2.1.7. Rhizostomeae

Within Rhizostomeae, *Mastigias papua* (a member of the Mastigiidae family), called spotted or lagoon jellyfish, was studied [41] and a member of the Cepheidae family, *Cotylorhiza tuberculata*, known as fried egg jellyfish, was investigated for its microbiome [42,43].

The first study of the microbiome of Rhizostomeae was on the *Cotylorhiza tuberculata* gastric cavity, adopting a culture-independent molecular approach in combination with classical culturing of aerobic heterotrophs [42]. However, no body compartment or life stage specificity was tested or comparison with the diversity of ambient seawater communities made. Both the culturing and culture-independent approaches revealed similar results: reduced diversity of the gastric cavity-associated microbial community, with four major groups of microorganisms detected. The dominant bacteria were

*Spiroplasma*, with the closest relative *S. poulsonii* (*Spiroplasmataceae, Mollicutes*), followed by *Thalassospira* (*Rhodospirillaceae, Alphaproteobacteria*), affiliated with the fish pathogen of the genus *Tenacibaculum* (with the closest relative *T. soleae*), and *Synechococcus*. Cortes-Lara et al. [42] suggested that bacteria affiliated with *Spiroplasma, Thalassospira*, and *Tenacibaculum* might be part of the jellyfish's digestive system, while *Synechococcus* could have been ingested by *C. tuberculata*. Within the culturable fraction, almost 80% of the bacteria were *Vibrionaceae* (*V. xuii* and *V. harveyi*). However, even though Cortes-Lara et al. [42] reported high similarity of the microbiomes among the studied specimens, one should note that this study was based on only four individuals sampled at just one location in one season. Thus, these conclusions might not be representative of the entire population or the jellyfish species.

A subsequent study [43], also focusing on the gastric microbiome of *C. tuberculata*, this time using a shotgun metagenomic approach, revealed the entire genomic repertoire of the dominant members of the bacterial community. Applying fluorescence *in situ* hybridization using fluorescently labeled oligonucleotide probes to target specific bacterial groups and epifluorescence microscopy (CARD-FISH) allowed for localization and identification of specific bacteria within adult medusae. Viver et al. [43] proposed a simple model of microbial-animal digestive associations and hypothesized on the role of the microbiome of *C. tuberculata* in jellyfish ecology. *C. tuberculata* individuals were collected during the same sampling campaign and location as in the study by Cortes-Lara et al. [42], but over an additional two consecutive years to account for interannual community variability.

One of the most important findings of this study was that the microbiome of the gastric cavity of *C. tuberculata* was dominated by eukaryotic cells (*Onychodromopsis*-like and *Symbiodinium* cells) and their infecting bacteria. In line with the previous study [42], low diversity of the gastric cavity-associated microbial community was found, dominated by a few bacterial species, such as a *Simkania*-like lineage of the phylum *Chlamydia*, *Tenacibaculum*-like, *Spiroplasma*-like, and *Mycoplasma*-like bacteria. The association between the jellyfish and its low-diversity microbiome was temporarily stable and possibly related to food ingestion and protection from pathogens. Microscopic analysis contradicted to some extent the results of the metagenome analysis, emphasizing the importance of coupling microscopy-based methods with omic approaches to study the jellyfish-associated microbiome. Based on their findings, Viver et al. [43] proposed three candidate taxa: *Simkania*-like lineage, *Candidatus* Syngnamydia medusae sp.nov. (affiliated with the candidate genus "*Candidatus* Syngnamydia" [59], in particular *Candidatus* Syngnamydia salmonis [60]), *Spiroplasma*-like bacteria, *Candidatus* Medusoplasma gen.nov, *Candidatus* Medusoplasma mediterranei sp.nov., and *Tenacibaculum*-like bacteria, *Candidatus* Tenacibaculum medusae.

Within Rhizostomeae, the microbiome of *Mastigias cf. papua* was also studied [41]. This study employed 16S rRNA gene 454 pyrosequencing to examine the bacterial community associated with adult medusae collected during three sampling campaigns at three sampling locations during one season. Major groups of the microbiome of *Mastigias cf. papua* were *Gammaproteobacteria* (with *Endozoicimonaceae* being most abundant), followed by *Mollicutes*, *Spirochaetes*, and *Alphaproteobacteria* (with the orders *Kiloniellales* and *Rhodobacterales*).
