3.6.3. Tenericutes

Within the *Tenericutes* phylum, the class *Mollicutes* has frequently been associated with jellyfish. In particularly, in *A. aurita* it has been suggested that *Mollicutes* is a potential endosymbiont [35,36]. In addition, *Mollicutes* was detected in both studied representatives of Rhizostomeae, in the gastric cavity of *Cotylorhiza tuberculata* [34,43] and associated with the Mastiigiidae family. *Mollicutes* was also reported as part of the microbiome of Cubozoa [41] and frequently associated with ctenophores [44,45]. Most of the detected bacteria within the *Mollicutes* class were affiliated with either the *Spiroplasmataceae* or *Mycoplasmataceae* family. The unique characteristics of *Mollicutes* are the lack of a cell wall, small size and simple cell structure, reduced genome, and simplified metabolic pathways, all indicative of a parasitic lifestyle corresponding to their preferred habitat in jellyfish, the gastric cavity, and their suggested endosymbiotic relationship with jellyfish. These bacteria are widespread commensals or pathogens of humans, mammals, reptiles, fish, plants, and arthropods and have been reported also in cnidarians, such as corals [131,132], and are known for their antimicrobial resistance, all characteristic with the potential to be exploited for biotechnology. Their frequent detection as part of the jellyfish microbiome indicates that they are important members of the jellyfish-associated microbial consortium. However, it is important to note that these microorganisms are also one of the most common sources of cross-contamination in academic and biopharmaceutical production laboratories. For the *Spiroplasma*-like bacteria identified as the dominant microbes in the gastric cavity of *C. tuberculata*, the estimated genome was smaller than any other currently known genome of *Spiroplasmas*, which might be indicative of their intracellular lifestyle as predicted anaerobic fermenters. The apparently fit status of the analyzed jellyfish suggests that the intracellular *Spiroplasma*-like bacteria are commensals of *C. tuberculata* rather than pathogens [43]. Features of these bacteria with biotechnological potential are summarized in Table 2; however, the full biotechnological potential of *Mollicutes* associated with jellyfish remains to be explored as a major problem in research with these bacteria is the difficulty of cultivating them in vitro.

### 3.6.4. Minor Members of the Jellyfish Microbiome

*Betaproteobacteria* that were detected as a part of jellyfish microbiome affiliated with *Burkholderia*, *Achromobacter*, and *Cupriavidus* [37,40,44] were previously associated with degradation of PAHs, plastics, and xenobiotics in the marine environment [88–90]. Direct application of these features in biotechnology is apparent (Table 2). *Spirochaetes* found in association with Rhizostomeae, in particular with the Mastigiidae family, and with Cubozoa are known for their unique morphology, with tightly coiled spirals, and their motility. They are very widespread in marine habitats, but not much is known about their ecological role or their biotechnological potential. However, they might play an important role in the gu<sup>t</sup> microbiota of marine animals. *Spirochaetes* were reported in two jellyfish species in one stud, in which both species were collected at the same sampling location, an Indonesian marine lake [41]. This raises questions about a location-specific pattern and the methodological approach used by this particular study [41]. In any case, these bacteria include aerobic and anaerobic species, well-known pathogens (syphilis and Lyme disease), and mutualists, for example, inhabiting the guts of cows and termites ([41] and references therein), all features with biotechnological potential. Also, *Actinobacteria* were not common in the jellyfish microbiome; however, they were found in association with *A. aurita* [35,37] and with *M. leidyi* [26,45]. *Actinobacteria* is a bacterial taxon with the greatest biotechnological potential known [133]. *Firmicutes* probably represents a minor part of the jellyfish microbiome as well. However, since they are otherwise rarely recorded as part of natural marine microbial assemblages, their presence within the jellyfish microbiome might indicate that they play an important role in this consortium. They were recorded in association with Cubozoa [41] and with different ctenophores [45]. The biotechnological applicability of these bacteria (e.g., in remediation of polluted marine sediments, as probiotics in aquaculture) and their features (e.g., pathogenicity, resistance to high temperatures, irradiation, desiccation, wide range of fermentation pathways, source of toxins) are known (reviewed in Reference [6]). Within *Chlamydiae*, *Simkania*-like bacteria were found to be the most dominant microorganism in the gastric cavity of *C. tuberculata*. Based on their metagenome, Viver et al. [43] speculated that they are putative endosymbionts of a ciliate that was probably symbiotic with the jellyfish. These authors speculated that the ciliate might be involved in controlling the free-living microbial population within the gastric cavity through grazing, therefore reducing competition between the host and the specific bacterial population for food (i.e., copepods). Furthermore, the authors speculated that the role of the *Simkania*-like endosymbiont of a ciliate, for which metabolic modelling predicts an aerobic heterotrophic lifestyle, could be understood as a nested symbiosis (supported by size and genetic repertoire exhibiting a versatile lifestyle) or even potential pathogenic capability (supported by the repertoire of genes indicative

of virulence factors, and their relationship could be mutually beneficial for the host), features with biotechnological potential.

### **4. Conclusions and Future Research Directions**

Different taxonomic groups of jellyfish were studied for their associated microbiomes; but this review also reveals that the vast diversity of jellyfish as hosts remains to be explored (Figure 1). For instance, to our knowledge, not a single member of the Coronatae order was examined for its microbiome, while within Rhizostomeae, an entire suborder of Daktyliophorae remains unexplored and within Semaeostomeae, some members of the Drymonematidae family were never investigated for their microbiome. The WoRMS recognizes 69 genera of Scyphozoa comprising 191 species that populate different habitats, from tropical to polar regions, thus potentially harboring different microbes with unexplored biotechnological potential (Figure 1). Also, the Hydrozoa orders Actinulidae, Limnomedusae, Narcomedusae, and Trachymeduase remain to be investigated, and within Ctenophora several orders of the Tentaculata class remain to be explored.

**Figure 1.** Relative amounts of investigated species in gelatinous taxa. Number of species per taxon was assembled from World Register of Marine Species (WoRMS) database (accessed December 2018). In the Hydrozoa class, only the species with a pelagic stage in their lifecycle were considered, following the species list of Reference [134].

Based on existing data, we tried to depict some basic characteristics of the jellyfish microbiome. It seems that the jellyfish microbiome is distinct from the bacterial community of the ambient water, comprising bacteria known for their preference for a surface-attached lifestyle and in association with marine organisms. This implies a certain degree of specialization of the microbiome of jellyfish, which are potentially generalists or possibly generalist symbionts. To some extent, it appears that the microbiome is jellyfish species-specific. However, in some instances, there is evidence that the jellyfish microbiome also depends on the background microbial community in the ambient water, possibly for recruiting members. This would also sugges<sup>t</sup> a horizontal transmission of the microbiome to the outer body parts of the jellyfish. Accordingly, the microbiome associated with the outer body parts seems to exhibit a higher degree of similarity to the bacterial community in the ambient water and is also more diverse and variable than the microbiome associated with the inner compartments. It seems that the relationship between the microbiome of the inner body compartments and the jellyfish could be symbiotic, and that in this case, the mechanism of transmission could be vertical. The microbiomes of different life stages of jellyfish seem to vary, indicating a significant restructuring of the microbiome from the benthic to the pelagic stage of the jellyfish life cycle.

Our review of different methodological approaches used to study the jellyfish microbiome and the difficulties in comparing the available datasets calls for the establishment of more standardized and holistic sampling and analytic approaches. Also, as currently all hypotheses on the role and function of the jellyfish microbiome are rather speculative, we believe that metatranscriptomic, metaproteomics, and metabolomics should be applied, coupled with state-of-the-art microscopy techniques, to study the microbiome and the mechanisms underlying the associations to provide insight into the potential roles the microbiome might play in the ecology of jellyfish. Furthermore, we propose that investigations should be scaled down to the molecular level, i.e., the level at which microbial-mediated processes take place. Finally, regarding the biotechnological potential of the jellyfish-associated microbiome, the features of specific bacteria found to be associated with jellyfish that have potential application in blue biotechnology are, in fact, known characteristics of these bacterial strains or their closest relatives isolated from other marine organisms or substrates. To the best of our knowledge, no comprehensive study of biotechnological potential of the jellyfish-associated bacteria has been conducted to date. With this review, we provide insight into the jellyfish-associated microbiome and highlight its biotechnological potential, hoping to draw the attention of the blue biotechnology sector to explore jellyfish as a potentially grea<sup>t</sup> source of biotechnologically interesting microbes and the compounds they produce. Altogether, this will allow to fully exploiting the biotechnological potential locked in the jellyfish microbiome association.

**Author Contributions:** Conceptualization, T.T., A.M., V.T.; Investigation, T.T. and V.T.; Writing—original draft preparation, T.T.; Writing—review and editing, T.T., V.T., A.M., K.K., T.K., G.J.H.; Visualization, T.T., T.K., and K.K., Supervision, V.T. and G.J.H., Project administration, T.T., G.J.H., V.T.; Funding acquisition, T.T., G.J.H., V.T.

**Funding:** T.T. received funding from the European Union's Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie gran<sup>t</sup> agreemen<sup>t</sup> No 793778. V.T. and K.K. were funded by the Ministry of Higher Education, Science and Technology of the R Slovenia (ARRS P1-0237). T.K. was funded by project "Raziskovalci-2.0-NIB-529024" (contract number C3330-17-529024) by the Ministry of Higher Education, Science and Technology of the R Slovenia. G.J.H. was supported by the Austrian Science Fund (FWF) project ARTEMIS (project number: P 28781-B21).

**Acknowledgments:** T.T. would like to acknowledge Monika Bright for valuable discussions regarding some basic concepts discussed in the review.

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
