Morphological and Phylogenetic Study of Protococcidians Sheds Light on the Evolution of Epicellular Parasitism in Sporozoa (Apicomplexa), with the Description of Eleutheroschizon planoratum sp. nov

Round 1

Reviewer 1 Report

Dear Authors,

This is a very nice and carefully prepared study. The scientific content has a very high quality. The methods are superb. The microphotographs are excellent, real eye-catchers. I have recognized only some minor issues. All are marked and commented in the PDF attached.

However, I do not believe that the ITS2 models are correct.  I would say that helix II might have been assembled correctly in both species. Helix III might have been correctly folded in E. planoratum, while helix I in E. duboscqi. It is extremely unlikely that helix I is branched in one species, while helix III in another species. I strongly recommend to perform homology modeling and to find constraints that will made both helices to be straight (with some bulges). Homology modeling is often more important than the thermodynamic free Gibbs energy calculated by the program, as the secondary structure is under a complex folding control during transcription. The constrain information for helices I and III can be directly inserted in the box at http://www.unafold.org/mfold/applications/rna-folding-form.php

Homology modeling could be performed using RNAstructure ver. 6.0.1 (http://rna.urmc.rochester.edu/RNAstructureWeb).

How the hybridization 5.8-28S stem of the ITS2 was found? This is crucial and often a very complicated step. I use R2DT (Sweeney et al., 2021) to be sure. Please provide your strategy in M & M.

Despite this criticism, the single CBC found in helix II seems to be real (as this helix appears to be folded correctly in both species).

Comments for author File: Comments.pdf

English is basically fine, but there are quite many small issues. I have marked most of them in the PDF attached. However, I would recommend to check it by a professional.

Author Response

We thank the reviewers for their generous comments on the manuscript and have edited the manuscript to address their concerns (Please see the attachment and the revised manuscript). We have also changed Figure 5, added the graphical abstract and two new supplement figures (Figure S3-S4). Our manuscript has been checked by Prof. Valentina Kuznetsova (ORCID 0000-0001-8386-5453), a colleague fluent in English writing.

Reviewer #1:

This is a very nice and carefully prepared study. The scientific content has a very high quality. The methods are superb. The microphotographs are excellent, real eye-catchers. I have recognized only some minor issues. All are marked and commented in the PDF attached.

Response: We thank Reviewer #1 for in-depth reviewing. We have revised the manuscript following the recommendations and hope that the reviewer finds this satisfactory.

However, I do not believe that the ITS2 models are correct.  I would say that helix II might have been assembled correctly in both species. Helix III might have been correctly folded in E. planoratum, while helix I in E. duboscqi. It is extremely unlikely that helix I is branched in one species, while helix III in another species. I strongly recommend to perform homology modeling and to find constraints that will made both helices to be straight (with some bulges). Homology modeling is often more important than the thermodynamic free Gibbs energy calculated by the program, as the secondary structure is under a complex folding control during transcription. The constrain information for helices I and III can be directly inserted in the box at http://www.unafold.org/mfold/applications/rna-folding-form.php

Homology modeling could be performed using RNAstructure ver. 6.0.1 (http://rna.urmc.rochester.edu/RNAstructureWeb).

Response: The predicted 5.8S-LSU contacts are now presented as required (see Figure S3). We agree with Reviewer #1 that homology is more important for modeling than the computed Gibbs energy. This approach confirms the forked helix I in E. planoratum (see Figure S4). Unfortunately, none of the approaches, as well as the programs recommended by Reviewer #1, made it possible to construct a convincing model of helix III. We assume that this is due to weak pairing and real dynamics between alternative folds. At least the alignment of the primary structures is consistent with the hypothesis of independent expansions in the two species at different locations in their helix III region (Figure S4). Whether additional nucleotides form branching stems or extended internal bulges is questionable. Due to insufficient sampling of ITS2 from Eleuteroschizon spp., we cannot test the proposed models against covariances.

How the hybridization 5.8-28S stem of the ITS2 was found? This is crucial and often a very complicated step. I use R2DT (Sweeney et al., 2021) to be sure. Please provide your strategy in M & M.

Response: We have described our strategy in Results.

We mapped the complementary regions of 5.8S rRNA and LSU rRNA and thus localized the ITS2 region (Figure S3). Like some gregarines [27], Eleutheroschizon spp. have only three helices in the secondary structure of their ITS2 molecules. The first helix differs in the two species in the number of nucleotides. Based on the similar nucleotide motif in both species, we localized the target site of the expansion in the inner loop, resulting in the unusual branched helix I in E. planoratum (Figure S4). The second helix has the typical U-U mismatch [28]. The third helix is the longest and most variable; close values of initial ΔG were calculated for alternative folds of helix III. We chose variants with a similar arrangement of the conserved motif (Figure S4), but this choice suggests independent nucleotide extensions in the helix III in the two Eleutheroschizon species. Helix III seems to be partially unpaired due to the large number of mismatches and the small number of strong G-C pairs. Two CBCs were found in both Eleutheroschizon spp.: one in helix I and one in helix II (Figure 5). Considering only the most conserved part, one or two more CBCs are likely in helix III.

Please see the attachment and the revised manuscript.

Despite this criticism, the single CBC found in helix II seems to be real (as this helix appears to be folded correctly in both species).

Response: We have changed Figure 5. Please see the attachment and the revised manuscript.

peer-review-29868418.v1.pdf

Comments on the Quality of English Language

Response: We have made the necessary corrections.

English is basically fine, but there are quite many small issues. I have marked most of them in the PDF attached. However, I would recommend to check it by a professional.

Response: Our manuscript has been checked by Prof. Valentina Kuznetsova (ORCID 0000-0001-8386-5453), a colleague fluent in English writing. Please see the attachment and the revised manuscript.

Yours sincerely,

Gita Paskerova.

Author Response File: Author Response.pdf

Reviewer 2 Report

A well-written manuscript, good quality data, clearly presented with no unnecessary distractions or overinterpretations. The methods employed are appropriate and well executed. Great electron micrographs. This is the work of a highly skilled team with an excellent understanding of the systematics of these parasites. Great work overall. I don't have any objections.

PS: at line 412 the word 'secrets' perhaps meant 'secretions'?

Author Response

Reviewer #2: A well-written manuscript, good quality data, clearly presented with no unnecessary distractions or overinterpretations. The methods employed are appropriate and well executed. Great electron micrographs. This is the work of a highly skilled team with an excellent understanding of the systematics of these parasites. Great work overall. I don't have any objections.

Response: We thank Reviewer #2 for generous comments.

PS: at line 412 the word 'secrets' perhaps meant 'secretions'?

Response: Done. Please see the attachment and the revised manuscript.

Yours sincerely,

Gita Paskerova.

Author Response File: Author Response.pdf