several possible effects on the host by these these possible drivers of the species specificity are not mutu- bacterial symbionts. We use the word Bsymbiont^ here to ally exclusive and could have different influences on shaping encompass the full range of fitness effects from mutualism the microbiome of each species. For example, E. trisetosus, to pathogenesis. For example, Wolbachia, an and like all his congeners, has dorsal sculptured cuticular plates endosymbiont from the order Rickettsiales, can in- [49]. This sculpture is composed of holes and pits that can duce reproductive phenotypes in its hosts, including changing provide a sheltered environment for some bacteria. Bacteria reproductive modes and induction of parthenogenesis [74]. attached to the Echiniscus cuticle have been already noted by Parthenogenesis is a type of reproduction that is common in [71]. It is possible also to hypothesize that different species of tardigrades [75], and one of the tardigrade species the Echiniscus could host different bacteria based on (E. trisetosus) in which we found Rickettsiales symbionts is their different cuticular ornamentations. In species with probably parthenogenetic [41]. It is therefore possible to hy- smooth cuticle (like all of the other examined species), differ- pothesize that in the population of E. trisetosus used in this ent diets and production of microbiota-selecting macromole- study, as well as in other tardigrade species, parthenogenetic cules like AMPs could be the major factors influencing the reproduction might be induced by the infection of difference between different species. However, our lack of endosymbionts. knowledge on the exact dietary regime of tardigrades and on The presence of symbiotic bacteria in anhydrobiotic hosts their eventual production of AMPs makes it difficult to pro- such as tardigrades means that these symbionts must also en- pose a more specific hypothesis. The diversity of different dure desiccation to survive. Endosymbiotic bacteria have ex- microbial communities we found does not seem to correlate tremely reduced genomes, both in terms of base pairs and with the phylogeny of the sampled tardigrades. The statistical gene content [76, 77], and therefore, it is possible that genes power of the permutation test used for detecting Blomberg’s involved in desiccation tolerance have also been lost. phylogenetic signal is good when the number of analyzed Symbiotic bacteria present in tardigrades could exploit species is higher than 20 [61]. As consequence of the small tardigrade-produced protective molecules (such as proteins number of sampled tardigrade species, in this case only six, and metabolites) to survive desiccation stress. In support of the non-significant result of this test should not be considered this hypothesis, nuclear-encoded proteins are targeted to bac- definitive. terial symbionts in other systems (e.g., in aphids [78]), and Boothby et al. [13] found that tardigrade-specific proteins (cy- Tardigrades Harbor Putative Endosymbionts toplasmic abundant heat soluble proteins, CASH) can increase desiccation tolerance in heterologous systems such as bacteria We identified OTUs affiliated with the order Rickettsiales in and yeast up to 100-fold. Identifying the function of these tardigrades. Only a few bacterial orders are characterized ex- putative endosymbionts as well as the relative fitness effects clusively by an intracellular lifestyle (both mutualists and of the symbiosis on both host (tardigrade) and endosymbionts pathogens), and Rickettsiales (class Alphaproteobacteria) is will lead to new frontiers in tardigrade biology. one of them [72]. Six common OTUs detected in tardigrades Finally, as the mitochondria arose from the class belonged to Rickettsiales, both by classification or by infer- Alphaproteobacteria [79] and consequently show sequence ence on a phylogenetic tree. Three of them (166, 246, 1352) similarity with them, many mitochondrial genes (and numts, could be transient pathogens of tardigrades since they were i.e., nuclear mitochondrial DNA segments) in organisms as- not significantly associated with animals and were present in sociated with these bacteria may have been erroneously iden- variable abundances among the different replicates of the tified as Alphaproteobacterial homologs or vice versa, poten- same tardigrade species. In contrast, the other three OTUs tially leading to mistakes in the correct taxonomic assignment (6, 7, 30) were found strongly associated with different tardi- of these genes [80]. Thus, the presence of these Rickettsiales grade species (with variable abundances; see BResults^). We (Alphaproteobateria) is another aspect to be considered in fu- consider two explanations for these different abundances ture tardigrade genome sequencing projects and HGT-related among tardigrade species. First, it is possible that OTUs 6, studies. 7, and 30 are able to infect different tardigrade species, but with lower effectiveness in some of them. Second, despite the lack of direct evidence of the preying on Conclusions heterotardigrades, it is possible that the sequences of OTU6 and OTU7 found in P. areolatus were derived from food These data offer the first insights into the composition of wild items, as Paramacrobiotus includes species with a and cultured tardigrade microbiomes, including six different The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome... 479 species spanning a fraction of the diversity in the phylum 3. Goldstein B, King N (2016) The future of cell biology: emerging – Tardigrada and of the different habitats colonized by model organisms. Trends Cell Biol 26(11):818 824 4. Horikawa DD, Kunieda T, Abe W et al (2008) Establishment of a limnoterrestrial tardigrades. Just like other ecdysozoans, tardi- rearing system of the extremotolerant tardigrade Ramazzottius grades possess a microbiome distinct from their environment, varieornatus: a new model animal for astrobiology. Astrobiology including potential endosymbiotic bacteria, as observed in 8(3):549–556 mites [81], insects [82], [32], and nematodes 5. Jönsson KI, Rabbow E, Schill RO et al (2008) Tardigrades survive exposure to space in low Earth orbit. Curr Biol 18(17):R729–R731 [33]. Tardigrade microbiomes are under the influence of both 6. Rebecchi L, Altiero T, Guidetti R et al (2009) Tardigrade resistance environmental factors and host species. In contrast, the influ- to space effects: first results of experiments on the LIFE-TARSE ence of microbes on host biology (e.g., tardigrade feeding mission on FOTON-M3 (September 2007). Astrobiology 9(6): – behavior, cryptobiotic abilities, reproductive modes) is still 581 591 7. Rebecchi L, Altiero T, Cesari M et al (2011) Resistance of the almost completely unknown. This aspect of tardigrade biolo- anhydrobiotic eutardigrade Paramacrobiotus richtersi to space gy could be uncovered through manipulation of the microbi- flight (LIFE–TARSE mission on FOTON-M3). J Zool Syst Evol ota composition in culture and subsequent characterization of Res 49(s1):98–103 symbiont genomes. 8. Persson D, Halberg KA, Jørgensen A et al (2011) Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit. J Zool Syst Evol Res 49(s1):90–97 Acknowledgments We thank Kristian Hassel (Norwegian University of 9. Guidetti R, Rizzo AM, Altiero T, Rebecchi L (2012) What can we Science and Technology, Trondheim, Norway) and Renzo Rabacchi learn from the toughest animals of the Earth? Water bears (Museo Civico di Ecologia e Storia Naturale di Marano sul Panaro, (tardigrades) as multicellular model organisms in order to perform Modena, Italy) for the taxonomic determination of mosses in sample S7 scientific preparations for lunar exploration. Planet Space Sci 74(1): and lichens in samples S4 and S5, respectively. We thank K. Ingemar 97–102 Jönsson (Kristianstad University, Kristianstad, Sweden) for kindly pro- 10. Rebecchi L (2013) Dry up and survive: the role of antioxidant viding sample S6 and Mauro Mandrioli (University of Modena and defences in anhydrobiotic organisms. J Limnol 72(1s):8 Reggio Emilia, Modena, Italy) for providing some laboratory reagents. 11. Wang C, Grohme MA, Mali B et al (2014) Towards decrypting We additionally wish to thank Kathy B. Sheehan and MaryAnn Martin cryptobiosis—analyzing anhydrobiosis in the tardigrade (Indiana University, USA) for their support during laboratory work. using transcriptome sequencing. PLoS Finally, we thank the anonymous reviewers and the reviewer Diane One 9(3):e92663 Nelson for the constructive suggestions in order to improve the 12. Kondo K, Kubo T, Kunieda T (2015) Suggested involvement of manuscript. PP1/PP2A activity and de novo gene expression in anhydrobiotic survival in a tardigrade, ,bychemicalgenetic Authors’ Contributions This work is a part of the Ph.D. thesis of M.V. approach. PLoS One 10(12):e0144803 M.V. and R.G. designed and conceived the experiments. M.V. and M.C. 13. Boothby TC, Tapia H, Brozena AH et al (2017) Tardigrades use performed the laboratory work. M.V. and I.G.N. analyzed the data. intrinsically disordered proteins to survive desiccation. Mol Cell I.G.N., L.R., and R.G. provided reagents, instruments, and funds. M.V. 65(6):975–984 wrote the first draft of the manuscript. M.V.,I.G.N., M.C., L.R., and R.G. 14. Garey JR, Schmidt-Rhaesa A (1998) The essential role of Bminor^ participated in revising the manuscript. phyla in molecular studies of animal evolution. Am Zool 38(6): 907–917 Funding Information This research was supported by the Italian 15. Gabriel WN, McNuff R, Patel SK et al (2007) The tardigrade BProgramma Nazionale Ricerche in Antartide (PNRA)–Ministero Hypsibius dujardini, a new model for studying the evolution of dell’Istruzione dell’Università e della Ricerca (MIUR)^ as part of the development. Dev Biol 312(2):545–559 project BEvolutive and phylogeographic history of Antarctic organisms 16. Edgecombe GD, Legg DA (2014) Origins and early evolution of and responses by ecosystems to climatic and environmental changings^ arthropods. Paléo 57(3):457–468 (PdR 2013 B1/01) and by the BBando per il finanziamento di azioni di 17. 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