2.1.4. Ulmaridae

One of the most studied jellyfish is *Aurelia aurita*, also known as moon jellyfish. To our knowledge, it is the only jellyfish species from this family that has been investigated for its associated microbiome, and at the same time the most comprehensively investigated of all jellyfish species. Nevertheless, we have to point out the unclear taxonomy of genus *Aurelia*. Mayer [46] and Kramp [47] described 12 and 6 *Aurelia* species, respectively, based on morphological characteristics of the medusa; later only *A. aurita* and *A. labiata* were recognized as distinct species [48]. Despite more than 100 years of *Aurelia* research, the taxonomy of this genus is still unclear [49], and recent molecular analysis indicated that *A. aurita* is represented by several cryptic species [50,51]. The World Register of Marine Species (WoRMS) currently (as of 17 December 2018) recognizes nine *Aurelia* species. It is therefore not always clear which of these species are investigated for their associated microbiome, but for this review we will retain the species name given by the authors of the respective articles.

The first and most detailed study of the microbiome of *A. aurita* was performed by Weiland-Bräuer et al. [35]. These authors investigated the microbiome associated with different life stages of *A. aurita* (polyp, strobila, ephyrae, juvenile, and adult medusae), investigated different compartments of the adult medusae (mucus versus gastric cavity), and compared the microbiome of the polyp stage of specimens from different geographic locations. In this study, fluorescence in situ hybridization (FISH) was used to determine the distribution of specific groups of bacteria on the polyps. The taxonomic composition of the microbiome of *A. aurita* was assessed using the next generation sequencing technique 454 pyrosequencing of the V1–V2 hypervariable region of the 16S rRNA gene.

While several interesting findings were reported in the study of Weiland-Brauer et al. [35], here we highlight only those most important for our review. Different stages of the strobilation event, strobila and ephyra, and juvenile medusa harbored a similar microbiome, which significantly differed from the microbiome of the non-strobilating perennial polyp. At the same time, the richness of the bacterial community was approximately the same among all examined life stages. The microbiome analysis revealed that *Gammaproteobacteria*, *Alphaproteobacteria*, *Bacteroidetes*, and *Actinobacteria* dominate all life stages, with different relative contributions of each bacterial group depending on the stage. Based on 16S rRNA gene amplicon sequencing and FISH coupled with confocal laser scanning microscopy, the authors reported that the entire epithelial surface of the polyp stage of *A. aurita* was covered by bacteria. They proposed that colonization of the polyp occurs on the mucus coating its

epithelial surface, mainly by *Gammaproteobacteria* (mainly *Crenothrix*), while bacteria detected inside and between cells of the polyp tissue were most likely a novel *Mycoplasma* strain (class *Molicutes*), suggested to be potential endosymbionts of *A. aurita* polyps. However, sequencing approaches failed to detect this strain in polyps. Other dominant colonizers of polyps were *Bacteroidetes* (in particular *Lacinutrix*, a member of the *Flavobacteriaceae* family) and *Alphaproteobacteria* (dominated by *Phaeobacter*, a member of the *Rhodobacteriaceae* family). The next developmental phase of *A. aurita*, the strobilating polyp, was associated with *Gammaproteobacteria* (*Crenotrichaceae* and *Vibrionaceae*) and *Actinobacteria* (*Nocardiaceae*). Newly released ephyrae were dominated by *Gammaproteobacteria* (*Crenotrichaceae* and *Pseudoalteromonadaceae*), *Alphaproteobacteria* (*Rhodobacteraceae*) and *Actinobacteria* (*Microbacteriaceae*). Juvenile medusae were dominated by *Alphaproteobacteria* (*Rhodobacteraceae*), *Flavobacteriaceae*, and *Gammaproteobacteria* (*Vibrionaceae*). The differences in bacterial community composition between gastric cavity and umbrella mucus were also determined, revealing that both compartments were dominated by unclassified *Mycoplasma*. A small number of bacterial sequences from the gastric cavity and mucus were affiliated with *Rhodobacteraceae* (*Alphaproteobacteria*). Bacterial diversity differed between the gastric cavity and mucus, with mucus exhibiting greater variability in richness. The drawback of this study is the relatively low number of samples analyzed. One should be careful in extrapolating these results to the entire *A. aurita* taxonomic group (or even beyond), in particularly, since this study also shows the effect of the host's natural environment on the composition of jellyfish-associated microbiome.

The other two available studies of the microbiome of *A. aurita*, from the North Atlantic Ocean [36] and the Northern Adriatic [37], investigated only the adult medusa stage. Both Daley et al. [36] and Kos Kramar et al. [37] constructed and sequenced bacterial 16S rRNA gene clone libraries, which means that compared to the next-generation sequencing approach used by Weiland-Bräuer et al. [35], the number of sequences obtained and analyzed was lower, hence the entire diversity of bacteria was most likely not captured, particularly that of the rare community members. At the same time, the bacterial 16S rRNA gene clone libraries resulted in long and good-quality sequences, allowing the classification to lower taxonomic levels. However, even these two studies using clone libraries cannot be compared entirely, as they used different bacterial primers to amplify different regions of 16S rRNA. Besides, Kos Kramar et al. [37] also applied a culturing approach to investigate the cultivatable part of the jellyfish-associated microbial community.

Daley et al. [36] analyzed seven adult specimens, all collected during two sampling days in spring of the same year. In contrast, Kos Kramar et al. [37] collected 20 adult individuals during two sampling events in the same year, one in spring and one in early summer, investigating changes of the *A. aurita*-associated microbiome in relation to the age of the jellyfish population. Each jellyfish sample pool was then split in half for a culture-based and culture-independent approach to analyze the composition of the *A. aurita*-associated microbial community [37]. For both studies, it is difficult to tell whether the analyzed sample pool is truly representative of the studied jellyfish population, but both sampling designs did not account for interannual variability or spatial patchiness.

The conclusions drawn from these two studies differ. While Daley et al. [36] showed that *Aurelia* is associated with a consortium of bacteria composed of *Mycoplasmatales* (*Tenericutes*, *Mollicutes*) and many unclassified bacteria, several of them distantly related to *Mycoplasma*, in line with the results of Weiland-Brauer et al. [35], Kos Kramar et al. [37] did not detected any bacteria affiliated with this taxonomic group. Also, while in the North Atlantic *A. aurita* harbored only few *Gammaproteobacteria* (including *Psychrobacter* spp.), *A. aurita* collected in the Northern Adriatic were dominated by the gammaproteobacterial families *Vibrionaceae*, *Pseudoalteromonadaceae*, *Xanthomonadaceae*, and *Pseudomonadaceae*. Within the *Alphaproteobacteria*, different families were associated with Aurelia from the two studied systems: in the North Atlantic *Rickettsiales*, and in the Northern Adriatic *Rhodobacteraceae* (mostly *Phaeobacter* and *Ruegeria*). In the Northern Adriatic, *Betaproteobacteria* and *Actinobacteria* were found associated with *A. aurita*, but were not detected in *A.* *aurita* from the North Atlantic while *Cyanobacteria* were associated with *A. aurita* in both the Northern Adriatic and the North Atlantic.

The body part-specificity of the *A. aurita* microbiome was also studied in the Northern Adriatic [37]. *Betaproteobacteria* dominated in the gastral cavity, while *Alphaproteobacteria* and *Gammaproteobacteria* dominated in the "outer" body parts, mostly ex-umbrella mucus, in accordance with Weiland-Brauer et al. [35]. Bacterial strains associated with polycyclic aromatic hydrocarbons (PAHs) and plastic degradation, such as *Stenotrophomonas, Pseudomonas, Burkholderia, Achromobacter*, and *Cupriavidus* [52–56], were present in *A. aurita*'s gastric cavity, indicating an adaptation to anthropogenic pollution. Furthermore, the bacterial community associated with *A. aurita* changed at the transition from the peak to the senescent phase of the jellyfish bloom, and was characterized by an increase of *Gammaproteobacteria*, especially *Vibrionaceae* and *Alteromonadaceae* [37]. Those authors speculated on the different roles that *Vibrionaceae* might play during different stages of jellyfish, from possible commensal opportunistic visitors at the peak of jellyfish bloom to consumers of moribund jellyfish biomass at the end of the jellyfish lifespan. This is in accordance with studies of the degradation of jellyfish biomass by ambient microbial communities, where *Vibrionaceae* were found to dominate the jellyfish biomass-degrading assemblages [25–29]. Furthermore, the authors suggested that *Vibrionaceae* could be exploiting the nutrient-rich niche provided by *A. aurita*. Under proper conditions, such as a disturbed defense mechanism of the jellyfish and elevated water temperatures at the end of jellyfish bloom, upregulating the determinants of *Vibrio*'s virulence, such as motility, resistance to antimicrobial compounds, hemolysis, and cytotoxicity [57], they could outcompete other bacteria and become highly dominant in *A. aurita*.
