Hydra is an evolutionarily ancient multicellular organism which has been used as a model system in developmental biology. Previously, a study has shown that microbes or microbial factors may play an important role in asexual reproduction by budding of hydra. However, no attempts were made to identify the microbes associated with hydra during budding. In the present study, we attempted to explore the microbial community structure of budding hydra using Ion Torrent PGM. Our study showed notable differences in the microbiota of budding and non-budding hydra polyps. Differences in many bacterial taxa were also observed when microbiota of sexually matured male polyps was compared with non-reproductive polyps. The present study brings out significant differences in the microbial community structure of hydra during reproductive stages. Elucidating the possible role(s) of microbes in the reproduction of hydra may provide an interesting insight into the evolution of interactions between microbes and multicellular animals.

The microbiota associated with a host organism is known to aid many physiological processes including nutrition, immunity, resistance against pathogen, reproduction, and development^[2]. In fact, host microbiota interaction is one of the significant events in evolution and occurs in organisms ranging from single-celled amoeba to humans. Many organisms have developed specialised mechanisms that assure persistence of the symbiosis in the host^[3]. Hydra, an evolutionarily ancient multicellular organism, also harbours microbes which are believed to aid several vital processes such as immunity, cellular composition, proliferation and budding^[4]. This association appears to be species-specific and is believed to be controlled by species-specific anti-microbial peptides called arminins^[5]. Although Fraune and Bosch (2007)^[6] showed that hydra polyps maintained in the laboratory for long periods of time exhibit similarities in microbial composition with their counterparts from the wild, a recent study suggested the influence of external environment on the microbial community structure of laboratory and wild populations of Hydra vulgaris Ind-Pune^[7].

The role of microbes in the major life processes of hydra was demonstrated by Rahat and Dimentman^[8] who observed a reduction in budding in hydra grown in a germ-free medium. Further, on inoculation of non-sterile medium budding resumed, suggesting a role of microbes and/or microbial factors in the event.

Although a lot has been speculated about the role of bacteria in budding in hydra, there are no reports on either the microbial compositions of budding polyps or their possible role in budding. The present study is the first step in understanding the differences in microbial composition of hydra polyps during non-reproductive stage as compared to budding or asexual reproductive stage.

Samples studied by us showed a total of four bacterial phyla (Fig. 1A). Among them phylum Proteobacteria was highly abundant (82%–90%) followed by Bacteroidetes (3%–9%) and Firmicutes (3%–8%). These three phyla contributed more than 97% of the total bacterial community. All the samples in the present study were found to be dominated by Proteobacteria. Compared to NR polyps, AR polyps exhibited less abundance of Proteobacteria, though this remains the most abundant phylum. Such high abundance of Proteobacteria was also seen in the laboratory-maintained and wild populations of hydra^[7]. Interestingly, Franzenburg et al.^[9] found newly hatched hydra to be abundant in bacteria belonging to phylum Bacteroidetes. We also found an increase in the abundance of Bacteroidetes in budding as compared to non-budding polyps suggesting a change in the microbial composition of hydra during asexual reproduction.

Principle coordinates analysis (PCoA) plots also showed predominant differences in the microbial composition of NR and AR polyps (Fig. 1B) suggesting the possible influence of budding on the microbiota of hydra or vice versa. The shift in the composition of microbes was also reflected in an altered abundance of different bacterial groups (Table 1). Although microbes belonging to Oxalobacteraceae are the routinely found microbes in hydra, studies in the past have suggested that high abundance of Oxalobacteraceae in hydra polyps inoculated with foreign microbiota^[5]. The precise role of Oxalobacteraceae cannot be ascertained with the available data but an increased abundance of microbes belonging to Oxalobacteraceae clearly suggests a change in the microbial composition of hydra during reproduction. Likewise, Pseudomonadaceae, Sinobacteraceae, Rhodobacteraceae, and Cytophagaceae also showed increased abundance in the budding polyps. Microbes belonging to Rhizobiales, on the other hand, showed higher abundance in the non-budding polyps.

Fraune et al.^[10] found a correlation between the cellular composition of hydra and its microbial community structure. Disturbance in cellular composition of hydra leads to a change in its microbial community structure. The budding event is characterised by changes in the cellular composition of hydra, especially it is associated with a significant rise in the number of interstitial cells and its derivatives^[11]. This could be the possible reason for alteration in the microbiota of hydra during budding. It has been speculated that bacteria or factors derived from bacteria may be necessary for epithelial homeostasis and proliferation leading to budding in hydra^[12][10]. In this study, we have shown for the first time that there is a significant change in the microbial composition of hydra polyps during budding. Our data demonstrate changes in several bacterial groups during budding, which could be directly or indirectly influencing the budding process itself. Moreover, Fraune et al.^[10] showed that removal of interstitial cells is associated with the decrease of microbes belonging to Proteobacteria. Based on this study, the increase in the abundance of bacteria belonging to Proteobacteria during budding of hydra observed by us could be due to an increase in interstitial cells.

As is the case for budding polyps, there is no information about the microbial community structure of hydra when it attains sexual maturity. Fortunately, we came across sexually mature male hydra (SMH) in our lab. The sexual maturity can be easily confirmed visibly by occurrence of motile sperm in the testes. When the microbial composition of male polyps was studied; to our surprise, we found significant differences in its microbial composition as compared to both non-budding (non-gonad-bearing) polyps and budding polyps (Fig. 1A and B, Table 1). At the deeper taxonomic level, SMH polyps showed a high prevalence of Oxalobacteraceae, Sinobacteraceae, Rhizobiales, Comamonadaceae, and Methylophilaceae. Franzenburg et al.^[9] have shown the temporal variation of microbial community structure of hydra during embryogenesis suggesting the possible influence of microbiota on the development of hydra. In fact, like many other organisms, hydra is also considered as the metaorganism wherein there is a continuous interaction between the host and its microbiota. The present observation on changing microbiota during asexual reproduction and sexual maturity strongly indicates a role of microbes in reproduction in hydra. It may be mentioned that despite several attempts we were unable to get any sexual matured female polyps.

Studies in the past have shown that microbiota not only provides fitness benefit to the host but also plays a significant role in its reproduction^[13]. To our knowledge, this is the first study reporting clear changes in microbial content during asexual reproduction and sexual maturity indicating microbial influence over a major life event. Our findings provide a framework for undertaking detailed studies to elucidate the role(s) of associated microbes in budding in hydra. Studies to explore possible cross-kingdom signalling as a mode of communication between the host and the associated microbes during important life events would be highly rewarding.

In the present study, our analysis is limited to the stage at which there is the first sign of the budding. The exploration of microbial community structure at different stages of budding would shed more light on its role in budding of hydra. In the present study, we have explored the microbial community structure of reproducing males. However similar analysis in reproducing females remains to be carried out.

It has been shown that budding occurs under favourable conditions and is directly proportional to feeding^[14]. However, the precise signalling molecule(s) involved in this phenomenon is yet to be identified. Studies have shown that metabolites of arachidonic acid (AA) play an important role in tentacle regeneration, cell proliferation and budding in hydra^[15]. Especially, 12 HETEs (hydroxy-eicosatetraenoic acids), metabolites of lipoxygenase activity, are known to induce budding in hydra^[15]. The observed increase in the abundance of order Proteobacteria during budding seems relevant as the members of this order are known to produce lipoxygenase enzymes^[16]. Further, AA and its metabolites are also known to induce chemotaxis, cell proliferation, regulation of enzymatic activity and cAMP formation^[17]. Budding in hydra is initiated by movement of cells at the point of budding, followed by differentiation of interstitial cells into the bud region. In this regard, AA and its metabolites could be key triggers for budding. Although the presence of AA and its metabolites have already been shown in hydra polyps^[17], their source is not known. Artemia larvae used as a feed for Hydra are a rich source of AA^[18]. It is likely that bacteria belonging to phylum Proteobacteria help in the breakdown of AA present in Artemia to produce its metabolites such as HETEs that result in the induction of budding. Suppression of budding in hydra on starvation^[12] supports this conjecture. AA and its metabolites play a key role in the metamorphosis of Hydractinia echinata^[19] and in starfish, these are known to control egg maturation^[20]. These studies also point towards the role of symbiotic bacteria in the reproduction of host. However, further detailed studies are required to elucidate the precise mechanism involved.

A previous study has indicated possible influence of environmental factors on the microbiota associated with hydra^[7]. For the present study, we have used data of non-reproductive adults from the previous study^[7] for comparisons with both budding and sexually reproducing hydra.

All the hydra polyps were grown under identical conditions (such as temperature 18°C and pH (8.0)). Polyps were visually inspected and divided into two groups, based on the presence or absence of buds. Polyps showing first visible signs of budding were considered as Asexually Reproductive Adults (AR) while polyps without buds were considered as Non-Reproductive Adults (NR).

DNA was extracted from five polyps from each sample (i.e. AR and NR) using UltraClean® Tissue and Cells DNA isolation Kit by MO-BIO (USA). Primers specific to the V3 region (314F and 518R) of bacterial 16S rRNA gene mentioned in^[21] were used to explore microbiota associated with hydra. Obtained amplicons were purified using 1.5X Agencourt AMPure XP Beads (Beckman Coulter, Inc., Brea, CA). The fragment size distribution and DNA concentration were checked using a Bioanalyzer 2100 (Agilent Technologies, USA). These amplicons were clonally amplified through emulsion PCR using an Ion Xpress Template 200 Kit (Life Technologies, USA). Amplicon libraries were sequenced using Ion Torrent PGM platform (Life Technologies, USA) using 316 chip.

All the good quality sequences were trimmed and filtered using Mothur^[22] based on the following criteria: average quality score–20, maximum length–150 bp, minimum length–130 bp, maximum homopolymer–6 and maximum ambiguity–0. QIIME software package^[23] was used to perform all the downstream analysis. Briefly, eukaryotic sequences were removed from datasets and then sub-sampled. Obtained sequences were clustered into OTUs using UCLUST at 97% cut-off. Taxonomic assignment was done using the RDP classifier with a confidence value of 0.8.

This study was funded by extramural grants from Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India and Emeritus Scientist grant from Council for Scientific and Industrial Research (CSIR), New Delhi to SG and a DST-PURSE grant to SSG. YSS wishes to thank Department of Biotechnology (DBT), Government of India for financial support.

We would like to acknowledge Mr. Somak P. Chowdhury for his help during sequencing.

The authors declare no conflicts of interest.

Not Applicable.

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