Wolbachia Induces Structural Defects Harmful to Drosophila simulans Riverside Spermiogenesis

Round 1

Reviewer 1 Report

The manuscript of Maria Giovanna Riparbelli and coauthors describes the effects of Wolbachia on the spermatogenesis of Drosophila simulans.

By using transmission electron microscopy, they report several defects in spermiogenesis which include a reduced number of sperm tails accompanied and ultrastructural defects in elongating spermatids. They propose that these alteratiins could be the result of the mechanical stress induced by the presence of the bacteria during the process of spermatid elongation.

Overall, the data reported in the manuscript are novel and support the conclusions.

However, there are several typos and grammar errors that need to be addressed. Here is a list:

1)    Lines 62-64: “Rather, the bacteria that inhabits in high numbers almost all the spermatocytes and early spermatids [29,30], raises the question if their density can interfere with the proper dynamics of the germ 64 cells.”

2)    Lines 114-116: “Spermiogenesis is a complex process that transform round immotile spermatids in thin elongated moving sperm by a dramatic reorganization of the cytoplasmic organelles.” Should be changed to: Spermiogenesis is a complex process which transforms round immotile spermatids in thin elongated moving sperm by a dramatic reorganization of the cytoplasmic organelles.”

3)    Lines 116-118: ”The basal body, inherited by the cilium-like regions of the primary spermatocytes, nucleates the axoneme that  acquire” should be changed to: ”The basal body, inherited by the cilium-like regions of the primary spermatocytes, nucleates the axoneme that  acquires”

4)    Lines 266-269: “When the individualization process fails the cysts contained axonemes interspersed within large cytoplasmic areas; moreover these abnormal cysts appeared larger than the properly individualized cysts” should be changed to: “When the individualization process fails the cysts contains axonemes interspersed within large cytoplasmic areas; moreover, these abnormal cysts appear larger than the properly individualized cysts”

5)    Lines 285-288 “Improperly individualized cysts may contain normal mature sperm consisting of the axoneme and the mitochondrial derivative along with abnormal sperm that had only the axoneme or sperm in which the axoneme was lost and only the major mitochondrial derivative was found” This sentence is not clear and should be adjusted.

6)    Lines 321-32 : “The radial spokes  of the axoneme are no longer detectable and the central tubule pairs lost their usual position at the center of the axoneme.”  should be changed to “The radial spokes  of the axoneme are no longer detectable and the central tubule pairs lose their usual position at the center of the axoneme.”

7)    Lines 324-325: “Thus, the axonemes increase their diameter and lost their circular arrangement.” “Thus, the axonemes increase their diameter and lose their circular arrangement.”

 

The quality of the English is very good. I suggested minor changes in the comments to the authors.

Author Response

We thank the Reviewer for his/her positive evaluation of our manuscript. Here is the point-by-point answer to the Reviewer’s comments:

 

1)    Lines 62-64: “Rather, the bacteria that inhabits in high numbers almost all the spermatocytes and early spermatids [29,30], raises the question if their density can interfere with the proper dynamics of the germ 64 cells.”

Thanks, corrected

 

2)    Lines 114-116: “Spermiogenesis is a complex process that transform round immotile spermatids in thin elongated moving sperm by a dramatic reorganization of the cytoplasmic organelles.” Should be changed to: Spermiogenesis is a complex process which transforms round immotile spermatids in thin elongated moving sperm by a dramatic reorganization of the cytoplasmic organelles.”

Thanks, corrected as suggested

 

3)    Lines 116-118: ”The basal body, inherited by the cilium-like regions of the primary spermatocytes, nucleates the axoneme that  acquire” should be changed to: ”The basal body, inherited by the cilium-like regions of the primary spermatocytes, nucleates the axoneme that  acquires”

Thanks, corrected

 

4)    Lines 266-269: “When the individualization process fails the cysts contained axonemes interspersed within large cytoplasmic areas; moreover these abnormal cysts appeared larger than the properly individualized cysts” should be changed to: “When the individualization process fails the cysts contains axonemes interspersed within large cytoplasmic areas; moreover, these abnormal cysts appear larger than the properly individualized cysts”

Thanks, corrected

 

5)    Lines 285-288 “Improperly individualized cysts may contain normal mature sperm consisting of the axoneme and the mitochondrial derivative along with abnormal sperm that had only the axoneme or sperm in which the axoneme was lost and only the major mitochondrial derivative was found” This sentence is not clear and should be adjusted.

 

Thanks, corrected

6)    Lines 321-32 : “The radial spokes  of the axoneme are no longer detectable and the central tubule pairs lost their usual position at the center of the axoneme.”  should be changed to “The radial spokes  of the axoneme are no longer detectable and the central tubule pairs lose their usual position at the center of the axoneme.”

We rephased the sentence as suggested

 

7)    Lines 324-325: “Thus, the axonemes increase their diameter and lost their circular arrangement.” “Thus, the axonemes increase their diameter and lose their circular arrangement.”

Thanks, corrected

 

Reviewer 2 Report

Riparbelli et al. extended their previous observations regarding Wolbachia impact on spermiogenesis. They performed an ultrastructural analysis of sperm development in Wolbachia-infected Drosophila simulans and observed several structural defects such as mitochondrial and axoneme abnormalities, insufficient number of the germ cells within the cysts and incomplete individualization during sperm maturation. The paper is well written, and results are well presented. I have a couple of comments and one major concern for authors to address before I could provide my approval for publication.

 

L96 – Testes from both pupae and adults were dissected. However, it is not clear whether the results presented in the paper are from pupae or adult testes? If both, please indicate which ones are which and whether authors observed same defects across both stages or not. If not, what were the differences?

 

My major concern is the uninfected testes are not shown in parallel to infected testes to make sure the observed defects are not artifacts due to internal host physiological changes. Did authors test uninfected testes to rule it out?

 

What is the magnitude of defects shown here? For example, what percentage of cysts per testes show abnormalities?

 

Will there be similar defects, few or no defects in uninfected testes compared to those in infected testes? Quantification data from infected vs uninfected males along with microscopy visuals will help addressing this.

 

L400-403 Authors have not shown any visual or quantifying data on sperm shape defects in infected vs uninfected testes. That comparison will help justifying this study’s data with previous published ones and the % differences.

The manuscript is well written. 

Author Response

We thank the reviewer for his/her positive comments which allowed us to improve the manuscript Here is the point-by-point answer to the Reviewer’s comments

 

L96 – Testes from both pupae and adults were dissected. However, it is not clear whether the results presented in the paper are from pupae or adult testes? If both, please indicate which ones are which and whether authors observed same defects across bot h stages or not. If not, what were the differences?

Thank you very much for your kind suggestions! We agree that it is not clear which stages of development have been analysed. Therefore, we tried to clarify this point by adding a new paragraph (l.132-135). Moreover, we add to the figure explanation the developmental stage of the samples examined.

 

My major concern is the uninfected testes are not shown in parallel to infected testes to make sure the observed defects are not artifacts due to internal host physiological changes. Did authors test uninfected testes to rule it out?

The observation of the Reviewer is right! It was important to refer to uninfected flies as a comparison. Therefore, we add the new Figure 8 and the new section (3.6) to describe spermiogenesis progression in the bacteria free Drosophila simulans watsonville strain. Within section 3.6 we highlight the differences observed during the spermiogenesis of infected and uninfected Drosophila simulans strains.

 

What is the magnitude of defects shown here? For example, what percentage of cysts per testes show abnormalities?

According to the questions raised by the Reviewer we add a new paragraph (l.136-140). Moreover we add the new Figure 2A that is a cross section showing different levels of spermatids within the same cyst. From this low magnification picture it is possible to see that Wolbachia has a different localization along the spermatids.

 

Will there be similar defects, few or no defects in uninfected testes compared to those in infected testes? Quantification data from infected vs uninfected males along with microscopy visuals will help addressing this.

The new section (3.6) and the new Figure8 could represent the answer to the correct observations of the Reviewer.

 

L400-403 Authors have not shown any visual or quantifying data on sperm shape defects in infected vs uninfected testes. That comparison will help justifying this study’s data with previous published ones and the % differences.

Accordingly, we add the quantitative data requested (l.499-503).

 

Reviewer 3 Report

 

This descriptive ultrastructural study follows up on a study from the same group in Mechanisms of Development (2007) that had used both fluorescence and electron microscopy to describe the development and maturation of sperm cysts in Drosophila simulans DSR infected with Wolbachia bacteria that cause cytoplasmic incompatibility (CI). In the original paper, defects in maturing spermatids were described that are similar to what is described here, although the new descriptions are more extensive.

 

Overall, this work is technically satisfactory as far as it goes, but it could have been done more quantitatively and with comparisons to a D. simulans DSW infected line that does not cause CI. The latter would have allowed a more definitive dissociation of CI from spermiogenesis defects, which the authors infer (and which I also think is correct) but could have been established experimentally. A recent paper from the Bordenstein group (PLoS Biology 2022) suggests that defects in spermiogenesis ARE linked to CI, so a DSR versus DSW comparison would have been very helpful here.

 

Specific comments:

 

-Throughout the paper, it would have been nice to provide more quantitative data, noting what percentage of cysts or spermatids examined had the abnormalities described (ideally, comparing to uninfected or DSW). These numbers could replace comments such as “It is not uncommon to find within the same cyst spermatids that had one axoneme normally associated with two mitochondrial derivatives together with spermatids containing two axonemes but only two mitochondria, and spermatids with two axonemes and only one mitochondrion (Figure 2C,D).” [How common? What fraction had two axonemes and two mitochondria and what fraction two axonemes and only one mitochondria?]

 

-I found the ciliary cap section hard to follow, including exactly what it is in the figures. This could be improved, perhaps by providing a color marking of the cap structure. In general, referring in the main text to the arrowheads and arrows in the figures would make it easier to follow than the currently favored reference only in the legends. More extensive structure highlights in the figures might also be justified.

 

-Pg. 14: “these proteins [CifA and CifB] are released in the germ cell cytoplasm by temperate bacteriophages WO [25,26,54]” I know of no evidence of phage being the vehicle for CifA/B delivery to the germ cell cytoplasm. If I missed this, please elaborate.

 

-I wonder if a cartoon figure at the end that summarizes the observed cytological defects might be helpful?

The manuscript is very readable, but English grammar will need corrections in quite a few places.

Author Response

We thank the reviewer for his/her positive comments which allowed us to improve the manuscript Here is the point-by-point answer to the Reviewer’s comments

 

Overall, this work is technically satisfactory as far as it goes, but it could have been done more quantitatively and with comparisons to a D. simulans DSW infected line that does not cause CI. The latter would have allowed a more definitive dissociation of CI from spermiogenesis defects, which the authors infer (and which I also think is correct) but could have been established experimentally. A recent paper from the Bordenstein group (PLoS Biology 2022) suggests that defects in spermiogenesis ARE linked to CI, so a DSR versus DSW comparison would have been very helpful here.

Thanks! This suggestion is right and we had to analyse the uninfected strain. Accordingly we add the new section 3.6 and the relative new Figure8 to analyse the spermiogenesis in the bacteria free Drosophila simulans watsonville and thus make a comparison with the infected strain object of the present study.

 

Throughout the paper, it would have been nice to provide more quantitative data, noting what percentage of cysts or spermatids examined had the abnormalities described (ideally, comparing to uninfected or DSW). These numbers could replace comments such as “It is not uncommon to find within the same cyst spermatids that had one axoneme normally associated with two mitochondrial derivatives together with spermatids containing two axonemes but only two mitochondria, and spermatids with two axonemes and only one mitochondrion (Figure 2C,D).” [How common? What fraction had two axonemes and two mitochondria and what fraction two axonemes and only one mitochondria?]

Thank you for your suggestion. We agree that not including quantitative data was a major problem. Therefore, we add the required quantitative data together with the revised version of the manuscript.

 

I found the ciliary cap section hard to follow, including exactly what it is in the figures. This could be improved, perhaps by providing a color marking of the cap structure. In general, referring in the main text to the arrowheads and arrows in the figures would make it easier to follow than the currently favored reference only in the legends. More extensive structure highlights in the figures might also be justified.

Accordingly with the observations of the Reviewer we partially modified section 3.3 and added additional labels to Figure5 to better explain the ciliary cap structure. Moreover, we added in Figure 8D the ciliary cap of the uninfected Drosophila strain for comparison.

 

-Pg. 14: “these proteins [CifA and CifB] are released in the germ cell cytoplasm by temperate bacteriophages WO [25,26,54]” I know of no evidence of phage being the vehicle for CifA/B delivery to the germ cell cytoplasm. If I missed this, please elaborate.

Thank you very much for your suggestions! We rephased the sentence as follow: “Since the genes coding for Cifa and Cifb proteins are associated with the temperate bacteriophage…”

 

-I wonder if a cartoon figure at the end that summarizes the observed cytological defects might be helpful?

We agree that a cartoon figure could be useful, but we have serious problem to edit such cartoon. Please, accept our apologies but we don't have the possibility to do that.

 

Round 2

Reviewer 2 Report

Authors have successfully addressed my concerns. 

Author Response

We thank the Reviewer for his helpful suggestions that improved the manuscript.

Reviewer 3 Report

 

The authors have sharpened their analysis substantially with greater attention to quantification where possible. I had mistakenly thought the D. simulans DSW strain was a Wolbachia-infected line that did not suffer from CI –it is actually uninfected– so my hope that the link of CI to sperm morphological defects could be analyzed was misplaced. However, it is still a good control to have in general, so I welcome the addition of the data with this strain.

 

One more small point about the Discussion regarding the presence of cifA/B in WO prophage: I do not see the logic of why the presence of these genes in a prophage is relevant to the question of how the proteins might make their way into noninfected cysts. The same question, which I agree is relevant, would still hold even if these genes were outside of a prophage insertion in the bacterial genome.

 

Otherwise, I feel the response of the authors to my original critique is satisfactory.

Very minor English editing will be needed.

Author Response

We  preferred to remove the paragraph regarding the presence of cifA/B in the WO prophage from the last part of the Discussion.

We would also like to thanks the Reviewer for the helpful suggestions that improved the manuscript.