The Life Cycle of Aurelia aurita Depends on the Presence of a Microbiome in Polyps Prior to Onset of Strobilation

ABSTRACT Aurelia aurita’s intricate life cycle alternates between benthic polyp and pelagic medusa stages. The strobilation process, a critical asexual reproduction mechanism in this jellyfish, is severely compromised in the absence of the natural polyp microbiome, with limited production and release of ephyrae. Yet, the recolonization of sterile polyps with a native polyp microbiome can correct this defect. Here, we investigated the precise timing necessary for recolonization as well as the host-associated molecular processes involved. We deciphered that a natural microbiota had to be present in polyps prior to the onset of strobilation to ensure normal asexual reproduction and a successful polyp-to-medusa transition. Providing the native microbiota to sterile polyps after the onset of strobilation failed to restore the normal strobilation process. The absence of a microbiome was associated with decreased transcription of developmental and strobilation genes as monitored by reverse transcription-quantitative PCR. Transcription of these genes was exclusively observed for native polyps and sterile polyps that were recolonized before the initiation of strobilation. We further propose that direct cell contact between the host and its associated bacteria is required for the normal production of offspring. Overall, our findings indicate that the presence of a native microbiome at the polyp stage prior to the onset of strobilation is essential to ensure a normal polyp-to-medusa transition. IMPORTANCE All multicellular organisms are associated with microorganisms that play fundamental roles in the health and fitness of the host. Notably, the native microbiome of the Cnidarian Aurelia aurita is crucial for the asexual reproduction by strobilation. Sterile polyps display malformed strobilae and a halt of ephyrae release, which is restored by recolonizing sterile polyps with a native microbiota. Despite that, little is known about the microbial impact on the strobilation process's timing and molecular consequences. The present study shows that A. aurita’s life cycle depends on the presence of the native microbiome at the polyp stage prior to the onset of strobilation to ensure the polyp-to-medusa transition. Moreover, sterile individuals correlate with reduced transcription levels of developmental and strobilation genes, evidencing the microbiome's impact on strobilation on the molecular level. Transcription of strobilation genes was exclusively detected in native polyps and those recolonized before initiating strobilation, suggesting microbiota-dependent gene regulation.

In this article Jensen and colleagues explore the impact of the complex microbiome on strobilation of the jellyfish Aurelia aurita. They find that the presence of the native microbiome is required at the polyp stage for normal strobilation and ephyra release, and that introduction of the microbiome at subsequent timepoints is insufficient for metamorphosis. This paper is well thought-out with very nice figures. Understanding of host-microbe interactions in complex mutualisms is an important topic and this paper advances that understanding. I have some comments (see attached) that I think should be addressed, but consider them fairly minor.
In this article, Jensen and colleagues explore the impact of the complex microbiome on strobilation of the jellyfish Aurelia aurita. They find that the presence of the native microbiome is required at the polyp stage for normal strobilation and ephyra release, and that introduction of the microbiome at subsequent timepoints is insufficient for metamorphosis. This paper is well thought-out with very nice figures. Understanding of host-microbe interactions in complex mutualisms is an important topic and this paper advances that understanding. I have some comments (see below) that I think should be addressed, but consider them fairly minor. General Comments: 1) I'd briefly explain around line 142 that the inoculum is from filtered homogenate. 2) I liked your experiment demonstrating that cell-cell contact is required. My question building on this is whether you think tissue colonization by the bacteria is required, or whether you think bacteria in the water would be sufficient? 3) For lines 302-303 I would note that Kerwin et al. 2019 in mBio did find that chloramphenicol negatively impacted development of the Hawaiian bobtail squid embryo, but that other antibiotics did not, and that paper was looking at microbiome functionality. Not asking you to cite it, but wanted to point it out. :) I was a little confused by your description of the antibiotics tested in lines 451-458 -which were tested singly, and did those not work sufficiently? Given the known toxicity of chloramphenicol that you cite, it may have been better not to include that. I think though that since your recolonized sterile polyps were fine, the antibiotic treatment wasn't a problem. 4) The NCBI BioProject ID did not appear when searched -assume this will be public by publication. 5) Did you do a glycerol control since your inoculum were frozen with glycerol? I'm assuming not, in which case you should note that somewhere and the reasoning behind that decision. what do the 2nd and 3rd grey images represent? A description in the legend or in your text (around line 133) would be helpful (I see you get to this later on pg 9, but I think a brief description early on would still be helpful). The NC and SC abbreviations should be defined in the legend. Does the R1 ephyra not have a question mark because that was studied in your last paper? I was unclear why the inducer was shown outside of the box for R1 and why that was different with it being inside the box in R2/R3. Also why is it R1 and not R1i if the inducer was used? Should it say inducer instead of inductor in the figure? 2) I really liked your color scheme for Figure 2 and the way you organized the taxa. For the bolded genera it would be good to define what your threshold was for "relative high abundance". The scroll over with mouse click functionality wasn't working for me but I assume that will be checked in the proof stage. I was surprised to see that the alpha diversity of the recolonized polyps was so much higher. This should be discussed moredo you have a hypothesis? It doesn't seem like you should be able to strongly increase in richness between what's being inoculated and what's being colonized -unless you suspect an increase in richness (which at least to me doesn't seem super apparent). You later call the diversity comparable (lines 327-329) which seems at odds with your data. Visually comparing the inoculum and the recolonized polyps I wondered if any taxa were missing -might be helpful to underline or otherwise indicate taxa present in the inoculum but missing in recolonized, or alternatively present in native but missing in recolonized. 3) Figure 3 is really nicely done -great job! 4) For part A of Figure 4 are you inducing to get ephyra in the SC, R2, R3 conditions? Or do you still get some ephyra, just a smaller percentage? The percentages in the ephyra boxes should be mentioned in the legend. For the late strobila, where is the color coming from in NC and R1? It makes it a little hard to compare to the SC and R2 since those appear more as silhouettes, but maybe that's just what they look like? (Sorry I'm not familiar with what they should look like in this species.) 5) Figure 5 is really nice -great presentation of complex data! How do you explain the Dear authors, this is an interesting study, however, the manuscript needs a lot of work; the good news -there is nothing that can't be fixed (see comments in the manuscript file). The original title is misleading, general concepts of the jellyfish lifecycle are either not well understood or not precisely worded, the abstract is confusing and lacks any description of methods, there is a lack of understanding which parts belong into which section of the paper, referencing is done in a sloppy way -it's not enough to reference a study, it also needs to be put into context. There are conclusions that are not supported by the results. There is an overuse of specific words, e.g. the word crucial appears about 15 times. While the results are worth publishing, the importance of the study is generally overstated and the wording is disproportionally strong and often exaggerated to a point where it does not reflect the findings. The shortcomings of the study need to be elaborated on (e.g. husbandry conditions). Many sentences are highly confusing, thoughts need to be ordered and sentences reworded. There are grammatical and style issues and I do recommend a proper revision of the English, if not by a native speaker at least by an online grammar and punctuation checker. I feel that the manuscript has been submitted either prematurely, or in a rush, which is kind of disrespectful to the reviewers, please consider this in your future submissions. For more details see the manuscript file.
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In this article, Jensen and colleagues explore the impact of the complex microbiome on strobilation of the jellyfish Aurelia aurita. They find that the presence of the native microbiome is required at the polyp stage for normal strobilation and ephyra release, and that introduction of the microbiome at subsequent timepoints is insufficient for metamorphosis. This paper is well thought-out with very nice figures. Understanding of host-microbe interactions in complex mutualisms is an important topic and this paper advances that understanding. I have some comments (see below) that I think should be addressed, but consider them fairly minor.
General Comments: 1) I'd briefly explain around line 142 that the inoculum is from filtered homogenate.
2) I liked your experiment demonstrating that cell-cell contact is required. My question building on this is whether you think tissue colonization by the bacteria is required, or whether you think bacteria in the water would be sufficient? 3) For lines 302-303 I would note that Kerwin et al. 2019 in mBio did find that chloramphenicol negatively impacted development of the Hawaiian bobtail squid embryo, but that other antibiotics did not, and that paper was looking at microbiome functionality. Not asking you to cite it, but wanted to point it out. :) I was a little confused by your description of the antibiotics tested in lines 451-458 -which were tested singly, and did those not work sufficiently? Given the known toxicity of chloramphenicol that you cite, it may have been better not to include that. I think though that since your recolonized sterile polyps were fine, the antibiotic treatment wasn't a problem. 4) The NCBI BioProject ID did not appear when searched -assume this will be public by publication. 5) Did you do a glycerol control since your inoculum were frozen with glycerol? I'm assuming not, in which case you should note that somewhere and the reasoning behind that decision. A description in the legend or in your text (around line 133) would be helpful (I see you get to this later on pg 9, but I think a brief description early on would still be helpful). The NC and SC abbreviations should be defined in the legend. Does the R1 ephyra not have a question mark because that was studied in your last paper? I was unclear why the inducer was shown outside of the box for R1 and why that was different with it being inside the box in R2/R3. Also why is it R1 and not R1i if the inducer was used? Should it say inducer instead of inductor in the figure? 2) I really liked your color scheme for Figure 2 and the way you organized the taxa. For the bolded genera it would be good to define what your threshold was for "relative high abundance". The scroll over with mouse click functionality wasn't working for me but I assume that will be checked in the proof stage. I was surprised to see that the alpha diversity of the recolonized polyps was so much higher. This should be discussed moredo you have a hypothesis? It doesn't seem like you should be able to strongly increase in richness between what's being inoculated and what's being colonized -unless you suspect an increase in richness (which at least to me doesn't seem super apparent). You later call the diversity comparable (lines 327-329) which seems at odds with your data. Visually comparing the inoculum and the recolonized polyps I wondered if any taxa were missing -might be helpful to underline or otherwise indicate taxa present in the inoculum but missing in recolonized, or alternatively present in native but missing in recolonized. 3) Figure 3 is really nicely done -great job! 4) For part A of Figure 4 are you inducing to get ephyra in the SC, R2, R3 conditions? Or do you still get some ephyra, just a smaller percentage? The percentages in the ephyra boxes should be mentioned in the legend. For the late strobila, where is the color coming from in NC and R1? It makes it a little hard to compare to the SC and R2 since those appear more as silhouettes, but maybe that's just what they look like? (Sorry I'm not familiar with what they should look like in this species.) 5) Figure 5 is really nice -great presentation of complex data! How do you explain the difference in expression between the native and R1 at the polyp stage? The figure seems to contrast with your results at lines 253-254? 6) Figure 6 refers to A/B/C but these labels aren't present in the figure itself. For B in the legend you don't mention the inducer but the figure shows it -I'd reword to make the legend clear. You need to define the scale bars in the legend. In line 95 should be The expression, not These expression. 5) In line 97 should be "and is expressed" not "and be expressed". 6) In line 265 should it be "path" instead of "faith"? Or maybe "fate"? 7) Sentence starting The abundance in lines 331-334 needs to be revised for clarity. 8) Sentence starting Examples of in lines 341-342 is quite vague and could be revised to briefly provide the actual examples. 9) I don't think oyster should be italicized in line 369 -might actually be better to provide the specific species. 10) Line 369 has a typo -should be whether, not wether. 11) Line 481 -revise sentence Review for manuscript entitled Asexual reproduction of Aurelia aurita depends on the presence of a balanced microbiome at polyp stage by Nadin Jensen, Nancy Weiland-Bräuer, Shindhuja Joel, Cynthia Maria Chibani, Ruth Anne Schmitz considered for publication at Microbiology Spectrum journal.
The research of host-microbiota interactions is very up-to-date and in particularly this study as it brings new insights in novel model metaorganism, i.e., jellyfish, which is unlike other systems, e.g., corals, hydrozoans, etc, less studied metazoan host-microbiota system. Being ecologist my major concern, as always with model organisms that have been capture/kept in the lab for a long period of time, is the actual accuracy or relevance of results for ecology of this organisms. In my modest opinion these systems, like the case here, when organisms have been kept in the lab for over 10 years, are far away from their natural representatives. Else, study is very well designed, conducted and analysis is very appropriate and paper well structured (however a bit long and repetitive in some places and hence could be more condensed and streamlined). Hence, I suggest this paper can be accepted with minor revision. Please see my specific comments below.

Some minor comments:
Abstract: Line 23: Yet, the timing and molecular consequences of the microbiome during the 24 strobilation process had not been investigated. Is strangely formulated -the timing and molecular consequences of the microbiome-please re-write. For isolation of A. aurita-associated microbiota, 10 polyps, 10 early strobilae (5 days 407 post-induction), and 10 late strobilae (9 days post-induction) were transferred into 408 500 µl sterile ASW and washed three times to remove transient bacteria. The animals were collectively homogenized with a motorized pestle (KONTES, DWK Life Science, Wertheim, Germany) and filtered through a 3.1 µm filter (Lab Logistic group, Meckenheim, Germany) to remove eukaryotic cells. This is not clear to me. Did you combine/pool microbiota from all this different 'hosts' to obtain inoculum or you kept it separately for each 'host'? Depending on what exactly you did in my opinion affects the outcome substantially.
Line 412: Aliquots (105 cells/ml) of the filtrate were immediately frozen in liquid nitrogen and stored at -80 °C in the presence of 10 % glycerol. These frozen stocks served as inoculum for all recolonization experiments presented here. I wonder how many of cells lost their viability via this process -maybe also specific taxa…did you maybe check the effect on microbial community?
Line 482: Recolonization of sterile polyps, early and late strobila was conducted by addition of 10 µl of the thawed inoculum to ASW (105 cells/ml and per animal, see above), with fresh ASW containing inoculum applied the next day, so that the animals were exposed to the inoculum for 48 h. A control of inoculum was incubated for 48 h in ASW only. At this point I started to think about viable/dead cells within inoculum and then I realized that you used STYo0 to count -which allows you to distinguish live/dead cells, however maybe not all readers know that so would be nice to specify this somewhere..else makes you wonder how much viability of cells was affected by your freezing/thawing of inoculum, hence also affecting your community composition,

Results:
Line 149: what about alpha diversity of whole community?
Line 152 (and other cases): when you talk about relative abundance one needs to be aware that of course this depends on total cell abundance, e.g., one taxa can increase in relative abundance, but overall cell abundance (cells/mL) is lower and this is important!, also an increase/decrease can be due to decrease/increase of other taxa…it can give a wrong impression! Since I understood you used SYTO9 to distinguish between live and dead and made an effort to count cells on polyps etc., why not making an effort to take this actual cell numbers into account Line 156-157: sentence not clear, please re-write: Overall, the microbiota of recolonized polyps more strongly resembled that of native polyps than bacteria maintained in artificial seawater (ASW) in absence of animals.
Line 216-216: The individual administration of antibiotics also reduced the native bacterial community monitored by a decrease of 30 -80 % in CFU/ml. This is confusing to me: when you talk about CFU/mL I assume only cultivable part of community was assessed? But to my understanding this was not the case. Please explain.
Line 270-272: For this, native polyps with their diverse, healthy microbiome were incubated together with sterile polyps, but separated by a 0.2 µm filter that allowed the transfer of metabolites, but not of bacteria. If we are picky we would take into account the fact that some really small bacteria can escape 0.2 um filtration! Discussion: Line 287: Our results not only illustrate how important a native microbiome is for the asexual reproduction of A. aurita, but also demonstrated that the generation of offspring is only possible when the microbiome is present before the animal enters the strobilation process. Just a general thought: I cannot image where and when in natural scenario microbiome would not be present before animal enters strobilation process, so not really sure, from ecological perspective, why this I even relevant. Maybe discussion on this aspect could be elaborate.
Line 332-334: The abundance of Vibrio and Colwellia is increasing in recolonized polyps, whereas ASW shifted the bacterial population to a significantly structure accompanied with a lower α-diversity. This is a very strange sentence, I don't understand what is meant here. Please correct.
Line 336-337: During a reassembly of the microbiota on the host's mucus, interactions between host and bacteria as well as amongst bacteria ensure a specific microbial community pattern of Aurelia. How can you possibly know this? Did you test other jellyfish species and compare species-specificity of associated microbiota?
sterile polyps with a polyp-native microbiome can correct this defect. Here we 23 investigated the precise timing necessary for recolonization as well as the host-24 associated molecular processes involved. We deciphered that a natural microbiota 25 had to be present in polyps prior to the onset of strobilation in order to ensure normal 26 asexual reproduction and a successful polyp-to-jellyfish transition, meaning that 27 adding microbiota to sterile polyps after the onset of strobilation failed to restore the 28 normal strobilation process. The absence of a microbiome was associated with 29 decreased transcription of developmental and strobilation genes; these were 30 exclusively observed for native polyps and sterile polyps that were recolonized before 31 the initiation of strobilation. Direct cell contact between the host and its associated 32 bacteria was also required for the normal production of offspring. Overall, our findings 33 indicate that the presence of a native microbiome at the polyp stage prior to the 34 onset of strobilation is essential for a normal polyp-to-jellyfish transition. 35

37
All multicellular organisms are associated with microorganisms that play a 38 fundamental role in the health and fitness of the host. Notably, the native microbiome 39 of the Cnidarian Aurelia aurita is crucial for the asexual reproduction of the jellyfish. 40 Sterile polyps display malformed strobilae and a halt of ephyrae release that can be 41 restored by recolonizing sterile polyps with a native microbiota. Despite that, little is 42 known about the strobilation process's timing, molecular consequences, and 43 microbial impact. The present study shows that the life cycle of A. aurita depends on 44 the presence of the native microbiome at the polyp stage prior to the onset of 45 strobilation to ensure the polyp-to-jellyfish transition. Moreover, sterile individuals 46 correlate with reduced transcription levels of developmental and strobilation genes, 47 evidencing the microbiome's impact on strobilation on the molecular level.  An animal host together with its associated natural microbiota forms a unit that is 54 defined as a metaorganism (1,2). The complex functional interplay between a host 55 and its microbiota has a crucial impact on the physiology and proper development of 56 the host (3)(4)(5), as well as on the host metabolism (6-8), the immune system (9), 57 morphogenesis (10), reproduction (11)(12)(13)(14), and environmental adaption (15)(16)(17)(18). 58 Metaorganismal studies allow for the unravelling of mechanisms by which a host-59 associated microbial community affects these processes. Changes in the abundance 60 and diversity of or the complete absence of a specific microbiota can be associated 61 with autoimmune diseases (19), obesity (20), metabolic disorders (21)(22)(23), and 62 cancer (24,25). However, the constitution and functionality of a host-associated 63 microbiome depends on environmental conditions (26,27), such as the life stage 64 (28), and the physiology of the host (1). A variety of hosts can serve as 65 metaorganism models, but research on invertebrate-microbiota interactions, in 66 particular, broadens the concept of the metaorganism and enables the 67 4 disentanglement of fundamental mechanisms of host-microbe interactions as well as 68 their underlying regulatory principles (2). Recently, we established the moon jellyfish 69 Aurelia aurita, one of the most extensively studied Scyphozoans 30, 3, as a model for 70 metaorganism research (11,28,29). The advantage of jellyfish as model hosts lies in 71 their simple body structure, which consists of only two tissue layers and an acellular 72 layer: the endoderm, the ectoderm, and the mesoglea separating them (32). The 73 surfaces of both epithelia are subject to microbial colonization and interaction. 74 Despite its simple body structure, the jellyfish is distinguished by a complex life cycle. segments are then released as single ephyrae (33)(34)(35)(36). In a previous study, we 82 identified distinct bacterial community patterns for A. aurita's individual life stages, 83 pointing to the significance of microorganisms in the polyp-to-jellyfish transition (28).

84
At the molecular level, hormonal and neuronal signals act as nuclear hormone 85 receptors activate the metamorphosis (33,37). It has been demonstrated that several 86 proteins, including transcriptional regulators, participate in the induction of 87 strobilation. They include the retinoic X receptor (RxR), whose differential regulation 88 correlates with strobilation (33,38,39), and the transcription-regulating proteins de 89 novo methyl transferase (DNMT) and patched (Ptc), which are also implicated in the 90 induction of strobilation (33,(40)(41)(42). Moreover, the bone morphogenetic protein 91 (BMP) is engaged in the differentiation processes of the lobes and the gastrovascular 92 system, whereas Wnt1 is a morphogen for the localization involved in the anterior-93 5 posterior axis formation in metazoans (43)(44)(45). The molecular machinery of A. aurita 94 metamorphosis further includes the differential expression of the strobilation-specific 95 genes CL112, CL355, CL390, and CL631 (41,42). The expression of these four 96 genes is strongly upregulated in polyps that are about to enter the strobilation phase.  (41,42). Thus, CL112, CL355, CL390, and CL631 seem to 107 play a role in starting strobilation in A. aurita. 108 We recently described how in the absence of a native microbiota, the fitness of A. 109 aurita is severely impaired, and its asexual reproduction is halted (11). Recolonizing   Here, we aimed to identify the developmental stage(s) apt for the recolonization of 116 sterile animals in order to restore asexual reproduction, and to gain insights into the 117 role of the microbiota in this process. Sterile animals were recolonized with native The microbiota used as inoculum to recolonize sterile polyps was isolated from native    Table S1).

176
The gene expression analysis was extended with the developmental genes DNMT, 177 RxR, BMP, Wnt1, and Ptc. These genes produced similar expression patterns over     In order to exclude the possibility that the inducer, required for strobilation, had been 236 degraded or modified by the added bacteria, treatments R2 and R3 were repeated 237 with constant addition of the inducer. However, these R2i and R3i treatments did not 238 result in normal development (Fig. S2), as they also produced deformed strobilae and 239 severely deformed ephyrae. The deformation was thus unlikely caused by insufficient 240 inducer availability or activity., but rather by the timing of recolonization at a time   (Fig. 6). From this, we conclude that a direct contact between host and 268 bacteria is needed and only that leads to a healthy strobilation process. During this 269 direct contact a host receptor might directly perceive a signal, either due to the 270 contact itself or via a bacterial product that is produced in response to the contact.   Aurelia polyps harbor a specific bacterial community with a high diversity that 309 undergoes significant restructuring during the polyp-to-jellyfish transition (28,52). The 310 specific microbial community is assumed to be essential for the sessile life stage 311 (28). In return, the bacteria likely benefit from the polyp's mucus as a source of 312 nutrients (28,53,54). We observed that polyps with naturally associated native 313 microbiota and sterile polyps after recolonization harbored more similar microbial 314 communities in terms of diversity than either of them with the bacterial population in 315 the surrounding artificial seawater (Fig. 2), which corroborates with many other    Paired-end raw read files were processed using QIIME 2 (version qiime2-2021.2 414 (78)). The 16S rDNA sequences were denoised using DADA2 via q2-dada2. Quality fresh ASW containing inoculum applied the next day, so that the animals were   to account for multiple comparisons. These calculations were performed in R.

Acknowledgments
This work was conducted with the financial support of the DFG as part of the CRC1182 "Origin and function of metaorganisms" (Project B2).
For next-generation deep sequencing, we thank Sven Künzel and colleagues from the Department for Evolutionary Genetics of the Max Planck Institute for Evolutionary Biology.
We are further grateful for the fruiting discussions and support during the writing process by Trudy M. Wassenaar.

Data availability statement
All data generated during this study are included in this published article (and its supplemental file).
Sequence data were deposited under the NCBI BioProject PRJNA896887 comprising locus tag prefixes SAMN31571268 to SAMN31571288.

Competing Interests Statement
The authors declare no competing interests.        Revision of the manuscript "Asexual reproduction of Aurelia aurita depends on the presence of a balanced microbiome at polyp stage" by Nadin Jensen, Nancy Weiland-Bräuer, Shindhuja Joel, Cynthia Chibani, and Ruth Schmitz-Streit (Subject: Spectrum00262-23) We thank the three independent reviewers for the review process. We are pleased that the reviews were generally positive. We carefully addressed all questions in the point-by-point response below (reviewer-specific comments are highlighted in italics, compared to our answers in regular font) and revised the original manuscript according to the reviewers' recommendations. We have marked the changes in the manuscript with different colors for each reviewer (Reviewer 1, yellow; Reviewer 2, green; and Reviewer 3, turquoise).
Point-by-point responses: Response to Reviewer #1

In this article Jensen and colleagues explore the impact of the complex microbiome on strobilation of the jellyfish Aurelia aurita. They find that the presence of the native microbiome is required at the polyp stage for normal strobilation and ephyra release, and that introduction of the microbiome at subsequent timepoints is insufficient for metamorphosis. This paper is well thought-out with very nice figures. Understanding of host-microbe interactions in complex mutualisms is an important topic and this paper advances that understanding. I have some comments (see attached) that I think should be addressed, but consider them fairly minor.
We thank the reviewer for the review of our manuscript. We are pleased that only minor comments were raised and are thankful for the impression that we submitted a "well thought-out paper with very nice figures". Nevertheless, we are happy to answer the raised questions. Changes in the manuscript were accordingly highlighted in yellow.
General Comments:

1) I'd briefly explain around line 142 that the inoculum is from filtered homogenate.
Answer: We thank the reviewer for this valuable comment. We added the information in line 171 (track change version).

2) I liked your experiment demonstrating that cell-cell contact is required. My question building on this is whether you think tissue colonization by the bacteria is required, or whether you think bacteria in the water would be sufficient?
Answer: We thank the reviewer for his/her interest in our experiment. Indeed we propose that tissue colonization is required. In the meantime, we conducted preliminary experiments focusing on required tissue colonization for regular progeny output. We, therefore, incubated one sterile and one native polyp together in 1 mL sterile ASW for 72 h without a separating membrane in close vicinity before inducing strobilation with the chemical inducer. However, strobilation and ephyrae release failed. We further incubated sterile polyps in native ASW. Here, polyps were crucially diminished, as shown in a previous publication (Weiland-Bräuer et al., 2020, mBio). Consequently, we assume that tissue colonization of the polyp epithelium by key bacteria is required for the onset of strobilation.
Those results have to be further verified and will be included in a different manuscript

3) For lines 302-303 I would note that Kerwin et al. 2019 in mBio did find that chloramphenicol negatively impacted development of the Hawaiian bobtail squid
embryo, but that other antibiotics did not, and that paper was looking at microbiome functionality. Not asking you to cite it, but wanted to point it out. :) I was a little confused by your description of the antibiotics tested in lines 451-458 -which were tested singly, and did those not work sufficiently? Given the known toxicity of chloramphenicol that you cite, it may have been better not to include that. I think though that since your recolonized sterile polyps were fine, the antibiotic treatment wasn't a problem.
Answer: We thank the reviewer for her/his thoughts on antibiotic use. Indeed, we established an antibiotic mixture to generate sterile polyps by using different concentrations and mixtures of antibiotics. First, all antibiotics were singly tested; however, no single antibiotic could remove all bacteria, although cell numbers were reduced. Further, we tested different combinations of antibiotics, but again, 16S rRNA PCR revealed that some bacteria were unaffected by the mix. Finally, only the mixture of chloramphenicol, neomycin, ampicillin, streptomycin, rifampicin (each 50 mg/L), and 60 mg/L spectinomycin resulted in germ-free polyps. We are aware that antibiotics can have potential side effects on the host, as shown by Kerwin et al. 2019 for chloramphenicol. However, other antibiotics did not affect the embryo, as pointed out, and chloramphenicol did not affect adult squids. We now included the respective reference in the revised manuscript (line 351). All used antibiotics (single and combination) showed the same morphological deformations of A. aurita polyps independently of their target sites. Besides, recolonization led to a normal phenotype. We thus assume that neither chloramphenicol nor other antibiotics affect the polyp and conclude that a manipulated microbiota causes the observed defects.

4)
The NCBI BioProject ID did not appear when searched -assume this will be public by publication.
Answer: We thank the reviewer for this important comment. The reviewer is correct; we allow publicity of the dataset after the publication of the manuscript.

5) Did you do a glycerol control since your inoculum were frozen with glycerol? I'm assuming not, in which case you should note that somewhere and the reasoning behind that decision.
Answer: We thank the reviewer for bringing up this control. We proved the viability of cells after several freeze-thaw cycles without a significant loss of cell numbers due to the use of glycerol as a cryoprotectant. A glycerol-only control was conducted before the experimental start. A glycerol stock was prepared like the inoculum (10 % glycerol). Using the same concentrations as in the recolonization experiment (10 µl in 1 ml ASW), we incubated native and sterile polyps for 1 d to 7 d in the presence of glycerol. Polyp phenotypes and survival rates were not affected; thus, glycerol was rated neglectable. In the revised version, we have included this information in the Material&Methods part. 2) I really liked your color scheme for Figure 2 and  Answer: We thank the reviewer for the comments to improve the Figure. Particularly, a scroll-over function would be achievable and implemented in the published version. The bold font, highlighting specific genera, resulted from notably changed genera comparing treatments. However, the taxa are already sorted by decreasing abundance. Since the message was misleading, we removed the bold font to highlight those genera. The threshold for showing taxa is over 1 % relative abundance; this information is now included in the figure legend. Taxa below this threshold are grouped as "others". Within this group "others", genera present in native polyps but missing in recolonized polyps are hidden. We changed the Figure and legend accordingly.
We were also surprised by the alpha diversity within recolonized polyps and have the following hypothesis. The diversity is higher in recolonized polyps than in native ones but with less variation. We depict only a snapshot of the microbial composition after 48 h of recolonization and assume that the microbiota adapts with time (> 48 h), resulting in higher variation due to biological replicates. In total, 160 more ASVs were detected in recolonized polyps than in native ones, explaining the higher alpha diversity. However, none of those ASVs represent taxa not present in native polyp communities. Moreover, 122 ASVs were present in native but not in recolonized polyps among those representatives of Alpha-and Gamma-Proteobacteria, Bacilli, Bacteroida, and others. Those ASVs are suggested as irrelevant to the reproduction success. The higher relative abundance in recolonized polyps might be explained by a high number of ASVs that are outcompeted over time. In a future study, we plan to analyze the microbial community of recolonized polyps at different time points (long-term experiment).

3) Figure 3 is really nicely done -great job!
Answer: We thank the reviewer for taking our efforts into account. Figure 4 Answer: We thank the reviewer for raising those questions. All polyps, native and sterile, were induced for the onset of strobilation. Animals were recolonized at different time points (polyp, early and late strobila). All animals formed strobilae and released ephyrae; however, indicated treatments showed malformed phenotypes and consequently malformed and significantly less formed ephyrae. Exclusively, native and recolonized polyps showed regular strobilation and ephyrae release with the expected numbers. The respective percentages of released ephyrae of the different later stages are depicted in the box compared to the native polyp (which is set to 100 %), now mentioned in the figure legend of the revised version. We agree with the reviewer that different treatments are hard to compare based on their appearance; however, the brownish color of prolonged polyps and strobilae is a sign of regular strobilation. In 1980, a study by van den Branden described that the brown-red color of strobilae and ephyrae is formed in the animal and is melanin accompanied by a smaller fraction of a protein-bound brown pigment. These pigments appear as soon as strobilation is started and disappear after the liberation of ephyrae from the strobila. The information was added in the revised manuscript. Figure 5 is really nice -great presentation of complex data! How do you explain the difference in expression between the native and R1 at the polyp stage? The Figure  seems to contrast with your results at lines 253-254?

5)
Answer: We thank the reviewer for the valuable comment. Indeed, a difference between native polyps and recolonized ones was detected for CL355. The differences between CL112, CL390, and CL631 were non-significant, although the color code in part A is a bit misleading. That the differences are not significant is more visible in part B of the Figure, where the expression pattern over time for one gene is exemplarily shown -indicating no difference compared to the native polyp. Since the function of the CL genes has not been elucidated, we can only speculate that the slightly different community patterns of native and recolonized polyps cause the difference in expression patterns of CL355. We assume that the CL genes are differentially expressed during strobilation and might have different functions and importance throughout the process, revealing a variance in the effects of the treatments. In lines 295 ff., we describe the transcription profile of sterile polyps, which contrasts with native and recolonized ones. Similarly, recolonization after strobilation onset impaired gene expression of CL genes. Answer: We apologize for the incomplete Figure. We added the labels, the symbol for the inducer, and the scale bars. The legend was completed with the scale bar definitions. Answer: We thank the reviewer for reviewing our manuscript and the ecological perspective brought in. We agree with the reviewer that one should be careful with conclusions from studies with model organisms kept in the lab. We carefully went through our manuscript and toned down conclusions on ecological relevance. Nevertheless, we believe that model studies are essential to gain first insights into aspects of physiology, development, and ecology. We are pleased to answer the raised minor comments below, addressed in the revised manuscript accordingly (see marked passages in green).

Some minor comments:
Abstract: Line 23: Yet, the timing and molecular consequences of the microbiome during the 24 strobilation process had not been investigated. Is strangely formulated -the timing and molecular consequences of the microbiome-please re-write.
Answer: We thank the reviewer for the comment. The abstract was extensively changed due to addressing the comments of Reviewer 3, including the mentioned phrase. Answer: We thank the reviewer for asking about details on microbiota generation. We used a pool of 10 carefully washed animals for each life stage, not mixing the different stages. Those animals from one developmental stage were homogenized, the homogenate filtered to remove eukaryotic cells, and the microbial cell numbers were counted to set the concentration. Life stage-specific generated microbiota stocks were stored and used separately. Based on the life stage-specific microbial community patterns, we aimed to exclude life stage-specific community effects for the recolonization experiment, including R2/R3, shown in Figure 4C. We now rephrased the sentence in lines 485 ff. for a better understanding in the revised version. Answer: We thank the reviewer for this valuable comment. We checked the effect of several freeze/thaw cycles on the viability of cells in initial experiments by counting the cell numbers and colony-forming units. No significant loss of viability was detected even after three cycles. The community analysis of the inoculum (see Fig. 2) represents the snapshot of the inoculum after thawing before the use within recolonization experiments. Here, 85 ASVs present in the community of native polyps were not detected in the inoculum, suggesting that they got lost during preparation or lost viability after thawing (non-cultivable strains missed in cfu counts). Nevertheless, those microbes probably play a minor role in the considered asexual reproduction of A. aurita since the recolonized polyps reproduced like the native polyps. The affected taxa are grouped within "others". Answer: We thank the reviewer for the comment. All analyses refer to whole communities of the specified samples. Alpha diversity is plotted in Fig. 2B for the microbiome of native polyps, the generated microbiota of polyps used as inoculum in recolonization experiments, the recolonized polyp, and the inoculum incubated in ASW. Interestingly, the alpha diversity is higher in the recolonized polyp. We elaborated and suggested potential reasons for this question above (reviewer 1, comment to Figure 2).
Line 152 (and other cases): when you talk about relative abundance one needs to be aware that of course this depends on total cell abundance, e.g., one taxa can increase in relative abundance, but overall cell abundance (cells/mL) is lower and this is important!, also an increase/decrease can be due to decrease/increase of other taxa…it can give a wrong impression! Since I understood you used SYTO9 to distinguish between live and dead and made an effort to count cells on polyps etc., why not making an effort to take this actual cell numbers into account Answer: We thank the reviewer for the idea of accounting for the overall living cell abundance for the detected cell numbers. In consultation with bioinformaticians, we abstain from this recommendation. Since relative species abundance refers to how common or rare a species is relative to other species in a given location or community, the percentages can give insights into trends. However, they can not be calculated with detected cell numbers (due to the bioinformatic determination of species abundance). Theoretically calculating with living cell numbers might be misleading within a complex microbiota, as shown for A. aurita. Future studies should implement the detection of relevant key taxa by fluorescence in situ hybridization using taxon-specific probes. This would enable monitoring cell counts and localization in space and time.
Line 156-157: sentence not clear, please re-write: Overall, the microbiota of recolonized polyps more strongly resembled that of native polyps than bacteria maintained in artificial seawater (ASW) in absence of animals.
Answer: We apologize for the misunderstanding. We rephrased the sentence in the revised manuscript (lines 183 ff.).
Line 216-216: The individual administration of antibiotics also reduced the native bacterial community monitored by a decrease of 30 -80 % in CFU/ml. This is confusing to me: when you talk about CFU/mL I assume only cultivable part of community was assessed? But to my understanding this was not the case. Please explain.
Answer: We thank the reviewer for the valuable comment. For all (single and combination) antibiotic treatments and community reduction determination, we proved the potential sterility of animals by colony-counting the cultivable part of the microbiota in the first step. Secondly, non-sterile samples were not further analyzed. However, exclusively those samples without any detectable CFU (assumed to be sterile) were used for 16S rRNA amplicon sequencing. Sterility was generally verified by amplification failure.
Line 270-272: For this, native polyps with their diverse, healthy microbiome were incubated together with sterile polyps, but separated by a 0.2 µm filter that allowed the transfer of metabolites, but not of bacteria. If we are picky we would take into account the fact that some really small bacteria can escape 0.2 um filtration! Answer: We agree with the reviewer that very small microbes can escape 0.22 µm filtration. However, the potentially remaining microbes could not restore the impaired fitness effect of sterile polyps, indicating no crucial effect on the strobilation process.

Discussion:
Line 287: Our results not only illustrate how important a native microbiome is for the asexual reproduction of A. aurita, but also demonstrated that the generation of offspring is only possible when the microbiome is present before the animal enters the strobilation process. Just a general thought: I cannot image where and when in natural scenario microbiome would not be present before animal enters strobilation process, so not really sure, from ecological perspective, why this I even relevant. Maybe discussion on this aspect could be elaborate.
Answer: We thank the reviewer for the ecological point of view. We did not intend to discuss the importance and presence of microbes in general or even the occurrence of sterile surfaces in nature. We wanted to express that microbes, specifically key taxa and their related functions, are of enormous importance for the development of A. aurita at the specified time point (development stage). The only way to analyze that in experiments is to remove the microbes and elucidate the effects on asexual reproduction artificially. The presence of the specific polyp microbiome is crucial before the onset of strobilation for regular progeny output. We have therefore rephrased the sentence accordingly to avoid misunderstandings: the bacterial impact is required for normal offspring generation before the animal enters strobilation.
The bacterial impact is required before entering the strobilation process.
Line 332-334: The abundance of Vibrio and Colwellia is increasing in recolonized polyps, whereas ASW shifted the bacterial population to a significantly structure accompanied with a lower α-diversity. This is a very strange sentence, I don't understand what is meant here. Please correct.
Answer: We apologize for the misunderstanding. In lines 308 ff., we describe a conducted experiment. Native and sterile polyps were separated by a membrane, not allowing for cell but metabolite transfer. Since sterile polyps did not restore the impaired strobilation phenotype, we assume that direct contact with bacteria (e.g., a T6SS, or vesicles) is essential for regular strobilation. We agree with the reviewer that these are just preliminary results; however, those results point to a specific mechanistic interaction that has to be validated in future studies. Thus, the discussion section further includes our ideas on this potential interaction.
Lines 319-324: This belongs in the discussion part.
Answer: At this point, we would stay true to our writing style and make no changes.

Discussion:
Line 328: Again, you have only assessed strobilation which is ONLY ONE possible way of asexually reproduction in A. aurita. Others are budding, podocyst formation and many more. See Vagelli (2007) New observations on the asexual reproduction of Aurelia aurita (Cnidaria, Scyphozoa) with comments on its life cycle and adaptive significance.
Answer: We apologize for not being precise. We changed the unprecise wording of asexual reproduction to asexual reproduction by strobilation throughout the manuscript.
Line 347-352: Neglect means -fail to care for properly. Don't you agree that this is inappropriately strong wording? Those scientists were simply investigating another topic, not neglecting anything. Also, their work was done at a time long before the holobiont concept and /or microbiomes were a thing. You should simply say that it's the first time the microbiome has been considered in this context.
Answer: We apologize for the incorrect wording and agree with the reviewer. We changed the sentence accordingly.
Line 355: Only the ones that are visible to you.
Answer: We apologize for the misleading wording. We toned the sentence down.
Line 361: You are only mentioning one here. If you want to say "for other invertebrates", you need to mention more examples and references. Also, I would prefer to see references from organisms closer related to jellyfish, or at least marine invertebrates. A lot of research has been done on corals; can you find an example related to coral?
Answer: We agree with the reviewer on including more studies and references. We included an example of corals to support our statement (lines 369-371).
Line 372: What's the relevance of this quote? What are these proteins important for?
Answer: We thank the reviewer for the comment. The reference to the expression of mucus regulatory proteins in mice is an example of differential gene expression in native and sterile animals. Furthermore, mucus components are crucial in the interplay between the microbiota and the host in early-branching metazoans since mucus components are essential in developing the epithelial barrier as part of the innate immune defense.
Line 379: Lee at al. Is not about A. aurita but Chrysaora plocamia. Either remove or correct to "Some jellyfish polyps harbor …" or "Polyps from some jellyfish species, such as A. aurita and Chrysaora plocamia harbor …"