Being Safe, but Not Too Safe: A Nudibranch Feeding on a Bryozoan-Associated Hydrozoan

Round 1

Reviewer 1 Report

This is an excellent piece of work. I really do not have any problems with the narrative or underlying science. My only critical comment has to do with the title. I do not really think the "Being safe, but not too safe:" part is necessary. The theoretically protective nature of the bryozoan host is not really part of the narrative, and so the title distracts a bit from otherwise fantastic work.

Author Response

R: This is an excellent piece of work. I really do not have any problems with the narrative or underlying science. My only critical comment has to do with the title. I do not really think the "Being safe, but not too safe:" part is necessary. The theoretically protective nature of the bryozoan host is not really part of the narrative, and so the title distracts a bit from otherwise fantastic work.

A: We are very grateful to the reviewer for the positive comments. Regarding the title, the ‘safe but not too safe’ relates to both the possible protective action of the bryozoan skeleton (that has now been discussed a bit more) but also to the possible protection exerted by the hydrozoan towards its host, something that is discussed in the ‘Discussion’ section. Therefore, we would like to keep the title as it is.

 

Reviewer 2 Report

The paper presents additional genetic data and anatomical characters of the newly described nudibranch species Sakuraeolis marhe Fernández-Simón & Moles, 2023, currently known only from the Maldives and the Philippines, and intriguing data on its preying on bryozoan associated hydrozoan species. This is really the first well-documented record of a nudibranch feeding on a bryozoan associated hydrozoan, and it will widen our knowledge on the biology of this interesting group of molluscs.

The manuscript is clear, relevant for the field and structured appropriately. Cited literature is present-day, adequate and sufficient, covering all main questions. Methods are mostly appropriate for the objective, so the results are reproducible. Illustrations on a whole are of good quality and corroborate the text. Conclusions mostly consistent with the arguments presented.

 

The main complaints concern too brief and insufficient description of methods mainly dealing with material manipulations and examinations (mentioned below):

 

Lines 74-76: “A fragment of the bryozoan colony, together with associated hydrozoans and preying nudibranchs, was collected…” – it was a living material or it fixed immediately after collection? If fixed – how it was fixed?

Lines 78-80: “…organisms were identified morphologically…” – the organisms were fresh or fixed prior to examination? This could cause ethics questions.

Line 88: “…cerata were analyzed under a Zeiss Axioskop 40 compound microscope…” – what magnification and contrast method was applied?

Line 89: “… to identify the nematocyst types.” – 1) identification and comparison of nematocytes implies measurement of nematocytes, 2) how do you identify the nematocytes – according to classification proposed in what reference?

Lines 93 and 95: ‘...using the primers in Palumbi…”, “…the primers in Colgan…” – the primers according to Palambi…

 

The Results section should be subdivided into several subsections with own subtitles: Anatomy, Molecular phylogeny, Cnidae...

 

Lines 155-156: “…and further confirmed the morphological identification. The hydrozoan…” – there were no morphological descriptions for hydrozoan…

Line 157: “…perfectly matched deposited…” – in further descriptions the homology between mollusc sequences are measured in percent – it is better to use the same for hydroid data.

Lines 159-160: “…and it resulted 93.44% homologous with one S. enosimensis sequence (GB: HQ010538) from…” – 1) it resulted to be … 2) the mentioned sequence number do not correcpond to any on the Figure 3.

Lines 170-171: “…species, together with two Facelinidae sp. 2 sequences, again similarly to Cunha et…” – ypu compare species with seuqences (slang) and with Cinha...    …similarly to the date from…

Line 181: “…but not on Celleporaria zooids.” – How do you confirm it?

Lines 187-189: “Only large size stenoteles … despite being common in the Zanclea hydrorhiza [33]” – I recommend to rewrite the sentence to clarify what are you data and what are the data from the reference you compare your data with.

Lines 199-202: “Additionally, the characterization of the internal anatomical diagnostic features (i.e. the buccal 200 apparatus and the reproductive system) were reported for the first time, complementingthe original description of the species.” – It is better to move to the comclusion.

Lines 227-228: “…polyps, namely large size stenoteles and, very rarely, euryteles.” – You compare the types, but to be strict – one need to compare of the nematocytes sizes.

 

Remarks on the figures.

Figure 1, a-b – what is the substance at the bottom centre(left)?

Figure 1, “(d) Signs of predation of S. marhe (white arrowhead) on Zanclea sp. 2 polyps (consumed and partially consumed polyps indicated by black arrowheads).” – the picture is not very convincing…  May be larger magnification will help.

 

Figure 2. a, c, f-i – lack scale bar.

Figure 2. f-i – images are not of good quality, partly diffuse image.

 

Figure 3 – it shows maximum likelihood 16S phylogenetic reconstruction. It should be better to show the reconstructions based on the sequences of the other genes from the work.

 

Figure 4, d – the quality of the image is low, so the illustration is unconvincing.

 

Author Response

R: The paper presents additional genetic data and anatomical characters of the newly described nudibranch species Sakuraeolis marhe Fernández-Simón & Moles, 2023, currently known only from the Maldives and the Philippines, and intriguing data on its preying on bryozoan associated hydrozoan species. This is really the first well-documented record of a nudibranch feeding on a bryozoan associated hydrozoan, and it will widen our knowledge on the biology of this interesting group of molluscs.

The manuscript is clear, relevant for the field and structured appropriately. Cited literature is present-day, adequate and sufficient, covering all main questions. Methods are mostly appropriate for the objective, so the results are reproducible. Illustrations on a whole are of good quality and corroborate the text. Conclusions mostly consistent with the arguments presented.

The main complaints concern too brief and insufficient description of methods mainly dealing with material manipulations and examinations (mentioned below):

A: We are very grateful to the reviewer for the useful comments and suggestions. We have added details about the materials and methods and followed all reviewer’s requests as listed below.

 

R: Lines 74-76: “A fragment of the bryozoan colony, together with associated hydrozoans and preying nudibranchs, was collected…” – it was a living material or it fixed immediately after collection? If fixed – how it was fixed?

Lines 78-80: “…organisms were identified morphologically…” – the organisms were fresh or fixed prior to examination? This could cause ethics questions.

A: We first observed the interaction between the living organisms at the stereo microscope, the organisms were subsequently anesthetized with menthol crystals and finally fixed in 99% EtOH for molecular analyses and 10% formalin for morphological analyses. We added this information in the materials and methods section.

 

R: Line 88: “…cerata were analyzed under a Zeiss Axioskop 40 compound microscope…” – what magnification and contrast method was applied?

A: Added.

 

R: Line 89: “… to identify the nematocyst types.” – 1) identification and comparison of nematocytes implies measurement of nematocytes, 2) how do you identify the nematocytes – according to classification proposed in what reference?

A: Software and literature used were added.

 

R: Lines 93 and 95: ‘...using the primers in Palumbi…”, “…the primers in Colgan…” – the primers according to Palambi…

A: Modified. We also added information of the primer pairs used and the PCR protocols.

 

 

R: The Results section should be subdivided into several subsections with own subtitles: Anatomy, Molecular phylogeny, Cnidae...

A: Modified.

 

R: Lines 155-156: “…and further confirmed the morphological identification. The hydrozoan…” – there were no morphological descriptions for hydrozoan…

A: Thank you for the observation. We have now added a short description of the hydrozoan morphology in the Results section.

 

R: Line 157: “…perfectly matched deposited…” – in further descriptions the homology between mollusc sequences are measured in percent – it is better to use the same for hydroid data.

A: Added.

 

R: Lines 159-160: “…and it resulted 93.44% homologous with one S. enosimensis sequence (GB: HQ010538) from…” – 1) it resulted to be … 2) the mentioned sequence number do not correcpond to any on the Figure 3.

A: We corrected the error by replacing the accession number. We erroneously referred to another identical sequence obtained from the same individual and therefore not included in the tree.

 

R: Lines 170-171: “…species, together with two Facelinidae sp. 2 sequences, again similarly to Cunha et…” – ypu compare species with seuqences (slang) and with Cinha...    …similarly to the date from…

A: Modified.

 

R: Line 181: “…but not on Celleporaria zooids.” – How do you confirm it?

A: We observed the intact zooids after the passage of the nudibranch, contrarily to the eaten hydrozoans. We specified this in the Results section.

 

R: Lines 187-189: “Only large size stenoteles … despite being common in the Zanclea hydrorhiza [33]” – I recommend to rewrite the sentence to clarify what are you data and what are the data from the reference you compare your data with.

A: Modified.

 

R: Lines 199-202: “Additionally, the characterization of the internal anatomical diagnostic features (i.e. the buccal 200 apparatus and the reproductive system) were reported for the first time, complementingthe original description of the species.” – It is better to move to the comclusion.

A: Modified.

 

R: Lines 227-228: “…polyps, namely large size stenoteles and, very rarely, euryteles.” – You compare the types, but to be strict – one need to compare of the nematocytes sizes.

A: We added that both the type and size of kleptocnodae were concordant with those of Zanclea sp. 2 nematocysts.

 

R: Figure 1, a-b – what is the substance at the bottom centre(left)?

A: It looks like an alga partially overgrowing the bryozoan colony.

 

R: Figure 1, “(d) Signs of predation of S. marhe (white arrowhead) on Zanclea sp. 2 polyps (consumed and partially consumed polyps indicated by black arrowheads).” – the picture is not very convincing…  May be larger magnification will help.

A: We increased the magnification to better show the predated hydrozoan polyps.

 

R: Figure 2. a, c, f-i – lack scale bar.

A: We added scale bars for all images in Figure 2.

 

R: Figure 2. f-i – images are not of good quality, partly diffuse image.

A: We agree that the quality is not very good, but this is due to the very small size of the individual and therefore also of the buccal structures. However, we believe that the figures of the radular structures (the only ones to date available on this species) still are informative, confirming the molecular identification of the species and giving an essential support to the phylogenetic relationships mainly at the genus taxonomic level. This aspect is not trivial if considering the uncertainties on the definition the genera belonging to the polyphyletic Facelinidae Family. We hope that the reviewer will understand our point of view.

 

R: Figure 3 – it shows maximum likelihood 16S phylogenetic reconstruction. It should be better to show the reconstructions based on the sequences of the other genes from the work.

A: We run the phylogenetic analysis for the H3 region as well and the resulting tree has been included as supplementary material. We provided the H3 sequence mostly because the H3 is the nuclear marker most used in nudibranch phylogeny. However, this marker is weakly informative at the genus and species level and therefore the obtained tree is not well resolved and doesn’t add much to our results.  

 

R: Figure 4, d – the quality of the image is low, so the illustration is unconvincing.

A: We agree that the quality of this image is not the best, but this is the only available picture of an eurytele found in the cnidosac. The kleptocnida is with no doubt an eurytele and therefore we feel that it should be kept in the image.

 

Reviewer 3 Report

This is an interesting manuscript that describes a new feeding mode of a cladobranch nudibranch on symbiotic hydrozoan polyps in a bryozoan colony. The species interaction is very interesting: evidently, the nematocytes that are supposed to protect the bryozoan host are ultimately used to protect the nudibranch from predation. How cool!

The manuscript is well written, and the figures are nice overall.  However, there is one glaring issue: you generated only 16S sequence data whereas Cunha et al. generated a COI sequence for S. marhe. Consequently, the genetic identity of the nudibranch in your study has not been established.  I would strongly suggest generating a COI sequence for direct comparison with the sequence from Cunha et al (OQ207003) and generating a COI tree in addition to the 16S tree. Your ID of S. marhe is probably correct, but since da Cunha did not examine the internal anatomy, a direct genetic comparison would be the easiest way to establish that you actually observed the same species. If you cannot generate a COI sequence, you should change the name to Sakuraeolis cf. marhe. Additionally, Sakuraeolis seems to be a paraphyletic genus, as suggested by your 16S analysis, as well as the COI analysis by Cunha et al. The focus of your manuscript is ecological; I would therefore not expect a taxonomic revision, or even lengthy discussion of the validity of the genus, but you should at least briefly mention it when discussing your phylogenetic tree.

Starting in line 156, you repeatedly talk about the degree of “homology” between different sequences. The term is inappropriate here because it implies an evolutionary context. The correct term would be “percent identity”.

In Fig. 1d, I cannot see what the white arrowhead is pointing to. I just see a blurry structure. Is that the nudibranch in a different plane of focus? 

I believe that there should be additional scale bars for Fig. 2 e-i. Figs. 2 e and f might have the same scale bar, but the other images look like they were taken at higher magnification. Please check this. 

Author Response

R: This is an interesting manuscript that describes a new feeding mode of a cladobranch nudibranch on symbiotic hydrozoan polyps in a bryozoan colony. The species interaction is very interesting: evidently, the nematocytes that are supposed to protect the bryozoan host are ultimately used to protect the nudibranch from predation. How cool!

A: We are grateful to the reviewer for the positive and constructive comments that helped to improve the quality of the manuscript. The text was modified according to almost all suggestions, as listed below.

 

R: The manuscript is well written, and the figures are nice overall. However, there is one glaring issue: you generated only 16S sequence data whereas Cunha et al. generated a COI sequence for S. marhe. Consequently, the genetic identity of the nudibranch in your study has not been established. I would strongly suggest generating a COI sequence for direct comparison with the sequence from Cunha et al (OQ207003) and generating a COI tree in addition to the 16S tree. Your ID of S. marhe is probably correct, but since Cunha did not examine the internal anatomy, a direct genetic comparison would be the easiest way to establish that you actually observed the same species. If you cannot generate a COI sequence, you should change the name to Sakuraeolis cf. marhe.

A: We thank the Reviewer for this comment. We understand the reviewer's doubt but after considering several aspects we still continue to be sure that the identification is correct. In fact, three main points should be considered. First of all, the 16S mitochondrial molecular marker is broadly considered as informative and diagnostic at the species level in nudibranchs and as shown in the 16S phylogenetic tree (mostly including sequences from the same specimens analyzed in the COI tree of Cunha et al.), our sequence shows the same relationships and phylogenetic position of the S. marhe COI sequences.

Second, our specimens were collected at approximately the same time and from the exact same locality where the S. marhe holotype was obtained (Faafu Atoll, Central Maldives); in the same way they were found on the same substrate (bryozoan associated with hydrozoans). These facts mean that they are sympatric and share the same ecology and trophic behavior. Third, it is true that there are no data on the internal anatomy of S. marhe from the original description, but results of our anatomical examinations confirms this species as congeneric, with an external morphology perfectly matching that of S. marhe original description.

One last point should be taken into consideration, that is, the negative effect that would follow if we increased the confusion in the taxonomy of this group that is intrinsically characterized by a controversial and unclear systematics. Considering that there is no evidence that questions the correct identification of these individuals, we think it is more correct and useful to keep the name of the species as reported in the text. Furthermore, we believe that it would be preferable to avoid the use of cf., especially to allow other researchers interested in the topic to use the additional anatomical information (that should have included also in the original description) provided with our work. We really hope that the reviewer will understand our point of view.

 

R: Additionally, Sakuraeolis seems to be a paraphyletic genus, as suggested by your 16S analysis, as well as the COI analysis by Cunha et al. The focus of your manuscript is ecological; I would therefore not expect a taxonomic revision, or even lengthy discussion of the validity of the genus, but you should at least briefly mention it when discussing your phylogenetic tree.

A: We have added in the discussion a brief paragraph about the non-monophyly of Sakuraeolis and the need of an integrative systematic assessment of the genus.

 

R: Starting in line 156, you repeatedly talk about the degree of “homology” between different sequences. The term is inappropriate here because it implies an evolutionary context. The correct term would be “percent identity”.

A: Modified.

 

R: In Fig. 1d, I cannot see what the white arrowhead is pointing to. I just see a blurry structure. Is that the nudibranch in a different plane of focus?

A: We modified the figure also following the suggestion of the other reviewer.

 

R: I believe that there should be additional scale bars for Fig. 2 e-i. Figs. 2 e and f might have the same scale bar, but the other images look like they were taken at higher magnification. Please check this.

A: Thanks for pointing out this shortcoming giving us the opportunity to correct it. We added the scale bars to the figure.

Reviewer 4 Report

The work by Maggioni and colleagues presents novel data on anatomy, genetics and feeding biology of the recently described species Sakuraeolis marhe Fernández-Simón & Moles, 2023. It is the first detailed report of the nudibranch mollusc preying on the bryozoan-associated hydrozoan of the genus Zanclea.

The beauty of this work lies in the thorough observation and the variety of methods used.

The authors rightly state that many researchers assumed the interaction between the nudibranch and its potential prey species based only on their co-occurrence. Contrary to this approach, in the present work, Maggioni and colleagues perform in vivo observations to prove that the nudibranch Sakuraeolis marhe feeds on Zanclea. The authors also used morphological and genetic methods to identify three invertebrate species.

I have only minor concerns regarding the methodology description and presentation of the results.

‘Material and Methods’ section

p 2, line 79 ‘the organisms were identified morphologically and genetically’

— As I understand, the bryozoan was identified only morphologically. However, this phrase leads one to think that all three species (the nudibranch, the bryozoan and the hydrozoan) were identified both morphologically and genetically. Please, specify which animals were identified by both methods and which — by using only morphological characters.

p 2, lines 83-84 ‘biological tissues’

— ‘soft tissues’ may sound better.

‘Results’ section

p 3, lines 128-131 ‘The teeth show a degree of intraindividual variability … (Figure 2g—i)’

— The described characters are not clearly visible on these microphotographs. A schematic overview of old and new teeth having different morphology would be a good addition.

p 3, line 134 ‘the deferent duct’

— the term ‘vas deferens’ will be more appropriate as it is commonly used in the anatomical descriptions of the nudibranch reproductive system.

p 6, lines 183-184 ‘… identified as stenoteles (Figure 4a—c)’

— the type of kleptocnidae is not clearly recognizable in figures 4a and 4b. Please, leave reference only to figure 4c.

p 6, line 184 ‘… holotrichous euryteles (Figure 2d)’

— incorrect reference. Please, change it to figure 4d.

Figure 2

Please, add a scale bar to figure 2c.

Based on the above, I recommend accepting this paper after minor revisions.

Author Response

R: The work by Maggioni and colleagues presents novel data on anatomy, genetics and feeding biology of the recently described species Sakuraeolis marhe Fernández-Simón & Moles, 2023. It is the first detailed report of the nudibranch mollusc preying on the bryozoan-associated hydrozoan of the genus Zanclea.

The beauty of this work lies in the thorough observation and the variety of methods used.

The authors rightly state that many researchers assumed the interaction between the nudibranch and its potential prey species based only on their co-occurrence. Contrary to this approach, in the present work, Maggioni and colleagues perform in vivo observations to prove that the nudibranch Sakuraeolis marhe feeds on Zanclea. The authors also used morphological and genetic methods to identify three invertebrate species.

I have only minor concerns regarding the methodology description and presentation of the results.

A: We thank the reviewer for the positive comments. We have addressed all the comments as listed below.

 

R: ‘Material and Methods’ section

p 2, line 79 ‘the organisms were identified morphologically and genetically’

— As I understand, the bryozoan was identified only morphologically. However, this phrase leads one to think that all three species (the nudibranch, the bryozoan and the hydrozoan) were identified both morphologically and genetically. Please, specify which animals were identified by both methods and which — by using only morphological characters.

A: We added this information.

 

R: p 2, lines 83-84 ‘biological tissues’

— ‘soft tissues’ may sound better.

A: Modified.

 

R: ‘Results’ section

p 3, lines 128-131 ‘The teeth show a degree of intraindividual variability … (Figure 2g—i)’

— The described characters are not clearly visible on these microphotographs. A schematic overview of old and new teeth having different morphology would be a good addition.

A: We thank the reviewer for this suggestion. It would be beautiful to add this kind of information but considering that we have only two specimens we believe that in this case it would not be representative of the intraspecific variability of the species but would risk being misleading and not strictly in line with the focus of the work. However, it is a very interesting field and often underestimated by the experts of this group of mollusks. In fact, almost in all the descriptions of the radula that are recently made and published, the shape of a single tooth is reported as if it were representative of all the teeth of the radula even if there is a variability between them. For this reason, since we don't have more samples to analyze, we still prefer to point out that we have noticed a variability between the teeth but without going into the merits. However, we really appreciate the sensitivity and attention of the reviewer in this regard.

 

R: p 3, line 134 ‘the deferent duct’

— the term ‘vas deferens’ will be more appropriate as it is commonly used in the anatomical descriptions of the nudibranch reproductive system.

A: Modified.

 

R: p 6, lines 183-184 ‘… identified as stenoteles (Figure 4a—c)’

— the type of kleptocnidae is not clearly recognizable in figures 4a and 4b. Please, leave reference only to figure 4c.

A: Modified.

 

R: p 6, line 184 ‘… holotrichous euryteles (Figure 2d)’

— incorrect reference. Please, change it to figure 4d.

A: Modified.

 

R: Figure 2

Please, add a scale bar to figure 2c.

Based on the above, I recommend accepting this paper after minor revisions.

A: We added scale bars for all images in Figure 2.

Round 2

Reviewer 3 Report

I still think the paper would be stronger if you included the COI sequence, but I understand that there are sometimes limitations.