Cnidarian-microbe interactions are ecologically important (Pitt et al., 2005; Tinta et al., 2012; Glasl et al., 2016; Röthig et al., 2017; Webster and Reusch, 2017), facilitating energy acquisition and potentially contributing to the nutrition (Lesser et al., 2004; Rädecker et al., 2015; Röthig et al., 2021), health (Bourne et al., 2016; Hernandez-Agreda et al., 2017), immunity (Bosch, 2013), and defense of the hosts (Ritchie, 2006; Krediet et al., 2012; Glasl et al., 2016). A surface mucus layer (SML) covers the Cnidarian body and protects their live tissues from physical, chemical, and biological impacts (Brown and Bythell, 2005). Due to its chemical composition, the SML sustains a diverse microbial community of Bacteria, Archaea, Microalgae, and Fungi, many of which are host specific (Rohwer et al., 2002; Lampert et al., 2008; Carlos et al., 2013; Cleary et al., 2016). Considering the sparse distribution of nutrients in oligotrophic waters, where many Cnidarians develop, the SML represents a rich substrate that stimulates bacterial growth (Hansson and Norrman, 1995; Wild et al., 2004; Bythell and Wild, 2011). The composition of Cnidaria-associated bacterial communities is dynamic, displaying seasonal and geographic variations (Morrow et al., 2012; Sharp et al., 2017), particularly in short-term studies (Yang et al., 2017). However, the mechanisms that structure and regulate this community remain poorly understood.

As in corals, some Scyphozoan jellyfishes contain symbiotic dinoflagellate algae, like species in the genera Cassiopea, Mastigias, Linuchae, and Phyllorhiza (reviewed in Djeghri et al., 2019). The medusae complete their energetic demands with photosynthetic products supplied by the symbiotic algae, which also contribute to the production of the SML (Hofmann and Kremer, 1981; McCloskey et al., 1994; Freeman et al., 2016). Jellyfish may also lose their symbionts and bleach after heat and light stress (McGill and Pomoroy, 2008; Newkirk et al., 2018). The disruption of the symbiosis can affect the SML secretion and modify its biochemical properties (Lee et al., 2016; Rivera-Ortega and Thomé, 2018), which may be followed by changes in the associated bacteria, as occurs with corals (Bourne et al., 2007; Glasl et al., 2016; Wright et al., 2019). Symbionts can contribute significantly to mucus production in symbiotic corals (Brown and Bythell, 2005). A fraction of the carbon translocated to hosts gets lost from the association as dissolved and particulate organic carbon (mucus); this loss represents, for example, between 5% and 50% in 48 h incubations in the coral Stylophora pistillata (Tremblay et al., 2012), or 20-45% of daily net photosynthate in corals (as reviewed by Brown and Byhtell, 2005). But unlike corals, soft-body Cnidarians can directly use the energy from the translocation of organic carbon from the algal symbionts for biomass increase and mucus production. Environmental variations that affect the photosynthesis of symbionts can thus affect mucus production. Therefore, soft-body symbiotic Cnidarians like the jellyfish C. xamachana and the sea anemone Exaiptasia diaphana, represent an opportunity to directly study the interaction of algal symbionts with the animal host, and with the commensal/opportunistic bacteria that live in the mucus layer. But unlike E. diaphana, C. xamachana maintains a closer symbiotic relationship (McGill and Pomoroy, 2008; Voolstra, 2013; Freeman et al., 2016).

The Bacteria-Cnidaria-Symbiodiniaceae must be studied as consortia since the exchange and recycling of fundamental nutrients influence its health and stability (Bourne et al., 2016; Webster and Reusch, 2017; Goulet et al., 2020). However, such exchange can be affected by bleaching, disease, and colonization by opportunistic pathogens (Matthews et al., 2020). Shifts in the bacterial community following the loss of symbionts involving stress, environmental changes, or seasonal fluctuations can be complex, considering that the Bacteria-Symbiodiniaceae association is just beginning to be studied. Some studies have postulated the existence of bacteria closely associated with the surface layer of the symbiont that can help it tolerate physiological stress (as ROS) or variations in environmental conditions (Matthews et al., 2020; Garrido et al., 2021; Maire et al., 2021). A model system such as the jellyfish C. xamachana may prove helpful to define the role of algal symbionts as drivers of the bacterial community in symbiotic medusae.

Cassiopea medusae are Scyphozoans (Order Rhizostomeae) common in shallow waters of tropical and subtropical ecosystems like mangroves, seagrasses, and coral reefs (Niggl et al., 2010; Ohdera et al., 2018). The planktonic medusae shift from a benthic polyp stage (Colley and Trench, 1983), which may bring changes in the associated microbial community. Also, the medusa stage moves with marine currents, potentially affecting its distribution and microbiome. Cassiopea jellyfish increase their abundance and size in areas where human population density is high (Stoner et al., 2011). Nutrient loading can lead to blooms of epibenthic jellyfish which can have consequences to the ecology of the impacted area, competing with other filter-feeding consumers, and locking the biomass that can alter community structure and ecosystem function. Further, metamorphosis of the jellyfish larvae occurs only after the symbiont has been collected by the polyp and temperatures rise above 20°C (Fitt and Costley, 1998). With global change advancing, temperatures that used to limit the reproduction of the jellyfish to the summer months might be surpassed during colder months and exacerbate the density of these jellyfish (Aljbour et al., 2019). A limited number of studies have focused on the bacterial community of symbiotic Rhizostomeae jellyfishes. For example, the bacterial community of C. xamachana held in captivity was studied, showing the dominance of two families, Moxarellaceae and Pseduomonadaceae (Röthig et al., 2021). In the abundant Pacific jellyfish Mastigias papua, the associated bacterial community displayed high variation within the species, low diversity, and the highest bacterial abundance related to members of the Endozoicomonadaceae family (Cleary et al., 2016).

Among non-symbiotic Rhizostomeae jellyfishes, the moon jellyfish Aurelia aurita is one of the best-studied. Its bacterial community is similar throughout its pelagic developmental stages, only the polyp stage being different (Weiland-Bräuer et al., 2015). Also, reports indicate differences associated with geographic location (Kramar et al., 2019). After a jellyfish bloom, in the senescence phase, the bacterial community of A. aurita was characterized by an increase in Gammaproteobacteria, specifically Vibrionaceae and Alteromonadaceae (Kramar et al., 2019). Among other species studied, Cyanea lamarckii displayed significant differences in the bacterial community of the umbrella in contrast to other body parts (Hao et al., 2019). The authors also assessed the bacterial community in different life stages that in general, increased in diversity from larval to adult stages. A recent study of four jellyfish species that form large blooms indicated species-specific differences in their bacterial communities (Peng et al., 2021); Vibrio was the bacterial genus dominant in Aurelia coerulea, as in other Aurelia species (Weiland-Bräuer et al., 2015). Mycoplasma dominated the Rhopilema esculentum and Nemopilema nomurai jellyfishes, but in Cyanea nozakii its bacterial community was equally abundant in Sphingomonas, Phyllobacterium, and Ralstonia (Peng et al., 2021).

Cassiopea xamachana is a benthic jellyfish that lives in ecologically obligate symbiosis with the photosynthetic dinoflagellate Symbiodinium microadriaticum (Colley and Trench, 1983; Lampert, 2016). The adult medusa rests on the sea bottom, their convex umbrella and oral arms facing upwards to allow the capture of light by their symbionts (Lampert, 2016; Ohdera et al., 2018). The colonization by algal symbionts supports the transition from polyp to adult medusa (Hofmann et al., 1996; Newkirk et al., 2018). The relative simplicity of culturing polyps and adults of C. xamachana in laboratory conditions, makes this symbiotic jellyfish an ideal model system to study the mechanisms that regulate the colonization of the host by their symbionts. However, it is not clear how much C. xamachana depends on its algal symbionts, nor if a decrease in algal symbionts can restructure its associated bacterial community. In this study, we characterized the bacterial community of the SML of this symbiotic jellyfish while in captivity, contrasting summer and winter seasons, and the effect of a significant loss of symbionts (bleaching).

Cassiopea xamachana jellyfish naturally harbor symbiotic algae and associate with a surface bacterial community. We have studied this bacterial community by comparing symbiotic and bleached medusas in two seasons. Bleached medusas were obtained by additions of a labile sugar mix ( Figure 1 ) (Pogoreutz et al., 2017). The disruption of the symbiotic relationship in corals by increased organic carbon load is well established, including the expression of bacterial virulence genes and the formation of a hypoxic layer on the coral surface (Vega-Thurber et al., 2009). Elevated levels of dissolved organic carbon (DOC) (e. g. 25 mg L^-1) have also been shown to accelerate bacterial growth rates associated with the SML of corals, potentially disrupting the balance of the microbial community (Kline et al., 2006). However, an experimental study showed that the bacterial community from coral fragments treated with enriched DOC levels (e. g. 10 mg L^-1), remained stable after 28 days, did not show an increase in virulent bacteria, but caused bleaching of the coral fragments (Pogoreutz et al., 2017); these authors also observed the proliferation of diazotrophs. By using a lower concentration of DOC (0.3 mg L^-1) we also induced bleaching of the jellyfish but perhaps with milder effects over the SML-associated bacterial community ( Figure 2 ). It is not clear if moderate additions of labile carbon can promote the abundance of specific bacterial groups that may lead to changes in key members of this community (Kline et al., 2006; Vega-Thurber et al., 2009). For example, Vibrio species highly increase their abundance during coral bleaching events and become dominant (Bourne et al., 2007; Tout et al., 2015; McDevitt-Irwin et al., 2017), possibly due to a reduction of the antibiotic activity of bacteria found in the mucus of corals (Ritchie, 2006), and the ability of Vibrio to utilize a wide variety of sugars (Lee et al., 2016). Also, a study of the jellyfish Aurelia aurita reported an increase in Vibrionaceae bacteria at the end of the jellyfish bloom (Kramar et al., 2019). Further, bacterial groups associated with corals that potentially fix nitrogen (diazotrophs) are common and diverse. These may include Cyanobacteria, Alphaproteobacteria, and Deltaproteobacteria species (Olson et al., 2009). Although our results showed the occurrence of these bacteria, to better distinguish diazotrophs a specific study of nifH genes would have been necessary, considering that such genes correlate with the presence of algal symbionts (Olson et al., 2009).

Bleached medusas showed a lower diversity of bacterial taxa ( Figure 3 ), consistent with reports in stressed corals (McDevitt-Irwin et al., 2017). Medusas sampled during the summer showed a decrease of bacterial diversity after 28 days of incubation but during winter, the bacterial diversity of medusas remained without significant change over time ( Figures 3 , 4 ). This contrasts with results reported by Pogoreutz et al. (2017) in corals exposed to high DOC, where they observed an overall decrease of bacterial diversity over time. Our results indicated the dominance of Gammaproteobacteria, Bacteroidia, and Alphaproteobacteria in all mucus samples ( Figure S3 ). Jellyfish species from Indonesia, like Mastigias papua and Tripedalia cystophora, were also characterized by the higher abundance of Gammaproteobacteria class in bleached medusas and the abundant Endozoicomonas genus (Cleary et al., 2016). However, Rhizosotma pulmo, an endemic and common jellyfish of the Mediterranean Sea, has a completely different bacterial community to the one we observed in C. xamachana, even at the Phylum level (Cleary et al., 2016). Kelly et al. (2014) found that algae-dominated coral reefs from the Pacific, correlate with higher abundances of Gammaproteobacteria (such as Alteromonadales, Psuedomonadales, and Vibrionales), Betaproteobacteria, and Bacteriodetes; while reefs with high coral cover, correlate with higher abundances of Alphaproteobacteria (such as Rhodobacteriales and Sphongomonadales). Similar results have been obtained for symbiotic and non-symbiotic reef invertebrates, the former hosting Gammaproteobacteria and the latter Alphaproteobacteria (Bourne et al., 2013). These same bacterial classes have been identified in the gastric cavity of other non-symbiotic marine jellyfish and have been reported with potential in bioremediation, the degradation of polycyclic aromatic hydrocarbons, and the synthesis of antimicrobial compounds (Tinta et al., 2019).

In the present study, the initial abundance of Endozoicomonas ( Figure 5 ) was consistent with previous studies that report a high frequency of this genus and with the highest abundance among reef-forming corals (Bayer et al., 2013; Glasl et al., 2016; Neave et al., 2017a; Pogoreutz et al., 2017). However, Shiu et al. (2020) indicated that the relative abundance of Endozoicomonas usually decreases with heat-induced coral bleaching and perhaps positively correlates with Symbiodiniaceae abundance. It remains unclear whether this phenomenon of decreased Endozoicomonas abundance is caused by temperature stress or a decreased abundance of Symbiodiniaceae. In this respect, our results also suggest a correlation of Endozoicomonas with the presence of symbionts, and in the absence of heat stress. Additionally, Endozoicomonas has been recognized as an important, common bacterial genus, extremely flexible and diverse, associated with a high variety of marine organisms (Morrow et al., 2012; Forget and Juniper, 2013; Katharios et al., 2015; Neave et al., 2017b). Even though their role is not clearly defined yet, it has been proposed that some of their functions are related to nutrient acquisition and recycling of carbon and nitrogen (Morrow et al., 2015), antibiotic production, and the structuring of the microbiota (Bayer et al., 2013; Jessen et al., 2013; Rua et al., 2014). Endozoicomonas has also been found in high abundances and associated with other non-symbiotic marine jellyfish, being considered for its possible application in biotechnology and as a candidate for studies in probiotic applications (Mansson et al., 2011; Deering et al., 2016; Viver et al., 2017; Tinta et al., 2019).

The experimental bleaching of C. xamachana was difficult to achieve. We tried different bleaching methods (low temperature, menthol, DCMU addition) with poor success; medusas either lost a considerable amount of mass or died. The addition of sugars successfully caused the loss of algal symbionts and, even though the animals lost mass (≈ 30%), they were kept alive and active. Sugar addition has the downside of altering the associated bacterial community, increasing diazotroph abundance (Pogoreutz et al., 2017), which we may have noted as an increase in the relative abundance of Vibrio ( Figures 4 , 5 ). However, the relative abundance of Vibrio also increases with environmental deterioration and bleaching (Vega-Thurber et al., 2009; Kelly et al., 2014). A different bleaching method could help distinguish between these two factors (lack of symbionts and sugar addition) but is methodologically challenging. Considering that (1) we used a low sugar concentration for the experimental bleaching of the medusas, and (2) our results showed a significant effect of the loss of symbionts on the associated bacterial community, we may suggest it is plausible that algal symbionts may influence the structuring of the associated bacterial community.

The bacterial community of medusas like C. xamachana and other symbiotic model organisms has only begun to be studied over the past few years. Some studies have attempted to demonstrate cooperation between the associated microorganisms, and that the host may be able to select them (Fraune et al., 2015; Har et al., 2015). In our study the bleaching stress was unavoidable but there is still a lack of experimental evidence linking specific factors and the increase of Vibrio during bleaching. Overall, studies about the functional capabilities of the bacterial community of C. xamachana, can help us identify specific roles of certain members of this community that change in response to a loss of symbionts. Similar studies have identified other dominant families like Moraxellaceae and Pseudomonadaceae in C. xamachana when the nitrogen conditions are not limiting for the algal symbiont (Röthig et al., 2021). The associated bacteria participate in the elimination of dissolved inorganic nitrogen (DIN) to overcome the nitrogen limitation of the symbiotic algae, a mechanism that has been proposed to help stabilize the host-algae relationship (Röthig et al., 2021). However, functional studies of bacterial members will further help understand how biological factors, like alterations of the algal symbionts, may interact with this community and the key role of the photosynthetic activity of Symbiodiniaceae in the maintenance of the bacterial community, as previously reported (Littman et al., 2010).

Some bacterial members in the mucus in C. xamachana were enriched for specific taxa at each season ( Figure 6 ). Studies confirm that seasonal fluctuations include changes in the physiology of the associated host, in the months of low sea temperatures (winter); it has been documented those corals such as Astrangia poculata decrease their metabolic functions, retract their polyps, and feed less, losing biomass in consequence (Grace, 2017). As temperatures increase, the metabolic activity of the holobiont resumes, as occurs in tropical corals (Mouchka et al., 2010; Zaneveld et al., 2016; Sharp et al., 2017). In summer, the coral holobiont consumes more food and possibly supplies more substrates that promote bacterial growth following a restructuring of the bacterial members (Burmester et al., 2017; Grace, 2017). The variation in certain bacterial groups by seasonality suggests that the bacterial communities undergo a succession, that is, changes in the bacterial community can occur during the colder winter months, followed by a restructuring of the bacterial community in summer. Lastly, the genetic variation of the host may also play a role. According to several studies (Arai, 2001; Holland et al., 2004; Arai et al., 2017; Ohdera et al., 2018) molecular phylogenetic analyses indicate individuals of Cassiopea analyzed from the same geographic area corresponding with different clades.

The addition of sugars was confirmed to have considerable potential as a method for rapid and effective bleaching in C. xamachana. This method can broaden the study of symbiont-related questions like carbon metabolism and limitation, dissociation of host and algae, conditions for reestablishing this relationship, or capacities for symbiont cooperation. Endozoicomonas, Tenacibaculum, and 21other bacterial genera were abundant in medusas at day-0; however, it is not clear if DOC addition, symbiont loss, or both, were significant in provoking the shift of the bacterial community in the bleached condition, increasing the abundance of Vibrio. Seasonal fluctuations did not cause significant variations in dominant bacterial members; such changes were better associated with symbiont presence. However, given the low number of replicates we used, and the natural variation of the bacteria associated with the medusae, probably also dependent on a genetic variation of the host, more studies will be needed to ascertain this claim. At the class level, the bacteria in the mucus of C. xamachana were similar to bacterial classes found in other tropical, symbiotic Rhizostomeae jellyfish, in the model organism Exaiptasia anemone, and in reef-building corals, mainly dominated by Gammaproteobacteria and Alphaproteobacteria. Future studies with this model organism can enlighten the role of symbionts in driving the stability of the associated bacterial community. Such studies may include seasonal fluctuations and bleaching events, triggered by seawater temperature, nutrient levels, and climate change, all associated with diseases and mortality in symbiotic corals. Also, studies in other regions where C. xamachana distributes, can help us identify stable and functional associations of bacteria that may contribute to the fitness of this jellyfish.

The original contributions presented in the study are included in the article/ Supplementary Material and the NCBI (https://www.ncbi.nlm.nih.gov/) under accession no. PRJNA810909. Further inquiries can be directed to the corresponding author/s.

NC, PT contributed to the conception and design of the study. NC, DC-G organized the databases. NC, DC-G performed the statistical analysis. NC wrote the first draft of the manuscript. PT, JG-M wrote and edited sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

This work was supported by PAPIIT-DGAPA-UNAM [grant IN204318] to PET. ICML-UNAM provided funds for open access publication fees.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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