2.1.10. Ctenophora

The WoRMS lists over 200 Ctenophora species, but only a few have been examined for their associated microbial communities: among the *Tentaculata* class, *Mnemiopsis leidyi* [26,44,45,61], *Bolinopsis infundibulum* [45], and *Pleurobrachia pileus* (*Cydippida* order) [45], and only one species of the *Nuda* class, *Beroe ovata* [44,45].

Although Ctenophora are known to be parasitized by a variety of eukaryotes, including amoebae, dinoflagellates, and sea anemones [62–65], probably the first report on bacteria associated with Ctenophora was published by Moss et al. [61]. They observed rod-shaped bacteria in the ciliary structure (g-cilium) of the food grove of the lobate ctenophore *Mnemiopsis mccradyi* (now accepted as *Mnemiopsis leidyi* morphotype) that they described for the first time. Later, bacterial communities associated with *Mneniopsis leidyi* and its natural predator *Beroe ovata* were studied using terminal restriction fragment length polymorphism (T-RFLP), cloning, and sequencing of 16S rRNA genes by Daniels and Breitbart [44]. These authors found that the composition of bacterial communities associated with Ctenophora varied over time (sampling over three consecutive years and different seasons at the same location). *M. leidyi*-associated bacterial communities exhibited some degree of seasonal specificity, with families of *Proteobacteria* and *Bacteroides* being associated with specific months, while no such pattern was observed for *Beroe ovate*-associated bacterial communities. In general, the diversity of the Ctenophora-associated bacterial community was lower than the diversity in ambient seawater assemblages and included some known pathogens of sea anemones. *M. leidyi* was dominated by *Marinomonas* sp. (*Oceanospirillales, Gammaproteobacteria*), previously described in corals and sponges, which were not detected in the water column or in the microbiome of *Beroe ovata*, while the microbiome of *B. ovata* was dominated by *Rhodospirillaceae* (*Alphaproteobacteria*), followed by *Tenacibaculum aiptasiae* a5, originally described as a pathogen of sea anemones, which was not detected in *M. leidyi*. Interestingly, both ctenophores harbored cultured representatives of *Alphaproteobacteria* capable of hydrocarbon degradation, *Thalassospira*, and *Alcanivorax*. *M. leidyi* also contained *Nisaea*, a bacterial genus known to mediate denitrification and nitrite reduction. In both ctenophores, *Mycoplasma* and *Spiroplasma* sp., representatives of the phylum *Tenericutes* were present in low abundance, but were not detected in the surrounding water. Generally, many sequences associated with *M. leidyi* were closely related to bacteria previously described in various marine invertebrates, including corals, sponges, sea anemones, and bivalves, but also some *Betaproteobacteria*, previously reported from freshwater.

Dinasquet et al. [26] investigated the response of bacterioplankton to the presence of *Mnemiopsis leidyi* and analyzed the microbiota of its mesoglea and gastral space. As this analysis supplemented some core experiments and hence was not the main aim of the paper, only one sample of each compartment was analyzed. The resolution of the microbial community composition was high; however, as sequencing of 16S rRNA bacterial genes was performed with next generation sequencing technology 454 pyrosequencing. The gu<sup>t</sup> community was different from the mesoglea community and dominated by *Bacteroidetes* (*Flavobacteriaceae*, with the most dominant bacteria affiliated with *Tenacibaculum*) and *Alphaproteobacteria* (*Rhodobacteraceae*), but also contained *Cyanobacteria* and *Actinobacteria*. Since the examined individuals were starved prior to the analysis, these bacteria could be part of the gu<sup>t</sup> microbiome and were most likely not ingested with prey.

Hao et al. [45] investigated the microbiomes of several species of *Ctenophora*: *M. leidyi, Beroe* sp., *B. infundibulum*, and *Pleurobrachia pileus* collected from Helgoland Roads in the German Bight. Ctenophores were collected three times per week for two consecutive years, 10 individuals of each species each time (in total 496 specimens were analyzed), accounting for seasonal and interannual variability [45]. Total prokaryotic genomic DNA was extracted and analyzed using ARISA and 16S rRNA amplicon pyrosequencing. The four ctenophore species harbored species-specific bacterial communities. The largest difference in microbiome composition was found between *M. leidyi*, a highly invasive species in the region, and the other three species. The seasonal variability in microbiome composition was only analyzed for *M. leidyi*, as it was the only species present throughout all sampling seasons. The *M. leidyi* microbiomes collected in the summer clustered together, clearly separated from the microbiome composition associated with *M. leidyi* collected in autumn and winter. Altogether four major bacterial phyla were detected, *Proteobacteria, Tenericutes, Actinobacteria*, and *Firmicutes*, with *Proteobacteria* being the dominant phylum in all four ctenophore species. While *M. leidyi* and *P. pileus* were dominated by *Gammaproteobacteria*, *Beroe* sp. was dominated by *Alphaproteobacteria*, and *B. infundibulum* harbored roughly equal numbers of *Alpha*and *Gammaproteobacteria*. The major groups of *Alphaproteobacteria* in *Beroe* sp. were affiliated with *Rhodospirillaceae* (*Thalassospira*). The microbiome of *B. infundibulum* was dominated by *Oceanospirillaceae* (*Marinomonas*), while in *P. pileus*, *Pseudoalteromonadaceae* and *Moraxellaceae* were most abundant. *Beroe* and *M. leidyi* were also associated with a small percentage of *Vibrionaceae*.

### *2.2. Critical Overview of Methodological Approaches Used to Study Jellyfish Microbiome*

To depict the characteristics of the microbiome systems of jellyfish, the available datasets and the conclusions of different studies need to be compared. The methodologies used were different, making it occasionally difficult to directly compare the outcomes of these studies. Of particular relevance is that the taxonomic composition of the microbial communities was determined by applying different methods, but also different sampling approaches were used that might affect the relevance of the resulting data. The purpose here is not to criticize individual methodological approaches applied by different studies, since many of them used state-of-the-art techniques at the time they were performed. Rather, we want to point out that direct comparisons frequently cannot be made and general conclusions cannot be drawn when only limited comparable datasets are available.

Considering microscopy-based methods of enumerating microbes, the methodological spectrum spans from light and electron microscopy to epifluorescence and confocal laser scanning microscopy. Early studies applied light and electron microscopy to detect jellyfish-associated microbes and describe their morphology and location within the jellyfish host [31,33,38]. Later, epifluorescence and confocal laser scanning microscopy combined with specific oligonucleotide probes labeled with specific fluorochromes were applied, allowing localization of specific microbial taxa at varying phylogenetic resolutions ranging from kingdom- to strain-specific within specific jellyfish life stages and body compartments [35,43].

The range of techniques available to determine the phylogenetic composition of jellyfish microbiomes spans from culturing to culture-independent molecular biology-based techniques. Classical culturing, mostly applied in early studies, provides insight into only the cultivatable part of the microbial community, which presumably currently represents only 1–5% of the total community [66]. The first culture-independent techniques applied were DNA fingerprinting techniques, such as ARISA, DGGE, and T-RFLP (Table 1). These techniques allow recording of differences in the microbial community structure with varying resolution at low cost, but cover only those populations with a DNA content of 0.1–1% of the total community DNA. These fingerprinting techniques offer only limited possibility to identify the phylogeny of specific members of the microbial community. For example, the DGGE method has high resolution, in terms of detecting 1 bp differences among individual sequences and allows many samples to be analyzed and compared concurrently. However, to obtain information on specific phylogenetic groups of the bacterial community, one has to excise individual DGGE bands—each representing, in theory, different bacterial species/groups and clone and sequence them. In this process, especially the band-excision step is critical, as many bands are not separated well enough, which means that it is not possible to excise some and cross-contamination can occur during the procedure. Clone libraries using the 16S rRNA gene were also widely used as a culture-independent technique to study the phylogenetic composition of individual microbial communities. This approach allowed for in-depth phylogenetic analysis, as the obtained sequences were usually of good quality and long, allowing for classification even to the genus level. This approach, however, does not sufficiently describe the diversity of a given population, as it fails to detect the rare community members. Despite all these limitations and shortcomings, we believe that the early sequencing efforts provided valuable insights into dominant groups of bacteria associated with jellyfish, contributing important information to our understanding of jellyfish microbiomes. Due to these shortcomings and the labor-intensive nature of these methods, next-generation sequencing (NGS) techniques have been applied over the last decade to study the composition of jellyfish microbiomes with high resolution and efficiency. Next generation sequencing approaches have been rapidly developing since they were first introduced to marine microbial ecology by Sogin et al. [67], describing the rare biosphere using 454 pyrosequencing. Currently, the most frequently used NGS platform in microbial ecology is the Illumina MiSeq or HiSeq. Although the resulting data in different studies can be compared most easily, almost all publications on jellyfish microbiome (Table 1) have used different prokaryotic DNA extraction protocols, and different kits to construct DNA libraries, and frequently, different regions of the 16S rRNA gene were amplified with different sets of primers. All of these limits the comparability of the available sequencing datasets.

Nearly all of the studies conducted so far performed only bacterial 16S rRNA gene amplicon sequencing to obtain information on the phylogenetic diversity of the jellyfish microbiome. Therefore, hypotheses on the function and role of the microbiome associated with jellyfish and the relationships and interactions within host–microbiome associations are merely speculations based on our knowledge of specific microorganisms. The one exception to this is the study on the gastric cavity of *Cotylorhiza tuberculata*, in which a metagenome approach was used providing insight into the genetic potential of the microbiome [43]. To our knowledge no study on the metatranscriptome, metaproteome, or metabolome level of the jellyfish microbiome has been performed so far.

Studies have analyzed the jellyfish microbiome at different levels, investigating the degree of microbiome specialization, species specificity of the microbiome, jellyfish life stage and body compartment specificity, and jellyfish population specificity (Table 1). However, to our knowledge, a holistic approach covering all possible aspects in a single study has never been used. For example, the degree of specialization was addressed only occasionally by comparing the degree of similarity between the jellyfish microbiome and ambient water microbial communities [26,35,36,41,44]. Concurrent analyses of the jellyfish microbiome and the host's natural background, i.e., the pool of ambient water microbial community, would also provide insights into possible transmission mechanisms of the jellyfish microbiome (i.e., horizontal vs. vertical transmission).

Another important point to consider is the fact that most of the studies examined different jellyfish species collected from diverse marine systems, where jellyfish are continuously present or occur seasonally. A comparison of microbiomes associated with the same jellyfish species collected from different marine systems would enable us to understand the population specificity of the associated microbiome. However, this comparison is currently only possible for a few jellyfish families, i.e., Cyaneidae, Pelagiidae, Phialellidae, and Bolinopsidae, and in one single case at the genus level, i.e., for *Aurelia aurita* (Table 1). In addition, there are several studies on different jellyfish species from the same system, which allow us to speculate on the effect of the microbial community of the ambient water on the jellyfish microbiome [34,36,38,39,41,44,45]. The spatial patchiness and variability within specific systems have rarely been addressed [40,43], as mostly jellyfish collected from a single sampling location were analyzed. Studies have rarely accounted for the variability within jellyfish populations, as most are based on only a small number of specimens. Furthermore, only few studies have accounted for the interannual [40,43] and seasonal [37,44,45] variability of jellyfish–microbe associations. With a single exception [42,43], no studies of specific jellyfish species from the same marine system are available where different groups of researchers have studied them. Consequently, no critical assessment of the reproducibility of the results can be made. Most of the studies were performed on jellyfish collected from their natural habitat, usually during bloom conditions, but some studies were conducted using jellyfish grown in the laboratory [35,40].

Jellyfish were collected and analyzed at different stages of their life cycle, but this comprehensive approach was applied only rarely [35,39,40]. To our knowledge, there is just a single study that investigated the microbial community associated with each life stage of a specific jellyfish (*Aurelia aurita*, [35]). However, even in this case, not all body compartments were analyzed, and in the future, the methodological approach should be improved to avoid possible cross-contamination.

### **3. Characteristics of the Jellyfish-Associated Microbiome**

The screening and critical overview of all published studies to date on the jellyfish-associated microbiome revealed some potential characteristics and patterns of jellyfish-associated microbial communities.

### *3.1. What is the Degree of Specialization of the Jellyfish-Associated Microbiome?*

Only a few attempts have been made to investigate whether there is a jellyfish-specific microbiome. Are these microorganism generalists and thus also present in the water column or on organic detrital particles? Are they generalist symbionts (i.e., can be found in association with other similar organisms) or specialists (i.e., are found only in association with jellyfish)?

The approach applied by these studies was to compare the diversity of the jellyfish-associated microbiome with the bacterial community in the ambient water. The conclusion from these studies is that the jellyfish-associated bacterial community is significantly different in terms of composition and lower in diversity than the bacterial community in the ambient water. This was reported in all the studies on *A. aurita*'s microbiome from the North Atlantic coastal waters [36], Kiel Bight, Baltic Sea, English Channel, North Sea [35], and Northern Adriatic Sea [37]. In addition, this was also reported for the ctenophore *M. leidyi*, although only in one study so far [44].

The jellyfish-associated microbiome is frequently also found associated with other host organisms, such as corals, sponges, sea squirts, and sea horses, which sometimes also share similar lifestyle, morphological, and/or biochemical characteristics [37,40,41]. This indicates a certain degree of specialization of the jellyfish-associated microbiome, suggesting that the jellyfish microbiota could be symbiotic generalists. Some studies concluded that at least certain members of the jellyfish-associated microbiome are specific for certain jellyfish taxa. For example, a comparison of the microbiomes of *Chrysaora* and *Aurelia* revealed a certain number of shared bacterial species, suggesting that they are specific for scyphozoans in general [40]. However, one should be careful when drawing general conclusions from a limited number of investigations performed with different methods. There is

evidence, however, that some members of jellyfish-associated microbiomes are rarely present in ambient water microbial assemblages and are more frequently found in association with similar organisms, probably due to their ability to attach and thrive under the specific environmental conditions (e.g., high viscosity, high nutrient, and low oxygen concentrations, presence of toxins and other antimicrobial compounds) that these organisms have to offer.

The observed low-diversity microbiome of jellyfish is in contrast to the trend observed for corals and sponges, where the diversity of associated microbiomes is usually higher than that in the ambient water [11,68–70]. One possible explanation is the production of antimicrobial compounds by jellyfish or their associated microbiomes. As discussed by Cleary et al. [41], *A. aurita* produces an antimicrobial peptide, named aurelin, that is active against Gram-positive and -negative bacteria [71]. Likewise, extracts of *Cassiopeia* spp. showed strong antimicrobial activity against bacteria [72]. Antibacterial polyketides were also isolated from a fungal symbiont (*Paecilomyces variotii*) of the jellyfish *Nemopilema nomurai* [73]. Thus, survival in this specific environment might be possible for only some bacterial taxa, leading to reduced diversity within the jellyfish-associated microbiome. In conclusion, the degree of specialization of the jellyfish-associated microbiome needs to be further studied, expanding to different taxonomic groups of jellyfish, marine systems, and biogeographic provinces.

### *3.2. Is the Jellyfish-Associated Microbiome Jellyfish Population-Specific?*

The fact that studied jellyfish were collected in different marine systems ranging from the North Atlantic Ocean to marine lakes in Indonesia (Table 1) might help to determine whether the microbiome is specific to certain jellyfish populations.

Jellyfish are known to be able to inhabit very different marine systems around the world, from shallow coastal seas to deep-sea environments, and, even more, seem to be able to quickly acclimatize to changing physical conditions (e.g., temperature, salinity [74,75]) and adapt to emerging features of marine habitats such as lack of predators/competitors caused by overfishing [76] or use of increasing marine sprawl, such as platforms as a surface for their polyp phase [77]. This could also be reflected in their associated microbiome. From the research conducted so far, the question of whether there is population specificity of the microbiome can only be addressed for *A. aurita*, as it was analyzed from different marine systems (North Atlantic [36], Kiel Bight, Baltic Sea, Southern English Channel, North Sea [35], and Northern Adriatic Sea [37]), and for *M. leidyi* from its native environment, Tampa Bay (Florida, USA) [44], and to areas where it was recently introduced, such as Gullmar fjord (Sweden) [26] and Helgoland Roads (German Bight) [45]. Interestingly, the results for the two jellyfish phyla contrast. Whereas in scyphozoan *A. aurita* the microbiome seems to be population-specific and hence varies with sample location, the ctenophore *M. leidyi* harbored a similar microbiota regardless of the sample location (e.g., *Marinomonas* was detected in ctenophore from all studied systems).

If we assume that the jellyfish-associated microbiome is determined to some extent by the environment, many more jellyfish ecosystems should be explored to better understand the selective pressure. In particular, one would expect differences in the microbiome of open-ocean (or even deep-sea) vs. coastal jellyfish, which are subjected to different degrees of anthropogenic impact. In particular, coastal environments are common entry points of pathogenic bacteria into marine ecosystems via coastal runoff, wastewater treatment plant discharge, or other kinds of human activities. Furthermore, as pathogenic bacteria are known to prefer organic-rich environments and are capable of surface attachment, they could easily hitchhike with bypassing jellyfish or, even more likely, attach to and thrive on polyps commonly found on biofouling at pillars in ports, on platforms, or on other structures. As known filter feeders, some of the organisms that dominate in biofouling communities, such as mussels and oysters, can accumulate pathogens and microbes that produce toxic compounds, which could be transmitted to polyps and from there to adult medusae, which can drift large distances. In this way, jellyfish can be seen as vectors for pathogens and other allochthonous bacteria from coastal to open water. This could be important in light of the recently modelled dynamics of jellyfish populations showing that a population of jellyfish can drift far away from its source polyp population area [77]. Another important unexplored aspect of the transmission route of the jellyfish microbiome is ballast water, potentially introducing invasive/nonnative species of both jellyfish and microbiota into new marine environments.

### *3.3. Is there a Jellyfish Taxa-Specific Microbiome?*

Several studies have compared the composition of microbial communities associated with different jellyfish taxa collected from the same environment (Table 1). In a study on an Indonesian marine lake, representatives from different medusozoan classes were collected, i.e., a jellyfish from the Mastigiidae family, representative of the Scyphozoa class, and a jellyfish from the Tripedaliidae family, representative of the Cubozoa class [41]. This study indicated that the microbiome is jellyfish taxa-specific [41]. Another study performed on scyphozoan jellyfish of the order Semaeostomeae, *Cyanea lamarckii*, a representative of Cyaneidae family, and *Chrysaora hysoscella*, a representative of Pelagiidae family, both collected in the German Bight, showed that there was a clear difference between the microbiome associated with the life stages of the two species, indicating specificity of the microbiome for certain jellyfish taxa [39]. In a study on two species of Cyaneidae, *Cyanea capillata* and *Cyanea lamarckii*, collected in Scottish coastal waters, their microbiomes were found to be species-specific [38].

### *3.4. Is the Jellyfish-Associated Microbiome Specific to Different Life Stages?*

Selecting jellyfish as a host seems to be an advantage or survival strategy for at least some bacteria. However, due to dramatic changes of lifestyle in the life cycle of jellyfish (benthic/attached vs. pelagic/swimming), morphology (i.e., surface architecture), and (bio)chemical characteristics (i.e., different expression patterns of antimicrobial compounds during different life stages), its associated microbiome needs both resiliency and plasticity. Each life stage might represent a unique niche allowing for specific bacteria to grow, and consequently, life stage specificity of the microbiome might be possible. From a jellyfish perspective, different life stages are associated with different necessities and requirements, which could drive corresponding shifts in the structure of associated microbiomes, as hypothesized by Lee et al. [40].

Microbial communities associated with different life stages of specific jellyfish species have been only rarely investigated [35,39,40]. Comparing the results of these studies, it seems that the composition of the jellyfish-associated microbiome changes with the life stage, particularly, during the transition from the benthic to the pelagic stages. The observed shifts in the community composition during the different developmental stages raise questions on the functional role of the microbiome in general and the development of jellyfish, as discussed by Weiland-Brauer et al. [35]. Certain members of the microbiome might have specific metabolic functions, which might play a role during specific development stages of the jellyfish [35]. However, available data on the diversity of the microbiome associated with different jellyfish life stages provide information only on the genetic potential or potential metabolism of specific bacterial groups. Hence, one can only speculate on the functions of specific bacteria in different life stages of jellyfish.

Our current knowledge of the bacterial colonization of scyphozoans or other jellyfish larvae, the establishment of the microbiome, and the compositional stability at different life stages, especially during strobilation, is limited. In jellyfish, however, bacteria have been shown to be important for larval settlement: it was shown that the settlement of pedal stolons of scyphopolyps of *A. aurita* was induced by *Micrococcaceae*, presumably via its effective substances acylgalactosidyldiglyceride and monogalactosidyldiglyceride [78,79], while in *Cassiopea andromeda*, swimming buds and planulae were induced to settle and metamorphose by a compound released from a *Vibrio* species during growth [80–83]. Regulation of the jellyfish life cycle, in particular the transition from one life stage to another and the induction of metamorphosis, is still only poorly understood. Recently, three major studies shed light on the regulatory mechanisms of the life cycle of jellyfish. One study focused on transcriptome profiling of the life stages of *A. aurita* [84], another study focused specifically on molecules critical in controlling the polyp-to-jellyfish transition [85], and the third study determined the genome of *A. aurita* [86], representing the first fully sequenced genome of the medusa stage of a cnidarian and providing important insight into the evolution of animal complexity. These analyses of life stage transcriptomes showed that different transcript expression profiles are related to each specific life stage and that not only shifts in ambient temperature, but also other signaling factors could initiate and regulate the strobilation and metamorphosis processes. It was shown that the nuclear hormone receptors, including the retinoic acid signaling cascade, are core elements of the regulation machinery of the life cycle of *A. aurita*, identifying strobilation inducer and precursor, novel CL390 protein, and its minimal pharmacophore, 5-methoxy-2-methylindole [85]. We argue that additional strobilation regulators might be present, and it is tempting to speculate on the role that ambient or associated microbes play in this process, especially as this was not tested in any of the studies conducted so far and the exclusive production of these compounds by the host has not been shown. One might speculate that similar or antagonistic molecules are produced by the jellyfish-associated microbiome in particular, since it was shown that bacteria by themselves or via their extracellular vesicles induce metamorphosis in marine invertebrates ([87] and references therein). The identification of strobilation and proliferation agents also has direct biotechnological application, as it might represent an avenue to control jellyfish blooms [85].

In addition, it was speculated that for the benthic life stage, the associated microbiome is particularly important for producing vitamins, amino acids, and secondary metabolites and possibly for inducing subsequent developmental stages [35]. Among bacteria associated with *A. aurita* polyps, *Phaeobacter* is known as a producer of compounds that inhibit fouling of surfaces of the host and substrates in general by interfering with cues for the settlement of invertebrate larvae and spores of algae [88–90]. Also, the polyp-associated bacteria of the genus *Rhodococcus*, the only exclusively marine genus within *Actinobacteria*, produce extracellular enzymes and are an exceptionally rich source of secondary metabolites, particularly compounds with the potential to serve as novel antibiotics and anticancer drugs (reviewed in Reference [91]). Polyp-associated bacteria of the genus *Vibrio* are known to produce quorum-sensing signals, and their antagonistic behavior was proposed to stimulate the settlement of spores of other organisms [88,92]. In a study of *Chrysaora plocamia*, polyps were proposed to form the base for a plethora of asexual reproduction strategies, and it is likely that they represent a reservoir of microbial members essential for the initiation, development, and survival of the subsequent life forms [40], while the authors hypothesized that the microbiota associated with *C. plocamia* podocyst are conserved within the podocyst capsule, where they help to sustain the viability of podocysts and potentially facilitate the activation of excystment [40].

Taking all this information together, it seems that a major restructuring of the polyp-associated microbiome takes place at the transition from the benthic to the pelagic stage, also in terms of reduced diversity. Different pelagic stages seem to be more similar to each other than to the benthic stage. The observed changes could be a response to different lifestyles, metabolisms, morphologies and biochemical characteristics of jellyfish, but could also be due to different environmental conditions that the microbiome experiences in the benthic vs. pelagic stage of the host.

### *3.5. Is the Jellyfish-Associated Microbiome Body Part-Specific?*

In jellyfish, different types of cells are present in different body compartments [93]. Body compartments are characterized by different morphological and biochemical features, as well as by their degree of contact with the ambient water. Thus, different body compartments might be preferentially colonized by specific microbes.

Few studies have investigated the compartment specificity of the jellyfish microbiome. In *A. aurita*, the differences between the microbiomes of the mucus and the gastric cavity were investigated [35]; in *Cyanea lamarckii* and *Chrysaora hysoscella*, the composition of the microbiomes of the tentacles, umbrella, mouth arm, and gonads was studied [39]; and in ctenophore *M. leidyi*, differences in the

microbiome composition between the gu<sup>t</sup> and tissue were investigated [26]. In other studies, jellyfish were analyzed as whole individuals, even when they were dissected prior to analysis (Table 1).

### 3.5.1. Microbiome Associated with Outer Body Parts and Its Potential Role

For all investigated species, compartment specificity of the jellyfish-associated microbiome has been reported, with outer body parts, e.g., umbrella and mucus, usually harboring more diverse, rich, and variable microbial communities than the inner body compartments, e.g., gonads and gastric cavity [26,35,37,39]. The degree of similarity between the microbiomes associated with outer body compartments and ambient water bacterial assemblages was higher than that between the microbiomes of inner body compartments. The jellyfish's outer body compartments are in direct contact with microbial communities of the ambient water, thus the microbiome of the outer compartments is probably directly recruited from those communities, indicating the possibility of horizontal transfer of the jellyfish's ecto-microbiome.

The epidermis and gastrodermis of the jellyfish contain numerous types of unicellular mucus-producing gland cells, leading to the formation of a thin, constantly renewed mucus layer covering the entire external surface of the medusa [94,95]. Under conditions such as stress and moribundity, but also during reproduction and digestion, mucus release rates are higher than under nonstress conditions [95]. Mucus contains toxins and nematocysts, thus serves as an important chemical defense mechanism of jellyfish and plays a major role in surface cleansing [95,96]. Jellyfish also produce toxins and antimicrobial compounds, such as the peptide aurelin in the mesoglea of *A. aurita* [71]. The mesoglea is an extracellular matrix situated between the epidermal and gastrodermal layers [97] containing collagen and collagen-like proteins associated with mucopolysaccharides [98,99]. Secreted mucus and the mesoglea are mainly composed of proteins, lipids, and, to a lesser extent, carbohydrates in different ratios [100,101], representing an attractive niche for bacteria, especially those with a competitive advantage and specialized for settling from the ambient water. The specific physiochemical characteristics of mucus and the physiology of the host represent selective pressure and determine the abundance and diversity of metabolically active bacteria [102]. Hence, one can hypothesize that jellyfish can actively or passively select their bacterial associates. Whether bacteria directly adhere to external cell layers of jellyfish or are only associated with the thin mucus layer remains to be resolved.

Based on the two studies that actually focused on the composition of the microbiome associated with jellyfish mucus, *Gammaproteobacteria*, particularly *Pseudoalteromonas* and *Vibrio*, are abundant, but to some extent also *Alphaproteobacteria* (*Phaeobacter*, *Rugeria*, and *Roseovarius*) [35,37]. These bacteria were previously recognized as important players in the host defense against pathogens and fouling organisms from the surrounding seawater [88,92,103,104] because of their ability to produce antimicrobial compounds when attached to live or inert surfaces [90,104–107]. Considering that the host would recruit microbes that are beneficial for its development or contribute to its well-being [92], one might speculate that the mucus-associated microbiome serves as a defense mechanism to protect the jellyfish from hostile microbes and other organisms in the ambient water. It was even proposed that the antimicrobial compounds and even toxins found in jellyfish mucus could originate from the associated microbiome (by Schuett and Doepke [38], who found tetrodotoxin-producing bacteria on jellyfish tentacles). At the same time, the mucus-inhabiting microbiome could benefit from constant nutrient input and/or other compounds from the host.

### 3.5.2. Microbiome Associated with Inner Body Compartments and Its Potential Role

The inner body parts of jellyfish, such as the gonads and gastric cavity, are isolated from the surrounding environment and have different morphological and biochemical characteristics, allowing for development of very specific bacterial groups that are otherwise rarely found in the ambient water. This is in line with reports on lower diversity, richness, and variability of the microbiome of the inner body compartments of jellyfish [26,35,37,39]. Reduced microbial diversity has been found in the gastric

cavity of *Cotylorhiza tuberculata* [42,43]. Since a considerable fraction of the bacterial community present in the inner compartments of jellyfish is rarely or never found in the ambient water, these bacteria may have lost the ability to live independently and may be acquired by the host via vertical transmission as parental heritage through reproductive cells and larvae [43]. Based on metagenomic analysis, the role of these bacteria is possibly related to food digestion and protection from pathogens [43].

### *3.6. The Composition, Potential Role, and Biotechnological Potential of the Jellyfish-Associated Microbiome*

Overall, it is not surprising that the jellyfish-associated microbiome is a consortium of bacteria (see below) that prefer a particle-attached lifestyle, are capable of degrading complex organic compounds, and are known to be found in association with other marine organisms. These bacteria are known for commensalism, symbiosis, and parasitism, or are even pathogens of marine organisms. Frequently, they are capable of producing quorum-sensing signal molecules, antagonistic compounds, and/or factors that interfere with quorum sensing of other microbes (which involves sensing the abundance of other bacteria, expressing of virulence factors, and interfering with the chemical communication of other bacteria, exhibiting antagonistic behavior). All of these features have either a direct or indirect application in blue biotechnology (Table 2). However, so far there has been no comprehensive study investigating the biotechnological potential of bacteria associated with jellyfish. Therefore, the listed features (Table 2) are based on the literature on the biotechnological potential of these bacterial strains or their closest relatives isolated from other marine organisms or substrates. Bacteria associated with jellyfish are also known for their diverse metabolisms and involvement in the cycling of carbon, nitrogen, sulfur, and phosphorus, which prompted several authors to speculate on their role in supplying specific compounds to jellyfish, implying that there is a symbiotic relationship between jellyfish and the associated microbiome (discussed in detail below). For example, it was proposed that nitrifying bacteria could play an important role in the life cycle of jellyfish [40], as they are known to harbor key enzymes involved in the conversion of ammonia to hydroxylamine and further to nitric oxide, the latter known as an important messenger molecule to regulate metamorphosis in marine invertebrates [108], regulate swimming of the jellyfish *Aglantha digitale* [109], and facilitate the discharge of nematocytes in the sea anemone *Aiptasia diaphana* [110]. All of these features have the potential to be exploited by blue biotechnology (Table 2). Several jellyfish-associated bacteria were previously also associated with the processing of more peculiar substances, such as PAHs, plastics, and xenobiotics found in the ocean, with possible benefits for the host [37,40]. For example, the presence of PAH-degrading bacteria agrees with the high tolerance of *A. aurita* to crude oil exposure and its ability to accumulate PAHs [111], suggesting that the PAH-degrading microbial community associated with *Aurelia* facilitates the survival of jellyfish in polluted coastal systems. This also agrees with the findings of Kos Kramar et al. [37], who detected PAH and plastic-degrading bacteria within the gastric cavity of *A. aurita* collected in the Northern Adriatic. The potential of these microbes and/or the compounds they produce to be used for biotechnological applications is obvious (Table 2). Marine archaea are known for their biotechnological potential (reviewed in References [112,113]); however, only one study so far made an attempt to investigate the archaeal community potentially associated with jellyfish [42]. However, amplification of archaeal 16S rRNA failed [42].

The most frequently detected members of the jellyfish microbiome are affiliated with the following representatives: *Alpha-* and *Gammaproteobacteria*, *Bacteroidetes*, *Tenericutes*, and *Cyanobacteria*. Among the less frequently, but still repeatedly reported were bacteria affiliated with *Betaproteobacteria*, *Spirochaetes*, *Actinobacteria*, *Firmicutes*, *Chlamyidiae*, *Chloroflexi*, *Planctomycetes*, *Nitrospirae*, and *Nitrospinae*. As we have shown that the associated microbiome is probably not exclusively associated with jellyfish and is to some extent affected by the physiochemical boundary conditions, the term "transient microorganisms" would probably best describe the jellyfish-associated microbiome [114].


**Table 2.** Overview of dominant bacteria found to be associated with jellyfish and their attributed features with biotechnological potential.



### 3.6.1. Gamma- and Alphaproteobacteria

Within the *Proteobacteria*, *Gammaproteobacteria* are reported to be associated with every comprehensively studied jellyfish taxon so far, where they either dominated or at least represented a very substantial part of the jellyfish-associated microbiome. The exception to this seems to be the jellyfish gu<sup>t</sup> microbiome, where either only a small fraction of *Gammaproteobacteria* was detected [42,43] or none at all [26]. Within *Gammaproteobacteria*, different families were regularly reported as a part of jellyfish-associated microbiome: *Vibrionaceae*, *Pseudoalteromonadaceae*, *Alteromonadaceae*, *Oceanospirillaceae*, *Shewanellaceae*, *Crenotrichaceae*, *Methylococcalaceae*, *Endozoicimonadaceae*, *Moraxellaceae*, *Xanthomonadaceae*, and *Legionelaceae*. In general, *Gammaproteobacteria* in marine environments are associated with the ability to attach to surfaces and to degrade high-molecular-weight compounds. Within this class, there are many readily culturable bacteria, such as *Vibrio*, *Alteromonas*, *Pseudoalteromonas*, *Marinomonas*, *Shewanella*, and *Oceanospirillum* [115]. This suggests that most of the jellyfish-associated *Gammaproteobacteria* could be readily cultured, facilitating their possible exploitation for biotechnological applications (Table 2). Furthermore, these bacteria usually exhibit rapid growth and a feast-or-famine lifestyle with quorum-sensing playing an important role. In addition, many of these bacteria are found in biofilms and/or in association with other marine organisms (as symbionts or pathogens) and occupy micro-niches with specific environmental conditions (i.e., temperature, oxygen, nutrient availability). All of the listed features have direct and/or indirect potential for application in biotechnology (Table 2). For example, *Marinomonas* was detected as an abundant member of the microbiome of the ctenophore *M. leidyi* in several marine systems. In terms of their biotechnological potential, these bacteria contain multifunctional polyphenol oxidases that are able to oxidize a wide range of substrates, are producers of antibacterial compounds, and are involved in biodegradation processes [116,117].

*Marinomonas* also contain genes for the breakdown of dimethylsulfoniopropionate, indicative of their role in the cycling of sulfur [44]. *Pseudoalteromonas* is known to produce a variety of highly bioactive compounds, including extracellular enzymes, exopolysaccharides, and compounds involved in antimicrobial antifouling, with algicidal activity and various pharmaceutically relevant activities [118]. *Vibrio* is known as a producer of quorum-sensing signals and for its antagonistic behavior, but also for its proposed role in stimulating the settlement of spores of other organisms [119].

Within the jellyfish-associated *Alphaproteobacteria*, representatives of the *Rhodospirillaceae*, *Rhodobacteriaceae*, and *Kiloniellaceae* families and the order *Rhizobiales* are documented (Table 2). Among them, members of *Rhodobacteriaceae* can easily be cultivated and are commonly found in association with living organisms and detrital particles, in sediment and microbial mats, playing an important role in carbon and sulfur cycles. The *Phaeobacter* genus of the *Rhodobacteriaceae* family is known as a producer of inhibitory compounds that prevent or inhibit fouling of surfaces by interfering with cues for the settlement of invertebrate larvae or spores of algae [88–90], a feature with potential for biotechnological application. One of the most reported *Rhodospirillaceae* associated with jellyfish are bacteria affiliated with *Thalassospira* [42,44,45]. Bacteria of the *Thalassospira* genus might be involved in carbon cycling by providing an additional source of fixed carbon for jellyfish and exhibit chemotaxis to phosphate (as suggested in Reference [44]). Bacteria from this genus were found in microbial consortia degrading aromatic hydrocarbons [120,121] as part of the microbiome of sabellids (Polychaeta, Annelida) in crude oil enrichments with potential production of biosurfactants [122], features with biotechnological potential. Bacteria of the *Kiloniellaceae* family are also producers of antibiotic compounds [123], with direct application in biotechnology.

### 3.6.2. Bacteroidetes, Flavobacteria, Flavobacteriaceae

Jellyfish-associated bacteria of the *Bacteroidetes* phylum are affiliated with the *Flavobacteriaceae* family, and were found in association with Medusozoa, within which with Semaeostomeae, in particular with Ulmaridae (*A. aurita*, [35,36]), with Pelagiidae (*P. noctiluca* and *C. plocamia*, [33,40]), with Rhizostomeae (*C. tuberculata*, [42,43]), and with Ctenophora, where they were not always

detected in the same species, suggesting that their presence within the ctenophore microbiome might depend on food and/or the environment [26,44,45]. *Flavobacteriaceae* were also detected in all studied Hydrozoa from different systems. Therefore, *Flavobacteria* represent an important part of the jellyfish-associated microbiome, as they are widespread within jellyfish. However, the association seems to be dependent on the host's natural environment to some degree. This bacterial group is known to be easily cultivated and its most distinctive properties are gliding motility and the expression of various extracellular hydrolytic enzymes to degrade complex organic materials, with potential application in biotechnology (Table 2). Some members are pathogenic and some psychrophilic, both characteristics with biotechnological potential. They can be found in the human gu<sup>t</sup> and, in sewage-polluted waters, but also in seawater, where they persist for a long time, thus were proposed as indicators of water quality [124], also with potential biotechnological application. Among the most frequently reported members of the *Flavobacteria* found in association with different jellyfish species are bacteria affiliated with the genus *Tenacibaculum*. *Tenacibaculum maritimum* is a known fish pathogen that causes tenacibaculosis, a disease considered to be an important threat to aquaculture worldwide [125]. *Tenacibaculum maritimum* was the first specific bacterium associated with jellyfish to be extensively studied, after the first report that it might infect fish gills damaged by jellyfish venom [126]. Consequently, 20 years ago, jellyfish, in particular *Phialella quadrata*, *Cyanea capillata*, and *Pelagia noctiluca*, were recognized as a possible cause of mass mortality of fish farmed in sea cages [30,127,128]. Transmission of this bacterium via the ambient water and direct transmission from host to host have been proposed as possible routes of infection, in addition to ingestion through food [129]. However, as the survival of *T. maritimum* in seawater is rather limited [130], its natural reservoir remains unclear. Ferguson et al. [32] provided the first evidence of *Phialella quadrata* carrying filamentous bacteria affiliated with *T. maritimum* and proposed that jellyfish is a vector and a carrier of this fish pathogen. These authors suggested that the presence of this proteolytic enzyme-producing bacterium in the jellyfish mouth could support the pre-digestion of the prey of jellyfish and that it could be specific to jellyfish, playing an important role in both immune defense and their nutrition [32]. In a subsequent study, the presence of *T. maritimum* in the mouth of *Pelagia noctiluca* was shown [33]. Supported by the results of both studies, Delannoy et al. [33] proposed that some cnidarians might represent a natural host for *T. maritimum*. However, the environmental reservoir of *T. maritimum* has not been determined yet. Also, its survival in seawater and the suitable niche for its growth in ambient water remain unclear. Quantitative real-time PCR was applied to detect *T. maritimum* in both of the tested species, *Phialella quadrata* and *Muggiaea atlantica* [34]. Bacteria affiliated with the genus *Tenacibaculum* were also found in association with *Cotylorhiza tuberculata* [42,43], with hydromedusa (*N. bachei* [36]), and with the ctenophores *M. leidyi* [26] and *B. ovata* [44]. Based on the large number of genes indicative of carbohydrate and protein metabolism, it has been suggested that *Tenacibaculum*-like bacteria are polymer degraders in jellyfish and their high abundance in the mesogleal axis of the gastric filaments indicate their role in the digestion of ingested food items such as copepods [43]. The possibility for the biotechnological application of this bacterial species as a diagnostic tool and beyond is obvious (Table 2).
