A Structural Analysis of Host–Parasite Interactions in Achatina fulica (Giant African Snail) Infected with Angiostrongylus cantonensis

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Comment to “Comprehensive structural analysis of host-parasite interactions in Achatina fulica (giant African snail) infected with Angiostrongylus cantonensis”

The study is an interested contribution to host-parasite interaction aspects of the two taxa. Histological section alone and coupled with SEM provided additional information about the immune response of the snail. In my opinion the most interesting aspects about “beads” should be emphasized more, by providing sizes of these particles. Recent studies addressed extracellular vesicles and other kind of particles produced by living organisms as a central factor in cell-cell communication, even at inter-kingdom level. Moreover, i suggest to mitigate the correlation between this study and the one-health approach, that is not so direct and clear.

Minor concerns:

I suggest to remove or at least mitigate the mention of the study within the One Health Approach, because no additional aspect then directly interested host-parasite have been taken into account by the authors. At least they could stress the zoonotic potential, but one-health is a multidisciplinary approach not very well here addressed. IN this regard, at line 46 authors could include some case reports or incidence values in humans.

Lines 138-139 please rephrase avoiding to repeat “tissue”

Line 140: “nematode larvae and host tissue” please be more specific: cite the parasite species and specify the tissue

Figure 3A-C and lines 177-178: “… the granuloma surrounded by a cluster of beads, which are distributed in the boundary region of the larvae”. What’s the size of these particles? you should mention. Have you any clue if these particles may be related to extracellular vesicles? Apoptotic bodies? knowing the size might assist a better speculation about these particles. I suggest to change beads with particles.

Author Response

Rebuttal letter

Reviewer 1:

Minor concerns:

I suggest to remove or at least mitigate the mention of the study within the One Health Approach, because no additional aspect then directly interested host-parasite have been taken into account by the authors. At least they could stress the zoonotic potential, but one-health is a multidisciplinary approach not very well here addressed.

Reply: Thank you for your suggestion; it has been incorporated into the text.

 

IN this regard, at line 46 authors could include some case reports or incidence values in humans.

Reply: Thank you for your suggestion; it was included in the text.

Lines 138-139 please rephrase avoiding to repeat “tissue”

Line 140: “nematode larvae and host tissue” please be more specific: cite the parasite species and specify the tissue
Reply:  These sentences, as recommended by the Reviewer 3, have been removed.

Figure 3A-C and lines 177-178: “… the granuloma surrounded by a cluster of beads, which are distributed in the boundary region of the larvae”. What’s the size of these particles? you should mention. Have you any clue if these particles may be related to extracellular vesicles? Apoptotic bodies? knowing the size might assist a better speculation about these particles. I suggest to change beads with particles.

Reply: Thank you for your valuable suggestion. We have incorporated the change in the name of the structure and conducted measurements on these spherical particles. Furthermore, we propose in the paper that these particles may be classified as microvesicles, considering their size, thereby enhancing our results and discussion. Your suggestion has undoubtedly enriched the content of the paper. We have incorporated the methodology of measurements into the SEM description as per your suggestion.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

I recommend to the author that, when citing the bibliography in the text, they include the publication years, as in Brockelman et al. (year). While I understand there is a dedicated section for references, incorporating the publication date in the text enhances readability for the reader. 

The research showcases both originality and relevance through its utilization of scanning electron microscopy (SEM) to explore the structural organization of host tissue infected by A. cantonensis. While similar studies have been conducted with other nematode species, this approach proves particularly significant within the One Health framework, shedding light on the intricate dynamics of zoonosis and host-parasite interactions.

Notably, the study addresses a significant gap in experimental data related to A. cantonensis infection in the African giant snail. The methodology employed is not only adequate but also rigorous, effectively substantiating their hypothesis.

The conclusions drawn from the research align seamlessly with the presented evidence. Third-stage larvae of A. cantonensis, intricately associated with granulomas, are observed in various regions of A. fulica, providing valuable insights into the host-parasite relationship.

Moreover, the work is commendably supported by a well-selected array of references. The inclusion of references spanning from the early discovery of the parasite to current research enhances the depth and credibility of the study.

In addition, the tables and figures presented in the research are accurate and contribute effectively to the overall clarity of the findings. This comprehensive approach not only advances our understanding of A. cantonensis but also sets a commendable standard for future research in this field.

Author Response

Rebuttal letter

Reviewer 2:
I recommend to the author that, when citing the bibliography in the text, they include the publication years, as in Brockelman et al. (year). While I understand there is a dedicated section for references, incorporating the publication date in the text enhances readability for the reader.

Reply: We agree, but this format is the journal’s policy.

 

The research showcases both originality and relevance through its utilization of scanning electron microscopy (SEM) to explore the structural organization of host tissue infected by A. cantonensis. While similar studies have been conducted with other nematode species, this approach proves particularly significant within the One Health framework, shedding light on the intricate dynamics of zoonosis and host-parasite interactions.

Notably, the study addresses a significant gap in experimental data related to A. cantonensis infection in the African giant snail. The methodology employed is not only adequate but also rigorous, effectively substantiating their hypothesis.

The conclusions drawn from the research align seamlessly with the presented evidence. Third-stage larvae of A. cantonensis, intricately associated with granulomas, are observed in various regions of A. fulica, providing valuable insights into the host-parasite relationship.


Moreover, the work is commendably supported by a well-selected array of references. The inclusion of references spanning from the early discovery of the parasite to current research enhances the depth and credibility of the study.

In addition, the tables and figures presented in the research are accurate and contribute effectively to the overall clarity of the findings. This comprehensive approach not only advances our understanding of A. cantonensis but also sets a commendable standard for future research in this field.

 

Reply: Thank you for recognizing our research on A. cantonensis infection. Your positive comments on our methodology, conclusions, and comprehensive approach are truly appreciated. We are grateful for the acknowledgment of our contribution to filling a critical gap in experimental data and setting a standard for future research. Your support motivates us to continue exploring host-parasite interactions. Thank you.

 

Author Response File: Author Response.pdf

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Title

Remove word 'comprehensive', as this suggests all aspects have been investigated which is not true since only 1 timepoint and a few methods were employed. Comprehensive suggests live imaging and immunological tracing over time with possible RNA or protein analyses answering fundamental biological questions about parasitism in this intermediate host, which was not the case.

Abstract

Line 17 = "around the world, and experimental" change to "around the world. Experimental" Sentence is too long and should be split.

Introduction

Line 44 - "however with reports" should say "however there are reports"

Line 50 - "one of the responsible for the spread of A. cantonensis" should read "one of the intermediate hosts responsible for the spread of A. canonensis".

Line 64-65 "the deepening of the knowledge of the host-parasite relationship in zoonotic diseases is a useful tool to enhance effectiveness of public health. " This doesn't make sense and the point is quite weak.

Line 67-88 - This section needs to be condensed significantly. This text should be in the discussion not the introduction, but a short statement about why the study is novel and answers a question about host-parasite interactions is important.

Methods

Line 107 - "diaphanization" not "diafanizacion"

Results

Line 138 - heeds sub-heading such as 'histopathology'

Line 138-139 - remove "As expected, the tissue of the non-infected snail showed a regular and organized structures of the tissue." and "In our results,". You need to point to the differences between uninfected and infected tissues where relevant, not make one blanket statement at the start of the results.

Line 145 - "The tissue of the infected snails (Figure 1) did not show" - you need to expand on Figure 1 in the text and point out what you saw in each figure part or groups of figure parts. This is a figure with

Line 146 - "control group (not infected)" can be updated to "uninfected".

WHERE ARE THE CONTROLS IN THE DATA SHOWN?

References

Line 375 - "1. 1." All references are numbered twice! Poor formatting.

Comments on the Quality of English Language

See above comments, numerous areas where English can be improved but overall it is understandable.

Author Response

Rebuttal letter

Reviewer 3:

 

Title
Remove word 'comprehensive', as this suggests all aspects have been investigated which is not true since only 1 timepoint and a few methods were employed. Comprehensive suggests live imaging and immunological tracing over time with possible RNA or protein analyses answering fundamental biological questions about parasitism in this intermediate host, which was not the case.
Reply: Thank you for your comment; we have revised the title as suggested and removed the word “comprehensive”.

 
Abstract
Line 17 = "around the world, and experimental" change to "around the world. Experimental" Sentence is too long and should be split.
Reply: Done.

Introduction
Line 44 - "however with reports" should say "however there are reports"

Reply: Done.

Line 50 - "one of the responsible for the spread of A. cantonensis" should read "one of the intermediate hosts responsible for the spread of A. canonensis".
Reply: Done.

Line 64-65 "the deepening of the knowledge of the host-parasite relationship in zoonotic diseases is a useful tool to enhance effectiveness of public health. " This doesn't make sense and the point is quite weak.

Reply: We made the changes you suggested.

Line 67-88 - This section needs to be condensed significantly. This text should be in the discussion not the introduction, but a short statement about why the study is novel and answers a question about host-parasite interactions is important.
Reply: We revised the sentence, condensing the information.

Methods
Line 107 - "diaphanization" not "diafanizacion"
Reply: Thank you, the word has been corrected.

Results
Line 138 - heeds sub-heading such as 'histopathology'
Reply: Ok, it has been included in the result section.

 

Line 138-139 - remove "As expected, the tissue of the non-infected snail showed a regular and organized structures of the tissue." and "In our results,". You need to point to the differences between uninfected and infected tissues where relevant, not make one blanket statement at the start of the results.

Reply: This sentence was removed as recommended by different reviewers.

 

Line 145 - "The tissue of the infected snails (Figure 1) did not show" - you need to expand on Figure 1 in the text and point out what you saw in each figure part or groups of figure parts. This is a figure with…

Reply: Thank you for your suggestion; the description of Figure 1 has been enhanced in the results text. Please see lines 134-145 in the revised version of the paper.  

 

Line 146 - "control group (not infected)" can be updated to "uninfected".

Reply: Done.

WHERE ARE THE CONTROLS IN THE DATA SHOWN?

Reply: Thank you for the observation, and we sincerely appreciate and understand this request. We typically incorporate control groups in our laboratory experiments; however, for this particular study, we lack images of control snails. The primary focus of our article's results was to provide a detailed analysis of the granuloma structure, specifically concentrating on its characteristics rather than exploring the broader impact on the host tissue or incorporating various experimental groups.

References
Line 375 - "1. 1." All references are numbered twice! Poor formatting.

Reply: It has been modified.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have worked a lot with the intent to follow suggestions of the reviewers and I really appreciated the efforts. 

Most of the updates upgraded the manuscript and contributed to a better analysis and discussion. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Materials and methods needs more detail, for instance what series of xylene was used for line 108, if so.

Figure 1 - add additional histochemical labels to identify different parts of the tissue present for context.

Figure 1 - needs control snails for comparrison

Unable to continue due to the lack of controls used in the study.

Comments on the Quality of English Language

Abstract is really poorly written and confusing in places. The writing needs to be clear and to the point. For instance:

"The knowledge of the host-parasite relationship within the One Health Approach is relevant, since zoonosis are closely intertwined in this complex mechanisms of wild/human hosts, parasite adaption and dispersion." This sentence makes no sense. I get the idea of the writing but it really needs to be rewritten along with the whole abstract.

Line 30 - adaption  is not a word in English. Adaptation is a word.

Some formatting errors in the publication, please check these before resubmission.

Introduction in general ok but the formatting and English language needs some improvement in areas.

 

Author Response

Points to consider in the new version (marked version):

Marked in yellow: Improvements to the text and changes following suggestions from reviewers.

Marked in red: new information, new bibliography and other points presented by the reviewers.

Review 1: Materials and methods need more detail, for instance what series of xylene was used for line 108, if so.

Query: The protocol was changed and improved – lines 106-138

 

Review 1: Figure 1 - add additional histochemical labels to identify different parts of the tissue present for context.

Query: In Figure 1 the slides were stained with HE only. In figure 2, slide B was stained with Masson's trichrome (nucleus in dark blue, muscle fibers, connective tissues in red and collagen fibers in blue), slide C with Gomori reticulin (reticuline fibers in black), slide D with PAS (carbohydrates in red, nucleus in blue) and slides E-F with Alcian Blue pH 1.0 and Alcian Blue pH 2.5 (sulfated mucosubstances in dark blue and neuter mucosubstances in red), respectively. The information was included in the figure legend – page 5.

 

Review 1: Figure 1 - needs control snails for comparison. Unable to continue due to the lack of controls used in the study.

Query: We use control groups in our laboratory, but unfortunately, we do not have images of control snails in this experiment. Our objective in the results of this article was to detail the structure of the granuloma, without exploring the general impact on the rest of the host tissue and without different experimental groups.

 

Review 1: Abstract is really poorly written and confusing in places. The writing needs to be clear and to the point. For instance:
"The knowledge of the host-parasite relationship within the One Health Approach is relevant, since zoonosis are closely intertwined in this complex mechanisms of wild/human hosts, parasite adaptation and dispersion." This sentence makes no sense. I get the idea of the writing but it really needs to be rewritten along with the whole abstract.

Query: Thanks for the comments, the abstract was rewritten and improved. Page 1.

Review 1: Line 30 - adaption is not a word in English. Adaptation is a word.

Query: Ok, it was corrected. Line 25.

Review 1: Some formatting errors in the publication, please check these before resubmission.

Query: Ok, the paper was totally revised including by expert English native professional. 

Review 1: Introduction in general ok but the formatting and English language needs some improvement in areas.

Query: Ok, the English was revised by a native.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Lopes-Torres et al. attempted to understand the tissue mechanisms associated with the defense of the giant African snail Achatina fulica against Angiostrongylus cantonensis, the nematode causing eosinophilic meningitis. The topic is timely and important as the immunobiology of intermediate snail hosts is somewhat neglected.

Although the authors present good histological images and tried to apply quite an innovative methodology (SEM imaging of histological sections), they do not actually present a sufficient amount of novel data. Considering this a descriptive study, I would expect at least examination at more timepoints to evaluate the dynamics of the host reaction.

 

MAJOR ISSUES

(1) The introduction is completely missing some information on the pathology of Angiostrongylus in snails. I admit there is not much literature, but the authors neglect even the existing few papers (https://doi.org/10.1016/j.jip.2014.10.001, https://doi.org/10.1186/s13071-018-2710-2, https://doi.org/10.1186/s13071-016-1961-z, https://doi.org/10.1007/BF00380596).

(2) The authors state that "the granulomas may serve to protect the parasite against attack by the molluscan immune defense system" (l. 28-29). Do they have any mechanistic evidence for this? Have they considered the possibility that the cells present in the granuloma can harm the larvae? Or prevent nutrient uptake from the snail tissues? This conclusion seems quite strong to me, yet not evidenced properly.

(3) Also, do authors see a difference between "granuloma" used in their manuscript (and very often in the field of vertebrate pathology) and "encapsulation", which is considered one of the innate immune mechanisms in invertebrates? Please, comment on that.

(4) The discussion actually does not discuss the results but rather presents the background for the topic (which would be more suitable for the introduction part). Of note, the first reference to the author's original data (l. 199-200) appears after 30 lines of the general "literature review". And the conclusion is that the authors obtained similar results corresponding to the previously cited publications. This brings me back to the already-mentioned lack of novelty.

(5) The perilarval space is extensively discussed (l. 211-219) but I miss the most likely explanation of its origin - the fixation artifact arising from the different reactions of the host and larval tissues after immersion into the fixative. The authors discuss the possibility that the space could be filled with parasite proteases, but their own Fig. 3D lacks the reactivity with the parasite antigens in the perilarval space (contradicting the hypothesis).

 

MINOR ISSUES

The first part of the abstract (l. 15-21) is somewhat confusing. Many topics are mentioned in rather random order, lacking the text flow.

I would suggest specifying the "new approaches" (l. 25) if they should be the “novelty core” of the study.

I guess that the parasite cannot "present a multifocal inflammatory process..." (l. 25-26). I suggest rewording.

The conclusions (l. 29-34) are rather general and speculative.

I do not understand the use of "Lissachatina" as a keyword as "Achatina" is largely used throughout the manuscript.

"Rattus" should be in italics (l. 39). The same for "Achatina fulica" (l. 165).

The authors state "countries" but show the names of continents (l. 43-44).

What do you mean by "until posture" (l. 78)?

What do you mean by "per cutanus x [contra, versus?] per ors" (l. 81)?

What do you mean by "intact morphological aspect" (l. 119)?

The description "superficial topography of the granuloma" (l. 157) could be misleading as it comes from SEM analysis of the cross-section.

The area of origin of Fig. 3B should be outlined in Fig. 3A for better orientation.

I do not understand the purpose of Fig. 3D.

What do you mean by "responsible for widespread INFECTION OF this parasite" (l. 48-19)? Do you mean "prevalence"? It should be clarified/reworded.

I miss the information on whether uninfected snails were processed as a control group (l. 79-81).

 

"Brockelman et al." does not correspond to ref. [39] (page 8, 1st paragraph). 

Comments on the Quality of English Language

I suggest moderate language editing; some specific examples are shown above.

Author Response

Points to consider in the new version (marked version):

Marked in yellow: Improvements to the text and changes following suggestions from reviewers.

Marked in red: new information, new bibliography and other points presented by the reviewers.

Review 2

Review 2: Lopes-Torres et al. attempted to understand the tissue mechanisms associated with the defense of the giant African snail Achatina fulica against Angiostrongylus cantonensis, the nematode causing eosinophilic meningitis. The topic is timely and important as the immunobiology of intermediate snail hosts is somewhat neglected.

Query: Thanks, we improved the introduction and discussion with new references. The new reference numbers are marked in red on the paper.

Review 2: Although the authors present good histological images and tried to apply quite an innovative methodology (SEM imaging of histological sections), they do not actually present a sufficient amount of novel data. Considering this a descriptive study, I would expect at least examination at more timepoints to evaluate the dynamics of the host reaction.

Query: Our objective in this work was a detailed description of the tissue of the African snail Achatina fulica infected by Angiostrongylus cantonensis. We introduced new methodologies to improve the description of general morphology, but we do not have other infected tissues with different times. On the other hand, we suggest that this publication can serve as an incentive for other new works in this line of research and be used as a methodological reference. 

 

MAJOR ISSUES

1. The introduction is completely missing some information on the pathology of Angiostrongylus in snails. I admit there is not much literature, but the authors neglect even the existing few papers (https://doi.org/10.1016/j.jip.2014.10.001, https://doi.org/10.1186/s13071-018-2710-2, https://doi.org/10.1186/s13071-016-1961-z, https://doi.org/10.1007/BF00380596).

Query: These references were added in the introduction (Red marked)

(2) The authors state that "the granulomas may serve to protect the parasite against attack by the molluscan immune defense system" (l. 28-29). Do they have any mechanistic evidence for this? Have they considered the possibility that the cells present in the granuloma can harm the larvae? Or prevent nutrient uptake from the snail tissues? This conclusion seems quite strong to me, yet not evidenced properly.

Query: Our suggestion or inference of the protection of the parasite is based on the description of the perfect morphological structure of the nematode larva within the host tissue. Indicating that the nematode is still alive in the host tissue. This characteristic is very important to maintain the biological cycle of Angiostrongylus cantonensis in mammals using the Achatina fulica as an intermediate host. It is an event observed in natural and experimental infection as previously published (Tunholi-Alves et al., 2015; Coaglio et al., 2018). We include this inference and host-parasite relationship in our discussion line 237-239. 

References:

Tunholi-Alves, V.M.; Tunholi, V.M.; Amaral, L.S.; Mota, E. M.; Júnior, A.M.; Pinheiro, J.;  Garcia, J. (2015). Biochemical profile of Achatina fulica (Mollusca: Gastropoda) after infection by different parasitic loads of Angiostrongylus cantonensis (Nematoda, Metastrongylidae). J. Invert. Pathol. 2015, 124, 1-5.

Coaglio, A. L.; Ferreira, M.A.N.D.; Santos Lima, W.; Jesus Pereira, C.A. Identification of a phenoloxidase-and melanin-dependent defence mechanism in Achatina fulica infected with Angiostrongylus vasorum. Parasit. Vectors, 2018, 11, 1-8.

 

(3) Also, do authors see a difference between "granuloma" used in their manuscript (and very often in the field of vertebrate pathology) and "encapsulation", which is considered one of the innate immune mechanisms in invertebrates? Please, comment on that. 

Query: The term granuloma is used by many authors (Chuah et al. 2013; Tunholi-Alves et al., 2015; Coaglio et al., 2018; Barçante et al., 2020; Alberto-Silva et al., 2022 ) as evolutionary structure throughout both vertebrate and invertebrate species as a protective mechanism to destroy or encapsulate parasites forms. In the present paper, the A. fulica do not destroy the nematode but form granulomas to prevent the diffusion of antigens from larval stages, such as proteases that are harmful to host tissues.

References:

Chuah, C.; Jones, M.K.; Burke, M.L.; Owen, H.C.; Anthony, B.J.; McManus, D.P. Ramm, G.A.; Gobert, G.N. Spatial and temporal transcriptomics of Schistosoma japonicum-induced hepatic granuloma formation reveals novel roles for neutrophils. J. Leukoc. Biol. 2013;94, 353–65.

Tunholi-Alves, V.M.; Tunholi, V.M.; Amaral, L.S.; Mota, E. M.; Júnior, A.M.; Pinheiro, J.;  Garcia, J. (2015). Biochemical profile of Achatina fulica (Mollusca: Gastropoda) after infection by different parasitic loads of Angiostrongylus cantonensis (Nematoda, Metastrongylidae). J. Invert. Pathol. 2015, 124, 1-5.

Coaglio, A. L.; Ferreira, M.A.N.D.; Santos Lima, W.; Jesus Pereira, C.A. Identification of a phenoloxidase-and melanin-dependent defence mechanism in Achatina fulica infected with Angiostrongylus vasorum. Parasit. Vectors, 2018, 11, 1-8.

Alberto-Silva, A.C.; Garcia, J.S.; Mota, E.M.; Martins, F.G.; Pinheiro, J.; Mello-Silva, C.C.Reproductive alterations of Biomphalaria glabrata (Say, 1818) infected with Angiostrongylus cantonensis (Chen, 1935) and exposed to Euphorbia milii var. hislopii latex. Braz. J. Biol., 2022, 82, 1-13.

 

(4) The discussion actually does not discuss the results but rather presents the background for the topic (which would be more suitable for the introduction part). Of note, the first reference to the author's original data (l. 199-200) appears after 30 lines of the general "literature review". And the conclusion is that the authors obtained similar results corresponding to the previously cited publications. This brings me back to the already-mentioned lack of novelty.

Query: Thanks for review. We changed our discussion and improved our conclusion in host-parasite relationship contribution, exploring the new methodology SEM.

 

(5) The perilarval space is extensively discussed (l. 211-219) but I miss the most likely explanation of its origin - the fixation artifact arising from the different reactions of the host and larval tissues after immersion into the fixative. The authors discuss the possibility that the space could be filled with parasite proteases, but their own Fig. 3D lacks the reactivity with the parasite antigens in the perilarval space (contradicting the hypothesis).

Query: We can withdraw the comment from the discussion associated with ES products, however it was previously published by other authors. Our hypothesis is more associated with the resilience of the host tissue, promoted by the movement of the larva within the encapsulation. The assertion of ES proteins associated with this tissue-host interaction in the perilarval region is not directly linked to the perilarval space (attributed by us to larval movement), but the reaction of ES products in the general area of encapsulation, and its can be observed in Figures 3C and D in the page 6.

 

MINOR ISSUES

-The first part of the abstract (l. 15-21) is somewhat confusing. Many topics are mentioned in rather random order, lacking the text flow.

Query: Ok, it was corrected.

 

-I would suggest specifying the "new approaches" (l. 25) if they should be the “novelty core” of the study.

Query: Ok, it was rewritten, please see lines 26-28 in the Abstract.

 

-I guess that the parasite cannot "present a multifocal inflammatory process..." (l. 25-26). I suggest rewording.

Query: Ok, it was rewritten.

 

-The conclusions (l. 29-34) are rather general and speculative.

Query: Ok, it was rewritten.

 

-I do not understand the use of "Lissachatina" as a keyword as "Achatina" is largely used throughout the manuscript.

Query: Currently, A. fulica is included in the subgenus Lissachatina. Based on nepionic spirals, Bequaert (1950) placed the species of the genus Achatina from Central and West Africa into the subgenus Achatina and the species from East Africa into its new subgenus Lissachatina. Mead (1995) provided additional support for the differentiation of subgenera based on comparative anatomical studies of the reproductive tracts. Recently, some authors have treated Lissachatina as a genus; however, no published taxonomic work is currently available to support this treatment. Therefore, we prefer to use Achatina (Lissachatina) fulica Bowdich, 1822.

Reference:

Mead, A.R. Anatomical studies reveal new phylogenetic interpretations in Lissachatina (Pulmonata: Achatinidae). J. Molluscan Stud., 1995, 61, 257-273.

 

-"Rattus" should be in italics (l. 39). The same for "Achatina fulica" (l. 165).

Query: Ok, it was corrected. (line 42)

 

-The authors state "countries" but show the names of continents (l. 43-44).

Query: Ok, it was rewritten. “….than 30 countries in these continents.” (Line 45)                                                                                                                 

 

-What do you mean by "until posture" (l. 78)?

Query: It was modified to “The necropsy was performed 37 days after the infection.” (Lines 104-105)

 

-What do you mean by "per cutanus x [contra, versus?] per ors" (l. 81)?

Query: It was removed.

 

-What do you mean by "intact morphological aspect" (l. 119)?

Query: The sentence was modified to “…space without morphological changes.” (line 152)

 

-The description "superficial topography of the granuloma" (l. 157) could be misleading as it comes from SEM analysis of the cross-section.

Query: Yes, correct. We changed the description. Please see lines 181-184 and 190-193.

 

-The area of origin of Fig. 3B should be outlined in Fig. 3A for better orientation.

Query: Ok, thanks. We add the indication in the figure 3A.

 

-I do not understand the purpose of Fig. 3D.

Query: This figure is very important, it highlights the protective role of the granuloma formation, showing the nematode antigens marked only on the larva and inside the granuloma, without marking on the outside.

 

-What do you mean by "responsible for widespread INFECTION OF this parasite" (l. 48-19)? Do you mean "prevalence"? It should be clarified/reworded.

Query: We said that the spread of A. fulica caused the spread of A. cantonensis, an emerging parasite in new countries outside the Asian continent. 

 

-I miss the information on whether uninfected snails were processed as a control group (l. 79-81).

 Query: We use control groups in our laboratory, but unfortunately, we do not have images of control snails in this experiment. Our objective in the results of this article was to detail the structure of the granuloma, without exploring the general impact on the rest of the host tissue and without different experimental groups. 

 

-"Brockelman et al." does not correspond to ref. [39] (page 8, 1st paragraph). 

Query: The reference numbers were corrected (red marked).

 

Comments on the Quality of English Language

I suggest moderate language editing; some specific examples are shown above.

Query: The English was revised by a native.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the rewritten form of the Introduction and Discussion. However, I still miss the novelty of this descriptive study. Which data/findings (not methodologies) are original (not found in other studies), and how do they move the field forward? What are newly addressed biological questions? For example, the authors state: "multiscale images and the integrative results can be help to understand the host-parasite mechanisms" (l. 191-192)... what are the new mechanisms uncovered (not only hypothesized) in the study?

Comments on the Quality of English Language

It was improved, thanks.

Author Response

Dear Reviewer 2,

I extend my gratitude once again for your meticulous review and the constructive criticism you provided regarding the scientific contributions of our paper. We are confident that this work will make a valuable addition to the field of parasitology, invertebrate host-nematode studies, and other related areas.

While it is true that some of the results presented in our work have been previously discussed in other papers, it's essential to note that in many instances, these previous studies did not involve experiments with the same host-parasite combination. Moreover, our study goes beyond previous work by presenting detailed images and solid evidence of the granuloma's role in maintaining the balance between nematodes and snails. While previous research has hypothesized the granuloma's protective function in larvae recovery following experimental infection, our experience provides a more comprehensive view. We delve into the structural aspects of the granuloma, the preservation of the nematode, the integrity of the host tissue surrounding the granuloma, and the encapsulation of nematode excretory-secretory (ES) products within the granuloma structure.

We have diligently incorporated the valuable input from the reviewer, including the modification of the paper's title, with the aim of enhancing the paper's overall impact. We sincerely hope that these revisions have brought us closer to the final version deserving of publication approval.

Best regards, Eduardo Torres  

Author Response File: Author Response.pdf